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Cloning and expression of a transcription factor activator protein-1 member identified from the swimming crab Portunus trituberculatus.

ABSTRACT: Transcription activator proteins are regulatory proteins that bind to the promoter regions of target genes. Transcription activator protein-1 (AP-1) regulates numerous genes related to the immune system, apoptosis, and proliferation. In this study, the full-length cDNA of AP-1 from Portunus trituberculatus (PtAP-1) was identified by expressed sequence tag analysis and cDNA-end rapid amplification. The gene is 1183 bp and encodes a 256-amino acid protein with a predicted molecular mass and isoelectric point of 28.96 kDa and 8.90, respectively. PtAP-1 showed the highest expression level in the gonad tissue and the lowest expression level in blood, hemocyte, muscle, hepatopancreas, and gill, during the first 6 h of low-salinity stimulation (10%). Additionally, we observed steady decreases in PtAP-1 mRNA expression in the gill, but at 12 h, expression was initially upregulated, followed by a significant decrease until restoration to baseline levels at 48 h. Additionally, Vibrio alginolyticus challenge resulted in significant upregulation of PtAP-1 expression in the first 6 h, which was maintained at high levels for 48 h. From 48 to 72 h, we observed decreases in PtAP-1 levels, although they remained significantly higher than those detected at baseline. These results suggested that PtAP-1 is involved in the immune response and osmoregulation of crustaceans.

SUBMITTER: Wang H

PROVIDER: S-EPMC6237694 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Molting is one of the most important biological processes of crustacean species, and a number of molecular mechanisms facilitate this complex procedure. However, the understanding of the immune mechanisms underlying crustacean molting cycle remains very limited. This study performed transcriptome sequencing in hemolymph and hepatopancreas of the swimming crab (Portunus trituberculatus) during the four molting stages: post-molt (AB), inter-molt (C), pre-molt (D), and ecdysis (E). The results showed that there were 78,572 unigenes that were obtained in the hemolymph and hepatopancreas of P. trituberculatus. Further analysis showed that 98 DEGs were involved in immunity response of hemolymph and hepatopancreas, and most of the DEGs participated in the process of signal transduction, pattern recognition proteins/receptors, and antioxidative enzymes system. Specifically, the key genes and pathway involved in signal transduction including the GPCR126, beta-integrin, integrin, three genes in mitogen-activated protein kinase (MAPK) signaling cascade (MAPKKK10, MAPKK4, and p38 MAPK), and four genes in Toll pathway (Toll-like receptor, cactus, pelle-like kinase, and NFIL3). For the pattern recognition proteins/receptors, the lowest expression level of 11 genes was found in the E stage, including C-type lectin receptor, C-type lectin domain family 6 member A and SRB3/C in the hemolymph, and hepatopancreatic lectin 4, C-type lectin, SRB, Down syndrome cell adhesion molecule homolog, Down syndrome cell adhesion molecule isoform, and A2M. Moreover, the expression level of copper/zinc superoxide dismutase isoform 4, glutathione peroxidase, glutathione S-transferase, peroxiredoxin, peroxiredoxin 6, and dual oxidase 2 in stage C or stage D significantly higher than that of stage E or stage AB. These results fill in the gap of the continuous transcriptional changes that are evident during the molting cycle of crab and further provided valuable information for elucidating the molecular mechanisms of immune regulation during the molting cycle of crab.

Project description:The development of Portunus trituberculatus egg cells is directly related to the nutritional status of the fertilized egg, which affects the key production stages of offspring hatching. Vitellogenin plays a key role in the nutrient supply required for the development of the egg cells. The c-Jun N-terminal kinase (JNK) is an important member of the mitogen-activated protein kinase (MAPK) superfamily and plays an important role in cell proliferation, transformation, differentiation, and apoptosis. At present, there are no reports on the involvement of the JNK signaling pathway in the reproductive regulation of P. trituberculatus. In this study, rapid amplification of complementary DNA ends amplification technology was used to clone the full length of JNK complementary DNA, which has a length of 2094 bp, including an open reading frame (ORF) of 1266 bp encoding a 421-amino acid protein. The protein includes the S_TKC conserved domain with a TPY phosphorylation site, which is a typical feature of the JNK gene family. Observing tissue sections found the oocytes in the inhibitor group developed slowly, while the oocytes in the activated group showed accelerated development. Meanwhile, Portunus trituberculatus JNK and vitellogenin (Vg) genes exhibited the same trend in the hepatopancreas and ovaries, and the expression of the SP600125 group was downregulated (P < 0.05), while the anisomycin group was upregulated (P < 0.05). In addition, JNK enzyme activity and vitellin (Vn) content in the ovarian tissue showed that the JNK activity of the SP600125 group decreased, while activity increased in the anisomycin group. The accumulation of Vn content in the SP600125 group decreased, and that in the anisomycin group increased. In summary, after injection with inhibitor or activator, the JNK signaling pathway of P. trituberculatus was inhibited or activated, the accumulation of Vn in the ovary was reduced or increased, and ovarian development was inhibited or accelerated, respectively. These results indicated that the JNK signaling pathway is involved in the regulation of Vg synthesis and ovarian development in P. trituberculatus. The results of this study further add to the knowledge of the breeding biology of P. trituberculatus and provide a theoretical reference for the optimization of breeding techniques in aquaculture production systems.

Project description:Carbohydrate is the cheapest source of energy among the three major nutrient groups, an appropriate amount of carbohydrates can reduce feed cost and improve growth performance, but carnivorous aquatic animals cannot effectively utilize carbohydrates. The objectives of the present study are aimed at exploring the effects of dietary corn starch levels on glucose loading capacity, insulin-mediated glycemic responses, and glucose homeostasis for Portunus trituberculatus. After two weeks of feeding trial, swimming crabs were starved and sampled at 0, 1, 2, 3, 4, 5, 6, 12, and 24 hours, respectively. The results indicated that crabs fed diet with 0% corn starch exhibited lower glucose concentration in hemolymph than those fed with the other diets, and glucose concentration in hemolymph remained low with the extension of sampling time. The glucose concentration in hemolymph of crabs fed with 6% and 12% corn starch diets reached the peak after 2 hours of feeding; however, the glucose concentration in hemolymph of crabs fed with 24% corn starch attained the highest value after 3 hours of feeding, and the hyperglycemia lasted for 3 hours and decreased rapidly after 6 hours of feeding. Enzyme activities in hemolymph related to glucose metabolism such as pyruvate kinase (PK), glucokinase (GK), and phosphoenolpyruvate carboxykinase (PEPCK) were significantly influenced by dietary corn starch levels and sampling time. Glycogen content in hepatopancreas of crabs fed with 6% and 12% corn starch first increased and then decreased; however, the glycogen content in hepatopancreas of crabs fed with 24% corn starch significantly increased with the prolongation of feeding time. In the 24% corn starch diet, insulin-like peptide (ILP) in hemolymph reached a peak after 1 hour of feeding and then significantly decreased, whereas crustacean hyperglycemia hormone (CHH) was not significantly influenced by dietary corn starch levels and sampling time. ATP content in hepatopancreas peaked at 1 h after feeding and then decreased significantly in different corn starch feeding groups, while the opposite trend was observed in NADH. The activities of mitochondrial respiratory chain complexes I, II, III, and V of crabs fed with different corn starch diets significantly increased first and then decreased. In addition, relative expressions of genes related to glycolysis, gluconeogenesis, glucose transport, glycogen synthesis, insulin signaling pathway, and energy metabolism were significantly affected by dietary corn starch levels and sampling time. In conclusion, the results of the present study reveal glucose metabolic responses were regulated by different corn starch levels at different time points and play an important role in clearing glucose through increased activity of insulin, glycolysis, and glycogenesis, along with gluconeogenesis suppression.

Project description:BACKGROUND:The swimming crab Portunus trituberculatus is one of the most commonly farmed crustaceans in China. As one of the most widely known and high-value edible crabs, it crab supports large crab fishery and aquaculture in China. Only large and sexually mature crabs can provide the greatest economic benefits, suggesting the considerable effect of reproductive system development on fishery. Studies are rarely conducted on the molecular regulatory mechanism underlying the development of the reproductive system during the mating embrace stage in this species. In this study, we used high-throughput sequencing to sequence all transcriptomes of the P. trituberculatus reproductive system. RESULTS:Transcriptome sequencing of the reproductive system produced 81,688,878 raw reads (38,801,152 and 42,887,726 reads from female and male crabs, respectively). Low-quality (quality <20) reads were trimmed and removed, leaving only high-quality reads (37,020,664 and 41,021,030 from female and male crabs, respectively). A total of 126,188 (female) and 164,616 (male) transcripts were then generated by de novo transcriptome assembly using Trinity. Functional annotation of the obtained unigenes revealed that a large number of key genes and some important pathways may participate in cell proliferation and signal transduction. On the basis of our transcriptome analyses and as confirmed by quantitative real-time PCR, a number of genes potentially involved in the regulation of gonadal development and reproduction of P. trituberculatus were identified: ADRA1B, BAP1, ARL3, and TRPA1. CONCLUSION:This study is the first to report on the whole reproductive system transcriptome information in stage II of P. trituberculatus gonadal development and provides rich resources for further studies to elucidate the molecular basis of the development of reproductive systems and reproduction in crabs. The current study can be used to further investigate functional genomics in this species.

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Cloning and expression of a transcription factor activator protein-1 member identified from the swimming crab Portunus trituberculatus.

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