Functional Analysis And Screening Of Interacting Proteins Of VaERD15 Gene In Cold Resistance In Chinese Wild Vitis Amureusis

Grape is one of the most important fruit trees in the world.At present,the widely cultivated grape varieties in the world belong to Vitis vinifera L.,which has good quality but poor cold resistance,and its growth and productivity are strongly affected by low temperature.China is one of the original centers of grapes with abundant wild grape resources.Vitis amurensis is the most cold-resistant species in Vitis,which can tolerate-40low temperature and is a very important cold-resistant gene resource.It is of great significance that explore the genes related to cold resistance of Vitis amurensis and study their functions to understand the molecular mechanism of cold resistance and to improve the cold resistance of Vitis vinifera L.by molecular means.In our previous study,a c DNA library of the cold-resistant genes from Chinese wild Vitis amurensis was constructed after cold stress.Subsequently,the VaERD15 gene was cloned and transferred into Arabidopsis thaliana.It was proved that over-expression VaERD15 can improve the cold resistance of transgenic Arabidopsis thaliana.On this basis,the VaERD15 gene from Vitis amurensis acc.'Shuangyou'?cold-resistant?was cloned to analyze its function.The main results of this study are as follows:1. According to the VaERD15 sequence obtained from the transcriptome database of Vitis amurensis acc.'Shuangyou',the c DNA of'Shuangyou'was used as the template for the coding region PCR amplification.The CDS of VaERD15 was obtained,which is 507 bp and encode 169 amino acids and contains a PAM2 domain.Sequence analysis showed that the VaERD15 from'Shuangyou'has 99%homology with Vv ERD15 from'Red Globe',with only one base mutation.It indicated that the cold resistance of grape regulated by ERD15gene was mainly related to its promoter.The result of subcellular localization showed that VaERD15 was localized in the nucleus and cell membrane.After low temperature treatment at 4?,ERD15 had different responses to low temperature in'Red Globe'and'Shuangyou'.The expression of ERD15 induced by low temperature in'Shuangyou'was more significant than that in'Red Globe'.The expression of ERD15 in'Shuangyou'was 20.3 times that in'Red Globe'.2. The promoter which was the upstream 999 bp of ERD15 CDS and its deletion fragments were cloned from'Shuangyou'and'Red Globe',respectively.Then,GUS fusion expression vectors with the promoter and its deletion fragments were constructed respectively,which were instantly transformed into Nicotiana tabacum leaves by employing the agrobacterium GV3101,and the expression activity was analyzed after 4?,ABA,SA and PEG treatment.The results showed that P_VaERD15?VaERD15/p999?and P_{Vv ERD15}?Vv ERD15/p999?as well as each fragment can respond to 4?,ABA,SA and PEG.Affected by the type and number of cis-acting elements contained in the promoter,the activity of P_VaERD15,P_{Vv ERD15}and each deletion fragment was inconsistent.The activity of P_VaERD15and P_{Vv ERD15}under 4?and ABA treatment were higher than those of SA and PEG treatment,indicating that they were more affected by low temperature and ABA stress than SA and PEG.P_VaERD15,P_{Vv ERD15}and their deletion fragments were all induced by ABA,suggesting that their response to low temperature may be related to ABA signaling pathway.The first?VaERD15/p682 and Vv ERD15/p685?and second?VaERD15/p456 and Vv ERD15/p459?deletion fragments of the promoters in the'Shuangyou'and'Red Globe'may contain unknown cis-acting elements in action,so that the activity of the P_VaERD15and P_{Vv ERD15}as well as first deletion fragment were higher than those in the second and third?VaERD15/p276 and Vv ERD15/p290?deletion fragments.3. The interaction proteins including xyloglucan endotransglucosylase/hydrolase protein 7 ?XTH7?,glutamine synthetase?GS?,40S ribosomal protein 23?RPS23?,protein disulfide-isomerase LQY1?LQY1?of VaERD15 were obtained by yeast two-hybrid technique.The results of bimolecular fluorescence complementation?Bi FC?showed that VaERD15 can interact with Va XTH7,VaGS,Va RPS23 and Va LQY1.4. To further study the response of VaERD15 interaction proteins to cold stress,qRT-PCR was used to analyze the expression patterns of XTH7,GS,RPS23 and LQY1 in'Shuangyou'and'Red Globe'under 4?cold stress.The results showed that the expression levels of all four genes changed after cold stress,but XTH7 and GS were more obviously induced by low temperature than RPS23 and LQY1,indicating that XTH7 and GS were more closely related to cold resistance.Therefore,it is speculated that these genes may co-regulate grape responses to low temperature stress by interacting with VaERD15.