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

Abstract

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

CLC Number:

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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Construction of Eukaryotic Expression Vector and Bioinformatics Analysis of Circadian CLOCK Gene in Goats

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