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

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.

[A strategy for identifying six species of Sect.Cruciata(Gentiana) in Gansu using DNA barcode sequences].

Located in the transition zone between the Qinghai-Tibet Plateau and the Loess Plateau, Gansu province is one of the distribution centers of Sect. Cruciata, Gentiana (Gentianaceae) in China. Six species in the section, G. crassicaulis, G. straminea, G. siphonantha, G. officinalis, G. dahurica and G . macrophylla, are native to Gansu. In this paper, samples of 6 species and Halenia elliptica (outgroup) were collected. Nuclear DNA ITS, chloroplast DNA matK, rbcL, rpoC1, trnL (UAA) intron, psbA-trnH, atpB-rbcL, trnS (GCU)-trnG (UCC), rpl20-rps12 and trnL (UAA)-trnF (GAA) were sequenced from these samples. Based on the sequence analyses, high intragenomic polymorphisms were detected in ITS regions of G. crassicaulis, G. straminea, G. siphonantha, G. officinalis and G. dahurica, and they showed incomplete concerted evolution. A methodological study to identifying such close-related species as G. macrophylla, G. officinalis and G. dahurica was carried out based on the special genotypes. The results showed that 7 cp DNA sequence fragments could be used to identify G. crassicaulis, G. straminea and G. siphonantha. With nr ITS genotype II,III and IV of G. dahurica, the species can be distinguished from the close-related G. officinalis using 12 cloned sequences in a sample (with statistical significance).The cp DNA sequences of G. macrophylla were classified into two genotypes, and with genotype II, the species can be distinguished from the close-related G. officinalis and G. dahurica using 6 test samples each(with statistical significance). Furthermore, DNA barcode sequences were determined for all 6 species in Gansu. Also, the studies provide some basic data for analyses of genetic diversity and identification of Gentiana species.

[DNA-based identification of Gentiana robusta and related species].

The alpine plant Gentiana robusta is an endemic species to the Sino-Himalayan subregion. Also, it is one of the original plants used as traditional Tibetan medicine Jie-Ji. We sequence the nuclear ribosomal internal transcribed spacer (ITS) regions, matK, rbcL, rpoC1, trnL (UAA), psbA-trnH, atpB-rbcL, trnS( GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF( GAA) fragments of cp DNA in both G. robusta and such relative species as G. straminea, G. crassicaulis and G. waltonii. With Halenia elliptica as the outgroup, molecular systematic analysis reveals that G. robusta is a natural hybrid. G. straminea is the mother of hybrids, but the father is not very clear. In addition, the molecular markers for distinguishing G. robusta from the parental species or closely related species are identified, respectively. Our studies may provide valuable reference for the species identifications of medicinal plants with complex genetic backgrounds.

[Studies on genetic diversity of three Tibetan herbs].

The genetic diversity of three Tibetan herbs, i. e., Sang-Di, E-Dewa and Ye-Xingba (Tibetan names), was studied based on the field collection, specimen identification and DNA sequence analysis. Swertia hispidicalyx, Gentiana lhassica and Scrophularia dentata, as the original plants of the three Tibetan herbs, were collected and identified. The regions of ITS, matK, rbcL, rpoC1, trnL(UAA), psbA-trnH, atpB-rbcL, trnS (GCU)-trnG(UCC), rpl20-rps12, trnL(UAA)-trnF(GAA) and nadl 2nd intron were amplified and sequenced. The ITS regions of S. hispidicalyx and S. dentata were cloned and sequenced, and the sequences were classified into different genotypes. All the sequences were analyzed and compared with those of closely related species. Our studies may provide reference for the genetic diversity analysis and molecular identification of the three Tibetan herbs.

[Study on Embryonic Development of Four Species of Gentiana (Gentianaceae)].

OBJECTIVE: To study the embryonic development of Gentiana straminea, G. robusta, G. crassicaulis and G. tibetica. METHODS: The seed germination rates, length and width of embryos, starch grains and chloroplasts were observed and analyzed by statistic software. RESULTS: The seed germination rates of the four species were all high. The increase of the length of embryo depended mainly on the increase of the length of hypocotyl. Starch grains were stored in cells and chloroplasts appeared in cotyledons. The process of germination was divided into eight periods. CONCLUSION: The embryo growth characteristics of the four species are recognized, and the results can be used to study the in situ conservation, genetic breeding and cultivation of Sect. Cruciata.

[In situ investigation to three dimensional structures of Chinese medicines seeds].

This paper is aimed to microscopic identification of traditional Chinese medicines (TCMs) using an in situ imaging method. In this study, two kinds of Zingiberaceae seeds, Amomi Rotundus Fructus and Alpiniae Katsumadai Semen, were investigated by synchrotron radiation in-line X-ray phase-contrast computed tomography (IXPCT) imaging method. The results showed that the microstructures of these Zingiberaceae seeds could be clearly obtained from the virtual slices information in different observing angles. It proves that IXPCT is an effective imaging method, which can provide the imaging information for the microscopic identification of the intact TCMs in situ and non-destructively.

[Identification of medicinal plants used as Tibetan traditional medicine Chuan-Bu based on nrDNA markers].

OBJECTIVE: To identify the common Tibetan herb Chuan-Bu. METHOD: Local herbalists were visited to observe which plants were being used as Chuan-Bu. Samples of the indigenous plants were collected at the same time. Leaf materials were collected from field surveys. Total genomic DNA was extracted from silica gel-dried leaf samples. The PCR products were purified and directly sequenced. RESULT: As the origin of Chuan-Bu in Tibet autonomous region was authenticated, two species were determined, i. e. Euphorbia stracheyiand E. wallichii. Also, based on our earlier research, the origin of Chuan-Bu in Gansu province, is from E. kansuensis. The sequences of ITS1 for E. stracheyi and E. wallichii were 261 bp in size, and 221 bp in ITS2, respectively. The size of the 5.8S coding region was 164 bp for all species examined in the genus. Especially, there was a heterozygous locus in ITS1 (C/G; position 72) for E. stracheyi. The nucleotide divergence between sequences of the 6 species in pairwise comparisons was calculated and the result showed that the variable site could be detected in each pairwise comparison of sequences. Also, there were 8 point mutations in the 5.8S coding region. CONCLUSION: nrDNA ITS sequences can be used as the molecular markers to identify the Tibetan herb Chuan-Bu and such Traditional Chinese Medicines from the same genus Euphorbia as E. lathyris, E. humifusa and E. pekinensis.