Search

In this study, the expression of four reference genes (β-actin, EF1-α, GAPDH, and SubF0), two prophenoloxidase genes (proPO1 and proPO2), and the distribution of phenoloxidase (PO) activity in eight tissues (carapace, cuticle, hemolymph, hepatopancreas, muscle, pereiopods, pleopods, and uropods) of black tiger shrimp (Penaeus monodon) were evaluated. According to the geNorm and NormFinder algorithms, the most stable reference gene or the best combinations of reference genes for the evaluation of proPO1 and proPO2 expression are SubF0 for cuticle, muscle, and uropods, EF1-α for hemolymph and pereiopods, EF1-α + SubF0 for carapace, β-actin + SubF0 for hepatopancreas, and β-actin + GAPDH for pleopods. In the hemolymph and uropods, proPO1 expression was significantly stronger than that of proPO2 (433 and 5 times higher, respectively). However, in hepatopancreas, proPO2 expression was significantly stronger than proPO1 (36 times higher). Trypsin cleaves the proPO zymogen into active PO in most of the studied tissues, except hepatopancreas. PO activity (units/mg protein) following trypsin activation varied among the tissues in the following order: uropods > hemolymph > pleopods > pereiopods > hepatopancreas > carapace > cuticle > muscle. The results indicate that proPO1 is dominant in hemolymph, whereas proPO2 is dominant in hepatopancreas. Altogether, these results contribute to the elucidation of the phenoloxidase function in P. monodon. [ABSTRACT FROM AUTHOR]

This study aimed to characterize and determine the distribution of proteases in various anatomical parts of Penaeus monodon using mainly electrophoresis on sodium dodecyl sulfate (SDS) polyacrylamide gels impregnated with casein (casein-SDS-PAGE) and specific protease inhibitors. Twelve caseinolytic bands designated A-L, with estimated molecular masses of 11.4-49.6 kDa were detected by SDS-PAGE, and were most abundant in the hepatopancreas. Among the 12 bands, E, J, K and L were predominant, A, G and H were identified as chymotrypsin-like proteases, D, E, I, J, K and L were identified as trypsin-like proteases, and B, C and F were identified as serine proteases. The proteases were most active at 50°C and pH 7.0. The proteolytic and trypsin-like activities of Penaeus monodon were most abundant in hepatopancreas extracts and least abundant in extracts from the haemolymph. Trypsin-like proteases in the hepatopancreas extract were found to activate prophenoloxidase (proPO) to form phenoloxidase (PO). Résumé: Cette étude vise à caractériser et à déterminer la distribution des protéases dans diverses parties anatomiques de Penaeus monodon , en utilisant principalement la technique de l'électrophorèse en gel de polyacrylamide contenant du dodécylsulfate de sodium (SDS) imprégné de caséine (casein SDS-PAGE) et des inhibiteurs spécifiques de protéase. La technique de l'électrophorèse SDS-PAGE révèle l'existence de douze bandes d'activité caséinolytique, désignées A-L et de poids moléculaire variant de 11,4 à 49,6 KDa. Ces bandes sont les plus abondantes dans l'hépatopancréas. Sur les douze bandes révélées, quatre ont été trouvées prédominantes à savoir E, J, K et L. Les bandes A, G et H ont été identifiées comme des protéases de type chymotrypsine, les bandes D, E, I, J, K et L comme des protéases de type trypsine et les bandes B, C et F des comme des protéases à sérine. La meilleure activité protéasique a été observée à 50°C et à pH 7,0. Les activités protéolytiques et celles de type trypsine de Penaeus monodon ont été trouvées plus abondantes dans l'hépatopancréas et moins abondantes dans l'hémolymphe. La formation de la phénoloxydase (PO), dans l'hépatopancréas, est due à l'activation de la prophénoloxydase (proPO) par des protéases de type trypsine. [ABSTRACT FROM AUTHOR]

The expression stability of four potential reference genes (EF1‐α, β‐actin, GAPDH and SubF0) was evaluated to normalize the expression of six immune‐related genes (proPO1, proPO2, MnSOD, PAP, Rab7 and NOS) in different tissues of black tiger shrimp (Penaeus monodon) following melanin injection and using the geNorm and NormFinder algorithms. The stability rankings of the reference genes were as follows: EF1‐α > β‐actin > SubF0 > GAPDH in the muscle, GAPDH > EF1‐α > SubF0 > β‐actin in the carapace and EF1‐α > GAPDH > β‐actin > SubF0 in the haemolymph. To normalize the expression of six immune‐related genes of P. monodon in different tissues under the effect of melanin injection, EF1‐α and GAPDH alone were the most suitable reference genes for the muscle and carapace, respectively, whereas the combination of EF1‐α + β‐actin was the most suitable for the haemolymph. The expression of these six immune‐related genes in P. monodon was upregulated in the haemolymph, downregulated in the carapace and nearly unchanged in the muscle of the melanin‐injected group compared to that in the control group. Our results provide reference genes for reverse transcription quantitative polymerase chain reaction to analyse the protective effects of melanin in black tiger shrimp. [ABSTRACT FROM AUTHOR]