Effects Of Different Fatty Acids On The Expression Of Fatty Acid Banding Protein Related Genes In The Hepatopancreas Of Chinese Mitten Crab Eriocheir Sinensis

The Chinese Mitten Crab(Eriocheir sinensis)also known as crab genus,Crustacea,Decapoda,Grapsidae,Eriocheir sinensis,which has become a major economic crab in China.Lipid in fish,shrimp and crab and other aquatic animal growth,play a very important role in the physiological activity,while the hepatopancreas is the Chinese Mitten Crab center on the structure of lipid metabolism,lipid content,has the important influence function and biochemical composition of fatty acids.Fatty acid banding protein FABP and its distribution with the fatty acid requirements of the organization and participation function has a close relationship.Research shows that fewer types of differentiation of FABP but is involved in a variety of animal.The physiological process,the current research of the ligand affinity and function and understood.Based on the existing research results,from Chinese mitten crab hepatopancreas FABP3 was cloned by using RT-PCR technology,FABP9 and FABP10 genes,Construction of prokaryotic expression vector by means of molecular biology,the expression and purification of Es-FABP3,9 and 10 of three kinds of soluble protein and the protein as materials,using the steady-state fluorescence spectra of Eriocheir sinensis and FABP fatty acids and hydrophobic ligand affinity analysis,and through in vitro quantitative fluorescence PCR single fat acid on the regulation of Es-FABP3,9 and 10.Through the above research of Chinese mitten crab FABP and fatty acid affinity,fatty acid metabolism related genes FABP3,FABP9 and FABP10 in different fatty acids under the control of the interaction relationship,at the same time on the differentiation and function of crustacean animal FABP deeply and provide the basic information.The main research results and the conclusion is as follows:1.Prokaryotic expression and purification of FABP3,FABP9,FABP10Through the NCBI GenBank database query protein nucleotide sequence,according to three kinds of fatty acid banding protein FABP3,FABP9 and FABP10 nucleotide sequence to design primers,upstream and downstream of the restriction sites were inserted into Bam HI and Nde I.Extraction of total RNA from Chinese mitten crab hepatopancreas,receive cDNA by RT-PCR Technology.As a template for TA cloning in order to get the genes FABP3,FABP9 and FABP10 with restriction sites,and then the amplified products were recovered and purified.Electrophoretic analysis of the band is about 400 bp,which is consistent with the expected results.The target gene with pMD-19 T vector we obtain are used to the connection and then PCR identification and sequencing.Recovery of fragments after enzyme digestion of the correct sequence of the plasmid.The gene insert into pET-15 b and construction of prokaryotic expression vector pET-15b-FABP3,pET-15b-FABP9 and pET-15b-FABP10.These three plasmids were transformed into Host Escherichia coli E.coli BL21(DE3).The successful expression of the target protein were induced by IPTG.SDS-PAGE indicated that the protein molecular weight was 15 KD,and In accordance with the expected result.The fusion protein have His tags,expression and purification of FABP3,FABP9 and FABP10 after nickel ion affinity chromatography,these,as materials for subsequent experiments.2.FABP3,FABP9,FABP10 with different fatty acid affinity analysisGet Eriocheir sinensis fatty acid banding protein FABP3,FABP9 and FABP10 recombinant soluble protein,were studied the combination of characteristics of FABP3,FABP9 and FABP10 with different fatty acid of Chinese mitten crab by steady-state fluorescence technique.Using ANS as a fluorescent marker,through the emission spectra by steady-state fluorescence study combined of fatty acids and three kinds of FABP.The FABP3,FABP9,and FABP10 with different fatty acid affinities were calculated by calculating the dissociation constants.The results showed that FABP3 has a high affinity with long-chain fatty acids,and the affinity of FABP9 with different fatty acids decreases with the extension of carbon chain,and the affinity of FABP10 is higher than that of short chain fatty acids.Therefore,we hypothesized that FABP3,FABP9 and FABP10 have different affinities with different fatty acids in relation to the function of FABP and the different structures of them.3.The effect of different fatty acids in hepatopancreas on the expression of FABP3,FABP9 and FABP10This paper adopts the technology of in vitro,The purpose of this study was to study the effect of different fatty acid levels on the expression of fatty acid banding protein genes FABP3,FABP9 and FABP10 in the hepatopancreas of Chinese mitten crab.In vitro culture of hepatopancreas tissue,three concentrations of Palm Acid(PA),oleic acid(OA),linoleic acid(LA),arachidonic acid(ARA),DHA and EPA,respectively,50?m/L,100?m/L and 250?m/L,added into the culture medium.After 24 hours of culture in vitro,by adopting the technology of Real-Time PCR,research expression changes of organizational genes FABP3,FABP9 and FABP10 under the effect of different kinds of fatty acids.Results show,in the medium,which added 50 ?m/L,100 ?m/L,250 ?m/L DHA,EPA and LA,promoting the expression of FABP9 gene after 24 h.Under the concentration of DHA was 100 ?m/L,the expression level of FABP9 gene was the highest;The expression of FABP3 and FABP10 were not significantly affected by the addition of three concentrations of DHA.In the group of LA,the highest expression of FABP9 was in 250 ?m/L.The expression of FABP3 and FABP10 genes were not significantly affected by the addition of three concentrations of DHA.There was no significant difference in the expression of three genes FABP9,FABP10 and FABP3 in three kinds of concentrations of ARA,OA and LA after 24 h.The expression levels of FABP3,FABP9 and FABP10 were different in the various type and concentration of fatty acids,which may be related to the physiological period of the experimental animals and the need for fatty acids.