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中国生物工程杂志

CHINA BIOTECHNOLOGY
中国生物工程杂志
中国生物工程杂志  2024, Vol. 44 Issue (4): 14-22    DOI: 10.13523/j.cb.2308002
研究报告     
解脂耶氏酵母中Anillin家族蛋白的鉴定与功能*
邬欢1,**(),吴浩明2
1 华中科技大学同济医学院附属武汉儿童医院 武汉 430016
2 武汉轻工大学生命科学与技术学院 武汉 430023
Characterization of Anillin-related Protein and Its Function in Yarrowia lipolytica
WU Huan1,**(),WU Haoming2
1 Wuhan Children’s Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430016, China
2 School of Life Science and Technology, Wuhan Polytechnic University, Wuhan 430023, China
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摘要：

目的:鉴定解脂耶氏酵母中Anillin家族蛋白,探究其对Septin细胞骨架组织的调控功能。方法:将模式生物酿酒酵母Anillin家族蛋白Bud4序列在解脂耶氏酵母数据库中比对,找到潜在的Anillin家族蛋白;从解脂耶氏酵母基因组中扩增其序列,将其与绿色荧光蛋白(green fluorescent protein,GFP)融合表达,通过荧光显微镜观察其胞内定位;对酿酒酵母细胞中潜在的Anillin家族蛋白进行过量表达,观察其对细胞形态和Septin组织的影响,从而确定其调控Septin组织的功能。结果:通过解脂耶氏酵母数据库中序列比对,找到两个潜在的Anillin家族蛋白,分别将其命名为YlBud4A(数据库中蛋白编号YALI0D11880)和YlBud4B(数据库中蛋白编号YALI0B07623),其C端与该家族蛋白成员同源性高,含有保守的DUF1709和PH结构域;与酿酒酵母Bud4类似,YlBud4B可以定位在芽颈,在芽体较大细胞中呈明显的双环结构,而YlBud4A没有芽颈定位;在酿酒酵母细胞中过量表达YlBud4B导致细胞产生显著的芽体变长细胞形态,Septin的正常组织被破坏,在变长的芽体中呈圆形结构。结论:解脂耶氏酵母YlBud4B与Anillin家族蛋白在序列和功能上都具有同源性,其可以与Septin细胞骨架相互作用,在调控Septin组织过程中发挥功能。
关键词： 解脂耶氏酵母;  Anillin;  细胞骨架;  细胞形态;  Septin    
Abstract:

Objective: To identify the anillin-related protein in Yarrowia lipolytica and to study its function in septin organization. Methods: We identified the anillin-related protein in Y. lipolytica by aligning the protein sequence of Saccharomyces cerevisiae Bud4 in the Y. lipolytica genome database. The sequences of these anillin-related proteins were amplified and fused with green fluorescent protein GFP to observe their intracellular localization. In the model organism budding yeast, these anillin-related proteins of Y. lipolytica were overexpressed and their effects on cell morphology and septin organization were determined to investigate their function in this process. Results: Two potential anillin-related proteins, named YlBud4A (protein number YALI0D11880 in the database) and YlBud4B (protein number YALI0B07623), were identified by sequence comparison in the Y. lipolytica genome database, both of which were conserved in their C-terminal containing DUF1709 and PH domain. YlBud4A had no bud neck localization, whereas YlBud4B localized to the bud neck as a double ring in medium and large bud cells, a localization pattern identical to Bud4. Overexpression of YlBud4B in budding yeast caused elongated buds and septin disruption, which clustered into dots. Conclusion: YlBud4B may interact with the septins and it is likely to be the functional homolog of anillin-related protein whereas YlBud4A is not.
Key words: Yarrowia lipolytica    Anillin    Cytoskeleton    Cell morphology    Septin
收稿日期: 2023-08-02 出版日期: 2024-04-30
ZTFLH:  Q81  
基金资助: * 国家自然科学基金(31901797);湖北省卫生健康科研基金(WJ2019H379);武汉市卫生健康科研基金(WX19Q32)
通讯作者: ** 电子信箱:wuh@whu.edu.cn   
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邬欢
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引用本文:

邬欢, 吴浩明. 解脂耶氏酵母中Anillin家族蛋白的鉴定与功能*[J]. 中国生物工程杂志, 2024, 44(4): 14-22.

WU Huan, WU Haoming. Characterization of Anillin-related Protein and Its Function in Yarrowia lipolytica. China Biotechnology, 2024, 44(4): 14-22.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2308002    或    https://manu60.magtech.com.cn/biotech/CN/Y2024/V44/I4/14

菌株 基因型 来源
YEF473A(Saccharomyces cerevisiae) a his3-Δ200 leu2-Δ1 lys2-801 trp1-Δ63 ura3-52 参考文献[23]
YEF4603(Saccharomyces cerevisiae) a his3-Δ200 leu2-Δ1 lys2-801 trp1-Δ63 ura3-52 shs1Δ∷kanMX 参考文献[23]
JGY881(Saccharomyces cerevisiae) a his3 leu2 lys2 trp1 ura3 CDC3-GFP:LEU2 本研究
DH5α(Escherichia coli) F-endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoRnupG Φ80dlacZΔM15Δ (lacZYA-argF) U169, hsdR17 ( r K - m K +), λ- TaKaRa, Japan
表1  研究中所用菌株
名称 序列(5'→3')
YlBUD4A-4F CGCGGATCCATGACGGTCGAGTCTGGACGTCTTTTC
YlBUD4A-5F CGCGGATCCATGCTGCGTCAAGCTCCCGTGGCTG
YlBUD4A-6F CCGGAATTCTCCTTCTCTGAGGGAC
YlBUD4A-8F CGCGGATCCATGTCTGCCCCCTCGTCGCCGC
YlBUD4A-8R ACCCATCGATTCACTTCATAGTGTTCTTCTC
YlBUD4B-1F CCGGAATTCATGCACAGACACACTGCCAGCC
YlBUD4B-2F CCGGAATTCATGAGACACGATAGAGGCCGCCTGTTC
YlBUD4B-1R ACCCAAGCTTTCAGAGCCCACTTCCCTCACTG
表2  研究中所用主要引物
图1  解脂耶氏酵母Anillin家族蛋白的鉴定和结构分析 A:解脂耶氏酵母YlBud4A和YlBud4B、白色念珠菌Int1以及酿酒酵母Bud4蛋白的结构域示意图 B:4个蛋白C端区域的同源性比对
图2  YlBud4A和YlBud4B全长及片段的表达水平和蛋白大小 A:解脂耶氏酵母YlBud4A和YlBud4B的蛋白区段示意图 B:YlBud4A和YlBud4B蛋白区段的Western blot分析
图3  YlBud4A、YlBud4B全长及片段的胞内定位 将质粒pBG2-YlBUD4A、pBG2-YlBUD4B和pBG2-YlBUD4B-C2转入酿酒酵母菌株YEF473A,观察这些细胞中的荧光信号。标尺:5 μm
图4  过量表达YlBud4B导致细胞芽体变长 将质粒pEGKT426和pEGKT426-YlBUD4B转入野生型菌株YEF473A和shs1Δ菌株YEF4603,在40倍显微镜视野下观察细胞形态。 标尺:5 μm
图5  过量表达YlBud4B导致Septin组织絮乱 将质粒pEGKT426和pEGKT426-YlBUD4B转入菌株JGY881(Cdc3-GFP),观察septin组织情况。Cdc3-GFP指示septin定位。标尺:5 μm
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