Primary Characteristcs And Function Study Of LPL In Epithelial Cell In Xinong Saanen Dairy Goat

Lipoprotein lipase (LPL) catalyses the hydrolysis of the triacylglycerol component ofcirculating chylomicrons, very low density lipoproteins and milk, thereby providingnon-esterified fatty acids and2-monoacylglycerol for tissue utilisation. This study usedreverse transcription PCR and RACE techniques, and cloned the full-length cDNA sequencesof Xinong Saanen goat LPL gene; Used real-time fluorescence quantitative polymerase chainreaction (RT-qPCR) technology to detect the gene expression level in10organizations ofXinong Saanen sheep during the dry period; By constructing adenovirus RNA interferencevector, detected the function of LPL gene on lipid metabolism in the dairy goat mammarygland epithelial cells; Added the LPL lipase inhibitor Orlistat to dairy goat mammary glandepithelial cells,found that LPL activity was inhibited and expressions level of lipidmetabolism related genes changed. The main findings are as follows:1. Cloned full-length cDNA sequences of Xinong Saanen goat LPL gene and the lengthwas3555bp (GenBank accession Number: JQ670882), including142bp of5’UTR,1437bpof the CDS and3’UTR of1976bp, the gene encoded478amino acids. Squence analysisrevealed that the LPL gene similarity of goats and cattle, sheep, mice and pigs compared,nucleotide were98%,98%,88%,91%and85%, The amino acid similarity were98%,98%,91%,92%and88%,respectively; protein structure analysis showed that the LPL proteinmolecular weight was53.38kDa, isoelectric point (pI) was8.72, the formula wasC2390H3708N652O700S19.2. Using RT-qPCR detected the LPL expression levels in Ten organizations of XinongSaanen dairy goat, the highest relative expression levels of LPL was in fat (456.8), followedby heart (18.359) and lungs (16.521), LPL expression levels in breast tissue was father low，the expression of LPL in the liver and muscle were just detectable.3. Successfully cloned three shRNA targeted at LPL gene into pENTR/CMV-GFP/U6shuttle vector, and by interacting with pDsRed1-C1-LPL red fluorescent protein vectors andselected a relatively effective shRNA sequence, The virus was packaged and amplified inHEK293cells and the titer reached109U/mL. The virus multiplicity of infection in Mammary epithelial cells was measured (MOI=200), LPL expression was detected byRT-qPCR, the expression levels of ACACA, FASN and HSL were upregulated,while theexpression levels of PRLR, of SREBP GPR41were downregulated.4. Mammary epithelial cells was treated with Orlistat (10μmol/L), RT-qPCR resultsshowed that LPL mRNA expression decreased, the mRNA expression level of ACACA,FASN HSL, PRLR decreased, while GPR41and SREBP mRNA expression wereupregulated significantly.In conclusion, the obtained LPL cDNA sequence is highly homologic with cattle,sheep,human, mouse and porcine. Tissue distribution examination showed LPL is highly expressedin adipose tissue, heart, lung tissues, relatively low in breast tissue. Adenovirusvector-mediated LPL interference experiment in mammary epithelial cells lead to theupregulation of lipid metabolism related gene, such as ACACA, FASN, HSL and soon.Treated mammary epithelial cells with Orlistat to inhibit LPL activity, ACACA, FASN,HSL, PRLR expereeion levels were decreased,while GPR41and SREBP increased. Theseresults laid the foundation for further study of LPL gene function.