Construction of Yeast One-Hybrid Library for Screening of G-box Binding Proteins

Objective: In order to screen binding proteins of G-box,an important element in chalcone synthase(CHS) promoter region,and find transcriptional regulators of CHS gene.Methods: Matchmaker Gold Yeast One-Hybrid Library Screening System was employed in this study.Bait yeast strain was constructed by synthesiz ing oligonucleotides containing three tandem copies of G-box core sequences and integrating it into the genome of yeast.The cDNA for hypocotyls of turnip(Brassica rapa L.subsp.rapa Tsuda) was synthesized via SMART tech nology and co-transformed into bait yeast strain with pGADT7-Rec vector,one-hybrid cDNA library was simultane ously constructed and screened directly in yeast as a result of in vivo plasmid recombination.cDNA inserts in pos itive clones was amplified by yeast colony PCR and analyzed through NCBI Blast after sequencing.Results: Based on the experiments,we screened 2.52×106 yeast clones and got 94 positive clones.Colony PCR amplifica tion products were 0.4~2.0 kb in length and proteins encoded by them were inferred by NCBI Blast analysis.Con clusion: The results showed that the construction of yeast one-hybrid library was successful,candidate proteins for G-box binding proteins were acquired by preliminary library screening,which laid the foundation for researches on CHS expression regulation.