| Maize is an important food crop and a major subject of genetic research.The maize endosperm cells undergo sequential cell cycle activities such as acytokinetic mitosis,normal mitosis and endoreduplication after double fertilization.It is a model system for cell from mitotic to endoreduplication.Endoreduplication involves one or several rounds of nuclear DNA synthesis without chromosomal and cellular division,which leads to cell polyploidization.From the point of view of corn grain development,the stage of endoreduplication and the rapid expansion of grain are in complete synchronization,which may be a key driving force for grain expansion and closely related to the establishment and yield of grains.Meanwhile,studies have shown that the occurrence of endoreduplication is related to the stress resistance of plants and is the result of evolution of organisms under adverse conditions.Therefore,the cloning and identification of genes related to reduplication in maize can not only provide a new way for cultivate high-yielding corn varieties from the perspective of cell cycle regulation,but also provide genetic resources for the cultivation of maize stress-resistant varieties,which is of great significance for corn breeding.However,endoreduplication process in maize is not currently well understood.Here,total 12 of putative candidate genes were identified as members of SIAMESE-RELATED(ZmSMR)gene family in the Zea mays L through screening the GDB maize database.The basic bioinformatics and expression analysis were performed for ZmSMR gene family.Then,two members of the ZmSMR gene family,the ZmSMR4(KY946768)and ZmSMR7(KY946769)genes,were isolated by RT-PCR,and the functions of ZmSMR4 and ZmSMR7 genes were analyze at the gene,transcription,and protein levels through a series of experiments including expression analysis,subcellular localization,prokaryotic expression,phenotype identification and others.In addition,the transgenic Arabidopsis phenotype was analyzed at the cellular level by cellular dynamic analysis,statistical analysis of the degree of endoreduplication.Finally,the possible signal transduction induced by the prokaryotic expression system of E.coli,which was consistent with the expected target protein,and laid the foundation for further study of its function from the pathways and mechanisms involved in the ZmSMR4 and ZmSMR7 genes were preliminary studied by q RT-PCR and protein interaction techniques.The major results are as following:1.Analysis of bioinformatics and expression characteristics of the ZmSMR gene family,Total 12 genes were obtained and named as ZmSMR1-ZmSMR12 by searching,merging,and deleting programs in database,and the basic information of each member of the ZmSMR gene family was analyzed by the bioinformatics software.The analysis results showed that all ZmSMR gene family members are low molecular weight proteins;all the ZmSMR protein sequences exhibited modest sequence similarity to the SIAMESE gene from Arabidopsis,and contain 5 conserved functional domains;The 12 ZmSMR genes were located on 7chromosomes of maize;35 SMR genes from maize,rice and Arabidopsis can be divided into five major groups: I,II,III,IV and V,and members of the maize SMR gene family are divided into the first and fifth categories,the analysis of RNA-seq data suggested that the maize SMR gene family was tissue-specific expression and most ZmSMR genes might be associated with the transition from mitosis to endoreduplication because the expression levels of most ZmSMR genes were upregulated in endosperm cells during the phase of switching to an endoreduplication cell cycle.2.Cloning and expression analysis of ZmSMR4 and ZmSMR7 genes: In this study,two maize endoreduplication-related genes,ZmSMR4 and ZmSMR7,were successfully cloned by RT-PCR and submitted to the NCBI database.The expression patterns of ZmSMR4 and ZmSMR7 genes were analyzed by q RT-PCR.The results showed that ZmSMR4 and ZmSMR7 genes had tissue-specific expression;the expression of ZmSMR4 and ZmSMR7 genes not only was induced by drought stress,high salinity,low temperature and high temperature abiotic stresses,but also by hormone like ABA that associated with biological stress.It was suggested that the ZmSMR genes may be involved in many signal transduction pathways;the expression levels of ZmSMR4 and ZmSMR7 genes were significantly up-regulated on the 10 th day after pollination(DAP)and then decreased gradually,suggesting that the ZmSMR genes might be associated with the transition from mitosis to endoreduplication.And,the expression level of ZmSMR4 gene showed a distinct peak at 35 days after pollination,suggesting that the ZmSMR4 gene may also play a role in activating the biological processes of seed dormancy and dehydration.3.Prokaryotic expression analysis and subcellular localization of ZmSMR4 and ZmSMR7 proteins: In this study,the proteins of ZmSMR4 and ZmSMR7 were successfully protein level.Subcellular localization results showed that ZmSMR4 was localized on the nucleus and ZmSMR7 was localized on the nucleus and cell membrane.4.Function analysis of ZmSMR4 and ZmSMR7 gene: two genes,ZmSMR4 and ZmSMR7,were transformed into Arabidopsis sim mutants and expressed under the control of the GL3 promoter.The results showed that both ZmSMR4 and ZmSMR7 genes were able to rescue the multicellular trichome phenotype of Arabidopsis sim mutants,indicating that the ZmSMR4 and ZmSMR7 genes are homologue gene of SIAMESE gene in maize,and has similar functions with SIAMESE gene.The ZmSMR4 and ZmSMR7 genes were transformed into Arabidopsis thaliana by Agrobacterium-mediated inflorescence inoculation.the transgenic Arabidopsis showed that the ZmSMR4 and ZmSMR7 genes not only affected the growth and development of the transgenic Arabidopsis,but also were related to the resistance.The transgenic Arabidopsis plants overexpressing ZmSMR4 and ZmSMR7 genes not only displayed enhanced leaf margin serrations and shorter plant growth period,but also showed several interesting breeding phenotypes,such as early blossoming and fuller seeds.In addition,The transgenic Arabidopsis plants showed resistance phenotypes under abiotic stresses.5.Phenotypic analysis of transgenic Arabidopsis and investigation of the mechanism of ZmSMR4 and ZmSMR7 genes: This study demonstrated that the abnormal phenotype of transgenic Arabidopsis mainly caused by the cell division was inhibited and the endoreduplication level was promoted through a series of experiment including cell kinetic analysis,nuclear DNA ploidy detection and statistical analysis of endoreduplication degree.The interaction analysis between ZmSMR and CDKs though yeast two-hybrid assay indicated that ZmSMR gene may promote the transition from mitosis to endoreduplication by inhibiting the activity of cyclin-dependent kinase associated with cell division... |
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