【作者】 赵曌；

【导师】 张力群；

【作者基本信息】 中国农业大学 ， 植物病理学， 2014， 博士

【摘要】 生防假单胞菌2P24(Pseudomonas fluorescens2P24)分离自山东小麦全蚀病自然衰退土壤,对多种病原菌引起的土传病害都具有良好的生防效果,产生抗生素2,4-二乙酰基间苯三酚(2,4-diacetylphloroglucinol,2,4-DAPG)是其主要的防病机制。2,4-DAPG由phlACBD基因簇合成,该基因簇的表达受多种调控因子在不同水平的直接或间接调控,其中阻遏蛋白RsmA/E是重要的转录后调控因子。本研究从phlA基因的启动子分析入手,构建了phlA基因启动子转录报告质粒体系,并通过筛选影响RsmA/E的上游调控因子,进一步分析抗生素2,4-DAPG合成调控因子的调控机制。首先,对2,4-DAPG合成基因簇的第一个基因phlA的启动子进行分析,根据分析结果,将该启动子序列截断为11个含有不同长度phlA启动子的片段,并与p970km转录报告质粒连接,构建成11个phlA基因启动子转录报告质粒,分别检测各个启动子在2P24菌株胞内的活性,发现phlA基因启动子中存在多个转录调控元件。再将这些启动子转录报告质粒分别电击进入实验室前期研究的转录调控因子突变菌株中,以探究这些转录调控因子在phlA基因启动子上的转录调控作用位点。实验结果显示上述不同转录调控因子的作用位点基本都与PhlF蛋白的作用位点相同,均作用于含有pho位点的序列481-530中。故进一步选取GacA因子为代表,探究其调控phlA基因转录的机制,结果表明GacA是通过影响了PhlF蛋白的转录调控作用从而表现出与之作用位点相同。故推测PhlF是影响phlA基因转录最主要的直接调控因子,而其他转录调控因子很可能是通过影响PhlF因子从而调控phlA基因转录。其次,本文将分别含有rsmA基因和rsmE基因转录报告质粒的菌株2P24作为出发菌株进行转座子Tn5随机突变,在大约3万个突变体中,筛选到了86个对调控蛋白基因转录影响较大的突变菌株,其中,18个突变菌株中报告质粒的活性升高,即突变体中被破坏的基因对rsmA/rsmE基因表达有负调控作用；68个突变菌株中报告质粒的活性降低,即突变体中被破坏的基因对rsmA/rsmE基因表达有正调控作用。被破坏的基因中,sacB基因编码一种核糖核酸酶R蛋白,能够负调控rsmA基因的转录。进一步测定vacB基因在菌株2P24中的功能发现其影响菌株2P24抗生素的产生、群体感应信号的生成及游动性等多种生理性状,表明其可能是调控菌株2P24生防能力的一个重要调控因子。另外,本研究对实验室前期筛选得到的一个负调控抗生素产生的周质蛋白DsbA的调控机制进行进一步研究发现,DsbA是在转录后水平负调控phl4基因的表达,这种调控是通过同时在转录水平和转录后水平正调控小蛋白RsmA的表达来实现的,从而最终影响了2,4-DAPG的产量。更多还原

【Abstract】 Pseudomonas fluorescens2P24is a biocontrol agent that isolated from suppressive soil of wheat take-all disease in Shandong Province. It has remarkable biocontrol activity against plant diseases caused by soilborne pathogens. The polyketide metabolite2,4-diacetylphloroglucinol (2,4-DAPG) plays a major role in the biological control of diseases by this strain. Biosynthesis proteins of2,4-DAPG in strain2P24were encoded by a genetic locus phlACBD, whose expression was under the control of a number of transcriptional and post-transcriptional regulators. Among these regulators, RsmA and RsmE are important post-transcriptional repressors. In this study, we first analysed the promoter of phlA gene and constract the transcriptional report plasmid system of phlA gene promoter. Then, we screened the regulatory factors of rsmA/rsmE genes to further study the regulatory mechanism of the factors affecting the production of2,4-DAPG.In the first part, the promoter of the first gene phlAinphlA CBD operon was analysed and cut to11different length fragment. Then these fragments containing different length promoters of phlA gene were linked to plasmid p970Km to form11transcriptional report plasmids of phlA gene. These report plasmids were imported into the mutants of six regulator genes and the wild-type2P24by electroporation to explore the possible binding sites on phlA promoter by these regulators. The results indicated that the pho site which is bound by PhlF repressor was the major acting site of all the six regulators. Then, the regulatory mechanism of GacA was selected for further study. The results suggested that low production of2,4-DAPG could not relieve the repression by PhlF on pho site in gacA gene mutant. Therefore we speculated that PhlF was the major transcriptional regulator ofphlA gene and the other transcriptional regulators seemed to regulate the phlA transcription through affecting the function of PhlF.In the second part of this work, the strains with rsmA or rsmE transcriptional reporter were subjected to the random transposon mutagenesis. About86mutants were selected from-30000insertion colonies. Among these86mutants, the rsmA/E transcriptions in18mutants were increased while the other68mutants were decreased. A mutant defective in the vacB gene, which encoded the Ribonuclease R, drastically increased the rsmA gene expression. The effect of VacB on other biocontrol characters implied that VacB was a globe regulator in strain2P24.In the last part of this work, we studied the regulatory mechanism of DsbA on the production of2,4-DAPG. DsbA is a major periplasmic disulfide-bond-forming protein. The dsbA mutant produced more2,4-DAPG compared with the wild strain. Genetic evidences showed that DsbA did not affect transcription of the phlA gene but observably increased the phlA gene translation. Further study showed that the DsbA probably affected the2,4-DAPG production by promoting the expression of repression protein RsmA at both transcriptional level and translation level while this transcriptional regulation seemed to be indirect.更多还原