CDNA Cloning And Prokaryotic Expression Of The Floral Scent Gene BSMT3 For Petunia Hybrida

Karyotype analysis of Petunia hybrida showed that the karyotype formulas was 2n=14=8m+6sm. All the chromosomes were median or submedian centromeric, with no SAT. The karyogram was belonged to the type of 2A, and it was midrange symmetric kernel. A new cDNA (GenBank DQ494491) was cloned by RT-PCR from the total RNA of Petunia hybrida petal and the RT-PCR products were subcloned into the pMD18-T vector, the recombinant plasmid was transformed into DH5a. the correct colonies was confirmed by restriction enzyme digestion and sequencing analysis. Its sequence was 1105bp in length and encoded a mature peptide with 357 amino acids. It is different from other reported Petunia hybrida. The bioinformatics softwares DNAtools 5.1 and DNAman5.1 were employed to analyze the difference of protian sequence from other reported Petunia hybrida and constructed phylogenetic tree.The BSMT3 cDNA fragment was recloned into the expression vector pGEX4T-1 in EcoRI and SalI sites. The resulting plasmid pGEX4T-1- BSMT (pGBSMT) was transformed into DH5αE. coli cells. The correct colonies was confirmed by PCR, then extracted their plasmid. The plasmid was confirmed by restriction enzyme digestion of EcoRI and SalI. The result showed the The size of bond revealed on the agarosegel is about 1110 base pairs (bp), equivalent to the length of the BSMT3. Then BamHI enzyme cut pGBSMT, the result showed that the size of bond is about 680bp, equivalent to the expectant size.The plasmid pGBSMT was transformed into BL21(DE3) and picked the correct colonies. The fusion protein GST-BSMT was produced by induction of 0.1 mmol/L isopropyl-D-thiogalactoside (IPTG) and expression situation was assayed by SDS-PAGE. A new protein band was found in SDS-PAGE with molecular mass of about 60.6 KDAa which is consisted of a 40.6 KDAa protein deduced from the BSMT3 gene sequence and GST (26 KDAa). The fusion protein was purified by GST laminar analysis and SDS-PAGE showed the fusion protein was not degradated. The fusion protein was recovered from polyacrylamid gel and used to immunize the rabbit.The titer of rabbit anti-BSMT3 serum from immunized rabbit was 1: 12800. The titer of rabbit anti-pGH serum was determined through indirect ELISA (Enzyme Linked Immunosorbent Assay) with the rabbit anti-BSMT3 serum being and recovered BSMT3 being antigen. Preparated paraffin section of Petunia hybrida organization, BSMT3 gene mainly expressed in corolla and flower tube by immunohistochemical localization.