四川盆地生态区苏云金芽胞杆菌cry基因的鉴定及新型模式cry基因的克隆

Characterization of insecticidal crystal Protein cry gene of Bacillus thuringiensis from soil of Sichuan Basin and cloning of novel holotype cry gene

摘要:

摘要：【目的】为了明确四川盆地生态区土壤中苏云金芽胞杆菌cry 基因资源情况，进一步克隆出新型的杀虫晶体蛋白基因。【方法】本研究主要通过菌株晶体形状的光学显微镜及扫描电镜观察、PCR-RFLP技术鉴定cry基因型法、杀虫晶体蛋白的SDS-PAGE分析和菌株生物活性测定等方法对此地区菌株进行研究。【结果】从四川盆地不同生态区采集2650份土壤样品中分离了791株苏云金芽胞杆菌。PCR-RFLP鉴定结果表明：此地区的苏云金芽胞杆菌主要含有cry1，cry2，cry3，cry4/10，cry9，cry30和 cry40 等7种cry基因类型；含cry1基因的菌株最丰富，共有21种不同cry1型基因组合；从中发现了新型模式基因，并采用Tail-PCR技术获得了其中3个基因的全长序列，被国际苏云金芽胞杆菌杀虫晶体蛋白基因命名委员会命名为cry54Aa1、cry30Fa1和cry30Ga1。通过生物活性测定，发现对鳞翅目和双翅目害虫具毒力的菌株。未鉴定出基因型的80个菌株的伴胞晶体SDS-PAGE分析表明：这些菌株均有40～130 kDa蛋白表达，极有可能含新型的杀虫蛋白基因。【结论】研究结果充分体现了四川盆地生态区苏云金芽胞杆菌资源的多样性及特殊性，所蕴含的杀虫蛋白基因在农业生产上具有重要意义和应用前景。

Abstract:

Abstract：【Objective】In order to systematically investigate the cry gene resources from Bacillus thuringiensis (Bt) in different ecological regions in Sichuan Basin and further clone novel cry genes.【Methods】We used the methods of microscopic and scanning electron microscopic observation, PCR-restriction fragment length polymorphism (PCR-RFLP) analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and test of insecticidal activities to research Bt strains collected in this basin.【Results】We screened 791 Bt isolates from 2650 soil samples, and found cry1, cry2, cry3, cry4/10, cry9, cry30, and cry40-type genes in this basin. Strains containing cry1 genes were the most abundant in our collection (66%), and 21 different cry1-type gene combinations were found. Furthermore, several novel holotype cry genes were found and the full-length sequences of 3 novel cry genes were designated as cry54Aa1, cry30Fa1, and cry30Ga1 by B. thuringiensis Pesticidal Crystal Protein Nomenclature Committee. The results of insecticidal activities showed that Sichuan Basin harbored Bt isolates with insecticidal activities. SDS-PAGE assay of 80 strains without PCR products indicated that these strains may harbor potentially novel cry proteins gene.【Conclusion】The diversity and particularity of cry gene resources in Sichuan Basin have important meanings in theories and practices.