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OA北大核心CSTPCD

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 tritub erculatus 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.