Ligand Binding Characteristics of the Chemosensory Protein AipsCSP2 from Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae)

doi:10.16409/j.cnki.2095-039x.2021.01.015

Abstract

Abstract: To clarify the expression of AipsCSP2 gene in male and female adult tissues of Agrotis ipsilon (Hufnagel), we investigate the ligand binding characteristics of the AipsCSP2 and explore its functions. According to the pheromone gland transcriptome data, the AipsCSP2 gene was cloned and analyzed by bioinformatics and phylogenetic analysis. The expression level of the gene in different tissues of male and female adult was determined by qPCR. The recombinant protein was expressed and purified with prokaryotic expression system. Finally, the binding ability of this protein to the sex pheromone components of A. ipsilon and other Lepidoptera insects, as well as the volatiles of the main host plants were determined by fluorescence competitive binding assay. Results showed that the open reading frame (ORF) of AipsCSP2 gene was 360 bp and it encoded 119 amino acids. It has four conserved cysteines in the amino acid sequence and is a typical chemosensory protein. The tissue expression patterns showed that it was specifically high expressed in the female adult pheromone gland and male adult accessory gland, low expressed in other tissues. Fluorescent competitive binding assay results showed that AipsCSP2 has a very strong binding ability to cis-11-hexadecylaldehyde, the precursor of A. ipsilon sex pheromone cis-11-hexadecylacetate, with Ki of 1.48 μmol/L. It also has a strong binding ability to alcoholic and aldehydic sex pheromone of other Lepidoptera insects, such as tetradecanol, cis-9-hexadecaldehyde and cis-7-hexadecaldehyde, with Ki of 1.10 μmol/L, 0.63 μmol/L and 1.51 μmol/L. It has a moderate ability to the sex pheromone components of A. ipsilon cis-7-dodecanoacetate and cis-8-dodecanoacetate. The binding ability to the volatiles of the main host plants of A. ipsilon was weak or not. It is suggested that AipsCSP2 may be involved in the synthesis and recognition of sex pheromone in A. ipsilon, and play an important role in reproductive activities.

Key words: Agrotis ipsilon, chemosensory protein, AipsCSP2, tissue expression, prokaryotic expression, fluorescent competitive binding assay

CLC Number:

S433.8

RAO Fuqiang, SU Xu, LI Zibo, GENG Ting, ZHANG Yongjun, SONG Ping, GU Shaohua. Ligand Binding Characteristics of the Chemosensory Protein AipsCSP2 from Agrotis ipsilon (Hufnagel) (Lepidoptera: Noctuidae)[J]. Chinese Journal of Biological Control, 2021, 37(3): 486-494.

References

[1] 苏旭, 张永军, 耿亭, 等. 小地老虎化学感受蛋白AipsCSP8的表达及配体结合特性分析[J]. 昆虫学报, 2019, 62(12):1369-1378.
[2] 郭海琴. 小地老虎的发生与防治技术研究[J]. 种子科技, 2017, 35(9):117-118.
[3] 张行国, 贾艺凡, 温洋, 等. 粘虫、小地老虎和棉铃虫三种鳞翅目害虫上灯行为节律研究[J]. 应用昆虫学报, 2017, 54(2):190-197.
[4] 安兴奎. 绿盲蝽气味受体的鉴定、表达及功能分析[D]. 北京:中国农业科学院, 2019.
[5] 袁远, 高熹, 喜超, 等. 椰子织蛾CSP基因的克隆与序列分析[J]. 江苏农业科学, 2019, 47(3):43-46.
[6] 王桂荣, 吴孔明, 郭予元. 编码棉铃虫化学感受蛋白cDNA的克隆及序列分析[J]. 中国农业科学, 2005, 38(11):2233-2239.
[7] Angeli S, Ceron F, Scaloni A, et al. Purification, structural characterization, cloning and immunocytochemical localization of chemoreception proteins from Schistocerca gregaria[J]. European Journal of Biochemistry, 1999, 262(3):745-754.
[8] 李慧, 顾天滋, 陈昌宇, 等. 仁扇舟蛾化学感受蛋白cDNA的克隆、序列分析及时空表达[J]. 林业科学, 2018, 54(4):67-75.
[9] 李玲. 沙葱萤叶甲嗅觉相关蛋白的鉴定及功能研究[D]. 呼和浩特:内蒙古农业大学, 2019.
[10] Liu Y L, Guo H, Huang L Q, et al. Unique function of a chemosensory protein in the proboscis of two Helicoverpa species[J]. Journal of Experimental Biology, 2014, 217(10):1821-1826.
[11] Jin X, Brandazza A, Navarrini A, et al. Expression and immunolocalisation of odorant-binding and chemosensory proteins in locusts[J]. Cellular & Molecular Life Sciences:CMLS, 2005, 62(10):1156-1166.
[12] Zhang G, Chen J, Wang F, et al. Characterization and levels of expression of sensory neuron membrane proteins in the adult citrus fruit fly (Diptera:Tephritidae)[J]. Journal of Insect Science, 2018, 18(6):1-8.
[13] Hua J F, Zhang S, Cui J J, et al. Functional characterizations of one odorant binding protein and three chemosensory proteins from Apolygus lucorum (Meyer-Dur) (Hemiptera:Miridae) legs[J]. Journal of Insect Physiology, 2013, 59(7):690-696.
[14] Gong D P, Zhang H J, Zhao P, et al. Identification and expression pattern of the chemosensory protein gene family in the silkworm, Bombyx mori[J]. Insect Biochemistry and Molecular Biology, 2007, 37(3):266-277.
[15] Sabatier L, Jouanguy E, Dostert C, et al. Pherokine-2 and -3:Two Drosophila molecules related to pheromone/odor-binding proteins induced by viral and bacterial infections[J]. European Journal of Biochemistry, 2003, 270(16):3398-3407.
[16] 李竹梅. 华山松大小蠹化学感受蛋白和味觉受体基因功能研究[D]. 杨凌:西北农林科技大学, 2018.
[17] Gu S H, Wu K M, Guo Y Y, et al. Identification of genes expressed in the sex pheromone gland of the black cutworm Agrotis ipsilon with putative roles in sex pheromone biosynthesis and transport[J]. BMC Genomics, 2013, 14(1):636.
[18] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^−ΔΔC_T method[J]. Methods, 2001, 25(4):402-408.
[19] 谷少华. 小地老虎性信息素通讯的分子和细胞机制[D]. 北京:中国农业科学院, 2013.
[20] Zhou J J, Kan Y, Antoniw J, et al. Genome and EST analyses and expression of a gene family with putative functions in insect chemoreception[J]. Chemical Senses, 2006, 31(5):453-465.
[21] 张玉, 杨斌, 王桂荣. 昆虫嗅觉相关可溶性蛋白的研究进展[J]. 环境昆虫学报, 2019, 41(2):229-240.
[22] Mckenna M P, Hekmat-Scafe D S, Gaines P, et al. Putative Drosophila pheromone-binding proteins expressed in a subregion of the olfactory system[J]. Journal of Biological Chemistry, 1994, 269(23):16340-16347.
[23] 孙可可. 苹果小吉丁虫化学感受蛋白基因的克隆、表达及功能分析[D]. 杨凌:西北农林科技大学, 2018.
[24] 徐浩智, 游银伟, 张龙. 昆虫化学感受蛋白及其功能研究进展[J]. 农业生物技术学报, 2015, 23(1):118-125.
[25] Yoshizawa Y, Sato R, Tsuchihara K, et al. Ligand carrier protein genes expressed in larval chemosensory organs of Bombyx mori[J]. Insect Biochemistry and Molecular Biology, 2011, 41(8):542-562.
[26] Gong L, Luo Q, Rizwan-Ul-Haq M, et al. Cloning and characterization of three chemosensory proteins from Spodoptera exigua and effects of gene silencing on female survival and reproduction[J]. Bulletin of Entomological Research, 2012, 102(5):600-609.