Two copies of 4-(cytidine 5'-diphospho)-2-C-methyl-d-erythritol kinase (CMK) gene in Ginkgo biloba: molecular cloning and functional characterization
Two copies of 4-(cytidine 5'-diphospho)-2-C-methyl-d-erythritol kinase (CMK) gene in Ginkgo biloba: molecular cloning and functional characterization
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DOI：
10.1007/s00425-008-0794-1
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发表时间：
2008-11-01
期刊：
影响因子：
4.3
通讯作者：
Kim, Soo-Un
中科院分区：
文献类型：
--
作者：
Kim, Sang-Min;Kim, Yeon-Bok;Kim, Soo-Un
4-(胞苷5‘-二磷酸)-2-C-甲基-d-赤藓糖醇激酶(CMK或YchB)是2-C-甲基-d-赤藓糖醇4-磷酸途径的第四个酶，在三磷酸腺苷的存在下，使4-(胞苷5’-二磷酸)-2-C-甲基-d-赤藓糖醇的2-羟基磷酸化。从银杏中克隆了两个编码CMK的同源基因(GbCMK1和GbCMK2)。用ychB基因敲除突变株Escherichia coliNMW29进行互补分析，证实了两种同工酶的活性。GbCMKs在银杏胚根和子叶中的转录谱表明，GbCMK1在两个器官中的转录水平相似，而GbCMK2在银杏内酯合成的胚根中的转录水平较高。光和茉莉酸甲酯处理可选择性地增加胚根中GbCMK2的转录丰度。这些转录本的差异诱导模式表明，GbCMK1和GbCMK2分别与初级和次级代谢有很高的相关性。在拟南芥和烟草瞬时表达系统中，两种同工酶的转运肽都将融合的绿色荧光蛋白(GFP)运送到叶绿体中；有趣的是，GbCMK1的转运肽除了将GFP蛋白运送到叶绿体外，还将GFP蛋白运送到细胞质和细胞核中。
4-(Cytidine 5'-diphospho)-2-C-methyl-d-erythritol kinase (CMK or YchB), the fourth enzyme of the 2-C-methyl-d-erythritol 4-phosphate pathway, phosphorylates the 2-hydroxyl group of 4-(cytidine 5'-diphospho)-2-C-methyl-d-erythritol in the presence of ATP. Two isogenes encoding CMK (GbCMK1 and GbCMK2) were cloned and characterized from Ginkgo biloba. The activities of both isozymes were confirmed by complementation assay using Escherichia coli NMW29, a ychB knock-out mutant. The transcript profiles of GbCMKs in the radicles and the cotyledons of the cultured Ginkgo biloba embryos demonstrated that the transcript levels of GbCMK1 were similar in both organs, whereas that of GbCMK2 was predominantly high in the ginkgolide-synthesizing radicles. Selective increases in the transcript abundance of GbCMK2 in the radicles, induced by light and methyl jasmonate treatments, were observed. These differential induction patterns of the transcripts imply GbCMK1 and GbCMK2 respectively have high correlations with the primary and the secondary metabolisms. The transit peptides of both isozymes delivered the fused green fluorescent protein (GFP) into the chloroplast in the Arabidopsis and the Nicotiana transient expression systems; interestingly, the transit peptide of GbCMK1 delivered the GFP protein into the cytosol and the nucleus in addition to the chloroplasts.