Functional Analysis Of Cyclin Cyc9 And Its Related Proteins From Tetrahymena Thermophila

Cell recognition is required for sexual reproduction of different organisms.The recognition is regulated at different levels and evolved different mechanism from yeast to ciliate,and exhibited more specific mechanism in higher plant and mammalian cells.Sexual reproduction of unicellular eukaryotes begins with the recognition of different mating types cells.The different regulatory factors and membrane proteins participate in the cellular recognition.The protozoan Tetrahymena thermophila has seven different pairing types,and different mating cells are randomly produced by gene rearrangement mechanisms during sexual reproduction.When different mating type cells are paired,the cell morphology changes,the paired cells form a stable binding region,and the micronuclei begin meiosis and gamete fusion.At present,sex determination and meiosis mechanisms make a great progress in T.thermophila,but the molecular mechanism of cell recognition is less clear.In the study,a cyclin-specific cyclin gene CYC9 was identified.Function of Cyc9 and Cyc9-involved protein was systematically analyzed.The main results are as follows:1.Specific expression of CYC9 during starvation and sexual reproduction.Based on the T.thermophila genomic database?http//: www.ciliate.org?and the functional genomic database?http://tfgd.ihb.ac.cn?,CYC9?TTHERM₀0940290?was identified,full length is 1915 bp and has an open reading frame of 1218 bp encoding 405 amino acids.Cyc9 contained a cyclic protein box consisting of 95 aa in the middle and a lysine-rich region at the N-terminus.Microarray expression profiling and real-time fluorescence quantitative analysis showed that CYC9 was not expressed during the growth phase,and expressed during starvation and the sexual reproduction stage.The recombinant plasmid p XS75CYC9 was constructed.The plasmid was transformed into wild-type cell and mutants were screened and identifed.Immunofluorescence stainning showed that Cyc9 p mainly localized in the cytoplasm.2.CYC9 knockout inhibits cellular recognition of different mating cells.The plasmid p Neo4CYC9 was constructed and transformed into different mating types B2086 and Cu428 of Tetrahymena cells.The CYC9 knockout mutant cells ?CYC9-B and ?CYC9-C were obtained by Paromomycin resistance screening and phenotypic assorment.Different mating type ?CYC9 mutant cells failed to recognition each other and formed pair cells.Furthermore,cellular morphology of ?CYC9 mutant is abnormal when different mating type cells were mixed,cell polarity prominent structure was failed to induced.Then,the p Bsr CYC9 recombinant plasmid was constructed and transformed into ?CYC9-B and ?CYC9-C mutants.Expression of CYC9 was recovered in the rescued mutants.The different mating type mutants also recovered recognition and paired.Furthermore,the mutants finished sexual development.The results indicate that CYC9 is required for cell recognition and sexual development.3.Identification and analysis of CYC9 related genes.The total RNA of wild type and ?CYC9 mutant cell lines at different developmental stages were extracted and differential transcriptome analysis was performed.Loss of expression of CYC9 resulted in a 10-fold decrease in the expression level of the sex-determining gene MTA6 and a decrease in MTB6 expression by about 20-fold.In addition,the expression of the cyclin-dependent protein kinase catalytic subunit CKS1 reduced by about 35-fold.To further understand the factors involved in the regulation of the Cyclin Cyc9 p regulatory pathway,the co-expressed gene CDK19 was also screened by the Tetrahymena functional genomic database?http://tfgd.ihb.ac.cn?.4.The koockout of CDK19,CKS1,which is associated with CYC9 interferes with cell recognition pairing.The knockout plasmids p Neo4CDK19 and p Neo4CKS1 were constructed and transformed into different mating types Cu427 and Cu428 of Tetrahymena cells.CDK19 and CKS1 knockout mutant cells were obtained by Paromomycin resistance screening and phenotypic assortment.Paired observations of different mating CDK19 knockout mutant cell lines showed that cells failed to recognition each other.Cell pair also significantly decreased in CKS1 knockdown mutants.These results indicated that Cdk19 and Cks1 are also involved in the regulation of recognition of mating cells,Cyc9,Cdk19,and Cks1 function in the same signal pathway.In this study,the function of the cyclin gene CYC9 and its related factors were analyzed in T.thermophila.The CYC9 was not expressed in the growing stage and highly expressed in the early stage of starvation and sexual development stage.Cyc9 localized in the cytoplasm.CYC9,CKS1,and CDK19 are required for the recognition of different mating type cells of Tetrahymena.The knockout of CYC9 also inhibits the expression of sex-determining genes MTA6 and MTB6.The results indicate that cyclins and related proteins are involved in the regulation of cell recognition during the sexual reproduction in Tetrahymena.