Cloning and analysis of MADS-box genes expressed in Poa annua MD-2 apices during floral meristem formation

Poa annua MD-2 is a day-neutral, annual grass. It takes approximately 25 to 35 days to flower under controlled conditions. MADS-box genes are involved in many aspects of flower morphogenesis, such as determination of flowering time, floral meristem identity and floral organ identity. The purpose of this study was to isolate MADS-box genes expressed in P. annua MD-2 floral meristems. Degenerate primers based on the MADS-box sequences of maize and rice were used to amplify a fragment of approximately 170 bp from MD-2 genomic DNA that was cloned into a pBluescript II SK(+) vector. Three plasmids contained identical MADS sequences of 169 bp. The cloned fragment was named PaMADS. mRNA from 5mg MD-2 meristem tissue at the panicle primordia initiation stage was isolated by using paramagnetic beads and first-strand cDNA was synthesized on the beads. A poly-C tail was then added. The cDNA was amplified for 20 cycles using primers containing either poly T or poly G and a restriction site. The products were cloned into a lambda Zap II vector. 320,000 plaques of the cDNA library were screened with PaMADS and two full-length MADS-box genes, PaMADS1 and PaMADS2, were isolated. PaMADS1 is 1159 bp in length and encodes a 258 aa protein which has 59% homology with AGL6 from Arabidopsis thaliana. The MADS-box motif of PaMADS1 is identical with Zag3 from maize and OsMADS6 from rice, and 98% identical with AGL6. Within the K-box, PaMADS1 has 94 to 95% homology with ZAG3 and Osmads6, but only has 64% homology with AGL6. PaMADS2 is currently being sequenced. Northern blot analysis indicated that PaMADS1 is a floral-specific gene and is expressed at panicle primordia initiation and pre-pollen stages. Southern hybridization indicated that there are many copies of MADS-box genes in both P. annua and P. pratensis.