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引用本文:杨子平,李辉亮,郭冬,彭世清.巴西橡胶树胶乳酵母双杂交cDNA表达文库的构建及分析[J].热带生物学报,2013,4(4):303-307.
巴西橡胶树胶乳酵母双杂交cDNA表达文库的构建及分析
Construction and Analysis of Latex cDNA Library for A Yeast Two hybrid Library
  
DOI:
中文关键词: 橡胶树  酵母双杂交  cDNA文库
英文关键词: Hevea brasiliensis  yeast two hybrid  cDNA library
基金项目:国家自然科学基金项目(31170634)和中央级公益性科研院所基本科研业务费专项资金资助项目(ITBB110205)
作者单位
杨子平 1.海南大学 农学院海南 海口 5702282. 中国热带农业科学院 热带生物技术研究所/农业部热带作物生物学与遗传资源利用重点实验室海南 海口571101 
李辉亮 2. 中国热带农业科学院 热带生物技术研究所/农业部热带作物生物学与遗传资源利用重点实验室海南 海口571101 
郭冬 2. 中国热带农业科学院 热带生物技术研究所/农业部热带作物生物学与遗传资源利用重点实验室海南 海口571101 
彭世清 2. 中国热带农业科学院 热带生物技术研究所/农业部热带作物生物学与遗传资源利用重点实验室海南 海口571101 
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中文摘要:
      提取巴西橡胶树胶乳总RNA,并纯化mRNA。采用特异引物(oligo-dT) 反转出第一链DNA,经LD PCR合成第二链DNA。将ds cDNA和经SmaⅠ线性化的pGADT7 Rec质粒共转化Y187酵母菌,构建酵母双杂交cDNA表达文库。结果表明,转化单菌落个数为2.8×107;文库滴度为4.92×107·mL-1;插入片段大小主要分布于500~2 000 bp间;重组率为96%。实验数据表明该文库能满足后续的杂交筛选。
英文摘要:
      Total RNA was extracted from laticifers of Hevea brasiliensis, and total RNA was used to purify mRNA with MACHERY NAGEL Purification of poly(A)RNA kit. The first strand cDNA was synthesized by reverse transcription of mRNA with SMART oligo dT technique, and LD PCR was performed to synthesize double strand cDNA. The double strand cDNA was then purified by running through a CHROMA SPINTM TE 400 column of clontech to select with ds cDNA molecules >200 bp. Purified ds cDNA and lineared pGADT7 Rec were co transformed into Y187 yeast strain to construct a yeast two hybrid cDNA library of Hevea brasiliensis laticifers. The detection showed that the library contained 2.8×107 independent clones, and that the titer of library was 4.92×107·mL-1. The sizes of most inserts ranged from 500 to 2 000 bp in this library, and the recombination rate was 96%. These results showed that the library was suitable for yeast mating and can be used to screen interaction proteins.
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