Analysis Of The Role Of Two MicroRNA Responsive To Gibberellin And Its Target Genes In Grape Flower Development

MicroRNAs(miRNAs),as an important growth regulator,are also involved in gibberellic acid(GA)signaling,revealing much relationship between miRNAs and GA in various plant responses.In this study,the effects of two responsive to gibberellin miRNA and their target genes in grape flower development were analyzed.The main results are as follows:1.It was found that grapevine miR061(VvmiR061)is a GA3 responsive miRNA.in this study,grapevine REV(VvREV)and HOX32(VvHOX32),two target genes of VvmiR061,were predicted,verified,and cloned;homologous conservation was analyzed in various plants.The expression profiles of both VvmiR061 and its target genes(VvREV and VvHOX32)under GA3 treatment were detected by qRT-PCR during grapevine flower and berry development.Results revealed that GA3 treatment has up-regulated the transcription of VvREV and VvHOX32,while it down-regulated the expression of VvmiR061.The function of VvmiR061 in cleaving target genes VvREV and VvHOX32 was diminished by GA3 treatment during flower developmental process.The results of this study exhibited the importance of VvmiR061 in regulating flower development and GA3 signaling pathway and also contributed some to the knowledge of small RNA-mediated regulation in grape.2.The constructed pCAMBIA1300-VvREV/VvHOX32 expression vector were transformed into tobacco—mediated leaf disc transformation method.Transgenic tobacco showed plant height,flowering phenomenon.The expression of flowering related genes in transgenic tobacco were detected,and the expression of NtFT gene in transgenic tobacco was higher than that of wild type,and the NtFLC gene was lower than that of wild type tobacco.The expression of NtFLC gene in transgenic tobacco was lower than that of wild type VvHOX32.These results indicate that VvREV and VvHOX32 regulate flower development by regulating the expression of FT and FLC genes.3.A MYB transcription factor gene of gibberelic acid(GA)signaling pathway,designated as VvGAMYB was cloned using homology-based cloning and reverse transcription-PCR(RT-PCR)from Vitis vinifera L.'Wink'.The open reading frame of VvGAMYB was 1680bp,encoding a protein of 559 amino acids.VvGAMYB also contained the conserved motifs Boxl,Box2 and Box3,which are typical structures in the GAMYB family.The VvGAMYB protein was mainly localized in the nucleus of onion epidermal cell.The expression of VvGAMYB and its downstream gene VvLFY increased during the development of grape flower.The GA3 treatment promoted the expression of VvGAMYB and VvLFY,which promoted the development of grape flower.VvGAMYB was the target gene of VvmiR159,and its expression was regulated by VvmiR159.The VvmiR159 could negatively regulate expression of VvGAMYB by cleavage.The expression of VvmiR159 and its target gene VvGAMYB showed a negative correlation,and its expression was reduced by GA treatment,which also reduced the cleavage of VvGAMYB.These results suggest that VvGAMYB is involved in the flowering process of grape through the GA flowering pathway.In this process,the role of GA in inhibiting the VvmiR159 cleavage of VvGAMYB and thus up regulation of VvLFY gene expression is involved in the regulation of flowering process.