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.

Identification, cloning and expression of the insecticidal protein genes from Bacillus thuringiensis isolate Rpp39

Objective] We characterized strain Rpp39 isolated from the soil of Sichuan, China. A type of cry2Aa gene was cloned and expressed in E. coli. Methods] We characterized strain Rpp39 by scanning electron microscope, PCR-RFLP identification and SDS-PAGE analysis. We cloned cry2Aa gene by PCR clone. We constructed the recombinant plasmid pET-2Aa, and transformed E.coli.BL21(DE3) to express by IPTG. We determined the toxicity of expressed products to the larvae of Plutella xylostella and Chilo supperssalis through bioassay. Results] The strain Rpp39 contained three types parasporal crystals, diamond, squareness and round, observed under the scanning electron microscope. SDS-PAGE analysis showed that two kinds of molecular mass of insecticidal crystal proteins, one was about 130kD and the other 60 kDa, were expressed in isolate Rpp39. The strain contained cry1Aa, cry1Ab, cry1Ac, cry1Ia and cry2Aa gene, analyzed by the PCR-RFLP method. One cry2Aa-type gene of Rpp39 was cloned and designated as cry2Aa12 by Bacillus thuringiensis Insecticidal Crystal Proteins Nomenclature Committee. Sequence analysis revealed that this gene contained an open reading frame of 1902 nucleotides encoding a protein of 634 amino acids. Compared with Cry2Aa1 protein, Cry2Aa12 protein has shown as high as 99.7% amino acid homology. We amplified the full open reading frame sequence of the cry2Aa12 gene by use of a pair of PCR primers P3/P4 designed according to its DNA sequence, and the PCR product was inserted into the Nde I / BamH I site of E. coli expression vector pET-30a to obtain the recombinant plasmid pET-2Aa. The result of SDS-PAGE proved that Cry2Aa12 could be expressed as 65 kDa protein in E. coli BL21(DE3) strain induced by IPTG. We found that Cry2Aa12 was highly toxic to the larvae of Plutella xylostella and Chilo supperssalis through bioassay, with LC50 as 5.4 mg/mL and 22.3 mg/mL, respectively. Conclusion] Strain Rpp39 and cry2Aa12 gene cloned from strain Rpp39 were came from Sichuan ecological condition in China. They could be affluent in the resources of strain and gene. It has important significance to accumulate the resource.