红花花青素合成酶基因的克隆及表达分析

Cloning and Expression Analysis of Anthocyanidin Synthase in Safflower

根据红花转录物测序结果中得到的中间序列,采用RT-PCR和RACE方法从红花花瓣中克隆到1个ANS基因的全长cDNA,该基因全长序列1 226 bp,具有完整的开放阅读框(ORF),共1 050 bp,编码349个氨基酸。生物信息学软件分析显示,该基因编码的蛋白理论分子量约为82.27 kDa,等电点为5.09,序列里含有典型的加尾信号序列AATAA和Poly(A)。保守结构域预测表明,该基因编码的蛋白具有典型的ANS蛋白功能结构域,其保守结构域中含有铁离子及2-O-酮戊二酸结合位点。结合其他物种的ANS基因构建系统树表明,红花ANS基因与其他物种氨基酸具有一定的同源性,其中与芍药的亲缘关系最近。应用实时荧光定量PCR分析表明,ANS基因在红花的初花期和盛花期的表达量最高。

In this study, the full-length cDNA sequence of anthocyanidin synthase （ANS） gene was cloned from flowers of Carthamus tinctorius L. （safflower） by RT-PCR and RACE techniques according to the sequences of transcriptome in safflower. The full-length cDNA of the ANS was 1 226 bp and included a whole open reading frame of 1 050 bp, encoding a polypeptide of 349 amino acids. The putative protein of the gene showed predicted molecular weight of 82.27 kDa with a theoretical pI of 5.09, and this gene contains typical AATAA tail signal sequence and Poly（A）. The conserved structural domain analysis showed that it had the typical functional domains of ANS protein, containing 2-oxoglutarate and iron ion combination sites. Safflower ANS had high homology with other species according to the blasting and phylogenetic analysis, which indicated that safflower ANS was more related to ANS from Paeonia lactiflora Pall. Real-time PCR results indicated that relative expression of ANS gene was highest in early flowering period and blooming period.