Cloning And Functional Analysis Of The Promoter Of PMADS9 Gene From The Petunia Hybrida Vilm

MADS-box genes are a class of homeotic genes widely distributed in plants and the expression of MADS-box genes are discoveried in different tissues, organisms, stages of growth and development.They belong to an ancient gene family and are a kind of important regulatory factors during the growth and development of plant. At present,the research of MADS-box genes regulatory mechanism has become a hot topic in the field of plant molecular biology. PMADS9 gene of Petunia is a member of AGL15 subfamily which might regulate flowering time, inhibit floral organ senescence,and promote the formation of somatic embryos.By RACE technology and YADE method,we had cloned coding region of PMADS9(GenBank accession number:DQ418547) from RNA Petunia flower buds and obtained 1053bp promoter sequence (GenBank accession number:FJ798977) upstream the 5'translation start site(ATG) from DNA of leaves. Then designing primers at both ends of the sequence and using PCR method, we got the full-length sequences of PMADS9 gene,5861bp (GenBank accession number EU338501). RACE analysis revealed that the gene has at least four transcription start sites(TSS),two of which located within the first exon of the coding region.In this study,we continued to amplify the upstream unknown promoter region of PMADS9 gene,and obtained a longer 800bp sequence.The preliminary studies of the promoter functions are great significant for understanding the characteristics of expression regulation of PMADS9 gene,as well as more in-depth studying the molecular mechanisms of floral organ development. In this paper,the main experimental results are as follows:1.On this basis of 1053bp promoter sequence,we used hiTAIL-PCR technology to amplify the unstream unknown promoter sequence and obtained a longer 800bp fragment.Through the comparion and splice of the new sequence and the original 1053bp by SeqMan software, we ultimately acquired the 1853bp promoter sequence of PMADS9 gene.2.Cis-regulatory elements of the PMADS9 promoter sequence predicted by PLACE and PlantCARE online analysis software are related with seed and pollen development and environmental response.Analysis of promoter sequences from AGL15-clade MADS-box genes by FootPrinter,showed that very conserved RY-repeat motifs were exist amony them,and the conversation of promoters between So lanaceae and the selected 5 species of Rosids is higher than that between Brassicaceae and the same selected species even though So lanaceae is less closely related to Rosids than Brassicaceae.Furthermore,the results also suggested that regions of 200～400bp and 800～1000bp upstream of the ATG were functionally important.3.According to the cis-regulatory elements locations on the promoter,we designed different primers and used PCR method to obtain the 5'end deletion fragments of PMADS9 promoter.New restructuring expression vectors were constructed by replacing the CaMV35S promoter of pCAMBIA1305.1 with the 6 deletion fragments respectively to drive the expression of the reporter gene GUSpus..The new vector was transferred into Agrobacterium to infect leaf disks of Petunia.The results of GUS staining and PCR identification indicated that the 6 promoter fragments were successfully transferred into the receptor plants.4.After transgenic plants flowering,the analysis results of GUS histochemical staining showed that the full-length promoter sequence(1853bp) can start GUS gene high express in stamens,pistils and ovarys.On the contrary,GUS activity was not detected in the stems,leaves,bracts and calyxes.This is consistent with the previous results of PMADS9 gene expression analysis.Among the different deletion fragments,there no significant difference in expression patterns.The shortest deletion promoter fragment(298bp) was able to start the GUS reporter gene expression in transgenic plants,indicating that it contained a variety of basic regulatory elements required for promoter.