鲫TLR9基因克隆及生殖因素对其在肠道表达的影响

Molecular cloning of TLR9 gene in crucian carp(Carassius auratus L.) and evaluation of the influence of reproductive maturity on its expression in gut

为揭示Toll样受体家族基因TLR9在鲫(Carassius auratus L.,缩写为Ca)免疫过程中的作用,本研究利用RT-PCR和RACE技术克隆获得了鲫TLR9(CaTLR9)基因cDNA,并就注射外源性人绒毛膜促性激素(hCG)和不同性腺周期对该基因在鲫肠道表达的影响进行了分析。结果表明,CaTLR9 cDNA全长3 509 bp,包含3 195 bp的开放阅读框(ORF)、72 bp的5′非编码区和242 bp的3′非编码区。ORF编码1 064个氨基酸组成的蛋白质。该蛋白包含19个氨基酸组成的信号肽,15个富含亮氨酸的重复结构域(LRR),1个跨膜区和1个TIR结构域。系统进化分析表明,CaTLR9与其他鲤科鱼类同源性较高,与鲤(Cyprinus carpio)相似度为92%,其次是草鱼(Ctenopharyngodon idella)和斑马鱼(Danio rerio),分别为83%和79%。实时荧光定量PCR结果表明,CaTLR9在鲫脾中表达量最高,其次为肾和鳃,其他组织的表达量均较低。鲫肠道TLR9在非繁殖期的表达水平明显低于繁殖期,注射适当剂量的外源hCG可显著上调鲫肠道TLR9的表达。本研究对揭示鲫非特异性免疫能力调节机制,提高鲫健康养殖水平具有一定参考价值。

TLR9, a member of the Toll-like receptor family, plays a key role in the innate immune system by identify-ing the DNA CpG motif of bacteria and viruses. Using rapid amplification of cDNA ends （RACE）, we cloned the full length TLR9 cDNA sequence of crucian carp （Carassius auratus L., Abbr. Ca）. We then evaluated the effects of ex-ogenous hCG and gonad development on CaTLR9 expression using real-time PCR. The CaTLR9 cDNA was 3 509 bp in length with a 3′-untranslated region （242 bp）, a 5′-untranslated region （72 bp）, and a 3 195 bp open reading frame （ORF）. The ORF of CaTLR9 cDNA encodes a 1 064 amino acid protein with a signal peptide of 19 amino acids, 15 leucine rich repeat （LRR） motifs, one transmembrane zone, and one Toll/IL-1 receptor （TIR） domain. The amino acid sequence of CaTLR9 possessed 92%, 83%and 79%identity with the TLR9s of Cyprinus carpio, Ctenopharyngodon idella and Danio rerio, respectively. The expression level of TLR9 was highest in the spleen of crucian carp, followed by the kidney and gill. The level of expression was low in the remaining tissues. The expression of TLR9 was higher during the breeding season than the non-breeding season. Additionally, injections of exogenous hCG significantly up-regulated the expression of TLR9 in the intestine of crucian carp. Our results suggest that TLR9 is a new candidate target gene for investigating non-specific immunity in crucian carp and improving the health of cultured fish.