植物内生蜡样芽孢杆菌M22 Mn-SOD基因的克隆及在大肠杆菌中表达

Molecular cloning of two Mn superoxide dismutase genes from Bacillus cereus M22 and their expression in Escherichia coli

通过PCR和反向PCR,从蜡样芽孢杆菌M22中扩增到2条长度为1 322 bp和1 395 bp的基因片段(GenBank登录号为AY540749和AY468485),分别含657 bp和627 bp的开放阅读框,各自编码218个和208个氨基酸残基的功能蛋白。2个蛋白氨基酸序列同源性为46％,均不含信号肽。与其它锰超氧化物歧化酶序列同源性高,保守氨基酸残基类型及位置与其它Mn-SOD相同,可确定2个基因均为蜡样芽孢杆菌Mn-SOD基因。分别将2个基因的开放阅读框插入载体pET-22b(+)构建表达质粒pET-sodA,转化大肠杆菌BL21(DE3),IPTG诱导蛋白表达。SDS-PAGE显示融合蛋白相对分子质量分别为28 kD和27 kD。转入pET-sodA的SOD缺陷型大肠杆菌QC779转化子恢复在10μmol/L paraquat的LB平板上生长的能力。活性电泳显示,M22的Mn-SOD在QC779中成功表达。

Two complete sequences of Mn-containing superoxide dismutase (Mn-SOD) genes, sodA1 and sodA2, were cloned from endophytic Bacillus cereus M22 by using direct PCR and inverse PCR (IPCR). Nucleate sequencing revealed that the open reading frame of sodA1 and sodA2 consisted of 218 and 208 amino acids, respectively. The deduced proteins, which had the highly conserved Mn-SOD motif, shared high identity with the previously isolated members of Mn-SOD family. However, SODA1 and SODA2 had a lower identity to each other. These two SOD genes were inserted into the expression vector pET-22b (+) and the recombinant proteins were expressed in E. coli BL21(DE3) at a high amount as the soluble protein. Transformation of pET-sodA into E. coli QC779, a double mutant devoid of Mn-SOD and Fe-SOD activities, recovered the SOD deficiency and the transformant could grow on the LB plate containing 10 μmol/L paraquat. SODAI and SODA2 were sensitive to potassium cyanide (KCN) and hydrogen peroxide (H2O2) indicated that they used manganese (Mn) as a cofactor.