过表达周期蛋白Cyc28影响四膜虫的有性生殖进程

Overexpression of Cyc28 Affects Sexual Reproduction in Tetrahymena thermophila

真核生物的细胞周期通过连续的激活和失活特定的周期蛋白/周期蛋白依赖性激酶复合物活性进行调控。嗜热四膜虫含有34种周期蛋白,有性生殖期特异表达的周期蛋白Cyc2和Cyc17在四膜虫小核减数分裂中发挥重要功能。本研究从嗜热四膜虫中鉴定出一种新的周期蛋白CYC28(TTHERM_00082190)基因,预测编码266个氨基酸。实时荧光定量PCR表明,CYC28在有性生殖时期特异表达,且在4 h表达水平最高。通过同源重组构建获得MTT1启动子调控下的HA-CYC28突变体细胞。免疫荧光定位表明,HA-Cyc28定位在细胞质和凋亡的亲本大核中。分别构建CYC28敲除突变株和RNA干扰细胞株,对CYC28敲减突变体细胞的分析发现,营养生长和有性生殖期突变细胞发育正常。然而,过表达株Cyc28突变体引起原核染色体排列异常,原核不能完成有丝分裂形成配子核,有性生殖进程终止。结果表明,Cyc28参与细胞的有性生殖进程,它的正常表达和降解对原核有丝分裂的完成是必需的。

The eukaryotic cell cycle is controlled by the successive activation and inactivation of specific CDK / cyclin complexes activity. There are 34 cyclins in Tetrahymena thermophila. Meiotic specifically expressed cyclins Cyc2 and Cyc17 play key roles in Tetrahymena meiosis progress. In this study,a novel meiosis-specifically expressed CYC28（ TTHERM_00082190） gene was identified,which encodes 266 amino acids. CYC28 is specifically expressed during conjugation stage and peaked at 4 h as determined by real-time PCR. MTT1-HA-CYC28 mutants were created by homologous recombination.Immunostaining showed that HA-Cyc28 localized in the cytoplasm during early stage of sexual reproduction,and localized in the apoptotic parental macronuclei in later conjugation stage. CYC28 knockout construct p Neo4-Δ-CYC28 and CYC28 knockdown construct p CYC28 hp CYH were created,respectively. Furthermore,different CYC28 knockdown mutants were obtained by gene knockout or RNAi. Tetrahymena sexual development progress is normal in the CYC28 knockdown mutant strains.However,overexpression of Cyc28 led to abnormal pronuclei and failed to form gametic nuclei by mitosis.The Tetrahymena sexual reproduction progress was aborted. The results showed that Cyc28 was involved in the sexual reproduction,its normal expression and degradation was required for pronuclear mitosis and gametic nuclei formation.