BmNPV侵染家蚕中肠组织转录组分析及BmTret1-like基因的功能鉴定

发布时间：2018-04-01 21:34

本文选题：家蚕核型多角体病毒　切入点：转录组分析　出处：《江苏科技大学》2017年硕士论文

【摘要】：由家蚕核型多角体病毒(Bombyx mori.Nucleopolyhedrovirus,BmNPV)引起的血液型脓病是一种急性传染病,一旦感染,就会给养蚕业造成巨大损失。培育抗性品种和筛选抗性基因是蚕业研究工作者不断探讨的课题。本实验以研究室育成的对BmNPV有高抗性的家蚕品种和常规品种为素材,对感染病毒后家蚕抗性品种和常规品种中中肠组织的转录组数据进行分析,获得可能参与家蚕抗病毒机制中的差异基因。以筛选出的家蚕促海藻糖转运因子样(Bombyx mori facilitated trehalose transporter Tret1-like,BmTret1-like)基因为研究对象,对其进行基因克隆及基因表达分析,通过RNAi技术初步探讨BmTret1-like基因与家蚕抗病毒的关系。主要研究结果如下:1、BmNPV侵染家蚕中肠组织转录组分析我们以家蚕抗性品种BYN和常规品种BY中肠组织为材料构建文库,共得到12.82Gb Clean Date(BY品种得到6.40Gb Clean Date,BYN品种得到6.42Gb Clean Date),样品Q30碱基百分比均不小于86.03%。将Clean Date与参考基因组序列比对,可得到序列在参考基因组中的位置信息及样品特有的序列信息。在识别单碱基错配,查找潜在单核苷酸多态性位点时样品BY比对出57 789个SNP位点,BYN比对出58725个SNP位点,2个文库中SNP位点分布在基因区的比例达到60%以上。将Mapped Reads拼接后与原基因组注释信息比较,共发掘出788个新基因。在BmNPV侵染家蚕后蚕体内参与抗病机理调节的基因较多。在筛选到的496个差异表达基因中,244个基因发生上调,252个基因发生下调,其中有486个基因被注释。对unigenes进行GO分类分析可知属细胞组件中细胞和细胞膜占主要部分;属生物进程中代谢过程、单组织进程、细胞进程和生物调节所包含的基因较多;属分子功能中具有催化活性和结合功能的基因数目较多。KEGG信号通路分析可知有142个基因被注释。在COG数据库分析中发现差异表达基因主要与氨基酸、碳水化合物、无机离子的代谢与运输有关。家蚕感染BmNPV病毒后体内出现复杂的基因表达调控,有多条通路参与抗病毒机制的调节。在与参考基因比对中有大部分基因被定位在单一通路上,这些基因的特征及其抗病机理的影响需要进一步研究。2、家蚕BmTret1-like基因的克隆及表达分析通过家蚕抗性品种BYN和常规品种BY中肠组织克隆BmTret1-like基因,扩增得到1272bp片段。将该基因与家蚕基因组中的同源序列比较可以看出其同源基因在染色体中分布并不均匀,主要分布在27连锁群上的最多。将该基因与其他物种构建系统发生树可以看出BmTret1-like基因与白纹伊蚊、火红蚁、红鳍东方渶及意蜂亲缘关系较近,与黑腹果蝇、秀隐线虫等亲缘关系较远。BmTret1-like基因在家蚕各个组织中均表达,没有表现出组织特异性。在中肠组织中,添加病毒后蚕体内BmTret1-like基因表达量都有所下调;在脂肪体组织中,添加病毒后蚕体内BmTret1-like基因在抗性品种和非抗品种中表现出不同的调节趋势,在非抗性品种中除12h时间点外均表现出上调趋势,而抗性品种中都表现出下调趋势。3、利用RNAi实验初步探索BmTret1-like基因功能根据已知片段设计合成BmTret1-like基因的dsRNA,用带有荧光基团的BV病毒穿刺抗性品种及常规品种,其中抗性品种一组注射dsRNA,一组直接穿刺病毒。结果被RNAi的抗性品种中观察到荧光,只穿刺病毒的抗性品种中未观察到荧光,常规品种中荧光效果明显。这说明对BmTret1-like基因RNAi后家蚕体内的抗病毒机制受到影响,说明BmTret1-like基因确实参与家蚕抗病毒机制的调节。
[Abstract]:From Bombyx mori nuclear polyhedrosis virus (Bombyx, mori.Nucleopolyhedrovirus, BmNPV) blood pus disease is an acute infectious disease, once the infection will cause huge loss. Commercial sericultural resistance breeding and screening of resistance genes are constantly explore the sericultural research workers subject. In this experiment, laboratory incubation of silkworm BmNPV high resistant varieties and conventional varieties as material, analysis of transcriptome data after infection of midgut tissues of silkworm varieties and conventional varieties were obtained, may be involved in the antiviral mechanism of the difference of silkworm gene in silkworm. The screened promoting trehalose transporter (Bombyx Mori facilitated trehalose transporter like Tret1-like, BmTret1-like) gene the object of study, carries on the gene cloning and gene expression analysis by RNAi of BmTret1-like gene and silkworm disease Toxic relationship. The main results are as follows: 1, BmNPV infected midgut transcriptome analysis in our silkworm varieties BYN and conventional varieties BY midgut was constructed by 12.82Gb Clean library, Date (BY 6.40Gb Clean Date BYN varieties, varieties of 6.42Gb Clean Date, Q30) samples are not less than base percentage 86.03%. Clean Date with a reference genome sequence alignment, sequence information can be obtained in specific sequences in the reference genome location information and samples. In recognition of single base mismatch, find potential single nucleotide polymorphism samples of BY showed that 57789 SNP loci, BYN showed that 58725 SNP loci, 2 SNP loci in the library located in the gene region of the proportion reached 60%. Mapped Reads splicing with the original genome annotation information, unearthed 788 new genes. In BmNPV infected silkworm Bombyx mori after reference Gene regulation and more resistant mechanism. In 496 different screening expressed genes, 244 genes up-regulated and 252 genes were down regulated, of which 486 genes were annotated. GO classification analysis of cell and cell membrane that belongs to cell components accounted for the main part of unigenes is in the process of biological metabolism; a single organization, process, process and biological regulation gene more cells contained; catalytic activity and binding function of the gene number.KEGG pathway analysis of 142 genes were annotated with molecular function. In the database of COG found in the analysis of differentially expressed genes and amino acids, carbohydrates, inorganic ion metabolism and transport related the regulation of gene expression. The complex body of Silkworm Infected with BmNPV virus, the regulation of multiple pathways in antiviral mechanism. In comparison with the reference gene in a large part of the base Because is located in a single pathway, further study of.2 need to influence the characteristics of these genes and the mechanism of disease resistance, cloning and expression analysis of BmTret1-like gene of Bombyx mori by cloning the BmTret1-like gene of Bombyx mori varieties BYN and conventional varieties of BY midgut tissue, amplified 1272bp fragment. The homologous sequences of this gene in the silkworm genome and its comparison homologous genes are not distributed uniformly in chromosomes, most are mainly distributed in 27 linkage groups. The gene with other species phylogenetic tree can be seen BmTret1-like gene and Aedes albopictus, red ants, near t.rubripes Ying and Italian bee genetic relationship, and Drosophila melanogaster, show hidden nematodes were expressed distantly related.BmTret1-like gene in different tissues of silkworm, showing no tissue specificity. In the midgut, add the virus of silkworm BmTret1-like Because the expression were decreased; in fat body, added after virus BmTret1-like gene in silkworm and non resistant varieties showed different trends in the regulation of resistant varieties, in non resistant varieties except 12h time point showed upward trend, while the resistant varieties showed a downward trend in.3, RNAi experiment preliminary exploration of the function of BmTret1-like gene fragment of BmTret1-like gene was synthesized according to the known design using dsRNA with BV virus resistant varieties with puncture fluorescent group and conventional varieties, including resistant varieties was injected with dsRNA, a group of direct puncture virus. Results have been observed by fluorescent RNAi in resistant varieties, only resistant varieties were not observed in the virus puncture the fluorescence, fluorescence effect of conventional varieties is obvious. This indicates that the effect of antiviral mechanism in silkworm by BmTret1-like RNAi gene, BmTret1-like gene is the argument And the regulation of the anti-virus mechanism of silkworm.

【学位授予单位】：江苏科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S884.51

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