Cloning and Prokaryotic Expression of Transcription Factor StWRKY57 Gene in Potato

doi:10.7668/hbnxb.20191222

Abstract

Abstract: In order to explore the function of StWRKY57 (PGSC0003DMT400072958) in potato, a full-length cDNA sequence of the homologous StWRKY57 gene in Solanum tuberosum PB06 named StWRKY57-PB was cloned by RT-PCR and bioinformatics methods were used to analyze the sequence characteristic of StWRKY57-PB. For prokaryotic expression and purify, the prokaryotic expression vector of StWRKY57-PB was constructed based on pGEX-KG, then fusion protein was induced in Escherichia coli strains. The results showed that the StWRKY57-PB gene contained a 981 bp open reading frame(ORF), and the coding protein belonged to the Ⅱ c class of the WRKY transcription factor family, containing 326 amino acid residues with a WRKY(WRKYGQK) domain and a zinc-finger-like motif(C-X₄-C-X₂₃-H-X₁-H). StWRKY57-PB was determined structurally to be 16.56% a-helix, 13.50% extended strand, 7.98% β-turn and 61.96% random coil. Compared with StWRKY57(XP_006348711.1), there were 4 amino acid mutations in StWRKY57-PB. Multiple alignment and phylogenetic tree analyses indicated that StWRKY57-PB showed the next-closest relationship with SlWRKY57 with 93% similarity and was farthest to AtWRKY57. StWRKY57-PB protein was stably expressed in three Escherichia coli strains of Rosetta, BL21 and Tuner with 0.5 mmol/L IPTG. Fusion protein of GST-StWRKY57-PB was purified through GST SefiroseTM resin, and then Western Blot was carried to verify the successful expression and purification of the protein. It is of great significance to further determine the activity and biological function of this protein in vitro.

Key words: Potato, StWRKY57, GST tag, Sequence analysis, Prokaryotic expression

CLC Number:

S532.03
Q78

HU Yueqing, WANG Ruozhong, HUANG Zhigang, XIAO Langtao. Cloning and Prokaryotic Expression of Transcription Factor StWRKY57 Gene in Potato[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(1): 10-17. doi: 10.7668/hbnxb.20191222.

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