Polyclonal Antibody Preparation, Eukaryotic Expression and Subcellular Localization of Frankliniella occidentalis FKBP6 Protein
WANG Xin1, ZHENG Li-Min2, ZHAO Xing-Yue3, MIAO Shu-Yue4, YE Qian2, ZHANG Yu2, SHI Xiao-Bin2, ZHANG De-Yong1,2,*, CHEN Jian-Bin2,*
1 Longping Branch, College of Biology, Hunan University, Changsha 410125, China; 2 Institute of Plant Protection, Hunan Academy of Agricultural Sciences, Changsha 410125, China; 3 College of Horticulture and Plant Protection, Inner Mongolia Agricultural University, Hohhot 010084, China; 4 Tobacco Research Institute, Henan Academy of Agricultural Sciences, Xuchang 461000, China
Abstract：Frankliniella occidentalis is an invasive pest that harms global agricultural production. Exploring the biological functions of immunophilins FK506-binding protein 6 (FKBP6) of F. occidentalis can provide a new target for the prevention and control of F. occidentalis. In this study, FKBP6 (GenBank No. XP_026292958.1) of F. occidentalis was analyzed through bioinformatics, the sequence of FKBP6 gene (GenBank No. XM_026437173.1) was cloned, expression of FKBP6 protein was induced through prokaryotic cells, and the polyclonal antibody was prepared. Then, the protein was expressed by the Bac-to-Bac baculovirus system, the polyclonal antibody and its subcellular localization were detected through Western blot and immunofluorescence (IF) labeling. The amino acid sequence alignment and phylogenetic tree analysis showed that FKBP6 proteins among 20 insect species had conserved regions, but they were only highly related among species. FKBP6 protein in Sf9 cells was expressed through baculovirus systems, and then IF and Western blot dection showed that polyclonal antibodies specifically bound to FKBP6 protein. Furthermore, subcellular localization results showed that FKBP6 was localized in the cytoplasm. This study provides molecular conditions for the study of the biological function of FKBP6.
[1] 陈建斌, 刘勇, 郑立敏, 等. 2017. 番茄斑萎病毒及其与传播介体西花蓟马互作的研究进展[J]. 贵州农业科学, 45(04): 54-58. (Chen J B, Liu Y, Zheng L M, et al.2017. Research progress on Tomato spot wilt virus and its interaction with transmission mediator western blossom thrips[J]. Guizhou Agricultural Sciences, 45(04): 54-58.) [2] 杜霞, 吴阔, 刘霞, 等. 2020. 云南省马铃薯病毒及蓟马优势种发生趋势[J]. 中国农业科学, 53(3): 551-562. (Du X, Wu K, Liu X, et al.2020. The occurrence trends of dominant species of potato viruses and thrips in Yunnan province[J]. Scientia Agricultura Sinica, 53(3): 551-562.) [3] 潘慧, 赵忠豪, 章松柏, 等. 2018. 番茄斑萎病毒核衣壳蛋白N及膜多糖蛋白Gn的多克隆抗体制备及应用[J]. 福建农业学报, 33(09): 963-968. (Pai H, Zhao Z H, Zhang S B, et al.2018. Preparation and application of polyclonal antibodies against nucleocapsid protein N and membrane polysaccharide protein Gn of Tomato spot wilt virus[J]. Journal of Fujian Agriculture, 33(09): 963-968.) [4] 张友军, 吴青君, 徐宝云, 等. 2003. 危险性外来入侵生物—西花蓟马在北京发生危害[J]. 植物保护, 29(04): 58-59. (Zhang Y J, Wu Q J, Xu B Y, et al.2003. Dangerous alien invasive organisms-West flower thrips harm in Beijing[J]. Plant Protection, 29(04): 58-59.) [5] 赵忠豪, 潘慧, 刘勇, 等. 2020. SRBSDV P8蛋白的多克隆抗体制备及其应用[J]. 杂交水稻, 35(03): 71-75. (Zhao Z H, Pai H, Liu Y, et al.2020. Preparation and application of polyclonal antibodies against Southern rice black-streaked dwarf virus P8 protein[J]. Hybrid Rice, 35(03): 71-75.) [6] Badillo-Vargas I E, Chen Y, Martin K M, et al.2019. Discovery of novel thrips vector proteins that bind to the viral attachment protein of the plant Bunyavirus Tomato spotted wilt virus[J]. Journal of Virology, 93(21): e00699-19. [7] Chatterji U, Bobardt M, Selvarajah S, et al.2009. The isomerase active site of cyclophilin A is critical for hepatitis C virus replication[J].The Journal of Biological Chemistry, 284(25): 16998-17005. [8] Ghartey-Kwansah G, Li Z, Feng R, et al.2018. Comparative analysis of FKBP family protein: Evaluation, structure, and function in mammals and Drosophila melanogaster[J]. BMC Developmental Biology, 18(1): 7. [9] Gollan P J, Bhave M, Aro E M, 2012. The FKBP families of higher plants: Exploring the structures and functions of protein interaction specialists[J]. FEBS Letters, 586(20): 3539-3547. [10] He Z, Guo J F, Reitz S R, et al.2020. A global invasion by the thrip, Frankliniella occidentalis: Current virus vector status and its management[J]. Insect Scicence, 27(4): 626-645. [11] Kasai H, Kawakami K, Yokoe H, et al.2015. Involvement of FKBP6 in Hepatitis C virus replication[J]. Scientific Report, 5(1): 16699. [12] Kaul A, Stauffer S, Berger C, et al.2009. Essential role of cyclophilin A for Hepatitis C virus replication and virus production and possible link to polyprotein cleavage kinetics[J]. PLOS Pathogens, 5(8): e1000546. [13] Kirk W D J, Terry L I.2003. The spread of the Western flower thrips Frankliniella occidentalis (Pergande)[J]. Agricultural and Forest Entomology, 5(4): 301-310. [14] Liu Z, Yang F, Robotham J M, et al.2009. Critical role of cyclophilin A and its prolyl-peptidyl isomerase activity in the structure and function of the Hepatitis C virus replication complex[J]. Journal of Virology, 83(13): 6554-6565. [15] Tamborindeguy C, Bereman M S, DeBlasio S, et al.2013. Genomic and proteomic analysis of Schizaphis graminum reveals cyclophilin proteins are involved in the transmission of Cereal yellow dwarf virus[J]. PLOS ONE, 8(8): e71620. [16] Wang X, Zhou Y, Guan J, et al.2022. FKBP39 controls the larval stage jh activity and development in drosophila melanogaster[J]. Insects, 13(4): 330. [17] Wu S Y, Tang L D, Zhang X R, et al.2018. A decade of a thrips invasion in China: Lessons learne[J]. Ecotoxicology, 27(7): 1032-1038. [18] Xiol J, Cora E, Koglgruber R, et al.2012. A role for Fkbp6 and the chaperone machinery in piRNA amplification and transposon silencing[J] Molecular Cell, 47(6): 970-979. [19] Yang F, Robotham J M, Nelson H B, et al.2008. Cyclophilin A is an essential cofactor for hepatitis C virus infection and the principal mediator of cyclosporine resistance in vitro[J] Journal of Virology, 82(11): 5269-5278.
