泥蚶TLR3基因的生物学特性及表达分析  

Identification of TgTLR3 and Its Immune Response to Hypoxia and Sulfide in Tegillarca granosa

作　　者：杨正宽 张秀梅 姜璞源 夏飞宇 徐颖 盘钰峰 杨扬 王一航 YANG Zhengkuan;ZHANG Xiumei;JIANG Puyuan(School of Fisheries of Zhejiang Ocean University,Zhoushan 316022;Laboratory for Marine Fisheries Science and Food Production Processes,Pilot National Laboratory for Marine Science and Technology,Qingdao 266071,China)

机构地区：[1]浙江海洋大学水产学院,浙江舟山316022 [2]青岛海洋科学与技术试点国家实验室海洋渔业科学与食物产出过程功能实验室,山东青岛266071

出　　处：《浙江海洋大学学报（自然科学版）》2024年第3期183-192,共10页Journal of Zhejiang Ocean University：Natural Science

基　　金：浙江省基础公益研究计划项目(LQ22C190004);浙江省重点研发计划项目(2021C02047)。

摘　　要：为探究泥蚶Toll样受体3(TLR3)基因的生物学特性及其对缺氧和硫化物胁迫的响应,克隆了泥蚶TgTLR3核心片段序列,并运用实时荧光定量PCR技术检测了TgTLR3基因在血淋巴、鳃、肝胰腺、外套膜、闭壳肌、斧足等组织的分布情况以及在缺氧和硫化物胁迫下血淋巴中的表达模式。结果显示,泥蚶TgTLR3开放阅读框2634 bp,编码877个氨基酸。其中,49~593位为胞外富含亮氨酸重复序列,跨膜域为658~680位,720~865位为白细胞介素受体1结构域,蛋白质分子量为99.4 kD,等电点为7.76。氨基酸同源性比对发现,泥蚶TgTLR3氨基酸序列与毛蚶相似度高达80.05%。系统进化树显示,TgTLR3与毛蚶首先聚为一支,表明与毛蚶亲缘关系最近。TgTLR3在泥蚶各组织中均有表达,其中血淋巴表达量最高。缺氧胁迫48~96 h后,血淋巴中TgTLR3显著升高;添加硫化物后,TgTLR3在48 h显著低于缺氧组但在96 h仍显著高于对照。上述研究结果表明,TgTLR3基因在泥蚶应对缺氧和硫化物胁迫的免疫调控中可能发挥着重要作用。研究结果为探究软体动物TLR3的功能及其响应缺氧及硫化物胁迫的免疫机制提供了基础数据。In order to investigate the biological characteristics and immunoregulatory effects of the Toll-like receptor 3(TLR3)on hypoxia and sulfide exposure in the blood clam Tegillarca granosa,we cloned the core sequence of TgTLR3 and examined mRNA the expression level of TgTLR3 in haemolymph,gill,hepatopancreas,mantle,adductor muscle,foot and haemolymph.The mRNA level of TgTLR3 in haemolymph was also examined when exposed to hypoxia and sulfide using quantitative real-time fluorescence PCR(qRT-PCR).The results showed that the TgTLR3 has an open reading frame(ORF)of 2634 bp which encodes 877 amino acids.Among them,the position of 49-593 represents extra cellular leucine-rich repeats(LRRs),at position of 658-680 and 20-865 represent the transmembrane domain and the interleukin receptor 1 structural domain(TIR),respectively.The molecular weight(Mw)and putative isoelectric point(pI)of TgTLR3 were 99.4 kD and 7.76,respectively.The amino acid homology of T.granosa comparison showed that the TgTLR3 sequence was most similar to that of Anadara sativa,with the identity and similarity of 80.05%.The phylogenetic tree analysis showed that TgTLR3 was clustered with A.sativa and was the closest relative to A.sativa.TgTLR3 was expressed in all tissues and it was most expressed in the haemolymph.After hypoxia exposure,TgTLR3 in the haemolymph was significantly up-regulated at 48-96 h.After the combined hypoxia and sulfide exposure,TgTLR3 was significantly lower than the hypoxia group at 48 h but it was still significantly higher than the control group at 96 h.The findings suggest that TgTLR3 might play an important role in the immune response to hypoxia and sulfide in T.granosa.This study provides a basis data for investigating for the function of TLR3 and the immune mechanism in response to hypoxia and sulfide stresses in mollusc.

关 键 词：泥蚶 Toll样受体3(TLR3) 克隆 缺氧 硫化物 

分 类 号：Q959.215[生物学—动物学]