Prokaryotic expression analysis of aluminum associated receptor-like protein kinase AhPRK4 in peanut (Arachis hypogaea)

Abstract

The pollen receptor-like protein kinase (PRK) family, an LRR receptor-like protein kinase, not only plays a role in pollen development and fertilization, but also plays a role in stress response. Based on the analysis of transcriptome data that generated in our previous study, we found that AhPRK4 was an aluminum-responsive gene. To explore the role of AhPRK4 in response to aluminum stress, we analyzed the expression of AhPRK4 by qRT-PCR in ‘ZH2’ (Al-sensitive) and ‘99-1507’ (Al-tolerant), clarified the protein structure and genetic relationship of AhPRK4 by sequence analysis, phylogenetic tree construction and other genetic analysis, constructed the recombinant plasmid by homologous recombination, obtained the intracellular domain recombinant protein of AhPRK4 by prokaryotic expression technology and determined the activity of the recombinant protein by incubation with phosphorylated antibodys. The results were as follows: (1) The transcription level of AhPRK4 was up-regulated after different aluminum treatments time and different aluminum concentrations, indicating that AhPRK4 was an aluminum inducible gene. (2) The AhPRK4 protein had 673 amino acids with transmembrane domain, signal peptide and phosphorylation active sites, belonging to the LRR-III protein kinase family. (3) The GST-AhPRK4-CD recombinant protein was induced in vitro and verified by Western Blot. And the recombinant protein had phosphorylated on both serine/threonine and tyrosine residues, but had no significant auto-phosphorylation activity. In conclusion, AhPRK4 is an aluminum responsive gene, which participates in the regulation of short-term aluminum stress and is phosphorylated in vitro.

Keywords

• peanut (arachis hypogaea)
• aluminum stress
• pollen receptor-like protein kinase
• expression analysis
• prokaryotic expression