油果实中查尔酮合成酶基因PsCHS的克隆表达分析及其启动子的分离

Cloning,Expression Analysis and Promoter Isolation of Chalcone-synthase Gene from Fruits of Nane,Prunus salicina Lindl.var.cordata

从成熟果实均一化全长cDNA文库中分离了编码CHS基因的全长cDNA序列,命名为PsCHS,根据其序列设计引物,采用Genome Walking方法从基因组DNA中分离获得PsCHS基因上游的调控序列,命名为PsCHSp,PsCHS基因全长1 442bp,其中ORF 1 176bp,编码392个氨基酸;采用APA-Walking技术,获得该基因的5ˊ端调控区,经在线软件预测,启动子序列含有典型的结构特征元件TATA-box和CAAT-box,还包含光响应元件、厌氧诱导元件、胚乳表达相关元件、MYB结合位点以及激素响应元件;RT-PCR结果显示,PsCHS基因在果实发育的前期表达量较高,花后40d表达量最高,随后开始下降,果实成熟期表达量较低。分离获得的PsCHS基因属于查尔酮合成酶基因家族成员之一,查尔酮合成酶(Chalcone synthase,CHS,EC 2.3.1.74)是类黄酮合成途径中的一个重要酶,该基因可能对类黄酮的合成起到调控作用。

Chalcone-synthase（CHS,EC 2.3.1.74）is an important enzyme involved inflavonoids synthesis pathway in plants.This study aimed to investigate the gene structure and expression profile of CHS gene in the fruits of nane（Prunus salicina Lindl.var.cordata）.A full-length cDNA sequence harboring a CHS gene,named PsCHS,was successfully separated from a normalized full-length cDNA library of matured nane fruits.The up-stream promoter sequence of PsCHSwas separated by genome walking strategy using primer pair designed byPsCHSsequence.The length of PsCHS was 1 442 bp with ORF of 1 176 bp and deduced amino acid of 392 aa.From the prediction by an online software,the promoter sequence of PsCHS have typical structure element TATA-box and CAAT-box,photon-response element,anaerote-induced element,endosperm-related element, MYB-bingding element and hormone-response element,etc.RT-PCR indicated that PsCHShad higher expression level at the earlier stage of development of the fruit,especially 40 dafter blossom,then decreased to a lower level at the maturing stage.PsCHSseperated in this study was a member of the CHS gene family.Since CHS is a key enzyme involved in flavonoid biosynthesis pathway,PsCHS might act with a regulatory role in biosynthesis of flavonoids.