葡萄miR159s靶基因的鉴定及其应答GA在果实不同组织的调控作用

doi:10.3864/j.issn.0578-1752.2019.16.011

摘要/Abstract

摘要：

【目的】鉴定葡萄miR159家族成员及其靶基因,明确葡萄miR159家族成员及其靶基因应答外源赤霉素（GA）在无核葡萄果实不同组织发育过程中的作用。【方法】以‘白罗莎里奥’葡萄（Rosario Bianco）为试材,利用miR-RACE和PCR技术克隆鉴定VvmiR159a/b/c的成熟体及前体序列;通过PsRNATarget软件预测VvmiR159s的靶基因,利用生物信息学软件对其进行系统进化及保守结构域分析;通过启动子作用元件分析预测VvmiR159s及其靶基因的潜在功能;采用RLM-RACE和PPM-RACE验证VvmiR159s对靶基因的裂解作用;利用qRT-PCR鉴定其应答外源GA在葡萄果实不同组织发育过程中的时空表达模式。【结果】从‘白罗莎里奥’葡萄果实中克隆获得VvmiR159a/b/c的成熟体序列、前体序列并折叠发夹结构。鉴定到VvmiR159s的四条靶基因（VvGAMYB、VvMYB48、VvAIX15、VvGRAS-l）,其中VvmiR159s与VvGAMYB匹配程度最高,与VvAIX15匹配程度最低。靶基因系统进化及保守结构域分析显示,4条靶基因与其他物种同源基因具有较高同源性。VvmiR159s前体基因及其靶基因启动子的激素响应元件均以赤霉素和水杨酸响应元件为主,表明其可能主要通过应答这两种激素参与调控葡萄的生长发育过程。VvmiR159s在不同组织中的表达具有时空特异性,其中VvmiR159c在果皮和果肉中的表达趋势不同,在果皮中VvmiR159a/b/c与VvAIX15的表达水平呈负相关;而在果肉中,VvmiR159a/b与VvGAMYB和VvAIX15的表达模式相反。此外,在葡萄果皮、果肉中GA处理可显著下调VvmiR159a/c的表达,但却在幼果果肉中上调VvmiR159b的表达。表明葡萄miR159家族成员在果实不同组织中通过不同模式应答GA信号参与果实发育的调控。【结论】葡萄miR159家族含有VvmiR159a/b/c 3个成员;3个成员均可切割VvGAMYB、VvMYB48、VvAIX15及VvGRAS-l 4个靶基因;VvmiR159a/b/c及其4个靶基因可能以不同的GA应答模式参与调控葡萄果皮与果肉的发育。

关键词: 葡萄, miR159, 靶基因, GA, 进化特性, 基因表达

Abstract:

【Objective】The main objective of the present research was to identify the miR159 family members and their target genes from grapevine genome, to validate the role of VvmiR159 family members and their target genes in response to exogenous GA in the development of different tissues of seedless grape berries. 【Method】The mature and precursor sequences of VvmiR159a/b/c were cloned and identified by miR-RACE and PCR techniques from grapevine cv. ‘Rosario Bianco’. The target genes of VvmiR159s were predicted by PsRNATarget software, and the phylogenetic and conserved domain analysis was performed by bioinformatics tools. The potential functions of VvmiR159s and their target genes were predicted by cis-elements analysis of their promoters. RLM-RACE and PPM-RACE verified the cleavage roles of four target genes by VvmiR159a/b/c, and the qRT-PCR method was used to detect their temporal and spatial expression patterns in different tissues of grape berry induced by exogenous GA application 【Result】The mature and precursor sequences of VvmiR159a/b/c were cloned from the ‘Rosario Bianco’ and further folded into the typical hairpin structures. Four target genes (VvGAMYB, VvMYB48, VvAIX15, and VvGRAS-1) of VvmiR159s were identified, of which VvmiR159s strongly matched with VvGAMYB the most and VvAIX15 was weak. Phylogenetic evolution and conserved domain analysis of target genes showed that the four target genes had high homology with other species. All VvmiR159s and four target genes had the main hormone related cis-elements responsive to GA and SA, indicating that they might be involved in the regulation of grape berry growth and development mainly by responding to corresponding hormones. The expression of VvmiR159s in different tissues was spatial-temporal-specific. The expression of VvmiR159c was deviated in berry pericarp and flesh, and the expression level of VvmiR159a/b/c was negatively correlated with VvAIX15 in the pericarp, however in flesh, VvmiR159a/b was contradictory to VvGAMYB and VvAIX15 expression pattern. In addition, GA treatment VvmiR159a/c was significantly down-regulated in grape pericarp and flesh, but VvmiR159b up-regulated in young fruit flesh. These results indicated that different members of the VvmiR159 family participated in the regulation of fruit development through different modes of response to GA signaling in different tissues of the fruit. 【Conclusion】The grape miR159 family contains three members of VvmiR159a/b/c; all of three members can cleave four target genes: VvGAMYB, VvMYB48, VvAIX15 and VvGRAS-l; VvmiR159a/b/c and their four target genes may participate in the regulation of grape pericarp and flesh development in different GA-responsive modes.

Key words: grape, miR159, target gene, GA, evolutionary characteristic, gene expression

张文颖, 韩旭, 朱旭东, 解振强, 纠松涛, 黄雨晴, 贾海锋, 房经贵, 王晨. 葡萄miR159s靶基因的鉴定及其应答GA在果实不同组织的调控作用[J]. 中国农业科学, 2019, 52(16): 2858-2870.

ZHANG WenYing, HAN Xu, ZHU XuDong, XIE ZhenQiang, JIU SongTao, HUANG YuQing, JIA HaiFeng, FANG JingGui, WANG Chen. Identification of the Target Genes of VvmiR159s and Their Regulation in Response to GA in Different Tissues of Grape Berry[J]. Scientia Agricultura Sinica, 2019, 52(16): 2858-2870.

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链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2019.16.011

https://www.chinaagrisci.com/CN/Y2019/V52/I16/2858

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引物名称 Primer name	引物序列 Primer Sequence	引物用途 Use
VvmiR159a-F	CCATGGAATCTTTTTCCGGACACTACAACA	VvmiR159a前体序列的扩增 Amplification of VvmiR159a precursor
VvmiR159a-R	ACTAGTATCCCACCCGGATTTTGAAATAA	VvmiR159a前体序列的扩增 Amplification of VvmiR159a precursor
VvmiR159b-F	CCATGGTGTTATTGGGTTTGGGTCATCTCA	VvmiR159b前体序列的扩增 Amplification of VvmiR159b precursor
VvmiR159b-R	ACTAGTACCCAGATTAACAATACAAAAGGTC	VvmiR159b前体序列的扩增 Amplification of VvmiR159b precursor
VvmiR159c-F	CCATGGCAGTCAGTAATGCAAGCCACA	VvmiR159c前体序列的扩增 Amplification of VvmiR159c precursor
VvmiR159c-R	ACTAGTAGCTGCAGTTTTGTTGGGTTT	VvmiR159c前体序列的扩增 Amplification of VvmiR159c precursor
Actin-F	TACAATTCCATCATGAAGTGTGATG	Actin 内参引物 Actin primer
Actin-R	TTAGAAGCACTTCCTGTGAACAATG	Actin 内参引物 Actin primer
VvmiR159a qRT-F	CTTCTCCGAGGTCATGCACA	VvmiR159a 定量RT-PCR引物 Real-time PCR of VvmiR159a
VvmiR159a qRT-R	ATGGGAGCTCCTTTACGCTC	VvmiR159a 定量RT-PCR引物 Real-time PCR of VvmiR159a
VvmiR159b qRT-F	TTGCCATTCCAACACTCATC	VvmiR159b 定量RT-PCR引物 Real-time PCR of VvmiR159b
VvmiR159b qRT-R	TTTCTGGCCTTGGAGTGAAG	VvmiR159b 定量RT-PCR引物 Real-time PCR of VvmiR159b
VvmiR159c qRT-F	GCTCCCTTCAATCCAAACAA	VvmiR159c 定量RT-PCR引物 Real-time PCR of VvmiR159c
VvmiR159c qRT-R	TTATGGATCCCACAGCCCTA	VvmiR159c 定量RT-PCR引物 Real-time PCR of VvmiR159c
VvGAMYB qRT-F	GAAATGGAATCCCCCTGTTT	VvGAMYB 定量RT-PCR引物 Real-time PCR of VvGAMYB
VvGAMYB qRT-R	TCCTTGTTGGTAGGGTCAGG	VvGAMYB 定量RT-PCR引物 Real-time PCR of VvGAMYB
VvMYB48 qRT-F	TTCTCAGGAACTGGGGAATG	VvMYB48 定量RT-PCR引物 Real-time PCR of VvMYB48
VvMYB48 qRT-R	AAAGAAGCTGTCCGGAGTCA	VvMYB48 定量RT-PCR引物 Real-time PCR of VvMYB48
VvAIX15 qRT-F	GTGCCCAAAGGCTATGTTCC	VvAIX15 定量RT-PCR引物 Real-time PCR of VvAIX15
VvAIX15 qRT-R	TCTTCCCTGCATGGGATTGT	VvAIX15 定量RT-PCR引物 Real-time PCR of VvAIX15
VvGRAS-l qRT-F	TTGGAGGCCTTAGAAGGGGA	VvGRAS-l 定量RT-PCR引物 Real-time PCR of VvGRAS-l
VvGRAS-l qRT-R	ACAGTGAGCTTTAGGCGAGG	VvGRAS-l 定量RT-PCR引物 Real-time PCR of VvGRAS-l

基因名称 Gene name	靶基因编号 Target Acc.	靶区 Target regions	靶片段 Target aligned fragment	作用方式 Interaction mode	注释 Annotation
VvGAMYB	VIT_213s0067g01630	CDS:956-976	TGGAGCTCCCTTCACTCCAAT	Cleavage	GAMYB转录因子 Transcription factor GAMYB
VvMYB48	VIT_215s0021g02040	CDS:97-117	TGGAGCTCCATTCGCTCCAAA	Cleavage	MYB48转录因子 Transcription factor MYB48
VvAIX15	VIT_203s0038g01150	CDS:152-172	TGAAGCACCCTTCATTCCAAA	Cleavage	生长素诱导蛋白X15 Auxin-induced protein X15
VvGRAS-l	VIT_200s0226g00070	CDS:6-26	CAGAACTCTCTTCACTCCAAA	Cleavage	GRAS家族转录因子 GRAS family transcription factor