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Hyoscyamine and scopolamine, belonging to medicinal tropane alkaloids (MTAs), are potent anticholinergic drugs. Their industrial production relies on medicinal plants, but the levels of the two alkaloids are very low in planta. Engineering the MTA's production is an everlasting hot topic for pharmaceutical industry. With understanding the MTA's biosynthesis, biotechnological approaches are established to produce hyoscyamine and scopolamine in an efficient manner. Great advances have been obtained in engineering MTA's production in planta. In this review, we summarize the advances on the biosynthesis of MTAs and engineering the MTA's production in hairy root cultures, as well in plants. The problems and perspectives on engineering the MTA's production are also discussed. [ABSTRACT FROM AUTHOR]

Real-time quantitative PCR (RT-qPCR) is an important technique for studying gene expression analysis, but accurate and reliable results depend on the use of a stable reference gene. This study proposes to test the expression stability of candidate reference genes in the callus of Saussurea laniceps, a unique Tibetan medicinal plant. Based on the S. laniceps callus transcriptome, eleven candidate reference genes, including TUA2, TUB3, TUB8, TIF3B1, TIF3H1, ELF5A, PP2AA2, UEV1D, UBL5, UBC36, and SKIP1), were validated for RT-qPCR normalization in the callus under abiotic stress (salt, cold, and UV) and hormone treatments (abscisic acid, MeJA, and salicylic acid). The stability of the candidate genes was evaluated in all the samples of S. laniceps. Comprehensive analysis of all samples showed that the best reference genes were UBC36 and UBL5. ELF5A and TIF3B1 were ranked as the most stable genes in the sample sets under abiotic stress. For hormone stimulation, UBC36 and TIF3H1 genes had the best stability. This study provides useful guidelines and a starting point for reference gene selection for expression analysis using RT-qPCR techniques in S. laniceps. [ABSTRACT FROM AUTHOR]

Przewalskia tangutica is a traditional medicinal plant from Tibet used for the analgesic effect from the tropane alkaloids (TAs) produced by the plant. Its roots have the highest yield of hyoscyamine in all plant species and so have been overharvested becoming an endangered medicinal plant species. Metabolic engineering is a good way to improve the yield of TAs in plants. In our study, two functionally distinct tropinone reductases genes, PtTRI and PtTRII, were cloned from P. tangutica and the functional divergence were characterized. The enzyme kinetics of PtTRI and PtTRII were investigated. The phylogenetic analysis classified them into different clades: PtTRI and PtTRII were in the clade of tropine‐forming reductase and pseudotropine‐forming reductase, respectively. We found PtTRI to be expressed in the roots but less in leaves, whereas PtTRII was expressed in the roots at higher levels than in the leaves. The kinetic parameters (Km, Vmax, and Kcat) were analyzed using purified recombinant enzymes at their optimum pH. Enzymatic analysis results showed that tropinone is a better substrate for PtTRII compared with PtTRI, suggesting that PtTRII might be a potential gene target for TA biosynthesis engineering. Compared with the reported TRIs, PtTRI exhibited a higher affinity for tropinone. [ABSTRACT FROM AUTHOR]

Polygonatum cirrhifolium is a rare and endangered Tibetan medicinal plant. In this paper, resource status of P. cirrhifolium from 13 counties of Tibet Plateau was Investigated by combining field Investigation with laboratory analysis, and sustainable utilization countermeasures were analyzed. Results showed that (1) distribution area sequence of P. cirrhifolium in each county wets Gongbo'gvamda County > Chayu County > Lang County > Milin County > Maizhokunggar County > Zuogong County > Mangkang County > Jiali County > Dlngqing County > Duilongdeqing County > Baqing County = Suo County = Nimu County. Its total distribution area was 458 723.81 hm², and It was not distributed in Baqing County, Nimu County and Suo County. (2) Average dry weight per plant of P. cirrhifolium medicinal part In each county had the bellow sequence; Duilongdeqing County > Gongbo'gvamda County > Maizhokunggar County > Zuogong County > Dlngqing County > Lang County > Mangkang County > Milin County >Jiali County >Chayu County > Baqing County = Nimu County = Suo County, and it reached 46.48 g/plant in Duilongdeqing County. (3) Biomass of P. cirrhifolium medicinal part in each county had the bellow sequence; Duilongdeqing County >Gongbo'gvamda County >Maizhokunggar County > Lang County > Dlngqing County > Zuogong County > Jiali County > Mangkang County > Milin County > Chayu County > Nimu County = Suo County = Baqing County, and It reached 300.52 kg/hm² in Duilongdeqing County. (4) Reserve amount sequence of P. cirrhifolium in each county was Gongbo'gvamda County > Lang County > Maizhokunggar County > Chayu County > Zuogong County > Dingqing County > Milin County > Jiali County > Mangkang County > Duilongdeqing County > Baqing County = Nimu County = Suo County, and total reserve amount was 42 293.961. (5) At medicinal function aspect, P. cirrhifolium Is sweet, and has the effects of keeping fit, nourishing vitality, regenerating teeth and delaying senility. (6) For resource status of P. cirrhifolium, It is suggested establishing resource protection region and constructing germplasm resource bank; establishing plantation park and perfecting cultivation and planting techniques; deeply processing and extending Industry chain of Tibetan medicine; updating concept and rationally developing and using. [ABSTRACT FROM AUTHOR]

Mirabilis himalaica is an endangered medicinal plant species in the Tibetan Plateau. The two genes respectively encoding chalcone synthase ( MhCHS) and chalcone isomerase ( MhCHI) were isolated and characterized from M. himalaica. The sequence analysis revealed that the two genes were similar with their corresponding homologous genes in other plants. The tissue profiles showed that both MhCHS and MhCHI had higher expression levels in roots than in stems and leaves. Transgenic hairy root cultures respectively with overexpressing MhCHS and MhCHI were established. The genomic PCR detection confirmed the authority of transgenic hairy root lines, in which either MhCHS or MhCHI expression levels were much higher than that in non-transgenic hairy root line. Finally, the HPLC detection results demonstrated that the rotenoid contents in MhCHS/ MhCHI-transformed hairy root lines were enhanced. This study provided two candidate genes that could be used to genetic engineering rotenoid biosynthesis in M. himalaica and an alternative method to produce rotenoid using transgenic hairy root cultures. [ABSTRACT FROM AUTHOR]

Saussurea medusa is an important traditional Tibetan medicinal plant in China. In this study, we assembled the complete chloroplast (cp) genome of S. medusa. The complete S. medusa chloroplast genome is a circular molecular structure of 152,257 bp in length with coding GC 37.93%, consisting of two inverted repeats (25,204 bp) separated by a large single-copy region (83,334 bp) and a small single-copy region (18,515 bp). The complete chloroplast genome of S. medusa contained 130 genes, including 87 protein-coding genes, 35 tRNA genes, and eight rRNA genes. Phylogenetic analysis shows that S. medusa is most closely related to Saussurea inversa and Saussurea pseudoleucoma. The complete chloroplast genome sequence of S. medusa facilitates the phylogenetic studies of Asteraceae. [ABSTRACT FROM AUTHOR]

Mirabilis himalaica is an important Tibetan medicinal plant in China. However, it has become a rare and class I endangered Tibetan medicine plant. Therefore, the use of callus to propagate germplasm resources is of great significance. We found that the flavonoid content of M. himalaica callus increased continuously with the extension of UV-B treatment. Multi-omics profiles were used to reveal the co-expression patterns of gene networks of flavonoid metabolism in M. himalaica callus during UV-B radiation. Results showed that five medicinal metabolics, including geranin, eriodictyol, astragalin, isoquercetin, pyrotechnic acid, and one anthocyanin malvide-3-O-glucoside were identified. The transcriptome data were divided into 46 modules according to the expression pattern by WGCNA (weighted gene co-expression network analysis), of which the module Turquoise had the strongest correlation with six target metabolites. We found that seven structural genes and twenty-five transcription factors were related to the metabolism of flavonoid synthesis, among which the structural genes CHI , C4H and UGT79B6 had strong co-expression relationships with the 6 target metabolites. WRKY42, WRKY7, bHLH128 and other transcription factors had strong co-expression relationships with multiple structural genes. Consequently, these findings suggest callus grown under UV-B treatment could be an effective alternative medical resource of M. himalaica , which is valuable for conservation and usage of this wild and endangered plant. • Six key medicinal metabolic were identified in M. himalaica callus by UV-B radiation. • WGCNA was used to analyze the relationship between gene modules and metabolites. • CHI , C4H and UGT79B6 had strong co-expression relationship with 6 target metabolites. • WRKY42, WRKY7, bHLH128 had strong co-expression relationships with structural genes. [ABSTRACT FROM AUTHOR]

Atropa belladonna is one of the most important plant sources for producing pharmaceutical tropane alkaloids (TAs). T1 progeny of transgenic A. belladonna , in which putrescine N -methyltransferase (EC. 2.1.1.53) from Nicotiana tabacum (NtPMT) and hyoscyamine 6β-hydroxylase (EC. 1.14.11.14) from Hyoscyamus niger (HnH6H) were overexpressed, were established to investigate TA biosynthesis and distribution in ripe fruits, leaves, stems, primary roots and secondary roots under field conditions. Both NtPMT and HnH6H were detected at the transcriptional level in transgenic plants, whereas they were not detected in wild-type plants. The transgenes did not influence the root-specific expression patterns of endogenous TA biosynthetic genes in A. belladonna . All four endogenous TA biosynthetic genes ( AbPMT , AbTRI , AbCYP80F1 and AbH6H ) had the highest/exclusive expression levels in secondary roots, suggesting that TAs were mainly synthesized in secondary roots. T1 progeny of transgenic A. belladonna showed an impressive scopolamine-rich chemotype that greatly improved the pharmaceutical value of A. belladonna . The higher efficiency of hyoscyamine conversion was found in aerial than in underground parts. In aerial parts of transgenic plants, hyoscyamine was totally converted to downstream alkaloids, especially scopolamine. Hyoscyamine, anisodamine and scopolamine were detected in underground parts, but scopolamine and anisodamine were more abundant than hyoscyamine. The exclusively higher levels of anisodamine in roots suggested that it might be difficult for its translocation from root to aerial organs. T1 progeny of transgenic A. belladonna , which produces scopolamine at very high levels (2.94–5.13 mg g −1 ) in field conditions, can provide more valuable plant materials for scopolamine production. [ABSTRACT FROM AUTHOR]

[Display omitted] • A tyrosine aminotransferase gene (RcTAT) was cloned from Rhodiola crenulata. • RcTAT catalyzes the formation of 4-hydroxyphenylpyruvate from tyrosine. • Suppression of RcTAT significantly enhances the salidroside production. Rhodiola crenulata is a traditional Tibetan medicinal plant mainly distributed in high altitude areas of Tibet. As the main extract of R. crenulata , salidroside has important medicinal activities in enhancing the body's immunity and resisting microwave radiation by influencing cell metabolism. Tyrosine is the starting amino acid precursor for salidroside biosynthesis. As an aromatic amino acid, tyrosine participates not only in salidroside biosynthesis, but also in other metabolite biosynthesis which might compete against salidroside biosynthetic pathway. In this study, we functionally identified a tyrosine aminotransferase in R. crenulata (RcTAT) that could convert tyrosine into 4-hydroxyphenylpyruvate (4-HPP). The Km and Vmax values of RcTAT for tyrosine were respectively 0.21 mM and 0.17 μmol/min.mg under 9.0 pH and 39 °C. RNAi-mediated suppression of RcTAT expression markedly increased salidroside production in hairy root culture of R. crenulata. In summary, RcTAT is a key gene involved in the metabolic pathway that competes against salidroside biosynthesis, and suppressing its expression is a promising way to elevate salidroside production in planta. [ABSTRACT FROM AUTHOR]

• Glyphosate is used for screening transgenic plants of Atropa belladonna. • Homozygous transgenic plants overexpressing HnH6H and G2-EPSPS are obtained. • Homozygous transgenic plants have high-yield scopolamine and glyphosate resistance. Atropa belladonna , one of the most important medicinal plants in China, is used to produce anticholinergic tropane alkaloids. Weeds severely reduce the agricultural productivity of A. belladonna in fields, and the low content of high-value scopolamine in planta limits its economic value. In this study, homozygous lines of A. belladonna , with glyphosate resistance and high yield of scopolamine, were developed using metabolic engineering technology for the first time. Two homozygous transgenic lines (T2GH03 and T2GH06), harboring G2-EPSPS and HnH6H , were established and tested in the field. Glyphosate resistance assays demonstrated that the two homozygous transgenic lines could resist commercial recommended dose of glyphosate. Alkaloid analysis showed that the transgenic lines produced more scopolamine than wild-type plants. T2GH03 and T2GH06 produced scopolamine at the levels of 5.45 mg/g dry weight (DW) and 7.04 mg/g DW respectively in leaves, and 1.06 mg/g DW to 1.07 mg/g DW respectively in roots. Compared with wild-type plants, the scopolamine contents of homozygous transgenic lines increased by over 10 folds in leaves and nearly 5 folds in roots. To sum up, Atropa belladonna's new varieties with glyphosate resistance and high-yield scopolamine are developed, which are highly valuable for industrial production of scopolamine. [ABSTRACT FROM AUTHOR]

• A root-expressed SAUR gene (AbSAUR1) was cloned from Atropa belladonna. • Overexpression of AbSAUR1 significantly increased the biomass of Atropa belladonna. • Overexpression of AbSAUR1 did not reduce biosynthesis of tropane alkaloids. • The yield of TAs was significantly improved in AbSAUR1 -overexpressing plants. Tropane alkaloids (TAs), including hyoscyamine, anisodamine and scopolamine, are widely used as anticholinergic drugs in the clinic. At present, the production of TAs is completely dependent on extraction from medicinal plants of Solanaceae. Atropa belladonna , commonly known as deadly nightshade, is a major commercial source of TAs. Previous studies have focused on increasing TA content by overexpressing TA biosynthesis genes. However, research on increasing TA yield by increasing the biomass of A. belladonna has not been reported. In this study, a Small Auxin Up-regulated RNA (SAUR) gene from A. belladonna (AbSAUR1) had been cloned for the first time. Based on heat map and tissue expression analysis, it was demonstrated that AbSAUR1 was specifically expressed in roots. Compared to control plants, transgenic lines of A. belladonna overexpressing AbSAUR1 showed a number of phenotypic changes, including increased plant height, thickened stems, increased number of branches, leaves and roots. Meanwhile, the biomass of AbSAUR1 -overexpressing plants also increased significantly, with fresh weight and dry weight being 3.79- and 3.20-fold higher than control plants, respectively. On average, there was no significant change in TA content, but the production of hyoscyamine, anisodamine and scopolamine was slightly enhanced. The overall yield of TAs was significantly improved, reaching 3.55 times that of the control. In summary, the AbSAUR1 gene can be employed as an effective molecular tool to increase the yield of TAs by increasing the biomass of A. belladonna. [ABSTRACT FROM AUTHOR]