VvmiR164s-VvNAC100作用模块鉴定及其在葡萄子房发育过程中响应赤霉素的表达分析  

Identification of the VvmiR164s-VvNAC100 Action Module and Analysis of Their Expressions Responsive to Exogenous GA During Grape Ovary Development

作　　者：王霏 肖迎珂 宣旭娴 张晓雯 刘菲 查紫仙 代梦瞳 王西成[2] 吴伟民[2] 房经贵[1] 王晨[1] WANG Fei;XIAO YingKe;XUAN XuXian;ZHANG XiaoWen;LIU Fei;ZHA ZiXian;DAI MengTong;WANG XiCheng;WU WeiMin;FANG JingGui;WANG Chen(School of Horticulture,Nanjing Agricultural University,Nanjing 210095;Jiangsu Academy of Agricultural Sciences,Nanjing 210014)

机构地区：[1]南京农业大学园艺学院,南京210095 [2]江苏省农业科学院,南京210014

出　　处：《中国农业科学》2023年第10期1966-1981,共16页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2018YFD1000106);国家自然科学基金面上项目(31972373);江苏省种业振兴“揭榜挂帅”项目(JBGS(2021)086);江苏省高等学校优势学科项目(PAPD)。

摘　　要：【目的】鉴定葡萄miR164s(VvmiR164a/b/c/d)及其靶基因,明确VvmiR164s及其靶基因应答外源赤霉素在葡萄单性结实过程中的调控作用。【方法】以‘魏可’葡萄(Vitis vinifera L. Wink)为试材,利用miR-RACE、PCR、RLM-RACE与PPM-RACE、qRT-PCR及生物信息学等技术,分析VvmiR164s-VvNAC100模块应答外源赤霉素在葡萄单性结实过程中的时空表达特征及其潜在功能。【结果】赤霉素在花前处理‘魏可’葡萄,可诱导其单性结实,导致葡萄的无核。克隆鉴定了VvmiR164a/b/c/d的精确序列,预测到其4条靶基因VvNAC100-1、VvNAC100-2、VvNAC098、VvNAC021,结合匹配程度与前期数据,在此重点分析靶基因VvNAC100-2,并将其命名为VvNAC100。克隆鉴定靶基因VvNAC100,验证其裂解位点,并对其开展蛋白进化、染色体定位及结构分析。VvNAC100裂解位点位于miRNA 5′端的第9位与第11位,裂解频度为17/20与11/20,定位于葡萄的Chr14上,编码363个氨基酸,含有一个NAM结构域,且其定位于细胞核。VvNAC100蛋白与其他物种氨基酸序列保守性较高,功能相似,其中与辣椒、烟草等物种亲缘关系较近。VvMIR164a/b/c/d的启动子与其靶基因VvNAC100的启动子均包含多种激素作用元件,表明其可能通过响应不同的激素来参与葡萄生长发育的调控。RT-qPCR结果显示,随着葡萄子房的发育,VvmiR164b的表达水平呈下降趋势,其靶基因VvNAC100在子房发育前期呈上升的表达趋势,具有一定的负相关,而VvmiR164a/c/d与VvNAC100表达模式相似,呈现一定的正相关;GA处理后,VvmiR164a/c/d在葡萄子房单性结实过程中的表达极显著地上升,进而也显著抑制了VvNAC100在这一时期的表达,从而促进了VvmiR164a/c/d-VvNAC100表达水平的负相关;但VvmiR164b在GA处理后表现出下降的趋势,且其与VvNAC100的表达水平呈现一定的正相关,表明GA处理增强了VvmiR164a/c/d对VvNAC100的负调控,减弱了VvmiR164b对VvNAC100的负调控作用。【结�【Objective】The aim of this study was to identify grapevine miR164s(VvmiR164a/b/c/d)and their target genes,and to elucidate the regulatory roles of VvmiR164s and their target genes during exogenous GA-induced grape parthenocarpic process.【Method】Using‘Wink’grapes(Vitis vinifera L.Wink)as the test material,miR-RACE,PCR,RLM-RACE and PPM-RACE,qRT-PCR and bioinformatics were used to analyze the spatio-temporal expression of VvmiR164s-VvNAC100 module in response to exogenous GA and its potential functions during grape parthenocarpic process.【Result】Gibberellin application on‘Wink’grapes before flowering induced parthenocarpy,leading to seedless berries.The precise sequence of VvmiR164a/b/c/d was cloned and identified,and four of its target genes were predicted,including VvNAC100-1,VvNAC100-2,VvNAC098,and VvNAC021.Combining the degree of match with the comprehensive analysis of the previous data,this work focused on the analysis of the target gene VvNAC100-2,which was named as VvNAC100.The VvNAC100 cleavage site was located at position 9 and position 11 of the 5′end of miRNA,with a cleavage frequency of 17/20 and 11/20,respectively,and was localized on Chr14,encoding 363 amino acids and containing a NAM.The VvNAC100 protein is highly conserved in amino acid sequence and functionally similar to other species,among which it is more closely related to species such as pepper and tobacco.The promoters of VvMIR164a/b/c/d and its target gene VvNAC100 both contain multiple hormone-acting elements,suggesting that it might be involved in the regulation of grape growth and development by responding to mult-hormones.The RT-qPCR results showed that the expression level of VvmiR164b tended to decrease as the grape ovary developed,while its target gene VvNAC100 showed an increasing expression trend in the early stage of ovary development with a certain negative correlation.On the other hand,VvmiR164a/c/d showed a similar expression pattern with VvNAC100 with a certain positive correlation.However,after GA treatme

关 键 词：葡萄 赤霉素 单性结实 VvmiR164s VvNAC 

分 类 号：S663.1[农业科学—果树学]