玉米大斑病菌（Setosphaeria turcica）候选效应分子预测及交配型基因位点克隆与分析

发布时间：2018-02-04 19:09

本文关键词： 玉米大斑病菌效应分子交配型基因位点生物信息学　出处：《沈阳农业大学》2017年硕士论文　论文类型：学位论文

【摘要】：玉米大斑病(Nothern leaf blight of corn)是我国玉米产区重要叶部病害之一,由大斑刚毛座腔菌[Setosphaeria turcicaa(Pass.)Leonard et Suggs]侵染引起,其致病力是影响流行的关键因素。目前已有学者在细胞壁降解酶、黑色素、营养元素、毒素、及生理分化等因素对其致病力影响方面进行了研究,在效应分子和交配型基因位点方面尚无深入研究。本文旨在利用生物信息学方法预测玉米大斑病菌候选效应分子,为进一步研究效应分子在病原菌与寄主互作中的作用奠定基础;克隆并分析玉米大斑病菌交配型基因位点,明确其在我国主要玉米产区的分布,探究病原菌有性生殖过程中交配型基因发挥的作用,为玉米大斑病菌致病机理的探究及防治提供理论依据。1.玉米大斑病菌效应分子预测与分析。本文根据玉米大斑病菌Setosphaeriaa turcicaa 28A全基因组信息,利用生物软件从玉米大斑病菌中11698条蛋白序列中预测候选效应分子,再通过对上述蛋白半胱氨酸含量、信号肽长度及冗余性分析,最终获得60个符合条件的候选效应分子,其中有24条蛋白序列在数据库中没有任何与之同源的序列。对预测出的60个候选效应因子功能分析结果表明,3个效应因子具有预测功能,其余57个效应因子功能未知。2.玉米大斑病菌株分离鉴定。从我国主要玉米产区黑龙江、吉林、辽宁、四川、云南等省份共采集并分离获得的99个菌株中鉴定出73个玉米大斑病菌(Setosphaeria turcica)菌株,其余为8个枝孢霉菌(Cladosporium tenuissimum)菌株,6个玉米生离蠕孢菌(Bipolaris zeicola)菌株,6个互隔交链孢霉菌(Alternaria alternata)菌株,5个极细链格孢菌(Alternaria tenuissima)菌株,1个芸薹链格孢菌(Alternariabrassicae)菌株。对已分离菌株进行系统发育分析,发现玉米大斑病菌(S.turcica)菌株、互隔交链孢霉菌(A.alternata)菌株、玉米生离蠕孢菌(B.zeicolaa)菌株亲缘关系较近,构成一个亚枝;互隔交链孢霉菌(A.alternata)菌株,芸薹链格孢菌(A.brassicae)菌株,极细链格孢菌(A.tenuissima)菌株亲缘关系相对较近,构成一个亚枝;枝孢霉菌(C.tenuissimum)菌株构成一个小分支,亲缘关系与其它真菌相对较远。3.玉米大斑病菌交配型基因位点克隆与生物信息学分析。本文克隆了玉米大斑病菌的StM471-1/StM4T1-2基因位点,全长为12382bp和12284bp,分别包含1个MAT基因,基因位点侧翼序列分别包含三个可编码蛋白和三个螺旋结构,且位于不同交配型基因位点的同一基因相似性均高达98%以上。两交配型基因位点核心区分别编码379个和344个氨基酸,其中StM4T1-1-1编码保守α结构域,StM4T1-2-1编码保守HMG盒子结构域,交配型基因通过自身保守结构域在真菌有性生殖中发挥作用。其侧翼序列包含的GAP、ORF1、Bgl-1蛋白可能在功能上与玉米大斑病菌的有性生殖过程协同作用。4.玉米大斑病菌交配型鉴定。玉米大斑病菌生殖方式为异宗配合,存在两种交配型,为Mating-type 1-1(M4T1-1)和Mating-type 1-2(M4T1-2)。供试73个玉米大斑病菌菌株中,MAT1-1菌株占49.3%;M4T1-2菌株占43.8%,M4T1-1:M4T1-2比率接近1:1;部分菌株中同时检测到MAT1-1和 M4T1-2。
[Abstract]:Northern Corn Leaf Blight (Nothern leaf blight of corn) is one of China's important corn leaf disease, the spot bristles seat cavity bacteria [Setosphaeria (Pass.) turcicaa Leonard et Suggs] caused by infection, its pathogenicity is a key factor affecting the epidemic. At present, scholars in the cell wall degrading enzymes, melanin, nutrient elements, toxin, effects and physiological factors on the differentiation of pathogenicity was studied, there is no in-depth study on effect of molecular and mating type loci. This paper aims to use bioinformatics prediction setosphaeriaturcica candidate effectors methodology, which lays a foundation for further study on the molecular effects of pathogen host interaction in the role of; cloning and analysis of setosphaeriaturcica mating type loci, the distribution in the main maize producing areas in China, play the mating type genes of pathogenic bacteria of importance in the process of sexual reproduction, To provide theoretical basis for the prediction and analysis of.1. of setosphaeriaturcica effector molecules to explore prevention and treatment of setosphaeriaturcica pathogenesis. According to setosphaeriaturcica Setosphaeriaa turcicaa 28A genome information prediction candidate effectors from 11698 protein sequences of setosphaeriaturcica in using biological software, and through the protein the content analysis of signal peptide length and redundancy, eventually won the 60 eligible candidate effectors, of which 24 protein sequence has no homologous sequences in the database. The results show that the predicted 60 candidate effector function analysis, the 3 effect factor has the forecast function, the remaining 57 effect factor of unknown function.2. setosphaeriaturcica isolate. The main maize producing areas in Heilongjiang, Jilin, Liaoning, Sichuan, Yunnan and other provinces were collected and separated Identified 73 setosphaeriaturcica 99 strains obtained in (Setosphaeria turcica) for the rest of the 8 strains of Cladosporium (Cladosporium tenuissimum) strains, 6 maize leaves Helminthosporium (Bipolaris zeicola) strains, 6 Alternaria fungus (Alternaria alternata) strains, 5 very fine Alternaria (Alternaria tenuissima) 1 strains of Alternaria brassicae (Alternariabrassicae) strains. On isolated strains for phylogenetic analysis, found that corn leaf blight (S.turcica) strains of Alternaria fungi (A.alternata) strains of maize leaves Helminthosporium (B.zeicolaa) near the strain relationship the relationship, constitute a sub branch; Alternaria fungi (A.alternata) strains of Alternaria brassicae (A.brassicae) strains from Alternaria tenuissima (A.tenuissima) strains relatively close genetic relationship, constitute a sub branch; Cladosporium (C.tenuissimum) strains. A small branch, the phylogenetic relationship with other fungi relatively distant.3. setosphaeriaturcica mating type loci cloning and bioinformatics analysis. The cloned StM471-1/StM4T1-2 gene locus setosphaeriaturcica, was 12382bp and 12284bp respectively, including 1 MAT gene loci flanking sequences respectively contains three can encoding protein and three helix, the same gene in different mating type loci and the similarity were as high as 98%. Two mating type loci were core region encoding 379 and 344 amino acids, including StM4T1-1-1 encoding StM4T1-2-1 encoding alpha conserved domain, a conserved HMG box domain by mating type genes the conserved domain in the sexual reproduction of fungi play a role. The flanking sequences included GAP, ORF1, Bgl-1 protein may be the process of sexual reproduction of bacteria and exserohilumturcicum in function coordination Mating type identification of pathogen.4. maize. Setosphaeriaturcica reproduction is heterothallic, there are two kinds of mating type, Mating-type 1-1 (M4T1-1) and Mating-type 1-2 (M4T1-2). 73 tested isolates, MAT1-1 strains accounted for 49.3%; M4T1-2 was 43.8%, the ratio of M4T1-1:M4T1-2 to 1:1; some strains detected for MAT1-1 and M4T1-2.

【学位授予单位】：沈阳农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S435.131.4

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