家蚕ML家族基因的鉴定及病原诱导表达特征
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摘要
MD-2相关脂类识别蛋白(MD-2-related lipid-recognition,ML)家族是一类广泛分布于动物、植物和真菌的含有ML单一结构域的蛋白家族,在先天免疫、脂类识别及代谢过程中发挥重要作用。通过同源检索在家蚕基因组数据库中比对获得4个ML家族成员基因,包括BmNPC2、BmML-1、BmML-2和BmEr-16,克隆其cDNA序列,生物信息学分析表明家蚕ML家族蛋白具有典型的ML结构域和信号肽,并至少含有4个保守的半胱氨酸残基。系统进化分析显示BmNPC2与黑脉金斑蝶的Promoting以及烟草天蛾的MsML-1亲缘关系较近,BmML-2、BmEr-16以及BmML-1与果蝇的NPC2家族蛋白亲缘关系较近。qRT-PCR结果显示ML家族基因在家蚕各组织中均有转录,其中在脂肪体和马氏管中的表达量相对较高。将大肠埃希菌以及球孢白僵菌、黑胸败血芽孢杆菌、核型多角体病毒和家蚕微孢子虫等家蚕病原通过注射或添食家蚕5龄幼虫进行免疫诱导,qRT-PCR结果显示家蚕ML家族成员在不同病原诱导之后均有不同程度的上调表达,推测ML家族成员可能参与到宿主对入侵病原物的免疫过程。
MD-2 related lipid-recognition( ML) proteins comporise a conserved domain protein family widely distributed in animals,plants and fungi,which play important roles in innate immunity,lipid-recognition and metabolism. In this stu-dy,four ML family genes including Bm NPC2,Bm ML-1,Bm ML-2 and Bm Er-16 were obtained by homologous similarity search in the Bombyx mori genome database. Bioinformatical analysis showed that ML family proteins of B. mori had typical ML domains and signal peptides,and contained at least 4 conserved cysteine residues. Phylogenetic analysis showed that Bm NPC2 had closer relationship with Promoting protein of Danaus plexippus and Ms ML-1 of Manduca sexta,while Bm ML-2,Bm Er-16 and Bm ML-1 had closer relationship with NPC2 family protein of Drosophila. Real-time quantitative PCR results showed that the ML genes were expressed in all tissues,among which fat body and Malpighian tubule had higher expression levels. After the 5 th instar larvae inoculated or fed with Escherichia coli,Beauveria bassiana,Bacillus bombyseptieus,Bombyx mori nucleopolyhedrovirus and Nosema bombycis,real-time quantitative PCR detection showed that ML member genes of silk-worm were up-regulated differently after pathogen induction, which indicated that ML family members might play a role in the innate immunity of silkworm.
MD-2 related lipid-recognition( ML) proteins comporise a conserved domain protein family widely distributed in animals,plants and fungi,which play important roles in innate immunity,lipid-recognition and metabolism. In this stu-dy,four ML family genes including Bm NPC2,Bm ML-1,Bm ML-2 and Bm Er-16 were obtained by homologous similarity search in the Bombyx mori genome database. Bioinformatical analysis showed that ML family proteins of B. mori had typical ML domains and signal peptides,and contained at least 4 conserved cysteine residues. Phylogenetic analysis showed that Bm NPC2 had closer relationship with Promoting protein of Danaus plexippus and Ms ML-1 of Manduca sexta,while Bm ML-2,Bm Er-16 and Bm ML-1 had closer relationship with NPC2 family protein of Drosophila. Real-time quantitative PCR results showed that the ML genes were expressed in all tissues,among which fat body and Malpighian tubule had higher expression levels. After the 5 th instar larvae inoculated or fed with Escherichia coli,Beauveria bassiana,Bacillus bombyseptieus,Bombyx mori nucleopolyhedrovirus and Nosema bombycis,real-time quantitative PCR detection showed that ML member genes of silk-worm were up-regulated differently after pathogen induction, which indicated that ML family members might play a role in the innate immunity of silkworm.
引文
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[2] MIYAKE K.Innate recognition of lipopolysaccharide by CD14 and toll like receptor 4-MD-2:unique roles for MD-2[J].Int Immunopharmacol,2003,3(1):119-128
[3] YANG R B,MARK M R,Gray A,et al.Toll-like receptor-2 mediates lipopolysaccharide-induced cellular signalling[J].Nature,1998,395(6699):284-288
[4] SHIMAZU R,AKASHI S,OGATA H,et al.MD-2,a molecule that confers lipopolysaccharide responsiveness on Toll-like receptor 4[J].Exp Med,1999,189(11):1777-1782
[5] MILLER Y I,VIRIYAKOSOL S,BINDER C J,et al.Minimally modified LDL binds to CD14,induces macrophage spreading via TLR4/MD-2,and inhibits phagocytosis of apoptotic cells[J].J Biol Chem,2003,278(3):1561-1568
[6] FRIEDLAND N,LIOU H L,LOBEL P,et al.Structure of a cholesterol-binding protein deficient in Niemann-Pick type C2 disease[J].Proc Natl Acad Sci USA,2003,100(5):2512-2517
[7] SHI X Z,ZHONG X,YU X Q.Drosophila melanogaster NPC2 proteins bind bacterial cell wall components and may function in immune signal pathways[J].Insect Biochem Mol Biol,2003,42:545-556
[8] BERGER A C,VANDERFORD T H,GERNERT K M,et al.Saccharomyces cerevisiae Npc2p is a functionally conserved homologue of the human Niemann-Pick disease type C 2 protein,hNPC2[J].Eukaryot Cell,2005,4:1851-1862
[9] HUANG X,WARREN J T,BUCHANAN J A,et al.Drosophila Niemann-Pick type C-2 genes control sterol homeostasis and steroid biosynthesis:a model of human neurodegenerative disease[J].Development,2007,134(20):3733-3742
[10] ADACHI T,ISHII K,MATSUMOTO Y,et al.Niemann-Pick disease type C2 protein induces triglyceride accumulation in silkworm and mammalian cell lines[J].Biochem J,2014,459(1):137-147
[11] LI Z H,PAN G Q,MA Z G,et al.Comparative proteomic analysis of differentially expressed proteins in the Bombyx mori fat body during the microsporidia Nosema bombycis infection[J].J Invertebr Pathol,2017,149:36-43
[12] KANAYA T,KOBAYASHI J.Purification and characterization of an insect haemolymph protein promoting in vitro replication of the Bombyx mori nucleopolyhedrovirus[J].J Gen Virol,2000,81(4):1135-1141
[13] BAO Y Y,TANG X D,LV Z Y,et al.Gene expression profiling of resistant and susceptible Bombyx mori strains reveals nucleopolyhedrovirus-associated variations in host gene transcript levels[J].Genomics,2009,94(2):138-145
[14] 雷妍圆,吕利华,何余容.不同接种方式下球孢白僵菌对小菜蛾的致病力[J].植物保护,2010,36(6):142-146
[15] 李俊,张婷,张媛媛,等.39K基因对家蚕核型多角体病毒(BmNPV)复制和转录的影响[J].农业生物技术学报,2016,24(3):416-425
[16] 周成,潘国庆,万永继,等.家蚕微孢子虫N. bombycis分离纯化方法的优化[J].蚕学通讯,2002,22(1):7-9
[17] LIVAK K J,SCHMITTGENT 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] AO J,LING E,RAO X,et al.A novel ML protein from Manduca sexta may function as a key accessory protein for lipopolysaccharide signaling[J].Mol Immunol,2008,45(10):2772-2781
[19] MIYAKE S,YAMANO Y,MORISHIMA I.Promoting protein,a silkworm hemolymph protein promoting in vitro replication of nucleopolyhedrovirus,binds to β-glucans[J].Biosci Biotechnol Biochem,2005,69(10):2012-2014