Bioimformation Analysis And Mutant Investigation Of Fis-class Protein In Oryza Sativa

Rice is one of the most important crops of the world,which is a model of monocotyledon plant as well.Endoperm which is the main edible part of rice takes a crucial role in the embryo development.FIS-class genes(FIE,FIS2,MEA) in Arabidopsis are polycomb genes closely relative to endoperm-independent development. The polycomb protein group(PcG) genes(FIE1,FIE2,Mez1,Mez2) in maize are also concerned with the regulation of endosperm.The research on PcG genes that is helpful to understand the mechanism of endosperm development in cereal has a potential value in breeding.In this study,bioimformation method was used to search the homologs in rice by FIS-class genes of Arabidopsis.The sequences imformation,the structures of the proteins, the similarity and phylogeny tree of homologous proteins were analysed.Then we analysed their origion and functions.The phenotype of the mutants of OsEz1,OsEz3 and OsEMF2B was observed by the method of reverse genetics.With RT-PCR and microarray data,the expression of the PcG genes was analysised for further research on them.The main results were as follows:1.Arabidopsis FIS proteins were used in TBLASTN search of the rice nucleic acid database for the homologous genes.And every FIS gene got two homologs with different origion,OsFIE2A and OsFIE2B for FIE in Arabidopsis,OsEMF2A and OsEMF2B for FIS2,while OsEz1 and OsEz3 for MEA.2.Result of sequences alignment done by Clustalw2 was used for the phylogeny tree constructed by MEGA4.0.Evolvement ralationship between OsEz1 and Mez1 is more closer as OsEz3 and Mez3.OsEMF2B and ZmFIE2 of maize which are belong to monocotyledon branch of the classⅠin the phylogeny tree may origion from the same ancestor,while OsEMF2A protein belongs to classⅡ.It indicated that the two EMF may have different roles in rice.3.OsFIE2A and OsFIE2B,OsEMF2A and OsEMF2B,OsEz1 and OsEz3 genes in rice have no similarity in introns and upstream sequence,that suggusts different expreesion models.Microarray data and RT-PCR revealed that the exression profiles of OsFIE2A and OsFIE2B,OsEMF2A and OsEMF2B,OsEz1 and OsEz3 are different. OsFIE2B was detectable only in the endosperm in wild type,and its expression strengthened gradually from 3 to 7 DAP.The transcript of other genes was detectable at all stages of every tissue.The expression level of OsEz1,OsFIE2B and OsEMF increased gradually from 3 to 7 DAP.The expression level of OsEz3 and OsFIE2A in the endosperm reached the maximum at 4-5 DAP.Microarray data indicated that the expression of OsFIE2A and OsEMF2A is much stronger in anther than any other tissues.4.The result of newcpgreport and cpgplot program showed that OsEz1 and OsFIE2A are imprinting genes as both get 3 CpG islands in the gene region and upstream.It implied OsEz1 and OsFIE2A are imprinting genes.5.Phenotype of Osemf2b mutant:Osemf2b heterozygote mutant was smaller than wild plant,and got less inflorescence branches and flowerlets;some of the flowerlets at the top of the inflorescence were atrophic and whity;flowerlets were abnomal,the pistils may composed more than one carpel,or the ovaries expanded before pollination. Homozygous mutant didn't blossom and bear fruit.Thus,OsEMF2B gene could affect the development of the flower organs,leading to no seed or lower seed-setting rate.6.Phenotype of Osez1 mutant:the flowerlets of Osez1 heterozygote mutant got extra glumes and/or carpels;the proportion of ovary expand-independent in an ear before pollination from 3%to 47%;anther and clinandrium developed with defect;the average field seed-setting rate of the mutant was 53%;the endosperm of the mutant proliferate independently after self-pollination,and the embyo grew dysplasia, stagnating in the pear-shape period.So we speculated that OsEz1 may be inbolved in a several reproductive processes of rice,the impact of flowerlet development,the regulation of embryo and endosperm development,and the maintenance of normal development of anther.