Study On Reciprocal Regulation Mechanism Of MIH And EcR Genes In The Molting Process Of Chinese Mitten Crab,Eriocheir Sinensis

Chinese mitten crab(Eriocheir sinensis)is one of the most economically important aquatic animals in China.Molting is a vital biological phenomenon of crustaceans,which is closely related to the growth and leg regeneration of Chinese mitten crab.In this study,the transcriptomic variation of hepatopancreas during different molting stages were analyzed,then after the differential expressed ecdysone receptor(EcR)gene had been selected,an attempt was made to focus on the molt-inhibiting hormone(MIH)gene which has antagonism with EcR.The profiles of spatiotemporal expression and reciprocal regulation mechanism of the two genes were invetstigated during the molting process.The main results are as follows: 1.Study on gene expression characteristics of Chinese mitten crab Eriocheir sinensis during the molting cycleIn this study,RNA-Seq was used to investigate transcriptomic profiles of the hepatopancreas and differentially expressed genes were identified in one molting cycle of Chinese mitten crab(Eriocheir sinensis).A total of 97,398 transcripts were assembled with 31,900 transcripts annotated.Transcriptomic comparison revealed 1,189 genes differentially expressed amongst different molting stages.According to these genetic differential expression genes,a pattern associated with energy metabolism and physiological regulation during a molting cycle was established.It turned out that differentially expressed genes are mainly linked to energy consumption at postmolt stage which is referred to as a stage to keep cellular homeostasis,shell induration and recovery.Comparatively,genetic differential expression at intermolt stage are mainly connected with carbohydrates,lipids metabolism and biosynthetic processes.It plays a crucial role in crab’s growth and development due to the function of energy storage.At premolt stage,a preparation stage for upcoming molting,genes are highly expressed not only to response to the stimulation of steroid hormone,but also strongly related to lipids synthesis and immune system development.The expression profiles of twelve functional genes detected via RNA-Seq were corroborated through qPCR assay.The result showed that the expression of EcR gene at premolt stage is significantly higher than it at postmolt stage.In this study,the energy variation during the molting process of Chinese mitten crab had been revealed by the sequence analysis results of the crab’s hepatopancreas.Moreover,differentially expressed genes was found among different molting stages and EcR was selected as an important molting related gene which provided theoretical basis for further work on the mechanism of crab’s molting process.2.Selection of appropriate reference genes for qPCR in Chinese mitten crab,Eriocheir sinensisAccuracy of the qPCR highly depends on the stability of the reference gene used for data normalization.Generally,steadiness of reference genes were evaluated for precise qPCR results.In this study,stability of expression of the ten candidate reference genes including three different reference genes(UBE,α-TUB and β-ACTIN)selected according to the transcriptome data from chapter 2 and seven genes(AK、GST、HSP90、 S27、VATB 、 GAPDH and NaK)originated from NCBI were evaluated in different developmental stages,tissues,and molting stages of E.sinensis by applying the commonly used software geNorm,NormFinder and BestKeeper.It is suggested that UBE and S27 are the most stable reference genes under different experimental conditions.The commonly used reference genes β-ACTIN and GAPDH were most unstable in different developmental stages.Normally,two or three different reference genes were selected for data standardization so that more accurate results of gene expression could be obtained.In order to validate the suitability of reference genes,the expression of EcR gene was analyzed among different molting stages by using different reference genes,and the results presented that the expression level of EcR was elevated using the least stable reference gene GST,which was much higher compared with using three most stable reference genes.The stable reference genes identified in this study provide valuable resources for the following gene expression study of E.sinensis and give reference to reference gene selection for gene expression study in crustaceans.3.The spatiotemporal expression of EcR and MIH genes in Chinese mitten crab,Eriocheir sinensis during the molting cycleIn this study,the protein expression of EcR and MIH genes in different tissues and different molting stages were analyzed by Western blot.It is because of the fact that much more effort were put onto the study of EcR and MIH expression on mRNA level than it on protein level.The results showed that the protein expression of EcR gene was significantly higher at the premolt stage than that at the postmolt,while the protein expression of MIH showed the opposite trend,which was higher at the postmolt.The MIH protein was highly expressed in eyestalk,and also expressed in muscle and heart;the EcR protein was highly expressed in epidermis and muscle.The EcR and MIH genes were also analyzed by fluorescence immunohistochemistry.The fluorescence signal intensity of MIH at promolt stage was remarkably lower than it at postmolt stage,and mainly detected in the intercellular substance.While the fluorescence signal intensity of EcR gene at premolt stage was higher than it at the postmolt.At the premolt,it was mainly detected in the nucleus,while at the postmolt stage it was mainly enriched in the cytoplasm.In addition,the ecdysteroid hormone levels at the whole molting stage was measured by ELISA.It is illustrated that the concentration of ecdysteroid hormone fluctuate regularly,and molting would started when EH content reached 12 IU/mL.4.Reciprocal regulation mechanism of EcR and MIH genes in Chinese mitten crab,Eriocheir sinensisIn this study,the molecular interaction mechanism of EcR and MIH genes was studied by RNAi and injecting the antibody and recombinant protein of MIH gene.It turned out that the expression of EcR and MIH genes could be successfully knocked down by double-stranded RNA injection in vivo.The RNA interference system was preliminarily established in this study.When EcR gene was knocked down,the expression level of MIH gene decreased accordingly.But when MIH gene was knocked down,the expression level of EcR gene was increased.The expression of MIH gene was significantly reduced by injecting the antibody of MIH protein within 48 h,while the expression of EcR gene notably dropped in 12 h after injection and then dramatically rose after 24 h and 48 h.There was a sharp increase during the period of 48 h after injecting the recombinant protein of MIH,while it showed an obvious decrease in the expression of EcR gene.Moreover,it is demonstrated that knocking down MIH gene can remarkably accelerate the molting process;and knocking down the EcR gene has an effect on delaying molting and increases the mortality;injecting the MIH recombinant protein could delay molting and lead to a large number of crabs’ death.