Identification And Functional Analysis Of RA68 And OsUBP1 Related To Rice Flower Development

One cDNA whose corresponding mRNA was preferentially accumulated in rice florets was isolated by PCR-mediated RNA subtraction hybridization and RACE strategy. This gene, termed RA68 encoded a protein with proline- and threonine- rich motif. RA68 consists of three exons and two introns and is localized in chromosome 2. Southern blot and sequence analysis revealed that RA68 existed as one copy in the rice genome. The putative RA68 protein contained 219 amino acid residues with a putative signal peptide and two domains. The N-terminal domain is hydrophilic and rich in pralines and threonines embedded with PTPTSYG motif. The C-terminal domain is hydrophobic. In Situ hybridization showed that RA68 was expressed in the inner and outer layer of coleoptiles and the epidermis of young leaf primordial in the buds at the vegetative stage. At the reproductive stage, RA68 was expressed in the inflorescence meristem, the tip of rachis branch, spikelet primordia, macrospore sac and pollen grains. RA68 protein was localized at the nucleus using GFP localization. The heading date of transgenic antisense RA68 rice was delayed 30d than the wild type. All these results showed that RA68 may be a floral meristem identity gene in rice and function in the flowering transition. OsUBP1was isolated by RACE and RT-PCR, the putative protein consists of UBP domain (Cys Box and His Box) and TopVIA domain. RT-PCR showed that multiple different transcripts were produced by alternative splicing and the expression of OsUBPI was spatio-temporally regulated. Different transcripts presented in leaves, roots, spikelets and buds. Besides the canonical splice sites of GT-AG, several cases of splice sites with GC-AG, CT-AC, TT-GA, GT-CA and CT-GA dinucleotides at the splice junctions were observed. The key domain of OsUBPI, Cys Box, was produced by the GC-AG alternative splicing. OsUBPI and OsSPO11-1 locaterice; RA68; flowering transition; floral meristem identity gene; In Situ hybridizationat the same locus of chromosome 11. In Situ hybridization showed that OsUBP1 mRNA expressed in the tapetum, pollen grains, macrospore sac and the vascular bundle at the bottom of spikelets. Most of the antisense OsUBPi rice plants were sterile. All these suggested that OsUBPi may be involved in the reproduction of rice.