七彩红竹中类黄酮-3-O-葡萄糖基转移酶基因的克隆及功能分析

摘要

类黄酮-3-O-葡萄糖基转移酶(Flavonoid-3-O-glucosyltransferase,3GT)是花青苷(Anthocyanins)生物合成途径中的关键酶,它主要负责将不稳定的花色素转变为稳定的花色素苷,虽然目前已经从其他植物中克隆获得类黄酮-3-O-葡萄糖基转移酶,但是对竹子中的类黄酮-3-O-葡萄糖基转移酶并不清楚.该文以产生花青素的七彩红竹(Indosasa hispida McClure cv．Rainbow)为材料,首先通过3GT 的同源比对后设计3GT基因特异引物,获得3 GT基因片段；然后运用RT-PCR 及 RACE 技术从七彩红竹茎中克隆得到完整的3 GT基因(Ih3GT).结果表明：Ih3GT 基因的 cDNA 全长序列为1730 bp,含有1个1425 bp 的开放阅读框(ORF),编码474个氨基酸；系统进化分析显示,七彩红竹3GT 与其他禾本科植物的3GT 聚类到同一个分支；该基因推断的蛋白与水稻(Oryza sativa)3GT蛋白的相似性为69％,与二穗短柄草(Brachypodium dis-tachyon)3 GT的相似性为67％；经氨基酸序列比对,推断七彩红竹3 GT含有糖基转移酶基因家族特有的结构域PSPG-box；半定量PCR的结果显示,七彩红竹3GT 基因在微红的幼茎中大量表达,而在其他组织中并不表达,说明Ih3GT具有组织表达特异性.该结果为今后深入研究七彩红竹花色苷的形成机理、鉴定 Ih3GT酶活性以及利用Ih3GT基因培育竹子新品种奠定了基础.%Flavonoid-3-O-glucosyltransferase (3GT)is the key enzyme in anthocyanins biosynthesis,which trans-forms unstable anthocyanidins into stable anthocyanins.The function of Flavonoid-3-O-glucosyltransferase in bam-boo is unclear,although many flavonoid-3-O-glucosyltransferases from other plant had been reported,so far.In-dosasa hispida McClure cv.Raln bow is of few bamboo species which can produce anthocyanins in clum.Therefore,I .hispida McClure cv.Raln bow is very important material to reveal the function of Flavonoid-3-O-glucosyltrans-ferase in bamboo and the mechanism of anthocyanins biosynthesis in bamboo.Therefore,it is first step to clone 3GT gene from I.hispida McClure cv.Raln bow.First,gene special primers of 3GT were obtalned based on the homology analysis of reported flavonoid-3-O-glucosyltransferase,and the RNA was extracted from yound red clum which pro-duce anthocyanins part in I .hispida McClure cv.Raln bow by Trizol method.And then gene fragment of I .hispida McClure cv.Ralnbow was cloned with special primers of 3GT.According to the obtalned fragment sequence,the primers were designed,which were used in rapid amplification of cDNA ends (RACE).Next,the 3′end and 5′end sequence of 3GT were obtalned by RACE,the full length gene of Ih3GT was assembled by ATG software.Finally, the full length gene of Ih3GT was cloned from I .hispida McClure cv.Ralnbow by reverse transcription-polymerase chaln reaction (RT-PCR).ORF analysis program was used to confirm open reading frame,MEGA software was used to construct phylogenetic tree.the DNAman software was used in Homology analysis,and semi RT-PCR was applied in gene expression profile.The results showed that the cDNA sequence of Ih3GT consisted of 1 425 bp open reading frame (ORF)which encodes 474 amino acid,Ih3GT and 3GT from Poaceae were grouped in same clade,the deduced protein of 3GT from I.hispida McClure cv.Raln bow shared 69% identities with 3GT of Oryza sativa and shared 67% identities with 3GT of Brachypodiumdistachyon.Homology analysis showed that deduced Ih3GT protein had a glycosyltransferase signature domaln PSPG-box.Expression profiling with semi RT-PCR analysis revealed that Ih3GT was expressed in young red culm and was not expressed in old culm,old leaf,young leaf and shoot.This im-plied that the expression of Ih3GT from I.hispida McClure cv.Raln bow showed obvious tissue specificity.This study will provide useful information to reveal mechanism of anthocyanins biosynthesis in I .hispida McClure cv. Ralnbow in future.Ih3GT gene can be transformed into Eschera coli,and heterologous expression obtaln Ih3GT pro-tein and detect the enzyme activity of Ih3GT,and it can also be transformed intoArabidopsisthalianaorOryzasati-va,the function of Ih3GT will be confirmed by heterologous expression.The obtalned Ih3GT also can been implied in bamboo breeding or other horticultural plants by genetic engineering.