茶树CsANS基因及其启动子的克隆与生物信息学分析

摘要

采用RACE技术,获得茶树武夷奇种C18叶片花青素合成途径中的花青素合成酶(ANS)基因的cDNA全长序列.采用染色体步移技术获得该基因的启动子序列.采用实时荧光定量PCR技术检测该基因在不同遮光处理下的表达动态.结果表明,CsANS全长cDNA为1000 bp,其中ORF(Open Reading Frame)为957 bp,编码320个氨基酸,含有DIOX-N和20G-Fell-Oxy保守功能结构域;分离得到CsANS基因上游调控序列1010 bp,其含启动子核心元件TATA-box及ACE、GT1-motif、Sp1(光响应元件)、circadian(生物钟相关元件)等与花青素合成途径相关的重要顺式作用元件.荧光定量PCR分析表明,该基因在全光照处理(CK)时表达量较高,75%遮光时表达量较低.说明该基因的表达受光照强弱的控制.%The full length cDNA sequence of anthocyanin synthase gene(CsANS)was cloned from 'Wuyi qizhong C18' (Camellia sinensis) using rapid amplification of cDNA ends(RACE)technology. The promoter of CsANS was isolated using Genome Walking technology. The gene expression of ANS under different shading treatments was analyzed by the real-time PCR. The results showed that the full length cDNA of CsANS was 1000 bp, and it contained a 957 bp open reading frame (ORF), which encoding a protein of 320 amino acid, including two conservative functional domains, DIOX-N and 20G-Fell-Oxy. A sequence containing 1010 bp was isolated and found to be the promoter of CsANS, which contained many cis-acting elements related to anthocyanin biosynthetic pathway, such as the core promoter element TATA-box and light responsive element (including ACE, GT1-motif and Sp1), circadian (cis-acting regulatory element involved in circadian control). The real-time PCR analysis revealed that the gene was highly expressed under the sunshine (CK), while lowly expressed under the 75% shading. This study reveals that the expression of CsANS was regulated by light intensity.