Analysis Of The Role Of Grape Vv-miR156s And Its Targeted VvSBPs In Grape Berry Development And Ripening

MicroRNAs(miRNAs)are a class of endogenous non-coding small RNA molecules of about 21-25 nt in size that exist in plants and animals.It regulates the expression of target gene by specifically matching the mRNA sequence.SBP-box is a plant-specific transcription factor.Studies in Arabidopsis found that members of SPLs were targeted by miR156s,thus playing an important role in plant growth and development.This study analyzed the mechanism of microRNA mediated grape transcription factor SBP-box regulating fruit development and maturation.The main findings are as follows:1.The qRT-PCR and miR-RACE techniques were used to clone the mature and precursor sequences of the members of Vv-miR156s from 'Wink',and the similarity of the mature sequences was analyzed.Vv-miR156s could be divided into five categories:Vv-miRl 56a,Vv-miR156b/c/d,Vv-miRl 56e,Vv-miR156f/g/i,and Vv-miR156h.In comparison with the sequence of 'Pinot Noir' grapes in miRBase,it was found that the precursor sequences of the two grape varieties are the same,but the mature sequences are different,and this difference is due to the displacement of the mature sequence at the 5' end.Bioinformatics analysis of members of the Vv-miR156s family 'of Wink' revealed that their mature sequences are highly conserved,the precursors have a stable secondary stem-loop structure,and the precursor genes are evenly distributed in the six chromosome in grape.Evolutionary characteristics are consistent with the location information,that is,sequences who are located on the same chromosome are grouped together.Prediction of Target gene results indicate that most target genes of Vv-miR156s belong to members of the transcription factor SBP-box.2.To better understand the function diverse of Vv-miR156s targeted genes and non targeted genes in grape berry development and ripening process,we systematically analyze the bioinformatics of SBP-box genesobtained from grape transcription factor database(http://planttfdb.cbi.pku.edu.cn/analysis).Totally 19 SBP-box genes were found in transcription factor database,among which 10 members are predicted as the targeted genes of Vv-miR156s.The results show that the members are unevenly distributed on the 13 chromosomes of grapes,encoding 115-981 amino acid.The molecular weight of the protein varies from 13KD to 109KD,most of which are alkaline,hydrophilic,unstable protein and contain a complete,highly conserved SBP domain.The secondary structures are alpha-helices and random coils,and are mainly located in the nucleus.Phylogenetic analysis shows that SBP-box proteins can be clustered into three clusters.Analysis of chip data shows that the expression of VvSBPs genes shows tissue-and organ-specific patterns.Most of the SBP genes highly express in certain stages of growth and development in vegetative organs,flowers and berry skins,pulps,and seeds.In addition,all SBP-box members contain elements related to grape berry development and ripening and the different number of each type of elements.qRT-PCR results indicated that nine members(included the 5 predicted targeted ones)probably promote berry ripening process and nearly seven members(included the 5 predicted targeted ones),the second subclass,promote the growth and development of grape berry but inhibit the berry ripening process.It is speculated that the SBP gene plays an important role in different stages of fruit development.3.Validated the cleavage products,sites and frequencies of Vv-miR156a,b/c/d,e,f/g/i and their respective targets(Vv-SBP2,9,10,16)were validated in diverse stages of grape berries by our developed PPM-RACE and modified RLM-RACE together with qRT-PCR.With berry development and ripening,expression of Vv-miR156a,b/c/d,e and f/g/i exhibited an overall increasing trend,while their targets showed opposite trends at some specific corresponding stages.Additionally,ABA and NAA promoted or repressed the expression of Vv-miR156/target VvSBPs to some extent before grape berry ripening.The results indicate that,under the targeting of Vv-miR156s,VvSBP2 and VvSBP16 may positively regulate fruit coloration and maturation,whereas VvSBP9 and VvSBP10 may inhibit fruit coloration and maturation.4.The VvSBP9 gene was cloned and transformed into tobacco using agrobacterium-mediated leaf disc method.Transgenic tobacco showed higher plant height,thicker stems,longer internodes,and striped petals.Through tissue-specific expression analysis of wild-type and transgenic tobacco NtSBP9,it was found that overexpression of VvSBP9 gene can up-regulate the expression of NtSBP9 in stems and flowers.Analysis of anthocyanin-related gene expression found that expression level of NtCHI and NtCHS of tobacco with VvSBP9 overexpression is lower than that of wild type.This study shows that VvSBP9 regulates tobacco anthocyanin accumulation by inhibting the expression of CHI and CHS genes.