Analysis On PhGRP Promoter Deletion Mutation And Its Binding With Transcription Factors

Petunia(Petunia hybrid),as the first widely used bedding plants,its research direction has been extended to multiple areas and has become an important model plant.As the most important part in gene regulation,promoters play a critical role in plant growth and response to the external environment,therefore,more and more energy were put into the promoter cloning and functional studies.Looking for anther/pollen-specific promoter,we can inhibit and destroy these specific genes which may lead to male sterility and which is one of the important methods to create male sterile using genetic engineering technology.Transcription factors are a very important class of proteins which play a significant role in enhancing or inhibiting genes.We have cloned the full-length of promoter PhGRP.In this study,we further construct the 5' deletion vectors and homologously transformed to petunia and heterologously transformed to Arabidopsis thaliana to find the key cis-acting elements.In addition,we use the yeast one-hybrid to verify the candidate transcription factors obtained from the Yeast one-hybrid library,for further research on PhGRP promoter regulatory networks.1.The construction of PhGRP promoter 5' deletion vectors and genetic transformation.Three 5' deletion fragments of PhGRP promoter fused with GUS reporter gene,pPhGRP-690::GUS,pPhGRP-509::GUS and pPhGRP-206::GUS are constructed successfully and transformed into petunia and Arabidopsis thaliana.7,7,11 transgenic petunia positive lines are obtained,respectively and 8,11,16 transgenic arabidopsis positive lines.pPhGRP-1220::GUS transformation obtain 20 petunia positive transgenic plants and 6 arabidopsis positive transgenic plants.The results of histochemical GUS staining indicate that pPhGRP upstream of the start codon-690bp to-1220bp is a key segment of the promoter specifically expressed in the anther,which comprise a core of pollen/anther-specific elements GTGA.2.Yeast one-hybrid experimental results show the existence of pPhGRP interaction with PhMYC2 and PhY10 two transcription factors in vitro.