Mutant Morphological Characteristics And Expressional Analysis Of OsRBR1 Gene In Rice

The retinoblastoma susceptibility protein was first discovered as the tumor suppressor.Its core function is the regulation of cell cycle progression by RB/E2F mode.RB is the key gene which regulates cell proliferation,differentiation and apoptosis,thus involved in the formation of organs.Retinoblastoma-related(RBR)is the RB homology in higher plants,and they have similar sequences and domains.RBR is a small gene family in plants,and its main function is to regulate cell cycle progression.In addition,RBR is also involved in the control of cell size,apoptosis of cells,gametophyte development and maintainance of balance between stem cell proliferation and differentiation.There are two RBR genes(OsRBR1/2)in rice,but their specific function is not clear.Our team found one kind of mutant from rice breeding materials,which is related to floral organ development in rice.The fine mapping and sequence analysis revealed the deletion of 28bp fragments-at 11th exon in OsRBR1 gene,which lead to the early termination of transcription.OsRBR1 gene was identified as a candidate gene,and the mutant was named Osrbrl-1.We analyzed the mutant morphological characteristics and expression of OsRBR1 gene in rice using resin sections,scanning electron microscopy(SEM),Q-PCR and in situ hybridization.The results are as follows:1.Phenotype characteristics of Osrbr1-1Resin section analysis revealed that mature spikelets' phenotypes of Osrbr1-1 have significant variation compared to wild-type.All of lodicules,stamens,pistil are degenerated.The floral meristem differentiates into glume-like organs.The number of the vascular bundles greatly increased in the palea and lemma of Osrbr1-1.Lemma and palea shortened,making the hook shaped structure disappear resulting to partially open glume.SEM showed that the developments of palea primordium and lemma primordium are the same between Osrbr1-1 and wild-type flowers.Lodicules primordium,stamens primordium and pistil primordium of the mutant did not differentiate.Glume-like primordium was produced continuously due to multiple spikelets primordium formation.The mutant spikelets completely lost the floral meristem determinacy.This effect resulted to bad development of floral organs.2.Expressional analysis of OsRBR1We analyzed the expression levels of OsRBR1 using Quantitative Real Time PCR(Q-PCR)in the root,stem,leaf and panicle of rice.The results are as follows:there was moderate expression in the root,stem and panicle but very high expression levels at 10cm leaf,however hardly no expression was found at 30cm leaf.We infer that the OsRBR1 gene is more active in the basal region,where cell divisions are expected to occur.Since the gene may influence the development of floral organ,we analyzed OsRBR1 expression of the spikelets using in situ hybridization.The expression levels of OsRBR1 gene were found to be high in the early floral primordial of wild-type,but decrease gradually on further development of floral organ.This demonstrated that OsRBR1 plays an important role in the early development of flower.In addition we analyzed the expressional levels of OsRBR2 and compared to OsRBR1.The results indicated that the expressional levels of OsRBR1 and OsRBR2 were significantly different in root and panicle.In the vegetative organs the OsRBR2 was constitutively expressed at higher level than the OsRBR.There was no expression of OsRBR2 in panicle.This pattern suggests that the two RBR genes have different function.3.Relationship between OsRBR1 with other floral organ identity genesWe analyzed the expressional levels of a few flower developmental key genes in wild-type and Osrbr1-1 by Q-PCR.We found the expressional levels of B-class and C-class gene OsMADS2/4/16/1//3/58 were down-regulated in mutants,and C-class gene OsMADS3/58 was significantly down-regulated.These results demonstrated that OsMADS1 and OsMADS6 might determine rice flower organ development by regulating expression of these genes.In conclusion,OsRBR1 might determine rice floral organ development by regulating expression of these genes.4.Subcellular localization analysisA GFP and OsRBR1 fusion expressional vector was constructed to transform the young onion epidermal cells by Agrobacterium.The results showed that the fusion protein was expressed specially in the nucleus,while the signals did not express in the cytoplasm.Therefore OsRBR1 protein belongs to the nuclear localization protein.