vvi-miR172s及其靶基因响应赤霉素调控葡萄果实发育的作用分析

[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...

Function Analysis of vvi-miR172s and Their Target Genes Response to Gibberellin Regulation of Grape Berry Development

【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-miR172a/b/c/d were cloned and identified by miR-RACE and PCR techniques from grapevine cv. Rosario Bianco. The target genes of vvi-miR172s 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-miR172s. 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-miR172s and target genes in different tissues of grape berry induced by exogenous GA₃ application. 【Result】The mature and precursor sequences of vvi-miR172a/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-miR172s 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 VvRAP2-3 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-miR172c and VvRAP2-1 had the hormone related cis-elements response to GA₃, suggesting that they might be involved in the regulation of grape berry development by responding to corresponding hormones. Microarray data analysis revealed that the expression patterns of vvi-miR172c and target genes were tissue or orGA₃n specific. RT-qPCR analysis showed that vvi-miR172a/b/d showed a decrease expression trend in pericarp, while VvRAR2-1 exhibited a reverse expression trend to the former, indicating that vvi-miR172a/b/d neGA₃tively regulated VvRAR2-1. However, in flesh, VvAP2 was contradictory to vvi-miR172d, with the increased expression during grape development, suggesting that there was a significant neGA₃tive correlation between vvi-miR172d and VvAP2. Interestingly, GA₃ treatment promoted the neGA₃tive regulation of vvi-miR172d on VvRAP2-1 and induced the neGA₃tive regulation of vvi-miR172c on VvAP2/VvRAP2-1/VvRAP2-3. 【Conclusion】VvAP2, VvRAP2-1, VvRAP2-2, and VvRAP2-3 were the real target genes of vvi-miR172s. Among the vvi-miR172 family, vvi-miR172 a/b/d might mediate VvRAP2-1 regulation of pericarp development, whereas vvi-miR172d might mediate VvAP2 involved in the development of grape flesh. vvi-miR172c/d and their target genes might participate in the regulation of grape pericarp and flesh development in response to exogenous GA.