芹菜品种‘六合黄心芹’ AgCCoAOMT基因的克隆及表达特性分析

摘要

According to transcriptome database of Apium graveolens Linn. in Apiaceae, caffeoyl-CoA-O-methyltransferase gene was cloned from total RNA in leaf of A. graveolens ‘Liuhe Huangxinqin’ , which was named as AgCCoAOMT gene. Sequence analysis result shows that AgCCoAOMT gene contains an open reading frame ( ORF) with length of 726 bp, 241 amino acids are encoded. The protein encoded by this gene is AgCCoAOMT, its theoretical relative molecular mass is 27 010, and theoretical isoelectric point is pI 5. 35. In AgCCoAOMT protein, percentage of acidic amino acids is higher than that of basic amino acids, and percentage of aliphatic amino acids is about three times of that of aromatic amino acids. This protein belongs to hydrophobic protein and contains a conserved AdoMet MTases domain, meaning that AgCCoAOMT protein belongs to AdoMet MTases superfamily. Tertiary structure of AgCCoAOMT protein includes many a-helix and β-turn, its consistency with that of CCoAOMT protein from Medicago sativa Linn. is 84. 58%. Analysis results of multiple alignment and phylogenetic tree show that AgCCoAOMT protein has high conservation. And it has the nearest phylogeny with CCoAOMT protein from the same family species Ammi majus Linn. and Petroselinum crispum ( Mill.) Nyman ex A. W. Hill, also, has nearer phylogeny with CCoAOMT protein from Nelumbo nucifera Gaertn. in Nymphaeaceae. qRT-PCR determination result shows that AgCCoAOMT gene can express in root, stem, petiole and leaf blade of ‘Liuhe Huangxinqin’ , and its relative expression in leaf blade is extremely significantly higher than that of other tissues (P<0. 01), meaning that expression of this gene has obvious tissue specificity. There is obvious difference in relative expression of this gene in leaf blade at different growth and development stages, its relative expression at commodity stage ( the sixty-fifth day after sowing ) is extremely significantly higher than that at seedling stage ( the twenty-fifth day after sowing) and vigorous growth stage ( the forty-fifth day after sowing ) . It is suggested that AgCCoAOMT gene is related to leaf senescence and lignin content increasing of ‘Liuhe Huangxinqin ’ , and has high conservation during evolutionary process.%根据伞形科( Apiaceae)植物芹菜( Apium graveolens Linn.)的转录组数据库,从芹菜品种‘六合黄心芹’(‘Liuhe Huangxinqin’)叶片总RNA中克隆获得咖啡碱-辅酶A-O-甲基转移酶基因,命名为AgCCoAOMT基因。序列分析结果表明：AgCCoAOMT基因包含1个长度726 bp的开放阅读框( ORF),编码241个氨基酸；该基因编码的蛋白质为AgCCoAOMT,其理论相对分子质量为27010,理论等电点pI 5.35；在AgCCoAOMT蛋白中,酸性氨基酸所占比例高于碱性氨基酸,脂肪族氨基酸所占比例约为芳香族氨基酸的3倍；该蛋白属于疏水性蛋白,并含有1个保守的AdoMet MTases结构域,说明AgCCoAOMT蛋白属于AdoMet MTases超级家族；AgCCoAOMT蛋白的三级结构包含多个a-螺旋和β-折叠,与紫花苜蓿( Medicago sativa Linn.) CCoAOMT蛋白三级结构的一致性为84.58%。多重比对和系统树分析结果表明：AgCCoAOMT蛋白有较高的保守性；该蛋白与同科植物大阿米芹( Ammi majus Linn.)和欧芹﹝Petroselinum crispum ( Mill.) Nyman ex A. W. Hill〕CCoAOMT蛋白的进化关系最近,与睡莲科( Nymphaeaceae)植物莲( Nelumbo nucifera Gaertn.) CCoAOMT蛋白的进化关系较近。 qRT-PCR检测结果表明：AgCCoAOMT基因能够在‘六合黄心芹’的根、茎、叶柄和叶片中表达,且在叶片中的相对表达量极显著(P<0.01)高于其他组织,说明该基因的表达具有明显的组织特异性；不同生长发育阶段该基因在叶片中的相对表达量有明显差异,其在商品期(播种后第65天)的相对表达量极显著高于幼苗期(播种后第25天)和生长旺盛期(播种后第45天)。研究结果显示：AgCCoAOMT基因与‘六合黄心芹’叶的老化和木质素含量升高有关,且在进化过程中具有较高的保守性。