吕丁丁 侯成香 耿涛 黄玉霞 覃光星 高坤 郭锡杰

摘要：【目的】阐明家蚕（Bombyx mori）抗菌肽对球孢白僵菌感染的应答模式，为揭示家蚕及鳞翅目昆虫抗真菌机制提供参考依据。【方法】利用实时荧光定量PCR检测家蚕被球孢白僵菌感染后抗菌肽基因cecropinA、gloverin2、lebocin1/2和lysozyme的表达情况，比较4种抗菌肽在家蚕脂肪体、血淋巴、马氏管和中肠中不同时间点的表达差异。【结果】经球孢白僵菌诱导后，cecropinA基因在家蚕脂肪体中的最高上调表达倍数为10.2倍，在血淋巴中的最高上调表达倍数为13.2倍，在马氏管中感染初期显著上调表达（P<0.05，下同），在中肠中感染后期顯著上调表达；gloverin2基因在家蚕脂肪体中出现2次上调表达过程，最高上调表达倍数为15.7倍，在血淋巴中感染前期呈上调表达，在马氏管中略呈下调表达，中肠中的gloverin2基因在接种后16～48 h呈显著上调表达，最高上调表达倍数为6.7倍；lebocin1/2基因在家蚕脂肪体和血淋巴中的最高上调表达倍数分别为19.4和22.4倍，在马氏管中仅在接种后16 h呈显著上调表达，在中肠中则是接种后24～48 h呈显著上调表达；lysozyme基因在家蚕脂肪体中显著上调表达，在血淋巴和马氏管中感染初期也呈显著上调表达，但在中肠中的表达无明显规律。【结论】cecropinA、gloverin2、lebocin1/2和lysozyme基因在球孢白僵菌侵染家蚕的过程中均不同程度地被诱导上调表达，尤其在家蚕脂肪体和血淋巴中较明显，由此推测这4种抗菌肽在抵御球孢白僵菌的侵染过程中发挥重要作用。

关键词： 家蚕；球孢白僵菌；抗菌肽；表达水平

中图分类号： S884.31 文献标志码：A 文章编号：2095-1191（2016）11-1952-06

Abstract：【Objective】The present study was conducted to evaluate response pattern of antimicrobial peptides in Bombyx mori against Beauveria bassiana infection in order to provide beneficial reference for researches on antifungal mechanism of B. mori and other lepidoptera insects. 【Method】The expression patterns of antimicrobial peptide genes cecropinA， gloverin2， lebocin1/2 and lysozyme in B. mori after being infected with B. bassiana were detected using real-time fluorescence quantitative PCR. The differential expressions of the four antimicrobial peptide genes in fat body， hemolymph， malpighian tubule and midgut at different time points were compared. 【Result】After being induced by B. bassiana， the expression of cecropinA was up-regulated by 10.2 times to the maximum in fat body and 13.2 times to maximum in hemolymph. The expression of cecropinA was also significantly up-regulated in malpighian tubule at early stage of the infection（P<0.05， the same below）， and in midgut at middle-late stages of the infection. Up-regulated expression of gloverin2 occurred twice in fat body and the maximum re-regulated expression multiple was 15.7 times. The expression of gloverin2 up-regulated in hemolymph at earlier stage but down-regulated in malpighian tubule. It also up-regulated in mindgut after 16-48 hours of infection， with maximum up-regulated multiples reaching 6.7 times. The expression of lebocin1/2 up-regulated in fat body by 19.4 times to utmost limit and by 22.4 times to the maximum in hemolymph. It also up-regulated significantly in malpighian tubule after 16 hours of infection and in midgut after 24-48 hours of infection. The expression of lysozyme significantly up-regulated in fat body， and also significantly up-regulated in hemolymph and malpighian tubule at earlier stage of infection. But the expression in midgut showed no obvious regulation. 【Conclusion】The expressions of antimicrobial peptide genes cecropinA， gloverin2， lebocin1/2 and lysozyme up-regulated to different extents after B. mori infected by B. bassiana， especially in fat body and hemolymph of B. mori. The results suggest that cecropinA， gloverin2， lebocin1/2 and lysozyme may play important roles in B. mori antifungal process against B. bassiana infection.

Key words： Bombyx mori； Beauveria bassiana； antimicrobial peptide； expression characteristics

0 引言

【研究意义】家蚕（Bombyx mori）的先天性免疫系统包括细胞免疫应答和体液免疫应答。体液免疫应答主要是合成和释放若干抗微生物肽分子，而这些抗菌肽（Antimicrobial peptides，AMPs）均产生于脂肪体、血细胞及上皮组织（吕鸿声，2008）。家蠶抗菌肽在正常情况下不表达或呈低水平的组成型表达，一旦有外界微生物入侵，即在体内迅速合成与释放，参与免疫反应。白僵病是危害最严重、病势最急的一种家蚕真菌性传染病（吕鸿声等，1990），研究家蚕抗菌肽对球孢白僵菌（Beauveria bassiana）感染的应答模式，对阐明家蚕对白僵菌的体液免疫机制及其防治具有重要意义。【前人研究进展】自1981年Boman和Steiner在惜古比天蚕（Hyatophora cecropia）中发现可诱导抗微生物肽以来，目前已从家蚕中分离鉴定出多种可诱导抗菌肽，如cecropins、defensins、lebocins、gloverins等（Yamano et al.，1994；Chowdhury et al.，1995；Cheng et al.，2006；Kaneko et al.，2007， 2008）。当昆虫受到免疫刺激时，溶菌酶（lysozymes）在其体内含量迅速升高，并参与免疫应答，因此也常被视为免疫分子（吕鸿声，2008）。……