多星韭AwANSs基因的克隆与表达分析  

Cloning and Expression Analysis of AwANS Genes in Allium wallichii

作　　者：彭婷 林颖 谭圆圆 饶英 黄覃 张文娥[1] 汪波[4] 田瑞丰 刘国锋 PENG Ting;LIN Ying;TAN Yuan-yuan;RAO Ying;HUANG Qin;ZHANG Wen-e;WANG Bo;TIAN Rui-feng;LIU Guo-feng(College of Agriculture,Guizhou University,Guiyang 550025;Department of Botany,Guangzhou Institute of Forestry and Landscape Architecture,Guangzhou 510405;College of Life Science&Technology,Huazhong Agricultural University,Wuhan 430070;College of Plant Science&Technology,Huazhong Agricultural University,Wuhan 430070;Human Resources Development Center of the Ministry of Agriculture and Rural Affairs/China Association of Agricultural Science Societies,Beijing 100125)

机构地区：[1]贵州大学农学院,贵阳550025 [2]广州市林业和园林科学研究院,广州510405 [3]华中农业大学生命科学技术学院,武汉430070 [4]华中农业大学植物科学技术学院,武汉430070 [5]农业农村部人力资源开发中心中国农学会,北京100125

出　　处：《生物技术通报》2025年第3期230-239,共10页Biotechnology Bulletin

基　　金：贵州省科技计划项目(黔科合基础-ZK[2022]一般095);国家自然科学基金项目(32060365);华中农业大学自主创新基金(2662022YJ019)。

摘　　要：【目的】多星韭(Allium wallichii)是极具特色的高山花卉,花青苷合成酶(ANS)是类黄酮代谢途径下游合成花青苷的关键酶,研究ANS基因在紫花和白花多星韭花色形成中发挥的作用。【方法】采用p H示差法检测多星韭紫花和白花不同发育时期总花青苷含量;利用RT-PCR技术克隆紫花和白花Aw ANS基因,并对其进行表达模式分析。【结果】紫花多星韭花朵总花青苷含量随花发育逐渐增加,在S4达到峰值,白花花瓣中均检测不到花青苷含量。从紫花中克隆到2条ANS基因序列(Aw ANS^(pa)和Aw ANS^(pb)),CDS长度均为1074 bp,编码357个氨基酸;从白花中克隆到5条ANS序列(Aw ANS^(wa)、Aw ANS^(wb)、Aw ANS^(wc)、Aw ANS^(wd)、Aw ANS^(we)),Aw ANS^(wa)和Aw ANS^(wd)CDS长度分别为1074 bp和1077 bp,分别编码357、358个氨基酸,另外3条序列出现大片段缺失导致蛋白翻译提前终止。系统进化树分析显示Aw ANSs与洋葱亲缘关系最近。RT-q PCR分析显示,紫花Aw ANSs在花瓣中表达量最高,在根、雌蕊和果实中表达量极低;Aw ANSs在紫花多星韭的表达随花发育而逐渐升高,在S5达到峰值;而在白花中几乎检测不到Aw ANSs表达。【结论】Aw ANSs的表达具有明显的时空表达特异性,在花瓣中表达量最高。与紫花相比,白花多星韭的花瓣中不积累花青素,其Aw ANSs基因在整个花发育过程中也几乎不表达,表明Aw ANSs基因对多星韭紫色花的形成起着重要作用。【Objective】Allium wallichii is a characteristic alpine flower.Anthocyanidin synthase(ANS)is a key enzyme in the anthocyanin biosynthesis,which lies downstream of flavonoid biosynthesis.This study is to investigate the role of ANS in the flower color formation of purple and white A.wallichii.【Method】pH-differential method was used to determine the total anthocyanin contents at different developmental stage of flowers.The coding sequences of AwANS genes were cloned by RT-PCR.The sequence alignments and expression patterns of AwANSs were also analyzed.【Result】The total anthocyanin contents of purple flowers increased gradually with flower development and peaked at S4,while those of white flowers could barely be measured.Two sequences of AwANS(AwANSpa and AwANSpb)were cloned from white A.wallichii flowers and both of them had the CDS sequence length of 1074 bp CDS with 357 amino acids.However,there were five sequences of AwANS(AwANS^(wa),AwANS^(wb),AwANS^(wc),AwANS^(wd),and AwANS^(we))cloned from purple A.wallichii flowers.Among them,AwANS^(wa) and AwANS^(wd) were 1074 bp and 1077 bp in CDS length,encoding two proteins with 357 and 358 amino acids,respectively.The protein translations of the remaining three sequences were terminated prematurely because of large fragment deletion.Phylogenetic analysis indicated that AwANSs were the closest to A.cepa.qRT-PCR analyses indicated that AwANSs in purple A.wallichii flowers had the highest expressions in blooming flowers and had the lowest expressions in the roots,pistils and fruits.The expression of AwANSs enhanced gradually with flower development and reached the peak at S5 in purple A.wallichii flowers.However,AwANSs expression were almost undetectable in white flowers.【Conclusion】The expressions of AwANSs have obvious spatial and temporal specificity and are the highest in flowers.Compared with purple A.wallichii,anthocyanins could not be detected and AwANSs are barely expressed throughout flower development in white flowers,suggesting AwANSs might play a

关 键 词：多星韭 花青苷合成酶 基因克隆 时空表达分析 花青苷 

分 类 号：R73[医药卫生—肿瘤]