凡纳滨对虾易位子相关蛋白α亚基(TRAPα)基因解析及其与WSSV抗性的关联  

Analysis of the translocon-associated protein alpha subunit(TRAPα)gene and association study of TRAPαwith WSSV resistance in Litopenaeus vannamei

作　　者：王晶晶 李旭鹏[2,3] 薛倩 曹宝祥 栾生[2,3] 罗坤[2] 隋娟 代平[2,3] 曹家旺[2] 谭建 陈宝龙[2] 傅强 孔杰 高焕 孟宪红[2,3] WANG Jingjing;LI Xupeng;XUE Qian;CAO Baoxiang;LUAN Sheng;LUO Kun;SUI Juan;DAI Ping;CAO Jiawang;TAN Jian;CHEN Baolong;FU Qiang;KONG Jie;GAO Huan;MENG Xianhong(College of Marine Science and Fisheries,Jiangsu Ocean University,Key Laboratory of Marine Biological Resources and Environment of Jiangsu Province,Lianyungang 222005,China;State Key Laboratory of Mariculture Biobreeding and Sustainable Goods,Yellow Sea Fisheries Research Institute,Chinese Academy of Fishery Sciences,Qingdao 266071,China;Laboratory for Marine Fisheries Science and Food Production Processes,Qingdao Marine Science and Technology Center,Qingdao 266237,China)

机构地区：[1]江苏海洋大学海洋科学与水产学院,江苏省海洋生物资源与环境重点实验室/江苏省海洋生物技术重点实验室,江苏连云港222005 [2]中国水产科学研究院黄海水产研究所,海水养殖生物育种与可持续产出全国重点实验室,山东青岛266071 [3]青岛海洋科技中心海洋渔业科学与食物产出过程功能实验室,山东青岛266237

出　　处：《中国水产科学》2024年第1期1-13,共13页Journal of Fishery Sciences of China

基　　金：国家重点研发计划项目(2022YFF1000304);国家自然科学基金项目(32172960);国家现代农业产业技术体系资助项目(CARS-48);中央级公益性科研院所基本科研业务费专项经费项目(2020TD26);湛江市海洋装备与海洋生物揭榜挂帅制人才团队项目(2021E05032)。

摘　　要：本研究旨在探讨凡纳滨对虾(Litopenaeusvannamei)易位子相关蛋白α亚基(translocon-associatedproteinalpha,TRAPα)基因特征及其在抗白斑综合征病毒(white spot syndrome,WSSV)中的作用。通过PCR和Sanger测序技术,获得凡纳滨对虾TRAPα的开放阅读框(openreadingframe,ORF)序列,将该基因命名为Lv-trapα,并进行生物信息学分析。采用real-time PCR分析Lv-trapα基因在健康凡纳滨对虾和感染WSSV不同时间点的凡纳滨对虾肝胰腺、鳃、肌肉、眼柄中的Lv-trapα表达水平。同时,利用重亚硫酸氢盐测序技术(bisulfite sequencing PCR,BSP)检测健康凡纳滨对虾和感染WSSV后96 h的凡纳滨对虾肝胰腺组织中Lv-trapα基因上游DNA序列的甲基化水平。结果显示,Lv-trapα的ORF全长873 bp,共编码290个氨基酸,预测相对分子质量为32466.4,理论等电点为4.45。多序列比对发现TRAPα蛋白的保守性较高。Lv-trapαDNA序列中有8个单核苷酸多态性位点(single nucleotide polymorphism,SNP),其中1个SNP位点处于外显子区域且属于错义突变,其余7个SNP位点处于内含子区域。real-time PCR结果显示,Lv-trapα基因在凡纳滨对虾肝胰腺、鳃、肌肉、眼柄中均有表达,且在感染WSSV后显著上调表达(P<0.05)。值得注意的是,在感染WSSV后96 h,体内病毒含量不同的凡纳滨对虾肝胰腺中Lv-trapα的表达水平差异显著,高病毒含量组中Lv-trapα的表达水平显著高于低病毒含量组(P<0.05),提示Lv-trapα表达水平和WSSV复制水平存在正相关性。BSP结果显示,Lv-trapα基因上游1个CpG位点(存在于NCBI数据库NW_020872863.1第360336-360337 nt位置)的甲基化水平和Lv-trapα表达水平呈负相关,该CpG位点的甲基化水平和凡纳滨对虾体内WSSV病毒含量也呈负相关。本研究可为深入研究凡纳滨对虾抗WSSV的分子机制和抗病分子育种提供理论参考。This study aims to explore the genetic characteristics of the translocon-associated protein alpha subunit(TRAPα)of Litopenaeus vannamei and its role in resistance to white spot syndrome virus(WSSV).The ORF(open reading frame)sequence of TRAPαfrom L.vannamei was obtained through PCR and Sanger sequencing techniques.The gene was named Lv-trapαand bioinformatics analysis was conducted.Real-time PCR was used to analyze the expression level of the Lv-trapαgene in the hepatopancreas,gills,muscle,and eyestalk of healthy L.vannamei and those infected with WSSV at different time points.Meanwhile,bisulfite sequencing PCR(BSP)was used to detect the methylation level of the upstream DNA sequence of the Lv-trapαgene in the hepatopancreas of healthy L.vannamei and those infected with WSSV after 96 h.The results showed that the ORF of Lv-trapαwas 873 bp in length,encoding 290 amino acids.The predicted relative molecular mass was 32466.4 and theoretical isoelectric point was 4.45.Multiple sequence alignment with various species including Penaeus chinensis,Procambarus clarkii,and Portunus trituberculatus revealed that conservation of the TRAPαprotein was relatively high.Through PCR and Sanger sequencing techniques,eight single nucleotide polymorphism(SNP)sites were found in the Lv-trapαDNA sequence,among which one SNP site was located in the exon region and belonged to a missense mutation,while the other seven SNP sites were located in intron regions.Real-time PCR showed that the Lv-trapαgene was expressed in the hepatopancreas,gills,muscle,and eyestalk of L vannamei,and its expression was significantly up-regulated(P<0.05)after infection with WSSV.Notably,96 h post infection with WSSV,the expression level of Lv-trapαin the hepatopancreas of L.vannamei with different internal viral loads showed significant differences.The expression level of Lv-trapαin the HPC group was significantly higher than that in the LPC group(P<0.05),suggesting that the expression level of Lv-trapαwas positively correlated with the replication

关 键 词：凡纳滨对虾 白斑综合征病毒(WSSV) TRAP 表达水平 SNP DNA甲基化 

分 类 号：S917[农业科学—水产科学]