荸荠肉桂酸4-羟基化酶基因的克隆及其在鲜切荸荠黄化过程中的表达分析

Cloning and Expression Analysis of Cinnamic Acid 4-Hydroxylase Gene during Yellowing of Fresh-cut Chinese Water-chestnut

肉桂酸4-羟基化酶(cinnamic acid 4-hydroxylase,C4H)是苯丙烷代谢途径中的关键酶,在植物黄酮类物质合成代谢过程中发挥重要作用。鲜切荸荠贮藏过程中由于黄酮类物质积累而导致的黄化现象很可能离不开C4H的参与。本研究利用RACE技术在荸荠中克隆得到C4H基因cDNA全长,命名为Cw C4H(GenBank登录号:MG719237),该序列长度为1736 bp,编码一条由505个氨基酸组成的多肽,分子量为57.826 kD。Cw C4H包含细胞色素P450结构域,其二级结构以α-螺旋为主。多重比对结果表明,CwC4H具有该基因家族特有的保守结构域并与其它植物的C4H蛋白有很高的相似性。系统进化分析表明CwC4H与高粱C4H蛋白亲缘关系较近。荧光定量PCR分析表明,鲜切荸荠黄化过程中Cw C4H表达量快速上升,水杨酸处理抑制了鲜切荸荠的黄化和CwC4H的表达。

Cinnamic acid 4-hydroxylase(C4 H),which is a key enzyme in phenylpropanoid pathway,plays a vital role in the biosynthesis of flavonoids in plants.C4 H may play an important role in surface etiolation of fresh-cut Chinese water-chestnut caused by flavonoids accumulation.In this study,the full-length cDNA of C4 H in Chinese water-chestnut was cloned using the RACE methods,and it was named CwC4 H(GenBank accession number MG719237).The sequence consists of 1736 bp,and encoding a polypeptide of 505 amino acids with a molecular weight of 57.826 kD.CwC4 H contained four functional domains.Alpha helix was the maximum structural part in the protein secondary structure of CwC4 H.The multiple sequence alignment showed that CwC4 H had the characteristic conservative domains of the C4 H family,and was highly homologous to other C4 H proteins from different species.Phylogenetic tree analysis indicated that Cw C4 H was very closely related to C4 H protein of Sorghum bicolor.Real-time quantitative PCR(qRT-PCR)analysis revealed that the expression level of CwC4 H increased dramatically during surface etiolation of Chinese water chestnut.However,salicylic acid treatment significant inhibited surface yellowing and the expression of CwC4 H.