Gene Cloning And Expression Analysis Of Chitin-Binding Proteins From The Cuticle Of Macrobrachium Nipponense

The cuticle of crustaceans,covering the soft body parts and serving as the protective shield,limits their growth.So the crustaceans must undergo periodic shedding and replacement of the cuticle during ecdysis.Chitin-binding proteins,the main component of cuticle,are closely related to the formation of the new cuticle during the molt cycle.Up to now,the results about chitin-binding proteins has not been reported in Macrobrachium nipponense.Our previous study demonstrated that the duration of molting cycle of the juvenile prawn M.nipponense was shortened and the endocuticle thickness of carapace was significantly increased by KK-42 treatment,which suggested that the expression level and sequential character of MnCBPs genes may be one of the vital effects during the molt cycle.In order to investigate the possible relationship between MnCBPs and ecdysis and the molecular mechanism of KK-42 action,the part sequence of MnCBPs genes were firstly cloned from pereiopod cuticular tissue of M.nipponense and the spatio-temporal expression of the MnCBPs genes as well as the effects of KK-42 on the gene expressions were investigated using Real-time PCR.About 600 prawns with body length of 3.0 ± 0.5 cm were randomly divided into two groups: KK-42 treatment group and control one.The prawns were soaked for 1 min in KK-42 solution at a concentration of 1.95 × 10-4 mol/L(KK-42 treatment)or 0(control),respectively.Then,the pereiopod,gastroenterology and hepatopancreas were collected at different time points after the KK-42 treatment for the research of expression of MnCBPs genes.Five partial fragments of MnCBPs genes encoding chitin-binding proteins containing the Rebers-Riddiford consensus sequence were first obtained from pereiopod cuticular tissue of M.nipponense,which were named MnCBP-1,MnCBP-2,MnCBP-3,MnCBP-4 and MnCBP-5(GenBank accession number: KU316937,KU356775,KU356774,KU306737 and KU356773).The length of each sequence was 1396 bp?624 bp?378 bp?834 bp and 403 bp,respectively.The similarity of the deduced amino acid sequence for MnCBP-1,MnCBP-2,MnCBP-3,MnCBP-4 and MnCBP-5 was separately 44 % to Daphnia pulex,62 % to Procambarus clarkia,75 % to P.clarkia,48 % to Penaeus japonicas and 48 % to Artemia franciscana.In pereiopod cuticular tissue,the expressions of MnCBP-1 and MnCBP-5 increased continuously in premolt,while that of MnCBP-2,MnCBP-3 and MnCBP-4 exhibited decrease in early premolt stage D0-2 but rose in late premolt stage D3-4 compared with the intermolt.All of the MnCBPs genes from the hepatopancreas expressed highly in premolt.In gastroenterology,the changes of MnCBP-1,MnCBP-2,MnCBP-3 and MnCBP-5 mRNA levels were not statistically significant.The expression of MnCBP-4 was not detected at different stages of the molt cycle.After the KK-42 treatment,the changes of expression pattern of MnCBPs genes were different.All of the MnCBPs genes expression were significantly up-regulated in pereiopod cuticular tissue,but down-regulated in hepatopancreas,and increased just in some stages of the molt cycle in gastroenterology.In conclusion,our present results indicate that the relationship between MnCBPs gene expression levels with the molt cycle exists,and KK-42 treatmen can significantly up-regulate the MnCBPs gene expression in pereiopod cuticular tissue.