Mapping-based Cloning And Functional Analysis Of SUPERWOMAN3 Genes In Rice(Oryza Sativa L.)

Rice（Oryza sativa L.）,a monocotyledonous plant and a model plant of the perennialfamily,is one of the most important food crops in the world.Floral organ development,including its characteristics,size,and number,affects the morphology and number of seeds,which determines the yield and quality of rice in turn.Therefore,the molecular mechanism of rice floral organ development has been of great significance.At the end of the 20th century,scientists proposed floret/spikelet development models and mechanisms based on dicotyledonous plants such as Arabidopsis and Antirrhinum.Because of the differentiation of monocotyledonous and dicotyledonous plants,these models and mechanisms were not fully applicable to monocotyledonous plants such as rice.In this study,we identified a superpistillate mutant superwoman3（spw3）from the EMS（ethyl methane sulfonate）mutagenesis library of XD 1B（Indica）.Phenotypic variant analysis,based mapping of SPW3,and functional analysis will then be described.The main results are as follows:1.Phenotypic analysis of spw3 mutantsIn order to clarify the biological function of SPW3 gene,we analyzed the floral development of spw3 mutant and wild type in detail.Compared with wild type XD 1B,the most obvious phenotype of spw3 mutant was that nearly half of the stamen organs were homologous to pistil,and the anther was composed of parenchyma cells and vascular bundles,resembling an ovary.In addition,the mutation also showed abnormal development of other spikelet or floral organs:most of the sterile lemmas were elongated like lemma,and contained multiple vascular bundle tissues;Palea partially degenerated or elongated like lemma,without obvious palea margin tissue;Most of the lodicules elongated and palea lamellar,with cell layer similar in structure to palea.These changes indicated that SPW3 gene could regulate the development of floral organs and sterile lemma.2.Identification and transgenic confirmation of SPW3spw3 was crossed with sterile line 56S,and the F₁generation plants showed normal phenotype.After self-crossing,mutant phenotype plants appeared in F₂generation population.The separation ratio between normal plants and mutant plants was 3∶1.The results indicated that the spw3 mutant character was controlled by a single recessive gene.By BSA method,the SPW3 gene was located between simple sequence repeat markers M1 and M2 on the X chromosome,and was further located between two SNP markers with 2 and 1 recombination,respectively,at a physical distance of about 86.0kb.There were 14 completely open reading frames（ORFs）in the localization interval,and it was found that base substitution of T-C occurred in the fifth exon of the eighth ORF in the spw3 mutant interval,resulting in the conversion of the encoding amino acid from leucine to proline.The mutant phenotype was restored in the genetic complementary plants,which was consistent with that of wild type spikelet.The results showed that ORF8 was SPW3 gene.3.Expression pattern analysis of SPW3 geneIn order to determine the expression pattern of SPW3,q PCR and in situ hybridization were used to detect the expression of SPW3 in wild type plants.q RT-PCR analysis found that SPW3 was expressed in all tissues of rice plants,with the highest expression in inflorescence and the lowest expression in root.During the development of spikelet,SPW3 can be detected in the spikelet<0.5cm at the initial stage of organ primordium,and the expression of SPW3 is highest in the spikelet 1～2cm.In situ hybridization showed that the expression signal of SPW3 was detected in the palea and lemma primordium,and then it was clearly expressed in the lodicule primordium,stamen primordium,and pistil primordium.The SPW3 expression pattern was consistent with the spw3 mutant phenotype.4.SPW3 gene encodes a deoxyhypusin hydroxylase DOHHThe SPW3 gene encodes a deoxyhypusin hydroxylase DOHH which is responsible for the modification of the eukaryotic initiation factor 5A（e IF5A）.DOHH belongs to the HEAT-repeat protein family which is a highly conserved protein.Until now,these proteins have been poorly studied,especially in botany.Prediction results from the SMART protein analysis website indicated that the protein encoded by SPW3 had an E-Z type HEAT repeat region.Phylogenetic analysis showed that SPW3 proteins in different species have high homology,and SPW3 homologous proteins from monocotyledon and dicotyledon form a small branch respectively.5.SPW3 protein localized in the cytoplasm and nucleus without transcriptional activation activityThe results of subcellular localization and GFP fluorescence observation of SPW3-GFP fusion protein in transgenic plants showed that SPW3 protein was localized in the nucleus and cytoplasm.The yeast self-activation assay showed that SPW3 protein had no self-activation activity.6.SPW3 gene regulates floral development characteristic genesThe expression patterns of genes related to floral organ development were analyzed by q RT-PCR and in situ hybridization.In spw3 mutant,the pistil characteristic development gene DL was significantly up-regulated,and the expression signal was detected in some stamens.The expression of Os MADS2,Os MADS4 and Os MADS16,which regulate the development of lodicule and stamen,were substantially down-regulated,and the signals were only detected in a few stamens.These results suggest that SPW3 could be the upstream of Os MADS16 and affect the development of floral organs by activating class B genes.