本文選題：草魚(yú) + 草魚(yú)呼腸孤病毒��； 參考：《西北農(nóng)林科技大學(xué)》2017年碩士論文

【摘要】：草魚(yú)(Ctenopharyngodon idella)是我國(guó)重要的淡水“四大家魚(yú)”品種之一,其養(yǎng)殖業(yè)發(fā)展面臨著各類(lèi)病原微生物的威脅,以草魚(yú)呼腸孤病毒(Grass carp reovirus,GCRV)引起的草魚(yú)出血病危害最為嚴(yán)重。水環(huán)境的特殊性及病毒性疾病的高發(fā)性和低可控性極大地增加了藥物治療和預(yù)防的難度。免疫基因遺傳多態(tài)性研究致力于尋找疾病關(guān)聯(lián)的多態(tài)性位點(diǎn),單核苷酸多態(tài)性(single nucleotide polymorphisms,SNPs)位點(diǎn)等重要分子標(biāo)記的發(fā)掘增加了以此為基礎(chǔ)構(gòu)建高密度遺傳連鎖圖譜及實(shí)現(xiàn)大規(guī)模良種選育的可能性。Toll樣受體7(Toll-like receptor 7,TLR7)和Toll樣受體8(Toll-like receptor 8,TLR8)是定位于胞內(nèi)膜組分的TLRs成員,兩者均參與草魚(yú)抗GCRV先天免疫應(yīng)答,然而其調(diào)控機(jī)制尚不明確。本研究探尋了草魚(yú)TLR7、TLR8基因(CiTLR7、CiTLR8)的SNPs并進(jìn)行了基于每個(gè)SNP位點(diǎn)的個(gè)體基因分型;依據(jù)分型數(shù)據(jù)進(jìn)行了SNPs與GCRV易感/抗性性狀的關(guān)聯(lián)分析、連鎖不平衡分析、單體型分析及兩基因SNP-SNP互作分析。此外,本研究還檢測(cè)了CiTLR7/CiTLR8基因的表達(dá)量水平,并進(jìn)行了mRNA表達(dá)量與表型關(guān)聯(lián)位點(diǎn)基因型的關(guān)聯(lián)分析。基于基因不同區(qū)域SNPs位點(diǎn)所進(jìn)行的生物信息學(xué)分析,用于探尋關(guān)聯(lián)及非關(guān)聯(lián)位點(diǎn)可能的作用機(jī)制。本研究獲得的主要結(jié)果包括:1.檢測(cè)到CiTLR7基因的11個(gè)可靠的SNPs以及CiTLR8基因中8個(gè)可靠的SNPs。2.基于CiTLR7基因SNPs的GCRV易感/抗性性狀關(guān)聯(lián)分析發(fā)現(xiàn):CiTLR7基因內(nèi)含子區(qū)的820 A/G位點(diǎn)和編碼區(qū)(coding sequence,CDS)1726 A/G位點(diǎn)的基因型頻率在易感組和抗性組間存在顯著差異(P0.05)。3.基于CiTLR8基因SNPs的GCRV易感/抗性性狀關(guān)聯(lián)分析發(fā)現(xiàn):CiTLR8基因3’非翻譯區(qū)(untranslated region,UTR)內(nèi)的4062 A/T位點(diǎn)的基因型和等位基因頻率在易感組和抗性組間存在顯著差異(P0.05);4168 C/T位點(diǎn)的上述分布存在極顯著差異(P0.01)。4.CiTLR7基因已知的11個(gè)SNPs未形成與GCRV易感/抗性表型相關(guān)的單體型結(jié)構(gòu);而CiTLR8基因已知的8個(gè)SNPs存在與GCRV易感/抗性表型顯著或極顯著相關(guān)的單體型結(jié)構(gòu)(4062 A-4178 T、4062 T-4178 T、4062 A-4231 G、4062 T-4231 G;P0.05/0.01),表明SNPs連鎖可能在抗GCRV免疫應(yīng)答中發(fā)揮重要作用。5.GCRV感染后,抗性組草魚(yú)脾臟組織中TLR7和TLR8基因的mRNA表達(dá)量均極顯著高于易感組(P0.01),說(shuō)明兩基因在草魚(yú)脾臟組織抗GCRV免疫應(yīng)答中發(fā)揮正調(diào)控作用。6.基因間SNPs互作分析發(fā)現(xiàn),CiTLR7(-758 A/C和820 A/G)和CiTLR8(1559C/T和4168 C/T)之間存在極顯著的SNPs位點(diǎn)互作效應(yīng),說(shuō)明這兩個(gè)基因完成抗病毒免疫應(yīng)答可能涉及SNPs間的互作。本研究所獲得的草魚(yú)免疫基因SNPs為日后抗病分子育種提供了分子標(biāo)記,且基于SNPs的分析為CiTLR7和CiTLR8抗病機(jī)理的研究提供了新的思路。
[Abstract]:Grass carp grass carp (Ctenopharyngodon idella) is one of the most important freshwater "four domestic fish" species in China. The development of grass carp breeding industry is threatened by various pathogenic microorganisms. Grass carp haemorrhage caused by grass carp reovirus Grass carp reovirusGCRV is the most serious.The particularity of water environment and the high incidence and low controllability of viral diseases greatly increase the difficulty of drug treatment and prevention.The study of genetic polymorphisms of immune genes is devoted to the search for polymorphic loci associated with diseases.The discovery of important molecular markers, such as single nucleotide polymorphismsms (SNPs), increased the possibility of constructing high-density genetic linkage maps and achieving large-scale breeding of improved varieties. Toll-like receptor 7(Toll-like receptor 7 TLR7 and Toll like receptor 8(Toll-like receptor 8 TLR8) were identified.Is a member of the TLRs located in the endocytic component,Both participate in the innate immune response of grass carp to GCRV, but its regulatory mechanism is not clear.In this study, the SNPs of TLR7 and TLR8 gene of grass carp TLR7 and CiTLR8) were explored and the individual genotyping based on each SNP locus was carried out, and the association analysis between SNPs and GCRV susceptibility / resistance traits and linkage disequilibrium analysis were carried out according to the typing data.Haplotype analysis and SNP-SNP interaction analysis of two genes.In addition, the expression level of CiTLR7/CiTLR8 gene was detected, and the correlation analysis between mRNA expression and phenotypic association loci genotypes was carried out.The bioinformatics analysis based on SNPs loci in different regions of genes is used to explore the possible mechanism of action of related and non-related loci.The main results of this study are as follows: 1: 1.11 reliable SNPs of CiTLR7 gene and 8 reliable SNPs. 2 of CiTLR8 gene were detected.The analysis of GCRV susceptibility / resistance traits based on CiTLR7 gene SNPs showed that the genotype frequencies of 820A / G and 1726A / G loci in the intron region of the GCRV CiTLR7 gene were significantly different between the susceptible group and the resistant group (P0.05. 3).Association analysis of GCRV susceptibility / resistance traits based on CiTLR8 gene SNPs found that the genotype and allele frequencies of 4062 A / T locus in 3 'untranslated region UTRs of the GCRV gene were significantly different between susceptible and resistant groups.There was a very significant difference in the distribution between 11 SNPs of CiTLR7 gene and P0.01G. 4. Haplotypes associated with susceptibility / resistance phenotype of GCRV were not formed.However, 8 known SNPs of CiTLR8 gene have haplotype structure 4062 A-4178 Tn4062 T-4178 Tn4062 A-4231 GN 4062 T-4231 P 0.05 / 0.01, which indicates that SNPs linkage may play an important role in the immune response to GCRV. 5. GCRV may play an important role in the response to GCRV infection.The mRNA expression of TLR7 and TLR8 genes in the spleen of grass carp in resistant group was significantly higher than that in susceptible group, indicating that the two genes play a positive role in regulating the immune response to GCRV in the spleen of grass carp.SNPs interaction analysis showed that there was a significant SNPs site interaction between CiTLR71-758A / C and 820A / G) and CiTLR8(1559C/T and 4168 C / T, suggesting that the completion of antiviral immune response between the two genes might involve the interaction between SNPs.The immune gene SNPs of grass carp obtained in this study provides molecular markers for future molecular breeding of disease resistance, and the analysis based on SNPs provides a new idea for the study of the mechanism of disease resistance of CiTLR7 and CiTLR8.
【學(xué)位授予單位】：西北農(nóng)林科技大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：S943

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