The Research Of Over Expression And RNA Interference Of ELOVL6Gene In Dairy Goat Mammary Epithelial Cell

Elongase of very long chain fatty acids6(ELOVL6) is the rate-limiting enzyme in theelongation cycle in mammals that catalyzes the elongation of long-chain saturated andmonounsaturated fatty acids with12–16carbons to18carbons. In this study, we clonedELOVL6coding sequences (CDS) by RT-PCR. We constructed recombinant adenovirusover-expression and RNA interference vector for ELOVL6, packaged high titer recombinantadenovirus. We incubated dairy goat mammary epithelial cells(GMEC) with recombinantadenovirus, and examined the effects of ELOVL6alteration on composition of fatty acid, andaccumulation of triglyceride (TG) and lipid droplets in cells. This study provides theexperimental foundation for the function study of ELOVL6in goat mammary fatty acidmetabolism. The main results of this study were as follows:(1) The CDS of ELOVL6gene was795bp (GenBank No. KF667508), encoding264amino acids. Compared with Bos taurus, Mus musculus and Homo sapiens, the nucleic acidsequence homology of dairy goat ELOVL6was97%,90%and93%, and the amino acidsequence homology was99%,92%and95%, respectively. The protein structure analysisusing TMpred showed that5transmembrane helixes were speculated in ELOVL6protein,and there was a conserved histidine box (HWYHH) in between the second and the thirdtransmembrane helix. The ELOVL6protein had strong hydrophobic properties analyzed byProtScale.(2) Tissue expression analysis showed that the ELOVL6gene was extensively expressedin dairy goat. The expression of ELOVL6gene was most abundant in adipose tissue, followedby small intestine, and minimum in heart.(3) We linked the ELOVL6gene into shuttle vector pAdTrack-CMV, the recombinantplasmid linearized by PmeⅠ was transformed intoE. coli BJ5183competent cell containingthe backbone vector pAdEasy-1to construct vector pAd-HA-ELOVL6by homologousrecombination. And then, pAd-HA-ELOVL6was transfected into HEK293cells forrecombinant adenovirus packaging and proliferation. The titer of recombinant adenoviruscontaining ELOVL6gene was108U/mL. After incubated with recombinant adenovirus for48h,the mRNA levels of ELOVL6gene was increased by about200-fold in GMEC. Theover-expression of ELOVL6gene led to decrease C16-FA, and increase C18-FA, elongation index for C16-FA, total desaturation index, and TG content, but had no influence onaccumulation of lipid droplets in GMEC.(4) According to goat ELOVL6nucleotide sequence, we designed four shRNA(shRNA-620, shRNA481, shRNA360, shRNA306) targeting different sequence of ELOVL6gene. After annealing, shRNA were inserted into shuttle vector pENTR/CMV-GFP/U6, andrecombined with pAd/PL-DEST to construct vector pAdEasy-shRNA by LR homologousrecombination. And then, pAdEasy-shRNA was transfected into HEK293cells forrecombinant adenovirus packaging and proliferation. The titer of recombinant adenovirusexpressed shRNA-620, shRNA481, shRNA360, shRNA306was109U/mL,109U/mL,108U/mL,109U/mL respectively. The recombinant adenovirus(expressing shRNA481sequence)infecting GMEC for48h, we found that mRNA levels of ELOVL6gene decreased by about80%. The knockdown of ELOVL6gene led to increase C16-FA, and decrease C18-FA,elongation index for C16-FA, total desaturation index, and TG content, but had no influenceon accumulation of lipid droplets in GMEC.In conclusion, the CDS of dairy goat ELOVL6gene was cloned, and this gene catalyzedthe elongation of C16-FA to C18-FA. Expression alteration of ELOVL6gene affected TGcontent significantly, but had no effect on the accumulation of lipid droplets in GMEC. Theresults of this study provide experimental basis for the function research of ELOVL6gene.