本文關(guān)鍵詞：水稻白葉枯病菌3個(gè)GGDEF結(jié)構(gòu)域蛋白的功能鑒定　出處：《中國(guó)農(nóng)業(yè)科學(xué)院》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 水稻白葉枯病菌 環(huán)二鳥苷酸 GGDEF結(jié)構(gòu)域 毒性 運(yùn)動(dòng)性 生物膜形成 胞外多糖產(chǎn)生

【摘要】：水稻白葉枯病菌(Xanthomonas oryzae pv.oryzae,Xoo)不僅是一種重要的植物病原細(xì)菌,也是研究植物—微生物互作的模式病原微生物。本論文旨在研究Xoo中環(huán)二鳥苷酸(c-di-GMP)信號(hào)途徑的功能。利用Xoo與水稻互作的模式系統(tǒng),通過分析基因缺失突變體的表型,研究可能編碼鳥苷酸環(huán)化酶(DGC)GGDEF結(jié)構(gòu)域的基因(PXO_00466、PXO_02019、PXO_03378)的功能。首先通過無標(biāo)記突變技術(shù),獲得了3個(gè)單基因缺失突變體(?PXO_00466、?PXO_02019、?PXO_03378)。接下來通過不同組合方式,構(gòu)建了3個(gè)雙基因缺失突變體(?PXO_00466/?PXO_02019、?PXO_00466/?PXO_03378和?PXO_02019/?PXO_03378)以及1個(gè)三基因缺失突變體(?PXO_00466/?PXO_02019/?PXO_03378)。利用這些基因缺失突變體,研究了它們?cè)谡{(diào)控Xoo毒性方面的作用。為了檢測(cè)這些基因突變體在Xoo毒性方面的變化,將單、雙以及三基因缺失突變體接種了水稻葉片,測(cè)量了水稻病斑長(zhǎng)度。結(jié)果顯示,與Xoo野生型相比,所有突變都導(dǎo)致病斑長(zhǎng)度增加。通常認(rèn)為c-di-GMP合成酶(DGC)缺失會(huì)導(dǎo)致細(xì)菌細(xì)胞中相應(yīng)c-di-GMP水平的降低,從而誘導(dǎo)毒性增強(qiáng)。我們的研究結(jié)果與此推論是一致的。在游動(dòng)性實(shí)驗(yàn)中,發(fā)現(xiàn)3個(gè)單基因突變體都呈現(xiàn)游動(dòng)性增強(qiáng);1個(gè)雙基因突變體(?PXO_00466/?PXO_02019)和1個(gè)三基因突變體也表現(xiàn)游動(dòng)性增強(qiáng),而另外2個(gè)雙基因突變體(?PXO_00466/?PXO_03378和?PXO_02019/?PXO_03378)則表現(xiàn)游動(dòng)性減弱。檢測(cè)突變體中生物膜形成的變化,發(fā)現(xiàn)除?PXO_03378外,其他突變體的生物膜水平都有所增加。最后也檢測(cè)了突變體中胞外多糖產(chǎn)生的變化,發(fā)現(xiàn)除了單基因突變體?PXO_03378、雙基因突變體?PXO_00466/?PXO_03378和?PXO_02019/?PXO_03378的胞外多糖減少外,其他突變體的胞外多糖均有所增加。綜合分析以上結(jié)果,發(fā)現(xiàn)PXO_00466和PXO_02019基因缺失常導(dǎo)致的表型變化相同,而且在二者的雙基因突變體中有些表型還有疊加效應(yīng),說明它們是可能編碼了有功能的DGC,負(fù)調(diào)控了Xoo游動(dòng)性和胞外多糖產(chǎn)生。而PXO_03378基因可能具有c-di-GMP受體的功能,介導(dǎo)了部分PXO_00466和PXO_02019調(diào)控的c-di-GMP信號(hào)途徑。下一步將通過生化手段進(jìn)一步解析這些基因編碼蛋白的活性。本論文結(jié)果為研究Xoo中其它GGDEF結(jié)構(gòu)域的功能提供了借鑒意義。
[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.
【學(xué)位授予單位】：中國(guó)農(nóng)業(yè)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S435.111.47
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本文編號(hào)：1372751