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Chinese Journal Of Biological Control ›› 2020, Vol. 36 ›› Issue (1): 79-86.DOI: 10.16409/j.cnki.2095-039x.2020.01.005

• RESEARCH REPORTS • Previous Articles     Next Articles

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

BU Yunfei, LIU Tian, CHEN Lei, YANG Qing   

  1. 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

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

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