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

doi:10.16409/j.cnki.2095-039x.2020.01.005

中国生物防治学报 ›› 2020, Vol. 36 ›› Issue (1): 79-86.DOI: 10.16409/j.cnki.2095-039x.2020.01.005

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

卜云飞, 刘田, 陈磊, 杨青

大连理工大学生命科学与技术学院, 大连 116024

收稿日期:2019-03-05 出版日期:2020-02-08 发布日期:2020-02-26
通讯作者: 刘田,副教授,E-mail:tianliu@dlut.edu.cn
作者简介:卜云飞,硕士研究生,E-mail:byf@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 of Cuticular Protein CPR9 of Ostrinia furnacalis and its Interaction with Chitin

BU Yunfei, LIU Tian, CHEN Lei, YANG Qing

School of Life Science and Biotechnology, Dalian University of Technology, Dalian 116024, China

Received:2019-03-05 Online:2020-02-08 Published:2020-02-26

摘要/Abstract

摘要： 亚洲玉米螟Ostrinia furnacalis是我国玉米最重要的害虫，了解亚洲玉米螟表皮形成的机制将有助于对其开展生物防治。表皮蛋白和几丁质是构成昆虫表皮的两种主要成分，因此研究两者的相互作用对于阐明昆虫表皮形成机制具有关键意义。CPR9是亚洲玉米螟体壁表皮转录组中相对丰度最高的表皮蛋白基因。序列分析发现CPR9属于CPR家族中RR-1亚家族的表皮蛋白，含有R&R结构域，可能是通过结合几丁质来发挥功能。为了研究CPR9与几丁质的相互作用，本研究利用pET-SUMO载体实现了SUMO-CPR9融合蛋白的可溶表达，并通过SUMO蛋白酶处理及两次金属螯合亲和层析纯化了CPR9，平均收率为5 mg/L。几丁质结合试验发现，CPR9对不同类型的几丁质的结合具有选择性。CPR9对α-几丁质结合能力很强，对胶体几丁质结合能力较强，但不与β-几丁质结合。脱乙酰几丁质（壳聚糖）结合试验发现，CPR9能与脱乙酰几丁质结合并使其发生团聚。圆二色光谱分析表明，CPR9在结合脱乙酰几丁质后，其二级结构中平行β折叠和β转角比例上升，反平行β折叠及无规卷曲比例下降。本研究发现了CPR家族表皮蛋白与不同类型几丁质的结合存在特异性，而且在结合过程中会发生结构变化，有助于进一步了解昆虫表皮的形成机制。

关键词: 昆虫表皮, 表皮蛋白, 几丁质结合, 结构变化

Abstract: Asian corn borer (Ostrinia furnacalis) is the most important pest of corn in China. Understanding the mechanism of cuticle formation in O. furnacalis can help the biological control of this pest. Because cuticular protein and chitin are major components of insect cuticle, investigation of the interaction between them is crucial for elucidating the mechanism of insect cuticle formation. CPR9 is the most abundant cuticular protein gene in the integument transcriptome of O. furnacalis. Sequence analysis revealed that CPR9 belongs to the cuticular protein RR-1 subfamily of CPR family and contains a R&R domain, possibly functioning by binding to chitin. To study the interaction between CPR9 and chitin, pET-SUMO vector was used to express SUMO-CPR9, the soluble fusion protein. Then CPR9 was obtained by SUMO protease treatment and two steps of metal-chelating affinity chromatography with a yield of 5 mg/L. Chitin binding assay revealed that CPR9 is selective for the binding of different types of chitin. CPR9 has strong binding ability to α-chitin and moderate binding ability to colloid chitin, but does not bind to β-chitin. The deacetylated chitin (chitosan) binding assay revealed that CPR9 can bind and induce coacervation of deacetylated chitin. As revealed by circular dichroism spectroscopy, when bound to deacetylated chitin, CPR9 showed increased proportions of parallel β-sheet and β-turn and decreased proportions of anti-parallel β-sheet and random coil. These results indicate that CPR family cuticular proteins bind different forms of chitin with specificity and experience conformational changes during binding, which provides further insights into the mechanism of insect cuticle formation.

Key words: insect cuticle, cuticular protein, chitin-binding, structural changes

中图分类号:

S435.132

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.

BU Yunfei, LIU Tian, CHEN Lei, YANG Qing. Expression, Purification of Cuticular Protein CPR9 of Ostrinia furnacalis and its Interaction with Chitin[J]. Chinese Journal Of Biological Control, 2020, 36(1): 79-86.

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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

Expression, Purification of Cuticular Protein CPR9 of Ostrinia furnacalis and its Interaction with Chitin

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