桂花查尔酮异构酶Of CHI基因的克隆与表达分析

摘要

Chalcone isomerase CHI is a key enzyme in the biosynthesis pathway of anthocyanin .In order to understand the synthesis mechanism of anthocyanin in O .f ragrans ,we detected the anthocyanin content of three O .f ragrans cultivars in this study . The results showed that :(1 ) the anthocyanin content in‘Chenghongdangui’ was the highest ,while lower in ‘Jingui’ and‘Zaoyingui’ .(2) The full‐length cDNA sequenceof OfCHI(1069bp)wasobtainedbyusingRACEandRT‐PCRmethods.OfCHIencodedade‐duced protein of 248 amino acids ,with the relative molecular weight of 26 .85 kD and isoelectric point of 6 .34 .(3) Multiple sequence alignment showed that O .f ragrans had 68 .13% ,65 .86% and 63 .53% simi‐larity with Camellia nitidissima ,Punica granatum and Lonicera j aponica ,respectively .OfCHI contained the active sites of ChI protein :Thr47 ,Tyr108 ,Met115 and Ser192 .Phylogenetic tree analysis showed that OfCHI shared the same origin with other species ,and had the closest relationship with Oleaeuropaea OeCHI .(4) OfCHI expression was detected in different cultivars and tissues using qRT‐PCR .The re‐sults showed that OfCHI expression level was the highest in‘Chenghongdangui’ ,while lower in‘Jingui’ and‘Zaoyingui’ .OfCHI expression was detected in flowers ,stems and leaves of ‘Chenghongdangui’ ,‘Jingui’ and‘Zaoyingui’ ,and showed the same expression trend with the highest expression level in leav‐es .The study provides not only theoretical basis for the synthesis mechanism of anthocyanin in osman‐thus ,but also gene patent for developing new osmanthus cultivars of different flower color .%查尔酮异构酶CHI是花青素苷合成途径中的关键酶。为了解桂花花青苷的合成机理，该研究对3个桂花品种的花青苷含量进行了测定。结果显示：（1）‘橙红丹桂’花中的花青苷含量最高，‘金桂’和‘早银桂’中花青苷含量较低。（2）利用RACE和RT‐PCR方法获得了桂花查尔酮异构酶基因（OfCHI）的全长cDNA序列1069 bp ，该基因编码248个氨基酸，相对分子量为26．85 kD ，等电点为6．34。（3）多重序列比对显示，OfCHI与金花茶Cn‐CHI、忍冬LjCHI、石榴PgCHI的相似性分别为68．13％、65．86％和63．53％，氨基酸序列中含有CHI蛋白的活性位点Thr47、Tyr108、Met115以及 Ser192；系统进化树分析表明，OfCHI与其他物种起源相同，而与油橄榄OeCHI亲缘关系最近。（4）利用qRT‐PCR对不同桂花品种、不同组织中OfCHI的表达量检测结果显示，OfCHI在‘橙红丹桂’花中表达量最高，在‘金桂’和‘早银桂’中表达量较低；OfCHI在‘橙红丹桂’、‘金桂’和‘早银桂’的花、茎、叶中均有表达，且表达趋势相同，均为叶中表达量最高。该研究为揭示桂花青素苷的合成机理奠定了理论基础，并为培育不同花色的桂花新品种提供了基因专利。