Map-based Cloning And Functional Analysis Of EL1 Gene In Rice (Oryza Sativa L.ssp. Indica)

Rice(Oryza sativa L.) is not only one of the most important food crops in the world but also a model plant for study of the molecular developmental biology in monocots.Research on rice reproductive mechanism is significant both in theory and in human agriculture.In rice,most of genes involved in floral development were isolated by screening the cDNA or DNA library with the probes homologous to sequences of dicot plants,and their functions were identified by the altered morphology of floral organs in transgenic plant.The acquisition and research on the rice mutants related to floral development play an important role in understanding the function and interaction of genes in reproductive process,especially in floral development.In this study,we isolated a gene,ELONGATED LODICULES 1(EL1) involved in the flower development in rice,we have completed the investigation of the anatomical structure of floral organs,genetic analysis,fine mapping and further analyzed its functions.Our results may provide some information on the molecular mechanism of the controlling of floral organ development in plants.The results as follows:1.Phenotypic Observation and Cytological Analysis of el1The rice el1 mutant was found from a spontaneous mutation in an indica maintainer line Jin 2B. In the whorl 1 of el1 flower,palea/lemma is in the shape of crook "S".In whorl 2 the lodicules were elongated and two stamens were transformed into lodicule-like organs.In the whorl 3 of el1 flower, stamens number decreased.In the whorl 4 of el1 flower,lodicules and/or pistils number increased. Because the phenotypic traits have not been reported,temporarily designated as el1(elongated lodicules 1) mutant.2.Genetic Analysis and Preliminary Mapping of EL1The genetic analysis was conducted on the F₁ hybrids and F₂ populations obtained from 4 crosses.The F₁ population of four crosses showed wild-type phenotype.In the four F₂ populations, all the segregation rates of wild-type and el1 mutation plants fit the ratio of 3:1.Therefore,the el1 mutant phenotype is controlled by a single recessive gene.We used the SSR marker to analyze one F₂ populations(el1×602) of crosses,and in this cross, the gene EL1 was mapped between RM1232 and RM5389 on chromosome 1 with respective genetic distance 7.3 and 4.5 cM.3.Fine Mapping of EL1 and Filtrating the Candidate GenesThe gene EL1 was mapped between(RM128) PS2C8 and RM1152 with genetic distances 0.83 cM and 0.79 cM(physical distant:645Kb) by new developmental SSR,InDel,STS marker.In this region,a prominent candidate gene,OsMADS32,which is a transcription factor with MADS domain. This domain defined function has been well established in the regulation of flower development in higher plants,was identified.So we filtrated OsMADS32 was the candidate gene of EL1.4.Determining the Candidate Gene of EL1 and Cloning AnalysisSequence Analysis of OsMADS32:We sequenced the cDNA and DNA of OsMADS32 of mutant plant and wild-type plant.We compared the OsMADS32 transcripts and found a single nucleotide T deletion resided at the el1 mutated allele,causing a premature translation stop. Furthermore,the results showed OsMADS32 was the candidate gene of EL1.Digestion Analysis of the Candidate Gene:An original Rsal restriction site was identified and experimentally confirmed in the wild-type allele,while in the mutant allele the Rsal restriction site was abolished due to the T deletion.We amplified a 200bp sequence(including mutant locus) and digested the mutant plants population,but none of them was able to be catalyzed.Taken together, these results indicated that the candidate gene that we isolated was the candidate gene.5.Temporal and Spatial Expression Patterns of EL1EL1 was specifically expressed in panicle:To determine the temporal and spatial expression patterns of EL1 gene,semi-quantitative RT-PCR and quantitative PCR were used to detect the expression of EL1 in root,stem,leaf and panicle,and the results showed that EL1 was only specifically expressed in panicle.The in situ hybridization of EL1:While the floral organ initiation,EL1 transcripts were detected in all flower primordia and spikelet apical meritems.After floral organ initiation,EL1 was abundantly expressed in the lodicules,stamens and pistils stranger than in palea/lemma.6.Functional Analysis of the Candidate GeneRNA Interference of EL1:We constructed the RNA interference vector.Construct was introduced into embryo callus of rice cv.Zhonghuall(Oryza sativa L.ssp.japonica) by Agrobacterium-mediated T-DNA transfer.The phenotype of positive plants were the same as mutants',furthermore,the results confirmed the function of El1 in rice flower development.Overexpression of EL1:We constructed the over expression vector of El1 by 35S promotor and achieved transgenic positive plants.the palea of the ransgenic positive plants become short and narrow but the inner three whorls organs have not changed.7.Phylogeny Analysis of EL1 geneA phylogenetic tree was constructed using the main MADS-box genes,the results showed that EL1 was clustered with two wheat MADS-box genes(TaAGL14 and TaAGL15,respectively).Based on the gene sequence,it is a unique branch in grass.8.Expression Analysis of Genes Involved in Flower DevelopmentWe chose 11 genes involved in fllower development,and the expression did not detect significantly changes in all the genes between el1 mutant and wild-type.The result preliminary indicated the uniqueness of EL1 gene in flower development in rice...