一种遗传转化方法在海洋红酵母(<em>Rhodotorula mucilaginosa</em>)中的应用

doi:10.13523/j.cb.20160611

中国生物工程杂志

2016, Vol. 36

Issue (6): 81-86 DOI: 10.13523/j.cb.20160611

技术与方法

一种遗传转化方法在海洋红酵母(Rhodotorula mucilaginosa)中的应用

孙文怡^1,2,4, 张素芳², 林心萍³, 栾雨时¹

1. 大连理工大学生命科学与技术学院大连 116024;
2. 中国科学院大连化学物理研究所大连 116023;
3. 大连工业大学食品学院大连 116034;
4. 吉林师范大学生命科学学院四平 136000

Application of a Transformation in Rhodotorula mucilaginosa Genetic Engineering

SUN Wen-yi^1,2,4, ZHANG Su-fang², LIN Xin-ping³, LUAN Yu-shi¹

1. School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China;
2. Dalian Institute of Chemical Physics, CAS, Dalian 116023, China;
3. School of Food Science and Technology, Dalian Polytechnic University, Dalian 116034, China;
4. School of Life Sciences, Jilin Normal University, Siping 136000, China

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摘要：

海洋红酵母作为一种产油微生物富含油脂和类胡萝卜素,但由于没有合适的遗传操作方法,无法对细胞反应器进行理性改进,阻碍了它的进一步发展。首先利用农杆菌介导转化成功构建了海洋红酵母的遗传操作平台。参考完成基因注释的圆红冬孢酵母基因组信息,分离其甘油醛-3-磷酸脱氢酶启动子,以基于圆红冬孢酵母密码子优化的潮霉素作为筛选标记,构建载体转化至农杆菌AGL1中,利用农杆菌介导转化成功得到潮霉素抗性表型正确的转化子,通过表型验证,基因型验证以及Western blot在蛋白水平验证,鉴定了hyg基因的有效表达,证明了外源抗性基因成功导入海洋红酵母菌株中,实现了海洋红酵母新的转化方法的建立。

关键词： 海洋红酵母; 农杆菌介导转化; 潮霉素

Abstract:

Rhodotorula mucilaginosa is an oleaginous ocean yeast that of great interest for both lipid and carotenoids production. However, rationally engineering of this cell factory is impeded due to the absence of efficient and reliable genetic tools. Agrobacterium-mediated transformation (ATMT) was successfully developed for R. mucilaginosa. First, the promoter of glyceraldehyde-3-phosphate dehydrogenase (GPD) was identified by referring to the completely annotated genome information of Rhodosporidium toruloides. Then, the codon optimized hygromycin (hyg) gene under GPD was used as both selection marker and functional gene through ATMT transformation in R. mucilaginosa, the integration was confirmed by phenotype, genotype and Western blot analysis in protein level. The results provided a practical method for functional integration and expression of hyg genes in R. mucilaginosa, which would facilitate the development of genetic tools.

Key words: Rhodotorula mucilaginosa Hygromycin Agrobacterium-mediated transformation

收稿日期: 2015-12-24 出版日期: 2016-03-16

ZTFLH:

Q789

基金资助:

国家自然科学基金面上项目(31370128)资助项目

通讯作者: 孙文怡 E-mail: sunwenyi@dicp.ac.cn

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引用本文:

孙文怡, 张素芳, 林心萍, 栾雨时. 一种遗传转化方法在海洋红酵母(Rhodotorula mucilaginosa)中的应用[J]. 中国生物工程杂志, 2016, 36(6): 81-86.

SUN Wen-yi, ZHANG Su-fang, LIN Xin-ping, LUAN Yu-shi. Application of a Transformation in Rhodotorula mucilaginosa Genetic Engineering. China Biotechnology, 2016, 36(6): 81-86.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.20160611 或 https://manu60.magtech.com.cn/biotech/CN/Y2016/V36/I6/81

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