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三疣梭子蟹(Portunus trituberculatus)CTL基因SNP位点E4-205 C/T与抗溶藻弧菌相关性分析_^*
郝贵杰^1,2, 林锋³, 雷宁³, 黄爱霞³, 崔雁娜³, 沈亚芳³, 张海琪①³, 文菁⁴
1. 岭南师范学院生命科学与技术学院广东湛江 524048;2. 农业农村部淡水渔业健康养殖重点实验室浙江省鱼类健康与营养重点实验室浙江省淡水水产研究所浙江湖州 313001;3.农业农村部淡水渔业健康养殖重点实验室浙江省鱼类健康与营养重点实验室浙江省淡水水产研究所浙江湖州;4.岭南师范学院生命科学与技术学院广东湛江

摘要:

C-型凝集素(C-type lectin, CTL)是甲壳类动物体液免疫中重要的免疫因子之一。而溶藻弧菌感染三疣梭子蟹(Portunus trituberculatus)过程中CTL基因的表达和抗病机制尚不明确。为初步阐明CTL基因SNP E4-205 C/T位点与抗溶藻弧菌感染相关的分子机制,对不同基因型梭子蟹进行溶藻弧菌感染,通过绝对定量方法分析不同基因型梭子蟹肝胰腺、肌肉组织中溶藻弧菌的复制情况。发现溶藻弧菌感染后12 h内梭子蟹肝胰腺、肌肉组织中C/C组细菌数量显著高于T/T组,结果表明T/T组梭子蟹抗感染能力显著高于C/C组。进一步对该位点非同义突变(ACT-ATT)导致的一个氨基酸改变(Thr-Ile)的两种蛋白进行体外重组表达,并对两种重组蛋白CTL-ATT及CTL-ACT进行活性分析。发现两种重组蛋白对溶藻弧菌生长均具有一定的抑制作用,CTL-ATT的抑菌活性显著高于CTL-ACT。重组蛋白CTL-ATT与PAMPs和溶藻弧菌结合活性均高于CTL-ACT,两种蛋白与PAMPs和溶藻弧菌的结合活性具有浓度依赖性。结果表明三疣梭子蟹CTL基因参与机体感染溶藻弧菌后的免疫应答过程,其多态性与抗溶藻弧菌感染存在一定的关联性,三疣梭子蟹CTL基因SNP E4-205 C/T位点为T/T基因型时的抗溶藻弧菌感染能力强于C/C型。

关键词: 三疣梭子蟹(Portunus trituberculatus) 溶藻弧菌单核苷酸多态性凝集素高分辨率溶解曲线

DOI：10.11693/hyhz20230100019

分类号:

基金项目:国家自然科学基金项目, 42076087号

附件

SNP SITE E4-205 C/T OF CTL GENE AND ITS ASSOCIATION WITH VIBRIO ALGINOLYTICUS RESISTANCE IN PORTUNUS TRITUBERCULATUS

HAO Gui-Jie^1,2, LIN Feng³, LEI Ning³, HUANG Ai-Xia³, CUI Yan-Na³, SHEN Ya-Fang³, ZHANG Hai-Qi³, WEN Jing⁴

1. Life Science and Technology School, Lingnan Normal University, Zhanjiang 524048, China;2. Key Laboratory of Healthy Freshwater Aquaculture, Ministry of Agriculture and Rural Affairs, Key Laboratory of Fish Health and Nutrition of Zhejiang Province, Institute of Freshwater Fisheries, Huzhou 313001, China;3.Key Laboratory of Healthy Freshwater Aquaculture,Ministry of Agriculture and Rural Affairs,Key Laboratory of Fish Health and Nutrition of Zhejiang Province,Institute of Freshwater Fisheries;4.Life Science and Technology School,Lingnan Normal University

Abstract:

C-type lectin (CTL) is one of the important immune factors in humoral immunity of crustaceans. However, the expression of CTL gene and disease-resistant mechanism in Portunus trituberculatus infected by Vibrio alginolyticus are not clear. In order to clarify the molecular mechanism related to the anti-V. alginolyticus infection at the site of CTL gene SNP E4-205 C/T, the different genotypes of P. trituberculatus infected with V. alginolyticus and analyzed the replication of V. alginolyticus in hepatopancreas and muscle tissues of P. trituberculatus by absolute quantitative method. The results showed that the number of bacteria in group C/C in hepatopancreas and muscle tissues within 12h after infection with V. alginolyticus was significantly higher than that in group T/T. The anti-infection ability of P. trituberculatus in group T/T was significantly higher than that in group C/C. Furthermore, the two proteins of one amino acid change (Thr-Ile) caused by non-synonymous mutation (ACT-ATT) at this site were recombinantly expressed and purified in vitro, and the activities of the two recombinant proteins CTL-ATT and CTL-ACT were analyzed. The results showed that the two recombinant proteins both inhibited the growth of V. alginolyticus, and the antibacterial activity of CTL-ATT was significantly higher than that of CTL-ACT. The binding activities of PAMPs and V. alginolyticus of recombinant protein CTL-ATT were higher than those of CTL-ACT, and the binding activities of two proteins to PAMPs and V. alginolyticus were concentration-dependent. The results of this study indicated that the TCL gene of P. trituberculatus was involved in the immune response process after the body was infected with V. alginolyticus, and its polymorphism had a certain correlation with anti-V. alginolyticus infection. When the site of SNP E4-205 of the CTL gene was the T/T genotype, the anti-V. alginolyticus infection ability of P. trituberculatus was higher than that of C/C type.

Key words: Portunus trituberculatus Vibrio alginolyticus Single nucleotide polymorphism Lectins High resolution dissolution curve