| Cell cultivation in vitro is an essential basis for gene molecular function studies in mammals and aquatic invertebrates.Chinese mitten crab(Eriocheir sinensis)is a typical molting crustacean,but its cell cultivation technology has been weakly conducted,which has high limited the further studies on gene molecular functions.In this study,methods and conditions were screened and optimized to culture hematocyte in vitro of Chinese mitten crab.Then,metazoan target of rapamycin(mTOR)was used as a target to investigate its expression difference and response level to ds RNAi in hematocytes between in vivo and in vitro.The aims of this study are to evaluate the feasibility of cultured hematocytes in studying gene functions in Chinese mitten crab,and to investigate the biological function of mTOR gene on molting and growth in this species.The main results were presented as follows:1.Primary cultivation and relative function gene detection of hematocyte in Chinese mitten crabIn order to investigate optimal culture condition in vitro,such as medium,osmotic pressure and serum concentration,this chapter analyzed cultivation efficiencies of hemocytes in Chinese mitten crab from five different culture medium(1 x L-15,2 x L-15,3 x L-15,sf-900 and TC-100 medium),osmotic pressures(original osmotic pressure,569?803?991?1215 m Osm/kg)and serum concentrations(0%,5%,10%,15%,20%).In addition,we also detected the relative gene expression in the cultured hemocytes.The result showed that the TC-100 was the best medium according to morphological profiles and survival rate of cultured hemocytes.Furthermore,the best cultivation conditions were determined with osmotic pressure of 991 m Osm/kg and 0% bovine serum,which showed the best morphological profile and more than 50% of survival rates after 10 days cultivation.Expression of the three house-keeping gene(?-actin,S27 and UBE)in Chinese mitten crab were steadily detected in hemocytes in continuously 10 days cultured.In generally,this study indicated that hemocytes cultured in vitro could be used in the further molecular function studies in Chinese mitten crab.2.Gene cloning and expression analysis of mTOR in tissue in vivo and cultured hematocyte in Chinese mitten crabIn order to validate feasibility of gene function studies for cultured hematocyte and investigate function of mTOR gene in molting and growth in Chinese mitten crab,gene cloning was conducted and expression profiles were detected in the 12 tissues(eyestalk,liver,pancreas,heart,gill,stomach,intestine,walking foot,blood,epidermis,thoracic ganglion)on the four molting stages(pre-molting stage,molting stage,postmolting stage and inter-molting stage).Meanwhile,expression levels of mTOR were compared between the cultured hematocyte and blood in vivo.The results showed that the length of mTOR gene is 7026 bp of ORF with 1449 bp of 5' UTR and 1677 bp of 3'UTR,which encodes 2342 deduced amino acids.The expression profiles of mTOR gene were detected in all of the 12 tissues on the 4 molting stages,while the high expression level were observed in the tissues of hepatopancreas,intestines,thoracic ganglia and blood.The highest expression levels were found in the leg muscles,claw muscles and chest muscles at the inter-molting,but the lowest were observed at the molting stage,which implied that the expression levels of mTOR was low at the promolting and molting stages where the muscle was at an atrophic state and was high at the post-molting and inter-molting stages where the muscle was at developing state,indicating mTOR would plays a positive role in regulating muscle growth.The expression level of cultured hematocytes was the similar as that of blood in vivo,demonstrating the cultured hematocytes can be used in gene function studies in Chinese mitten crab.3.Study on RNA interference effect of mTOR gene in vivo and in vitroIn order to investigate RNA interference effect of mTOR gene between in vivo and in vitro,and further explore molecular function of mTOR gene in growth and molting processes of Chinese mitten crab,RNAi technology was employed to evaluate expression of mTOR gene between hematocytes in vivo and in vitro.Meanwhile,the expression characteristics of growth-related genes(P38,IGF2),molting-related genes(ECR,RXR,MIH)and lipids metabolism-related genes(LP,CPT,FABP)were detected in muscle and hepatopancreas during one-molting cycle.Compared with the control group,it was found that molting interval was significantly longer and special growth rate was also significantly lower(P<0.05),but survival rate and hepatopancreas index were decreased with no significant difference(P>0.05)when the mTOR gene was interfered.The expression of the molting-related genes obviously decreased in muscle and hepatopancreas,as well as significantly decreased for the three growth-related genes,but significantly increased for the three metabolism-related genes(P<0.05)in knock-down crabs.However,the survival rates was significantly lower in cultured hematocytes than that in hematocytes in vitro when mTOR was knocked down. |
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