ISOLATION AND CHARACTERIZATION OF FLORAL HOMEOTIC GENE OM1 IN AN ORCHID ARANDA DEBORAH

Abstract

The complementary DNA (cDNA) made to the poly(A)+RNA of leaves, inflorescences and mature flowers in an orchid (Aranda Deborah) were synthesized. Three phage expression libraries were constructed in lambda-ZAP II® (Stratagene, USA). One gene, termed oml, successfully isolated from mature flower cDNA library by utilizing a 1.0 kb agamous (ag) cDNA fragment from Arabidopsis revealed an open reading frame (ORF) of750 nucleotides coding for a protein of250 amino acids. This gene also contains a short 5' non-translated region of26 nucleotides and 3' non-coding region of 161 nucleotides which terminates with a polyadenylated tract. The sequence comparison between the OM1 protein and various floral homeotic gene products showed two domains of relatively high homology. The alignment analysis of the MADS box domains among the orchid OM1, the Arabidopsis AGIA and AG, the petunia FBP1 and FBP2, the snapdragon DEF A and tomato 1M5 exhibited %%, 77%, 66%, 95%, 59% and 93% amino acid identity respectively. In addition to the MADS box, the OM I protein shared a second conserved domain with AGIA, AG, FBPI, FBP2, TMS and DEF A proteins. This domain is designated K box, because of its similarity to the coiled-coil domain of human type II keratin. RNA blot analysis indicated that oml mRNA was detectable in the mature flower but not in inflorescence buds, flower buds and other vegetative organs. Further RNA blot analysis for the expression pattern of oml gene in different floral organs showed that its transcriptional activity was relatively strong in the petals and sepals, but not in the column. These results suggest that the oml gene is exclusively expressed in the mature flower and is probably not an early floral homeotic gene. For expression of oml gene in E coli, the oml gene was PCR-amplified using designed primers and subsequently subcloned into pGEX-2T® expression vector (Pharmacia, USA). The resultant recombinants were used to transform E coli JM 101. The oml gene in pGEX-2T was expressed as a protein fused to the Glutathione-S-Transferase (GST). This fusion protein migrated at 58 KDa on 14% SDS-PAGE. The fusion protein was then used to immunize rabbit and/or mice to raise polyclonal antiserum against OM1 protein. To further study translational activity of the oml gene, the crude nuclear protein was extracted from young inflorescence buds, flower buds, mature flowers, roots, sterns, leaves, sepals, petals and columns and analysed in immunoblot experiments. The results showed that translational activity of oml gene can only be detected in the petals and sepals of mature flower, but not in other organs. This was in agreement with the results of RNA blot.