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

Crustaceans have a rigid cuticle,which is also called exoskeleton,covering the soft body for protection from the environment.Crustaceans periodically take off the old cuticle and produce new cuticle for growth and development.The cuticle is mainly composed of chitin,protein and calcium carbonate,in which cuticle protein（CPs）combined with chitin to control the cuticle calcification.As the main structural protein,CPs plays an extremely important role in the formation of the new cuticle.The preliminary study has found that KK-42 can significantly shorten the molting cycle of juvenile M.nipponense,increase the thickness of the endocuticle in the cuticle,suggesting that CPs may be one of the target molecules for KK-42 action.In theory,the expression level of CPs and its timing changes will affect the thickness of the cuticle,and thus affect the process of molting cycle.This study was based on transcriptome sequencing,and then confirmed that gene sequence contains chitin_bind₄ domain by NCBI Conserved Domain Search.We used qRT-PCR to quantitatively study the temporal and spatial expressions of MnCPs gene and their changes after KK-42 treatment.The aim is to understand the relationship between MnCPs and molt cycle,and to clarify that KK-42 shortened the molting period.About 500 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 uropod,pleopods and pereiopod were collected at different time after KK-42 treatment for the research of expression of MnCPs genes.Three genes were named according to the order in which our laboratory found the CP gene,they were called MnCP-6,MnCP-7and MnCP-8（GenBank accession number: KY399478,KY399479 and KY411918）.Using CutProtFam-Pred to predict that MnCP-6 and MnCP-7 sequences contain the RR-2 domain,which encodes the cuticle protein of the hardened part of M.nipponense.The length of each sequence was 373 bp、739 bp and 1183 bp.The similarity of the deduced amino acid sequence for MnCP-6,MnCP-7 and MnCP-8 was separately 73% to Hyalella azteca,70% to Hyalella azteca and 43% to acyrthosiphon pisum.The transcriptional levels of the above three MnCPs showed significant differences at different stages of the molting cycle and in different tissues.The uropod transcript level as control,in stage C,D0-2 and D3-4,MnCP-6 expression in pereiopods > pleopods;In stage A,expression in pereiopods < pleopods.MnCP-7 expression level in pleopods at stage C and stage A was less than that in pereiopods,and was similar in three cuticle tissues mentioned above at stage D0-2,and was the highest in pereiopods at stage D3-4.The expression of MnCP-8 was the highest in stage C,D0-2 and D3-4 in pereiopods,at stage C the expression of MnCP-8 was the highest in pleopods.KK-42 treatment significantly up-regulated the expression of the genes,in uropod the expression of MnCP-6 gene significantly increased in C and D3-4,and the expression of MnCP-7 gene in pereiopods increased in stage C,and then decreased at stage D0-2.In stages D3-4 and A,the expression of MnCP-8 in pleopods increased first and then decreased.