草鱼TLR7和TLR8基因单核苷酸多态性与草鱼出血病的关联研究

发布时间：2018-04-16 08:40

  本文选题：草鱼 + 草鱼呼肠孤病毒　； 参考：《西北农林科技大学》2017年硕士论文

【摘要】：草鱼(Ctenopharyngodon idella)是我国重要的淡水“四大家鱼”品种之一,其养殖业发展面临着各类病原微生物的威胁,以草鱼呼肠孤病毒(Grass carp reovirus,GCRV)引起的草鱼出血病危害最为严重。水环境的特殊性及病毒性疾病的高发性和低可控性极大地增加了药物治疗和预防的难度。免疫基因遗传多态性研究致力于寻找疾病关联的多态性位点,单核苷酸多态性(single nucleotide polymorphisms,SNPs)位点等重要分子标记的发掘增加了以此为基础构建高密度遗传连锁图谱及实现大规模良种选育的可能性。Toll样受体7(Toll-like receptor 7,TLR7)和Toll样受体8(Toll-like receptor 8,TLR8)是定位于胞内膜组分的TLRs成员,两者均参与草鱼抗GCRV先天免疫应答,然而其调控机制尚不明确。本研究探寻了草鱼TLR7、TLR8基因(CiTLR7、CiTLR8)的SNPs并进行了基于每个SNP位点的个体基因分型;依据分型数据进行了SNPs与GCRV易感/抗性性状的关联分析、连锁不平衡分析、单体型分析及两基因SNP-SNP互作分析。此外,本研究还检测了CiTLR7/CiTLR8基因的表达量水平,并进行了mRNA表达量与表型关联位点基因型的关联分析。基于基因不同区域SNPs位点所进行的生物信息学分析,用于探寻关联及非关联位点可能的作用机制。本研究获得的主要结果包括:1.检测到CiTLR7基因的11个可靠的SNPs以及CiTLR8基因中8个可靠的SNPs。2.基于CiTLR7基因SNPs的GCRV易感/抗性性状关联分析发现:CiTLR7基因内含子区的820 A/G位点和编码区(coding sequence,CDS)1726 A/G位点的基因型频率在易感组和抗性组间存在显著差异(P0.05)。3.基于CiTLR8基因SNPs的GCRV易感/抗性性状关联分析发现:CiTLR8基因3’非翻译区(untranslated region,UTR)内的4062 A/T位点的基因型和等位基因频率在易感组和抗性组间存在显著差异(P0.05);4168 C/T位点的上述分布存在极显著差异(P0.01)。4.CiTLR7基因已知的11个SNPs未形成与GCRV易感/抗性表型相关的单体型结构;而CiTLR8基因已知的8个SNPs存在与GCRV易感/抗性表型显著或极显著相关的单体型结构(4062 A-4178 T、4062 T-4178 T、4062 A-4231 G、4062 T-4231 G;P0.05/0.01),表明SNPs连锁可能在抗GCRV免疫应答中发挥重要作用。5.GCRV感染后,抗性组草鱼脾脏组织中TLR7和TLR8基因的mRNA表达量均极显著高于易感组(P0.01),说明两基因在草鱼脾脏组织抗GCRV免疫应答中发挥正调控作用。6.基因间SNPs互作分析发现,CiTLR7(-758 A/C和820 A/G)和CiTLR8(1559C/T和4168 C/T)之间存在极显著的SNPs位点互作效应,说明这两个基因完成抗病毒免疫应答可能涉及SNPs间的互作。本研究所获得的草鱼免疫基因SNPs为日后抗病分子育种提供了分子标记,且基于SNPs的分析为CiTLR7和CiTLR8抗病机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.
【学位授予单位】：西北农林科技大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S943

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