摘要
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 IspE and lspH unigene from Lonicera japonica and its substitutes, and analyze their gene se- quence, protein properties and transcriptional level. Method： IspE and IspH unigene ware obtained from the transcriptome dataset of L. japonica. Full-length cDNA of IspE and IspH were cloned from buds of L. japonica, L. japonica var. chinensis, L. hypoglaaca and L. dasystyla using RT-PCR technology, and named as LJlspE, LHIspE, LJCIspE, and LDIspE, LJlspH, LJCIspH, LHIspH and LDI- spH, respectively. And we also predicted the structure and function of IspE and IspH proteins. Result： IspE contained an open reading frame that consisted of 1 221 bp, encoding one polypeptide with 422 amino acids. A complete open reading frame of IspH gene consis- ted of 1 380 bp and encoded 459 amino acids. Both IspE and IspH ware non-secreted proteins and localized in the chloroplast. Tran- scripted level of IspE and IspH in bud of L. japonica, L. hypoglauca and L. dasystyla was not significantly difference, but their tran- scripted level in L. japonica var. chinensis was significantly higher than that in L. japonica. Conclusion ： The clone of IspE and IspH will help for further research on the synthesis of terpenes, aroma and color.
出处
《中国中药杂志》
CAS
CSCD
北大核心
2013年第1期32-36,共5页
China Journal of Chinese Materia Medica
基金
国家自然科学基金项目(81001605)
中药制药过程新技术国家重点实验室开放基金项目(SKL2010M0101)
