Effects of rrm1 on NNV Resistance Revealed by RNA-seq and Gene Editing

Abstract

Viral nervous necrosis (VNN) disease caused by the nervous necrosis virus (NNV) is a major disease, leading to a huge economic loss in aquaculture. Previous GWAS and QTL mapping have identified a major QTL for NNV resistance in linkage group 20 in Asian seabass. However, no causative gene for NNV resistance has been identified. In this study, RNA-seq from brains of Asian seabass fingerlings challenged with NNV at four time points (5, 10, 15 and 20 days post-challenge) identified 1228, 245, 189 and 134 DEGs, respectively. Eight DEGs, including rrm1, were located in the major QTL for NNV resistance. An association study in 445 survived and 608 dead fingerlings after NNV challenge revealed that the SNP in rrm1 were significantly associated with NNV resistance. Therefore, rrm1 was selected for functional analysis, as a candidate gene for NNV resistance. The expression of rrm1 was significantly increased in the gill, liver, spleen and muscle, and was suppressed in the brain, gut and skin after NNV challenge. The rrm1 protein was localized in the nuclear membrane. Overexpression of rrm1 significantly decreased viral RNA and titer in NNV-infected Asian seabass cells, whereas knock-down of rrm1 significantly increased viral RNA and titer in NNV-infected Asian seabass cells. The rrm1 knockout heterozygous zebrafish was more susceptible to NNV infection. Our study suggests that rrm1 is one of the causative genes for NNV resistance and the SNP in the gene may be applied for accelerating genetic improvement for NNV resistance.

Genomic resources and their applications in aquaculture of Asian seabass (Lates calcarifer)

“…37 Another gene associated with NNV resistance is rrm1 (ribonucleotide reductase catalytic subunit M1). 79 After NNV challenge, rrm1 expression was dramatically enhanced in the gill, liver, spleen and muscle, while it was repressed in the brain, stomach and skin. In NNV-infected Asian seabass cells, over-expression of rrm1 decreased viral RNA and titre, but knocking down of rrm1 boosted viral RNA and titre.…”

“…These genes were identified by the use of QTL mapping and GWAS for NNV resistance. 37,69,78,79 For example, gab3 was cloned and described after detecting a significant QTL for NNV resistance on linkage group (LG) 20. 35 Its cDNA is 2165 bp long, having a 1674-bp open reading frame (ORF) that encodes 557 AAs.…”

“…In Asian seabass, ESTs and transcriptomes have been generated to address various important biological questions related to aquaculture, such as immune response to pathogens and parasites, 32,54,79,[92][93][94] responses to various stresses [95][96][97][98] and feeds, 99 growth differences and temperature adaptation 100 (Table 3). The first set of 11,431 high-quality cDNA sequences were obtained by sequencing ESTs from nine normalized cDNA libraries using Sanger sequencing and bioinformatics analysis, and was reported in 2011.…”

“…[142][143][144] Although the knowledge of some known genes found in model organisms, human and livestock can be applied in genome editing in aquaculture, it is still essential to identify DNA sequence variants in causative genes for important traits in aquaculture species. 1,79,142 In Asian seabass, a few genes related to NNV and iridovirus were identified and characterized, such as gab3 (GRB2 associated binding protein 3), rrm1 (ribonucleotide reductase catalytic subunit M1) and snx2 (sorting nexin 2). 37,40,79 In Asian seabass, the gab3 cDNA (complementary DNA) is 2165 bp long, with an ORF (open reading frame) of 1674 bp that encodes 557 amino acids.…”

“…1,79,142 In Asian seabass, a few genes related to NNV and iridovirus were identified and characterized, such as gab3 (GRB2 associated binding protein 3), rrm1 (ribonucleotide reductase catalytic subunit M1) and snx2 (sorting nexin 2). 37,40,79 In Asian seabass, the gab3 cDNA (complementary DNA) is 2165 bp long, with an ORF (open reading frame) of 1674 bp that encodes 557 amino acids.…”