Cloning and Construction of Yeast Two-hybrid Bait Vector of Wheat Ta-SKP2A Gene

doi:10.7668/hbnxb.2016.02.004

Abstract

Abstract: SKP2A is an F-box protein that regulates the proteolysis of cell cycle transcription factors.In order to understand the role of SKP2A in the interaction between wheat and leaf rust pathogen, a full-length sequence of Ta-SKP2A was firstly obtained from wheat cv.China Spring, and ligated with pGBKT7 vector.Recombinant vector was transformed into yeast Y187 competent cell, further detected its self-activated activity and toxicity effect for yeast cells.Results showed that Ta-SKP2A was 1 146 bp and encoded a polypeptide of 382 amino acids.The ORF with restriction enzyme sites of BamH I and EcoR I was inserted into expression vector pGBKT7.The results showed that the bait vector pGBKT7-Ta-SKP2A was successfully constructed by sequencing.The recombinant bait plasmid grew well on SD/-Trp plate, which showed that the bait plasmid was not toxic to yeast cell.The yeast strains containing bait plasmid couldn't grow on SD/-Leu/-Trp, SD/-Leu/-Trp/-His and SD/-Trp/-Leu/-His/-Ade plate, so the bait plasmid didn't have transcriptional activation.A recombinant bait plasmid with Ta-SKP2A was successfully constructed, which would lay foundation for screening for targeted protein interacted with Ta-SKP2A, and would help to understand its function in the interaction of wheat and leaf rust pathogen.

Key words: Wheat, F-box gene Ta-SKP2A, Construction of bait vector, Toxicity detection, Transcriptional activation

CLC Number:

Q784

XU Yuan, LI Lingxian, WEI Chunru, WEI Xinyan, YU Xiumei, LIU Daqun. Cloning and Construction of Yeast Two-hybrid Bait Vector of Wheat Ta-SKP2A Gene[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2016, 31(2): 17-22. doi: 10.7668/hbnxb.2016.02.004.

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http://www.hbnxb.net/EN/Y2016/V31/I2/17

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