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  本文關(guān)鍵詞：噻枯唑防治水稻白葉枯病和柑橘潰瘍病的作用機制研究　出處：《南京農(nóng)業(yè)大學(xué)》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 噻枯唑 硫氨素 胞外多糖 柑橘潰瘍病

【摘要】：噻枯唑是我國目前用來防治黃單胞菌屬致病細(xì)菌引起的水稻白葉枯病(Xanthomonas oryzae pv. oryzaae),水稻細(xì)菌性條斑病(Xanthomonas oryzae pv.oryzicola)和柑橘潰瘍病(Xanthomonas citri pv. citri)的常用殺細(xì)菌劑。水稻白葉枯病是由水稻黃單胞菌引起的一種在全世界水稻種植地區(qū)十分常見并且會造成嚴(yán)重經(jīng)濟(jì)損失的的細(xì)菌性病害。除了利用品種抗性作為控制水稻白葉枯病流行危害的關(guān)鍵措施以外,在病害發(fā)生初期采用殺菌劑進(jìn)行化學(xué)防治是重要的應(yīng)急措施。由于在水稻上長期頻繁使用,已經(jīng)在田間檢測到抗藥性菌株。噻枯唑抗型菌株表現(xiàn)活體抗藥性,而在離體條件下不表現(xiàn)抗藥性。1.噻枯唑敏感性菌株ZJ173與抗性菌株2-1-1轉(zhuǎn)錄組分析本文以噻枯唑敏感性菌株ZJ173和在藥劑處理條件的水稻上篩選到的ZJ173抗藥性突變體2-1-1為研究對象,首先研究了這兩個菌株的生物學(xué)表型差異,包括致病力,生長能力,胞外多糖產(chǎn)量,生物膜形成,游動性以及碳源利用率。其次對這兩個菌株進(jìn)行高通量轉(zhuǎn)錄組測序,篩選出表達(dá)量有顯著性差異的基因并進(jìn)行功能分析。結(jié)果表明,ZJ173與2-1-1的致病力與生長能力沒有顯著性差異,但是2-1-1的胞外多糖產(chǎn)量顯著高于ZJ173,生物膜產(chǎn)量高于ZJ173,細(xì)胞游動性弱于ZJ173。與這一結(jié)果對應(yīng)的是,在轉(zhuǎn)錄組得到的差異基因中,也包含了與細(xì)胞游動與趨化性相關(guān)的基因。這說明噻枯唑抗性菌株可能具有更強的細(xì)胞聚集能力,形成生物膜結(jié)構(gòu)來抵抗外界化學(xué)藥劑的傷害。同時,在分析對碳源利用率后發(fā)現(xiàn),ZJ173需要從外界環(huán)境中吸收的碳水化合物種類與數(shù)量均顯著多于2-1-1,轉(zhuǎn)錄差異表達(dá)基因中也包含了與三羧酸循環(huán)以及琥珀酸合成相關(guān)的基因,這說明2-1-1菌體內(nèi)的碳水化合物代謝活力高于ZJ173。以上結(jié)果表明,噻枯唑抗性機制可能與菌體的游動聚集能力以及碳水化合物代謝有關(guān)。2.硫氨素合成途徑相關(guān)基因thiG對Xoo的致病性與藥敏性的影響thiG基因是硫氨素合成途徑中的重要基因,主要負(fù)責(zé)硫氨素的噻唑環(huán)的合成。在上一章的研究中發(fā)現(xiàn)在轉(zhuǎn)錄組測序得到的差異表達(dá)基因中有硫氨素合成途徑的相關(guān)基因。Xoo的thiG基因缺失突變體表現(xiàn)致病力下降,其生長速率在不含硫氨素的培養(yǎng)基中下降,在含有硫氨素的培養(yǎng)基中和水稻葉片組織中生長速率恢復(fù)野生菌株的水平。在離體條件下,thiG缺失突變體對噻枯唑藥敏性增強。這些結(jié)果說明thiG突變體需要從外界環(huán)境中吸收硫氨素來維持自身正常生長,我們推測thiG缺失突變體可能同時吸收與硫氨素結(jié)構(gòu)相似的噻枯唑,從而導(dǎo)致低濃度藥劑對突變體的抑菌效果優(yōu)于野生型菌株。同時,與野生型菌株ZJ173相比,thiG基因的缺失突變體細(xì)胞的聚集能力會增強,包括生物膜產(chǎn)量增加,而細(xì)胞游動性和遷移能力降低,這些結(jié)果說明,可能由于thiG缺失突變體在水稻上的侵染和擴散能力下降,所以表現(xiàn)致病力下降。雙組份調(diào)控系統(tǒng)rpfC和rpfG編碼的蛋白質(zhì)可以通過第二信使cyclic di-GMP調(diào)控細(xì)胞的聚集,研究結(jié)果顯示rpfC和rpfG基因的表達(dá)量在thiG缺失突變體中表現(xiàn)下調(diào)。突變體的胞外多糖產(chǎn)量雖然沒有發(fā)生明顯變化,但是與胞外多糖合成與分泌的相關(guān)基因,像gumD,gumE,gumH和gumM,均出現(xiàn)了上調(diào)表達(dá),而這四個gum基因同時也逆向調(diào)控生物膜的形成,進(jìn)一步證明突變體的細(xì)胞聚集能力增強與胞外多糖生物合成相關(guān)。這些結(jié)果說明硫氨素合成途徑中的噻唑環(huán)合成基因thiG對Xoo的致病力和細(xì)胞聚集能力有至關(guān)重要的作用。3.Xoo的胞外多糖合成相關(guān)gum基因家族對噻枯唑藥敏性影響水稻白葉枯病菌(Xoo)的胞外多糖作為一種重要的致病因子,與病原菌的侵染,吸附以及聚集都密切相關(guān),并且可以保護(hù)菌體抵抗外界不良環(huán)境的干擾。本文研究發(fā)現(xiàn),噻枯唑敏感性菌株ZJ173和抗藥性菌株2-1-1的胞外多糖產(chǎn)量存在很大差異。通過構(gòu)建ZJ173與2-1-1各個胞外多糖合成基因簇gum基因家族的缺失突變體,在水稻上分別測定各個突變體對噻枯唑的藥敏性。結(jié)果表明,在水稻上ZJ173的突變體由于致病力下降,在噻枯唑未處理與處理后病斑長度沒有顯著性差異,因此不能判斷噻枯唑是否對突變體發(fā)揮抑菌效果。然而,2-1 -1的gum突變體2-1-1△gumC和2-1-1△gumI在活體條件下的致病力沒有降低,同時對噻枯唑表現(xiàn)出抗性水平下降,說明gumC和gumI基因的缺失會提高抗性菌株2-1-1的藥敏性。綜上所述,Xoo缺失gum基因后的噻枯唑藥敏性發(fā)生不同變化,由于gum基因簇在胞外多糖合成與分泌中負(fù)責(zé)的功能不同,gum基因缺失不同程度地改變了 Xoo的胞外多糖產(chǎn)量和結(jié)構(gòu),并直接影響對噻枯唑的藥敏性。4.噻枯唑抑制柑橘潰瘍病菌的生長以及誘導(dǎo)柑橘防衛(wèi)基因的表達(dá)柑橘潰瘍病是由Xanthomonas citri pv. citri (Xcc)引起的一種在柑橘種植地區(qū)廣泛存在的并造成嚴(yán)重經(jīng)濟(jì)損失的細(xì)菌性病害。在本研究中,我們證實了噻枯唑通過抑制Xcc的生長和誘導(dǎo)寄主的抗病反應(yīng),可以有效的控制柑橘潰瘍病。噻枯唑處理柑橘會誘導(dǎo)致病相關(guān)基因(PR1, PR2, CHI和RpRd1)以及NPR基因(NPR1,NPR3和NPR4)的上調(diào)表達(dá),尤其是在處理初期。另外,我們發(fā)現(xiàn)噻枯唑會誘導(dǎo)類黃酮合成途徑中的標(biāo)志性基因CitCHS和CitCHI以及水楊酸合成途徑中的關(guān)鍵基因PAL的表達(dá)。而且,噻枯唑還可以誘導(dǎo)防衛(wèi)反應(yīng)的啟動效應(yīng)的誘導(dǎo)基因AZI1的上調(diào)表達(dá)。綜上所述,噻枯唑處理柑橘后誘導(dǎo)的防衛(wèi)反應(yīng)可能與SA信號傳導(dǎo)途徑和啟動效應(yīng)有關(guān),同時這一研究為以后在柑橘潰瘍病的化學(xué)防治方面提供了一種新的選擇。
[Abstract]:Saikuzuo in China is currently used in the treatment of Xanthomonas leaf blight of rice caused by pathogenic bacteria (Xanthomonas oryzae pv. oryzaae), rice bacterial leaf streak (Xanthomonas oryzae pv.oryzicola) and Citrus Canker (Xanthomonas citri pv. citri) commonly used bactericide. Rice bacterial leaf blight is a bacterial disease caused by Xanthomonas Oryza, which is very common in rice growing areas all over the world and will cause serious economic losses. In addition to using variety resistance as a key measure to control the epidemic of rice bacterial blight, it is an important emergency measure to use fungicides for chemical control in the early stage of disease. Drug resistant strains have been detected in the field because of the long-term use of rice on the rice. Saikuzuo resistant strains showed in vivo resistance, and in vitro expression of resistance. 1. Saikuzuo sensitive strain ZJ173 and strain 2-1-1 transcriptome analysis based on sensitivity of Saikuzuo strain ZJ173 and treatment conditions in the pharmaceutical rice screened ZJ173 resistant mutants of 2-1-1 as the research object, firstly study the phenotype differences among the two strains, including pathogenicity, growth ability, exopolysaccharide yield, biological film forming, motility and utilization ratio of carbon source. Secondly, the two strains were sequenced by high flux transcriptome, and the genes with significant differences in expression were screened and functional analysis was carried out. The results showed that there was no significant difference in pathogenicity and growth ability between ZJ173 and 2-1-1, but the yield of exo polysaccharide in 2-1-1 was significantly higher than that in ZJ173, and the yield of biofilm was higher than that of ZJ173, and the motility of cells was weaker than that of ZJ173. In correspondence with this result, in the differential genes of the transcriptional group, it also contains genes associated with cellular movement and chemotaxis. This shows that Saikuzuo resistant strains may have stronger ability of cell aggregation, formation of biofilm structure to resist external chemical damage. At the same time, in the analysis of the utilization ratio of carbon source, ZJ173 need carbohydrates type and quantity of absorption from the external environment were significantly higher than 2-1-1. The gene also contains three carboxylic acid cycle and succinic acid synthesis related gene expression transcription differences, indicating that the carbohydrate metabolism activity of 2-1-1 in ZJ173 cell. The above results show that the resistance of Saikuzuo and possible mechanisms of cell aggregation ability and carbohydrate metabolism on swimming. 2., the influence of thiG gene on the pathogenicity and drug sensitivity of Xoo is thiG, which is an important gene in the thiosynthesis pathway. It is mainly responsible for the synthesis of thiazoles thiazole ring. In the previous chapter, we found that there are genes associated with thiin synthesis pathway in the differentially expressed genes that are sequenced in the transcriptional group. The thiG gene deletion mutant of Xoo showed a decrease in pathogenicity, and its growth rate decreased in the medium without sulfur ammonia. The growth rate of the wild strain was restored in the medium containing thionine and the tissue of rice leaves. In vitro, thiG deletion mutant of Saikuzuo drug sensitivity enhancement. These results indicate that thiG mutants need to absorb sulfur ammonia from the environment always maintain their normal growth, we hypothesized that thiG deletion mutant may also absorb and sulfur ammonia element structure similar to Saikuzuo, resulting in bacteriostasis effect is better than that of the wild type strain of low concentration of pesticides on mutants. At the same time, compared with the wild type strain ZJ173, aggregation ability of thiG gene deletion mutant cells can be enhanced, including biofilm production increased, and cell motility and migration ability decreased, these results suggest that thiG may be due to the deletion mutant in rice infection and diffusion capacity decreased, so the decline in pathogenicity. Two component regulatory system rpfC and rpfG encoded protein can regulate cell aggregation through second messenger cyclic di-GMP. The results showed that the expression of rpfC and rpfG gene was down regulated in thiG deficient mutants. Mutants of exopolysaccharide production although did not change significantly, but the genes related with extracellular polysaccharide synthesis and secretion of gumE, like gumD, gumH and gumM, were up-regulated, and four of the gum gene was also reverse regulation of biofilm formation, further evidence of mutant cell aggregation ability and exopolysaccharide biosynthesis. These results indicate that the thiazole ring synthesis gene thiG in the thiazine synthesis pathway plays a vital role in the pathogenicity and cell aggregation of Xoo. Xanthomonas oryzae pv.oryzae 3.Xoo exopolysaccharides biosynthesis gum gene family of Saikuzuo drug sensitivity (Xoo) is an important pathogenic factor for the extracellular polysaccharide, infection and pathogenic bacteria, adsorption and aggregation are closely related, and can protect the cell against the interference of the external environment is not good. This study found that the sensitivity of Saikuzuo resistant strains strains ZJ173 and 2-1-1 of the exopolysaccharide yield differences. Through the construction of ZJ173 deletion mutant and 2-1-1 various extracellular polysaccharide biosynthesis gene cluster of gum gene family, drug sensitivity of each mutant to Saikuzuo were detected in rice. The results showed that ZJ173 in rice mutants due to pathogenicity in decline, Saikuzuo untreated and treated lesion length had no significant difference, therefore cannot judge whether to play the inhibitory effect of mutant saikuzuo. However, the pathogenicity of gum mutant 2-1 -1 2-1-1 gumC and 2-1-1 Delta gumI in vivo did not decrease, at the same time, Saikuzuo exhibits resistance decreased, indicating deletion of gumC gene and gumI gene will increase the susceptibility of 2-1-1 resistant strains. In summary, Saikuzuo drug sensitivity of Xoo gene deletion of gum after the changes, because the gum gene cluster in the extracellular polysaccharide synthesis and secretion in different functions, gum gene deletion difference Xoo extracellular polysaccharide production and structure, and a direct impact
【學(xué)位授予單位】：南京農(nóng)業(yè)大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.111.47;S436.66

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