Identification, cloning and characterization of an extracellular signal-regulated kinase (ERK) from Chinese shrimp, Fenneropenaeus chinensis.

Extracellular signal-regulated kinase (ERK) is a serine/threonine-specific protein kinase, which participates in signaling transduction pathways that control intracellular events, including resumption of meiosis, embryogenesis, cell differentiation, cell proliferation, cell death and response to radiation. Some virus species evolved the ability to hijack the host cell ERK signaling transduction pathway for viral replications and gene expressions. To obtain a better understanding of ERK, we cloned a cDNA encoding ERK from the muscle of Fenneropenaeus chinensis (FcERK). The FcERK contained a 1098 bp open reading frame (ORF) encoding a protein of 365 amino acid residues with a conserved phosphorylation motif TEY in the kinase activation loop. Pair-wise and multiple sequence alignment revealed that ERK is highly conserved across taxa. The FcERK gene expressions in the hepatopancreas and gill were noticeably higher than the expression observed in the muscle. A challenge test was performed to reveal the responses of FcERK in different tissues to white spot syndrome virus (WSSV) infection. Post WSSV challenge, the FcERK expression in the gill significantly increased during the early stage of the viral infection, the FcERK expression in the muscle increased later than that in the gill, and the FcERK expression in the hepatopancreas significantly decreased. The FcERK gene expression profile accorded with the results that the virus primarily infects tissues originating from the ectoderm, with less infection of the tissues originating from the mesoderm, and hardly any infection in the tissues originating from the entoderm. Two single nucleotide polymorphisms (SNPs) were identified in the FcERK gene, involving C/T transition. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps were obtained using a high-resolution melting (HRM) method. In the two groups, the MAFs of both sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distributions of both mutation sites between the two groups were not significantly different. These results lead to a better understanding of the molecular mechanisms of the host-virus interaction and provide useful information for disease control.

Li X ，Meng X ，Kong J ，Luo K ，Luan S ，Cao B ，Liu N ，Pang J ，Shi X ... -  《-》

Molecular cloning and characterization of a cathepsin B gene from the Chinese shrimp Fenneropenaeus chinensis.

Cathepsin B is a unique member of the cathepsin superfamily, which acts as both an endopeptidase and peptidyl-dipeptidase. To obtain a better understanding of this enzyme, we cloned a cDNA encoding cathepsin B from the muscle of Fenneropenaeus chinensis (FcCB). FcCB contained a 996-bp open reading frame (ORF) encoding a protein of 331 amino acid residues with a putative signal peptide and a propeptide_C1 at the N-terminal, a glutamine oxyanion hole and active site cysteine, histidine and asparagine residues. A region from residue 79 to 327 conferred the peptidase activity of FcCB. Pair-wise and multiple sequence alignment with 17 other organisms, including ten different vertebrate species, five different invertebrate species and two different plant species, indicated that the signal peptide and the propeptide_C1 at the N-terminal of FcCB were less conserved than the mature protein, except when compared with Penaeus monodon, Litopenaeus vannamei and Marsupenaeus japonicas, all of which belong to the genus Penaeus. The expression of FcCB in the hepatopancreas was higher than that in the gill. The expression of FcCB in the gill was higher than that in the muscle. A challenge test was performed to reveal the responses of FcCB in different tissues to white spot syndrome virus (WSSV) infection, which causes serious economic losses in the shrimp farming industry. The FcCB gene expressions in the ectoderm, mesoderm and entoderm were not the same prior to WSSV infection, but at 6 h after WSSV challenge, the FcCB expression in the gill, hepatopancreas and muscle was up-regulated, suggesting that FcCB might be involved in the immune response to WSSV. Three single nucleotide polymorphisms (SNPs) were identified in the FcCB gene, involving C/T transitions, which are known as mutation hot spots. Notably, the three SNPs constituted a haplotype that can be used as an indicator of the haplotype block. The SNP genotypes of two groups of shrimps, respectively comprising 96 WSSV-resistant shrimps and 96 WSSV-susceptible shrimps, were obtained using a high-resolution melting (HRM) method. Associated factors, including observed heterozygosity (Ho), expected heterozygosity (He), minor allele frequency (MAF) and P-values for the deviation from Hardy-Weinberg equilibrium (HWE), were obtained. For the association analysis with WSSV resistance, the P-values were calculated using Pearson's chi-square test. In the two groups, the MAFs of all sites were greater than 0.05, and no site departed significantly (P < 0.05) from HWE. The genotype distribution of the C-984T mutation site between the two groups was not significantly different. These results lead to a better understanding of the molecular mechanisms of the host-virus interaction and provide useful information for solving the WSSV problem.

Li X ，Meng X ，Kong J ，Luo K ，Luan S ，Cao B ，Liu N ，Pang J ，Shi X ... -  《-》

Isolation and expression analysis of an MAPKK gene from Fenneropenaeus chinensis in response to white spot syndrome virus infection.

Mitogen-activated kinase kinase (MAPKK) is an important gene involved in the host-virus interaction process. To obtain a better understanding of MAPKK in the interaction process between the Chinese shrimp Fenneropenaeus chinensis and white spot syndrome virus (WSSV), we cloned the sequence of an MAPKK cDNA from F. chinensis (FcMAPKK) and investigated the effect of FcMAPKK on WSSV infection. The results showed that the FcMAPKK gene contained a 1227 bp open reading frame (ORF), which encoded a highly conserved protein with a serine/threonine protein kinase catalytic (S_TKc) domain. The deduced amino acid sequence of FcMAPKK shared identities between 11.9 and 92.6% with MAPKKs from vertebrate, invertebrate, plant and fungus species. The FcMAPKK was expressed in all the examined tissues in the normal F. chinensis. FcMAPKK expression level was highest in the hepatopancreas where it was approximately 2.6-fold the expression level in the gill, and lowest in the muscle where it was approximately 0.3-fold the expression level in the hepatopancreas. The FcMAPKK expression levels in the muscle, gill, and hepatopancreas were all changed post WSSV challenge. The FcMAPKK expression was significantly (P < 0.01) up-regulated in the muscle of F. chinensis at 48 h post WSSV infection. The WSSV began to replicate quickly in the normal F. chinensis at 48 h post infection, while the WSSV replication in the U0126-treated F. chinensis could be significantly (P < 0.05) inhibited. The results suggested that FcMAPKK might be involved in the WSSV infection process, and hijacking of FcMAPKK might be required for WSSV replication in F. chinensis.

Li X ，Kong J ，Meng X ，Luo K ，Luan S ，Cao B ，Liu N ... -  《-》

cDNA cloning and expression analysis of a phosphopyruvate hydratase gene from the chinese shrimp Fenneropenaeus chinensis.

Li X ，Meng X ，Luo K ，Luan S ，Cao B ，Kong J ... -  《-》

Molecular cloning and characterization of a cDNA encoding extracellular signal-regulated kinase from Litopenaeus vannamei.

Extracellular signal-regulated kinase (ERK) is a serine/threonine-specific kinase, which is activated by downstream signaling molecules of cellular activation, cytokine and chemokine stimulation and various other stimuli. Here we cloned an ERK gene from Litopenaeus vannamei and designated it as lverk. The lverk cDNA contained an open reading frame of 1098 bp encoding 365 amino acids. LVERK had a conserved TEY motif and serine/threonine protein kinase (S_TKc) domain, and close phylogenetic relationship to Penaeus monodon and Marsupenaeus japonicus ERK. Immunofluorescence staining analysis showed that following serum stimulation LVERK was located in cytoplasm and nucleus, but phospho-LVERK was prominently in nucleus, suggesting conserved ERK signaling module occurred in shrimp cells. Then during the white spot syndrome virus (WSSV) infection, LVERK and phospho-LVERK increased at the early stage of infection. Once silencing of lverk in vivo, the replication of WSSV was obviously inhibited. Moreover, treatment of mitogen-activated protein kinase kinase inhibitor in vitro could result in reduction of WSSV proliferation and delay of viral early gene transcription. Our results indicated a role of LVERK involved in WSSV infection. Understanding how WSSV influences ERK signaling pathway to dismantle an effective immune response may lead to insight into pathogenic progression and possible disease control.

Shi H ，Yan X ，Xu X ，Ruan L ... -  《-》