Functional Analysis Of Grape Ovule Specific Transcription Factor VvNAC26

As one of the most important and common horticultural crops,grapevine(Vitis vinifera)is not only used in fresh food,but also widely used in juice making,drying and wine making,which has high nutritional and economic value.And seedless varieties of grape have gradually become the main trend and breeding direction of fresh grape production and consumption all over the world because of their unique and excellent commodity characters,such as consumption and pleasant taste.However,there are few related genes in grape seedless traits reported in the literature,and the research on the formation mechanism and gene regulation network of seedless traits needs to be improved.Therefore,using transgenic technology and yeast two-hybrid technology to find out grape seedless related genes and their interaction genes can provide new ideas and methods for the study of seedless mechanism,which is of great significance for the establishment of new seedless grape germplasm resources.Based on transcriptome and genomic data of our research group,VvNAC26 as a seedless candidate genes has been found,and it’s expression level in different stages of ovule development was significantly different,which suggested that VvNAC26 may be involved in the ovule development of grape.Based on this,the expression analysis,gene sequence analysis,promoter activity,gene function and interaction genes of VvNAC26 genes in different nucleated seedless grapes were studied.The main results were as follows:1.Expression pattern analysis of VvNAC26 gene.The result of VvNAC26 expression levels in root,stem,leave,blossom,tendril and fruit of the seedless cultivar ‘Thompson Seedless’ and seeded cultivar ‘Red Globe’ shows that expression can be detected in all of these organs in both cultivars.And it has similar patterns of expression in both cultivars,High expression of VvNAC26 be found in fruit.But there was no significant difference in the expression of leaf,flower and fruit in the two varieties.In addition,four seed grape cultivars(Kyoho,Red Globe,Jumeigui,Zuijinxiang)and four seedless grape cultivars(Thompson Seedless,Flame Seedless,Bixiang Seedless,and Crimson Seedless)be selected to verify the expression pattern of VvNAC26.Compared with the seeded cultivars,VvNAC26 showed higher expression in the four seedless cultivars during early ovule development at 20,27,34 DAF.After 41,49 DAF,the expression of VvNAC26 in Red Globe and Jumeigui increased,but remained lower than that in Thompson Seedless,Bixiang Seedless,and Crimson Seedless.2.Cloning and sequence analysis of VvNAC26 gene.The 849 bp gene sequence was amplified from the ovule c DNA mixture of nucleated grape and seedless grape.Gene sequence analysis showed that VvNAC26 gene had no base sequence difference among different grape varieties.The cloned VvNAC26 c DNA located on chromosome 1,containing three exons and two introns,was 849 bp long and would encode a protein of 282 amino acids.And VvNAC26 belonging to the NAP subfamily,with high sequence homology(66.43% and 62.81%,respectively)with soybean Gm NAC1 gene and chickpea Car NAC3 gene.Subcellular localization analysis found that VvNAC26 gene played a role in the nucleus.3.Cloning and activity analysis of promoter.According to the promoter sequence information of VvNAC26 gene in ‘Pinot Noir’,primers were designed and 1500 bp long promoter sequences were obtained from seeded grape ‘Red Globe’ and seedless grape‘Thompson Seedless ’.Promoter sequence analysis revealed four single-base differences in promoter sequences with a length of about 1500 bp of ‘Red Globe’ and ‘Thompson Seedless ’ but the number of promoter elements was the same as the species cis-acting elements,which is associated with plant hormones.Promoter activity analysis found no difference in promoter activity at the same sequence length,but promoter activity varied with length.These results suggest that different lengths of promoter fragments may contain potential cis-acting elements with the function of positive and negative regulation of promoter activity change.4.Construction and genetic transformation of VvNAC26 overexpression vector.Constructing VvNAC26 overexpression vector,heterologous transformed tomato found that the solid volume of tomato fruit in transgenic line became smaller,the color was darker,and the leaf color was darker.Microsection of fruits and seeds revealed that the pericarp cells of VvNAC26 transgenic tomato fruit were smaller than controls and the cells of the cotyledon of VvNAC26 seeds were arranged more closely and the cells were narrow.And the number of fruit and seed cells became smaller.The gene quantitative analysis showed that the expression level of tomato gene Sl NAP1、Sl NAC2 and the Sl WEE1 did not change,but the expression level of ethylene and ABA related genes increased.5.Screening of interacting proteins.Autoactivation and toxicity testing suggested that p GBKT7-NAC26 bait protein does not autonomously activate and not toxic when expressed in yeast.The yeast two-hybrid assay found five candidate interacting proteins,and the interaction between VvNAC26 and VvMADS9 was further verified by BiFC assy.