白僵菌诱导家蚕抗菌肽enbocin1基因的表达特征及产物的抗真菌活性

Expression Patterns of Antimicrobial Peptide Gene enbocin1 in Bombyx mori Induced by Beauveria bassiana and Its Antifungal Activity

家蚕的抗菌肽家族基因enbocin1是蚕体免疫系统相关基因。为解析家蚕对病原真菌感染免疫应答反应的分子机制,采用实时荧光定量PCR检测抗菌肽基因enbocin1在家蚕5龄幼虫感染球孢白僵菌(Beauveria bassiana)后的时空表达模式,结果显示:enbocin1主要在幼虫脂肪体中显著上调表达,而且持续至感染诱导后30 h,感染后8、30 h的表达水平分别上调了约44倍和6倍;在马氏管、体壁中该基因的诱导表达差异不显著,在血淋巴中几乎检测不到该基因的表达。进一步用原核表达系统成功表达并纯化获得了高纯度的重组家蚕抗菌肽enbocin1,体外抑菌试验表明其对白僵菌具有较强的抑菌活性,且重组enbocin1蛋白浓度与抑菌活性之间具有量效关系。用重组enbocin1蛋白制备了效价达1∶30 000的多克隆抗体,可用于进一步深入研究enbocin1的功能及其作用机制。研究结果提示抗菌肽enbocin1在家蚕抵御真菌感染的过程中可能具有重要作用。

Antimicrobial peptide gene enbocin1 is an immune system-related gene in silkworm,Bombyx mori. For better understanding of the molecular mechanisms of silkworm in response to the infection of pathogenic fungus,the spatial and temporal expression patterns of enbocin1 gene were detected by real-time fluorescence quantitive PCR after the 5th instar silkworm larvae were infected by Beauveria bassiana. The result showed that the expressional level of enbocin1 gene was up-regulated significantly and mainly in fat body and kept at a high level till 30 h post infection（ hpi）,and the expression levels at 8 and 30 hpi were up-regulated by 44-and 6-folds respectively. The expressional level of enbocin1 had little change in Malpighian tube and integument after infection,and was almost undetectable in hemolymph. Then,the recombinant enbocin1 protein was successfully expressed with prokaryotic expression system and highly purified. Antifungal test in vitro verified that the recombinant enbocin1 protein had high antifungal activity against B. bassiana in a dose-effect manner. Furthermore,polyclonal antibody against the recombinant enbocin1 was prepared with a titer of 1 ∶ 30 000,which could be used for further study of the function and functional mechanism of enbocin1. These results suggest that antimicrobial peptide enbocin1 may play an important role in response of silkworm to fungal infection.