Cold Resistance Function Analysis Of The VaCP17 Gene In Chinese Wild Vitis Amureusis

Grape is one of the oldest angiosperms on earth,grape fruits and its processed products have rich nutritional,medicinal and economic value,so it is widely cultivated all over the world.Among them,the excellent varieties of Vitis vinifera L.has the largest cultivation area,but its cold resistance is poor,which greatly limits its cultivation in cold areas.Vitis amurensis Rupr.is the most cold-resistant species in the genus.It is of great significance to explore the cold-resistant genes in Vitis amurensis,explore its gene function and mechanism and improve cold resistance of Vitis vinifera by methods of molecular breeding.In this study,on the basis of the cold resistance transcription factor gene VaDREB obtained in the previous research of our group,and the VaDREB interaction protein VaCP17 screened by yeast two-hybrid.The following studies were carried out:（1）VaCP17 gene cloning,protein subcellular localization and tissue specificity analysis.Furthermore,by transferring VaCP17 gene into Arabidopsis thaliana,the cold resistance function of transgenic rabidopsis thaliana was analyzed from the changes of phenotype,physiological and biochemical indexes and expression of cold related genes;（2）The promoter fragments of CP17 gene were isolated from V.amurensis acc.‘Shuangyou’and V.vinifera cv.‘Red Globe’,and GUS fusion expression vector were constructed and transformed into Nicotiana benthamiana’s leaves,and the expression activities of the promoters were analyzed after 4℃treatment.（3）VaCP17 interaction proteins were screened by yeast two-hybrid technique to reveal the molecular mechanism of cold resistance of VaCP17.The main results are as follows:1.Based on the sequence fragment of VaCP17 gene,the cDNA of coding region of V.murensis acc.‘Shuangyou’was amplified by PCR.The CDS sequence of VaCP17 is 1404bp and encodes 467 amino acids,which contains three conserved domains,Inhibitor＿I29,Peptidase＿C1A and GRAN.The sequence alignment results showed that it had 99%homology and 5 base mutations with the CP17 of‘Pinot Noir’.The VaCP17 was transformed into Arabidopsis protoplasts and found that it located on the cell membrane.Tissue analysis revealed that CP17 has different expression specifity in different organs of V.amurensis acc.‘Shuangyou’and V.vinifera cv.‘Red Globe’,among which the expression was highest in the stem of‘Shuangyou’and in the flower of‘Red Globe’.2.By constructing an overexpression vector,VaCP17 was transformed into Arabidopsis to obtain T3 transgenic plants;under-6℃cold stress,the leaves wilting phenomenon of wild-type Arabidopsis was earlier than that of transgenic lines,and its electrical conductivity and malondialdehyde were higher than those of transgenic lines,the proline,soluble sugar content and SOD,POD and CAT activities were lower than those of transgenic lines.The relative expression of cold resistance related genes（CBF1,CBF2,CBF3,RD29A,COR15A,KIN1,NCED3,AOC1 and JAZ10）in the transgenic plants were determined after treating at 4℃for 48 hours.The results showed that the expression of these genes were up-regulated.3.The promoter fragments P_VaCP17and P_VvCP17of CP17 gene were cloned from‘Shuangyou’and‘Red Globe’.The analysis of cis-elements showed that both P_VaCP17and P_VvCP17sequences contained cis-elements related to stresses,such as,ERE,ABRE,MBS,etc.But there were differences in types and quantities.Accordingly,the P_VaCP17and P_VvCP17promoters were constructed into the GUS fusion vector and transiently transformed into Nicotiana benthamiana.The GUS protein activity was quantitatively analyzed after low temperature treatment at 4℃.The results showed that both P_VaCP17and P_VvCP17sequences were induced by low temperature at 4℃,but the GUS activity of P_VaCP17was stronger.4.The proteins NAC domain-containing protein 72（NAC72）,Calmodulin-7（CAM7）and Dehydration inducing protein 19（Di19）interacting with VaCP17 were screened by yeast two-hybrid.Bimolecular fluorescence complementary revealed that VaCP17interact with VaNAC72,VaCAM7 and VaDi19 in vivo.