Construction and Analysis of Latex cDNA Library for A Yeast Two-hybrid Library

Key words: 
• Hevea brasiliensis /
• yeast two-hybrid /
• cDNA library

Abstract: 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 SPIN^TM TE-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 × 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.