昆虫学报 ›› 2021, Vol. 64 ›› Issue (7): 781-789.doi: 10.16380/j.kcxb.2021.07.002

• 研究论文 • 上一篇    下一篇

小菜蛾溶菌酶Pxlys的基因克隆及其 重组蛋白的抗菌活性分析

宁燕夏, 苏月华, 杨梅*   

  1. (福建师范大学生命科学学院, 福州 350117)
  • 出版日期:2021-07-20 发布日期:2021-08-02

cDNA cloning of lysozyme Pxlys of Plutella xylostella (Lepidoptera: Plutellidae) and the analysis of antibacterial activity of its recombinant protein

NING Yan-Xia, SU Yue-Hua, YANG Mei*   

  1.  (College of Life Sciences, Fujian Normal University, Fuzhou 350117, China)
  • Online:2021-07-20 Published:2021-08-02

摘要:

 【目的】本研究旨在通过研究小菜蛾Plutella xylostella溶菌酶的功能,进一步认识小菜蛾的免疫防御机理,为小菜蛾的生物防治提供新的思路。【方法】利用RACE技术克隆小菜蛾溶菌酶基因。构建原核表达载体pET-29a-Pxlys,利用原核表达系统表达并用镍柱亲和层析纯化重组蛋白Pxlys。利用牛津杯法检测重组蛋白Pxlys对停滞棒杆菌Corynebacterium stationis、藤黄微球菌Micrococcus luteus、金黄色葡萄球菌Staphyloccocus aureus、大肠杆菌Escherichia coli、志贺氏菌Shigella sp.、沙门氏菌Salmonella sp.和苏云金芽胞杆菌Bacillus thuringiensis的抑菌活性,并利用扫描电子显微镜观察重组蛋白Pxlys对停滞棒杆菌和大肠杆菌的溶菌特征。【结果】克隆获得开放阅读框长423 bp的小菜蛾溶菌酶基因Pxlys(GenBank登录号: MN702780)序列,它编码140个氨基酸,相对分子质量为15.79 kD。抑菌试验表明,重组蛋白Pxlys不仅对革兰氏阳性细菌的停滞棒杆菌、藤黄微球菌和金黄色葡萄球菌有较强的抑菌活性(抑菌圈直径分别为20.0±1.1, 19.0±0.5和16.5±0.5 mm),而且对革兰氏阴性细菌大肠杆菌、志贺氏菌和沙门氏菌也有抑菌活性(抑菌圈直径分别为16.3±0.5, 15.0±0.5和14.0±1.1 mm),重组蛋白Pxlys对革兰氏阳性细菌比对革兰氏阴性细菌表现出更强的抑菌活性。另外,重组蛋白Pxlys还表现出对苏云金芽胞杆菌的抑菌活性。扫描电子显微镜下,经重组蛋白Pxlys处理过的停滞棒杆菌和大肠杆菌的溶菌特征不同。【结论】Pxlys具有广谱的抗微生物活性,其对革兰氏阳性细菌和革兰氏阴性细菌的抑菌机理可能存在不同。研究结果为深入研究小菜蛾免疫防御系统提供基础。

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