关键词: 葡萄, vvi-miR160s, VvARF18, GA, 种子发育

Abstract:

【Objective】This study was performed to investigate the roles and the modes responsive to gibberellin (GA) of the vvi-miR160 family and its target genes in the development of Wink grape seed. 【Method】miR-RACE, RT-qPCR, bioinformatics and RLM-RACE were employed to identify vvi-miR160s and its target gene, and to analyze their modes responsive to GAof spatio-temporal expression and potential functions. 【Result】GA treatment before flowering strongly inhibited the ovule and seed development of Wink grape and induced grape seedless berries with high efficiency, and the seedless rate of berries reached 99.8%. The precursor gene sequence (501 bp) and mature sequence of vvi-miR160s were cloned and identified, which were highly conserved across different plant species. The mature sequences of vvi-miR160s were used as queries to predict the target gene VvARF18. The cleavage sites with 9/17 being their cleavage frequency of vvi-miR160s on VvARF18 were detected between the 10th and 11th sites by RLM-RACE and PPM-RACE, which proved that VvARF18 was the true target gene of vvi-miR160s. VvARF18 encoded 683 amino acids, and a nuclear localization signal existed at positions 398-411, while the protein sub-cellular was localized on the nucleus. The homology of VvARF18 with other in other species was highly conserved. The VvARF18 protein was closely related to tea, tobacco, plum and other species. The number of elements and their order were the same across different species, and the genes structures were similar. The VvARF18 promoter contained four types of cis-elements, which possessed more hormone-related cis-elements. RT-qPCR analysis showed that vvi-miR160c/d/e showed a ‘V’-shaped expression trend with the development of grape berries, and the lowest expression levels were found during the stone-hardening stage. VvARF18 exhibited an opposite expression trend to the former, with the highest expression during stone-hardening stage, indicating that vvi-miR160c/d/e negatively regulates VvARF18, but there was no significant negative correlation between vvi-miR160a/b and VvARF18 expression levels. GA treatment significantly up-regulated the expression of vvi-miR160a/b in the development of grape hardcore seeds, and also conspicuously inhibited the expression of VvARF18 in the corresponding period. The expression levels between vvi-miR160a/b and VvARF18 under GA treatments showed the typical negative correlation, indicating that GA treatment promoted the negative regulation of vvi-miR160a/b on VvARF18; reversely, GA weakened the negative regulation of vvi-miR160c/d/e on VvARF18. 【Conclusion】Among the vvi-miR160 family, vvi-miR160c/d/e may mediated VvARF18 regulation of seed development during specific stages of grape seed development, whereas vvi-miR160a/b may mediated VvARF18, which might be mainly involved in the regulation of GA-induced grape seedless berry development.

Key words: grape, vvi-miR160s, VvARF18, gibberellin (GA), seed development