小地老虎化学感受蛋白AipsCSP2配体结合特性分析

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

中国生物防治学报 ›› 2021, Vol. 37 ›› Issue (3): 486-494.DOI: 10.16409/j.cnki.2095-039x.2021.01.015

小地老虎化学感受蛋白AipsCSP2配体结合特性分析

饶福强^1,2, 苏旭^1,2, 李仔博², 耿亭³, 张永军², 宋萍¹, 谷少华⁴

1. 河北农业大学植物保护学院, 保定 071000;
2. 中国农业科学院植物保护研究所/植物病虫害生物学国家重点实验室, 北京 100193;
3. 农业农村部廊坊作物有害生物科学观测实验站, 廊坊 065000;
4. 中国农业大学昆虫学系, 北京 100193

收稿日期:2020-05-27 出版日期:2021-06-08 发布日期:2021-02-23
通讯作者: 宋萍,副教授,E-mail:songpingbaoding@126.com;谷少华,副教授,E-mail:gushaohua@cau.edu.cn
作者简介:饶福强,硕士研究生,E-mail:raofuqiang@foxmail.com。
基金资助:
国家自然科学基金（31772164，31972338，31772176）；国家重点研发计划（2019YFD0300103）

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

RAO Fuqiang^1,2, SU Xu^1,2, LI Zibo², GENG Ting³, ZHANG Yongjun², SONG Ping¹, GU Shaohua⁴

1. College of Plant Protection, Agricultural University of Hebei, Baoding 071000, China;
2. State Key Laboratory for Biology of Plant Diseases and Insect Pests/Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
3. Langfang Experimental Station of Crop Pests Science, MOA, Langfang 065000, China;
4. College of Plant Protection, China Agricultural University, Beijing 100193, China

Received:2020-05-27 Online:2021-06-08 Published:2021-02-23

摘要/Abstract

摘要： 为了明确小地老虎化学感受蛋白基因AipsCSP2在雌、雄成虫各组织中的表达情况，解析AipsCSP2蛋白的配体结合特性并探讨其功能。本文基于小地老虎性腺转录组数据，利用PCR技术克隆AipsCSP2基因，并进行生物信息学和系统进化分析；采用qPCR技术测定该基因在小地老虎雌、雄成虫不同组织中的表达水平；利用原核表达技术体外表达并纯化AipsCSP2蛋白；最后采用荧光竞争性结合试验测定该蛋白与小地老虎和其他鳞翅目昆虫性信息素组分以及主要寄主植物挥发物的结合能力。结果显示AipsCSP2基因开放阅读框（open reading frame，ORF）长度为360 bp，编码119个氨基酸，氨基酸序列中具有4个保守半胱氨酸位点，是典型的化学感受蛋白；组织表达谱结果显示AipsCSP2基因在雌成虫性腺和雄成虫附腺中特异高表达，在其他组织中表达量较低；荧光竞争性结合试验结果表明，AipsCSP2蛋白和小地老虎性信息素组分顺-11-十六碳乙酸酯的合成前体顺-11-十六碳醛有着极其强的结合能力，Ki值为1.48 μmol/L，此外AipsCSP2蛋白和其他鳞翅目昆虫的醇类和醛类性信息素如十四烷醇、顺-9-十六碳醛、顺-7-十六碳醛也有着极其强的结合能力，Ki值分别为1.10 μmol/L、0.63 μmol/L和1.51 μmol/L，和小地老虎性信息素组分顺-7-十二碳乙酸酯、顺-8-十二碳乙酸酯的结合能力为中等，与小地老虎主要寄主植物挥发物结合能力较弱或不结合。推测AipsCSP2蛋白可能主要参与了小地老虎性信息素的合成与识别过程，并在生殖活动中起着重要的作用。

关键词: 小地老虎, 化学感受蛋白, AipsCSP2, 组织表达分析, 原核表达, 荧光竞争性结合试验

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

中图分类号:

S433.8

饶福强, 苏旭, 李仔博, 耿亭, 张永军, 宋萍, 谷少华. 小地老虎化学感受蛋白AipsCSP2配体结合特性分析[J]. 中国生物防治学报, 2021, 37(3): 486-494.

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.

参考文献

[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.

小地老虎化学感受蛋白AipsCSP2配体结合特性分析

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

PDF

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 5

编辑推荐

Metrics

本文评价

联系我们

[1]	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.
[2]	井维霞, 黄欣蒸, 刘丹凤, 张强, 寇俊凤, 曲爱军, 张永军, 郭予元. 棉花转甲基酶基因GhBSMT1的克隆、原核表达及表达谱分析[J]. 中国生物防治学报, 2017, 33(4): 463-471.
[3]	刘晓垒1,2 , 王品舒2 , 束长龙2 , 王秀丽1 , 郝再彬1 , 张永军2 , 宋福平2 . 四株对小地老虎有活性的苏云金芽胞杆菌的毒力评价[J]. , 2012, 28(2): 198-204.
[4]	钟玉林1 ; 刘长海2 ; 郑哲民3 . 人工移植索线虫怀卵雌虫防治玉米害虫[J]. , 2001, 17(2): 57-59.
[5]	陈国生1 ; 汪义慰1 ; 李国强1 ; 橝传发2,3 ; 陈炳阳2,3 ; 金扬生2,3 ; 李汪兆2,3 . 六索线虫防治小地老虎的小区试验[J]. , 1989, 5(3): 125-126.