柞蚕抗菌肽cecropin B的结构和抑菌活性及基因表达分析  

Analysis on Structure,Antibiotic Activity and Gene Expression of Antimicrobial Peptide cecropin B from Antheraea pernyi

作　　者：叶博 赵振军[1,2] 李佩佩 岳冬梅[1,2] 张波 王林美[1,2] 李树英 范琦[1,2] Ye Bo;Zhao Zhenjun;Li Peipei;Yue Dongmei;Zhang Bo;Wang Linmei;Li Shuying;Fan Qi(Liaoning Ocean and Fisheries Science Research Institute,Dalian Liaoning 116023,China;Dalian Institute of Biotechnology,Liaoning Academy of Agricultural Sciences,Dalian Liaoning 116024,China)

机构地区：[1]辽宁省海洋水产科学研究院,辽宁大连116023 [2]辽宁省农业科学院大连生物技术研究所,辽宁大连116024

出　　处：《蚕业科学》2019年第5期660-668,共9页ACTA SERICOLOGICA SINICA

基　　金：辽宁省自然科学基金指导计划项目(No.20170540512);辽宁省科学事业公益研究基金项目(No.20180013)

摘　　要：抗菌肽对昆虫抵御病原微生物的侵染具有重要作用。使用在线软件分析成熟的柞蚕抗菌肽cecropin B高级结构,并与鳞翅目其他昆虫抗菌肽进行聚类分析。利用巴斯德毕赤酵母体外表达柞蚕cecropin B,并测试重组柞蚕cecropin B的抑菌活性。采用qRT-PCR方法检测3个不同品种柞蚕蛹在柞蚕肠球菌、柞蚕核型多角体病毒和柞蚕微孢子虫感染情况下,体内cecropinB基因的转录表达情况。结果显示,柞蚕cecropin B成熟肽的N端和C端分别有1个α螺旋结构,聚类分析将柞蚕cecropin B归为昆虫抗菌肽cecropin B亚型;柞蚕cecropin B在巴斯德毕赤酵母工程菌中得到体外表达,重组柞蚕cecropin B具有体外抑菌活性;柞蚕肠球菌感染的不同品种柞蚕蛹的cecropinB基因转录表达水平均上调,柞蚕核型多角体病毒或柞蚕微孢子虫感染的不同品种柞蚕蛹的cecropinB基因转录表达水平有明显差异,提示柞蚕cecropin B在柞蚕抵抗不同病原微生物的感染过程中可能具有不同的作用。Antimicrobial peptide plays an important role in protecting insects from pathogen infection. Therefore,tertiary structure of mature cecropin B from Antheraea pernyi was predicated and phylogenetic tree of cecropins from Antheraea pernyi and other Lepidoptera insects were analyzed using online software in this study. Then,A. pernyi cecropin B was expressed in vitro by Pichia pastoris expression system and its antibiotic activity was also assayed. Furthermore,transcription levels of A. pernyi cecropin B in tussah pupa of 3 varieties were detected by qRT-PCR after they were infected with Enterococcus pernyi,Antheraea pernyi nucleopolyhedrovirus(ApNPV)and Nosema pernyi,respectively. The results showed that N terminal and C terminal on the mature peptide of A. pernyi cecropin B each contained a α-helical structure. A. pernyi cecropin B was classified into antimicrobial peptide cecropin B subtype of insects according to phylogenetic analysis. Besides,recombinant A. pernyi cecropin B was expressed in vitro with antibiotic activity in the P. pastoris expression system. Transcription levels of A. pernyi cecropin B gene in all tussah pupa were up-regulated after infected with Enterococcus pernyi. However,significant difference was found in transcription level of A.pernyi cecropin B gene between varieties infected with ApNPV and N. pernyi. These results indicated that A. pernyi cecropin B may function differently in the defending process of A. pernyi against different pathogen infection.

关 键 词：柞蚕 抗菌肽cecropin B 巴斯德毕赤酵母表达系统 病原微生物 实时荧光定量PCR 

分 类 号：S885.1[农业科学—特种经济动物饲养] S881.24[农业科学—畜牧兽医]