Map-based Cloning And Functional Analysis Of Two Floral Organ Development Genes In Rice (Oryza Sativa L.)

Rice(Oryza sativa L.),the model plant of monocots,is one of the very important crops in the world.The flower development and floral organ development may play a significant role in rice yield,which influences food security.With the development of molecular biology research techniques,as well as the increase in the number of floral organ mutants identification,the characteristics of the flower development is becoming more clear.In this study,two mutants in floral organ osmads34-t and dfl(deformed flower)were obtained by EMS(Ethylmethylsulfone).We did the systematic study for expression and function of OsMADS34 and DF1 genes,including phenotype characteristics,microscopic observation and gene mapping.The main results were as follows:1?OsMADS34 gene:(1)Phenotype of floral organ:The osmads34-t mutant shows elongated sterile lemmas,and the morphology and the number of components of the four whorls of floral organs were investigated,finding that these organs were normal.SEM analysis revealed that the epidermis of sterile lemmas had few protrusions and trichomes.(2)Change in grains:The number of spikelets on primary branches and the secondary branch were decreased,but the number of primary branche was increased.Both the kilo-grain weight and the weight of kilo-brown rice were remarkably reduced in the osmads34-t mutant.The osmads34-t grains,on secondary branches,were obviously different from that of the wild type.Interestingly,osmads34-t grains on secondary branches were shorter in length,but equal in width.(3)OsMADS34 gene map based cloning:Using 1326 F2 recessive individuals,we narrowed down this gene to a 143 kilobase(Kb)regions between SSR marker RM5813 and Y7.In this region,LOC_Os03g54170 encodes a predicted OsMADS34 protein with the conserved MADS-box domain.This gene has four nucleotide(GGAT)deletion in the genomic sequence of osmads34-t,which leads to a frameshift and premature translational termination.(4)Expression pattern of OsMADS34 and protein localization:OsMADS34 was expressed in all tissues and organs examined,including roots,culms,leaves,sheaths,panicles,rudimentary glumes,sterile lemmas,lemmas and paleae,with strong expressions detected in young panicles and reproductive organ.GUS signals were observed in all of the tissues examined,and the strongest signals detected in young panicles.The subcellular localization suggested that OsMADS34 is a nuclear protein that may function as a transcription factor.2?DF1 gene:(1)Phenotype of floral organ:The hull of dfl flowers seemed not open and the stamens were enclosed in the lemma and palea.The grain hulls were not closed at one side in the dfl mutant at maturation stage.Compared with the wild type,cell numbers and cell sizes were affected in df1 hulls,and the hulls became thicker.The lodicules of df1 florets were elongated and green features were found in some dfl lodicules.There was no abnormity presentation in the number and identity of stamens and pistils in df1 florets.(2)Change in grains:The grain and brown grain in size,1000-grain weight and weight of 1000 brown grain were markedly decreased in dfl mutant.The setting percentage and pollen viability of the df1 mutant were affected by the thick hulls and abnormal lodicules.(3)DF1 gene map based cloning:Using all 869 F2 recessive individuals,DF1 was finally narrowed down within a 65 kb region.In this region,a gene that encoded a DUF640 domain transcription factor with a single nucleotide T deletion in this region,resulted in coding frameshift and premature translational termination.(4)Expression pattern of DF1 and protein localization:DF1 exhibited more abundant expressions in intemodes,young panicles,lemmas,paleae,and lodicules than those in other tissues or organs examined.DF1-GFP fusion protein were detected exclusively in the nucleus of rice protoplasts,which suggested that DF1 encoded a nuclear protein and may act as a transcription factor.Combined OsMADS34 and DF1 analysis,we drawed following conclusion:Sterile lemma and lemma have highly homology.The rice palea was a fusion organ of the mrp(marginal regionof palea)and bop(body of palea).MADS-box genes or other flower related genes may affect grain size in rice.