| Chinese mitten crab(Eriocheir sinensis), commonly known as river crab, is an important economic animal with an important position in the aquaculture industry. In this study, based on culture biology and molecular biology, phenotypic observation and gene expression of molting and growth-related genes were conducted in the laboratory conditions. Meanwhile, the dynamic changes of molting hormone and molting-related genes in different molting stage within one molting cycle were investigated. Furthermore, prokaryotic expression profiles of molt-inhibiting hormone(MIH) genes and influence of its recombinant protein on endogenous gene expression were also discussed. The results were indicated: Part1. Observation on Individual Molting, Growth and Their Association Analysis with Relative Gene Expression in Eriocheir sinensisMolting is an essential biological process for the growth and development of crustaceans, however, the molecular mechanism of molting on growth regulation was less studied. In this study, observation of molting and growth performance were individually conducted under the indoor laboratory condition, and gene expression level of two molting related genes(ecdysteroid receptor gene, EcR; retinoid X receptor,RXR), and one grwoth related gene(myostatin,MSTN) were investigated using quantitative RT-PCR method. The total of 60 juvenile crabs in each male and female groups were reared in 120 water closets of 60 L with 1 individual in each closet for two molting times. The growth traits(body weight, carapace length, carapace width, and merus length of second foot) were measured for each crab were measured at the initial stocking stage, on the fifth day after the first molting and the second day after the second molting. Meantime, muscle and hepatoparncreas tissues were immediately collected and stored in-80℃ refrigerator condition after growth traits measurements at the second molting for further gene expression analysis. The correlation analysis were conducted between the expression level of the three genes(EcR, RXR and MSTN) and growth traits using SPSS software. A sharp gaining of body weight were found after molting and followed by a slow and continuous increment in post-molting stage. Condition factor could be used as an index indicating the next molting will be initiated when it accumulated over 60%. Significant negative correlation were found between MSTN expression level and the growth rate of carapace length(r =﹣0.450, P<0.05) and carapace width(r =﹣0.410,P<0.05), but significantly positive correlation were detected between MSTN expression level and condition factor(r = 0.450,P<0.05). There was no significant correlation between the EcR, RXR genes expression level and the rate of weight increment, carapace length, and carapace width increment. Generally, higher expression of MSTN indicated lower rate of weight increment, and higher expression of EcR, RXR indicated higher rate of weight increment. This research indicated a regular pattern on the growth performance, and condition factor measuring nutrient substance content could be used as an indicator showing the initiation of the next molting, and the gene expression of EcR, RXR, and MSTN were correlated with the growth traits in the juvenile Chinese mitten crab. Part2. Analysis of relative hormonal changes and gene expression at different molting stages during the molt cycle of Eriocheir sinensisMolting is an important biological process in the growth and development stages of Eriocheir sinensis, The study on endocrine hormone and related gene expression in different molting stages of Eriocheir sinensis provide certain basis for the research of crab’s precocity, regeneration. In this study, we observed themorphological characteristics and detected the Ecdysone content level at different molting stages during the molt cycle by Elisa. Quantitative RT-PCR method were used to detect the relative expression of the MIH、EcR gene. The results indicated that Ecdysone contentlevel and EcR gene relative expression at pre-molting stage reach a peak and significantly higher than other molting stages, while MIH gene expression is opposite. So we concluded, when the Ecdysone content level and EcR gene expression reach the highest peak, molting will be started. The correlation analysis were conducted among ecdysone content level, the relative expression of the two genes(EcR and MIH) and growth traits using SPSS software. We found that with the rise of ecdysone content level and the EcR relative expression, the weight of the crab increase, but with the rise of MIH gene relative expression, the weight decreased. So, MH、EcR with a high concentration can effectiviely promote weight gain, but MIH with a high concentration can not make weight gain. Part3. Prokaryotic Expression of Molt-Inhibiting Hormone Gene and Effect of Its Exogenous Recombinant Protein on Endogenous Gene Expression in Eriocheir SinensisMIH(molt inhibiting hormone) is an essential hormone in regulating molting and growth of crustacean, which is produced in eyestalk of crustacean. However, it is very difficult to obtain MIH content in single individual because separation and purification of this protein require large numbers of samples, which limits the physiology and biochemical study of MIH gene. Thus, an alternative way is to use prokaryotic expression for conducting large amount of recombinant proteins. By now, the influence of the exogenous recombinant MIH protein on the endogenous MIH gene expression of Chinese mitten crab has been remained unclear. In this study, a series of experiments were conducted to identify the optimal conditions for prokaryotic expression of MIH gene. Its exogenous recombinant protein was firstly used via injecting into the eyestalk of Chinese mitten crab for observing the expression profiles of endogenous MIH gene. The experiments conditions included different concentration of IPTG(Isopropy-β-D-thiogalactopyranoside)(0.5 mmo L/L, 1.0 mmoL/L, 2 mmoL/L, and 4 mmo L/L), different induction time(0 h, 2 h, 4 h, and 6 h), and different induction temperature(27℃ and 37℃). The results indicated that the highest amount of content of recombinant MIH protein were obtained under the conditions of IPTG with concentration of 1.0mmol/L for 6h at 27℃. Then the recombinant protein from the induced bacterial expression(pET-28a-MIH) were injected into living crabs. It was found that the expression level of MIH gene in eyestalk was significantly higher after 4h injection than those of PBS control group(P <0.05), while no significant difference was found among 8h, 24 h injection and PBS control groups(P >0.05). Furthermore, the expression level of endogenous MIH gene in eyestalk was significant higher after 4 h than that after 8 h and 24 h(P <0.05), while there were no significant difference among the 4 h, 8 h, and 24 h with injection of PBS(P>0.05). Our results indicated exogenous MIH recombination protein could shortly promoted the expression of endogenous MIH protein, and then partly restrain the molting in crab. This result provided insight into the molting mechanism of crab and made a foundation for antibody preparation in future. |
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