Molecular characterization of a p38 MAPK from Litopenaeus vannamei and its expression during the molt cycle and following pathogen infection.

The p38 mitogen-activated protein kinase (MAPK), a serine/threonine-specific protein kinase, has been reported to be involved in innate immunity, development and muscle differentiation. To explore the function of p38 in shrimp, partial cDNA sequence of p38 in Litopenaeus vannamei (designated as Lv-p38) was characterized and the expression of Lv-p38 in hepatopancreas of the shrimp after being infected with Vibrio parahaemolyticus and in muscle of the shrimp at different molt stages was detected by quantitative RT-PCR in this study. The results showed that the open reading frame of Lv-p38 was 1098 bp and encoded a protein of 365 amino acids. The protein of Lv-p38 which showed close phylogenetic relationship to Marsupenaeus japonicus p38 had a conserved TGY motif and serine/threonine protein kinase (S_TKc) domain. The expression of Lv-p38 was detected in all tested tissues, especially in the hepatopancreas and muscle. The expression of Lv-p38 in the hepatopancreas was different from that of the control at the 24th hour after the injection of V. parahaemolyticus and in the muscle significantly increased at stage C but decreased at other stages during molt, illustrating that Lv-p38 could be involved in pathogen infection and the molt cycle of shrimp. In conclusion, we identified Lv-p38 and studied its role in pathogen infection and molting, which might facilitate our understanding of the function of p38 in innate immunity and growth during molt of shrimp.

He S ，Qian Z ，Yang J ，Wang X ，Mi X ，Liu Y ，Hou F ，Liu Q ，Liu X ... -  《-》

Characterization of the putative farnesoic acid O-methyltransferase (LvFAMeT) cDNA from white shrimp, Litopenaeus vannamei: Evidence for its role in molting.

Hui JH ，Tobe SS ，Chan SM 《PEPTIDES》

Molecular characterization and function of a p38 MAPK gene from Litopenaeus vannamei.

p38 mitogen-activated protein kinases (MAPKs) are broadly expressed from yeasts to mammals, and are involved in the regulation of cells responsible to various extracellular stimuli. In this study, a p38 MAPK gene (designated as Lvp38) from Litopenaeus vannamei, was cloned and characterized. It contained the conserved structures of a Thr-Gly-Tyr (TGY) motif and a substrate-binding site, Ala-Thr-Arg-Trp (ATRW). The tissue distribution patterns showed that Lvp38 was widely expressed in all examined tissues, with the highest expression in hemocytes, nerves, and intestines. Quantitative real-time PCR revealed that Lvp38 was upregulated in gills and hemocytes after infection with the Gram-negative Vibrio alginolyticus and the Gram-positive Staphylococcus aureus. Reporter gene assays indicated that Lvp38 activated the expression of antimicrobial peptides (AMPs) of Drosophila and shrimp. Knockdown of Lvp38 by RNA interference (RNAi) resulted in a higher mortality of L. vannamei under V. alginolyticus and S. aureus infection, as well as a reduction in the expression of three shrimp AMP genes, namely, PEN4, crustin, and ALF2. Taken together, our data indicated that Lvp38 played a role in defending against bacterial infections.

Yan H ，Zhang S ，Li CZ ，Chen YH ，Chen YG ，Weng SP ，He JG ... -  《-》

MKK4 from Litopenaeus vannamei is a regulator of p38 MAPK kinase and involved in anti-bacterial response.

LvMKK4, a homologue of the mammalian mitogen-activated protein kinase kinase 4 (MKK4), was isolated and identified from Litopenaeus vannamei in the present study. The full-length cDNA of LvMKK4 is 1947 bp long, with an open reading frame (ORF) of 1185 bp encoding a putative protein of 388 amino acids. LvMKK4 contains several characteristic domains such as D domain, SIAKT motif and kinase domain, all of which are conserved in MAP kinase kinase family. Like mammalian MKK4 but not Drosophila MKK4, LvMKK4 could bind to, phosphorylate and activate p38 MAPK, which provided some insights into the signal transduction mechanism of MKK4-p38 cascade in invertebrates. Our real-time PCR data indicated that LvMKK4 was ubiquitously expressed in all tested tissues and extraordinarily abundant in muscle. Dual luciferase reporter assays in Drosophila S2 cells revealed that LvMKK4 activated the transcription of antimicrobial peptide genes (AMPs), including Drosophila Attacin A, Drosomycin, and shrimp Penaeidins. Additionally, LvMKK4 was up-regulated in both intestine and hepatopancreas by a variety of inflammatory stimuli including LPS, Vibrio parahaemolyticus, Staphhylococcu saureus, Poly (I: C) and white spot syndrome virus. Furthermore, RNAi-mediated knockdown of LvMKK4 enhanced the sensitivity of L. vannamei to V. parahaemolyticus infection. These findings suggested that LvMKK4 played an important role in anti-bacterial response and could be a potential target for inflammation treatment.

Wang S ，Yin B ，Li H ，Xiao B ，Lǚ K ，Feng C ，He J ，Li C ... -  《-》

Two types of calmodulin play different roles in Pacific white shrimp (Litopenaeus vannamei) defenses against Vibrio parahaemolyticus and WSSV infection.

Calmodulin (CaM) plays an important role in calcium-dependent signal transduction pathways. In the present study, two alternative splicing isoforms of CaM (named LvCaM-A and LvCaM-B) cDNA were cloned from the Pacific white shrimp, Litopenaeus vannamei. LvCaM-A was of 1101 bp, including a 5'-terminal untranslated region (UTR) of 70 bp, a 3'-terminal UTR of 581 bp and an open reading frame (ORF) of 450 bp encoding a polypeptide of 149 amino acids with a calculated molecular weight (Mw) of 17 kDa and pI of 4.41. LvCaM-B was 689 bp, including a same 5'-UTR of 70 bp, a 3'-terminal UTR of 109 bp and an ORF of 510 bp encoding a polypeptide of 169 amino acids with a calculated Mw of 19 kDa and pI of 4.36. Both LvCaM-A and LvCaM-B contained 4 conservative EF-hand motifs. Quantitative real-time reverse transcription PCR analysis revealed LvCaM-A to be expressed in most shrimp tissues, with the predominant expression in nerve and the weakest expression in heart. However, LvCaM-B expression level was much weaker than those of LvCaM-A in all the tested tissues with main expression in hepatopancreas. The expression of LvCaM-A and LvCaM-B after challenge with Vibrio parahaemolyticus and WSSV were tested in hemocytes, hepatopancreas and nerve. The results indicated that LvCaM-A and LvCaM-B transcripts could be significantly induced in hemocytes and hepatopancreas respectively by injection with V. parahaemolyticus. The highest expression of LvCaM-A was in the hemocytes with 2.3 times (at 48 h) greater expression than in the control (p < 0.05). However, sharp down-regulation of both LvCaM-A and LvCaM-B were detected in nerve after Vibrio- and WSSV injection (p < 0.05). These results suggested that CaM might play an important role in shrimp's defense against pathogenic infection.

Ji PF ，Yao CL ，Wang ZY 《-》