Functional Characteration Of One Prawn Lipocalin Gene (Macrobrachium Rosenbergii)

The giant fresh water prawn Macrobrachium rosenbergii is widely cultured economical shrimp. In Japan, United States, Israel, Southeast Asia and also southeast China coast, the prawns are widely farmed, thus this research material is easy to get. Their variety body colors reflect different characteristics for different gender and developmental stages. The color shift also strongly correlated with the developmental molt and reproductive molt. Thus, to start with the color shifts, investigation of the relationship of growth, reproduction and coloration of the crustacean should have applied value. Here, we study one lipocalin family gene MLC of prawn M. rosenbergii in this point of view.First, we designed degenerate primers, and obtained a fragment of lipocalin family member MCL(Macrobrachium rosenbergii lipocalin). An existing shrimp cDNA library was screened with the gene specific probe to a get a full length MLC cDNA sequence. The cDNA was found to contain a5bp5'untranslated region (UTR),609bp open reading frame (ORF) and112bp3'UTR with a consensus polyadenylation signal (AATAAA). The ORF of the cDNA was conceptually translated into a203amino acid s precursor. The sequence contained a probable signal peptide with the most likely site of cleavage between the Ala17and Asp18aminoacid.Secondly, we monitored the spatial and temporal distributions of the mRNA by using Northern Blotting analysis.Our results demonstrated that this gene expresses abundantly in the subepidermal adipose tissue, while faintly in the hepatopancreas and central nervous system. However, no signal was detected in other tissues including muscle, gill and ovary. Its expression levels in subepidermal adipose tissue during various stages of maturation as well as through the whole molting cycle showed that prawn lipocalin is involved in sexual maturation, as the maximal level was observed just aftermolt.Third, knock-down of the MLC gene by RNA interference (RNAi) was performed to clarify the biological function of prawn MLC. RNAi-mediated knock-down of MLC was highly efficient in decreasing MLC protein levels and resulted in a shift in prawn body color from blue to orange-red over the entire carapace. These results suggested that MLC plays a role in the regulation of coloration through its association with astaxanthin.Fourth, in an in vitro binding assay, MLC specifically associated with astaxanthin and molt hormone (20-hydroxyecdysone). The in vitro binding rates were1:0.29and1:0.12respectively. This suggests that MLC may also be involved in the regulation of molting in crustacean.In summary, we believe that MLC protein regulate the prawn coloration process through binding astaxanthin as its ligand. The result of MLC expression in the central nervous system and in vitro binding to the molt hormone20-HE infer that MLC may also participate in molting process.