The Screening And Identification Of Proteins Interacting With The Promoter Of WZY2 Gene In Wheat Dehydrin

Dehydrin as a type of stress response protein plays an important role in plant growth and development.When the plant is subjected to abiotic stresses such as low temperature,drought,high temperature and high salt,a highly hydrophilic protein can be produced which has a specific protective effect on cellular nucleic acids,intracellular proteins and membrane structures,which stabilizes the cell membrane system and many the structure of macromolecules.The high expression of dehydrin can increase the stress resistance of plants.There will be great significance to study the stress resistance mechanism of crops.In the study,WZY2,The dehydrin promoter was isolated from Zhenyin 1 cultivar of Triticum aestivum.The recombinant plasmid pAbAi-WZY2 was constructed and transformed into the E.coli.Using the method of homologous recombination,Construction of yeast bait vector(recombinant Y1H-pAbAi-WZY2),After self-activation verification,the wheat cDNA library was screened and its interaction with transcription factors was identified.Use online software to analyze and predict the properties of transcription factors.Real time quantitive PCR was used to demonstrate the expression of transcription factors under different abiotic stresses.We cloned the dehydrin WZY2 gene promoterand the full-length of promoter is 633 bp(Without ATG).The WZY2 gene promoter was predicted and the results showed that the cis-acting elements related to stress were ABA response elements(ABREs),anaerobic specific inducing elements(GC-motifs),low temperature response elements(LTREs),and methyl jasmonate.Response elements(TGACG motifs);Elements associated with photoresponse: AT1 motifs,G-boxes,GATA motifs,TGG motifs.Determined the background expression level of the yeast bait vector,17 transcription factors were selected from the wheat cDNA library.After verification by transactivation,There are 3 transcription factors that interact with the promoter elements WZY2.They are bHLH49-like(351),Zincfinger A20/AN1(407),and bHLH47-like(467),respectively.Online analysis and prediction of transcription factors indicate that: The CDS sequence of the bHLH49-like(351)gene is 783 bp and encodes 260 amino acids.Relative molecular mass: 28.85 ku,theoretical isoelectric point: 7.78.The CDS of the AN1-like Zinc finger(407)gene is 486 bp long and encodes 161 amino acids.Relative molecular mass: 17.74 ku,theoretical isoelectric point: 8.51.The CDS of the bHLH47-like(467)gene is 780 bp long and encodes 259 amino acids.Relative molecular mass: 29.15 ku,theoretical isoelectric point: 6.62.These transcription factors are highly hydrophilic and belong to hydrophilic proteins.Real-time quantitative PCR results showed that WZY2 generally showed an upward trend under different stresses.The three transcription factors obtained based on the WZY2 promoter all showed an increasing trend.It is speculated that WZY2 expression may be regulated by these transcription factors,but the results need to be further verified.The bHLH49-like(351),AN1-like Zinc finger(407),and WZY2 can all response to PEG,ABA and cold stress and were ABA dependent.However,bHLH47-like(467)can only respond to cold stress and is a non-ABA dependent transcription factor.According to the above study,The wheat dehydrin WZY2 gene promoter can bind to related transcription factors,thereby activating downstream reporter gene expression.To control the network of counter-revolutionary mechanisms.The study laid a theoretical foundation for the gene regulation of wheat drought tolerance.