茉莉花香气相关基因JsDXS及其启动子的克隆与表达分析

Cloning and Expression Analysis of Deoxyoxylulose-5-phosphate Synthase Gene Related to Aroma from Jasminum sambac and Isolation of Its Promoter

以双瓣茉莉花瓣为材料,采用RT-PCR和RACE技术相结合的方法,获得茉莉花香气形成限速酶即5–磷酸脱氧木酮糖合成酶(Deoxyoxylulose-5-phosphate synthase,DXS)基因(命名为JsDXS);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技术检测该基因在花瓣发育过程中的表达动态。结果表明,JsDXS全长cDNA为2 505 bp,其中ORF(Open Reading Frame)为2 145 bp,编码714个氨基酸,含有TPP-DXS、TPP-PYR-DXS-TK-like和Transketolase C等保守功能结构域;分离得到JsDXS基因上游调控序列745 bp,其含启动子核心元件TATA-box及T/GBOXATPIN2(茉莉酮酸酯相关元件)、CIACADIANLELHC(生物钟相关元件)、MYCCONSENSUSAT(低温相关元件)等与花香形成相关的重要顺式作用元件;荧光定量PCR分析表明,该基因在18:00时表达量较低,随着时间的推移逐渐上调,到夜间22:00时表达量达到最高,逐渐又出现下调的趋势,直至凌晨2:00时表达量降到最低,说明该基因的表达受生物钟的控制。

The full length cDNA sequence of Deoxyoxylulose-5-phosphate synthase gene(JsDXS)was cloned from Jasminum sambacby using RT-PCR and rapid amplification of cDNA ends(RACE)method. According to the 2 505 bp sequence,the promoter of JsDXS was isolated using Genome Walking Method. The gene expression pattern in blooming was analyzed by the real-time PCR. Gene structure analysis showed that the full length cDNA contained a 2 145 bp open reading frame(ORF),encoding a protein of 714 amino acid. There were several conserved domains,such as TPP-DXS,TPP-PYR-DXSTK-like,Transketolase C,etc. One 745 bp sequence was found to be the promoter of JsDXS,which contained many cis-acting elements, such as TATA-box, T/GBOXATPIN2( related to jasmonate signaling),MYCCONSENSUSAT(related to cold stress),CIACADIANLELHC(necessary for circadian expression). The real-time PCR analysis revealed that the gene expression at 18:00 was low and started to increase and reached a maximum at 22:00,and then started to decrease and reached a minimum at 2:00. This phenomenon perhaps illustrated that the expression of JsDXS was regulated by circadian.