多星韭AwANSs基因的克隆与表达分析

doi:10.13560/j.cnki.biotech.bull.1985.2024-1004

生物技术通报 ›› 2025, Vol. 41 ›› Issue (3): 230-239.doi: 10.13560/j.cnki.biotech.bull.1985.2024-1004

• 研究报告 • 上一篇

多星韭AwANSs基因的克隆与表达分析

彭婷¹^,³(), 林颖², 谭圆圆¹, 饶英¹, 黄覃³, 张文娥¹, 汪波⁴, 田瑞丰⁵, 刘国锋²()

^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

收稿日期:2024-10-15 出版日期:2025-03-26 发布日期:2025-03-20
通讯作者: 刘国锋，男，博士，研究方向：园林植物遗传育种；E-mail: gfliu@mail.hzau.edu.cn
作者简介:彭婷，女，博士，研究方向：园林植物遗传育种；E-mail: tpeng@mail.hzau.edu.cn
基金资助:
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)

Cloning and Expression Analysis of AwANS Genes in Allium wallichii

PENG Ting¹^,³(), LIN Ying², TAN Yuan-yuan¹, RAO Ying¹, HUANG Qin³, ZHANG Wen-e¹, WANG Bo⁴, TIAN Rui-feng⁵, LIU Guo-feng²()

^1.College of Agriculture, Guizhou University, Guiyang 550025
^2.Department of Botany, Guangzhou Institute of Forestry and Landscape Architecture, Guangzhou 510405
^3.College of Life Science & Technology, Huazhong Agricultural University, Wuhan 430070
^4.College of Plant Science & Technology, Huazhong Agricultural University, Wuhan 430070
^5.Human Resources Development Center of the Ministry of Agriculture and Rural Affairs/China Association of Agricultural Science Societies, Beijing 100125

Received:2024-10-15 Published:2025-03-26 Online:2025-03-20

摘要/Abstract

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pH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AwANS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（AwANS^pa 和AwANS^pb ），CDS长度均为1 074 bp，编码357个氨基酸；从白花中克隆到5条ANS序列（AwANS^wa 、AwANS^wb 、AwANS^wc 、AwANS^wd 、AwANS^we ），AwANS^wa 和AwANS^wd CDS长度分别为1 074 bp和1 077 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AwANSs与洋葱亲缘关系最近。RT-qPCR分析显示，紫花AwANSs在花瓣中表达量最高，在根、雌蕊和果实中表达量极低；AwANSs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AwANSs表达。结论 AwANSs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AwANSs基因在整个花发育过程中也几乎不表达，表明AwANSs基因对多星韭紫色花的形成起着重要作用。

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

Abstract:

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 (AwANS^pa and AwANS^pb ) were cloned from white A. wallichii flowers and both of them had the CDS sequence length of 1 074 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 1 074 bp and 1 077 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 key role in the flower color formation of purple A. wallichii.

Key words: Allium wallichii, anthocyanidin synthase, gene cloning, spatio-temporal expression analyses, anthocyanins

彭婷, 林颖, 谭圆圆, 饶英, 黄覃, 张文娥, 汪波, 田瑞丰, 刘国锋. 多星韭AwANSs基因的克隆与表达分析[J]. 生物技术通报, 2025, 41(3): 230-239.

PENG Ting, LIN Ying, TAN Yuan-yuan, RAO Ying, HUANG Qin, ZHANG Wen-e, WANG Bo, TIAN Rui-feng, LIU Guo-feng. Cloning and Expression Analysis of AwANS Genes in Allium wallichii[J]. Biotechnology Bulletin, 2025, 41(3): 230-239.

图/表 7

图1 紫花和白花多星韭花发育的5个阶段

Fig. 1 Five flower developmental stages of purple and white Allium wallichii

表1 本研究所用引物

Table 1 Primer sequences used in this study

引物名称 Primer name	序列 Primer sequence（5′-3′）	用途 Usage
AwANSs-Fw	AACTACAACATTACCTACAAACACAA	cDNA克隆
AwANSs-Rev	TGTAGACGAACCAGGCTCAA	cDNA克隆
AwANSs-qFw	TCACACCACCACCTGCAAGAGT	RT-qPCR
AwANSs-qRev	CGGATGAGCGAAGGTCGGTTATTG	RT-qPCR
AwTUB2-qFw	GAAAATCCGAGAAGAATACCCAGAC	RT-qPCR
AwTUB2-qRev	TGTCGTAAAGGGCTTCGTTGTC	RT-qPCR

图2 多星韭不同花发育时期的总花青苷含量数据结果为3次生物学重复的平均值±标准误，采用Duncan多重检验，不同字母表示差异显著性（P<0.05），下同。“nd”表示“未检测到”

Fig. 2 Total anthocyanin contents at different developmental stages of flowers in A. wallichiiData are as mean±SE of three independent biological replicates. According to the Duncan multiple test, the different letters above each column indicate significant differences at 0.05 level. The same below. “nd” indicates that the total anthocyanin content is not detected

图3 AwANSs基因扩增M代表分子量标记，P1代表紫花多星韭，W1、W2代表白花多星韭

Fig. 3 Amplification of AwANSsM refers to marker. P1 indicates purple A. wallichii. W1 and W2 indicate white A. wallichii

图4 多星韭（A. wallichii）与其他物种ANS序列比对同源比对的各序列为水稻OsANS（CAA69252.1）、拟南芥AtANS（AT4G22880）、郁金香TgANS（USH59355.1）、洋葱AcANSL（ABM66367.1）。黑色线条标记为2-酮戊二酸双加氧酶家族保守结构域，*和#分别表示亚铁离子结合位点氨基酸和2-酮戊二酸结合位点氨基酸

Fig. 4 Sequence alignment of ANSs from A. wallichii and other speciesAwANSs were aligned with other ANSs from Oryza sativa (OsANS: CAA69252.1), Arabidopsis thaliana (AtANS: AT4G22880), Tulipa gesneriana (TgANS: USH59355.1), and Allium cepa (AcANSL: ABM66367.1). Conserved domain 2OG-Fe Ⅱ_Oxy is indicated by the solid line. Conserved residues that are required for ferrous-iron coordination and 2-oxoglutarate binding are marked by * and #, respectively

图5 多星韭AwANSs与其他物种ANS的系统进化树Nt：烟草；Ph：矮牵牛；Sl：番茄；Dc：香石竹；Sli：滨雪轮；Tc：可可；Ao：药葵；Md：苹果；Fv：野草莓；Rc：中国月季；Mi：紫罗兰；At：拟南芥；Lc：垂花百合；Tg：郁金香；Is：溪荪；Os：水稻；Zm：玉米；Lch：中国石蒜；Ac：洋葱；Aw：多星韭

Fig. 5 Phylogenetic analysis of AwANSs from A. wallichii and ANS from other speciesNt: Nicotiana tabacum; Ph: Petunia×hybrida; Sly: Solanum lycopersicum; Dc: Dianthus caryophyllus; Sli: Silene littorea; Tc: Theobroma cacao; Ao: Althaea officinalis; Md: Malus domestica; Fv: Fragaria vesca subsp. Vesca; Rc: Rosa chinensis; Mi: Matthiola incana; At: Arabidopsis thaliana; Lc: Lilium cernuum; Tg: Tulipa gesneriana; Is: Iris sanguinea; Os: Oryza sativa; Zm: Zea mays; Lch: Lycoris chinensis; Ac: Allium cepa; Aw: Allium wallichii

图6 AwANSs基因的时空表达分析

Fig. 6 Spatio-temporal expression analysis of AwANSs

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多星韭AwANSs基因的克隆与表达分析

Cloning and Expression Analysis of AwANS Genes in Allium wallichii

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