关键词:  家蚕, 抗菌肽, 细菌性肠道病, qRT-PCR, SAAP-148, 抑菌效果

Abstract: 【Aim】Bacterial intestinal disease is one of the main diseases of Bombyx mori. The purpose of this study is to explore the inhibitory effects of the broad-spectrum antimicrobial peptide SAAP-148 on the pathogenic bacteria of bacterial intestinal disease in B. mori, and to provide references for further use of antimicrobial peptides instead of antibiotics to prevent bacterial diseases in B. mori.【Methods】The expression levels of antimicrobial peptide genes in the midgut of healthy and bacterial intestinal disease-infected individuals of the day-4 5th instar larvae of B. mori were detected by qRT-PCR. The bacterial flora in the midgut of the day-4 5th instar larvae of B. mori infected with bacterial intestinal disease was isolated, and the phylogenetic tree was constructed by maximum likelihood method. The pathogenicity of the eight isolated bacteria and four common pathogenic bacteria of bacterial disease including Enterococcus faecalis, Bacillus bombyseptieus, B. thuringiensis and Serratia marcescens, to the healthy 5th instar larvae of B. mori was determined by feeding method (1×10¹⁰ and 1×10¹⁴ CFU/mL bacterial suspensions) and puncture inoculation method (2×10⁸ and 2×10⁹ CFU/mL bacterial suspensions). The inhibitory effects of the antimicrobial peptide SAAP-148 on the isolated pathogenic bacteria of intestinal disease and other common pathogenic bacteria of bacterial disease such as B. bombyseptieus were analyzed by agar plate diffusion method.【Results】The expression levels of the detected antibacterial peptide genes of B. mori in the midgut of the day-4 5th instar larvae of B. mori infected with bacterial intestinal disease were significantly up-regulated as compared with those of the healthy individuals, indicating that the antibacterial peptides in B. mori play a certain role in resisting bacterial intestinal disease. A total of eight pathogenic bacterial species (Mammaliicoccus sciuri, Aeromonas hydrophila, Enterobacter cancerogenus, E. bugandensis, E. hormaechei, Citrobacter freundii, Klebsiella pneumoniae and K. variicola) were isolated from the midgut of the day-4 5th instar larvae of B. mori infected with bacterial intestinal disease. The puncture results showed that the eight bacterial species were pathogenic. Among the eight bacterial species, M. sciuri, A. hydrophila, E. cancerogenus and E. bugandensis had strong pathogenicity, and E. hormaechei, C. freundii, K. pneumoniae and K. variicola were less pathogenic. The antibacterial peptide SAAP-148 had in vitro inhibitory effects on the eight isolated pathogenic bacteria and other four common pathogenic bacteria of B. mori bacterial disease, and the inhibitory effect increased with the increase of SAAP-148 concentration.【Conclusion】The antibacterial peptide SAAP-148 not only has obvious inhibitory effects on the pathogenic bacteria that may cause bacterial intestinal disease of B. mori, but also has inhibitory effects on the pathogenic bacteria that cause black chest sepsis, prodigiotic sepsis, bacterial poisoning of B. mori, and E. hormaechei which has produced resistance to ampicillin. SAAP-148 can be used as an ideal antibacterial agent to prevent the occurrence of bacterial disease of B. mori.

Key words: Bombyx mori, antibacterial peptide, bacterial intestinal disease, qRT-PCR, SAAP-148, antibacterial effects