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【目的】假单胞菌株M18(Pseudomonassp.M18)是从甜瓜根际土壤中分离获得的一株对多种植物病原菌具有显著拮抗作用的菌株,在菌群传感(quorumsensing)系统的调控下,能分泌吩嗪-1-羧酸(PCA)以及多种吩嗪(phz)类衍生物的抗真菌物质。全局性因子GacA是M18菌株吩嗪类物质的合成与菌群传感系统的重要调控因子,本文将就GacA对上述两者的调控做进一步研究。【方法】PCR基因扩增和测序研究M18菌株中PCA合成基因簇,运用RT-PCR及构建phzA-lacZ转录融合手段研究M18菌株中两个phz基因簇各自的转录特征以及受全局性因子GacA的调控作用,运用翻译融合手段进一步研究M18菌株中GacA对菌群传感系统的调控作用。【结果】M18菌株的染色体中存在着两个PCA合成基因簇phzA1-G1和phzA2-G2,与铜绿假单胞菌株(P.aeruginosa)PAO1的一致性达99%。但是,M18菌株phzA2-G2基因簇下游的非编码区与PAO1菌株不同,存在着三段单位长度为144 bp的重复序列;在野生菌株M18中,phzA1-G1的转录水平明显高于phzA2-G2,GacA促进phzA1-G1转录,抑制phzA2-G2转录,区别性地调控两个phz基因簇的转录,GacA在整体上抑制phz基因簇的转录,与PAO1菌株中,GacA对phz的调控方式相反;gacA基因突变对菌群传感系统中lux家族基因中的lasI表达量无显著影响,但正调控las系统下游的rhlI表达量,GacA对phz基因簇表达的调控部分通过菌群传感系统实现。【结论】在M18菌株和PAO1菌株中,GacA对吩嗪类产物合成的调控方式相反,对菌群传感系统的调控也存在着差异性,这些差异可能是两个菌株在各自不同生境下长期进化的结果。
【Objective】 Pseudomonas sp.M18 is a strain isolated from muskmelon rhizosphere soil and has a significant antagonism against various plant pathogens. Under the control of the quorum sensing system, Anti-fungal substances capable of secreting phenazine-1-carboxylic acid (PCA) and various phenazine derivatives. The global factor GacA is an important regulator of the synthesis of phenazine and the bacterial flora sensing system of M18 strain. In this article, we will further investigate the regulation of GacA on both of them. 【Method】 PCR amplification and sequencing were used to study PCA gene cluster in M18 strain. RT-PCR and phzA-lacZ transcriptional fusion were used to study the transcriptional characteristics of the two phz gene cluster in M18 strain, Regulatory role, the use of translational fusion means to further study M18 strains GacA regulation of bacterial sensing system. 【Result】 The results showed that there are two PCA synthetic gene clusters phzA1-G1 and phzA2-G2 in the chromosome of M18 strain, which is 99% identical to PAO1 of P. aeruginosa. However, the non-coding region downstream of the phzA2-G2 gene cluster of M18 strain was different from that of PAO1 strain in that there were three repeat sequences with a unit length of 144 bp. The phzA1-G1 transcriptional level in wild strain M18 was significantly higher than that in phzA2-G2 , GacA promoted phzA1-G1 transcription, inhibited phzA2-G2 transcription, differentially regulated the transcription of two phz gene clusters, and GacA inhibited phz gene cluster transcription as a whole. In the PAO1 strain, GacA reversed phz regulation; The mutation of gacA gene had no significant effect on the expression of lasI in lux family genes in the bacterial sensing system. However, the regulation of rhlI expression in the downstream of las system was regulated by gacA gene. The regulation of phz gene cluster by GacA was partially realized by the bacterial sensor system. 【Conclusion】 In M18 strain and PAO1 strain, GACA regulates the synthesis of phenazine products in the opposite way, and there are also differences in the regulation of the bacterial sensing system. These differences may be due to the long-term The result of evolution.