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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 F(杨帆) ;  Zhang SF(张素芳) ;  Zhao ZB(赵宗保)
会议名称: 12th Asia Pacific Confederation of Chemical Engineering Congress
会议日期: 2008-08-03
出版日期: 2008-08-03
会议地点: 中国
通讯作者: 赵宗保
部门归属: 十八室
主办者: 中国化工学会
摘要: Oleaginous yeast Rhodosporidium toruloides is an excellent microbial lipid producer using carbohydrates as the feedstock [1]. Therefore, it is important to develop molecular biology tools to further manipulate this microorganism. Extraction of high-quality RNA from R. toruloides is particular challenging due to high level of polysaccharides, lipids and other secondary metabolites [2]. To obtain an optimal protocol for RNA extraction from R. toruloides, four methods, including guanidine thiocyanate [3], glass beads-hot acid phenol, glass beads-TRIzol, and modified RNAiso, were evaluated. RNA quality was assessed using UV absorbance (A260/A280 and A260/A230), agarose gel electrophoresis, reverse transcription (RT)-PCR reactions, and Gel-Pro analyzer 4.5. Large differences in RNA yield and quality among protocols were found. The optimum method was modified RNAiso method, where RNA was isolated using liquid nitrogen-RNAiso with salt precipitation and the addition of PVP andβ-mercaptoethanol. This method consistently recovered RNA in good quality with high yield. Around 500 μg total RNA per gram of cells was obtained with an average purity measured as A260/A280 of 1.80. Construction of full-length cDNA library with the RNA sample afforded a library with a titer of 105 cfu/ml. Electrophoresis gel analysis indicated the fragments ranged from 500 bp to 1.5 kb, with the average size of 1000 bp. Random picking clones showed that the recombination rate was 80%. These results showed that the cDNA library of R. toruloides was in high quality, and could be used to screen the genes related to lipid accumulation.
语种: 中文
内容类型: 会议论文
URI标识: http://cas-ir.dicp.ac.cn/handle/321008/113192
Appears in Collections:中国科学院大连化学物理研究所_会议论文

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Recommended Citation:
Yang F,Zhang SF,Zhao ZB. HIGH-QUALITY RNA PREPARATION FROM RHODOSPORIDIUM TORULOIDES AND cDNA LIBRARY CONSTRUCTION THEREWITH[C]. 见:12th Asia Pacific Confederation of Chemical Engineering Congress. 中国. 2008-08-03.
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