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cry1Ah1基因是本实验室克隆的具有自主知识产权的模式基因,对鳞翅目害虫水稻二化螟等具有高毒力,具有较好的应用前景。为提高cry1Ah1基因在水稻中的表达量,探讨密码子使用频率对基因表达的影响,依据水稻密码子使用频率设计5种不同的优化方案,提高GC含量并去除剪切信号等不稳定因素后合成cry1Ah1基因杀虫活性区域。优化后的基因在大肠杆菌Rosetta(DE3)中正常表达了65 kDa蛋白,表达蛋白对2龄小菜蛾和水稻二化螟初孵幼虫都具有良好的杀虫活性。优化的基因转化水稻日本晴后,PCR阳性率达到87%以上,实时荧光定量RT-PCR和ELISA分析表明全部采用最高频率密码子的优化方案效果最好,Cry1Ah蛋白平均表达量占可溶性蛋白的0.104%。
The cry1Ah1 gene is a model gene with independent intellectual property rights cloned in our laboratory and has high virulence to Lepidoptera pests, such as rice borer, and has a good application prospect. In order to improve the expression of cry1Ah1 gene in rice and to explore the effect of codon usage on gene expression, five different optimization schemes were designed according to the frequency of codon usage in rice, GC content was increased, and unstable factors such as shear signal were removed to synthesize cry1Ah1 gene insecticidal activity region. The optimized gene expressed 65 kDa protein in E.coli Rosetta (DE3), and the expressed protein had good insecticidal activity against the 2nd instar larvae and the 2nd instar larvae. The PCR-positive rate of the optimized gene was above 87% after transfered by Nipponbare. Real-time fluorescent quantitative RT-PCR and ELISA analysis showed that all the optimized programs with the highest frequency codon were the best, the average expression of Cry1Ah protein accounted for 0.104% of the soluble protein .