山羊生物钟基因CLOCK真核表达载体的构建和生物信息学分析

doi:10.16431/j.cnki.1671-7236.2021.12.001

摘要/Abstract

摘要： 试验旨在构建山羊昼夜运动输出周期蛋白(circadian locomotor output cycles kaput,CLOCK)基因真核表达载体,系统分析山羊CLOCK蛋白的生物学特性。从山羊卵巢组织中提取总RNA,反转录成cDNA后经PCR扩增山羊CLOCK基因CDS区序列,并以同源重组的方式将其连接至pcDNA3.1-Puro-N-3HA载体;经PCR、酶切和测序鉴定后,将阳性质粒命名为pcDNA3.1-3HA-gCLOCK;将pcDNA3.1-Puro-N-3HA和pcDNA3.1-3HA-gCLOCK质粒分别转染至HEK293T细胞中,通过实时荧光定量PCR和Western blotting检测山羊CLOCK基因的表达效果,并对山羊CLOCK基因进行系统的生物信息学分析。结果显示,山羊CLOCK基因CDS区片段长2 538 bp,将其与线性化的pcDNA3.1-Puro-N-3HA载体重组连接并通过酶切和测序鉴定后,成功构建了pcDNA3.1-3HA-gCLOCK真核表达载体;实时荧光定量PCR和Western blotting检测结果显示,pcDNA3.1-3HA-gCLOCK转染组CLOCK基因在mRNA和蛋白水平的表达量均极显著高于pcDNA3.1-Puro-N-3HA对照组(P<0.01)。生物信息学分析结果表明,山羊CLOCK基因CDS区序列与绵羊、牛和马的相似性分别为99.4%、98.7%和95.6%。山羊CLOCK蛋白是一种不稳定蛋白,具有一定的亲水性,无跨膜区和信号肽。二级结构由α-螺旋、延伸链、β-转角和无规则卷曲组成;三级结构与小鼠和人的CLOCK蛋白相比具有极高的相似性。本研究成功构建了山羊生物钟基因CLOCK真核表达载体,并进行了生物信息学分析,为进一步研究山羊CLOCK基因的生物学功能及山羊生物钟的转录调控机制提供了材料。

关键词: 山羊; CLOCK基因; 真核表达载体; 生物信息学分析

Abstract: This study was aimed to construct the eukaryotic expression vector of circadian locomotor output cycles kaput (CLOCK) gene in goats, and systematically analyze the biological characteristics of CLOCK protein in goats.Total RNA extracting from ovary tissues in goats was reverse transcribed into cDNA, the coding sequence (CDS) of CLOCK gene in goats was amplified by PCR using the cDNA template.PCR product was ligated to pcDNA3.1-Puro-N-3HA vector by homologous recombination.The recombinant plasmid was identified by PCR, restriction enzyme digestion and sequencing, and the positive plasmid was named pcDNA3.1-3HA-gCLOCK.The pcDNA3.1-Puro-N-3HA and pcDNA3.1-3HA-gCLOCK plasmids were transfected into HEK293T cells, respectively.The expression of CLOCK gene was examined by Real-time quantitative PCR and Western blotting analysis.Meanwhile, several bioinformatics analysis methods were performed systematically on CLOCK gene in goats.The results showed that the length of CLOCK gene in goats were 2 538 bp, the PCR fragment was linked to the linearized pcDNA3.1-Puro-N-3HA and identified by restriction enzyme digestion and sequencing, the eukaryotic expression vector of pcDNA3.1-3HA-gCLOCK was successfully constructed.Real-time quantitative PCR and Western blotting results showed that the expression of mRNA and protein of CLOCK gene in goats transfected with pcDNA3.1-3HA-gCLOCK in HEK293T cells were extremely significantly higher than that in pcDNA3.1-Puro-N-3HA group (P<0.01).Bioinformatics analysis showed that the similarity of CLOCK gene CDS sequence in goats with Ovis aries, Bos taurus and Equus caballus was 99.4%, 98.7% and 95.6%, respectively.CLOCK protein in goats was an unstable protein with certain hydrophilicity, and no transmembrane region and signal peptide was observed.The secondary structure of CLOCK protein consisted mainly of alpha helix, extended chain, beta turn and random coil, and the tertiary structure was highly similar to CLOCK protein in mouse and human.In this study, the eukaryotic expression vector of CLOCK gene in goats was successfully constructed, and bioinformatics analysis was carried out, which provided materials for the further study on the biological functions of CLOCK gene and the transcriptional regulation mechanism of circadian clock system in goats.

Key words: goats; CLOCK gene; eukaryotic expression vector; bioinformatics analysis

中图分类号:

赵泓淙, 高登科, 江海圳, 李雅婷, 李丹, 马白荣, 靳亚平, 陈华涛. 山羊生物钟基因CLOCK真核表达载体的构建和生物信息学分析[J]. 中国畜牧兽医, 2021, 48(12): 4327-4338.

ZHAO Hongcong, GAO Dengke, JIANG Haizhen, LI Yating, LI Dan, MA Bairong, JIN Yaping, CHEN Huatao. Construction of Eukaryotic Expression Vector and Bioinformatics Analysis of Circadian CLOCK Gene in Goats[J]. China Animal Husbandry and Veterinary Medicine, 2021, 48(12): 4327-4338.

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