Functional Analysis Of Cyclin Cyc28 In Tetrahymena Thermophila

The eukaryotic cell cycle is controlled by the successive activation and inactivation of specific CDK/cyclin complexes activity.In mammals,cyclinA and cyclinB dephosphorylated the target protein,promoting transformation of metaphase to anaphase,cyclinD terminates G1 phase by phosphorylating Rb protein,promotes transformation of G1/S.Different organisms contain different types of cyclin,ciliate protozoa shows more diversity of cyclins,showing that there is more subtle mechanism in ciliate.Tetrahymena thermophila has two nucleus,the maronucleus is polyploid and transcriptionally active during the vegetative growth,but the mironucleus is diploid and transcriptionally silent during the vegetative growing stage.Tetrahymena has two reproductive methods:asexual reproduction and sexual reproduction.During sexual reproduction,micronucleus produces four pronucleus by meiosis,of which three degradation,the other one produces two gametic nucleus by mitosis.gametes nuclear produces zygote by exchange and fusion,zygote produces new macronuclei and new micronuclei through mitosis,then cell completes sexual reproduction.Tetrahymena thermophila contains 34 cyclins,in which CYC2,CYC17 and CYC28 are specifically expressed during meiosis,Cyc2 regulates the initiation of micronucleus meiosis,and Cyc17 regulates the separation of homologous chromosomes,but the function of Cyc28 is not clear,the study identified a meiotic specifically expressed gene CYC28,the results are as follows:1.Bioinformatics analysis of CYC28 Basing on the Tetrahymena genome database and the functional genome database,we found that CYC28（TTHERM₀0082190）gene was 1174 bp,the open reading frame was 801 bp,which encodes 266 amino acids.RT-PCR showed sequence of CYC28 was correct.Sequence alignment analysis showed that there was a conservedcyclin box at the N-terminus,consisting of 121 amino acids,and a breakdown box,consisting of 9 amino acid residues.Cyclin sequence analysis showed that Cyc28 may belong to the cyclinB3 subfamily.Microarray and RT-PCR analysis showed CYC28 didn’t express during vegetative growth and starvation,but express at 4 h during conjugation.2.Localization and expression analysis of HA-Cyc28.In order to study the function of Cyc28,we constructed the expression vector pXS75-CYC28 with HA tag.The vector was triggered by MTT1 promoter,transformed into cells.Immunofluorescent localization showed that Cyc28 was localized in the cytoplasm at early stage of sexual reproduction,localized in on the old apoptotic nucleus at 8 h.Immunoblotting showed that HA-Cyc28 was up-regulated at 2 h,down-regulated at 4 h and 8 h,and degradation after pair separated.3.Knockdown CYC28 had no effect on sexual reproduction.For further analyze the function of Cyc28 in the process of sexual reproduction from Tetrahymena thermophila,we first constructed the knockout vector pNeo4-Δ-CYC28,and the knockdown cells were screened by homologous recombination.Compared with wild-type cells,knockdown CYC28 didn’t affect sexual reproduction process.As failing to obtain knockout cells,we constructed the interference plasmid pCYC28 hpCYH,regulated by Cd2+.The interference plasmid was transformed into Tetrahymena by gene gun,and the mutant cells were obtained by actinomycin resistance.RT-PCR detected and obtained stable strains,but the development of nuclei was not affected,indicating that knockdown Cyc28 had no significant effect on the process of sexual reproduction.4.Overexpression of Cyc28 affects sexual reproduction.The CYC28 expression level of mutants was 10 times that of wild type.Compared with the wild type,the sexual reproduction process was abnormal.45.5% cells didn’t present condensed chromosomes,but present chromosomes depolymerized micronuclei at 3.5 h during the first meiosis;At 4h,36.5% ofcells remained at the first meiosis,31.5% of cells proceeded to the second meiotic division,but micronuclei is loose;At 6h,most of the cells failed to form gametic nuclei by mitosis;At 8h,45.3% of the cells did not form new macronucleus.At 12 h,there was no cell that containing two macronuclei and a micronucleus in cells.The results showed overexpression of Cyc28 led to abnormal pronuclei and failed to form gametic nuclei by mitosis.In this study,we first identified the cyclin gene CYC28,qRT-PCR showed CYC28 was specifically expressed in sexual stage.Knockdown CYC28 didn’t affect cell development,but over-express CYC28 affected micronucleus mitosis during sexual reproduction,indicating it’s necessary for sexual reproduction that normal expression and degradation of Cyc28.Further,we could obtain knockout cells,reveal its specific function during sexual reproduction in Tetrahymena thermophila,which may perfect the regulation mechanism of cyclin in ciliate protozoa.