Gene Cloning Of Chitin-Binding Proteins From Macrobrachium Nipponense And Induction Effect Of KK-42 On Expression

The requirement for growth and development of Macrobrachium nipponense needs to be met by molting periodically.As one of the main components of the cuticle,chitin-binding proteins（CBPs）control the formation and calcification of animal exoskeletons,which is closely related to the molting cycle.The previous researches in our lab have found that imidazole derivative KK-42 could shorten significantly the duration of molting cycle of the juvenile prawn M.nipponense.In order to understand the relationship between expression patterns of M.nipponense chitin-binding proteins（MnCBPs）and differences in cuticular tissues and investigate the possible mechanism of KK-42 shortening the duration of molting cycle,three genes containing chitin_bind₄ conserved domain called MnCBP-12,MnCBP-13 and MnCBP-14（on the basis of discovery orders in our lab）were first cloned in carapace from M.nipponense,according to the result from cuticular tissues transcriptome sequencing,and genbank accession number were KY065119,KY126405 and MG729398,respectively.The full length cDNA of MnCBP-12 was 604 bp by RACE and encoded a putative protein of 122 amino acid residues.Amino acid sequence alignment showed that MnCBP-12 had 61%similarity with a cuticle protein（P81576.1）from Cancer pagurus and phylogenetic analysis showed that a cuticle protein（XP 020712733.1）from Ceratitis capitata was the most closely related to MnCBP-12.However,the other two sequences had only partial cDNA fragments,MnCBP-13had 50%similarity with a cuticle protein（ABM54465.1）from Portunus pelagicus.MnCBP-14 had 52%similarity with a cuticle protein（AQT26399.1）from M.nipponense.Through histological observation,we also found that it was difficult to completely separate the muscle from the epidermis in the tail fan by human operation.In addition,through real-time PCR analysis,the expressions of MnCBP-12 in the tail fan,residuum of intact tail fan getting rid of muscle,muscle of tail fan and muscle of abdominal at stage intermolt（C）,early premolt(D_0-2)and late premolt(D_3-4)were analyzed.The result showed that the expression of MnCBP-12 in muscle was negligible,and the results of pleopod and pereiopod were consistent with the tail fan（the results were not shown）.Therefore,the cuticular tissues could be replaced by the entire appendage tissues,which did not affect the overall expression trends of MnCBPs.RNA was extracted from the stage C,D_0-2,D_3-4 and postmolt（A）of healthy juvenile M. nipponense（body length 3.5±0.5 cm）in the carapace,tail fan,pleopod and pereiopod（the latter three could be collectively referred to as appendages）which had different degrees of calcification and thicknesses.Real-time PCR technique was used to analyze the relative expression levels of MnCBP-12,MnCBP-13 and MnCBP-14 in different stages of the molting cycle and 0,3,6,12,24 and 48 hours after KK-42 treatment.The result showed MnCBP-12 mRNA expression level was highest in carapace during stage D_3-4 and A,however,in the other three appendage tissues,consistent with each other,were the highest expression during stage A.And MnCBP-13 mRNA expression level was highest during stage D_3-4 in carapace,tail fan and pleopod,while during stage D_0-2 in pereiopod.Besides,MnCBP-14 mRNA expression level was highest during stage D_3-4 in carapace,while during stage A in tail fan and pleopod（P<0.05）,and the change of expression level was not obvious in pereiopod.Collectively,it suggests that the expressions of MnCBPs derived from epithelial cells from four cuticular tissues are related to the molting cycle.MnCBP-12 and MnCBP-14 have likely an important role in forming the parallel sheets of new exocuticle and endocuticle,while MnCBP-13 has likely an important role in forming those of new epicuticle and exocuticle.And their differential expressions in different cuticular tissues is one of the possible reasons why cuticle has different structures.Furthermore,the expression characteristics of MnCBP-12,MnCBP-13 and MnCBP-14 in the four cuticular tissues tended to be consistent after KK-42 treatment,because all the expression levels were generally higher than those of the control group at stage C and D_0-2,while lower at stage D_3-4 and A.Thus,KK-42 can significantly up-regulate the expression of MnCBPs during stage C and D_0-2,which is one of the possible reasons why KK-42 can shorten the duration of molting cycle.