Pmr1基因缺失对粟酒裂殖酵母细胞有性生殖和有丝分裂的影响及其分子机制研究*

目的:pmr1基因编码P型钙转运ATP酶Pmr1,参与维持细胞壁完整性和调控胞质分裂。以粟酒裂殖酵母为模式细胞,探究pmr1缺失后对细胞有性生殖及细胞分裂中肌动蛋白环精细动力学的影响,揭示pmr1缺失后细胞异常生长过程中的关键基因和代谢通路。方法:通过细胞生长速率测定、产孢统计、绿色荧光蛋白标记肌动蛋白和活细胞成像的方法,检测pmr1缺失对细胞有丝分裂和有性生殖的影响;采用RNA-Seq对野生型菌株和pmr1Δ菌株测序和生物信息学分析,并进行qRT-PCR验证。结果:pmr1缺失后细胞生长减慢,分裂期细胞长度减小,子囊孢子长度增加,且肌动蛋白环的形成时间增加。RNA测序结果显示,mfm1、mfm2和mat1-Mc下调,错配修复通路cdc1和exo1上调以及糖酵解/糖异生途径pgi1、pfk1和dld1下调是引起pmr1Δ孢子长度增加的主要因素;糖酵解/糖异生途径tdh1、pgk1下调,以及脂肪酸合成代谢途径fas1、fas2、cut6和lcf1下调导致了pmr1Δ分裂期细胞长度减小;hsp9上调是影响pmr1Δ收缩环形成时间增加的关键基因。qRT-PCR实验证实,pmr1缺失后关键基因的表达趋势与RNA-Seq结果一致。结论:pmr1缺失后,粟酒裂殖酵母细胞中错配修复通路、糖酵解/糖异生途径及脂肪酸合成代谢途径发生障碍,导致细胞及孢子形态均异常,且肌动蛋白环形成受阻,细胞增殖减缓。

Effects of pmr1 Gene Deletion on Sexual Reproduction and Mitosis of Fission Yeast Cells and Its Molecular Mechanism

Objective: The pmr1 gene encodes P-type calcium-transporting ATPase Pmr1, which is involved in maintaining cell wall integrity and regulating cytokinesis. In this study, fission yeast was used as a model cell to explore the effects of pmr1 deletion on the sexual reproduction and dynamics of actin rings during cell mitosis, and to reveal the key genes and metabolic pathways of abnormal cell mitosis after pmr1 deletion. Methods: The effects of pmr1 deletion on cell mitosis and sexual reproduction were detected by cell growth rate measurement, spore production observation and statistics, green fluorescent protein-labeled monitoring and living cell imaging; The wild-type and pmr1Δ strain were sequenced by RNA-Seq and analyzed by bioinformatics, and verified by qRT-PCR. Results: The pmr1 deletion could slow down cell growth, decrease cell length in mitosis, increase length of sexually reproductive ascospore, and increase formation time of actin ring. RNA-sequencing results revealed that down-regulation of mfm1, mfm2 and mat1-Mc, up-regulation of cdc1 and exo1 in the mismatch repair pathway, and down-regulation of pgi1, pfk1 and dld1 in the glycolysis/gluconeogenesis pathway are the main factor of the increased spore length in the pmr1Δ; Meanwhile, the down-regulation of tdh1 and pgk1 in the glycolysis/gluconeogenesis pathway and the down-regulation of fas1, fas2, cut6 and lcf1 in the fatty acid anabolic pathway also led to the decreased cell length in mitosis of pmr1Δ; The up-regulation of hsp9 is the key gene that affected the formation time of actin ring in pmr1Δ. qRT-PCR experiments confirmed that the expression trends of key genes after pmr1 deletion were consistent with the RNA-Seq results. Conclusion: After pmr1 deletion, the mismatch repair pathway, glycolysis/gluconeogenesis pathway and fatty acid anabolic pathway are hindered in fission yeast cells, resulting in abnormal cell and spore morphology, obstruction of actin ring formation, and slowing down of cell proliferation.