本文關(guān)鍵詞： 貉 僵化癥 畢氏腸微孢子蟲(chóng) 附紅細(xì)胞體 比較轉(zhuǎn)錄組 比較蛋白組　出處：《吉林農(nóng)業(yè)大學(xué)》2017年博士論文　論文類(lèi)型：學(xué)位論文

【摘要】：貉子是我國(guó)廣泛養(yǎng)殖的珍貴的毛皮動(dòng)物之一,近幾年出現(xiàn)的貉僵化癥,又稱(chēng)僵貉病,嚴(yán)重影響?zhàn)B殖貉經(jīng)濟(jì)效益。本試驗(yàn)首先以河北某地的典型僵化癥貉為研究對(duì)象,從僵貉臨床癥狀、血液生理生化指標(biāo)、感染源確診和疾病治療方面對(duì)貉僵化病產(chǎn)生的影響及影響程度進(jìn)行了探索性的初步研究,并調(diào)查了我國(guó)養(yǎng)貉主產(chǎn)區(qū)微孢子蟲(chóng)、附紅細(xì)胞體和僵化病的流行情況。然后利用高通量測(cè)序技術(shù)對(duì)僵貉與正常貉的肝臟、脾臟進(jìn)行比較轉(zhuǎn)錄組、蛋白組研究,取得了以下發(fā)現(xiàn):1、針對(duì)河北某地的僵化貉樣品,利用常規(guī)病理剖檢、膠體金試紙條、PCR、細(xì)菌分離培養(yǎng)、動(dòng)物回歸和血常規(guī)和血液生化檢測(cè),并利用相應(yīng)藥物治療,排除冠狀病毒、犬瘟熱病毒、細(xì)小病毒、水貂阿留申病毒、貉藍(lán)狐源阿留申病毒和遺傳原因,綜合確診該場(chǎng)為以附紅細(xì)胞體為主由附紅細(xì)胞體和畢氏腸微孢子蟲(chóng)混合感染導(dǎo)致僵化。2、利用巢氏PCR調(diào)查我國(guó)養(yǎng)貉主產(chǎn)區(qū)發(fā)現(xiàn)貉畢氏腸微孢子蟲(chóng)總陽(yáng)性率為22.30%,感染率最高的地區(qū)為河北省陽(yáng)性率為40.74%,公貉陽(yáng)性率為27.10%,母貉陽(yáng)性率為17.33%;DNA測(cè)序結(jié)果發(fā)現(xiàn)鑒定了六個(gè)不同的ITS區(qū),包括四個(gè)已知的基因型(D,CHN-DC1,NCF2和CHN-F1)和兩個(gè)新的基因型(NCR1和NCR2),系統(tǒng)發(fā)育分析顯示所有六種基因型屬于組1-人獸共患病組,表明貉是人類(lèi)畢氏腸微孢子蟲(chóng)感染的潛在來(lái)源,應(yīng)注意貉畢氏微孢子對(duì)人類(lèi)公共健康的威脅。3、采用血液滴片檢測(cè)方法檢測(cè)了我國(guó)養(yǎng)貉主產(chǎn)區(qū)貉血液樣品,發(fā)現(xiàn)附紅細(xì)胞體陽(yáng)性率為62.29%,感染率最高的地區(qū)為河北省陽(yáng)性率為68.64%,母貉和公貉附紅細(xì)胞體感染率分別為61.16%和63.38%。應(yīng)引起我們對(duì)附紅細(xì)胞體大面積感染引起的免疫抑制問(wèn)題關(guān)注。4、調(diào)查統(tǒng)計(jì)7省區(qū)38個(gè)貉養(yǎng)殖場(chǎng)發(fā)現(xiàn)貉僵化癥總體陽(yáng)性率為4.71%,各省區(qū)之間差異不顯著,不同省區(qū)內(nèi)各個(gè)養(yǎng)殖場(chǎng)之間差異明顯,發(fā)生率高的比低的場(chǎng)子相差18.4倍,僵化癥發(fā)生率與管理水平呈相反趨勢(shì),管理好的場(chǎng)子貉僵化癥發(fā)生率低。5、應(yīng)用比較轉(zhuǎn)錄組學(xué)技術(shù)對(duì)僵化癥貉和正常貉的肝臟和脾臟2個(gè)器官的14個(gè)樣品進(jìn)行研究,共得到1,571,229個(gè)轉(zhuǎn)錄本和290568個(gè)unigenes,通過(guò)與Nr數(shù)據(jù)庫(kù)進(jìn)行比對(duì)注釋,發(fā)現(xiàn)貉與狗相似性最高達(dá)到61.62%,符合已知分類(lèi)學(xué)地位。僵貉脾臟相對(duì)于正常貉的轉(zhuǎn)錄組有1842個(gè)基因上調(diào)和876個(gè)基因下調(diào),僵貉肝臟相對(duì)于正常貉有1392個(gè)基因表達(dá)上調(diào)和604個(gè)基因表達(dá)下調(diào),其相關(guān)基因可以作為僵化診斷治療及預(yù)防的候選基因。6、對(duì)僵貉脾臟比較轉(zhuǎn)錄組Pathway富集分析,發(fā)現(xiàn)共涉及六大分類(lèi)42個(gè)通路,聚類(lèi)發(fā)現(xiàn),僵貉與正常貉脾臟差異基因Pathway富集顯著性最高的為代謝通路和PI3K/Akt信號(hào)通路。對(duì)僵貉肝臟比較轉(zhuǎn)錄組Pathway富集分析,發(fā)現(xiàn)共涉及六大分類(lèi)41個(gè)通路。聚類(lèi)發(fā)現(xiàn),僵貉與正常貉肝臟差異基因Pathway富集顯著性最高的為環(huán)境信息中的加工信號(hào)轉(zhuǎn)導(dǎo)途徑PI3K-Akt信號(hào)通路、代謝通路、ECM受體相互作用通路。7、對(duì)僵化貉和正常貉的脾臟進(jìn)行比較蛋白質(zhì)組學(xué)研究,共鑒定出2,469個(gè)蛋白,其中1,484個(gè)蛋白可以被定量,僵化貉脾臟中相對(duì)于正常貉正常貉有23個(gè)蛋白表達(dá)上調(diào),33個(gè)蛋白表達(dá)下調(diào)。上調(diào)差異主要是細(xì)胞骨架成分涉及生物粘附和細(xì)胞粘附過(guò)程中的酶活性調(diào)節(jié)等。下調(diào)差異蛋白主要是MHC蛋白復(fù)合物涉及免疫系統(tǒng)、免疫反應(yīng)等功能。KEGG分析發(fā)現(xiàn)上調(diào)差異基因共涉及25個(gè)通路,下調(diào)差異基因共涉及2個(gè)通路。8、對(duì)僵化貉和正常貉的肝臟進(jìn)行比較蛋白質(zhì)組學(xué)研究,總共鑒定出有2,025個(gè)蛋白,其中有1,282個(gè)蛋白可以被精確定量,僵化貉肝臟中相對(duì)于正常貉有93個(gè)蛋白表達(dá)上調(diào),119個(gè)蛋白表達(dá)下調(diào)。上調(diào)差異基因共涉及1個(gè)通路,下調(diào)差異基因共涉及16個(gè)通路。在僵化貉與正常貉差異蛋白分子功能富集分析排名前三位的是氧化還原酶活性、鐵離子結(jié)合、血紅素結(jié)合,與機(jī)體感染附紅細(xì)胞體后內(nèi)呼吸和氧氣運(yùn)輸相關(guān)功能分子關(guān)系較大。
[Abstract]:Raccoon is one of the precious fur animal extensively cultivated in China, in recent years the raccoon rigid disease, also known as stiff raccoon disease, seriously affect the economic benefits. The raccoon dog breeding first in a typical Hebei province sclerosis raccoon as the research object, from the dead raccoon clinical symptoms, blood physiological and biochemical indexes, the to explore the preliminary study of the effect on treatment of infection disease produces rigid raccoon source diagnosis and disease and the influence, and the survey of the raccoon dog producing microsporidia, Eperythrozoon and epidemic situation of rigid disease. Then use high-throughput sequencing of normal liver and stiff raccoon raccoon, spleen comparative transcriptome, proteome research, made the following findings: 1 in Hebei, a rigid raccoon sample, using routine pathological examination, colloidal gold strip, PCR, bacterial culture, animal regression and blood routine and blood biochemical detection, and use The corresponding drug treatment, exclusion of coronavirus, canine distemper virus, parvovirus, mink Aleutian disease virus, blue fox raccoon source Aleutian disease virus and genetic causes, comprehensive diagnosis of the field to Eperythrozoon dominated by Eperythrozoon and Pichia intestinal microsporidia infection leads to mixed rigid.2, China's main producing areas of the raccoon dog survey found raccoon dog intestinal microsporidia Pichia total positive rate was 22.30% by nested PCR, the highest infection rate in the area for the positive rate of Hebei province was 40.74%, the positive rate of male raccoon was 27.10%, the positive rate of mother raccoon was 17.33%; DNA were found in six different ITS regions were identified, including four known genotypes (D, CHN-DC1, NCF2 and CHN-F1) and two new genotypes (NCR1 and NCR2), phylogenetic analysis showed that all six genotypes belonging to the group of 1- Amphixenosis group, that is the human intestinal raccoon Pichia microsporidia infection potential sources, should pay attention to the raccoon dog Bi Shiwei The spores of human public health threat detection.3, China's main producing areas of the raccoon dog with blood dripping raccoon blood sample detection method, it was found that the positive rate of Eperythrozoon infection rate was 62.29%, the highest positive rate of Hebei province is 68.64%, and female raccoon dog male raccoon dog Eperythrozoon infection rate was 61.16% and 63.38%. should cause us to a large area of Eperythrozoon infection induced immunosuppression on.4, investigation and statistics of 7 provinces in 38 raccoon farms that raccoon sclerosis total positive rate was 4.71%, there was no significant difference between different provinces, different provinces between the various farms significantly higher incidence than low a difference of 18.4 times, the incidence rate of sclerosis and management level had the opposite trend, the incidence rate of low.5 manage place raccoon sclerosis, using comparative transcriptome of raccoon dog and raccoon normal sclerosis of the liver and spleen of 2 organs 14 The sample of a total of 1571229 transcripts and 290568 unigenes, comparing notes with the Nr database, and found that the highest similarity of raccoon dog reached 61.62%, consistent with the known taxonomic status. Compared with the normal group of stiff raccoon spleen transcription of 1842 genes of the raccoon and 876 genes were down regulated compared with normal liver, stiff raccoon raccoon dog there are 1392 up-regulated genes and 604 downregulated genes, the gene can be used as candidate genes for.6 diagnosis and treatment and prevention of the rigid and stiff of raccoon comparative transcriptome Pathway enrichment analysis were found, involving a total of six classification 42 paths, clustering found stiff raccoon and normal raccoon dog Pathway gene significantly enriched spleen the highest metabolic pathway and PI3K/Akt pathway. The liver transcriptome stiff raccoon Pathway enrichment analysis found, involving a total of six classification 41 pathways. Clustering, stiff and normal raccoon dog Raccoon dog liver Pathway gene significantly enriched the highest processing of environmental information in the signal transduction pathway of PI3K-Akt signaling pathway, metabolic pathway, ECM receptor interaction pathway.7, the rigid and normal spleen raccoon raccoon comparative proteomics study, 2469 proteins were identified, of which 1484 proteins can be quantitative, rigid Raccoon in spleen compared with normal raccoon normal raccoon expression of 23 proteins, 33 proteins expression down. Upregulation of differences are mainly components of the cytoskeleton involved enzyme activity process of biological adhesion and cell adhesion in the regulation. The difference is mainly MHC down-regulation of protein protein complexes involved in the immune system, immune response function.KEGG analysis found that 25 pathways involving a total of up-regulated differential gene expression, downregulation of genes involving a total of 2 path.8 of normal liver and rigid raccoon raccoon comparative proteomics study, total discrimination Out of 2025 proteins, including 1282 proteins can be accurately quantified relative to normal liver in rigid raccoon raccoon expression of 93 protein, 119 protein down regulated. 1 pathways involving upregulation of different genes, 16 down regulated pathways involving genes. The rigid differential proteins and normal raccoon raccoon dog the functional enrichment analysis of the top three is the oxidoreductase activity, ferric ion binding, heme binding, and respiratory infection of Eperythrozoon in oxygen transport function of molecular relationship is larger.

【學(xué)位授予單位】：吉林農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S858.92

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