Cloning And Functional Analysis Of β VPE Promoter From Grape

Programmed cell death (PCD) involved in the process of ovule abortion which happensin stenospermocarpy grapes, Vacuolar processing enzyme (VPE) is a key enzyme inmediating and executing programmed cell death in plants, and βVPE, the seed-type VPE, hasthe relationgship with the development of the seed. Rencent researches have indicated thatβVPE gene shows two different expression patterns between seed and seedless grapes ovuledevelopment process, and might be concerned with ovule abortion. Considering the importantrole of promoters in the regulation of gene expression, we studied the promoter of βVPE genefrom grapes to explore its impact on gene expression. In this study, we cloned and analyzedthe βVPE genes and promoters from different seed and seedless grape varieties. Then,constructed the GUS fusion vectors harboring βVPE promoters from Thompson Seedless andPinot Noir, and also constructed the expression vectors of different deletion βVPE promotersfrom Thompson Seedless. Using floral dip (agrobacterium-mediated transformation) weobtained the transgenic Arabidopsis of all prompter fragments. We also analysed the activityand tissue specificity of all prompter fragments by GUS staining and quantitative detection ofGUS activity. Experimental results as follows:1. The βVPE gDNA sequences from6different seed and seedless grape varieties wereobtained and analysed. The similarity of6βVPE gDNA sequences was96.72%, and all the6sequences contained9exons and8introns. The main ORF sequence differences wereinsertion/deletion (indel) mutations of individual base. The result indicated that βVPE gDNAsequences from6different seed and seedless grape varieties were highly conserved.Meanwhile, there was a insertion/deletion of36bp in the3’UTR (untranslated region). TheβVPE promoter sequences from12different seed and seedless grape varieties were obtainedand analysed, and the similarity of12βVPE ptomoter sequences was94.56%. We found thatthere were insertion/deletion of7fragments in12βVPE promoter sequences, and the7fragment differences might not be related to seed or seedless of grape varieties. On the whole,we considered that the βVPE promoter sequences from different grape varieties wereconserved.2. The Cis-acting elements prediction of βVPE promoter from Thompson Seedlessshowed that βVPE promoter not only had the typical core promoter elements such as TATA-BOX and CAAT-BOX, but also had several regulatory elements related to seedspecificity, like RY-repeat-motif,(CA)n element, E-BOX, G-BOX,-300element,GCN4-motif, Skn-1-motif, SEF1and so on. GUS activity analysis of seeds in transgenicArabidopsis thaliana of βVPE promoters from Thompson Seedless and Pinot Noir showedthat the two βVPE promoters both had expression activity, and no significant differencebetween Pinot Noir and Thompson Seedless. We considered that βVPE promoter might havelimited impact on the expression diffrences between Pinot Noir and Thompson Seedless.3. Tissue specificity analysis indicated that the βVPE promoter from grapes onlyexpressed in seed. The results showed that the promoter had no GUS activity in the root, stem,inflorescence, leaf, Silique except the seed. Considering no blue in inflorescences wasobserved after GUS staining, we thought that the weak expression of the βVPE promoter ininflorescences examined by quantitative detection of GUS activity was caused by ovules insome inflorescences. GUS staining in seed and seedling of transgenic Arabidopsis thaliana ofthe βVPE promoter from Thompson Seedless showed that it expreseed in embryo (cotyledon,hypocotyl and suspensor), but not in the leaf, stem and root of seedling. These resultsindicated that the βVPE promoter from grape was seed specific promoter.4. Deletion analysis of βVPE promoter from Thompson Seedless showed that p0and p1were both expressed specifically in seeds, not in roots, stems, inflorescences, leaves andSiliques. In the transgenic Arabidopsis thaliana of p2, GUS activity was not only detected inseeds but also in leaves and Siliques, and the similar situation occurred in the transgenicArabidopsis thaliana of p3and p4. It supposed that there was some regulatory element orsequence related to seed specificity and determined seed-specific expression in the lostsequence between p1and p2(-1302~-1041bp). Every different deletion βVPE promoters hadexpression activity in seeds. The expression levels of p0, p2and p4were similar andsignificantly lower than p1and p3, and p3(-875~-1bp) was the highest. The results aboveshowed that there might be some regulatory element or sequence to enhance the expressionactivity in the lost framents between p1and p2(-1302~-1041bp), p3and p4(-875~-701bp);while in the deleted sequence between p0and p1(-1697~-1302bp), p2and p3(-1041~-875bp), an opposite phenomenon was observed. We speculated that there were regulatory elementor sequence to reduce the expression activity, and SEF1-motif, E-BOX and Skn-1-motif mightbe invoved in the decrease of activity.