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

Abstract

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

CLC Number:

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.

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

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