棉花黄萎病菌高效基因敲除体系的建立与Thit功能的研究

发布时间：2018-01-06 15:19

本文关键词：棉花黄萎病菌高效基因敲除体系的建立与Thit功能的研究　出处：《石河子大学》2016年博士论文　论文类型：学位论文

【摘要】：目的:棉花黄萎病是由大丽轮枝菌引起的危害极其严重的土传维管束真菌病害,每年给我国棉花生产造成极大的经济损失。该病用药剂难于防治,成为棉花生产发展的瓶颈。提高棉花对大丽轮枝菌的抗病性是亟待解决的问题。然而,大丽轮枝菌致病机理的研究仍停留在初步探索阶段,病程关键基因的研究也较少。同时,大丽轮枝菌野生型菌株基因打靶效率低,阻碍了大丽轮枝菌病程关键基因的研究。因此,建立高效的大丽轮枝菌基因敲除体系分析病程关键基因的功能,并利用RNAi技术沉默寄主体内大丽轮枝菌病程关键基因可为防治棉花黄萎病提供技术支撑和理论基础。方法:本研究利用In-Fusion克隆技术构建同源重组载体和农杆菌介导的大丽轮枝菌遗传转化的方法建立高效的大丽轮枝菌基因敲除体系。并利用分子生物学、细胞生物学和质谱-色谱等技术方法研究大丽轮枝菌质膜蛋白(硫胺素转运蛋白)的功能,同时利用Gateway技术构建RNAi载体,转化本生烟。结果和结论:1.利用同源重组和大量的转化子筛选获得大丽轮枝菌ΔVd Ku70缺陷突变体,对其进行功能分析,结果表明ΔVd Ku70缺陷突变体菌株与野生型菌株相比,其形态特征(菌落直径、生长率、孢子产量)和致病力基本一致。选择三个潜在的毒素基因Vd2-Od E2、Vd Fre B、Vd Fre6作为敲除的靶基因分析ΔVd Ku70缺陷突变体菌株的同源重组频率。结果显示ΔVd Ku70缺陷突变体菌株的基因打靶效率相比野生型提高了22.8%-34.7%,显著提高了基因打靶效率,实现了靶基因的精确打靶,为大丽轮枝菌致病关键靶基因的研究提供了实验背景菌株,建立了高效大丽轮枝菌基因敲除突变体的转化体系。2.利用同源重组和转化子筛选获得大丽轮枝菌硫胺素转运蛋白基因敲除突变体菌株,命名为ΔVd Thit。ΔVd Thit突变体菌株的形态学特征(菌落直径、生长率、孢子产量、孢子萌发率)分析结果表明:ΔVd Thit突变体菌株的生长能力和生殖能力表现出显著缺陷。ΔVd Thit突变体菌株的逆境胁迫结果表明:ΔVd Thit突变体菌株对逆境胁迫(紫外照射、活性氧压力、高渗透压)的敏感性增强。3.以本氏烟为寄主检测ΔVd Thit突变体的毒力,结果发现寄主植株的感病程度显著降低,并利用q PCR检测寄主体内的真菌生物量及寄主茎段的离体培养,结果显示其毒力显著降低。同时从细胞生物学方面利用荧光显微镜观察VdΔThit-GFP菌株入侵和定殖过程,结果显示在寄主入侵过程中出现显著的缺陷。通过HPLC-MS和GC-MS探索ΔVd Thit突变体毒力降低的机制,结果显示ΔVd Thit突变体菌株中丙酮酸代谢中间产物(乙酰辅酶A和乙偶姻)显著降低,可能是毒力降低和逆境胁迫敏感性增强的重要原因之一。4.通过补充外界不同浓度的硫胺素,ΔVd Thit突变体菌株的形态特征(菌落直径、产孢量)与毒力得到部分恢复。同时利用q PCR分析ΔVd Thit突变体菌株中硫胺素从头合成途径基因的表达,结果显示从头合成途径中两个关键基因(Vd Sti35和Vdthi11)表达上调,且随着外界硫胺素浓度的升高,两个关键基因(Vd Sti35和Vdthi11)表达量更高。表明硫胺素转运蛋白的主动运输途径是大丽轮枝菌硫胺素来源的主要途径,从头合成途径仅是辅助途径,暗示硫胺素转运蛋白是机体不可缺少的蛋白因子。5.大丽轮枝菌不同生长时期和寄主入侵过程中硫胺素转运蛋白基因的表达模式分析结果表明,硫胺素转运蛋白在大丽轮枝菌生长发育和入侵、定殖过程中起着不可替代的作用。同时本研究利用Gateway技术,构建硫胺素转运蛋白基因的三段靶基因片段的RNAi载体,转化本生烟,分别获得多个株系的转大丽轮枝菌硫胺素转运蛋白基因的靶标区段的本生烟植株,分别命名为RNAi-Vd Thit-1、RNAi-Vd Thit-2、RNAi-Vd Thit-3。对获得靶标区段的本生烟植株进行抗病性实验,结果表明:获得硫胺素转运蛋白靶标基因的本生烟植株对大丽轮枝菌的抗病性与野生型相比显著增强,尤其是转化获得的靶标区段Vd Thit-1基因片段的本生烟植株的抗病性最强,对大丽轮枝菌的抗病性表现为高抗等级。综上研究结果表明:本研究获得大丽轮枝菌ΔVd Ku70缺失突变体,提高基因打靶效率,为大丽轮枝菌致病关键靶基因的研究提供实验背景菌株。探索了大丽轮枝菌硫胺素转运蛋白的致病机理,为研究大丽轮枝菌致病机制奠定理论和实验基础。
[Abstract]:Objective: to cotton Verticillium wilt is the harm caused by Verticillium dahliae and serious soil borne vascular fungal diseases, each year causing great economic losses to cotton production in China. The disease drugs are difficult to control, become a bottleneck in the development of cotton production. Improve the disease resistance of Cotton Verticillium dahliae is a problem to be solved. However, the research of Verticillium dahliae pathogenic mechanism is still in the initial stage of exploration, less research course of key genes. At the same time, Verticillium dahliae strains of wild type gene targeting low efficiency, hinder the research of Verticillium dahliae by key course. Therefore, the establishment of Verticillium dahliae gene knockout system analysis of course gene functions, and use RNAi technology to silence the host V.dahliae course key genes can provide technical support and theoretical basis for the prevention and control of Cotton Verticillium wilt. Methods: This study used In-Fusion cloning and construction of homologous recombinant vector and Agrobacterium mediated genetic transformation of Verticillium dahliae method to establish efficient gene knockout system. And the use of molecular biology, cell biology and GC-MS techniques of Verticillium dahilae membrane protein (thiamine transporter) function, at the same time using Gateway technology to construct RNAi vector transformation nicotianabenthamiana. Results and conclusion: 1. by homologous recombination and a large number of transformants of Verticillium dahliae Ku70 Delta Vd mutant screening and functional analysis of the results showed that, compared to Ku70 Vd mutant strain and wild-type strain, its morphological characteristics (colony diameter. The growth rate and spore yield) and pathogenicity are basically the same. The selection of three potential toxin gene Vd2-Od E2, Vd Fre B, Vd Fre6 as the target gene in the analysis on Vd Ku70 mutant strains with defects The source of recombination frequency. The results showed that the gene targeting efficiency Vd Ku70 mutant strain compared to the wild type increased 22.8%-34.7%, significantly increased the gene targeting efficiency, to achieve the precise targeting of target genes and provide experimental background for the study of Verticillium dahliae strains pathogenic key target genes, established a highly efficient gene of Verticillium dahliae knockout mutants of.2. transformation system by homologous recombination and transformant Verticillium dahilae thiamine transporter gene knockout mutant strains, named as morphological characteristics of delta Vd Delta Vd Thit. Thit mutant strain (colony diameter, growth rate, sporulation, spore germination rate) analysis results showed that the growth ability and reproductive ability a Vd Thit mutant strains showed significant defects. A Vd Thit mutant strain stress results showed that a Vd Thit mutant strain of stress (UV radiation, Active oxygen pressure, osmotic pressure) sensitivity to enhance.3. benthamiana host detection for virulence Delta Vd Thit mutant, the disease degree of host plant was significantly reduced, and the use of in vitro culture of Q PCR detection in host fungal biomass and host stems, the results showed that the toxicity was significantly reduced. At the same time from the field of cell biology using fluorescence microscopy Vd Delta Thit-GFP strains invasion and colonization process, results showed that there was significant defects in the host intrusion process. Through HPLC-MS and GC-MS to explore the mechanism of reducing Vd virulence of the Thit mutant, showed that pyruvate metabolism Delta Vd Thit mutant intermediate products (acetyl coenzyme A and B I marriage) decreased significantly, may be one of the important reasons to reduce the stress sensitivity and virulence of.4. enhanced by adding different concentrations of thiamine outside, a Vd Thit mutant strain form State characteristics (colony diameter, sporulation and virulence) was partially recovered. At the same time using the Q PCR analysis of gene expression Vd Thit mutant strain of thiamine in de novo synthesis, the results show that two key genes in the de novo pathway (Vd Sti35 and Vdthi11) expression, and increased with the increasing concentration of thiamine outside two. Key genes (Vd Sti35 and Vdthi11) showed high expression amount. Active transport of thiamine transporter protein pathway is the main way to Verticillium dahilae thiamine sources, de novo synthesis is the only auxiliary way, suggesting that thiamine transport protein expression patterns of thiamine transporter gene invasion protein factor.5. of Verticillium dahliae in different growth periods and the host body indispensable in thiamine transporter. The results showed that the growth and invasion of Verticillium dahliae, the colonization process plays an irreplaceable At the same time. This study uses Gateway technology, RNAi vector three gene fragments to construct thiamine transporter gene, transformation of the smoke, the smoke section plant target multiple lines were to Verticillium dahilae thiamine transporter gene, named RNAi-Vd Thit-1, RNAi-Vd Thit-2, RNAi-Vd Thit-3. resistance experiment of nicotianabenthamiana plants obtained target sections showed that the obtained nicotianabenthamiana plants thiamine transporter target gene of Verticillium dahliae resistance was significantly enhanced compared with the wild type, especially the strongest resistance of nicotianabenthamiana plant target segment Vd Thit-1 fragments into the the resistance to Verticillium dahliae was high resistant level. The research results show that: the study of Verticillium dahliae Vd Ku70 deletion mutant, improve gene targeting efficiency, as Dahlia To provide experimental background strains of Verticillium pathogenicity of key target genes. To explore the pathogenic mechanism of Verticillium dahliae thiamine transporter, lay a theoretical and experimental basis for study on the pathogenic mechanism of Verticillium dahliae.

【学位授予单位】：石河子大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S435.621.2

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