稻瘟菌MgCYP51B基因稀有密码子和mRNA二级结构分析与表达优化

The analysis of rare codons and secondary structure of mRNA for the optimization expression about Magnaporthe oryzae CYP51B

在大肠杆菌中异源表达稻瘟菌CYP51(P450-14DM,MgCYP51B)基因,通过对MgCYP51B进行跨膜区预测和同源模建分析,并截短稻瘟菌CYP51B基因,构建了9种不同的重组表达质粒以实现蛋白表达.结果表明:只有pET28-MgB-83在3种宿主菌中实现了表达,且在BL21(DE3)pLysS中表达量最大.通过对稀有密码子和mRNA翻译起始区二级结构进行分析,发现稀有密码子和mRNA翻译起始区二级结构稳定性对MgCYP51B蛋白的表达都有影响,适用于稀有密码子表达的Rossetta(DE3)并不适用于MgCYP51B基因的最优表达.同时只有翻译起始区二级结构自由能最低的pET28-MgB-83才能实现表达.本研究实现了稻瘟菌MgCYP51B基因在大肠杆菌中的异源表达,证实密码子的偏爱性和翻译起始区二级结构稳定性影响MgCYP51B蛋白表达.

We achieved the heterologous expression of Magnaporthe oryzae CYP51B gene in E.coli.By the anaysis of transmembrane domain and homology modeling,the different lengths of MgCYP51B gene were truncated,nine recombinant expression plasmids were successfully constructed to achieve the heterologous expression.The results showed that only the pET28-MgB-83 could express in different host strains and the amount of protein expression was the largest in the BL21 （DE3）pLysS.The analysis showed that rare codons and secondary structure stability of mRNA translation initiation region could affect the expression of MgCYP51B.The E.coli Rossetta （DE3） which is good for expression of rare codons is not suitable for the optimal expression of MgCYP51B,while only the pET28-MgB-83 which has the lowest free energy of mRNA structure could successfully express the recombined proteins.We successfully achieved the heterologous expression of Magnaporthe oryzae CYP51B gene in E.coli,and vertified the effect of rare codons and stability of mRNA secondary structure of the translation initiation region on the expression of MgCYP51B.