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

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。

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

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 PsCHS was separated by genome walking strategy using primer pair designed byPsCHS sequence.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 PsCHS had higher expression level at the earlier stage of development of the fruit,especially 40 d after blossom,then decreased to a lower level at the maturing stage. PsCHS seperated 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.