Abstract Arginine kinase (AK, L-arginine N-phosphotransferase, EC 2.7.3.3) is a phosphotransferase that plays a critical role in cellular energy metabolism in invertebrates such as insect, crustacean and some unicellular organisms. It catalyzes the reversible transfer of the phosphoric group of adenosine triphosphate (ATP) to arginine yielding adenosine diphosphate (ADP) and phosphoarginine. In order to better understand the role of AK gene and its regulatory mechanism during the cytochrome P450 (CYP6B6) expression of cotton bollworm(Helicoverpa armigera), the H. armigera AK (HarmAK) cDNA was cloned from midgut by RT-PCR on the basis of yeast one-hybrid results. The fragment digested by double enzymes was linked to a prokaryotic expression vector pET28a to construct the recombinant expression plasmid pET28a-HarmAK, and then converted into Escherichia coli BL21 competent cells. The fusion protein His-HarmAK was induced to express by isopropyl-β-D-thiogalactoside (IPTG), and purified by Ni2+ affinity chromatography. SDS-polyaerylamide gel electrophoresis (SDS-PAGE) and Western blot analysis were used to examine the fusion protein. The fusion protein activity was determined by the pH-spectrophotometric assay. qRT-PCR was used to test HarmAK expression level in both developmental stages of the cotton bollworm and different tissues of the 6th instar larvae. The results of sequencing and sequence analysis showed that the ORF of the HarmAK gene was 1 068 bp, encoding 355 amino acid residues and the predicted molecular weight and isoelectric point was 39.8 kD and 5.76, respectively. And the protein was deduced to have no signal peptide and transmembrane helices and abundant secondary structure. Besides, the HarmAK was monomer, which had 2 ligands including 1 L-arginine and 1 ADP. The recombinant pET28a-HarmAK expressed a soluble protein after IPTG induction. SDS-PAGE and Western blot analysis indicated that the fusion protein, purified using Ni2+ affinity chromatography, had the predicted size and higher purity. And the activity of the fusion protein was (5.5±0.85) μmol/(min·mg) protein by the pH-spectrophotometric assay, which showed that the fusion His-HarmAK was able to catalyze the phosphorylation of L-arginine. Expression profile results showed that HarmAK expressed in both different tissues and developmental stages. The HarmAK expression level was higher in midgut, head and integument than that of fat body. The amount of HarmAK was the highest in the 1st instar larvae and gut of 6th instar larvae, and then decreased to lower level from 2nd instar larvae to adult. Our results will provide very useful information for using HarmAK as a new molecular target to control H. armigera.
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