Function Analysis And Expression Regulation Mechanism Of Bovine Elongation Of Very Long Chain Fatty Acids Protein 7

Bovine milk lipids,which plays an essential role on providing the basic nutritional requirements for human,is necessary for the absorption of lipidic solubility nutriments.Meanwhile,it also effects the further processing of dairy products.With the development of bovine whole genome technicals and researches,scientists identified a group of bovine lipogenesis related genes.The modification of fatty acids composition has became a hot research field.In the pathway of milk lipids metabolism,α-linolenic acid,which is the substrate of polyunsaturated fatty acids,can be extended and converted into the bioactive lipids.In the extension reaction of ALA,bovine elongation of very long chain fatty acids protein 7 is the rate-limited enzyme.With the development of molecular biology techniques,it became evident that phenotypic characteristics can be affected by the DNA transcriptional regulation.In order to regulate the synthesis of LC-PUFAs in bovine milk,it is essential to investigate the role and the regulation of ELOVL7 in the ruminant mammary gland.The studies on the function and expression regulation of ELOVL7 show the potential application value and provide new thoughts on the molecular breeding engineering.In present research,we constructed the tissue profiles of bovine ELOVL7 with the technology of molecular cloning and real-time PCR.The over-expression of bovine ELOVL7 and cell culture were utilized to demonstrate the detail function of ELOVL7 in bovine mammary epithelial cells(b MECs).The promoter analysis,protein pull-down assay,RNA interference and western blot were used to identify the transcriptional regulation of bovine ELOVL7 in b MECs.Moreover,bioinformatics analyses,dual-luciferase reporter assays,binding sites mutation,over expression and RNA interference assay of mi RNAs,were conducted to explore the post-transcriptional regulation of bovine ELOVL7.The results can be summarized as follow:1.The coding region of bovine ELOVL7 was 846 base pair.According to the analysis of the phylogenetic trees,ELOVL7 and ELOVL1 located at the same branch location,which indicated that they diverged from a common ancestor.Bovine ELOVL7 has a closer evolutionary relationship with goat ELOVL7,but far from the phylogenetic location of chick and duct.The topology prediction of transmembrane protein indicated that ELOVL7 has seven trans-membrane regions and located on the membrane of endoplasmic reticulum.2.The tissue profile of bovine ELOVL7 was detected.Nine tissues,including heart,liver,spleen,lung,kidney,intestine,stomach,skeletal muscle and abdominal fat,were collected for real-time PCR.The results demonstrated that bovine ELOVL7 exhibited high endogenous expression in a broad range of tissues except skeletal muscle.Expressions in kidney and abdominal fat of bull were high,whereas expressions in intestine and stomach of cow were high.3.The function of bovine ELOVL7 was identified in b MECs.With the over-express of bovine ELOVL7,the accumulation of lipids increased in b MECs.Moreover,with the addition of exogenous α-linolenic acid,overexpress of bovine ELOVL7 induced more lipids synthesis in b MECs.4.The 5’-flanking region of bovine ELOVL7 promoter was amplified and analyzed.Neither a TATA box nor a CAAT box was found on bovine ELOVL7 promoter.However,the online program Meth Primer revealed two predicted Cp G islands within the promoter region of the bovine ELOVL7 and several GC-rich regions were located at the second proximal Cp G island.The prediction of transcriptional factor binding sites showed that there are lots of positive and dominant transcriptional factors regulated the activating the promoter of bovine ELOVL7,including GR,C/EBP,SREBP,SP1 and PPAR direct repeat 1.5.The corn promoter and transcriptional factor of bovine ELOVL7 promoter were identified.Promoter analysis of bovine ELOVL7 promoter,including bioinformatics analyses,dual-luciferase reporter assays were used to locate the corn promoter,which is proved between-143 bp and-128 bp.Protein pull-down assay,western bolt assay,over expression and RNA interference assay,have independently and synthetically demonstrated that transcription factor Sp1(SP1)specifically interacted with the GC-box at-143 to-128 base pair on bovine ELOVL7 promoter.The binding of SP1 activated the transcription of bovine ELOVL7,resulting in the accumulation of lipid droplets in b MECs.6.The post-transcription regulation of bovine ELOVL7 was explored.Bioinformatics analyses and dual-luciferase reporter assays identified that bta-mi R-7 bound to the front of bovine ELOVL7 3`UTR,and suppressed the expression of bovine bovine ELOVL7,which lead to the decrease of triglyceride content.In the present research,we independently and synthetically the function of bovine ELOVL7 and the regulation metabolism of bovine ELOVL7,including transcription and post-transcription regulation.Our research provide the new technology pathway to improve the quality of milk and modify the composition of fatty acid in milk.