西花蓟马clip丝氨酸蛋白酶基因的鉴定与表达分析
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摘要
本研究旨在鉴定西花蓟马的clip丝氨酸蛋白酶(clip-domian serine proteases,Fo CSPs)基因,分析其被球孢白僵菌侵染后的表达模式,为研究clip丝氨酸蛋白酶在西花蓟马先天免疫反应中的作用奠定基础。共鉴定出西花蓟马9个clip丝氨酸蛋白酶基因Fo CSPs,每个Fo CSPs编码的氨基酸序列都在氮端有一个发夹(clip)区域,在碳端有一个具有催化功能的丝氨酸蛋白酶区域,催化区域有3个保守的氨基酸,即组氨酸(His),天冬氨酸(Asp)和色氨酸(Ser)。系统进化树分析表明,Fo CSP215与褐飞虱NITRY19位于同一进化支上,Fo CSP261和Fo CSP236与烟草天蛾Ms POAEO,斯氏按蚊Ag CLIP和金小蜂Nv SP16P聚为一支,其余6个Fo CSPs聚为一支,并与烟草天蛾5个Ms HPs聚成的分支汇合在一起。球孢白僵菌侵染后,成虫和若虫Fo CSPs的相对表达量均为上调表达,但程度不同。对于同一基因,在若虫体内的最高相对表达量高于成虫。总之,Fo CSPs可能参与到西花蓟马应对球孢白僵菌侵染的免疫反应中,是西花蓟马生物防治的潜在靶标。
The study is to identify the clip-domain serine protease genes in Frankliniella occidentalis(FoCSPs), to analyse the express pattern infected by the fungus Beauveria bassiana, and to investigate the function of FoCSPs in innate immunity of F. occidentalis. Nine FoCSPs genes with clip domain were identified in F. occidentalis. Each FoCSP contains a clip domain at the N-terminus and a catalytic domain at the C-terminus. The catalytic domains characterized by 3 conserved amino acids, namely His, Asp and Ser. A phylogenetic tree analysis showed that FoCSP215 and NITRY19 from Nilaparvata lugens were in the same evolutionary group. FoCSP261, FoCSP236, Manduca sexta Ms POAEO, Anopheles gambiae Ag CLIP and Manduca sexta Nv SP16 P in evolution were clustered into one clade. The remaining 6 FoCSPs clustered in one clade, which converge with another branch consisting of 5 Ms HPs in M. sexta. After the fungal infection of B. bassiana, the expression level of all FoCSPs in F. occidentalis was up-regulated, although the degrees were different. For each FoCSP, the higher expression level was monitored in infected nymph than in infected adults. In conclusion, FoCSPs might be involved in the immune response to B. bassiana, and therefore could be a potential target for the biological control of F. occidentalis.
The study is to identify the clip-domain serine protease genes in Frankliniella occidentalis(FoCSPs), to analyse the express pattern infected by the fungus Beauveria bassiana, and to investigate the function of FoCSPs in innate immunity of F. occidentalis. Nine FoCSPs genes with clip domain were identified in F. occidentalis. Each FoCSP contains a clip domain at the N-terminus and a catalytic domain at the C-terminus. The catalytic domains characterized by 3 conserved amino acids, namely His, Asp and Ser. A phylogenetic tree analysis showed that FoCSP215 and NITRY19 from Nilaparvata lugens were in the same evolutionary group. FoCSP261, FoCSP236, Manduca sexta Ms POAEO, Anopheles gambiae Ag CLIP and Manduca sexta Nv SP16 P in evolution were clustered into one clade. The remaining 6 FoCSPs clustered in one clade, which converge with another branch consisting of 5 Ms HPs in M. sexta. After the fungal infection of B. bassiana, the expression level of all FoCSPs in F. occidentalis was up-regulated, although the degrees were different. For each FoCSP, the higher expression level was monitored in infected nymph than in infected adults. In conclusion, FoCSPs might be involved in the immune response to B. bassiana, and therefore could be a potential target for the biological control of F. occidentalis.
引文
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[22]朱洋铿,方琦,胡萃,等.菜粉蝶丝氨酸蛋白酶基因Pr-SP1的克隆及其表达谱分析[J].昆虫学报,2011,54(8):859-868.
[23]Jang I H,Chosa N,Kim S H,et al.A Sp?tzle-processing enzyme required for toll signaling activation in Drosophila,innate immunity[J].Developmental Cell,2006,10(1):45-55.
[2]An C J,Jiang H B,Kanost M R.Proteolytic activation and function of the cytokine sp?tzle in innate immune response of a lepidopteran insect,Manduca sexta[J].FEBS Journal,2010,277(1):148-162.
[3]Krem M M,Cera E D.Evolution of enzyme cascades from embryonic development to blood coagulation[J].Trends in Biochemical Sciences,2002,27(2):67-74.
[4]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.
[5]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.
[6]Perona J J,Craik C S.Structural basis of substrate specificity in the serine proteases[J].Protein Science,1995,4(3):337-360.
[7]陈建平,赵萍,董照明,等.参与家蚕免疫反应的clip丝氨酸蛋白酶家族基因分析[J].蚕业科学,2012,38(2):241-248.
[8]李玉欣,元希武,韩琦,等.家蚕丝氨酸蛋白酶Bm HP14基因的克隆与表达模式分析[J].蚕业科学,2012,38(5):814-821.
[9]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.
[10]杨松等,黄复生,段建华.斯氏按蚊丝氨酸蛋白酶基因克隆及其与约氏疟原虫卵囊黑化关系的研究[J].中国人兽共患病学报,2005,21(12):1059-1063.
[11]张友军,吴青君,徐宝云,等.危险性外来入侵有害生物——西花蓟马在北京发生危害[J].植物保护,2003,29(4):58-59.
[12]Faria M,Wraight S P.Biological control of Bemisia tabaci with fungi[J].Crop Protection,2001,20(9):767-778.
[13]李娟,吴圣勇,王晓青,等.防治西花蓟马的球孢白僵菌菌株筛选及耐热性测定[J].中国生物防治学报,2015,31(6):845-852.
[14]李银平,雷仲仁,王海鸿.对西花蓟马高效的球孢白僵菌菌株筛选及产孢特性研究[J].中国生物防治学报,2013,29(2):219-226.
[15]An C J,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,2009,84(29):19716-19726.
[16]Ross J,Jiang H,Kanost M R,et al.Serine proteases and their homologs in the Drosophila melanogaster genome:an initial analysis of sequence conservation and phylogenetic relationships[J].Gene,2003,304(3):117-131.
[17]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.
[18]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.
[19]Jiang H,Wang Y,Gu Y,et al.Molecular identification of a bevy of serine proteinases in Manduca sexta hemolymph[J].Insect Biochemistry and Molecular Biology,2005,35(8):931-943.
[20]Povelones M,Bhagavatula L,Yassine H,et al.The CLIP-domain serine protease homolog SPCLIP1 regulates complement recruitment to microbial surfaces in the malaria mosquito Anopheles gambiae[J].PLo S Pathogens,2013,9(9):e1003623.
[21]Veillard F,Troxler L,Reichhart J M.Drosophila melanogaster clip-domain serine proteases:structure,function and regulation[J].Biochimie,2016,122:255-269.
[22]朱洋铿,方琦,胡萃,等.菜粉蝶丝氨酸蛋白酶基因Pr-SP1的克隆及其表达谱分析[J].昆虫学报,2011,54(8):859-868.
[23]Jang I H,Chosa N,Kim S H,et al.A Sp?tzle-processing enzyme required for toll signaling activation in Drosophila,innate immunity[J].Developmental Cell,2006,10(1):45-55.