杀虫基因Cry1 Ab的合成、表达及其抗虫性鉴定

摘要

The objective of this study was to modify the coding regions of wild Bacillus thuringiensis Cry1Ab, and then to construct its prokaryotic expression vector and the protein expression in E. coli and insect assay with A-sian corn borer. The coding regions of wild Bacillus thuringiensis Cry1Ab was optimized and modified according to plant preferred codons and frequency,and then to synthesize the modified gene (Cry1Ab). Analysis of the DNA se-quences revealed that the modified gene was 1 848 bp in length and had 66% percent homology with the native cry1Ab gene,and G+C content was raised from 37. 3% to 63. 04%. The modified Cry1Ab gene were artificially synthesized and ligated into prokaryotic expression vector pET28b( +) and transformed into E. coli BL21(DE3). SDS-PAGE analysis and about 70 kDa specific fusion protein was produced. Results of insect assay with Asian corn borer showed the expression products of Cry1Ab protein in E. coli had a toxicity to Asian corn borer. Its mortality rate is 93. 33% and the growth of the survival larvae were seriously inhibited. We would like to recommend the modified Cry1Ab gene as the candidate for biotechnological crop development.%对苏云金芽孢杆菌Cry1Ab野生型基因的编码区序列进行改造,以获得密码子优化的抗虫基因,构建原核表达载体后,将重组载体转入大肠杆菌进行诱导表达并对诱导蛋白进行抗虫试验。首先根据植物密码子的偏好性及使用频率,优化、改造并人工合成了Cry1Ab基因,序列分析表明：改造后的Cry1Ab基因全长1848 bp,与原始序列同源性为66%,G+C含量由原来的37.34%提高到63.04%。将改造后的Cry1Ab基因与原核表达载体pET28b(+)连接,转化大肠杆菌BL21(DE3),阳性菌液进行诱导表达后,SDS-PAGE分析结果显示： Cry1Ab蛋白在BL21(DE3)细胞中得到高效表达,分子量为70 kDa。喂虫试验结果表明：诱导表达的蛋白对玉米螟具有很强的毒性,幼虫死亡率达到93．33%,而且,存活的玉米螟生长发育也受到明显抑制。该抗虫基因可以作为培育转基因抗虫作物的候选基因。