Functional Analyses And Interaction Protein Screening Of CBS1/2

There are a large number of genes involved in regulating the asymmetrical cell division of microspore,the formation of reproductive cell,the division of reproductive cell,the process of male germ cell differentiation and formation of sperm cells during male gametophyte development in angiosperms.However,little is known about the molecular mechanisms regulating gene expression in reproductive cell and sperm cells.Previous studies have shown that the expression of LGC1(LILY GENERATIVE CELL-SPECIFIC 1)is repressed by the Germline Restrictive Silencer Factor(GRSF)in somatic cells.The expression of DUO1 is regulated by the positive activation elements of promoter in sperm cells.The cis-regulatory element Male Gamete Selective Activation(MGSA)site was identified in the promoter of an isopentenyl transferase gene(PzIPT1)of Plumbago zeylanica,which controls the specific expression of PzIPT1 in sperm cells.More regulatory mechanisms controlling gene expression in sperm cells still need to be further explored.Interacting proteins of MGSA,named as CBS(cis-regulatory element-binding protein in sperm cells),were screened in mature pollen of Arabidopsis thaliana by yeast one-hybrid in previous studies.In this study,to elucidate the biological functions of CBS1 and CBS2,promoter sequences of these two genes were cloned to create trandgenic plants of pCBS1::GFP,pCBS1::GUS,pCBS2::GFP and pCBS2::GUS.Expression analyses found that CBS1 and CBS2 express in sperm cells and no signals were observed in vegetative cell of Arabidopsis mature pollen.Phenotypic analyses revealed that cbs1,cbs2,cbs1 cbs1 ls and cbs2 cbs2 ls display no obvious defects regarding vegetative growth,inflorescence development and pollen activity.However,some pollen grains of mutants with disordered nuclei were identified through DAPI staining identified some.Because of possible functional redundancy,high-order mutants will be created to further reveal the biological functions of these CBS genes.Yeast two-hybrid(Y2H)experiments were then performed to screen for interacting proteins of CBS1 and CBS2.Eighty interacting proteins of CBS1 were identified and interactions of ten of them were confirmed.A total of 147 protein-coding clones showing interactions with CBS2 were identified,and sixteen of them showed confirmed interactions with CBS2.Expression patterns,subcellular localization and putative functions of these identified interaction proteins were further analyzed with the help of online databases,which provides a basis for further study of regulatory mechanisms of gene expression in sperm cells.In addition,two promoter fragments of PzIPT1 were used to create transgenic materials ?761-HYG-GFP and ?130-HYG-GFP.Homozygous transgenic lines with single T-DNA insertion were used for screening of genetic modifiers by activation tagging.Those T1 generation mutants with enhanced GFP signals in the ?130-HYG-GFP background or decreased GFP signals in the ?761-HYG-GFP background were selected for further investigation to identify more factors involved in regulation of gene expression in sperm cells.