摘要
【目的】miR172是植物生长发育的重要调节因子,阐释vvi-miR172s及其靶基因应答赤霉素在葡萄果实不同组织发育过程中的作用,从miRNA角度认识GA调控葡萄果实发育的作用机制。【方法】以‘白罗莎里奥’葡萄为材料,以miR-RACE和PCR技术克隆vvi-miR172a/b/c/d的成熟体和前体序列,由psRNA Target软件预测vvi-miR172s的靶基因;利用RLM-RACE技术验证vvi-miR172s剪切靶基因的裂解作用及其作用位点;采用生物信息学软件对靶基因、靶蛋白进行系统进化、序列结构分析及亚细胞定位预测;通过在线软件PLANTCARE进行启动子作用元件分析;通过qRT-PCR和芯片数据分析vvi-miR172s和靶基因在不同器官、不同发育阶段的基因表达图谱,以及应答GA3在果实不同组织中的时空表达模式。【结果】克隆鉴定了vvi-miR172a/b/c/d的成熟体和前体序列,预测到VvAP2、VvRAP2-1、VvRAP2-2和VvRAP2-3四个靶基因,验证到裂解产物及其裂解位点,证明它们为vvi-miR172s的真实靶基因。序列结构分析显示,VvAP2、VvRAP2-1、VvRAP2-2、VvRAP2-3均含有10个外显子和9个内含子,其motif元件的种类和数量相似,均含有两个排列顺序相近的AP2蛋白结构域,表明其结构和功能具有一定的保守性;且VvAP2、VvRAP2-1和VvRAP2-2与杨树的亲缘关系较近,VvRAP2-3与大豆具有较高的同源性;靶基因蛋白二级结构均为α-螺旋,可进一步折叠为稳定的三级结构;4个靶蛋白亚细胞定位主要位于细胞核内。启动子作用元件分析发现vvi-miR172c和VvRAP2-1中含有赤霉素响应元件,表明它们可能响应赤霉素调控葡萄生长发育;芯片数据分析显示vvi-miR172c和靶基因的表达模式具有组织或器官特异性,且它们之间呈现一定的负相关,表明它们之间存在负调控作用;RT-qPCR结果显示,随着葡萄果实的发育,果皮中vvi-miR172a/b/d呈下降的表达趋势,而靶基因VvRAP2-1的表达模式相反,表明vvi-miR172a/b/d对VvRAP2-1负调�
【Objective】 miR172 is an important regulator of plant growth and development. The purpose of this research was to clarify the roles and modes response to gibberellin(GA) of the miR172 family and its target genes in development of different tissues of grape berry. 【Method】The mature and precursor sequences of vvi-miR172 a/b/c/d were cloned and identified by miR-RACE and PCR techniques from grapevine cv. Rosario Bianco. The target genes of vvi-miR172 s were predicted by psRNA Target software, and the phylogenetic, structure analysis and subcellular localization were performed by bioinformatics tools. RLM-RACE verified the cleavage roles of four target genes by vvi-miR172 s. The cis-elements analysis of their promoters was predicted by PLANTCARE software. The qRT-PCR method was used to detect the temporal and spatial expression patterns of vvi-miR172 s and target genes in different tissues of grape berry induced by exogenous GA3 application.【Result】The mature and precursor sequences of vvi-miR172 a/b/c/d were cloned, and four target genes(VvAP2, VvRAP2-1, VvRAP2-2 and VvRAP2-3) were identified from grape genome. The cleavage sites of vvi-miR172 s on target genes were detected by RLM-RACE, which proved that VvAP2, VvRAP2-1, VvRAP2-2 and VvRAP2-3 were the true target genes. Gene structure analysis result showed that all target genes contained 10 exons, 9 introns and 2 AP2 domains. The type and number of motif elements were similar, indicating that gene structures were highly conserved. Phylogenetic analysis showed that VvAP2, VvRAP2-1, and VvRAP2-2 were closer to poplar, and Vv RAP2-3 had high homology with soybean. The secondary structure prediction of target proteins indicated that they existed in the form of alpha-helix and further folded into the stable tertiary structure. Subcellular localization results showed that all target proteins were mainly located in the nucleus. Both vvi-miR172 c and VvRAP2-1 had the hormone related cis-elements response to GA3, suggesting that they might be involved in the r
作者
宣旭娴
盛子璐
解振强
黄雨晴
巩培杰
张川
郑婷
王晨
房经贵
XUAN XuXian;SHENG ZiLu;XIE ZhenQiang;HUANG YuQing;GONG PeiJie;ZHANG Chuan;ZHENG Ting;WANG Chen;FANG JingGui(College of Horticulture,Nanjing Agricultural University,Nanjing 210095;Jiangsu Vocational College of Agriculture and Forestry,Jurong 212499,Jiangsu)
出处
《中国农业科学》
CAS
CSCD
北大核心
2021年第6期1199-1217,共19页
Scientia Agricultura Sinica
基金
国家重点研发计划(2018YFD1000106)
江苏省自然科学基金面上项目(BK20181318)
国家自然科学基金面上项目(31972373)
大学生国家创新创业计划(S20190012)。
