柞蚕空胴病病原菌基因组、侵染后柞蚕蛋白质组及柞蚕免疫Toll通路研究
发布时间：2018-07-26 12:09
【摘要】：柞蚕(Antheraea pernyi)是我国重要的资源昆虫之一,在食品、服装纺织、化妆品、医疗保健等行业具有广泛的用途。我国柞蚕茧年产量在8x104t以上,约占全球总产量的90%,具有较高的经济价值,柞蚕产业已成为蚕区主要的经济来源之一。同时,柞蚕作为典型的鳞翅目昆虫,整个幼虫期均在野外生长发育,其生物学规律及病理过程与农林业的鳞翅目害虫相似性较高,可作为模式昆虫进行研究,为害虫防控提供参考。柞蚕空胴病(Empty-gut disease)是影响柞蚕茧产量的主要病害之一,全国柞蚕产区均有分布且危害严重。然而,当下柞蚕空胴病的研究基础仍十分薄弱,有必要进行数据积累及系统研究。因此,开展柞蚕空胴病病原菌、病原与宿主互作关系及宿主免疫防御机制等方面的研究对于了解柞蚕空胴病的致病机理及开发针对性防控技术等具有重要意义,从而保障柞蚕产业的健康与可持续发展。本研究采用高通量测序技术对柞蚕空胴病病原菌进行了基因组测序及分析；采用iTRAQ技术对添食柞蚕空胴病病原菌后柞蚕与健康柞蚕血淋巴进行了比较蛋白质组学研究；通过RACE及RT-PCR技术鉴定了柞蚕Toll通路关键内源配体蛋白Spatzle基因,构建柞蚕Spatzle蛋白的原核表达载体,并制备多克隆抗体；采用半定量RT-PCR技术研究了柞蚕先天免疫Toll通路中关键基因的时空表达谱,并通过添食不同种类外源微生物,采用实时荧光定量PCR技术分析了Toll通路关键基因的表达量变化。本文研究内容将极大程度的丰富柞蚕空胴病病原菌的核苷酸数据,为从基因组层面明确其分类学地位及挖掘致病因子等功能基因奠定了基础；通过查明柞蚕空胴病病原菌侵染后柞蚕体内蛋白质变化情况,为从蛋白质层面揭示病原与宿主之间的互作奠定了基础；探讨Toll通路在柞蚕免疫过程中的作用,为阐明柞蚕先天免疫机制奠定了基础。研究结果在柞蚕空胴病发病机理的揭示、抗病品种的选育及柞蚕空胴病防控技术的开发等方面具有重要的参考价值。研究结果如下：1.柞蚕空胴病病原菌基因组大小约3.09 Mb, GC含量为38.35%,含有3 153个编码基因,平均长度为854 bp,有2916个基因被注释到NR数据库,1537个基因被注释到COG数据库,1577个基因被注释到GO数据库,1487个基因被注释到KEGG数据库,1242个基因被注释到Swiss-Prot数据库,2812个基因被注释到TrEMBL数据库,且有21个、39个和1个基因分别注释到PHI、VFDB及ARDB数据库,有2929个基因被注释到至少1个数据库,仅有224个基因(7.1%)未被注释到任何数据库,有130个分泌蛋白；共鉴定97个非编码RNA,包括65个tRNAs、19个rRNAs及13个small RNAs (sRNAs);含有108个散在重复序列(Interspered Repeat)和223个串联重复序列(Tandem Repeat);同时,基因组中含有9个基因岛(GIs)、平均长度为14058 bp,3个前噬菌体、平均长度为37 430 bp,没有规律成簇的间隔短回文重复(CRISPR);柞蚕空胴病病原菌基因组核苷酸序列已提交至NCBI,登录号：LPVT00000000;基于基因组层面构建的进化树表明柞蚕空胴病病原菌属于肠球菌,为柞蚕肠球菌Enterococcus pernyt。2.利用iTRAQ技术进行健康柞蚕及柞蚕肠球菌侵染后柞蚕的血淋巴蛋白质组学研究,共鉴定2206个柞蚕蛋白质及33个柞蚕肠球菌蛋白质。柞蚕肠球菌蛋白质功能注释结果表明,共有129个蛋白质被注释到3个GO本体中24个条目；在GO生物学过程本体中,共有49个蛋白质被注释10个GO条目；在GO细胞组分本体中,共有43个蛋白质被注释9个GO条目；在GO分子功能本体中,共有37个蛋白质被注释5个GO条目,其中,注释为结合(binding)功能的蛋白质数量最多,有17个,占45.95%；其次,为催化活性(catalytic activity)有14个,占37.84%。此外,共有35个蛋白质被注到15个COG功能分类中,28个蛋白质注释到43条代谢通路中。柞蚕蛋白质功能注释结果表明,共有7495个蛋白质被注释到3个GO本体中51个条目；在GO生物学过程本体中,共有3495个蛋白质被注释22个GO条目；在GO细胞组分本体中,共有2438个蛋白质被注释15个GO条目；在GO分子功能本体中,共有1562个蛋白质被注释14个GO条目,其中,注释为催化活性(catalytic activity)功能的蛋白质数量最多,有692个,占44.30%；其次为结合(binding)功能,有611个,占39.12%。此外,共有1445个蛋白质被注到24个COG功能分类中,1783个蛋白质注释到295条代谢通路中。蛋白质定量分析表明,柞蚕肠球菌侵染后柞蚕与健康柞蚕血淋巴蛋白质相比,差异表达蛋白质有305个,其中上调蛋白104个、下调蛋白201个；GO富集分析表明,有67个差异蛋白富集到细胞组分本体的76个GO条目,其中,显著富集于胞外区、胞外间隙及胞外组分等9个GO条目(P-Value≤0.05);119个差异蛋白富集到分子功能本体100个GO条目,其中,25个GO条目的富集结果具有显著性(P-Value≤0.05),主要具有各种酶活性调节功能,包括肽酶、内肽酶、水解酶、糖苷酶、单加氧酶、单酚单加氧酶、羧肽酶、氧化还原酶等,此外,还富集于糖胺聚糖结合、肽聚糖结合、细胞表面结合和细菌细胞表面结合等结合功能；103个差异蛋白富集到生物过程本体352个GO条目,其中,27个GO条目的富集结果具有显著性(P-Value≤0.05),主要参与宿主防御、先天免疫、免疫系统、黑色素合成及应激等相关生物学过程,此外,还富集于氨基聚糖分解、酪氨酸代谢、有机羟基化合物生物合成与代谢、次生代谢产物合成与代谢、白三烯代谢与合成、肽聚糖代谢与降解等生物学过程；Pathway富集分析表明,共有213个差异表达蛋白质富集于175个代谢通路,差异蛋白显著富集于阿米巴病、精氨酸和脯氨酸代谢、细胞粘附分子、胞外基质与受体互作、幽门螺杆菌感染中的上皮细胞信号转导、粘多糖降解、利什曼病、白细胞跨内皮迁移、溶酶体、疟疾、吞噬体、蛋白质消化与吸收等23个代谢通路(P-value≤0.05)。3.克隆到的柞蚕Spatzle基因(ApSPZ)全长1065 bp,开放阅读框(ORF)为777bp,编码258个aa,等电点(PI)为8.53,分子质量(Mw)为29.71 KDa。该蛋白为核蛋白,且属于分泌蛋白,在第22 aa和第23 aa位置之间存在信号肽位点。柞蚕Spatzle蛋白与烟草天蛾Spatzle蛋白相似性最高,达到40%,与家蚕Spatzle、果蝇Spatzle相似性分别为33.15%和13.58%。柞蚕Spatzle蛋白是Spatzle家族中SPZl类群的一员。成功构建柞蚕Spatzle基因的重组表达载体pET30a-ApSPZ,诱导表达的融合蛋白与预测理论分子量相当,大小约29 kD,融合蛋白大多以包涵体的形式存在于细胞质中。以柞蚕重组Spatzle蛋白为抗原,制备兔多克隆抗体,抗血清效价大于121 K,纯度较好,浓度为0.2 mg·ml-1,特异性较好,灵敏度较高,稀释1 000倍下仍可检测出16 ng的柞蛋Spatzle蛋白。4.在柞蚕4个发育时期中,Toll通路6个基因在卵期仅有Spatzle和MyD88有表达；在幼虫期除Toll基因未被检测到外,其他5个基因均有表达；而在蛹期均有表达,且表达量普遍较高；在成虫期,Spatzle、Cactus及dorsalA有表达。说明先天免疫Toll通路主要在柞蚕蛹期及幼虫期发挥作用,同时,Spatzle基因在所有时期均表达,说明Spatzle在柞蚕生活史中发挥重要作用。在不同组织中,Toll通路所有基因在马氏管和脂肪体中均有表达，且相对表达量均较高,说明这些基因在柞蚕先天免疫系统发挥重要的作用；仅MyD88基因在中肠中有表达,其他基因在中肠中均未见表达,说明Toll通路不是柞蚕中肠内主要的免疫机制。柞蚕Toll通路基因在柞蚕肠球菌及柞蚕微孢子虫诱导情况下表达量均发生了显著”性上调,且对柞蚕微孢子虫相对于柞蚕肠球菌存在明显的滞后,而在大肠杆菌诱导下未发生显著性变化。柞蚕Toll通路主要是针对革兰氏阳性菌和真菌的先天免疫通路,而革兰氏阴性菌不能激活Toll通路。
[Abstract]:Tussah (Antheraea pernyi) is one of the most important resource insects in China. It is widely used in food, clothing, textile, cosmetics, medical care and other industries. The annual yield of tussah cocoon in China is above 8x104t, accounting for about 90% of the total output of the world. The tussah industry has become one of the main economic sources of the silkworm area. As a typical Lepidoptera insect, silkworm grew and developed throughout the larval stage. Its biological and pathological processes were similar to that of Lepidoptera pests in agroforestry. It can be used as a model insect to study and provide reference for pest control. Empty-gut disease is one of the main diseases affecting the yield of tussah cocoon. However, the research foundation of the Antheraea pernyi empty carcass disease is still very weak. It is necessary to carry out the data accumulation and systematic research. Therefore, the research on the pathogenic bacteria of the tussah empty carcass, the relationship between the pathogen and the host and the host immune defense mechanism are important for the understanding of the pathogenesis of the tussah empty carcass disease. And the development of the targeted prevention and control technology is of great significance, thus ensuring the health and sustainable development of the tussah industry. The genome sequencing and analysis of the pathogenic bacteria of the tussah empty carcass disease were carried out by high throughput sequencing technology. The iTRAQ technique was used to compare the blood lymph of Tussah and healthy tussah after the pathogens of the tussah empty carcass disease. The key endogenous ligand protein Spatzle gene of tussah Toll pathway was identified by RACE and RT-PCR technology. The prokaryotic expression vector of tussah Spatzle protein was constructed and polyclonal antibody was prepared. The spatio-temporal expression profile of the key genes in the innate Toll pathway of tussah was studied by semi quantitative RT-PCR technology. The changes in the expression of key genes in the Toll pathway were analyzed by real time fluorescence quantitative PCR technology. The contents of this paper will enrich the nucleotide data of the pathogenic bacteria of the tussah empty carcass disease to a great extent, which lays the foundation for identifying the taxonomic status and digging out the pathogenic factors of the pathogenic factors from the genome level. The protein change of Tussah Silkworm Infected by the pathogen of tussah empty carcass disease was found, which laid the foundation for revealing the interaction between the pathogen and the host from the protein level, and the role of the Toll pathway in the immune process of the tussah silkworm, which laid the foundation for clarifying the innate immune mechanism of tussah silkworm. The results are as follows: 1. the genome size of 1. tussah empty carcass disease pathogens is about 3.09 Mb, the GC content is 38.35%, the 3153 encoding genes are contained, the average length is 854 BP, 2916 genes are annotated to the NR database and 1537 genes. Annotated to the COG database, 1577 genes were annotated to the GO database, 1487 genes were annotated to the KEGG database, 1242 genes were annotated to the Swiss-Prot database, 2812 genes were annotated to the TrEMBL database, and 21, 39 and 1 were annotated to the PHI, VFDB, and ARDB databases, and 2929 genes were annotated to at least 1. According to the library, only 224 genes (7.1%) were not annotated to any database and had 130 secretory proteins, and 97 non coded RNA were identified, including 65 tRNAs, 19 rRNAs and 13 small RNAs (sRNAs); 108 were scattered in the repeat sequence (Interspered Repeat) and 223 series repeats (Tandem Repeat); meanwhile, the genome contains 9 gene islands. GIs), with an average length of 14058 BP, 3 pre phages with an average length of 37430 BP, and no regular cluster interval short palindrome repeat (CRISPR); the genome nucleotide sequence of the pathogenic bacteria of the Antheraea pernyi empty carcass was submitted to NCBI, the login number: LPVT00000000; the phylogenetic tree based on the genome level indicated that the pathogen of the tussah empty carcass disease belonged to Enterococcus, A total of 2206 tussah proteins and 33 Antheraea pernyi Enterococcus proteins were identified by iTRAQ technique using iTRAQ technology in the haemolymph proteomics of tussah silkworm and tussah Enterococcus by iTRAQ technology. A total of 129 proteins were annotated to 3 GO copies of the protein function annotation of tussah Enterococcus. 24 entries in the body; in the biological process ontology of GO, 49 proteins were annotated with 10 GO entries; in the GO cell component body, a total of 43 proteins were annotated and 9 GO entries; in the GO molecular function body, 37 proteins were annotated and 5 GO entries, of which the number of protein binding (binding) functions was the most, There were 17, 45.95%, and 14 catalytic activity, accounting for 37.84%., and 35 proteins were injected into 15 COG functional classifications, 28 proteins were annotated to 43 metabolic pathways. The Antheraea pernyi protein functional annotation indicated that 7495 proteins were annotated to 51 entries in 3 GO bodies; and in GO birth. A total of 3495 proteins were annotated with 22 GO entries in the physical process ontology; in the GO cell component body, a total of 2438 proteins were annotated with 15 GO entries; in the GO functional body, 1562 proteins were annotated with 14 GO entries, of which the number of proteins with the function of catalytic activity (catalytic activity) was the largest and 69. 2, accounting for 44.30%, followed by binding function, 611, accounting for 39.12%., 1445 proteins were injected into 24 COG functional categories, 1783 proteins were annotated to 295 metabolic pathways. Protein quantitative analysis showed that the difference expressed protein of Tussah and healthy tussah after the infection of tussah Enterococcus 305, of which 104 proteins were up and 201 down regulated proteins; GO enrichment analysis showed that 67 differential proteins were enriched in 76 GO entries of the cell component body, of which 9 GO entries (P-Value < 0.05) were enriched in extracellular domain, extracellular space and extracellular group, and 119 differential proteins were enriched in 100 GO entries of molecular functional ontology, and 25 The enrichment results of GO were significant (P-Value < 0.05), mainly with various enzyme activity regulating functions, including peptidase, endopeptidase, hydrolase, glucosidase, monooxygenase, monooxygenase, carboxypeptidase, oxidoreductase and so on. In addition, it was enriched in glycosaminoglycan binding, peptidoglycan binding, cell surface binding and bacterial cell surface junction. The 103 differential proteins are enriched in the 352 GO entries of biological process ontology, of which 27 GO strips have significant enrichment results (P-Value < 0.05), mainly involved in the related biological processes of host defense, innate immunity, immune system, melanin synthesis and stress, in addition to aminoglycan decomposition and tyrosine generation. The biological processes of biosynthesis and metabolism of organic hydroxyl compounds, synthesis and metabolism of secondary metabolites, metabolism and synthesis of leukotrienes, metabolism and degradation of peptidoglycan, Pathway enrichment analysis showed that 213 differential proteins were enriched in 175 metabolic pathways, and differential proteins were significantly enriched in amielac, arginine and proline Metabolism, cell adhesion molecules, extracellular matrix and receptor interaction, epithelial cell signal transduction in Helicobacter pylori infection, mucopolysaccharide degradation, leishmaniasis, leucocyte transendothelial migration, lysosome, malaria, phagocyst, protein digestion and absorption, and other 23 metabolic pathways (P-value < 0.05).3. cloned in the tussah Spatzle gene (ApSPZ) full length of 1065 BP, the open reading frame (ORF) is 777bp, encoding 258 AA, the isoelectric point (PI) is 8.53, the molecular mass (Mw) is 29.71 KDa., and the protein is a nuclear protein, and it belongs to the secretory protein, and there is a signal peptide site between twenty-second AA and twenty-third AA. The highest similarity between the tussah Spatzle protein and the Spatzle protein of the tobacco moth is 40%, with the silkworm Spatzle, fruit fly E similarity is 33.15% and 13.58%. of tussah Spatzle protein is a member of SPZl group in Spatzle family. The recombinant expression vector pET30a-ApSPZ of tussah Spatzle gene is successfully constructed. The induced fusion protein is equivalent to the predicted molecular weight, about 29 kD, and most of the fusion protein exists in the cytoplasm in the form of inclusion body. Tussah is in the form of Antheraea pernyi. The recombinant Spatzle protein of the silkworm was used to prepare the rabbit polyclonal antibody. The antiserum titer was more than 121 K, the purity was better, the concentration was 0.2 mg. Ml-1, the specificity was better, the sensitivity was higher, the 16 ng of the tussah egg Spatzle protein.4. could still be detected in the 4 development period of tussah, and the 6 genes of Toll pathway were only Spatzle and MyD88 in the egg stage. The other 5 genes were expressed in the larval stage except for the Toll gene, and the expression was expressed in the pupal stage, and the expression was generally high. In the adult stage, Spatzle, Cactus and dorsalA were expressed. It indicated that the congenital immune Toll pathway played a role in the pupal stage and the larval stage of the tussah, while the Spatzle gene was expressed at all times. It shows that Spatzle plays an important role in the life history of tussah. In different tissues, all genes in the Toll pathway are expressed in martensitic tube and fat body, and the relative expression is high, indicating that these genes play an important role in the innate immune system of tussah. Only the MyD88 gene is expressed in the midgut, and the other genes are not in the midgut. The expression of Toll pathway was not the main immune mechanism in the midgut of Antheraea pernyi. The expression of tussah Toll pathway gene in the induction of tussah Enterococcus and tussah microspore were significantly up-regulated, and there was a significant lag between the tussah microspore and the tussah Enterococcus, but it did not occur significantly under the induction of Escherichia coli. Sexual variation. The Toll pathway of tussah is mainly directed against the innate immune pathway of gram positive bacteria and fungi, while gram negative bacteria can not activate the Toll pathway.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S885.1
，
本文编号：2145964
[Abstract]:Tussah (Antheraea pernyi) is one of the most important resource insects in China. It is widely used in food, clothing, textile, cosmetics, medical care and other industries. The annual yield of tussah cocoon in China is above 8x104t, accounting for about 90% of the total output of the world. The tussah industry has become one of the main economic sources of the silkworm area. As a typical Lepidoptera insect, silkworm grew and developed throughout the larval stage. Its biological and pathological processes were similar to that of Lepidoptera pests in agroforestry. It can be used as a model insect to study and provide reference for pest control. Empty-gut disease is one of the main diseases affecting the yield of tussah cocoon. However, the research foundation of the Antheraea pernyi empty carcass disease is still very weak. It is necessary to carry out the data accumulation and systematic research. Therefore, the research on the pathogenic bacteria of the tussah empty carcass, the relationship between the pathogen and the host and the host immune defense mechanism are important for the understanding of the pathogenesis of the tussah empty carcass disease. And the development of the targeted prevention and control technology is of great significance, thus ensuring the health and sustainable development of the tussah industry. The genome sequencing and analysis of the pathogenic bacteria of the tussah empty carcass disease were carried out by high throughput sequencing technology. The iTRAQ technique was used to compare the blood lymph of Tussah and healthy tussah after the pathogens of the tussah empty carcass disease. The key endogenous ligand protein Spatzle gene of tussah Toll pathway was identified by RACE and RT-PCR technology. The prokaryotic expression vector of tussah Spatzle protein was constructed and polyclonal antibody was prepared. The spatio-temporal expression profile of the key genes in the innate Toll pathway of tussah was studied by semi quantitative RT-PCR technology. The changes in the expression of key genes in the Toll pathway were analyzed by real time fluorescence quantitative PCR technology. The contents of this paper will enrich the nucleotide data of the pathogenic bacteria of the tussah empty carcass disease to a great extent, which lays the foundation for identifying the taxonomic status and digging out the pathogenic factors of the pathogenic factors from the genome level. The protein change of Tussah Silkworm Infected by the pathogen of tussah empty carcass disease was found, which laid the foundation for revealing the interaction between the pathogen and the host from the protein level, and the role of the Toll pathway in the immune process of the tussah silkworm, which laid the foundation for clarifying the innate immune mechanism of tussah silkworm. The results are as follows: 1. the genome size of 1. tussah empty carcass disease pathogens is about 3.09 Mb, the GC content is 38.35%, the 3153 encoding genes are contained, the average length is 854 BP, 2916 genes are annotated to the NR database and 1537 genes. Annotated to the COG database, 1577 genes were annotated to the GO database, 1487 genes were annotated to the KEGG database, 1242 genes were annotated to the Swiss-Prot database, 2812 genes were annotated to the TrEMBL database, and 21, 39 and 1 were annotated to the PHI, VFDB, and ARDB databases, and 2929 genes were annotated to at least 1. According to the library, only 224 genes (7.1%) were not annotated to any database and had 130 secretory proteins, and 97 non coded RNA were identified, including 65 tRNAs, 19 rRNAs and 13 small RNAs (sRNAs); 108 were scattered in the repeat sequence (Interspered Repeat) and 223 series repeats (Tandem Repeat); meanwhile, the genome contains 9 gene islands. GIs), with an average length of 14058 BP, 3 pre phages with an average length of 37430 BP, and no regular cluster interval short palindrome repeat (CRISPR); the genome nucleotide sequence of the pathogenic bacteria of the Antheraea pernyi empty carcass was submitted to NCBI, the login number: LPVT00000000; the phylogenetic tree based on the genome level indicated that the pathogen of the tussah empty carcass disease belonged to Enterococcus, A total of 2206 tussah proteins and 33 Antheraea pernyi Enterococcus proteins were identified by iTRAQ technique using iTRAQ technology in the haemolymph proteomics of tussah silkworm and tussah Enterococcus by iTRAQ technology. A total of 129 proteins were annotated to 3 GO copies of the protein function annotation of tussah Enterococcus. 24 entries in the body; in the biological process ontology of GO, 49 proteins were annotated with 10 GO entries; in the GO cell component body, a total of 43 proteins were annotated and 9 GO entries; in the GO molecular function body, 37 proteins were annotated and 5 GO entries, of which the number of protein binding (binding) functions was the most, There were 17, 45.95%, and 14 catalytic activity, accounting for 37.84%., and 35 proteins were injected into 15 COG functional classifications, 28 proteins were annotated to 43 metabolic pathways. The Antheraea pernyi protein functional annotation indicated that 7495 proteins were annotated to 51 entries in 3 GO bodies; and in GO birth. A total of 3495 proteins were annotated with 22 GO entries in the physical process ontology; in the GO cell component body, a total of 2438 proteins were annotated with 15 GO entries; in the GO functional body, 1562 proteins were annotated with 14 GO entries, of which the number of proteins with the function of catalytic activity (catalytic activity) was the largest and 69. 2, accounting for 44.30%, followed by binding function, 611, accounting for 39.12%., 1445 proteins were injected into 24 COG functional categories, 1783 proteins were annotated to 295 metabolic pathways. Protein quantitative analysis showed that the difference expressed protein of Tussah and healthy tussah after the infection of tussah Enterococcus 305, of which 104 proteins were up and 201 down regulated proteins; GO enrichment analysis showed that 67 differential proteins were enriched in 76 GO entries of the cell component body, of which 9 GO entries (P-Value < 0.05) were enriched in extracellular domain, extracellular space and extracellular group, and 119 differential proteins were enriched in 100 GO entries of molecular functional ontology, and 25 The enrichment results of GO were significant (P-Value < 0.05), mainly with various enzyme activity regulating functions, including peptidase, endopeptidase, hydrolase, glucosidase, monooxygenase, monooxygenase, carboxypeptidase, oxidoreductase and so on. In addition, it was enriched in glycosaminoglycan binding, peptidoglycan binding, cell surface binding and bacterial cell surface junction. The 103 differential proteins are enriched in the 352 GO entries of biological process ontology, of which 27 GO strips have significant enrichment results (P-Value < 0.05), mainly involved in the related biological processes of host defense, innate immunity, immune system, melanin synthesis and stress, in addition to aminoglycan decomposition and tyrosine generation. The biological processes of biosynthesis and metabolism of organic hydroxyl compounds, synthesis and metabolism of secondary metabolites, metabolism and synthesis of leukotrienes, metabolism and degradation of peptidoglycan, Pathway enrichment analysis showed that 213 differential proteins were enriched in 175 metabolic pathways, and differential proteins were significantly enriched in amielac, arginine and proline Metabolism, cell adhesion molecules, extracellular matrix and receptor interaction, epithelial cell signal transduction in Helicobacter pylori infection, mucopolysaccharide degradation, leishmaniasis, leucocyte transendothelial migration, lysosome, malaria, phagocyst, protein digestion and absorption, and other 23 metabolic pathways (P-value < 0.05).3. cloned in the tussah Spatzle gene (ApSPZ) full length of 1065 BP, the open reading frame (ORF) is 777bp, encoding 258 AA, the isoelectric point (PI) is 8.53, the molecular mass (Mw) is 29.71 KDa., and the protein is a nuclear protein, and it belongs to the secretory protein, and there is a signal peptide site between twenty-second AA and twenty-third AA. The highest similarity between the tussah Spatzle protein and the Spatzle protein of the tobacco moth is 40%, with the silkworm Spatzle, fruit fly E similarity is 33.15% and 13.58%. of tussah Spatzle protein is a member of SPZl group in Spatzle family. The recombinant expression vector pET30a-ApSPZ of tussah Spatzle gene is successfully constructed. The induced fusion protein is equivalent to the predicted molecular weight, about 29 kD, and most of the fusion protein exists in the cytoplasm in the form of inclusion body. Tussah is in the form of Antheraea pernyi. The recombinant Spatzle protein of the silkworm was used to prepare the rabbit polyclonal antibody. The antiserum titer was more than 121 K, the purity was better, the concentration was 0.2 mg. Ml-1, the specificity was better, the sensitivity was higher, the 16 ng of the tussah egg Spatzle protein.4. could still be detected in the 4 development period of tussah, and the 6 genes of Toll pathway were only Spatzle and MyD88 in the egg stage. The other 5 genes were expressed in the larval stage except for the Toll gene, and the expression was expressed in the pupal stage, and the expression was generally high. In the adult stage, Spatzle, Cactus and dorsalA were expressed. It indicated that the congenital immune Toll pathway played a role in the pupal stage and the larval stage of the tussah, while the Spatzle gene was expressed at all times. It shows that Spatzle plays an important role in the life history of tussah. In different tissues, all genes in the Toll pathway are expressed in martensitic tube and fat body, and the relative expression is high, indicating that these genes play an important role in the innate immune system of tussah. Only the MyD88 gene is expressed in the midgut, and the other genes are not in the midgut. The expression of Toll pathway was not the main immune mechanism in the midgut of Antheraea pernyi. The expression of tussah Toll pathway gene in the induction of tussah Enterococcus and tussah microspore were significantly up-regulated, and there was a significant lag between the tussah microspore and the tussah Enterococcus, but it did not occur significantly under the induction of Escherichia coli. Sexual variation. The Toll pathway of tussah is mainly directed against the innate immune pathway of gram positive bacteria and fungi, while gram negative bacteria can not activate the Toll pathway.
【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S885.1
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本文编号：2145964
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