山羊INSIG1基因超表达对乳腺上皮细胞中脂质合成的影响

doi:10.11843/j.issn.0366-6964.2016.09.008

Abstract

Abstract:

The aim of this study was to construct an insulin-induced gene-1 (INSIG1) recombinant adenovirus overexpression vector of Xinong Saanen dairy goat，and determine its effect on lipid synthesis in goat mammary epithelial cells (GMEC)，to lay the foundation for its functional study in regulating lipid synthesis in goat mammary gland.The primers were designed according to the INSIG1 sequence in GenBank (accession number：JQ665439)，then its coding sequences (CDS) was cloned by PCR.The gene fragments were inserted into shuttle vector pAdTrack-CMV to obtain pAdTrack-CMV-INSIG1 vector.After being linearized by PmeⅠ，pAdTrack-CMV-INSIG1 vector was transformed into Escherichia coli BJ5183 competent cells to obtain the recombinant adenovirus overexpression vector pAdEasy-INSIG1 by homologous recombination.Next，the pAdEasy-INSIG1 vector，linearized by Pac Ⅰ，was transfected into 293A cells for viral packaging and amplification.LaSRT was used for titer assay.Finally，after infecting the goat mammary gland epithelial cells(GMEC) with Ad-INSIG1 recombinant adenovirus，qRT-PCR was used to measure the mRNA expression of INSIG1 and genes related to lipid synthesis，and the GPO-Trinder enzyme reaction was used to measure the cellular triacylglycerol (TAG) content.The result showed that INSIG1 overexpression recombinant adenovirus vector was successfully constructed，and the recombinant adenovirus Ad-INSIG1 with a high titer of 2×108 U•mL^-1was obtained.Compared with Ad-GFP infected group，the mRNA expression of INSIG1 increased by about 500-fold after GMEC incubated with Ad-INSIG1 for 48 h.No obvious changes were observed on the mRNA expression of sterol regulatory element binding protein 1 (SREBP1) and SREBP cleavage-activating protein (SCAP)，however，there was a significant decrease in the expression genes related to fatty acid de novo synthesis and desaturasion：acetyl-CoA carboxylas α (ACCα)，fatty acid synthase (FASN) and stearoyl-CoA desaturase 1 (SCD1) (P<0.05).Among the 3 key genes involved in TAG synthesis，the transcript abundance of glycerol-3-phosphate acyltransferase (GPAM) and diacylgycerol acyltransferase 2 (DGAT2) were significantly decreased (P<0.05)，and 1-acyl-sn-glycerol-3-phosphate acyltransferase 6 (AGPAT6) had no obvious change.Meanwhile，the mRNA expression of adipose triacylglycerol lipase (ATGL)，an enzyme catalyzes the initial step of TAG hydrolysis，also decreased significantly (P<0.05).No significant difference was found in the cellular TAG content between 2 groups.In conclusion，INSIG1 can inhibit the expression of genes related to lipid synthesis in GMEC，and may have a regulatory function on lipid synthesis in goat mammary gland.

Key words: GMEC, INSIG1, overexpression, lipid synthesis

CLC Number:

S827
S813.3

WANG Miao，LUO Jun，XU Hui-fen，ZHU Jiang-jiang，HE Qiu-ya，YAO Da-wei，SHI Huai-ping. Effect of INSIG1 Overexpression on the Lipid Synthesis in Goat Mammary Gland Epithelial Cells[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2016, 47(9): 1806-1816.

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Effect of INSIG1 Overexpression on the Lipid Synthesis in Goat Mammary Gland Epithelial Cells

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