Cold Resistance Function Analysis And Molecular Mechanism Of VvCOLD1 Gene In Grape

Low temperature not only affect normal growth of grapevine plants but also affect yield and quality grapevine.Therefore,identifying cold resistant relative genes in grapevine has great research significance.Previous studies have found that GPCR-type-G-proteins are mainly involved in low temperature response and height development regulation of plants.No report showed the role of VvCOLD1 protein in cold signal pathway.This study cloned VvCOLD1 gene in grapevine,analyzed the tissue specificity of VvCOLD1 gene expression and its expression pattern under low temperature and ABA treatment,explored cold resistance function by overexpressing VvCOLD1 gene in Arabidopsis thaliana and verified the transcription factors regulating VvCOLD1 and the proteins interacting with VvCOLD1.The upstream regulators of VvCBFs expression were screened and verified by yeast one hybrid and dual-luciferase assay.We also analyzed the expression pattern of the transcription factors regulating VvCBFs gene under low temperature stress.The five main results of this experiment are as follows:1.Cloning and expression analysis of VvCOLD1 gene in grape.VvCOLD1 gene was cloned from Thompson Seedless and Beibinghong grapevine varieties.The nucleotide length was 1407 bp and encoded 468 amino acids.The 187 th amino acid of COLD1 protein in Thompson Seedless and Beibinghong was methionine(MET),which was consistent with that in chilling-sensitive indica rice.Real-time quantitative PCR results indicated that VvCOLD1 was expressed in roots,stems,leaves,flowers and tendrils of Thompson Seedless and Beibinghong grapevine.The expression level was the highest in the mature leaves of Beibinghong grapevine.After 4℃ low temperature treatment,the expression of VvCOLD1 in Thompson Seedless and Beibinghong grapevine was down-regulated within 0.5 h.Then,the expression of the gene was up-regulated and peaked at 3 h.And VvCOLD1 could be inhibited by ABA stress in Thompson seedless and Beibinghong grapevine.2.Cold resistance function analysis of VvCOLD1 gene in grapevine.The VvCOLD1 overexpression vector was constructed and overexpressing line of VvCOLD1-OE in Arabidopsis thaliana were generated by the experiment of genetic transformation.And,we carried out freeze phenotype analysis and q RT-PCR analysis on overexpressing line.After-10℃ freezing stress treatment,most of the VvCOLD1 overexpressing plants had survived.However,almost all of the atgtg1 and atgtg2 mutant seedlings had died compared with the wild-type plants.After 48 h of 4°C treatment,the q RT-PCR assay indicated the expression levels of cold-responsible genes CBF1、CBF2、COR47 and COR15 were significantly higher in the overexpression lines compared with the wild-type plants.In addition,the expression levels of these genes were lower in the atgtg1 and atgtg2 mutant lines compared with the wildtype plants.3.VvCOLD1 interacted with G protein α subunit Vv Gα protein in grapevine.The CDS sequence of Vv Gα and VvCOLD1 gene was cloned from Vitis vinifera cv.Thompson Seedless by PCR amplification.The yeast two-hybrid and bimolecular fluorescence complementation assay indicated that VvCOLD1 interacted with the Vv Gα protein.The q RT-PCR assay indicated Vv Gα was expressed in roots,stems,leaves,flowers and tendrils and could be induced obviously by low temperature.4.The cold-resistant transcription factors VvCBFs negatively regulate VvCOLD1 gene expression in grapevine.The promoter of VvCOLD1 gene and the CDS sequence of VvCBFs transcription factor were cloned from Vitis vinifera cv.Thompson Seedless by PCR amplification.The cis-acting element prediction and analysis of VvCOLD1 promoter revealed that the promoter region of VvCOLD1 gene contains CRT/DRE element which CBF transcription factor could bind to.Yeast one-hybrid assay indicated that the VvCBF1 and VvCBF4 interacted with VvCOLD1 promoter.Dual luciferase assay indicated that VvCBF1,VvCBF2,VvCBF3 and VvCBF4 could inhibit VvCOLD1 promoter activity.5.The transcription factor regulating VvCBFs gene expression were screened and identified.The promoter of VvCBFs gene and the CDS sequence of candidate transcription factor were cloned from Vitis vinifera cv.Thompson Seedless by PCR amplification.Yeast one-hybrid and dual luciferase assay indicated that:(1)Vv MYB24 and Vv MYB44 interacted with the VvCBF1 promoter,Vv MYB24 could activate VvCBF1 promoter activity under the condition of 22℃;(2)Vv ZAT10,Vv ZAT11,Vv MYB24,Vv MYB44,VvCBF1 and VvCBF4 interacted with VvCBF2 promoter.Vv ZAT10,Vv MYB24 and Vv MYB44 could inhibit VvCBF2 promoter activity,Vv ZAT11 and VvCBF4 could activate VvCBF2 promoter activity.(3)Vv ZAT11,Vv MYB24,Vv MYB44 and Vv WRKY75 interacted with VvCBF3 promoter.Vv MYB24 and Vv ZAT12 could inhibit VvCBF3 promoter activity;(4)Vv ZAT10、Vv ZAT12、Vv MYB24 and Vv MYB44 interacted with VvCBF4 promoter.Vv ZAT10 and Vv ZAT12 could activate VvCBF4 promoter activity,and Vv MYB24 and Vv MYB44 could inhibit VvCBF4 promoter activity.In addition,the q RT-PCR assay indicated the expression levels of Vv ZAT10、Vv ZAT11、Vv ZAT12、Vv MYB24 and Vv MYB44 were induced after 4°C low temperature treatment.