The Functional Study Of Drought-Responsive Protein WZY2 And WZY3-1

Water supply is extremely vital for plant growth and development.Drought stress caused by water deficiency is a main factor limiting the plant distributions and threatening the food security.As one of the major food crops,wheat always undergoes abiotic stresses such as drought and extreme temperatures in its life cycle.Thus,the discoveries of abiotic stress-related gene will definitely provide significant guidance in abiotic stress tolerance improvement in wheat.LEA(Late embryogenesis abundant)protein family is a group of proteins that positively regulate abiotic stress tolerance in plant.LEA proteins are divided into seven subfamilies according to their conserved motifs.These proteins are rich in lysine,histidine and glycine,and are highly hydrophilic and thermostabe.According to revent studies,LEA proteins are widely located in cell nucleus and cytoplasm.In this study,we analyzed the functions of a LEA2 gene WZY2 in RNAi-mediated transgenic wheat(WZY2-RNAi)and summarized the DEGs(Differentially expressed genes)in wild-type(WT)and WZY2-RNAi wheat.Through NMR(Nuclear magnetic resonance)and CD(Circular dichroism)methods,we explored the protein structure variations under PEG(Polyethylene glycol),cold,heat and metal ions treatments.Also,the RNA-Seq was conducted to discover the molecular mechanisms and new LEA genes in wheat cultivar Zhengyin 1.The main results are as follows:1.Under drought stress,the contents of water,chlorophyll,and proline were decreased in WZY2-RNAi leaves than that of WT.The activities of SOD(Superoxide dismutase)and POD(Peroxidase)were also reduced,and the accumulation of MDA(malondialdehyde)is increased in WZY2-RNAi.Through RNA-Seq in WT and WZY2-RNAi under drought stress,we discovered 1214 DEGs,with 427 upregulated and 787 downregulated.These DEGs were mainly enriched in “abiotic stimuli response” and “photosynthesis” in GO(Gene ontology)terms.In addition,the DEGs also mainly enriched in “photosynthesis” and “MAPK signal pathway” in KEGG(Kyoto Encyclopedia of Genes and Genomes)terms.2.Through NMR and CD spectrum methods,we confirmed the disordered properties of WZY2 and WZY3-1 in solution state.In WZY2,the proportion of disordered structure was above 60% under room temperature.However,the proportion of disordered structure of WZY2 continued to decrease under PEG,temperature and metal ions treatments,with the dramatically increased in ?-helixstructure.In truncation assay of WZY2,the proportion of ?-helix structure increased only with the present of K-segment under treatments mentioned above.The whole WZY3-1 also exhibited similar structural variation trends with WZY2.3.RNA-Seq data were summarized to further investigate the gene expression patterns of Wheat “Zhengyin 1”under drought stress.As a result,5948 DEGs were identified,with 2693 genes upregulated and 3255 genes down-regulated.In GO terms,the DEGs are mainly enriched in “Chloroplast organization”,“Metabolic process”,“Photosynthesis”,“Abiotic stimuli response”,“Osmotic stress response”;“Thylakoid”.And the DEGs were also significantly enriched in “Starch and sucrose metabolism”,“Peroxisome”,and “Porphyrin and chlorophyll metabolism”,in KEGG terms.We also confirmed 15 new LEA genes through BLAST and motif search in RNA-Seq,and cloned the ORF(Open reading frame)of a LEA gene WZY2-2 within the RNA-Seq data.Our results indicated WZY2-2 is inducible to drought,cold and heat stress,and the WZY2-2 protein was mainly localized in cytoplasm.