Preparation of GmWRKY50 Protein Antibody and Analysis of Its Protein Level Expression

doi:10.7668/hbnxb.20192501

Abstract

Abstract: To elucidate the mechanism of the transcription factor GmWRKY50 in the interaction between soybean and Soybean mosaic virus (SMV),the full-length coding region of GmWRKY50 gene was cloned by PCR using RNA of soybean leaves as template,the similar amino acid sequences of GmWRKY50 in different species were compared by MEGA 7.0 and phylogenetic tree analysis was carried out.The recombinant plasmid pColdⅡ- GmWRKY50 was constructed through prokaryotic expression technology.After transformation of Escherichia coli BL21,the GmWRKY50 recombinant protein with His tag was induced by IPTG.The recombinant protein was purified by Ni-NTA column,and the purified recombinant protein was collected for polyclonal antibody preparation.Western Blotting was used to detect the expression level changes of GmWRKY50 before and after Soybean mosaic virus infection.The results showed that the CDS of GmWRKY50 gene was 495 bp in length,encoding 165 amino acids,and contained a WRKY domain at the N terminal,belonging to WRKY DNA-binding domain protein.Sequence alignment and phylogenetic tree analysis showed that soybean GmWRKY50 had the closest homologous relationship with Arabidopsis AtWRKY50. When IPTG concentration was 0.5 mmol/L and induction time was 4 h,the induced effect of recombinant protein was good,and the target protein fused with His protein tag was obtained by nickel ion affinity purification,which has high solubility.The polyclonal antibody prepared with fusion protein as antigen could specifically bind GmWRKY50,GmWRKY50 was up-regulated by SMV infection in incompatible combinations.This study lays the foundation for further investigation of the function of transcription factor GmWRKY50 in soybean resisting SMV infection.

Key words: Soybean, GmWRKY50, Prokaryotic expression, Protein purification, Western Blotting

CLC Number:

Q78
S565.03

FAN Huifen, SUN Tianjie, SU Weihua, XIAO Fuming, ZHANG Jie, WANG Dongmei. Preparation of GmWRKY50 Protein Antibody and Analysis of Its Protein Level Expression[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2021, 36(6): 28-34. doi: 10.7668/hbnxb.20192501.

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