【作者】 田李；

【导师】 戴小枫；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2011， 博士

【摘要】 大丽轮枝菌(Verticillium dahliae)是引起棉花黄萎病的病原菌,在世界范围内造成重大经济损失。已有研究表明大丽轮枝菌的分泌蛋白即毒素蛋白是造成棉花黄萎、落叶和减产的主要因素之一。因此构建大丽轮枝菌基因敲除体系(实验平台)和分泌蛋白预测体系(生物信息平台),并在此基础上对潜在的毒性分泌蛋白(secreted protein)和分泌系统蛋白(secretion system protein)的编码基因进行功能研究,将为揭示大丽轮枝菌致病机理,寻找防治黄萎病的生物学靶点提供思路。构建了高效的大丽轮枝菌基因敲除体系。具体从以下几方面进行了体系的优化:(1)融合PCR和Gateway技术相结合高效构建基因敲除载体;(2)利用农杆菌介导法转化大丽轮枝菌;(3)使用在T-DNA之间加入致死基因HSVtk的双元载体,使T-DNA随机插入转化子在添加5-氟脱氧尿苷的培养基上不能存活,从而通过“反向筛选”提高基因敲除的效率。使用上述平台对大丽轮枝菌腺嘌呤合成酶基因和几丁质合成酶基因进行基因敲除验证,基因敲除转化子在总转化子中的比例分别达到87%和44%。建立了预测大丽轮枝菌基因组范围内所有分泌蛋白的计算方法。利用已公布的大丽轮枝菌(V. dahliae VdLs.17)全基因组序列,组合使用生物信息学软件SignalP、TargetP、TMHMM、Big-pi和PROSITE进行计算,预测得到VdLs.17基因组内共有922个分泌蛋白。利用上述分泌蛋白的预测方法,在本实验室两株不同致病性生理型Vd114(弱毒性)和Vd991(强毒性)基因组全测序的基础上,进行比较分析后,得到6个强毒力菌株Vd991特有的分泌蛋白基因,推测它们可能在强致病性型Vd991的致病机制中发挥了重要作用。为验证其生物学功能,通过本文已建立的大丽轮枝菌基因敲除体系,分别得到了上述6个蛋白编码基因在Vd991中缺失的突变体菌株。但是表型分析发现,6个突变体菌株无论从生长速率,产孢量和致病性方面都与野生型菌株Vd991没有明显的变化。其原因可能是由于大丽轮枝菌分泌的诸多毒性蛋白可能共同参与了生物学毒性的产生,缺失一个在功能上冗余的分泌蛋白基因后,不会产生容易识别的表型改变。这一初步结果暗示,较难找到单一的分泌蛋白基因做为防治黄萎病的生物学靶点。LHS(Lumenal Hsp Seventy,内质网Hsp70家族成员)蛋白近年来被发现在稻瘟病菌蛋白分泌中扮演了重要的角色。本研究在Vd991中克隆了其同源基因VdLHS。缺失VdLHS的大丽轮枝菌突变体菌株△Lhs产生的分泌蛋白总量仅有野生型Vd991的24%,并且几乎丧失了对寄主植物军棉1号的侵染能力。因此分泌系统蛋白VdLHS可调控大丽轮枝菌多种蛋白的分泌,在大丽轮枝菌的致病性中起了重要作用。这一初步研究和发现,为防治棉花黄萎病提供了一个潜在的生物学靶点。更多还原

【Abstract】 Verticillium dahliae is a soil-borne plant pathogen responsible forVerticillium wilt diseases in temperate and subtropical regions, collectively they affect over 200 hosts, including many economically important crops such as cotton. It is reported that V. dahliae produce secreted cell wall degrading enzymes and other extracellular phytotoxins molecules that induce host cell death. So, developing an efficient method of gene knockout system in Verticillium dahliae and perform functional analysis for potential secreted toxin encoding genes by knockout experiment would help clarify the infection mechanism of this fungal pathogen.By using fusion PCR, we constructed gene knockout vectors. By using Agrobacterium tumefaciens-mediated transformation and applying a herpes simplex virus thymidine kinase (HSVtk) gene in T-DNA as a conditional lethal gene to counter-select against ectopic transformants, we developed an efficient method to select gene knockout transformants. Gene knockout frequency for ADE4 and ChsV was 87% and 44%, respectively.The softwares SignalP, TargetP, TMHMM, Big-pi and PROSITE were combined to predict the sectetome of Verticillium dahliae VdLs.17. Finally, we identified 922 possible secreted proteins in Verticillium dahliae VdLs.17 secretome. This study carried out a detailed comparative analysis of genes encoding secreted proteins between Vd114 and Vd991 which show different virulence towards cotton to discover potential Lineage-Specific secreted protein coding genes in Vd991. Finally, we identified 6 possible Vd991 specific secreted proteins. We constructed mutants for each specific proteins in Vd991. But mutants showed similar phenotype with Vd991. One reason is that gene redundancy can mask the phenotype of singal loss-of-function mutant. So, we further analysis the secretion system protein in Vd991.A molecular chaperone of the lumenal hsp seventy gene (VdLHS), which plays a role in the first step of extracellular protein secretion by importing them into the endoplasmic reticulum (ER) and subsequent protein folding, was disrupted via Agrobacterium tumefaciens-mediated transformation in Vd991. The total amount of secreted extracellular phytotoxins is significantly reduced in the△Lhs culture filtrate when culturing the fungus in Czapek’s medium to imitate the phytotoxin secreting ability in planta. The mutants were severely impaired in virulence on cotton Junmian 1. Results of this study suggest that VdLHS plays important roles in protein secretion and may provide potential novel targets for controlling the devastating disease.更多还原