马缨杜鹃查尔酮合酶的基因克隆及表达分析

Cloning and expression analysis of chalcone synthase gene from Rhododendron delavayi

关键词:  花青素苷, 马缨杜鹃, 查尔酮合酶, 酶活检测, 遗传转化

Key words:  anthocyanin, Rhododendron delavayi, chalcone synthase, enzyme assay, genetic transformation

摘要:

为探究查尔酮合酶(CHS)在马缨杜鹃(Rhododendron delavayi)花色形成中的作用,该研究以马缨杜鹃为材料,克隆了RdCHS2编码区全长,并对其组织表达情况、体外催化活性及体内功能进行了解析。结果表明:(1)RdCHS2的编码序列(CDS)全长为1 170 bp,编码389个氨基酸,与其他植物CHS归为一类,具有CHS典型的保守基序。(2)基因表达分析显示,RdCHS2在花葶中表达最高,在雄蕊中表达最低,随开花进程的推进其表达量呈逐渐上升趋势。(3)酶活检测显示,RdCHS2能够分别催化丙二酰辅酶A与对香豆酰辅酶A生成柚皮素查尔酮,以及催化丙二酰辅酶A与肉桂酰辅酶A生成松属素查尔酮。(4)拟南芥tt4突变体互补实验显示,RdCHS2可成功恢复拟南芥tt4突变体子叶与下胚轴中花青素苷的合成。该研究证实,RdCHS2具有典型CHS活性,可参与花青素苷的合成,为今后进一步明确该基因的具体功能奠定了基础。

Abstract:

To elucidate the role of chalcone synthase(CHS)in flower color formation of Rhododendron delavayi, the full-length coding region of RdCHS2 was cloned from R. delavayi, and its expression profile, catalytic activity in vitro and biological function in vivo were analyzed. The results were as follows:(1)The full-length coding sequence(CDS)of RdCHS2 was 1 170 bp, encoding 389 amino acids. Phylogenetic analysis showed that RdCHS2 was grouped into the same clade with CHS from other plants and had typical conserved motifs of CHS.(2)Gene expression analysis showed that the expression of RdCHS2 was the highest in scapes and the lowest in stamens, and its expression increased gradually as flowering progressed.(3)Enzymatic assays confirmed that RdCHS2 catalyzed the synthesis of naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA, as well as catalyzed the synthesis of pinocembrin chalcone from malonyl-CoA and cinnamoyl-CoA.(4)Complementation experiments in Arabidopsis thaliana tt4 mutant demonstrated that RdCHS2 successfully restored anthocyanin biosynthesis in cotyledons and hypocotyls of A. thaliana tt4 mutant. These results show that RdCHS2 exhibits typical CHS activity and participates in the biosynthesis of anthocyanin, which lays a foundation for further clarifying the specific function of this gene.