梅PmARF17克隆及其在花发育中与内源激素的调控模式

doi:10.3864/j.issn.0578-1752.2021.13.013

摘要/Abstract

摘要：

【目的】分析梅PmARF17的生物学功能,探究梅花发育进程中其表达丰度与内源激素动态变化的关系,为梅花发育的调控研究提供依据。【方法】以梅品种‘大嵌蒂’为试材,克隆PmARF17,利用生物信息学软件分析基因结构、系统进化及其与其他物种同源蛋白的差异;亚细胞定位确定PmARF17蛋白在细胞中作用的部位;以梅品种‘大嵌蒂’和‘龙眼’不同发育阶段的花芽、叶芽、花器官为试材,利用qRT-PCR检测PmARF17时空表达模式,通过UPLC法测定IAA、GA₃、ABA、ZT含量的动态变化,并与PmARF17的表达进行相关性分析;克隆PmARF17启动子,分析启动子的顺式作用元件,利用瞬时表达解析PmARF17与GA₃的调控模式。【结果】从梅品种‘大嵌蒂’中克隆得到PmARF17,系统进化树分析表明PmARF17蛋白与其他植物的ARF蛋白序列高度同源;亚细胞定位表明其作用于细胞核和细胞膜上;qRT-PCR表达和内源激素含量的相关性分析表明,PmARF17的表达与IAA含量的变化趋势没有明显的相关性。PmARF17在雌蕊完好花芽中的表达水平相对不完全花芽显著上调,而GA₃含量与PmARF17的表达趋势一致。ABA和ZT含量总体上与PmARF17的表达呈相反的趋势,表明两者可能抑制PmARF17的表达。PmARF17启动子含有GA顺式元件,且具有启动活性和组织表达特异性,在花瓣、雄蕊及根部特异表达。【结论】 PmARF17可能是梅花发育的正调控基因,促进梅雌蕊的正常发育。PmARF17的表达可能受到GA₃的正调控,其可能通过作用于雄蕊和花瓣,进而影响梅的雌蕊发育进程。

关键词: 梅, 生长素响应因子, 雌蕊发育, 基因表达, 内源激素, 瞬时表达

Abstract:

【Objective】The purposes of this study were to analyze the biological functions of PmARF17gene and to explore the regulation pattern between the expression of PmARF17 and plant endogenous hormones, which could provide the basis for studying the regulatory mechanism of pistil abortion inPrunus mume. 【Method】 PmARF17 gene was isolated from the cultivar Daqiandi of Prunus mume, and the gene structure, phylogeny and homology with other species were analyzed by bioinformatics software. Subcellular localization determined the position of PmARF17 protein in the cell. qRT-PCR was used to detect the spatiotemporal expression pattern of PmARF17in different stages of flower buds, leaf buds, flower buds with different pistil morphology, and different mature floral organs of the varieties Daqiandi and Longyan. The contents of IAA, GA₃, ABA and ZT in flower buds, leaf buds, pistil morphology flower buds and different mature flower organs of Daqiandi and Longyan were determined by UPLC method, and the correlation analysis was conducted with the qRT-PCR expression ofPmARF17.Cloning, element analysis and transient expression of PmARF17 promoter were performed to study the regulation pattern between PmARF17 and GA₃. 【Result】Phylogenetic tree analysis showed PmARF17 protein sequence was highly conserved with ARF protein of other plants. Subcellular localization experiment showed PmARF17 protein was located on the nucleus and cell membrane. The correlation analysis between qRT-PCR expression and endogenous hormone content showed the expression of PmARF17 had no obvious similarity with the change trend of IAA content in different flower samples. The expression of PmARF17 in complete pistil was significantly up-regulated compared to incomplete pistil, and GA₃ content was consistent with the expression trend ofPmARF17. The content of ABA and ZT in different flower samples and the expression of PmARF17 showed an overall opposite trend, indicating that they might inhibit the expression of PmARF17. The PmARF17promoter contained GAcis-element and had promoter activity and tissue expression specificity, specifically expressed in petals, stamens and roots. 【Conclusion】PmARF17 might be a positive regulation gene of flower development, and it might promote pistil development of Prunus mume. The expression of PmARF17 might be positively regulated by GA₃, which affected the pistil development of Prunus mume by acting on stamens and petals.

Key words: Prunus mume, auxin response factor, pistil development, gene expression, endogenous hormones, transient expression

李艳林,SHAHID Iqbal,侍婷,宋娟,倪照君,高志红. 梅PmARF17克隆及其在花发育中与内源激素的调控模式[J]. 中国农业科学, 2021, 54(13): 2843-2857.

LI YanLin,SHAHID Iqbal,SHI Ting,SONG Juan,NI ZhaoJun,GAO ZhiHong. Isolation of PmARF17 and Its Regulation Pattern of Endogenous Hormones During Flower Development in Prunus mume[J]. Scientia Agricultura Sinica, 2021, 54(13): 2843-2857.

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引物名称Primer name	引物序列 Primer Sequence (5′-3′)	引物用途 Primer use
PmARF17gene-F	GCAGACATGAAACCTCCTCCTATGC	PmARF17基因扩增 Amplification of PmARF17 gene
PmARF17gene-R	CAAGCCATTGTATCATTGCTATCC	PmARF17基因扩增 Amplification of PmARF17 gene
PmARF17 GFP-F	GGAAGATCTATGGGGGGTCTAATCGATC	亚细胞定位 Subcellular localization
PmARF17 GFP-R	GGAAGATCTTCCTTGTAAGCAGCTTTCC	亚细胞定位 Subcellular localization
PmARF17qPCR-F	TTAGTGGCAGTCAGGATG	实时荧光定量PCR qRT-PCR
PmARF17qPCR-R	GCAGTTGAGGTTGAGTTG	实时荧光定量PCR qRT-PCR
Actin-F	TGAAGCATACACCTATGATGATGAAG	内参引物 Actin primer
Actin-R	CTTTGACAGCACCAGTAGATTCC	内参引物 Actin primer
PmARF17 promoter-F	GAGATGCCTAACACTCTAAAGTC	PmARF17启动子扩增 Amplification of PmARF17promoter
PmARF17 promoter-R	CCTCCGTTGCACTGTTCAGATCG	PmARF17启动子扩增 Amplification of PmARF17promoter
Promoter-ApaI-F	GGGGCCCGAGATGCCTAACACTCTAAAGTC	瞬时表达试验 Transient expression
Promoter-SmaI-R	CCCCCGGGGTGGAAATAAACAAAAATTGTA	瞬时表达试验 Transient expression