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当前位置: 首页 > 文章 > 巴西橡胶树胶乳酵母双杂交cDNA表达文库的构建及分析 热带生物学报 2013,4 (4) 303-307
Position: Home > Articles > Construction and Analysis of Latex cDNA Library for A Yeast Two-hybrid Library Journal of Tropical Biology 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

作  者：
杨子平;李辉亮;郭冬;彭世清
单  位：
海南大学农学院;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
关键词：
橡胶树;酵母双杂交;cDNA文库
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LDPCR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。
译  名：
Construction and Analysis of Latex cDNA Library for A Yeast Two-hybrid Library
作  者：
YANG Ziping;LI Huiliang;GUO Dong;PENG Shiqing;College of Agronomy,Hainan University;Ministry of Agriculture Key Laboratory of Biology and Genetic Resources of Tropical Crops Institute of Tropical Bioscience and Biotechnology,Chinese Academy of Tropical Agricultural Sciences;
关键词：
Hevea brasiliensis;;yeast two-hybrid;;cDNA library
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 SPINTMTE-400 column of clontech to select with ds cDNA molecules > 200 bp. Purified ds cDNA and lineared pGADT7-Rec were cotransformed 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 × 107independent 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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