茶树肉桂酸4-羟基化酶基因的克隆及表达分析

摘要

Cinnamate4-hydroxylase(C4H)isa key enzyme in thephenylpropanoid pathwayin tea plant.The gene caninfluencethebiosynthesisofsecondary metabolitessuch aslignin and flavonoids.ThecDNAfull-length ofC4H genewascloned fromtea plantbyrapid amplification of cDNA endswith a 1518bpopen readingframeencodinga proteinof 505amino acids.Thededucedproteinmolecular weightwas58.15kD anditstheoretical isoelectric point was9.29.A 1840bp promoter sequencewasisolatedby genomewalking method.Thepromoterregionnot onlyhas the basictranscriptional elementsof TATA-box and CAAT-box, but alsohasmanypotentialinducibleand tissue-specificcis-actingelements.Quantitative RT-PCR analysisshowed that theCsC4Hgeneexpressed in bud, leaf, stem and root.The gene was cloned intothe expression vectorpYES-DEST52 foreukaryoticexpressionin Saccharomyces cerevisiaeWAT11.Theenzyme reaction productsweredetected by LC-MSmethod. The results indicated thatcinnamic acidwaspara-hydroxylatedbytarget proteinstogeneratep-coumaric acid.%肉桂酸4-羟基化酶（Cinnamate 4-hydroxylase, C4H）是茶树苯丙烷代谢途径中的关键酶，能够影响木质素和类黄酮等次级代谢物的生物合成。本文采用5′-RACE 技术，克隆了茶树肉桂酸4-羟基化酶基因（CsC4H）的全长序列，其中开放阅读框长1518 bp，编码505个氨基酸，推测蛋白分子量为58.15 kD，理论等电点为9.29。利用基因组步移技术克隆得到该基因上游1840 bp的启动子序列。该启动子区域除了分布有TAAT-box和CAAT-box等基本转录元件外，还存在多个诱导型和组织特异型的顺式作用元件。实时荧光定量PCR结果表明该基因在芽、叶、茎、根中都有表达。将该基因重组至表达载体pYES-DEST52上，并在酿酒酵母 WAT11中进行真核表达。利用 LC-MS方法检测酶反应产物，结果表明目的蛋白能够催化肉桂酸生成p-香豆酸。