Bt杀虫基因与Bt转基因抗虫植物研究进展

苏云金杆菌是一类非常重要的昆虫病原体,它能产生特异性的杀虫结晶蛋白,对农业上和生物医学上的许多有害的昆虫有毒杀作用,这些害虫包括鳞翅目、双翅目、鞘翅目、膜翅目、螨类和线虫。近三十年来,以苏云金杆菌为基础的生物杀虫剂已在世界范围内商业化用于防治重要经济作物的害虫。近年来有关Bt基因的遗传、分子生物学和基因工程已取得显著进展。本文对苏云金杆菌杀虫晶体蛋白基因的分类和杀虫机理及用该类基因构建的工程转基因植物研究状况作一简要综述,同时对Bt基因工程存在的潜在问题和解决途径作了简单的探讨。     

Bt杀虫基因与Bt转基因抗虫植物研究进展

苏云金杆菌是一类非常重要的昆虫病原体,它能产生特异性的杀虫结晶蛋白,对农业上和生物医学上的许多有害的昆虫有毒杀作用,这些害虫包括鳞翅目、双翅目、鞘翅目、膜翅目、螨类和线虫。近三十年来,以苏云金杆菌为基础的生物杀虫剂已在世界范围内商业化用于防治重要经济作物的害虫。近年来有关Ｂｔ基因的遗传、分子生物学和基因工程已取得显著进展。本文对苏云金杆菌杀虫晶体蛋白基因的分类和杀虫机理及用该类基因构建的工程转基因植物研究状况作一简要综述,同时对Ｂｔ基因工程存在的潜在问题和解决途径作了简单的探讨。     

抗虫基因的抗虫原理及其应用现状和展望

对目前植物抗虫基因工程中广泛应用的几个抗虫基因的抗虫原理及应用现状作了概述,并讨论了可能出现的问题和相应的对策。     

转苏云金芽胞杆菌杀虫晶体蛋白基因抗虫植物的研究进展与商品化

本文综述了转ICPs基因植物从ICPs在植物中低表达,具杀虫效应到高表达而进行田间应用试验的研究进展及转ICPs抗虫植物的商品化现状。     

转Bt基因作物对蚯蚓影响研究进展

转苏云金杆菌杀虫蛋白(Bt)基因作物的商品化种植可能对土壤生态系统产生不利影响是近10年来颇有争议的问题.转Bt基因作物可通过多种方式向土壤中释放苏云金杆菌杀虫蛋白即Bt蛋白,从而引起土壤生物和生态系统基本功能的变化;蚯蚓可加快动植物残体的降解,促进有机质的分解和矿化,与其他土壤生物相比,蚯蚓对某些污染物更敏感.本文从研究中用到的蚯蚓种类、采用的实验方式、研究的科学问题等方面综述了转Bt基因作物对土壤动物蚯蚓影响的研究进展,并对转Bt基因作物对土壤动物蚯蚓影响研究的发展趋势进行了展望,旨在为转Bt基因作物对非靶标土壤动物的影响提供参考,进而为全面评价转Bt基因作物对土壤生态系统的影响提供依据.     

苏云金杆菌抗虫基因cryI Ac 转化辣椒的研究

通过农杆菌介导法将杀虫结晶蛋白基因cryIAc导入到辣椒子叶外植体中,经卡那霉素筛选、组织化学法检测GUS活性以及PCR、southern杂交分析证实cryIAc基因已整合进辣椒核基因组中。     

苏云金杆菌抗虫基因cryIAc转化辣椒的研究

通过农杆菌介导法将杀虫结晶蛋白基因cryIAc导入到辣椒子叶外植体中,经卡那霉素筛选、组织化学法检测GUS活性以及PCR、Southern杂交分析证实cryIAc基因已整合进辣椒核基因组中。     

抗虫转基因欧洲黑杨的培育*

用带有35 S-Ω-Bt-NOS嵌合基因的双元载体的农杆菌LBA 4404转化欧洲黑杨的叶片外植体,共获得54株转化再生植株。用这些再生植株对杨尺蠖(Apcchimia cinerarius)进行毒力测定,昆虫校正死亡率在80一96％的再生植株占总测定植株的15％。部分再生植株对舞毒娥进行测定,表明在5～9天内校正死亡率高达100％,存活昆虫的生长和发育也明显地受到抑制。根据苗木在苗圃中高生长和昆虫校正死亡率采用重心聚类法进行分析,初步选出高生长良好和杀虫率居中的3株再生植株。以选出的植株为主进行了PCR及PCR产物的South—ern blot和再生植株DNA Southern blot测定,结果证明Bt基因已插入到这些植株的DNA上,并表达出苏云金杆菌杀虫蛋白的杀虫活性。     

苏云金杆菌杀虫蛋白的多样性

苏云金杆菌是生物防治中应用最为广泛的一种杀虫剂,其杀虫蛋白具有广泛的多样性。本文就苏云金杆菌杀虫蛋白的基因、基因分布、杀虫蛋白结构以及作用机制的多样性进行了概述。     

Isolation, characterization and expression of a novel vegetative insecticidal protein gene of Bacillus thuringiensis

Twenty-four serovars of Bacillus thuringiensis (Bt) were screened by polymerase chain reaction to detect the presence of vegetative insecticidal protein gene (vip)-like sequences by using vip3Aa1-specific primers. vip-like gene sequences were identified in eight serovars. These genes were cloned and sequenced. The deduced amino acid sequence of the vip3Aa14 gene from Bacillus thuringiensis tolworthi showed considerable differences as compared to those of Vips reported so far. The vip3Aa14 gene from Bt tolwarthi was expressed in Escherichia coli using expression vector pET29a. The expressed Vip3Aa14 protein was found in cytosolic supernatant as well as pellet fraction, but the protein was more abundant in the cytosolic supernatant fraction. Both full-length and truncated (devoid of signal sequence) Vips were highly toxic to the larvae of Spodoptera litura and Plutella xylostella. Truncation of Vip3Aa14 protein at N-terminus did not affect its insecticidal activity.     

Effective bacterial insecticidal proteins against coleopteran pests: A review

Coleoptera, the order of insects commonly referred to as beetles, are able to survive in various environments, and thus, comprise the largest order in the animal kingdom. Coleopterans mainly include coprophagous and phytophagous lineages, and many species of the latter lineage are serious pests. In addition to traditional chemical methods, biocontrol measures using various bacterial insecticidal proteins have also gradually been developed to control these insect pests. In this review, we summarized the possible coleopteran‐pest‐specific bacteria and insecticidal proteins that have been reported in the literature thus far and have provided a comprehensive overview and long‐term guidance for the control of coleopteran pests in the future.     

苏云金芽孢杆菌cry基因研究进展

从cry基因的分类、cry基因与转座因子的关系、cry基因的表达调控以及cry基因的作用机理等 4个方面综述了cry基因的研究进展 ,并简要展望了其研究和应用前景。     

苏云金芽胞杆菌Rpp39杀虫晶体蛋白基因的鉴定及cry2Aa12基因的克隆表达

[目的]本研究的目的是分析从四川生态条件下分离的苏云金芽胞杆菌Rpp39菌株的特性,从分子水平上揭示该菌株对鳞翅目高毒力的原因;进一步从中分离克隆cry2Aa基因,并对其进行初步的表达研究.[方法]本研究主要采用扫描电镜观察、PCR-RFLP鉴定法和SDS-PAGE分析法研究菌株的特性;采用PCR直接克隆法克隆cry2Aa全长基因,并亚克隆到原核表达载体pET-30a中,构建重组表达质粒pET-2Aa,再转入受体菌E.coli.BL21(DE3)中进行诱导表达;采用室内生物测定法测定表达产物对小菜蛾和水稻二化螟的毒力.[结果]经扫描电镜观察菌株Rpp39主要产生菱形、方形和圆形3种伴胞晶体;SDS-PAGE分析表明主要产生130 kDa和60 kDa左右2种蛋白;经PCR-RFLP鉴定,该菌株含有cry1Aa、cry1Ab、cry1Ac、cry1Ia和cry2Aa五类杀虫晶体蛋白基因;1种cry2Aa类杀虫晶体蛋白全长基因被克隆,序列分析显示该基因的开放阅读框(ORF)为1902 bps,编码由634个氨基酸组成的蛋白质,氨基酸序列与Cry2Aa1蛋白同源性为99.7%,被国际Bt杀虫晶体蛋白基因命名委员会命名为cry2Aa12.重组表达质粒pET-2Aa在E.coli BL21(DE3)中,经IPTG诱导能正常表达,SDS-PAGE电泳验证含有65 kDa表达蛋白.生物活性测定表明表达的包涵体蛋白对小菜蛾和二化螟具有杀虫活性,LC50分别为5.4 μg/mL和22.3μg/mL.[结论]菌株Rpp39及从中分离克隆的cry2Aa12基因来自四川生态条件,丰富了菌株及基因的资源,在资源积累方面具有重要意义.     

苏云金芽孢杆菌杀虫晶体蛋白超量表达的机制

杀虫晶体蛋白是苏云金芽孢杆菌主要杀虫成分,进一步提高杀虫晶体蛋白的表达量是苏云金芽杆菌高效工程菌构建的主要途径。本文讨论了ｃｒｙ基因启动子活性、ｍＲＮＡ稳定性、不同ｃｒｙ基因间的协同表达发及伴了孢晶体的形成等几个方面在转录水平或转录后水平上对杀虫晶体蛋白表达的影响。     

Cry1Ac基因载体构建及其在枯草芽孢杆菌中的杀虫活性表达

根据苏云金芽孢杆菌Bacillus thuringiensis HD-73基因Cry1Ac和枯草芽孢杆菌Bacillus subtilis木糖诱导型启动子PxylR序列, 分别设计2对特异引物Cry1Ac F/R和Pxy F/R,扩增获得了完整的启动子PxylR和Cry1Ac基因序列,进一步以上述产物混合物为模板,以Pxy F/Cry1Ac R作引物进行重迭PCR,获得了载体PxylR-Cry1Ac,经SphⅠ和BamHⅠ完全酶切后,将PxylR-Cry1Ac插入大肠杆菌-苏云金芽孢杆菌穿梭载体pHT315,重组表达质粒pCry1Ac315转化枯草芽孢杆菌感受态细胞。工程菌株质粒酶切电泳分析、SDS-PAGE电泳分析和杀虫生物活性测定结果证实了Cry1Ac基因的导入及其在枯草芽孢杆菌JAAS01D中的有效表达。     

苏云金芽孢杆菌及其杀虫晶体蛋白 作用机制的研究进展

综合叙述了苏云金芽胞杆菌Bacillus thuringiensis和杀虫晶体蛋白的作用机制及在不同水平上解释这些机制的一些流行模型和有关亚分子结构的作用。     

A proteomic analysis approach to study insecticidal crystal proteins from different strains of Bacillus thuringiensis

We obtained and compared a new cry2Ac6 gene from Bacillus wuhanensis 140, and Bacillus thuringiensis (Bt) subsp. kurstaki 4.0718 and B.t. kurstaki XL004 that share a similar genetic background but occupy different ecological niches. Using a proteomic approach and function-based activity profiling, we systemically identified the insecticidal crystal proteins (ICPs) from the three Bt species, which were found to be mainly distributed at pH 4–7 on two-dimensional electrophoresis (2DE) gels by PDQuest software. The proteins that exhibited a significant difference in expression were excised, digested in-gel and identified by MALDI-TOF-MS. Thirty-three differently expressed proteins were identified from the three Bt strains. The Cry2Ac6, Cry1Ab16, CryIG, CryH2, CryI, CryINA67-1 and CryI⁺ crystal protein mixture from B.t. wuhanensis 140, Cry1Ac, Cry2Aa and endotoxin delta1 from B.t. kurstaki 4.0718 were further analyzed by bioinformatics analysis. Two common proteins were founded in three strains, the heat shock proteins (HSP60) and the translation elongation factor Tu, which help with protein refolding and prevent protein degradation. The different enzymes of metabolism, including glutamate racemase, chemotaxis protein histidine kinase and related kinases pyruvate dehydrogenase complex E1orE3 were identified. Some protein spots could not be identified. The results indicate that each Bt strain has unique ICPs as well as some common proteins related to ICPs formation, and that the virulence of Bt strains is closely related to the expression of specific ICPs.     

Diversity of Colombian strains of Bacillus thuringiensis with insecticidal activity against dipteran and lepidopteran insects

AIM: To evaluate the genetic and molecular diversity and insecticidal activity of Bacillus thuringiensis isolates from all the natural regions of Colombia. METHODS AND RESULTS: A total of 445 isolates from a collection of B. thuringiensis were characterized. The parasporal crystal morphology that was most abundant was bipyramidal (60%). Almost 10% of the isolates were toxic to Spodoptera frugiperda and 5.6% against Culex quinquefasciatus larvae. cry gene content determined by PCR indicated that 10.6% of the isolates contained cry1 genes and 1.1% contained cry2, cry4 or cry11 genes. Protein content of the parasporal crystal was determined by SDS-PAGE; 25 and 18 different protein profiles were found in isolates active against S. frugiperda and C. quinquefasciatus, respectively. CONCLUSIONS: Bacillus thuringiensis presents great genetic and molecular diversity even in isolates from the same soil sample. Moreover, the diversity and activity of the isolates might have a relationship with the geographical origin of the samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The results obtained here indicate that some of the B. thuringiensis isolates characterized in this study are potential control agents that could be used in programmes against mosquitoes and S. frugiperda.