苏云金芽孢杆菌Ⅱ类转座因子的应用与研究进展

苏云金杆菌的转座因子分为两类：Ⅰ类,插入序列;Ⅱ类,转座子．在结构上,这些转座因子与苏云金芽孢杆菌杀虫晶体蛋白（ICPs）基因相联系,并且这些具有转座活性的转座因子在ICPs基因的转移和变异上起着重要作用．对苏云金芽孢杆菌的Ⅱ类转座因子即转座子的结构、作用机制、应用与研究进展进行了综述．     

苏云金芽胞杆菌转座因子的转座机理及其与杀虫晶体蛋白基因的关系

各种苏云金芽胞杆菌在杀虫毒力和杀虫谱上有很大差异。研究表明,这种特异性的杀虫毒力与存在于苏云金芽胞杆菌内的转座因子有密切关系,不同类型的转座因子其转座方式各异,总的来说可分为３种,即同源重组、转座重组和特异位点重组。这种转座过程的发生往往伴随着苏云金芽胞杆菌杀虫晶体蛋白的变异,这在基因工程菌的构建和杀虫多样性的研究上有着重要意义。     

苏云金芽胞杆菌转座因子的类群和结构

苏云金芽胞杆菌的活性成分主要是杀虫晶体蛋白（ICPs）。已经证明,大多数编码这些蛋白的基因定位于质粒上,在结构上总是与插入序列和转座子相联系。这些具有转座活性的流动因子参与了杀虫晶体蛋白基因的转移,在苏云金芽胞杆菌ICPs基因的变异性上起着极其重要的作用。本文系统介绍苏云金芽胞杆菌转座因子的分类及结构等研究进展,为有目的地利用转座因子提供参考。     

紫外线诱变苏云金芽孢杆菌最佳条件探索

为寻找高毒性的苏云金芽孢杆菌(BT菌)菌株,我们用紫外线对苏云金芽孢杆菌的库尔斯塔克亚种进行诱变,再对得到的突变菌株进行筛选,得到高毒性的菌株。本文设计实验,先对苏云金芽孢杆菌进行生长曲线的测定和表征,再通过控制变量法对苏云金芽孢杆菌进行外线诱变,最后总结出最佳的诱变条件。     

利用PCR技术直接鉴定苏云金芽孢杆菌杀虫晶体蛋白基因型的快速方法

根据PCR程序中热变性温度可使菌体裂解,释放出DNA的原理,利用苏云金芽孢杆菌杀虫晶体蛋白不同基因型的特异混合引物,对苏云金芽孢杆菌营养体直接进行PCR分析。根据不同基因型的特异扩增产物片段的分子量大小便可直接确定该苏云金芽孢杆菌杀虫晶体蛋白的基因型。本文对本室筛选天然菌株和苏云金芽孢杆菌克隆菌株分别进行了cryl基因的PCR分析。表明,该法结果可靠快速易行,在方法学上,为苏云金芽孢杆菌菌株鉴定、新菌株的筛选和基因型分析提供了极大的方便。     

苏云金芽孢杆菌毒蛋白发掘方法的研究进展

自上世纪初发现苏云金芽孢杆菌对昆虫有杀虫活性以来,如何发掘利用苏云金芽孢杆菌的杀虫晶体蛋白服务于农业生产和人类的卫生防控就成为一个重要课题。从早期的分离菌株使用菌体的复合物喷施到使用分子手段转化植物特定表达,人类在苏云金芽孢杆菌杀虫晶体蛋白的利用精准度上在不断提高,但随之而来就是抗性的产生。因此不断发掘可使用的新的苏云金芽孢杆菌杀虫晶体蛋白基因资源就是一个很重要的话题。本综述就苏云金芽孢杆菌杀虫晶体蛋白基因的发掘方法研究做一系统论述。     

我国森林土壤中苏云金芽孢杆菌生态分布的研究

从我国8个森林立地带(寒温带、中温带、暖温带、北亚热带、中亚热带、南亚热带、高原亚热带、热带)所属的13个自然保护区,采集了0—5cm土层林下土壤样品384个.测定了土壤pH、水分和养分.从中分离观察芽孢杆菌菌落1873个,分离出苏云金芽孢杆菌79株,并对其所属亚种进行了初步鉴定.其平均出土率和分离率分别为14.32%和4.21%.研究了芽孢杆菌和苏云金芽孢杆菌在森林土壤中生态分布的规律及苏云金芽孢杆菌对6种昆虫的室内毒力测定,从中筛选出不少的高效菌株.为研究苏云金芽孢杆菌在我国森林生态系中资源的保护、开发和利用,具有重要意义.     

苏云金芽孢杆菌HS66的转座因子分析

移动遗传因子普遍存在于苏云金芽孢杆菌(Bt)基因组中,而且大部分位于Bt毒素基因两侧附近,与Bt毒素基因进化和转移密切相关。本研究完成了一株对鳞翅目昆虫表现出很强的杀虫活性的Bt菌株HS66的基因组序列草图测定,并利用本地BLAST软件和ISFinder软件,进行了插入序列和转座子序列的查找及基本信息的汇总,结果表明Bt HS66基因组序列含有丰富的插入序列和转座子序列。转座因子分析是整个Bt HS66基因组分析过程中的重要的一环,后续工作中若定位移动因子在染色体及质粒序列的位置,对解析转座因子功能,帮助预测相关杀虫基因信息,以及全面理解Bt HS66基因组将有极大的帮助。     

苏云金芽孢杆菌双组份信号转导系统的生物信息学分析

苏云金芽孢杆菌(Bacillus thuringiensis)能产生杀虫晶体蛋白等多种活性成分,是目前应用最广泛的微生物杀虫剂。本文采用生物信息学方法,系统分析了由本实验室完成全基因组测序的苏云金芽孢杆菌YBT-1520、CT-43和BMB171 3个菌株的双组分信号转导系统(Two-componentsignal transduction system,TCS)的分布、结构及功能,并初步构建了部分TCS的调控网络关系图。本研究旨在为深入研究苏云金芽孢杆菌的生长、代谢以及毒力因子的表达与调控,全面了解伴孢晶体的形成机制开辟新的研究方向。     

单宁对苏云金芽孢杆菌库斯塔克变种杀棉铃虫效力的作用(英文)

本文报道棉铃虫、苏云金芽孢杆菌库斯塔克变种和单宁三者间的相互作用。苏云金芽孢杆菌芽孢晶体混合粉以6个不同的浓度（0％,0．005％,0．01％,0．015％,0．02％,0．025％湿重）分别加入含0．025％单宁和不含单宁的人工饲料中。初孵幼虫分别在上述饲料中饲养48h后,转入相应的不含苏云金芽孢杆菌的人工饲料上饲养,直到留存的幼虫化蛹。结果表明,单宁与苏云金芽孢杆菌混用可提高苏云金芽孢杆菌对棉铃虫的毒力,LD50降低45％,并可显著抑制该虫的生长发育,但二者在抑制生长发育方面无交互作用。选择取食试验显示,苏云金芽孢杆菌对五龄幼虫有显著的抑食性,但单宁无此作用,单宁与苏云金芽孢杆菌混用不会增强抑食性,但单宁能抑制苏云金芽孢杆菌菌落的生长,浓度高于15mg／100ml还能抑制芽孢的萌发。     

Efficient transformation of Bacillus thuringiensis requires nonmethylated plasmid DNA.

The transformation efficiency of Bacillus thuringiensis depends upon the source of plasmid DNA. DNA isolated from B. thuringiensis, Bacillus megaterium, or a Dam- Dcm- Escherichia coli strain efficiently transformed several B. thuringiensis strains, B. thuringiensis strains were grouped according to which B. thuringiensis backgrounds were suitable sources of DNA for transformation of other B. thuringiensis strains, suggesting that B. thuringiensis strains differ in DNA modification and restriction. Efficient transformation allowed the demonstration of developmental regulation of cloned crystal protein genes in B. thuringiensis.     

Fate of Bacillus thuringiensis strains in different insect larvae

In favorable conditions Bacillus thuringiensis spores germinate and vegetative cells multiply, whereas in unfavorable conditions Bacillus thuringiensis sporulates and produces insecticidal crystal proteins. The development of B. thuringiensis strains was investigated in the larvae of insects belonging to the orders Lepidoptera and Diptera. Bacillus thuringiensis strains able to kill the insects did not always multiply in cadavers. Strains with no specificity to kill the insect sometimes multiplied when the insects were killed mechanically. These results indicate that some insect larvae represent an environment that favors the germination of B. thuringiensis spores and the multiplication of vegetative cells; however, there was no correlation between the toxin specificity and the specificity of the host.     

Homologous and heterologous transcription of the Cry+-plasmid in Bacillus thuringiensis

The possibility of homologous and heterologous transception of Cry+ plasmids in Bacillus thuringiensis is demonstrated. Cry+ plasmids from crystal bearing strain of Bacillus thuringiensis were transferred into acrystalline strain belonging to H5 serotype by mutual incubation. The donor strain was previously marked by the transmissive plasmid pAM beta 1 coding for erythromycin and lincomycin resistance. The transcipients having acquired the ability to synthesize delta-endotoxin were referred to H5 serotype due to their phenotype. By analogous method Cry+ plasmid was transferred from Bacillus thuringiensis to Bacillus cereus. Bacillus cereus strain GP7 was used as a recipient strain resistant to tetracycline. The presence of delta-endotoxin in transcipients was confirmed by bioprobes and immunoenzyme assay. To prove the transfer of Cry+ plasmid the plasmid profiles of the parent strains and transcipients have been analyzed. The formation of cellular contacts during mutual incubation of Bacillus thuringiensis and Bacillus cereus strains was demonstrated by electron microscopic study of ultrafine cuts.     

土壤来源的五个苏云金芽孢杆菌新亚种的鉴定

从中国土壤中分离的大量苏云金芽孢杆菌菌株中鉴定出H42、H43、H56、H60及H62等5种新H血清型,并进行了形态、培养特征、生化反应、晶体蛋白质成分及毒力特性等项检测鉴定,鉴定了5个苏云金芽孢杆菌新亚种： Bacillus thuringiensis subsp. jinghongiensis (H42), B.thuringiensis subsp. guiyangiensis (H43),B.thuringiensis subsp. rongseni(H56),B.thuringiensis subsp. pingluonsis(H60)及B.thuringiensis subsp. zhaodongensis(H62) 。毒力生物测定证明5个新亚种的代表菌株对棉铃虫(Heliothis armigera)\,小菜蛾(Plutella xylostella)\,柳蓝叶甲(Plagiodera versicolora)幼虫均无毒力。H42、H43、H56、H60对埃及伊蚊(Aedes aegypti)\,斑须按蚊(Anopheles stephensi)及尖音库蚊(Culex pipiens)亦均无毒;H62对埃及伊蚊无毒,但对尖音库蚊与斑须按蚊有低毒。     

Comparative analysis of the 16S to 23S ribosomal intergenic spacer sequences of Bacillus thuringiensis strains and subspecies and of closely related species.

Bacillus thuringiensis spacer regions between the 16S and 23S rRNAs were amplified with conserved primers, designated 19-mer and 23-mer primers. A spacer region of 144 bp was determined for all of 6 B. thuringiensis strains, 7 B. thuringiensis subspecies, and 11 B. thuringiensis field isolates, as well as for the closely related species Bacillus cereus and Bacillus anthracis. Computer analysis and alignment of nucleotide sequences identified three mutations and one deletion in the intergenic spacer region (ISR) of B. thuringiensis subsp. kurstaki HD-1 when compared with ISR sequences from other subspecies. The same differences were identified between the ISR of B. thuringiensis strains and the ISR of B. cereus and B. anthracis. These minor differences do not seem to be sufficient to allow the design of a species-specific oligonucleotide probe.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Transfer of the toxin protein genes of Bacillus sphaericus into Bacillus thuringiensis subsp. israelensis and their expression.

The genes encoding the toxic determinants of Bacillus sphaericus have been expressed in a nontoxic and a toxic strain of Bacillus thuringiensis subsp. israelensis. In both cases, the B. sphaericus toxin proteins were produced at a high level during sporulation of B. thuringiensis and accumulated as crystalline structures. B. thuringiensis transformants expressing B. sphaericus and B. thuringiensis subsp. israelensis toxins did not show a significant enhancement of toxicity against Aedes aegypti, Anopheles stephensi, and Culex pipiens larvae.     

Persistence of Bacillus thuringiensis subsp. kurstaki in Urban Environments following Spraying

Bacillus thuringiensis subsp. kurstaki is applied extensively in North America to control the gypsy moth, Lymantria dispar. Since B. thuringiensis subsp. kurstaki shares many physical and biological properties with Bacillus anthracis, it is a reasonable surrogate for biodefense studies. A key question in biodefense is how long a biothreat agent will persist in the environment. There is some information in the literature on the persistence of Bacillus anthracis in laboratories and historical testing areas and for Bacillus thuringiensis in agricultural settings, but there is no information on the persistence of Bacillus spp. in the type of environment that would be encountered in a city or on a military installation. Since it is not feasible to release B. anthracis in a developed area, the controlled release of B. thuringiensis subsp. kurstaki for pest control was used to gain insight into the potential persistence of Bacillus spp. in outdoor urban environments. Persistence was evaluated in two locations: Fairfax County, VA, and Seattle, WA. Environmental samples were collected from multiple matrices and evaluated for the presence of viable B. thuringiensis subsp. kurstaki at times ranging from less than 1 day to 4 years after spraying. Real-time PCR and culture were used for analysis. B. thuringiensis subsp. kurstaki was found to persist in urban environments for at least 4 years. It was most frequently detected in soils and less frequently detected in wipes, grass, foliage, and water. The collective results indicate that certain species of Bacillus may persist for years following their dispersal in urban environments.