特异性杀虫基因Bt cry3A在桑粒肩天牛幼虫两种肠道常驻内生菌中的转化和表达

Transformation and expression of specific insecticide gene Bt cry3A in resident endogenetic bacteria isolated from Apriona germari (Hope) larvae intestines

【目的】将特异性杀虫毒蛋白基因Btcry3A转入桑粒肩天牛(Apriona germari Hope,Ag)幼虫肠道常驻内生菌中,构建能在天牛幼虫肠道中定殖并表达特异性杀虫基因Bt cry3A的工程菌。【方法】以传统方法和16S rDNA分子生物学分析等方法分离、鉴定Ag幼虫肠道优势的常驻内生菌,从中筛选出适合转化的候选菌株。利用电转化技术将含有对鞘翅目昆虫具专一性毒力Bt cry3A基因的Escherichia coli-Bacillus thuringiensis穿梭表达质粒pHT305a和pHT7911分别转入Ag幼虫肠道常驻内生菌短短芽孢杆菌(Brevibacillus brevis Ag12,Ag12)和苏云金芽孢杆菌(Bacillus thuringiensis Ag13,Ag13)中。【结果】从Ag幼虫肠道共分离获得18个不同种的可培养细菌菌株,并从中选取菌株Ag12和Ag13作为出发菌株转入Bt cry3A基因。经质粒稳定性试验、转化子生长特性测试、伴胞晶体电镜检测、毒蛋白SDS-PAGE分析、工程菌定殖性分析以及生物毒力测试,结果显示cry3A基因已经成功转入Ag幼虫的常驻内生菌短短芽孢杆菌和苏云金芽孢杆菌中,并且工程菌Ag12-305a、Ag13-305a、Ag12-7911和Ag13-7911都能在天牛幼虫肠道内稳定生长、繁殖并表达分子量约65kDa的伴孢晶体杀虫蛋白Cry3A。【结论】Bt cry3A基因已成功转入桑粒肩天牛幼虫肠道优势常驻内生菌中,获得了四株能在桑粒肩天牛幼虫肠道内定殖,并能表达目的杀虫基因Bt cry3A的转基因工程菌。

[Objective] Transforming the specific insecticidal gene Bt cry3A into the dominant resident endogenetic bacteria in intestines of Apriona germari （Hope） larvae to construct transgenic bacteria that can colonize and express the insecticidal gene Bt cry3A perfectly in intestines of Apriona germari （Hope） larvae. [Method] We isolated and identified the dominant resident endogenetic bacteria by traditional methods and molecular method based of 16S rDNA analysis. Two Escherichia coli - Bacillus thuringiensis shuttle plasmid pHT305a and pHT7911 which contained specific insecticidal gene Bt cry3A were transformed into two resident endogenetic bacteria Brevibacillus brevis Agl2 and Bacillus thuringiensis Agl3 isolated from A. germari larvae intestines respectively by electro-transformation. [Results] Eighteen species of bacteria have isolated and identified from Apriona germari larvae intestines and two of them （Brevibacillus brevis Agl2 and Bacillus thuringiensis Agl3） were selected as starting bacteria to recieve the Bt cry3A. The 4 transgenic engineering strains Ag 12-7911, Ag 12-305a, Ag 13-7911 and Ag 13-305a were obtained successfully and validated by testing the plasmid stability in recombinants, transformants vegetal properties, crystal poisonous protein observation, expressional protein SDS-PAGE. The Bt cry3A gene had been transformed into Brevibacillus brevis and Bacillus thuringiensis. Both bioassay and examination of the engineering strains in intestines after feeding them to larvae showed that all these transformant strains （Brevibacillus brevis Ag12-305a, Bacillus thurigiensis Ag13-305a, Brevibacillus brevis Ag12-7911 and Bacillus thurigiensis Ag13-7911） could colonize and express 65 kDa protoxin in intestines of A. germari larvae and had insecticidal activity. [Conclusion] We obtained four transgenic bacteria that can colonize and express the target insecticide gene Bt cry3A in A. germari larvae. They may be developed as a new insecticide.