Su Juanjuan, Su Jianguo, Shang Xueying, Wan Quanyuan, Chen Xiaohui, Rao Youliang
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
College of Animal Science and Technology, Northwest A&F University, Yangling 712100, China.
Fish Shellfish Immunol. 2015 Mar;43(1):1-12. doi: 10.1016/j.fsi.2014.12.005. Epub 2014 Dec 13.
Toll-like receptor 8 (TLR8), a prototypical intracellular member of TLR family, is generally linked closely to antiviral innate immune through recognizing viral nucleic acid. In this study, 5'-flanking region of Ctenopharyngodon idella TLR8 (CiTLR8), 671bp in length, was amplified and eight SNPs containing one SNP in the intron, three SNPs in the coding region (CDS) and four SNPs in the 3'-untranslated region (UTR) were identified and characterized. Of which 4062 A/T was significantly associated with the susceptibility/resistance to GCRV both in genotype and allele (P < 0.05), while 4168 C/T was extremely significantly associated with that (P < 0.01) according to the case (susceptibility)-control (resistance) analysis. Following the verification experiment, further analyses of mRNA expression, linkage disequilibrium (LD), haplotype and microRNA (miRNA) target site indicated that 4062 A/T and 4168 C/T in 3'-UTR might affect the miRNA regulation, while the exertion of antiviral effects of 4062 A/T might rely on its interaction with other SNPs. Additionally, the high-density of SNPs in 3'-UTR might reflect the specific biological functions of 3'-UTR. And also, the mutation of 747 A/G in intron changing the potential transcriptional factor-binding sites (TFBS) nearby might affect the expression of CiTLR8 transcriptionally or post-transcriptionally. Moreover, as predicted, the A/G transition of the only non-synonymous SNP (3846 A/G) in CDS causing threonine/alanine variation, could shorten the length of the α-helix and ultimately affect the integrity of the Toll-IL-1 receptor (TIR) domain. The functional mechanism of 3846 A/G might also involve a threonine phosphorylation signaling. This study may broaden the knowledge of TLR polymorphisms, lay the foundation for further functional research of CiTLR8 and provide potential markers as well as theoretical basis for resistance molecular breeding of grass carp against GCRV.
Toll样受体8（TLR8）是TLR家族典型的细胞内成员，通常通过识别病毒核酸与抗病毒天然免疫密切相关。本研究扩增了草鱼TLR8（CiTLR8）的5'侧翼区，长度为671bp，鉴定并表征了8个单核苷酸多态性（SNP），其中内含子中有1个SNP，编码区（CDS）中有3个SNP，3'非翻译区（UTR）中有4个SNP。根据病例（易感性）-对照（抗性）分析，其中4062 A/T在基因型和等位基因水平上均与对草鱼呼肠孤病毒（GCRV）的易感性/抗性显著相关（P < 0.05），而4168 C/T与之极显著相关（P < 0.01）。经过验证实验后，对mRNA表达、连锁不平衡（LD）、单倍型和微小RNA（miRNA）靶位点的进一步分析表明，3'UTR中的4062 A/T和4168 C/T可能影响miRNA调控，而4062 A/T抗病毒作用的发挥可能依赖于其与其他SNP的相互作用。此外，3'UTR中高密度的SNP可能反映了3'UTR的特定生物学功能。并且，内含子中747 A/G的突变改变了附近潜在的转录因子结合位点（TFBS），可能在转录或转录后影响CiTLR8的表达。此外，如预测的那样，CDS中唯一的非同义SNP（3846 A/G）的A/G转换导致苏氨酸/丙氨酸变异，可缩短α-螺旋长度并最终影响Toll-IL-1受体（TIR）结构域的完整性。3846 A/G的功能机制可能还涉及苏氨酸磷酸化信号传导。本研究可能拓宽对TLR多态性的认识，为CiTLR8的进一步功能研究奠定基础，并为草鱼抗GCRV抗性分子育种提供潜在标记及理论依据。
