Identification And Expression Of DREB Transcription Factor Family And Functional Verification Of VaDREB Gene

Our country is the world’s largest producer and consumer of fresh grape,grape industry is developing rapidly,promoting farmers’ sustainable income.However,grapes of china are mainly distributed in arid or semi-arid areas of 25°～ 45° north latitude,and have poor adaptability to low temperature arid climate in the main planting area,which is very easy to cause cold damage and freezing damage.Low temperature injury often causes different degrees of damage to grape flower buds,branches and vines,root system,resulting in the decline of grape quality and yield,resulting in huge economic losses,which greatly limits the sustainable development of our grape industry.DREB(dehydration responsive element binding protein)is one of the important transcription factors in plant growth regulation,which plays an important role in regulating the expression of stress-related genes and improving the adaptability of plants to stress.In this study,the DERB gene family of grape was identified and bioinformatics analyzed based on grape genome database.In addition,two DREB genes that respond to low temperature were screened out by low temperature treatment of ‘Beibing Hong’(Vitis amurensis Rupr.)and ‘Thompson seedless’(Vitis vinifera L.),overexpression vectors were constructed and allogeneically transformed into Arabidopsis for cold resistance function study,providing candidate genes for cold resistance breeding of grape and theoretical basis for cold resistance research of other species.The main results obtained are as follows:1.Identification of the DREB gene family in the grape.Based on the grape genome database(PN40024.V2),53 grape DREBs were identified and named according to their chromosomal localizations(VvDREB1-VvDREB53).Through phylogenetic with grape and Arabidopsis DREB genes,grape VvDREBs genes were divided into six subgroups(A1-A6).Collinearity analysis and Ka/Ks calculations indicated that tandem and segmental duplication events were the main reasons for the expansion of the grape DREB family,and that most of the DREB genes in grapes were selected for purification.Through gene structure and conserved motif prediction,the evolutionary relationship of DREB family genes in grape were analyzed.The predictive analysis of promoter cis-acting elements of grape DREB family genes showed that there were photo-responsive elements,plant hormone response elements,anaerobic response elements,drought response elements and low-temperature response elements on the promoters of these grape DREB family genes,these abundant promoter elements indicate that DREB family genes in grape may have various biological functions.2.Expression analysis of the DREB gene family in the grape.After cryogenic treatment of ‘Beibinghong’(V.amurensis Rupr.)and ‘Thompson Seedless’(V.vinifera L.),semi-quantitative PCR detection of 53 grape DREB genes showed that 28 grape DREB genes were responsive to cold stress.Among them,12 DREB genes were selected for real-time PCR experiments,and it was found that VvDREB6 was significantly induced in the early stage of cold treatment and VaDREB37 in the late stage of cold treatment,and these two genes may play a key role in the response of grapes to low temperature stress.3.Cloning and functional study of VaDREB6.Using ‘Beibinghong’ cDNA as template,the full-length 726 bp coding sequence of VaDREB6 was amplified,encoding 241 amino acids and subcellular localization in the nucleus.The overexpression vector was constructed and the VaDREB6 gene was overexpressed in Arabidopsis.After cold treatment,the wilting degree of leaves of three transgenic Arabidopsis strains were significantly lower than that of wild type Arabidopsis,the relative electrical conductivity and malondialdehyde content of leaves were significantly lower than that of wild type Arabidopsis,and the activity of antioxidant enzymes SOD(Superoxide Dismutase),CAT(Catalase)and POD(Peroxidase)were significantly higher than that of wild type Arabidopsis,the survival rate of Arabidopsis strains were significantly higher than that of wild Arabidopsis thaliana after a week of normal growth conditions.After cold treatment,the expression levels of AtCBF3,AtRD29A,AtCOR15 and AtCOR47 of transgenic Arabidopsis were significantly higher than those of wild-type Arabidopsis at the critical time point of cold stress response.These results indicated that VaDREB6 significantly enhanced the cold resistance of transgenic Arabidopsis strains.4.Cloning and functional study of VaDREB37.Using ‘Beingbinghong’ cDNA as template,the full-length 714 bp coding sequence of VaDREB37 was amplified,encoding237 amino acids,and subcellular localization in the nucleus and cytoplasm.The overexpression vector was constructed and the VaDREB37 gene was overexpressed in Arabidopsis.After cold treatment,the wilting degree of leaves of transgenic Arabidopsis strains were significantly higher than that of wild-type Arabidopsis,the relative conductivity and malondialdehyde content of the leaves were significantly higher than that of wild-type Arabidopsis,and the antioxidant enzymes SOD,CAT,and POD were significantly lower than that of wild-type Arabidopsis,and the survival rate was significantly lower than that of transgenic Arabidopsis after restoration.After cold treatment,the expression levels of AtCBF1,AtCBF2,AtCOR15 and AtCOR47 of transgenic Arabidopsis were significantly lower than those of wild-type Arabidopsis at the critical time point of cold stress response.These results indicate that VaDREB37 significantly reduces the cold resistance of transgenic Arabidopsis strains.