Regulation Of Cold Tolerance By The Transcription Factor VaMYC2 In Chinese Wild Vitis Amureusis

Grapevine is one of the most important fruit crops in the world and is widely cultivated in both hemispheres.Its berries are rich in sugar,organic acids,minerals,vitamins,amino acids,resveratrol and other nutrients,which is a kind of fruit with high nutritional value,medicinal value and economic value.Vitis vinifera L.is the most widely cultivated grape species in the world,with good quality but poor cold tolerance.Another species,Vitis amurensis Rupr.native to China,has the strongest cold tolerance,which vines can withstand the low temperature of-40℃,and is an important cold tolerance gene resource.In our previous studies,a cold-tolerant transcription factor Va CBF6 was obtained in Chinese wild V.amurensis by transcriptome sequencing analysis,and then its interacting protein Va MYC2 was screened out by yeast two-hybrid.On this basis,Va MYC2 gene was cloned from V.amurensisand and overexpressed in Vitis vinifera cv.‘Thompson Seedless’ to study its function under cold stress.By verifying the interaction proteins and target genes of Va MYC2,the molecular mechanism of Va MYC2 regulating grape cold tolerance was preliminarily revealed.The main results are as follows:1.The primers were designed according to the sequence of Vv MYC2 gene(accession number: XM＿002280217)published on NCBI website,we cloned the Va MYC2 gene from V.amurensis acc.‘Shuangyou’ and found that its CDS sequence was 1992 bp,encoding663 amino acids and containing b HLH conserved domain,which indicated that Va MYC2 belongs to b HLH transcription factor family.By means of sequence alignment,we found that the base sequences of Va MYC2 was 99.50% similar to Vv MYC2,with 10 base differences.2.Tissue-specific expression analysis showed that MYC2 was expressed in various tissues of V.amurensis acc.‘Shuangyou’ and V.vinifera cv.‘Red Globe’,and the expression level in ‘Shuangyou’ was significantly higher than that in ‘Red Globe’,and the highest expression level of MYC2 gene was found in the stem and inflorescence of‘Shuangyou’.3.The expression of Va MYC2 was induced by low temperature(-1℃),Me JA and ETH,but inhibited by ABA.In addition,Va MYC2 transcription factor localizes in the nucleus and has transcriptional activation activity.4.By constructing a plant overexpression vector and overexpressing Va MYC2 gene in V.vinifera cv.‘Thompson Seedless’,three transgenic grape lines were obtained by detections of q RT-PCR and Western blot.After 41 h of low temperature stress at-1℃,it was found that compared with the wild type(WT),the transgenic lines showed less freezing injury,lower electrolyte leakage and malondialdehyde content,but higher contents of soluble sugar,soluble protein and proline,as well as antioxidant enzyme activities(SOD,POD and CAT).Meanwhile,the expression levels of some cold tolerance-related genes(CBF1,CBF2,CBF3,KIN2,COR15 and P5CS)were significantly increased in transgenic lines.These indicated that overexpressing Va MYC2 improved the cold tolerance of transgenic ‘Thompson Seedless’ grapes.5.Through yeast two-hybrid assay and bimolecular fluorescence complementation assay,it was confirmed that Va MYC2 interacts with Va JAZ1 and Va JAZ7,respectively.Through yeast one-hybrid assay and dual luciferase assay,it was found that Va MYC2 can bind and activate the promoters of Va CBF1 and VaP5CS.