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目的:克隆金银花类药用植物4-二磷酸胞苷-2-C-甲基赤藓糖激酶(4-diphosphocytidyl-2-C-methyl-D-erythritol ki-nase,IspE)和4-羟基-3-甲基-2-邻苯基二磷酸还原酶(4-hydroxy-3-methylbut-2-enyl diphosphate reductase,IspH)基因,并对其基因序列、蛋白特性和转录活性进行分析、比较。方法:从忍冬Lonicera japonica转录组测序结果中分析获得了IspE,IspH基因。分别以忍冬、红白忍冬L.japonica var.chinensis、红腺忍冬L.hypoglauca和水忍冬L.dasystyla新鲜花蕾为材料,利用RT-PCR技术克隆获得了4种金银花类药用植物IspE和IspH基因的全长cDNA。运用生物信息学分析软件,预测编码蛋白的结构和功能,并通过RT-PCR检测IspE和IspH在忍冬、红白忍冬、红腺忍冬、水忍冬花蕾中的转录情况。结果:金银花类药用植物IspE基因含有1个完整的开放阅读框,长度为1 221 bp,编码406个氨基酸;IspH含有一个完整的开放阅读框,长度为1 380 bp,编码459个氨基酸。IspE和IspH均为非分泌蛋白,均定位于叶绿体中。RT-PCR分析结果表明在忍冬、红腺忍冬和水忍冬的花蕾中IspE,IspH基因的转录水平没有显著差异,但红白忍冬花蕾中IspE,IspH基因的转录水平均显著高于忍冬。结论:克隆获得忍冬、红白忍冬、红腺忍冬和水忍冬中IspE,IspH基因,并证实了其在不同金银花类药用植物中的表达,为进一步研究IspE,IspH基因对萜类化合物生物合成和花香气以及颜色的影响奠定了基础。
Objective: To clone 4-diphosphocytidyl-2-C-methyl-D-erythritol ki-nase (IspE) and 4-hydroxy- 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (IspH) gene and its gene sequence, protein characteristics and transcriptional activity were analyzed and compared. Methods: The IspE and IspH genes were obtained from the Lonicera japonica transcriptome sequencing results. The honeysuckle honeysuckle, L.japonica var. Chinensis, L.hypoglauca and L.dasystyla were respectively cloned by RT-PCR. The IspE and IspH genes of four honeysuckle medicinal plants Full-length cDNA. Bioinformatics analysis software was used to predict the structure and function of the encoded protein. The transcriptional status of IspE and IspH in honeysuckle, honeysuckle honeysuckle, honeysuckle honeysuckle, water honeysuckle bud were detected by RT-PCR. Results: The IspE gene of the honeysuckle medicinal plant contains a complete open reading frame with a length of 1 221 bp encoding 406 amino acids. IspH contains a complete open reading frame with a length of 1 380 bp encoding 459 amino acids. IspE and IspH are non-secreted proteins, both located in the chloroplast. The results of RT-PCR analysis showed that the transcript levels of IspE and IspH genes in honeysuckle, honeysuckle and honeysuckle were not significantly different, but the transcript levels of IspE and IspH in honeysuckle honeysuckle were significantly higher than those in honeysuckle. Conclusion: The IspE and IspH genes of Lonicera japonica, Lonicera japonica, Lonicera macranthoides and Lonicera japonica were cloned and confirmed to be expressed in different honeysuckle medicinal plants. To further study the effects of IspE and IspH on terpenoid biosynthesis And floral aroma as well as the impact of color laid the foundation.