Studies On The Detection Of Nosema Pernyi And Transcriptome And Proteome Of Infected Midgut In Anthcraea Pernyi

Candidate: Jiang Yiren Supervisor: Prof. Duan Yuxi Prof. Qin LiThe 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 about7×10⁴t, 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^-1for 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 was1:10⁴and3mg·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]ï¼›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.