| Chinese mitten crab(Eriocheir sinensis)is one of the important aquatic economic species in China.It is widely farmed throughout the country,with a total national production of 750,000 tons in 2017.Hepatopancreas is the main place for the storage and metabolism of lipids,usually containing 30-80% fat,which is a good model for studying the lipid metabolism of crustaceans.The lipid metabolism mechanism of aquatic animals has been a research focus in recent years,and the lipid metabolism of crab has also made great progress.However,there are few reports on the genes related to fatty acid ?-oxidation and cholesterol metabolism of E.sinensis.Therefore,in terms of fatty acid ?-oxidation key genes,this study verified the ORFs of three CPT subtypes of crab,analyzed the molecular characteristics and tissue expression patterns of these genes,and explored the effects of dietary fish oil replacement on the expression of the three CPT isoforms in the hepatopancreas during gonadal development.In terms of key genes of cholesterol metabolism,the ORF of SOAT1 was verified,and then the molecular characteristics and tissue expression patterns of the gene were analyzed.Then the changes of SOAT1 expression and cholesterol content in the main tissues of different ovarian development stages were studied.Finally,the yeast transformation method was used to explore the the function of SOAT1.The research results are as follows:1.Cloning and expression of CPT genesThe carnitine palmitoyltransferase(CPT)family includes CPT 1 and CPT 2 that transport long-chain fatty acids into the mitochondrial compartment for ?-oxidation.In this study,three isoforms(CPT 1?,CPT 1? and CPT 2)of the CPT family were cloned from Chinese mitten crab(E.sinensis)and their complete coding sequence(CDS)was obtained.Sequence analysis revealed deduced amino acid sequences of 915,775 and 683 amino acids,respectively.Gene expression analysis revealed a broad tissue distribution for all three isoforms,with high CPT 1? and CPT 2 mRNA levels in the hepatopancreas of males and females.In males,CPT 1? was highly expressed in gill,heart,brain ganglia and muscle,while in females,CPT 1?-mRNA levels were relatively high in muscle,hepatopancreas and ovary tissue.The effects of dietary fish oil replacement on the expression of the three CPT isoforms in the hepatopancreas during gonadal development were investigated using five experimental diets formulated with replacement of 0,25,50,75 and 100% fish oil by 1:1 rapeseed oil: soybean oil.The results showed that Diets 2# and 5# yielded higher CPT 1? and CPT 2 mRNA expression in males(P < 0.05),while in females,expression of all three CPT isoforms increased then declined in the hepatopancreas with increasing dietary fish oil replacement.The observed changes in CPT gene expression varied in different isoforms and gender,suggesting the three CPT genes might play different roles in fatty acid ?-oxidation in E.sinensis.2.Cloning and expression analysis of SOAT1 geneAcyl-CoA: sterol acyltransferases(SOATs)include SOAT1 and SOAT2 that catalyze the esterification of cholesterol and long-chain fatty acyl-CoA to cholesterol esters in the endoplasmic reticulum.This study cloned the CDS of SOAT1 based on the genome data of E.sinensis.The results showed that the open reading frame of SOAT1 contains 1242 nucleotides,encodes 413 amino acids,with a molecular weight of 48.5 kDa,and has one signal peptide.The predicted protein has nine transmembrane domains.In addition,the amino acid sequence of SOAT1 was similar to that of Daphnia magna by 51%.The results of Q-PCR showed that SOAT1 was widely expressed in various tissues,and the expression level was highest in the hepatopancreas,followed by the ovary.With the development of ovary,the expression level of SOAT1 in hepatopancreas showed a downward trend,while the expression level of ovary gradually increased.3.Changes in cholesterol levels during different ovarian developmental stagesIn order to investigate the change rules of cholesterol content in the ovary development of E.sinensis and its relationship with the expression of SOAT1,we collected samples of ovary,hepatopancreas and hemolymph at different stages of ovarian development,and measured the contents of free cholesterol(FC)and total cholesterol(TC).The results showed that the TC content of ovary increased gradually with the development of ovary,and the CE content of III-V stages were significantly higher than that of I-II stages(P < 0.05).The contents of TC and CE in hepatopancreas showed a trend of increasing first and then decreasing.Similar to that in hepatopancreas,the trends of TC and CE content in hemolymph also increased first and then decreased.4.Functional study of SOAT1SOAT1 catalyzes the binding of free cholesterol and long-chain fatty acids to form cholesterol esters in cell,which is an extremely important enzyme involved in the absorption,transport and storage of cholesterol.To further identify the gene function of E.sinensis SOAT1,we performed gene function identification by Saccharomyces cerevisiae expression system.The PCR and reverse transcription QPCR results of the yeast plasmid before and after the SOAT1 gene transfer indicated that the SOAT1 could be transcribed in the yeast system.TLC results showed that the accumulation of small amounts of triglycerides,indicating that the SOAT1 of E.sinensis had the ability to restore the yeast H1246 to synthesize triglycerides. |
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