山羊NR1D1基因真核表达载体的构建及生物信息学分析

Construction of a Eukaryotic Expression Vector of Goat NR1D1 Gene and its Bioinformatics Analysis

本研究旨在构建山羊核受体NR1D1基因的真核表达载体,以探究其在山羊生物钟系统中的功能。从山羊卵巢组织提取总RNA,反转录合成cDNA后通过PCR扩增山羊NR1D1基因的编码序列(Coding Sequence,CDS)。将获得的目的片段与酶切线性化的真核表达载体pcDNA3.1-Puro-N-3HA进行同源重组连接,把PCR、酶切和测序鉴定正确的重组质粒命名为pcDNA3.1-3HA-g NR1D1。将pcDNA3.1-Puro-N-3HA质粒和pcDNA3.1-3HA-g NR1D1重组质粒转染至HEK293T细胞,通过实时荧光定量PCR(qPCR)和Western Blotting技术检测重组质粒的过表达效果,同时对NR1D1基因进行生物信息学分析。结果显示:pcDNA3.1-3HA-g NR1D1真核表达载体构建成功,且与转染pcDNA3.1-Puro-N-3HA组相比,pcDNA3.1-3HA-g NR1D1真核表达载体转染组中NR1D1基因在mRNA和蛋白表达水平均显著升高;山羊NR1D1基因CDS区长度为1851bp,共编码616个氨基酸;山羊NR1D1蛋白为不稳定蛋白,不含跨膜结构和信号肽,且该蛋白的三级结构与人和小鼠高度相似。本研究成功构建了山羊NR1D1基因的真核表达载体,在mRNA和蛋白水平验证了其在HEK293T细胞的过表达效果,为进一步研究山羊NR1D1基因的生物学功能提供了前期基础。

This study aimed to construct a eukaryotic expression vector of goat nuclear receptor gene NR1D1, and explore the function of NR1D1 gene in goat circadian clock system. Total RNA extracting from goat ovarian tissues was reverse transcribed into cDNA, the coding sequence(CDS) fragment of goat NR1D1 gene was amplified by PCR using the cDNA template. The targeted fragment was connected with the linearized eukaryotic expression vector pcDNA3.1-Puro-N-3HA by homologous recombination, and the correct recombinant plasmid identified by PCR, restriction enzyme digestion, and sequencing was named pcDNA3.1-3HA-g NR1D1. The pcDNA3.1-Puro-N-3HA and pcDNA3.1-3HA-g NR1D1 recombinant plasmids were transfected into HEK 293T cells, and the overexpression effect of the recombinant plasmid was detected by real-time quantitative PCR(qPCR) and Western Blotting. Meanwhile, goat NR1D1 gene was analyzed by different kinds of bioinformatics software. The results showed that the pcDNA3.1-3HA-g NR1D1 eukaryotic expression vector was successfully constructed. In comparison with the control group, the expression of the NR1D1 gene in the experimental group was significantly increased at both the mRNA and protein levels. The CDS of the goat NR1D1 gene was 1 851 bp in length and encoded 616 amino acids. The goat NR1D1 protein was an unstable protein without transmembrane structure and signal peptide. The tertiary structure of the goat NR1D1 protein was highly similar to that of human and mouse. In this study, the eukaryotic expression vector of goat NR1D1 gene was successfully constructed, and its overexpression efficiency in HEK293T cells was validated at mRNA and protein levels, which laid the foundation for further research on the biological functions of goat NR1D1 gene.