Drought-resistance Functional Verification And Regulatory Factors Screening Of VyP5CR Gene From Chinese Wild Vitis Yeshanensis

Grape（Vitis vinifera L.）is an important fruit crop with large planting area in the world.The cultivated area and yield of grape in China are among the top in the world,and the arid and semi-arid regions in northwest China are the most important grape producing areas in China.The low rainfall and insufficient soil moisture have seriously affected the growth and development of grape,resulting in a significant decline in grape quality and yield,and restricting the further development of grape industry.Therefore,improving drought tolerance of grapes is an important objective in grape breeding.China is one of the origin centers of Vitis in the world and is rich in wild grape resources.Among them,Vitis yeshanensis accession‘Yanshan-1’is originated from Tashan,north of Shanhaiguan,Hebei province of China,and has strong drought resistance,which is a very important drought resistant gene resource.Research and utilize of this germplasm resource is of great significance to excavate its drought resistance gene and breed new varieties of drought-resistant grape through molecular breeding to solve the production problems.In a previous study,we carried out a transcriptome sequencing for the drought-resistant grapevine‘Yanshan-1’and the drought-sensitive grapevine‘He’an（♀）’under drought stress,and obtained a differentially expressed gene,i.e.L-pyrrolline-5-carboxylate reductase（P5CR）,which is a key gene for proline synthesis.The heterologous expression of VyP5CR gene in Arabidopsis was found to enhance drought resistance of plants.On this basis,in order to verify the drought tolerance function of VyP5CR gene in grapevine,it was overexpressed in V.vinifera cultivar‘Thompson Seedless’.In addition,upstream transcription regulatory factors of VyP5CR were screened by yeast one-hybrid and luciferase assays.These results will provide a foundation for further revealing the drought resistance mechanisms of VyP5CR gene,and for its application in molecular breeding of drought resistance in grape.The main results obtained are as follows:1.It was found that VyP5CR located in cell membranes and the cytoplasm by instantaneously transferred of VyP5CR gene into Arabidopsis protoplasts and subcellular localization analysis;followed,CaMV35S-VyP5CR-GFP was co-localized with AtSYP121（AT1G04750）,a protein located in the Golgi and plasma membrane,and AtVAMP721（AT3G11820）,a protein located in the cell membranes and endomembranes of Arabidopsis,and found that VyP5CR overlapped with AtSYP121 and with AtVAMP721,indicating VyP5CR localization in Golgi,endomembranes and plasma membrane.2.According to real-time quantitative PCR（qRT-PCR）analysis,the relative expression levels of P5CR gene in different tissues of roots,stems,young leaves,mature leaves,inflorescence,young berries and tendrils from drought-resistant grapevine V.yeshanensis acc.‘Yanshan-1’and drought-sensitive V.riparia acc.‘He’an（♀）’were detected.The results showed that P5CR was expressed in all tissues of‘Yanshan-1’and‘He’an（♀）’,and its expression was the highest in roots.3.The potted plants of V.yeshanensis acc.‘Yanshan-1’was treated with exogenous hormones（ABA,MeJA and SA）and cold stress（–1℃）,and the relative expression of VyP5CR was analyzed by qRT-PCR at different stress time points.The results showed that,within 1 h of ABA treatment,the expression of VyP5CR was rapidly up-regulated,then decreased by 4 h and 8 h,increased again at 12 h.MeJA induced the expression of VyP5CR,while SA,on the contrary,inhibited the expression of VyP5CR at some time points.VyP5CR was induced by the treatment with–1℃,but the expression of VyP5CR was not significantly up-regulated until 48 h after cold treatment.In summary,VyP5CR gene can be induced by exogenous hormones ABA,MeJA and SA and low temperature in addition to drought induction.4.The VyP5CR gene was transformed into V.vinifera cv.‘Thompson Seedless’through the Agrobacterium-mediated method,and OE-17,OE-23,and OE-75 three transgenic grape lines were obtained by Kan resistance screening,transcription（qRT-PCR detection）and protein level（Western blot detection）identification.The potted seedlings of wild-type（WT）and three transgenic grape lines were treated with drought stress.The results showed that compared with WT,transgenic lines were phenotypically more tolerant to drought,and they wilted much less than the WT lines under drought stress,with smaller stomatal apertures and longer taproots.Furthermore,transgenic lines had lower electrolyte leakage,malondialdehyde（MDA）,H₂O₂and O₂^-content,but higher leaf relative water content（leaf RWC）,proline content,peroxidase（POD）,superoxide dismutase（SOD）and catalase（CAT）enzyme activities and more stable photosynthetic efficiency in terms of physiological and biochemical indexes.In addition,under drought stress,the overexpression of VyP5CR in transgenic lines induced the up-regulated expression of some drought-resistant genes（DREB2A,ERD14,KIN2,RD22,RD29B,NAC72,NCED1,P5CS and OAT）.These results indicates that overexpression of VyP5CR gene could improve the drought tolerance of transgenic grape.5.Through yeast one-hybrid and luciferase activity assays,we found that MADS-box transcription factors VyAGL42 and VyMADS23 could directly bind to the promoter of the VyP5CR gene and activate its transcription.Therefore,VyAGL42 and VyMADS23 might synergistically regulate drought resistance of grape with VyP5CR.