越橘VcANS基因的克隆及表达分析

【目的】从越橘果实中克隆花色素合成酶（ANS）基因cDNA，为研究越橘花色素苷合成的分子机制奠定基础。【方法】以越橘（Vaccinium spp.）品种“北陆”（V.corymbosum L.）为试材，并以Illumina测序文库（NCBI登录号：SRA046311）中差异表达的Unigene 38125片段为基础，利用cDNA末端快速扩增（RACE）技术获得花色素合成酶基因全长cDNA，利用DNAMAN和MEGA软件进行多序列比对和系统进化分析，探讨ANS基因在不同组织和器官中的相对表达量及其与对应花色素苷含量的关系。【结果】成功克隆了越橘花色素合成酶基因序列，命名为VcANS，GenBank登录号为JN654701。序列分析结果表明，VcANS全长1 424 bp，包含93 bp的5′非编码区、248 bp的3′非编码区和1个长度为1 083 bp编码360个氨基酸的开放阅读框，该基因编码的蛋白具有ANS家族普遍存在的2酮戊二酸和Fe²⁺依赖的氧化酶超家族的保守结构域。序列比对和系统进化分析表明，VcANS与杜鹃花科植物亲缘关系最近。在越橘果实发育的不同阶段，VcANS相对表达量的变化与花色素苷含量的变化趋势具有一致性。【结论】获得了越橘花色素合成酶基因全长，推测其对越橘花色素苷的形成起调控作用。

Molecular cloning and expression analysis of VcANS gene in blueberry

【Objective】 In this study,a full-length cDNA for the anthocyanin synthase(ANS) gene from blueberry fruit was cloned,aiming to clarify the molecular mechanism of anthocyanin biosynthesis.【Method】 Using blueberry(Vaccinium spp.) cultivar ’Northland’(V.corymbosum L.) as material,a full-length cDNA sequence of ANS was obtained from skin of the blueberry using RT-PCR and RACE based on the differentially expressed unigene 38125 from the Illumina sequencing library(NCBI accession No.SRA046311).Using DNAMAN and MEGA software for multiple sequence alignment and phylogenetic analysis,the relationship between the relative expression of VcANS and anthocyanin content was investigated in different tissues and organs.【Result】 The cDNA sequence was named VcANS(GenBank accession No.JN654701).Sequence analyses indicated that VcANS was 1 424 bp in full length,containing a 5′-untranslated region(5′-UTR) of 93 bp,a 3′-UTR of 248 bp,and an opening reading frame(ORF) of 1 083 bp encoding a protein of 360 amino acids,which possessed the conserved structural domain of the 2-oxoglutarate and Fe2+-dependent oxygenase superfamily.Sequence alignment and phylogenetic tree analyses showed that VcANS had the closest phylogenetic relationship with the Ericaceae.The relative expression of VcANS had significant consistency with the content of anthocyanins.【Conclusion】 A full-length anthocyanin synthase gene was obtained by using RT-PCR and RACE techniques,which will lay the molecular biology foundation for further research on the molecular mechanism of anthocyanin biosynthesis and new varieties breeding of blueberry.