全萼秦艽转录组中环烯醚萜类相关基因挖掘及验证  

Transcriptome analysis and validation of key genes involved in biosynthesis of iridoids in Gentiana lhassica

作　　者：康恒 赵志礼[1] 倪梁红[1] 李尉涛 赵淑娟[1] 刘铜华 KANG Heng;ZHAO Zhi-li;NI Liang-hong;LI Wei-tao;ZHAO Shu-juan;LIU Tong-hua(Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Tibetan Traditional Medical College,Lhasa 850000,China;Beijing University of Chinese Medicine,Beijing 100029,China)

机构地区：[1]上海中医药大学,上海201203 [2]西藏藏医药大学,西藏拉萨850000 [3]北京中医药大学,北京100029

出　　处：《中国中药杂志》2021年第18期4704-4711,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金面上项目(82073959,81173654);藏医药区域协同创新中心项目(2018XTCX005)。

摘　　要：国产龙胆科龙胆属Gentiana植物广泛分布有龙胆苦苷等环烯醚萜苷类活性成分,具有重要的药用价值。在前期工作基础上,对藏族药"解吉那保"(■)基原植物之一全萼秦艽G.lhassica转录组进行测序分析,分别构建其根、茎、叶、花转录组数据库,挖掘可能参与编码环烯醚萜苷类生物合成通路中关键酶的unigene,应用qRT-PCR技术验证AACT、DXS、MCS、HDS、IDI、GPPS、GES、G10H、7-DLNGT、7-DLGT、SLS 11个基因在根、茎、叶、花中的表达量,测定根、茎、叶、花中龙胆苦苷、马钱苷酸含量。(1)共得到76486条unigene,平均长度为852 bp。(2)335条unigene参与到19个KEGG次生代谢标准通路中,其中,条数最多的通路为苯丙素生物合成,有75条;没有注释到异黄酮生物合成通路。(3)171条unigene参与编码环烯醚萜类化合物的生物合成代谢通路中的27个关键酶;值得注意的是,1-脱氧-D-葡萄糖-5-磷酸还原异构酶(DXR)基因表达量非常高。(4)qRT-PCR与RNA-Seq数据计算结果基本吻合,实验涉及的11个基因皆为地上部位(茎、叶、花)的相对表达量高于地下部位(根)。(5)龙胆苦苷、马钱苷酸总量的HPLC测定值均为地上部位(茎、叶、花)大于地下部位(根),差异明显。该研究可为藏族药"解吉那保"客观、准确的品种鉴定,种质资源评价,探讨龙胆科药用植物次生产物累积规律及高山濒危物种保护等提供基础科学资料。As the main chemical constituents,iridoids are widely distributed within Gentiana,Gentianaceae,with promising bioactivities.Based on the previous work,the transcriptome of G.lhassica,an original plant of Tibetan herb"Jieji Nabao",was sequenced and analyzed in this study,and the transcriptome databases of roots,stems,leaves,and flowers were constructed so as to explore unigenes that may encode the key enzymes in the biosynthetic pathway of iridoids.Then,qRT-PCR was used to validate the relative expression levels of 11 genes named AACT,DXS,MCS,HDS,IDI,GPPS,GES,G10H,7-DLNGT,7-DLGT,and SLS in roots,stems,leaves,and flowers.Also,the total contents of gentiopicroside and loganic acid were determined by HPLC,respectively.The results are as follows:(1)a total of 76486 unigenes with an average length of 852 bp were obtained;(2)335 unigenes were involved in 19 stan-dard secondary metabolism pathways in KEGG database,with phenylpropanoid biosynthesis having the maximum number(75 unigenes),and no isoflavone biosynthetic pathway was annotated;(3)171 unigenes participatedin 27 key enzymes encoding in the biosynthetic pathway of iridoids,and 1-deoxy-D-xylulose-5-phosphate reductoisomerase(DXR)gene was highly expressed;(4)qRT-PCR results were approximately consistent with RNA-Seq data and the relative expression levels of the 11 genes were higher in the aboveground parts(stem,leaf,and flower)than in the underground part(root);(5)the total contents of gentiopicroside and loganic acid were higher in the aboveground parts(stem,leaf,and flower)than in the underground part(root),and the difference was significant.This study provides basic scientific data for accurate species identification,evaluation of germplasm resources,research on secondary pro-duct accumulation of medicinal plants within Gentianaceae,and protection of endangered alpine species.

关 键 词：全萼秦艽 转录组 环烯醚萜类 代谢通路 QRT-PCR HPLC 

分 类 号：R29[医药卫生—民族医学]