Expression Analysis,Cloning Of Grape Seedless Genes And Functional Analysis Of VvMADS46

Grape(Vitis vinifera L.)is a kind of important economic fruit in the world,which is widely cultivated both in our country and abroad.Seedless grape species eat conveniently,taste great,also with a lot of nutrition,leading a favor for consumers and owning a broad market.Consumption of seedless table grapes in the world has a rising trend these years,those countries with great grape production gradually pay more attention for seedless grape research,including our country.Seedless grapes breeding is one of the major objectives present and a short future,so grape seedless related genes exploitation has become an important aspect for scientific research.This research is based on the previous seedless and seed grape hybrids transcription genome analysis of our research team,27 genes were selected and finished three seed grapes and three seedless grapes ovule different developmental period expression analysis,which is necessary for grape seedless regulation genes exploitation.Furthermore,five ovule different expression genes were cloned,then analyzed by DNAman after sequencing.Additionally,the gene VvMADS46 was constructed the subcellular localization and overexpression vectors,then infected onion surface cells and tomato cotyledons for further gene function research.Main results:1.27 grape seedless related genes were analyzed by semi-quantitative PCR in three seed grape and three seedless grape materials of different developmental periods of the ovule.20genes(HB1,GRAS,AP2-2,HB2,MYB1,MYB2,NAC26,GPDC8,SCD1,MADS46,TLP2,SCD2,KAN1,P450-1,NAC2,Knlike3,VVSTM,KAN1-2,GH3-1,TLP1)in seedless grape ovules showed higher quantity of expression and 7 genes(NAC86,DDM,C2H2-1,C2H2-2,NAC41,GH3-9,GSLG)expression quantity in seed grape ovules were high.This result is benefit for grape seedless genes screening and regulation analysis.2.Five ovule different expression genes: MYB1,MYB2,NAC26,NAC41 and HB1 were cloned in seedless grape ‘Thompson Seedless' and seed grape ‘Red Globe'.Gene length:1686bp,1090 bp,1054bp,1055 bp and 1657 bp.These five genes sequences all show great similarity in both two grape materials,indicating gene sequences is not the main effect for gene different expression.3.VvWRKY46 expression was analyzed in ‘Thompson Seedless' and ‘Red Globe'different tissues and organs.The results showed that VvMADS46 expressed in all different tissues and organs of seedless grape ‘Thompson Seedless' and seed grape ‘Red Globe'.However,a higher expression was detected in inflorescence,flowers and fruits.At the same time,VvMADS46 cDNA sequences were cloned in seedless grape ‘Thompson Seedless' and seed grape ‘Red Globe'.The open reading frame is 618 bp,encoding 206 amino acids.Furthermore,the subcellular localization and overexpression vectors were constructed,then infecting onion surface cells and tomato cotyledons.In transgenic tomato lines the tomato seeds decreased significantly,indicating the possible function of VvMADS46 in grape seedless regulation.Fluorescence observation showed that VvMADS46 located in the nucleus.Besides,the VvMADS46 promoters were cloned in seedless grape ‘Thompson Seedless' and seed grape‘Red Globe'.Sequences alignment and Plantcare analysis found that a lack of endosperm developmental regulation element in seedless grape ‘Thompson Seedless',which may associated with grape seedless regulation.