Molecular cloning of N-acetyl-β-D-glucosaminidase (NAGase) gene and the effect of KK-42 on NAGase gene in Macrobrachium nipponense

In order to further explore the molecular mechanism that KK-42 shortens the molt cycle of the juvenile prawn Macrobrachium nipponense, the full-length cDNA sequence of NAGase, the rate-limiting enzyme in chitin catabolism, was cloned from carapace by RACE technique. The relative expression of NAGase mRNA and its activity in cuticular tissue were determined before and after KK-42 treatment. Sequence analysis showed that the full length NAGase cDNA was 2 536 bp, encoding 617 amino acids. Homology analysis indicated that NAGase was less conserved and had the highest similarity of 68% to that from Litopenaeus vannamei. Phylogenetic analysis showed that the amino acid sequence of NAGase from M. nipponense was clustered into one major group with that from Portunus trituberculatus, L. vannamei, and Fenneropenaeus chinensis. The NAGase sequence from L. vannamei and F. chinensis shared more similarities with each other, M. nipponense belonged to a separate branch. The mRNA concentration of cuticular NAGase in control group peaked at premolt D₀ stage. Once treated by KK-42, the mRNA content increased significantly, with a 253% rise in D₄ phase at 3 h, as well as a 226% or 187% rise in C or D₀ phase at 6 h. The activity of NAGase rose gradually from C to D₄. KK-42 treatment could cause significant increase of NAGase activity in C and D₀ stages, especially in C stage, during which the activity increased by 11.26, 5.99 and 7.15 folds, respectively, at 3, 6 and 12 h. The results above suggest that the induction effect of KK-42 on the cuticular NAGase of M. nipponense may be one of the molecular mechanisms to shorten the molt cycle.