水芹咖啡酰辅酶A-O-甲基转移酶基因的克隆与表达特性分析

Cloning and expression characteristics analysis of caffeoyl-CoA O-methyltransferase gene in Oenanthe javanica

[目的]本文旨在研究水芹咖啡酰辅酶A-O-甲基转移酶基因(OjCCoAOMT)的序列及其表达特性,了解该基因在水芹木质素代谢中的作用。[方法]利用RT-PCR技术从水芹中克隆OjCCoAOMT基因,用生物信息学方法对获得的序列进行分析,并利用实时荧光定量PCR技术研究该基因在水芹不同组织以及不同贮藏条件下的表达。[结果]序列分析结果表明:OjCCoAOMT基因包含1个长度为726 bp的开放阅读框(ORF),编码241个氨基酸,蛋白相对分子质量为27.111×10~3,理论等电点为5.38。OjCCoAOMT蛋白属于疏水性蛋白,无序化比例为12.45%,空间结构主要由8个α-螺旋和7个β-折叠组成。多重比对与系统树分析结果表明:OjCCoAOMT蛋白与同科植物欧芹的进化关系最近。实时荧光定量PCR显示,在‘八卦洲水芹’和‘南选八卦洲紫水芹’的叶片及叶柄中OjCCoAOMT基因的表达量差异显著。在室温(25℃)、低温(4℃)和室温黑暗(25℃) 3种贮藏条件下OjCCoAOMT基因表达量差异较大,低温(4℃)下表达量最高。2个品种的基因表达量也具有显著差异。[结论]OjCCoAOMT蛋白有较高的保守性。OjCCoAOMT基因在水芹中的表达具有明显的组织特异性和材料差异性,同时在低温(4℃)条件下表达量最高。

[Objectives] The study aimed to investigate the sequence and expression characteristics of caffeoyl-CoA O -methyltransferase gene in Oenanthe javanica(OjCCoAOMT) and to understand its role in lignin metabolism.[Methods] The OjCCoAOMT gene was cloned from O.javanica using RT-PCR technique. The bioinformatics methods were used to analyze putative amino acid sequence and RT-qPCR method was used to study the expression profiles of OjCCoAOMT gene in different tissues of O.javanica and under different storage conditions.[Results] Sequence analysis result showed that OjCCoAOMT gene contained an open reading frame(ORF)with length of 726 bp,encoded 241 amino acids. The relative molecular mass of OjCCoAOMT protein was 27.111×10 3. Theoretical isoelectric point was 5.38. The protein belonged to hydrophobic protein and the ratio of its unordered sequence was 12.45%. Tertiary structure of OjCCoAOMT protein mainly consisted of eight α-helix and seven β-sheet. The analysis results of multiple alignment and phylogenetic tree showed that OjCCoAOMT had the nearest phylogeny with CCoAOMT protein from the same family species Petroselinum crispum . RT-qPCR analysis showed that the expression profiles of OjCCoAOMT gene were significantly different in different tissues(leaf blade and petiole)of ‘Baguazhoushuiqin’and‘Nanxuanbaguazhouzishuiqin’. The expression levels of OjCCoAOMT gene were different under various storage conditions,such as room temperature(25℃),low temperature(4℃)and darkness at room temperature(25℃). The expression level was peak at 4℃. Moreover,significant differences also existed between two varieties.[Conclusions] OjCCoAOMT protein has high conservation. The relative expression level of OjCCoAOMT gene has tissue and material specialties under different treatments. Moreover, OjCCoAOMT gene has high expression level under the treatment of 4℃.