VvmiR396及其靶基因VvGRF1在葡萄种子发育过程中的表达分析  

Expression analysis of VvmiR396 and its target gene VvGRF1 during grape seed development

作　　者：张晓雯 宣旭娴 白云赫 王霏 吴伟民[2] 王壮伟[2] 董天宇 张川[1] 房经贵[1] 王晨[1] ZHANG Xiaowen;XUAN Xuxian;BAI Yunhe;WANG Fei;WU Weimin;WANG Zhuangwei;DONG Tianyu;ZHANG Chuan;FANG Jinggui;WANG Chen(College of Horticulture,Nanjing Agricultural University,Nanjing 210095,China;Insititute of Polomogy,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

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

出　　处：《南京农业大学学报》2023年第5期862-874,共13页Journal of Nanjing Agricultural University

基　　金：国家重点研发计划项目(2018YFD1000106);国家自然科学基金项目(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]本文旨在鉴定葡萄miR396家族成员(VvmiR396)及其靶基因VvGRF1,探究VvmiR396-VvGRF1在种子败育/发育中的作用模式。[方法]通过miR-RACE、RLM-RACE、PPM-RACE、烟草共转化活体验证等方法鉴定VvmiR396及其靶基因,利用生物信息学分析其潜在功能与进化保守性,利用RT-qPCR方法检测VvmiR396在有核和无核品种中的时空表达差异及VvmiR396-VvGRF1相关性差异。[结果]与葡萄品种‘魏可’相比,‘京可晶’花后28 d时种子发育被严重抑制,出现败育且种子萎缩,而‘魏可’种子正常发育。对VvmiR396a、VvmiR396b、VvmiR396c、VvmiR396d的前体及其成熟体序列进行鉴定,其前体均可形成典型的茎环结构,成熟体序列均位于茎环的5′臂上。VvmiR396与烟草、番茄等物种的同源序列亲缘关系较近,而与小麦的亲缘关系较远。预测到VvmiR396的8条潜在靶基因,利用RLM-RACE、PPM-RACE技术体外鉴定到VvGRF1和VvGRF10是VvmiR396的真实靶基因,且VvmiR396对VvGRF1的裂解位点及频度为10^(th)(19/22、5/25)/11 th(2/22、2/25),对VvGRF10的裂解位点及频度为9^(th)(17/21、3/20)/11 th(2/21、1/20);进一步利用烟草共转化体系对它们的裂解作用进行活体验证,发现VvmiR396对VvGRF1的裂解作用强于VvGRF10。同源保守性分析发现:VvGRF1与胡杨、毛白杨、荷花、烟草等物种的亲缘关系较近,且不同物种的GRF1蛋白均含有QLQ、WRC两个结构域;VvMIR396(VvmiR396的前体基因)和VvGRF1的启动子上都有多种激素(生长素、赤霉素、脱落酸等)响应元件。定量结果表明:在‘京可晶’种子败育过程中,VvmiR396的表达量逐渐升高,且在败育关键期(花后42 d)表达量最高,而VvGRF1呈现相反表达模式;在有核品种‘魏可’种子发育中,VvmiR396表达量呈下降趋势,在花后42 d表达量最低,靶基因的表达模式相反,表明VvmiR396可能介导VvGRF1正调控葡萄种子败育,而负调控种子的发育。进一步相关性分析发现,‘京�[Objectives]The paper aimed to identify the members of VvmiR396 family(VvmiR396)and their target genes,and to investigate the mode of action of VvmiR396-VvGRF1 in seed abortion/development.[Methods]Identification of VvmiR396 and its target genes was performed by miR-RACE,RLM-RACE,PPM-RACE,and in vivo validation of tobacco co-transformation,and their potential functions and evolutionary conservation were analyzed,and the temporal and spatial expression profiles of VvmiR396 and their targets in seeded and non-seeded varieties were detected,and their expressional correlation was analyzed.[Results]Compared to‘Wink’grapes,the seed development of‘Jingkejing’grapes was severely inhibited at 28 days after flowering,with abortion and seed atrophy,while the former seeds developed normally.The precursors of VvmiR396a,VvmiR396b,VvmiR396c and VvmiR396d and their mature sequences were identified,all of which could form a typical stem-loop structure,and their mature sequences were all located on the 5′arm of the stem-loop.VvmiR396 had a close relationship with the homologous sequences of tobacco and tomato,while it had a farther relationship with wheat.Eight potential target genes of VvmiR396 were predicted,and VvGRF1 and VvGRF10 were identified as the true target genes of VvmiR396 in vitro using RLM-RACE and PPM-RACE techniques,and the cleavage sites and frequencies of VvmiR396 for VvGRF1 were 10 th(19/22,5/25)/11^(th)(2/22,2/25),cleavage sites and frequency for VvGRF10 were 9^(th)(17/21,3/20)/11^(th)(2/21,1/20).The cleavage roles of VvmiR396 on their target genes were further verified in vivo,and it was found that the cleavage role of VvmiR396 on VvGRF1 was stronger than that of VvGRF10.Homology conservation analysis revealed that VvGRF1 was closely related to species such as Populus euphratica,Populus tomentosa,Nelumbo nucifera,and Nicotiana tabacum,and GRF1 protein contained QLQ and WRC domains across different plant species.There were various hormone(gibberellin,auxin,abscisic acid,etc.)response elements on the p

关 键 词：葡萄 VvmiR396 GRF 假单性结实 种胚败育 

分 类 号：S663.1[农业科学—果树学]