Jasmonate- and abscisic acid-activated AaGSW1-AaTCP15/AaORA transcriptional cascade promotes artemisinin biosynthesis in Artemisia annua.

Artemisinin, a sesquiterpene lactone widely used in malaria treatment, was discovered in the medicinal plant Artemisia annua. The biosynthesis of artemisinin is efficiently regulated by jasmonate (JA) and abscisic acid (ABA) via regulatory factors. However, the mechanisms linking JA and ABA signalling with artemisinin biosynthesis through an associated regulatory network of downstream transcription factors (TFs) remain enigmatic. Here we report AaTCP15, a JA and ABA dual-responsive teosinte branched1/cycloidea/proliferating (TCP) TF, which is essential for JA and ABA-induced artemisinin biosynthesis by directly binding to and activating the promoters of DBR2 and ALDH1, two genes encoding enzymes for artemisinin biosynthesis. Furthermore, AaORA, another positive regulator of artemisinin biosynthesis responds to JA and ABA, interacts with and enhances the transactivation activity of AaTCP15 and simultaneously activates AaTCP15 transcripts. Hence, they form an AaORA-AaTCP15 module to synergistically activate DBR2, a crucial gene for artemisinin biosynthesis. More importantly, AaTCP15 expression is activated by the multiple reported JA and ABA-responsive TFs that promote artemisinin biosynthesis. Among them, AaGSW1 acts at the nexus of JA and ABA signalling to activate the artemisinin biosynthetic pathway and directly binds to and activates the AaTCP15 promoter apart from the AaORA promoter, which further facilitates formation of the AaGSW1-AaTCP15/AaORA regulatory module to integrate JA and ABA-mediated artemisinin biosynthesis. Our results establish a multilayer regulatory network of the AaGSW1-AaTCP15/AaORA module to regulate artemisinin biosynthesis through JA and ABA signalling, and provide an interesting avenue for future research exploring the special transcriptional regulation module of TCP genes associated with specialized metabolites in plants.

Overexpression of blue light receptor AaCRY1 improves artemisinin content in Artemisia annua L. .

Artemisinin, an effective antimalarial compound, is isolated from the medicinal plant Artemisia annua L. However, because of the low content of artemisinin in A. annua, the demand of artemisinin exceeds supply. Previous studies show that the artemisinin biosynthesis is promoted by light in A. annua. Cryptochrome1 (CRY1) is involved in many processes in the light response. In this study, AaCRY1 was cloned from A. annua. Overexpressing AaCRY1 in Arabidopsis thaliana cry1 mutant resulted in blue-light-dependent short hypocotyl phenotype and short coleoptile under blue light. Yeast two-hybrid and subcellular colocalization showed that AaCRY1 interacted with AtCOP1 (ubiquitin E3 ligase CONSTITUTIVE PHOTOMORPHOGENIC1). Overexpression of AaCRY1 in transgenic A. annua increased the artemisinin content. When AaCRY1 was overexpressed in A. annua driven by the CYP71AV1 (cytochrome P450 dependent amorpha-4,11-diene 12-hydroxylase) promoter, the artemisinin content was 1.6 times higher than that of the control. Furthermore, we expressed the C terminal of AaCRY1(CCT) involved a GUS-CCT fusion protein in A. annua. The results showed that the artemisinin content was increased to 1.7- to 2.4-fold in GUS-CCT transgenic A. annua plants. These results demonstrate that overexpression of GUS-CCT is an effective strategy to increase artemisinin production in A. annua.

The roles of AaMIXTA1 in regulating the initiation of glandular trichomes and cuticle biosynthesis in Artemisia annua.

The glandular secretory trichomes (GSTs) on Artemisia annua leaves have the capacity to secrete and store artemisinin, a compound which is the most effective treatment for uncomplicated malaria. An effective strategy to improve artemisinin content is therefore to increase the density of GSTs in A. annua. However, the formation mechanism of GSTs remains poorly understood. To explore the mechanisms of GST initiation in A. annua, we screened myeloblastosis (MYB) transcription factor genes from a GST transcriptome database and identified a MIXTA transcription factor, AaMIXTA1, which is expressed predominantly in the basal cells of GST in A. annua. Overexpression and repression of AaMIXTA1 resulted in an increase and decrease, respectively, in the number of GSTs as well as the artemisinin content in transgenic plants. Transcriptome analysis and cuticular lipid profiling showed that AaMIXTA1 is likely to be responsible for activating cuticle biosynthesis. In addition, dual-luciferase reporter assays further demonstrated that AaMIXTA1 could directly activate the expression of genes related to cuticle biosynthesis. Taken together, AaMIXTA1 regulated cuticle biosynthesis and prompted GST initiation without any abnormal impact on the morphological structure of the GSTs and so provides a new way to improve artemisinin content in this important medicinal plant.

Transcriptome Analysis of Genes Associated with the Artemisinin Biosynthesis by Jasmonic Acid Treatment under the Light in Artemisia annua.

Artemisinin is a sesquiterpene lactone endoperoxide extracted from a traditional Chinese medicinal plant Artemisia annua. Artemisinin-based combination therapies (ACTs) are recommended as the best treatment of malaria by the World Health Organization (WHO). Both the phytohormone jasmonic acid (JA) and light promote artemisinin biosynthesis in A. annua. Interestingly, we found that the increase of artemisinin biosynthesis by JA was dependent on light. However, the relationship between the two signal pathways mediated by JA and light remains unclear. Here, we collected the A. annua seedlings of 24 h continuous light (Light), 24 h dark treatment (Dark), 4 h MeJA treatment under the continuous light conditions (Light-MeJA-4h) and 4 h MeJA treatment under the dark conditions (Dark-MeJA-4h) and performed the transcriptome sequencing using Illumina HiSeq 4000 System. A total of 266.7 million clean data were produced and assembled into 185,653 unigenes, with an average length of 537 bp. Among them, 59,490 unigenes were annotated and classified based on the public information. Differential expression analyses were performed between Light and Dark, Light and Light-MeJA-4h, Dark and Dark-MeJA-4h, Light-MeJA-4h, and Dark-MeJA-4h, respectively. Furthermore, transcription factor (TF) analysis revealed that 1588 TFs were identified and divided into 55 TF families, with 284 TFs down-regulated in the Dark relative to Light and 96 TFs up-regulated in the Light-MeJA-4h relative to Light. 8 TFs were selected as candidates for regulating the artemisinin biosynthesis and one of them was validated to be involved in artemisinin transcriptional regulation by Dual-Luciferase (Dual-LUC) assay. The transcriptome data shown in our study offered a comprehensive transcriptional expression pattern influenced by the MeJA and light in A. annua seedling, which will serve as a valuable resource for further studies on transcriptional regulation mechanisms underlying artemisinin biosynthesis.

A basic leucine zipper transcription factor, AabZIP1, connects abscisic acid signaling with artemisinin biosynthesis in Artemisia annua.

Artemisinin is a sesquiterpenoid especially synthesized in the Chinese herbal plant, Artemisia annua, which is widely used in the treatment of malaria. Artemisinin accumulation can be enhanced by exogenous abscisic acid (ABA) treatment. However, it is not known how ABA signaling regulates artemisinin biosynthesis. A global expression profile and phylogenetic analysis as well as the dual-LUC screening revealed that a basic leucine zipper family transcription factor from A. annua (namely AabZIP1) was involved in ABA signaling to regulate artemisinin biosynthesis. AabZIP1 had a higher expression level in the inflorescences than in other tissues; ABA treatment, drought, and salt stress strongly induced the expression of AabZIP1. Yeast one-hybrid assay and electrophoretic mobility shift assay (EMSA) showed that AabZIP1 bound to the ABA-responsive elements (ABRE) in the promoter regions of the amorpha-4,11-diene synthase (ADS) gene and CYP71AV1, which are two key structural genes of the artemisinin biosynthetic pathway. A mutagenesis assay showed that the C1 domain in the N-terminus of AabZIP1 was important for its transactivation activity. Furthermore, the activation of ADS and CYP71AV1 promoters by AabZIP1 was enhanced by ABA treatment in transient dual-LUC analysis. The AabZIP1 variant with C1 domain deletion lost the ability to activate ADS and CYP71AV1 promoters regardless of ABA treatment. Notably, overexpression of AabZIP1 in A. annua resulted in significantly increased accumulation of artemisinin. Our results indicate that ABA promotes artemisinin biosynthesis, likely through 1 activation of ADS and CYP71AV1 expression by AabZIP in A. annua. Meanwhile, our findings reveal the potential value of AabZIP1 in genetic engineering of artemisinin production.

Type 2C phosphatase 1 of Artemisia annua L. is a negative regulator of ABA signaling.

The phytohormone abscisic acid (ABA) plays an important role in plant development and environmental stress response. Additionally, ABA also regulates secondary metabolism such as artemisinin in the medicinal plant Artemisia annua L. Although an earlier study showed that ABA receptor, AaPYL9, plays a positive role in ABA-induced artemisinin content improvement, many components in the ABA signaling pathway remain to be elucidated in Artemisia annua L. To get insight of the function of AaPYL9, we isolated and characterized an AaPYL9-interacting partner, AaPP2C1. The coding sequence of AaPP2C1 encodes a deduced protein of 464 amino acids, with all the features of plant type clade A PP2C. Transcriptional analysis showed that the expression level of AaPP2C1 is increased after ABA, salt, and drought treatments. Yeast two-hybrid and bimolecular fluorescence complementation assays (BiFC) showed that AaPYL9 interacted with AaPP2C1. The P89S, H116A substitution in AaPYL9 as well as G199D substitution or deletion of the third phosphorylation site-like motif in AaPP2C1 abolished this interaction. Furthermore, constitutive expression of AaPP2C1 conferred ABA insensitivity compared with the wild type. In summary, our data reveals that AaPP2C1 is an AaPYL9-interacting partner and involved in the negative modulation of the ABA signaling pathway in A. annua L.

[Comparative study on substituting hedyseri radix for astragali radix in serum containing yiqiyangxue prescription on aged mice spleen lymphocyte proliferation and anti-oxidant effect].

OBJECTIVE: By substituting Hedyseri Radix for Astragali Radix in Yiqiyangxue prescription, to compare the effects of both serum containing medicine on aged mice spleen lymphocyte proliferation and anti-oxidant effect. METHOD: After using the same dose of Hedyseri Radix to replace Astragali Radix in Yiqiyangxue prescription, the best concentration of serum containing medicine,the best incubation time and the effects of ConA-induced spleen lymphocyte proliferation were determined by MTY method. Use reagent kits to detect the activity of SOD, MDA and ROS levels in aged mice spleen lymphocytes and IL-2 level in culture supernatant fluid of spleen lymphocytes. RESULTS: Both serum containing medicine can enhance the proliferation of aged mice spleen lymphocytes. The best concentration of serum containing medicine was 40% and the incubation time was 72 h. The serum containing Yiqiyangxue of Hedyseri Radix prescription acted more effective than that of Astragali Radix on the enhancement of proliferation. Both serum containing medicine showed similar effects on increasing SOD activity, IL-2 level and decreasing MDA and ROS level. Moreover,serum of Hedyseri Radix was superior in the enhancement of proliferation, IL-2 and the reduction of ROS level. CONCLUSION: Both serum containing medicine of Hedyseri Radix and Astragali Radix generate the same effect of anti-aging and enhancement of proliferation.

Induction and flow cytometry identification of tetraploids from seed-derived explants through colchicine treatments in Catharanthus roseus (L.) G. Don.

The tetraploid plants of Catharanthus roseus (L.) G. Don was obtained by colchicine induction from seeds explants, and the ploidy of the plants was identified by flow cytometry. The optimal treatment is 0.2% colchicine solution treated for 24 hours, and the induction rate reaches up to 30%. Comparing with morphological characteristics and growth habits between tetraploids and the control, we found that tetraploids of C. roseus had larger stoma and more branches and leaves. HPLC analysis showed tetraploidization could increase the contents of terpenoid indole alkaloids in C. roseus. Thus, tetraploidization could be used to produce higher alkaloids lines for commercial use. QRT-PCR results showed that the expression of enzymes involved in terpenoid indole alkaloids biosynthesis pathway had increased in the tetraploid plants. To our knowledge, this was the first paper to explore the secondary metabolism in autotetraploid C. roseus induced by colchicine.

Molecular cloning and characterization of 4-hydroxyphenylpyruvate dioxygenase gene from Lactuca sativa.

Vitamin E has been found to be associated with an important antioxidant property in mammals and plants. In photosynthetic organisms, the enzyme 4-hydroxyphenylpyruvate dioxygenase (HPPD; E.C. 1.13.11.27) plays an important role in the vitamin E biosynthetic pathway. The full-length cDNA encoding HPPD was isolated from Lactuca sativa L. by rapid amplification of cDNA ends (RACE). The cDNA, designated as LsHPPD, was 1743 base pairs (bp) long containing an open reading frame (ORF) of 1338 bp encoding a protein of 446 amino acids. Sequence analysis indicated that LsHPPD shared high identity with HPPD from Medicago truncatula L. Real-time fluorescent quantitative PCR (qPCR) analysis revealed that LsHPPD was preferentially expressed in mature leaves compared with other tissues and that the LsHPPD expression was sensitive to high light and drought stress treatments. Transient expression of LsHPPD via agroinfiltration resulted in 12-fold increase in LsHPPD mRNA expression level and 4-fold enhancement in α-tocopherol content compared with the negative control. A decrease in chlorophyll content and inhibition of photosystem II were observed during stress treatments and agroinfiltration.

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkalo id accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkalo id accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.(AU)

Examination of camptothecin and 10-hydroxycamptothecin in Camptotheca acuminata plant and cell culture, and the affected yields under several cell culture treatments.

Camptothecin and its derivatives are monoterpenoid indole alkaloids exhibiting significant anti-tumor actions. With the aim of improving the production of these pharmaceuticals, the contents of camptothecin and 10-hydroxycamptothecin in different tissues including roots, stems, leaves, young flower buds, opening flowers, fading flowers and seeds from Camptotheca acuminata, were investigated. The young flower buds had the highest alkaloid concentrations (camptothecin, 2.46 mg/g of dry weight; 10-hydroxycamptothecin, 1.41 mg/g of dry weight). Callus showed lower concentrations but it should also be considered as a potential source of these pharmaceuticals. In the present study, the growth rate of Camptotheca acuminata cells in culture did not correlate with contents of camptothecin and 10-hydroxycamptothecin. Alkaloid accumulation by cells under various treatments (heavy metal ions, UV-B), methyl-jasmonate, abscisic acid, salicylic acid and hydrogen peroxide was examined, and the most notable effects appeared in the cells induced by UV-B light (which showed an 11-fold increase in camptothecin concentration) and by salicylic acid (which showed a 25-fold increase in 10-hydroxycamptothecin concentration). These results are significant in the context of the production of both pharmaceuticals.

[Analysis of catharanthine content and agronomic traits in Catharanthus roseus].

Catharanthine content and agronomic traits in major Catharanthus roseus varieties were analyzed. It was found that there existed great difference in catharanthine content and agronomic traits among the varieties. Catharanthine content was the highest in variety Pacifica Polka Dot (PPD), reaching 3.79 mg g(-1) dry leaf weight, and the lowest in variety Cooler Pink (CP) with only 0.9 mg g(-1) dry leaf weight. Correlation existed in certain extent between catharanthine content and agronomic traits in C. roseus. Path analysis showed that among all the agronomic traits analyzed, internodal distance positively affected catharanthine content at significant level (P<0.05), with the path coefficient being 1.473. This study provides useful information for high-catharanthine content C. roseus introduction and breeding.

Promotion of nicotine biosynthesis in transgenic tobacco by overexpressing allene oxide cyclase from Hyoscyamus niger.

Plant secondary metabolites are a wide variety of low-molecular weight compounds whose productions are often enhanced in response to both biotic and abiotic stresses. Many of the responses are mediated by a class of hormones, named as jasmonates. In jasmonate biosynthetic pathway of plants, allene oxide cyclase (AOC, EC 5.3.99.6) catalyzes the most crucial step. Here a heterologous AOC gene from Hyoscyamus niger L. (black henbane), named HnAOC (GenBank accession No. AY708383), was overexpressed in Nicotiana tabacum cv. Petit Havana to investigate the consequence on nicotine content. This study revealed that the transcription of HnAOC in tobacco resulted in overexpression of nicotine biosynthetic pathway genes and higher yield of nicotine, with the maximum of 4.8-fold over control. Therefore, it indicated that without the cost of extrinsic hormones, genetic manipulation of jasmonate biosynthetic pathway genes could be an alternative approach in metabolic engineering for the production of valuable secondary metabolites, which were induced by jasmonates.

Molecular cloning, expression profiling and functional analyses of a cDNA encoding isopentenyl diphosphate isomerase from Gossypium barbadense.

Gossypol, a type of plant defence sesquiterpenoid phytoalexin, is synthesized from the MEP (2C-methyl-D-erythritol 4-phosphate) and MVA (mevalonate) pathway in the isoprenoid biosynthetic system. The key step is the isomerization of IPP (isopentenyl diphosphate) to DMAPP (dimethylallyl diphosphate), which is catalysed by IPI (IPP isomerase; EC 5.3.3.2). A full-length cDNA encoding IPI (designated GbIPI) was cloned from Gossypium barbadense by RACE (rapid amplification of cDNA ends). The full-length cDNA of GbIPI was 1205 bp and contained a 906 bp ORF (open reading frame) encoding a protein of 302 amino acids, with a predicted molecular mass of 34.39 kDa and an isoelectric point of 6.07. Amino acid sequence analysis revealed that the GbIPI has a high level of similarity to other IPIs. Southern-blot analysis revealed that GbIPI belongs to a small gene family. Expression analysis indicated that GbIPI expression is highest in stems, followed by leaves, and is lowest in roots, and that the expression of GbIPI could be induced by Verticillium dahliae Kleb, MeJA (methyl jasmonate) and SA (salicylic acid). The functional colour assay indicated that GbIPI could accelerate the accumulation of beta-carotene in Escherichia coli transformants. The cloning and functional analysis of GbIPI will be useful in increasing understanding of the role of IPI in isoprenoid biosynthesis at the molecular level.

Molecular cloning, characterization and expression of a jasmonate biosynthetic pathway gene encoding allene oxide cyclase from Camptotheca acuminata.

AOC (allene oxide cyclase; EC 5.3.99.6), an essential enzyme in jasmonic acid and its methyl ester biosynthesis, was cloned from Camptotheca acuminata (named as CaAOC), a native medicinal plant species in China. CaAOC had significant similarity at the amino-acid level with AOCs from other plant species. Comparison between the sequences of the full-length cDNA and genomic DNA of CaAOC revealed that the genomic DNA of CaAOC contained an 89-bp intron and a 240-bp intron. Southern-blot analysis indicated that CaAOC was a multiple-copy gene, and real-time quantitative PCR analysis showed that CaAOC was expressed constitutively in all organs tested, with the highest expression level in leaves. The results from treatment experiments using different signalling components, including methyl jasmonate, abscisic acid, salicylic acid and H(2)O(2), revealed that expression of CaAOC had a prominent diversity. Heavy metal (copper) significantly enhanced CaAOC expression, whereas wounding (induced by UV-B) was not so effective.

Molecular cloning, expression profiling and functional analysis of a DXR gene encoding 1-deoxy-D-xylulose 5-phosphate reductoisomerase from Camptotheca acuminata.

As the second enzyme of the non-mevalonate terpenoid pathway for isopentenyl diphosphate biosynthesis, DXP reductoisomerase (DXR, EC: 1.1.1.267) catalyzes a committed step of the MEP pathway for camptothecin (CPT) biosynthesis. In order to understand more about the role of DXR involved in the CPT biosynthesis at the molecular level, the full-length DXR cDNA sequence (designated as CaDXR) was isolated and characterized for the first time from a medicinal Nyssaceae plant species, Camptotheca acuminata. The full-length cDNA of CaDXR was 1823 bp containing a 1416 bp open reading frame (ORF) encoding a polypeptide of 472 amino acids. Comparative and bioinformatic analyses revealed that CaDXR showed extensive homology with DXRs from other plant species and contained a conserved transit peptide for plastids, an extended Pro-rich region and a highly conserved NADPH binding motif in its N-terminal region owned by all plant DXRs. Phylogenetic analysis indicated that CaDXR was more ancient than other plant DXRs. Tissue expression pattern analysis revealed that CaDXR expressed strongly in stem, weak in leaf and root. CaDXR was found to be an elicitor-responsive gene, which could be induced by exogenous elicitor of methyl jasmonate. The functional color complementation assay indicated that CaDXR could accelerate the biosynthesis of carotenoids in the Escherichia coli transformant, demonstrating that DXP reductoisomerase plays an influential step in isoprenoid biosynthesis.

Molecular cloning and characterization of the yew gene encoding squalene synthase from Taxus cuspidata.

The enzyme squalene synthase (EC 2.5.1.21) catalyzes a reductive dimerization of two farnesyl diphosphate (FPP) molecules into squalene, a key precursor for the sterol and triterpene biosynthesis. A full-length cDNA encoding squalene synthase (designated as TcSqS) was isolated from Taxus cuspidata, a kind of important medicinal plants producing potent anti-cancer drug, taxol. The full-length cDNA of TcSqS was 1765 bp and contained a 1230 bp open reading frame (ORF) encoding a polypeptide of 409 amino acids. Bioinformatic analysis revealed that the deduced TcSqS protein had high similarity with other plant squalene synthases and a predicted crystal structure similar to other class I isoprenoid biosynthetic enzymes. Southern blot analysis revealed that there was one copy of TcSqS gene in the genome of T. cuspidata. Semiquantitative RT-PCR analysis and northern blotting analysis showed that TcSqS expressed constitutively in all tested tissues, with the highest expression in roots. The promoter region of TcSqS was also isolated by genomic walking and analysis showed that several cis-acting elements were present in the promoter region. The results of treatment experiments by different signaling components including methyl-jasmonate, salicylic acid and gibberellin revealed that the TcSqS expression level of treated cells had a prominent diversity to that of control, which was consistent with the prediction results of TcSqS promoter region in the PlantCARE database.

Ganodermataceae: natural products and their related pharmacological functions.

The objective of this paper is to review the natural products and the pharmacological functions of Ganodermataceae family. Presently, studies on the bioactive components of Lingzhi are focused on polysaccharides and triterpenes/triterpenoids compounds. New Ganoderma polysaccharides, including their molecular weights, glycosyl residue compositions, glycosyl linkage and branches, are summarized in this paper. Also presented are new types of triterpenes and their characteristics from Lingzhi. Taking Ganoderma lucidum as an example, we reviewed its pharmacological functions in anti-tumor and immune-modulating activities for treating hypoglycemosis, hepatoprotection, and the effect on blood vessel system. Based on the advances in Lingzhi research in the past few decades, both G. lucidum and G. sinense are considered as the representative species of medicinal mushroom Lingzhi in China. Until 2001, G. tsugae was only advised to be used as the materials of the health products. The biologically-active components related to pharmacological functions of these three species were studied more than other Ganodermataceae family species; however, which have been used in less modern folk medicine.

Molecular cloning and characterization of a glutathione S-transferase gene from Ginkgo biloba.

Glutathione S-transferases (GSTs) play an important role in the response of plants to changing environmental conditions. Here, we report the cloning of the GST gene for GST from Ginkgo biloba, a native medicinal plant species in China, by rapid amplification of cDNA ends (RACE). The full-length cDNA (designated as GbGST) was 1008 bp and contained a 684 bp open reading frame (ORF) encoding a polypeptide of 228 amino acids. The genomic sequence of GbGST was also obtained. Semi-quantitative RT-PCR analysis revealed that GbGST expressed in all tested tissues of G. biloba, including leaf, root and stem and the expression of GbGST could be induced by UV, MJ and drought treatments, suggesting that GbGST was potentially involved in plant's stress tolerance. To our knowledge, this is the first GST cDNA cloned from Ginkgoaceae. Based on comparative analyses of amino acid sequence, phylogeny, predicted three-dimensional structure together with the gene structure, the GbGST should be classified into the tau class.