中国中药杂志

2016, v.41(06) 1021-1026

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灵芝DNA甲基转移酶(GlMET1)基因克隆及原核表达诱导分析
Cloning and prokaryotic expression analysis of DNA methyltransferas 1 ( MET1) in Ganoderma lucidum

查良平;王亚君;刘爽;赵玉洋;袁媛;黄璐琦;
ZHA Liang-ping;WANG Ya-jun;LIU Shuang;ZHAO Yu-yang;YUAN Yuan;HUANG Lu-qi;College of Pharmacy,Chengdu Universityof Traditional Chinese Medicine;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;National Center for Nanoscience and Technology;

摘要(Abstract):

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 key enzyme in the process of DNA methylation,playing an important role in regulation of gene expression in vivo. According to the Ganoderma lucidum transcriptome data,a full-length c DNA sequence of MET1 from G. lucidum was cloned for the first time,the Gen Bank registration number is KU239998,and we conducted a comprehensive bioinformatics analysis of the genetic characteristics and spatial structure. The prokaryotic expression analysis showed that E. coli[p ET28a( +)-Gl MET1] in BL21( DE3) could induce objective protein,shaking the culture at 16 ℃ until the host bacterium( A600) was approximately 0. 8,and added IPTG to finally concentration of 0. 2 mmol·L~(-1),and then the optimal expression of Gl MET1 recombinant protein was accumulated for the induction time of 20 h. The real-time PCR results showed that the expression levels of Gl MET1 had obvious differences among varieties of G. lucidum. During the maturity stage,the expression levels of Gl MET1 were lower than that in juvenile stage,the results showed that with the growth of G. lucidum,the expression levels of Gl MET1 were on the decline. The research provided an important basis for studying the mechanism of DNA methyltransferase thoroughly.

关键词(KeyWords): 灵芝;甲基化;甲基转移酶;基因克隆;原核表达
Ganoderma lucidum;methylation;methyltransferas;gene cloning;prokaryotic expression

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基金项目(Foundation): 国家杰出青年科学基金项目(81325023);; 国家高技术研究发展计划(863)项目(SS2014AA022201)

作者(Author): 查良平;王亚君;刘爽;赵玉洋;袁媛;黄璐琦;
ZHA Liang-ping;WANG Ya-jun;LIU Shuang;ZHAO Yu-yang;YUAN Yuan;HUANG Lu-qi;College of Pharmacy,Chengdu Universityof Traditional Chinese Medicine;State Key Laboratory Breeding Base of Dao-di Herbs,National Resource Center for Chinese Materia Medica,China Academy of Chinese Medical Sciences;National Center for Nanoscience and Technology;

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