酵母亚细胞结构分离效率评估菌株的构建 <sup>*</sup>

doi:10.13523/j.cb.2006053

中国生物工程杂志

2020, Vol. 40

Issue (10): 10-23 DOI: 10.13523/j.cb.2006053

技术与方法

酵母亚细胞结构分离效率评估菌株的构建 ^*

胡妍,李辉,何承文,朱婧,谢志平(

)

上海交通大学生命科学技术学院微生物代谢国家重点实验室上海 200240

Construction of a Yeast Strain for the Evaluation of Subcellular Fractionation

HU Yan,LI Hui,HE Cheng-wen,ZHU Jing,XIE Zhi-ping(

)

State Key Laboratory of Microbial Metabolism, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai 200240, China

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摘要：

背景: 真核细胞依赖其亚细胞结构高效地完成复杂的生化反应。尽管目前有一些亚细胞结构分离技术,但却缺乏评估这些分离技术的简单、有效方法。目的: 构建可以用来检测亚细胞结构分离效率的酿酒酵母菌株。方法: 通过传统的分子生物学与细胞生物学方法以酿酒酵母为背景构建了亚细胞结构分离效率评估菌株,并进行可行性检测。首先,将酵母各细胞器、自噬体及质膜的标记蛋白进行分组,用不同的蛋白标签分别标记每组蛋白。然后,以标记蛋白-GFP/RFP作为对照组通过免疫荧光法检测蛋白标签对标记蛋白的亚细胞定位是否有影响。最后,通过非连续性密度梯度离心验证该检测菌株能否用来检测亚细胞结构的分离效率。结果: 成功构建了酵母亚细胞结构分离效率评估菌株,该菌株标记了大多数酵母细胞亚细胞结构。挂标签的标记蛋白仍然定位在各自标记蛋白对应的亚细胞结构。非连续性密度梯度离心后,酵母各个亚细胞结构的标记蛋白均可以被检测到。结论: 该酵母亚细胞结构分离效率评估菌株是检测各亚细胞结构分离结果的方便工具,对今后酿酒酵母细胞生物学的研究有潜在的应用价值。

关键词： 酿酒酵母; 细胞器; 自噬体; 质膜; 亚细胞结构分离

Abstract:

Background: An eukaryotic cell relies on its cellular organelles and organelles derived compartments to perform complicated biochemical reactions efficiently. One valid way to reveal the efficient cooperation between each organelle is to isolate distinct organelles. Although several techniques are developed to separate organelles, there is almost no easy method to assess the isolation process. Goal: Construct a strain of Saccharomyces cerevisiae to evaluate the efficiency of subcellular fractionation. Methods: A detection strain of Saccharomyces cerevisiae was constructed by traditional molecular biological and cell biological methods. One soluble protein and ten membrane proteins, chosen to represent subcellular compartments as well as plasma membrane, were grouped and labeled with different epitope tags in one strain. Immunofluorescence results were compared with fluorescent protein fusions to evaluate the impact of epitope tags on the localization of these proteins. Finally, density gradient centrifugation was used to illustrate the usability of the detection strain. Results: The detetion strain labeling all major subcellular compartments of Saccharomyces cerevisiae was constructed successfully. All epitope tagged organelle markers localized to the intended subcellular sites. Each compartment of Saccharomyces cerevisiae could be detected after density gradient centrifugation. Conclusion: The detection strain is a convenient tool for the evaluation of subcellular fractionation results. It is potentially useful for future research of Saccharomyces cerevisiae cell biology.

Key words: Yeast Organelles Autophagosome Plasma membrane Subcellular fractionation

收稿日期: 2020-06-28 出版日期: 2020-11-10

ZTFLH:

Q819

基金资助: * 国家自然科学基金(91754110);国家自然科学基金(91957104)

通讯作者: 谢志平 E-mail: zxie@sjtu.edu.cn

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引用本文:

胡妍,李辉,何承文,朱婧,谢志平. 酵母亚细胞结构分离效率评估菌株的构建 ^*[J]. 中国生物工程杂志, 2020, 40(10): 10-23.

HU Yan,LI Hui,HE Cheng-wen,ZHU Jing,XIE Zhi-ping. Construction of a Yeast Strain for the Evaluation of Subcellular Fractionation. China Biotechnology, 2020, 40(10): 10-23.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2006053 或 https://manu60.magtech.com.cn/biotech/CN/Y2020/V40/I10/10

表1 酿酒酵母亚细胞结构标记蛋白分组

表2 质粒及其线性化酶切位点

表3 大质粒及其线性化酶切位点

表4 菌株列表

图1 YOT027 Western blot检测结果图

图2 免疫荧光与荧光蛋白共定位图

图3 免疫荧光条件测试

图4 YOT027密度梯度离心结果图

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