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目的:为研究冬凌草二萜类合成的相关基因,在冬凌草转录组信息数据的基础之上,以冬凌草无菌苗为研究材料,克隆冬凌草二萜类合成的关键酶1-脱氧木酮糖-5-磷酸还原异构酶(l-deoxy-D-lxyluloses-phosphatereduetoisomerase,DXR)基因。方法:采用逆转录PCR技术克隆冬凌草DXR基因,实时荧光定量PCR法分析其组织表达模式。结果:DXR c DNA基因全长1 500 bp,DXR基因开放阅读框为1 422 bp,编码473个氨基酸组成的蛋白质序列,理论相对分子质量为51.39 k Da,等电点为6.09,是一种亲水性蛋白。DXR在茎中表达量相对较高,在愈伤组织中表达量最低。结论:研究结果为深入研究冬凌草DXR酶的活性和功能及为冬凌草二萜类化合物的生物合成机制、优良基因挖掘奠定基础。
OBJECTIVE: To study the related genes of diterpenoid synthesis of Rubescens indica L., based on the information of Rubescens indica transcriptome, L-deoxy-xylulose-5-phosphate reductoisomerase (l-deoxy-D-lxyluloses-phosphatereduetoisomerase, DXR) gene. Methods: DXR gene was cloned by reverse transcription polymerase chain reaction (RT-PCR) and its expression pattern was analyzed by real-time fluorescence quantitative PCR. RESULTS: The full-length DXR cDNA was 1 500 bp in length and 1 422 bp in open reading frame of DXR gene, encoding a protein sequence of 473 amino acids. The theoretical relative molecular mass was 51.39 kDa and the isoelectric point was 6.09. Aqueous protein. DXR expression in the stem is relatively high, the lowest expression in callus. CONCLUSION: The results of this study are to further study the activity and function of DXR enzyme in Rubia cordyceflora and to lay a foundation for the biosynthesis mechanism of diterpenoids and the search of good genes.