陆川猪GPR1基因真核表达载体构建及组织表达谱分析

doi:10.16431/j.cnki.1671-7236.2018.08.002

摘要/Abstract

摘要：

试验旨在构建陆川猪G蛋白偶联受体1（G protein-coupled receptor 1，GPR1）基因真核表达载体，并对其组织表达谱进行分析。采用RT-PCR技术从10周龄陆川猪皮下脂肪组织中扩增出GPR1基因CDS区后，使用常规分子克隆手段构建含GPR1基因片段的真核表达载体pEGFP-N1-GPR1，利用双酶切和测序对重组质粒pEGFP-N1-GPR1进行鉴定，并以脂质体法将重组质粒转染3T3-L1细胞24 h后观察细胞荧光表达情况。收集所转染3T3-L1细胞并提取其总RNA，实时荧光定量PCR进一步检测GPR1真核表达载体表达情况；提取6头10周龄陆川猪心脏、肝脏、脾脏、肺脏、肾脏、背最长肌、皮下脂肪总RNA，实时荧光定量PCR检测GPR1基因mRNA在陆川猪各组织中的表达量。结果表明，陆川猪GPR1基因CDS全长1 068 bp，成功将其连接至pEGFP-N1真核表达载体，重组表达载体pEGFP-N1-GPR1质粒和空载pEGFP-N1质粒所转染3T3-L1细胞均能表现出绿色荧光，且空白对照组并未表现出绿色荧光。实时荧光定量PCR结果证实，GPR1基因在重组质粒试验组的表达量极显著高于空载质粒组（P<0.01）。GPR1基因在10周龄陆川猪肝脏中表达量最高，在心脏、脾脏、肺脏、肾脏、皮下脂肪中均有表达，在背最长肌中几乎不表达。本试验成功构建了真核表达载体pEGFP-N1-GPR1，并获得了GPR1基因组织表达谱，为进一步研究GPR1基因对陆川猪脂肪沉积的影响提供参考。

关键词: 陆川猪; GPR1基因; pEGFP-N1载体; 表达谱

Abstract:

This study was aimed to construct the eukaryotic expression vector of G protein-coupled receptor 1 (GPR1) gene in Luchuan pigs,and analyze its tissue expression profile.The CDS region of GPR1 gene was amplified by RT-PCR from the subcutaneous adipose tissue of 10-week-old Luchuan pig,and the eukaryotic expression vector (pEGFP-N1-GPR1) containing the GPR1 gene fragment was constructed.After identification of the recombinant plasmid pEGFP-N1-GPR1 by double digestion and sequencing,it was transfected into 3T3-L1 cells by lipofectamine.Moreover,the fluorescence expression of 3T3-L1 cells was observed by fluorescence microscope after 24 hours and transfected 3T3-L1 cells were collected to extract total RNA.Meanwhile,the expression of GPR1 eukaryotic expression vector was further detected by Real-time PCR.Total RNA was extracted from heart,liver,spleen,lung,kidney,longissimus dorsi and subcutaneous fat of six 10-week-old Luchuan pigs to detect the expression of GPR1 gene mRNA in these tissues.The results indicated that the CDS sequence of GPR1 gene in Luchuan pig was 1 068 bp.The recombinant plasmid pEGFP-N1-GPR1 and the empty vector pEGFP-N1 transfected 3T3-L1 cells all showed green fluorescence,while the blank control group did not display green fluorescence.Real-time PCR result confirmed that the expression level of GPR1 gene in experimental group was extremely significantly higher than that of control group (P<0.01).The expression level of GPR1 gene in liver of 10-week-old Luchuan pig was the highest,and followed by lung,kidney,subcutaneous fat,heart and spleen,but was almost absent in longissimus dorsi.In conclusion,recombined vector pEGFP-N1-GPR1 was successfully constructed,and the tissue expression profile of GPR1 gene was appeared,which laid a foundation for further research about the molecular effect of GPR1 gene on fat deposition in Luchuan pig.

Key words: Luchuan pig; GPR1 gene; pEGFP-N1 vector; expression profile

中图分类号:

谢婉, 何剑雄, 夏攀洁, 吴延军, 焦迪, 陈宝剑, 关志惠, 兰干球, 郭亚芬, 谢炳坤. 陆川猪GPR1基因真核表达载体构建及组织表达谱分析[J]. 《中国畜牧兽医》, 2018, 45(8): 2049-2056.

XIE Wan, HE Jianxiong, XIA Panjie, WU Yanjun, JIAO Di, CHEN Baojian, GUAN Zhihui, LAN Ganqiu, GUO Yafen, XIE Bingkun. Eukaryotic Expression Vector Construction and Tissue Expression Profile Analysis of GPR1 Gene in Luchuan Pig[J]. , 2018, 45(8): 2049-2056.

参考文献

[1] BARNEA G,STRAPPS W,HERRADA G,et al.The genetic design of signaling cascades to record receptor activation[J].Proceedings of the National Academy of Sciences,2008,105(1):64-69.
[2] ERNST M C,ISSA M,GORALSKIK B,et al.Chemerin exacerbates glucose intolerance in mouse models of obesity and diabetes[J].Endocrinology,2010,151(5):1998-2007.
[3] ZHANG X,XIAO Q,TIAN K,et al.Identification of three novel splicing variants and expression analysis of chicken GPR1 gene[J].Biomedicine Research International,2017,2017:1074054.
[4] 黄艳娜,兰干球,蒋钦杨.广西陆川猪和巴马香猪的分子生物学研究进展[J].中国畜牧兽医, 2014,41(11):79-84.HUANG Y N,LAN G Q,JIANG Q Y.Research progress on molecular biology of Guangxi Luchuan pigs and Bama miniature pigs[J].China Animal Husbandry & Veterinary Medicine,2014,41(11):79-84.(in Chinese)
[5] 黄雅琼,江永强,梁文全,等.国家级保护品种陆川猪的发展现状[J].黑龙江畜牧兽医,2013,7:43-45.HUANG Y Q,JIANG Y Q,LIANG W Q,et al.Development status of national protected breed——Luchuan pig[J].Heilongjiang Animal Science and Veterinary Medicine,2013,7:43-45.(in Chinese)
[6] 王迪,杨曙明,刘潇威.G蛋白偶联受体在不同表达系统中高水平表达的研究进展[J].生命科学研究,2012,16(6):545-550.WANG D,YANG S M,LIU X W.Advances in high-level expression of G protein-coupled receptors in different expression systems[J].Life Science Research,2012,16(6):545-550.(in Chinese)
[7] ROURKE J L,DRANSE H J,SINAL C J.CMKLR1 and GPR1 mediate chemerin signaling through the RhoA/ROCK pathway[J].Molecular and Cellular Endocrinology,2015,417(5):36-51.
[8] 廖红海,林亚秋,朱江江,等.山羊GPR1基因的克隆及表达特征分析[J].中国畜牧杂志,2016,52(11):5-10.LIAO H H,LIN Y Q,ZHU J J,et al.Molecular cloning and expression characteristics of GPR1 gene of goat[J].Chinese Journal of Animal Husbandry,2016,52(11):5-10.(in Chinese)
[9] DE HEHAU O,DEGROOT G N,IMBAULT V,et al.Signaling properties of chemerin receptors CMKLR1,GPR1 and CCRL2[J].PLoS One,2016,11(10):e0164179.
[10] 吴孟水,宁翠利,刘宽芝.Chemerin的研究进展[J].医学综述,2016,22(11):2102-2106.WU M S,NING C L,LIU K Z.The research progress of Chemerin[J].Medical Recapitulate,2016,22(11):2102-2106.(in Chinese)
[11] HUANG J,ZHANG J,LEI T,et al.Cloning of porcine Chemerin,ChemR23 and GPR1 and their involvement in regulation of lipogenesis[J].BMB Reports,2010,43(7):491-498.
[12] 田小飞,马玮娟,方光光,等.干预GPR1通路对实验性小鼠脂肪累积的影响[J].生物化学与生物物理进展,2015,42(5):457-467.TIAN X F,MA W J,FANG G G,et al.The effects of experimental mice adipose accumulation by targeting GPR1 pathway[J].Progress in Biochemistry and Biophysics,2015,42(5):457-467.(in Chinese)
[13] BUECHLER C.Chemerin in liver diseases[J].Endocrinology & Metabolic Syndrome,2014,3(4):144-150.
[14] EENST M C,SINAL C J.Chemerin:At the crossroads of inflammation and obesity[J].Trends in Endocrinology & Metabolism,2010,21(11):660-667.
[15] YANG Y L,REN L R,SUN L F,et al.The role of GPR1 signaling in mice corpus luteum[J].Journal of Endocrinology,2016,230(1):55-65.
[16] 袁曼曼,张涛,张向前,等.京海黄鸡G蛋白偶联受体1基因生物信息学及组织表达分析[J].中国畜牧兽医, 2017,44(8):2215-2225.YUAN M M,ZHANG T,ZHANG X Q,et al.Bioinformatics and tissue expression analysis of G protein-coupled receptor 1 gene in Jinghai Yellow chicken[J].China Animal Husbandry & Veterinary Medicine,2017,44(8):2215-2225.(in Chinese)
[17] ROURKE J L,MURUGANANDAN S,DRANSE H J,et al.GPR1 is an active Chemerin receptor influencing glucose homeostasis in obese mice[J].Journal of Endocrinology,2014,222(2):201-215.
[18] 孙俊丽,潘天彪,黄敏瑞,等.陆川猪的繁殖性能研究[J].广西农业科学,2007,38(6):688-690.SUN J L,PAN T B,HUANG M R,et al.Study on reproductive performance of Luchuan pigs[J].Guangxi Agricultural Sciences,2007,38(6):688-690.(in Chinese)
[19] 沈君叶,俞英,王茜,等.母猪繁殖力性状影响因素分析及遗传参数估计[J].遗传,2012,34(5):591-596.SHEN J Y,YU Y,WANG X,et al.Factors analysis and genetic parameter estimation of female reproductive traits in pigs[J].Hereditas,2012,34(5):591-596.(in Chinese)
[20] 黄锦亮.猪PPARγ2基因肌肉超表达真核表达载体制备以及在转基因小鼠中的功能验证[D].武汉:华中农业大学,2012.HUANG J L.Construction of swine muscle-specific overexpression PPARγ2 eukaryotic-expressing vectors and the function verification in transgenic mice[D].Wuhan:Huazhong Agricultural University,2012.(in Chinese)
[21] 孔庆然,刘忠华.外源基因在转基因动物中遗传和表达的稳定性[J].遗传,2011,33(5):504-511.KONG Q R,LIU Z H.Inheritance and expression stability of transgene in transgenic animals[J].Hereditas,2011,33(5):504-511.(in Chinese)
[22] 张志岐,束刚,方心灵,等.G蛋白偶联受体介导游离脂肪酸的信号通路及生理功能[J].中国生物化学与分子生物学报,2009,25(9):789-795.ZHANG Z Q,SHU G,FANG X L,et al.Signal pathway and physiological functions of free fatty acids mediated by G protein-coupled receptors[J].Chinese Journal of Biochemistry and Molecular Biology,2009,25(9):789-795.(in Chinese)
[23] 魏星灿.猪脂肪分解通路中主要基因的网络构建[D].保定:河北农业大学,2014.WEI X C.The network construction of main genes of pig lipolysis pathways[D].Baoding:Hebei Agricultural University,2014.(in Chinese)