CN104744574A - 对玉米螟有杀虫活性的Bt蛋白及其应用 - Google Patents
对玉米螟有杀虫活性的Bt蛋白及其应用 Download PDFInfo
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Abstract
本发明提供了对玉米螟有杀虫活性的Bt蛋白及其应用,所述蛋白质来源于苏云金芽胞杆菌,该蛋白选自Cry1Bb蛋白或Cry1Be蛋白中的至少一种。本发明提供的对玉米螟高毒力的几种Bt蛋白,将为抗玉米螟转基因作物的制备提供新的基因来源,并有效延缓抗性产生。
Description
技术领域
本发明涉及生物技术领域,具体涉及几种对玉米螟具有杀虫活性的Bt蛋白及应用。
背景技术
玉米螟(Pyrausta nubilalis)属于鳞翅目,螟蛾科,是玉米的主要虫害。主要分布于北京、东北、河北、河南、四川、广西等地。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。玉米螟,可危害玉米植株地上的各个部位,使受害部分丧失功能,降低籽粒产量。
此外,由于气候变暖、种植模式的改变导致病虫害加重,使得玉米螟危害的形势非常严峻,防治问题刻不容缓。而化学防治所需人力物力颇多,因此生物防治是解决当前问题的最佳途径。
苏云金芽胞杆菌(Bacillus thuringiensis)产生的伴胞晶体蛋白是主要的杀虫成分,已有研究发现大多数晶体蛋白对鳞翅目、双翅目、鞘翅目、膜翅目、同翅目、直翅目、食毛目多种昆虫,以及一些线虫、螨类和原生动物具有特异性的杀虫活性。
为防治玉米螟对农作物带来的危害,寻找对东方粘虫和玉米螟高毒力的Bt蛋白可为防治东方粘虫和玉米螟生物灾害提供多种杀虫资源,这对我国的生物防治有着十分重要的意义。此外,也将为我国抗虫转基因作物的研究提供优良的候选基因。
此外,目前已有将对玉米螟有活性的Bt蛋白应用到防治玉米螟上,导致玉米螟对一些Bt蛋白产生抗性,因此对玉米螟有活性的新的Bt蛋白资源的挖掘,为防治这类害虫提供丰富的候选蛋白,也将对延缓抗性产生具有重要的意义。
发明内容
本发明的一个目的是针对现有技术的不足,提供了几种对玉米螟高毒力的Bt蛋白,以克服对玉米螟防治资源的匮乏。
为实现上述目的,本发明采用如下技术方案:
本发明提供了对玉米螟有杀虫活性的Bt蛋白,所述蛋白质来源于苏云金芽胞杆菌,所述蛋白选自Cry1Bb蛋白或Cry1Be蛋白中的至少一种。
本发明一个优选的方面,如下序列组成的上述Bt蛋白:其中,Cry1Bb蛋白是如下1)或2):
1)序列表中序列1所示的氨基酸序列组成的蛋白;
2)在序列表中序列1的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸组成的且具有1)相同功能的蛋白;
Cry1Be蛋白是如下3)或4):
3)序列表中序列2所示的氨基酸序列组成的蛋白;
4)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸组成的且具有3)相同功能的蛋白。
本发明的另一个目的是提供了含有上述Bt蛋白的应用。
本发明一个优选的方面,上述组合物中的Bt蛋白含量为10-70%,优选30-50%,特别优选40%。
进一步地,所述的Bt蛋白在制备玉米螟杀虫剂上的应用。
更近进一步地,所述的Bt蛋白在制备抗玉米螟转基因植物上的应用。
本发明再一个目的是提供了含有上述Bt蛋白的杀虫剂或生物制剂。
本发明还提供了含有上述Bt蛋白的转基因植物。
本发明提供的防治玉米螟的Bt蛋白,可为防治玉米螟的生物灾害提供多种杀虫资源,这对我国的生物防治有着十分重要的意义。此外,也将为我国抗玉米螟转基因作物的研究提供优良的候选基因,因此有望对其进行多基因转基因作物的研发,有望治理具有一定抗药性的害虫。上述蛋白也将为抗虫转基因作物的研发提供新的基因来源。
附图说明
图1为Cry1Ab、Cry1Ac、Cry2Ab、Cry1Ah、Cry1Ca蛋白的SDS-PAGE电泳结果图;其中从左到右依次是分子标记物,泳道1:BSA,泳道2:Cry 1Ab蛋白,泳道3:Cry 1Ac蛋白,泳道4:Cry 1Ca蛋白,泳道5:Cry 2Ab蛋白,泳道6:空白对照无晶体突变株HD73-。
图2为Cry1Ba、Cry1Bb、Cry1Be、Cry9Aa、Cry9Eb、Cry9Ee、Vip3Aa11蛋白的SDS-PAGE电泳结果图;从左到右依次是分子标记物,泳道1:BSA,泳道2:Cry1Ba蛋白,泳道3:Cry1Bb蛋白,泳道4:Cry1Be蛋白,泳道5:Cry9Aa蛋白,泳道6:Cry9Eb蛋白,泳道7:Cry9Ee蛋白,泳道8:Vip3Aa11蛋白,泳道9:pEB空载体。
具体实施方式
以下实施例用于进一步说明本发明,但不应理解为对本发明的限制。在不背离本发明精神和实质的前提下,对本发明所作的修饰或者替换,均属于本发明的范畴。
本发明所用的菌株均来自中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室。
本发明所用到的培养基:
1/2LB液体培养基:0.5%胰蛋白胨、0.25%酵母提取物、0.5%氯化钠,pH7.0,121℃,灭菌20min;
LB液体培养基:1.0%胰蛋白胨、0.5%酵母提取物、1.0%氯化钠,121℃,灭菌20min。
实施例1
1蛋白的提取
1)Cry1Ab、Cry1Ac、Cry2Ab蛋白的提取
Cry1Ab、Cry1Ac、Cry2Ab蛋白均于苏云金芽孢杆菌中进行表达,采用1/2LB液体培养基、重复溶解的方法进行提取,具体操作参见:“贝壳状革耳菌和黄孢平革菌固体培养酶系比较”一文。
2)Cry1Bb、Cry1Be、Cry9Aa、Cry9Eb、Cry9Ee、Vip3A11蛋白的提取Cry1Bb、Cry1Be、Cry9Aa、Cry9Eb、Cry9Ee、Vip3A11蛋白均于大肠杆菌中进行表达,采用LB培养基、破碎离心的方法进行提取,具体操作参见:“贝壳状革耳菌和黄孢平革菌固体培养酶系比较”一文。但由于各个蛋白的最适表达条件不同,通过反复摸索发现:Cry1Bb在18℃,IPTG终浓度1.0mmol/L可溶组分表达量最高,30℃,IPTG终浓度0.1mmol/L时不可溶组分表达量最高;Cry1Be在30℃,IPTG终浓度为0.1mmol/L可溶组分表达量最高,30℃,IPTG终浓度为0.5mmol/L时,不可溶组分表达量最高;Cry9Ee在30℃,IPTG终浓度为0.5mmol/L时可溶组分表达量最高,18℃,IPTG终浓度为0.1mmol/L时不可溶组分表达量最高。
2、SDS-PAGE电泳分析及定量
杀虫晶体蛋白的SDS-PAGE分析:吸取蛋白样品进行制样,100℃煮沸10min,13000g离心10min,取上清点样,4%浓缩胶,8%分离胶,80V电泳20min,150V电泳直到胶底边缘。电泳结束后取出凝胶,进行脱色、染色及扫描图谱,扫描结果见图1和图2。SDS-PAGE结果显示,9种蛋白分子量与预期大小一致(Cry1Ab蛋白分子量大小为130.46kDa,Cry1Ac为133.16kDa,Cry1Ca为134.55kDa,Cry2Ab为70.63kDa。Cry1Ba为137kDa、Cry1Bb为137kDa、Cry1Be为136kDa、Cry9Aa为129kDa、Cry9Eb为130kDa、Vip3Aa11为89kDa)。
3、玉米螟室内生物活性测定-Bt蛋白初筛
使用人工饲料(中国农业科学院植物保护研究所迁飞害虫组提供)进行玉米螟生物活性测定,在此过程中将惯常使用的24孔板用培养皿进行替代,探索发现是用培养皿的效果更好,因为玉米螟约3~4龄会进行自相残杀,而生测采用初孵幼虫,7天统计死亡率时并未处出现自相残杀现象。
步骤如下:称取30g人工饲料放置于灭菌的培养皿中;加入待测样品溶液3mL,充分搅拌,混匀后平均分装于三个培养皿中;根据饲料的干湿程度室温放置一段时间,直到饲料表面没有水滴。每皿接入30头初孵幼虫,每个浓度重复3次,共处理90头试虫(接完虫子垫上三层卫生纸盖紧培养皿,用橡皮筋扎紧,以免虫子逃逸);将样品置于27℃光照培养箱培养,光周期为16:8,湿度70%-80%左右,每天观察饲料干湿程度,适当做出微调;培养7d调查死虫和活虫数,计算死亡率、校正死亡率,检测结果见表1:
表1Cry1Bb和Cry1Be蛋白对亚洲玉米螟生物活性初次测定结果
从上表中可见,对照组的玉米螟生长状况良好,而饲喂含有Cry1Bb、Cry1Be蛋白的玉米螟出现大幅度死亡,可见Cry1Bb、Cry1Be蛋白对玉米螟具有较好的杀虫活性。
4、玉米螟室内生物活性测定-Bt蛋白复筛
根据上述初筛的结果,选取对玉米螟高毒力的Cry1Bb、Cry1Be的两种蛋白,参照3的初筛方法。统计7日死亡率,最后使用spss软件计算(LC50),统计结果见表2:
表2亚洲玉米螟生物活性测定结果
蛋白种类 | 致死中浓度LC50 | 置信区间(95%) |
Cry1Bb | 0.461 | (0.010,1.583) |
Cry1Be | 24.013 | (10.040,80.034) |
Cry1Bx | 31.257 | (21.722,112.411) |
通过表3可见,Cry1Bb对玉米螟有非常好的杀虫活性,Cry1Be对玉米螟有较好的杀虫活性,上述两种Bt蛋白可用于防治玉米螟的危害上。
Claims (8)
1.对玉米螟有杀虫活性的Bt蛋白,所述蛋白质来源于苏云金芽胞杆菌,其特征在于,所述蛋白选自Cry1Bb蛋白或Cry1Be蛋白中的至少一种。
2.根据权利要求1所述的Bt蛋白,其特征在于,是如下序列组成的上述蛋白:其中,Cry1Bb蛋白是如下1)或2):
1)序列表中序列1所示的氨基酸序列组成的蛋白;
2)在序列表中序列1的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸组成的且具有1)相同功能的蛋白;
Cry1Be蛋白是如下3)或4):
3)序列表中序列2所示的氨基酸序列组成的蛋白;
4)在序列表中序列2的氨基酸序列经过取代和/或缺失和/或添加一个或几个氨基酸组成的且具有3)相同功能的蛋白。
3.一种包含权利要求1或2所述的Bt蛋白的应用。
4.根据权利要求3所述的应用,其特征在于,所述组合物中的所述蛋白质含量为10-70%,优选30-50%,特别优选40%。
5.根据权利要求3所述的应用,其特征在于,所述的Bt蛋白组合物在制备玉米螟杀虫剂上的应用。
6.根据权利要求3所述的应用,其特征在于,所述的Bt蛋白组合物在制备抗玉米螟转基因植物上的应用。
7.含有权利要求1或2所述的Bt蛋白的杀虫剂或生物制剂。
8.含有权利要求1或2所述的Bt蛋白的转基因植物。
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CN110144361A (zh) * | 2018-02-11 | 2019-08-20 | 中国种子集团有限公司 | Cry1Ab/Cry1AcZM基因的抗粘虫新用途 |
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CN106928329A (zh) * | 2017-03-06 | 2017-07-07 | 中国农业科学院植物保护研究所 | 一种新的杀虫蛋白及其核苷酸序列 |
CN106928329B (zh) * | 2017-03-06 | 2020-09-22 | 中国农业科学院植物保护研究所 | 一种新的杀虫蛋白及其核苷酸序列 |
CN110144361A (zh) * | 2018-02-11 | 2019-08-20 | 中国种子集团有限公司 | Cry1Ab/Cry1AcZM基因的抗粘虫新用途 |
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