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DNA甲基转移酶是DNA甲基化过程中最关键的酶,对生物体内的基因表达调节起着重要作用。研究根据灵芝转录组数据,通过全基因合成首次克隆得到灵芝DNA甲基转移酶Gl MET1,Genbank注册号为KU239998,并对其基因特性和空间结构进行全面的生物信息学分析。原核表达诱导分析表明p ET28a(+)-Gl MET1重组质粒在BL21(DE3)中能成功表达出目的蛋白,对其诱导条件进行优化,得出蛋白最合适的表达条件为:诱导温度为16℃,重组菌生长2.5 h(A600为0.8),IPTG浓度为0.2 mmol·L~(-1),诱导时间为12 h。实时荧光定量PCR结果表明不同品种灵芝Gl MET1的基因表达水平均有明显差异,且Gl MET1在成熟期的表达水平均低于幼年期,说明Gl MET1表达量随着灵芝的生长发育呈下降趋势。该研究结果为深入研究DNA甲基转移酶蛋白的作用机制奠定了基础。
DNA methyltransferase is the most critical enzyme in DNA methylation process and plays an important role in the regulation of gene expression in organisms. According to Ganoderma lucidum transcriptome data, Ganoderma lucidum DNA methyltransferase Gl MET1 was first cloned by whole-genome synthesis and GenBank accession number KU239998. The bioinformatics analysis was performed on its gene characteristics and spatial structure. The result of prokaryotic expression analysis showed that the recombinant protein of pET28a (+) - Gl MET1 could successfully express the target protein in BL21 (DE3), and the optimal conditions for its expression were obtained. The optimum conditions for the protein expression were: induction temperature 16 ℃ , The recombinant bacteria grew 2.5 h (A600 was 0.8), the concentration of IPTG was 0.2 mmol·L -1, and the induction time was 12 h. Real-time PCR results showed that the gene expression levels of Gl MET1 in different varieties of ganoderma lucidum were significantly different, and the expression level of Gl MET1 in mature stage was lower than that in infancy, indicating that Gl MET1 expression decreased with the growth and development of ganoderma lucidum. This study lays the foundation for further study on the mechanism of DNA methyltransferase.