Costruction、Identification And Expression Of Flax-Derived Fatty Acid Unsaturase Gene FAD3B Expression Vectors And Evaluation Of Reference Genes In Bovine Transfected Cells

Omega-3polyunsaturated fatty acids (ω-3PUFA) is very important to human health, but it can not be synthesized in mammalian animals. In order to have animals to express fatty acid unsaturase to improve ω-3polyunsaturated fatty acids, animal transgenic technology probably be effective and feasible. Fatty acid desaturase genes derived from nematodes, plants and fungi have been used for the study of transgenic animals. The present study obtained fatty acid desaturase gene from flax and constructed over-expression transgenic vectors. The expression of these vectors in cells, embryos and mice were identified.Experiment1:Cloning of flax FAD3B gene and constructed recombined eukaryotic over-expression vectors with different promoters.Flax(Linum usitatissimum Linn), common named flax, contains linolenic acid up to50.1%(oil plant database), has very strong flax ω-3fatty acid desaturase activity. In this experiment, flax fatty acid desaturase (FAD3B) gene was isolated by RT-PCR. Two recombined eukaryotic expression vectors pIRES2-AcGFP1-CMV-FAD3B and pIRES-AcGFP-CAG-FAD3B were constructed. PCR and restriction analysis of these recombinant vectors showed that these vectors were constructed correctly. Experiment2:Identifications of the expression vectors in bovine transgenic cells and the subsequent cloned embryos, and the transgenic miceThe two vectors were transfected to bovine fibroblast cells. We found that noculated with1×105cells in6-well plates and transfection after24hours, the (?)ransient transfection efficiency reached to30.71%. The FAD3B gene mRNA copy number in pIRES-AcGFP-CMV-FAD3B and pIRES-AcGFP-CAG-FAD3B transfected gene cells were1.53×105and1.36×105, respectively. The CMV promoter drived vector resulted in1.125times higher than the CAG promoter vector.After transfer of the transgenic cells to enucleated bovine oocytes the cloned embryos had similar development to the non-transgenic controls. Identification of the cloned blastocysts showed that FAD3B gene had obvious expression. The study provides a reliable experimental basis for future transgenic large domestic animals. Transgenic mice can be used as disease model, and verify the vector design at the individual level. The vector plasmids were microinjected into zygote pronuclei and eighty-four mice produced. Unfortunately, all of these mice were transgenic negative.Experiment3:Evaluation of reference genes and detection of candidate endogenous gene differentially expression in bovine transfected cellsDifferent over-expression levels of transfected cells were used as the experimental subjects. Nine candidate reference genes were evaluated by qRT-PCR, including ACTB、GAPDH、8S rRNA、UXT、PPP1R11、RPS15A、SF3A1、EEF1A2and HMBS. Three statistical algorithms geNorm, NormFinder and BestKeeper were used to rank the genes by their stability values. The overall rank ordering of candidate internal control genes was PPP1R11>EEF1A2>18S rRNA>RPS15A>GAPDH> HMBS>UXT>ACTB>SF3A1, PPP1R11and EEF1A2can be considered as most stable reference genes. Selection of stable control genes can normalize the expression level of gene, laid a solid foundation so as to elaborate the function of genes.Analysis of the associated genes in the regulatory networks is important for evaluation of transgene effectiveness and security in transgenic cells. We chose the transcription factors in lipid metabolic pathways and signaling pathways. After liposome transfection, G418screening and empty vector expression, the expression of transcription factors in lipid metabolic pathways significantly changed. The over expression of FAD3B led to the endogenous gene expression levels decreased, addition to the PLA2G4gene expression levels tend to inconsistent in two kinds of transgenic cells, which indicated the disorder of the cell regulatory networks in transfected cells.This study provides a kind of scientific data for a further investigation of transgenic animals using flax unsaturated fatty acid gene.