技术领域technical field
本发明涉及一种杀虫蛋白的用途，特别是涉及一种Cry1A.105蛋白质通过在植物中表达来控制粟灰螟为害植物的用途。The present invention relates to the use of an insecticidal protein, in particular to the use of a Cry1A.105 protein to control the plant damage caused by the pod borer by expressing it in plants.
背景技术Background technique
栗灰螟(Chilo infuscatellus)又名甘蔗二点螟，属于鳞翅目螟蛾科，广泛分布于东南亚、南亚及中国，在中国主要为害禾本科作物，如玉米、高粱、谷子和甘蔗等。粟灰螟为蛀茎害虫，植株受害后可造成枯心株。一般情况下，春谷区和春夏谷混播区发生重，夏谷区为害轻。Chestnut gray borer (Chilo infuscatellus), also known as sugarcane two-spot borer, belongs to Lepidoptera moth family. It is widely distributed in Southeast Asia, South Asia and China. In China, it mainly damages gramineous crops, such as corn, sorghum, millet and sugarcane. Millet ash borer is a stem-boring pest, which can cause dead plants after the plant is damaged. Under normal circumstances, the spring valley area and the spring-summer valley mixed-seeding area have heavy occurrences, and the summer valley area has light damage.
玉米是中国重要的粮食作物，随着全球温室效应的加强，近两年温度不断上升，虫害发生种类及数量都有所提高。粟灰螟曾经是华北地区和华南地区的主要害虫，主要为害高粱、谷子和甘蔗。但近年来，出现了大量关于粟灰螟为害玉米的报道，且该为害呈上升趋势。目前针对粟灰螟的为害，主要依靠农业防治、化学防治和物理防治。Corn is an important food crop in China. With the strengthening of the global greenhouse effect, the temperature has continued to rise in the past two years, and the types and numbers of insect pests have increased. The millet ash borer used to be the main pest in North China and South China, mainly harming sorghum, millet and sugarcane. However, in recent years, there have been a large number of reports about the damage to corn by the pod borer, and the damage is on the rise. At present, mainly rely on agricultural control, chemical control and physical control for the damage of millet borer.
农业防治是对整个农田生态系统中的多因素的综合协调管理，调控作物、害虫、环境因素、创造一个有利于作物生长而不利于粟灰螟发生的农田生态环境。因粟灰螟一般在土表下10cm的残茎中越冬，因此可以通过及时清理收获后的玉米、高粱、谷子和甘蔗的枯叶、残茎，以达到消灭越冬虫源的目的。同时可针对粟灰螟的食性较为单一，而采用轮作的方式以降低虫源密度，例如可与大豆、棉花、蔬菜等阔叶类作物进行轮种。但不同作物的经济效益的不同，轮作很可能造成农民收入的降低，而很难得到实行。Agricultural control is a comprehensive and coordinated management of multiple factors in the entire farmland ecosystem, regulating crops, pests, and environmental factors, and creating a farmland ecological environment that is conducive to crop growth but not conducive to the occurrence of the millet borer. Because the millet borer usually overwinters in the residual stems 10cm below the soil surface, the dead leaves and residual stems of corn, sorghum, millet and sugarcane after harvest can be cleaned in time to achieve the purpose of eliminating the source of overwintering insects. At the same time, in view of the monotonous feeding habits of the millet borer, the method of crop rotation can be adopted to reduce the density of insect sources. For example, it can be rotated with broad-leaved crops such as soybeans, cotton, and vegetables. However, the economic benefits of different crops are different, and crop rotation is likely to cause a reduction in farmers' income, so it is difficult to implement.
化学防治即农药防治，是利用化学杀虫剂来杀灭害虫，是粟灰螟综合治理的重要组成部分，它具有快速、方便、简单和高经济效益的特点，特别是粟灰螟大发生的情况下，是必不可少的应急措施。粟灰螟作为蛀茎害虫，对其的防治时期把握非常重要，用药最佳时期是卵孵盛期至幼虫蛀茎之前，否则高龄幼虫蛀入茎秆之后，将很难达到防治的目的。目前化学防治方法主要是药液喷雾和施撒毒土。但化学防治也有其局限性，如使用不当往往会导致农作物发生药害、害虫产生抗药性，以及杀伤天敌、污染环境，使农田生态系统遭到破坏和农药残留对人、畜的安全构成威胁等不良后果。Chemical control, that is, pesticide control, is the use of chemical insecticides to kill pests. It is an important part of the comprehensive management of the millet moth. It has the characteristics of fast, convenient, simple and high economic benefits. In this case, it is an essential emergency measure. As a stem-boring pest, it is very important to grasp the control period. The best period of medication is from the egg hatching period to before the larvae eat the stem. Otherwise, it will be difficult to achieve the purpose of prevention and control after the older larvae enter the stem. At present, chemical control methods are mainly liquid spraying and spreading poisonous soil. However, chemical control also has its limitations. Improper use will often lead to chemical damage of crops, resistance of pests, killing natural enemies, polluting the environment, destroying the farmland ecosystem, and posing threats to the safety of humans and animals due to pesticide residues. Adverse consequences.
物理防治主要根据害虫对环境条件中各种物理因素的反应，利用各种物理因素如光、电、色、温湿度等以及机械设备进行诱杀、辐射不育等方法来防治害虫。目前应用最广泛的是频振式杀虫灯诱杀，它利用害虫成虫的趋光性，近距离用光，远距离用波，引诱害虫靠近，对粟灰螟成虫的具有一定的防治效果；但是频振式杀虫灯需要每天及时清理高压电网上的污垢，否则会影响杀虫效果；并且在雷雨天不能开灯，在操作上还有电击伤人的危险；此外安装灯的一次性投入较大。Physical control is mainly based on the response of pests to various physical factors in the environment, using various physical factors such as light, electricity, color, temperature and humidity, etc., as well as mechanical equipment to trap and kill pests, radiation sterility and other methods to control pests. At present, the most widely used is the frequency vibration insecticidal lamp trapping and killing, which uses the phototaxis of the adult pests, uses light at a short distance, and uses waves at a long distance to lure the pests to approach, and has a certain control effect on the adults of the mites; but the frequency vibration Type insecticidal lamps need to clean the dirt on the high-voltage power grid in time every day, otherwise it will affect the insecticidal effect; and the lights cannot be turned on in thunderstorm days, and there is a risk of electric shock and injury during operation; in addition, the one-time investment of installing lights is relatively large .
为了解决农业防治、化学防治和物理防治在实际应用中的局限性，科学家们经过研究发现将来自于苏云金芽孢杆菌的编码杀虫蛋白的抗虫基因转入植物中，可获得一些抗虫转基因植物以防治植物虫害。Cry1A.105杀虫蛋白是众多杀虫蛋白中的一种，Cry1A.105蛋白是一个嵌合蛋白，它分别来自于Cry1Ab蛋白、Cry1Ac蛋白和Cry1Fa蛋白。In order to solve the limitations in the practical application of agricultural control, chemical control and physical control, scientists have discovered through research that the insect-resistant gene encoding the insecticidal protein from Bacillus thuringiensis is transferred into plants, and some insect-resistant transgenic plants can be obtained to control plant pests. Cry1A.105 insecticidal protein is one of many insecticidal proteins, and Cry1A.105 protein is a chimeric protein, which comes from Cry1Ab protein, Cry1Ac protein and Cry1Fa protein respectively.
Cry1A.105蛋白被昆虫摄入进入中肠，毒蛋白原毒素被溶解在昆虫中肠的碱性pH环境下。蛋白N-和C-末端被碱性蛋白酶消化，将原毒素转变成活性片段；活性片段和昆虫中肠上皮细胞膜上表面上受体结合，插入肠膜，导致细胞膜出现穿孔病灶，破坏细胞膜内外的渗透压变化及pH平衡等，扰乱昆虫的消化过程，最终导致其死亡。The Cry1A.105 protein is ingested into the midgut by insects, and the protoxin is dissolved in the alkaline pH environment of the insect midgut. The N- and C-terminals of the protein are digested by alkaline protease, and the protoxin is converted into an active fragment; the active fragment binds to the receptor on the upper surface of the insect midgut epithelial cell membrane, and inserts into the intestinal membrane, resulting in perforated lesions in the cell membrane, destroying the inner and outer membranes. Changes in osmotic pressure and pH balance, etc., disturb the digestive process of insects and eventually lead to their death.
已证明转Cry1A.105基因的植株可以抵抗玉米螟等鳞翅目(Lepidoptera)害虫的侵害，然而，至今尚无关于通过产生表达Cry1A.105蛋白的转基因植株来控制粟灰螟对植物危害的报道。It has been proved that plants transgenic with Cry1A.105 gene can resist Lepidoptera (Lepidoptera) pests such as corn borer. However, there is no report about controlling the damage to plants by producing transgenic plants expressing Cry1A.105 protein. .
发明内容Contents of the invention
本发明的目的是提供一种杀虫蛋白的用途，首次提供了通过产生表达Cry1A.105蛋白的转基因植株来控制粟灰螟对植物危害的方法，且有效克服现有技术农业防治、化学防治和物理防治等技术缺陷。The object of the present invention is to provide an application of an insecticidal protein, which provides for the first time a method for controlling the damage to plants by producing transgenic plants expressing the Cry1A.105 protein, and effectively overcomes the problems of agricultural control, chemical control and Technical defects such as physical prevention and control.
为实现上述目的，本发明提供了一种控制粟灰螟害虫的方法，包括将粟灰螟害虫至少与Cry1A.105蛋白接触。In order to achieve the above object, the present invention provides a method for controlling the pest of C. pombe, comprising at least contacting the pest of C. pombe with Cry1A.105 protein.
进一步地，所述Cry1A.105蛋白存在于至少产生所述Cry1A.105蛋白的宿主细胞中，所述粟灰螟害虫通过摄食所述宿主细胞至少与所述Cry1A.105蛋白接触。Further, the Cry1A.105 protein is present in at least the host cell that produces the Cry1A.105 protein, and the pest of C. pombe has at least contacted with the Cry1A.105 protein by ingesting the host cell.
更进一步地，所述Cry1A.105蛋白存在于至少产生所述Cry1A.105蛋白的细菌或转基因植物中，所述粟灰螟害虫通过摄食所述细菌或所述转基因植物的组织至少与所述Cry1A.105蛋白接触，接触后所述粟灰螟害虫生长受到抑制和/或导致死亡，以实现对粟灰螟危害植物的控制。Furthermore, the Cry1A.105 protein is present in bacteria or transgenic plants that at least produce the Cry1A.105 protein, and the pest Cry1A. .105 protein contact, after the contact, the growth of the pest of C. pombe borer is inhibited and/or death is caused, so as to realize the control of the damage to plants by C. pombe borer.
所述转基因植物可以处于任意生育期。The transgenic plants can be at any growth stage.
所述转基因植物的组织为叶片、茎秆、果实、雄穗、雌穗、花药或花丝。The tissue of the transgenic plant is leaf, stem, fruit, tassel, ear, anther or silk.
所述对粟灰螟危害植物的控制不因种植地点和/或种植时间的改变而改变。The control of the plants harmed by the pod borer does not change due to the change of planting place and/or planting time.
所述植物为玉米、高粱、谷子、甘蔗、水稻、小麦、大麦或燕麦。The plant is corn, sorghum, millet, sugar cane, rice, wheat, barley or oats.
所述接触步骤之前的步骤为种植含有编码所述Cry1A.105蛋白的多核苷酸的植物。The step preceding the contacting step is growing plants containing the polynucleotide encoding the Cry1A.105 protein.
优选地，所述Cry1A.105蛋白的氨基酸序列具有SEQ ID NO:1所示的氨基酸序列。所述Cry1A.105蛋白的核苷酸序列具有SEQ ID NO:2所示的核苷酸序列。Preferably, the amino acid sequence of the Cry1A.105 protein has the amino acid sequence shown in SEQ ID NO:1. The nucleotide sequence of the Cry1A.105 protein has the nucleotide sequence shown in SEQ ID NO:2.
在上述技术方案的基础上，所述植物还可以包括至少一种不同于编码所述Cry1A.105蛋白的核苷酸的第二种核苷酸。On the basis of the above technical solution, the plant may further include at least one second nucleotide different from the nucleotide encoding the Cry1A.105 protein.
进一步地，所述第二种核苷酸编码Cry类杀虫蛋白质、Vip类杀虫蛋白质、蛋白酶抑制剂、凝集素、α-淀粉酶或过氧化物酶。Further, the second nucleotide encodes Cry-type insecticidal protein, Vip-type insecticidal protein, protease inhibitor, lectin, α-amylase or peroxidase.
优选地，所述第二种核苷酸编码Cry2Ab蛋白。Preferably, said second nucleotide encodes a Cry2Ab protein.
进一步地，所述Cry2Ab蛋白的氨基酸序列具有SEQ ID NO:3所示的氨基酸序列。Further, the amino acid sequence of the Cry2Ab protein has the amino acid sequence shown in SEQ ID NO:3.
更进一步地，所述Cry2Ab蛋白的核苷酸序列具有SEQ ID NO:4所示的核苷酸序列。Furthermore, the nucleotide sequence of the Cry2Ab protein has the nucleotide sequence shown in SEQ ID NO:4.
可选择地，所述第二种核苷酸为抑制目标昆虫害虫中重要基因的dsRNA。Optionally, the second nucleotide is a dsRNA that suppresses an important gene in the target insect pest.
为实现上述目的，本发明还提供了一种Cry1A.105蛋白质控制粟灰螟害虫的用途。To achieve the above object, the present invention also provides a use of the Cry1A.105 protein to control the pest of the pod borer.
为实现上述目的，本发明还提供了一种产生控制粟灰螟害虫的植物的方法，包括向所述植物的基因组中引入编码Cry1A.105蛋白的多核苷酸序列。In order to achieve the above object, the present invention also provides a method for producing plants for controlling the pest of the pod borer, comprising introducing a polynucleotide sequence encoding the Cry1A.105 protein into the genome of the plant.
为实现上述目的，本发明还提供了一种产生控制粟灰螟害虫的植物繁殖体的方法，包括将由所述方法获得的第一植株与第二植株杂交，和/或取下由所述方法获得的植株上具有繁殖能力的组织进行培养，从而产生含有编码Cry1A.105蛋白的多核苷酸序列的植物繁殖体。In order to achieve the above object, the present invention also provides a method for producing plant propagules for controlling the pest of pomegranate borer, comprising crossing the first plant obtained by the method with the second plant, and/or removing the plant propagule obtained by the method. The tissue with reproductive ability on the obtained plant is cultured to produce plant propagule containing the polynucleotide sequence encoding Cry1A.105 protein.
为实现上述目的，本发明还提供了一种培养控制粟灰螟害虫的植物的方法，包括：To achieve the above object, the present invention also provides a method of cultivating a plant for controlling the pest of the pod borer, comprising:
种植至少一个植物繁殖体，所述植物繁殖体的基因组中包括编码Cry1A.105蛋白的多核苷酸序列；Planting at least one plant propagule, the genome of the plant propagule includes a polynucleotide sequence encoding Cry1A.105 protein;
使所述植物繁殖体长成植株；growing said plant propagules into plants;
使所述植株在人工接种粟灰螟害虫和/或粟灰螟害虫自然发生危害的条件下生长，收获与其他不具有编码Cry1A.105蛋白的多核苷酸序列的植株相比具有减弱的植物损伤和/或具有增加的植物产量的植株。The plant is grown under the condition of artificial inoculation with the pest of Cryopteris mori and/or the damage caused by the natural occurrence of the pest of Cryopteris arborescens, and the harvest has reduced plant damage compared with other plants that do not have the polynucleotide sequence encoding the Cry1A.105 protein and/or plants having increased plant yield.
本发明中所述的“植物繁殖体”包括但不限于植物有性繁殖体和植物无性繁殖体。所述植物有性繁殖体包括但不限于植物种子；所述植物无性繁殖体是指植物体的营养器官或某种特殊组织，其可以在离体条件下产生新植株；所述营养器官或某种特殊组织包括但不限于根、茎和叶，例如：以根为无性繁殖体的植物包括草莓和甘薯等；以茎为无性繁殖体的植物包括甘蔗和马铃薯(块茎)等；以叶为无性繁殖体的植物包括芦荟和秋海棠等。The "plant propagule" mentioned in the present invention includes but not limited to sexual plant propagule and plant vegetative propagule. The sexual propagules of plants include but not limited to plant seeds; the vegetative propagules of plants refer to the vegetative organs or certain special tissues of plants, which can produce new plants under in vitro conditions; the vegetative organs or certain special tissues A special tissue includes but not limited to roots, stems and leaves, for example: plants with roots as asexual propagules include strawberry and sweet potato, etc.; plants with stems as asexual propagules include sugarcane and potatoes (tubers) etc.; Propagate plants include aloe vera and begonias.
本发明中所述的“接触”，是指昆虫和/或害虫触碰、停留和/或摄食植物、植物器官、植物组织或植物细胞，所述植物、植物器官、植物组织或植物细胞既可以是其体内表达杀虫蛋白，还可以是所述植物、植物器官、植物组织或植物细胞的表面具有杀虫蛋白和/或具有产生杀虫蛋白的微生物。"Contact" in the present invention means that insects and/or pests touch, stay and/or feed on plants, plant organs, plant tissues or plant cells, and the plants, plant organs, plant tissues or plant cells can be The insecticidal protein is expressed in vivo, or the plant, plant organ, plant tissue or plant cell has the insecticidal protein and/or has a microorganism that produces the insecticidal protein.
本发明术语“控制”和/或“防治”是指粟灰螟害虫至少与Cry1A.105蛋白接触，接触后粟灰螟害虫生长受到抑制和/或导致死亡。进一步地，粟灰螟害虫通过摄食植物组织至少与Cry1A.105蛋白接触，接触后全部或部分粟灰螟害虫生长受到抑制和/或导致死亡。抑制是指亚致死，即尚未致死但能引起生长发育、行为、生理、生化和组织等方面的某种效应，如生长发育缓慢和/或停止。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。此外，含有编码Cry1A.105蛋白的多核苷酸序列的控制粟灰螟害虫的植物和/或植物种子，在人工接种粟灰螟害虫和/或粟灰螟害虫自然发生危害的条件下，与非转基因的野生型植株相比具有减弱的植物损伤，具体表现包括但不限于改善的茎秆抗性、和/或提高的籽粒重量、和/或增产等。Cry1A.105蛋白对粟灰螟的“控制”和/或“防治”作用是可以独立存在的，不因其它可“控制”和/或“防治”粟灰螟害虫的物质的存在而减弱和/或消失。具体地，转基因植物(含有编码Cry1A.105蛋白的多核苷酸序列)的任何组织同时和/或不同步地，存在和/或产生，Cry1A.105蛋白和/或可控制粟灰螟害虫的另一种物质，则所述另一种物质的存在既不影响Cry1A.105蛋白对粟灰螟的“控制”和/或“防治”作用，也不能导致所述“控制”和/或“防治”作用完全和/或部分由所述另一种物质实现，而与Cry1A.105蛋白无关。通常情况下，在大田，粟灰螟害虫摄食植物组织的过程短暂且很难用肉眼观察到，因此，在人工接种粟灰螟害虫和/或粟灰螟害虫自然发生危害的条件下，如转基因植物(含有编码Cry1A.105蛋白的多核苷酸序列)的任何组织存在死亡的粟灰螟害虫、和/或在其上停留生长受到抑制的粟灰螟害虫、和/或与非转基因的野生型植株相比具有减弱的植物损伤，即为实现了本发明的方法和/或用途，即通过粟灰螟害虫至少与Cry1A.105蛋白接触以实现控制粟灰螟害虫的方法和/或用途。The terms "control" and/or "prevention" in the present invention refer to that the pests of C. pombe are at least in contact with the Cry1A.105 protein, and the growth of the pests of C. pombe is inhibited and/or death is caused after the contact. Further, the pest of C. pombe borer comes into contact with at least the Cry1A.105 protein by feeding on plant tissue, and all or part of the pest of C. pombe borer is inhibited in growth and/or causes death after the contact. Inhibition refers to sublethal, that is, it is not lethal but can cause certain effects in growth and development, behavior, physiology, biochemistry and organization, such as slowing and/or stopping growth and development. At the same time, the plants should be normal in morphology and can be cultivated for consumption and/or production of the product under conventional methods. In addition, the plants and/or plant seeds containing the polynucleotide sequence encoding the Cry1A.105 protein for controlling the pest of C. pombe, under the condition of artificial inoculation of the pest of C. pombe and/or the natural occurrence of the pest of C. pom. The transgenic wild-type plants have reduced plant damage, and the specific performances include but are not limited to improved stalk resistance, and/or increased grain weight, and/or increased yield and the like. The "controlling" and/or "prevention" effect of the Cry1A.105 protein on the pod borer can exist independently, and will not be weakened and/or due to the existence of other substances that can "control" and/or "prevent" the pod borer pest or disappear. Specifically, any tissue of the transgenic plant (containing the polynucleotide sequence encoding the Cry1A.105 protein) simultaneously and/or asynchronously, exists and/or produces the Cry1A.105 protein and/or another agent capable of controlling the pest A substance, the presence of the other substance neither affects the "control" and/or "control" effect of the Cry1A.105 protein on the pod borer, nor can it lead to the "control" and/or "control" The action is completely and/or partly carried out by the other substance independently of the Cry1A.105 protein. Usually, in the field, the process of ingestion of plant tissues by P. pombe pest is short and difficult to be observed by naked eyes. Therefore, under the conditions of artificial inoculation and/or naturally occurring damage of P. pombe pest, such as transgenic Any tissue of the plant (containing the polynucleotide sequence encoding the Cry1A.105 protein) has a dead pest of the pod borer, and/or a growth-inhibited pest of the pod borer, and/or a non-transgenic wild-type Compared with the plant, the plant has reduced damage, that is to realize the method and/or use of the present invention, that is, to realize the method and/or use of controlling the pest of C. pombe borer by at least contacting the Cry1A.105 protein.
在本发明中，Cry1A.105蛋白在一种转基因植物中的表达可以伴随着一个或多个Cry类杀虫蛋白质和/或Vip类杀虫蛋白质的表达。这种超过一种的杀虫毒素在同一株转基因植物中共同表达可以通过遗传工程使植物包含并表达所需的基因来实现。另外，一种植物(第1亲本)可以通过遗传工程操作表达Cry1A.105蛋白质，第二种植物(第2亲本)可以通过遗传工程操作表达Cry类杀虫蛋白质和/或Vip类杀虫蛋白质。通过第1亲本和第2亲本杂交获得表达引入第1亲本和第2亲本的所有基因的后代植物。In the present invention, the expression of Cry1A.105 protein in a transgenic plant may be accompanied by the expression of one or more Cry-like insecticidal proteins and/or Vip-like insecticidal proteins. Such co-expression of more than one insecticidal toxin in the same transgenic plant can be achieved by genetically engineering the plant to contain and express the desired gene. In addition, one plant (the first parent) can express the Cry1A.105 protein through genetic engineering, and the second plant (the second parent) can express the Cry-like insecticidal protein and/or the Vip-like insecticidal protein through genetic engineering. Progeny plants expressing all the genes introduced into the first parent and the second parent are obtained by crossing the first parent and the second parent.
RNA干扰(RNA interference，RNAi)是指在进化过程中高度保守的、由双链RNA(double-stranded RNA，dsRNA)诱发的、同源mRNA高效特异性降解的现象。因此在本发明中可以使用RNAi技术特异性剔除或关闭目标昆虫害虫中特定基因的表达。RNA interference (RNA interference, RNAi) refers to the phenomenon of efficient and specific degradation of homologous mRNA induced by double-stranded RNA (double-stranded RNA, dsRNA), which is highly conserved during evolution. Therefore, RNAi technology can be used in the present invention to specifically knock out or shut down the expression of specific genes in target insect pests.
在分类系统上，一般主要根据成虫翅的脉序、连锁方式和触角的类型等形态特征，将鳞翅目分为亚目、总科、科等，而螟蛾科是鳞翅目中种类最多的科之一，全世界已发现1万种以上，仅中国记录就有几千条。大部分螟蛾科昆虫是农作物的害虫，多数以蛀茎形式为害，如二化螟和玉米螟。尽管粟灰螟与二化螟、玉米螟等同属于鳞翅目螟蛾科，除了在分类标准上存在相似性，在其它形态结构上则存在极大差异；就好比植物中的草莓与苹果一样(同属于蔷薇目蔷薇科)，它们都有花两性，辐射对称，花瓣5片等特征，但是其果实以及植株形态却是千差万别。而粟灰螟不管是从幼虫形态还是成虫形态上来看，都具有其独特的特征。如背部纵线，在农民中就有流传着“高粱玉米谷，背线三四五”，表示同属于螟蛾科的高粱条螟、玉米螟和粟灰螟在背线数量上就存在明显的差异。而背部纵线下就是背血管，背血管是昆虫循环器官的重要组成部分，内里充满了有昆虫“血液”之称的血淋巴。因此体表形态上看似细微的背线数量的差异，体现的却是背血管的差异，是昆虫循环系统上的差异。In the classification system, Lepidoptera is generally divided into suborders, superfamilies, families, etc. based on the morphological characteristics such as the venation sequence, linkage mode, and antennae types of adult wings, and the Lepidoptera has the most species in Lepidoptera. One of the most famous families, more than 10,000 species have been found in the world, and there are thousands of records in China alone. Most of the moths are pests of crops, mostly in the form of stem borers, such as the stem borer and the corn borer. Although millet borer, Chilo borer, and Corn borer belong to the family Lepidoptera, except for the similarity in taxonomic standards, there are great differences in other morphological structures; just like strawberries and apples in plants ( They all belong to the family Rosaceae (Rosaceae), they all have the characteristics of bisexual flowers, radial symmetry, and 5 petals, but their fruits and plant shapes are quite different. The millet borer has its own unique characteristics no matter in terms of larval form or adult form. For example, the vertical line on the back, there is a popular saying among farmers that "sorghum and corn grains, the top line is three, four, five", which means that there are obvious differences in the number of top lines of the sorghum borer, corn borer and millet borer which belong to the family Boreridae. difference. Under the longitudinal line of the back is the dorsal blood vessel. The dorsal blood vessel is an important part of the insect's circulatory organs, and it is filled with hemolymph, which is called the "blood" of insects. Therefore, the seemingly slight differences in the number of dorsal lines on the body surface reflect differences in the dorsal blood vessels, which are differences in the circulatory system of insects.
同属螟蛾科的昆虫不仅在形态特征上存在较大差异，同时在取食习性上，也存在差异。例如同为螟蛾科的二化螟主要为害水稻，极少为害其它禾本科作物。而粟灰螟未见有报道对水稻造成为害，更多的是对南方的甘蔗，北方的高粱、谷子与玉米造成为害。取食习性的不同，也暗示着体内消化系统所产生的酶和受体蛋白不同。而消化道中产生的酶是Bt基因起作用的关键点，只有能够与特异性Bt基因相结合的酶或受体蛋白，才有可能使得某个Bt基因对该害虫具有抗虫效果。越来越多的研究表明，同目不同科、甚至同科不同种的昆虫对同种Bt蛋白的敏感性表现不同。例如Vip3Aa基因对螟蛾科的二化螟(Chilosuppressalis)、亚洲玉米螟(Ostrinia furnacalis)都表现出了抗虫活性，但是对于同属螟蛾科的印度谷螟(Plodia interpunctella)以及欧洲玉米螟(Ostrinianubilalis)却没有抗虫效果。上述四种害虫均属于鳞翅目螟蛾科，但同种Bt蛋白对四种螟蛾科害虫表现出不同的抗性效果。尤其是欧洲玉米螟和亚洲玉米螟在分类上甚至同属于螟蛾科Ostrinia属(同目同科同属)，但是其对同种Bt蛋白的反应却是截然不同的，更加充分说明了Bt蛋白与昆虫体内酶和受体的相互作用方式是复杂且难以预料的。Insects belonging to the same family of Mothidae not only have great differences in morphological characteristics, but also in feeding habits. For example, Chilo borer, also belonging to the family Boreridae, mainly damages rice and rarely damages other gramineous crops. However, there is no report of the millet borer causing damage to rice, and more damage is caused to sugarcane in the south, sorghum, millet and corn in the north. The difference in feeding habits also implies that the enzymes and receptor proteins produced by the digestive system in the body are different. The enzymes produced in the digestive tract are the key points for Bt genes to function. Only enzymes or receptor proteins that can combine with specific Bt genes can make a certain Bt gene have an insect-resistant effect on the pest. More and more studies have shown that insects of the same order, different families, or even different species of the same family have different sensitivities to the same Bt protein. For example, the Vip3Aa gene showed insect resistance activity against Chilosuppressalis and Ostrinia furnacalis, but it was resistant to Indian meal moth (Plodia interpunctella) and European corn borer (Ostrinia furnacalis). ) has no anti-insect effect. The above four pests all belonged to Lepidoptera Boridae, but the same Bt protein showed different resistance effects to the four Bleridae pests. In particular, European corn borer and Asian corn borer even belong to the genus Ostrinia (the same order, the same family and the same genus) in classification, but their responses to the same Bt protein are completely different, which fully illustrates the relationship between Bt protein and The interactions between enzymes and receptors in insects are complex and unpredictable.
本发明中所述的植物、植物组织或植物细胞的基因组，是指植物、植物组织或植物细胞内的任何遗传物质，且包括细胞核和质体和线粒体基因组。The genome of a plant, plant tissue or plant cell in the present invention refers to any genetic material in a plant, plant tissue or plant cell, and includes nucleus, plastid and mitochondrial genome.
本发明中所述的多核苷酸和/或核苷酸形成完整“基因”，在所需宿主细胞中编码蛋白质或多肽。本领域技术人员很容易认识到，可以将本发明的多核苷酸和/或核苷酸置于目的宿主中的调控序列控制下。The polynucleotides and/or nucleotides described in the present invention form an entire "gene" that encodes a protein or polypeptide in a desired host cell. Those skilled in the art will readily recognize that the polynucleotides and/or nucleotides of the present invention can be placed under the control of regulatory sequences in the intended host.
本领域技术人员所熟知的，DNA典型的以双链形式存在。在这种排列中，一条链与另一条链互补，反之亦然。由于DNA在植物中复制产生了DNA的其它互补链。这样，本发明包括对序列表中示例的多核苷酸及其互补链的使用。本领域常使用的“编码链”指与反义链结合的链。为了在体内表达蛋白质，典型将DNA的一条链转录为一条mRNA的互补链，它作为模板翻译出蛋白质。mRNA实际上是从DNA的“反义”链转录的。“有义”或“编码”链有一系列密码子(密码子是三个核苷酸，一次读三个可以产生特定氨基酸)，其可作为开放阅读框(ORF)阅读来形成目的蛋白质或肽。本发明还包括与示例的DNA有相当功能的RNA。As is well known to those skilled in the art, DNA typically exists in double-stranded form. In this arrangement, one strand is complementary to the other and vice versa. As DNA replicates in plants other complementary strands of DNA are produced. Thus, the present invention includes the use of the polynucleotides exemplified in the Sequence Listing and their complements. "Coding strand" as commonly used in the art refers to the strand combined with the antisense strand. To express a protein in vivo, one strand of DNA is typically transcribed into a complementary strand of mRNA, which serves as a template for translation of the protein. mRNA is actually transcribed from the "antisense" strand of DNA. The "sense" or "coding" strand has a series of codons (a codon is three nucleotides, read three at a time to yield a specific amino acid) that can be read as an open reading frame (ORF) to form a protein or peptide of interest. The invention also includes RNAs that are functionally equivalent to the exemplified DNAs.
本发明中核酸分子或其片段在严格条件下与本发明Cry1A.105基因杂交。任何常规的核酸杂交或扩增方法都可以用于鉴定本发明Cry1A.105基因的存在。核酸分子或其片段在一定情况下能够与其他核酸分子进行特异性杂交。本发明中，如果两个核酸分子能形成反平行的双链核酸结构，就可以说这两个核酸分子彼此间能够进行特异性杂交。如果两个核酸分子显示出完全的互补性，则称其中一个核酸分子是另一个核酸分子的“互补物”。本发明中，当一个核酸分子的每一个核苷酸都与另一个核酸分子的对应核苷酸互补时，则称这两个核酸分子显示出“完全互补性”。如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在至少常规的“低度严格”条件下退火且彼此结合，则称这两个核酸分子为“最低程度互补”。类似地，如果两个核酸分子能够以足够的稳定性相互杂交从而使它们在常规的“高度严格”条件下退火且彼此结合，则称这两个核酸分子具有“互补性”。从完全互补性中偏离是可以允许的，只要这种偏离不完全阻止两个分子形成双链结构。为了使一个核酸分子能够作为引物或探针，仅需保证其在序列上具有充分的互补性，以使得在所采用的特定溶剂和盐浓度下能形成稳定的双链结构。In the present invention, the nucleic acid molecules or fragments thereof hybridize with the Cry1A.105 gene of the present invention under stringent conditions. Any conventional nucleic acid hybridization or amplification method can be used to identify the presence of the Cry1A.105 gene of the present invention. Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize to each other. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if two nucleic acid molecules exhibit perfect complementarity. In the present invention, two nucleic acid molecules are said to exhibit "complete complementarity" when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to be "complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.
本发明中，基本同源的序列是一段核酸分子，该核酸分子在高度严格条件下能够和相匹配的另一段核酸分子的互补链发生特异性杂交。促进DNA杂交的适合的严格条件，例如，大约在45℃条件下用6.0×氯化钠/柠檬酸钠(SSC)处理，然后在50℃条件下用2.0×SSC洗涤，这些条件对本领域技术人员是公知的。例如，在洗涤步骤中的盐浓度可以选自低度严格条件的约2.0×SSC、50℃到高度严格条件的约0.2×SSC、50℃。此外，洗涤步骤中的温度条件可以从低度严格条件的室温约22℃，升高到高度严格条件的约65℃。温度条件和盐浓度可以都发生改变，也可以其中一个保持不变而另一个变量发生改变。优选地，本发明所述严格条件可为在6×SSC、0.5％SDS溶液中，在65℃下与SEQ ID NO:2发生特异性杂交，然后用2×SSC、0.1％SDS和1×SSC、0.1％SDS各洗膜1次。In the present invention, a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize to a complementary strand of another matched nucleic acid molecule under highly stringent conditions. Suitable stringent conditions to promote DNA hybridization, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at about 45° C., followed by washing with 2.0× SSC at 50° C., are known to those skilled in the art. is well known. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed. Preferably, the stringent conditions of the present invention can be in 6×SSC, 0.5% SDS solution, at 65° C. to specifically hybridize with SEQ ID NO: 2, and then use 2×SSC, 0.1% SDS and 1×SSC , 0.1% SDS each washed once.
因此，具有抗虫活性并在严格条件下与本发明SEQ ID NO:2杂交的序列包括在本发明中。这些序列与本发明序列至少大约40％-50％同源，大约60％、65％或70％同源，甚至至少大约75％、80％、85％、90％、91％、92％、93％、94％、95％、96％、97％、98％、99％或更大的序列同源性。Therefore, a sequence having insect-resistant activity and hybridizing to SEQ ID NO: 2 of the present invention under stringent conditions is included in the present invention. These sequences are at least about 40%-50% homologous, about 60%, 65% or 70% homologous, even at least about 75%, 80%, 85%, 90%, 91%, 92%, 93% homologous to the sequences of the present invention %, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence identity.
本发明中所述的基因和蛋白质不但包括特定的示例序列，还包括保存了所述特定示例的蛋白质的杀虫活性特征的部分和/片段(包括与全长蛋白质相比在内和/或末端缺失)、变体、突变体、取代物(有替代氨基酸的蛋白质)、嵌合体和融合蛋白。所述“变体”或“变异”是指编码同一蛋白或编码有杀虫活性的等价蛋白的核苷酸序列。所述“等价蛋白”是指与权利要求的蛋白具有相同或基本相同的抗粟灰螟害虫的生物活性的蛋白。The genes and proteins described in the present invention not only include specific exemplary sequences, but also include parts and/or fragments (including compared with the full-length protein and/or terminal parts) that preserve the insecticidal activity characteristics of the specific exemplary proteins. deletions), variants, mutants, substitutions (proteins with substituted amino acids), chimeras and fusion proteins. The "variant" or "variation" refers to the nucleotide sequence encoding the same protein or encoding an equivalent protein with insecticidal activity. The "equivalent protein" refers to a protein that has the same or substantially the same biological activity as the claimed protein against the pest of pomella arborescens.
本发明中所述的DNA分子或蛋白序列的“片段”或“截短”是指涉及的原始DNA或蛋白序列(核苷酸或氨基酸)的一部分或其人工改造形式(例如适合植物表达的序列)，前述序列的长度可存在变化，但长度足以确保(编码)蛋白质为昆虫毒素。The "fragment" or "truncation" of a DNA molecule or protein sequence in the present invention refers to a part of the original DNA or protein sequence (nucleotide or amino acid) or its artificially modified form (such as a sequence suitable for plant expression) ), the aforementioned sequences may vary in length, but are of sufficient length to ensure that the (encoded) protein is an insect toxin.
使用标准技术可以修饰基因和容易的构建基因变异体。例如，本领域熟知制造点突变的技术。又例如美国专利号5605793描述了在随机断裂后使用DNA重装配产生其它分子多样性的方法。可以使用商业化核酸内切酶制造全长基因的片段，并且可以按照标准程序使用核酸外切酶。例如，可以使用酶诸如Bal31或定点诱变从这些基因的末端系统地切除核苷酸。还可以使用多种限制性内切酶获取编码活性片段的基因。可以使用蛋白酶直接获得这些毒素的活性片段。Genes can be modified and genetic variants readily constructed using standard techniques. For example, techniques for making point mutations are well known in the art. As another example, US Patent No. 5605793 describes methods for generating additional molecular diversity using DNA reassembly following random fragmentation. Fragments of full-length genes can be produced using commercially available endonucleases, and exonucleases can be used according to standard procedures. For example, nucleotides can be systematically excised from the ends of these genes using enzymes such as Bal31 or site-directed mutagenesis. Genes encoding active fragments can also be obtained using a variety of restriction enzymes. Active fragments of these toxins can be obtained directly using proteases.
本发明可以从B.t.分离物和/或DNA文库衍生出等价蛋白和/或编码这些等价蛋白的基因。有多种方法获取本发明的杀虫蛋白。例如，可以使用本发明公开和要求保护的杀虫蛋白的抗体从蛋白质混合物鉴定和分离其它蛋白。特别地，抗体可能是由蛋白最恒定和与其它B.t.蛋白最不同的蛋白部分引起的。然后可以通过免疫沉淀、酶联免疫吸附测定(ELISA)或western印迹方法使用这些抗体专一地鉴定有特征活性的等价蛋白。可使用本领域标准程序容易的制备本发明中公开的蛋白或等价蛋白或这类蛋白的片段的抗体。然后可以从微生物中获得编码这些蛋白的基因。The present invention can derive equivalent proteins and/or genes encoding these equivalent proteins from B.t. isolates and/or DNA libraries. There are many ways to obtain the pesticidal protein of the present invention. For example, antibodies to the pesticidal proteins disclosed and claimed herein can be used to identify and isolate other proteins from a mixture of proteins. In particular, antibodies may be elicited from the part of the protein that is the most constant and most different from other B.t. proteins. These antibodies can then be used to specifically identify equivalent proteins with characteristic activities by immunoprecipitation, enzyme-linked immunosorbent assay (ELISA) or western blotting methods. Antibodies to the proteins disclosed in the present invention or equivalent proteins or fragments of such proteins can be readily prepared using standard procedures in the art. Genes encoding these proteins can then be obtained from microorganisms.
由于遗传密码子的丰余性，多种不同的DNA序列可以编码相同的氨基酸序列。产生这些编码相同或基本相同的蛋白的可替代DNA序列正在本领域技术人员的技术水平内。这些不同的DNA序列包括在本发明的范围内。所述“基本上相同的”序列是指有氨基酸取代、缺失、添加或插入但实质上不影响杀虫活性的序列，亦包括保留杀虫活性的片段。Due to the redundancy of the genetic code, many different DNA sequences can encode the same amino acid sequence. It is well within the level of skill in the art to generate such alternative DNA sequences encoding identical or substantially identical proteins. These different DNA sequences are included within the scope of the present invention. The "substantially identical" sequence refers to a sequence that has amino acid substitutions, deletions, additions or insertions but does not substantially affect the insecticidal activity, and also includes fragments that retain insecticidal activity.
本发明中氨基酸序列的取代、缺失或添加是本领域的常规技术，优选这种氨基酸变化为：小的特性改变，即不显著影响蛋白的折叠和/或活性的保守氨基酸取代；小的缺失，通常约1-30个氨基酸的缺失；小的氨基或羧基端延伸，例如氨基端延伸一个甲硫氨酸残基；小的连接肽，例如约20-25个残基长。The substitution, deletion or addition of the amino acid sequence in the present invention is a routine technique in the art, and such amino acid changes are preferably: small characteristic changes, that is, conservative amino acid substitutions that do not significantly affect the folding and/or activity of the protein; small deletions, Typically deletions of about 1-30 amino acids; small amino- or carboxy-terminal extensions, eg, amino-terminal extensions of a methionine residue; small linker peptides, eg, about 20-25 residues in length.
保守取代的实例是在下列氨基酸组内发生的取代：碱性氨基酸(如精氨酸、赖氨酸和组氨酸)、酸性氨基酸(如谷氨酸和天冬氨酸)、极性氨基酸(如谷氨酰胺、天冬酰胺)、疏水性氨基酸(如亮氨酸、异亮氨酸和缬氨酸)、芳香氨基酸(如苯丙氨酸、色氨酸和酪氨酸)，以及小分子氨基酸(如甘氨酸、丙氨酸、丝氨酸、苏氨酸和甲硫氨酸)。通常不改变特定活性的那些氨基酸取代在本领域内是众所周知的，并且已由，例如，N.Neurath和R.L.Hill在1979年纽约学术出版社(Academic Press)出版的《Protein》中进行了描述。最常见的互换有Ala/Ser，Val/Ile，Asp/Glu，Thu/Ser，Ala/Thr，Ser/Asn，Ala/Val，Ser/Gly，Tyr/Phe，Ala/Pro，Lys/Arg，Asp/Asn，Leu/Ile，Leu/Val，Ala/Glu和Asp/Gly，以及它们相反的互换。Examples of conservative substitutions are those that occur within the following groups of amino acids: basic amino acids (such as arginine, lysine, and histidine), acidic amino acids (such as glutamic acid and aspartic acid), polar amino acids ( such as glutamine, asparagine), hydrophobic amino acids (such as leucine, isoleucine, and valine), aromatic amino acids (such as phenylalanine, tryptophan, and tyrosine), and small molecules Amino acids (such as glycine, alanine, serine, threonine, and methionine). Those amino acid substitutions which generally do not alter a particular activity are well known in the art and have been described, for example, by N. Neurath and R. L. Hill, "Protein", 1979, Academic Press, New York. The most common interchanges are Ala/Ser, Val/Ile, Asp/Glu, Thu/Ser, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Tyr/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, and their opposite interchanges.
对于本领域的技术人员而言显而易见地，这种取代可以在对分子功能起重要作用的区域之外发生，而且仍产生活性多肽。对于由本发明的多肽，其活性必需的并因此选择不被取代的氨基酸残基，可以根据本领域已知的方法，如定点诱变或丙氨酸扫描诱变进行鉴定(如参见，Cunningham和Wells，1989，Science244：1081-1085)。后一技术是在分子中每一个带正电荷的残基处引入突变，检测所得突变分子的抗虫活性，从而确定对该分子活性而言重要的氨基酸残基。底物-酶相互作用位点也可以通过其三维结构的分析来测定，这种三维结构可由核磁共振分析、结晶学或光亲和标记等技术测定(参见，如de Vos等，1992，Science 255：306-312；Smith等，1992，J.Mol.Biol 224:899-904；Wlodaver等，1992，FEBS Letters 309：59-64)。It will be apparent to those skilled in the art that such substitutions can be made outside of regions important to the function of the molecule and still result in an active polypeptide. Amino acid residues essential for the activity of the polypeptides of the present invention, and therefore selected not to be substituted, can be identified according to methods known in the art, such as site-directed mutagenesis or alanine scanning mutagenesis (see, for example, Cunningham and Wells , 1989, Science 244: 1081-1085). The latter technique introduces mutations at every positively charged residue in the molecule, and detects the anti-insect activity of the resulting mutant molecules, so as to determine the amino acid residues that are important for the activity of the molecule. Substrate-enzyme interaction sites can also be determined by analysis of their three-dimensional structure by techniques such as nuclear magnetic resonance analysis, crystallography or photoaffinity labeling (see, e.g., de Vos et al., 1992, Science 255 : 306-312; Smith et al., 1992, J. Mol. Biol 224:899-904; Wlodaver et al., 1992, FEBS Letters 309:59-64).
在本发明中，Cry1A.105蛋白包括但不限于序列1，与序列1所示的氨基酸序列具有一定同源性的氨基酸序列也包括在本发明中。这些序列与本发明序列类似性/相同性典型的大于60％，优选的大于75％，更优选的大于80％，甚至更优选的大于90％，并且可以大于95％。也可以根据更特定的相同性和/或类似性范围定义本发明的优选的多核苷酸和蛋白质。例如与本发明示例的序列有49％、50％、51％、52％、53％、54％、55％、56％、57％、58％、59％、60％、61％、62％、63％、64％、65％、66％、67％、68％、69％、70％、71％、72％、73％、74％、75％、76％、77％、78％、79％、80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、97％、98％或99％的相同性和/或类似性。In the present invention, the Cry1A.105 protein includes but is not limited to Sequence 1, and amino acid sequences having certain homology with the amino acid sequence shown in Sequence 1 are also included in the present invention. The similarity/identity of these sequences to the sequences of the invention is typically greater than 60%, preferably greater than 75%, more preferably greater than 80%, even more preferably greater than 90%, and may be greater than 95%. Preferred polynucleotides and proteins of the invention can also be defined in terms of more specific identity and/or similarity ranges. For example, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79% , 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96 %, 97%, 98% or 99% identity and/or similarity.
在本发明中，产生所述Cry1A.105蛋白的转基因植物包括但不限于MON89034转基因玉米事件和/或包含MON89034转基因玉米事件的植物材料(如在CN101495635A所描述的)或者MON87751转基因大豆事件和/或包含MON87751转基因大豆事件的植物材料(如在USDA APHIS非管制状态申请13-337-01p所描述的)，其均可以实现本发明的方法和/或用途，即通过粟灰螟害虫至少与Cry1A.105蛋白接触以实现控制粟灰螟害虫的方法和/或用途。本领域技术人员所理解的，使上述转基因事件中的Cry1A.105蛋白在不同植物中表达亦能实现本发明的方法和/或用途。更具体地，所述Cry1A.105蛋白存在于至少产生所述Cry1A.105蛋白的转基因植物中，所述粟灰螟害虫通过摄食所述转基因植物的组织至少与所述Cry1A.105蛋白接触，接触后所述粟灰螟害虫生长受到抑制和/或导致死亡，以实现对粟灰螟危害植物的控制。In the present invention, the transgenic plants producing the Cry1A.105 protein include but are not limited to MON89034 transgenic maize event and/or plant material comprising MON89034 transgenic maize event (as described in CN101495635A) or MON87751 transgenic soybean event and/or Plant material comprising the MON87751 transgenic soybean event (as described in the USDA APHIS non-regulatory status application 13-337-01p), which can realize the method and/or use of the present invention, that is, through the Cry1A. 105 Proteins are contacted to realize the method and/or use of controlling the pest of pomella pod borer. Those skilled in the art understand that the method and/or use of the present invention can also be realized by expressing the Cry1A.105 protein in the above transgenic event in different plants. More specifically, the Cry1A.105 protein is present in a transgenic plant that at least produces the Cry1A.105 protein, and the pest Cry1A.105 contacts at least the Cry1A.105 protein by ingesting the tissue of the transgenic plant, contacts The latter pests are inhibited in growth and/or lead to death, so as to realize the control of damage to plants by the pod borer.
本发明中所述调控序列包括但不限于启动子、转运肽、终止子，增强子，前导序列，内含子以及其它可操作地连接到所述Cry1A.105蛋白的调节序列。The regulatory sequences in the present invention include but not limited to promoters, transit peptides, terminators, enhancers, leader sequences, introns and other regulatory sequences operably linked to the Cry1A.105 protein.
所述启动子为植物中可表达的启动子，所述的“植物中可表达的启动子”是指确保与其连接的编码序列在植物细胞内进行表达的启动子。植物中可表达的启动子可为组成型启动子。指导植物内组成型表达的启动子的示例包括但不限于，来源于花椰菜花叶病毒的35S启动子、玉米Ubi启动子、水稻GOS2基因的启动子等。备选地，植物中可表达的启动子可为组织特异的启动子，即该启动子在植物的一些组织内如在绿色组织中指导编码序列的表达水平高于植物的其他组织(可通过常规RNA试验进行测定)，如PEP羧化酶启动子。备选地，植物中可表达的启动子可为创伤诱导启动子。创伤诱导启动子或指导创伤诱导的表达模式的启动子是指当植物经受机械或由昆虫啃食引起的创伤时，启动子调控下的编码序列的表达较正常生长条件下有显著提高。创伤诱导启动子的示例包括但不限于，马铃薯和西红柿的蛋白酶抑制基因(pinⅠ和pinⅡ)和玉米蛋白酶抑制基因(MPI)的启动子。The promoter is a promoter that can be expressed in plants, and the "promoter that can be expressed in plants" refers to a promoter that ensures the expression of the coding sequence linked to it in plant cells. A promoter expressible in plants may be a constitutive promoter. Examples of promoters directing constitutive expression in plants include, but are not limited to, the 35S promoter derived from cauliflower mosaic virus, the corn Ubi promoter, the promoter of the rice GOS2 gene, and the like. Alternatively, the promoter expressible in plants may be a tissue-specific promoter, i.e., the promoter directs expression of the coding sequence at higher levels in some tissues of the plant, such as in green tissues, than in other tissues of the plant (which can be determined by routine RNA assays), such as the PEP carboxylase promoter. Alternatively, the promoter expressible in plants may be a wound-inducible promoter. A wound-inducible promoter or a promoter directing a wound-induced expression pattern means that when a plant is subjected to mechanical or insect-induced wounds, the expression of the coding sequence under the regulation of the promoter is significantly increased compared with normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, the promoters of the potato and tomato protease inhibitors (pinI and pinII) and the maize proteinase inhibitor (MPI).
所述转运肽(又称分泌信号序列或导向序列)是指导转基因产物到特定的细胞器或细胞区室，对受体蛋白质来说，所述转运肽可以是异源的，例如，利用编码叶绿体转运肽序列靶向叶绿体，或者利用‘KDEL’保留序列靶向内质网，或者利用大麦植物凝集素基因的CTPP靶向液泡。The transit peptide (also known as secretion signal sequence or targeting sequence) is to direct the transgene product to a specific organelle or cell compartment. For the receptor protein, the transit peptide can be heterologous, for example, using the encoding chloroplast transport The peptide sequences target the chloroplast, or the endoplasmic reticulum using the 'KDEL' retention sequence, or the vacuole using the CTPP of the barley lectin gene.
所述前导序列包含但不限于，小RNA病毒前导序列，如EMCV前导序列(脑心肌炎病毒5’非编码区)；马铃薯Y病毒组前导序列，如MDMV(玉米矮缩花叶病毒)前导序列；人类免疫球蛋白质重链结合蛋白质(BiP)；苜蓿花叶病毒的外壳蛋白质mRNA的不翻译前导序列(AMV RNA4)；烟草花叶病毒(TMV)前导序列。The leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a poty virus group leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin heavy chain binding protein (BiP); untranslated leader of coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader.
所述增强子包含但不限于，花椰菜花叶病毒(CaMV)增强子、玄参花叶病毒(FMV)增强子、康乃馨风化环病毒(CERV)增强子、木薯脉花叶病毒(CsVMV)增强子、紫茉莉花叶病毒(MMV)增强子、夜香树黄化曲叶病毒(CmYLCV)增强子、木尔坦棉花曲叶病毒(CLCuMV)、鸭跖草黄斑驳病毒(CoYMV)和花生褪绿线条花叶病毒(PCLSV)增强子。The enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancers, Scrophulariaceae mosaic virus (FMV) enhancers, carnation weathering ring virus (CERV) enhancers, cassava vein mosaic virus (CsVMV) enhancers , Mirabilis Mosaic Virus (MMV) Enhancer, Night Scent Yellow Leaf Curl Virus (CmYLCV) Enhancer, Multan Cotton Leaf Curl Virus (CLCuMV), Commelina Yellow Mottle Virus (CoYMV) and Peanut Chlorotic Streak Flower Leaf virus (PCLSV) enhancer.
对于单子叶植物应用而言，所述内含子包含但不限于，玉米hsp70内含子、玉米泛素内含子、Adh内含子1、蔗糖合酶内含子或水稻Act1内含子。对于双子叶植物应用而言，所述内含子包含但不限于，CAT-1内含子、pKANNIBAL内含子、PIV2内含子和“超级泛素”内含子。For monocot applications, the introns include, but are not limited to, the maize hsp70 intron, the maize ubiquitin intron, the Adh intron 1, the sucrose synthase intron, or the rice Act1 intron. For dicot applications, such introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.
所述终止子可以为在植物中起作用的适合多聚腺苷酸化信号序列，包括但不限于，来源于农杆菌(Agrobacterium tumefaciens)胭脂碱合成酶(NOS)基因的多聚腺苷酸化信号序列、来源于蛋白酶抑制剂Ⅱ(pinⅡ)基因的多聚腺苷酸化信号序列、来源于豌豆ssRUBISCO E9基因的多聚腺苷酸化信号序列和来源于α-微管蛋白(α-tubulin)基因的多聚腺苷酸化信号序列。The terminator may be a suitable polyadenylation signal sequence that functions in plants, including, but not limited to, the polyadenylation signal sequence derived from the nopaline synthase (NOS) gene of Agrobacterium tumefaciens , the polyadenylation signal sequence from the protease inhibitor Ⅱ (pinⅡ) gene, the polyadenylation signal sequence from the pea ssRUBISCO E9 gene and the polyadenylation signal sequence from the α-tubulin gene Polyadenylation signal sequence.
本发明中所述“有效连接”表示核酸序列的联结，所述联结使得一条序列可提供对相连序列来说需要的功能。在本发明中所述“有效连接”可以为将启动子与感兴趣的序列相连，使得该感兴趣的序列的转录受到该启动子控制和调控。当感兴趣的序列编码蛋白并且想要获得该蛋白的表达时“有效连接”表示：启动子与所述序列相连，相连的方式使得得到的转录物高效翻译。如果启动子与编码序列的连接是转录物融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得得到的转录物中第一翻译起始密码子是编码序列的起始密码子。备选地，如果启动子与编码序列的连接是翻译融合并且想要实现编码的蛋白的表达时，制造这样的连接，使得5’非翻译序列中含有的第一翻译起始密码子与启动子相连结，并且连接方式使得得到的翻译产物与编码想要的蛋白的翻译开放读码框的关系是符合读码框的。可以“有效连接”的核酸序列包括但不限于：提供基因表达功能的序列(即基因表达元件，例如启动子、5’非翻译区域、内含子、蛋白编码区域、3’非翻译区域、聚腺苷化位点和/或转录终止子)、提供DNA转移和/或整合功能的序列(即T-DNA边界序列、位点特异性重组酶识别位点、整合酶识别位点)、提供选择性功能的序列(即抗生素抗性标记物、生物合成基因)、提供可计分标记物功能的序列、体外或体内协助序列操作的序列(即多接头序列、位点特异性重组序列)和提供复制功能的序列(即细菌的复制起点、自主复制序列、着丝粒序列)。The "operably linked" in the present invention refers to the linkage of nucleic acid sequences, which allows one sequence to provide the required function for the linked sequence. The "operably linked" in the present invention can be linking a promoter with a sequence of interest, so that the transcription of the sequence of interest is controlled and regulated by the promoter. "Operably linked" when a sequence of interest encodes a protein and expression of that protein is desired means that a promoter is linked to said sequence in such a way that the resulting transcript is efficiently translated. If the junction of the promoter and coding sequence is a transcript fusion and expression of the encoded protein is desired, the junction is made such that the first translation initiation codon in the resulting transcript is that of the coding sequence. Alternatively, if the junction of the promoter and coding sequence is a translational fusion and expression of the encoded protein is to be achieved, the junction is made such that the first translation initiation codon contained in the 5' untranslated sequence is fused with the promoter linked in such a way that the resulting translation product is in-frame with the translational open reading frame encoding the desired protein. Nucleic acid sequences that may be "operably linked" include, but are not limited to: sequences that provide gene expression function (i.e., gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, polynucleotides adenylation sites and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (i.e. T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide selection Sequences for sexual function (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scoreable marker function, sequences that facilitate sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences) and sequences that provide Sequences of replication function (ie bacterial origin of replication, autonomously replicating sequences, centromere sequences).
本发明中所述的“杀虫”或“抗虫”是指对农作物害虫是有毒的，从而实现“控制”和/或“防治”农作物害虫。优选地，所述“杀虫”或“抗虫”是指杀死农作物害虫。更具体地，目标昆虫是粟灰螟害虫。"Insecticidal" or "insect-resistant" in the present invention refers to being toxic to crop pests, so as to achieve "control" and/or "control" crop pests. Preferably, the "insecticide" or "insect resistance" refers to killing crop pests. More specifically, the target insect is the pest of pod borer.
本发明中Cry1A.105蛋白对粟灰螟害虫具有毒性。本发明中的植物，特别是谷子、甘蔗、高粱和玉米，在其基因组中含有外源DNA，所述外源DNA包含编码Cry1A.105蛋白的核苷酸序列，粟灰螟害虫通过摄食植物组织与该蛋白接触，接触后粟灰螟害虫生长受到抑制和/或导致死亡。抑制是指致死或亚致死。同时，植物在形态上应是正常的，且可在常规方法下培养以用于产物的消耗和/或生成。此外，该植物可基本消除对化学或生物杀虫剂的需要(所述化学或生物杀虫剂为针对Cry1A.105蛋白所靶向的粟灰螟害虫的杀虫剂)。植物材料中杀虫晶体蛋白(ICP)的表达水平可通过本领域内所描述的多种方法进行检测，例如通过应用特异引物对组织内产生的编码杀虫蛋白质的mRNA进行定量，或直接特异性检测产生的杀虫蛋白质的量。In the present invention, the Cry1A.105 protein is toxic to the pest of the pod borer. Plants in the present invention, especially millet, sugarcane, sorghum and corn, contain exogenous DNA in their genome, and described exogenous DNA comprises the nucleotide sequence of coding Cry1A. Exposure to the protein inhibits the growth and/or causes death of the pod borer pest. Inhibition means lethal or sublethal. At the same time, the plants should be normal in morphology and can be cultivated for consumption and/or production of the product under conventional methods. In addition, the plants can substantially eliminate the need for chemical or biological insecticides (that is, insecticides directed against the Cry1A.105 protein-targeted pest of the pod borer). Expression levels of insecticidal crystal proteins (ICPs) in plant material can be detected by a variety of methods described in the art, such as by quantification of mRNA encoding insecticidal proteins produced in tissues using specific primers, or by direct specificity. The amount of pesticidal protein produced was measured.
可以应用不同的试验测定植物中ICP的杀虫效果。本发明中目标昆虫主要为粟灰螟。Different assays can be used to determine the insecticidal efficacy of ICPs in plants. The target insects in the present invention are mainly the pod borer.
本发明中，所述Cry1A.105蛋白可以具有序列表中SEQ ID NO:1所示的氨基酸序列。除了包含Cry1A.105蛋白的编码区外，也可包含其他元件，例如编码选择性标记的蛋白质。In the present invention, the Cry1A.105 protein may have the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing. In addition to the coding region comprising the Cry1A.105 protein, other elements may also be included, such as proteins encoding selectable markers.
此外，包含编码本发明Cry1A.105蛋白的核苷酸序列的表达盒在植物中还可以与至少一种编码除草剂抗性基因的蛋白质一起表达，所述除草剂抗性基因包括但不限于，草胺膦抗性基因(如bar基因、pat基因)、苯敌草抗性基因(如pmph基因)、草甘膦抗性基因(如EPSPS基因)、溴苯腈(bromoxynil)抗性基因、磺酰脲抗性基因、对除草剂茅草枯的抗性基因、对氨腈的抗性基因或谷氨酰胺合成酶抑制剂(如PPT)的抗性基因，从而获得既具有高杀虫活性、又具有除草剂抗性的转基因植物。In addition, the expression cassette comprising the nucleotide sequence encoding the Cry1A.105 protein of the present invention can also be expressed in plants together with at least one protein encoding a herbicide resistance gene, and the herbicide resistance gene includes but not limited to, Glufosinate-resistant genes (such as bar gene, pat gene), dichloride-resistant genes (such as pmph gene), glyphosate-resistant genes (such as EPSPS gene), bromoxynil (bromoxynil)-resistant genes, sulfonate Urea resistance gene, resistance gene to herbicide palapat, resistance gene to cyanamide or resistance gene of glutamine synthetase inhibitor (such as PPT), so as to obtain both high insecticidal activity and Genetically modified plants for herbicide resistance.
本发明中，将外源DNA导入植物，如将编码所述Cry1A.105蛋白的基因或表达盒或重组载体导入植物细胞，常规的转化方法包括但不限于，农杆菌介导的转化、微量发射轰击、直接将DNA摄入原生质体、电穿孔或晶须硅介导的DNA导入。In the present invention, exogenous DNA is introduced into plants, such as introducing the gene or expression cassette or recombinant vector encoding the Cry1A.105 protein into plant cells. Conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, micro-ejection Bombardment, direct DNA uptake into protoplasts, electroporation, or whisker silicon-mediated DNA introduction.
本发明提供了一种杀虫蛋白的用途，具有以下优点：The invention provides a kind of application of insecticidal protein, which has the following advantages:
1、内因防治。现有技术主要是通过外部作用即外因来控制粟灰螟害虫的危害，如农业防治、化学防治和物理防治；而本发明是通过植物体内产生能够杀死粟灰螟的Cry1A.105蛋白来控制粟灰螟害虫的，即通过内因来防治。1. Prevention and treatment of internal causes. The prior art is mainly to control the damage of the pest of the pod borer through external effects, that is, external causes, such as agricultural control, chemical control and physical control; while the present invention controls the pest by producing Cry1A.105 protein that can kill the pod borer in the plant The pest of millet ash borer is controlled through internal causes.
2、无污染、无残留。现有技术使用的化学防治方法虽然对控制粟灰螟害虫的危害起到了一定作用，但同时也对人、畜和农田生态系统带来了污染、破坏和残留；使用本发明控制粟灰螟害虫的方法，可以消除上述不良后果。2. No pollution and no residue. Although the chemical prevention and control method used in the prior art has played a certain role in controlling the harm of the pod borer pest, it has also brought pollution, damage and residue to people, livestock and farmland ecosystems; using the present invention to control the pod borer pest The method can eliminate the above-mentioned adverse consequences.
3、全生育期防治。现有技术使用的控制粟灰螟害虫的方法都是阶段性的，而本发明是对植物进行全生育期的保护，转基因植物(Cry1A.105蛋白)从发芽、生长，一直到开花、结果，都可以避免遭受粟灰螟的侵害。3. Prevention and treatment during the whole growth period. The methods used in the prior art to control the pests of the pod borer are staged, but the present invention protects the plants throughout their growth period, and the transgenic plants (Cry1A.105 protein) germinate, grow, bloom and bear fruit. can avoid the attack of millet moth.
4、全植株防治。现有技术使用的控制粟灰螟害虫的方法大多是局部性的，如叶面喷施；而本发明是对整个植株进行保护，如转基因植物(Cry1A.105蛋白)的叶片、茎秆、果实、雄穗、雌穗、花药或花丝等都是可以抵抗粟灰螟侵害的。4. Whole plant control. Most of the methods used in the prior art to control the pests of the pod borer are local, such as foliage spraying; and the present invention protects the whole plant, such as the leaves, stems, and fruits of transgenic plants (Cry1A.105 protein). .
5、效果稳定。现有技术使用的频振式杀虫灯不仅需要每天及时清理高压电网的污垢，而且在雷雨天不能使用；本发明是使所述Cry1A.105蛋白在植物体内进行表达，有效地克服了频振式杀虫灯的效果受外界因素影响的缺陷，且本发明转基因植物(Cry1A.105蛋白)的防治效果在不同地点、不同时间、不同遗传背景也都是稳定一致的。5. The effect is stable. The frequency vibration type insecticidal lamp used in the prior art not only needs to clean up the dirt of the high-voltage power grid in time every day, but also cannot be used in thunderstorm days; the present invention expresses the Cry1A.105 protein in plants, effectively overcoming the frequency vibration The effect of the type insecticidal lamp is affected by external factors, and the control effect of the transgenic plant (Cry1A.105 protein) of the present invention is also stable and consistent at different locations, different times, and different genetic backgrounds.
6、简单、方便、经济。现有技术使用的频振式杀虫灯的一次性投入较大，且操作不当还有电击伤人的危险；本发明只需种植能够表达Cry1A.105蛋白的转基因植物即可，而不需要采用其它措施，从而节省了大量人力、物力和财力。6. Simple, convenient and economical. The one-time investment of the frequency-vibrating insecticidal lamp used in the prior art is relatively large, and there is a risk of electric injury if the operation is improper; the present invention only needs to plant transgenic plants that can express the Cry1A. Other measures, thus saving a lot of manpower, material and financial resources.
7、效果彻底。现有技术使用的控制粟灰螟害虫的方法，其效果是不彻底的，只起到减轻作用；而本发明转基因植物(Cry1A.105蛋白)可以造成初孵粟灰螟幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，都是明显的发育不良，且已停止发育，在田间自然环境中无法存活，而转基因植物大体上只受到轻微损伤。7. The effect is thorough. The method used in the prior art to control the pest of C. pombe borer is incomplete and only plays a role in mitigating; while the transgenic plant (Cry1A.105 protein) of the present invention can cause a large number of deaths of newly hatched C. cinerea larvae, and The developmental progress of a small number of surviving larvae was greatly inhibited. After 3 days, the larvae were basically still in the newly hatched state, and they were all obviously stunted and had stopped developing. Minor damage.
下面通过附图和实施例，对本发明的技术方案做进一步的详细描述。The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.
附图说明Description of drawings
图1为本发明杀虫蛋白的用途的含有Cry1A.105核苷酸序列的重组克隆载体DBN01-T构建流程图；Fig. 1 is the construction flowchart of the recombinant cloning vector DBN01-T containing the Cry1A.105 nucleotide sequence for the purpose of the insecticidal protein of the present invention;
图2为本发明杀虫蛋白的用途的含有Cry1A.105核苷酸序列的重组表达载体DBN100745构建流程图；Fig. 2 is a construction flow chart of the recombinant expression vector DBN100745 containing the Cry1A.105 nucleotide sequence for the use of the insecticidal protein of the present invention;
图3为本发明杀虫蛋白的用途的转基因玉米植株接种粟灰螟的叶片损伤图；Fig. 3 is the leaf damage diagram of the transgenic corn plant inoculated with the insecticidal protein of the present invention;
图4为本发明杀虫蛋白的用途的转基因甘蔗植株接种粟灰螟的叶片损伤图；Fig. 4 is the leaf damage diagram of the transgenic sugarcane plant inoculated with the insecticidal protein of the present invention;
图5为本发明杀虫蛋白的用途的转基因高粱植株接种粟灰螟的叶片损伤图；Fig. 5 is the leaf damage diagram of the transgenic sorghum plant inoculated with the insecticidal protein of the present invention;
图6为本发明杀虫蛋白的用途的转基因谷子植株接种粟灰螟的叶片损伤图。Fig. 6 is a diagram of leaf injury of transgenic millet plants inoculated with the insecticidal protein of the present invention inoculated with P. argentina.
具体实施方式detailed description
下面通过具体实施例进一步说明本发明杀虫蛋白的用途的技术方案。The technical scheme of the use of the insecticidal protein of the present invention is further illustrated below through specific examples.
第一实施例、基因的获得和合成The first embodiment, the acquisition and synthesis of genes
1、获得核苷酸序列1. Obtain the nucleotide sequence
Cry1A.105杀虫蛋白质的氨基酸序列(1177个氨基酸)，如序列表中SEQ IDNO:1所示；编码相应于所述Cry1A.105杀虫蛋白质的氨基酸序列(1177个氨基酸)的Cry1A.105核苷酸序列(3534个核苷酸)，如序列表中SEQ ID NO:2所示。The amino acid sequence (1177 amino acids) of the Cry1A.105 insecticidal protein, as shown in SEQ ID NO: 1 in the sequence listing; the Cry1A.105 nucleus corresponding to the amino acid sequence (1177 amino acids) of the Cry1A.105 insecticidal protein encoded Nucleotide sequence (3534 nucleotides), as shown in SEQ ID NO:2 in the sequence listing.
编码Cry2Ab杀虫蛋白质的氨基酸序列(634个氨基酸)，如序列表中SEQID No:3所示；编码相应于所述Cry2Ab杀虫蛋白质的氨基酸序列(634个氨基酸)的Cry2Ab核苷酸序列(1905个核苷酸)，如序列表中SEQ ID NO:4所示。The amino acid sequence (634 amino acids) of encoding Cry2Ab insecticidal protein, as shown in SEQID No:3 in the sequence table; The Cry2Ab nucleotide sequence (1905) of encoding corresponding to the amino acid sequence (634 amino acids) of described Cry2Ab insecticidal protein nucleotides), as shown in SEQ ID NO:4 in the sequence listing.
2、合成上述核苷酸序列2. Synthesize the above nucleotide sequence
所述Cry1A.105核苷酸序列(如序列表中SEQ ID NO:2所示)和所述Cry2Ab核苷酸序列(如序列表中SEQ ID NO:4所示)由南京金斯瑞生物科技有限公司合成；合成的所述Cry1A.105核苷酸序列(SEQ ID NO:2)的5’端还连接有NcoI酶切位点，所述Cry1A.105核苷酸序列(SEQ ID NO:2)的3’端还连接有HindIII酶切位点；合成的所述Cry2Ab核苷酸序列(SEQ ID NO:4)的5’端还连接有NcoI酶切位点，所述Cry2Ab核苷酸序列(SEQ ID NO:4)的3’端还连接有SpeI酶切位点。The Cry1A.105 nucleotide sequence (as shown in SEQ ID NO: 2 in the sequence listing) and the Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 4 in the sequence listing) were provided by Nanjing GenScript Co., Ltd. synthesis; the 5' end of the synthetic Cry1A.105 nucleotide sequence (SEQ ID NO: 2) is also connected with an NcoI restriction site, and the Cry1A.105 nucleotide sequence (SEQ ID NO: 2 ) is also connected with a HindIII restriction site at the 3' end; the 5' end of the synthesized Cry2Ab nucleotide sequence (SEQ ID NO: 4) is also connected with a NcoI restriction site, and the Cry2Ab nucleotide sequence (SEQ ID NO: 4) is also connected with a SpeI restriction site at the 3' end.
第二实施例、重组表达载体的构建及重组表达载体转化农杆菌The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium
1、构建含有Cry1A.105基因的重组克隆载体1. Construction of a recombinant cloning vector containing the Cry1A.105 gene
将合成的Cry1A.105核苷酸序列连入克隆载体pGEM-T(Promega，Madison，USA，CAT：A3600)上，操作步骤按Promega公司产品pGEM-T载体说明书进行，得到重组克隆载体DBN01-T，其构建流程如图1所示(其中，Amp表示氨苄青霉素抗性基因；f1表示噬菌体f1的复制起点；LacZ为LacZ起始密码子；SP6为SP6RNA聚合酶启动子；T7为T7RNA聚合酶启动子；Cry1A.105为Cry1A.105核苷酸序列(SEQ ID NO:2)；MCS为多克隆位点)。The synthesized Cry1A.105 nucleotide sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T , its construction process is as shown in Figure 1 (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ initiation codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase initiation sub; Cry1A.105 is the Cry1A.105 nucleotide sequence (SEQ ID NO: 2); MCS is the multiple cloning site).
然后将重组克隆载体DBN01-T用热激方法转化大肠杆菌T1感受态细胞(Transgen，Beijing，China，CAT：CD501)，其热激条件为：50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组克隆载体DBN01-T)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)，在表面涂有IPTG(异丙基硫代-β-D-半乳糖苷)和X-gal(5-溴-4-氯-3-吲哚-β-D-半乳糖苷)的氨苄青霉素(100毫克/升)的LB平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上生长过夜。挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，氨苄青霉素100mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒：将菌液在12000rpm转速下离心1min，去上清液，沉淀菌体用100μl冰预冷的溶液I(25mM Tris-HCl，10mM EDTA(乙二胺四乙酸)，50mM葡萄糖，pH8.0)悬浮；加入200μl新配制的溶液II(0.2M NaOH，1％SDS(十二烷基硫酸钠))，将管子颠倒4次，混合，置冰上3-5min；加入150μl冰冷的溶液III(3M醋酸钾，5M醋酸)，立即充分混匀，冰上放置5-10min；于温度4℃、转速12000rpm条件下离心5min，在上清液中加入2倍体积无水乙醇，混匀后室温放置5min；于温度4℃、转速12000rpm条件下离心5min，弃上清液，沉淀用浓度(V/V)为70％的乙醇洗涤后晾干；加入30μl含RNase(20μg/ml)的TE(10mM Tris-HCl，1mM EDTA，PH8.0)溶解沉淀；于温度37℃下水浴30min，消化RNA；于温度-20℃保存备用。Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 competent cells (Transgen, Beijing, China, CAT: CD501) by heat shock method, and the heat shock conditions were: 50 μl Escherichia coli T1 competent cells, 10 μl plasmid DNA (recombinant Cloning carrier DBN01-T), 42 ° C water bath for 30 seconds; 37 ° C shaking culture for 1 hour (shaking at 100 rpm speed), coated with IPTG (isopropylthio-β-D-galactoside) and X -gal (5-bromo-4-chloro-3-indole-β-D-galactoside) ampicillin (100 mg/L) LB plates (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10g/L, agar 15g/L, adjust the pH to 7.5 with NaOH) and grow overnight. Pick white colonies and culture them in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, adjust the pH to 7.5 with NaOH) at a temperature of 37°C overnight. Extract the plasmid by alkaline method: centrifuge the bacterial solution at 12000rpm for 1min, remove the supernatant, and use 100 μl of ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylenediaminetetraacetic acid), 50mM glucose , pH8.0) suspension; add 200 μl freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix, put on ice for 3-5min; add 150 μl ice-cold Solution III (3M potassium acetate, 5M acetic acid), mixed thoroughly immediately, placed on ice for 5-10min; centrifuged at 4°C and 12000rpm for 5min, added 2 times the volume of absolute ethanol to the supernatant, and mixed After uniformity, place at room temperature for 5 minutes; centrifuge at 4°C and 12,000 rpm for 5 minutes, discard the supernatant, wash the precipitate with ethanol with a concentration (V/V) of 70%, and dry it; add 30 μl of RNase (20 μg/ml) TE (10mM Tris-HCl, 1mM EDTA, pH 8.0) to dissolve the precipitate; in a water bath at 37°C for 30min to digest the RNA; store at -20°C for later use.
提取的质粒经AhdI和XhoI酶切鉴定后，对阳性克隆进行测序验证，结果表明重组克隆载体DBN01-T中插入的所述Cry1A.105核苷酸序列为序列表中SEQ ID NO:2所示的核苷酸序列，即Cry1A.105核苷酸序列正确插入。After the extracted plasmid was identified by digestion with AhdI and XhoI, the positive clones were sequenced and verified, and the results showed that the Cry1A.105 nucleotide sequence inserted in the recombinant cloning vector DBN01-T was shown in SEQ ID NO: 2 in the sequence table The nucleotide sequence, that is, the Cry1A.105 nucleotide sequence is correctly inserted.
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Cry2Ab核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN02-T，其中，Cry2Ab为Cry2Ab核苷酸序列(SEQ ID NO:4)。酶切和测序验证重组克隆载体DBN02-T中所述Cry2Ab核苷酸序列正确插入。According to the above-mentioned method for constructing the recombinant cloning vector DBN01-T, the synthesized Cry2Ab nucleotide sequence was connected into the cloning vector pGEM-T to obtain the recombinant cloning vector DBN02-T, wherein Cry2Ab was the Cry2Ab nucleotide sequence (SEQ ID NO: 4). Enzyme digestion and sequencing verified the correct insertion of the Cry2Ab nucleotide sequence in the recombinant cloning vector DBN02-T.
2、构建含有Cry1A.105基因的重组表达载体2. Construction of a recombinant expression vector containing the Cry1A.105 gene
用限制性内切酶NcoI和HindIII分别酶切重组克隆载体DBN01-T和表达载体DBNBC-01(载体骨架：pCAMBIA2301(CAMBIA机构可以提供))，将切下的Cry1A.105核苷酸序列片段插到表达载体DBNBC-01的NcoI和HindIII位点之间，利用常规的酶切方法构建载体是本领域技术人员所熟知的，构建成重组表达载体DBN100745，其构建流程如图2所示(Kan：卡那霉素基因；RB：右边界；Ubi：玉米Ubiquitin(泛素)基因启动子(SEQ ID NO:5)；Cry1A.105：Cry1A.105核苷酸序列(SEQ ID NO:2)；Nos：胭脂碱合成酶基因的终止子(SEQID NO:6)；Hpt：潮霉素磷酸转移酶基因(SEQ ID NO:7)；LB：左边界)。Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (CAMBIA institutions can provide)) were digested with restriction endonucleases NcoI and HindIII respectively, and the excised Cry1A.105 nucleotide sequence fragment was inserted into Between the NcoI and HindIII sites of the expression vector DBNBC-01, it is well known to those skilled in the art to construct the vector by using the conventional enzyme digestion method, and construct the recombinant expression vector DBN100745, and its construction process is as shown in Figure 2 (Kan: Kanamycin gene; RB: right border; Ubi: corn Ubiquitin (ubiquitin) gene promoter (SEQ ID NO: 5); Cry1A.105: Cry1A.105 nucleotide sequence (SEQ ID NO: 2); Nos : terminator of nopaline synthase gene (SEQ ID NO: 6); Hpt: hygromycin phosphotransferase gene (SEQ ID NO: 7); LB: left border).
将重组表达载体DBN100745用热激方法转化大肠杆菌T1感受态细胞，其热激条件为：50μl大肠杆菌T1感受态细胞、10μl质粒DNA(重组表达载体DBN100745)，42℃水浴30秒；37℃振荡培养1小时(100rpm转速下摇床摇动)；然后在含50mg/L卡那霉素(Kanamycin)的LB固体平板(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，琼脂15g/L，用NaOH调pH至7.5)上于温度37℃条件下培养12小时，挑取白色菌落，在LB液体培养基(胰蛋白胨10g/L，酵母提取物5g/L，NaCl 10g/L，卡那霉素50mg/L，用NaOH调pH至7.5)中于温度37℃条件下培养过夜。碱法提取其质粒。将提取的质粒用限制性内切酶NcoI和HindIII酶切后鉴定，并将阳性克隆进行测序鉴定，结果表明重组表达载体DBN100745在NcoI和HindIII位点间的核苷酸序列为序列表中SEQ ID NO:2所示核苷酸序列，即Cry1A.105核苷酸序列。The recombinant expression vector DBN100745 was transformed into Escherichia coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 μl Escherichia coli T1 competent cells, 10 μl plasmid DNA (recombinant expression vector DBN100745), 42 ° C water bath for 30 seconds; 37 ° C shaking Cultivate for 1 hour (shaking on a shaking table at 100rpm rotating speed); /L, adjust the pH to 7.5 with NaOH) and culture at 37°C for 12 hours, pick white colonies, and in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, Kanamycin 50 mg/L, adjust the pH to 7.5 with NaOH) and cultivate overnight at a temperature of 37°C. The plasmid was extracted by alkaline method. The extracted plasmid was identified after digestion with restriction endonucleases NcoI and HindIII, and the positive clones were sequenced and identified. The results showed that the nucleotide sequence between the NcoI and HindIII sites of the recombinant expression vector DBN100745 was SEQ ID in the sequence table The nucleotide sequence shown in NO:2 is the Cry1A.105 nucleotide sequence.
按照上述构建重组表达载体DBN100745的方法，将NcoI和SpeI酶切重组克隆载体DBN02-T切下的所述Cry2Ab核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100744。酶切和测序验证重组表达载体DBN100744中的核苷酸序列含有为序列表中SEQ ID NO:4所示核苷酸序列，即Cry2Ab核苷酸序列，所述Cry2Ab核苷酸序列可以连接所述Ubi启动子和Nos终止子。According to the method for constructing the recombinant expression vector DBN100745 described above, the Cry2Ab nucleotide sequence excised from the recombinant cloning vector DBN02-T by NcoI and SpeI was inserted into the expression vector DBNBC-01 to obtain the recombinant expression vector DBN100744. Enzyme digestion and sequencing verify that the nucleotide sequence in the recombinant expression vector DBN100744 contains the nucleotide sequence shown in SEQ ID NO: 4 in the sequence table, that is, the Cry2Ab nucleotide sequence, and the Cry2Ab nucleotide sequence can be connected to the Ubi promoter and Nos terminator.
按照上述构建重组表达载体DBN100745的方法，将NcoI和HindIII、NcoI和SpeI分别酶切重组克隆载体DBN01-T和DBN02-T切下的所述Cry1A.105核苷酸序列和Cry2Ab核苷酸序列插入表达载体DBNBC-01，得到重组表达载体DBN100029。酶切和测序验证重组表达载体DBN100029中的核苷酸序列含有为序列表中SEQ ID NO:2和SEQ ID NO:4所示核苷酸序列，即Cry1A.105核苷酸序列和Cry2Ab核苷酸序列，所述Cry1A.105核苷酸序列和所述Cry2Ab核苷酸序列可以连接所述Ubi启动子和Nos终止子。According to the above-mentioned method for constructing the recombinant expression vector DBN100745, insert the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence excised from the recombinant cloning vector DBN01-T and DBN02-T respectively by NcoI and HindIII, NcoI and SpeI The expression vector DBNBC-01 was used to obtain the recombinant expression vector DBN100029. Enzyme digestion and sequencing verify that the nucleotide sequence in the recombinant expression vector DBN100029 contains the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 4 in the sequence listing, namely the Cry1A.105 nucleotide sequence and the Cry2Ab nucleoside acid sequence, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence can be connected to the Ubi promoter and the Nos terminator.
3、重组表达载体转化农杆菌3. Transformation of recombinant expression vector into Agrobacterium
对己经构建正确的重组表达载体DBN100745、DBN100744和DBN100029用液氮法转化到农杆菌LBA4404(Invitrgen，Chicago，USA，CAT：18313-015)中，其转化条件为：100μL农杆菌LBA4404、3μL质粒DNA(重组表达载体)；置于液氮中10分钟，37℃温水浴10分钟；将转化后的农杆菌LBA4404接种于LB试管中于温度28℃、转速为200rpm条件下培养2小时，涂于含50mg/L的利福平(Rifampicin)和100mg/L的卡那霉素(Kanamycin)的LB平板上直至长出阳性单克隆，挑取单克隆培养并提取其质粒，用限制性内切酶AhdI和XhoI对重组表达载体DBN100745、DBN100744和DBN100029酶切后进行酶切验证，结果表明重组表达载体DBN100745、DBN100744和DBN100029结构完全正确。The correctly constructed recombinant expression vectors DBN100745, DBN100744 and DBN100029 were transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 μL Agrobacterium LBA4404, 3 μL plasmid DNA (recombinant expression vector); placed in liquid nitrogen for 10 minutes, and warm water bath at 37°C for 10 minutes; inoculate the transformed Agrobacterium LBA4404 in an LB test tube and cultivate it for 2 hours at a temperature of 28°C and a rotation speed of 200rpm, and spread it on Contain 50 mg/L of Rifampicin (Rifampicin) and 100 mg/L of Kanamycin (Kanamycin) on the LB plate until a positive monoclonal grows, pick the monoclonal culture and extract its plasmid, use restriction endonuclease AhdI and XhoI digested the recombinant expression vectors DBN100745, DBN100744 and DBN100029 for enzyme digestion and verification. The results showed that the structures of the recombinant expression vectors DBN100745, DBN100744 and DBN100029 were completely correct.
第三实施例、转基因植株的获得The third embodiment, the acquisition of transgenic plants
1、获得转基因玉米植株1. Obtaining transgenic corn plants
按照常规采用的农杆菌侵染法，将无菌培养的玉米品种综31(Z31)的幼胚与第二实施例中3所述的农杆菌共培养，以将第二实施例中2构建的重组表达载体DBN100745、DBN100744和DBN100029中的T-DNA(包括玉米Ubiquitin基因的启动子序列、Cry1A.105核苷酸序列、Cry2Ab核苷酸序列、Hpt基因和Nos终止子序列)转入到玉米染色体组中，获得了转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株；同时以野生型玉米植株作为对照。According to the commonly used Agrobacterium infection method, the immature embryos of the aseptically cultured corn variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 3 in the second embodiment, so that the The T-DNA in recombinant expression vectors DBN100745, DBN100744 and DBN100029 (including the promoter sequence of maize Ubiquitin gene, Cry1A.105 nucleotide sequence, Cry2Ab nucleotide sequence, Hpt gene and Nos terminator sequence) were transferred to maize chromosome In the group, the corn plants transferred to Cry1A.105 nucleotide sequence, the corn plant transferred to Cry2Ab nucleotide sequence and the corn plant transferred to Cry1A.105-Cry2Ab nucleotide sequence were obtained; as comparison.
对于农杆菌介导的玉米转化，简要地，从玉米中分离未成熟的幼胚，用农杆菌悬浮液接触幼胚，其中农杆菌能够将Cry1A.105核苷酸序列、Cry2Ab核苷酸序列和Cry1A.105-Cry2Ab核苷酸序列传递至幼胚之一的至少一个细胞(步骤1：侵染步骤)，在此步骤中，幼胚优选地浸入农杆菌悬浮液(OD660＝0.4-0.6，侵染培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖68.5g/L、葡萄糖36g/L、乙酰丁香酮(AS)40mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L，pH5.3))中以启动接种。幼胚与农杆菌共培养一段时期(3天)(步骤2：共培养步骤)。优选地，幼胚在侵染步骤后在固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖20g/L、葡萄糖10g/L、乙酰丁香酮(AS)100mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、琼脂8g/L，pH5.8)上培养。在此共培养阶段后，可以有一个选择性的“恢复”步骤。在“恢复”步骤中，恢复培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)中至少存在一种己知抑制农杆菌生长的抗生素(头孢霉素)，不添加植物转化体的选择剂(步骤3：恢复步骤)。优选地，幼胚在有抗生素但没有选择剂的固体培养基上培养，以消除农杆菌并为侵染细胞提供恢复期。接着，接种的幼胚在含选择剂(潮霉素)的培养基上培养并选择生长着的转化愈伤组织(步骤4：选择步骤)。优选地，幼胚在有选择剂的筛选固体培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、潮霉素50mg/L、2,4-二氯苯氧乙酸(2,4-D)1mg/L、植物凝胶3g/L，pH5.8)上培养，导致转化的细胞选择性生长。然后，愈伤组织再生成植物(步骤5：再生步骤)，优选地，在含选择剂的培养基上生长的愈伤组织在固体培养基(MS分化培养基和MS生根培养基)上培养以再生植物。For Agrobacterium-mediated transformation of maize, briefly, immature immature embryos were isolated from maize and contacted with a suspension of Agrobacterium capable of transforming the Cry1A.105 nucleotide sequence, the Cry2Ab nucleotide sequence and The Cry1A.105-Cry2Ab nucleotide sequence is delivered to at least one cell of one of the immature embryos (step 1: infection step), during which the immature embryos are preferably immersed in an Agrobacterium suspension (OD660 =0.4-0.6, Infection medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g/L, acetosyringone (AS) 40mg/L, 2,4-dichlorophenoxy Acetic acid (2,4-D) 1mg/L, pH5.3)) to initiate inoculation. The immature embryos were co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, immature embryos are cultured on solid medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L after the infection step. , 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH5.8). After this co-cultivation phase, there can be an optional "recovery" step. In the "recovery" step, recovery medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/ L. At least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium exists in the plant gel (3 g/L, pH 5.8), and no selection agent for plant transformants is added (step 3: recovery step). Preferably, immature embryos are cultured on solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the inoculated immature embryos are cultured on a medium containing a selection agent (hygromycin) and the growing transformed callus is selected (step 4: selection step). Preferably, the immature embryos are cultured on solid medium for selection with selective agents (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, hygromycin 50mg/L, 2,4-dichlorobenzene Oxyacetic acid (2,4-D) 1 mg/L, Phytogel 3 g/L, pH 5.8) resulted in selective growth of transformed cells. Then, the callus regenerates into plants (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on solid medium (MS differentiation medium and MS rooting medium) to regenerated plants.
筛选得到的抗性愈伤组织转移到所述MS分化培养基(MS盐4.3g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、6-苄基腺嘌呤2mg/L、潮霉素50mg/L、植物凝胶3g/L，pH5.8)上，25℃下培养分化。分化出来的小苗转移到所述MS生根培养基(MS盐2.15g/L、MS维他命、干酪素300mg/L、蔗糖30g/L、吲哚-3-乙酸1mg/L、植物凝胶3g/L，pH5.8)上，25℃下培养至约10cm高，移至温室培养至结实。在温室中，每天于28℃下培养16小时，再于20℃下培养8小时。The resistant callus obtained by screening was transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, hygromycetes 50 mg/L vegetal, 3 g/L phytogel, pH 5.8), cultured and differentiated at 25°C. Differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, plant gel 3g/L , pH 5.8), cultivated at 25°C to a height of about 10 cm, and moved to the greenhouse for cultivation until firm. In the greenhouse, culture was carried out at 28°C for 16 hours and at 20°C for 8 hours every day.
2、获得转基因高粱植株2. Obtaining transgenic sorghum plants
参考Molecular Biology and Genetic Engineering ISSN 2053-5767的高粱转化方法。收集高粱品种APKI的种子，并用清水冲洗数次；浸泡于tween-20浸润液中5分钟；之后用双蒸水悬浮清洗，并在通风橱中干燥；种子表面用70％(v/v)乙醇消毒30秒，紧接着用0.1％(w/v)HgCl2消毒6分钟；再用双蒸水清洗5-6次；将种子铺于含有MS基础固体培养基(pH5.8)的培养皿中，将培养皿摆放于温度为24±2℃、相对湿度为70％、光周期(光/暗)为12:12的培养间中；3-5天后，种子发芽，取茎尖外植体浸泡于农杆菌中30分钟；取出浸泡后的外植体摆放于已灭菌的滤纸上；黑暗条件下共培养72小时；愈伤组织用含有500mg/L头孢霉素的无菌水清洗3-5次；将清洗后的愈伤组织转移至诱导培养基上培养7天；再转移至筛选培养基上2-3周，重复筛选3次；抗性愈伤被转移至再生培养基上；再生出叶片等，将小苗移至生根培养基上，待生根后移栽至温室中。培养基配方参考Molecular Biology and Genetic Engineering ISSN 2053-5767，其中筛选剂根据本发明中转基因载体所用，更换为潮霉素。由此获得了转入Cry1A.105核苷酸序列的高粱植株、转入Cry2Ab核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株；同时以野生型高粱植株作为对照。Refer to the sorghum transformation method of Molecular Biology and Genetic Engineering ISSN 2053-5767. Collect the seeds of sorghum variety APKI and rinse them several times with water; soak them in tween-20 infiltration solution for 5 minutes; then suspend and wash them with double distilled water, and dry them in a fume hood; wash the surface of the seeds with 70% (v/v) ethanol Disinfect for 30 seconds, followed by disinfection with 0.1% (w/v) HgCl2 for 6 minutes; wash with double distilled water for 5-6 times; spread the seeds in a petri dish containing MS basic solid medium (pH5.8) , place the culture dish in a culture room where the temperature is 24±2°C, the relative humidity is 70%, and the photoperiod (light/dark) is 12:12; after 3-5 days, the seeds germinate, and the shoot tip explants are taken Soak in Agrobacterium for 30 minutes; take out the soaked explants and place them on sterilized filter paper; co-culture for 72 hours under dark conditions; callus is washed with sterile water containing 500 mg/L cephalosporin for 3 -5 times; the cleaned callus was transferred to the induction medium for 7 days; then transferred to the selection medium for 2-3 weeks, and the selection was repeated 3 times; the resistant callus was transferred to the regeneration medium; After the leaves are regenerated, the seedlings are moved to the rooting medium, and after rooting, they are transplanted into the greenhouse. For the formula of the medium, refer to Molecular Biology and Genetic Engineering ISSN 2053-5767, wherein the screening agent is replaced with hygromycin according to the transgenic vector in the present invention. Thus, the sorghum plants transferred to the Cry1A.105 nucleotide sequence, the sorghum plant transferred to the Cry2Ab nucleotide sequence and the sorghum plant transferred to the Cry1A.105-Cry2Ab nucleotide sequence were obtained; at the same time, wild-type sorghum plants were used as control.
3、获得转基因甘蔗植株3. Obtaining transgenic sugarcane plants
转化方法主要参考广西大学2012级硕士李粲学位论文第22页至24页。取甘蔗顶端新生茎节，去掉蔗梢和叶鞘，留下茎尖生长锥及心叶茎段。在超净工作台上，用75％(v/v)酒精棉球对表面进行擦拭消毒，用已灭菌的镊子小心剥去心叶外层，取中间5—7cm长的心叶段，横切成厚度约3mm的薄片接种于诱导培养基上，温度26℃条件下，黑暗培养20天。挑选生长情况良好的愈伤组织转移到新的MS培养基中预培养4天，再用于转化试验；转化时，在超净工作台中将待侵染的愈伤组织用已灭菌的镊子夹出，放在干净的滤纸上面静置2小时，至表面完全干燥，稍有收缩；将干燥的甘蔗愈伤组织放入侵染液中浸泡30分钟，同时放在摇床上缓慢摇动；将愈伤组织捞出并转移到干净的滤纸上，在超净工作台中完全吹干，直至愈伤组织表面干燥、无水膜。把愈伤组织块切成0.6*0.6cm的小块，之后转移到含有100μmol/L乙酰丁香酮(AS)的MR固体培养基中，温度23℃暗培养3天；把侵染后的愈伤组织夹出，置于滤纸上在超净工作台上吹干，直到材料表面干爽后，将材料转移到含有500mg/L头孢霉素和潮霉素筛选的分化培养基中；每隔2周更换一次培养基，期间把被污染的愈伤组织剔除，当幼苗长约3cm高的时候，转移到含有潮霉素筛选剂的生根培养基中诱导生根。由此获得了转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry2Ab核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株；同时以野生型甘蔗植株作为对照。The conversion method mainly refers to pages 22 to 24 of Li Can’s dissertation for the 2012 master of Guangxi University. Take the new stem node at the top of the sugarcane, remove the tip and leaf sheath, and leave the growth cone of the stem tip and the stem section of the heart leaf. On the ultra-clean workbench, wipe and disinfect the surface with 75% (v/v) alcohol cotton balls, carefully peel off the outer layer of the heart leaf with sterilized tweezers, take the middle 5-7cm long heart leaf segment, and horizontally Thin slices cut to a thickness of about 3mm were inoculated on the induction medium, and cultured in the dark for 20 days at a temperature of 26°C. Select the callus with good growth and transfer it to the new MS medium for pre-cultivation for 4 days, and then use it for the transformation test; when transforming, use sterilized forceps to clamp the callus to be infected in the ultra-clean workbench out, put it on a clean filter paper and let it stand for 2 hours until the surface is completely dry and shrinks slightly; put the dried sugarcane callus in the invasion solution for 30 minutes and shake it slowly on a shaker; the callus The tissue was removed and transferred to a clean filter paper, and dried completely in an ultra-clean workbench until the surface of the callus was dry and without a water film. Cut the callus into small pieces of 0.6*0.6cm, then transfer to MR solid medium containing 100 μmol/L acetosyringone (AS), and culture in the dark at 23°C for 3 days; the infected callus Clip out the tissue, put it on filter paper and blow dry on the ultra-clean workbench until the surface of the material is dry, then transfer the material to the differentiation medium containing 500mg/L cephalosporin and hygromycin selection; replace every 2 weeks A culture medium, during which the polluted callus was removed, and when the seedlings were about 3 cm high, they were transferred to the rooting medium containing hygromycin selection agent to induce rooting. Thus obtained the sugarcane plant transferred to the Cry1A.105 nucleotide sequence, the sugarcane plant transferred to the Cry2Ab nucleotide sequence and the sugarcane plant transferred to the Cry1A.105-Cry2Ab nucleotide sequence; control.
4、获得转基因谷子植株4. Obtain transgenic millet plants
转化方法参考河北农业大学2012级硕士王寒玉学位论文第9页至第10页。将成熟种子在0.1％(v/v)Tween-20溶液中浸泡后，用70％(v/v)乙醇清洗，然后转移到0.1％(w/v)HgCl2溶液中，最后用灭菌水清洗2-3次。将经过灭菌处理的种子转移到MS培养基上，温度25℃暗培养2-3天。待种子茎尖长到4-6mm时，在无菌条件下将茎尖转移到愈伤诱导培养基上。For the conversion method, refer to pages 9 to 10 of the thesis of Wang Hanyu, a 2012 master of Hebei Agricultural University. After the mature seeds were soaked in 0.1% (v/v) Tween-20 solution, washed with 70% (v/v) ethanol, then transferred to 0.1% (w/v)HgCl2 solution, and finally rinsed with sterilized water Wash 2-3 times. Transfer the sterilized seeds to MS medium, and culture in dark at 25°C for 2-3 days. When the shoot tip of the seed grows to 4-6 mm, the shoot tip is transferred to the callus induction medium under aseptic conditions.
茎尖愈伤组织的诱导：将诱导后的茎尖愈伤组织浸取农杆菌菌悬液30分钟，取出茎尖愈伤组织放于已灭菌滤纸上，吸出多余菌液，转移到共培养培养基上(MS+100mol/L AS+2,4-D)，温度28℃黑暗中共培养2-4天。之后将共培养后的愈伤组织转到MS愈伤诱导培养基(2,4-D 4.5μmol/L、2.25μmol/L Kn、头孢霉素500mg/L)上，温度25℃暗培养，每两周对愈伤进行一次继代。幼穗在愈伤诱导培养基上培养5周后，选取结构致密的黄白色愈伤组织转移到分化培养基上进行分化，分化培养基中加入筛选剂潮霉素。约五周后，愈伤形成节状结构，将有节状结构的愈伤转移到MS分化生根培养基(噻二唑苯基脲(TDZ)4.5μmol/L、蔗糖120μmol/L)上。由此获得了转入Cry1A.105核苷酸序列的谷子植株、转入Cry2Ab核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株；同时以野生型谷子植株作为对照。Induction of shoot apex callus: Soak the induced shoot apex callus in the Agrobacterium suspension for 30 minutes, take out the shoot apex callus and put it on the sterilized filter paper, suck out the excess bacteria liquid, and transfer it to the co-culture On the culture medium (MS+100mol/L AS+2,4-D), co-culture at 28°C in the dark for 2-4 days. Afterwards, the co-cultured calli were transferred to MS callus induction medium (2,4-D 4.5 μmol/L, 2.25 μmol/L Kn, cephalosporin 500 mg/L), cultured in the dark at 25°C, The callus was subcultured every two weeks. After the young ears were cultured on the callus induction medium for 5 weeks, the yellow-white callus with compact structure was selected and transferred to the differentiation medium for differentiation, and the screening agent hygromycin was added to the differentiation medium. After about five weeks, the calli formed nodular structures, and the calli with nodular structures were transferred to MS differentiation rooting medium (thiadiazole urea (TDZ) 4.5 μmol/L, sucrose 120 μmol/L). Thus obtained the millet plant transferred to the Cry1A.105 nucleotide sequence, the millet plant transferred to the Cry2Ab nucleotide sequence and the millet plant transferred to the Cry1A.105-Cry2Ab nucleotide sequence; control.
第四实施例、用TaqMan验证转基因植株The fourth embodiment, using TaqMan to verify transgenic plants
分别取转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株的叶片约100mg作为样品，用Qiagen的DNeasy Plant Maxi Kit提取其基因组DNA，通过Taqman探针荧光定量PCR方法检测Cry1A.105基因和Cry2Ab基因的拷贝数。同时以野生型玉米植株作为对照，按照上述方法进行检测分析。实验设3次重复，取平均值。About 100 mg of leaves of corn plants transferred to the Cry1A.105 nucleotide sequence, the corn plant transferred to the Cry2Ab nucleotide sequence, and the corn plant transferred to the Cry1A.105-Cry2Ab nucleotide sequence were taken as samples, and Qiagen's DNeasy Plant Maxi Kit extracted its genomic DNA, and detected the copy number of Cry1A.105 gene and Cry2Ab gene by Taqman probe fluorescent quantitative PCR method. At the same time, wild-type maize plants were used as a control, and detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.
检测Cry1A.105基因和Cry2Ab基因拷贝数的具体方法如下：The specific method for detecting the copy number of Cry1A.105 gene and Cry2Ab gene is as follows:
步骤11、分别取转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株和野生型玉米植株的叶片各100mg，分别在研钵中用液氮研成匀浆，每个样品取3个重复；Step 11, taking the leaves of the corn plant transferred to the Cry1A.105 nucleotide sequence, the corn plant transferred to the Cry2Ab nucleotide sequence, the corn plant transferred to the Cry1A.105-Cry2Ab nucleotide sequence, and the wild-type corn plant 100mg each, grind into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;
步骤12、使用Qiagen的DNeasy Plant Mini Kit提取上述样品的基因组DNA，具体方法参考其产品说明书；Step 12, using the DNeasy Plant Mini Kit of Qiagen to extract the genomic DNA of the above sample, the specific method refers to its product manual;
步骤13、用NanoDrop 2000(Thermo Scientific)测定上述样品的基因组DNA浓度；Step 13, measure the genomic DNA concentration of above-mentioned sample with NanoDrop 2000 (Thermo Scientific);
步骤14、调整上述样品的基因组DNA浓度至同一浓度值，所述浓度值的范围为80-100ng/μl；Step 14, adjusting the genomic DNA concentration of the above samples to the same concentration value, the concentration value ranges from 80-100ng/μl;
步骤15、采用Taqman探针荧光定量PCR方法鉴定样品的拷贝数，以经过鉴定已知拷贝数的样品作为标准品，以野生型玉米植株的样品作为对照，每个样品3个重复，取其平均值；荧光定量PCR引物和探针序列分别是：Step 15, using the Taqman probe fluorescent quantitative PCR method to identify the copy number of the sample, using the sample with known copy number after identification as a standard, and using the sample of the wild-type corn plant as a control, each sample was repeated 3 times, and the average Value; Fluorescence quantitative PCR primer and probe sequences are respectively:
以下引物和探针用来检测Cry1A.105核苷酸序列：The following primers and probes were used to detect the Cry1A.105 nucleotide sequence:
引物1：GCGCATCCAGTTCAACGAC如序列表中SEQ ID NO:8所示；Primer 1: GCGCATCCAGTTCAACGAC as shown in SEQ ID NO:8 in the sequence listing;
引物2：GTTCTGGACGGCGAAGAGTG如序列表中SEQ ID NO:9所示；Primer 2: GTTCTGGACGGCGAAGAGTG as shown in SEQ ID NO: 9 in the sequence listing;
探针1：TGAACAGCGCCCTGACCACCG如序列表中SEQ ID NO:10所示；Probe 1: TGAACAGCGCCCTGACCACCG as shown in SEQ ID NO: 10 in the sequence listing;
以下引物和探针用来检测Cry2Ab核苷酸序列：The following primers and probes were used to detect the Cry2Ab nucleotide sequence:
引物3：CTGATACCCTTGCTCGCGTC如序列表中SEQ ID NO:11所示；Primer 3: CTGATACCCTTGCTCGCGTC as shown in SEQ ID NO: 11 in the sequence listing;
引物4：CACTTGGCGGTTGAACTCCTC如序列表中SEQ ID NO:12所示；Primer 4: CACTTGGCGGTTGAACTCCTC as shown in SEQ ID NO: 12 in the sequence listing;
探针2：CGCTGAGCTGACGGGTCTGCAAG如序列表中SEQ ID NO:13所示；Probe 2: CGCTGAGCTGACGGGTCTGCAAG as shown in SEQ ID NO: 13 in the sequence listing;
PCR反应体系为：The PCR reaction system is:
所述50×引物/探针混合物包含1mM浓度的每种引物各45μl，100μM浓度的探针50μl和860μl 1×TE缓冲液，并且在4℃，贮藏在琥珀试管中。The 50X primer/probe mix contained 45 μl of each primer at a concentration of 1 mM, 50 μl of a probe at a concentration of 100 μM and 860 μl of 1X TE buffer, and was stored in amber tubes at 4°C.
PCR反应条件为：The PCR reaction conditions are:
利用SDS2.3软件(Applied Biosystems)分析数据。Data were analyzed using SDS2.3 software (Applied Biosystems).
实验结果表明，Cry1A.105核苷酸序列、Cry2Ab核苷酸序列和Cry1A.105-Cry2Ab核苷酸序列均己整合到所检测的玉米植株的染色体组中，而且转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株均获得了单拷贝的转基因玉米植株。The experimental results show that the Cry1A.105 nucleotide sequence, the Cry2Ab nucleotide sequence and the Cry1A.105-Cry2Ab nucleotide sequence have all been integrated into the genome of the maize plant detected, and the Cry1A.105 nucleotide sequence has been transferred into A single-copy transgenic maize plant was obtained from the maize plant of sequence, the maize plant of Cry2Ab nucleotide sequence and the maize plant of Cry1A.105-Cry2Ab nucleotide sequence.
按照上述用TaqMan验证转基因玉米植株的方法，对转基因高粱植株、转基因甘蔗植株和转基因谷子植株进行检测分析。实验结果表明，Cry1A.105核苷酸序列、Cry2Ab核苷酸序列和Cry1A.105-Cry2Ab核苷酸序列均己分别整合到所检测的高粱植株、甘蔗植株和谷子植株的染色体组中，而且转入Cry1A.105核苷酸序列的高粱植株、转入Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105核苷酸序列的谷子植株、转入Cry2Ab核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株均获得了单拷贝的转基因植株。Transgenic sorghum plants, transgenic sugarcane plants and transgenic millet plants were detected and analyzed according to the above-mentioned method of using TaqMan to verify transgenic maize plants. The experimental results show that the Cry1A.105 nucleotide sequence, the Cry2Ab nucleotide sequence and the Cry1A.105-Cry2Ab nucleotide sequence have been integrated into the genomes of the detected sorghum plants, sugarcane plants and millet plants respectively, and the transgenic Sorghum plant with Cry1A.105 nucleotide sequence, sorghum plant with Cry2Ab nucleotide sequence, sorghum plant with Cry1A.105-Cry2Ab nucleotide sequence, sugarcane plant with Cry1A.105 nucleotide sequence , sugarcane plants transferred to Cry2Ab nucleotide sequence, sugarcane plants transferred to Cry1A.105-Cry2Ab nucleotide sequence, millet plants transferred to Cry1A.105 nucleotide sequence, millet plants transferred to Cry2Ab nucleotide sequence and millet plants transferred with Cry1A.105-Cry2Ab nucleotide sequence obtained single-copy transgenic plants.
第五实施例、转基因植株的抗虫效果检测The fifth embodiment, detection of insect resistance effect of transgenic plants
将转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株；转入Cry1A.105核苷酸序列的高粱植株、转入Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株；转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株；转入Cry1A.105核苷酸序列的谷子植株、转入Cry2Ab核苷酸序列的谷子植株，转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株；相应的野生型玉米植株、高粱植株、甘蔗植株和谷子植株，以及经Taqman鉴定为非转基因的玉米植株、高粱植株、甘蔗植株和谷子植株对粟灰螟进行抗虫效果检测。Corn plants transferred to Cry1A.105 nucleotide sequence, corn plants transferred to Cry2Ab nucleotide sequence, corn plants transferred to Cry1A.105-Cry2Ab nucleotide sequence; transferred to Cry1A.105 nucleotide sequence Sorghum plants, sorghum plants transferred to Cry2Ab nucleotide sequences, sorghum plants transferred to Cry1A.105-Cry2Ab nucleotide sequences; sugarcane plants transferred to Cry1A.105 nucleotide sequences, Cry2Ab nucleotide sequences transferred Sugarcane plants, sugarcane plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence; millet plants transferred to the Cry1A.105 nucleotide sequence, millet plants transferred to the Cry2Ab nucleotide sequence, transferred to the Cry1A.105-Cry2Ab nucleus Millet plants with nucleotide sequences; corresponding wild-type corn plants, sorghum plants, sugarcane plants and millet plants, and non-transgenic corn plants, sorghum plants, sugarcane plants and millet plants identified by Taqman have insect resistance effects detection.
1、转基因玉米植株的抗虫效果检测1. Detection of insect resistance effect of transgenic corn plants
分别取转入Cry1A.105核苷酸序列的玉米植株、转入Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株、野生型玉米植株和经Taqman鉴定为非转基因的玉米植株(展开嫩叶)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将玉米叶片剪成约1cm×2cm的长条状，取1片剪后的长条状叶片放入圆形塑料培养皿底部的保湿滤纸上，每个培养皿中放10头粟灰螟(初孵幼虫)，虫试培养皿加盖后，在温度22-26℃、相对湿度70％-80％、光周期(光/暗)0:24的条件下放置3天后，根据粟灰螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry1A.105核苷酸序列的共3个转化事件株系(S1、S2和S3)，转入Cry2Ab核苷酸序列的共3个转化事件株系(S4、S5和S6)，转入Cry1A.105-Cry2Ab核苷酸序列的共3个转化事件株系(S7、S8和S9)，经Taqman鉴定为非转基因的(NGM1)共1个株系，野生型的(CK1)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表1和图3所示。The corn plants transferred to the Cry1A.105 nucleotide sequence, the corn plants transferred to the Cry2Ab nucleotide sequence, the corn plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence, the wild-type corn plants and those identified as The fresh leaves of non-transgenic corn plants (opened young leaves) are washed with sterile water and the water on the leaves is blotted dry with gauze, then the corn leaves are cut into strips of about 1cm×2cm, and one piece is cut Put the strip-shaped blades of the long strip leaves on the moisture-retaining filter paper at the bottom of the circular plastic culture dish, put 10 millet moths (newly hatched larvae) in each culture dish, and after the worm test culture dish is covered, at a temperature of 22-26 ℃, After standing for 3 days under the conditions of relative humidity 70%-80% and photoperiod (light/dark) 0:24, the total score of resistance (full score 300 points): total score=100×mortality rate+[100×mortality rate+90×(number of newly hatched worms/total number of inoculated worms)+60×(number of newly hatched-negative control worms/total number of inoculated worms)+10×( Number of negative control insects/total number of inoculated insects)]+100×(1-leaf damage rate). A total of 3 transformation event strains (S1, S2 and S3) transferred into the Cry1A.105 nucleotide sequence, a total of 3 transformation event strains (S4, S5 and S6) transferred into the Cry2Ab nucleotide sequence, transferred into A total of 3 transformation event lines (S7, S8 and S9) of the Cry1A.105-Cry2Ab nucleotide sequence, a total of 1 strain identified as non-transgenic (NGM1) by Taqman, and a total of wild type (CK1) Strains; 3 strains were selected from each strain for testing, and each strain was repeated 6 times. The results are shown in Table 1 and Figure 3.
表1、转基因玉米植株接种粟灰螟的抗虫实验结果Table 1. Insect resistance experiment results of transgenic corn plants inoculated with P. argentina
表1的结果表明：转入Cry1A.105核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株对粟灰螟均具有较好的杀虫效果，粟灰螟的平均死亡率均在80％以上，其抗性总分也均在280分左右；而经Taqman鉴定为非转基因的玉米植株和野生型玉米植株的抗性总分一般在20分左右。The results in Table 1 show that: the corn plants transferred to the Cry1A.105 nucleotide sequence and the corn plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence all have a good insecticidal effect on P. The average mortality rate is above 80%, and the total resistance score is about 280 points; while the total resistance score of non-transgenic maize plants and wild-type maize plants identified by Taqman is generally around 20 points.
图3的结果表明：与野生型玉米植株相比，转入Cry1A.105核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株可以造成粟灰螟初孵幼虫的大量死亡，且对极少量存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，同时表现出极弱的生命力，且转入Cry1A.105核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株大体上只受到极轻微损伤，肉眼几乎无法辨别出粟灰螟的取食痕迹，其叶片损伤率均在3％以下。The result of Fig. 3 shows: compare with wild-type corn plant, the corn plant that transfers Cry1A.105 nucleotide sequence and the corn plant that transfers Cry1A.105-Cry2Ab nucleotide sequence can cause the larvae of newly hatched larvae A large number of larvae died, and the development progress of a very small number of surviving larvae was greatly inhibited. After 3 days, the larvae were basically still in the newly hatched state, and at the same time showed extremely weak vitality, and the corn plants and transgenic plants with the Cry1A.105 nucleotide sequence The maize plants with the Cry1A.105-Cry2Ab nucleotide sequence were generally only slightly damaged, and the feeding traces of the millet moth could hardly be discerned with the naked eye, and the leaf damage rate was below 3%.
转入Cry2Ab核苷酸序列的玉米植株对粟灰螟则没有表现出控制效果，无论是死亡率、叶片损伤率、幼虫发育进度，还是抗性总分，与经Taqman鉴定为非转基因的玉米植株和野生型玉米植株相比，均未表现出差异。The corn plants transferred with the Cry2Ab nucleotide sequence did not show any control effect on the pod borer, whether it was mortality rate, leaf damage rate, larval development progress, or the total resistance score, which was different from the non-transgenic corn plants identified by Taqman. Neither showed a difference compared to wild-type maize plants.
由此证明转入Cry1A.105核苷酸序列的玉米植株和转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株都显示出高抗粟灰螟的活性，这种活性足以对粟灰螟的生长产生不良效应从而使其在田间得以控制。同时通过控制粟灰螟的钻蛀为害，也有可能降低玉米上病害的发生，极大的提高玉米的产量及品质。It is thus proved that the corn plants transferred to the Cry1A.105 nucleotide sequence and the corn plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence all show high activity against the pod borer. Adverse effects are thereby controlled in the field. At the same time, by controlling the borer damage of the millet borer, it is also possible to reduce the occurrence of diseases on corn and greatly improve the yield and quality of corn.
2、转基因甘蔗植株的抗虫效果检测2. Detection of insect resistance effect of transgenic sugarcane plants
分别取转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株、野生型甘蔗植株和经Taqman鉴定为非转基因的甘蔗植株(展开嫩叶)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将甘蔗叶片剪成约1cm×2cm的长条状，取1片剪后的长条状叶片放入圆形塑料培养皿底部的保湿滤纸上，每个培养皿中放10头粟灰螟(初孵幼虫)，虫试培养皿加盖后，在温度22-26℃、相对湿度70％-80％、光周期(光/暗)0:24的条件下放置3天后，根据粟灰螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry1A.105核苷酸序列的共3个转化事件株系(S10、S11和S12)，转入Cry2Ab核苷酸序列的共3个转化事件株系(S13、S14和S15)，转入Cry1A.105-Cry2Ab核苷酸序列的共3个转化事件株系(S16、S17和S18)，经Taqman鉴定为非转基因的(NGM2)共1个株系，野生型的(CK2)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表2和图4所示。The sugarcane plants transferred to the Cry1A.105 nucleotide sequence, the sugarcane plants transferred to the Cry2Ab nucleotide sequence, the sugarcane plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence, the wild-type sugarcane plants and those identified as Wash the fresh leaves of non-transgenic sugarcane plants (tender leaves) with sterile water and dry the water on the leaves with gauze, then cut the sugarcane leaves into strips of about 1cm×2cm, take 1 piece and cut it Put the strip-shaped blades of the long strip leaves on the moisture-retaining filter paper at the bottom of the circular plastic culture dish, put 10 millet moths (newly hatched larvae) in each culture dish, and after the worm test culture dish is covered, at a temperature of 22-26 ℃, After standing for 3 days under the conditions of relative humidity 70%-80% and photoperiod (light/dark) 0:24, the total score of resistance was obtained according to the three indicators of developmental progress, mortality rate and leaf damage rate of P. Score=100×mortality+[100×mortality+90×(number of newly hatched worms/total number of worms)+60×(number of newly hatched-negative control worms/total number of worms)+10×(number of negative control worms/ The total number of infested insects)]+100×(1-leaf damage rate). A total of 3 transformation event lines (S10, S11 and S12) transferred into the Cry1A.105 nucleotide sequence, a total of 3 transformation event lines (S13, S14 and S15) transferred into the Cry2Ab nucleotide sequence, transferred into A total of 3 transformation event lines (S16, S17 and S18) of the Cry1A.105-Cry2Ab nucleotide sequence, a total of 1 strain identified as non-transgenic (NGM2) by Taqman, and a total of wild type (CK2) Strains; 3 strains were selected from each strain for testing, and each strain was repeated 6 times. The results are shown in Table 2 and Figure 4.
表2的结果表明：转入Cry1A.105核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株对粟灰螟均具有较好的杀虫效果，粟灰螟的平均死亡率均在80％以上，其抗性总分也均在280分左右；而经Taqman鉴定为非转基因的甘蔗植株和野生型甘蔗植株的抗性总分一般在20分以下。图4的结果表明：与野生型甘蔗植株相比，转入Cry1A.105核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株可以造成粟灰螟初孵幼虫的大量死亡，且对极少量存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，同时表现出极弱的生命力，且转入Cry1A.105核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株大体上只受到极轻微损伤，肉眼几乎无法辨别出粟灰螟的取食痕迹，其叶片损伤率均在3％以下。The results in Table 2 show that: the sugarcane plants transferred to the Cry1A.105 nucleotide sequence and the sugarcane plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence all have a better insecticidal effect on C. The average mortality rate is above 80%, and the total resistance score is about 280 points; while the total resistance score of non-transgenic sugarcane plants and wild-type sugarcane plants identified by Taqman is generally below 20 points. The result of Fig. 4 shows: compared with the wild-type sugarcane plant, the sugarcane plant that transfers the Cry1A.105 nucleotide sequence and the sugarcane plant that transfers the Cry1A.105-Cry2Ab nucleotide sequence can cause the death of newly hatched larvae of P. A large number of larvae died, and the development progress of a very small number of surviving larvae was greatly inhibited. After 3 days, the larvae were basically still in the newly hatched state, and at the same time showed extremely weak vitality, and the sugarcane plants and transgenic plants with the Cry1A. The sugarcane plants with the Cry1A.105-Cry2Ab nucleotide sequence were generally only slightly damaged, and the feeding traces of the millet moth could hardly be discerned with the naked eye, and the leaf damage rate was below 3%.
转入Cry2Ab核苷酸序列的甘蔗植株对粟灰螟则没有表现出控制效果，无论是死亡率、叶片损伤率、幼虫发育进度，还是抗性总分，与经Taqman鉴定为非转基因的甘蔗植株和野生型甘蔗植株相比，均未表现出差异。The sugarcane plants transferred with the Cry2Ab nucleotide sequence did not show any control effect on the pod borer, whether in terms of mortality rate, leaf damage rate, larval development progress, or total resistance score, which was different from the sugarcane plants identified as non-transgenic by Taqman. None showed differences compared to wild-type sugarcane plants.
表2、转基因甘蔗植株接种粟灰螟的抗虫实验结果Table 2. Insect resistance test results of transgenic sugarcane plants inoculated with P. argentina
由此证明转入Cry1A.105核苷酸序列的甘蔗植株和转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株都显示出高抗粟灰螟的活性，这种活性足以对粟灰螟的生长产生不良效应从而使其在田间得以控制。同时通过控制粟灰螟的钻蛀为害，也有可能降低甘蔗上病害的发生，极大的提高甘蔗的产量及品质。This proves that the sugarcane plants that have been transferred to the Cry1A.105 nucleotide sequence and the sugarcane plants that have been transferred to the Cry1A.105-Cry2Ab nucleotide sequence all show high activity against the mites, which is sufficient for the growth of the mites. Adverse effects are thereby controlled in the field. At the same time, by controlling the borer damage of the millet ash borer, it is also possible to reduce the occurrence of diseases on sugarcane, and greatly improve the yield and quality of sugarcane.
3、转基因高粱植株的抗虫效果检测3. Detection of insect resistance effect of transgenic sorghum plants
分别取转入Cry1A.105核苷酸序列的高粱植株、转入Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株、野生型高粱植株和经Taqman鉴定为非转基因的高粱植株(展开嫩叶)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将高粱叶片剪成约1cm×2cm的长条状，取1片剪后的长条状叶片放入圆形塑料培养皿底部的保湿滤纸上，每个培养皿中放10头粟灰螟(初孵幼虫)，虫试培养皿加盖后，在温度22-26℃、相对湿度70％-80％、光周期(光/暗)0:24的条件下放置3天后，根据粟灰螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分(满分300分)：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry1A.105核苷酸序列的共3个转化事件株系(S19、S20和S21)，转入Cry2Ab核苷酸序列的共3个转化事件株系(S22、S23和S24)，转入Cry1A.105-Cry2Ab核苷酸序列的共3个转化事件株系(S25、S26和S27)，经Taqman鉴定为非转基因的(NGM3)共1个株系，野生型的(CK3)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表3和图5所示。Sorghum plants transferred to Cry1A.105 nucleotide sequence, sorghum plants transferred to Cry2Ab nucleotide sequence, sorghum plants transferred to Cry1A.105-Cry2Ab nucleotide sequence, wild-type sorghum plants and identified as Fresh leaves of non-transgenic sorghum plants (opened young leaves), rinsed with sterile water and blotted dry with gauze, then cut sorghum leaves into strips of about 1cm×2cm, took 1 piece and cut it Put the strip-shaped blades of the long strip leaves on the moisture-retaining filter paper at the bottom of the circular plastic culture dish, put 10 millet moths (newly hatched larvae) in each culture dish, and after the worm test culture dish is covered, at a temperature of 22-26 ℃, After standing for 3 days under the conditions of relative humidity 70%-80% and photoperiod (light/dark) 0:24, the total score of resistance (full score 300 points): total score=100×mortality rate+[100×mortality rate+90×(number of newly hatched worms/total number of inoculated worms)+60×(number of newly hatched-negative control worms/total number of inoculated worms)+10×( Number of negative control insects/total number of inoculated insects)]+100×(1-leaf damage rate). A total of 3 transformation event lines (S19, S20 and S21) transferred into the Cry1A.105 nucleotide sequence, a total of 3 transformation event lines (S22, S23 and S24) transferred into the Cry2Ab nucleotide sequence, transferred into A total of 3 transformation event lines (S25, S26 and S27) of the Cry1A.105-Cry2Ab nucleotide sequence, a total of 1 strain identified as non-transgenic (NGM3) by Taqman, and a total of wild type (CK3) Strains; 3 strains were selected from each strain for testing, and each strain was repeated 6 times. The results are shown in Table 3 and Figure 5.
表3、转基因高粱植株接种粟灰螟的抗虫实验结果Table 3. The results of the insect resistance experiment of transgenic sorghum plants inoculated with P. argentina
表3的结果表明：转入Cry1A.105核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株对粟灰螟均具有较好的杀虫效果，粟灰螟的平均死亡率均在90％左右，其抗性总分也均在280分以上；而经Taqman鉴定为非转基因的高粱植株和野生型高粱植株的抗性总分一般在20分左右。The results in Table 3 show that: the sorghum plants transferred to the Cry1A.105 nucleotide sequence and the sorghum plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence all have a better insecticidal effect on P. The average mortality rate is about 90%, and the total resistance score is above 280 points; while the total resistance score of non-transgenic sorghum plants and wild-type sorghum plants identified by Taqman is generally around 20 points.
图5的结果表明：与野生型高粱植株相比，转入Cry1A.105核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株可以造成粟灰螟初孵幼虫的大量死亡，且对极少量存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，同时表现出极弱的生命力，且转入Cry1A.105核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株大体上只受到极轻微损伤，肉眼几乎无法辨别出粟灰螟的取食痕迹，其叶片损伤率均在3％以下。The results in Figure 5 show that: compared with wild-type sorghum plants, the sorghum plants transferred to the Cry1A.105 nucleotide sequence and the sorghum plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence can cause the death of newly hatched larvae of P. A large number of larvae died, and the development progress of a very small number of surviving larvae was greatly inhibited. After 3 days, the larvae were basically still in the newly hatched state, and at the same time showed extremely weak vitality, and the sorghum plants and transgenic plants with the Cry1A. The sorghum plants with the Cry1A.105-Cry2Ab nucleotide sequence were generally only slightly damaged, and the feeding traces of the millet moth could hardly be discerned with the naked eye, and the leaf damage rate was below 3%.
转入Cry2Ab核苷酸序列的高粱植株对粟灰螟则没有表现出控制效果，无论是死亡率、叶片损伤率、幼虫发育进度，还是抗性总分，与经Taqman鉴定为非转基因的高粱植株和野生型高粱植株相比，均未表现出差异。The sorghum plants transferred with the Cry2Ab nucleotide sequence did not show any control effect on the militaris militaris, whether in terms of mortality rate, leaf damage rate, larval development progress, or total resistance score, compared with non-transgenic sorghum plants identified by Taqman. None showed differences compared to wild-type sorghum plants.
由此证明转入Cry1A.105核苷酸序列的高粱植株和转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株都显示出高抗粟灰螟的活性，这种活性足以对粟灰螟的生长产生不良效应从而使其在田间得以控制。同时通过控制粟灰螟的钻蛀为害，也有可能降低高粱上病害的发生，极大的提高高粱的产量及品质。This proves that the sorghum plants that have been transferred to the Cry1A.105 nucleotide sequence and the sorghum plants that have been transferred to the Cry1A.105-Cry2Ab nucleotide sequence have shown high activity against the mites, and this activity is sufficient to have an effect on the growth of the mites. Adverse effects are thereby controlled in the field. At the same time, by controlling the borer damage of the millet ash borer, it is also possible to reduce the occurrence of diseases on the sorghum, and greatly improve the yield and quality of the sorghum.
4、转基因谷子植株的抗虫效果检测4. Detection of insect resistance effect of transgenic millet plants
分别取转入Cry1A.105核苷酸序列的谷子植株、转入Cry2Ab核苷酸序列的谷子植株、转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株、野生型谷子植株和经Taqman鉴定为非转基因的谷子植株(展开嫩叶)的新鲜叶片，用无菌水冲洗干净并用纱布将叶片上的水吸干，然后将谷子叶片剪成约1cm×2cm的长条状，取1片剪后的长条状叶片放入圆形塑料培养皿底部的保湿滤纸上，每个培养皿中放10头粟灰螟(初孵幼虫)，虫试培养皿加盖后，在温度22-26℃、相对湿度70％-80％、光周期(光/暗)0:24的条件下放置3天后，根据粟灰螟幼虫发育进度、死亡率和叶片损伤率三项指标，获得抗性总分：总分＝100×死亡率+[100×死亡率+90×(初孵虫数/接虫总数)+60×(初孵-阴性对照虫数/接虫总数)+10×(阴性对照虫数/接虫总数)]+100×(1-叶片损伤率)。转入Cry1A.105核苷酸序列的共3个转化事件株系(S28、S29和S30)，转入Cry2Ab核苷酸序列的共3个转化事件株系(S31、S32和S33)，转入Cry1A.105-Cry2Ab核苷酸序列的共3个转化事件株系(S34、S35和S36)，经Taqman鉴定为非转基因的(NGM4)共1个株系，野生型的(CK4)共1个株系；从每个株系选3株进行测试，每株重复6次。结果如表4和图6所示。The millet plants transferred to the Cry1A.105 nucleotide sequence, the millet plant transferred to the Cry2Ab nucleotide sequence, the millet plant transferred to the Cry1A.105-Cry2Ab nucleotide sequence, the wild-type millet plant and those identified by Taqman as For the fresh leaves of non-transgenic millet plants (open young leaves), rinse them with sterile water and dry the water on the leaves with gauze, then cut the leaves of millet into strips of about 1cm×2cm, take 1 piece and cut it Put the strip-shaped blades of the long strip leaves on the moisture-retaining filter paper at the bottom of the circular plastic culture dish, put 10 millet moths (newly hatched larvae) in each culture dish, and after the worm test culture dish is covered, at a temperature of 22-26 ℃, After standing for 3 days under the conditions of relative humidity 70%-80% and photoperiod (light/dark) 0:24, the total score of resistance was obtained according to the three indicators of developmental progress, mortality rate and leaf damage rate of P. Score=100×mortality+[100×mortality+90×(number of newly hatched worms/total number of worms)+60×(number of newly hatched-negative control worms/total number of worms)+10×(number of negative control worms/ The total number of infested insects)]+100×(1-leaf damage rate). A total of 3 transformation event lines (S28, S29 and S30) transferred into the Cry1A.105 nucleotide sequence, a total of 3 transformation event lines (S31, S32 and S33) transferred into the Cry2Ab nucleotide sequence, transferred into A total of 3 transformation event lines (S34, S35 and S36) of the Cry1A.105-Cry2Ab nucleotide sequence, a total of 1 strain identified as non-transgenic (NGM4) by Taqman, and a total of wild type (CK4) Strains; 3 strains were selected from each strain for testing, and each strain was repeated 6 times. The results are shown in Table 4 and Figure 6.
表4、转基因谷子植株接种粟灰螟的抗虫实验结果Table 4. Insect resistance experiment results of transgenic millet plants inoculated with P. argentina
表4的结果表明：转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株对粟灰螟均具有较好的杀虫效果，粟灰螟的平均死亡率均在90％左右，其抗性总分也均在280分以上；而经Taqman鉴定为非转基因的谷子植株和野生型谷子植株的抗性总分一般在20分左右。The results in Table 4 show that: the millet plants transferred to the Cry1A.105 nucleotide sequence and the millet plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence all have good insecticidal effects on P. The average mortality rate is about 90%, and the total resistance score is above 280 points; while the total resistance score of non-transgenic millet plants and wild-type millet plants identified by Taqman is generally around 20 points.
图6的结果表明：与野生型谷子植株相比，转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株可以造成粟灰螟初孵幼虫的大量死亡，且对极少量存活幼虫发育进度造成极大的抑制，3天后幼虫基本仍处于初孵状态，同时表现出极弱的生命力，且转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株大体上只受到极轻微损伤，肉眼几乎无法辨别出粟灰螟的取食痕迹，其叶片损伤率均在5％以下。The result of Fig. 6 shows: compared with wild-type millet plant, the millet plant that transfers Cry1A.105 nucleotide sequence and the millet plant that transfers Cry1A.105-Cry2Ab nucleotide sequence can cause the larvae of newly hatched larvae of P. A large number of larvae died, and the developmental progress of a very small number of surviving larvae was greatly inhibited. After 3 days, the larvae were basically still in the newly hatched state, and at the same time showed extremely weak vitality. The millet plants with the Cry1A.105-Cry2Ab nucleotide sequence were generally only slightly damaged, and the feeding traces of the millet moth could hardly be discerned with the naked eye, and the leaf damage rate was below 5%.
转入Cry2Ab核苷酸序列的谷子植株对粟灰螟则没有表现出控制效果，无论是死亡率、叶片损伤率、幼虫发育进度，还是抗性总分，与经Taqman鉴定为非转基因的谷子植株和野生型谷子植株相比，均未表现出差异。The millet plants transferred with the Cry2Ab nucleotide sequence showed no control effect on the millet moth, whether it was the mortality rate, leaf damage rate, larval development progress, or the total resistance score, compared with the non-transgenic millet plants identified by Taqman. Compared with wild-type millet plants, no difference was shown.
由此证明转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株都显示出高抗粟灰螟的活性，这种活性足以对粟灰螟的生长产生不良效应从而使其在田间得以控制。同时通过控制粟灰螟的钻蛀为害，也有可能降低谷子上病害的发生，极大的提高谷子的产量及品质。This proves that the millet plants transferred to the Cry1A.105 nucleotide sequence and the millet plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence all show high activity against the mites, and this activity is sufficient for the growth of the milia. Adverse effects are thereby controlled in the field. At the same time, by controlling the borer damage of millet ash borer, it is also possible to reduce the occurrence of diseases on millet and greatly improve the yield and quality of millet.
上述实验结果还表明转入Cry1A.105核苷酸序列的玉米植株、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105核苷酸序列的高粱植株、转入Cry1A.105-Cry2Ab核苷酸序列的高粱植株、转入Cry1A.105核苷酸序列的甘蔗植株、转入Cry1A.105-Cry2Ab核苷酸序列的甘蔗植株、转入Cry1A.105核苷酸序列的谷子植株和转入Cry1A.105-Cry2Ab核苷酸序列的谷子植株对粟灰螟的控制/防治显然是因为植物本身可产生Cry1A.105蛋白，所以，本领域技术人员熟知的，根据Cry1A.105蛋白对粟灰螟的相同毒杀作用，可产生类似的可表达Cry1A.105蛋白的转基因植株能够用于控制/防治粟灰螟的危害。本发明中Cry1A.105蛋白包括但不限于具体实施方式中所给出氨基酸序列的Cry1A.105蛋白，同时转基因植株还可以产生至少一种不同于Cry1A.105蛋白的第二种杀虫蛋白质，如Vip类蛋白、Cry类蛋白。The above experimental results also show that the corn plants transferred to the Cry1A.105 nucleotide sequence, the corn plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence, the sorghum plants transferred to the Cry1A.105 nucleotide sequence, the Cry1A. Sorghum plant with 105-Cry2Ab nucleotide sequence, sugarcane plant with Cry1A.105 nucleotide sequence, sugarcane plant with Cry1A.105-Cry2Ab nucleotide sequence, millet with Cry1A.105 nucleotide sequence Plants and millet plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence control/prevent and control of the pomegranate moth obviously because the plant itself can produce the Cry1A.105 protein, so, as is well known to those skilled in the art, according to the Cry1A.105 protein With the same poisonous effect on C. miliflora, similar transgenic plants expressing Cry1A.105 protein can be produced, which can be used to control/prevent the damage of C. milifolia. The Cry1A.105 protein in the present invention includes but is not limited to the Cry1A.105 protein of the amino acid sequence given in the specific embodiment, and the transgenic plant can also produce at least one second insecticidal protein different from the Cry1A.105 protein, such as Vip-like proteins, Cry-like proteins.
综上所述，本发明杀虫蛋白的用途通过植物体内产生能够杀死粟灰螟的Cry1A.105蛋白来控制粟灰螟害虫；与现有技术使用的农业防治方法、化学防治方法和物理防治方法相比，本发明对植物进行全生育期、全植株的保护以防治粟灰螟害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。In summary, the use of the insecticidal protein of the present invention controls the pest of C. pombe through the production of Cry1A.105 protein capable of killing C. pombe in plants; it is different from the agricultural control methods, chemical control methods and physical control methods used in the prior art Compared with the method, the present invention protects the plants during the whole growth period and the whole plant to prevent and control the damage of the pod borer pest, and has no pollution, no residue, stable and thorough effect, simple, convenient and economical.
最后所应说明的是，以上实施例仅用以说明本发明的技术方案而非限制，尽管参照较佳实施例对本发明进行了详细说明，本领域的普通技术人员应当理解，可以对本发明的技术方案进行修改或者等同替换，而不脱离本发明技术方案的精神和范围。Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The scheme shall be modified or equivalently replaced without departing from the spirit and scope of the technical scheme of the present invention.
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| CN201410806943.3ACN104522033B (en) | 2014-12-22 | 2014-12-22 | The use of insecticidal protein |
| PCT/CN2015/092007WO2016101684A1 (en) | 2014-12-22 | 2015-10-15 | Uses of insecticidal protein |
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| CN201410806943.3ACN104522033B (en) | 2014-12-22 | 2014-12-22 | The use of insecticidal protein |
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| CN104824010B (en)* | 2015-05-20 | 2018-06-19 | 北京大北农科技集团股份有限公司 | The purposes of insecticidal proteins |
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| WO2016101684A1 (en) | 2016-06-30 |
| CN104522033A (en) | 2015-04-22 |
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| PB01 | Publication | ||
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| CB02 | Change of applicant information | Address after:100080 Zhongguancun street, Beijing, No. 14, layer 27, Applicant after:Beijing Dabeinong Technology Group Co., Ltd. Applicant after:BEIJING DBN BIOTECH CO., LTD. Address before:100080 Zhongguancun street, Beijing, No. 14, layer 27, Applicant before:Beijing Dabeinong Technology Group Co., Ltd. Applicant before:Biotechnology Center of Beijing Dabeinong Technology Group Co., Ltd. | |
| COR | Change of bibliographic data | ||
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| TR01 | Transfer of patent right | Effective date of registration:20200907 Address after:100193 No. 2, West Old Summer Palace Road, Beijing, Haidian District, Institute of atomic energy, Chinese Academy of Agricultural Sciences, building 49 Patentee after:BEIJING DABEINONG BIOTECHNOLOGY Co.,Ltd. Address before:100080, 14, Zhongguancun Avenue, 27, Beijing, Haidian District Co-patentee before:BEIJING DABEINONG BIOTECHNOLOGY Co.,Ltd. Patentee before:BEIJING DABEINONG TECHNOLOGY GROUP Co.,Ltd. |