华癸根瘤菌7653R中RND型外排泵家族基因的共生功能研究

发布时间：2018-08-07 19:40

【摘要】：虽然细菌外排泵的研究历史较长,其涉及的药物运输功能研究主要集中在病原菌中,根瘤菌中外排泵的研究较少,外排泵系统相关基因在根瘤菌共生固氮中的功能表型和作用机制也值得深入研究。本文研究了华癸中慢生根瘤菌7653R中RND家族基因的功能。获得了如下实验结果:一方面,我们基于前期工作中的根瘤菌7653R转录组数据,分析发现MCHK_0866、MCHK_0867两个基因在共生条件下明显上调表达,这两个基因表达产物组成一个RND型外排泵。对外排泵编码基因及候选调控基因在基因组上的组织结构进行了分析。采用测定OD600观察菌株生长曲线的变化。通过测定最低抑菌浓度检测菌株的药物敏感性,RT-PCR检测目的基因经特定物质处理后表达量的变化。通过细菌单杂交系统初步检测外排泵的转录调控。结果显示,MCHK_0866和MCHK_0867所编码蛋白共同组成一个RND家族射流泵。缺失该外排泵后,细菌生长曲线在稳定期OD600数值降低,对萘啶酸、四环素和SDS的敏感性发生变化,萘啶酸处理细菌后两个基因的表达量增加。下游属于TetR转录因子家族的基因MCHK_0869表达产物作用于MCHK_0867的启动子区域。该外排泵与萘啶酸的运输有关,缺失后自生生长受到影响,表达受到下游转录因子的调控。MCHK_0867插入失活后对结瘤能力和固氮酶活影响较大,与双突变菌株共生表型相似。另一方面,本文选取另外3个在共生根瘤中表达水平无显著变化的RND家族基因,分别构建了其基因插入失活突变株。对突变菌株进行了共生表型实验。结果发现MCHK_0811和MCHK_3948基因失活后,对结瘤能力和根瘤固氮酶活有一定的影响;MCHK_6517失活后对共生表型没有影响。上述结果为后续研究根瘤菌外排泵中与共生固氮相关基因的作用机制,提供了研究材料和工作基础。
[Abstract]:Although the research of bacterial efflux pump has a long history, the research on drug transport function is mainly concentrated in pathogenic bacteria, and the research on Rhizobium efflux pump is less. The functional surface and mechanism of efflux pump system related genes in symbiotic nitrogen fixation of rhizobia are also worthy of further study. The function of RND family genes in Rhizobium sinensis 7653R was studied. The results are as follows: on the one hand, based on the data of 7653R transcriptome of Rhizobium, we found that the expression of MCHK0866MCHK0867 gene was upregulated obviously under symbiotic condition, and the two gene expression products constituted a RND type efflux pump. The tissue structure of external pump coding gene and candidate regulatory gene in genome were analyzed. OD600 was used to observe the change of growth curve. The drug sensitivity of the strain was detected by RT-PCR. The expression of target gene was detected by the treatment of specific substances. The transcriptional regulation of efflux pump was detected by single hybridization system. The results showed that MCHK0866 and MCHK_0867 encoded protein constitute a RND family jet pump. When the efflux pump was absent, the bacterial growth curve decreased at the stable stage of OD600, and the sensitivity to naphthalic acid, tetracycline and SDS changed, and the expression of the two genes increased after NAA treatment. The downstream MCHK_0869 expression products belonging to the TetR transcription factor family act on the promoter region of MCHK_0867. The efflux pump was related to the transport of naphthalic acid, and its expression was regulated by the downstream transcription factor. MCHK0867 had a great effect on nodule ability and nitrogenase activity after insertion and inactivation, which was similar to the symbiotic phenotype of the double mutant strain. On the other hand, three other RND family genes with no significant change in expression level in symbiotic nodule were selected, and their gene insertion inactivated mutants were constructed respectively. The symbiotic phenotype of mutant strain was tested. The results showed that the inactivation of MCHK_0811 and MCHK_3948 genes had no effect on nodule ability and nodule nitrogenase activity after inactivation of MCHK6517. These results provide a basis for further study on the mechanism of symbiotic nitrogen-fixing genes in rhizobia efflux pump.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：Q933

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