蜡样芽孢杆菌M22Fe-SOD基因克隆及其分析

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

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10μmol/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M 22sodF基因后,突变体抗氧化能力未显著降低。

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.