技术领域technical field
本发明涉及一种控制害虫的方法，特别是涉及一种用在植物中表达的Cry1A.105蛋白来控制二点委夜蛾危害植物的方法。The invention relates to a method for controlling pests, in particular to a method for controlling plant damage to plants by using Cry1A.105 protein expressed in plants.
背景技术Background technique
二点委夜蛾（Athetis lepigone）属鳞翅目夜蛾科，为杂食性害虫，目前发现其主要危害玉米作物，主要分布于我国黄淮海夏玉米种植区，在境外主要分布于日本、朝鲜、俄罗斯、欧洲等地。二点委夜蛾主要在玉米气生根处的土壤表层处危害玉米根部，咬断玉米地上茎杆或浅表层根，受危害的玉米田轻者玉米植株东倒西歪，重者造成缺苗断垄，玉米田中出现大面积空白地，危害严重地块甚至毁种。Athetis lepigone belongs to Lepidoptera Noctuidae and is an omnivorous pest. At present, it is found that it mainly harms corn crops. It is mainly distributed in the summer corn planting areas of my country's Huanghuaihai, and abroad mainly in Japan, North Korea, Russia, Europe and other places. The two-spot moth mainly harms the corn roots at the soil surface of the aerial roots of corn, and bites off the corn stems or superficial roots of the corn. A large area of blank land appears, seriously endangering plots and even destroying species.
玉米是中国主要的粮食作物，2011年7月9日，中央电视台新闻联播首次报道二点委夜蛾在中国的发生情况，2011年秋季至2012年5月31日，国家玉米产业体系病虫防控研究室通过多次田间调查，发现2012年度二点委夜蛾的越冬种群数量较大，虫口基数偏高，一代幼虫种群密度大，在黄淮海夏玉米苗期有继续暴发为害的可能。为了防治二点委夜蛾，人们目前主要采用的防治方法有：农业防治和化学防治。Corn is the main food crop in China. On July 9, 2011, CCTV News Network reported for the first time the occurrence of Noctuid moth in China. From the autumn of 2011 to May 31, 2012, the National Corn Industry System Disease and Pest Prevention Through several field surveys, the control laboratory found that the number of overwintering populations of Spodoptera spp. in 2012 was large, the population base was high, and the population density of the first generation larvae was high. There is a possibility of continued outbreaks and damage in the seedling stage of Huanghuaihai summer corn. In order to prevent and control Spodoptera spp., the control methods that people mainly adopt at present are: agricultural control and chemical control.
农业防治是对整个农田生态系统中多个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二点委夜蛾发生的农田生态环境，如及时清除玉米苗基部麦秸、杂草等覆盖物至远离植株的玉米大行间并裸露出地面，以便于后续用药剂能直接接触到二点委夜蛾。因农业防治必须服从作物布局和增产的要求，应用有一定的局限性，不能作为应急措施，在二点委夜蛾爆发时就显得无能为力。Agricultural prevention and control is the comprehensive and coordinated management of multiple factors in the entire farmland ecosystem, regulating crops, pests, and environmental factors to create a farmland ecological environment that is conducive to crop growth but not conducive to the occurrence of spodoptera, such as timely removal of corn seedlings Cover the base with wheat straw and weeds to the large corn rows far away from the plants and expose the ground, so that the subsequent application of pesticides can directly contact the spodoptera. Because agricultural control must obey the requirements of crop distribution and yield increase, its application has certain limitations, and it cannot be used as an emergency measure, and it is powerless when the two-pointed armyworm breaks out.
化学防治即农药防治，是利用化学杀虫剂来杀灭害虫，是二点委夜蛾综合治理的重要组成部分，它具有快速、方便、简便和高经济效益的特点，特别是二点委夜蛾大发生的情况下，是必不可少的应急措施，它可以一定程度地在二点委夜蛾造成危害前降低虫口密度。目前化学防治方法主要有毒饵法、毒土法、灌药法和喷施农药等。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；且因二点委夜蛾喜潮湿、阴暗环境，一般躲藏于麦秸等覆盖物或者土表之下，使得化学农药很难直接接触二点委夜蛾虫体而使得防治效果不佳。Chemical control, that is, pesticide control, is the use of chemical insecticides to kill pests. It is an important part of the comprehensive control of the two-pointed moth. It is fast, convenient, simple and high-economic benefits, especially In the case of moth outbreaks, it is an essential emergency measure, which can reduce the population density to a certain extent before the second-point moths cause harm. At present, the chemical control methods mainly include poisonous bait method, poisonous soil method, irrigation method and spraying pesticide and so on. 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; and because Spodoptera moths prefer humid and dark environments, they generally hide under coverings such as wheat straw or under the soil surface, making it difficult for chemical pesticides to directly contact Spodoptera moths, resulting in poor control effects.
为了解决农业防治和化学防治在实际应用中的局限性，科学家们经过研究发现将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，是由苏云金芽孢杆菌库斯塔基亚种（Bacillus thuringiensis subsp.kurstaki，B.t.k.）产生的伴孢结晶蛋白。In order to solve the limitations of agricultural control and chemical control in practical application, scientists have discovered through research that by transferring insect-resistant genes encoding insecticidal proteins into plants, some insect-resistant transgenic plants can be obtained to prevent plant pests. Cry1A.105 insecticidal protein is one of many insecticidal proteins, and it is a parasporal crystal protein produced by Bacillus thuringiensis subsp. kurstaki (B.t.k.).
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、棉铃虫、小地老虎等鳞翅目（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二点委夜蛾对植物危害的报道。It has been proved that plants transgenic for Cry1A.105 can resist Lepidoptera pests such as corn borer, cotton bollworm, and cutworm. Report on plant damage caused by noctuid moths.
发明内容Contents of the invention
本发明的目的是提供一种控制害虫的方法，首次提供了通过产生表达Cry1A.105蛋白的转基因植株来控制二点委夜蛾对植物危害的方法，且有效克服现有技术农业防治和化学防治等技术缺陷。The purpose of the present invention is to provide a method for controlling pests, and for the first time provides a method for controlling the damage of Spodoptera spp. to plants by producing transgenic plants expressing Cry1A.105 protein, and effectively overcomes the prior art agricultural control and chemical control and other technical defects.
为实现上述目的，本发明提供了一种控制二点委夜蛾害虫的方法，包括将二点委夜蛾害虫与Cry1A.105蛋白接触。To achieve the above object, the present invention provides a method for controlling the pest of Spodoptera spp., comprising contacting the pest of Spodoptera spp. with Cry1A.105 protein.
进一步地，所述Cry1A.105蛋白存在于产生所述Cry1A.105蛋白的植物细胞中，所述二点委夜蛾害虫通过摄食所述植物细胞与所述Cry1A.105蛋白接触。Further, the Cry1A.105 protein exists in the plant cells that produce the Cry1A.105 protein, and the Cry1A.105 protein comes into contact with the Cry1A.105 protein by ingesting the plant cells.
更进一步地，所述Cry1A.105蛋白存在于产生所述Cry1A.105蛋白的转基因植物中，所述二点委夜蛾害虫通过摄食所述转基因植物的组织与所述Cry1A.105蛋白接触，接触后所述二点委夜蛾害虫生长受到抑制并最终导致死亡，以实现对二点委夜蛾危害植物的控制。Furthermore, the Cry1A.105 protein exists in the transgenic plant that produces the Cry1A.105 protein, and the Cry1A.105 protein is in contact with the Cry1A.105 protein by feeding on the tissue of the transgenic plant. The growth of the above-mentioned spodoptera pests is inhibited and eventually leads to death, so as to realize the control of plants damaged by the spodoptera.
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可以为根、叶片、茎秆、雄穗、雌穗、花药或花丝。The tissue of the transgenic plant may be root, leaf, stem, tassel, ear, anther or filament.
所述对二点委夜蛾危害植物的控制不因种植地点的改变而改变。The control of the plants harmed by the second-spotted armyworm does not change due to the change of the planting site.
所述对二点委夜蛾危害植物的控制不因种植时间的改变而改变。The control of plants harmed by the second-spotted armyworm does not change due to changes in planting time.
所述植物可以为玉米。The plant may be corn.
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，所述植物还可以产生至少一种不同于所述Cry1A.105蛋白的第二种核苷酸。On the basis of the above technical solution, the plant can also produce at least one second nucleotide different from the Cry1A.105 protein.
进一步地，所述第二种核苷酸可以编码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 may encode Cry-type insecticidal protein, Vip-type insecticidal protein, protease inhibitor, lectin, α-amylase or peroxidase.
优选地，所述第二种核苷酸可以编码Cry2Ab蛋白或Vip3A蛋白。Preferably, the second nucleotide can encode Cry2Ab protein or Vip3A protein.
更进一步地，所述第二种核苷酸包括SEQ ID NO:3或SEQ ID NO:4所示的核苷酸序列。Further, the second nucleotide comprises the nucleotide sequence shown in SEQ ID NO:3 or 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蛋白质控制二点委夜蛾害虫的用途。In order to achieve the above object, the present invention also provides a use of Cry1A.105 protein to control the pest of Spodoptera exigua.
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.
二点委夜蛾（Athetis lepigone）与小地老虎（Agrotis ypsilon Rottemberg）同属鳞翅目夜蛾科，尽管其侵害部位和形态上相近，但是二点委夜蛾与小地老虎在生物学上是清晰的、截然不同的两个物种，至少存在以下主要区别：Athetis lepigone and Agrotis ypsilon Rottemberg belong to the family Noctuidae of the order Lepidoptera. Although their infestation sites and shapes are similar, Athetis lepigone and Agrotis ypsilon Rottemberg are biologically related. Two distinct and distinct species with at least the following major differences:
1、食性不同。二点委夜蛾除严重威胁夏玉米外，还对花生和大豆也造成危害；而小地老虎为杂食性害虫，是对农、林木幼苗危害很大的地下害虫，除了危害玉米、高粱、粟等作物外，在东北主要危害落叶松、红松、水曲柳、核桃楸等苗木，在南方危害马尾松、杉木、桑、茶等苗木，在西北危害油松、沙枣、果树等苗木。1. Food habits are different. In addition to seriously threatening summer corn, the two-spot moth also causes damage to peanuts and soybeans; and the small cutworm is an omnivorous pest, which is a great underground pest to agricultural and forest seedlings. In addition to harming corn, sorghum, millet In addition to other crops, it mainly harms larch, Korean pine, ash, walnut and other seedlings in the northeast, masson pine, Chinese fir, mulberry, tea and other seedlings in the south, and damages Chinese pine, Chinese date, fruit trees and other seedlings in the northwest.
2、分布区域不同。目前二点委夜蛾主要在黄淮海夏玉米区的河北、山东、河南、山西、江苏、安徽共6省47个地（市）297个县（市、区）暴发危害；而小地老虎在我国以雨量丰富、气候湿润的长江流域和东南沿海发生量大，东北地区多发生在东部和南部湿润地区。2. The distribution area is different. At present, noctuid moths of the Second Point Committee are mainly in 297 counties (cities, districts) in 47 places (cities) in 6 provinces of Hebei, Shandong, Henan, Shanxi, Jiangsu, and Anhui in the Huanghuaihai summer corn region; In my country, the Yangtze River Basin with abundant rainfall and humid climate and the southeast coast have a large amount of occurrence, and the northeast region mostly occurs in the eastern and southern humid areas.
3、为害习性不同。二点委夜蛾在河北省部分夏玉米，尤其以小麦套播的玉米田发生重，主要以幼虫躲在玉米幼苗周围的碎麦秸下或在2-5厘米的表土层危害玉米苗，一般一株有虫1-2头，多的达10-20头；在玉米幼苗3-5叶期的地块，幼虫主要咬食玉米茎基部，形成3-4毫米圆形或椭圆形孔洞，切断营养输送，造成地上部玉米心叶萎蔫枯死；在玉米苗较大（8-10叶期）的地块幼虫主要咬断玉米根部，包括气生根和主根，造成玉米倒伏，严重者枯死。危害株率一般在1%-5%，严重地块达15%-20%。而小地老虎1-2龄幼虫昼夜均可群集于幼苗顶心嫩叶处取食危害；3龄后分散，幼虫行动敏捷、有假死习性、对光线极为敏感、受到惊扰即卷缩成团，白天潜伏于表土的干湿层之间，夜晚出土从地面将幼苗植株咬断拖入土穴、或咬食未出土的种子，幼苗主茎硬化后改食嫩叶和叶片及生长点，食物不足或寻找越冬场所时，有迁移现象。3. Different damage habits. Spodoptera moths occur heavily in some summer corn in Hebei Province, especially in cornfields inter-sowed with wheat. The larvae mainly hide under the broken wheat straw around the corn seedlings or damage the corn seedlings in the top soil layer of 2-5 cm. There are 1-2 worms per plant, and up to 10-20 worms; in the 3-5 leaf stage of corn seedlings, the larvae mainly bite the base of the corn stem, forming 3-4mm circular or oval holes, cutting off nutrients Transportation causes the aboveground corn heart leaves to wilt and die; in plots with large corn seedlings (8-10 leaf stage), larvae mainly bite off the corn roots, including aerial roots and taproots, causing corn lodging and severe cases to die. The rate of harmful plants is generally 1%-5%, and it is 15%-20% in serious plots. However, the 1-2 instar larvae of the cutworm can gather at the young leaves at the top of the seedlings day and night to eat and harm; after the 3rd instar, they disperse, the larvae are agile, have the habit of feigning death, are extremely sensitive to light, and curl up into balls when disturbed. It lurks between the dry and wet layers of the topsoil during the day, unearths at night and bites off the seedlings from the ground and drags them into the soil hole, or bites the unearthed seeds. After the main stem of the seedlings hardens, they eat young leaves, leaves and growth points. There is insufficient food or Migration occurs when looking for wintering places.
4、形态特征不同。4. Different morphological characteristics.
1）卵的形态不同：二点委夜蛾的卵成馒头状，上有纵脊，初产黄绿色，后土黄色；而小地老虎的卵亦成馒头形，具纵横隆线，初产乳白色，渐变黄色，孵化前卵一顶端具黑点。1) The shape of the eggs is different: the eggs of Spodoptera spp. are in the shape of steamed buns, with vertical ridges on them, and the first lay is yellow-green, and later the eggs are earthy yellow; while the eggs of cutworms are also in the shape of steamed buns, with vertical and horizontal ridges, and the eggs of the first lay Milky white, gradually changing to yellow, with black spots on the top of the eggs before hatching.
2）幼虫的形态不同：二点委夜蛾的老熟幼虫体长20mm左右，体色灰黄色，头部褐色，幼虫14-18mm长，黄灰色或黑褐色，比较明显的特征是个体节有一个倒三角的深褐色斑纹，腹部背面有两条褐色背侧线，到胸节消失；而小地老虎幼虫圆筒形，老熟幼虫体长37-50mm，头部褐色，具黑褐色不规则网纹，体灰褐至暗褐色，体表粗糙、布大小不一而彼此分离的颗粒，背线、亚背线及气门线均黑褐色，前胸背板暗褐色，黄褐色臀板上具两条明显的深褐色纵带，胸足与腹足黄褐色。2) The shape of the larvae is different: the mature larvae of Spodoptera bispotentum have a body length of about 20mm, a grayish yellow body color, and a brown head. The larvae are 14-18mm long, yellowish gray or dark brown. An inverted triangular dark brown stripe, two brown dorsal lines on the back of the abdomen, disappearing to the thoracic segment; while the larvae of the cutworm are cylindrical, the mature larvae are 37-50mm long, the head is brown, with dark brown irregular nets Stretch, grayish brown to dark brown body, rough body surface, cloth of different sizes and separate particles, topline, sub-topline and valve line are all dark brown, pronotum dark brown, yellowish brown buttocks with two Obvious dark brown longitudinal bands, thoracic and abdominal legs yellowish brown.
3）蛹的形态不同：二点委夜蛾的蛹长10mm左右，化蛹初期淡黄褐色，逐渐变为褐色，老熟幼虫入土做一丝质土茧包被内化蛹；而小地老虎的蛹长18-24mm，赤褐有光，口器与翅芽末端相齐，均伸达第4腹节后缘，腹部第4-7节背面前缘中央深褐色，且有粗大的刻点，两侧的细小刻点延伸至气门附近，第5-7节腹面前缘也有细小刻点，腹末端具短臀棘1对。3) The shape of pupae is different: the pupae of Spodoptera spp. is about 10mm long, light yellowish brown in the early stage of pupation, and gradually turn brown, mature larvae burrow into the soil and make a silky soil cocoon to pupate inside; The pupa is 18-24mm long, reddish brown and shiny, the mouthparts are aligned with the end of the wing buds, both extend to the rear edge of the 4th abdominal segment, the center of the front edge of the back of the 4th-7th abdominal segment is dark brown, and there are thick punctures, The small punctures on both sides extend to the vicinity of the stigma, and there are also small punctures on the front edge of the ventral segment 5-7, and there is a pair of short gluteal spines at the end of the abdomen.
4）成虫的形态不同：二点委夜蛾成虫体长10-12mm，翅展20mm，雌虫会略大于雄虫，头、胸、腹灰褐色，前翅灰褐色，有暗褐色细点，内线、外线暗褐色，环纹为一黑点，肾纹小，有黑点组成的边缘，外侧中凹，有一白点，外线波浪型，翅外缘有一列黑点，后翅白色微褐，端区暗褐色，腹部灰褐色，雄蛾外生殖器的抱器瓣端半部宽，背缘凹，中部有一钩状突起，阳茎内有刺状阳茎针；而小地老虎成虫体长17-23mm、翅展40-54mm，头、胸部背面暗褐色，足褐色，前足胫、跗节外缘灰褐色，中后足各节末端有灰褐色环纹，前翅褐色，前缘区黑褐色，外缘以内多暗褐色，基线浅褐色，黑色波浪形内横线双线，黑色环纹内有一圆灰斑，肾状纹黑色具黑边、其外中部有一楔形黑纹伸至外横线，中横线暗褐色波浪形，双线波浪形外横线褐色，不规则锯齿形亚外缘线灰色、其内缘在中脉间有三个尖齿，亚外缘线与外横线间在各脉上有小黑点，外缘线黑色，外横线与亚外缘线间淡褐色，亚外缘线以外黑褐色，后翅灰白色，纵脉及缘线褐色，腹部背面灰色。4) The shape of the adults is different: the body length of the adult moth is 10-12mm, and the wingspan is 20mm. The female is slightly larger than the male. The outer line is dark brown, the ring pattern is a black spot, the kidney pattern is small, there is a black spot on the edge, the outer side is concave, and there is a white spot, the outer line is wavy, there is a row of black spots on the outer edge of the wing, the hind wing is white and slightly brown, and the terminal area Dark brown, gray-brown abdomen, half of the genitalia of the male moth is wide, the dorsal edge is concave, there is a hook-like protrusion in the middle, and there are thorn-like penis needles in the penis; while the body length of the adult cutworm is 17-23mm , Wingspan 40-54mm, head and back of thorax dark brown, legs brown, forefoot tibia and outer edge of tarsus grayish brown, middle and hind legs with grayish brown rings at the end of each segment, forewing brown, anterior region dark brown, outer edge Dark brown inside, light brown base line, double black wavy inner horizontal line, a round gray spot inside the black ring, black kidney-shaped stripe with black edge, a wedge-shaped black stripe in the outer middle extending to the outer horizontal line, middle horizontal Line dark brown wavy, double-line wavy outer transverse line brown, irregular zigzag subouter border line gray, its inner margin has three sharp teeth between the midvein, subouter border line and outer transverse line on each vein Small black spots, black outer margin line, light brown between outer transverse line and sub-outer margin line, dark brown outside sub-outer margin line, hindwing grayish white, longitudinal vein and margin line brown, abdomen back gray.
5、生长习性和发生规律不同。二点委夜蛾幼虫一共6龄，幼虫期约18天，幼虫的抗逆性较强；二点委夜蛾成虫有两个明显的蛾峰：7月初前出现第1个蛾峰，7月中下旬到8月上中旬出现第2个蛾峰；成虫具有较强的生殖能力：平均单雌产卵量可达300-500粒，产卵可持续3-7天，而卵的孵化率接近100%；棉田倒茬玉米田比重茬玉米田发生严重，麦糠麦秸覆盖面积大比没有麦秸麦糠覆盖的严重，播种时间晚比播种时间早的严重，田间湿度大比湿度小的严重，二点委夜蛾喜阴湿环境，往往躲藏在麦秸或土中，给喷施农药造成了极大的不便。而小地老虎一年发生3-4代，老熟幼虫或蛹在土内越冬；早春3月上旬成虫开始出现，一般在3月中下旬和4月上中旬会出现两个发蛾盛期；成虫白天不活动，傍晚至前半夜活动最盛，喜欢吃酸、甜、酒味的发酵物和各种花蜜，并有趋光性，幼虫共分6龄，1、2龄幼虫先躲伏在杂革或植株的心叶里，昼夜取食，这时食量很小，为害也不十分显著；3龄后白天躲到表土下，夜间出来为害；5、6龄幼虫食量大增，每条幼虫一夜能咬断菜苗4-5株，多的达l0株以上；幼虫3龄后对药剂的抵抗力显著增加；3月底到4月中旬是第1代幼虫危害的严重时期；发生世代从10月到第2年4月都见发生和危害；西北地区二到三代，长城以北一般年二到三代，长城以南黄河以北年三代，黄河以南至长江沿岸年四代，长江以南年四到五代，南亚热带地区年六至七代；无论年发生代数多少，在生产上造成严重危害的均为第一代幼虫；南方越冬代成虫二月份出现，全国大部分地区羽化盛期在3月下旬至4月上、中旬，宁夏、内蒙古为4月下旬；小地老虎成虫多在下午3时至晚上10时羽化，白天潜伏于杂物及缝隙等处，黄昏后开始飞翔、觅食，3-4天后交配、产卵；卵散产于低矮叶密的杂草和幼苗上、少数产于枯叶、土缝中，近地面处落卵最多，每雌产卵800-1000粒、多达2000粒；卵期约5天左右，幼虫6龄、个别7-8龄，幼虫期在各地相差很大，但第一代约为30-40天；幼虫老熟后在深约5cm土室中化蛹，蛹期约9-19天；高温对小地老虎的发育与繁殖不利，因而夏季发生数量较少，适宜生存温度为15℃-25℃；冬季温度过低，小地老虎幼虫的死亡率增高；凡地势低湿，雨量充沛的地方，发生较多；头年秋雨多、土壤湿度大、杂草丛生有利于成虫产卵和幼虫取食活动，是第二年大发生的预兆；但降水过多，湿度过大，不利于幼虫发育，初龄幼虫淹水后很易死亡；成虫产卵盛期土壤含水量在15-20%的地区危害较重；沙壤土，易透水、排水迅速，适于小地老虎繁殖，而重黏土和沙土则发生较轻。5. The growth habits and occurrence rules are different. There are a total of 6 instars of the larvae of the two-pointed moth, and the larval period is about 18 days. The larvae have strong resistance to stress; The second moth peak appears from the middle to late August to the first and middle ten days of August; adults have strong reproductive ability: the average number of eggs laid by a single female can reach 300-500, and the egg laying can last for 3-7 days, and the hatching rate of eggs is close to 100%; Inverted stubble corn fields are more serious than heavy crop corn fields in cotton fields, wheat bran coverage is more serious than wheat bran coverage, late sowing time is more serious than early sowing time, field humidity is more serious than low humidity, two points Spodoptera moths like damp and damp environments, and often hide in wheat straw or soil, causing great inconvenience to spraying pesticides. The small cutworm produces 3-4 generations a year, and mature larvae or pupae overwinter in the soil; adults begin to appear in early March in early spring, and generally there will be two moth moth peaks in mid-to-late March and early-to-mid April; Adults are inactive during the day, and are most active in the evening to the first half of the night. They like to eat sour, sweet, and wine-flavored fermented products and various nectars, and are phototaxis. The larvae are divided into 6 instars. In the heart leaves of the plant, it feeds day and night. At this time, the food intake is very small, and the damage is not very obvious; after the 3rd instar, it hides under the surface soil during the day, and comes out to do damage at night; the food intake of the 5th and 6th instar larvae increases greatly, and each larva can bite overnight. Broken vegetable seedlings are 4-5 plants, most of which are more than 10 plants; the resistance of larvae to pesticides increases significantly after the 3rd instar; the end of March to mid-April is a serious period of damage to the first generation of larvae; the occurrence of generations is from October to the second Occurs and harms in April every year; two to three generations in Northwest China, two to three generations in the north of the Great Wall, three generations in the south of the Great Wall and north of the Yellow River, four generations in the south of the Yellow River to the Yangtze River, and four to five generations in the south of the Yangtze River , six to seven generations per year in subtropical regions; no matter how many generations occur in a year, it is the first-generation larvae that cause serious damage to production; the overwintering adults appear in February in the south, and the peak eclosion period in most parts of the country is from late March to late March. Early and mid-April, and late April in Ningxia and Inner Mongolia; adults of cutworms mostly emerge from 3:00 pm to 10:00 pm, hide in sundries and crevices during the day, and start flying and looking for food after dusk, 3-4 Mating and laying eggs after days; eggs are scattered on low-leaved weeds and seedlings, a few in dead leaves and soil crevices, the most eggs are dropped near the ground, and each female lays 800-1000 eggs, up to 2000 Granules; the egg stage is about 5 days, the larvae are 6 instars, and some are 7-8 instars. The larval stage varies greatly in different places, but the first generation is about 30-40 days; after the larvae are mature, they will melt in a soil chamber about 5cm deep Pupa, the pupal period is about 9-19 days; high temperature is not good for the development and reproduction of small cutworms, so the number of occurrences in summer is small, and the suitable survival temperature is 15°C-25°C; if the temperature is too low in winter, the mortality rate of small cutworm larvae Increased; where the terrain is low and humid, there are more occurrences; the first year of autumn rain, high soil humidity, and overgrown weeds are conducive to the oviposition of adults and larvae feeding activities, which is a harbinger of large occurrences in the second year; but excessive precipitation If there is too much humidity, it is not conducive to the development of larvae, and the first instar larvae are easy to die after being flooded; the area where the soil moisture content is 15-20% in the peak oviposition period of adults is more harmful; the sandy loam soil is easy to permeate and drain quickly, suitable for Small cutworms reproduce, while heavy clay and sandy soils produce lighter ones.
综合上述，可确定二点委夜蛾与小地老虎是两种害虫，且亲缘关系较远，无法交配产生后代。Based on the above, it can be determined that the two-pointed moth and the cutworm are two kinds of pests, and their relationship is far away, so they cannot mate to produce offspring.
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和PNA（肽核酸）。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 (codons are 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 the protein or peptide of interest. The present invention also includes RNA and PNA (peptide nucleic acid) that are functionally equivalent to the exemplified DNA.
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. Appropriate stringent conditions to promote DNA hybridization, for example, treatment with 6.0× sodium chloride/sodium citrate (SSC) at approximately 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 with SEQ ID NO: 2 specific hybridization, 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, the sequence having insect-resistant activity and hybridizing with 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 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二点委夜蛾害虫的生物活性的蛋白。The genes and proteins described in the present invention not only include specific example sequences, but also include parts and/or fragments (compared with the full-length protein and/or terminal parts) that preserve the insecticidal activity characteristics of the specific example 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 having the same or substantially the same biological activity against Spodoptera exigua pests as the claimed protein.
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 long enough 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. 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 that 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 in Protein, Academic Press, New York, 1979. 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，Science255：306-312；Smith等，1992，J.Mol.Biol224:899-904；Wlodaver等，1992，FEBS Letters309：59-64）。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 thus 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Cry1A.105蛋白，或者与上述蛋白的氨基酸序列具有至少70%同源性且对二点委夜蛾具有杀虫活性的杀虫片段或功能区域。In the present invention, the Cry1A.105 protein includes but is not limited to the Cry1A.105 protein, or an insecticidal fragment or functional region that has at least 70% homology to the amino acid sequence of the above-mentioned protein and has insecticidal activity against Spodoptera exigua .
因此，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。Therefore, 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 present 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% identical and/or similar.
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, 35S promoter derived from cauliflower mosaic virus, maize ubi promoter, rice GOS2 gene promoter 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 called secretory signal sequence or targeting sequence) directs the transgene product to a specific organelle or cellular compartment. The transit peptide can be heterologous to the receptor protein, for example, using a gene encoding a 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 potyvirus 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 that are functional for replication (i.e., bacterial origins of replication, autonomously replicating sequences, centromere sequences).
本发明中所述的“杀虫”是指对农作物害虫是有毒的。更具体地，目标昆虫是二点委夜蛾害虫。The term "insecticide" in the present invention means that it is toxic to crop pests. More specifically, the target insect is the pest of Spodoptera exigua.
本发明中Cry1A.105蛋白对二点委夜蛾害虫具有毒性。本发明中的植物，特别是玉米，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，二点委夜蛾害虫通过摄食植物组织与该蛋白接触，接触后二点委夜蛾害虫生长受到抑制并最终导致死亡。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蛋白所靶向的二点委夜蛾害虫的杀虫剂）。The Cry1A.105 protein of the present invention is toxic to the pest of Spodoptera spp. Plants in the present invention, especially maize, contain exogenous DNA in its genome, and described exogenous DNA comprises the nucleotide sequence of coding Cry1A.105 protein, and the pests of the two-spotted armyworm come into contact with the protein by ingesting plant tissues , the growth of Spodoptera moth pests is inhibited after exposure and eventually leads to death. 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 against the Cry1A.105 protein-targeted Spodoptera pest.
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。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 application of specific primers to quantify mRNA encoding insecticidal proteins produced in tissues, 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二点委夜蛾。Different assays can be used to determine the insecticidal efficacy of ICPs in plants. In the present invention, the target insects are mainly Spodoptera spp.
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，例如编码第二种杀虫核苷酸、编码选择性标记的蛋白质或赋予除草剂抗性的蛋白质的编码区。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 a coding region encoding a second pesticidal nucleotide, a protein encoding a selectable marker, or a protein that confers herbicide resistance.
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 resistance genes (e.g. bar gene, pat gene), bendichlor resistance gene (e.g. pmph gene), glyphosate resistance gene (e.g. EPSPS gene), bromoxynil (bromoxynil) resistance gene, sulfonate ureide resistance gene, resistance gene to herbicide palapat, resistance gene to cyanamide or resistance gene to glutamine synthetase inhibitors (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.
本发明提供了一种控制害虫的方法，具有以下优点：The invention provides a method for controlling pests, 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二点委夜蛾害虫的危害，如农业防治和化学防治；而本发明是通过植物体内产生能够杀死二点委夜蛾的Cry1A.105蛋白来控制二点委夜蛾害虫的，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 Spodoptera spp. pests through external effects, that is, external causes, such as agricultural control and chemical control; and the present invention is to produce Cry1A.105 protein that can kill Spodoptera spp. in plants. The control of the two-spot moth pest is to prevent and control 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二点委夜蛾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；使用本发明控制二点委夜蛾害虫的方法，可以消除上述不良后果。2. No pollution and no residue. Although the chemical control methods used in the prior art have played a certain role in controlling the harm of the two-spot moth pest, they have also brought pollution, destruction and residue to people, livestock and farmland ecosystems; use the present invention to control two-spot The method of eliminating the pests of the armyworm can eliminate the above-mentioned adverse consequences.
3、全生育期防治。现有技术使用的控制二点委夜蛾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，都可以避免遭受二点委夜蛾的侵害。3. Prevention and treatment during the whole growth period. The methods used in the prior art to control the pests of Spodoptera spp. are staged, but the present invention protects the plants throughout their growth period, and the transgenic plants (Cry1A.105 protein) start from germination, growth, flowering, As a result, all of them can avoid being attacked by the two-spot moth.
4、全植株防治。现有技术使用的控制二点委夜蛾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）的根、叶片、茎秆、雄穗、雌穗、花药、花丝等都是可以抵抗二点委夜蛾侵害的。4. Whole plant control. Most of the methods used in the prior art to control the pests of Spodoptera exigua are localized, such as foliar spraying; while the present invention protects the whole plant, such as the roots, leaves, and leaves of transgenic plants (Cry1A. The stalks, tassels, ears, anthers, filaments, etc. are all resistant to the attack of the two-spotted moth.
5、效果稳定。现有技术使用的喷施农药的方法需要直接喷施到作物表面，容易造成喷施不均匀或漏喷等情况；本发明是使所述Cry1A.105蛋白在植物体内进行表达，表达量基本上稳定一致，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 method of spraying pesticides used in the prior art needs to be directly sprayed on the surface of crops, which is likely to cause uneven spraying or missed spraying; the present invention expresses the Cry1A.105 protein in plants, and the expression level is basically Stable and consistent, 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。由于二点委夜蛾特殊的隐蔽发生与危害特征，导致对其危害的监测和防治较为困难，大大地增加了种植成本；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. Due to the special concealed occurrence and harm characteristics of Spodoptera spp., it is difficult to monitor and control its harm, which greatly increases the cost of planting; the present invention only needs to plant transgenic plants that can express the Cry1A.105 protein, and There is no need to adopt other measures, thereby saving a lot of manpower, material resources and financial resources.
7、效果彻底。现有技术使用的控制二点委夜蛾害虫的方法，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）可以造成初孵二点委夜蛾幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，都是明显的发育不良，且已停止发育，很难再对玉米造成危害，而转基因植物大体上只受到轻微损伤。7. The effect is thorough. The methods used in the prior art to control the pests of Spodoptera spp. are incomplete and only play a role in mitigating; while the transgenic plant (Cry1A.105 protein) of the present invention can cause the larvae of Spodoptera sp. larvae to hatch. A large number of deaths and great inhibition of the developmental progress of a small number of surviving larvae are obvious stunting, and have stopped developing, and it is difficult to cause harm to the corn, while the transgenic plants generally suffer only slight 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为本发明控制害虫的方法的含有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 of the method for controlling pests of the present invention;
图2为本发明控制害虫的方法的含有Cry1A.105核苷酸序列的重组表达载体DBN100032构建流程图；Fig. 2 is the construction flowchart of the recombinant expression vector DBN100032 containing the Cry1A.105 nucleotide sequence of the method for controlling pests of the present invention;
图3为本发明控制害虫的方法的转基因玉米植株接种二点委夜蛾的叶片损伤图。Fig. 3 is a diagram of leaf damage of transgenic corn plants inoculated with Spodoptera sp. in the method for controlling pests of the present invention.
具体实施方式Detailed ways
下面通过具体实施例进一步说明本发明控制害虫的方法的技术方案。The technical scheme of the method for controlling pests of the present invention is further illustrated below through specific examples.
第一实施例、Cry1A.105基因的获得和合成The first embodiment, the acquisition and synthesis of Cry1A.105 gene
1、获得Cry1A.105核苷酸序列1. Obtain the nucleotide sequence of Cry1A.105
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 is shown in SEQ ID NO: 1 in the sequence listing; the Cry1A.105 core encoding the amino acid sequence (1177 amino acids) corresponding to the Cry1A.105 insecticidal protein Nucleotide sequence (3534 nucleotides), as shown in SEQ ID NO:2 in the sequence listing.
2、获得Cry2Ab和Vip3A核苷酸序列2. Obtain the nucleotide sequences of Cry2Ab and Vip3A
编码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:3所示；编码Vip3A杀虫蛋白质的氨基酸序列（789个氨基酸）的Vip3A核苷酸序列（2370个核苷酸），如序列表中SEQ ID NO:4所示。The Cry2Ab nucleotide sequence (1905 nucleotides) encoding the amino acid sequence (634 amino acids) of the Cry2Ab insecticidal protein, as shown in SEQ ID NO:3 in the sequence listing; the amino acid sequence (789 nucleotides) encoding the Vip3A insecticidal protein amino acid) Vip3A nucleotide sequence (2370 nucleotides), as shown in SEQ ID NO:4 in the sequence listing.
3、合成上述核苷酸序列3. 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）、所述Cry2Ab核苷酸序列（如序列表中SEQ ID NO:3所示）和所述Vip3A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:3）的5’端还连接有NcoI酶切位点，所述Cry2Ab核苷酸序列（SEQ ID NO:3）的3’端还连接有SpeI酶切位点；合成的所述Vip3A核苷酸序列（SEQ ID NO:4）的5’端还连接有ScaI酶切位点，所述Vip3A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), the Cry2Ab nucleotide sequence (as shown in SEQ ID NO: 3 in the sequence listing) and the Vip3A nucleotide The sequence (shown as SEQ ID NO: 4 in the sequence listing) was synthesized by Nanjing KingScript Biotechnology Co., Ltd.; the 5' end of the synthesized Cry1A.105 nucleotide sequence (SEQ ID NO: 2) is also connected with NcoI restriction site, the 3' end of the Cry1A.105 nucleotide sequence (SEQ ID NO: 2) is also connected with a HindIII restriction site; the synthesized Cry2Ab nucleotide sequence (SEQ ID NO: 3 ) is also connected with a NcoI restriction site at the 5' end, and the 3' end of the Cry2Ab nucleotide sequence (SEQ ID NO: 3) is also connected with a SpeI restriction site; the synthetic Vip3A nucleotide sequence The 5' end of (SEQ ID NO: 4) is also connected with a ScaI restriction site, and the 3' end of the Vip3A nucleotide sequence (SEQ ID NO: 4) is also connected with a SpeI restriction site.
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 shown in Figure 1 (among them, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter 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，NaCl10g/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，NaCl10g/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150μl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（4M醋酸钾，2M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. The heat shock conditions were: 50 μl E. coli T1 competent cells, 10 μl plasmid DNA (recombinant Cloning vector DBN01-T), 42°C water bath for 30 seconds; 37°C shaking culture for 1 hour (shaking at 100rpm), the surface was coated with IPTG (isopropylthio-β-D-galactoside) and X -gal (5-bromo-4-chloro-3-indole-β-D-galactoside) ampicillin (100 mg/L) on 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 overnight at 37°C in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, adjust pH to 7.5 with NaOH) . Extract the plasmid by alkaline method: centrifuge the bacterial solution at 12000rpm for 1min, remove the supernatant, and use 100μl ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylenediaminetetraacetic acid), 50mM glucose , pH8.0) suspension; add 150μl 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 150μl ice cold Mix solution III (4M potassium acetate, 2M acetic acid) immediately and thoroughly, place on ice for 5-10min; centrifuge at 4°C and 12000rpm for 5min, add 2 times the volume of absolute ethanol to the supernatant, and mix 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 containing RNase (20 μg/ml) TE (10mM Tris-HCl, 1mM EDTA, pH8.0) to dissolve the precipitate; bathe in water at 37°C for 30min to digest RNA; store at -20°C for later use.
提取的质粒经EcoRI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 digested and identified by EcoRI 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:3）。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: 3). Enzyme digestion and sequencing verified the correct insertion of the Cry2Ab nucleotide sequence in the recombinant cloning vector DBN02-T.
按照上述构建重组克隆载体DBN01-T的方法，将合成的所述Vip3A核苷酸序列连入克隆载体pGEM-T上，得到重组克隆载体DBN03-T，其中，Vip3A为Vip3A核苷酸序列（SEQ ID NO:4）。酶切和测序验证重组克隆载体DBN03-T中所述Vip3A核苷酸序列正确插入。According to the above-mentioned method for constructing the recombinant cloning vector DBN01-T, the synthetic Vip3A nucleotide sequence was connected into the cloning vector pGEM-T to obtain the recombinant cloning vector DBN03-T, wherein Vip3A was the Vip3A nucleotide sequence (SEQ ID NO: 4). Enzyme digestion and sequencing verified the correct insertion of the Vip3A nucleotide sequence in the recombinant cloning vector DBN03-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DBN100032，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）；PMI：磷酸甘露糖异构酶基因（SEQ ID NO:7）；LB：左边界）。Recombinant cloning vector DBN01-T and expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA institutions)) 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 DBN100032, and its construction process is shown in Figure 2 (Kan: Kanamycin gene; RB: right border; Ubi: maize 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); PMI: phosphomannose isomerase gene (SEQ ID NO:7); LB: left border).
将重组表达载体DBN100032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DBN100032），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，NaCl10g/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，NaCl10g/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DBN100032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 DBN100032 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 DBN100032), 42 ° C water bath for 30 seconds; 37 ° C shaking Cultivate for 1 hour (shaking on a shaker at 100 rpm); 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, kana Mycin 50 mg/L, adjust the pH to 7.5 with NaOH) and cultivate overnight at 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 DBN100032 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DBN100032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DBN100076。酶切和测序验证重组表达载体DBN100076中的核苷酸序列含有为序列表中SEQ ID NO:2和SEQ ID NO:3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 DBN100032, 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 DBN100076. Enzyme digestion and sequencing verify that the nucleotide sequence in the recombinant expression vector DBN100076 contains the nucleotide sequences shown in SEQ ID NO: 2 and SEQ ID NO: 3 in the sequence listing, namely the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence acid sequence, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence can be connected to the Ubi promoter and the Nos terminator.
按照上述构建重组表达载体DBN100032的方法，将NcoI和HindIII、ScaI和SpeI分别酶切重组克隆载体DBN01-T和DBN03-T切下的所述Cry1A.105核苷酸序列和Vip3A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Vip3A核苷酸序列，所述Cry1A.105核苷酸序列和所述Vip3A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 DBN100032, insert the Cry1A.105 nucleotide sequence and the Vip3A nucleotide sequence excised from the recombinant cloning vector DBN01-T and DBN03-T respectively by NcoI and HindIII, ScaI 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 Vip3A nucleotide sequence acid sequence, the Cry1A.105 nucleotide sequence and the Vip3A 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DBN100032、DBN100076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DBN100032、DBN100076和DBN100029酶切后进行酶切验证，结果表明重组表达载体DBN100032、DBN100076和DBN100029结构完全正确。Transform the correctly constructed recombinant expression vectors DBN100032, DBN100076 and DBN100029 into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) with liquid nitrogen method, and the transformation conditions are: 100 μL Agrobacterium LBA4404, 3 μL plasmid DNA (recombinant expression vector); put 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. Contain 50mg/L rifampicin (Rifampicin) and 100mg/L kanamycin (Kanamycin) on the LB plate until a positive single clone grows, pick the single clone and culture it and extract its plasmid, use restriction endonuclease AhdI and XhoI digested the recombinant expression vectors DBN100032, DBN100076 and DBN100029 for enzyme digestion and verification. The results showed that the structures of the recombinant expression vectors DBN100032, DBN100076 and DBN100029 were completely correct.
第三实施例、转入Cry1A.105基因的玉米植株的获得及验证The third embodiment, the acquisition and verification of corn plants transferred to the Cry1A.105 gene
1、获得转入Cry1A.105基因的玉米植株1. Obtaining corn plants transferred to the Cry1A.105 gene
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DBN100032、DBN100076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、Vip3A核苷酸序列、PMI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Cry1A.105-Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Vip3A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 maize variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 3 in the second example, so that the T-DNA in recombinant expression vectors DBN100032, DBN100076 and DBN100029 (including the promoter sequence of maize Ubiquitin gene, Cry1A.105 nucleotide sequence, Cry2Ab nucleotide sequence, Vip3A nucleotide sequence, PMI gene and Nos terminator sequence ) into the maize genome, and obtained the maize plant with the Cry1A.105 nucleotide sequence, the maize plant with the Cry1A.105-Cry2Ab nucleotide sequence and the Cry1A.105-Vip3A nucleotide sequence maize plants; meanwhile, wild-type maize plants were used as controls.
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/或Vip3A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、琼脂8g/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（甘露糖）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5g/L、甘露糖12.5g/L、2,4-二氯苯氧乙酸（2,4-D）1mg/L、琼脂8g/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 / or the Vip3A 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-dichlorophenoxyacetic acid ( 2,4-D) 1mg/L, pH 5.3)) 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, agar 8g/L, pH 5.8), there is at least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium, 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 (mannose) and selected for growing transformed calli (step 4: selection step). Preferably, the immature embryos are cultured on a selective solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 5g/L, mannose 12.5g/L, 2,4-dichlorobenzene Oxyacetic acid (2,4-D) 1mg/L, agar 8g/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、甘露糖5g/L、琼脂8g/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、琼脂8g/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, mannose 5g/L, agar 8g/L, pH5.8), 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, agar 8g/L, pH5 .8) above, cultivate at 25°C to a height of about 10cm, and move to the greenhouse to cultivate 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、用TaqMan验证转入Cry1A.105基因的玉米植株2. Using TaqMan to verify the maize plants transferred to the Cry1A.105 gene
分别取转入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和转入Cry1A.105-Vip3A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、Cry2Ab基因和Vip3A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, corn plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence, and corn plants transferred to the Cry1A.105-Vip3A nucleotide sequence were taken as samples , Genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy numbers of Cry1A.105 gene, Cry2Ab gene and Vip3A gene were detected by Taqman probe fluorescence 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基因拷贝数的具体方法如下：The specific method for detecting the copy number of Cry1A.105 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Cry1A.105-Cry2Ab核苷酸序列的玉米植株、转入Cry1A.105-Vip3A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, respectively take the corn plant transferred to the Cry1A.105 nucleotide sequence, the corn plant transferred to the Cry1A.105-Cry2Ab nucleotide sequence, the corn plant transferred to the Cry1A.105-Vip3A nucleotide sequence and the wild type Each 100 mg of corn plant leaves was ground into a homogenate in a mortar with liquid nitrogen, and each sample was taken in 3 replicates;
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, use Qiagen's DNeasy Plant Mini Kit to extract the genomic DNA of the above sample, and refer to its product manual for specific methods;
步骤13、用NanoDrop2000（Thermo Scientific）测定上述样品的基因组DNA浓度；Step 13, using NanoDrop2000 (Thermo Scientific) to measure the genomic DNA concentration of the above sample;
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（CF1）：GCGCATCCAGTTCAACGAC如序列表中SEQ ID NO:8所示；Primer 1 (CF1): GCGCATCCAGTTCAACGAC as shown in SEQ ID NO: 8 in the sequence listing;
引物2（CR1）：GTTCTGGACGGCGAAGAGTG如序列表中SEQ ID NO:9所示；Primer 2 (CR1): GTTCTGGACGGCGAAGAGTG as shown in SEQ ID NO: 9 in the sequence listing;
探针1（CP1）：TGAACAGCGCCCTGACCACCG如序列表中SEQ ID NO:10所示；Probe 1 (CP1): 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（CF2）：CTGATACCCTTGCTCGCGTC如序列表中SEQ ID NO:11所示；Primer 3 (CF2): CTGATACCCTTGCTCGCGTC as shown in SEQ ID NO: 11 in the sequence listing;
引物4（CR2）：CACTTGGCGGTTGAACTCCTC如序列表中SEQ ID NO:12所示；Primer 4 (CR2): CACTTGGCGGTTGAACTCCTC as shown in SEQ ID NO: 12 in the sequence listing;
探针2（CP2）：CGCTGAGCTGACGGGTCTGCAAG如序列表中SEQ IDNO:13所示；Probe 2 (CP2): CGCTGAGCTGACGGGTCTGCAAG as shown in SEQ ID NO: 13 in the sequence listing;
以下引物和探针用来检测Vip3A核苷酸序列：The following primers and probes were used to detect the Vip3A nucleotide sequence:
引物5（VF1）：ATTCTCGAAATCTCCCCTAGCG如序列表中SEQ ID NO:14所示；Primer 5 (VF1): ATTCTCGAAATCTCCCCCTAGCG as shown in SEQ ID NO: 14 in the sequence listing;
引物6（VR1）：GCTGCCAGTGGATGTCCAG如序列表中SEQ ID NO:15所示；Primer 6 (VR1): GCTGCCAGTGGATGTCCAG as shown in SEQ ID NO: 15 in the sequence listing;
探针3（VP1）：CTCCTGAGCCCCGAGCTGATTAACACC如序列表中SEQID NO:16所示；Probe 3 (VP1): CTCCTGAGCCCCGAGCTGATTAACACC as shown in SEQID NO: 16 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860μl1×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 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、Cry1A.105-Cry2Ab核苷酸序列和Cry1A.105-Vip3A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Cry1A.105-Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Vip3A核苷酸序列的玉米植株均获得了含有单拷贝Cry1A.105基因的转基因玉米植株。The experimental results showed that the Cry1A.105 nucleotide sequence, the Cry1A.105-Cry2Ab nucleotide sequence and the Cry1A.105-Vip3A nucleotide sequence had all been integrated into the genome of the maize plant detected, and the Cry1A. The 105 nucleotide sequence maize plant, the maize plant transformed with the Cry1A.105-Cry2Ab nucleotide sequence and the maize plant transformed with the Cry1A.105-Vip3A nucleotide sequence all obtained a transgene containing a single copy of the Cry1A.105 gene corn plant.
第四实施例、转基因玉米植株的抗虫效果检测The fourth embodiment, detection of insect resistance effect of transgenic corn plants
将转入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-Vip3A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对二点委夜蛾进行抗虫效果检测。Corn plants transferred to the Cry1A.105 nucleotide sequence, corn plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence, corn plants transferred to the Cry1A.105-Vip3A nucleotide sequence, wild-type corn plants and Maize plants identified as non-transgenic by Taqman were tested for their resistance to Spodoptera exigua.
分别取转入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-Vip3A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（V3-V4期）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，然后将玉米叶片去除叶脉，同时剪成约1cm×4cm的长条状，取2片剪后的长条状叶片放入圆形塑料培养皿底部的滤纸上，所述滤纸用蒸馏水润湿，每个培养皿中放10头人工饲养的二点委夜蛾（初孵幼虫），虫试培养皿加盖后，在温度25-28℃、相对湿度70%-80%、光周期（光/暗）16：8的条件下放置3天后，根据二点委夜蛾幼虫发育进度、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个株系（S1、S2和S3），转入Cry1A.105-Cry2Ab核苷酸序列的共3个株系（S4、S5和S6），转入Cry1A.105-Vip3A核苷酸序列的共3个株系（S7、S8和S9），经Taqman鉴定为非转基因的（NGM）共1个株系，野生型的（CK）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 Cry1A.105-Cry2Ab nucleotide sequence, the corn plants transferred to the Cry1A.105-Vip3A nucleotide sequence, the wild type corn plant and The fresh leaves of corn plants (V3-V4 stage) identified as non-transgenic by Taqman were washed with sterile water and the water on the leaves was blotted dry with gauze, and then the corn leaves were removed from the veins and cut into approximately 1cm×4cm Long strips, take 2 strips of cut long leaves and put them on the filter paper at the bottom of the circular plastic petri dish, the filter paper is moistened with distilled water, put 10 artificially reared Spodoptera spp. in each petri dish (Newly hatched larvae), after the worm test petri dish was covered, it was placed for 3 days under the conditions of temperature 25-28°C, relative humidity 70%-80%, and photoperiod (light/dark) 16:8. The total score of resistance was obtained from the three indicators of developmental progress, mortality rate and leaf damage rate of noctuid moth larvae: total score=100×mortality+[100×mortality+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 worms/total number of inoculated worms)]+100×(1-leaf damage rate). A total of 3 strains (S1, S2 and S3) transferred to the Cry1A.105 nucleotide sequence, a total of 3 strains (S4, S5 and S6) transferred to the Cry1A.105-Cry2Ab nucleotide sequence, transferred to A total of 3 strains (S7, S8 and S9) with Cry1A.105-Vip3A nucleotide sequence, a total of 1 strain identified as non-transgenic (NGM) by Taqman, a total of 1 wild-type strain (CK) ; Select 3 strains from each strain to test, 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二点委夜蛾的抗虫实验结果Table 1. The results of the insect resistance experiment of transgenic corn plants inoculated with Spodoptera sp.
表1的结果表明：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和转入Cry1A.105-Vip3A核苷酸序列的玉米植株的生测总分均在280分左右或以上；而经Taqman鉴定为非转基因的玉米植株和野生型玉米植株的生测总分一般在130分左右。The results in Table 1 show that: the growth of corn plants transferred to Cry1A.105 nucleotide sequence, the corn plant transferred to Cry1A.105-Cry2Ab nucleotide sequence and the corn plant transferred to Cry1A.105-Vip3A nucleotide sequence The total score of the test is around 280 points or above; while the total score of the biometric test of the non-transgenic corn plants and wild-type corn plants identified by Taqman is generally around 130 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、转入Cry1A.105-Cry2Ab核苷酸序列的玉米植株和转入Cry1A.105-Vip3A核苷酸序列的玉米植株可以造成二点委夜蛾初孵幼虫的大量死亡，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，且转入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和转入Cry1A.105-Vip3A核苷酸序列的玉米植株大体上只受到极轻微损伤，叶片上仅为极少量针孔状损伤，其叶片损伤率均在2%以下。The results in Figure 3 show that: compared with wild-type maize plants, the maize plants that have been transferred to the Cry1A.105 nucleotide sequence, the maize plants that have been transferred to the Cry1A.105-Cry2Ab nucleotide sequence, and the Cry1A.105-Vip3A nuclear The corn plants with the nucleotide sequence can cause a large number of deaths of newly hatched larvae of Spodoptera bispotentum, and greatly inhibit the development progress of a small number of surviving larvae. The corn plants with the nucleotide sequence, the corn plants with the Cry1A.105-Cry2Ab nucleotide sequence and the corn plants with the Cry1A.105-Vip3A nucleotide sequence were generally only slightly damaged. A small amount of pinhole damage, the leaf damage rate is below 2%.
由此证明转入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和转入Cry1A.105-Vip3A核苷酸序列的玉米植株都显示出高抗二点委夜蛾的活性，这种活性足以对二点委夜蛾的生长产生不良效应从而使其得以控制。This proves that the maize plant transferred into the Cry1A.105 nucleotide sequence, the maize plant transferred into the Cry1A.105-Cry2Ab nucleotide sequence and the maize plant transferred into the Cry1A.105-Vip3A nucleotide sequence all show high resistance The activity of Spodoptera spp., which is sufficient to have adverse effects on the growth of Spodoptera so that it can be controlled.
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和转入Cry1A.105-Vip3A核苷酸序列的玉米植株对二点委夜蛾的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蛋白对二点委夜蛾的相同毒杀作用，可产生类似的可表达Cry1A.105蛋白的转基因植株能够用于防治二点委夜蛾的危害。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 plant transferred to the Cry1A.105 nucleotide sequence, the corn plant transferred to the Cry1A.105-Cry2Ab nucleotide sequence, and the corn plant transferred to the Cry1A.105-Vip3A nucleotide sequence are more effective at two points. The control of Spodoptera is obviously because the plant itself can produce Cry1A.105 protein, so, as is well known to those skilled in the art, according to the same poisonous effect of Cry1A.105 protein on Spodoptera bispotae, similar expressible Cry1A can be produced. The transgenic plants with .105 protein can be used to prevent and control the harm of the spodopteryx moth. 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，本发明控制害虫的方法通过植物体内产生能够杀死二点委夜蛾的Cry1A.105蛋白来控制二点委夜蛾害虫；与现有技术使用的农业防治方法和化学防治方法相比，本发明对植物进行全生育期、全植株的保护以防治二点委夜蛾害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。In summary, the method for controlling pests of the present invention controls the pests of Spodoptera bispotentum by producing the Cry1A.105 protein capable of killing Spodoptera bispotentum in plants; it is different from the agricultural control methods and chemical control methods used in the prior art In comparison, the present invention protects the whole growth period and the whole plant to prevent and control the damage of the pest of the spodopteryx moth, and has no pollution and 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.
| Application Number | Priority Date | Filing Date | Title |
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| CN201310594217.5ACN103636653B (en) | 2013-11-21 | 2013-11-21 | Pest control method |
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