周期蛋白Cyc2的过量表达及突变抑制嗜热四膜虫有性生殖发育

Overexpression and Mutation of Cyclin 2 Inhibit Sexual Reproduction in Tetrahymena thermophila

周期蛋白在细胞增殖过程中呈现周期性表达变化,不同的周期蛋白通过结合周期蛋白激酶(cyclin-dependent kinase,CDKs)及靶向特定蛋白质参与细胞有丝分裂和减数分裂过程的精确调控。嗜热四膜虫有性生殖期特异表达的B3型周期蛋白Cyc2(cyclin 2,Cyc2)对减数分裂的启始是必需的,但Cyc2蛋白的分子调控机制并不清楚。本研究通过0.1μg/mL和0.3μg/mL镉离子诱导突变细胞株OE-CYC2-B2086和OE-CYC2-CU428中CYC2基因在金属硫蛋白1基因(metallothionein gene 1,MTT1)启动子调控下上调表达。实时荧光定量PCR检测突变株OE-CYC2-B2086和OE-CYC2-CU428中CYC2的转录水平分别上调7.8倍和9.8倍。细胞有性生殖发育进程的荧光显微观察发现CYC2的表达上调并不影响有性生殖前期减数分裂的启始,但是干扰四膜虫有性生殖后期中新大核和新小核的正确形成。同时突变株OE-CYC2-B2086和OE-CYC2-CU428交配后,在镉离子诱导下不能产生有性生殖后代,但是该突变株分别和两种不同野生型细胞或CYC2敲除的突变细胞株交配能够恢复产生3%,15%或32%的有性生殖后代,有性生殖发育异常程度与CYC2的表达水平成正相关。进一步突变Cyc2第312位磷酸化位点丝氨酸为丙氨酸,获得CYC2单位点突变细胞株CYC2-S312A-B和CYC2-S312A-C。丝氨酸单位点突变阻止了四膜虫有性生殖期小核减数第1次分裂起始。结果表明周期蛋白2的表达水平和磷酸化修饰影响了不同阶段细胞核的功能,周期蛋白2对四膜虫有性生殖发育的正常进行是必需的。

Cyclins undergo a cycle of synthesis and degradation in each cell cycle and play key roles in both mitosis and meiosis by activating its cyclin-dependent kinase (Cdk) partners and directing it to specific target proteins. In Tetrahymena thermophila, Cyclin Cyc2 is required for initiation of micronuclear meiosis during the sexual reproduction stage, but the molecular function of Cyc2 is not well understood. In this study, Cyc2 was overexpressed in the mating mutants OE-CYC2-B2086 and OE-CYC2-CU428 under 0.1 μg/mL Cd2+ and 0.3 μg/mL Cd2+ induction, respectively. Expression levels of CYC2 increased 7.8 times and 9.8 times under 0.1 μg/mL Cd2+ and 0.3 μg/mL Cd2+ induction, respectively. Overexpression of Cyc2 had no effect for initiation of micronuclear meiosis during the early sexual reproduction stage, but affected formation and differentiation of the new macronucleus and new micronucleus during the late sexual reproduction stage. The mating between mutants OE-CYC2-B2086 and OE-CYC2-CU428 under 0.1 μg/mL Cd2+ and 0.3 μg/mL Cd2+ failed to produce progeny. However, the mutant phenotype was rescued by WT or CYC2 knockout cells and 3%, 15%, and 32% progenies were produced, respectively. The abnormal phenotype was dose-dependent for expression levels of CYC2. Furthermore, Ser312 of Cyc2 was mutated into alanine and the mutants CYC2-S312A-B and CYC2-S312A-C were created. The mutation of Ser312 of Cyc2 led to the developmental blockage at meiotic prophase I during the early conjugation stage. These results showed that expression levels and phosphorylation of Cyc2 affect nuclear development during different sexual stages, and Cyc2 is required for sexual reproduction in Tetrahymena.