Research On The Stress Tolerance Function Of VaCP17 And VaSAP15 Genes From Chinese Wild Vitis Amurensis Rupr.

Grape（Vitis spp.）is one of the most important fruit crops in the world.European grape（V.vinifera L.）is widely cultivated in the world with good quality but poor stress tolerance.Low-temperature and salt stress are the main stress factors affecting the growth and development of grapes,which seriously restrict the development of the grape industry.China is one of the original producing areas of grapes and has abundant wild grape resources.Among them,V.amurensis Rupr.,which is cold-tolerant and salt-tolerant,is a very important stress-resistant gene resource.In our previous studies,two genes,cysteine protease gene VaCP17 and stress-associated protein gene VaSAP15,were isolated from V.amurensis accession‘Shuangyou’after cold stress,and then the cold-tolerance function of VaCP17 in Arabidopsis thaliana and VaSAP15 in V.vinifera cultivar‘Thompson Seedless’was analyzed,respectively.On this basis,this study further verified the cold-tolerance function of VaCP17 in‘Thompson Seedless’and screened its upstream regulatory factors;analyzed the salt-tolerance function of VaSAP15 in‘Thompson Seedless’and revealed the upstream and downstream regulatory pathways with VaSAP15 as the hub,which laid a foundation for finally clarifying the molecular mechanism of cold and salt tolerance of V.amurensis and carrying out molecular breeding of grape stress tolerance.The main results are as follows:1.VaCP17 plays a positive role in the cold tolerance of grapes.Subcellular localization and bioinformatics analysis of VaCP17 presented that it is a plasma membrane-localized RD21 protein,and its N-terminus has a transmembrane structure and a signal peptide.Real-time quantitative PCR（RT-qPCR）results portrayed that VaCP17 responded to low-temperature（-1℃）,abscisic acid（ABA）,methyl jasmonate（MeJA）,and salicylic acid（SA）.The cold tolerance function of VaCP17 was verified by stable overexpression of VaCP17 in non-cold-resistant V.vinifera cv.‘Thompson Seedless’and its transient silencing in extremely cold-resistant V.amurensis accession‘Shuangyou’leaves.The results showed that under cold stress（-1℃）,compared with the wild type（WT）,the‘Thompson Seedless’grape plants stably overexpressing VaCP17 improved cold tolerance by reducing relative electrolyte leakage（REL）and malondialdehyde（MDA）accumulation,maintaining chlorophyll content and increasing SOD,POD,and CAT activities,and this process was accompanied by the significant up-regulation of stress-related genes（VvKIN2,VvRD29B and VvNCDE1）.However,the transient silencing of VaCP17 mediated by RNA interference exposed opposite physiological and biochemical responses compared with the‘Shuangyou’leaves carrying the empty vector,resulting in increased damage to‘Shuangyou’leaves.2.Transcription factor VaNAC72 positively regulates VaCP17 by recruiting interaction protein VaCBF2 or VaCP17.By analyzing the cis-acting elements of the promoter of VaCP17（ProVaCP17）,the grape NAC72 transcription factor recognition site（CACGTG）was identified.Yeast one-hybrid（Y1H）,Dual luciferase reporter assay（DLR）,transient overexpression of VaNAC72 in‘Shuangyou’leaves,and RT-qPCR analysis showed that VaNAC72,an interacting protein of VaCP17,could bind to ProVaCP17 and promote the expression of VaCP17.RT-qPCR analysis exhibited that VaNAC72 was also up-regulated by low-temperature,ABA,MeJA,and SA.The analysis of the cold tolerance function of VaNAC72 showed that under cold stress（-1℃）,the leaves of‘Shuangyou’overexpressing VaNAC72 exposed the same physiological and biochemical responses as those of the overexpressing VaCP17 grape plants compared with the‘Shuangyou’leaves carrying the empty vector.Subsequently,Yeast two-hybrids（Y2H）,Bimolecular fluorescence complementation（BiFC）,and Luciferase complementation assay（LCA）were used to confirm that VaNAC72 can form homodimers or heterodimers with itself or VaCBF2 in the cell nucleus.RT-qPCR analysis displayed that VaCBF2 could respond to low-temperature.Further co-expression analysis demonstrated that VaNAC72 could synergistically enhance the activity of ProVaCP17 with VaCBF2 and VaCP17,respectively.3.Overexpression of VaSAP15 enhances the salt tolerance of transgenic‘Thompson Seedless’grape.Through subcellular localization and bioinformatics analysis of VaSAP15,it was shown that VaSAP15 is a hydrophilic protein with cell nuclear and membrane localization containing the AN1 domain,and amino acid sequence alignment disclosed that VaSAP15 is homologous to the encoded protein of At SAP12 induced by salt and low-temperature stresses.The expression profile of VaSAP15 was analyzed by RT-qPCR,and the results showed that it was induced by salt（100 m M Na Cl）,low-temperature,and ABA.The wild-type and VaSAP15overexpressing‘Thompson Seedless’grape potted plants were irrigated with 100 m M Na Cl solution for 16 d to determine the relevant physiological and biochemical indicators.The results manifested that compared with the WT,the chlorophyll content,leaf relative water content,shoot length,and root length of VaSAP15-overexpression plants increased,the proline content and antioxidant enzyme（SOD and CAT）activities increased,and the REL level,MDA content,and reactive oxygen species level decreased,indicating that overexpression of VaSAP15 significantly improved the salt tolerance of transgenic‘Thompson Seedless’.RT-qPCR analysis exhibited that this process was accompanied by a significant up-regulation of stress-related genes（VvDREB2A,VvRD22,VvP5CS,and VvRD29B）.4.The transcription factor VaZAT10 positively regulates the expression of VaSAP15.The protein encoded by VaSAP15 gene degrades VaCOP9 through the 26S protease system,and VaZAT10 inhibits the expression of VaCOP9.A C₂H₂ zinc finger protein named VaZAT10 was identified by analyzing the cis-acting elements of the VaSAP15 promoter（ProVaSAP15）and its potential upstream transcription factors.Subsequently,Y1H,DLR,RT-qPCR,and tissue-specific analysis confirmed that VaZAT10 could mediate the up-regulated expression of VaSAP15.By analyzing the expression pattern of VaZAT10,it was shown that the gene was also induced by salt,low-temperature,and ABA to up-regulate its expression.Furthermore,Y2H,LCA,BiFC,molecular docking,and Co-immunoprecipitation confirmed that VaSAP15could interact with VaCOP9 in vivo and in vitro.Protein degradation experiments illustrated that the abundance of VaCOP9 could be inhibited by VaSAP15.In addition,RT-qPCR analysis of the expression of VaCOP9 under salt stress displayed that it was down-regulated after treatment.Pearson correlation analysis,Y1H,DLR,and RT-qPCR analysis confirmed that the transcription factor VaZAT10 can negatively regulate the expression of VaCOP9.