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| cDNA Cloning and Prokaryotic Expression of Chemosensory Protein AzanCSP3 from the Agrilus zanthoxylumi |
| YANG Ping, GONG Xue-Fang, CHEN Di, GUO Li, WANG Yan-Lai, LV Shu-Jie, XIE Shou-An* |
| Forestry College, Northwest A & F University, Yangling 712100, China |
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Abstract Agrilus zanthoxylumi is an important trunk-boring pest on the Zanthoxylum bungeanum, which lays a foundation for understanding the mechanism of olfactory perception and studying the role of chemosensory proteins (CSPs) in its chemical sensory system. Based on the transcriptome sequencing results of the team, cloning of the cDNA of AzanCSP3; and the signal peptide, isoelectric point, molecular mass, and three-dimensional structure of the signal peptide were predicted by using online tools (Expasy; Signal P4.1; Swiss model); the homology was compared by using BLAST, the phylogenetic tree was constructed using a neighbor-joining method with MEGA 10.0 software, and the affinity between AzanCSP3 and CSPs from other 11 Coleoptera species was analyzed. The correct recombinant plasmid of prokaryotic expression vector pET28a(+) was ligated and cloned into Escherichia coli BL21 competent cells and expressed by isopropyl β-D-thiogalactoside (IPTG), the expression of the target protein was analyzed by SDS-PAGE, and using Western blot to detect whether the expressed protein was the target fusion protein. The results showed that the cDNA sequence of a chemosensory protein of A. zanthoxylumi was cloned and named as AzanCSP3. Consisted with the results of the transcriptome data, the full-length cDNA was 846 bp, contained a complete open reading frame of 396 bp, encoding 132 amino acids, the molecular weight of the mature protein was 15.038 kD, the isoelectric point was 9.16, and the signal peptide with 28 amino acids at the N-terminus was predicted. The three-dimensional structure of AzanCSP3 had 6 conserved α-helices and formed a hydrophobic binding cavity with typical characteristics of the chemosensory protein family. The amino acid sequence alignment indicated that AzanCSP3 had higher homology with other Coleoptera CSP sequences, and had the highest amino acid sequence homology with AmalCSP4, which was 87.79%, phylogenetic results indicated that AzanCSP3, AmalCSP4 and AplaCSP3 were clustered with 100% confidence. Western blot showed that AzanCSP3 was successfully expressed in E.coli. The structural characteristics of chemosensory protein AzanCSP3 in A. zanthoxylumi were defined, which would lay a theoretical foundation for the in-depth study of chemosensory protein of A. zanthoxylumi and the rule of the relationship between A. zanthoxylumi and environmental chemical information.
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Received: 17 October 2019
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Corresponding Authors:
Correspondingauthor,shouanxie@163.com
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