亚洲玉米螟CPT1蛋白的表达、纯化和与几丁质的结合能力

doi:10.16409/j.cnki.2095-039x.2023.03.005

中国生物防治学报 ›› 2023, Vol. 39 ›› Issue (2): 462-470.DOI: 10.16409/j.cnki.2095-039x.2023.03.005

亚洲玉米螟CPT1蛋白的表达、纯化和与几丁质的结合能力

蒋望¹, 包涵¹, 陈金利¹, 刘田¹, 杨青^1,2,3

1. 大连理工大学生物工程学院, 大连 116024;
2. 中国农业科学院植物保护研究所, 北京 100193;
中国农业科学院农业基因组研究所, 深圳 518000

收稿日期:2020-02-18 发布日期:2023-04-13
通讯作者: 杨青,教授, E-mail:qingyang@dlut.edu.cn。
作者简介:蒋望,硕士, E-mail:jiangwang96@mail.dlut.edu.cn。
基金资助:
国家重点研发计划（2017YFD0200500，2018YFD0200100）；国家自然科学基金（31871959）

Expression, Purification and Characterization of Chitin-binding in Protein CPT1 from Ostrinia furnacalis

JIANG Wang¹, BAO Han¹, CHEN Jinli¹, LIU Tian¹, YANG Qing^1,2,3

1. School of Bioengineering, Dalian University of Technology, Dalian 116024, China;
2. Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
3. Agricultural Genomics Institute at Shenzhen, Chinese Academy of Agricultural Sciences, Shenzhen 518000, China

Received:2020-02-18 Published:2023-04-13

摘要/Abstract

摘要： 亚洲玉米螟Ostrinia furnacalis是为害我国主要农作物玉米的一种重要害虫。昆虫表皮在运动、抵御机械损伤及对抗外界环境压力等方面发挥关键性作用。Tweedle（CPT）家族表皮蛋白在形成昆虫表皮中发挥重要作用，研究CPT有助于开发新一代害虫防治方法。本研究在大肠杆菌系统中以包涵体的形式重组表达了OfCPT1蛋白，使用金属螯合层析进行纯化，复性得到了OfCPT1蛋白，收率为50 mg/L。几丁质结合试验发现，OfCPT1对α-几丁质、β-几丁质、壳聚糖、胶体几丁质和几丁质纳米微晶均有一定的结合能力，其中与几丁质纳米微丝结合能力最强。圆二色光谱结果表明，OfCPT1蛋白与壳聚糖结合后二级结构从以α-螺旋占主导变为以β-折叠占主导。本研究获得了亚洲玉米螟重组CPT蛋白并表征了它的部分生物化学性质。

关键词: 表皮蛋白, Tweedle, 几丁质结合, 杀虫剂靶标

Abstract: Asian corn borer, Ostrinia furnacalis, is an important agricultural insect pest that greatly impairs corn production in China. Insect cuticle plays key roles in protecting insects from locomotion, mechanical damage or environmental stresses. The cuticle proteins that belong to Tweedle (CPT) family are known to play important roles in insect cuticle formation. Investigation on CPT may help the development of new methods for insect pest control. In this study, OfCPT1, one of the CPTs derived from the Asian corn borer, was recombinantly expressed as inclusion bodies in Escherichia coli. The obtained OfCPT1 was purified by metal chelate chromatography and then renatured with a yield of 50 mg/L. Chitin binding assay showed that OfCPT1 had a strong binding ability with chitin nanofibers, but moderate binding to α-chitin, β-chitin, chitosan, colloid chitin, and chitin nanocrystal. The circular dichroism spectra of OfCPT1 before and after binding with chitosan, showed a structural change from α-helix dominance to β-sheet dominance. This work has accomplished the recombinant preparation of OfCPT1 and revealed its biochemical properties for the first time.

Key words: cuticular protein, Tweedle, chitin-binding protein, pesticide target

中图分类号:

S476

蒋望, 包涵, 陈金利, 刘田, 杨青. 亚洲玉米螟CPT1蛋白的表达、纯化和与几丁质的结合能力[J]. 中国生物防治学报, 2023, 39(2): 462-470.

JIANG Wang, BAO Han, CHEN Jinli, LIU Tian, YANG Qing. Expression, Purification and Characterization of Chitin-binding in Protein CPT1 from Ostrinia furnacalis[J]. Chinese Journal of Biological Control, 2023, 39(2): 462-470.

参考文献

[1] Vincent J F, Wegst U G. Design and mechanical properties of insect cuticle[J]. Arthropod Structure & Development, 2004, 33(3):187-199.
[2] Moussian B. Recent advances in understanding mechanisms of insect cuticle differentiation[J]. Insect Biochemistry and Molecular Biology, 2010, 40(5):363-375.
[3] Fukamizo T, Kramer K J. Mechanism of chitin oligisaccharide hydrolysis by the binary enzyme chitinase system in insect molting fluid[J]. Insect Biochemistry, 1985, 15(1):1-7.
[4] Chen P, Lin A Y, McKittrick J, et al. Structure and mechanical properties of crab exoskeletons[J]. Acta Biomaterialia, 2008, 4(3):587-596.
[5] Rinaudo M. Chitin and chitosan:Properties and applications[J]. Progress in Polymer Science, 2006, 31(7):603-632.
[6] Suksangpanya N, Yaraghi N A, Kisailus D, et al. Twisting cracks in Bouligand structures[J]. Journal of the Mechanical Behavior of Biomedical Materials, 2017, 76:38-57.
[7] Narkevicius A, Steiner L M, Parker R M, et al. Controlling the self-assembly behavior of aqueous chitin nanocrystal suspensions[J]. Biomacromolecules, 2019, 20(7):2830-2838.
[8] Pan P L, Ye Y X, Lou Y H, et al. A comprehensive omics analysis and functional survey of cuticular proteins in the brown planthopper[J]. Proceedings of the National Academy of Sciences of the United States of America, 2018, 115(20):5175-5180.
[9] Vannini L, Bowen J H, Reed T W, et al. The CPCFC cuticular protein family:Anatomical and cuticular locations in Anopheles gambiae and distribution throughout pancrustacea[J]. Insect Biochemistry and Molecular Biology, 2015, 65:57-67.
[10] Rebers J E, Riddiford L M. Structure and expression of a Manduca sexta larval cuticle gene homologous to Drosophila cuticle genes[J]. Journal of Molecular Biology, 1988, 203(2):411-423.
[11] Togawa T, Augustine D W, Emmons A C, et al. CPF and CPFL, two related gene families encoding cuticular proteins of Anopheles gambiae and other insects[J]. Insect Biochemistry and Molecular Biology, 2007, 37(7):675-688.
[12] Futahashi R, Okamoto S, Kawasaki H, et al. Genome-wide identification of cuticular protein genes in the silkworm, Bombyx mori[J]. Insect Biochemistry and Molecular Biology, 2008, 38(12):1138-1146.
[13] Cornman R S, Willis J H. Annotation and analysis of low-complexity protein families of Anopheles gambiae that are associated with cuticle[J]. Insect Molecular Biology, 2009, 18(5):607-622.
[14] Guan X, Middlebrooks B W, Alexander S, et al. Mutation of TweedleD, a member of an unconventional cuticle protein family, alters body shape in Drosophila[J]. Proceedings of the National Academy of Sciences of the United States of America, 2006, 103(45):16794-16799.
[15] Song T Q, Yang M L, Wang Y L, et al. Cuticular protein LmTwdl1 is involved in molt development of the migratory locust[J]. Insect Science, 2016, 23(4):520-530.
[16] Donitz J, Gerischer L, Hahnke S, et al. Expanded and updated data and a query pipeline for iBeetle-Base[J]. Nucleic Acids Research, 2018, 46(D1):D831-D835.
[17] Liang J, Wang T, Xiang Z, et al. Tweedle cuticular protein BmCPT1 is involved in innate immunity by participating in recognition of Escherichia coli[C]. 第十二届家(柞)蚕遗传育种暨良种繁育学术研讨会, 2016.
[18] Liang J, Wang T, Xiang Z, et al. Tweedle cuticular protein BmCPT1 is involved in innate immunity by participating in recognition of Escherichia coli[J]. Insect Biochemistry and Molecular Biology, 2015, 58:76-88.
[19] Alberti S, Gladfelter A, Mittag T. Considerations and challenges in studying liquid-liquid phase separation and biomolecular condensates[J]. Cell, 2019, 176(3):419-434.
[20] Ifuku S, Nogi M, Abe K, et al. Preparation of chitin nanofibers with a uniform width as alpha-chitin from crab shells[J]. Biomacromolecules, 2009, 10(6):1584-1588.
[21] Ifuku S, Saimoto H. Chitin nanofibers:preparations, modifications, and applications[J]. Nanoscale, 2012, 4(11):3308-3318.
[22] Aklog Y F, Nagae T, Izawa H, et al. Preparation of chitin nanofibers by surface esterification of chitin with maleic anhydride and mechanical treatment[J]. Carbohydr Polym, 2016, 153:55-59.
[23] 梁欣, 陈斌, 乔梁. 昆虫表皮蛋白基因研究进展[J]. 昆虫学报, 2014, 57(9):1084-1093.
[24] Lucie L, Peter S, Jonathan S, et al. Photosensitized cross-linking of tryptophan and tyrosine derivatives by rose bengal in aqueous solutions[J]. The Journal of organic chemistry, 2018, 83(18):10835-10844.
[25] Brunet P C J. The metabolism of the aromatic amino acids concerned in the cross-linking of insect cuticle[J]. Insect Biochemistry, 1980, 10(5):467-500.
[26] Islam S, Rahman Bhuiyan M A, Islam M N. Chitin and chitosan:structure, properties and applications in biomedical engineering[J]. Journal of Polymers and the Environment, 2017, 25(3):854-866.
[27] Anitha A S, Sowmya P T, Sudheesh K S, et al. Chitin and chitosan in selected biomedical applications[J]. Progress in Polymer Science, 2014, 39(9):1644-1667.
[28] Helge-Otto F, Andreas Z, Martin F, et al. Functional adaptation of crustacean exoskeletal elements through structural and compositional diversity:a combined experimental and theoretical study[J]. Bioinspiration & Biomimetics, 2016, 11(5):055006.
[29] 张鸿昊, 林乃波, 刘向阳. 蚕丝和蜘蛛丝多级结构对力学性能的影响[J]. 功能高分子学报, 2018, 31(6):501-512.
[30] Dong Z, Zhang W, Zhang Y, et al. Identification and characterization of novel chitin-binding proteins from the larval cuticle of silkworm, Bombyx mori[J]. Journal of Proteome Research, 2016, 15(5):1435-1445.

亚洲玉米螟CPT1蛋白的表达、纯化和与几丁质的结合能力

Expression, Purification and Characterization of Chitin-binding in Protein CPT1 from Ostrinia furnacalis

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 6

编辑推荐

Metrics

本文评价

联系我们

[1]	吴涵, 赵丹, 郭晓昌, 刘兆瑞, 郭巍, 毕扬. 甜菜夜蛾SeCDA8的基因克隆、蛋白表达及几丁质结合活性分析[J]. 中国生物防治学报, 2022, 38(6): 1508-1515.
[2]	刘晶, 包涵, 陈磊, 刘源涛, 刘田, 杨青. 双叉犀金龟表皮蛋白Td14144表达纯化及性质研究[J]. 中国生物防治学报, 2022, 38(2): 513-520.
[3]	卜云飞, 刘田, 陈磊, 杨青. 亚洲玉米螟表皮蛋白CPR9的表达纯化及与几丁质的相互作用[J]. 中国生物防治学报, 2020, 36(1): 79-86.
[4]	赵丹, 闫晓平, 陆秀君, 郭巍. 暗黑鳃金龟几丁质脱乙酰酶HpCDA5基因的细胞表达及活性分析[J]. 中国生物防治学报, 2018, 34(5): 686-692.
[5]	王晓冰, 邓登辉, 张坤, 郭江龙, 韩卫丽, 吴少英. 棉铃虫表皮蛋白基因的克隆、序列及表达分析[J]. 中国生物防治学报, 2018, 34(4): 527-532.
[6]	刘劲1,2 , 刘华2 , 袁京京2 , 刘峥2 , 杨秀芬2 , 张利莉1 , 邱德文2 , 曾洪梅2 . 格氏线虫表皮蛋白和分泌蛋白抑制昆虫免疫活性的比较[J]. , 2012, 28(3): 400-407.