苏云金芽孢杆菌杀虫晶体蛋白Cry1Ba3活性区的研究

 通过对Cry1Ba3蛋白序列的分析，以及与已知Cry蛋白比较，设计了6对特异性引物，PCR扩增获得6种不同长度的cry1Ba3基因片段。将这些基因片段克隆到pET-21b载体上，导入大肠杆菌BL21中进行诱导表达，最终得到6种不同长度的Cry1Ba3蛋白片段。采用浸叶法检测这些蛋白片段对小菜蛾的杀虫活性，研究结果表明含有第1-685位和含有第22-655位氨基酸的蛋白片段对小菜蛾的毒性，与全长Cry1Ba3蛋白相比，没有改变；含有第54-655位氨基酸的蛋白片段对小菜蛾的毒力明显降低；而含有第22-627位和含有第85-655位氨基酸的蛋白片段完全丧失了对小菜蛾的活性。上述结果表明Cry1Ba3蛋白对小菜蛾的最小活性区在第22-655位氨基酸之间。

Toxicity Analysis of Truncated Insecticidal Crystal Protein Cry1Ba3 from Bacillus thuringiensis

To determine the minimal active fragment of CrylBa3 protein, six fragments of cry1Ba3 gene with different lengths were amplified using PCR with different specific primers. These fragments were cloned into pET-21b vector at BamH I and Sal I restriction enzyme sites, and then introduced into E, coli BL21 strain. After induced with IPTG the proteins expressed by different fragments were analyzed by SDS-PAGE, and the results showed that all these truncated proteins could be expressed in E, coli BL21, Bioassay result against Plutella xylotella showed that the active fragment of Cry1Ba3 protein was located between amino acid No.22 and No. 655. As the insecticidal activity of the truncated protein containing amino acid No.54-655 was declined significantly, and No.22-627 and No.85-655 were lost completely, so the toxicity of fragment including amino acid No,22-655 was not changed with original Cry1Ba3,