Map-based Cloning And Functional Analysis Of MFS1Genes In Rice(Oryza Sativa L. Ssp. Indica)

Rice (Oryza sativa L.) is not only an important food crops in the world and a model plant in monocots but also an ideal object for studying flower development. Compaired with the model plants in dicots, rice bears a unique inflorescence consisting of spikelets to grass species, and has abundant genetic and genomic resources. So, reseach on rice reproductive development process would facilitate understanding of the molecular mechanism involved in spikelet development in rice and enrich knowledge of plant developmental biology for us. Meantime, it is very important to improve rice yields.In the study, we cloned a MULTI-FLORET SPIKELET1(MFS1) gene associated with spikelet development in rice. We did investgation of mfsl mutants, and analysed the expression patterns and functions of MFS1gene. The results as follows:1. Phenotypic observation of mfsl mutantsTwo mutants of rice, mfsl-1and mfsl-2, were identified from ethylmethane sulfonate (EMS)-treated Jinhui10(Oryza sativa L). Because mfsl-1showed more severe defects than mfsl-2, the main focus of our study was mfsl-1.The mfsl-1spikelet displayed degenerated the sterile lemma, the elongated rachilla, the extra lemma-like organ and the degraded palea. Additionally, mfsl-1flowers produced varied numbers of inner floral organs. Scanning Elctron Microscropy (SEM) showed that the sterile lemma and palea was degraded in mfsl-1, floral meristem was enlarged and the stamen development was not synchronous in a few cases.2. Genetic analysis and molecule mapping of MFSlWe employed3crosses to conduct the genetic analysis. The F1population showed wild-type phenotype and the segregation rates of wild-type and mfsl plants fit the ratio of3:1, suggesting that the MFS1gene was controlled by a single recessive gene. Next, we used the SSR markers and InDel markers to map the MFS1gene, the MFS1gene was finally mapped between Indell7and Indel24on chromosome5.3. Cloning and protein analysis of the MFS1geneSequencing of DNA and cDNA analysis found a single nucleotide substitution from C to T within a predicted AP2/ERF transcription factor (Os05g041760) in different positions of the two mfsl alleles, causing a Ala66to Val66substitution in mfs1-1and a Thr51to Ile51substitution in mfs1-2. So, we infer that the Os05g041760is the candidate gene. MFS1was shown to belong to a clade of unknown function in the AP2/ERF family, and localized in the nucleus.4. Expression patterns analysis of the MFS1geneSemi-quantitative RT-PCR and quantitative RT-PCR (qPCR) analysis exhibited that MFS1was universally expressed in various tissues including roots, stems, leaves and panicles, especially high in young panicle. In situ hybridization analysis showed MFS1was strong expressed in the meristems and sterile lemma primordium. The MFS1expression was reduced gradually as the floral organ started to develop.5. Function analysis of the MFS1geneThe MFS1complementation vector constructed and transformed into mfsl-1. we accuquired the transgenic positive plants and detected them GUS expression identification. In the transgenic plants, the mutant phenotypes were completely rescued in transgenic plants, suggesting that the Os05g41760is the MFS1gene.The MFS1interference vectors were constructed and transformed into Jinhui10(Oryza sativa L. ssp. indica) and Zhonghuall (Oryza sativa L. ssp. japonica). We accuquired the transgenic positive plants and detected them by PCR and GUS expression identification. In the transgenic plants, pleiotropic spikelet defects were similar to those of mfs1-1. The result further confirmed that the Os05g41760is the MFS1gene and the functions of the MFS1gene.6. MFS1regulates the expression of related floral development genesThe expression patterns showed the expression of A function gene OsMADS15was increased, the expressions of the AP2/ERF domian genes (FZP, SNB and OsIDSl) and the sterile lemma identity gene G1were decreased.