Expression Analysis and SNP Mining of the Parkin Co-Regulated Gene (PACRG) and parkin in Penaeus vannamei Against White Spot Syndrome Virus

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.

Keywords

• p. vannamei
• white spot syndrome virus
• parkin co-regulated gene
• parkin
• single nucleotide polymorphism