【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-trapα DNA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。更多还原

【Abstract】 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 TRAPα from L. vannamei was obtained through PCR and Sanger sequencing techniques. The gene was named Lv-trapα and bioinformatics analysis was conducted. Real-time PCR was used to analyze the expression level of the Lv-trapα gene 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-trapα gene in the hepatopancreas of healthy L. vannamei and those infected with WSSV after 96 h. The results showed that the ORF of Lv-trapα was 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 TRAPα protein was relatively high. Through PCR and Sanger sequencing techniques, eight single nucleotide polymorphism(SNP) sites were found in the Lv-trapα DNA 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-trapα gene 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-trapα in the hepatopancreas of L. vannamei with different internal viral loads showed significant differences. The expression level of Lv-trapα in the HPC group was significantly higher than that in the LPC group(P<0.05), suggesting that the expression level of Lv-trapα was positively correlated with the replication level of WSSV. Results of bisulfite sequencing technology showed that the methylation level of an upstream CpG site(located at position 360336-360337 in the NCBI database NW＿020872863.1) of the Lv-trapαgene was negatively correlated with the expression level of Lv-trapα, and the methylation level of this CpG site was also negatively correlated with the WSSV viral load in L. vannamei. This study provides a theoretical reference for in-depth research on the molecular mechanisms of L. vannamei resistance to WSSV and molecular-assisted breeding for disease-resistance.更多还原