葡萄COMT基因家族的鉴定与表达分析  

Identification and expression analysis of grape COMT gene family

作　　者：许雯雯 高换超 韩菲菲 李凯薇 贾山毅 李桂荣 XUWenwen;GAO Huanchao;HAN Feifei;LI Kaiwei;JIA Shanyi;LI Guirong(School of Horticulture and Landscape Architecture,Henan Institute of Science and Technology,Xinxiang 453003,Henan,China;Henan Province Engineering Research Center of Horticultural Plant Resource Utilization and Germplasm Enhancement,Xinxiang 453003,Henan,China)

机构地区：[1]河南科技学院园艺园林学院,河南新乡453003 [2]河南省特色园艺植物开发利用工程技术研究中心,河南新乡453003

出　　处：《果树学报》2023年第10期2061-2075,共15页Journal of Fruit Science

基　　金：河南省科技攻关项目(222102110199、232102111090)。

摘　　要：【目的】咖啡酸O-甲基转移酶(COMTs)是木质素合成过程中一种多功能酶,参与多种初生代谢和次生代谢途径,在植物木质素次生物质合成及植物抗逆胁迫反应中起着重要作用。旨在鉴定葡萄COMT基因家族成员,探究其对葡萄真菌病害胁迫的响应,为葡萄抗病育种提供基因资源。【方法】基于拟南芥COMT基因搜索葡萄COMT基因家族,运用生物信息学方法研究葡萄COMT蛋白质理化性质、基因染色体定位、motif分析和启动子顺式作用元件等;利用荧光定量PCR法分析COMT基因在抗病品种摩尔多瓦和感病品种夏黑葡萄上接种霜霉病的表达模式。【结果】从葡萄中鉴定出26个COMT基因,主要位于第10、12和15号染色体上;蛋白质分子质量差异较大,属于不稳定的两性蛋白;亚细胞定位显示其蛋白主要位于细胞质和细胞外。从构建的系统进化树中发现,该家族分为Ⅰ和Ⅱ2个亚组,在进化过程中比较保守,与拟南芥和水稻亲缘关系较近。启动子分析表明,葡萄COMT基因启动子包含丰富的植物激素响应和胁迫响应的顺式元件,接种葡萄霜霉病后,在抗病品种摩尔多瓦中,除COMT2基因外其余25条COMT基因在接种24 h后均显著上调,而在感病品种夏黑中只有63%的COMT基因出现显著上调,且抗病品种的表达量显著高于感病品种,其中以VvCOMT1、5、6、7、8、9和19上调最为显著。【结论】共鉴定了26个葡萄COMT基因家族成员,同时发现COMT基因在葡萄抗病品种中受霜霉病的强烈诱导,表明木质素在葡萄抗病中起重要作用,可为后期研究葡萄抗霜霉病分子机制提供候选基因。【Objective】Caffeic acid O-methyltransferase(COMTs)is a multifunctional enzyme in lignin synthesis.It participates in some primary and secondary metabolic pathways,and plays an important role in the synthesis of lignin secondary substances and the responses to stresses in plants.The European grape genome was used in the study to search for genes and proteins homologous to members of the COMT gene family,and identification of the final grape COMT gene family and analysis of their expression levels under grape downy mildew stress were carried out to provide a molecular basis and genetic resources for grape resistance breeding.【Methods】Firstly,the identified O-methyltransferase gene sequence was searched from the Arabidopsis database.Based on the Arabidopsis COMT gene,the grape COMT gene family was searched by BLASTP tool,and candidate genes of the VvCOMT family were obtained by search and comparison,and duplicate and redundant sequences were deleted.The gene structure,protein physicochemical properties,protein secondary structure,chromosome location and promoter cis-acting elements were obtained using various biological online analysis software;Moldova and Summer Black were selected as materials to inoculate the pathogen spores of grape downy mildew on the leaves respectively.The samples were taken at 0,6,24,48 h and 120 h after inoculation,and the expression patterns of the COMT gene family in Moldova and Summer Black after inoculation of downy mildew pathogen were analyzed by real-time fluorescence quantitative method.【Results】A grape COMT gene family consisting of 26 COMT members was obtained through the identification,and all COMT genes had a Methyltransfer_2.The C-terminal catalytic domain(PF00891)was named VvCOMT1-26 according to the chromosome position,84.6%of the grape COMTs had an amino acid length of over 300 aa,and their amino acid numbers ranged from 189 to 395.Among them,VvCOMT24(VIT_215s0048g02460)had the longest sequence with 395 amino acids,while VvCOMT3(VIT_208s0032g01130)has the shortes

关 键 词：葡萄霜霉病 咖啡酸-O-甲基转移酶 生物学分析 功能分析 

分 类 号：S663.1[农业科学—果树学]