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1.
苏云金芽胞杆菌SG3-7菌株是一株从秦岭山区灌木丛土壤中分离得到的产不规则伴胞晶体的野生菌,本研究对该菌株的伴胞晶体形态、杀虫基因的类型以及杀虫活性等方面进行研究。分析结果表明:SG3-7菌株的内生质粒携带有3个cry基因(cry2Ab、cry4A、cry9Ea),以及1个cyt1Aa型基因。其主要产生130 kDa和60 kDa分子量大小的伴胞晶体蛋白,对鳞翅目的甜菜夜蛾和菜青虫幼虫具有显著的杀虫活性,其LC_(50)分别为29.31μg·mL~(-1)和47.11μg·mL~(-1),但对棉铃虫幼虫无杀虫活性。 相似文献
2.
从河北省土壤分离出苏云金芽胞杆菌（Bacillus thuringiensis,Bt）MB-15菌株,利用室内生物活性测定的方法,与Bt标准菌株HD-1的杀虫毒力进行比较,结果发现该菌株胞晶混合液对棉铃虫(Helicoverpa armigera)、甜菜夜蛾(Spodoptera exigua)、小菜蛾(Plutella xylostella)、斜纹夜蛾(Spodoptera litura)和菜青虫(Pieris rapae)等5种鳞翅目蔬菜害虫的毒力均高于标准菌株Bt HD-1胞晶混合液。对发酵液各组分活性的分析发现,该菌株的上清液对胞晶混合物的杀虫活性有明显的增效作用。光学显微镜下观察该菌株伴胞晶体为菱形,SDS-PAGE分析显示其伴胞晶体主要由130.0 kDa和65.0 kDa 两种晶体蛋白组成。利用PCR-RFLP对其杀虫基因型进行鉴定,结果表明该菌株含有cry1Ac、cry2Aa、cry1I和vip3Aa基因。推断该菌株是一株对鳞翅目蔬菜害虫的防治具有潜在的商业开发价值的Bt野生株。 相似文献
3.
从河北省土壤分离出苏云金芽胞杆菌(Bacillus thuringiensis,Bt)MB-15菌株,利用室内生物活性测定的方法,与Bt标准菌株HD-1的杀虫毒力进行比较,结果发现该菌株胞晶混合液对棉铃虫(Helicoverpaarmigera)、甜菜夜蛾(Spodoptera exigua)、小菜蛾(Plutella xylostella)、斜纹夜蛾(Spodoptera litura)和菜青虫(Pieris rapae)等5种鳞翅目蔬菜害虫的毒力均高于标准菌株Bt HD-1胞晶混合液。对发酵液各组分活性的分析发现,该菌株的上清液对胞晶混合物的杀虫活性有明显的增效作用。光学显微镜下观察该菌株伴胞晶体为菱形,SDS-PAGE分析显示其伴胞晶体主要由130.0 kDa和65.0 kDa两种晶体蛋白组成。利用PCR-RFLP对其杀虫基因型进行鉴定,结果表明该菌株含有cry1Ac、cry2Aa、cry1I和vip3Aa基因。推断该菌株是一株对鳞翅目蔬菜害虫的防治具有潜在的商业开发价值的Bt野生株。 相似文献
4.
根据能形成伴胞晶体的特征，芽胞杆菌CTC菌株被鉴定为苏云金芽胞杆菌幕虫亚种。但该菌株明显不同于典型的苏云金芽胞杆菌，因为其伴胞晶体蛋白并不是由杀虫晶体蛋白基因编码，而是编码细胞表面S-层蛋白基因的产物，且伴胞晶体蛋白没有检测到对昆虫的毒性。本研究基于对CTC菌株及参照菌的16S-23S rRNA间隔区、编码S-层蛋白的基因进行扩增并测序，从系统发育水平检测到CTC菌株明显与苏云金芽胞杆菌幕虫亚种典型菌株T02存在差异，表明芽胞杆菌CTC菌株更接近蜡状芽胞杆菌。 相似文献
5.
模拟自然界中云斑天牛Batocera horsfieldi(Hope)成虫的生活状态,建立室内人工饲养及生测方法,对苏云金芽胞杆菌(Bt)的杀虫活性进行测定。将苏云金芽胞杆菌菌悬剂涂抹在野蔷薇枝条表面,饲喂云斑天牛成虫,20 d后统计死亡率、体质量变化及产卵数量。在室内测定了80株苏云金芽胞杆菌的活性,通过1次初筛和2次复筛,筛选到1株对云斑天牛成虫有较高致死率的菌株ZQ-51。该菌株处理云斑天牛成虫后平均死亡率为53.33%,平均体质量减少率和平均产卵减少率分别是5.67%和12.48%,与对照差异均极显著。从感染死亡的成虫肠道中重新分离了该菌株,并验证分离的菌株与ZQ-51具有相似的形态特征和杀虫活性,表明苏云金芽胞杆菌可通过云斑天牛取食进入肠道而发挥杀虫作用。 相似文献
6.
采用生物活性测定方法对实验室分离保存的73株Bt菌株进行杀棉铃虫幼虫的筛选,获得了3株对棉铃虫具有显著杀虫活性的菌株,其胞晶混合物对棉铃虫幼虫的LC50值为413. 43～574. 30μg·m L-1。对这3株Bt菌株研究发现,其产生的伴胞晶体类型包括菱形和方形,伴胞晶体蛋白编码基因的类型主要有cry1Aa、cry1Ab、cry1Ac、cry1Ia、cry2Ac、cry2Ah、cry4Ba、cry9Ea、cyt2Aa等,这些Bt菌株均表达约60 k Da和130k Da伴胞晶体蛋白。这3株Bt菌株的鉴定为棉铃虫的生物防治提供新的菌株资源和基因资源。 相似文献
7.
通过采用醋酸钠-抗生素筛选法从6个不同生境中筛选得到了苏云金芽孢杆菌新菌株W14、ZY1、3346、TMQ2、B31和P91,镜检可观察到其伴孢晶体呈小菱形和不规则形状.SDS-PAGE分析结果表明6个菌株表达的晶体蛋白分子量为130kDa,对其杀虫晶体蛋白的测定,结果发现这些菌株晶体蛋白含量均不高,仅为0.20%～0.29%,而B31则无晶体含量.同时对这6个Bt菌株进行了生物测定,发现所有菌株对棉铃虫杀虫活性均较低.由此可见,特殊生境获得的Bt菌株与常规菌株晶体含量不同,且晶体含量过低是其杀虫活性不高的根本原因. 相似文献
8.
【目的】分离和纯化嗜线虫致病杆菌北京变种（Xenorhabdus nematophila var. pekingensis）CB6菌株胞内和胞外杀虫蛋白，鉴定其蛋白种类。为进一步利用此类杀虫蛋白奠定基础。【方法】采用硫酸铵沉淀、DEAE Sepharose FF离子交换柱层析、Butyl Sepharose FF疏水柱层析和Sephacryl S-200 HR凝胶过滤对该类蛋白进行分离和纯化，采用Native-PAGE和SDS-PAGE技术对所纯化蛋白进行组分分析。【结果】获得达电泳纯的胞内杀虫蛋白E1和胞外杀虫蛋白E2。以2.58 μg•ml-1含量E1、以4.21 μg•ml-1含量E2喂饲棉铃虫初孵幼虫，对幼虫的生长抑制率分别达62.63％和97.9％。E1、E2经相同纯化参数处理获得的洗脱图相似，经native-PAGE和SDS-PAGE呈现出相似的单带电泳图谱。SDS-PAGE测得E1、E2表观分子量大于212 kD，该杀虫蛋白在60℃仍表现出较强的活性。电泳染色结果表明该类蛋白非糖蛋白也非酯蛋白。【结论】CB6菌株胞内杀虫蛋白E1和胞外杀虫蛋白E2可能是同一种（类）蛋白。 相似文献
9.
XS904菌株发酵条件的优化及其杀虫活性物质的理化特性 总被引：2，自引：0，他引：2
从浙江象山港海涂泥样中分离的海洋放线菌XS904菌株发酵液具有较强的杀虫活性。文章以XS904菌株发酵提取物的杀虫活性为指标，对该菌株的发酵条件如培养基装量、培养基初始pH值、发酵温度及发酵时间等进行优化，同时对具有杀虫活性的发酵产物的部分理化性质进行研究。结果表明：利用黄豆粉培养基液体发酵时，在培养基装量为25m1，初始pH7．5，温度30℃的条件下，XS904菌株通过5d发酵后得到的产物的杀虫活性最高；该杀虫活性成分极性较强，具有较好的pH值稳定性和热稳定性。利用Diaion HP2MGL大孔吸附树脂柱层析进行初步纯化，杀虫活性物质主要集中于乙醇和水的体积比为4：6的洗脱液中。 相似文献
10.
苏云金芽胞杆菌(Bacillus thuringiensis)能产生杀虫晶体蛋白(Insecticida Crystal Proteins, ICPs),对敏感昆虫有强烈毒性,而对高等动物和人无毒性。ICPs由cry或cyt基因编码,根据cry1I型基因设计引物,以Bt LB52菌株的质粒DNA为模板,扩增出了全长为2.1 kb的cry1I基因,其能通过表达载体pEB在大肠杆菌中高效表达为79.9 kDa的蛋白。经过AlginX软件分析该蛋白由712个氨基酸组成,分子量为79.9 kDa,等电点为6.54,为弱酸性蛋白质,NCBI Blast比对该蛋白的氨基酸序列与Cry1Ib3的相似性最高为98%,有12个氨基酸的差异。该基因已在GenBank中注册,登录号为ADK38579,并被国际基因命名委员会正式命名为cry1Ib6。它的表达产物对小菜蛾具有较高的毒力,LC50为1.196 μg/mL,为抗虫转基因植物研究提供了新的基因。 相似文献
11.
一株高毒苏云金杆菌的晶体蛋白组成及RAPD分析 总被引：1，自引：1，他引：1
筛选得到一株对摇蚊幼虫高毒的苏云金芽孢杆菌以色列亚种菌株(命名为SZ6),利用聚丙烯酰胺凝胶电泳(SDS-PAGE)和RAPD技术分析了其晶体蛋白和总DNA组成,结果表明:在晶体蛋白提纯中,固体培养基培养的菌株晶体蛋白比液体摇瓶培养的更易用双液相法提纯;晶体蛋白亚基在分子量上和标准菌株的晶体蛋白没有显著差异,都含有4条多肽(分子量分别为135、128、72和27KD),但RAPD分析结果显示,该毒株在DNA组成上,于多个位点处存在与标准菌株不同的结构。 相似文献
12.
[目的]研究苏云金芽孢杆菌LY30菌株对棉铃虫的毒性作用。[方法]初步比较LY30和HD-1菌株的形态特征、生理生化特性,并采用生物测定的方法,比较它们对棉铃虫初孵、二龄、三龄幼虫的毒效差异。[结果]LY30属于H3a3b3c血清型、kurstaki亚种,主要由135、65kD2种蛋白成分组成,在菌株生长形态、发酵培养特征、生理生化特性方面与生产菌株HD-1差别不大。LY30菌株发酵液对棉铃虫初孵、二龄、三龄幼虫的LC50分别为0.32、0.62、2.58μl/ml。[结论]LY30菌株对棉铃虫三龄以下的幼虫具有高毒力。 相似文献
13.
Bacillus thuringiensis (Bt) strain C002 contains crylAa, cry2Ab, cry1Ca insecticidal crystal genes and an unkown gene cryX, among which crylCa is located in a 6 -9 kb EcoR Ⅰ fragment of the chromosomal DNA. The total DNA and the plasmids DNA libraries of C002 were constructed in Bt-E. coli shuttle plasmid pHT315 by inserting 6 - 9 kb chromosomal and plasmid DNA fragments prepared respectively with EcoR Ⅰ complete and Sau3A Ⅰ partial digestion. On the basis of every 50 transformants pooled together from 5 - 10 tubes, the pools containing about 2 000 transformants from the plasmids DNA library and 400 transformants from the total DNA library were rapidly screened by PCR-RFLP. Clones containing crylAa, cryX, crylCa, and cry2Ab were isolated and named as pHT-1Aa, pHT-X, pHT-1Ca and pHT-2Ab respectively. Restriction analysis indicated that pHT-1Aa, pHT-1Ca and pHT-2Ab had the typical physical map of the homologous cry genes. Furthermore, each plasmid was transferred into Bt acrystalliferous strain cryB- by eletroporation. SDS-PAGE result showed that transformant of pHT-1Ca expressed 130 kDa protein and bioassay result proved its high toxicity against Spodotera exigua 1st instar larvae with 100% corrected motality. 相似文献
14.
Bt杀虫蛋白对甜菜夜蛾生长发育的影响 总被引：1，自引：0，他引：1
实验室内研究了Bt杀虫蛋白对甜菜夜蛾生长发育的影响。结果表明：甜菜夜蛾二龄幼虫取食含Bt杀虫蛋白浓度为0.5μg/g,1.0μg/g,2.0μg/g,4.0μg/g,8.0μg/g的人工饲料后,随着饲料中杀虫蛋白浓度增加,幼虫死亡率增加、幼虫发育历期延长、蛹的发育历期缩短、蛹重变轻、化蛹率降低。甜菜夜蛾四龄幼虫取食含Bt杀虫蛋白浓度为2.0μg/g,4.0μg/g,8.0μg/g,16.0μg/g的人工饲料后,幼虫死亡率、化蛹率、蛹的羽化率均无明显差异;而幼虫发育历期延长、蛹的发育历期缩短,蛹重减轻。杀虫蛋白浓度为8.0μg/g,16.0μg/g时,幼虫发育历期明显长于对照。杀虫蛋白浓度为16.0μg/g时, 蛹的发育历期和蛹重与对照差异显著 相似文献
15.
采用叶片浸蘸法测定 4株苏云金杆菌 (Bt)菌株对小地老虎 1龄幼虫的毒性 ,毒力回归式分别为 Y =11.6 36 1+2 2 .36 6 5X (Bt94 )、 Y =3.0 74 6 +15.5579X (Bt95)、 Y =8.0 4 0 0 +2 3.52 95X (Bt96 )、 Y =7.340 9+18.2 990 X (Bt97) .Bt对小地老虎 1- 3龄幼虫的毒杀效果随幼虫龄期上升呈显著下降 .Bt处理 3龄幼虫的死亡率仅在 0 - 5.0 0 % ,与对照无显著差异 .经初步观察 ,受测菌株对小地老虎有一定的亚致死效应 ,表现在幼虫对 Bt处理的叶片有拒食现象 ,幼虫增重明显低于对照 相似文献
16.
对美国白蛾高毒力苏云金杆菌菌株的筛选 总被引：1，自引：0，他引：1
为获得对美国白蛾(Hyphantria cunea(Drury))高毒力苏云金芽孢杆菌(Bacillus thuringiensis,Bt)菌株,本研究以43株Bt自然分离株为材料,经初步筛选和LC50测定得到了对美国白蛾幼虫高毒力菌株Bt S-19,在此基础上对该菌株的生物学特性进行了初步研究。结果表明,野生株间对美国白蛾活性差异明显,LC50测定结果经DPS软件分析,菌株Bt S-19活性最好,处理72 h后对初孵幼虫LC50值为1.8×105cells/mL,浓度对数回归方程为Y=1.082 9+0.744 4X,相关系数为0.988 8,明显好于标准菌株HD-1;对二龄、三龄、四龄和五龄幼虫72 h的LC50值分别为2.3×105、8.1×105、1.3×106和3.1×106cells/mL。光学显微镜下观察发现该菌株晶体为小菱形,生长对数期为2~5 h。推断该菌株是一株对美国白蛾有应用潜力的Bt野生株。 相似文献
17.
Effect of three insect-resistant maizes expressing Cry1Ie,Cry1Ab/Cry2Aj and Cry1Ab on the growth and development of armyworm Mythimna separata(Walker) 
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下载免费PDF全文 Three transgenic maize events(IE09 S034, Shuangkang 12–5 and C0030.3.5) produced Cry1 Ie, Cry1 Ab/Cry2 Aj and G10-EPSPS, Cry1 Ab and EPSPS, respectively, all of which target the Asian corn borer. The oriental armyworm Mythimna separata(Walker) is the secondary target. In this study, the effects of the three Bt maizes on the development and survival of armyworm were studied. The results showed that IE09 S034 had insecticidal activity against 1 st instar larvae, and the survival rate of armyworm fed with Bt maize for 10 days was 46.2%, significantly lower than that of the control. The larvae at 3 rd–6 th instar were more tolerant of the Bt toxin than the early instar larvae. However, Shuangkang 12-5 had good insecticidal activity against 1 st–5 th instar larvae. The mortality was nearly 100% when the larvae were fed with Shuangkang 12-5 before 3 rd instar, and the toxin had quick-acting efficacy. This event significantly inhibited the development of armyworm; that is, the larval duration of the 3 rd and 4 th instar larvae fed with Shuangkang 12-5 was prolonged by 4.5 and 3.0 days, respectively. The pupal weight and egg number were also significantly lower than those of the control. For C0030.3.5, it could control 1 st–5 th instar larvae effectively. The mortality rates were all over 50% if 1 st–3 rd larvae were fed with this event. The pupal weight of 4 th–6 th instar larvae fed with Bt maize were only 53.9, 56.8 and 54.6%, respectively, compared to that of the control. The number of eggs laid was significantly less than the control. The results indicate that all three transgenic maize events exhibit the potential to provide effective control of early instar larvae of armyworm, which can be commercialized in future to control lepidoptera pests such as Asian corn borer and armyworm. 相似文献
18.
[目的]筛选对苏云金芽孢杆菌(Bacillusthuringiensis,简称Bt)可湿性粉剂具有最佳增效作用的物质,并对增效作用显著的混配组合进行了增效作用的评定。[方法]以小菜蛾为生物测定昆虫,采用浸叶法测定了6种添加物对苏云金杆菌(Bacillusthuringiensis,Bt)可湿性粉剂的毒杀增效作用。[结果]研究发现0.1%ZnCl2、0.5%ZnCl2、1.0%ZnCl2、1.0%MgCl2、0.5%硼酸、1.0%硼酸、0.5%柠檬酸和1.0%柠檬酸对Bt可湿性粉剂表现出增效作用,其中0.5%硼酸对Bt可湿性粉剂的增效作用最强,增效比为17.2,1.0%ZnCl2次之,增效比为15.6。同时,杀虫速度的测定结果显示,0.5%硼酸的加入使Bt可湿性粉剂的致死中时缩短了约10h,加快了Bt的杀虫速度。此外,当将1.0%ZnCl2和0.5%硼酸分别与Bt可湿性粉剂溶液室温下混合放置15d后,两种混合物的毒力均没有明显的变化。[结论]硼酸作为Bt可湿性粉剂的增效因子,不但有利于增强Bt制剂的杀虫效果而且还可以提高其杀虫速度,从而在一定程度上克服了Bt制剂杀虫效果差和杀虫速度慢等实际问题。 相似文献
19.
《东北农业大学学报(英文版)》2017,(4):61-68
Vegetative Insecticidal Proteins(VIPs),a large family of insecticidal proteins,are produced from Bacillus thuringiensis (Bt) during the vegetative growth stage. VIPs represent the second generation of bio-insecticides that confer a wider insecticidal spectrum and have stronger activity.This work compared the geographical distribution of Bt strains and their vip3 genes in different climatic zones in China, the tropical (Hainan Province), subtropical (Guangxi Province) and temperate zones (Heilongjiang Province). A total of 156 Bt strains were isolated from 841 soil samples in Hainan Province tropical region, 356 Bt strains from 1 420 soil samples in Guangxi Province and 167 Bt strains from 1 010 soil samples in different geographical regions in Heilongjiang Province. Twenty-two out of 156 strains from tropical Hainan Province and two out of 356 from subtropical Guangxi Province were found to express vip3 genes,while vip3 genes were not expressed from temperate zone in Heilongjiang Province.Restriction Fragment Length Polymorphism(RFLP)was used to identify different types of vip 3 genes that were within the same family and three full-length vip3 genes were isolated.The genes cloned from Bacillus thuringiensis strain SL3 expressed in the transformed E.coli BL21 strain. Through SDS-PAGE, 88.6 ku insecticidal protein was expressed. The bioassays used two-instar larva of Lepidoptera insects (Spodoptera exigua and Agrotis ipsilon)were performed.The results of the bioassays showed that the protein strongly inhibited the body weight increasement on Spodoptera exigua and Agrotis ipsilon in a standard bioassay.Taken together,the results indicated that the distribution of Bt strains and vip3 genes had regional preference.Tropical and subtropical regions were the rich resources of Bt strains and vip3 genes compared with temperate region.These results would undoubtedly facilitate the studies of insecticidal proteins and expand the list of the pest-killing candidates to make fully use of the extremely rich microbial resources.The new vip 3 genes isolated in the current study might also help resolve the emerging insecticidal resistance problems. 相似文献