水稻白叶枯病菌3个GGDEF结构域蛋白的功能鉴定

发布时间：2018-01-03 06:22

  本文关键词：水稻白叶枯病菌3个GGDEF结构域蛋白的功能鉴定　出处：《中国农业科学院》2016年博士论文　论文类型：学位论文

  更多相关文章： 水稻白叶枯病菌 环二鸟苷酸 GGDEF结构域 毒性 运动性 生物膜形成 胞外多糖产生

【摘要】：水稻白叶枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)不仅是一种重要的植物病原细菌,也是研究植物—微生物互作的模式病原微生物。本论文旨在研究Xoo中环二鸟苷酸(c-di-GMP)信号途径的功能。利用Xoo与水稻互作的模式系统,通过分析基因缺失突变体的表型,研究可能编码鸟苷酸环化酶(DGC)GGDEF结构域的基因(PXO_00466、PXO_02019、PXO_03378)的功能。首先通过无标记突变技术,获得了3个单基因缺失突变体(?PXO_00466、?PXO_02019、?PXO_03378)。接下来通过不同组合方式,构建了3个双基因缺失突变体(?PXO_00466/?PXO_02019、?PXO_00466/?PXO_03378和?PXO_02019/?PXO_03378)以及1个三基因缺失突变体(?PXO_00466/?PXO_02019/?PXO_03378)。利用这些基因缺失突变体,研究了它们在调控Xoo毒性方面的作用。为了检测这些基因突变体在Xoo毒性方面的变化,将单、双以及三基因缺失突变体接种了水稻叶片,测量了水稻病斑长度。结果显示,与Xoo野生型相比,所有突变都导致病斑长度增加。通常认为c-di-GMP合成酶(DGC)缺失会导致细菌细胞中相应c-di-GMP水平的降低,从而诱导毒性增强。我们的研究结果与此推论是一致的。在游动性实验中,发现3个单基因突变体都呈现游动性增强;1个双基因突变体(?PXO_00466/?PXO_02019)和1个三基因突变体也表现游动性增强,而另外2个双基因突变体(?PXO_00466/?PXO_03378和?PXO_02019/?PXO_03378)则表现游动性减弱。检测突变体中生物膜形成的变化,发现除?PXO_03378外,其他突变体的生物膜水平都有所增加。最后也检测了突变体中胞外多糖产生的变化,发现除了单基因突变体?PXO_03378、双基因突变体?PXO_00466/?PXO_03378和?PXO_02019/?PXO_03378的胞外多糖减少外,其他突变体的胞外多糖均有所增加。综合分析以上结果,发现PXO_00466和PXO_02019基因缺失常导致的表型变化相同,而且在二者的双基因突变体中有些表型还有叠加效应,说明它们是可能编码了有功能的DGC,负调控了Xoo游动性和胞外多糖产生。而PXO_03378基因可能具有c-di-GMP受体的功能,介导了部分PXO_00466和PXO_02019调控的c-di-GMP信号途径。下一步将通过生化手段进一步解析这些基因编码蛋白的活性。本论文结果为研究Xoo中其它GGDEF结构域的功能提供了借鉴意义。
[Abstract]:Xanthomonas oryzae pv. oryzae Xoois is not only an important plant pathogen. It is also a model pathogenic microorganism that studies the interaction between plant and microorganism. The purpose of this paper is to study Xoo cyclic di-GMP (c-di-GMP). The function of signal pathway. The mode system of interaction between Xoo and rice. By analyzing the phenotypes of the deletion mutants, we studied the gene PXO _ 0 _ (00466) / PXO _ s _ (02019), which may encode the GGDEF domain of guanosine cyclidase (DGCU). The function of PXO _ (03378). First, three single-gene deletion mutants were obtained by using unlabeled mutation technique. PXO_00466,? PXO_02019,? PXO033780.Then, through different combinations, three double gene deletion mutants were constructed. PXO_00466/? PXO_02019,? PXO_00466/? PXO_03378 and? PXO_02019/? PXO03378) and a three-gene deletion mutant, PXO03378)? PXO_00466/? PXO_02019/? PXO033788.Using these gene deletion mutants, we studied their role in regulating Xoo toxicity. In order to detect the changes in Xoo toxicity of these gene mutants, we will single them. Two and three gene deletion mutants were inoculated with rice leaves and the length of diseased spot was measured. The results showed that compared with Xoo wild type. It is generally believed that the deletion of c-di-GMP synthase can lead to a decrease in the corresponding c-di-GMP level in bacterial cells. Our results are consistent with this inference. In the swimming experiment, we found that all of the three single gene mutants showed enhanced activity. A double gene mutant? PXO_00466/? PXO02019) and a three-gene mutants also showed increased mobility, while the other two double-gene mutants, PXO _ 02019) and a three-gene mutants, also showed increased mobility. PXO_00466/? PXO_03378 and? PXO_02019/? PXO _ 0 _ 3378) showed decreased mobility. The changes of biofilm formation in mutants were detected. Besides PXO_03378, the biofilm level of other mutants increased. Finally, we detected the changes of exopolysaccharide production in the mutants, and found that there were only single gene mutants. PXO03378, double gene mutants? PXO_00466/? PXO_03378 and? PXO_02019/? The exopolysaccharides of PXO_03378 decreased, but the exopolysaccharides of other mutants increased. It was found that PXO_00466 and PXO_02019 gene deletions often resulted in the same phenotypic changes, and some of the phenotypes in the two gene mutants also had superposition effects. These results suggest that they may encode functional DGCs, and negatively regulate the activity of Xoo and the production of extracellular polysaccharides, while the PXO_03378 gene may have the function of c-di-GMP receptor. Some of the c-di-GMP signaling pathways regulated by PXO_00466 and PXO_02019 are mediated. The next step will be to further elucidate the activity of these gene-encoded proteins by biochemical means. The results provide reference for studying the functions of other GGDEF domains in Xoo.
【学位授予单位】：中国农业科学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.111.47
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本文编号：1372751