Identification and Characterization of Certain Mads Box/spl Genes and Their Expression During Organogenesis in Petunia (Petunia Axillaris)”

Abstract

Petunia is an ornamental and considered as research model plants in molecular biology. In the present study, Publically available SRA data of leaf, bud and limb of Petunia axillaris downloaded from European Nucleotide Archive (ENA) for in-silico analysis. A total of 5,129 and 8,998 differentially expressed genes are found for Leaf vs. bud, Bud vs. Leaf respectively. MADS-box and SPL gene have ~60 and ~76 Amino acid DNA binding domain and classified as TFs family known to plays significant role during the development of floral organs in plants. Multiple sequence alignment analysis of these proteins indicated that FBP21, FBP22 and SPL genes shared highly conserved signature domain of their family MADS-box, a K-box domain and SBP domain respectively. The present study is also aim for 2-D and 3-D molecular modelling of FBP21, FBP22 and PaSPL genes using bioinformatics applications. The expression level of MADS box (FBP21 and FBP22) and semiquantitave expression of SPL genes (PaSPL6a, 6b, 6c, 6d, 9a, 9b and 9c) was worked out from five floral stage and leaf of Petunia. On the basis of PCR expression analysis, we revealed that FBP21 and FBP22 genes expressed in young flower bud and only FBP21 expressed in leaf tissues, suggesting that these genes are involved in floral transition and early flower development. PaSPL6a, b genes expression was detected in all five floral stage and leaf tissues. PaSPL6c and 6d were expressed in floral tissues but not detected in leaf sample. PaSPL9a, 9b and 9c expressed at higher level in all floral stage as compared with PaSPL6 genes and less detected in leaf sample. All PaSPL genes down regulated from initial flower to mature flower by 8 to 64 fold change. Complete coding region of MADS-box FBP21 and FBP22 were amplified by PCR from flower bud of Petunia axillaris and cloned into pGEMT cloning vector for sequencing. The sequence of FBP21and FBP22 contained an ORF of 648bp and 657bp encoding 215 and 218 amino acid residues respectively. The developed full FBP21 and FBP22 gene construct (pCAMBIA1302:FBP21 and pCAMBIA1302:FBP22 respectively) were successfully validated based on PCR amplification and restriction digestion assay for its structural integrity. The Agrobacterium mediated host cells transformation activity of the constructs 35S:FBP21:GFP and 35S:FBP22:GFP transgene is confirmed with successful GFP Expression in inoculated NB leaf.