Expression Pattern and Ligand Binding of Chemosensory Protein SlitCSP2 of Spodoptera litura (Lepidoptera: Noctuidae)

doi:10.16409/j.cnki.2095-039x.2022.01.018

Abstract

Abstract: The expression pattern, tissue localization and binding characteristics of nonvolatile compounds of SlitCSP2 were investigated for revealing the function of SlitCSP2 in chemical communication. The expression levels of SlitCSP2 in larval, pupal, male and female adult tissues were detected by RT-qPCR. The expression levels of SlitCSP2 protein in larval stages, and heads, antennae and legs of male adults were detected by Western blot. The distribution of SlitCSP2 in legs of male adults and larval stages were analyzed by immunohistochemistry. The binding characteristics of SlitCSP2 with 13 nonvolatile compounds were analyzed by fluorescence competition binding assay. RT-qPCR results showed that SlitCSP2 was expressed the highest in the third instar larvae, and gradually decreased from the third to the sixth instar larval stages. In adult stage, SlitCSP2 was expressed the highest in legs and thoraxes of male adults as well as in heads and wings of female adults. Moreover, the expression levels in legs, heads and thoraxes of male adults were significantly higher than those of female adults. Western blot results showed that SlitCSP2 protein was expressed higher in the third instar larvae and heads (without antennae). Immunohistochemistry results showed that SlitCSP2 was mainly distributed in heads, mouthparts and thoracic legs in larval stages. In male adults, SlitCSP2 was mainly distributed in anterior tarsus and the inside of the exoskeleton of legs. Fluorescence competitive binding experiments showed that SlitCSP2 had strong binding ability with tannin (K_i=1.77), anthocyanin (K_i=2.34) and folic acid (K_i=4.56). In this study, the expression characteristics of SlitCSP2 in larval stages and adult tissues were determined. Based on the results of immunolocalization and fluorescence competition, we speculate that SlitCSP2 plays an important role in host selection and detoxification in S. litura.

Key words: Spodoptera litura, SlitCSP2, prokaryotic expression, tissue expression, binding characteristics

CLC Number:

S188

WANG Yang, CHEN Junyuan, XU Rubing, WANG Fulian, LI Chuanren, TAN Xiaoling, DONG Xiaolin. Expression Pattern and Ligand Binding of Chemosensory Protein SlitCSP2 of Spodoptera litura (Lepidoptera: Noctuidae)[J]. Chinese Journal of Biological Control, 2022, 38(6): 1495-1507.

References

[1] Wang Q, Zhou J J, Liu J T, et al. Integrative transcriptomic and genomic analysis of odorant binding proteins and chemosensory proteins in aphids[J]. Insect Molecular Biology, 2019, 28(1): 1-22.
[2] Gu S H, Wang S Y, Zhang X Y, et al. Functional characterizations of chemosensory proteins of the alfalfa plant bug Adelphocoris lineolatus indicate their involvement in host recognition[J]. PLoS ONE, 2012, 7(8): e42871.
[3] Rogers M E, Sun M, Lerner M R, et al. Snmp-1, a novel membrane protein of olfactory neurons of the silk moth Antheraea polyphemus with homology to the CD36 family of membrane proteins[J]. Journal of Biological Chemistry, 1997, 272(23): 14792-14799.
[4] 周渊涛, 李玲, 庞保平, 等. 亚洲小车蝗触角转录组及化学感受蛋白基因表达谱分析[J]. 昆虫学报, 2019, 62(1): 21-32.
[5] 刘玉铃. 棉铃虫和烟青虫化学感受蛋白基因的克隆和表达[D]. 北京: 中国科学院大学, 2013.
[6] Briand L, Swasdipan N, Nespoulous C, et al. Characterization of a chemosensory protein (ASP3c) from honeybee (Apis mellifera L.) as a brood pheromone carrier[J]. European Journal of Biochemistry, 2002, 269(18): 4586-4596.
[7] Zhu J, Iovinella I, Dani F R, et al. Conserved chemosensory proteins in the proboscis and eyes of Lepidoptera[J]. International Journal of Biological Sciences, 2016, 12(11): 1394-1404.
[8] Bautista M A M, Bhandary B, Wijeratne A J, et al. Evidence for trade-offs in detoxification and chemosensation gene signatures in Plutella xylostella[J]. Pest Management Science, 2015, 71(3): 423-432.
[9] Xuan N, Guo X, Xie H Y, et al. Increased expression of CSP and CYP genes in adult silkworm females exposed to avermectins[J]. Insect Science, 2015, 22(2): 203-219.
[10] Liu G, Ma H, Xie H, et al. Biotype characterization, developmental profiling, insecticide response and binding property of Bemisia tabaci chemosensory proteins: role of CSP in insect defense[J]. PLoS ONE, 2016, 11(5): e0154706.
[11] 刘孝贺, 孙丽娜, 张怀江, 等. 昆虫化学感受蛋白研究进展[J]. 农学学报, 2020, 10(6): 22.
[12] 束炎南. 我国的大害虫——斜纹夜蛾[J]. 昆虫知识, 1959(3): 28-29.
[13] Armes N J, Wightman J A, Jadhav D R, et al. Status of insecticide resistance in Spodoptera litura in Andhra Pradesh, India[J]. Pesticide Science, 1997, 50(3): 240-248.
[14] 姚文辉. 斜纹夜蛾的生物学特性[J]. 华东昆虫学报, 2005, 14(2): 122-127.
[15] 章士美, 汪广. 斜纹夜蛾Spodoptera litura的初步考察[J]. 昆虫知识, 1959(3): 5-6.
[16] Subramanian S, Rabindra R J, Palaniswamy S, et al. Impact of granulovirus infection on susceptibility of Spodoptera litura to insecticides[J]. Biological Control, 2005, 33(2): 165-172.
[17] 刘保才, 王俊琪, 孙国语. 蔬菜病虫害化学防治中的 3R问题与科学使用农药[J]. 上海蔬菜, 2004(6): 68-69.
[18] 占胜利, 朱朝华, 邱庆军. 植物源农药与农产品质量安全[J]. 广西热带农业, 2008(3): 21-23.
[19] 邵仁志, 刘小安, 孙兰, 等. 中国植物源农药的研究进展[J]. 今日农药, 2017(7): 21-24.
[20] 李水清, 方宇凌, 张钟宁. 植物源昆虫拒食活性物质的研究和应用[J]. 昆虫知识, 2005, 42(5): 491-496.
[21] 刘金香, 钟国华, 谢建军, 等. 昆虫化学感受蛋白研究进展[J]. 昆虫学报, 2005, 48(3): 418-426.
[22] 张南, 陈巨龙, 仵均祥. 斜纹夜蛾半人工饲料的改进[J]. 西北农林科技大学学报(自然科学版), 2016, 44(4): 109-113.
[23] Livak K J, Schmittgen T D. Analysis of relative gene expression data using real-time quantitative PCR and the 2^-ΔΔCT method[J]. Methods, 2001, 25(4): 402-408.
[24] 赵静, 孙洋, 谭永安, 等. 甜菜夜蛾卵黄原蛋白多克隆抗体制备及其在不同发育时期蛋白表达[J]. 中国农业科学, 2017, 50(22): 4316-4324.
[25] 马菲. 舞毒蛾PBPs的免疫定位及与配体互作的关键位点[D]. 北京: 北京林业大学, 2014.
[26] 李燕华. 油料作物主要虫害发生规律[J]. 农村科技, 2005(1): 19-19.
[27] Gu S H, Wang S Y, Zhang X Y, et al. Functional characterizations of chemosensory proteins of the alfalfa plant bug Adelphocoris lineolatus indicate their involvement in host recognition[J]. PLoS ONE, 2012, 7: e42871.
[28] Ma L, Li Z Q, Bian L, et al. Identification and comparative study of chemosensory genes related to host selection by legs transcriptome analysis in the tea geometrid Ectropis obliqua[J]. PLoS ONE, 2016, 11(3): e0149591.
[29] 林美珍, 杨广, 尤民生. 3 种半翅目小型昆虫的疏水行为及其前跗节结构比较[J]. 福建农林大学学报(自然科学版), 2017, 46(2): 135-140.
[30] Dani F R, Michelucci E, Francese S, et al. Odorant-binding proteins and chemosensory proteins in pheromone detection and release in the silkmoth Bombyx mori[J]. Chemical senses, 2011, 36(4): 335-344.
[31] 张凌. 斜纹夜蛾CSP基因的克隆及功能研究[D]. 杭州: 中国计量大学, 2017.
[32] Lin X, Mao Y, Zhang L. Binding properties of four antennae-expressed chemosensory proteins (CSPs) with insecticides indicates the adaption of Spodoptera litura to environment[J]. Pesticide biochemistry and physiology, 2018, 146: 43-51.
[33] 潘娟, 商军, 黄士新. 钨酸钠-磷钼酸比色分光光度法测定饲料中的单宁[J]. 饲料工业, 2012(7): 51-55.
[34] 杨露, 谭会泽, 刘松柏, 等. 单宁的抗营养作用及其在畜禽营养中的研究进展[J]. 粮食与饲料工业, 2019(6): 53-56.
[35] 王国红, 刘兴平, 任琴, 等. 虫害松针的次生物质变化及对马尾松毛虫生长发育的影响[J]. 江西农业大学学报, 2008, 30(4): 581-585.
[36] 石萌. 儿茶素类化合物高效可食载体的开发[D]. 杭州: 浙江大学, 2016.
[37] Rathi J, Gopalakrishnan S. Insecticidal activity of aerial parts of Synedrella nodiflora Gaertn (Compositae) on Spodoptera litura (Fab.)[J]. Journal of Central European Agriculture, 2006, 7(2): 289-296.
[38] Su Q, Zhou Z, Zhang J, et al. Effect of plant secondary metabolites on common cutworm, Spodoptera litura (Lepidoptera: Noctuidae)[J]. Entomological Research, 2018, 48(1): 18-26.
[39] 黄敏燕, 李雪峰. 植物次生物质对斜纹夜蛾解毒酶活性的影响[J]. 基因组学与应用生物学, 2018, 37(8): 3495-3502.
[40] 詹忠浪, 周伟, 华娟, 等. 植物与昆虫互作中的化学响应[C]. 中国第九届植物化感作用学术研讨会论文摘要集, 2019.
[41] 夏悦昕. 亚洲玉米螟二氢叶酸还原酶基因克隆与生理功能分析[D]. 大连: 大连理工大学, 2014.