巴斯德毕赤酵母甘油转运体的发现及功能研究

doi:10.13523/j.cb.20170710

中国生物工程杂志

2017, Vol. 37

Issue (7): 48-55 DOI: 10.13523/j.cb.20170710

研究报告

巴斯德毕赤酵母甘油转运体的发现及功能研究

战春君^1,2,3, 李翔^1,2,3, 刘国强^1,2,3, 刘秀霞^1,2,3, 杨艳坤^1,2,3, 白仲虎^1,2,3

1. 江南大学粮食发酵工艺与技术国家工程实验室无锡 214122;
2. 江南大学工业生物技术教育部重点实验室无锡 214122;
3. 江南大学糖化学与生物技术教育部重点实验室无锡 214122

Identification of Glycerol Transporter in Pichia pastoris and Function Research

ZHAN Chun-jun^1,2,3, LI Xiang^1,2,3, LIU Guo-qiang^1,2,3, LIU Xiu-xia^1,2,3, YANG Yan-kun^1,2,3, BAI Zhong-hu^1,2,3

1. National Engineering Laboratory for Cereal Fermentation Technology, Jiangnan University, Wuxi 214122, China;
2. The Key Laboratory of Industrial Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China;
3. The Key Laboratory of Carbohydrate Chemistry and Biotechnology, Ministry of Education, School of Biotechnology, Jiangnan University, Wuxi 214122, China

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摘要： 目的：分离确认巴斯德毕赤酵母中甘油转运体并初步研究其功能。方法：通过生物信息学方法从NCBI数据库中查找可能的甘油转运体（gt1，GeneID：8197545），并通过DAS软件对其跨膜结构域进行预测。通过PCR方法扩增该基因并将其与EGFP融合克隆到pPICZ B及pRS424载体进行亚细胞定位；同时将其与pRS424载体连接后电转到粟酒裂殖酵母中进行异源表达确定其功能；通过同源重组敲除gt1基因并在不同培养基中培养测定aox1基因表达量。结果：生物信息学显示与酿酒酵母中已经证明的甘油转运体（sugar transporter 1，stl1）类似，GT1蛋白同样为具有疏水结构域的跨膜蛋白，同时亚细胞定位结果显示GT1蛋白定位于细胞膜上，包含有gt1基因的粟酒裂殖酵母可以在以甘油作为唯一碳源的培养基中生长而野生型不可以；在Δgt1突变体中aox1基因可以获得组成型表达。结论：分离并确认了巴斯德毕赤酵母中的甘油转运体GT1，并初步证明其与aox1基因甘油阻遏相关。

关键词： 巴斯德毕赤酵母; 甘油转运体; 亚细胞定位; 甘油阻遏; 同源重组

Abstract: Object Separation and identification glycerol transporter in Pichia pastoris (P. pastoris) and studing its function. Methods: the candidate glycerol transporter (gt1, GeneID:8197545) was found in NCBI database by bioinformatics, and transmembrance structure was predicted by DAS. Targeted gene fused with egfp was cloned into pPICZ B and pRS424 to study its location in cells; recombinant plasmid (gt1 + pRS424) was transformed into Schizosaccharomyces pombe (S. pombe) to study its function; In order to study the relationship between gt1 and aox1, gt1 was deleted by homologous recombination, and enzyme activity of AOX1 in wild and mutant were detected. Results: according to results of bioinformatics, it was found that GT1 share the same transmembrance regions with sugar transporter 1(stl1) which has been identified as a glycerol transporter in Saccharomuces cerevisiae (S. cerevisiae), results of subcellular localization showed that GT1 located on membrance, S. pombe transformed into gt1 could growth on glycerol medium, but wild type could not, which suggested that GT1 could absorbed glycerol from medium to support cells growth. Moreover, AOX1 achieved constitutive expression in Δgt1. Conclusion: glycerol transporter (gt1) in P. pastoris was separated and identified, and its function in glycerol repression was preliminarily clarified.

Key words: Glycerol repression Homologous recombination Subcellular localization Glycerol transporter Pichia pastoris

收稿日期: 2017-02-23 出版日期: 2017-07-25

ZTFLH:

Q815

基金资助: 国家自然科学基金项目（面上项目）（31570034）、江苏省自然科学基金项目（BK20150148）、中央高校基本科研业务费专项资金（JUSRP51401A）资助项目

通讯作者: 杨艳坤, 白仲虎 E-mail: yangyankun@jiangnan.edu.cn;baizhonghu@jiangnan.edu.cn

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引用本文:

战春君, 李翔, 刘国强, 刘秀霞, 杨艳坤, 白仲虎. 巴斯德毕赤酵母甘油转运体的发现及功能研究[J]. 中国生物工程杂志, 2017, 37(7): 48-55.

ZHAN Chun-jun, LI Xiang, LIU Guo-qiang, LIU Xiu-xia, YANG Yan-kun, BAI Zhong-hu. Identification of Glycerol Transporter in Pichia pastoris and Function Research. China Biotechnology, 2017, 37(7): 48-55.

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https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20170710 或 https://manu60.magtech.com.cn/biotech/CN/Y2017/V37/I7/48

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