發(fā)布時間：2018-04-01 21:34

  本文選題：家蠶核型多角體病毒　切入點：轉(zhuǎn)錄組分析　出處：《江蘇科技大學(xué)》2017年碩士論文

【摘要】：由家蠶核型多角體病毒(Bombyx mori.Nucleopolyhedrovirus,BmNPV)引起的血液型膿病是一種急性傳染病,一旦感染,就會給養(yǎng)蠶業(yè)造成巨大損失。培育抗性品種和篩選抗性基因是蠶業(yè)研究工作者不斷探討的課題。本實驗以研究室育成的對BmNPV有高抗性的家蠶品種和常規(guī)品種為素材,對感染病毒后家蠶抗性品種和常規(guī)品種中中腸組織的轉(zhuǎn)錄組數(shù)據(jù)進行分析,獲得可能參與家蠶抗病毒機制中的差異基因。以篩選出的家蠶促海藻糖轉(zhuǎn)運因子樣(Bombyx mori facilitated trehalose transporter Tret1-like,BmTret1-like)基因為研究對象,對其進行基因克隆及基因表達分析,通過RNAi技術(shù)初步探討B(tài)mTret1-like基因與家蠶抗病毒的關(guān)系。主要研究結(jié)果如下:1、BmNPV侵染家蠶中腸組織轉(zhuǎn)錄組分析我們以家蠶抗性品種BYN和常規(guī)品種BY中腸組織為材料構(gòu)建文庫,共得到12.82Gb Clean Date(BY品種得到6.40Gb Clean Date,BYN品種得到6.42Gb Clean Date),樣品Q30堿基百分比均不小于86.03%。將Clean Date與參考基因組序列比對,可得到序列在參考基因組中的位置信息及樣品特有的序列信息。在識別單堿基錯配,查找潛在單核苷酸多態(tài)性位點時樣品BY比對出57 789個SNP位點,BYN比對出58725個SNP位點,2個文庫中SNP位點分布在基因區(qū)的比例達到60%以上。將Mapped Reads拼接后與原基因組注釋信息比較,共發(fā)掘出788個新基因。在BmNPV侵染家蠶后蠶體內(nèi)參與抗病機理調(diào)節(jié)的基因較多。在篩選到的496個差異表達基因中,244個基因發(fā)生上調(diào),252個基因發(fā)生下調(diào),其中有486個基因被注釋。對unigenes進行GO分類分析可知屬細胞組件中細胞和細胞膜占主要部分;屬生物進程中代謝過程、單組織進程、細胞進程和生物調(diào)節(jié)所包含的基因較多;屬分子功能中具有催化活性和結(jié)合功能的基因數(shù)目較多。KEGG信號通路分析可知有142個基因被注釋。在COG數(shù)據(jù)庫分析中發(fā)現(xiàn)差異表達基因主要與氨基酸、碳水化合物、無機離子的代謝與運輸有關(guān)。家蠶感染BmNPV病毒后體內(nèi)出現(xiàn)復(fù)雜的基因表達調(diào)控,有多條通路參與抗病毒機制的調(diào)節(jié)。在與參考基因比對中有大部分基因被定位在單一通路上,這些基因的特征及其抗病機理的影響需要進一步研究。2、家蠶BmTret1-like基因的克隆及表達分析通過家蠶抗性品種BYN和常規(guī)品種BY中腸組織克隆BmTret1-like基因,擴增得到1272bp片段。將該基因與家蠶基因組中的同源序列比較可以看出其同源基因在染色體中分布并不均勻,主要分布在27連鎖群上的最多。將該基因與其他物種構(gòu)建系統(tǒng)發(fā)生樹可以看出BmTret1-like基因與白紋伊蚊、火紅蟻、紅鰭東方渶及意蜂親緣關(guān)系較近,與黑腹果蠅、秀隱線蟲等親緣關(guān)系較遠。BmTret1-like基因在家蠶各個組織中均表達,沒有表現(xiàn)出組織特異性。在中腸組織中,添加病毒后蠶體內(nèi)BmTret1-like基因表達量都有所下調(diào);在脂肪體組織中,添加病毒后蠶體內(nèi)BmTret1-like基因在抗性品種和非抗品種中表現(xiàn)出不同的調(diào)節(jié)趨勢,在非抗性品種中除12h時間點外均表現(xiàn)出上調(diào)趨勢,而抗性品種中都表現(xiàn)出下調(diào)趨勢。3、利用RNAi實驗初步探索BmTret1-like基因功能根據(jù)已知片段設(shè)計合成BmTret1-like基因的dsRNA,用帶有熒光基團的BV病毒穿刺抗性品種及常規(guī)品種,其中抗性品種一組注射dsRNA,一組直接穿刺病毒。結(jié)果被RNAi的抗性品種中觀察到熒光,只穿刺病毒的抗性品種中未觀察到熒光,常規(guī)品種中熒光效果明顯。這說明對BmTret1-like基因RNAi后家蠶體內(nèi)的抗病毒機制受到影響,說明BmTret1-like基因確實參與家蠶抗病毒機制的調(diào)節(jié)。
[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.

【學(xué)位授予單位】：江蘇科技大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S884.51

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