【摘要】：柞蠶(Antheraea pernyi)是我國(guó)重要的資源昆蟲(chóng)之一,在食品、服裝紡織、化妝品、醫(yī)療保健等行業(yè)具有廣泛的用途。我國(guó)柞蠶繭年產(chǎn)量在8x104t以上,約占全球總產(chǎn)量的90%,具有較高的經(jīng)濟(jì)價(jià)值,柞蠶產(chǎn)業(yè)已成為蠶區(qū)主要的經(jīng)濟(jì)來(lái)源之一。同時(shí),柞蠶作為典型的鱗翅目昆蟲(chóng),整個(gè)幼蟲(chóng)期均在野外生長(zhǎng)發(fā)育,其生物學(xué)規(guī)律及病理過(guò)程與農(nóng)林業(yè)的鱗翅目害蟲(chóng)相似性較高,可作為模式昆蟲(chóng)進(jìn)行研究,為害蟲(chóng)防控提供參考。柞蠶空胴病(Empty-gut disease)是影響柞蠶繭產(chǎn)量的主要病害之一,全國(guó)柞蠶產(chǎn)區(qū)均有分布且危害嚴(yán)重。然而,當(dāng)下柞蠶空胴病的研究基礎(chǔ)仍十分薄弱,有必要進(jìn)行數(shù)據(jù)積累及系統(tǒng)研究。因此,開(kāi)展柞蠶空胴病病原菌、病原與宿主互作關(guān)系及宿主免疫防御機(jī)制等方面的研究對(duì)于了解柞蠶空胴病的致病機(jī)理及開(kāi)發(fā)針對(duì)性防控技術(shù)等具有重要意義,從而保障柞蠶產(chǎn)業(yè)的健康與可持續(xù)發(fā)展。本研究采用高通量測(cè)序技術(shù)對(duì)柞蠶空胴病病原菌進(jìn)行了基因組測(cè)序及分析；采用iTRAQ技術(shù)對(duì)添食柞蠶空胴病病原菌后柞蠶與健康柞蠶血淋巴進(jìn)行了比較蛋白質(zhì)組學(xué)研究；通過(guò)RACE及RT-PCR技術(shù)鑒定了柞蠶Toll通路關(guān)鍵內(nèi)源配體蛋白Spatzle基因,構(gòu)建柞蠶Spatzle蛋白的原核表達(dá)載體,并制備多克隆抗體；采用半定量RT-PCR技術(shù)研究了柞蠶先天免疫Toll通路中關(guān)鍵基因的時(shí)空表達(dá)譜,并通過(guò)添食不同種類外源微生物,采用實(shí)時(shí)熒光定量PCR技術(shù)分析了Toll通路關(guān)鍵基因的表達(dá)量變化。本文研究?jī)?nèi)容將極大程度的豐富柞蠶空胴病病原菌的核苷酸數(shù)據(jù),為從基因組層面明確其分類學(xué)地位及挖掘致病因子等功能基因奠定了基礎(chǔ)；通過(guò)查明柞蠶空胴病病原菌侵染后柞蠶體內(nèi)蛋白質(zhì)變化情況,為從蛋白質(zhì)層面揭示病原與宿主之間的互作奠定了基礎(chǔ)；探討Toll通路在柞蠶免疫過(guò)程中的作用,為闡明柞蠶先天免疫機(jī)制奠定了基礎(chǔ)。研究結(jié)果在柞蠶空胴病發(fā)病機(jī)理的揭示、抗病品種的選育及柞蠶空胴病防控技術(shù)的開(kāi)發(fā)等方面具有重要的參考價(jià)值。研究結(jié)果如下：1.柞蠶空胴病病原菌基因組大小約3.09 Mb, GC含量為38.35%,含有3 153個(gè)編碼基因,平均長(zhǎng)度為854 bp,有2916個(gè)基因被注釋到NR數(shù)據(jù)庫(kù),1537個(gè)基因被注釋到COG數(shù)據(jù)庫(kù),1577個(gè)基因被注釋到GO數(shù)據(jù)庫(kù),1487個(gè)基因被注釋到KEGG數(shù)據(jù)庫(kù),1242個(gè)基因被注釋到Swiss-Prot數(shù)據(jù)庫(kù),2812個(gè)基因被注釋到TrEMBL數(shù)據(jù)庫(kù),且有21個(gè)、39個(gè)和1個(gè)基因分別注釋到PHI、VFDB及ARDB數(shù)據(jù)庫(kù),有2929個(gè)基因被注釋到至少1個(gè)數(shù)據(jù)庫(kù),僅有224個(gè)基因(7.1%)未被注釋到任何數(shù)據(jù)庫(kù),有130個(gè)分泌蛋白；共鑒定97個(gè)非編碼RNA,包括65個(gè)tRNAs、19個(gè)rRNAs及13個(gè)small RNAs (sRNAs);含有108個(gè)散在重復(fù)序列(Interspered Repeat)和223個(gè)串聯(lián)重復(fù)序列(Tandem Repeat);同時(shí),基因組中含有9個(gè)基因島(GIs)、平均長(zhǎng)度為14058 bp,3個(gè)前噬菌體、平均長(zhǎng)度為37 430 bp,沒(méi)有規(guī)律成簇的間隔短回文重復(fù)(CRISPR);柞蠶空胴病病原菌基因組核苷酸序列已提交至NCBI,登錄號(hào)：LPVT00000000;基于基因組層面構(gòu)建的進(jìn)化樹(shù)表明柞蠶空胴病病原菌屬于腸球菌,為柞蠶腸球菌Enterococcus pernyt。2.利用iTRAQ技術(shù)進(jìn)行健康柞蠶及柞蠶腸球菌侵染后柞蠶的血淋巴蛋白質(zhì)組學(xué)研究,共鑒定2206個(gè)柞蠶蛋白質(zhì)及33個(gè)柞蠶腸球菌蛋白質(zhì)。柞蠶腸球菌蛋白質(zhì)功能注釋結(jié)果表明,共有129個(gè)蛋白質(zhì)被注釋到3個(gè)GO本體中24個(gè)條目；在GO生物學(xué)過(guò)程本體中,共有49個(gè)蛋白質(zhì)被注釋10個(gè)GO條目；在GO細(xì)胞組分本體中,共有43個(gè)蛋白質(zhì)被注釋9個(gè)GO條目；在GO分子功能本體中,共有37個(gè)蛋白質(zhì)被注釋5個(gè)GO條目,其中,注釋為結(jié)合(binding)功能的蛋白質(zhì)數(shù)量最多,有17個(gè),占45.95%；其次,為催化活性(catalytic activity)有14個(gè),占37.84%。此外,共有35個(gè)蛋白質(zhì)被注到15個(gè)COG功能分類中,28個(gè)蛋白質(zhì)注釋到43條代謝通路中。柞蠶蛋白質(zhì)功能注釋結(jié)果表明,共有7495個(gè)蛋白質(zhì)被注釋到3個(gè)GO本體中51個(gè)條目；在GO生物學(xué)過(guò)程本體中,共有3495個(gè)蛋白質(zhì)被注釋22個(gè)GO條目；在GO細(xì)胞組分本體中,共有2438個(gè)蛋白質(zhì)被注釋15個(gè)GO條目；在GO分子功能本體中,共有1562個(gè)蛋白質(zhì)被注釋14個(gè)GO條目,其中,注釋為催化活性(catalytic activity)功能的蛋白質(zhì)數(shù)量最多,有692個(gè),占44.30%；其次為結(jié)合(binding)功能,有611個(gè),占39.12%。此外,共有1445個(gè)蛋白質(zhì)被注到24個(gè)COG功能分類中,1783個(gè)蛋白質(zhì)注釋到295條代謝通路中。蛋白質(zhì)定量分析表明,柞蠶腸球菌侵染后柞蠶與健康柞蠶血淋巴蛋白質(zhì)相比,差異表達(dá)蛋白質(zhì)有305個(gè),其中上調(diào)蛋白104個(gè)、下調(diào)蛋白201個(gè)；GO富集分析表明,有67個(gè)差異蛋白富集到細(xì)胞組分本體的76個(gè)GO條目,其中,顯著富集于胞外區(qū)、胞外間隙及胞外組分等9個(gè)GO條目(P-Value≤0.05);119個(gè)差異蛋白富集到分子功能本體100個(gè)GO條目,其中,25個(gè)GO條目的富集結(jié)果具有顯著性(P-Value≤0.05),主要具有各種酶活性調(diào)節(jié)功能,包括肽酶、內(nèi)肽酶、水解酶、糖苷酶、單加氧酶、單酚單加氧酶、羧肽酶、氧化還原酶等,此外,還富集于糖胺聚糖結(jié)合、肽聚糖結(jié)合、細(xì)胞表面結(jié)合和細(xì)菌細(xì)胞表面結(jié)合等結(jié)合功能；103個(gè)差異蛋白富集到生物過(guò)程本體352個(gè)GO條目,其中,27個(gè)GO條目的富集結(jié)果具有顯著性(P-Value≤0.05),主要參與宿主防御、先天免疫、免疫系統(tǒng)、黑色素合成及應(yīng)激等相關(guān)生物學(xué)過(guò)程,此外,還富集于氨基聚糖分解、酪氨酸代謝、有機(jī)羥基化合物生物合成與代謝、次生代謝產(chǎn)物合成與代謝、白三烯代謝與合成、肽聚糖代謝與降解等生物學(xué)過(guò)程；Pathway富集分析表明,共有213個(gè)差異表達(dá)蛋白質(zhì)富集于175個(gè)代謝通路,差異蛋白顯著富集于阿米巴病、精氨酸和脯氨酸代謝、細(xì)胞粘附分子、胞外基質(zhì)與受體互作、幽門螺桿菌感染中的上皮細(xì)胞信號(hào)轉(zhuǎn)導(dǎo)、粘多糖降解、利什曼病、白細(xì)胞跨內(nèi)皮遷移、溶酶體、瘧疾、吞噬體、蛋白質(zhì)消化與吸收等23個(gè)代謝通路(P-value≤0.05)。3.克隆到的柞蠶Spatzle基因(ApSPZ)全長(zhǎng)1065 bp,開(kāi)放閱讀框(ORF)為777bp,編碼258個(gè)aa,等電點(diǎn)(PI)為8.53,分子質(zhì)量(Mw)為29.71 KDa。該蛋白為核蛋白,且屬于分泌蛋白,在第22 aa和第23 aa位置之間存在信號(hào)肽位點(diǎn)。柞蠶Spatzle蛋白與煙草天蛾Spatzle蛋白相似性最高,達(dá)到40%,與家蠶Spatzle、果蠅Spatzle相似性分別為33.15%和13.58%。柞蠶Spatzle蛋白是Spatzle家族中SPZl類群的一員。成功構(gòu)建柞蠶Spatzle基因的重組表達(dá)載體pET30a-ApSPZ,誘導(dǎo)表達(dá)的融合蛋白與預(yù)測(cè)理論分子量相當(dāng),大小約29 kD,融合蛋白大多以包涵體的形式存在于細(xì)胞質(zhì)中。以柞蠶重組Spatzle蛋白為抗原,制備兔多克隆抗體,抗血清效價(jià)大于121 K,純度較好,濃度為0.2 mg·ml-1,特異性較好,靈敏度較高,稀釋1 000倍下仍可檢測(cè)出16 ng的柞蛋Spatzle蛋白。4.在柞蠶4個(gè)發(fā)育時(shí)期中,Toll通路6個(gè)基因在卵期僅有Spatzle和MyD88有表達(dá)；在幼蟲(chóng)期除Toll基因未被檢測(cè)到外,其他5個(gè)基因均有表達(dá)；而在蛹期均有表達(dá),且表達(dá)量普遍較高；在成蟲(chóng)期,Spatzle、Cactus及dorsalA有表達(dá)。說(shuō)明先天免疫Toll通路主要在柞蠶蛹期及幼蟲(chóng)期發(fā)揮作用,同時(shí),Spatzle基因在所有時(shí)期均表達(dá),說(shuō)明Spatzle在柞蠶生活史中發(fā)揮重要作用。在不同組織中,Toll通路所有基因在馬氏管和脂肪體中均有表達(dá)，且相對(duì)表達(dá)量均較高,說(shuō)明這些基因在柞蠶先天免疫系統(tǒng)發(fā)揮重要的作用；僅MyD88基因在中腸中有表達(dá),其他基因在中腸中均未見(jiàn)表達(dá),說(shuō)明Toll通路不是柞蠶中腸內(nèi)主要的免疫機(jī)制。柞蠶Toll通路基因在柞蠶腸球菌及柞蠶微孢子蟲(chóng)誘導(dǎo)情況下表達(dá)量均發(fā)生了顯著”性上調(diào),且對(duì)柞蠶微孢子蟲(chóng)相對(duì)于柞蠶腸球菌存在明顯的滯后,而在大腸桿菌誘導(dǎo)下未發(fā)生顯著性變化。柞蠶Toll通路主要是針對(duì)革蘭氏陽(yáng)性菌和真菌的先天免疫通路,而革蘭氏陰性菌不能激活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.
【學(xué)位授予單位】：沈陽(yáng)農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S885.1
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本文編號(hào)：2145964