In order to clarify the reproductive status, dynamic changes in mitosis interphase and the abnormal key expression genes and key metabolic pathways after uge1 gene deletion of Schizosaccharomyces pombe, live cell imaging was used to analyze sporulation and dynamic changes in mitosis interphase after uge1 gene deletion(Δuge1)combining with RNA sequencing(RNA-Seq)technology and bioinformatics analysis for wild-type and Δuge1 strain, and the expression of key genes was- DOI:10.3969/j.issn.1000-1565.2025.06.008uge1基因缺失对粟酒裂殖酵母生长生殖的影响及其分子机制青昕1,张丁1,余雯1,刘可1,丁祥2,侯怡铃1(1.西华师范大学 生命科学学院,西南野生动植物资源保护教育部重点实验室,四川 南充 637009;2.西华师范大学 环境科学与工程学院,四川 南充 637009)摘 要:为明确粟酒裂殖酵母uge1基因缺失后生殖情况、有丝分裂间期动力学变化及异常的关键表达基因和关键代谢通路,采用活细胞成像分析uge1基因缺失(Δuge1)后产孢情况及有丝分裂间期动力学变化,结合RNA测序(RNA-Seq)技术对野生型和Δuge1菌株测序并进行生物信息学分析,同时采用荧光定量PCR对关键基因的表达水平进行验证. 活细胞成像结果表明,uge1基因缺失导致酵母产4个正常孢子数的细胞减少,孢子直径增大,6%的Δuge1菌株出现细胞核解聚,微管束在细胞壁上的停留时间减少了(0.526±0.08)min. RNA测序结果显示,Δuge1菌株中mat2-Pc、mat2-Pi基因的上调表达与mfm1、mfm2基因的下调表达导致了粟酒裂殖酵母的异常生殖过程,aat1基因的表达量增加使纺锤体延伸时间变长,acp2和cdc8基因的过量表达则导致了肌动蛋白环收缩减慢. KEGG富集分析表明,脂肪酸合成代谢中fas1、fas2和cut6基因的表达下调干扰了脂肪酸合成代谢,促使核膜合成障碍,导致核解聚的发生. 研究结果揭示了粟酒裂殖酵母uge1基因缺失导致细胞生长生殖异常的关键表达基因和关键代谢通路,为深入研究uge1基因对细胞动力学影响的分子机制提供了一定的科学依据.关键词:uge1基因缺失;粟酒裂殖酵母;RNA测序;有性生殖;有丝分裂中图分类号:Q932 文献标志码:A 文章编号:1000-1565(2025)06-0623-14Effect of uge1 gene deletion on the growth and reproduction of Schizosaccharomyces pombe and its molecular mechanismQING Xin1, ZHANG Ding1, YU Wen1, LIU Ke1, DING Xiang2, HOU Yiling1(1. Key Laboratory of Southwest China Wildlife Resources Conservation, the Ministry of Education, College of Life Science, China West Normal University, Nanchong 637009, China; 2. College of Environmental Science and Engineering, China West Normal University, Nanchong 637009, China)Abstract: In order to clarify the reproductive status, dynamic changes in mitosis interphase and the abnormal key expression genes and key metabolic pathways after uge1 gene deletion of Schizosaccharomyces pombe, live cell imaging was used to analyze sporulation and dynamic changes in mitosis interphase after uge1 gene deletion(Δuge1)combining with RNA sequencing(RNA-Seq)technology and bioinformatics analysis for wild-type and Δuge1 strain, and the expression of key genes was- 收稿日期:2024-10-30;修回日期:2025-03-03 基金项目:四川省科技厅应用基础面上项目(2022NSFSC0107);四川省科技厅农业科技成果转化资金项目(2022NZZJ0003);四川省转移支付科技成果转移转化示范项目(22ZYZFSF0009;23ZHSF0082) 第一作者:青昕(1998—),女,西华师范大学在读硕士研究生,主要从事生物化学与分子生物学方向研究.E-mail:1026362669@qq.com 通信作者:侯怡铃(1983—),女,西华师范大学教授,主要从事生物化学与分子生物学方向研究.E-mail:yilingh128@163.com 第6期青昕等:uge1基因缺失对粟酒裂殖酵母生长生殖的影响及其分子机制河北大学学报(自然科学版) 第45卷also validated using fluorescence quantitative PCR. The results of live cell imaging showed that the deletion of the uge1 gene resulted in a decrease in the number of cells producing four normal spores in yeast, an increase in spore diameter. 6% Δuge1 strain exhibited nuclear depolymerization and the residence time of the microtubule bundle on the cell wall decreased by(0.526±0.08)min. RNA sequencing results showed that the up-regulated expression of mat2-Pc and mat2-Pi genes and the down-regulation of mfm1 and mfm2 genes in the Δuge1 strain led to the abnormal reproductive process of S.pombe. The increased expression of aat1 gene interfered with the extension of spindle, and the overexpression of acp2 and cdc8 genes led to the slow contraction of actin loop in Δuge1 strain. KEGG enrichment analysis showed that the down-regulation of fas1, fas2 and cut6 genes in fatty acid synthesis pathway interfered with the fatty acid synthesis, which promoted the disorder of nuclear membrane synthesis, and led to the occurrence of nuclear depolymerization. These results revealed the key genes and key metabolic pathways that the deletion of uge1 gene in S.pombe led to abnormal cell growth and reproduction, and provided a scientific basis for further research on the molecular mechanism of uge1 genes impact on cell dynamics.
[1] GRIGAITIS P, GRUNDEL D A J, VAN PELT-KLEINJAN E, et al. A computational toolbox to investigate the metabolic potential and resource allocation in fission yeast[J]. mSystems, 2022, 7(4):1-14. DOI:10.1128/msystems.00423-22.
[2] SUZUKI S, MATSUZAWA T, NUKIGI Y, et al. Characterization of two different types of UDP-glucose /-galactose 4-epimerase involved in galactosylation in fission yeast[J]. Microbiology, 2010, 156(3): 708-718. DOI:10.1099/mic.0.035279-0.
[3] MATSUZAWA T, HARA F, TANAKA N, et al. Ght2+ is required for UDP-galactose synthesis from extracellular galactose by Schizosaccharomyces pombe[J]. Appl Microbiol Biotechnol, 2013, 97(11): 4957-4964. DOI:10.1007/s00253-012-4637-4.
[4] DUDIN O, MERLINI L, BENDEZU' F O, et al. A systematic screen for morphological abnormalities during fission yeast sexual reproduction identifies a mechanism of actin aster formation for cell fusion[J]. PLoS Genet, 2017, 13(4): e1006721. DOI:10.1371/journal.pgen.1006721.
[5] 谭秀梅,丁祥,袁荣美,等.半乳糖代谢酶uge1基因的缺失导致裂殖酵母有丝分裂中细胞动力学的变化[J].华中师范大学学报(自然科学版), 2023, 57(2): 262-272. DOI:10.19603/j.cnki.1000-1190.2023.02.011.
[6] DESHPANDE G P, HAYLES J, HOE K L, et al. Screening a genome-wide S.pombe deletion library identifies novel genes and pathways involved in genome stability maintenance[J]. DNA Repair, 2009, 8(5): 672-679. DOI:10.1016/j.dnarep.2009.01.016.
[7] MOJARDÍN L, BOTET J, MORENO S, et al. Chromosome segregation and organization are targets of 5’-Fluorouracil in eukaryotic cells[J]. Cell Cycle, 2015, 14(2): 206-218. DOI:10.4161/15384101.2014.974425.
[8] HAN T X, XU X Y, ZHANG M J, et al. Global fitness profiling of fission yeast deletion strains by barcode sequencing[J]. Genome Biol, 2010, 11(6): R60. DOI:10.1186/gb-2010-11-6-r60.
[9] GANGULY A, GUO L, SUN L L, et al. Tdp1 processes chromate-induced single-strand DNA breaks that collapse replication Forks[J]. PLoS Genet, 2018, 14(8): e1007595. DOI:10.1371/journal.pgen.1007595.
[10] ZHANG X B, FANG Y, JAISENG W, et al. Characterization of tamoxifen as an antifungal agent using the yeast Schizosaccharomyces pombe model organism[J]. Kobe J Med Sci, 2015, 61(2): E54-E63.
[11] BROUSSARD A, FLORWICK A, DESBIENS C, et al. Human UDP-galactose 4’-epimerase(GALE)is required for cell-surface glycome structure and function[J]. J Biol Chem, 2020, 295(5): 1225-1239. DOI:10.1016/S0021-9258(17)49882-6.
[12] DAENZER J M I, SANDERS R D, HANG D, et al. UDP-galactose 4’-epimerase activities toward UDP-Gal and UDP-GalNAc play different roles in the development of Drosophila melanogaster[J]. PLoS Genet, 2012, 8(5): e1002721. DOI:10.1371/journal.pgen.1002721.
[13] MARÍN-QUÍLEZ A, DI BUDUO C A, BENITO R, et al. GALE variants associated with syndromic manifestations, macrothrombocytopenia, bleeding, and platelet dysfunction[J]. Platelets, 2023, 34(1): 2176699. DOI:10.1080/09537104.2023.2176699.
[14] DIAS COSTA F, FERDINANDUSSE S, PINTO C, et al. Galactose epimerase deficiency: Expanding the phenotype[J]. JIMD Rep, 2017, 37: 19-25. DOI:10.1007/8904_2017_10.
[15] 叶姿妤,丁祥,鲁艳,等.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[J].中国生物工程杂志, 2022, 42(12): 12-26. DOI:10.13523/j.cb.2204077.
[16] 舒锟,李西悦,刘选茹,等.杉木人工林土壤碳、氮、磷化学计量特征对短期氮添加的响应[J].云南大学学报(自然科学版), 2022, 44(4): 840-851. DOI:10.7540/j.ynu.20220081.
[17] TARICANI L, FEILOTTER H E, WEAVER C, et al. Expression of hsp16 in response to nucleotide depletion is regulated via the spc1 MAPK pathway in Schizosaccharomyces pombe[J]. Nucleic Acids Res, 2001, 29(14): 3030-3040. DOI:10.1093/nar/29.14.3030.
[18] HIROSE M, TOHDA H, GIGA-HAMA Y, et al. Interaction of a small heat shock protein of the fission yeast, Schizosaccharomyces pombe, with a denatured protein at elevated temperature[J]. J Biol Chem, 2005, 280(38): 32586-32593. DOI:10.1074/jbc.m504121200.
[19] NAIRN J, PRICE N C, KELLY S M, et al. Phosphoglycerate mutase from Schizosaccharomyces pombe: Development of an expression system and characterisation of three histidine mutants of the enzyme[J]. Biochim Biophys Acta BBA Protein Struct Mol Enzymol, 1996, 1296(1): 69-75. DOI:10.1016/0167-4838(96)00046-5.
[20] DENG J, WALTHER A. ATP-responsive and ATP-fueled self-assembling systems and materials[J]. Adv Mater, 2020, 32(42): 2002629. DOI:10.1002/adma.202002629.
[21] RYAN C J, ROGUEV A, PATRICK K, et al. Hierarchical modularity and the evolution of genetic interactomes across species[J]. Mol Cell, 2012, 46(5): 691-704. DOI:10.1016/j.molcel.2012.05.028.
[22] TODA T, SHIMANUKI M, YANAGIDA M. Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases[J]. Genes Dev, 1991, 5(1): 60-73. DOI:10.1101/gad.5.1.60.
[23] YABANA N, YAMAMOTO M. Schizosaccharomyces pombe map1+ encodes a MADS-box-family protein required for cell-type-specific gene expression[J]. Mol Cell Biol, 1996, 16(7): 3420-3428. DOI:10.1128/MCB.16.7.3420.
[24] KJAERULFF S, LAUTRUP-LARSEN I, TRUELSEN S, et al. Constitutive activation of the fission yeast pheromone-responsive pathway induces ectopic meiosis and reveals ste11 as a mitogen-activated protein kinase target[J]. Mol Cell Biol, 2005, 25(5): 2045-2059. DOI:10.1128/MCB.25.5.2045-2059.2005.
[25] STYRKÁRSDÓTTIR U, EGEL R, NIELSEN O. Functional conservation between Schizosaccharomyces pombe ste8 and Saccharomyces cerevisiae STE11 protein kinases in yeast signal transduction[J]. Mol Gen Genet, 1992, 235(1): 122-130. DOI:10.1007/BF00286189.
[26] ANVER S, ROGUEV A, ZOFALL M, et al. Yeast X-chromosome-associated protein 5(Xap5)functions with H2A.Z to suppress aberrant transcripts[J]. EMBO Rep, 2014, 15(8): 894-902. DOI:10.15252/embr.201438902.
[27] MERLINI L, DUDIN O, MARTIN S G. Mate and fuse: How yeast cells do it[J]. Open Biol, 2013, 3(3): 130008. DOI:10.1098/rsob.130008.
[28] JAKOCˇIŪNAS T, HOLM L R, VERHEIN-HANSEN J, et al. Two portable recombination enhancers direct donor choice in fission yeast heterochromatin[J]. PLoS Genet, 2013, 9(10): e1003762. DOI:10.1371/journal.pgen.1003762.
[29] KJAERULFF S, DAVEY J, NIELSEN O. Analysis of the structural genes encoding M-factor in the fission yeast Schizosaccharomyces pombe: Identification of a third gene, mfm3[J]. Mol Cell Biol, 1994, 14(6): 3895-3905. DOI:10.1128/mcb.14.6.3895.
[30] BLYTH J, MAKRANTONI V, BARTON R E, et al. Genes important for Schizosaccharomyces pombe meiosis identified through a functional genomics screen[J]. Genetics, 2018, 208(2): 589-603. DOI:10.1534/genetics.117.300527.
[31] LORENZ A, MEHATS A, OSMAN F, et al. Rad51/Dmc1 paralogs and mediators oppose DNA helicases to limit hybrid DNA formation and promote crossovers during meiotic recombination[J]. Nucleic Acids Res, 2014, 42(22): 13723-13735. DOI:10.1093/nar/gku1219.
[32] CAM H P, SUGIYAMA T, CHEN E S, et al. Comprehensive analysis of heterochromatin- and RNAi-mediated epigenetic control of the fission yeast genome[J]. Nat Genet, 2005, 37(8): 809-819. DOI:10.1038/ng1602.
[33] FUJITA I, YAMASHITA A, YAMAMOTO M. Dynactin and Num1 cooperate to establish the cortical anchoring of cytoplasmic dynein in S. pombe[J]. J Cell Sci, 2015, 128(8): 1555-1567. DOI:10.1242/jcs.163840.
[34] BERGSTRALH D T, DAWNEY N S, ST JOHNSTON D. Spindle orientation: A question of complex positioning[J]. Development, 2017, 144(7): 1137-1145. DOI:10.1242/dev.140764.
[35] PARDO M, NURSE P. The nuclear rim protein Amo1 is required for proper microtubule cytoskeleton organisation in fission yeast[J]. J Cell Sci, 2005, 118(Pt 8): 1705-1714. DOI:10.1242/jcs.02305.
[36] KOVAR D R, WU J Q, POLLARD T D. Profilin-mediated competition between capping protein and formin Cdc12p during cytokinesis in fission yeast[J]. Mol Biol Cell, 2005, 16(5): 2313-2324. DOI:10.1091/mbc.e04-09-0781.
[37] KOGA T, ONISHI M, NAKAMURA Y, et al. Sorting nexin homologues are targets of phosphatidylinositol 3-phosphate in sporulation of Schizosaccharomyces pombe[J]. Genes Cells, 2004, 9(6): 561-574. DOI:10.1111/j.1356-9597.2004.00744.x.
[38] IWAKI T, HOSOMI A, TOKUDOMI S, et al. Vacuolar protein sorting receptor in Schizosaccharomyces pombe[J]. Microbiology, 2006, 152(Pt 5): 1523-1532. DOI:10.1099/mic.0.28627-0.
[39] CHRISTENSEN J R, HOMA K E, MORGANTHALER A N, et al. Cooperation between tropomyosin and α-actinin inhibits fimbrin association with actin filament networks in fission yeast[J]. eLife, 2019, 8. DOI:10.7554/eLife.47279. DOI:10.7554/eLife.47279.
[40] NAKANO K, MABUCHI I. Actin-depolymerizing protein Adf1 is required for formation and maintenance of the contractile ring during cytokinesis in fission yeast[J]. Mol Biol Cell, 2006, 17(4): 1933-1945. DOI:10.1091/mbc.e05-09-0900.
[41] BERRO J, POLLARD T D. Synergies between Aip1p and capping protein subunits(Acp1p and Acp2p)in clathrin-mediated endocytosis and cell polarization in fission yeast[J]. Mol Biol Cell, 2014, 25(22): 3515-3527. DOI:10.1091/mbc.E13-01-0005.
[42] NAKANO K, MABUCHI I. Actin-capping protein is involved in controlling organization of actin cytoskeleton together with ADF/cofilin, profilin and F-actin crosslinking proteins in fission yeast[J]. Genes Cells, 2006, 11(8): 893-905. DOI:10.1111/j.1365-2443.2006.00987.x.
[43] BUTTRICK G J, MEADOWS J C, LANCASTER T C, et al. Nsk1 ensures accurate chromosome segregation by promoting association of kinetochores to spindle poles during anaphase B[J]. Mol Biol Cell, 2011, 22(23): 4486-4502. DOI:10.1091/mbc.E11-07-0608.
[44] TELEKAWA C, BOISVERT F M, BACHAND F. Proteomic profiling and functional characterization of post-translational modifications of the fission yeast RNA exosome[J]. Nucleic Acids Res, 2018, 46(21): 11169-11183. DOI:10.1093/nar/gky915.
[45] KINOSHITA N, GOEBL M, YANAGIDA M. The fission yeast dis3+ gene encodes a 110-kDa essential protein implicated in mitotic control[J]. Mol Cell Biol, 1991, 11(12): 5839-5847. DOI:10.1128/mcb.11.12.5839-5847.1991.
[46] WEICK E M, PUNO M R, JANUSZYK K, et al. Helicase-dependent RNA decay illuminated by a cryo-EM structure of a human nuclear RNA exosome-MTR4 complex[J]. Cell, 2018, 173(7): 1663-1677. DOI:10.1016/j.cell.2018.05.041.
[47] SCHWEIZER E, HOFMANN J. Microbial type I fatty acid synthases(FAS): Major players in a network of cellular FAS systems[J]. Microbiol Mol Biol Rev, 2004, 68(3): 501-517. DOI:10.1128/MMBR.68.3.501-517.2004.
[48] MAKAROVA M, GU Y, CHEN J S, et al. Temporal regulation of lipin activity diverged to account for differences in mitotic programs[J]. Curr Biol, 2016, 26(2): 237-243. DOI:10.1016/j.cub.2015.11.061.
[49] YANAGIDA M. Fission yeast cut mutations revisited: Control of anaphase[J]. Trends Cell Biol, 1998, 8(4): 144-149. DOI:10.1016/S0962-8924(98)01236-7. (
