The Research Of Overexpression And RNA Interference Of SREBP-1Gene On Mammary Epithelial Cell Of Xinong Saanen Goat

Sterol Regulatory Element Binding Protein-1gene, a central multifunctional nucleartranscriptional factor that in charge of fatty acid synthesis, through regulating the expressionand activity of a series of enzymes (including fatty acid synthase FASN, Acetyl-CoAcarboxylase ACC and stearoyl-CoA desaturase SCD) that paly important roles in the processof fatty acid synthesis, so as to regulate the synthesis of fatty acid. In this experiment, thewhole cDNA length of SREBP-1gene was cloned from Xinong Saanen dairy goat, and weconstructed the recombinant adenovirus overexpression and RNA interference vector. Hightiter of recombinant adenovirus was obtained after packaging and amplifying in HEK293cells. Then we infected goat mammary epithelial cells with recombinant adenovirus for48hand72h, detected the effects of SREBP-1overexpression and RNA interference on genesrelated to fatty acid metabolism, to establish foundation for further study of its important rolesin metabolism of fatty acid synthesis and lactation. The main results are as follows:(1) The whole cDNA length of SREBP-1gene is4058bp (GenBank accession No.JN790254) after cloned by RT-PCR and RACE from Xinong Saanen dairy goat(Caprahircus), which includes82bp of5′UTR,535bp of3′UTR, and3441bp of CDS region whichencoding1146amino acid. The similarity of CDS region of goat SREBP-1gene with Bostaurus, Sus scrofa, Homo sapiens and Mus musculus were97%,88%,86%,80%, amino acidsequence alignment results were97%,87%,85%,79%. Their similarity of5′UTR were96%,70%,41%,44%, and3′UTR's similarity were89%,64%,49%,34%. Physical characteranalysis results shows: the estimated weight of SREBP-1protein is120.96KDa, and theisoelectric point (pI) is8.58, the molecular formula of SREBP-1is C5349H8576N1522O1592S40。The total average hydrophily is-0.084. There is the conserved structure domain bHLH-LZexisted in the N terminal of bHLH-LZ transcription factor famliy.(2) By using RT-qPCR technology, we analyzed the tissue expression of SREBP-1gene,SREBP-1is expressed in all the detected tissues, liver has the most abundant expression, thenis the adipose tissue, small intestine, rumen, spleen and breast, and muscle has the minimumexpression among these tissues. (3) We constructed the target gene into adenovirus overexpression shuttle vectorpAdTrack-CMV-SREBP-1, and obtained the recombinant adenovirus vector pAdeasy-SREBP-1by usinghomologous recombination, transfected it into HEK293cell, Titer of recombinant adenovirus was109U/mL after packaging and amplifying in HEK293cells. By using dose gradient to infect mammaryepithelial cell, we found the optimal MOI (Multiplicity Of Infection) is200. Then we infected goatmammary epithelial cells with recombinant adenovirus and detected its effects on SREBP-1and fatty acidmetabolism related genes using RT-qPCR. The results shows that: compared with the control group,SREBP-1expression increased by about15times after infected for48h and30times after infected for72h.There were more obvious effects was observed after72h of infection, Fatty acid synthase (FASN) andAcetyl-CoA carboxylase (ACC) were upregulated by almost2times. The expression level of Peroxisomeproliferator activated receptorγ (PPARγ) increased by1.5times. Liver X receptorα (LXRα) and Adiposetriglyceride lipase (ATGL) upregulated by about1.2times compared with that of control. ButStearoyl-coenzyme A desaturas (SCD) had no obvious change.(4) Based on the sequence of SREBP-1gene, we designed four short hairpin RNA (shRNA) sequencetargeting different locus of SREBP-1gene: shRNA-1058,shRNA-1684,shRNA-2484,shRNA-3096, theninserted it into shuttle vector pENTR/CMV-GFP/U6. Co-transfected it and pDsRed1C1-SREBP-1intoHEK293cell to detect the effects of RNAi. We found that shRNA-2484and shRNA-3096sequencescauses an obvious interference effect. Then we recombined entry vectors(pENTR/CMV-GFP/U6-shRNA-2484/3096) and backbone vector (pAd/PL-DEST) to identify tworecombinant adenovirus vectors (pAD/PL-DEST/CMV-GFP/U6-2484/3096). Titer of recombinantadenovirus was108U/mL after packaging and amplifying in HEK293cells. By using dose gradient toinfect mammary epithelial cell, we found the optimal MOI (Multiplicity Of Infection) is200. We infectedgoat mammary epithelial cell with pAd-shRNA-2484/3096recombinant adenovirus, RT-qPCR resultsshows: after48h of infection, SREBP-1gene expression was decreased by about25%, FASN and ACC allhave a decrease more than40%, LXRα had a20%decrease, and SCD decreased by about13%, but ATGLexpression had no obvious change after infection for48h, and PPARγ expression increased by almost1.55times. After72h of infection, SREBP-1expression had no significant change.We cloned the whole cDNA length of SREBP-1gene and analyzed the basic feature of SREBP-1genesequence. Overexpression of SREBP-1gene in goat mammary epithelial cell lead to a significant increasein mRNA levels of FASN, ACC and PPARγ gene, and interference of this gene resulted in the decrease inmRNA levels of FASN, ACC, LXR and SCD gene. These results provide basis for the research of SREBP-1in the regulation of fatty acid metabolism in Xinong Saanen dairy goat.