Article(id=1297571131087672257, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1297570992835023717, articleNumber=null, orderNo=null, doi=10.13343/j.cnki.wsxb.20260146, pmid=null, cstr=null, oa=null, hot=null, price=null, onlineType=0, articleFormat=0, articleType=null, articleTypeStr=research-article, receivedDate=1771689600000, receivedDateStr=2026-02-22, revisedDate=null, revisedDateStr=null, acceptedDate=1776009600000, acceptedDateStr=2026-04-13, onlineDate=1787294665357, onlineDateStr=2026-08-21, pubDate=1785772800000, pubDateStr=2026-08-04, doiRegisterDate=null, doiRegisterDateStr=null, onlineIssueDate=1787294665357, onlineIssueDateStr=2026-08-21, onlineJustAcceptDate=null, onlineJustAcceptDateStr=null, onlineFirstDate=null, onlineFirstDateStr=null, sourceXml=null, magXml=null, createTime=1787294665357, creator=13701087609, updateTime=1787294665357, updator=13701087609, issue=Issue{id=1297570992835023717, tenantId=1146029695717560320, journalId=1192105938417971205, year='2026', volume='66', issue='8', pageStart='3681', pageEnd='4288', issueExtLink='null', onlineDate='null', pubDate='1785772800000', pubDateStr='2026-08-04', beforeIssueId=null, nextIssueId=null, price=null, status=1, issueComplete=1, articleOrder=1, issueType=-1, specialIssue=null, createTime=1787294632395, creator='13701087609', updateTime=1787294931551, updator='13701087609', preIssue=null, nextIssue=null, articleTotal=null, ext={EN=IssueExt(id=1297572247670124783, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1297570992835023717, language=EN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=), CN=IssueExt(id=1297572247670124784, tenantId=1146029695717560320, journalId=1192105938417971205, issueId=1297570992835023717, language=CN, specialIssueTitle=, coverIllustrator=null, specialIssueEditor=, specialIssueAbout=)}, issueFiles=null, downloadFileDto=null}, startPage=4215, endPage=4225, ext={EN=ArticleExt(id=1297571131494519746, articleId=1297571131087672257, tenantId=1146029695717560320, journalId=1192105938417971205, language=EN, title=Mechanism of exit from quiescence in
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[Objective] To explore genes related to the regulation of yeast spore germination, we previously screened for mutants defective in maintaining the quiescent state of spores. [Methods] Using the temperature-sensitive yeast mutant J33 with defective spore quiescence maintenance as the research material, heterozygous diploid strains were constructed through tetrad dissection and haploid fusion, and genetic segregation analysis was performed to clarify the genetic regulatory pattern of mutant phenotypes. The yeast genomic library complementation screening was applied to identify candidate mutant genes, and gene cloning and sequence alignment were conducted to confirm the mutation site. Furthermore, recombinant plasmids carrying wild-type and mutant TIP20 were constructed for functional complementation assays, and site-directed mutagenesis was used to verify the correlation between TIP20 mutation and strain phenotypes. [Results] Functional complementation experiments demonstrated that introduction of the wild-type TIP20 restored the growth at 37 ℃ and the normal spore germination phenotype of J33, while the mutant TIP20 and empty vector had no such effect. Site-directed mutagenesis analysis further confirmed that this single-base mutation was T1219G, a direct cause of the TS phenotype and defective spore germination of J33. [Conclusion] TIP20 is involved in the regulation of yeast spore germination. TIP20 encodes a subunit of the tethering complex involved in endoplasmic reticulum-Golgi retrograde transport. However, TIP20 is likely a multifunctional protein, and its mammalian homologue RINT1 has been reported to regulate the cell cycle checkpoint. Thus, TIP20 may also be involved in regulating the quiescent state of yeast cells.
, authors=Yibo LIU, Zhuo CHENG, Jia CHEN, Hideki Nakanishi, authorsList=Yibo LIU, Zhuo CHENG, Jia CHEN, Hideki Nakanishi, authorCompany=null, correspAuthors=Hideki Nakanishi, authorNote=null, correspAuthorsNote=
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【目的】 为探究酵母孢子萌发调控相关基因,前期筛选获得了孢子静止状态维持缺陷的突变体。 【方法】 以孢子静止期维持缺陷的温度敏感型酵母突变体J33为研究材料,通过四分体分离与单倍体融合构建杂合双倍体菌株,结合遗传分离分析明确突变表型的遗传调控模式;采用酵母基因组文库互补筛选技术定位突变候选基因,通过基因克隆、测序比对鉴定突变位点;进一步构建野生型及突变型TIP20重组质粒开展功能互补实验,结合基因定点突变技术验证目的基因突变与菌株表型的关联性。 【结果】 功能互补实验显示,导入野生型TIP20基因可恢复J33突变体在37 ℃下的生长能力及正常孢子萌发表型,而突变型TIP20基因和空载体无此效应。定点突变分析进一步证实,该单碱基突变是导致J33突变体温度敏感及孢子萌发缺陷的直接原因。 【结论】 TIP20参与调控酵母孢子萌发。已知TIP20编码拴系复合物亚基,该复合物参与内质网(endoplasmic reticulum, ER)-高尔基体逆行囊泡运输。然而,该蛋白可能具有多功能性,其哺乳动物同源蛋白RINT1可调控细胞周期检查点,因此TIP20可能也参与调控酵母细胞的静止状态。
, authors=刘意博, 成卓, 谌佳, 中西秀树, authorsList=刘意博, 成卓, 谌佳, 中西秀树, authorCompany=null, correspAuthors=中西秀树, authorNote=
作者贡献声明
刘意博:收集实验原始数据、图片绘制及数据分析,论文撰写及修改;成卓:辅助菌株筛选;谌佳:辅助数据收集;中西秀树:指导本实验及论文修改,数据核查,提供资金支持。
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Comparison of spore length and temperature sensitivity between mutant J33 and WT. A: Morphology of tetrad spores between J33 and WT; B: Comparison of ascus diameter between J33 mutant and WT (***: P<0.001); C: Dilution spot assay confirms the temperature sensitivity of the J33 mutant., figureFileSmall=Bynak77nniJZS7+Thm6nDw==, figureFileBig=+w16K31pXaUmv9669NOG+A==, tableContent=null), ArticleFig(id=1297571137022612462, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=CN, label=图1, caption=
J33突变体与野生型孢子长度和温度敏感性对比, figureFileSmall=Bynak77nniJZS7+Thm6nDw==, figureFileBig=+w16K31pXaUmv9669NOG+A==, tableContent=null), ArticleFig(id=1297571137215550447, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=EN, label=Figure 2, caption=
Analysis of spore staining after the first and third segregation of heterozygous tetrads. A: Diploid strains obtained from the first backcross of J33 with wild-type cells were subjected to sporulation [CFW staining patterns of tetrad spores from the temperature-sensitive mutant (J33), wild-type strain, and progeny from the first backcross were observed under a microscope]; B: The spores used in (A) were stained with CFW [The number of CFW-positive spores in asci containing four spores was counted, and asci were classified according to the number of CFW-positive spores. ****: P<0.000 1]; C: Diploid strains obtained from the third backcross of J33 with wild-type cells were subjected to sporulation [CFW staining patterns of tetrad spores from the temperature-sensitive mutant (J33), wild-type strain, and progeny from the third backcross were observed under a microscope]; D: The spores used in (C) were stained with CFW (The number of CFW-positive spores in asci containing four spores was counted, and asci were classified according to the number of CFW-positive spores. ****: P<0.000 1); E: J33 mutant, wild-type cells, and progeny from three rounds of backcrossing between J33 and wild-type cells were cultured individually at 30 °C or 37 °C; F: Progeny from three rounds of backcrossing between J33 and wild-type cells were subjected to sporulation (Spores were dissected from individual asci, and the four segregan TS from each ascus were cultured individually at 30 °C or 37 °C)., figureFileSmall=1fuQEl5NPqdJZfdvyK690A==, figureFileBig=zN++odiMSvoGT7PAcbikxA==, tableContent=null), ArticleFig(id=1297571137291047920, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=CN, label=图2, caption=
杂合四分体第1次分离与第3次分离后孢子着色分析, figureFileSmall=1fuQEl5NPqdJZfdvyK690A==, figureFileBig=zN++odiMSvoGT7PAcbikxA==, tableContent=null), ArticleFig(id=1297571137362351089, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=EN, label=Figure 3, caption=
Screening for mutant genes. A: Screening using a yeast genomic library was performed to find mutation in J33 mutant and a plasmid that could rescue the temperature sensitivity phenotype in J33 mutant was obtained; B: Schematic diagram of the DNA fragment found in the plasmid obtained by the screening., figureFileSmall=OndYG7Nc5Y9Z0F4EsSBYDQ==, figureFileBig=02/v9B1vIe0hiRbob/GXwg==, tableContent=null), ArticleFig(id=1297571137437848562, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=CN, label=图3, caption=
突变基因筛选, figureFileSmall=OndYG7Nc5Y9Z0F4EsSBYDQ==, figureFileBig=02/v9B1vIe0hiRbob/GXwg==, tableContent=null), ArticleFig(id=1297571137504957427, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=EN, label=Figure 4, caption=
Determination of mutation site. A: Complementation test of TIP20 in the J33 mutant [WT/TIP20G1219T: The corresponding site of the wild-type yeast TIP20 gene was site-directedly mutated from G to T; J33/TIP20 (TS): The mutant TIP20 gene was transformed into the J33 strain; J33/TIP20 (WT): The wild-type yeast TIP20 gene was transformed into the J33 strain. Wild-type yeast, J33 mutant, and the transformed strains were streaked on YPAD solid medium and incubated at 30 °C or 37 °C, respectively]; B: CFW staining of J33 mutant spores harboring TIP20 [Scale bar: 5 µm]; C: CFW staining of J33 mutant spores harboring TIP20T1219G [Scale bar: 5 µm]; D: Comparison of CFW staining levels of spores wild-type and J33 mutant transformed with empty vector, wild-type TIP20, TIP20 amplified from J33 mutant, or TIP20T1219G (***: P<0.001); E: Comparison of sporulation rates wild-type and J33 mutant transformed with empty vector, wild-type TIP20, TIP20 amplified from J33 mutant, or TIP20T1219G., figureFileSmall=INYsViqm/Jfa5IJRKwnZqQ==, figureFileBig=ObjU6uecNTvYvUC6an2hzA==, tableContent=null), ArticleFig(id=1297571137593037812, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=CN, label=图4, caption=
确定突变位点, figureFileSmall=INYsViqm/Jfa5IJRKwnZqQ==, figureFileBig=ObjU6uecNTvYvUC6an2hzA==, tableContent=null), ArticleFig(id=1297571137676923893, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=EN, label=Table 1, caption=
Strains used in this study
, figureFileSmall=null, figureFileBig=null, tableContent=
| Strain designation | Gene information | Source |
|---|
| AN120 | MATα/MATa trp1::hisG/trp1::hisG ARG4/arg4-NspI ura3/ura3 ho::LYS2/ho::LYS2 leu2/leu2 lys2/lys2 RME1/rme1::LEU2 his3∆SK/his3ΔSK | [12] |
| AN117-4B | MATa ho::LYS2 rme1::LEU2 ura3 leu2 trp1 his3ΔSKarg4-NspI lys2 | [15] |
| AN117-16D | MATa his3ΔSK lys2 ho::LYS2 ura3 leu2 trp1 | [15] |
), ArticleFig(id=1297571137739838454, tenantId=1146029695717560320, journalId=1192105938417971205, articleId=1297571131087672257, language=CN, label=表1, caption=
本研究所用菌株
, figureFileSmall=null, figureFileBig=null, tableContent=
| Strain designation | Gene information | Source |
|---|
| AN120 | MATα/MATa trp1::hisG/trp1::hisG ARG4/arg4-NspI ura3/ura3 ho::LYS2/ho::LYS2 leu2/leu2 lys2/lys2 RME1/rme1::LEU2 his3∆SK/his3ΔSK | [12] |
| AN117-4B | MATa ho::LYS2 rme1::LEU2 ura3 leu2 trp1 his3ΔSKarg4-NspI lys2 | [15] |
| AN117-16D | MATa his3ΔSK lys2 ho::LYS2 ura3 leu2 trp1 | [15] |
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