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【目的】自小麦全蚀病自然衰退土壤分离得到的荧光假单胞菌(Pseudomonas fluorescens)2P24,可防治多种由植物病原菌引起的土传病害。菌株2P24具有群体感应(quorum-sensing,QS)系统PcoI/PcoR,该系统影响生防菌2P24生物膜的形成以及其在小麦根围的定殖能力,从而影响2P24的生防能力。本文利用遗传学方法进一步研究了2P24中QS系统的调控途径。【方法】将QS系统信号合成基因pcoI的转录报告质粒p970Gm-pcoIp转入gacA基因突变菌株PM201中,再利用Tn5转座子对该菌株进行随机突变,筛选影响pcoI基因表达的调控因子。【结果】根据菌落颜色的变化筛选到2株突变菌株。Tn5插入位点和基因序列分析表明这2个突变体中Tn5破坏了同一个基因mvaT;设计引物利用PCR方法从2P24基因组中获得mvaT基因及其同源基因mvaV。转录融合报告实验表明:与野生菌株2P24相比,mvaT及mvaV突变体中pcoI基因的表达和N-乙酰高丝氨酸内酯的产量显著提高;HPLC试验表明mvaT和mvaV基因影响抗生素2,4-二乙酰基间苯三酚的合成。细菌双杂交试验证实,MvaT蛋白和MvaV蛋白在体内发生自身互作,这两个蛋白也可相互作用。【结论】以上结果表明mvaT和mvaV参与调控生防假单胞菌2P24的PcoI/PcoR群体感应系统,并可能影响其生防功能基因的表达。
【Objective】 Pseudomonas fluorescens 2P24, isolated from soils naturally degraded by wheat eclipses, can prevent many soil-borne diseases caused by plant pathogens. Strain 2P24 has a quorum-sensing (QS) system PcoI / PcoR, which affects the bioconjugation of Bioconjugate 2P24 biofilm and its colonization ability in wheat rhizosphere, and thus affects the biocontrol ability of 2P24. In this paper, the use of genetic methods to further study the regulation of 2P24 QS system approach. 【Method】 The transcriptional reporter plasmid p970Gm-pcoIp of the signal transduction gene pcoI of QS system was transferred into the gacA gene mutant PM201, and then the mutant was randomly selected by Tn5 transposon to screen the regulatory factors that affect the expression of pcoI. 【Result】 Two mutant strains were screened according to the change of colony color. Tn5 insertion site and gene sequence analysis showed that Tn5 in these two mutants destroyed the same gene mvaT. Primers were designed to obtain mvaT gene and its homologous gene mvaV from 2P24 genome by PCR. The results of transcriptional fusion assay showed that the expression of pcoI gene and the yield of N-acetyl homoserine lactone were significantly increased in mvaT and mvaV mutant compared with the wild strain 2P24. The results of HPLC showed that mvaT and mvaV genes affected the antibiotic 2,4-bis Synthesis of Acetyl Phloroglucinol. Bacterial two-hybrid assay confirmed that MvaT protein interacts with MvaV protein in vivo, and these two proteins can also interact with each other. 【Conclusion】 The above results indicate that mvaT and mvaV are involved in the regulation of the PcoI / PcoR population induction system of Pseudomonas aeruginosa 2P24 and may affect the expression of biocontrol genes.