Genetics and Breeding Center for Blunt Snout Bream, Ministry of Agriculture, Shanghai, 201306, China; Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai, 201306, China; National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China.
Genetics and Breeding Center for Blunt Snout Bream, Ministry of Agriculture, Shanghai, 201306, China; Key Laboratory of Freshwater Aquatic Genetic Resources, Ministry of Agriculture, Shanghai, 201306, China; National Demonstration Center for Experimental Fisheries Science Education, Shanghai Ocean University, Shanghai, 201306, China.
Fish Shellfish Immunol. 2022 Jan;120:451-457. doi: 10.1016/j.fsi.2021.12.010. Epub 2021 Dec 11.
N-ethyl-N-nitrosourea (ENU) selection is a useful technique to generate new mutations that may cause some functional changes in the gene. Through our previous genomic bulked segregant analysis (BSA), one single nucleotide polymorphism (SNP) at the 3' UTR of Toll interacting protein gene (TOLLIP) was identified in grass carp (Ctenopharyngodon idella) subjected to ENU-induced mutagenesis. We found that the overexpression of cid-miR-nov-1043 mimics significantly suppressed the luciferase activity of the TOLLIP 3' UTR, but TOLLIP mutation at the target site can decrease the binding affinity between the miRNA cid-miR-nov-1043 and TOLLIP 3' UTR, reducing the inhibition of TOLLIP mRNA transcription in grass carp subjected to ENU-induced mutagenesis. More importantly, we demonstrated that TOLLIP mRNA transcription levels in the gills, liver, kidney and the isolate white cells of the mutant grass carp were significantly (p < 0.01) higher than those in the corresponding tissues from the wild-type grass carp following infection with Grass Carp Reovirus (GCRV) for seven days, while the downstream gene of TOLLIP transforming growth factor β-activated kinase 1 (TAK1) and TAK1-binding protein 1 (TAB1), were higher expressed in wild-type grass carp. As a negative regulator in the pro-inflammatory pathway of NF-κB, TOLLIP inhibits the excessive inflammation in ENU grass carp after GCRV infection. Consistent with the TOLLIP expression, histopathological results demonstrated more severe inflammation in wild-type grass carp, compared to the TOLLIP mutant grass carp on the seventh day. Severe inflammation will lead to thoroughly infiltration of chloride and inflammatory cells in the gill filaments. This seriously hindered the exchange of oxygen, which ultimately disrupted blood circulation. Meanwhile, the survival rate of the mutant grass carp was significantly (p < 0.01) higher than that of the wild-type grass carp, indicating that the TOLLIP mutants showed strong anti-viral abilities. Our results revealed that an SNP in the TOLLIP 3' UTR may contribute to the suppression of serve inflammation subjected to ENU-induced mutagenesis following GCRV infection, which may be helpful for future resistant breeding development of grass carp.
N-乙基-N-亚硝脲（ENU）选择是一种产生新突变的有用技术，这些突变可能导致基因的某些功能改变。通过我们之前的基因组 bulked segregant analysis（BSA），在受到 ENU 诱变的草鱼（Ctenopharyngodon idella）中鉴定出 Toll 相互作用蛋白基因（TOLLIP）3'UTR 中的一个单核苷酸多态性（SNP）。我们发现cid-miR-nov-1043 的过表达显着抑制了 TOLLIP 3'UTR 的荧光素酶活性，但靶位点的 TOLLIP 突变会降低 miRNA cid-miR-nov-1043 与 TOLLIP 3'UTR 之间的结合亲和力，从而降低ENU 诱导的突变草鱼中 TOLLIP mRNA 转录的抑制作用。更重要的是，我们证明了在感染草鱼呼肠孤病毒（GCRV）七天后，突变草鱼鳃、肝、肾和分离白细胞中的 TOLLIP mRNA 转录水平显着（p<0.01）高于野生型草鱼相应组织中的转录水平，而 TOLLIP 的下游基因转化生长因子β激活激酶 1（TAK1）和 TAK1 结合蛋白 1（TAB1）在野生型草鱼中表达更高。TOLLIP 作为 NF-κB 促炎途径的负调节剂，抑制 GCRV 感染后 ENU 草鱼的过度炎症。与 TOLLIP 表达一致，组织病理学结果表明，与 TOLLIP 突变草鱼相比，野生型草鱼在第 7 天的炎症更严重。严重的炎症会导致鳃丝中氯离子和炎性细胞的彻底浸润。这严重阻碍了氧气的交换，最终破坏了血液循环。同时，突变草鱼的存活率显着（p<0.01）高于野生型草鱼，表明 TOLLIP 突变体表现出较强的抗病毒能力。我们的结果表明，TOLLIP 3'UTR 中的 SNP 可能有助于抑制 GCRV 感染后 ENU 诱导的突变草鱼的严重炎症，这可能有助于草鱼未来的抗性育种发展。
