向日葵大丽轮枝菌T-DNA突变体库的构建及微菌核形成和致病力相关基因的研究

发布时间：2018-03-19 14:38

  本文选题：向日葵黄萎病　切入点：大丽轮枝菌　出处：《内蒙古农业大学》2016年博士论文　论文类型：学位论文

【摘要】：向日葵黄萎病是由大丽轮枝菌(Verticillium dahlia Kleb.)侵染所致的一种严重危害向日葵生产的真菌病害,一旦发生很难得到有效的控制。微菌核是大丽轮枝菌主要的初侵染来源,在土壤中能够存活数十年。目前关于微菌核的形成机制和致病机理的研究还很少。本研究通过探究外源添加环腺苷酸(cAMP)对向日葵大丽轮枝菌的生物学特性及致病力的影响、构建大丽轮枝菌的T-DNA突变体库、分析T-DNA插入位点的侧翼序列和突变基因的功能,得出如下结论：1.外源添加cAMP能够显著提高向日葵大丽轮枝菌的产孢量,但对其生长速率、分生孢子萌发率、微菌核形成的数量、粗毒素分泌量以及致病力均有不同程度的抑制作用,其中以对向日葵微菌核形成的抑制作用最为明显。2.利用农杆菌介导的遗传转化体系,将带有潮霉素抗性标记和GFP报告基因的双元载体对大丽轮枝菌的分生孢子进行了遗传转化。通过潮霉素抗性标记的筛选,共获得了包含有800株阳性转化子的大丽轮枝菌的突变体库。3.随机挑取42株阳性转化子,对其菌落形态,菌丝生长速率,产孢量,粗毒素含量和致病力进行了研究。结果表明,42株阳性转化子中有4株转化子的菌落只产生白色气生菌丝,不能形成黑色的微菌核。相比对照,所测定的42株转化子的生长速率均有不同程度的降低；但是产孢量却表现出不同的变化趋势。粗毒素分泌量的测定结果表明,粗毒素分泌量升高的转化子有5株,而表现为下降趋势的转化子有27株。致病力测定的结果表明,致病力增强的转化子有4株,而28株转化子的致病力呈现降低的趋势,其余10个转化子的致病力与对照没有显著差异。4.利用hiTAIL-PCR的方法对20株突变体插入位点的右侧序列进行了扩增,共得到大小介于200-1200bp之间12个突变体插入位点的右侧序列。筛选到了2个与微菌核形成相关的候选基因Initiation-specific a-1,6-mannosyltransferase (VDAG_02820)和Aryl-alcohol dehydrogenase (VDAG_03323),通过功能预测分析将这两个基因命名为VdOCH1和VdAAD。5.通过同源重组的方法获得VdOCH1基因的敲除和互补突变体。功能分析结果表明,该基因不仅与大丽轮枝菌细胞壁的完整性有关,还能够调控病原菌的生长发育和微菌核的形成,并能影响大丽轮枝菌菌丝的穿透能力。此外,敲除VdOCHl基因能够降低病原菌的致病力。6.通过同源重组的方法获得VdAAD互补突变体。功能研究的结果表明,该基因不仅调控大丽轮枝菌的生长发育,而且调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.
【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.655

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