鸡TXNRD1基因真核表达载体的构建、组织表达和功能研究  

Construction of eukaryotic expression vector,tissue expression and function of TXNRD1 gene in chicken

作　　者：徐庶勋 魏志恒 姜新城 刘世昊 吴文平 王倚兰 邵芳[2] 顾志良[1] XU Shuxun;WEI Zhiheng;JIANG Xincheng;LIU Shihao;WU Wenping;WANG Yilan;SHAO Fang;GU Zhiliang(School of Biology and Food Engineering,Changshu Institute of Technology,Changshu 215500,China;The Second People’s Hospital of Nanjing Medical University,Changzhou 213003,China)

机构地区：[1]常熟理工学院生物与食品工程学院,江苏常熟215500 [2]南京医科大学附属常州市第二人民医院,江苏常州213003

出　　处：《黑龙江畜牧兽医》2025年第2期60-66,共7页Heilongjiang Animal Science And veterinary Medicine

基　　金：江苏省自然科学基金项目(BK20191476)。

摘　　要：为了探究硫氧还蛋白还原酶1(thioredoxin reductase1,TXNRD1)基因对鸡肝细胞脂类代谢相关基因表达的影响,试验以鸡的肝脏和脂肪组织cDNA为模板,PCR扩增鸡TXNRD1基因编码序列(CDS),测序鉴定后用生物信息学分析编码蛋白及其特性,构建鸡TXNRD1基因的真核表达载体,将其转染鸡肝癌细胞系(LMH)后对表达产物用Western-blot鉴定,采用实时荧光定量PCR检测LMH细胞中过表达TXNRD1基因对脂类代谢相关基因表达的影响,以及鸡不同组织TXNRD1基因的表达情况。结果表明:获得了鸡TXNRD1基因1497 bp的CDS片段,共编码499个氨基酸;该蛋白分子量为54818.7 u,理论等电点pI为6.03,不稳定系数为34.60(<40),为稳定蛋白,平均亲水性为-0.168(<0),为亲水蛋白质;构建真核重组表达载体gga-TXNRD1/pCMV-3Tag-9在LMH细胞中成功融合,并表达鸡TXNRD1-MYC;在LMH中过表达TXNRD1基因后LPL基因表达量显著下降(P<0.05),FASN基因表达量显著上升(P<0.05);TXNRD1基因在小肠、肾脏中表达量较高,在肝脏、腿肌、腺胃、胸肌表达量较低。说明TXNRD1基因对鸡肝脏脂类的合成具有促进作用。To investigate the effects of TXNRD1 gene on the expression of genes related to lipid metabolism in chicken liver cells,cDNA from chicken liver and adipose tissue was used as the template to amplify the CDS region of chicken TXNRD1 gene by PCR.The encoded protein and its properties were analyzed by bioinformatics after sequencing and identification,and the eukaryotic expression vector of chicken TXNRD1 gene was constructed.After transfected into LMH cells,the expression products were identified by Western-blot.The effects of overexpression of TXNRD1 gene on the expression of genes related to lipid metabolism in LMH cells and the expression of TXNRD1 gene in different tissues of chicken were detected by fluorescent quantitative PCR.The results showed that the CDS of chicken TXNRD1 gene was 1497 bp and encoded 499 amino acids.The molecular weight of TXNRD1 was 54818.7 u;the theoretical isoelectric point pI was 6.03,and the instability coefficient was 34.60(<40),which was a stable protein.Its average hydrophilic was-0.168(<0),which was a hydrophilic protein.The constructed eukaryotic recombinant expression vector gga-TXNRD1/pCMV-3Tag-9 was transfected into LMH cells to successfully express chicken TXNRD1-MYC.After overexpression of TXNRD1 gene in LMH,the expression of LPL gene decreased significantly(P<0.05),and the expression of FASN gene increased significantly(P<0.05).The expression of TXNRD1 gene was higher in small intestine and kidney,and lower in liver,leg muscle,glandular stomach and chest muscle.The results showed that TXNRD1 gene could promote the synthesis of lipids in chicken liver.

关 键 词：鸡 硫氧还蛋白还原酶1基因 真核表达载体 脂蛋白脂肪酶基因 脂肪酸合成酶基因 

分 类 号：S831.2[农业科学—畜牧学]