Cloning And Functional Analysis Of VvABCG20 Promoter From Grape

In this research,VvABCG20 gene promoters from different seed and seedless grape cultivars were cloned and analysed.Then,VvABCG20 promoters and deletion sequences were constructed with GUS fusion vectors.Analysing activity of each fragment promoter by instantaneous transformation tobacco leaves(agrobacterium-mediated transformation),and analyzing function of each fragment promoter under hormone treatment.We obtained the transgenic Arabidopsis of VvABCG20 prompter by floral dip.According GUS staining to analyse the activity and tissue specificity of VvABCG20 prompter.The results showed as follow:1.The VvABCG20 promoter sequences were obtained and analysed from 15 different seed and seedless grape cultivars.We found that 8 grape cultivars(seed and seedless)had two kinds of sequences(a and b),and the rest had only ‘b' sequence,‘a' sequences from Pinot Noir and Thompson Seedless were exactly the same,named P0,‘a'and ‘b' sequencedid not appeared regularly in the seed or seedless grapes.Sequences analysis showed that ‘b' had a fragment of 41 bp deletion than ‘a' at 802 bp,and the similarity between forecasting sequence and VvABCG20 ptomoter sequences from 15 grapes was over 97%.And in 23 VvABCG20 promoter sequences there were only 4 insertion/deletion fragments,and these might irrelevant to seed or seedless of grape.Furthermore we analysised single-nucleotide polymorphism(SNP)of 23 sequences from 15 grapes,the result showed that there were 66 variation sites and only one at 721 bp led to deletion of main Cis-acting elements deletion.Generally,we thought that the Vv ABCG20 promoter sequences were conserved which from different grape cultivars.2.Prediction of Cis-acting elements for VvABCG20 promoter from Thompson Seedless showed that VvABCG20 promoter had several regulatory elements related to seed specificity such as G-BOX,-300 element,Skn-1-motif,SEF1 and so on except the typical ones.Compared with the Cis-acting elements prediction of VvABCG20 promoter from Pinot Noir,it showed no difference in types and locations but in quantities.GUS activity analysis of leaves in infiltrated tobacco of VvABCG20 promoters showed that all the promoters all of them could drove GUS gene express,Pinot Noir promoter(P1)had the significant high activity than Thompson Seedless promoter(T1)and P0(P<0.01),and non-significance between P0 and T1.It might be some Cis-acting elements enhanced promoter activity in P1.3.According to the analysis results of cis acting element in ABCG20 gene promoter,we amplificated 5'-deletion fragment and each acquired five fragments from Pinot Noir(P1,P2,P3,P4,P5)and Thompson Seedless(T1,T2,T3,T4,T5).Hormone treatment analysis of VvABCG20 promoter and deletion fragments from Pinot Noir and Thompson Seedless showed that the ERE at-1350 bp of P0 and P1,-1348 bp of T1 could response to ETH and regulated GUS expression with negative regulation;The RY-motif might response to ETH and regulated GUS expression with positive regulation.In abiotic stress treatments with MeJA,it showed that BOX-?could response to MeJA treatment and enhanced expression activity.TGA-element at the end of each fragments in response to IAA could enhance expression activity.We speculated that some Cis-acting elements at upstream of-807 bp of Pinot Noir and-806 bp of Thompson Seedless could be induced by IAA and enhanced activity of promoter;ELI-box3 in P0 could enhance expression activity and be induced by ERE and IAA but MeJA.4.GUS staining analysis of VvABCG20 promoter of P0 showed that the CaMV 35 S promoter and VvABCG20 promoter both could drive expression of GUS gene in Arabidopsis thaliana,and no GUS activity could be found in WT plants.However,it needed a further testing on whether there was significant difference in the expression of each tissue or not.We speculated that it was possible that combination of specific transcription factors and promotes from grape were affected by heterologous plants Arabidopsis thaliana,or it might be the length of promoter we obtained was not sufficient.It needs a further verification on whether the VvABCG20 gene promoter was the seed specific promoter or not and which section related to seed specificity.