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学科主题: 物理化学
题名: High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith
作者: Yang, Fan1, 2;  Tan, Haidong1;  Zhou, Yongjin1, 2;  Lin, Xinping1, 2;  Zhang, Sufang1
通讯作者: 张素芳
关键词: RNA extraction ;  Rhodosporidium toruloides ;  Full-length cDNA library
刊名: MOLECULAR BIOTECHNOLOGY
发表日期: 2011-02-01
DOI: 10.1007/s12033-010-9322-1
卷: 47, 期:2, 页:144-151
收录类别: SCI
文章类型: Article
部门归属: 18
项目归属: 1816
产权排名: 1,1
WOS标题词: Science & Technology ;  Life Sciences & Biomedicine
类目[WOS]: Biochemistry & Molecular Biology ;  Biotechnology & Applied Microbiology
摘要: High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith
研究领域[WOS]: Biochemistry & Molecular Biology ;  Biotechnology & Applied Microbiology
英文摘要: Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer. Therefore, it is important to develop molecular biology tools to understand the basic mechanism for lipid accumulation and further manipulate the microorganism. High-quality RNA extraction from R. toruloides is particularly challenging due to high level of polysaccharides, lipids, and other secondary metabolites. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods were evaluated. Large difference in RNA yield and quality among these protocols was found. The optimum method was modified RNAiso procedure, where RNA was isolated using liquid nitrogen-RNAiso method with salt precipitation and the addition of beta-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 297 mu g total RNA per gram of cells was obtained with an average purity measured as A(260)/A(280) of 2.09. A titer of 10(5) cfu/ml could be harvested to construct a full-length cDNA library with the RNA sample in this quality. Electrophoresis gel analysis indicated the fragments ranged from 200 bp to 4.0 kb, with the average size of 1000 bp. Randomly picked clones showed the recombination efficiency at 80%. These results showed that RNA of R. toruloides was successfully extracted for the first time using the modified RNAiso method, and the cDNA library was appropriate for screening the genes related to lipid accumulation.
关键词[WOS]: LIPID-ACCUMULATION ;  OLEAGINOUS MICROORGANISMS ;  PLANT-TISSUES ;  EXTRACTION ;  RICH ;  POLYSACCHARIDES ;  PROTOCOL ;  CULTURE ;  BIOLOGY
语种: 英语
WOS记录号: WOS:000286378600006
Citation statistics: 
内容类型: 期刊论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/115574
Appears in Collections:中国科学院大连化学物理研究所_期刊论文

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作者单位: 1.CAS, Dalian Inst Chem Phys, Div Biotechnol, Dalian 116023, Peoples R China
2.Chinese Acad Sci, Grad Univ, Beijing 100049, Peoples R China

Recommended Citation:
Yang, Fan,Tan, Haidong,Zhou, Yongjin,et al. High-Quality RNA Preparation from Rhodosporidium toruloides and cDNA Library Construction Therewith[J]. MOLECULAR BIOTECHNOLOGY,2011,47(2):144-151.
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