| 王翠苹1, 赵 丹1, 李少雅1, 郭家萍1, 郭 巍1,2*, 陆秀君1.美国白蛾hcapn3基因的克隆及HcAPN3蛋白与Cry1Ac结合特性分析[J].植物保护,2016,42(2):62-67. |
| 美国白蛾hcapn3基因的克隆及HcAPN3蛋白与Cry1Ac结合特性分析 |
| Cloning of midgut aminopeptidase hcapn3 gene from Hyphantria cunea and binding to Cry1Ac toxin of HcAPN3 |
| 投稿时间：2015-02-05 修订日期：2015-04-16 |
| DOI： |
| 中文关键词: 美国白蛾 氨肽酶N HcAPN3 Gluzincin结构域 Ligand Blot分析 |
| 英文关键词:Hyphantria cunea aminopeptidase N HcAPN3 Gluzincin domain Ligand Blot analysis |
| 基金项目:国家现代农业产业技术体系(CARS-14); 国家自然科学基金(34711775); 北京农学院促进人才培养综合改革专项计划(BNRC&CC201404) |
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| 中文摘要: |
| 通过比对分析已知昆虫氨肽酶N (aminopeptidase N, APN)氨基酸序列并结合本实验室的美国白蛾(Hyphantria cunea)中肠iTRAQ结果, 设计了hcapn3基因特异引物, 获得hcapn3基因片段。通过RACE-PCR技术获得美国白蛾中肠氨肽酶N基因(hcapn3)全长序列(GenBank登录号为KJ013598)。Blastp分析表明, 获得的氨肽酶属于氨肽酶N3家族, 命名为HcAPN3。序列分析显示HcAPN3包括952个氨基酸残基, 具有典型的谷氨酸锌化氨肽酶(Gluzincin)结构域和羧基端(ERAP1_C)结构域。利用Bac to Bac表达系统在昆虫细胞中表达108 kDa的 HcAPN3蛋白。在原核表达系统中表达58 kDa 的Gluzincin结构域和49 kDa ERAP1_C的结构域蛋白。Ligand Blot分析结果显示, HcAPN3蛋白及Gluzincin结构域可与Cry1Ac蛋白特异性结合, 但ERAP1_C结构域未能与Cry1Ac结合。本研究首次克隆了美国白蛾氨肽酶基因并分析了HcAPN3与Cry1Ac的结合特性, 为下一步功能研究提供基础。 |
| 英文摘要: |
| The specific primers of aminopeptidase N gene were designed, and a fragment of hcapn3 was obtained by PCR method. A full-length gene (GenBank accession no. KJ013598) encoding APN of Hyphantria cunea was obtained by RACE-PCR method based on the sequences of published APNs from insects and iTRAQ of Hyphantria cunea midgut. Analysis of the protein sequences by NCBI Blastp revealed that it belonged to aminopeptidase N gene family 3, named HcAPN3. The primary structure of HcAPN3 contained 952 amino acids residues, a typical Gluzincin aminopeptidase family domain and ERAP1_C domain. The hcapn3 gene was successfully expressed in insect cells as secreted proteins (108 kDa) using recombinant baculoviruses (Bac to Bac system). Further, the Gluzincin aminopeptidase family domain and ERAP1_C-like C-terminal domain were expressed in E.coli as 58 kDa and 49 kDa proteins by SDS-PAGE analysis, respectively. In vivo Ligand-Blotting study demonstrated binding of Cry1Ac toxin to both HcAPN3 and peptidase Gluzincin family domain, not to ERAP1_C domain. The first identified aminopeptidase N from H. cunea may be a candidate receptor for Cry1Ac toxin. |
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