Clonging And Functional Analysis Of A Dehydrin Gene WZY2-1 In Wheat

Abiotic stress factors – cold,drought,salinity – severely limit plant growth and development as well as the yield of crops.Low temperatures,drought and salinity stress factors associated with plant cell dehydration.Dehydration stress factors can induce some of the cell response.Plant cell response to dehydration includes an accumulation of osmotic regulatory compounds including hydrophilic proteins such as dehydrins.Several physiological studies suggested that plant stress response have a positive relationship between the levels of accumulation of dehydrin transcripts.Wheat is main crop in china and the yield was limited by abiotic stress.Therefore,it is significant to study the stress tolerance of wheat to increase grain yield.In this study,we used wheat of Zhengyin NO.1 as plant material.We successfully isolated the complete c DNA sequence of WZY2-1 by using primers of F1 and R1.We analysed the expression of WZY2-1 gene in different stresses by q RT-PCR.We also studied the subcellular localization of WZY2-1 protein by using GFP fusion protein.In order to search the protective effect of WZY2-1 protein in E.coli,the WZY2-1 gene was transformed into E.coli strain and the WZY2-1 protein was expressed in E.coli strain BL21(DE3).The WZY2-1 protein was purified and the activity of Lactate dehydrogenase was measured in temperature stresses.The WZY2-1 gene was overexpressed in Arabidopsis thaliana that established the foundation for further studying the function and mechanism of dehydrin proteins in plants.The results of the experiment are as follows:1.In this study,we isolated a novel Kn-type dehydrin gene from wheat cultivar Zhengyin 1,which was designated WZY2-1.The ORF of WZY2-1 is 1740 bp,encoding 579 amino acids containing nine conserved K-fragments.2.Sequence alignment indicated that WZY2-1 had high homology to DHN5 gene in Hordeum vulgare.The WZY2-1 protein is predicted to be a highly-hydrophilic and disordered protein.3.The q RT-PCR assay indicated that the expression of WZY2-1 gene was induced by low temperature,PEG,and salt stresses rather than ABA.Thus,we conclude that WZY2-1 is an ABA-independent gene.4.The WZY2-1 protein expression was successful in E.coli strain BL21(DE3).We found that WZY2-1 protein can improve the tolerance to temperature,salt and osmotic stresses in E.coli.5.The p BI121-WZY2-1-GFP vector was constructed successfully,and the vector was transformed into onion epidermal cells.We found that the WZY2-1 protein was mainly localized in nucleus and cell membranes.6.The WZY2-1 protein was purified successfully,and we found that WZY2-1 protein can protect the activity of Lactate dehydrogenase in temperature stress.7.The p BI121-WZY2-1 vector was also constructed and transformed into Arabidopsis thaliana,We obtained transgenic plants by using antibiotic,genome DNA PCR and q RT-PCR.The T4 generation plants was finally obtained,and we found that WZY2-1 genes can improve drought tolerance of transgenic Arabidopsis thaliana...