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蛋白来控制桃蛀螟危害植物的方法。The invention relates to a method for controlling pests, in particular to a method for using Cry1A.105 protein expressed in plants to control the damage of plants by the peach borer borer.
背景技术Background technique
桃蛀螟（Conogethespunctiferalis）属鳞翅目螟蛾科，为杂食性害虫，除危害玉米、高粱等作物外，还危害桃、柿、板栗等果树，广泛分布于我国境内，北起黑龙江、内蒙古，南至台湾、海南、广东、广西、云南南缘，东接前苏联东境、朝鲜北境，西面自山西、陕西西斜至宁夏、甘肃后，折入四川、云南、西藏。危害玉米时，主要蛀食雌穗，也可蛀茎，受害株率达30%-80%；危害高粱时，初孵幼虫蛀入高粱幼嫩籽粒内，用粪便或食物残渣把口封住，在其内蛀害，吃空一粒又转一粒直至三龄前，三龄后吐丝结网缀合小穗中间留有隧道，在里面穿行啃食籽粒，严重的把高粱粒蛀食一空。此外还可蛀秆，危害情况类似玉米螟。The peach borer (Conogethes punctiferalis) belongs to the family Lepidoptera and is an omnivorous pest. In addition to harming crops such as corn and sorghum, it also harms peaches, persimmons, chestnuts and other fruit trees. It is widely distributed in my country, starting from Heilongjiang and Inner Mongolia in the north. It reaches Taiwan, Hainan, Guangdong, Guangxi, and the southern edge of Yunnan in the south, connects with the eastern border of the former Soviet Union and the northern border of Korea in the east, and turns into Sichuan, Yunnan, and Tibet in the west from Shanxi and Shaanxi westward to Ningxia and Gansu. When maize is harmed, it mainly eats the ears and stems, and the plant damage rate reaches 30%-80%. When harming sorghum, the newly hatched larvae eat into the young sorghum grains, and seal the mouth with feces or food residues. There is moth damage in it, eat one grain and turn another grain until the third age. After the third age, a tunnel is left in the middle of the spikelets, and the sorghum grains are severely eaten. . In addition, it can also bore stalks, and the damage is similar to corn borer.
玉米和高粱是中国重要的粮食作物，每年因桃蛀螟造成的粮食损失巨大，更甚者影响到当地人口的生存状况。为了防治桃蛀螟，人们通常采用的主要防治方法有：农业防治、化学防治和生物防治。Corn and sorghum are important food crops in China. The annual loss of food due to peach borer is huge, and even affects the living conditions of the local population. In order to control peach borer, the main control methods that people usually adopt are: agricultural control, chemical control and biological control.
农业防治是把整个农田生态系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桃蛀螟发生的农田生态环境。如利用处理桃蛀螟越冬寄主、拾毁落果和摘除虫果、改革耕作制度、种植抗桃蛀螟品种和种植诱集田等措施降低桃蛀螟的危害。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，在桃蛀螟爆发时就显得无能为力。Agricultural control is the 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 peach borer. For example, measures such as treating peach borer overwintering hosts, picking up fallen fruit and removing insect fruit, reforming farming systems, planting peach borer-resistant varieties, and planting trap fields to reduce the damage of peach borer. Because agricultural control must obey the requirements of crop layout and yield increase, its application has certain limitations, and it cannot be used as an emergency measure. It is powerless when peach borer breaks out.
化学防治即农药防治，是利用化学杀虫剂来杀灭害虫，是桃蛀螟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，特别是桃蛀螟大发生的情况下，是必不可少的应急措施，它可以在桃蛀螟造成危害前将其消灭。目前化学防治方法主要有化学诱杀、药液喷雾等。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。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 peach borer. Under such circumstances, it is an essential emergency measure, which can eliminate the peach borer before it causes damage. At present, chemical control methods mainly include chemical trapping and liquid spraying. 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.
生物防治是利用某些有益生物或生物代谢产物来控制害虫种群数量，以达到降低或消灭害虫的目的。其特点是对人、畜安全，对环境污染少，对某些害虫可达到长期控制的目的；但是效果常不稳定，并且不论桃蛀螟发生轻重均需同样投资进行。Biological control is the use of certain beneficial organisms or biological metabolites to control the population of pests in order to reduce or eliminate pests. Its characteristics are that it is safe for people and animals, has little environmental pollution, and can achieve the purpose of long-term control of some pests; but the effect is often unstable, and the same investment is required regardless of the severity of the occurrence of peach borer.
为了解决农业防治、化学防治和生物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（Bacillusthuringiensissubsp.kurstaki，B.t.k.）产生的伴孢结晶蛋白。In order to solve the limitations in the practical application of agricultural control, chemical control and biological control, 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桃蛀螟对植物危害的报道。It has been proved that the plants transgenic for Cry1A.105 can resist the attack of Lepidoptera pests such as corn borer, cotton bollworm, and cutworm. Reports on plant damage by borer borer.
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桃蛀螟对植物危害的方法，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 to plants by producing transgenic plants expressing Cry1A.105 protein, and effectively overcomes the prior art agricultural control and chemical control technologies defect.
为实现上述目的，本发明提供了一种控制桃蛀螟害虫的方法，包括将桃蛀螟害虫与Cry1A.105蛋白接触。To achieve the above object, the present invention provides a method for controlling the peach borer pest, comprising contacting the peach borer pest 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，所述桃蛀螟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 peach borer pest contacts 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，所述桃蛀螟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，接触后所述桃蛀螟害虫生长受到抑制并最终导致死亡，以实现对桃蛀螟危害植物的控制。Furthermore, the Cry1A.105 protein exists in the transgenic plant that produces the Cry1A.105 protein, and the peach borer pest contacts the Cry1A.105 protein by ingesting the tissue of the transgenic plant, and the resulting The growth of the peach borer pest is inhibited and eventually leads to death, so as to realize the control of the damage to plants by the peach 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.
所述对桃蛀螟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 peach borer does not change due to the change of the planting place.
所述对桃蛀螟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 peach borer does not change due to changes in planting time.
所述植物可以来自玉米、高粱、粟、向日葵、蓖麻、姜、棉花、桃、柿、核桃、板栗、无花果或松树。The plant may be from corn, sorghum, millet, sunflower, castor, ginger, cotton, peach, persimmon, walnut, chestnut, fig or pine.
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SEQIDNO:1所示的氨基酸序列。所述Cry1A.105蛋白的核苷酸序列具有SEQIDNO: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SEQIDNO:3或SEQIDNO:4所示的核苷酸序列。Furthermore, the second nucleotide includes 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蛋白质控制桃蛀螟害虫的用途。In order to achieve the above object, the present invention also provides a use of Cry1A.105 protein to control the peach borer pest.
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（RNAinterference，RNAi）是指在进化过程中高度保守的、由双链RNA（double-strandedRNA，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 (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.
桃蛀螟（Conogethespunctiferalis）与玉米螟（Ostrinianubilalis）同属鳞翅目螟蛾科，为杂食性害虫，尽管如此，桃蛀螟与玉米螟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：The peach borer (Conogethes punctiferalis) and the corn borer (Ostrinianubilalis) belong to the family Lepidoptera and are omnivorous pests. However, the peach borer and the corn borer are two clear and distinct species biologically, at least There are the following main differences:
1、食性不同。桃蛀螟不仅取食玉米等禾本科作物，同时也喜食桃、石榴、板栗、柿等果树；而玉米螟明显嗜好禾本科作物，最常危害高粱、玉米等。1. Food habits are different. The peach borer not only feeds on gramineous crops such as corn, but also eats fruit trees such as peaches, pomegranates, chestnuts, and persimmons; while the corn borer obviously prefers gramineous crops, and most often harms sorghum and corn.
2、分布区域不同。桃蛀螟遍布中国全境，在日本、朝鲜半岛、英国、澳大利亚等地也有分布。玉米螟包括亚洲玉米螟和欧洲玉米螟，其中亚洲玉米螟分布在中国东部及西南主要玉米、高粱产区；欧洲玉米螟主要分布在中国的新疆及欧洲、北美洲、西非及小亚细亚地区。从分布区域上来讲，桃蛀螟的分布区域较欧洲玉米螟和亚洲玉米螟都要广。2. The distribution area is different. The peach moth is distributed throughout China, and is also distributed in Japan, the Korean Peninsula, the United Kingdom, Australia and other places. The corn borer includes the Asian corn borer and the European corn borer. The Asian corn borer is distributed in the main corn and sorghum producing areas in eastern and southwestern China; the European corn borer is mainly distributed in Xinjiang, China, Europe, North America, West Africa and Asia Minor. In terms of distribution area, the distribution area of peach borer is wider than that of European corn borer and Asian corn borer.
3、为害习性不同。桃蛀螟为害高粱时，初孵幼虫蛀入高粱幼嫩籽粒内，用粪便或食物残渣把口封住，在其内蛀害，吃空一粒又转一粒直至三龄前；三龄后吐丝结网缀合小穗中间留有隧道，在里面穿行啃食籽粒，严重的把高粱粒蛀食一空；此外还可蛀秆；为害玉米时，主要蛀食雌穗，蛀入幼嫩籽粒后产生黏性粪便堵住蛀孔，在蛀孔内转粒为害；同时也可蛀茎、叶片、籽粒；桃蛀螟为害时常产生黏性粪便，增加了霉菌发生的概率，尤其是增加了黄曲霉的发生概率，影响了饲料加工。而玉米螟幼虫孵出后，先聚集在一起，然后在植株幼嫩部分爬行，开始危害；初孵幼虫，能吐丝下垂，借风力飘迁邻株，形成转株危害；幼虫多为五龄，三龄前主要集中在幼嫩心叶、雄穗、苞叶和花丝上活动取食，被害心叶展开后，即呈现许多横排小孔；四龄以后，大部分钻入茎秆；玉米螟可为害玉米植株地上的各个部位，叶片被幼虫咬食后，会降低其光合效率；雄穗被蛀，常易折断，影响授粉；苞叶、花丝被蛀食，会造成缺粒和秕粒；茎秆、穗柄、穗轴被蛀食后，形成隧道，破坏植株内水分、养分的输送，使茎秆倒折率增加，籽粒产量下降。各地的春、夏、秋播玉米都有不同程度受害，尤以夏播玉米最重。3. Different damage habits. When the peach borer damages sorghum, the newly hatched larvae eat into the young sorghum grains, seal their mouths with feces or food residues, and moth them inside, eat one grain and turn it to another until the third instar; after the third instar There is a tunnel in the middle of the spikelets combined with silk spinning and netting, and the grains are gnawed inside, and the sorghum grains are severely eaten; in addition, it can also be bored; when it is harmful to corn, it mainly eats the ears and penetrates into the young grains After that, viscous feces will be produced to block the boreholes, and the grains will be damaged in the boreholes; at the same time, it can also bore stems, leaves, and grains; the peach borer often produces viscous feces, which increases the probability of mold occurrence, especially the increase of yellow The occurrence probability of Aspergillus affects feed processing. After the corn borer larvae hatch, they first gather together, and then crawl on the young part of the plant, causing damage; the newly hatched larvae can spin and droop, and use the wind to float away to neighboring plants, causing damage to the plant; most of the larvae are fifth instar , before the third instar, it mainly concentrates on the young heart leaves, tassels, bracts and filaments to feed, and after the damaged heart leaves unfold, there are many horizontal rows of small holes; after the fourth instar, most of them penetrate into the stalk; maize Borers can damage all parts of the corn plant above the ground. After the leaves are bitten by larvae, their photosynthetic efficiency will be reduced; ; After the stalks, stalks, and cobs are eaten by moths, they form tunnels, destroying the transportation of water and nutrients in the plant, increasing the rate of stalk inversion, and reducing grain yield. Corn sown in spring, summer and autumn in various places suffered damage to varying degrees, especially corn sown in summer.
4、形态特征不同。4. Different morphological characteristics.
1）卵形态不同：桃蛀螟卵长0.6-0.7mm，卵面粗糙，密布细小圆形刻点或网状花纹，孵化前为橙红色，单粒散产于粗糙表面；而玉米螟卵成扁平椭圆形，孵化前端部附近出现小黑点，数十粒呈鱼鳞状不规则排列。1) Egg shapes are different: peach moth borer eggs are 0.6-0.7mm long, with rough egg surface, densely covered with small round punctures or net-like patterns, orange-red before hatching, and single grains are scattered on the rough surface; Flat oval, small black dots appear near the front end of hatching, and dozens of grains are irregularly arranged in the shape of fish scales.
2）幼虫形态不同：桃蛀螟幼虫体色多变，浅灰至暗红色，腹面多为淡绿色，头部暗褐色，体背暗红色，腹面淡绿色，前胸背板和臀板深褐色，各体节毛片明显，灰褐至黑褐色，第l-8腹节上各具8个，排成两列，前列6个较大，后列2个较小，3龄后雄性幼虫第5腹节背面出现2个暗褐色性腺；而玉米螟幼虫背部黄白色至淡暗红褐色，头和前胸背板深褐色，背线明显，两侧有较模糊的暗褐色亚背线，腹部1-8节有毛瘤两列，前列4个，后列2个。2) The larvae are different in shape: the body color of the peach moth larvae is changeable, from light gray to dark red, the ventral surface is mostly light green, the head is dark brown, the back of the body is dark red, the ventral surface is light green, the pronotum and rump are dark brown , each somites have obvious hairs, grayish brown to dark brown, 8 on each of the 1-8th abdominal segments, arranged in two rows, 6 larger in the front row, 2 smaller in the rear row, the 5th male larvae after the 3rd instar Two dark brown gonads appear on the back of the abdominal segment; while the back of the corn borer larvae is yellowish-white to light dark reddish brown, the head and pronotum are dark brown, the top line is obvious, and there are fuzzy dark brown sub-top lines on both sides. Section -8 has two rows of hair tumors, 4 in the front row and 2 in the back row.
3）蛹形态不同：桃蛀螟蛹初期为淡黄绿色，后变深褐色，头部、腹部第1-8节背面密布细小突起，第5-7节腹部前缘有1条由小齿状突构成的突起线，腹部末端有细长卷曲的钩刺6根；而玉米螟蛹黄褐色，腹背密布横皱纹，腹末有5-8根钩刺。3) The shape of the pupae is different: the early stage of the pupae is light yellow-green, and then it turns dark brown. The head and the back of the 1-8th segment of the abdomen are densely covered with small protrusions, and there is a small tooth-shaped protuberance on the front edge of the 5th-7th segment of the abdomen. The protruding lines formed by protrusions have 6 slender and curly barbs at the end of the abdomen; while the corn borer pupae are yellowish brown, with dense horizontal wrinkles on the abdomen and back, and 5-8 barbs at the end of the abdomen.
4）成虫形态不同：桃蛀螟成虫黄至橙黄色，胸、腹部及翅上具有很多黑色斑点，前胸两侧的被毛上各具1个黑点，雄蛾腹部第9节末端为黑色，甚为明显，较钝，有黑色毛丛，雌蛾腹部末端圆锥形，末节仅背面端部有极少的黑色鳞片；而玉米螟成虫前翅黄褐色，有两条褐色波状横纹，两纹之间有两条黄褐色短纹，后翅灰褐色，雌蛾翅色较雄蛾浅淡，前翅呈现黄色，内、外横线及斑纹不及雄蛾明显。4) The shape of the adults is different: the adults of the peach borer moth are yellow to orange yellow, with many black spots on the chest, abdomen and wings, and one black spot on the coat on both sides of the prothorax, and the end of the ninth segment of the abdomen of the male moth is black , very obvious, relatively blunt, with black tufts, the female moth has a conical abdomen end, and only a few black scales on the back end of the terminal segment; while the forewings of corn borer adults are yellowish brown, with two brown wavy horizontal stripes and two stripes There are two yellow-brown short stripes in between, the hind wings are gray-brown, the wings of female moths are lighter than those of male moths, the forewings are yellow, and the inner and outer horizontal lines and markings are less obvious than those of male moths.
5、生长习性和发生规律不同。桃蛀螟在辽宁年生1-2代，河北、山东、陕西3代，河南4代，长江流域4-5代，均以老熟幼虫在玉米、向日葵、蓖麻等残株内结茧越冬；在河南1代幼虫于5月下旬-6月下旬先在桃树上危害，2-3代幼虫在桃树和高粱上都能危害，第4代则在夏播高粱和向日葵上危害，以4代幼虫越冬，翌年越冬幼虫于4月初化蛹，4月下旬进入化蛹盛期，4月底-5月下旬羽化，越冬代成虫把卵产在桃树上；6月中旬-6月下旬1代幼虫化蛹，1代成虫于6月下旬开始出现，7月上旬进入羽化盛期，2代卵盛期跟着出现，这时春播高粱抽穗扬花，7月中旬为2代幼虫危害盛期；2代羽化盛期在8月上、中旬，这时春高粱近成熟，晚播春高粱和早播夏高粱正抽穗扬花，成虫集中在这些高粱上产卵，第3代卵于7月底8月初孵化，8月中、下旬进入3代幼虫危害盛期；8月底3代成虫出现，9月上中旬进入盛期，这时高粱和桃果已采收，成虫把卵产在晚夏高粱和晚熟向日葵上，9月中旬-10月上旬进入4代幼虫发生为害期，10月中、下旬气温下降则以4代幼虫越冬。在河南1代卵期8天，2代4.5天，3代4.2天，越冬代6天；1代幼虫历期19.8天，2代13.7天，3代13.2天，越冬代208天，幼虫共5龄；1代蛹期8.8天，2代8.3天，3代8.7天，越冬代19.4天；一代成虫寿命7.3天，2代7.2天，3代7.6天，越冬代10.7天。成虫羽化后白天潜伏在高粱田经补充营养才产卵，把卵产在吐穗扬花的高粱上，卵单产，每雌可产卵169粒，一穗产卵3-5粒，在四川宜宾从秋玉米抽雄至蜡熟阶段把卵产在雄穗、雌穗、叶鞘合缝处或叶耳正反面，百株卵量高达1729粒；幼虫者熟后在穗中或叶腋、叶鞘、枯叶处及高粱、玉米、向日葵秸秆中越冬；雨多年份发生重，近年来受全球温室效应的影响，华北及东北地区夏季雨水量大，使得桃蛀螟逐步成为华北地区的主要玉米害虫，但由于其食性杂，常在不同寄主间转移为害，且最爱以钻蛀果穗及茎秆为害，一般喷洒农药难以防治。而玉米螟的发生代数随纬度而有显著的差异：在中国，北纬45°以北1代，45°-40°2代，40°-30°3代，30°-25°4代，25°-20°5-6代。海拔越高，发生代数越少；在四川省一年发生2-4代，温度高、海拔低，发生代数较多，通常以老熟幼虫在玉米茎秆、穗轴内或高粱、向日葵的秸秆中越冬，次年4-5月化蛹，蛹经过10天左右羽化。成虫夜间活动，飞翔力强，有趋光性，寿命5～10天，喜欢在离地50厘米以上、生长较茂盛的玉米叶背面中脉两侧产卵，一个雌蛾可产卵350-700粒，卵期3-5天；玉米螟适合在高温、高湿条件下发育，冬季气温较高，天敌寄生量少，有利于玉米螟的繁殖，危害较重；卵期干旱，玉米叶片卷曲，卵块易从叶背面脱落而死亡，危害较轻。5. The growth habits and occurrence rules are different. Peach borer has 1-2 generations annually in Liaoning, 3 generations in Hebei, Shandong, and Shaanxi, 4 generations in Henan, and 4-5 generations in the Yangtze River Basin. They all overwinter as mature larvae cocooning in stumps such as corn, sunflower, and castor; In Henan, the 1st generation larvae first injure peach trees from late May to late June, the 2nd to 3rd generation larvae can injure both peach trees and sorghum, and the 4th generation injures summer sown sorghum and sunflower. The first generation larvae overwinter, and the next year the overwintering larvae pupate in early April, enter the peak pupation period in late April, emerge from the end of April to late May, and the overwintering adults lay eggs on peach trees; one generation from mid-June to late June The larvae pupate, and the first generation of adults begins to appear in late June, and enters the peak eclosion period in early July, followed by the second generation of eggs. The peak eclosion period is in early to mid-August, when the spring sorghum is nearly mature, and the late-sowed spring sorghum and early-sowed summer sorghum are heading and blooming. Adults concentrate on these sorghums to lay eggs. The third generation eggs hatch at the end of July and early August. In the middle and late August, the third generation of larvae will enter the peak period of damage; at the end of August, the third generation of adults will appear, and in the first and middle ten days of September, it will enter the peak period. From mid-September to early October, the 4th generation larvae will cause damage, and when the temperature drops in mid-to-late October, the 4th generation larvae will survive the winter. In Henan, the egg period of the first generation is 8 days, the second generation is 4.5 days, the third generation is 4.2 days, and the overwintering generation is 6 days; age; the pupal period of the first generation is 8.8 days, the second generation is 8.3 days, the third generation is 8.7 days, and the overwintering generation is 19.4 days; the adult life of the first generation is 7.3 days, the second generation is 7.2 days, the third generation is 7.6 days, and the overwintering generation is 10.7 days. After adult emergence, they lurk in the sorghum field during the day and lay eggs after supplementing nutrients. The eggs are laid on the sorghum that is producing ears and blooming flowers. The eggs are laid alone, and each female can lay 169 eggs, and one ear can lay 3-5 eggs. It was born in Yibin, Sichuan. From tasseling to wax ripening, autumn corn lays eggs in the joints of tassels, ears, leaf sheaths, or the front and back of leaf ears. The number of eggs per 100 plants is as high as 1729; larvae mature in ears or in leaf axils, leaf sheaths, and dead leaves. and overwinter in sorghum, corn, and sunflower stalks; heavy rainfall occurs in many years. In recent years, affected by the global greenhouse effect, North China and Northeast China have a large amount of summer rainfall, making the peach borer gradually become the main corn pest in North China. However, due to its It has miscellaneous feeding habits, often transfers damage among different hosts, and most likes to damage fruit ears and stems by borers, and it is generally difficult to control by spraying pesticides. However, the number of generations of corn borer varies significantly with latitude: in China, there is one generation north of 45° north latitude, two generations at 45°-40°, three generations at 40°-30°, four generations at 30°-25°, and four generations at 25°. °-20°5-6 generations. The higher the altitude, the fewer the generation generations; in Sichuan Province, there are 2-4 generations a year, the temperature is high and the altitude is low, the generation generation is more, usually mature larvae in corn stalks, cobs or sorghum, sunflower stalks Surviving the winter in the middle of the year, pupating in April-May of the following year, and the pupae emerge after about 10 days. Adults are nocturnal, have strong flying power, have phototaxis, and live for 5-10 days. They like to lay eggs on both sides of the midrib on the back of corn leaves that are more than 50 cm above the ground and grow luxuriantly. A female moth can lay 350-700 eggs. The period is 3-5 days; the corn borer is suitable for development under high temperature and high humidity conditions, the temperature is high in winter, and the amount of natural enemies parasitized is small, which is conducive to the reproduction of the corn borer, and the damage is serious; the egg stage is dry, the corn leaves are curled, and the egg mass is easy to come from. The back of the leaf falls off and dies, which is less harmful.
综合上述，可确定桃蛀螟与玉米螟是两种害虫，且亲缘关系较远，无法交配产生后代。Based on the above, it can be determined that peach borer and corn borer 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SEQIDNO: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 6.0X sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by washing with 2.0X 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 Wash each membrane once with %SDS.
因此，具有抗虫活性并在严格条件下与本发明SEQIDNO: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桃蛀螟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 that has the same or substantially the same biological activity against peach borer pest 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（AcademicPress）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 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deVos等，1992，Science255：306-312；Smith等，1992，J.Mol.Biol224:899-904；Wlodaver等，1992，FEBSLetters309：59-64）。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, which can be determined by techniques such as nuclear magnetic resonance analysis, crystallography, or photoaffinity labeling (see, e.g., deVos 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%同源性且对桃蛀螟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 protein and has insecticidal activity against Peach Borer.
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（AMVRNA4）；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（Agrobacteriumtumefaciens）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ssRUBISCOE9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 proteinase inhibitor II (pinⅡ) gene, the polyadenylation signal sequence from the pea ssRUBISCOE9 gene and the polyadenylation signal sequence from the α-tubulin gene glycosylation 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，目标昆虫是桃蛀螟害虫。The term "insecticide" in the present invention means that it is toxic to crop pests. More specifically, the target insect is the peach borer pest.
本发明中Cry1A.105蛋白对桃蛀螟害虫具有毒性。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，桃蛀螟害虫通过摄食植物组织与该蛋白接触，接触后桃蛀螟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蛋白所靶向的桃蛀螟害虫的杀虫剂）。The Cry1A.105 protein in the present invention is toxic to the peach borer pest. The plant in the present invention, especially corn, contains exogenous DNA in its genome, and described exogenous DNA comprises the nucleotide sequence of coding Cry1A.105 protein, and peach borer pest contacts with this protein by feeding on plant tissue, contacts The growth of the Peach Borer pest is inhibited 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 peach borer pest targeted by the Cry1A.105 protein.
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桃蛀螟。Different assays can be used to determine the insecticidal efficacy of ICPs in plants. The target insects in the present invention are mainly peach borer.
本发明中，所述Cry1A.105蛋白可以具有序列表中SEQIDNO: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桃蛀螟害虫的危害，如农业防治、化学防治和生物防治；而本发明是通过植物体内产生能够杀死桃蛀螟的Cry1A.105蛋白来控制桃蛀螟害虫的，即通过内因来防治。1. Prevention and treatment of internal causes. The prior art mainly controls the harm of the peach borer pest through external effects, that is, external causes, such as agricultural control, chemical control and biological control; while the present invention controls the pest by producing the Cry1A.105 protein that can kill the peach borer in plants. The peach borer pest 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桃蛀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；使用本发明控制桃蛀螟害虫的方法，可以消除上述不良后果。2. No pollution and no residue. Although the chemical control method used in the prior art has played a certain role in controlling the harm of the peach borer pest, it has also brought pollution, damage and residue to people, livestock and farmland ecosystems; using the present invention to control the peach 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桃蛀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，都可以避免遭受桃蛀螟的侵害。3. Prevention and treatment during the whole growth period. The methods used in the prior art to control the peach borer pest are staged, but the present invention protects the plants throughout their growth period, and the transgenic plants (Cry1A.105 protein) from germination, growth, to flowering and fruiting, can avoid the attack of peach borer.
4、全植株防治。现有技术使用的控制桃蛀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）的根、叶片、茎秆、雄穗、雌穗、花药、花丝等都是可以抵抗桃蛀螟侵害的。4. Whole plant control. Most of the methods used in the prior art to control peach borer pests are local, such as foliar spraying; while the present invention protects the whole plant, such as the roots, leaves, and stems of transgenic plants (Cry1A.105 protein). , tassels, ears, anthers, filaments, etc. are all resistant to peach borer damage.
5、效果稳定。现有技术使用的生物杀虫剂需要直接喷施到作物表面，因此造成有活性的结晶蛋白（包括Cry1A.105蛋白）在环境中被降解；本发明是使所述Cry1A.105蛋白在植物体内进行表达，有效地避免了生物杀虫剂在自然界不稳定的缺陷，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 biopesticides used in the prior art need to be sprayed directly on the crop surface, thus causing the active crystalline protein (including Cry1A.105 protein) to be degraded in the environment; the present invention is to make the Cry1A.105 protein in the plant The expression effectively avoids the defect that biopesticides are unstable in nature, 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。现有技术使用的生物杀虫剂在环境中易被降解，因此需要重复生产和重复应用，并为在农业生产上的实际应用带来困难，大大地增加了成本；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. The biopesticides used in the prior art are easily degraded in the environment, so repeated production and repeated application are required, which brings difficulties to the practical application in agricultural production and greatly increases the cost; the present invention only needs to be planted to express The transgenic plant of the Cry1A.105 protein is sufficient, without taking 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桃蛀螟害虫的方法，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）可以造成初孵桃蛀螟幼虫的大量死亡，且对小部分存活幼虫发育进度造成极大的抑制，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 method used in the prior art to control the Peach Borer pest 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 Peach Borer larvae, and The developmental progress of a small number of surviving larvae is greatly inhibited, all of which are obviously stunted and have stopped developing, and it is difficult to cause harm to 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桃蛀螟的叶片损伤图。具体实施方式Fig. 3 is a diagram of leaf damage of transgenic corn plants inoculated with Peach Borer in the method for controlling pests of the present invention. detailed description
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SEQIDNO: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SEQIDNO: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; Cry1A.105 nucleosides encoding the amino acid sequence (1177 amino acids) corresponding to the Cry1A.105 insecticidal protein acid 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SEQIDNO: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SEQIDNO: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 amino acids) encoding the Vip3A insecticidal protein The 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SEQIDNO:2所示）、所述Cry2Ab核苷酸序列（如序列表中SEQIDNO:3所示）和所述Vip3A核苷酸序列（如序列表中SEQIDNO: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（SEQIDNO:2）的5’端还连接有NcoI酶切位点，所述Cry1A.105核苷酸序列（SEQIDNO:2）的3’端还连接有HindIII酶切位点；合成的所述Cry2Ab核苷酸序列（SEQIDNO:3）的5’端还连接有NcoI酶切位点，所述Cry2Ab核苷酸序列（SEQIDNO:3）的3’端还连接有SpeI酶切位点；合成的所述Vip3A核苷酸序列（SEQIDNO:4）的5’端还连接有ScaI酶切位点，所述Vip3A核苷酸序列（SEQIDNO: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 sequence (as shown in SEQ ID NO: 3 in the sequence listing) Shown in the list as SEQ ID NO: 4) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; the 5' end of the synthesized Cry1A.105 nucleotide sequence (SEQ ID NO: 2) is also connected with an NcoI restriction site, and the The 3' end of the Cry1A.105 nucleotide sequence (SEQ ID NO: 2) is also connected with a HindIII restriction site; the 5' end of the synthesized Cry2Ab nucleotide sequence (SEQ ID NO: 3) is also connected with a NcoI restriction site point, the 3' end of the Cry2Ab nucleotide sequence (SEQ ID NO: 3) is also connected with a SpeI restriction site; the 5' end of the synthesized Vip3A nucleotide sequence (SEQ ID NO: 4) is also connected with a ScaI enzyme 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（SEQIDNO: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（25mMTris-HCl，10mMEDTA（乙二胺四乙酸），50mM葡萄糖，pH8.0）悬浮；加入150μl新配制的溶液II（0.2MNaOH，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（10mMTris-HCl，1mMEDTA，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 of ice-cold solution I (25mM Tris-HCl, 10mM EDTA (ethylenediaminetetraacetic acid), 50mM glucose, pH8 .0) to suspend; add 150 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 (4M potassium acetate, 2M acetic acid), mix thoroughly immediately, 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, mix well and room temperature Place 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 TE containing RNase (20 μg/ml) ( 10mM Tris-HCl, 1mM EDTA, PH8.0) to dissolve the precipitate; in a water bath at a temperature of 37°C for 30min to digest the RNA; to store at a temperature of -20°C for later use.
提取的质粒经EcoRV和XohI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SEQIDNO: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 EcoRV and XohI, 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 the nucleus shown in SEQIDNO:2 in the sequence table. The nucleotide sequence, that is, the Cry1A.105 nucleotide sequence was inserted correctly.
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（SEQIDNO: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（SEQIDNO: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 is connected into the cloning vector pGEM-T to obtain the recombinant cloning vector DBN03-T, wherein Vip3A is 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（SEQIDNO:5）；Cry1A.105：Cry1A.105核苷酸序列（SEQIDNO:2）；Nos：胭脂碱合成酶基因的终止子（SEQIDNO:6）；PMI：磷酸甘露糖异构酶基因（SEQIDNO: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: corn Ubiquitin (ubiquitin) gene promoter (SEQ ID NO: 5); Cry1A.105: Cry1A.105 nucleotide sequence (SEQ ID NO: 2); Nos: nopaline synthesis Terminator of enzyme 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SEQIDNO: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 is identified after restriction endonuclease NcoI and HindIII digestion, and positive clones are carried out sequencing identification, the result shows that the nucleotide sequence of recombinant expression vector DBN100032 between NcoI and HindIII sites is SEQIDNO in the sequence listing: The nucleotide sequence shown in 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SEQIDNO:2和SEQIDNO: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 SEQIDNO: 2 and SEQIDNO: 3 in the sequence table, that is, the Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence. The Cry1A.105 nucleotide sequence and the Cry2Ab nucleotide sequence can be connected with 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SEQIDNO:2和SEQIDNO: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 SEQIDNO: 2 and SEQIDNO: 4 in the sequence table, that is, the Cry1A.105 nucleotide sequence and the Vip3A nucleotide sequence. The Cry1A.105 nucleotide sequence and the Vip3A nucleotide sequence can be connected with 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DNeasyPlantMaxiKit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 DNeasyPlantMaxiKit, and the copy numbers of Cry1A.105 gene, Cry2Ab gene and Vip3A gene were detected 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DNeasyPlantMiniKit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 DNeasyPlantMiniKit to extract the genomic DNA of the above sample, and refer to its product manual for specific methods;
步骤13、用NanoDrop2000（ThermoScientific）测定上述样品的基因组DNA浓度；Step 13, using NanoDrop2000 (ThermoScientific) 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SEQIDNO: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SEQIDNO: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SEQIDNO: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SEQIDNO: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SEQIDNO: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SEQIDNO: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SEQIDNO: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SEQIDNO: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SEQIDNO:16所示；Probe 3 (VP1): CTCCTGAGCCCCGAGCTGATTAACACC as shown in SEQ ID 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（AppliedBiosystems）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桃蛀螟进行抗虫效果检测。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 Peach Borer.
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1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头人工饲养的桃蛀螟（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，根据桃蛀螟幼虫发育进度、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 In the shape of a long strip, take 1 strip-shaped leaf after cutting and put it on the filter paper at the bottom of a circular plastic petri dish, and the filter paper is moistened with distilled water, and put 10 artificially raised peach moths (primary peach borer) in each petri dish hatching larvae), after the insect 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. Progress, mortality and leaf damage rate are three indicators, and the total score of resistance is obtained: total score=100×mortality+[100×mortality+90×(number of newly hatched worms/total number of inoculated worms)+60×(newly hatched -number of negative control insects/total number of inoculated insects)+10×(number of negative control insects/total number of inoculated insects)]+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桃蛀螟的抗虫实验结果Table 1. The results of the insect resistance experiment of transgenic corn plants inoculated with Peach Borer
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分以上，部分可达满分300分；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30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 above 280 points, and some can reach the full score of 300 points; 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 30 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桃蛀螟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1%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 the newly hatched larvae of the peach moth borer, and greatly inhibit the development progress of a small number of surviving larvae. Maize plants with acid sequence, corn plants with Cry1A.105-Cry2Ab nucleotide sequence and corn plants with Cry1A.105-Vip3A nucleotide sequence were generally only slightly damaged, with only a few needles on the leaves. Hole damage, the leaf damage rate is below 1%.
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桃蛀螟的活性，这种活性足以对桃蛀螟的生长产生不良效应从而使其得以控制。This proves that the corn plants transferred to the Cry1A.105 nucleotide sequence, the corn plants transferred to the Cry1A.105-Cry2Ab nucleotide sequence and the corn plants transferred to the Cry1A.105-Vip3A nucleotide sequence all show high resistance to peach moth Borer activity sufficient to adversely affect the growth of the peach borer and thus allow it to 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桃蛀螟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蛋白对桃蛀螟的相同毒杀作用，可产生类似的可表达Cry1A.105蛋白的转基因植株能够用于防治桃蛀螟的危害。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。Above-mentioned experimental result also shows that the maize plant that transfers to Cry1A.105 nucleotide sequence, the maize plant that transfers to Cry1A.105-Cry2Ab nucleotide sequence and the maize plant that transfers to Cry1A.105-Vip3A nucleotide sequence are to peach borer The prevention and control of borer is obviously because the plant itself can produce Cry1A.105 protein. Therefore, those skilled in the art know that according to the same poisonous effect of Cry1A.105 protein on peach borer borer, a similar protein that can express Cry1A.105 can be produced. The transgenic plants can be used to prevent and control the damage of the peach borer. 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桃蛀螟的Cry1A.105蛋白来控制桃蛀螟害虫；与现有技术使用的农业防治方法、化学防治方法和生物防治方法相比，本发明对植物进行全生育期、全植株的保护以防治桃蛀螟害虫的侵害，且无污染、无残留，效果稳定、彻底，简单、方便、经济。In summary, the method for controlling pests of the present invention controls peach borer pests through the production of Cry1A.105 protein capable of killing peach borer in plants; it is different from the agricultural control methods, chemical control methods and biological control methods used in the prior art In comparison, the present invention protects the whole growth period and the whole plant of the plant to prevent and control the damage of the peach borer pest, and has no pollution and no residue, and the effect is stable and thorough, 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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| CN104522033B (en)* | 2014-12-22 | 2016-09-14 | 北京大北农科技集团股份有限公司 | The use of insecticidal protein |
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| CN102972426B (en)* | 2012-12-03 | 2014-07-09 | 北京大北农科技集团股份有限公司 | Pest control method |
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