陆川猪GPR1基因真核表达载体构建及组织表达谱分析

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。

Eukaryotic Expression Vector Construction and Tissue Expression Profile Analysis of GPR1 Gene in Luchuan Pig

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.