中国农业科学2017，Vol.50Issue(9)：1723-1733,11.DOI:10.3864/j.issn.0578-1752.2017.09.018
家蚕表皮蛋白BmCPAP3-G的表达特征及其与几丁质的结合特性
Expression Pattern and Chitin-Binding Mode Analyses of Cuticle Protein BmCPAP3-G in the Silkworm (Bombyx mori)
摘要
Abstract
[Objective]The objective of this study is to explore the expression pattern of BmCPAP3-G and the binding mode of BmCPAP3-G with chitin, which will lay a foundation for the research of the cuticle proteins of silkworm (Bombyx mori).[Method]The sequence features of CPAP motif cuticular proteins and the conserved domains of BmCPAP3-G were analyzed by bioinformatics methods. The recombinant proteins were expressed by prokaryotic expression and purified by Ni affinity chromatography. The protein was identified by 5800 MALDI-TOF/TOF mass spectrometry, and then was used to prepare the polyclonal antibodies. The chitin-binding activity of BmCPAP3-G was verified by chitin affinity chromatography. The spatial and temporal expression patterns of BmCPAP3-G were analyzed by semi-quantitative RT-PCR and western blot. The binding mode of the domains of BmCPAP3-G with chitin was detected by using chitin affinity chromatography. [Result]The BmCPAP3-G protein has a signal peptide consisting of 18 amino acids, the molecular weight of 27 kD and the isoelectric point of 4.82, the encoding gene located on the chromosome No.15. BmCPAP3-G protein has three ChtBD2 domains, in which cysteine and aromatic amino acid showed very high homology. The BmCPAP3-G was cloned, expressed and the active recombinant protein was purified. After being identified by mass spectrometer, the polyclonal antibody against BmCPAP3-G was prepared. The BmCPAP3-G was found to bind chitin by using chitin affinity chromatography. The spatial and temporal expression patterns of BmCPAP3-G were analyzed by semi-quantitative RT-PCR and western blot, revealing similar results at the transcriptional and protein levels. BmCPAP3-G was expressed highly in the head and cuticle, and minimally in the midgut, gonad, and silk gland. In the silk gland, BmCPAP3-G had a high expression level in the fourth molting and its expression decreased in the cuticle and silk gland as the fifth instar goes on. Active recombinant proteins domain_3, domain_1-2, domain_2-3 were successfully expressed and it was found that all the domain_3, domain_1-2, and domain_2-3 could bind to chitin in vitro, but their binding abilities were different. Individual domains could bind with chitin, and two domains showed stronger chitin binding capacity than the single domain. [Conclusion]BmCPAP3-G is a typical cuticular protein of CPAP family and may be involved in the degradation and formation process of chitin layer in the silk gland during molting stage. BmCPAP3-G could bind with chitin by a single ChtBD2 domain, and multiple domains in which makes it have stronger chitin binding capacity.关键词
家蚕/表皮蛋白/CPAP/表达谱/几丁质结合Key words
silkworm (Bombyx mori)/cuticular proteins/CPAP/expression pattern/chitin-binding引用本文复制引用
张薇薇,董照明,张艳,张晓璐,张守亚,赵萍..家蚕表皮蛋白BmCPAP3-G的表达特征及其与几丁质的结合特性[J].中国农业科学,2017,50(9):1723-1733,11.基金项目
国家自然科学基金(31530071,31472154)、西南大学博士基金项目(SWU116076) （31530071,31472154）
