桑树花青素合酶基因的克隆与信息学分析

Molecular Cloning and Information Analysis of ANS Genes Encoding Anthocyanin Synthases from Mulberry(Morus alba)

花青素合酶ANS是花青素合成过程中的一个关键限速酶。本文利用同源克隆、RT-PCR从桑椹中克隆出花青素合酶(MaANS)基因,并进行生物信息学分析和组织特异性表达分析。通过染色体步移法获得的MaANS基因的5′端和3′端,获得基因全长1535bp,由2个外显子和1个内含子组成,包含完整CDS区域为1077bp,编码358个氨基酸,与来源于草莓、甘薯、苹果、沙梨的ANS基因同源性均达到80%以上。MaANS编码的蛋白质属于2-酮戊二酸双加氧酶家族。MaANS在进化树中的位置与草莓最近,与苹果、沙梨次之。组织表达分析表明,MaANS基因在幼叶和成熟果实中高水平表达,表明该基因的表达具有组织特异性,为研究桑树果色的形成原因和表达调控奠定了基础。

Anthocyanidin synthase （ANS, leucoanthocyanidin oxygenase） is one of the critical enzymes in the biosynthesis of the anthocyanin. Anthocyanidin synthase gene fragment （designated as MaANS） was isolated from mulberry fruit （Moru alba） by RT-PCR based on homology cloning and genome walking technology. MaANS with the 5＇ and 3＇ was cloned by genome-walking. The full-length genomic sequence of MaANS is 1 535 bp, which consists of two exons and one intron. The coding region length was 1 077 bp, and their deduced protein consisted of 358 amino acid residues. Multiple alignments revealed that the nucleic acid of MaANS shared above 80% identity with that of Fragaria x ananassa, Ipomoea batatas, Malus domestica, and Pyrus pyrifolia. Structural analysis showed that the MaANS protein might belong to the 2 OG and Fe（II）-dependent oxygenase family. Phyloge- netic tree analysis revealed that MaANS was the most close with Fragaria x ananassa, then Malus domestica and Pyrus pyrifolia. Reverse transcription-PCR （RT-PCR） analyses of MaANS transcripts showed that it was abundantly expressed in the young leaves and ripened fruit. All research made an essential foundation for pathway and regulation of gene expression in anthocyanin biosynthesis in mulberry fruit.