Effect Of KK-42 On Gene Expression Of Three Chitin Binding Proteins In Macrobrachium Japonicus

Macrobrachium nipponense Chitin Binding Proteins?Mn CBPs?are the main structural proteins that make up the cuticle.The expressions of the genes coding Mn CBPs are closely related to the molting cycle.Due to the poor similarity among CBPs and relatively large temporal expression differences of Mn CBPs in different parts of the cuticle,it suggests that the functions of CBPs are not exactly the same in the molting cycle.Previous research in our laboratory has found that KK-42 can significantly shorten the period of molting cycle of juvenile M.nipponense after treatment.In order to preliminarily analyze the molecular mechanism of KK-42 treatment,three novel Mn CBPs containing chitin_bind₄ domain are selected according to the sequencing results of the transcriptome of cuticular tissues from M.nipponense,and the fragment sequences of c DNA are cloned from the carapace by PCR.The sequencing length are 362,431 and 1327 bp,respectively,and named as Mn CBP-6?Gen Bank:KY126106?,Mn CBP-7?Gen Bank:KY270879?and Mn CBP-8?Gen Bank:MG818722?on the basis of the order in the laboratory.Through bioinformatics analysis,it is found that these three Mn CBPs sequences all contain RR consensus.Multiple sequences alignment with other species finds that Mn CBP-6,Mn CBP-7 and Mn CBP-8respectively have the highest similarity with the Hyalella azteca?XP₀18014406.1?,Daphnia magna?JAN65546.1?and Aedes aegypti?XP₀21699126.1?,and the degrees are 60 %,52 % and 43 %,respectively.Real-Time PCR?RT-PCR?is used to analyse the relative expressions of Mn CBP-6,Mn CBP-7and Mn CBP-8 in carapace,tail fan,pleopod and pereiopod at stage intermolt?C?,early premolt?D0-2?,latepremolt?D3-4?and postmolt?A?four different stages of molting cucle from healthy juvenile M.nipponense?body length 3.0 ± 0.5 cm?and expression level changes of the three genes mentioned above after KK-42 treatment.The results show that the expression change trends of Mn CBP-6,Mn CBP-7 and Mn CBP-8 are related to the molting cycle,but the expression patterns of different parts of cuticular tissues are different.The expressions of Mn CBP-6 in carapace,tail fan and pereiopod tend to be consistent,which are all expressed highest at stage D3-4 and A,while at stage D0-2 and A in pleopod.The expression peaks of Mn CBP-7 in carapace and pereiopod are both at stage D3-4,while the peak in tail fan is at stage A,and in pleopod is at stage D0-2 and A.And the expressions of Mn CBP-8 in carapace,tail fan,pleopod and pereiopod are consistent,and the peaks of expressions are all at stage A.Collectively,the results suggest that Mn CBP-7 may be involved in the formation of new exocuticle and endocuticle,while Mn CBP-6 and Mn CBP-8 is mainly involved in the formation of new endocuticle.After KK-42 treatment,the expressions of Mn CBPs are significantly increased,especially at stage C,D0-2 or D3-4.And compared with the control group,the expression peak of each gene is moved forward.At stage C,the overall expressions of Mn CBP-6⁸ in carapace and tail fan and Mn CBP-8 in pleopod are significantly increased,especially the expression of Mn CBP-8 in the tail fan at 24 hours is 301 times higher than that in the control group.While the expressions of Mn CBP-6 7 in pleopod and Mn CBP-6⁸ in pereiopod are up-regulated at some time points.At stage D0-2,the expressions of Mn CBP-6in carapace,Mn CBP-6,Mn CBP-7 in tail fan and Mn CBP-6,Mn CBP-7 in pereiopod are up-regulated,especially the expression of Mn CBP-7 in tail fan at 3 hours is more 5200 times higher than that in the control group.While the expressions of Mn CBP-7,Mn CBP-8 in carapace and Mn CBP-8 in tail fan andpleopod are only up-regulated at some time point after treatment.At stage D3-4,the up-regulated effect occurs mainly in pleopod.The expressions of Mn CBP-6 and Mn CBP-7 are higher than that in the control group,the most obvious is that the expression of Mn CBP-7 is more 15,000 times than that of the control group at 6 hours,and the Mn CBP-8 increases only at 6 hours after treatment.It is speculated that KK-42 may indirectly induce the expressions of Mn CBPs by upregulating the concentration of molting hormone to make the expression peaks of Mn CBPs move forward,which results in accelerating formation of new cuticle.Therefore,this may be one of the reasons why KK-42 significantly shortened the period of the molting cycle.