Cloning Of Xuhuai Goat SCD1Gene, Subcellular Localization And The Preparation Of Transgenic Mice And Transgenic Sheep

Stearoyl-coenzyme A dehydrogenase (SCD) is a transmembrane protease which exists in the endoplasmic reticulum, is a particulate proteases belonging dehydrogenase family. The cDNA of SCD gene of mammalian was first found in the livers of mice. SCD have4kinds of isomer in mice, namely SCD1to SCD4, encoded355,358,359and352amino acids, respectively. Under normal dietary conditions, SCD1mRNA is highly expressed in white adipose tissue, brown adipose tissue, meibomian gland, Harder gland and preputial gland, and can be induced significantly by high carbohydrate in the liver and heart.SCD1is an important enzyme in fat metabolism, that is rate-limiting enzyme catalyzes unsaturated fatty acids synthesis from saturated fatty acids, which plays a central regulation role in fatty acid biosynthesis. The proportion of saturated fatty acids (SFAS) and unsaturated fatty acids (MUFAs) in lipids affect the lipoprotein metabolism, biofilm mobility and signal transduction, and thus associated with diabetes, athero-sclerosis, cancer, obesity, and many other diseases. Therefore, SCD1become a research focus in recent years. The study found that SCD1may affect the ratio of SFAs/MUFAs in milk, intramuscular fat deposition and composition, is a major candidate gene for improving the quality of milk and meat. Unsaturated fatty acid content of intramuscular fat increased also has an important impact on the improvement of the quality of meat and meat flavor, and unsaturated fatty acids are closely related with human health. The study cloned Xuhuai goat SCD1gene CDS and analyze its biological information, studied its expression in vitro and subcellular localization, finally through the testis injection mediated of SCD1gene preparation transgenic mice and transgenic sheep. The main research contents were as follows:1. SCD1gene cloned、analyze its biological information and construction of its recombinant eukaryotic expression vector. SCD1gene-specific primers were designed and synthesized. Xuhuai goat SCD1cDNA was first cloned by RT-PCR method from adipose tissue. The SCD1gene was cloned into pMD-19T vector. The digestion and sequencing results show that the pMD19-T-SCD1was constructed correctly. Then the SCD1gene was sub cloned into the eukaryotic expression vector pEGFP-C1to construct the recombinant fusion expression vector named pEGFP-SCDl. Digestion and sequencing results confirmed that the pEGFP-SCD1was constructed correctly. The result of bioinformatics analysis show that the coding sequence (CDS) length of Xuhuai goat SCD1gene was1074bp, encoding357amino acids, the protein molecular size was41.35kDa and isoelectric point was7.0. GenBank accession number was JX854036. There were high similarity of SCD1gene sequence and amino acid sequence between Xuhuai goat and Human, Rattus norvegicus, Mus, Ovis aries, Goat, Bos Taurus, Sus Scrofa, which reported on the GenBank.2. Studies of SCD1gene recombinant expression in vitro and subcellular localization. NIH-3T3cells were transfected with pEGFP-SCD1through polyethylene imine (PEI) and observed under inverted microscope48h after transfection and extracted total RNA. Then software prediction, mRNA was detected to identify the expression in NIH-3T3cells by RT-PCR. Software prediction showed that SCD1mainly expressed in cytoplasm; Recombinant SCD protein fluorescence in the cytoplasm, no fluorescence in the nucleus fluorescence were observed under inverted fluorescence microscope; which was consistent with the result software prediction. RT-PCR results showed that the recombinant expression vector was expressed successfully in NIH3T3cells.3. Preparation of transgenic mice carrying SCD1gene. Recombinant vector carried the SCD1of Xuhuai goat was transfered into male mice testicle by testicular injection,and then put male and female mice into one cage. After F1genetion gave birth, we detected the target gene on DNA and protein levels. At last,we got6.67%positive individuals. In the same way,we detected the F2generation by F1Positive generation, and got20%positive individuals. The results show:the biological function of the exogenous gene given full play in mice level.4. Preparation of transgenic sheep carrying SCD1gene.The basic idea reference to testicular injection to produce transgenic mice, testicular injection to produce transgenic sheep.