Cloning and Function Analysis of a Clip-domain Serine Protease Gene NlSCP4 in the Brown Planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)

doi:10.16409/j.cnki.2095-039x.2021.11.008

Abstract

Abstract: Clip-domain serine protease (CSP),a protein hydrolase with conserved structure and diverse functions,widely exists in different groups of insects and plays important physiological roles in insect growth,development and immunity.In this study,a CSP-encoding gene NlCSP4(GenBank accession No.OK018340) was successfully cloned and identified in the brown planthopper (BPH),Nilaparvata lugens.The full-length cDNA of NlCSP4 is 1911 bp,encoding 636 amino acids with the predicted molecular weight of 69.29 kD and isoelectric point of 9.02.NlCSP4 protein contains a predicted signal peptide consisting of 25 amino acid residues and a clip domain comprising 44 amino acid residues at the N-terminal region,and a typical Tryp_SPc domain at the C-terminus with three specific conserved catalytic sites,namely His,Asp and Ser.The phylogenetic analysis showed that NlCSP4 clustered together with other hemipteran CSPs but were quite distinct from those of lepidopterans in the neighbor-joining tree.The qRT-PCR results showed that the expression of NlCSP4 had obvious temporospatial characteristics.The transcript level of NlCSP4 in old nymphs (4^th and 5^thinstars) was significantly higher than those in the eggs and young nymphs (1^st,2^nd and 3^rd instars),and the highest expression level was observed in the female adults.NlCSP4 was expressed in the fat body,gut and ovary,with the highest expression level in the fat body of the female adults.The expression of NlCSP4 in BPH was significantly up-regulated after infection with the Gram-positive bacteria Staphylococcus aureus and the entomopathogenic fungus Metarhizium anisopliae within 72 h post injection.However,no obvious induction of NlCSP4 expression was observed when the BPH was challenged by the Gram-negative bacteria Escherichia coli in a short time (within 36 h post injection).Microinjection of NlCSP4 dsRNA could significantly inhibit the expression level of NlCSP4 in the 5^th-instar nymphs of BPH.The results of bioassay experiments showed that the survival rate and the ability to resist M.anisopliae infection in the 5^th-instar nymphs of BPH were both significantly decreased by RNAi-mediated knockdown of NlCSP4.Our preliminary results confirm that NlCSP4 plays key roles in host growth,development and immune defense,and can be used as an important potential target for RNAi-mediated control of BPH.

Key words: Nilaparvata lugens, clip-domain serine proteinase, gene cloning, gene expression, RNA interference

CLC Number:

Q966

WU Wei, CHENG Yiqing, WANG Zhengliang, YU Xiaoping. Cloning and Function Analysis of a Clip-domain Serine Protease Gene NlSCP4 in the Brown Planthopper,Nilaparvata lugens(Hemiptera:Delphacidae)[J]. Chinese Journal of Biological Control, 2022, 38(5): 1202-1212.

References

[1] Jiang H, Kanost M R. The clip-domain family of serine proteinases in arthropods[J]. Insect Biochemistry and Molecular Biology, 2000, 30(2):95-105.
[2] Veillard F, Troxler L, Reichhart J M. Drosophila melanogaster clip-domain serine proteases:Structure, function and regulation[J]. Biochimie, 2016, 122:255-269.
[3] Kanost M R, Jiang H. Clip-domain serine proteases as immune factors in insect hemolymph[J]. Current Opinion in Insect Science, 2015, 11:47-55.
[4] Chu Y, Liu Y, Shen D, et al. Serine proteases SP1 and SP13 mediate the melanization response of Asian corn borer, Ostrinia furnacalis, against entomopathogenic fungus Beauveria bassiana[J]. Journal of Iinvertebrate Pathology, 2015, 128:64-72.
[5] Yang W J, Chen C X, Yan Y, et al. Clip-domain serine protease gene (LsCLIP3) is essential for larval-pupal molting and immunity in Lasioderma serricorne[J]. Frontiers in Physiology, 2020, 10:1631.
[6] 唐琦,周倩,邱立鹏,等.丝氨酸蛋白酶在昆虫先天免疫中的功能和作用机制研究进展[J].蚕业科学, 2017, 43(3):502-508.
[7] Muta T, Hashimoto R, Miyata T, et al. Proclotting enzyme from horseshoe crab hemocytes. cDNA cloning, disulfide locations, and subcellular localization[J]. Journal of Biological Chemistry, 1990, 265(36):22426-22433.
[8] Lin H, Xia X, Yu L, et al. Genome-wide identification and expression profiling of serine proteases and homologs in the diamondback moth, Plutella xylostella(L.)[J]. BMC Genomics, 2015, 16:1054.
[9] Jang I H, Nam H J, Lee W J. CLIP-domain serine proteases in Drosophila innate immunity[J]. BMB Reports, 2008, 41(2):102-107.
[10] Mwangi S, Murungi E, Jonas M, et al. Evolutionary genomics of Glossina morsitans immune-related CLIP domain serine proteases and serine protease inhibitors[J]. Infection Genetics and Evolution, 2011, 11(4):740-745.
[11] Cao X, Gulati M, Jiang H. Serine protease-related proteins in the malaria mosquito, Anopheles gambiae[J]. Insect Biochemistry and Molecular Biology, 2017, 88:48-62.
[12] Liu H, Heng J, Wang L, et al. Identification, characterization, and expression analysis of clip-domain serine protease genes in the silkworm, Bombyx mori[J]. Developmental and Comparative Immunology, 2020, 105:103584.
[13] Cao X, He Y, Hu Y, et al. Sequence conservation, phylogenetic relationships, and expression profiles of nondigestive serine proteases and serine protease homologs in Manduca sexta[J]. Insect Biochemistry and Molecular Biology, 2015, 62:51-63.
[14] Cao X, Jiang H. Building a platform for predicting functions of serine protease-related proteins in Drosophila melanogaster and other insects[J]. Insect Biochemistry and Molecular Biology, 2018, 103:53-69.
[15] Zou Z, Lopez D L, Kanost M R, et al. Comparative analysis of serine protease-related genes in the honey bee genome:possible involvement in embryonic development and innate immunity[J]. Insect Molecular Biology, 2006, 15(5):603-614.
[16] Chen B, Kayukawa T, Jiang H, et al. DaTrypsin, a novel clip-domain serine proteinase gene up-regulated during winter and summer diapauses of the onion maggot, Delia antiqua[J]. Gene, 2005, 347(1):115-123.
[17] El Moussawi L, Nakhleh J, Kamareddine L, et al. The mosquito melanization response requires hierarchical activation of non-catalytic clip domain serine protease homologs[J]. PLoS Pathogens, 2019, 15(11):e1008194.
[18] Zhang X, Li M, El Moussawi L, et al. CLIPB10 is a terminal protease in the regulatory network that controls melanization in the African malaria mosquito Anopheles gambiae[J]. Frontiers in Cellular and Infection Microbiology, 2021, 10:585986.
[19] Liu H W, Wang L L, Meng Z, et al. A clip domain serine protease involved in moulting in the silkworm, Bombyx mori:cloning, characterization, expression patterns and functional analysis[J]. Insect Molecular Biology, 2017, 26(5):507-521.
[20] Sousa G L, Bishnoi R, Baxter R H G, et al. The CLIP-domain serine protease CLIPC9 regulates melanization downstream of SPCLIP1, CLIPA8, and CLIPA28 in the malaria vector Anopheles gambiae[J]. PLoS Pathogens, 2020, 16(10):e1008985.
[21] 赵倩倩,于洁,张吉良,等.西花蓟马clip丝氨酸蛋白酶基因的鉴定与表达分析[J].中国生物防治学报, 2017, 33(1):63-69.
[22] 于洁,王登杰,张林雅,等.参与烟粉虱免疫反应的clip丝氨酸蛋白酶基因分析[J].植物保护学报, 2016, 43(1):55-61.
[23] Ma L, Chen F, Wang W, et al. Identification of two clip domain serine proteases involved in the pea aphid's defense against bacterial and fungal infection[J]. Insect Science, 2020, 27(4):735-744.
[24] 陈春旭,许抗抗,杨洪,等.烟草甲clip丝氨酸蛋白酶基因的克隆及其对外源激素和免疫胁迫的表达响应[J].昆虫学报, 2019, 62(5):535-546.
[25] 吕进,祝增荣,娄永根,等.稻飞虱灾变和治理研究透析[J].应用昆虫学报, 2013, 50(3):565-574.
[26] Xue J, Zhou X, Zhang C X, et al. Genomes of the rice pest brown planthopper and its endosymbionts reveal complex complementary contributions for host adaptation[J]. Genome Biology, 2014, 15:521.
[27] Kumar S, Stecher G, Li M, et al. MEGA X:Molecular evolutionary genetics analysis across computing platforms[J]. Molecular Biology and Evolution, 2018, 35(6):1547-1549.
[28] 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.
[29] Tang Q Y, Zhang C X. Data processing system (DPS) software with experimental design, statistical analysis and data mining developed for use in entomological research[J]. Insect Science, 2013, 20(2):254-260.
[30] Bao Y Y, Qu L Y, Zhao D, et al. The genome-and transcriptome-wide analysis of innate immunity in the brown planthopper, Nilaparvata lugens[J]. BMC Genomics, 2013, 14:160.
[31] Bao Y Y, Qin X, Yu B, et al. Genomic insights into the serine protease gene family and expression profile analysis in the planthopper, Nilaparvata lugens[J]. BMC Genomics, 2014, 15(1):507.
[32] Wang L, Yang L, Zhou X S, et al. A clip domain serine protease stimulates melanization activation and expression of antimicrobial peptides in the chinese oak silkworm, Antheraea pernyi[J]. Journal of Asia-Pacific Entomology, 2018, 21(3):864-871.
[33] Lee K Y, Zhang R, Kim M S, et al. A zymogen form of masquerade-like serine proteinase homologue is cleaved during pro-phenoloxidase activation by Ca²⁺ in coleopteran and Tenebrio molitor larvae[J]. European Journal of Biochemistry, 2002, 269(17):4375-4383.
[34] Zhang H, Tang B, Lin Y, et al. Identification of three prophenoloxidase-activating factors (PPAFs) from an invasive beetle Octodonta nipae Maulik (Coleoptera:Chrysomelidae) and their roles in the prophenoloxidase activation[J]. Archives of Insect Biochemistry and Physiology, 2017, 96(4):e21425.
[35] An C, Ishibashi J, Ragan E J, et al. Functions of Manduca sexta hemolymph proteinases HP6 and HP8 in two innate immune pathways[J]. Journal of Biological Chemistry, 2009, 284(29):19716-19726.
[36] Jang I H, Chosa N, Kim S H, et al. A Spätzle-processing enzyme required for toll signaling activation in Drosophila innate immunity[J]. Developmental Cell, 2006, 10(1):45-55.
[37] Yang L, Lin Z, Fang Q, et al. The genomic and transcriptomic analyses of serine proteases and their homologs in an endoparasitoid, Pteromalus puparum[J]. Developmental and Comparative Immunology, 2017, 77:56-68.
[38] Liu S, Jaouannet M, Dempsey D A, et al. RNA-based technologies for insect control in plant production[J]. Biotechnology Advances, 2020, 39:10746.
[39] Wu J M, Zheng R E, Zhang R J, et al. A clip domain serine protease involved in egg production in Nilaparvata lugens:Expression patterns and RNA interference[J]. Insects, 2019, 10(11):378.
[40] Zheng R E, Ji J, Wu J, et al. PCE3 plays a role in the reproduction of male Nilaparvata lugens[J]. Insects, 2021, 12(2):114.
[41] Jin S F, Feng M G, Ying S H, et al. Evaluation of alternative rice planthopper control by the combined action of oil-formulated Metarhizium anisopliae and low-rate buprofezin[J]. Pest Management Science, 2011, 67:36-43.
[42] Tang J F, Liu X Y, Ding Y C, et al. Evaluation of Metarhizium anisopliae for rice planthopper control and its synergy with selected insecticides[J]. Crop Protection, 2019, 121:132-138.