Effects Of KK-42 On Up-regulation Of 3 CBPs In Cuticle From Macrobrachium Nipponense

The mechanism of crustacean's molting cycles has always been the focus of study among relevant researchers,and the forming of new cuticle is the critical factor of the progress of molting cycles.Chitin,the main composition of cuticles,is binding with chitin-binding proteins?CBPs?,which are significant structural proteins in crustacean cuticles synthesized and secreted in stages by epidermal epithelia,the target cells of molting hormone.Previous study shows that imidazole KK-42 could shorten the period of molting cycles of juvenile Macrobrachium nipponense and increase the thickness of endocuticle at stage D₃,which suggests that the change of cuticular thickness is relative to the progress of molting cycles and the temporal expression of CBPs in cuticle is one of the molecular mechanisms of the shortening of molting cycles.According to the data of cuticular transcriptome,we have cloned partial cDNA sequences of 3 CBPs from the cuticles of Macrobrachium nipponense?Mn CBPs?and studied the temporal and spatial expression characteristics of MnCBPs and the effects of KK-42 treatment on their expression characteristics by Quantitative Real-time PCR?q RT-PCR?in order to explore the relation between Mn CBPs and molting cycles and accumulate data for clarifying the mechanism of KK-42 for shortening molt cycles henceforth.We separated 400 juveniles Macrobrachium nipponense whose length is 3.0±0.5 cm into KK-42treatment and control groups which are soaked by a solution of 1.95×10^-44 mol/L KK-42 and the same solution only without KK-42 for 1 min respectively.Then we extracted total RNA of carapaces,tail fans,pleopods and pereiopods at stages C?D_0-2?D_3-4-4 and A from two groups for following researches.The results are as follows:3 partial cDNA sequences of MnCBPs have been cloned which are named MnCBP-9,MnCBP-10 and MnCBP-11,whose lengths are 1,143 bp,420 bp and 1,713 bp and Genebank accessions are MG871746,MG871747 and KY065118.Analysis of bioinformatics shows that 3 MnCBPs all contain chitin_bind₄domain.And the results of online BLAST suggest that MnCBP-9,MnCBP-10 and MnCBP-11 share 49%identity with Procambarus clarkii?BAM99303.1?,63%identity with Drosophila kikkawai?XP₀17017873.1?and 39%identity with Marsupenaeus japonicus?BAB13738.1?,respectively.The results of qRT-PCR indicate that the amounts of Mn CBP-9 expression reach their peaks in carapaces,tail fans and pleopods at stage D_3-4,which suggests MnCBP-9 is involved in the forming of new exocuticle.The expression peaks of MnCBP-10 in carapaces,tail fans and pereiopods are at stage D_3-4,A and D_0-2,respectively,while shows no expressed differences among stages in pleopods.The amounts of MnCBP-11 expression all reach their peaks in carapaces,tail fans and pereiopods at stage A,which suggests that Mn CBP-11 is involved in the forming of new endocuticle.KK-42 treatment can significantly up-regulate the expression levels of MnCBPs,especially at satge C,D_0-2-2 or D_3-4-4 and the peak values of each gene expression generally move forward compared with the control group,the results are as follows:?1?stage C:MnCBP-9 and Mn CBP-10 in carapaces and tail fans,MnCBP-11 in carapaces,tail fans and pereiopods.?2?stage D_0-2:all of gene expression levels are up-regulated except that of MnCBP-10 in pleopods and pereiopods.?3?stage D_3-4:MnCBP-9 in tail fans and pereiopods and Mn CBP-11 in tail fans,pleopods and pereiopods.Among them,the expression of MnCBP-9 in carapaces at time point of 6 h(stage D_0-2)increases exponentially,1,658 times higher than that of the control.Therefore,KK-42 can promote Mn CBPs expression levels and advance the peak values of each gene expression,the above-mentioned genes have their own variable expression patterns in different parts of cuticles:the peak values of MnCBP-9 and MnCBP-10 expression in carapaces move forward from stage D_3-4-4 to D_0-2-2 and the peak values of Mn CBP-11 expression in carapaces,tail fans and pereiopods are in advance from stage A to D_3-4-4 even D_0-2.This may accelerate the forming of the new cuticle and shorten the period of the molting cycle.The above results indicate that the expression levels of 3 MnCBPs are related to molting cycles.The expression characteristics of one gene vary among different parts of cuticle.KK-42 treatment significantly up-regulates the expressions of these 3 genes.