Function And Molecular Mechanisms Of The Transcription Factors VaMYB44 Form Vitis Amureusis In Response To Cold Stress

Grape is one of the most important fruit tree crops in the world.Vitis vinifera is a grape species widely cultivated throughout the world with excellent fruit quality but poor cold resistance.Cold injury is a prominent problem in grape production.Another species,V.amurensis,can resist temperatures as low as-40°C,so it is widely used to breed cold-tolerant grape varieties.It is important to study and explore the cold-resistant genes of Vitis amurensis for understanding its molecular mechanism of cold tolerance and grape molecular breeding.In our group previously studies,a differentially expressed cold-resistant transcription factor Va CBF6 gene was obtained from V.amurensis by transcriptome sequencing analysis for V.vinifera cv.‘Red Globe’(cold-sensitive)and V.amurensis acc.‘Shuangyou’(cold-resistant)after cold treatment,and then the Va ADC interactiing with Va CBF6 was screened.Furthermore,VaMYB44 interactiing with Va ADC was screened.On this basis,in order to reveal the role of VaMYB44 gene in plant cold resistance,we cloned the VaMYB44 gene from V.amurensis,and heterologus expressing it in Arabidopsis thaliana and V.vinifera cv.‘Thompson Seedless’.Meanwhile,the interaction proteins of VaMYB44 and the upstream regulatory factors of VaMYB44 gene were screened to preliminarily reveal the molecular mechanism of VaMYB44 regulating grape cold resistance.The main results obtained are as follows:1.VaMYB44 gene was cloned from the cold-tolerant V.amurensis acc.‘Shuangyou’,and the ORF of VaMYB44 was 942 bp and encoded a putative protein of 313 amino acids.The results of sequence analyzing showed that the N-terminus of VaMYB44 contains the conserved structural domains of R2 and R3,and VaMYB44 belongs to the R2R3-MYB transcription factor family.Subcellular localization of VaMYB44 indicated that VaMYB44 is localized in the cell nucleus,and VaMYB44 assumed the function of transcription activation in yeast,and its self-activating region was located in the C-terminus.VaMYB44 was expressed in various organs,with lower expression in the stems and young leaves.The expression of VaMYB44 was induced by ethylene,abscisic acid,and methyl jasmonate.Exposure of the cold-sensitive ‘Thompson Seedless’ and cold-resistant ‘Shuangyou’grapevines to cold stress(-1°C)resulted in differential expression of MYB44 in leaves with the former reaching 14 folds of the latter after 3 h of cold stress.2.By constructing a fusion expression vector,VaMYB44 was transformed into Arabidopsis and V.vinifera cv.‘Thompson Seedless’ to obtain transgenic Arabidopsis and transgenic grape plants.The results showed that compared with their wild-type,expression of VaMYB44 in Arabidopsis and ‘Thompson Seedless’ grape increased freezing damage;the transgenic plants had higher MDA content and electrolyte permeability,and lower activities of SOD,POD,and CAT;the expression levels of some cold-related genes(CBF1,CBF2,CBF3,RD29 A,NCED3 and KIN1)were decreased.3.Va MYC2 and Va TIFY5 A,two interacting proteins of VaMYB44,were screened by yeast two-hybrid assay.Then,the interaction relationship between VaMYB44 and its interacting proteins was verify in yeast and in plant by yeast two-hybrid assay and bimolecular fluorescence complementation assay.4.The promoter of the VaMYB44 gene was cloned from ‘Shuangyou’,and some stress-related cis-acting elements including ABRE,TATC-box,TCA-element,TGA-element,etc.are contained in the promoter of VaMYB44 gene.Va MYC2 can bind directly to and activate the promoter of VaMYB44 by yeast one-hybrid assay and dual luciferase reporter assay.