Functional Identification Of Genes Related To The Endogenous Taurine Synthesis Pathway And Their Regulatory Mechanisms In The Trachinotus Ovatus

As one of the important mariculture fish,the Trachinotus ovatus has a high economic value and is one of the main economically farmed marine fish along the southern coast of China.In recent years the cost of farming has gradually increased as the price of fishmeal and other feed ingredients has increased.While the addition of taurine to feed can alleviate the adverse effects of replacing fishmeal with vegetable protein in feed,the anabolism of endogenous taurine in Trachinotus ovatus trevally and its regulatory mechanism have not been elucidated yet.In this paper,we aimed to identify the functions of genes involved in the endogenous taurine synthesis pathway in Trachinotus ovatus and to investigate the regulatory mechanisms.Five key enzymes in the endogenous taurine synthesis pathway of pomfret trevally were examined in a culture experiment with different concentrations of exogenous taurine: Cystathionine β-synthetase(CBS),Cystathionine γ-lyase(CSE),Cysteine dioxygenase(CDO),Cysteine sulfinate decarboxylase(CSAD)and Cysteamine dioxygenase(ADO).In addition,the five enzymes were recombinantly expressed and their activity in the taurine synthesis pathway was measured in an Trachinotus ovatus muscle cell line as well as by high performance liquid chromatography.In order to investigate the regulatory mechanism of endogenous taurine synthesis in pomfret trevally,the core promoter regions and key binding sites of the relevant genes were identified using promoter activity analysis,point mutation and electrophoretic mobility shift assay(EMSA),and the regulatory mechanism of endogenous taurine synthesis in pomfret trevally was preliminarily elucidated.The main results were as follows.The main experimental results are as follows:1.The expression of the five related enzymes was verified in an ovine pompano muscle cell line and all showed a decreasing trend with increasing exogenous taurine content,with CDO and CSAD expression significantly decreasing.In addition,four key enzymes for taurine synthesis,CBS,CSE,CSAD and ADO,were successfully recombinantly expressed;the enzymatic activity of CBS was 1.712 μmol NADH/min and that of CSAD was 1.712 μmol NADH/min using an enzyme activity kit.The enzymatic activity of CBS was 1.712 μmol NADH/min,CSE was inactive,CDO was 227.232 U/mg,CSAD was 326.523 U/mg and ADO was 462.651 U/mg.The results of subcellular localization assays showed that all five key enzymes were localized in the cytoplasm and none in the nucleus.2.In this study,promoter deletions as well as point mutants were constructed,and analysis of promoter activity showed that the core promoter region of CBS was between CBS-P1 and CBS-P2(-2,000 bp to-1,165 bp),the core promoter region of CSE was CSE-P5(-296 bp to +1bp),the core promoter region of CDO was CDO-P3(-1 182 bp to +1 bp),and the ADO core promoter region is ADO-P2(-1 221 bp to +1 bp).By constructing point mutants,the possible binding sites for CBS were further identified as CBS-P1-1(-1,937 bp to-1,925 bp)and CBS-P1-8(-1,379 bp to-1,366 bp);the possible binding sites for CSE were CSE-P5-4(-179 bp to-169 bp),CSE-P5-6(-120 bp to-110 bp)and CSE-P5-10(-26 bp to-19 bp);the possible binding sites for CDO are CDO-P3-4(-763 bp to-724 bp)and CDO-P3-7(-369 bp to-359 bp);the possible binding sites for ADO are ADO-P2-1(-1,146 bp to-1,138 In addition,the specificity of hepatocyte nuclear factor 4α(HNF4α)and nuclear factor-kgene binding(NFKB)to the CDO promoter was verified by gel migration assays.The specific binding of CDO promoter was verified.3.To further elucidate the regulatory mechanism of HNF4α and NFKB on CDO,a vector containing the CDO core promoter fragment and eukaryotic expression vectors of HNF4α and NFKB genes were constructed and cotransfected into ovine pompano muscle cells,respectively,for double luciferase activity assay,and the results showed that HNF4α could significantly increase CDO core promoter activity,indicating that The results showed that HNF4α could significantly increase CDO core promoter activity,indicating that HNF4α has the function of positively regulating CDO gene expression.Also,in this study,it was found that the gene expression of both transcription factors HNF4αand NFKB decreased significantly with the increase of exogenous taurine concentration,further verifying their important regulatory role on taurine metabolism in Trachinotus ovatus.In summary,this study identified the endogenous taurine synthesis pathway in Trachinotus ovatus and identified the core promoter region of key enzymes through promoter activity analysis.Point mutation and EMSA experiments further confirmed the binding sites of transcription factors HNF4α and NFKB to the CDO core promoter.In ovine pompholyx,the main endogenous taurine synthesis pathway is the CDOCSAD pathway.At the cellular level,the expression of HNF4α and NFKB decreased when the exogenous taurine content increased,together with the expression of CDO;the cotransfection results indicated that the expression of the key enzyme CDO was positively regulated by the transcription factor HNF4α.The above results tentatively elucidated the regulatory mechanism of endogenous taurine synthesis-related enzymes in Trachinotus ovatus,and laid the foundation for subsequent feed supplementation and breeding improvement of Trachinotus ovatus using gene editing.