本文關(guān)鍵詞：棉花黃萎病菌高效基因敲除體系的建立與Thit功能的研究　出處：《石河子大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 大麗輪枝菌(Verticillium dahliae) 硫胺素轉(zhuǎn)運蛋白 抗病性 ΔVdKu70突變體 基因打靶效率

【摘要】：目的:棉花黃萎病是由大麗輪枝菌引起的危害極其嚴(yán)重的土傳維管束真菌病害,每年給我國棉花生產(chǎn)造成極大的經(jīng)濟損失。該病用藥劑難于防治,成為棉花生產(chǎn)發(fā)展的瓶頸。提高棉花對大麗輪枝菌的抗病性是亟待解決的問題。然而,大麗輪枝菌致病機理的研究仍停留在初步探索階段,病程關(guān)鍵基因的研究也較少。同時,大麗輪枝菌野生型菌株基因打靶效率低,阻礙了大麗輪枝菌病程關(guān)鍵基因的研究。因此,建立高效的大麗輪枝菌基因敲除體系分析病程關(guān)鍵基因的功能,并利用RNAi技術(shù)沉默寄主體內(nèi)大麗輪枝菌病程關(guān)鍵基因可為防治棉花黃萎病提供技術(shù)支撐和理論基礎(chǔ)。方法:本研究利用In-Fusion克隆技術(shù)構(gòu)建同源重組載體和農(nóng)桿菌介導(dǎo)的大麗輪枝菌遺傳轉(zhuǎn)化的方法建立高效的大麗輪枝菌基因敲除體系。并利用分子生物學(xué)、細(xì)胞生物學(xué)和質(zhì)譜-色譜等技術(shù)方法研究大麗輪枝菌質(zhì)膜蛋白(硫胺素轉(zhuǎn)運蛋白)的功能,同時利用Gateway技術(shù)構(gòu)建RNAi載體,轉(zhuǎn)化本生煙。結(jié)果和結(jié)論:1.利用同源重組和大量的轉(zhuǎn)化子篩選獲得大麗輪枝菌ΔVd Ku70缺陷突變體,對其進(jìn)行功能分析,結(jié)果表明ΔVd Ku70缺陷突變體菌株與野生型菌株相比,其形態(tài)特征(菌落直徑、生長率、孢子產(chǎn)量)和致病力基本一致。選擇三個潛在的毒素基因Vd2-Od E2、Vd Fre B、Vd Fre6作為敲除的靶基因分析ΔVd Ku70缺陷突變體菌株的同源重組頻率。結(jié)果顯示ΔVd Ku70缺陷突變體菌株的基因打靶效率相比野生型提高了22.8%-34.7%,顯著提高了基因打靶效率,實現(xiàn)了靶基因的精確打靶,為大麗輪枝菌致病關(guān)鍵靶基因的研究提供了實驗背景菌株,建立了高效大麗輪枝菌基因敲除突變體的轉(zhuǎn)化體系。2.利用同源重組和轉(zhuǎn)化子篩選獲得大麗輪枝菌硫胺素轉(zhuǎn)運蛋白基因敲除突變體菌株,命名為ΔVd Thit。ΔVd Thit突變體菌株的形態(tài)學(xué)特征(菌落直徑、生長率、孢子產(chǎn)量、孢子萌發(fā)率)分析結(jié)果表明:ΔVd Thit突變體菌株的生長能力和生殖能力表現(xiàn)出顯著缺陷。ΔVd Thit突變體菌株的逆境脅迫結(jié)果表明:ΔVd Thit突變體菌株對逆境脅迫(紫外照射、活性氧壓力、高滲透壓)的敏感性增強。3.以本氏煙為寄主檢測ΔVd Thit突變體的毒力,結(jié)果發(fā)現(xiàn)寄主植株的感病程度顯著降低,并利用q PCR檢測寄主體內(nèi)的真菌生物量及寄主莖段的離體培養(yǎng),結(jié)果顯示其毒力顯著降低。同時從細(xì)胞生物學(xué)方面利用熒光顯微鏡觀察VdΔThit-GFP菌株入侵和定殖過程,結(jié)果顯示在寄主入侵過程中出現(xiàn)顯著的缺陷。通過HPLC-MS和GC-MS探索ΔVd Thit突變體毒力降低的機制,結(jié)果顯示ΔVd Thit突變體菌株中丙酮酸代謝中間產(chǎn)物(乙酰輔酶A和乙偶姻)顯著降低,可能是毒力降低和逆境脅迫敏感性增強的重要原因之一。4.通過補充外界不同濃度的硫胺素,ΔVd Thit突變體菌株的形態(tài)特征(菌落直徑、產(chǎn)孢量)與毒力得到部分恢復(fù)。同時利用q PCR分析ΔVd Thit突變體菌株中硫胺素從頭合成途徑基因的表達(dá),結(jié)果顯示從頭合成途徑中兩個關(guān)鍵基因(Vd Sti35和Vdthi11)表達(dá)上調(diào),且隨著外界硫胺素濃度的升高,兩個關(guān)鍵基因(Vd Sti35和Vdthi11)表達(dá)量更高。表明硫胺素轉(zhuǎn)運蛋白的主動運輸途徑是大麗輪枝菌硫胺素來源的主要途徑,從頭合成途徑僅是輔助途徑,暗示硫胺素轉(zhuǎn)運蛋白是機體不可缺少的蛋白因子。5.大麗輪枝菌不同生長時期和寄主入侵過程中硫胺素轉(zhuǎn)運蛋白基因的表達(dá)模式分析結(jié)果表明,硫胺素轉(zhuǎn)運蛋白在大麗輪枝菌生長發(fā)育和入侵、定殖過程中起著不可替代的作用。同時本研究利用Gateway技術(shù),構(gòu)建硫胺素轉(zhuǎn)運蛋白基因的三段靶基因片段的RNAi載體,轉(zhuǎn)化本生煙,分別獲得多個株系的轉(zhuǎn)大麗輪枝菌硫胺素轉(zhuǎn)運蛋白基因的靶標(biāo)區(qū)段的本生煙植株,分別命名為RNAi-Vd Thit-1、RNAi-Vd Thit-2、RNAi-Vd Thit-3。對獲得靶標(biāo)區(qū)段的本生煙植株進(jìn)行抗病性實驗,結(jié)果表明:獲得硫胺素轉(zhuǎn)運蛋白靶標(biāo)基因的本生煙植株對大麗輪枝菌的抗病性與野生型相比顯著增強,尤其是轉(zhuǎn)化獲得的靶標(biāo)區(qū)段Vd Thit-1基因片段的本生煙植株的抗病性最強,對大麗輪枝菌的抗病性表現(xiàn)為高抗等級。綜上研究結(jié)果表明:本研究獲得大麗輪枝菌ΔVd Ku70缺失突變體,提高基因打靶效率,為大麗輪枝菌致病關(guān)鍵靶基因的研究提供實驗背景菌株。探索了大麗輪枝菌硫胺素轉(zhuǎn)運蛋白的致病機理,為研究大麗輪枝菌致病機制奠定理論和實驗基礎(chǔ)。
[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.

【學(xué)位授予單位】：石河子大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2016
【分類號】：S435.621.2

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相關(guān)期刊論文 前1條

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本文編號：1388425