本文選題：向日葵黃萎病　切入點：大麗輪枝菌　出處：《內蒙古農業(yè)大學》2016年博士論文　論文類型：學位論文

【摘要】：向日葵黃萎病是由大麗輪枝菌(Verticillium dahlia Kleb.)侵染所致的一種嚴重危害向日葵生產的真菌病害,一旦發(fā)生很難得到有效的控制。微菌核是大麗輪枝菌主要的初侵染來源,在土壤中能夠存活數(shù)十年。目前關于微菌核的形成機制和致病機理的研究還很少。本研究通過探究外源添加環(huán)腺苷酸(cAMP)對向日葵大麗輪枝菌的生物學特性及致病力的影響、構建大麗輪枝菌的T-DNA突變體庫、分析T-DNA插入位點的側翼序列和突變基因的功能,得出如下結論：1.外源添加cAMP能夠顯著提高向日葵大麗輪枝菌的產孢量,但對其生長速率、分生孢子萌發(fā)率、微菌核形成的數(shù)量、粗毒素分泌量以及致病力均有不同程度的抑制作用,其中以對向日葵微菌核形成的抑制作用最為明顯。2.利用農桿菌介導的遺傳轉化體系,將帶有潮霉素抗性標記和GFP報告基因的雙元載體對大麗輪枝菌的分生孢子進行了遺傳轉化。通過潮霉素抗性標記的篩選,共獲得了包含有800株陽性轉化子的大麗輪枝菌的突變體庫。3.隨機挑取42株陽性轉化子,對其菌落形態(tài),菌絲生長速率,產孢量,粗毒素含量和致病力進行了研究。結果表明,42株陽性轉化子中有4株轉化子的菌落只產生白色氣生菌絲,不能形成黑色的微菌核。相比對照,所測定的42株轉化子的生長速率均有不同程度的降低；但是產孢量卻表現(xiàn)出不同的變化趨勢。粗毒素分泌量的測定結果表明,粗毒素分泌量升高的轉化子有5株,而表現(xiàn)為下降趨勢的轉化子有27株。致病力測定的結果表明,致病力增強的轉化子有4株,而28株轉化子的致病力呈現(xiàn)降低的趨勢,其余10個轉化子的致病力與對照沒有顯著差異。4.利用hiTAIL-PCR的方法對20株突變體插入位點的右側序列進行了擴增,共得到大小介于200-1200bp之間12個突變體插入位點的右側序列。篩選到了2個與微菌核形成相關的候選基因Initiation-specific a-1,6-mannosyltransferase (VDAG_02820)和Aryl-alcohol dehydrogenase (VDAG_03323),通過功能預測分析將這兩個基因命名為VdOCH1和VdAAD。5.通過同源重組的方法獲得VdOCH1基因的敲除和互補突變體。功能分析結果表明,該基因不僅與大麗輪枝菌細胞壁的完整性有關,還能夠調控病原菌的生長發(fā)育和微菌核的形成,并能影響大麗輪枝菌菌絲的穿透能力。此外,敲除VdOCHl基因能夠降低病原菌的致病力。6.通過同源重組的方法獲得VdAAD互補突變體。功能研究的結果表明,該基因不僅調控大麗輪枝菌的生長發(fā)育,而且調控病原菌微菌核的形成能力、粗毒素分泌量和致病力。
[Abstract]:Sunflower Verticillium dahlia Kleb.is a kind of fungal disease that seriously endangers the production of sunflower, once it occurs, it is difficult to be effectively controlled. Microsclerotia is the main primary infection source of Rhizoctonia dahliae. There are few studies on the mechanism of microsclerotia formation and pathogenicity. The effects of exogenous adenylate cAMPon on the biological characteristics and pathogenicity of Rhizoctonia davidii were studied. The T-DNA mutants library was constructed, and the flanking sequence of T-DNA insertion site and the function of mutant gene were analyzed. The conclusion is as follows: 1. Exogenous addition of cAMP can significantly increase the sporulation, but increase the growth rate of Vibrio dahliensis. The rate of conidial germination, the number of microsclerotia, the secretion of crude toxin and the pathogenicity of sunflower microsclerotia were inhibited to some extent, among which the inhibition on the formation of sunflower microsclerotia was the most obvious. 2. The genetic transformation system mediated by Agrobacterium tumefaciens was used. The conidial spores of Rhizoctonia dahliensis were transformed with the double vector containing hygromycin resistance marker and GFP reporter gene, and screened by hygromycin resistance marker. A mutant library containing 800 positive transformants was obtained. 42 positive transformants were randomly selected for colony morphology, mycelium growth rate and sporulation. The content and pathogenicity of crude toxin were studied. The results showed that 4 of 42 positive transformants produced only white airborne hyphae, and could not form black microsclerotia. The growth rate of the 42 transformants was decreased to some extent, but the sporulation showed different trends. The results of crude toxin secretion showed that there were 5 transformants with higher crude toxin secretion. The results of pathogenicity test showed that there were 4 transformants with enhanced pathogenicity and 28 transformants with decreased pathogenicity. The pathogenicity of the other 10 transformants was not significantly different from that of the control. (4) hiTAIL-PCR method was used to amplify the right side sequence of the insertion site of 20 mutants. A total of 12 right sequences of insertion sites in the range of 200-1200 BP were obtained. Two candidate genes Initiation-specific a-1 6-mannosyltransferase VDAG02820 and Aryl-alcohol dehydrogenase VDAG03323 were screened. The two genes were named VdOCH1 and VDAG03323 by functional prediction analysis. VdAAD.5. the knockout and complementary mutants of VdOCH1 gene were obtained by homologous recombination. The gene is not only related to the integrity of cell wall, but also can regulate the growth and development of pathogenic bacteria and the formation of microsclerotia. Knockout of VdOCHl gene can reduce the pathogenicity of pathogenic bacteria. 6. VdAAD complementary mutants were obtained by homologous recombination. The results of functional studies showed that the gene not only regulated the growth and development of Verticillium dahliensis, but also decreased the pathogenicity of the pathogen. Moreover, the ability of microsclerotia formation, crude toxin secretion and pathogenicity were regulated.
【學位授予單位】：內蒙古農業(yè)大學
【學位級別】：博士
【學位授予年份】：2016
【分類號】：S435.655

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