关键词: 小菜蛾, 溶菌酶, RACE技术, 镍柱亲和层析, 牛津杯法, 抗微生物活性

Abstract: 【Aim】 This study aims to further understand the immune defense mechanism of Plutella xylostella and to provide new ideas for the biological control of P. xylostella by studying the function of lysozyme in P. xylostella. 【Methods】 RACE technique was used to clone lysozyme gene of P. xylostella. The prokaryotic expression vector pET-29a-Pxlys was constructed. The recombinant Pxlys was expressed by prokaryotic expression system and purified by nickel column affinity chromatography. The antibacterial activities of the recombinant Pxlys against Corynebacterium stationis, Micrococcus luteus, Staphyloccocus aureus, Escherichia coli, Shigella sp., Salmonella sp. and Bacillus thuringiensis were detected by Oxford cup method, and the lysis characteristics of the recombinant Pxlys against C. stationis and E. coli were observed under scanning electron microscope. 【Results】 The 423 bp ORF sequence of lysozyme gene Pxlys (GenBank accession number: MN702780) of P. xylostella was obtained by cloning. It encodes 140 aa with the relative molecular weight of 15.79 kD. The antibacterial activity test showed that the recombinant Pxlys had strong antibacterial activities not only against Gram-positive bacteria C. stationis, M. luteus and S. aureus, with the inhibition zone diameter of 20.0±1.1, 19.0±0.5 and 16.5±0.5 mm, respectively, but also against Gram-negative bacteria E. coli, Shigella sp. and Salmonella sp., with the inhibition zone diameter of 16.3±0.5, 15.0±0.5 and 14.0±1.1 mm, respectively. The recombinant Pxlys showed stronger antibacterial activity against Gram-positive bacteria than against Gram-negative bacteria. Besides, the recombinant Pxlys also showed the bactericidal activity against B. thuringiensis. Under scanning electron microscope, the lysis characteristics of the recombinant Pxlys against C. stationis and E. coli were different. 【Conclusion】 Pxlys has a broad-spectrum antimicrobial activity, and may have different antibacterial mechanisms against Gram-positive bacteria and Gramnegative bacteria. The results provide the basis for further understanding the immune defense system of P. xylostella. 

Key words: Plutella xylostella, lysozyme, RACE technique, nickel column affinity chromatography, Oxford cup method, antimicrobial activity