【作者】 梁士敏；

【导师】 许艳丽；

【作者基本信息】 东北林业大学 ， 微生物学， 2011， 硕士

【摘要】 环鸟苷二磷酸(e-di-GMP)是一种新发现的细菌的新第二信使,并发现了鸟苷酸环化酶(DGC)和磷酸二脂酶(PDE)分别控制其合成和水解,而GGDEF结构域和EAL或HD-GYP结构域分别是DGC和PDE的活性中心。在细菌的基因组中存在着大量的GGDEF、EAL和HD-GYP结构域,如38个结构域存在于铜绿假单胞杆菌中、98个结构域存在于沙雷杆菌中、53结构域个存在于霍乱弧菌中和29结构域个存在于大肠杆菌中。另外发现,细菌的多种生物学功能受c-di-GMP调控,如：毒性、运动性和运动性等。水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,简称Xoo)是重要的水稻致病菌。然而在其侵染水稻的过程中可能某些信号调控起着至关重要的作用,进而对其致病性具有决定性作用。但是,现在信号究竟是通过何种手段对Xoo的致病性及相关基因的表达进行调控的仍没有明确报道。多年研究发现,环鸟苷二磷酸(c-di-GMP)是一种新发现的、广泛存在于细菌中调控着细菌多种生物学功能的第二信使。水稻白叶枯病菌有25-31个GGDEF、EAL和HD-GYP结构域。然而,至今仍不清楚部分信号蛋白基因的生物学功能。本研究着重对c-di-GMP系统中重要的3个基因(PXO-02944、PXO-04753和PXO-03877)进行了分子鉴定和功能分析。通过对PXO-02944、PXO-04753和PXO-03877进行分子克隆、序列分析、缺失突变和互补及其表型特征的鉴定；利用RT-Q-PCR方法,定量测定了A02944xoo、△04753xoo和Δ03877xoo毒性相关基因的表达。研究目的在于从Xoo中鉴别出相应的结构域蛋白基因、揭示其生物学功能,为全面阐明c-di-GMP代谢调控机理提供科学依据。采用基因标记置换方法,分别构建推测了具有c-di-GMP合成活性的鸟苷酸环化酶活性基因(PXO-02944)和降解活性的磷酸二酯酶基因(PXO-04753和PXO-03877)缺失突变体。与PXO99A相比,△02944xoo胞外多糖和生物膜合成能力以及对水稻的毒性显著增强；△03877xoo的生物膜、胞外多糖形成能力和对水稻的致病性明显减弱；△04753对烟草的致敏性和生物膜形成显著降低。总之,本研究阐述了PXO-02944、PXO-04753和PXO-03877作为c-di-GMP信号途径应答调控因子,分别调控了Xoo的多种生物学功能及其相关基因的表达。更多还原

【Abstract】 Cyclic diguanylate (c-di-GMP) is found newly as a bacterial second messenger, and the same time, which is formed by diguanylate cyclases with GGDEF motifs that synthesize this second messenger; phosphodiesterases with EAL or HD-GYP motifs catabolize c-di-GMP. Many proteins with GGDEF, EAL or HD-GYP domains are existed in bacterial genomes,such as, there are 38 in Pseudomonas aeruginosa,98 in Serratia spp,53 in Vibrio cholerae, and 29 in E. coli. c-di-GMP has many key roles in regulation of a range of biochemical functions in virulence, biofilm formation, motility and environmental adaptation.Xanthomoans oryzae pv. oryzae (Xoo) is one of the most serious bacterial disease for rice. It mought contain that there was a signaling regulation network in Xoo which strictly control virulence gene expression through signal sensing, perception and transduction. However, at present, there was little new to be known about the virulence regulation network in Xoo. As reported,c-di-GMP regulated many kinds of complex physiological processes. There were 25-31 proteins with GGDEF, EAL or HD-GYP domains in Xoo, However,we still do not understood the functions of some proteins like as above. There are too many year research on c-di-GMP signaling.pathway and its functions in our laboratory. My research was focused on the identification and functional analysis of the new protein gene(PXO-02944、PXO-04753 and PXO-03877) that c-di-GMP signaling system in Xoo.PXO-02944、PXO-04753 and PXO-03877 were studied by gene cloning, sequence, deletion, complement and phenotype analysis; at the same time analyzing the expressions of the genes of EPS and T3SS inΔ02944xoo、Δ04753xoo andΔ03877xoo.My study aimed to identify the corresponding domain protein gene to revEAL its biological function from Xoo, so that providing the scientific basis for metabolic regulation mechanism of c-di-GMP.PXO-02944、PXO-04753 and PXO-03877 were investigated by gene deletion mutation using the marker exchange. Comparing to PXO99A, exopolysaccharide (EPS), biofilm production and virulence had a great ascent forΔ02944xoo; induction of hypersensitive responses (HR) in tobacco and biofilm formation reduced significantly forΔ04753xoo; theΔ03877xoo mutant demonstrated great deficiency in EPS, biofilm production and virulence. In general, That confirmed PXO-02944、PXO-04753 and PXO-03877 might act as important response regulators in c-di-GMP signaling system to be involved in regulation of many kinds of biological functions of Xoo.更多还原