甜樱桃黄酮醇合酶基因的克隆及其表达分析  

Cloning and expression analysis of PacFLS in sweet cherry(Prunus avium L. )

作　　者：柳爱玲[1] 沈欣杰[1] 刘芸[1] 袁华招[1] 张晓明[2] 李天红[1] 

机构地区：[1]中国农业大学农学与生物技术学院果树系,北京100193 [2]北京市农林科学院林业果树研究所,北京100093

出　　处：《中国农业大学学报》2013年第2期56-63,共8页Journal of China Agricultural University

基　　金：国家自然科学基金(31171938);公益性行业(农业)科研专项经费(201003021)

摘　　要：为了解黄酮醇合酶在甜樱桃类黄酮生物合成途径中的作用,根据其他物种已知的FLScDNA保守序列设计简并引物,采用RT-PCR和RACE的技术,从甜樱桃(Prunus avium.L)嫩叶中扩增获得FLScDNA全长,命名为PaFLS(GenBank登录号:JQ289290)。该基因cDNA全长为1 077bp,开放阅读框为1 014bp,编码337个氨基酸,分子质量为38ku,等电点pI为5.58。生物信息学分析表明PaFLS属于依赖2-酮戊二酸的双加氧酶家族(2OG-FeII_Oxy),该基因所编码的氨基酸序列与苹果、梨和草莓的同源性分别为99%、97%和77%。将该基因重组到表达载体pGEX4T-1中进行原核表达,电泳检测到一条约为65ku的融合蛋白(含27ku的GST标签)。组织时空表达分析表明,PaFLS在甜樱桃的花中表达量最高,这可能和黄酮醇参与花粉萌发及利于授粉有关。The flavonol synthase gene is an important gene involved in the fiavonoid biosynthesis pathway. A full-length cDNA sequence of FLS gene, PaFLS （GenBank accessionn number：JQ289290）, was cloned from the young leaves of Prunus avium L. by using RT-PCR and RACE method. The full-length cDNA of PaFLS was 1 077 bp with an open reading frame of 1 014 bp,which encoded a putative protein of 337 amino acids with a molecular weight of about 38 kDa and an isoelectric point of 5.58. GenBank conserved domain search and function analysis revealed that PaFLS belonged to the 2-oxoglutarate iron-dependent oxygenase （2OG-Fell_Oxy） family. Sequence analysis showed that the homologies of FLS protein from P, avium with Malus domestica, Pyrus communis and Fragaria ananassa were 99% ,97% and 77%, respectively. The prokaryotic expression system of pGEX-4T-1/PaFLS was constructed and transformed into E. coil BL21. The protein expression was induced by 0.5 mmol/L IPTG. A 65 kDa fusion protein was detected by SDS-PAGE. The expression levels of PaFLS in different tissues of plants were analyzed by quantitative real-time PCR. Higher level of transcription was observed in the flowers than that in the fruits, pholems and leaves, which might be associated with pollinator attraction and pollen germination.

关 键 词：甜樱桃 黄酮醇合酶基因 基因克隆 原核表达 

分 类 号：S661.4[农业科学—果树学]