核型多角体病毒侵染柞蚕中肠转录组及免疫相关基因分析

发布时间：2018-01-08 03:31

本文关键词：核型多角体病毒侵染柞蚕中肠转录组及免疫相关基因分析　出处：《沈阳农业大学》2016年博士论文　论文类型：学位论文

【摘要】：柞蚕(Antheraea pernyi Guerin-Meneville,1855)属大蚕蛾科柞蚕属的泌丝昆虫,是一种重要的经济昆虫,同时也是重要的模式昆虫。柞蚕幼虫全龄在野外柞园中饲养,其幼虫生长发育极易受到野外环境条件包括柞树叶质、气候环境因素以及多种病原微生物等影响,导致柞蚕病害的发生。研究表明,因柞蚕因病害造成的减产在20%-30%,柞蚕病害已成为制约柞蚕产业发展的主要影响因素之一。其中,柞蚕脓病是柞蚕生产上的主要病害,其病原为柞蚕核型多角体病毒(Antheraea pernyi nucleopolyhedro-virus, ApNPV),该病在世界养蚕业国家常有爆发,传染性极强,不易控制,在生产上危害最为严重,常造成巨大的经济损失。ApNPV感染既取决于自身的表型差异,又与宿主本身生理状况有关,是病原与宿主相互作用的过程。ApNPV入侵宿主体内后,利用宿主体内内环境中的营养物质完成自身的复制、增殖并释放。而柞蚕体内存在的免疫屏障面对病毒的入侵,会开启防御机制,通过细胞内的特异性变化来抵御病毒的增殖,这种变化首先体现在基因水平上,因此研究ApNPV感染后宿主基因的表达差异,可以从分子水平了解ApNPV侵染后宿主生理反应的分子生物学机制,对于阐明柞蚕被ApNPV感染后体内免疫相关基因的表达及调控模式具有重要意义,为进一步利用柞蚕免疫相关基因资源及利用分子生物学方法辅助抗病育种等奠定了基础。研究结果如下：1.柞蚕感染ApNPV后中肠转录组分析结果采用4.05×106多角体/mL的ApNPV对柞蚕3龄幼虫经口添食,提取经显微镜下确认ApNPV感染柞蚕幼虫中肠组织进行转录组分析,建立了ApNPV感染柞蚕的转录组数据库,为分析ApNPV感染后柞蚕基因表达规律提供了基础数据库。总共在ApNPV感染的柞蚕中肠中得到5,172个差异表达基因,包括2,183个上调基因和2,989个下调基因。生物信息学分析表明,筛选到参与柞蚕免疫反应的差异基因主要分为以下几类：与细胞凋亡相关的基因、热激蛋白、丝氨酸蛋白酶抑制剂、丝氨酸蛋白酶和细胞色素P450。对这些基因的分析为进一步探究宿主应对病毒感染的免疫分子机制提供了理论基础。2.柞蚕免疫相关基因—鸟苷三磷酸酶基因(ApGTPase)的克隆与表达在柞蚕感染ApNPV的转录组数据库中筛选到了表达量上调的柞蚕鸟苷三磷酸酶基因,利用生物信息学方法对该基因的氨基酸序列进行结构分析以及功能预测,半定量PCR技术检测了鸟苷三磷酸酶基因在柞蚕不同发育时期的表达谱,通过对柞蚕4龄幼虫分别经口添食柞蚕核型多角体病毒、柞蚕微孢子虫(Nosema pernyi)、柞蚕链球菌(Streptococcus pernyi)3种病原物,利用实时荧光定量PCR技术检测不同外源物刺激作用下,鸟苷三磷酸酶基因在不同时间段的相对表达量变化趋势,结果表明：柞蚕鸟苷三磷酸酶基因(ApGTPase)开放阅读框ORF长度1194 bp,编码397个氨基酸,推测其蛋白的等电点(pI)6.64,蛋白分子量(Mw)为44.6 kDa。与家蚕(Bombyx mori)、棉铃虫(Helicoverpa armigera)、柑橘凤蝶(Papilio xuthus)、黑腹果蝇(Drosophila melanogaster)的鸟苷三磷酸酶氨基酸序列相似度分别为95%,93%,93%,78%,表明该基因与同样来自鳞翅目昆虫的家蚕、棉铃虫和柑橘凤蝶的同源相似度较高,而与双翅目的黑腹果蝇相似度较低。在不同病原物诱导后的柞蚕中肠组织中鸟苷三磷酸酶基因的相对表达量有不同程度的升高,并普遍高于对照组(无菌水处理组)的相对表达水平,表明鸟苷三磷酸酶可能参与了柞蚕的免疫防御反应,可以作为柞蚕免疫基因资源利用。3.柞蚕免疫相关基因—脂肪酶基因(Aplipase)的克隆与表达分析根据柞蚕感染核型多角体病毒的转录组数据库,设计特异引物,克隆得到柞蚕脂肪酶基因(Aplipase),对该基因的氨基酸序列进行结构分析以及功能预测,半定量PCR技术构建了脂肪酶基因在柞蚕不同发育时期的表达谱；通过对柞蚕4龄幼虫分别经口添食柞蚕核型多角体病毒、柞蚕微孢子虫、柞蚕链球菌3种病原物,利用实时荧光定量PCR技术检测不同外源物刺激作用下,柞蚕脂肪酶基因在不同时间段的相对表达量变化趋势。结果表明克隆得到柞蚕脂肪酶基因(Aplipase)的cDNA序列,已知其开放阅读框ORF长度为1527bp,编码508个氨基酸。BLASTp比对可知,柞蚕脂肪酶基因与鳞翅目昆虫家蚕、大红斑蝶(Danaus plexippus)、柑橘凤蝶的脂肪酶基因同源序列相似度为79%左右。半定量PCR结果显示,脂肪酶基因在柞蚕5龄幼虫的脂肪体组织的表达水平较高；不同病原物经口添食柞蚕4龄幼虫,实时荧光定量PCR技术检测添毒后72 h内脂肪体中脂肪酶转录水平变化,柞蚕脂肪酶基因在柞蚕核型多角体病毒和白僵菌诱导条件下,转录水平变化趋势最显著,说明柞蚕脂肪酶基因在外源病原物的诱导下高表达,推测该基因产物与柞蚕的免疫防御有光,可以利用该基因或基因产物进行柞蚕免疫方面的研究。以上研究结果为深入理解ApNPV感染柞蚕后分子水平所发生的变化,揭示柞蚕-ApNPV之间的互作机制奠定了理论基础；对于更进一步研究柞蚕对不同病原物的免疫机制具有重要意义,参考免疫相关基因的表达水平可为抗性品种的选育提供理论指导。
[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.

【学位授予单位】：沈阳农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S885.1

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