柞蚕微孢子虫检测及感染柞蚕中肠转录组及蛋白质组学研究
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摘要
柞蚕（Antheraea pernyi Guérin-Méneville，1855）为鳞翅目大蚕蛾科的泌丝昆虫，是中国特有的生物资源，我国年产柞蚕茧约7×10~4t，占世界野蚕丝总产量的90%以上。柞蚕微孢子虫病是柞蚕生产上主要病害之一，致病病原物为柞蚕微孢子虫（Nosemapernyi），该病在我国柞蚕产区均有分布，近年来由于柞蚕微孢子虫病的发生及蔓延给柞蚕生产带来了严重的经济损失，已成为严重制约柞蚕产业发展的重要因素之一。因此，开展柞蚕微孢子虫病的侵染机制、病害的诊断检测及防治技术等研究对于保障柞蚕产业的健康发展具有重要意义。
本研究利用Percoll密度梯度离心技术分离纯化了柞蚕微孢子虫，通过感染微孢子虫的柞蚕幼虫血淋巴蛋白质SDS-PAGE电泳及质谱分析鉴定差异蛋白条带，分别采用RNA-Seq技术及iTRAQ技术对感染柞蚕微孢子虫的柞蚕幼虫中肠组织的转录组及定量蛋白质组学研究，分析了健康及患病样品的差异基因及差异蛋白，以期从柞蚕微孢子虫与柞蚕互作方面探讨柞蚕微孢子虫病的发病机制及微孢子虫侵染过程中的关键基因与蛋白，并进行柞蚕微孢子虫病检测技术研究。研究结果如下：
1．明确了柞蚕微孢子虫孢子的分离纯化技术，采用差速离心法进行柞蚕微孢子虫孢子的粗提，进而利用Percoll不连续密度梯度（Percoll浓度分别为25%、50%、75%、100%），15000r·min~(-1)离心30min，纯化效果可达到孢子纯净度95%以上。
2．分别采用间接竞争ELISA检测法、胶体金免疫层析检测法、PCR技术3种方法进行了柞蚕微孢子虫病检测研究。（1）PCR检测技术：筛选了适合柞蚕微孢子虫及柞蚕基因组DNA的提取方法—斑迹抽提法，针对微孢子虫SSU rRNA序列设计的3对引物均具有较好的柞蚕微孢子虫检测特异性，并选择引物N1/N2验证了检测灵敏度，最低检测量仅需要柞蚕微孢子虫DNA0.47ng，灵敏性较高。（2）间接竞争ELISA检测法：制备柞蚕微孢子虫多克隆抗体，效价为1:10~4、浓度为3mg·mL~(-1)，该方法检测灵敏度为1.6×105个·mL~(-1)孢子。（3）胶体金检测法：采用双抗夹心法与竞争法2种方法制备的胶体金试纸条检测时间均为10min，检测灵敏度为0.8×107个·mL孢子，且与柞蚕血淋巴无交叉反应，特异性较强，其中，双抗夹心法试纸条颜色明显、清晰、易于判定，更具有实用价值。
3．柞蚕5龄雌幼虫感染微孢子虫96h和144h后血淋巴蛋白质分别出现分子质量约28kD（AP28）和44kD（AP44）的2条蛋白条带相对于对照组染色加深的现象，对2个蛋白条带质谱分析共鉴定117个不重复蛋白，其中2个样品同时鉴定蛋白12个，AP28单独鉴定蛋白52个，AP44单独鉴定蛋白53个。AP28和AP44鉴定蛋白中参与到柞蚕免疫系统及刺激应答生物过程的无重复蛋白共有29个，其中AP28中包括：热激蛋白、泛素样蛋白、泛素结合酶E2、保幼激素环氧水解酶、微管结合蛋白、溶菌酶、ADP-核糖基化因子、防御蛋白、肽聚糖识别蛋白等15个；AP44中包括：蛋白酶、DRK、酚氧化酶原、真核翻译起始因子、类免疫球蛋白等10个；二者共有：热激蛋白21.4、抗菌肽等4个。
4．采用RNA-Seq技术研究了健康（SM1）和患柞蚕微孢子虫病（SM2）柞蚕中肠组织转录组，并对差异表达基因进行了统计分析。SM1和SM2样本得到的重叠群Contig数分别为56211和60126，平均长度为280nt和256nt，Unigene的数量分别为27496和30801，平均长度为485nt和406nt，将来自同一个物种的SM1和SM2的Unigene进行合并去冗余后得到所有Unigene的量为28000，平均长度为564nt，N50为722nt。对去冗余后的所有Unigene功能注释，16056个Unigenes注释到NCBI nr数据库；与COG数据库比对后共注释到11351个Unigenes，涉及25种功能；GO分析表明，21303个Unigenes被注释到50个GO条目中，其中有6947个基因与细胞组分相关，4081个基因发挥分子功能，10275个基因参与不同的生物学过程；10351个Unigenes共注释到242个代谢通路，其中代谢类通路最多，共1707个、占总量的16.49%。
按照FDR≤0.001且|log2Ratio|≥1条件，对SM2相对于SM1表达差异的Unigene进行筛选，得到差异表达基因8515个，其中上调基因为7150个，下调基因为1365个。差异表达基因的GO分析显示共有5704个DEGs注释到42个GO条目。DEGs的GO-CellularComponent显著富集分析表明，共有624个DEGs富集到细胞组成154个条目中，其中142个DEGs显著富集于4个条目（Q-value≤0.05），包括星状体、细胞骨架、纺锤体、细胞骨架组分；DEGs的GO-MolecularFunction显著富集分析显示，共有1047个差异基因富集于223个条目，分子功能富集于酶调节活性及结合功能，其中106个DEGs显著富集于6个条目（Q-value≤0.05），包括内肽酶抑制因子活性、内肽酶调节因子活性、肽酶抑制因子活性、肽酶调节因子活性、酶抑制因子活性、细胞骨架蛋白结合；DEGs的GO-BiologicalProcess富集分析显示，共有819个差异基因富集于873个条目，209个差异基因显著富集于11个生物过程条目（Q-value≤0.05），分别参与细胞生长调控、轴突导向、蛋白复合体亚基结构、神经元分化过程中细胞形态、大分子复合物解体、胞内大分子复合物解体、蛋白质复合物解体、胞内蛋白质复合物解体、微管解聚、微管聚合或解聚、蛋白解聚。DEGs的Pathway富集分析表明，共有3092个DEGs富集于231个代谢通路，其中2390个DGEs显著富集于34个代谢通路（Q-value≤0.05），主要参与到营养物质代谢与吸收、神经系统互作、病害及与免疫系统相关等通路。
5．以柞蚕转录组数据作为参考序列，利用iTRAQ技术对健康（M1）及患柞蚕微孢子虫病（M2）柞蚕中肠组织蛋白质进行了定量蛋白质组学研究，共鉴定蛋白质1987个。鉴定蛋白被注释到46个GO条目，注释到细胞组分本体中共有3642个蛋白，1779个蛋白发挥分子功能，包括11种功能分类，共有5003个蛋白质参与到25个生物学过程，其中与柞蚕病理及抗病生物过程相关的蛋白包括死亡63个、免疫系统过程38个、代谢过程800个、刺激应答289个；1474个柞蚕蛋白被COG功能分为23类，数量最多的是预测仅有全局功能，并有7个蛋白注释为防御机制功能，25个未知功能蛋白，同时发现功能序列在蛋白质组及转录组水平方面表达具有明显的差异；通过对柞蚕蛋白质Pathway分析发现共有1505条蛋白注释到241个代谢通路中，其代谢通路的蛋白最多，与转录组结果相符。
通过SM2相对于SM1蛋白表达相对定量比较，共发者表达差异蛋白203个，其中上调蛋白112个，下调蛋白91个。差异蛋白大量被注释到鳞翅目昆虫，其中上调蛋白52.68%、下调蛋白68.13%。差异蛋白GO富集分析表明，在细胞组分本体共有94个差异蛋白被富集到细胞组成73个条目，显著富集于胞外基质组分、胞外基质、胞外区组分、蛋白胞外基质、核糖核蛋白复合体、基膜、胞外区、肌节、横纹肌细丝、肌原纤维、连接丝体组分及连接丝体等组分（P-value≤0.05）；在分子功能本体共有118个差异蛋白被富集到67个分子功能条目，显著富集于结构分子活性、模式结合、多糖结合、糖类结合、糖胺聚糖结合、连接酶、碳碳键形成等分子功能条目（P-value≤0.05），并首次在柞蚕上发现2个具有糖胺聚糖等结合功能蛋白，蛋白ID分别为Unigene14322_All及Unigene3787_All；在生物学过程本体共有98个差异蛋白被富集到256个生物学过程条目，显著富集于分支链家族氨基酸代谢过程、基因表达、剪接体组装、大分子代谢过程负调控、戊糖代谢过程、肌肉系统过程及核小体组构等生物过程中（P-value≤0.05）。差异蛋白Pathway富集分析表明，共有154个差异蛋白富集于136个代谢通路，显著富集于胞外基质与受体互作通路、代谢通路、半乳糖代谢、甘油磷酸脂代谢、半胱氨酸和蛋氨酸代谢、α-亚麻酸代谢、赖氨酸生物合成、糖类消化和吸收、核糖体、淀粉和蔗糖代谢、剪接体、疟疾、扩张性及肥厚性心肌病等14个代谢通路，其中在胞外基质与受体间互作通路中，发现蛋白ID为Unigene21115_All、Unigene4217_All、Unigene22755_All、Unigene14322_All、 Unigene842_All、 Unigene7641_All、 Unigene10826_All、Unigene14191_All的发生了上调，Unigene3787_All发生了下调，这其中包含2个糖胺聚糖结合功能蛋白，即Unigene14322_All（2.42倍）及Unigene3787_All（0.387倍），表明柞蚕细胞胞外区是柞蚕微孢子虫侵染宿主过程中重要的区域。通过对差异蛋白及与其对应转录本的关联及聚类分析，发现差异蛋白及与其对应的转录本之间关联性为0.2404，聚类结果显示共有44个Unigenes反向相关，43个Unigenes正向相关。
Candidate: Jiang Yiren Supervisor: Prof. Duan Yuxi Prof. Qin Li
The Chinese oak silkworm (Antheraea pernyiGuérin-Méneville,1855) which belongs toLepidoptera: Saturniidae, is the most well known wild silkmoths for insect food and silkproduction. In China, annual output of tussah cocoon is about7×10~4t, the percentage of thatwith total output of wild silk in world is nearly90%, and the income from tussah rearing hasbecome the main economic source in most sericultural areas. But owing to great harm of themicrosporidiosis, some people engaged in sericulture have to give up tussah rearing. Nosemapernyi is the lethal pathogen of microsporidiosis in A. pernyi. Microsporidiosis has specificnegative effects on A. pernyi and is widely distributed in the main sericultural areas of China,there are much serious economic loss in the process of the rearing of A. pernyi in China everyyear. Now, the spread of this disease alarmed the sericulture, thus studies on the diseasebecame the focus of everyone's attention. Therefore, it’s very important to study thepathogensis, detection, and drug for prevention and treatment about microsporidiosis fordevelopment of A. pernyi.
This paper has studied the method of separate and purify the spores of N. pernyi using thecombined method of differential centrifugation and Percoll density gradient centrifugation,and identify the different protein straps of hemolymph from the female5thinstar larvae usingSDS-PAGE combined with LC-MS/MS. For clarifying the pathogensis of microspordiosis,finding the key genes or proteins in response to pathogen infection, and providing effectivetargets for detection and drug of microspordiosis, transcriptomics and quantitative proteomicsof midgut from the5thinstar larvae of healthy tussah and tussah infection by N. pernyi havebeen studied using RNA-Seq and iTRAQ, and the different expressions of mRNA and proteinfrom two samples have been quantified to discuss the interaction between A. pernyi and N.pernyi. Meanwhile, the detection methods for N. pernyi were studied, for establishing amethod to detect N. pernyi. The results were as follows:
1．The method of separate and purify the spores of N. pernyi has been ascertained. Thespores of N. pernyi can be purified by the combined method of differential centrifugation andPercoll density gradient centrifugation. The purity of spores centrifuged at15000r·min~(-1)for 30min in discontinuous density gradient25%,50%,75%,100%can reach to more than95%.
2．The three methods were used to detect the pathogen of microsporidiosis using thespores of N. pernyi as material.(1) PCR-based method for detection of N. pernyi: First, themethod of DNA extraction which adapts to both spores of N. pernyi and A. pernyi wasdetermined as Banji. Second, three sets of primers were designed by the reported conservedregions of microsporidian SSU rRNA, the specific DNA sequence was amplified from N.pernyi by PCR, and there is no PCR products in A. pernyi. Third, the primer pair N1/N2wasselected to investigate the sensitivity of detection of N. pernyi, the result showed thatprimer N1/N2generated an amplicon of600bp when the content of DNA from N. pernyispores was at least0.47ng. It was observed that PCR diagnosis of N. pernyi using the specificprimers provideds increased specificity and sensitivity.(2) Indirect competitive ELISA fordetection of N. pernyi: The polyclonal antibody against Nosema pernyi was prepared byimmunizing rabbits using the suspension containing spores of N. pernyi, titre andconcentration of antibody was1:10~4and3mg·mL~(-1), the sensitivity of this method was1.6×105spores·mL~(-1).(3) Gold Immunochromatographic Assay (GICA) for detection of N.pernyi: The colloidal gold test strips were prepared by the double antibody sandwich methodand the competition method. The working time of two test strips was both about10minutes，and the sensitivity of this method was0.8×107spores·mL~(-1). The methods were simple, highspecificity and no cross-reaction with the hemolymph of A. pernyi. Moreover, the doubleantibody sandwich method is worth of further study, because its result was clearer and morereliable than that of the competition method.
3．The protein straps whose molecular weight are about44kD and28kD in haemolymphof the female larvae fed with the spores of N. pernyi increased higher than that of the controlfrom96hours and144hours respectively. The two protein straps was identified byLC-MS/MS for achieve more comprehensive proteome profiles.117non-redundant proteinswere identified,52proteins belong to AP28alone,53proteins belong to AP44alone, and12proteins belong to both them.29proteins involved in the immune system and response tostimulus process have been annotated from all the identified proteins from both AP28andAP44. There were heat shock protein [Antheraea yamamai], ubiquitin-like protein[Antheraea yamamai], ubiquitin-conjugating enzyme E2isoform1[Bombyx mori],ubiquitin-conjugating enzyme E2I [Bombyx mori]；ubiquitin conjugating enzyme E2[Bombyxmori], juvenile hormone epoxide hydrolase [Manduca sexta], microtubule-associated proteinRP/EB family member3[Bombyx mori], lysozyme-like protein1[Antheraea mylitta], lysozyme precursor [Antheraea assama], ADP-ribosylation factor [Bombyx mori], putativedefense protein [Antheraea mylitta], RecName:Full=Putative defense protein2; Short=DFP-2;Flags:Precursor, peptidoglycan recognition protein-like protein [Antheraea mylitta],peptidoglycan recognition protein A [Samia cynthia ricini] and peptidoglycan recognitionprotein-like protein [Antheraea pernyi] in AP28alone. There were proteasome beta3subunit[Bombyx mori], proteasome zeta subunit [Bombyx mori], proteasome subunit alpha type6-A[Bombyx mori], DRK [Bombyx mori], tyrosine3-monooxygenase protein zeta polypeptide[Bombyx mori], RGS-GAIP interacting protein GIPC [Bombyx mori], prophenoloxidase[Manduca sexta], eukaryotic translation initiation factor6[Bombyx mori], hemolin[Antheraea pernyi] and loquacious [Bombyx mori] in AP44alone. And there were heat shockprotein hsp21.4[Bombyx mori], prophenoloxidase subunit2[Bombyx mori], attacin-likeprotein [Antheraea pernyi] and basic attacin [Antheraea pernyi] belonging to both them.
4．De novo transcriptomic analysis of midgut tissues from healthy tussah larvae (SM1)and tussah larvae infected by N. pernyi (SM2) has been studied by RNA sequencing(RNA-seq), and the differentially expressed genes (DEGs) between the two samples wereanalyzed.56211contigs (average length=280nt) and60126contigs (average length=256nt)were assemblied in SM1reads and SM2reads respectively,27496unigenes (averagelength=485nt) and30801unigenes (average length=406nt) were obtained by assemblingfrom SM1and SM2respectively.28000All-unigenes (N50=722nt) with a mean size of564nt were originated from SM1clean reads and SM2clean reads combined, and16056unigenes from All-unigenes were annotated to NCBI non-redundant (nr) nucleotide database.There were11351unigenes identified form All-unigenes with25COG categories by clusterof orthologous group (COGs) classification. Based on homologous genes,21303unigenesfrom All-unigenes were categorized into50Gene ontology (GO) terms consisting of threedomains: celluar componet (6947unigenes), molecular function (4081unigenes) andbiological process (10275unigenes). Pathway analysis revealed that there are10351unigenes associated with242pathways, and16.49%of them (1707unigenes) were annotatedto metabolic pathway.
A threshold value of FDR≤0.001and an absolute value of log2Ratio≥1were used to judgethe significance of the differences in gene expression. Based on these criteria,8515unigeneswere differentially expressed between SM1and SM2, including7150unigenes that wereup-regulated and1365unigenes that were down-regulated in SM2tissues. GO analysis ofdifferential expression genes (DEGs) showed that5704DEGs were annotated to42GO terms. GO-CellularComponent enrichment analysis showed that624DEGs were enriched to154terms, and142DEGs were significantly enriched to4GO terms (Q-value≤0.05),including aster, cytoskeleton, spindle and cytoskeletal part; GO-MolecularFunctionenrichment analysis showed that1047DEGs were enriched to223terms, mainly involved inenzyme regulator activity and binding, and106DEGs were significantly enriched to6GOterms (Q-value≤0.05), including endopeptidase inhibitor activity, endopeptidase regulatoractivity, peptidase inhibitor activity, peptidase regulator activity, enzyme inhibitor activity,cytoskeletal protein binding; GO-BiologicalProcess enrichment analysis showed that819DEGs were enriched to873terms, and209DEGs were significantly enriched to11GO terms(Q-value≤0.05), including regulation of cell, axon guidance, protein complex subunitorganization, cell morphogensis involved in neuron differentiation, macromolecular complexdisassembly, cellular macromolecular complex disassembly, protein complex disassembly,cellular protein complex disassembly, microtubule depolymerization, microtubulepolymerization or depolymerization and protein depolymerization. Pathway enrichmentanalysis revealed that there are3092DEGs enriched to231pathways, and there were2390DEGs significantly enriched to34pathways takeing the Q-vlaue≤0.05as a threshold, mainlyinvolved in protein metabolic, disease, immune system and so on.
5．Quantitative protemic analysis of midgut tissues from healthy tussah larvae (M1) andtussah larvae infected by N. pernyi (M2) has been studied by isobaric tag for relative andabsolute quantitation (iTRAQ) using the A. pernyi transcriptome as reference, and thesamples were correspondingly same as transcriptomic analysis. There were1987proteinsidentified in this study. The proteins were categorized into46GO terms consisting of threedomains: celluar componet (3642proteins), molecular function (1779proteins) including11functional categories and biological process (5003proteins) including some process involvedin pathology and resistence of A. pernyi, such as death (63proteins), immune system progress(38proteins), metabolic progress (800proteins), response to stimulus (289proteins).1474proteins were identified to23COG categories with COG classification, the cluster of generalfunctional prediction only occupied the highest number (246proteins,16.69%),7proteinsinvolved in defense mechanisms, and25proteins are function unknown. There weresignificant expression difference in functional sequences between transcriptome andproteome. Pathway analysis revealed that there were1505proteins associated with241pathways, and28.7percent of them (432proteins) were annotated to metabolic pathway, theresult was same as transcriptome.
203differentially expressed proteins were identified by relative quantitative analysis ofprotein expression of SM2to SM1. There were112up-regulated proteins and91down-regulated proteins in the differentially expressed proteins.52.68percent ofup-regulated and68.13percent of down-regulated proteins shared high homology with thoseof Lepidoptera insects. When processing GO enrichment analysis,94differntial expressionproteins were enriched to73GO terms in celluar component, and significantly enrichment toextracellular matrix part, extracellular matrix, extracellular region part, proteinaceousextracellular matrix, ribonucleoprotein complex, basement membrance, extracellular region,sarcomere, striated muscle thin filament, myofibril, contractile fiber part and contractile fiber(P-value≤0.05); In molecular founction,118differential expression proteins were enriched to67terms mainly associated with structural molecule activity and binding, and significantlyenrichment to pattern binding, polysaccharide binding, carbohydrate binding, structuremolecular activity, glycosaminoglycan binding and ligase activity, forming carbon-carbonbond (P-value≤0.05), moreover,2proteins associated with glycosaminoglycan bindingfunction were first discovered in A. pernyi, protein ID of them was Unigene14322_All andUnigene3787_All respectively. In biological process,98differtntial expression proteins wereenriched to256terms, and significantly enrichment to branched chain family amino acidmetabolic process, gene expression, spliceosome assembly, positive regulation ofmacromolecule metabolic process, pentose metabolic process, muscle system process andnucleosome organization (P-value≤0.05). Pathway enrichment analysis revealed that there are154proteins enriched to136pathways, and takeing the P-vlaue≤0.05as a threshold,14pathways were siginificantly enrichment, including ECM-receptor interaction, Galactosemetabolism, Ribosome, Starch and sucrose metabolism, Lysine biosynthesis, Metabolicpathways, Carbohydrate digestion and absorption, Glycerophospholipid metabolism, Malaria,Dilated cardiomyopathy, Hypertrophic cardiomyopathy (HCM), Cysteine and methioninemetabolism, alpha-Linolenic acid metabolism, Spliceosome.9proteins were significantlyenriched to ECM-receptor interaction pathway, including8up-regualted proteins (Protein ID:Unigene21115_All, Unigene4217_All, Unigene22755_All, Unigene14322_All,Unigene842_All, Unigene7641_All, Unigene10826_All and Unigene14191_All) and1down-regulated protein (Priotein ID: Unigene3787_All). Unigene14322_All (2.42times) andUnigene3787_All (0.387times) were the2proteins associated with glycosaminoglycanbinding function, it showed that extracellular region of cell from A. pernyi was the mainregion where N. pernyi infected A. pernyi. A high correction of differential expressed proteins and corresponding transcripts was not noted (γ=0.2404), presumably there waspost-transcriptional regulation. There were44unigenes involved in positive associationexpression in differential expression proteins and transcripts and43unigenes involved inpositive association expression in differential expression proteins and transcripts.
本研究利用Percoll密度梯度离心技术分离纯化了柞蚕微孢子虫，通过感染微孢子虫的柞蚕幼虫血淋巴蛋白质SDS-PAGE电泳及质谱分析鉴定差异蛋白条带，分别采用RNA-Seq技术及iTRAQ技术对感染柞蚕微孢子虫的柞蚕幼虫中肠组织的转录组及定量蛋白质组学研究，分析了健康及患病样品的差异基因及差异蛋白，以期从柞蚕微孢子虫与柞蚕互作方面探讨柞蚕微孢子虫病的发病机制及微孢子虫侵染过程中的关键基因与蛋白，并进行柞蚕微孢子虫病检测技术研究。研究结果如下：
1．明确了柞蚕微孢子虫孢子的分离纯化技术，采用差速离心法进行柞蚕微孢子虫孢子的粗提，进而利用Percoll不连续密度梯度（Percoll浓度分别为25%、50%、75%、100%），15000r·min~(-1)离心30min，纯化效果可达到孢子纯净度95%以上。
2．分别采用间接竞争ELISA检测法、胶体金免疫层析检测法、PCR技术3种方法进行了柞蚕微孢子虫病检测研究。（1）PCR检测技术：筛选了适合柞蚕微孢子虫及柞蚕基因组DNA的提取方法—斑迹抽提法，针对微孢子虫SSU rRNA序列设计的3对引物均具有较好的柞蚕微孢子虫检测特异性，并选择引物N1/N2验证了检测灵敏度，最低检测量仅需要柞蚕微孢子虫DNA0.47ng，灵敏性较高。（2）间接竞争ELISA检测法：制备柞蚕微孢子虫多克隆抗体，效价为1:10~4、浓度为3mg·mL~(-1)，该方法检测灵敏度为1.6×105个·mL~(-1)孢子。（3）胶体金检测法：采用双抗夹心法与竞争法2种方法制备的胶体金试纸条检测时间均为10min，检测灵敏度为0.8×107个·mL孢子，且与柞蚕血淋巴无交叉反应，特异性较强，其中，双抗夹心法试纸条颜色明显、清晰、易于判定，更具有实用价值。
3．柞蚕5龄雌幼虫感染微孢子虫96h和144h后血淋巴蛋白质分别出现分子质量约28kD（AP28）和44kD（AP44）的2条蛋白条带相对于对照组染色加深的现象，对2个蛋白条带质谱分析共鉴定117个不重复蛋白，其中2个样品同时鉴定蛋白12个，AP28单独鉴定蛋白52个，AP44单独鉴定蛋白53个。AP28和AP44鉴定蛋白中参与到柞蚕免疫系统及刺激应答生物过程的无重复蛋白共有29个，其中AP28中包括：热激蛋白、泛素样蛋白、泛素结合酶E2、保幼激素环氧水解酶、微管结合蛋白、溶菌酶、ADP-核糖基化因子、防御蛋白、肽聚糖识别蛋白等15个；AP44中包括：蛋白酶、DRK、酚氧化酶原、真核翻译起始因子、类免疫球蛋白等10个；二者共有：热激蛋白21.4、抗菌肽等4个。
4．采用RNA-Seq技术研究了健康（SM1）和患柞蚕微孢子虫病（SM2）柞蚕中肠组织转录组，并对差异表达基因进行了统计分析。SM1和SM2样本得到的重叠群Contig数分别为56211和60126，平均长度为280nt和256nt，Unigene的数量分别为27496和30801，平均长度为485nt和406nt，将来自同一个物种的SM1和SM2的Unigene进行合并去冗余后得到所有Unigene的量为28000，平均长度为564nt，N50为722nt。对去冗余后的所有Unigene功能注释，16056个Unigenes注释到NCBI nr数据库；与COG数据库比对后共注释到11351个Unigenes，涉及25种功能；GO分析表明，21303个Unigenes被注释到50个GO条目中，其中有6947个基因与细胞组分相关，4081个基因发挥分子功能，10275个基因参与不同的生物学过程；10351个Unigenes共注释到242个代谢通路，其中代谢类通路最多，共1707个、占总量的16.49%。
按照FDR≤0.001且|log2Ratio|≥1条件，对SM2相对于SM1表达差异的Unigene进行筛选，得到差异表达基因8515个，其中上调基因为7150个，下调基因为1365个。差异表达基因的GO分析显示共有5704个DEGs注释到42个GO条目。DEGs的GO-CellularComponent显著富集分析表明，共有624个DEGs富集到细胞组成154个条目中，其中142个DEGs显著富集于4个条目（Q-value≤0.05），包括星状体、细胞骨架、纺锤体、细胞骨架组分；DEGs的GO-MolecularFunction显著富集分析显示，共有1047个差异基因富集于223个条目，分子功能富集于酶调节活性及结合功能，其中106个DEGs显著富集于6个条目（Q-value≤0.05），包括内肽酶抑制因子活性、内肽酶调节因子活性、肽酶抑制因子活性、肽酶调节因子活性、酶抑制因子活性、细胞骨架蛋白结合；DEGs的GO-BiologicalProcess富集分析显示，共有819个差异基因富集于873个条目，209个差异基因显著富集于11个生物过程条目（Q-value≤0.05），分别参与细胞生长调控、轴突导向、蛋白复合体亚基结构、神经元分化过程中细胞形态、大分子复合物解体、胞内大分子复合物解体、蛋白质复合物解体、胞内蛋白质复合物解体、微管解聚、微管聚合或解聚、蛋白解聚。DEGs的Pathway富集分析表明，共有3092个DEGs富集于231个代谢通路，其中2390个DGEs显著富集于34个代谢通路（Q-value≤0.05），主要参与到营养物质代谢与吸收、神经系统互作、病害及与免疫系统相关等通路。
5．以柞蚕转录组数据作为参考序列，利用iTRAQ技术对健康（M1）及患柞蚕微孢子虫病（M2）柞蚕中肠组织蛋白质进行了定量蛋白质组学研究，共鉴定蛋白质1987个。鉴定蛋白被注释到46个GO条目，注释到细胞组分本体中共有3642个蛋白，1779个蛋白发挥分子功能，包括11种功能分类，共有5003个蛋白质参与到25个生物学过程，其中与柞蚕病理及抗病生物过程相关的蛋白包括死亡63个、免疫系统过程38个、代谢过程800个、刺激应答289个；1474个柞蚕蛋白被COG功能分为23类，数量最多的是预测仅有全局功能，并有7个蛋白注释为防御机制功能，25个未知功能蛋白，同时发现功能序列在蛋白质组及转录组水平方面表达具有明显的差异；通过对柞蚕蛋白质Pathway分析发现共有1505条蛋白注释到241个代谢通路中，其代谢通路的蛋白最多，与转录组结果相符。
通过SM2相对于SM1蛋白表达相对定量比较，共发者表达差异蛋白203个，其中上调蛋白112个，下调蛋白91个。差异蛋白大量被注释到鳞翅目昆虫，其中上调蛋白52.68%、下调蛋白68.13%。差异蛋白GO富集分析表明，在细胞组分本体共有94个差异蛋白被富集到细胞组成73个条目，显著富集于胞外基质组分、胞外基质、胞外区组分、蛋白胞外基质、核糖核蛋白复合体、基膜、胞外区、肌节、横纹肌细丝、肌原纤维、连接丝体组分及连接丝体等组分（P-value≤0.05）；在分子功能本体共有118个差异蛋白被富集到67个分子功能条目，显著富集于结构分子活性、模式结合、多糖结合、糖类结合、糖胺聚糖结合、连接酶、碳碳键形成等分子功能条目（P-value≤0.05），并首次在柞蚕上发现2个具有糖胺聚糖等结合功能蛋白，蛋白ID分别为Unigene14322_All及Unigene3787_All；在生物学过程本体共有98个差异蛋白被富集到256个生物学过程条目，显著富集于分支链家族氨基酸代谢过程、基因表达、剪接体组装、大分子代谢过程负调控、戊糖代谢过程、肌肉系统过程及核小体组构等生物过程中（P-value≤0.05）。差异蛋白Pathway富集分析表明，共有154个差异蛋白富集于136个代谢通路，显著富集于胞外基质与受体互作通路、代谢通路、半乳糖代谢、甘油磷酸脂代谢、半胱氨酸和蛋氨酸代谢、α-亚麻酸代谢、赖氨酸生物合成、糖类消化和吸收、核糖体、淀粉和蔗糖代谢、剪接体、疟疾、扩张性及肥厚性心肌病等14个代谢通路，其中在胞外基质与受体间互作通路中，发现蛋白ID为Unigene21115_All、Unigene4217_All、Unigene22755_All、Unigene14322_All、 Unigene842_All、 Unigene7641_All、 Unigene10826_All、Unigene14191_All的发生了上调，Unigene3787_All发生了下调，这其中包含2个糖胺聚糖结合功能蛋白，即Unigene14322_All（2.42倍）及Unigene3787_All（0.387倍），表明柞蚕细胞胞外区是柞蚕微孢子虫侵染宿主过程中重要的区域。通过对差异蛋白及与其对应转录本的关联及聚类分析，发现差异蛋白及与其对应的转录本之间关联性为0.2404，聚类结果显示共有44个Unigenes反向相关，43个Unigenes正向相关。
Candidate: Jiang Yiren Supervisor: Prof. Duan Yuxi Prof. Qin Li
The Chinese oak silkworm (Antheraea pernyiGuérin-Méneville,1855) which belongs toLepidoptera: Saturniidae, is the most well known wild silkmoths for insect food and silkproduction. In China, annual output of tussah cocoon is about7×10~4t, the percentage of thatwith total output of wild silk in world is nearly90%, and the income from tussah rearing hasbecome the main economic source in most sericultural areas. But owing to great harm of themicrosporidiosis, some people engaged in sericulture have to give up tussah rearing. Nosemapernyi is the lethal pathogen of microsporidiosis in A. pernyi. Microsporidiosis has specificnegative effects on A. pernyi and is widely distributed in the main sericultural areas of China,there are much serious economic loss in the process of the rearing of A. pernyi in China everyyear. Now, the spread of this disease alarmed the sericulture, thus studies on the diseasebecame the focus of everyone's attention. Therefore, it’s very important to study thepathogensis, detection, and drug for prevention and treatment about microsporidiosis fordevelopment of A. pernyi.
This paper has studied the method of separate and purify the spores of N. pernyi using thecombined method of differential centrifugation and Percoll density gradient centrifugation,and identify the different protein straps of hemolymph from the female5thinstar larvae usingSDS-PAGE combined with LC-MS/MS. For clarifying the pathogensis of microspordiosis,finding the key genes or proteins in response to pathogen infection, and providing effectivetargets for detection and drug of microspordiosis, transcriptomics and quantitative proteomicsof midgut from the5thinstar larvae of healthy tussah and tussah infection by N. pernyi havebeen studied using RNA-Seq and iTRAQ, and the different expressions of mRNA and proteinfrom two samples have been quantified to discuss the interaction between A. pernyi and N.pernyi. Meanwhile, the detection methods for N. pernyi were studied, for establishing amethod to detect N. pernyi. The results were as follows:
1．The method of separate and purify the spores of N. pernyi has been ascertained. Thespores of N. pernyi can be purified by the combined method of differential centrifugation andPercoll density gradient centrifugation. The purity of spores centrifuged at15000r·min~(-1)for 30min in discontinuous density gradient25%,50%,75%,100%can reach to more than95%.
2．The three methods were used to detect the pathogen of microsporidiosis using thespores of N. pernyi as material.(1) PCR-based method for detection of N. pernyi: First, themethod of DNA extraction which adapts to both spores of N. pernyi and A. pernyi wasdetermined as Banji. Second, three sets of primers were designed by the reported conservedregions of microsporidian SSU rRNA, the specific DNA sequence was amplified from N.pernyi by PCR, and there is no PCR products in A. pernyi. Third, the primer pair N1/N2wasselected to investigate the sensitivity of detection of N. pernyi, the result showed thatprimer N1/N2generated an amplicon of600bp when the content of DNA from N. pernyispores was at least0.47ng. It was observed that PCR diagnosis of N. pernyi using the specificprimers provideds increased specificity and sensitivity.(2) Indirect competitive ELISA fordetection of N. pernyi: The polyclonal antibody against Nosema pernyi was prepared byimmunizing rabbits using the suspension containing spores of N. pernyi, titre andconcentration of antibody was1:10~4and3mg·mL~(-1), the sensitivity of this method was1.6×105spores·mL~(-1).(3) Gold Immunochromatographic Assay (GICA) for detection of N.pernyi: The colloidal gold test strips were prepared by the double antibody sandwich methodand the competition method. The working time of two test strips was both about10minutes，and the sensitivity of this method was0.8×107spores·mL~(-1). The methods were simple, highspecificity and no cross-reaction with the hemolymph of A. pernyi. Moreover, the doubleantibody sandwich method is worth of further study, because its result was clearer and morereliable than that of the competition method.
3．The protein straps whose molecular weight are about44kD and28kD in haemolymphof the female larvae fed with the spores of N. pernyi increased higher than that of the controlfrom96hours and144hours respectively. The two protein straps was identified byLC-MS/MS for achieve more comprehensive proteome profiles.117non-redundant proteinswere identified,52proteins belong to AP28alone,53proteins belong to AP44alone, and12proteins belong to both them.29proteins involved in the immune system and response tostimulus process have been annotated from all the identified proteins from both AP28andAP44. There were heat shock protein [Antheraea yamamai], ubiquitin-like protein[Antheraea yamamai], ubiquitin-conjugating enzyme E2isoform1[Bombyx mori],ubiquitin-conjugating enzyme E2I [Bombyx mori]；ubiquitin conjugating enzyme E2[Bombyxmori], juvenile hormone epoxide hydrolase [Manduca sexta], microtubule-associated proteinRP/EB family member3[Bombyx mori], lysozyme-like protein1[Antheraea mylitta], lysozyme precursor [Antheraea assama], ADP-ribosylation factor [Bombyx mori], putativedefense protein [Antheraea mylitta], RecName:Full=Putative defense protein2; Short=DFP-2;Flags:Precursor, peptidoglycan recognition protein-like protein [Antheraea mylitta],peptidoglycan recognition protein A [Samia cynthia ricini] and peptidoglycan recognitionprotein-like protein [Antheraea pernyi] in AP28alone. There were proteasome beta3subunit[Bombyx mori], proteasome zeta subunit [Bombyx mori], proteasome subunit alpha type6-A[Bombyx mori], DRK [Bombyx mori], tyrosine3-monooxygenase protein zeta polypeptide[Bombyx mori], RGS-GAIP interacting protein GIPC [Bombyx mori], prophenoloxidase[Manduca sexta], eukaryotic translation initiation factor6[Bombyx mori], hemolin[Antheraea pernyi] and loquacious [Bombyx mori] in AP44alone. And there were heat shockprotein hsp21.4[Bombyx mori], prophenoloxidase subunit2[Bombyx mori], attacin-likeprotein [Antheraea pernyi] and basic attacin [Antheraea pernyi] belonging to both them.
4．De novo transcriptomic analysis of midgut tissues from healthy tussah larvae (SM1)and tussah larvae infected by N. pernyi (SM2) has been studied by RNA sequencing(RNA-seq), and the differentially expressed genes (DEGs) between the two samples wereanalyzed.56211contigs (average length=280nt) and60126contigs (average length=256nt)were assemblied in SM1reads and SM2reads respectively,27496unigenes (averagelength=485nt) and30801unigenes (average length=406nt) were obtained by assemblingfrom SM1and SM2respectively.28000All-unigenes (N50=722nt) with a mean size of564nt were originated from SM1clean reads and SM2clean reads combined, and16056unigenes from All-unigenes were annotated to NCBI non-redundant (nr) nucleotide database.There were11351unigenes identified form All-unigenes with25COG categories by clusterof orthologous group (COGs) classification. Based on homologous genes,21303unigenesfrom All-unigenes were categorized into50Gene ontology (GO) terms consisting of threedomains: celluar componet (6947unigenes), molecular function (4081unigenes) andbiological process (10275unigenes). Pathway analysis revealed that there are10351unigenes associated with242pathways, and16.49%of them (1707unigenes) were annotatedto metabolic pathway.
A threshold value of FDR≤0.001and an absolute value of log2Ratio≥1were used to judgethe significance of the differences in gene expression. Based on these criteria,8515unigeneswere differentially expressed between SM1and SM2, including7150unigenes that wereup-regulated and1365unigenes that were down-regulated in SM2tissues. GO analysis ofdifferential expression genes (DEGs) showed that5704DEGs were annotated to42GO terms. GO-CellularComponent enrichment analysis showed that624DEGs were enriched to154terms, and142DEGs were significantly enriched to4GO terms (Q-value≤0.05),including aster, cytoskeleton, spindle and cytoskeletal part; GO-MolecularFunctionenrichment analysis showed that1047DEGs were enriched to223terms, mainly involved inenzyme regulator activity and binding, and106DEGs were significantly enriched to6GOterms (Q-value≤0.05), including endopeptidase inhibitor activity, endopeptidase regulatoractivity, peptidase inhibitor activity, peptidase regulator activity, enzyme inhibitor activity,cytoskeletal protein binding; GO-BiologicalProcess enrichment analysis showed that819DEGs were enriched to873terms, and209DEGs were significantly enriched to11GO terms(Q-value≤0.05), including regulation of cell, axon guidance, protein complex subunitorganization, cell morphogensis involved in neuron differentiation, macromolecular complexdisassembly, cellular macromolecular complex disassembly, protein complex disassembly,cellular protein complex disassembly, microtubule depolymerization, microtubulepolymerization or depolymerization and protein depolymerization. Pathway enrichmentanalysis revealed that there are3092DEGs enriched to231pathways, and there were2390DEGs significantly enriched to34pathways takeing the Q-vlaue≤0.05as a threshold, mainlyinvolved in protein metabolic, disease, immune system and so on.
5．Quantitative protemic analysis of midgut tissues from healthy tussah larvae (M1) andtussah larvae infected by N. pernyi (M2) has been studied by isobaric tag for relative andabsolute quantitation (iTRAQ) using the A. pernyi transcriptome as reference, and thesamples were correspondingly same as transcriptomic analysis. There were1987proteinsidentified in this study. The proteins were categorized into46GO terms consisting of threedomains: celluar componet (3642proteins), molecular function (1779proteins) including11functional categories and biological process (5003proteins) including some process involvedin pathology and resistence of A. pernyi, such as death (63proteins), immune system progress(38proteins), metabolic progress (800proteins), response to stimulus (289proteins).1474proteins were identified to23COG categories with COG classification, the cluster of generalfunctional prediction only occupied the highest number (246proteins,16.69%),7proteinsinvolved in defense mechanisms, and25proteins are function unknown. There weresignificant expression difference in functional sequences between transcriptome andproteome. Pathway analysis revealed that there were1505proteins associated with241pathways, and28.7percent of them (432proteins) were annotated to metabolic pathway, theresult was same as transcriptome.
203differentially expressed proteins were identified by relative quantitative analysis ofprotein expression of SM2to SM1. There were112up-regulated proteins and91down-regulated proteins in the differentially expressed proteins.52.68percent ofup-regulated and68.13percent of down-regulated proteins shared high homology with thoseof Lepidoptera insects. When processing GO enrichment analysis,94differntial expressionproteins were enriched to73GO terms in celluar component, and significantly enrichment toextracellular matrix part, extracellular matrix, extracellular region part, proteinaceousextracellular matrix, ribonucleoprotein complex, basement membrance, extracellular region,sarcomere, striated muscle thin filament, myofibril, contractile fiber part and contractile fiber(P-value≤0.05); In molecular founction,118differential expression proteins were enriched to67terms mainly associated with structural molecule activity and binding, and significantlyenrichment to pattern binding, polysaccharide binding, carbohydrate binding, structuremolecular activity, glycosaminoglycan binding and ligase activity, forming carbon-carbonbond (P-value≤0.05), moreover,2proteins associated with glycosaminoglycan bindingfunction were first discovered in A. pernyi, protein ID of them was Unigene14322_All andUnigene3787_All respectively. In biological process,98differtntial expression proteins wereenriched to256terms, and significantly enrichment to branched chain family amino acidmetabolic process, gene expression, spliceosome assembly, positive regulation ofmacromolecule metabolic process, pentose metabolic process, muscle system process andnucleosome organization (P-value≤0.05). Pathway enrichment analysis revealed that there are154proteins enriched to136pathways, and takeing the P-vlaue≤0.05as a threshold,14pathways were siginificantly enrichment, including ECM-receptor interaction, Galactosemetabolism, Ribosome, Starch and sucrose metabolism, Lysine biosynthesis, Metabolicpathways, Carbohydrate digestion and absorption, Glycerophospholipid metabolism, Malaria,Dilated cardiomyopathy, Hypertrophic cardiomyopathy (HCM), Cysteine and methioninemetabolism, alpha-Linolenic acid metabolism, Spliceosome.9proteins were significantlyenriched to ECM-receptor interaction pathway, including8up-regualted proteins (Protein ID:Unigene21115_All, Unigene4217_All, Unigene22755_All, Unigene14322_All,Unigene842_All, Unigene7641_All, Unigene10826_All and Unigene14191_All) and1down-regulated protein (Priotein ID: Unigene3787_All). Unigene14322_All (2.42times) andUnigene3787_All (0.387times) were the2proteins associated with glycosaminoglycanbinding function, it showed that extracellular region of cell from A. pernyi was the mainregion where N. pernyi infected A. pernyi. A high correction of differential expressed proteins and corresponding transcripts was not noted (γ=0.2404), presumably there waspost-transcriptional regulation. There were44unigenes involved in positive associationexpression in differential expression proteins and transcripts and43unigenes involved inpositive association expression in differential expression proteins and transcripts.
引文
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