凡纳对虾parkin共调基因和parkin在抗白斑综合征病毒中的表达分析和SNP开发

Expression analysis and SNP mining of the parkin co-regulated gene (PACRG) and parkin in Penaeus vannamei against white spot syndrome virus

关键词:  凡纳对虾  白斑综合征病毒(WSSV)  parkin共调基因(PACRG)  parkin基因  SNP

Key words:  P. vannamei  White spot syndrome virus  parkin co-regulated gene  parkin  Single nucleotide polymorphism

摘要:

本实验室前期研究中基于全基因组关联分析(GWAS)方法筛选到抗白斑综合征病毒(WSSV)候选基因：parkin共调基因(PACRG)。PACRG与帕金森病相关基因parkin共用一个双向启动子，二者共同参与细胞自噬过程，从而在细胞保护方面发挥作用。本研究对凡纳对虾(Penaeus vannamei) PACRG和parkin在抗WSSV中的功能进行探讨，对其mRNA和氨基酸序列进行特征分析，利用real-time PCR技术检测对虾感染WSSV后不同时间、不同组织中PACRG和parkin的表达水平。通过荧光原位杂交技术(FISH)进行空间定位。利用PCR和Sanger测序技术获得单核苷酸多态性位点(SNP)并进行抗WSSV的关联分析。结果显示，PACRG开放阅读框(ORF)全长600 bp，编码199个氨基酸，预测包含一个ParcG结构域。parkin ORF序列全长1 653 bp，编码550个氨基酸，预测包含UBQ、IBR结构域和一个信号肽结构。与多物种进行同源序列比对发现，凡纳对虾PACRG氨基酸序列与日本对虾(Penaeus japonicus)的同源性高达89.70%；凡纳对虾parkin氨基酸序列与中国对虾(Penaeus chinensis)和斑节对虾(Penaeus monodon)的同源性高达93.45%。PACRG和parkin蛋白的保守性较高。感染WSSV后，PACRG和parkin在对虾肝胰腺、鳃、肌肉和眼柄中的表达水平发生显著变化，其中，眼柄中PACRG和parkin表现出极相似的组织表达模式。在肌肉中，PACRG mRNA和WSSV在空间位置上呈现高度重叠状态。结合上述结果，推测PACRG和parkin在凡纳对虾与WSSV的互作中发挥功能。在PACRG中筛选到2个SNP位点，在parkin中筛选到15个SNP位点，其中位于parkin非翻译区(UTR)的5个SNP位点与抗WSSV性状显著相关。本文为研究凡纳对虾抗WSSV的分子机制和抗病分子育种提供了理论依据和参考数据。

Abstract:

In a preliminary study conducted in our laboratory, the parkin co-regulated gene (PACRG) was identified as a candidate for white spot syndrome virus (WSSV) resistance using a genome-wide association approach. PACRG is genetically closely linked to the Parkinson´s disease-associated gene parkin, both of which are regulated by a bidirectional promoter. The PACRG and parkin genes have been found to interact with each other, associate with autophagy, and participate in cellular protection. Therefore, the functions of PACRG and parkin in WSSV resistance in Penaeus vannamei were investigated. The mRNA and amino acid sequences were analyzed, and the expression levels in shrimp infected with WSSV at different times and tissues were detected by real-time PCR. Spatial localization was performed using fluorescence in situ hybridization. PCR and Sanger sequencing were employed to obtain single nucleotide polymorphisms (SNPs) and conduct an association analysis of these SNPs with resistance to WSSV. Our findings illustrated that the complete open reading frame (ORF) sequence of PACRG was 600 bp, encoded 199 amino acids, and was predicted to contain the ParcG structural domain. The complete sequence of parkin mRNA was 2,329 bp, comprising a 1,653 bp ORF, 100 bp 5′-untranslated region (UTR), and a 576 bp 3′-UTR, encoding 550 amino acids. Parkin is predicted to contain UBQ and IBR structural domains and a signal peptide structure. Amino acid sequence alignment and phylogenetic tree analysis showed that the homology of PACRG between P. vannamei and Penaeus japonicus was the highest at 89.70% similarity. The phylogenetic relationship of P. vannamei was the closest to Penaeus chinensis and P. japonicus. Thus, PACRG may exhibit high evolutionary conservation. The parkin homology between P. vannamei and P. chinensis was the highest, with a similarity of 93.45%. It has been speculated that the parkin protein exhibits a high degree of evolutionary conservation. Herein, real-time PCR results suggested that PACRG and parkin were expressed in the hepatopancreas, gill, muscle, and eyestalk of healthy P. vannamei, with no significant difference. Following the challenge with WSSV, the PACRG and parkin expression levels in the hepatopancreas, gill, muscle, and eyestalk of P. vannamei were significantly altered. Post-WSSV infection for 48, 96, 192, and 228 h, the PACRG and parkin expression levels in the hepatopancreas of P. vannamei were significantly downregulated. At 48, 72, 96, 144, 192, and 228 h post-WSSV infection, PACRG expression in the gill of P. vannamei were significantly downregulated. However, at 48, 96, and 228 h post-WSSV infection, the parkin expression levels in the gill of P. vannamei were significantly upregulated. Post-WSSV infection at 96, 192, and 228 h, the PACRG and parkin expression levels in P. vannamei muscle were significantly upregulated. Post-WSSV infection, PACRG and parkin exhibited similar expression patterns in the eyestalk. The location of PACRG mRNAs mostly overlapped with the WSSV replication site in the shrimp muscle, suggesting that PACRG plays a functional role in the interaction between P. vannamei and WSSV. Two SNPs were identified within the ORF of the PACRG, One SNP was identified within the ORF of parkin, and one SNP was identified in the UTR of parkin. After conducting association analyses of these SNPs with WSSV resistance, SNPs located in the UTR of parkin-specific SNP3, SNP4, SNP5, SNP7, and SNP9 were significantly associated with resistance to WSSV. This study provides a theoretical reference for future research on the molecular mechanisms underlying P. vannamei’s resistance to WSSV.