葡萄COMT基因家族的鉴定与表达分析

Identification and expression analysis of grape COMT gene family

【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 shortest sequence with only 189 amino acids.The relative molecular weight of the proteins were between 21179.45(VvCOMT3)and 43521.88(VvCOMT24);The isoelectric points were distributed between 5.18(VvCOMT22)and 6.23(VvCOMT3),and the isoelectric points were all less than 7;The protein instability coefficients ranged from 27.21(VvCOMT23)to 43.30(VvCOMT5),among them 6 instability coefficients over 40 belonged to unstable proteins;All proteins with a hydrophilicity index between-0.158(VvCOMT16)and 0.079(VvCOMT7)were amphoteric proteins;The subcellular localization results showed that the final localization was in cytoplasm and extracellular space.By constructing the phylogenetic trees of the different species,it could be observed that the grape COMT gene was relatively conservative and highly similar in the evolutionary process,which was closely related to Arabidopsis and rice,and was farthest related to apple.From the prediction of the secondary structure of the proteins,it could be seen that the grape COMT gene family containedα-helix,β-corner,irregular curl and extended chain four configurations,the total proportion ofα-helix and irregular curl was 70%.The Motif analysis yielded 19 more conservative motifs,and each VvCOMT gene was distributed with 4-14 motifs,the motif 6 present in all COMT genes,indicating that the motif 6 was highly conservative.At the same time,it was found that the COMT genes in the same group contained the same motifs.For example,the closely related VvCOMTs in Group I contained motifs 5,7,8,17 and 19,while those in GroupⅡwere different.Most COMT genes contained motifs 13,14 and 16,while the VvCOMT4 only contained motifs 2,3,6 and 10.The difference in the motifs contained in different branches might be one of the reasons for functional differentiation in the evolution of the VvCOMT.It was found that 26 VvCOMT genes were irregularly distributed on 7 chromosome skeletons,among them the 12 chromosome had the most genes,including 13 VvCOMT genes.The promoter visualization showed that 227 cisacting elements were found in the grape COMT gene family,including 184 hormone related elements,19 fungal induction related elements and 29 defense and stress response related elements.By analyzing the expression of the COMT genes in the two cultivars after inoculation with downy mildew pathgen,it was found that 25 VvCOMTs were significantly upregulated under downy mildew stress in the resistant cultivar Moldova,with 42%of the COMT genes significantly upregulated at 48 h after inoculation,VvCOMT1,5,6,7,8,9 and 19 were most significantly upregulated after inoculation with the pathogen in the susceptible cultivar Summer Black.Among them,the VvCOMT2 was not significantly upregulated in both cultivars,indicating that it did not respond to the infection of downy mildew.In addition,the expression of the COMT genes in resistant cultivar was significantly higher than that in susceptible cultivars,indicating that the VvCOMT genes played a certain role in the process of resistance to downy mildew.【Conclusion】This study showed that the grape COMT gene family responded to grape downy mildew and played an important role in the process of resistance to downy mildew infection.