中华根瘤菌自体诱导物合成酶基因的筛选及其在大肠杆菌中的表达

Screening for autoinducer synthase gene in Sinorhizobium meliloti and analysis of the autoinducer produced by recombinant expression in Escherichia coli

摘要:

通过携带有mariner转座子的质粒pJZ290随机插入诱变中华根瘤菌(Sinorhizobium meliloti)建立突变子文库,并从中筛选到自体诱导物(autoinducer,AI)部分缺失突变株YW1。Arbitrary PCR扩增、DNA测序得到YW1基因组DNA中mariner转座子两端侧翼序列,经DNA序列拼接在GenBank上进行同源性分析后获得一个621bp的完整的开放阅读框(ORF),该ORF编码的酶具有206个氨基酸,与草木樨中华根瘤菌(Sinorhizobium medicae)WSM419的LuxI类自体诱导物合成酶(autoinducer synthase)TraI的同源性高达99%。因此,也将该基因命名为traⅠ。将该基因克隆到广宿主范围表达载体pYC12并在大肠杆菌Escherichia coli DH5α中成功表达,C18反相薄层层析(TLC)在阳性重组子培养上清中检测到四种自体诱导物分子,其中的两种正是AI缺失突变株YW1所缺失的AI,这些结果表明该traⅠ基因在苜蓿中华根瘤菌负责合成两种自体诱导物分子,为进一步研究其群体感应系统奠定了理论基础。

Abstract:

The plasmid pJZ290 containing mariner transposon was used to mutagenize Sinorhizobium meliloti and 3000 transposon insertion mutants were subsequently screened for autoinducer-deficient (AI-deficient) mutants. One AI-deficient mutant YW1 was obtained by quantitative activity assay and qualitative TLC detection. In an effort to characterize the transposon insertions of autoinducer-deficient mutant YW1, we performed an arbitrary PCR and sequencing techniques to identify insertion sites. The sequence analysis result showed that the transposon inserted between the 277th bp and the 278th bp of one 621bp ORF in autoinducer-deficient mutant YW1. The 612-bp ORF encodes a putative protein of 206 amino acids that is highly homologous (99% identity) to AHL-synthase traI of Sinorhizobium medicae WSM419. Cloned into the broad host range expression vectors pYC12 and transformed into Escherichia coli DH5α, the putative AI synthase gene was overexpressed in E. coli, and four different autoinducers could be detected in the supernatant of the positive recombinant strain by TLC, among which the two AHL molecules that were deficient in AI-deficient mutant YW1 could be found. All of these showed that the 621bp ORF was an AI synthase gene. This study paved the way of further studying quorum sensing systems in S. meliloti.