蜡样芽孢杆菌M22Fe-SOD基因克隆及其分析

 通过筛选蜡样芽孢杆菌M 22基因组文库, 得到约3.9 kb的基因组片段。BLAST结果显示:其中包含1条长度为915 bp、编码304个氨基酸的超氧化物歧化酶(SOD)基因, 其与Bacillus thuringiensis和Bacillus anthracis中的sodF基因同源性高达95%。此sodF基因能互补恢复大肠杆菌SOD缺陷型菌株QC871在10 μmol/L paraquat中的生长能力。对重组表达蛋白的抑制实验表明, 此SOD基因为Fe-SOD。利用pET-30b (+)构建表达载体pET-sodF并转化到大肠杆菌BL21(DE3)中, 经IPTG诱导后, SOD融合蛋白表达量显著增加。构建自杀载体p299-8, 同源重组突变蜡样芽孢杆菌M22 sodF基因后, 突变体抗氧化能力未显著降低。

Cloning and characterization of Fe-SOD gene from Bacillus cereus M22

3.9 kb genomic segment was screened from the genomic library of Bacillus cereus M22,a highly effective biocontrol agent of plant diseases.The BLAST result showed that it contained a 915 bp superoxide dismutase(SOD) gene,which encoded 304 amino acid residues and displayed 95% homology with the sodF gene of Bacillus thuringiensis and Bacillus anthracis.The sodF gene could strongly complement the Escherichia coli SOD-mutant,and the SOD inhibition assays indicated that the SOD protein was a Fe-SOD.Entire sodF open reading frame was cloned into plasmid pET-30b( ) and then transfered into E.coli BL21(DE3),and IPTG was added to induce overexpression of the SOD protein of the transformant.The sodF-mutant of B.cereus M22 was obtained by homologous recombination of the chromosomal copy of the gene with a suicide plasmid p299-8,and the mutant showed almost the same oxidant resistance as the wild type.