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Abstract A yeast one-hybrid library was constructed to screen the binding proteins of suspensor specific cis-element. In this study, five repeats of suspensor specific cis-element sequence(GAAAAGCGAA) were synthesized and ligated with reporter vector pHIS2.1 to construct the bait vector pHIS2.1-G564-5A. Total RNA was extracted from immature seeds of Arabidopsis thaliana, and the purified mRNA was used as template to synthesize ds cDNA with SMARTTMⅢ and CDSⅢ adapter. The purified ds cDNA, linearized pGADT7-Rec2 and pHIS2.1-G564-5A were simultaneously transformed into the yeast strain Y187 by LiAc-mediated method. The results showed that the co-transformation efficiency was 5.53×105 cfu/μg. After 3 times screening in SD/-Trp/-Leu/-His/ 100 mmol/L 3-AT medium, 276 positive clones were obtained and 142 of which were sequenced, whereafter the sequences were annotated by BLASTX against NCBI no-redundant protein database and 117 sequences were identified. Gene functional analysis showed that there were 14 proteins with DNA binding and 8 proteins with protein binding function, and this protein contained TATA binding protein 1(TBP1), Scaffold-associated region(SAR) DNA-binding protein, Ovule development protein, G-box binding factor 1(GBF1), and Histone H3. The results provide candidate genes for isolation and cloning transcription factors expressed specifically in suspensor.
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Received: 04 April 2011
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