Screening And Confirmation Of The Upstream Regulator Elements For ZmLTP3 Gene In Maize

Abiotic stresses are the major factors affecting the yield of agricultural crops.Researches on the molecular regulation mechanism of crop responding to abiotic stresses are of great importance to increase crop yield.Researches had showed that lipid transfer proteins are involved in many biological processes,such as synthesis and transport of wax,extension of cell wall,development of reproductive organs,abilities of plants to resist biological stress,the regulation of the degradation activity of pectin and so on.Whereas,researches about abiotic stress were rarely reported.Previous studies results showed that overexpression of ZmLTP3 gene in maize could improve the ability of drought and salt tolerance in maize.However,the mechanism of gene expression regulation of plant abiotic stress is still unknown.Through taking the promoter of ZmLTP3 gene as template amplifying different promoter sequence,then ligated those promoter sequence with the expression vector pGreen0029-kan-GUS.By using agrobacterium tumefactions infection method infect Arabidopsis thaliana,then through GUS staining method aim to ascertain the core sequence of the promoter in ZmLTP3.The core promoter fragment was connected with the pHIS2 vector to construct the bait plasmid,and the cDNA library was constructed with maize B73 material.Transcription factors were screened by yeast one-hybrid.method.cDNA sequence of the screened transcription factors was constructed into pET28a+ vector and expressed in the Transetta(DE3)strain.The following results were obtained:1.The plant expression vector containing different promoter fragments were constructed to infect Arabidopsis thaliana by agrobacterium tumefactions infection method,and leafs of Arabidopsis thaliana were analyzed by GUS staining.The leaves of transgenic Arabidopsis thaliana introduced into the second promoter sequence can be stained blue through GUS dye.Thus,the second amplified fragment was observed as the core sequence of 2mLTP3 gene promoter.2.The bait plasmid and library plasmid were transformed into yeast Y187 receptive cells by using yeast one-hybrid method.Then through screening,sequencing and sequence alignment,.indicated that the observed library plasmid sequence keep highest similarity with ZmLSDl.The result showed that the transcription factor that could interact with the core sequence of ZmLTP3 gene promoter was ZmLSDl1 Result showed that ZmLSD1 is exactly the one which can interact with the core sequence of ZmLTP3 gene promoter3.ZmLSD1 protein existing in inclusion body was induced by prokaryotic expression method.