乳白石蒜<em>LaDFR1</em>基因及启动子克隆与功能研究

doi:10.3969/j.issn.1674-7968.2023.07.006

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摘要乳白石蒜(Lycoris albiflora)新品种'梦幻少女'的渐变花色为石蒜属(Lycoris)特殊花色,二氢黄酮醇4-还原酶基因(dihydroflavonol 4-reductase, DFR)是乳白石蒜花瓣花色变化的关键酶基因。为进一步研究该基因的功能和作用,本研究利用 RT-PCR 和染色体步移技术(Genome walking)分别从乳白石蒜花瓣中克隆到LaDFR1 基因及启动子序列,对 LaDFR1 基因进行生物信息学和表达模式分析,并进一步在烟草(Nicotiana tabacum)中过表达 LaDFR1,研究其功能。结果表明,LaDFR1 cDNA 序列 OFR 长 1 161 bp,编码386 个氨基酸。LaDFR1 蛋白保守结构域具有 NADPH 和底物结合位点,属 NADB-Rossmann 超基因家族。序列比对和系统发育分析显示,乳白石蒜与百子莲(Agapanthus praecox)的 DFR 氨基酸序列一致性达到80.43%,聚为一个分支。qPCR 检测发现,LaDFR1 基因在乳白石蒜 3 个不同开花时期均有表达,开花第 1天表达量最低,随着开花时间的延长,表达量逐渐上升;LaDFR1 主要在花瓣中表达,其次是花丝,在花葶中表达最低。获得一条长 1 570 bp 的 LaDFR1 基因启动子序列,含有多个光照、脱落酸诱导、MYB 和 NAC 结合顺式作用元件,构建 LaDFR1pro: PBI121 整合载体瞬时转化烟草叶片,光照条件下较黑暗处理诱导的 GUS 活性更高,说明 LaDFR1 启动子具有光诱导活性。综上,本研究成功获得 LaDFR1 基因及其启动子序列,LaDFR1 可能参与乳白石蒜花瓣花青素苷的合成和积累,这一研究结果可为进一步研究乳白石蒜花色变化的调控及用基因工程进行花色改良提供靶基因。

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	朱熠辰
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	高燕会

关键词 ：乳白石蒜, 二氢黄酮醇4-还原酶(DFR), 基因克隆, 表达分析

Abstract：The new variety Lycoris albiflora 'Astro Girl' has gradually changing flower colors, which is a special flower color of Lycoris. Previous studies have found that the dihydroflavonol 4-reductase (DFR) genes are the key enzyme genes for the anthocyanins biosynthesis in the petals of Lycoris. In order to further study the DFR genes function, LaDFR1 and its promoter sequence were cloned by RT-PCR and Genome walking technology from petals in Lycoris chinensis×radiata 'Astro Girl' in this study. Bioinformatics and expression pattern analysis of LaDFR1 were carried out. The function of the LaDFR1 promoter was further investigated by overexpressing it in tobacco (Nicotiana tabacum) leaves. The results showed that, (1) LaDFR1 cDNA with 1 161 bp ORF was cloned, encoding 386 amino acids. The conserved domain of amino acid sequence of LaDFR1 had NADPH and substrate binding sites, which belonged to NADB Rossmann super gene family. Sequence alignment and phylogenetic analysis showed that the consistency of DFR amino acid sequence between L. chinensis×radiata 'Astro Girl' and Nelumbo nucifera was 80.43%. (2) LaDFR1 was mainly expressed in petals, followed by filaments, and the lowest expression in scape. The expression of LaDFR1 gene was the lowest in the first flowering day, and the expression increased gradually with the extension of flowering time. (3) LaDFR1 promoter sequence with 1 570 bp was obtained, which contained multiple light, abscisic acid (ABA) induction, MYB and NAC binding cis acting elements. The LaDFR1pro: PBI121 integration vector was constructed to transiently transform tobacco leaves. The GUS activity induced by light was higher than dark, which indicated that the LaDFR1 promoter had photoinduced activity. In summary, the LaDFR1 gene and its promoter sequence were successfully obtained in this study, which may be involved in the synthesis and accumulation of anthocyanins in the petals of L. chinensis×radiata 'Astro Girl'. This study can provide target genes for further study on the regulation of color changes in Lycoris albiflora and color improvement using genetic engineering.

Key words： Lycoris albiflora Dihydroflavonol 4-reductase (DFR) Gene clone Expression analysis

收稿日期: 2022-05-05

基金资助:浙江农林大学校级学生科研训练项目(2020KX0042); 浙江省“十四五”农业(花卉新品种选育)新品种选育重大科技专项(2021C02071-6); 浙江省自然科学基金(LZ22C160003); 国家自然科学基金(31670696)

通讯作者: *gaoyanhui408@126.com

引用本文:

朱熠辰, 薛惠敏, 周琪, 宗杰, 高燕会. 乳白石蒜LaDFR1基因及启动子克隆与功能研究[J]. 农业生物技术学报, 2023, 31(7): 1396-1404.
ZHU Yi-Chen,XUE Hui-Min,ZHOU Qi,ZONG Jie,GAO Yan-Hui . Cloning and Function Study of LaDFR1 Gene and Promoter from Lycoris albiflora. 农业生物技术学报, 2023, 31(7): 1396-1404.

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http://journal05.magtech.org.cn/Jwk_ny/CN/10.3969/j.issn.1674-7968.2023.07.006 或 http://journal05.magtech.org.cn/Jwk_ny/CN/Y2023/V31/I7/1396

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