Isolation And Functional Analysis Of Drought Stress Responsive Gene TaWNK In Wheat

Plants have suffered from biological stresses,such as pests and plant disease,and abiotic stresses,like drought,high temperature and high salt,resulting in a decline in crop yields and quality.Therefore,it is important to cultivate new resistant crops.In the long-term adaptive process in adversity,plants have developed a variety of mechanisms to deal with adversity,which mainly involves adverse signals transmission in plants.Among them,the reversible phosphorylation of protein kinase plays an essential role in signal identification and transmission between cells.Protein phosphorylation is defined as the process that protein kinase transfers phosphate group of ATP to the amino acid residues,divided into itself phosphorylation and substrate phosphorylation.It is a kind of mechanism which is widely existed in biology,and it involves almost all physiological and biochemical processes.Thisstudy cloned a protein kinase gene from wheat drought transcriptome belonging to With No Lysine Kinase(WNK)familyand was named as TaWNK.The molecular characteristics of TaWNK were clarified through preliminary bioinformatics analysis,subcellular localization and expression pattern analysis.And we screenedcDNA libraries in wheat by yeast two-hybrid systemand examined the interactions between proteins by yeast two-hybrid system and Bimolecular Fluorescence Complementation(BiFC).Furthermore,the resistance identification of TaWNK gene under different abiotic stress conditions was investigated by using transgenic arabidopsis resistance identification experiment.The main conclusions are as follows:(1)The total length of CDS(Coding Sequence)is 968 bp,including two exons and one intron(exons and introns distributed alternately),as well as 5 'noncoding region(3 bp)and 3' noncoding region(337 bp).And it encodes 322 amino acids,the molecular weight is about 36.35 kD and isoelectric point(PI)is 6.90.It contains a serine/threonine kinase conservative region,which shows that the gene is a member of the serine/threonine kinase family and belongs to the WNKs family.Secondary structure of its protein is composed of 9 alpha helixes and 3 beta structures,and the signal peptide is shown in the 6-7 position of the amino acid sequence.The signal peptide sequence is M****T.Phylogenetic tree and sequence conservative analysis showed that WNK gene was very conservative in plants.From the cis element analysis in its promoter region,we can guess TaWNK may involved in ABA signaling pathways,the change of the light cycle,endosperm expression and drought stress response,and may respond to temperature changes,involved in gibberellin signaling pathways.(2)TaWNK was located in cytoplasm.TaWNK gene was induced by the abiotic stresses,such as drought,high temperature,low temperature and high salt,and also induced by ABA and BR,which were some plant hormones.Thus,it can be guessed TaWNK may participate in a variety of signaling pathways.(3)Overexpression TaWNKArabidopsiscould promote the resistance of Arabidopsis to ABA,drought and NaCl,but can make the Arabidopsis be sensitive to low temperature.This shows that TaWNK may play a rolein the ABA signaling pathwayand drought,high salt,low temperature and other adversity stresses.(4)Yeast two hybrid system and BiFC were used to prove the interaction between Ta WNK and two candidate protein Ta C2DP1 and TaPROF1,respectively.And studies have shown that TaC2DP1 is dependent on the ABA stress signaling pathways,so we guess TaWNK gene also plays a certain role in the ABA signaling pathways.