枯草芽孢杆菌CAS15嗜铁素基因dhbC的克隆、表达及功能鉴定
Cloning, expression and functional analysis of the dhbC gene from the siderophore producing bacterium Bacillus subtilis CAS15
投稿时间:2008-09-15  
DOI:  
中文关键词:枯草芽孢杆菌, dhbC基因, 基因克隆, 序列分析, 原核表达, 基因敲除
英文关键词:Bacillus subtilis, dhbC gene, gene cloning, sequence analysis, prokaryotic expression, gene knockout
基金项目:中央级公益性科研院所基本科研业务费专项(Nos. 2008hzs1J013, 2008hzs1J014), 国家科技支撑计划(No. 2007BAD48B04), 公益性行业(农业)科研专项(No. nyhyzx07-033-2-3)资助。
作者单位E-mail
余贤美 山东农业大学植物保护学院, 泰安 271018
中国热带农业科学院 环境与植物保护研究所, 儋州 571737 
 
林超 中国热带农业科学院 环境与植物保护研究所, 儋州 571737
海南大学环境与植物保护学院, 儋州 571737 
 
郑服丛 中国热带农业科学院 环境与植物保护研究所, 儋州 571737
海南大学环境与植物保护学院, 儋州 571737 
 
贺春萍 中国热带农业科学院 环境与植物保护研究所, 儋州 571737  
张修国 山东农业大学植物保护学院, 泰安 271018 zhxg@sdau.edu.cn 
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中文摘要:
      通过PCR技术扩增得到dhbC基因, 对其进行序列分析发现, dhbC基因片段长为1197 bp, 预期编码398个氨基酸, 蛋白分子量大小为43.8 kD。将目的片段连接到表达载体pET-30a(+), 转化大肠杆菌Escherichia coli BL21(DE3)获得重组菌株BL21(DE3)/pET-30a-dhbC, 以IPTG在30oC诱导4 h实现高效表达, 获得一个分子量为48.8 kD的融合蛋白。重组蛋白可溶性分析结果表明: 融合蛋白主要为可溶性蛋白。Western blotting分析结果表明: 重组蛋白可与兔抗His-tag多克隆抗体发生特异性反应, 在48.8 kD处有特异条带, 与预期结果一致, 证明重组质粒中含有dhbC基因。通过同源重组的策略将dhbC基因敲除后重新导入, 验证了dhbC基因与嗜铁素的生物合成密切相关。
英文摘要:
      We amplified dhbC gene from the siderophore producing bacterium CAS15 by PCR. After ligated the PCR product to pMD18-T vector and then sequenced, we obtained a 1197 bp fragment. The blast result showed that the nucleotide acids of dhbC gene (Accession No. FJ194456) of CAS15 shared 99.7% identity with that of dhbC gene of Bacillus subtilis (GenBank Accession No. Z99120), and was predicted to encode a 43.8 kD polypeptide with 398 amino acid residues. We cloned the dhbC gene into expression vector pET-30a(+) and then transformed into Escherichia coli BL21(DE3) via calcium chloride transformation method, and obtained the recombinant E. coli BL21(DE3)/pET-30a-dhbC. Induced by 1 mmol/L IPTG, the fusion protein 6His-DhbC, a 48.8 kD polypeptide was successfully expressed mainly in soluble form in E. coli BL21(DE3), and the amount reached highest at 30oC for 4 h. According to the N-terminal fusion 6 His-tag, we purified the recombinant polypeptide by Ni2+ metal affinity chromatography and finally identified it by Western blotting. The result indicated that the recombinant DhbC had the antigenicity to rabbit anti-his-tag polyclonal antibody, which provides the basis for the study of practical utilization in production and the biocontrol mechanism of B. subtilis. Finally, we deleted dhbC gene by gene knockout and then retransformed it into the dhbC gene-delected mutant, which confirmed that dhbC gene play an important role in siderophore biosynthesis.
余贤美,林超,郑服丛,贺春萍,张修国.枯草芽孢杆菌CAS15嗜铁素基因dhbC的克隆、表达及功能鉴定[J].生物工程学报,2009,25(6):819~825
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