本文關(guān)鍵詞：核型多角體病毒侵染柞蠶中腸轉(zhuǎn)錄組及免疫相關(guān)基因分析　出處：《沈陽(yáng)農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 柞蠶 轉(zhuǎn)錄組測(cè)序 鳥苷三磷酸酶 脂肪酶

【摘要】：柞蠶(Antheraea pernyi Guerin-Meneville,1855)屬大蠶蛾科柞蠶屬的泌絲昆蟲,是一種重要的經(jīng)濟(jì)昆蟲,同時(shí)也是重要的模式昆蟲。柞蠶幼蟲全齡在野外柞園中飼養(yǎng),其幼蟲生長(zhǎng)發(fā)育極易受到野外環(huán)境條件包括柞樹葉質(zhì)、氣候環(huán)境因素以及多種病原微生物等影響,導(dǎo)致柞蠶病害的發(fā)生。研究表明,因柞蠶因病害造成的減產(chǎn)在20%-30%,柞蠶病害已成為制約柞蠶產(chǎn)業(yè)發(fā)展的主要影響因素之一。其中,柞蠶膿病是柞蠶生產(chǎn)上的主要病害,其病原為柞蠶核型多角體病毒(Antheraea pernyi nucleopolyhedro-virus, ApNPV),該病在世界養(yǎng)蠶業(yè)國(guó)家常有爆發(fā),傳染性極強(qiáng),不易控制,在生產(chǎn)上危害最為嚴(yán)重,常造成巨大的經(jīng)濟(jì)損失。ApNPV感染既取決于自身的表型差異,又與宿主本身生理狀況有關(guān),是病原與宿主相互作用的過程。ApNPV入侵宿主體內(nèi)后,利用宿主體內(nèi)內(nèi)環(huán)境中的營(yíng)養(yǎng)物質(zhì)完成自身的復(fù)制、增殖并釋放。而柞蠶體內(nèi)存在的免疫屏障面對(duì)病毒的入侵,會(huì)開啟防御機(jī)制,通過細(xì)胞內(nèi)的特異性變化來(lái)抵御病毒的增殖,這種變化首先體現(xiàn)在基因水平上,因此研究ApNPV感染后宿主基因的表達(dá)差異,可以從分子水平了解ApNPV侵染后宿主生理反應(yīng)的分子生物學(xué)機(jī)制,對(duì)于闡明柞蠶被ApNPV感染后體內(nèi)免疫相關(guān)基因的表達(dá)及調(diào)控模式具有重要意義,為進(jìn)一步利用柞蠶免疫相關(guān)基因資源及利用分子生物學(xué)方法輔助抗病育種等奠定了基礎(chǔ)。研究結(jié)果如下：1.柞蠶感染ApNPV后中腸轉(zhuǎn)錄組分析結(jié)果采用4.05×106多角體/mL的ApNPV對(duì)柞蠶3齡幼蟲經(jīng)口添食,提取經(jīng)顯微鏡下確認(rèn)ApNPV感染柞蠶幼蟲中腸組織進(jìn)行轉(zhuǎn)錄組分析,建立了ApNPV感染柞蠶的轉(zhuǎn)錄組數(shù)據(jù)庫(kù),為分析ApNPV感染后柞蠶基因表達(dá)規(guī)律提供了基礎(chǔ)數(shù)據(jù)庫(kù)。總共在ApNPV感染的柞蠶中腸中得到5,172個(gè)差異表達(dá)基因,包括2,183個(gè)上調(diào)基因和2,989個(gè)下調(diào)基因。生物信息學(xué)分析表明,篩選到參與柞蠶免疫反應(yīng)的差異基因主要分為以下幾類：與細(xì)胞凋亡相關(guān)的基因、熱激蛋白、絲氨酸蛋白酶抑制劑、絲氨酸蛋白酶和細(xì)胞色素P450。對(duì)這些基因的分析為進(jìn)一步探究宿主應(yīng)對(duì)病毒感染的免疫分子機(jī)制提供了理論基礎(chǔ)。2.柞蠶免疫相關(guān)基因—鳥苷三磷酸酶基因(ApGTPase)的克隆與表達(dá)在柞蠶感染ApNPV的轉(zhuǎn)錄組數(shù)據(jù)庫(kù)中篩選到了表達(dá)量上調(diào)的柞蠶鳥苷三磷酸酶基因,利用生物信息學(xué)方法對(duì)該基因的氨基酸序列進(jìn)行結(jié)構(gòu)分析以及功能預(yù)測(cè),半定量PCR技術(shù)檢測(cè)了鳥苷三磷酸酶基因在柞蠶不同發(fā)育時(shí)期的表達(dá)譜,通過對(duì)柞蠶4齡幼蟲分別經(jīng)口添食柞蠶核型多角體病毒、柞蠶微孢子蟲(Nosema pernyi)、柞蠶鏈球菌(Streptococcus pernyi)3種病原物,利用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)不同外源物刺激作用下,鳥苷三磷酸酶基因在不同時(shí)間段的相對(duì)表達(dá)量變化趨勢(shì),結(jié)果表明：柞蠶鳥苷三磷酸酶基因(ApGTPase)開放閱讀框ORF長(zhǎng)度1194 bp,編碼397個(gè)氨基酸,推測(cè)其蛋白的等電點(diǎn)(pI)6.64,蛋白分子量(Mw)為44.6 kDa。與家蠶(Bombyx mori)、棉鈴蟲(Helicoverpa armigera)、柑橘鳳蝶(Papilio xuthus)、黑腹果蠅(Drosophila melanogaster)的鳥苷三磷酸酶氨基酸序列相似度分別為95%,93%,93%,78%,表明該基因與同樣來(lái)自鱗翅目昆蟲的家蠶、棉鈴蟲和柑橘鳳蝶的同源相似度較高,而與雙翅目的黑腹果蠅相似度較低。在不同病原物誘導(dǎo)后的柞蠶中腸組織中鳥苷三磷酸酶基因的相對(duì)表達(dá)量有不同程度的升高,并普遍高于對(duì)照組(無(wú)菌水處理組)的相對(duì)表達(dá)水平,表明鳥苷三磷酸酶可能參與了柞蠶的免疫防御反應(yīng),可以作為柞蠶免疫基因資源利用。3.柞蠶免疫相關(guān)基因—脂肪酶基因(Aplipase)的克隆與表達(dá)分析根據(jù)柞蠶感染核型多角體病毒的轉(zhuǎn)錄組數(shù)據(jù)庫(kù),設(shè)計(jì)特異引物,克隆得到柞蠶脂肪酶基因(Aplipase),對(duì)該基因的氨基酸序列進(jìn)行結(jié)構(gòu)分析以及功能預(yù)測(cè),半定量PCR技術(shù)構(gòu)建了脂肪酶基因在柞蠶不同發(fā)育時(shí)期的表達(dá)譜；通過對(duì)柞蠶4齡幼蟲分別經(jīng)口添食柞蠶核型多角體病毒、柞蠶微孢子蟲、柞蠶鏈球菌3種病原物,利用實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)不同外源物刺激作用下,柞蠶脂肪酶基因在不同時(shí)間段的相對(duì)表達(dá)量變化趨勢(shì)。結(jié)果表明克隆得到柞蠶脂肪酶基因(Aplipase)的cDNA序列,已知其開放閱讀框ORF長(zhǎng)度為1527bp,編碼508個(gè)氨基酸。BLASTp比對(duì)可知,柞蠶脂肪酶基因與鱗翅目昆蟲家蠶、大紅斑蝶(Danaus plexippus)、柑橘鳳蝶的脂肪酶基因同源序列相似度為79%左右。半定量PCR結(jié)果顯示,脂肪酶基因在柞蠶5齡幼蟲的脂肪體組織的表達(dá)水平較高；不同病原物經(jīng)口添食柞蠶4齡幼蟲,實(shí)時(shí)熒光定量PCR技術(shù)檢測(cè)添毒后72 h內(nèi)脂肪體中脂肪酶轉(zhuǎn)錄水平變化,柞蠶脂肪酶基因在柞蠶核型多角體病毒和白僵菌誘導(dǎo)條件下,轉(zhuǎn)錄水平變化趨勢(shì)最顯著,說(shuō)明柞蠶脂肪酶基因在外源病原物的誘導(dǎo)下高表達(dá),推測(cè)該基因產(chǎn)物與柞蠶的免疫防御有光,可以利用該基因或基因產(chǎn)物進(jìn)行柞蠶免疫方面的研究。以上研究結(jié)果為深入理解ApNPV感染柞蠶后分子水平所發(fā)生的變化,揭示柞蠶-ApNPV之間的互作機(jī)制奠定了理論基礎(chǔ)；對(duì)于更進(jìn)一步研究柞蠶對(duì)不同病原物的免疫機(jī)制具有重要意義,參考免疫相關(guān)基因的表達(dá)水平可為抗性品種的選育提供理論指導(dǎo)。
[Abstract]:(Antheraea pernyi Guerin-Meneville, 1855 tussah) is a silk spinning insect Saturniidae Antheraea, is an important economic insect, but also an important model insect. All age in the field of tussah larvae reared in Oak Park, the growth of the larvae are vulnerable to environmental conditions including wild oak leaf, influence of climate and environment the factors and a variety of pathogenic microorganisms, resulting in the occurrence of tussah silkworm disease. The study shows that for tussah cause by disease reduction in 20%-30%, tussah silkworm disease has become one of the main factors restricting the development of tussah industry. Among them, tussah silkworm pus disease is the main disease of tussah production, the pathogen of Antheraea pernyi nucleopolyhedrovirus (Antheraea pernyi nucleopolyhedro-virus, ApNPV), the disease in sericultural countries often outbreak of highly contagious, not easy to control, in the production of the most serious hazards, often cause huge economic losses The loss of.ApNPV infection depends both on their phenotypic differences, but also related to the physiological status of host, pathogen and host interaction is the process of.ApNPV invading the body, the use of nutrients in the host to complete its replication, proliferation and release. And the presence of Antheraea pernyi immune barrier in the face of virus, will open the defense mechanism, through specific changes in cells to resist the virus proliferation, this change is reflected in the level of gene expression, so the study of ApNPV infection of host genes, molecular biology mechanism from molecular level understanding of ApNPV infected host physiological reaction, to clarify the significance of tussah related gene expression after in vivo ApNPV infection and regulation mode for the further use of silkworm immunity and using molecular biology method auxiliary resistance related gene resources Laid the foundation for breeding. The results are as follows: 1. tussah ApNPV infection midgut transcriptome analysis results using 4.05 * more than 106 /mL ApNPV angle of tussah 3 instar larvae of oral feeding, were extracted under the microscope to confirm ApNPV infection of tussah larvae midgut transcriptome analysis, established the transcriptome database of ApNPV infected silkworm the database provides a basis for the expression of ApNPV gene of Antheraea pernyi after infection. A total of 5172 differences in ApNPV infected tussah midgut expressed genes, including 2183 up-regulated genes and 2989 down regulated genes. Bioinformatics analysis showed that the difference in immune response to screening of tussah genes were mainly divided into the following categories: apoptosis related genes, heat shock protein, serine protease inhibitor, analysis of the genes of the serine protease and cytochrome P450. to further explore the host Molecular mechanism of immune response to viral infection provides a theoretical basis for.2. gene of Antheraea pernyi immune guanosine three phosphatase (ApGTPase) gene cloning and expression in ApNPV infected silkworm transcriptome database screened up-regulated the tussah guanosine three phosphatase gene, the amino acid sequence of the gene by Bioinformatics Method Structure Analysis and function prediction, semi quantitative PCR technique to detect the guanosine three phosphatase gene expression in different developmental stages of silkworm spectrum based on Tussah 4 instar larvae were orally feeding ApNPV, n.antheraeae (Nosema pernyi), Streptococcus pernyi (Streptococcus pernyi) 3 kinds of pathogens. Different exogenous stimulation using real-time fluorescence quantitative PCR detection, results show that guanosine three phosphatase gene relative expression in different time variation trend: Zhashui Silkworm guanosine three phosphatase gene (ApGTPase) ORF open reading frame length of 1194 BP, encoding 397 amino acids, that the isoelectric point of the protein (pI) 6.64, the molecular weight of the protein (Mw) and 44.6 kDa. (Bombyx mori), Bombyx mori (Helicoverpa armigera), cotton bollworm (Papilio xuthus), Papilio xuthus Drosophila melanogaster (Drosophila melanogaster) guanosine three amino acid phosphatase sequence similarity were 95%, 93%, 93%, 78%, and also from the silkworm gene of Lepidoptera, homologous high similarity of cotton bollworm and citrus swallowtail, and Diptera Drosophila melanogaster similarity is low. The guanosine phosphatase gene in three different pathogen induced by Antheraea pernyi in the midgut and higher expression level in different degree, and is generally higher than that of the control group (treated with sterile water group) showed that the relative expression levels of guanosine phosphate three enzymes may be involved in the immune response of Tussah Silkworm That can be used as tussah immune gene resources using.3. immune related gene of Antheraea pernyi lipase gene (Aplipase) cloning and expression analysis based on the transcriptome database of Antheraea pernyi nuclear polyhedrosis virus infection, specific primers were designed and cloned the lipase gene of Antheraea pernyi (Aplipase), forecast analysis of structure and function of the amino acid sequence of the gene, half quantitative PCR technology to construct the lipase gene expression in different developmental stages of the silkworm spectrum; the silkworm 4 instar larvae were orally feeding ApNPV, n.antheraeae, 3 kinds of pathogen Streptococcus pernyi, different exogenous stimulation using real-time fluorescence quantitative PCR detection of lipase gene in Antheraea pernyi. The relative expression change trend in different time. The results showed that the cloned Antheraea pernyi lipase gene (Aplipase) sequence of cDNA, known for its open reading frame The length of ORF is 1527bp, encoding 508 amino acids of.BLASTp than that of tussah lipase gene and Lepidoptera silkworm, monarch butterfly (Danaus plexippus), the lipase gene of Papilio xuthus homologous sequence similarity is 79%. Semi quantitative PCR results showed that adipose tissue lipase gene in Antheraea pernyi 5 instar larvae of a higher level; different pathogens by mouth feeding silkworm 4 instar larvae, lipase in transcriptional level within 72 h after adding the poison in fat body was detected by real-time fluorescence quantitative PCR, the induction conditions of lipase gene in Antheraea pernyi nucleopolyhedrovirus and white muscardine fungus, the most significant changes in transcript levels, indicating the lipase gene is highly expressed in silkworm induced by exogenous pathogens, speculated that the gene product and silkworm immune defense light, can be studied using the tussah immune genes or gene products. The above research Change results for understanding ApNPV infection after silkworm molecular level, revealing the interaction mechanism between tussah -ApNPV laid a theoretical foundation for further research; tussah plays an important role in the immune mechanism of pathogen, provide theoretical guidance for breeding the expression level of genes related to immunity for reference of resistant varieties.

【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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1 叢培東;丹東柞蠶生產(chǎn)與生態(tài)環(huán)境關(guān)系的探討[D];大連海事大學(xué);2009年

2 鄒昌瑞;柞蠶腸道菌群分析及產(chǎn)酶菌的篩選與鑒定[D];安徽農(nóng)業(yè)大學(xué);2011年

3 宋佳;微生物誘導(dǎo)柞蠶血淋巴溶菌酶基因表達(dá)及分析[D];沈陽(yáng)農(nóng)業(yè)大學(xué);2013年

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