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

The MADS-box genes were named after four of the originally cloned members- MCM1,AG,DEFA and SRF - which all possess a highly conserved 56 amino acid motif within their DNA-binding domains. MADS-box genes have been isolated from numerous eukaryotic organisms and encode transcription factors involved in growth and development.The MADS region linked specifically to the coalescent site of target gene and formed a homogenous or heterogenous dimer to regulate it,which can make different kinds of floral organs for many flowering plants.So they are important factors to control the development of floral organs and to make homeotic transformations.It is very useful to analyze the floral organ identity genes in order to understand the molecular model of plant floral organ development.The PMADS9 gene fragment is a member of AGLI5 subfamily and it has been newly discovered in petunia flowers.The PMADS9 and the AGL15 belong to MADS-box gene family together.But function of The PMADS9 gene has not been reported.At present,the PMADS9 gene fragment was amplified by meas of PCR using the Petunia hybrida young floral bud cDNA as template(GenBank accession number: DQ418547).On this basis using the meas of PCR we cloned the entire gene sequence of the PMADS9(GenBank accession number:EU338501).Now we have conducted the PMADS9 gene validation and analysis preliminarily and gained some results.In order to more fully study the PMADS9 gene,we continue to clone its gene promoter region.We believed that analyzing the PMADS9 gene promoter Contributed to further analysis and understanding the PMADS9 gene and further confirmed the function of the AGL15 subfamily gene and revealed the differences in the function of plant species.In this study,using multi-template "YADE" method amplified the PMADS9 gene promoter sequences.Y-shaped adaptor dependent extension(YADE)method is a useful tool to amplify the flankingsequence of a known DNA sequence,but its efficiency is frequently limited by the restriction sites around the known sequence.In this paper,we demonstrated that using multiple templates derived from several restrictions and ligations could dramatically increase the efficiency of YADE method and render it suitable for sequential amplification of flanking sequences.We collected the DNA from the Petunia leaf and cloned the Petunia PMADS9 gene promoter sequences(GenBank accession number:FJ798977).Through on-line analysis revealed that the promoter sequences not only had the general structural features but also had its own characteristics.For example:TATA-BOX,CAAT -BOX,ACGTA-BOX are all the general conservative promoter; G-BOX,Skn-1-motif,TCA are all specific cis-acting elements of the Petunia PMADS9 gene promoter.And in this experiment we framed cloning vector pMD-prom,transient expression vector pMD-prom-Gus and expression vector pCAMBIA1381Z-prom.Through biolistic transformation we preliminarily found that this sequences had promoter activity.