中华根瘤菌Sinorhizobium sp.1128自体诱导物合成酶基因的克隆及对生理功能的影响

Recombinant expression of an autoinducer synthase of Sinorhizobium sp.1128 in Escherichia coli

摘要:

【目的】从中华根瘤菌Sinorhizobium sp. 1128中克隆自体诱导物合成酶基因，从而研究该基因在Sinorhizobium sp. 1128群体感应系统中的作用。【方法】利用基因序列同源性比对以及分子克隆的方法，从中华根瘤菌Sinorhizobium sp. 1128中克隆自体诱导物合成酶基因；利用大肠杆菌异源表达、C18反相薄层层析(TLC)的方法研究该基因的特性；通过中间片段融合的方法缺失该基因，并通过结瘤实验研究该基因对Sinorhizobium sp. 1128生理功能的影响。【结果】以草木樨中华根瘤菌Sinorhizobium medicae WSM419自体诱导物合成酶基因Smed_1560序列设计引物，通过PCR扩增在Sinorhizobium sp. 1128中寻找到一新的自体诱导物合成基因，命名为traI2。该基因在大肠杆菌Escherichia coli DH5a中表达后能产生两种自体诱导物分子。在Sinorhizobium sp.1128中将该基因缺失，自体诱导物活性下降；回复突变后，自体诱导物活性得到恢复，结瘤实验结果表明该基因能影响根瘤菌的结瘤效率。【结论】中华根瘤菌Sinorhizobium sp. 1128群体感应系统是一个复杂的交互系统，它对结瘤的生理功能具有一定的影响。

Abstract:

[Objective] To explore the roles of quorum sensing system in establishing symbiosis between bacterium Sinorhizobium sp.1128 and its plant host Melilotus suaveolens Ledeb. [Methods] According to homologous analysis, we designed primers to amplify the autoinducer synthase encoding genes in Sinorhizobium sp.1128 according to Sinorhizobium medicae WSM419 genome sequences. The autoinducer synthase encoding genes were cloned into the expression vector of pYC12 and expressed in E. coli DH5a. Thin-layer chromatography ( TLC) assay was used to study their roles in autoinducer production. A duplicated inactivation of the gene was used to explore its function in plant nodulation. [Re-sults]Homologous analysis showed that at least three annotated acylated homoserine lactone (AHL) synthase genes ex-isted in Sinorhizobium medicae WSM419 genome. We cloned these three autoinducer synthase genes in Sinorhizobium sp.1128. One of these genes named traI2 was over expressed in E. coli DH5α. At least two different AHLs were produced by the recombinant strain. Disruption of traI2 reduced both the autoinducers (AI) activities and AHL production by TLC detection. Furthermore, the complementation of traI2 reverted the phenotype of AI activities. These findings demonstrate that traI2 was responsible for AI synthesis in Sinorhizobium sp.1128. More important, the traI2 deficient strains were defective in nodule formation on their host plant. [Conclusion] The quorum sensing circuits in Sinorhizobium sp.1128 may play an important role in symbiosis between plant and bacterium.