The Integration of the Metabolome and Transcriptome for Dendrobium nobile Lindl. in Response to Methyl Jasmonate.

Dendrobium nobile Lindl., as an endangered medicinal plant within the genus Dendrobium, is widely distributed in southwestern China and has important ecological and economic value. There are a variety of metabolites with pharmacological activity in D. nobile. The alkaloids and polysaccharides contained within D. nobile are very important active components, which mainly have antiviral, anti-tumor, and immunity improvement effects. However, the changes in the compounds and functional genes of D. nobile induced by methyl jasmonate (MeJA) are not clearly understood. In this study, the metabolome and transcriptome of D. nobile were analyzed after exposure to MeJA. A total of 377 differential metabolites were obtained through data analysis, of which 15 were related to polysaccharide pathways and 35 were related to terpenoids and alkaloids pathways. Additionally, the transcriptome sequencing results identified 3256 differentially expressed genes that were discovered in 11 groups. Compared with the control group, 1346 unigenes were differentially expressed in the samples treated with MeJA for 14 days (TF14). Moreover, the expression levels of differentially expressed genes were also significant at different growth and development stages. According to GO and KEGG annotations, 189 and 99 candidate genes were identified as being involved in terpenoid biosynthesis and polysaccharide biosynthesis, respectively. In addition, the co-expression analysis indicated that 238 and 313 transcription factors (TFs) may contribute to the regulation of terpenoid and polysaccharide biosynthesis, respectively. Through a heat map analysis, fourteen terpenoid synthetase genes, twenty-three cytochrome P450 oxidase genes, eight methyltransferase genes, and six aminotransferase genes were identified that may be related to dendrobine biosynthesis. Among them, one sesquiterpene synthase gene was found to be highly expressed after the treatment with MeJA and was positively correlated with the content of dendrobine. This study provides important and valuable metabolomics and transcriptomic information for the further understanding of D. nobile at the metabolic and molecular levels and provides candidate genes and possible intermediate compounds for the dendrobine biosynthesis pathway, which lays a certain foundation for further research on and application of Dendrobium.

[Effects of mycorrhizal planting on small molecular chemical components of Dendrobium officinale].

This study aimed to provide a scientific basis for the application of the mycorrhizal planting technology of Dendrobium officinale by investigating the effects of mycorrhizal planting on the fingerprints of D. officinale and the content of six chemical components. Seventeen samples of D. officinale under mycorrhizal and conventional planting were collected from four regions, such as Jinhua of Zhejiang. The HPLC fingerprints were established to evaluate the similarity of the samples. The content of six chemical components of the samples was determined by HPLC. There were 15 common peaks in the fingerprints, and five of them were identified by marker compounds, which were naringenin, 4,4'-dihydroxy-3,5-dimethoxybibenzyl, 3,4'-dihydroxy-5-methoxybibenzyl, 3',4-dihydroxy-3,5'-dimethoxybibenzyl(gigantol), and 3,4-dihydroxy-4',5-dimethoxybibenzyl(DDB-2). The similarities of the fingerprints of mycorrhizal and conventional planting samples and the control fingerprint were in the ranges of 0.733-0.936 and 0.834-0.942, respectively. The influences of mycorrhizal planting on fingerprints were related to planting regions, the germplasm of D. officianle, and the amount of fungal agent. The content of six chemical components in the samples varied greatly, and the content of DDB-2 was the highest, ranging from 69.83 to 488.47 µg·g~(-1). The mycorrhizal planting samples from Chongming of Shanghai and Taizhou of Jiangsu showed an increase in the content of 5-6 components, while samples from Zhangzhou of Fujian and Jinhua of Zhejiang showed an increase in the content of 1-2 components. The results showed that mycorrhizal planting technology did not change the chemical profile of small molecular chemical components of D. officinale, but affected the content of chemical components such as bibenzyls, which has a good application prospect.

Potential Specificity Between Mycorrhizal Fungi Isolated from Widespread Dendrobium spp. and Rare D. huoshanense Seeds.

In nature, orchid seed germination and seedling development depend on compatible mycorrhizal fungi. Mycorrhizal generalist and specificity affect the orchid distribution and rarity. Here, we investigated the specificity toward fungi in the rare D. huoshanense by mycorrhizal fungal isolation and symbiotic germination in vitro. Twenty mycorrhizal fungal strains were isolated from the roots of adult Dendrobium spp. (six and 12 strains from rare D. huoshanense and widespread D. officinale, respectively, and two strains from D. nobile and D. moniliforme, respectively) and 13 strains belong to Tulasnellaceae and seven strains belong to Serendipitaceae. Germination trials in vitro revealed that all 20 tested fungal strains can stimulate seed germination of D. huoshanense, but only nine strains (~ 50%) can support it up to the seedling stage. This finding indicates that generalistic fungi are important for early germination, but only a few can maintain a symbiosis with host in seedling stage. Thus, a shift of the microbial community from seedling to mature stage probably narrows the D. huoshanense distribution range. In addition, to further understand the relationship between the fungal capability to promote seed germination and fungal enzyme activity, we screened the laccase and pectase activity. The results showed that the two enzymes activities of fungi cannot be directly correlated with their germination-promoting activities. Understanding the host specificity degree toward fungi can help to better interpret the limited geographic distribution of D. huoshanense and provides opportunities for in situ and ex situ conservation and reintroduction programs.

Profiles of Cytokinins Metabolic Genes and Endogenous Cytokinins Dynamics during Shoot Multiplication In Vitro of Phalaenopsis.

Shoot multiplication induced by exogenous cytokinins (CKs) has been commonly used in Phalaenopsis micropropagation for commercial production. Despite this, mechanisms of CKs action on shoot multiplication remain unclear in Phalaenopsis. In this study, we first identified key CKs metabolic genes, including six isopentenyltransferase (PaIPTs), six cytokinin riboside 5' monophosphate phosphoribohydrolase (PaLOGs), and six cytokinin dehydrogenase (PaCKXs), from the Phalaenopsis genome. Then, we investigated expression profiles of these CKs metabolic genes and endogenous CKs dynamics in shoot proliferation by thidiazuron (TDZ) treatments (an artificial plant growth regulator with strong cytokinin-like activity). Our data showed that these CKs metabolic genes have organ-specific expression patterns. The shoot proliferation in vitro was effectively promoted with increased TDZ concentrations. Following TDZ treatments, the highly expressed CKs metabolic genes in micropropagated shoots were PaIPT1, PaLOG2, and PaCKX4. By 30 days of culture, TDZ treatments significantly induced CK-ribosides levels in micropropagated shoots, such as tZR and iPR (2000-fold and 200-fold, respectively) as compared to the controls, whereas cZR showed only a 10-fold increase. Overexpression of PaIPT1 and PaLOG2 by agroinfiltration assays resulted in increased CK-ribosides levels in tobacco leaves, while overexpression of PaCKX4 resulted in decreased CK-ribosides levels. These findings suggest de novo biosynthesis of CKs induced by TDZ, primarily in elevation of tZR and iPR levels. Our results provide a better understanding of CKs metabolism in Phalaenopsis micropropagation.

[Comparison and health risk assessment of mineral elements in stems and leaves of Dendrobium officinale cultivated with conventional method and mycorrhizal fungi].

This study aims to analyze the variation of the content of mineral elements in stems and leaves of Dendrobium officinale cultivated with conventional method and mycorrhizal fungi, which is expected to lay a basis for safety of stems and leaves of D. officinale. A total of 7 samples from Jiangsu, Fujian, Shanghai, and Zhejiang were collected, which were then cultivated with conventional method and mycorrhizal fungi, separately. The content of 17 mineral elements in stems and leaves was measured by inductively coupled plasma-mass spectrometry(ICP-MS), and the content changes of the mineral elements were analyzed. The health risks of Pb, Cd, Hg, and As in stems were assessed by target hazard quotient(THQ). The results showed that the content of polluting elements in stems and leaves of D. officinale was low, and the content in the plants cultivated with mycorrhizal fungi was reduced. The content of K, Ca, Mg, and P was high in stems and leaves of the species, suggesting that cultivation with mycorrhizal fungi improved the content of other elements irregularly. According to the THQ, the safety risk of stems of D. officinale cultivated with either conventional method or mycorrhizal fungi was low, particularly the D. officinale cultivated mycorrhizal fungi. The results indicated that cultivation with mycorrhizal fungi influenced the element content in stems and leaves of D. officinale. It is necessary to study the culture substrate, processing technology, and the mechanism of the increase or decrease in mineral elements of D. officinale in the future.

Recent advances and new insights in biosynthesis of dendrobine and sesquiterpenes.

Sesquiterpenes are one of the most diverse groups of secondary metabolites that have mainly been observed in terpenoids. It is a natural terpene containing 15 carbon atoms in the molecule and three isoprene units with chain, ring, and other skeleton structures. Sesquiterpenes have been shown to display multiple biological activities such as anti-inflammatory, anti-feedant, anti-microbial, anti-tumor, anti-malarial, and immunomodulatory properties; therefore, their therapeutic effects are essential. In order to overcome the problem of low-yielding sesquiterpene content in natural plants, regulating their biosynthetic pathways has become the focus of many researchers. In plant and microbial systems, many genetic engineering strategies have been used to elucidate biosynthetic pathways and high-level production of sesquiterpenes. Here, we will introduce the research progress and prospects of the biosynthesis of artemisinin, costunolide, parthenolide, and dendrobine. Furthermore, we explore the biosynthesis of dendrobine by evaluating whether the biosynthetic strategies of these sesquiterpene compounds can be applied to the formation of dendrobine and its intermediate compounds. KEY POINTS: • The development of synthetic biology has promoted the study of terpenoid metabolism and provided an engineering platform for the production of high-value terpenoid products. • Some possible intermediate compounds of dendrobine were screened out and the possible pathway of dendrobine biosynthesis was speculated. • The possible methods of dendrobine biosynthesis were explored and speculated.

Genome-wide analysis and prediction of genes involved in the biosynthesis of polysaccharides and bioactive secondary metabolites in high-temperature-tolerant wild Flammulina filiformis.

BACKGROUND: Flammulina filiformis (previously known as Asian F. velutipes) is a popular commercial edible mushroom. Many bioactive compounds with medicinal effects, such as polysaccharides and sesquiterpenoids, have been isolated and identified from F. filiformis, but their biosynthesis and regulation at the molecular level remains unclear. In this study, we sequenced the genome of the wild strain F. filiformis Liu355, predicted its biosynthetic gene clusters (BGCs) and profiled the expression of these genes in wild and cultivar strains and in different developmental stages of the wild F. filiformis strain by a comparative transcriptomic analysis. RESULTS: We found that the genome of the F. filiformis was 35.01 Mb in length and harbored 10,396 gene models. Thirteen putative terpenoid gene clusters were predicted and 12 sesquiterpene synthase genes belonging to four different groups and two type I polyketide synthase gene clusters were identified in the F. filiformis genome. The number of genes related to terpenoid biosynthesis was higher in the wild strain (119 genes) than in the cultivar strain (81 genes). Most terpenoid biosynthesis genes were upregulated in the primordium and fruiting body of the wild strain, while the polyketide synthase genes were generally upregulated in the mycelium of the wild strain. Moreover, genes encoding UDP-glucose pyrophosphorylase and UDP-glucose dehydrogenase, which are involved in polysaccharide biosynthesis, had relatively high transcript levels both in the mycelium and fruiting body of the wild F. filiformis strain. CONCLUSIONS: F. filiformis is enriched in a number of gene clusters involved in the biosynthesis of polysaccharides and terpenoid bioactive compounds and these genes usually display differential expression between wild and cultivar strains, even in different developmental stages. This study expands our knowledge of the biology of F. filiformis and provides valuable data for elucidating the regulation of secondary metabolites in this unique F. filiformis strain.

Do fungal associates of co-occurring orchids promote seed germination of the widespread orchid species Gymnadenia conopsea?

Interactions with mycorrhizal fungi have been increasingly recognized as one of the most important ecological factors determining the distribution and local abundance of orchids. While some orchid species may interact with a variety of fungal associates, others are more specific in their choice of mycorrhizal partners. Moreover, orchids that co-occur at a given site, often associate with different partners, possibly to avoid competition and to allow stable coexistence. However, whether differences in mycorrhizal partners directly affect seed germination and subsequent protocorm formation remains largely unknown. In this research, we used in vitro germination experiments to investigate to what extent seed germination and protocorm formation of Gymnadenia conopsea was affected by the origin and identity of fungal associates. Fungi were isolated from G. conopsea and three other co-occurring orchid species (Dactylorhiza viridis (Coeloglossum viride), Herminium monorchis, and Platanthera chlorantha). In total, eight fungal associates, belonging to Tulasnellaceae, Ceratobasidiaceae, and Serendipitaceae, were successfully isolated and cultured. While all eight fungal strains were able to promote early germination of G. conopsea seeds, only fungal strain GS2, a member of the Ceratobasidiaceae isolated from G. conopsea itself, was able to promote protocorm formation and subsequent growth to a seedling. Two other fungal strains isolated from G. conopsea only supported seed germination until the protocorm formation stage. The other five fungal strains isolated from the co-occurring orchid species did not support seed germination beyond the protocorm stage. We conclude that, although G. conopsea is considered a mycorrhizal generalist that associates with a wide range of fungi during its adult life, it requires specific fungi to promote protocorm formation and growth to a seedling.

Combined Metabolome and Transcriptome Analyses Reveal the Effects of Mycorrhizal Fungus Ceratobasidium sp. AR2 on the Flavonoid Accumulation in Anoectochilus roxburghii during Different Growth Stages.

Anoectochilus roxburghii is a traditional Chinese herb with high medicinal value, with main bioactive constituents which are flavonoids. It commonly associates with mycorrhizal fungi for its growth and development. Moreover, mycorrhizal fungi can induce changes in the internal metabolism of host plants. However, its role in the flavonoid accumulation in A. roxburghii at different growth stages is not well studied. In this study, combined metabolome and transcriptome analyses were performed to investigate the metabolic and transcriptional profiling in mycorrhizal A. roxburghii (M) and non-mycorrhizal A. roxburghii (NM) growth for six months. An association analysis revealed that flavonoid biosynthetic pathway presented significant differences between the M and NM. Additionally, the structural genes related to flavonoid synthesis and different flavonoid metabolites in both groups over a period of six months were validated using quantitative real-time polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The results showed that Ceratobasidium sp. AR2 could increase the accumulation of five flavonol-glycosides (i.e., narcissin, rutin, isorhamnetin-3-O-beta-d-glucoside, quercetin-7-O-glucoside, and kaempferol-3-O-glucoside), two flavonols (i.e., quercetin and isorhamnetin), and two flavones (i.e., nobiletin and tangeretin) to some degrees. The qRT-PCR showed that the flavonoid biosynthetic genes (PAL, 4CL, CHS, GT, and RT) were significantly differentially expressed between the M and NM. Overall, our findings indicate that AR2 induces flavonoid metabolism in A. roxburghii during different growth stages, especially in the third month. This shows great potential of Ceratobasidium sp. AR2 for the quality improvement of A. roxburghii.

Symbiotic and Asymbiotic Germination of Dendrobium officinale (Orchidaceae) Respond Differently to Exogenous Gibberellins.

Seeds of almost all orchids depend on mycorrhizal fungi to induce their germination in the wild. The regulation of this symbiotic germination of orchid seeds involves complex crosstalk interactions between mycorrhizal establishment and the germination process. The aim of this study was to investigate the effect of gibberellins (GAs) on the symbiotic germination of Dendrobium officinale seeds and its functioning in the mutualistic interaction between orchid species and their mycobionts. To do this, we used liquid chromatograph-mass spectrometer to quantify endogenous hormones across different development stages between symbiotic and asymbiotic germination of D. officinale, as well as real-time quantitative PCR to investigate gene expression levels during seed germination under the different treatment concentrations of exogenous gibberellic acids (GA3). Our results showed that the level of endogenous GA3 was not significantly different between the asymbiotic and symbiotic germination groups, but the ratio of GA3 and abscisic acids (ABA) was significantly higher during symbiotic germination than asymbiotic germination. Exogenous GA3 treatment showed that a high concentration of GA3 could inhibit fungal colonization in the embryo cell and decrease the seed germination rate, but did not significantly affect asymbiotic germination or the growth of the free-living fungal mycelium. The expression of genes involved in the common symbiotic pathway (e.g., calcium-binding protein and calcium-dependent protein kinase) responded to the changed concentrations of exogenous GA3. Taken together, our results demonstrate that GA3 is probably a key signal molecule for crosstalk between the seed germination pathway and mycorrhiza symbiosis during the orchid seed symbiotic germination.

Chemical Composition and Antioxidant Activity of Tuber indicum from Different Geographical Regions of China.

Tuber indicum, an endemic truffle species in eastern Asian, is an edible mushroom that is both an important export and widely distributed across China. Many existing studies on truffles focus on analyzing their taxonomy, population genetics, volatile organic compounds and artificial cultivation of the truffles, while little information is available about their nutrient composition and pharmacological activity, especially the relationship between chemical composition in ascocarps and their geographic distributions. This study presents a comprehensive investigation of the chemical composition of T. indicum, including free sugars, fatty acids, organic acids, phenolic acids, flavonoids, and polysaccharides, and tracks the antioxidant activity of T. indicum ascocarps collected from five geographical regions of four provinces in P. R. China: Hebei, Tibet, Yunnan, and Liaoning province. Our results showed that T. indicum collected from Qujing, Yunnan province, possessed the highest amount of free sugars (23.67 mg/g dw), total flavonoids (2.31 mg/g dw), total phenolics (4.46 mg/g dw) and the highest DPPH and ABTS radical-scavenging activities. The amount of water-soluble polysaccharides was the highest (115.24 mg/g dw) in ascocarps from Tibet, the total organic acids was the highest (22.073 mg/g dw) in ascocarps from Gongshan, and polyunsaturated fatty acids were most abundant in those from Hebei province. This study reveals that the quantity of chemical compounds in T. indicum varies by geographical origin. Detecting differences in chemical composition may provide important data for understanding the relationship between environmental factors and truffle formation, as well as quality evaluation of the commercial species T. indicum throughout China.

Two new tetracyclic triterpenoids from the endophytic fungus Hypoxylon sp. 6269.

Two new tetracyclic triterpenoids, integracide E (1) and isointegracide E (2), as well as three known secondary metabolites (i.e. integracide A (3), 2-deoxyintegracide A (4) and 2-deoxyintegracide B (5)), were isolated from mycelium of the endophytic fungus Hypoxylon sp. 6269. Structures were determined by a combination of 1D and 2D NMR techniques and mass spectrometry. All of the compounds were tested for their anti-HIV-1 integrase activities.

[Isolation and expression analysis of a hexose transporter gene DoHT1 in Dendrobium officinale].

In this study, RACE technology was employed to isolate the full length cDNA of DoHT1 in Dendrobium officinale, followed by bioinformatics analysis of the sequence characteristics. And the expression pattern of the gene was also analyzed by quantitative PCR. The full length cDNA of DoHT1 was 1 586 bp in length, containing a 1 536 bp ORF, which encoded a 511-aa protein with molecular weight of 56.18 kD and isoelectric point of 9.08. The deduced DoHT1 protein had the major facilitator superfamily conserved domain (22-483), SUGAR₋TRANSPORT₋1 (139-164), and SUGAR₋TRANSPORT₋2 (338-355), typical for sugar transporter; DoHT1, without a single peptide had 11 transmembrane regions, and was predicted to locate in the plasma membrane; DoHT1 had high identities (54.7%-80.7%) with HTs proteins from various plants. DoHT1 belonged to the MST (monosaccharide transporter) group of the evolutionary tree, and was closely related to the Phalaenopsis equestris. DoHT1 was differentially expressed in the three included organs. The transcripts were significantly the most abundant in the leaves with 19.36 fold than roots, then 1.82 fold in the stems than the roots. The identification and molecular characterization of the full length DoHT1 will be essential for further function study of the gene during the regulation of sugar metabolism of D. officinale.

[Interaction between endophytes and host plant and the role of endophytes in genuineness analysis of medicinal plant].

Research of plant development and metabolism has drawn lots of attention with the fast development of science of mycorrhizal biology, molecular biology and metabonomics technology. It has become one of hot fields in the study of endophytes and plant, which would affect plant 's metabolite composition. This would provide opportunity for appraising and modifying traits to medicinal plant, and would also perfect the tranditional standpoint on forming reason of medicinal plant genuineness. Here we provide a review of theory and mechanism, research and application of interaction between plant and endophyte. This review may enhance understanding of medicinal plant, and evaluating the quality of herbs in production.

[DNA marker-assisted selection of medicinal plants (â ) .Breeding research of disease-resistant cultivars of Panax notoginseng].

SSR is one of the most important molecular markers used in molecular identification and genetic diversity research of Dendrobium nobile. In order to enrich the library of SSR and establish a method for rapid identification of D. nobile, the SSR information was analyzed in the transcriptome of D. nobile. A total of 32 709 SSRs were obtained from the transcriptome of D. nobile, distributed in 26 742 unigenes with the distribution frequency of 12.90%. SSR loci occurred every 3 748 bp. Mono-nucleotide repeat was the main type, account for as much as 72.18% of all SSRs, followed by di-nucleotide (15.97%) and tri-nucleotide (11.19%). Among all repeat types, A/T was the predominant one followed by AG/CT. Finally a total of 62 157 primer pairs were designed for marker development. Randomly 20 pairs of primers were selected for PCR amplification, 17 amplified on clear and reproducible bands, the amplification rate was 85.0%.Thirteen pairs were polymorphic among the 3 Dendrobium plants. The results indicated that the unigenes generated from transcriptome sequencing in D. nobile can be used as effective source to develop SSR markers. The SSR loci in the transcriptome of D. nobile have the characteristics of type riches, high density and high potential of polymorphism, and these characteristics might applied in the study of molecular identification, genetic diversity and marker-assisted breeding of D. nobile and its closely related species.

Discovery and Characterization of a New Family of Diterpene Cyclases in Bacteria and Fungi.

Diterpene cyclases from bacteria and basidiomycete fungi are seldom studied. Here, we presented the identification and verification of EriG, a member of the UbiA superfamily, as the enzyme responsible for the cyclization of the cyathane skeleton in the mushroom Hericium erinaceum. Genome mining using the EriG protein sequence as a probe led to the discovery of a new family of ubiquitous UbiA-related diterpene cyclases in bacteria and fungi. We successfully characterized seven new diterpene cyclases from bacteria or basidiomycete fungi with the help of an engineered Escherichia coli strain and determined the structures of their corresponding products. A new diterpene with an unusual skeleton was generated during this process. The discovery of this new family of diterpene cyclases provides new insight into the UbiA superfamily.

[Molecular cloning and characterization of a novel DoSWEET1 gene from Dendrobium officinale].

SWEET (sugars will be eventually exported transporters) constitute a large and conserved gene family of sugar transporters in eukaryotes, which are important in the cellular metabolisms, growth and development, and plant-microbe interaction in plants. In the present study, a full length cDNA of SWEET encoding gene, designed as DoSWEET1 (GenBank accession No. KT957550), was identified in Dendrobium officinale using RT-PCR and RACE approaches. DoSWEET1 was 1 150 bp in length and encoded a 262-aa protein with a molecular weight of 29.18 kD and an isoelectric point of 9.49. The deduced DoSWEET1 protein contained seven transmembrane regions and two conserved MtN3-slv domains (11-94, 130-212）. Multiple sequence alignment revealed that DoSWEET1 had high identities 45%-54.6%） with SWEET proteins from various plants. A neighbor joining phylogenetic analysis suggests that DoSWEET1 belonged to the class Ⅱ subgroup of the SWEET evolutionary tree, and was closely related to rice OsSWEET13, OsSWEET14, and OsSWEET15. qPCR analysis demonstrated that DoSWEET1 gene was differentially expressed in the three included organs of D. officinale, and the expression was most abundant in the roots at 9.88 fold over that of the stems, followed by that of the leaves with 2.85 fold higher. In the 3rd symbiotic germinating seeds infected by Tulasnella sp., the transcipts were dramatically induced by 1 359.06 fold over that in the ungerniamted control seeds, suggesting a vital role of the gene in the D. officinale symbiotic germination process. Molecular cloning and characterization of the novel DoSWEET1 gene provides a foundation for the functional study of the gene in sugar translocation during the D. officinale symbiotic germination process.

[Advance in molecular biology of Dendrobium (Orchidaceae)].

With the development of molecular biology, the process in molecular biology research of Dendrobium is going fast. Not only did it provide new ways to identify Dendrobium quickly, reveal the genetic diversity and relationship of Dendrobium, but also lay the vital foundation for explaining the mechanism of Dendrobium growth and metabolism. The present paper reviews the recent process in molecular biology research of Dendrobium from three aspects, including molecular identification, genetic diversity and functional genes. And this review will facilitate the development of this research area and Dendrobium.

[Molecular cloning and characterization of two kind of heat-shock protein gene from Polyporus umbellatus].

With RT-PCR approaches, the full-length cDNA of two heat shock protein genes were cloned from total RNA of the Polyporus umbellatus sclerotium. The full open reading frame cDNA sequence of the Hsp90 was 2 091 bp, encoding 696 amino acid residues with a predicted molecular mass of 78.9 kDa. The full open reading frame cDNA sequence of the Hsp70 was 1 944 bp, encoding 647 amino acid residues with a predicted molecular mass of 70.5 kDa. The Hsp90 and Hsp70 protein contained the conservative structure domain, respectively. Phylogenetic analysis showed that Hsp90 and Hsp90 from Trametes versicolor were clustered into one group, Hsp70 and Hsp70 from Fistulina hepatica were clustered into one group. Real-time PCR analysis showed that, the expression of Hsp90 and Hsp70 in the infected part by Amillariella mellea was upregulated. The expression profiling of Hsp90 and Hsp70 showed same patterns underbiotic stress. The results indicate that these two genes may play an important role in response to Amillariella mellea infection.

[Effect of endophytic fungi on modernization of traditional Chinese medicine].

The modernization of traditional Chinese medicine (TCM) is a foundation of TCM to go abroad and get international recognition. It is the only way to revitalize the TCM industry. But in the process of it, we are facing various challenges: heavy metal contamination, low content of active ingredients, less innovation, the contradiction between resource utilization and protection, and so on. How to apply new technology and new theory of life science to solve these problems becomes an urgent matter. In recent years, the studies found that endophytic fungi played an irreplaceable positive role in the growth and development of herbal medicine, and had great impact on the quality of traditional Chinese medicine. Therefore this paper introduces the effect of endophytic fungi on genuine traditional Chinese medicines, cultivation of TCM, development and protection of TCM, et al, and explores its applicative prospect, providing new idea and new power for promoting the development of modernization of TCM.