烟草靶斑病菌内切多聚半乳糖醛酸酶基因endoPGs的克隆及表达特征分析

【目的】内切多聚半乳糖醛酸酶（endo polygalacturonases，endoPGs）是重要的致病因子之一。论文从烟草靶斑病菌（Rhizoctonia solani）强致病力菌株YC-9和弱致病菌株LF-2中克隆该致病基因，分析比较该基因的序列特征、进化关系和表达特性，研究其在与烟草互作过程的致病作用。【方法】通过比较GenBank中不同植物病原真菌的endoPGs序列设计简并引物，首先获得菌株YC-9及LF-2的endoPGs基因片段，再采用RACE技术克隆获得其cDNA全长序列；生物信息学分析比较其保守结构域及序列特征；采用MEGA 4.0软件构建endoPGs的系统进化树；通过real-time RT-PCR技术对强致病力菌株YC-9及弱致病力菌株LF-2的endoPGs与烟草K326互作的表达特性进行研究。【结果】克隆获得了烟草靶斑病菌强致病力菌株YC-9和弱致病力菌株LF-2的endoPGs的cDNA全长序列，分别命名为endoPG1和endoPG2，开放阅读框（ORF）长度均为1 086 bp，编码361个氨基酸；比较分析表明endoPG1和endoPG2的推测蛋白均具有PLNO3003基因家族保守结构域，其跨膜结构间存在差异；成功构建了该基因系统进化树，结果表明来自烟草靶斑病菌的endoPGs构成独立分支，且烟草靶斑病菌endoPG1及endoPG2同源关系最近；real-time RT-PCR表达特性分析结果显示，靶斑病菌endoPG1和endoPG2与烟草互作（接种）之后该基因的表达与未互作（不接种）的对照样品相比均呈明显上调趋势，同时endoPG1表达迅速且高于endoPG2。【结论】 烟草靶斑病菌强致病力菌株YC-9及弱致病力菌株LF-2的内切多聚半乳糖醛酸酶基因均具有PLNO3003基因家族的保守结构域，其推测蛋白的跨膜结构间存在差异，该推测蛋白与烟草靶斑病菌同源关系最近，且endoPG1和endoPG2的推测蛋白的同源性最高；内切多聚半乳糖醛酸酶基因的表达受与烟草互作的诱导，在不同致病力菌株中存在明显差异。

Cloning and Expression Analysis of the Endo Polygalacturonases Gene endoPGs in Rhizoctonia solani Causing Tobacco Target Spot

【Objective】 Endo polygalacturonases (endoPGs) is considered to be one of the important pathogenic factors. The objectives of this study are to clone and compare endoPGs from the strong pathogenic strain YC-9 and the weak pathogenic strain LF-2 from Rhizoctonia solani, analyze the sequence features, evolutionary relationships and expression characteristics of endoPG1 and endoPG2, and to provide a theoretical foundation for clarifying molecular mechanism of the pathogenicity. 【Method】 Degenerate primers were developed based on the sequence of different endoPGs from plant pathogens in GenBank, and partial cDNA fragments of the strains YC-9 and LF-2 were firstly acquired, then the full-length cDNA sequences were cloned through RACE techniques, and the conserved domains and sequence features of the genes were analyzed by bioinformatics’ methods. A phylogenetic tree was constructed using the MEGA 4.0 software, and the relative expression characters of endoPG1 and endoPG2 were also analyzed by real-time RT-PCR. 【Result】 Two endo polygalacturonases genes were cloned and named as endoPG1 and endoPG2. The genes possessed the conserved domains of PLNO3003 gene superfamily. The open reading frame (ORF) of the full length cDNA was 1 086 bp, encoding a protein of 361 amino acid residues. Differences between endoPG1 and endoPG2 were indicated by comparing the full-length cDNA sequences and transmembrane regions of prediction protein. The PLNO3003 subunit phylogenetic tree was constructed, which showed that the corresponding amino acid sequences of endoPGs from R. solani formed an independent branch, and that of endoPG1 and endoPG2 had the highest homology. Real-time RT-PCR demonstrated that the expression of endoPG1 and endoPG2 were more obviously up-regulated in hyphae of inoculated tobacco leaves than in hyphae of non-inoculated ones, and the expression quantity of endoPG1 was higher than that of endoPG2. 【Conclusion】 The full cDNA sequences of endoPG1 and endoPG2 were successfully cloned from tobacco target spot disease of R. solani, and both have conserved domains of PLNO3003 gene superfamily, and it has obviously differences between transmembrane regions of prediction protein of endoPG1 and endoPG2. Phylogenetic analysis showed that the corresponding amino acid sequences of endoPG1 and endoPG2 have a highest homology relationship. Finally the endoPG1 and endoPG2 gene expression can be obviously induced by interaction with tobacco and the strong and weak pathogenicity strains in YC-9 and LF-2 have obvious differences.