家蚕抗菌肽Attacin1对球孢白僵菌的抗菌活性

Antifungal Effect of Bombyx mori Antimicrobial Peptide Attacin1 on Beauveria bassiana

抗菌肽是宿主在免疫过程中产生的一类重要抗菌物质。通过实时荧光定量PCR检测发现家蚕幼虫在被球孢白僵菌(Beauveria bassiana)感染后抗菌肽Attacin1的编码基因BmAttacin1表达显著上调。克隆获得了BmAttacin1成熟蛋白的编码序列,利用原核表达系统成功表达并获得BmAttacin1蛋白。抑菌圈实验结果表明,BmAttacin1对革兰阴性菌大肠埃希菌(Escherichia coli)和球孢白僵菌有明显的抑菌效果,而对革兰阳性菌枯草芽孢杆菌(Bacillus subtilis)没有抑菌效果;进一步将BmAttacin1与球孢白僵菌分生孢子孵育,间接免疫荧光检测发现BmAttacin1能与球孢白僵菌分生孢子结合。在BmN培养细胞中过表达BmAttacin1基因后,家蚕其他7种抗菌肽基因也都上调表达,其中天蚕素类抗菌肽基因BmCecropinA的表达上调了10倍,BmCecropinB上调了9倍,表明BmAttacin1的表达可能对其他抗菌肽基因的表达具有协同增强作用。试验结果为深入研究BmAttacin1与球孢白僵菌的相互作用以及BmAttacin1在家蚕抵御白僵菌感染过程中发挥的作用奠定了一定的基础。

Antimicrobial peptides are a class of important antimicrobial complex produced by host during immune process.In this study,real-time fluorescence quantitative PCR(qRT-PCR)revealed that expression of BmAttacin1 gene of antimicrobial peptide Attacin1 was significantly up-regulated in silkworm larvae infected with Beauveria bassiana. Meanwhile,coding sequence of BmAttacin1 mature peptide was cloned and recombinant BmAttacin1 was successfully expressed in prokaryotic expression system. The expressed recombinant protein exhibited obvious antimicrobial activity against Gramnegative bacteria Escherichia coli and B. bassiana rather than Gram-positive bacteria Bacillus subtilis in bacteriostatic zone experiment. Furthermore,after incubation of BmAttacin1 with B. bassiana conidia,indirect immunofluorescence detection indicated that BmAttacin1 could be bound to B. bassiana conidia. When BmAttacin1 was over-expressed in BmN cell,gene expressions of other 7 antimicrobial peptides were up-regulated,among which antimicrobial peptide of cecropin genes BmCecropin A and BmCecropin B was upregulated for 10 folds and for 9 folds,indicating that expression of BmAttacin1 could synergistically enhance the gene expression of other antimicrobial peptides. These results laid a foundation for further study on the interaction between BmAttacin1 and B. bassiana and the function of BmAttacin1 against B. bassiana infection in silkworm.