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

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Sma Ⅰ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.

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 eDNA was synthesized by reverse transcription of mRNA with SMART oligo-dT technique, and LD-PCR was performed to synthesize double strand eDNA. 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 eDNA library of Hevea brasiliensis laticifers. The detection showed that the library contained 2.8 ×10^7 independent clones, and that the titer of library was 4.92 ×10^7 · 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 in- teraction proteins.