GA3介导miR171s及其靶基因VvSCLs调控葡萄种子发育的作用分析

目的]鉴定vvi-miR171s成员及其靶基因,明确vvi-miR171s成员及其靶基因在葡萄种子发育中的重要作用及其响应赤霉素(Gibberellin,GA)调控种子发育的表达模式.方法]以'魏可'葡萄('Wink')果实为试材,利用miR-RACE技术鉴定vvi-miR171a/b/e/f/g/h的成熟体序列;...

Function Analysis of GA3 Mediate miR171s and Its Target Genes VvSCLs in Grape Seed Development

【Objective】The main objective of the present research was to identify the vvi-miR171s and their target genes VvSCL6/15/22 from grapevine genome, and to confirm the role of vvi-miR171s and VvSCL6/15/22 in grape seed development process and their expression pattern in response to exogenous GA₃. 【Method】The mature sequences of vvi-miR171a/b/e/f/g/h were validated by miR-RACE technique from grape (Vitis vinifera L.) cv ‘Wink’. The potential target genes of vvi-miR171s were predicted by PsRNATarget software, and the chromosome localization, phylogenetic, gene structure, conserved domain and motifs analysis were performed by bioinformatics tools. The potential functions of vvi-miR171s and their target genes were predicted by promoters cis-elements analysis. RLM-RACE and PPM-RACE verified the cleavage roles of three target genes by vvi-miR171a/b/e/f/g/h, and the qRT-PCR method was used to detect their temporal expression patterns in grape seed and seedless grape induced by exogenous GA₃. 【Result】Six vvi-miR171s mature sequences in grape were determined and cloned, which were consistent of the sequences of miRBase. Based on this result, the target genes of them, including VvSCL6, VvSCL15 and VvSCL22, were prophesied. The target genes bioinformatics analysis showed evolutionary genetic distance between VvSCL6/22 genes and homologous genes in apple (Monodelphis domestica), peach (Prunus persica), and cherry (Prunus avium) is relatively closed, while VvSCL15 had the same genetic distance from the homologous genes in Arabidopsis, apple, peach and cherry. Meanwhile, the gene structure of VvSCLs was similar to their close genes in phylogenetic tree. All SCL protein sequences had GRAS domain, both element types, arrangement order of motifs. And 5 same motifs for GRAS domain were identified, indicating that the SCL protein structure was relatively conservative, with the conserved functionality. Subsequently, an array of gibberellin and Salicylic acid responsive cis-acting elements were identified in vvi-miR171s and target genes VvSCLs promoter sequences, indicating their possible involvement in the regulation of GA-responsive and SA-responsive grape growth and development. Moreover, the qRT-PCR analysis revealed that the expression of vvi-miR171a was significantly repressed by GA₃ in seed, while its target gene VvSCLs showed reciprocal expression pattern, particularly. There was a strong negative correlation between vvi-miR171a -VvSCL15 in the grape seed of CK and seed region of GA₃ treatment. Taken together, vvi-miR171a might the major member of vvi-miR171s in response to GA₃, additionally, it might involve in the grape berry seed development through negative regulating of VvSCL15 gene developmental in response to GA₃. 【Conclusion】vvi-miR171a/b/e/f/g/h were identified from grapevine ‘Wink’, and all of members could cleave three target genes VvSCL6/15/22. The vvi-miR171a-VvSCL15 might be the major member in response to gibberellin and involve in regulating grape seed development.