稻曲病菌T-DNA插入突变体B2510的插入位点分析及致病相关基因Uvt3277功能的初步研究

发布时间：2019-06-19 15:52

【摘要】：水稻稻曲病是一种水稻穗部病害,现已由次要病害上升为主要病害。稻曲病菌(Ustilaginoideavirens)是稻曲病的病原真菌,在穗部形成的稻曲球严重影响水稻品质和产量,稻曲球产生的毒素能对人畜健康产生危害。因此深入了解稻曲病的致病机理,可以为水稻稻曲病的防治提供理论依据。1、本文从稻曲病菌T-DNA插入突变体库中筛选到一株致病力减弱的突变菌株B2510,通过分析突变菌株B2510的生物学性状和T-DNA插入位点的侧翼基因,为稻曲病菌的分子致病机理的研究提供方法和理论依据。与野生型P1相比,突变菌株B2510丧失了产孢能力,生长速率在MM培养基上明显降低,而在PSA和TB3培养基中则没有显著差异。Southern杂交分析T-DNA的拷贝数,结果显示T-DNA以双拷贝形式插入到突变菌株B2510的基因组中。运用hiTAIL-PCR技术扩增T-DNA插入位点的侧翼序列,获得两个可能与致病性有关的基因UVC6TF和UVRASGAP。T-DNA分别插在基因UVC6TF的启动子区域和UVRASGAP的下游3'端,且稻曲基因组和T-DNA序列均未丢失。半定量RT-PCR分别分析两个基因在突变菌株B2510中的表达量,发现与P1相比均显著下降。因此推测这两个基因与稻曲病菌致病性相关,可能在某一阶段参与调控稻曲病菌在水稻上的致病过程。为进一步了解基因的功能,构建基因UVC6TF的沉默载体,并通过ATMT的方法导入稻曲病菌。获得的2个沉默转化子中UVC6TF的表达分别被抑制了 50%和90%,分析它们的产孢量、生长速率等生物学性状和致病力检测。结果发现该基因不影响菌株正常的生长发育,但是对分生孢子的产生过程有一定的调控作用。产孢能力降低,从而使其致病能力减弱。此外,T-DNA插入还破坏了基因UVRASGAP的表达,下一步计划则是对单个基因或是两个基因进行基因敲除、回补等方法进一步研究基因功能,对稻曲病菌致病过程的解析具有重要意义。2、真菌对铁离子的运输有着特殊的机制。铁转运蛋白参与细胞内铁离子的跨膜运输,有时也可作为二级调控影响基因的表达和机制,对保持体内铁离子的平衡和菌株致病因子的产生具有重要意义。Uvt3277是低亲和力铁转运蛋白,且有研究阐明可能与稻曲病菌的致病过程相关。本研究则对基因Uvt3277的功能进行进一步地验证。构建发夹结构沉默载体,用农杆菌介导转化的方法(ATMT)导入稻曲病菌中:分析沉默转化子的表型,来验证低铁转运蛋白在稻曲病菌的生长发育、致病性的关系。结果发现沉默菌株的生长速率与野生菌株P1相比有一定的降低,但基因沉默效率与生长速率之间没有明显的线性关系,说明基因Uvt3277可能协同其他调控因子影响稻曲病菌的生长发育。铁胁迫性测定发现在一定的Fe2+浓度下,沉默菌株能够利用Fe2+正常生长。另外,沉默菌株的致病能力都比P1强,表明铁转运蛋白在一定程度上影响到致病相关的负调控途径。同时酵母双杂交技术筛选出低铁转运蛋白的互作蛋白,从而进一步为稻曲病菌致病机理的研究提供理论基础。但是筛选到的候选蛋白太少,只得到两个疑似蛋白:ATP/ADP carrier(AAC)protein和SH3 domain-containing protein,因此还需进行验证或重新酵母双杂交筛选。
[Abstract]:Rice (Oryza sativa L.) is a disease of the ear part of rice, and is now the main disease of the secondary diseases. Utilaginoidea (Utilaginoidea) is a pathogenic fungus of rice-koji disease. The rice-koji balls formed in the ear part seriously affect the quality and yield of rice, and the toxin produced by rice-yeast can be harmful to the health of human and animal. Therefore, it is possible to provide a theoretical basis for the prevention and treatment of rice scab.1. A mutant strain B2510 with reduced pathogenicity is selected from T-DNA insertion mutant library. By analyzing the biological character of the mutant strain B2510 and the flanking gene of the T-DNA insertion site, the invention provides a method and a theoretical basis for the research on the molecular pathogenesis of the rice koji. The mutant strain B2510, compared to the wild type P1, lost the sporulation capacity and the growth rate was significantly reduced on the MM medium, while there was no significant difference in the PSA and TB3 medium. Southern blot analysis of the copy number of T-DNA results in the insertion of T-DNA into the genome of the mutant strain B2510 in a double-copy form. The flanking sequence of the T-DNA insertion site was amplified by using the hiTAIL-PCR technique to obtain two possible pathogenicity-related gene UVC6TF and UVRASGAP. T-DNA was inserted in the promoter region of the gene UVC6TF and the downstream 3 'end of the UVRASGAP, respectively, and both the rice-koji genome and the T-DNA sequence were not lost. The expression of the two genes in the mutant strain B2510 was analyzed by semi-quantitative RT-PCR. Therefore, it is presumed that the two genes are related to the pathogenicity of the rice-koji, and may be involved in the control of the pathogenic process of the rice-koji germs on the rice in a certain stage. In order to further understand the function of the gene, the silencing vector of the gene UVC6TF was constructed, and the rice koji was introduced by the method of ATMT. The expression of UVC6TF in the obtained 2 silent transformants was inhibited by 50% and 90%, respectively. The results show that the gene does not affect the normal growth and development of the strain, but has a certain regulation effect on the production process of the conidia. The ability to produce a spore is reduced, so that its pathogenic ability is reduced. In addition, that T-DNA insertion also destroy the expression of the gene UVRASGAP, and the next step is to further study the gene function of a single gene or two genes, such as gene knock-out and back-repair, which is of great significance to the analysis of the pathogenic process of the rice koji. The fungi have a special mechanism for the transport of iron ions. The iron transporter participates in the transmembrane transport of iron ions in the cell, and can also be used as the expression and mechanism of the secondary regulation and influence gene, which is of great significance for maintaining the balance of iron ions in the body and the generation of the pathogenic factors of the strain. Uvt3277 is a low-affinity iron transporter and has been shown to be related to the pathogenesis of rice-koji. This study further verifies the function of the gene Uvt3277. To construct the silencing vector of the hairpin structure, the method of Agrobacterium-mediated transformation (ATMT) was used to introduce the phenotype of the silencing transformant to verify the relationship between the growth and the pathogenicity of the low-iron transporter in the rice koji. The results showed that the growth rate of the silent strain is lower than that of the wild strain P1, but there is no obvious linear relation between the efficiency of the gene silencing and the growth rate. The determination of iron stress shows that the silent strain can grow with Fe2 + under a certain Fe2 + concentration. In addition, the pathogenic ability of the silent strain is higher than that of P1, indicating that the iron transporter has a certain degree of negative regulation of the disease. At the same time, the yeast two-hybrid technology is used to screen the interaction protein of the low iron transporter, thus further providing a theoretical basis for the research of the pathogenic mechanism of the rice koji. However, too few candidate proteins are screened, and only two suspected proteins are available: ATP/ ADP carrier (AAC) protein and SH3 domain-containing protein, and therefore, verification or re-yeast two-hybrid screening is required.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S435.111.4

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