干扰PTEN基因对奶山羊乳腺上皮细胞脂质合成相关基因的转录及脂肪酸组成的影响

doi:10.11843/j.issn.0366-6964.2020.04.022

摘要/Abstract

摘要： 旨在通过RNA干扰技术揭示蛋白酪氨酸磷酸酶（phosphatase and tensin homolog deleted on chromosome 10，PTEN）基因在奶山羊乳腺上皮细胞中对乳脂合成及脂肪酸组成的影响。利用RT-PCR方法扩增到西农萨能奶山羊乳腺组织中PTEN基因（GenBank登录号：MK158074.1）的CDS区，进行序列分析和不同泌乳时期差异转录分析。合成靶向该基因的siRNA，由RT-qPCR结果筛选出有效siRNA，将其转染至奶山羊的乳腺上皮细胞，进一步检测干扰该基因后对脂质合成相关基因转录水平及脂肪酸组成的影响。结果表明：本研究首次克隆到奶山羊（Capra hircus）PTEN基因的CDS区，全长为1 212 bp；经序列比对发现，山羊的PTEN基因核苷酸序列同牛（Bos taurus）、猪（Sus scrofa）和人（Homo sapiens）的相似度分别为99%、98%和97%，编码氨基酸序列相似性均在99%以上；蛋白质结构预测发现：其蛋白不存在跨膜结构域，亚细胞定位于细胞质中，N端具有保守的磷酸酶功能区；该基因在奶山羊泌乳盛期乳腺组织中的转录量较干奶期下降51.5%；合成的siRNA转染至乳腺上皮细胞后，成功筛选出理想的siRNA，干扰效率达到94%（P<0.01）；与对照组相比，干扰PTEN基因后显著上调SREBP1、FASN、ACACA、SCD1基因转录量（P<0.01或P<0.05），显著下调LPL、FABP3、ACOX1、CPT1B、GPAM、DGAT2和HSL基因转录量（P<0.01或P<0.05）。脂肪酸检测分析发现：干扰该基因能够显著上调C16：1的去饱和指数（P<0.05），但对C18：1的去饱和指数没有显著影响。综上所述，PTEN基因能够调控乳腺上皮细胞中脂质合成相关基因的转录及脂肪酸组成，在奶山羊乳脂代谢中发挥重要作用。

关键词: 奶山羊, 乳腺上皮细胞, PTEN, RNA干扰, 脂质代谢, 脂肪酸组成

Abstract: The aim of this study was to investigate the effect of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) gene on milk fat synthesis and fatty acid composition in goat mammary gland epithelial cells by RNA interference. The complete CDS region of PTEN gene (GenBank accession number:MK158074.1) was amplified from the mammary gland tissues of Xinong Saanen dairy goat by RT-PCR. Sequence analysis and expression analysis of different lactation stages were performed. Then the siRNA targeting the PTEN gene was synthesized and screened by RT-qPCR to further detect PTEN interference on the transcription level of lipid synthesis-related genes and fatty acid composition. Results showed that:the CDS region of Capra hircus PTEN gene was cloned for the first time, with a total length of 1 212 bp. Compared with cattle (Bos Taurus), porcine (Sus scrofa) and human (Homo sapiens), the nucleic acid sequence homology of dairy goat PTEN was 99%, 98% and 97%,and the amino acid sequence homology was all reach up to 99%. The protein structure analysis showed that there was no transmembrane structure in PTEN protein. Subcellular localization analysis showed PTEN located in the cytoplasm. N terminal of PTEN protein has a phosphatase catalytic domain. The transcription of PTEN gene decreased dramatically by 51.5% in peak lactation compared with dry period. By transfecting the synthesized siRNA into mammary epithelial cells,the ideal siRNA was successfully screened of which the interference efficiency reached 94% (P<0.01). Compared with the control group,the mRNA transcriptions of SREBP1, FASN, ACACA and SCD1 were significantly up-regulated with PTEN interference (P<0.01 or P<0.05),and the mRNA transcriptions of LPL, FABP3, ACOX1, CPT1B, GPAM, DGAT2 and HSL were significantly down-regulated (P<0.01 or P<0.05). Fatty acid analysis showed that PTEN interference significantly up-regulated the desaturation index of C16:1 (P<0.05), but didn't affect C18:1. In summary, PTEN can regulate the transcription of lipid synthesis-related genes and fatty acid composition in mammary epithelial cells,which play an important role in milk fat metabolism in dairy goat.

Key words: dairy goat, GMEC, PTEN, RNA interference, lipid synthesis, fatty acid composition

中图分类号:

S287
S813.3

姚大为, 马静, 陈丽丽, 王添祯, 孙欢, 宋文芹, 马毅. 干扰PTEN基因对奶山羊乳腺上皮细胞脂质合成相关基因的转录及脂肪酸组成的影响[J]. 畜牧兽医学报, 2020, 51(4): 851-860.

YAO Dawei, MA Jing, CHEN Lili, WANG Tianzhen, SUN Huan, SONG Wenqin, MA Yi. Effects of Interfering PTEN Gene on the Transcription of Lipid Synthesis-related Genes and Fatty Acid Composition in Goat Mammary Epithelial Cells[J]. Acta Veterinaria et Zootechnica Sinica, 2020, 51(4): 851-860.

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