Isolation and characterization of cDNA sequences encoding for MADS-box transcriptional factors from rice leaves

MADS-box genes are characterized by the conserved MADS domain and found in a wide range of eukaryotes including metazoans, fungi, slime mold, and green plants. Throughout plant evolution, MADS-box genes have been recruited as transcriptional regulators active in the development of diverse plant structures. In monocots, MADS-box genes have been identified in maize, rice, and orchids which play important roles during reproductive and vegetative developmental processes. Although rice MADS-box genes involved in flower and fruit development have been well characterized, rice MADS-box genes expressed in vegetative structures have yet to be explored. The aim of this project is to isolate and analyze the cDNA sequences encoding for putative MADS-box transcription factors from MR84 leaves. MR84 is currently the popular local cultivar which produced good quality and yield as well as confers resistance to Karah and Merah diseases in Malaysia. Besides that, this project also aimed to characterize leaf-expressed MADS-box genes in MR84 and to examine their expression in developing leaves. Poly-A+ RNA was isolated from developing leaves at different stages using oligo-dT magnetic beads, and first strand cDNA was synthesized directly on the beads. A degenerate 5' (forward) primer was designed against the conserved amino acid sequences in the MADS-box domain and an anchored dT18 as the reverse primer for the three-prime (3’)-RACE procedure. PCR using sscDNA as template resulted in an expected fragment size of approximately 1,013 bp. This PCR product was cloned in pGEM-T Easy plasmid vector and sequenced using forward and reverses universal M13 sequencing primers. The sequence analyses of the cloned product showed 94% similarity to other rice MADS-box cDNA. However, in order to obtain the full sequence, five-prime (5’)-RACE using inner 5'-RACE as forward primer and MADS-specific as reverse primer were used to obtain the remaining 284 bp of the cDNA. A BLAST search of the complete 1,297 bp full sequence confirmed that this was the MADS-box gene and it was designated as OsMADS_UMS1. The deduced amino acid sequence of OsMADS_UMS1contains the typical MADS-box cDNA namely, the M, I and K boxes as well as a C terminal variable domain. Phylogenetic analyses using the PHYUP software showed that OsMADS_UMS1 belongs to the class F (AGL20 or TM3) subfamily. Real-time RT-PCR analysis shows gradual mRNA steady-state levels increment during leaf development. This preliminary result indicates a possible role for OsMADS_UMS1 in leaf initiation and its further development. In the course of this study, another partial MADS-box cDNA was isolated designated OsMADS_UMS2. This cDNA was not further studied.