ABSTRACT
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 76 486 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.
Subject(s)
Gene Expression Profiling , Gene Expression Regulation, Plant , Gentiana/genetics , Iridoids , TranscriptomeABSTRACT
italic>Gentiana crassicaulis Duthie ex Burk. in Gentiana (Sect. Cruciata), Gentianaceae, is one of the original plants of both Gentianae Macrophyllae Radix and Tibetan herb Jie-Ji Na-Bao, which contain such bioactive iridoids as gentiopicroside, loganic acid and others. In this study, based on previous work, the transcriptome of G. crassicaulis was sequenced and analyzed to construct transcriptome databases of roots, stems, leaves and flowers. qRT-PCR verification was conducted for parts of unigenes that may be key enzymes in the pathway of iridoid biosynthesis. The results are as follows: ① a total of 159 534 unigenes were obtained, with an average length of 679 bp. According to the functional classification of GO, unigenes can be divided into 3 categories with 67 branches. The unigenes were aligned in the KOG database and were classified into 25 categories according to function. ② In the KEGG database, 215 unigenes were implicated in 20 standard secondary metabolism pathways. The analysis shows that 305 unigenes encoded 28 key enzymes in the pathway of iridoid biosynthesis, and their expression in different organs is different; and ③ qRT-PCR was approximately consistent with RNA-Seq results. The 7 annotated unigenes identified in this study, HMGS, DXS, MCS, GPPS, G10H, 7-DLNGT and STR, all had higher relative expression levels in the above-ground parts (stem, leaf and flower) than in the underground part (root). Iridoids are common active and index components of such traditional Chinese medicines as Qinjiao, Longdan, Dangyao, and Qingyedan, among others. Therefore, this work provides basic scientific data for further development including obtaining active components or intermediates through biotechnology, exploring the accumulation of effective components, evaluating the quality of different ecotype varieties, and identifying authentic biosynthesis pathways of medicinal materials.