Toll样受体3(SpTLR3)来自裂殖菌(S.普伦安蒂)被克隆和鉴定,本研究检测了SpTLR3基因的组织分布。此外,通过定量实时聚合酶链反应(qRT-PCR)分析脂多糖(LPS)攻击后S.prenanti的髓样分化因子88基因(SpMyD88)和7个TLR基因(SpTLR2、SpTLR3、SpTLR4、SpTLR18、SpTLR22-1、SpTLR22-2和SpTLR22-3)的相对mRNA表达水平。SpTLR3基因全长3097bp,完整编码序列(CDS)为2715bp,编码904个氨基酸。SpTLR3氨基酸序列与其他脊椎动物的TLR3序列具有43.94-100%的同一性;SpTLR3在所有8个检查的组织中表达;最高水平出现在肝脏中,显着高于所有其他组织(p&lt;0.05),其次是心脏和肌肉的水平。LPS在12或24h显著上调S.prenanti组织中的所有8个基因(p<0.05)。与PBS对照组相比,在LPS诱导后12小时,在SpTLR2或SpTLR3中没有发现显著的转录变化,但在24小时显著上调(p<0.001)。在24hLPS诱导后,在头肾SpTLR22基因中发现了最丰富的转录本,从高到低的水平,是SpTLR22-1(564倍),SpTLR22-3(508倍)和SpTLR22-2(351倍)。在这八个基因中,SpTLR4的表达上调程度最低。总的来说,头肾中的SpTLR4较早参与抗菌免疫应答,在LPS刺激后12小时,该水平升高,具有极显著意义(p<0.001),而其他七个基因在注射后24小时表达最显著。一起来看,结果表明,SpMyD88、SpTLR2、SpTLR3、SpTLR4、SpTLR18、SpTLR22-1、SpTLR22-2和SpTLR22-3参与了LPS刺激的天然免疫反应,基因的反应强度是器官特异性的,具有不同的动力学。我们的发现将有助于更全面地了解这些TLR基因在抗菌免疫中的作用。
Toll-like receptor 3 (SpTLR3) from Schizothorax prenanti (S. prenanti) was cloned and identified, and the tissue distribution of the SpTLR3 gene was examined in this study. Moreover, the relative mRNA expression levels of myeloid differentiation factor 88 gene (SpMyD88) and seven TLR genes (SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3) from S. prenanti after lipopolysaccharide (LPS) challenge were analyzed through quantitative real-time polymerase chain reaction (qRT-PCR). The full length of SpTLR3 gene is 3097 bp, and complete coding sequence (CDS) is 2715 bp, which encodes 904 amino acids. The SpTLR3 amino acid sequence shared 43.94−100% identity with TLR3 sequences from other vertebrates; SpTLR3 was expressed in all eight tissues examined; and the highest level appeared in the liver, which was significantly higher than in all other tissues (p < 0.05), followed by the levels in the heart and muscles. LPS significantly up-regulated all eight genes in the S. prenanti tissues at 12 or 24 h (p < 0.05). Compared with the PBS control group, no significant transcripts changes were found in SpTLR2 or SpTLR3 at 12 h after LPS induction, but they were significantly up-regulated at 24 h (p < 0.001). The most abundant transcripts were found in the head kidney SpTLR22 genes after 24 h LPS induction, with high to low levels, which were SpTLR22-1 (564-fold), SpTLR22-3 (508-fold) and SpTLR22-2 (351-fold). Among these eight genes, the expression level of SpTLR4 was the least up-regulated. Overall, SpTLR4 in the head kidney was involved in the antibacterial immune response earlier, and the level was increased at 12 h with extreme significance after LPS stimulation (p < 0.001), while the other seven genes were the most significantly up-regulated at 24 h post injection. Taken together, the results suggest that SpMyD88, SpTLR2, SpTLR3, SpTLR4, SpTLR18, SpTLR22-1, SpTLR22-2 and SpTLR22-3 participate in an innate immune response stimulated by LPS, and the response intensity of the genes was organ-specific, with differing kinetics. Our findings will contribute to a more complete understanding of the roles of these TLR genes in antibacterial immunity.