Isolation And Functional Analysis Of Malus Baccata (L.) Borkh. WRKY Transcription Factors MbWRKY50 And MbWRKY65

During natural growth,plants can be affected by harsh external environments such as cold,frost,drought and salt damage,which can hinder their growth and development.Malus baccata(L.)Borkh.is a common rootstock of the apple genus,which grows mainly in northeastern China and has strong resistance to low temperature and drought.The WRKY family of transcription factors is one of the largest families of transcription factors in plants,and several studies have found that WRKY proteins can efficiently bind to cis-elements of the W-box to activate or repress transcription of downstream target genes to regulate different abiotic and biotic stress responses and plant development,such phenomenon that has been systematically analyzed in Arabidopsis thaliana,tomato,soybean,and cucumber.Therefore,in this experiment,M.baccata was chosen as the material;two WRKY family genes,MbWRKY50 and MbWRKY65,were obtained by gene cloning technique,and to conduct resistance studies on these two genes,and the following conclusions were drawn:(1)The MbWRKY50 gene is 486 bp long and encodes 161 amino acids,MbWRKY50 protein has a theoretical molecular mass(MW)of 38.46 k Da,a theoretical isoelectric point(p I)of 5.27,and an average hydrophilicity coefficient of 0.661,and the MbWRKY50 amino acid sequence contains amino acid sequences and zinc finger motifs unique to the WRKY transcription factor family,phylogenetic tree analysis indicated the highest homology with the apple Md WRKY50protein;the MbWRKY65 gene is 822 bp long and encodes 273 amino acids,the MbWRKY65 protein has a theoretical molecular mass(MW)of 68.42 k Da,a theoretical isoelectric point(p I)of5.03,and an average hydrophilicity coefficient of 1.077,and the MbWRKY65 amino acid sequence contains amino acid sequences and zinc finger motifs unique to the WRKY transcription factor family,phylogenetic tree analysis indicated the highest homology with the apple Md WRKY65 protein;structural analysis of MbWRKY50 and MbWRKY65 proteins revealed their structural similarity to other WRKY family proteins;indicating that MbWRKY50 and MbWRKY65 are members of the WRKY gene family.(2)Subcellular localization using a GFP fusion protein expression assay showed that both proteins are localized to the nucleus,which is consistent with their function as transcription factors.(3)The results of real-time fluorescence quantitative PCR showed that MbWRKY50 and MbWRKY65 were expressed in young leaves,old leaves,roots and stems of M.baccata,but the expression level was slightly different in different parts.Five abiotic stresses,including cold,high-salt,dehydration,heat and ABA,were able to induce the expression of MbWRKY50 and MbWRKY65 genes,and both genes were more sensitive to cold and drought stress.(4)Expression vectors were constructed by homologous recombination technology and transferred into the model plant A.thaliana and horticultural crop tomato,were subjected to cold and drought stress treatment,determination of relevant physiological indicators in wild type(WT),empty load(UL)and transgenic A.thaliana and tomato under low temperature and drought treatments,respectively.The results showed that the transgenic plants were less damaged and had higher chlorophyll and proline contents,higher SOD,POD and CAT activities and lower malondialdehyde,hydrogen peroxide and superoxide anion radical contents and relative conductivity after stress treatment compared to WT and UL lines.It is indicated that the transgenic plants had greater tolerance to low temperature and drought stress.(5)To further analyze the role of MbWRKY50 and MbWRKY65 genes in low temperature and drought stress,expression analysis of several resistant genes downstream of transgenic A.thaliana and tomato was performed,revealed that the expression level of downstream stress-related genes was more significantly up-regulated in the transgenic A.thaliana and tomato relative to WT and UL lines under low temperature and drought treatments,suggesting that these two genes may go through ABA-induced pathway to activate the expression of downstream stress-related genes and thus improve the cold and drought tolerance of the transgenic plants.Therefore,it can be concluded from this study that MbWRKY50 and MbWRKY65 genes have a facilitative role in plant tolerance to low temperature and drought stresses.This discovery both provides a basis for the application of M.baccata in low temperature and arid environments and enriches the WRKY transcription factor family.