Acta Entomologica Sinica ›› 2020, Vol. 63 ›› Issue (7): 798-806.doi: 10.16380/j.kcxb.2020.07.003

• RESEARCH PAPERS • Previous Articles     Next Articles

Inhibition of the activation of prophenol oxidase and the expression of antimicrobial peptide genes by the serine protease inhibitor Bmserpin2 in Bombyx mori

LI Bing1,2, SUN Fan2, TAO Shan-Shan2, XIA Jia-Feng2, YE Chong-Jun2,*   

  1.  (1. Key Laboratory of Silkworm and Mulberry Genetic Improvement, Ministry of Agriculture, Zhenjiang, Jiangsu 212018, China; 2. Sericultural Research Institute, Anhui Academy of Agricultural Sciences, Hefei 230061, China)
  • Online:2020-07-20 Published:2020-07-29

Abstract: 【Aim】 Serine protease inhibitor family proteins are important protease inhibitors that regulate the autoimmune response in insects. This study aims to investigate the regulatory role of Bmserpin2 in two important autoimmune pathways, i.e., the prophenol oxidase (PPO) activation pathway and the TOLL pathway of antimicrobial peptides (AMPs) induced by gram-positive bacteria, in Bombyx mori. 【Methods】 Bmserpin2 gene fragment of B. mori was amplified by PCR, and the target protein expression was induced by prokaryotic expression system and purified by nickel column. The effect of Bmserpin2 on the activities of trypsin, chymotrypsin, elastase and protease K was determined after the reaction between the purified recombinant Bmserpin2 and the above proteases, respectively. The expression patterns of Bmserpin2 in the head, midgut, fat body, hemolymph, silk gland, and integument of the day-3 5th instar larvae of B. mori were detected by RT-qPCR. The recombinant Bmserpin2 was injected into the day-3 5th instar larvae of B. mori, and the effect of Bmserpin2 on the PPO activity in their hemolymphs was determined. After the day-3 5th instar larvae of B. mori was induced by Micrococcus luteus to produce antimicrobial peptides (AMP) and injected with Bmserpin2, the expression levels of AMP genes gloverin2 and moricin in their hemolymphs were detected by RT-qPCR. 【Results】 The recombinant plasmid was successfully constructed, and the target protein Bmserpin2 was expressed and purified. Bmserpin2 significantly inhibited the activities of trypsin and elastase, but had no significant effect on the activities of chymotrypsin and proteinase K, suggesting that Bmserpin2 has biological activity and catalytic specificity to different proteases. The expression levels of Bmserpin2 were the highest in the hemolymph and fat body of the 5th instar larvae of B. mori. Most importantly, Bmserpin2 was found to inhibit the PPO activity in the hemolymph of the 5th instar larvae of B. mori after injection of the recombinant Bmserpin2. AMP was produced in the 5th instar larvae of B. mori induced by M. luteus. The transcription levels of AMP genes gloverin2 and moricin in the hemolymph in the Bmserpin2 and M. luteus mixedly injected group was significantly down-regulated as compared with that in the M. luteus injected group. 【Conclusion】 Bmserpin2 may be involved in the immune pathway of the activation of PPO and the extracellular cascade reaction of TOLL pathway in B. mori.

Key words: Bombyx mori, autoimmune, serine protease inhibitor, prophenol oxidase, antimicrobial peptide, TOLL pathway