长筒石蒜花青素合成酶基因LlANS的克隆与表达分析  

Cloning and expression analysis of anthocyanidin synthase( LlANS) in Lycoris longituba

作　　者：王晰 丁文杰 李娅 刘家伟 王良桂[1] 岳远征[1] WANG Xi;DING Wenjie;LI Ya;LIU Jiawei;WANG Lianggui;YUE Yuanzheng(College of Landscape Architecture,Nanjing Forestry University,Key Laboratory of Landscape Architecture of Jiangsu Province,Nanjing 210037,China)

机构地区：[1]南京林业大学风景园林学院,江苏省风景园林重点实验室,江苏南京210037

出　　处：《河南农业大学学报》2018年第4期611-617,共7页Journal of Henan Agricultural University

基　　金：国家自然科学基金项目(31601785);江苏高校品牌专业建设工程资助项目(PPZY2015A063);南京林业大学科技创新基金项目(CX2016002).

摘　　要：以粉色系长筒石蒜(Lycoris longituba)花瓣为材料,基于前期获得的长筒石蒜转录组数据,通过序列分析结合聚合酶链式反应(Polymerase chain reaction,PCR)方法,成功克隆得到长筒石蒜Ll ANS基因的全长编码序列,并对其进行了生物信息学分析及亚细胞定位观察。结果表明,该基因开放阅读框(Open reading frame,ORF)全长为1 068 bp,编码355个氨基酸,蛋白质相对分子质量约为40.08 k D,理论等电点为5.58,属于酸性不稳定的亲水性蛋白;Ll ANS蛋白属于α-酮戊二酸依赖性双加酶家族,具有典型的花青素合成酶(Anthocyanidin synthase,ANS)蛋白功能结构域2OG-Fell-Oxy;系统进化树分析表明该基因与同属的石蒜和中国石蒜中ANS基因具有较高同源性,最高达97%;亚细胞定位观察显示Ll ANS蛋白定位于细胞核、细胞质及细胞膜中。此外,实时荧光定量分析证实Ll ANS基因在长筒石蒜粉色花花蕾期表达量较低,在盛花期的表达量最高。以上结论表明,Ll ANS作为长筒石蒜花色苷合成途径中的一个关键节点基因,对于长筒石蒜粉色花花色的形成起着极为重要的作用。In this study,based on the transcriptomic database of Lycoris longituba petals,the full-length coding sequence of LlANS gene from L.longituba was obtained by sequence analysis and polymerase chain reaction(PCR).Bioinformatics analysis and subcellular localization were also conducted.The result showed that the open reading frame(ORF)of LlANS was 1 068 bp which could encod 355 amino acids.The molecular weight of LlANS protein was about 40.08 kD and the theoretical isoelectric point was 5.58 indicating that LlANS protein belongs to theα-oxoglutarate-dependent dioxygenase family,which has the typical anthocyanidin synthase(ANS)protein functional domain 2OG-Fell-Oxy.Phylogenetic tree analysis showed that this gene had higher homology with the ANS gene in lycoris sinensis and Chinese lycoris sinensis,which could be as high as 97%.The result of subcellular localization indicated that LlANS was mainly located in nucleus,cytoplasm and cell membrane;In addition the real-time quantility reverse translation PCR(qRT-PCR)showed that the lowest expression level of this gene was in the primary blooming stage of L.longituba petals and the highest expression was in the full blooming stage.These results implied that LlANS could be a critical gene in the metabolic pathway of anthocyanin and might play an important role in the formation of pink flower.

关 键 词：长筒石蒜 花青素合成酶 基因克隆 亚细胞定位 表达分析 

分 类 号：S682.2[农业科学—观赏园艺]