葡萄全基因组DELLA蛋白基因家族鉴定及其应答外源赤霉素调控葡萄果实发育的特征

doi:10.3864/j.issn.0578-1752.2018.16.009

摘要/Abstract

摘要： 【目的】明确DELLA蛋白基因在葡萄基因组数据库中的数量、结构和组织表达差异，探究DELLA蛋白在葡萄赤霉素（GA）信号传导及葡萄无核果实发育机理中的作用机制。【方法】基于拟南芥、水稻等植物中的DELLA蛋白基因，利用HMMER程序和NCBI的CDD程序鉴定葡萄基因组中的DELLA蛋白基因；以‘白罗莎里奥’葡萄品种为试材，采用PCR技术克隆3个DELLA蛋白基因的cDNA全长序列；通过启动子作用元件分析预测其潜在功能；利用生物信息学软件对其染色体定位、基因结构、系统进化、理化特性、亚细胞定位及蛋白互作等进行分析；利用农杆菌介导的烟草瞬时表达技术分析DELLA蛋白的亚细胞定位情况；采用qRT-PCR方法检测葡萄DELLA蛋白基因应答GA在果皮、果肉、种子（或种子区）的时空表达特征。【结果】鉴定得到3个葡萄DELLA蛋白基因，克隆并验证其精确序列，分别命名为VvGAI1（VIT_201s0011g05260）、VvRGA（VIT_214s0006g00640）及VvSLR1（VIT_211s0016g04630），其染色体定位、开放阅读框（ORF）大小、编码氨基酸数量分别为：Chr1、1 773 bp、590个；Chr14、1 710 bp、569个；Chr11、1 599 bp、532个。基因结构分析表明其DNA序列均无内含子，只有1个外显子，基因结构高度保守。进化分析显示VvGAI1与VvRGA亲缘关系较近，被聚类为一组，而VvSLR1为另一组。3个基因的启动子均含有响应赤霉素和胚乳发育相关的作用元件，表明它们可能参与响应GA信号传导和胚乳发育过程。亚细胞定位结果显示3个DELLA蛋白均定位于细胞核。qRT-PCR结果显示除果皮中VvSLR1在近成熟期具有表达高峰外，其余均在幼果期高表达，且外源GA处理均不同程度降低了3个DELLA蛋白基因在葡萄果皮、果肉和种子区的表达，尤以种子区下调水平最为显著。蛋白互作分析表明3个DELLA蛋白均为葡萄GA信号传导的核心作用元件，均可能与GIDI1和SLY1互作参与葡萄GA信号传导。【结论】葡萄基因组中含有3个DELLA蛋白基因家族成员；不同物种间DELLA蛋白结构高度保守；GA可能通过负调控这3个成员参与葡萄果皮、果肉和种子区的发育，且3个成员均可能通过应答GA信号调控葡萄无核果实的发育。

关键词: 葡萄, DELLA, 亚细胞定位, 表达, 互作, 赤霉素

Abstract: 【Objective】The objectives of this study are to identify the DELLA protein family genes from grapevine (Vitis vinifera) genome, to know the profile of DELLA protein family such as gene number, gene structure and tissue expression in grape, and to explore the mechanism of DELLA protein in gibberellic acid (GA) signaling pathway and in seedless fruit development of grapevine.【Method】DELLA protein genes in grape genome were identified by HMMER and NCBI CDD software based on DELLAgenes from Arabidopsis and rice. The full-length cDNAs were obtained by clone techniques from grapevine cv. ‘Rosario Bianco’. Cis-elements of their promoters were identified to predict their potential functions. Their chromosomal localization, gene structures, phylogenetic analysis, physicochemical properties, subcellular localizations and protein interactions were analyzed by bioinformatics analysis softwares. Subcellular localization of DELLA proteins were observed by Agrobacterium-mediated transient expression in tobacco leaf. The qRT-PCR method was used to detect the temporal and spatial expression of DELLA protein genes in the grape berry pericarp, berry flesh and seed (or seed area) induced by exogenous GA.【Result】A total of 3 DELLA proteingenes were identified from grape genome, their precise sequences were cloned and verified, named VvGAI1 (VIT_201s0011g05260), VvRGA (VIT_214s0006g00640) and VvSLR1 (VIT_211s0016g04630), respectively. Their chromosomal localization, open reading frame (ORF), number of amino acid of DELLA genes were Chr1, 1 773 bp, 590; Chr14, 1 710 bp, 569; Chr11, 1 599 bp, 532, respectively. Gene structure analysis result showed that there no intron and only one exon, and gene structures were highly conserved. Phylogenetic analysis showed that VvGAI1 was closely related to VvRGA, and were clustered into one group, with VvSLR1 was another group. The promoters of the 3 genes all contained elements that were responsible for GA and endosperm development, suggesting that they might be involved in response to GA signaling and endosperm development. Subcellular localization result showed that all 3 grape DELLA proteins were located in the nucleus. The results of qRT-PCR showed that the expression of 3 DELLA genes, except for VvSLR1 in the berry pericarp had a peak of expression at near maturity, the rest were highly expressed in young fruit stage, and exogenous GA treatment all reduced the expression of three DELLA protein genes in the grape berry pericarp, berry flesh and seed area, especially in the seed area. Protein interaction analysis showed that all 3 DELLA proteins were the core components of GA signal transduction in grape, which may interact with GIDI1 and SLY1 in GA signal transduction.【Conclusion】DELLA protein gene family in grapevine contains 3 genes. Their structures among different species are highly conserved. GA may participate in the development of berry pericarp, berry flesh and seed area through negative regulation of these three members, and all 3 members may regulate the development of grapes seedless fruit by responding to GA.

Key words: grape, DELLA, subcellular location, expression, interaction, gibberellic acid (GA)

张文颖，王晨，朱旭东，马超，王文然，冷翔鹏，郑婷，房经贵. 葡萄全基因组DELLA蛋白基因家族鉴定及其应答外源赤霉素调控葡萄果实发育的特征[J]. 中国农业科学, 2018, 51(16): 3130-3146.

ZHANG WenYing, WANG Chen, ZHU XuDong, MA Chao, WANG WenRan, LENG XiangPeng, ZHENG Ting, FANG JingGui. Genome-Wide Identification and Expression of DELLA Protein Gene Family During the Development of Grape Berry Induced by Exogenous GA[J]. Scientia Agricultura Sinica, 2018, 51(16): 3130-3146.

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