Cloning and expression of three thaumatin-like protein genes from

Genes encoding thaumatin-like protein () are frequently found in fungal genomes. However, information ongenes inis still limited. In this study, threegenes were cloned from. The full-length coding sequence of,andwere 768, 759 and 561 bp long, respectively, encoding for 256, 253 and 187 amino acids. Phylogenetic trees showed that,andwere clustered with sequences fromand, respectively. The expression patterns of the threegenes were higher inwithinfection than in the sclerotia without. Furthermore, over-expression of three PuTLPs were carried out inBL21 (DE3) strain, and high quality proteins were obtained using Ni-NTA resin that can be used for preparation of specific antibodies. These results suggest that,andinmay be involved in the defense response toinfections.

Molecular cloning and characterization of four small GTPase genes from medicinal fungus Polyporus umbellatus / 药学学报

Four small GTPase genes which may be relative to sclerotial development were firstly cloned from medicinal fungus Polyporus umbellatus using rapid amplification of cDNA end PCR (RACE) method. The results showed that full-length cDNA of PuRhoA was 698 bp contained 585 bp ORF, which was predicted to encode a 194 amino acid protein with a molecular weight of 21.75 kD with an isoelectric point (pI) of 6.44; the full length cDNA of PuRhoA2 was 837 bp in length and encoded a 194 amino acid protein with a molecular weight of 21.75 kD and an isoelectric point (pI) of 6.33; the full length cDNA of Puypt1 was 896 bp in length and encoded a 204-aa protein with a molecular weight of 22.556 kD and an isoelectric point (pI) of 5.75; the full length cDNA of PuRas was 803 bp in length and encoded a 212-aa protein with a molecular weight of 23.821 kD and an isoelectric point (pI) of 5.2. There are fani acyl transferase enzyme catalytic site and myrcene-transferase enzyme catalytic site in PuRhoA1 while the PuRhoA2 only possess myrcene-transferase enzyme catalytic site. Puypt1 contains the Rab1-Ypt1 conserved domain of small GTPase family and PuRas contains the fani acyl transferase enzyme catalytic site. According to the phylogenetic analysis all these four small GTPase clustered with basidiomycete group. Quantitative real-time PCR analysis revealed that Puypt1, PuRas and PuRhoA1 transcripts were significantly higher in the beginning of sclerotial formation than that in the mycelia, whereas the transcripts levels of PuRhoA2 gene were particularly lower in sclerotia than that in mycelia, suggesting that these four genes might be involved in P umbellatus selerotial development.

Molecular characterization of a HMG-CoA reductase gene from a rare and endangered medicinal plant, Dendrobium officinale / 药学学报

The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) catalyzes the conversion of HMG-CoA to mevalonate in mavalonic acid pathway, which is the first committed step for isoprenoid biosynthesis in plants. However, it still remains unclear whether HGMR gene plays a role in the isoprenoid biosynthesis in Dendrobium officinale, an endangered epiphytic orchid species. In the present study, a HMGR encoding gene, designed as DoHMGR1 (GenBank accession JX272632), was identified from D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoHMGR1 was 2 071 bp in length and encoded a 562-aa protein with a molecular weight of 59.73 kD and an isoelectric point (pI) of 6.18. The deduced DoHMGR1 protein, like other HMGR proteins, constituted four conserved domains (63-561, 147-551, 268-383 and 124-541) and two transmembrane motifs (42-64 and 85-107). Multiple sequence alignment and phylogenetic analyses demonstrated that DoHMGR1 had high identity (67%-89%) to a number of HMGR genes from various plants and was closely related to Vanda hybrid cultivar, rice and maize monocots. Real time quantitative PCR (qPCR) analysis revealed that DoHMGR1 was expressed in the three included organs. The transcripts were the most abundant in the roots with 2.13 fold over that in the leaves, followed by that in the stems with 1.98 fold. Molecular characterization of DoHMGR1 will be useful for further functional elucidation of the gene involving in isoprenoid biosynthesis pathway in D. officinale.

Prokaryotic soluble expression, purification and function study of LEDGF/p75 protein / 药学学报

HIV-1 integrase (IN) is a key enzyme for the viral replication. The protein-protein interaction (PPI) between HIV-1 IN and a cellular cofactor lens epithelium-derived growth factor (LEDGF/p75) is a validated target for anti-HIV drug discovery. In order to build the platform for screening inhibitor against PPI between IN and LEDGF/p75, the vector containing the LEDGF/p75 protein cDNA was constructed and expressed in Escherichia coli and the function of the LEDGF/p75 protein was assayed. The LGDGF/p75 encoding gene optimized according to the preference codon usage of E. coli, was synthesized and cloned into the expression vector pGEX-4T-1 to form a recombined plasmid, then transformed into host cell E. coli BL21 (DE3). The recombined clones were identified and confirmed by BamH I/Sal I digestion and sequencing, the successfully recombined plasmid in the host cell was induced by IPTG and the condition of the expression was optimized. The expressed protein was purified by the Ni2+ affinity chromatography column and SDS-PAGE was used to analyze the molecular weight and specificity. In addition, ELISA assay was used to analyze the function of the recombinant protein. The recombinant LGDGF/p75 was soluble, and expressed highly and stably in E. coli. The protein was proved to enhance HIV-1 IN strand transfer activity in vitro by ELISA. It will be helpful to build the platform of screening inhibitors against PPI between IN and LEDGF/p75.

Isolation and differential expression of a novel MAP kinase gene DoMPK4 in Dendrobium officinale / 药学学报

Mitogen-activated protein kinases (MAPKs) are important signaling transduction components well conserved in eukaryotes and play essential roles in various physiological, developmental and hormonal responses in plant. In the present study, a MAPK gene, designated as DoMPK4 (GenBank accession No. JX297597), is identified from a rare endangered medicinal orchid species D. officinale using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK4 is 1 518 bp in length and encoded a 369 aa protein with a molecular weight of 42.42 kD and an isoelectric point of 5.55. DoMPK4 protein contained a serine/threonine protein kinase active site (158-170), a MAP kinase site (71-174), and eight conserved motifs. DoMPK4 had a transmembrane (214-232) but no signal peptide. Multiple sequence alignment showed that DoMPK4 shared high identities (74.9%-80.6%) with MAPK proteins from various plants. Phylogenetic analysis demonstrated that DoMPK4 belonged to group A of the MAPK evolutionary tree, and is closely related to monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK4 is differentially expressed among the five organs including leaf, stem, root, seed, and protocorm-like body (PLB). The transcription level of DoMPK4 is the highest in the PLBs with 17.65 fold, followed by seeds, roots, and stems with 5.84, 2.28, and 1.64 fold, respectively. The progressive enhancement of DoMPK4 transcripts in the developing PLBs compared to that in the germinating seeds, suggests a role of DoMPK4 during the development of embryogenic PLBs formation in D. officinale.

Molecular cloning and characterization of S-adenosyl-L-methionine decarboxylase gene (DoSAMDC1) in Dendrobium officinale / 药学学报

S-Adenosyl-L-methionine decarboxylase (SAMDC) is a key enzyme in the polyamines biosynthesis, thus is essential for basic physiological and biochemical processes in plant. In the present study, a full length cDNA of DoSAMDC1 gene was obtained from symbiotic germinated seeds of an endangered medicinal orchid species Dendrobium officinale, using the rapid amplification of cDNA ends (RACE)-PCR technique for the first time. The full length cDNA was 1 979 bp, with three open reading frames, i.e. tiny-uORF, small-uORF and main ORF (mORF). The mORF was deduced to encode a 368 amino acid (aa) protein with a molecular mass of 40.7 kD and a theoretical isoelectric point of 5.2. The deduced DoSAMDC1 protein, without signal peptide, had two highly conserved function domains (proenzyme cleavage site and PEST domain) and a 22-aa transmembrane domain (89-110). Multiple sequence alignments and phylogenetic relationship analyses revealed DoSAMDC1 had a higher level of sequence similarity to monocot SAMDCs than those of dicot. Expression patterns using qRT-PCR analyses showed that DoSAMDC1 transcripts were expressed constitutively without significant change in the five tissues (not infected with fungi). While in the symbiotic germinated seeds, the expression level was enhanced by 2.74 fold over that in the none-germinated seeds, indicating possible involvement of the gene in symbiotic seed germination of D. officinale.

Isolation, idetification and anti-HIV-1 integrase activity of culturable endophytic fungi from Tibetan medicinal plant Phlomis younghusbandii Mukerjee / 药学学报

A total of 52 endophytic fungi were isolated from roots and stems of Tibetan medicinal plant Phlomis younghusbandii Mukerjee. These fungal isolates were molecularly identified based on ITS sequnces and 28S sequences distributed to 12 genera, including Phoma, Chaetosphaeronema, Fusarium and Leptosphaeria, etc. Among them, the dominant genus was Phoma. Extracts of all strains were evaluated for anti-HIV-1 integrase activity by using soluable integrase expressed in E. coli BL21 (DE3). The results showed that seven samples from five fungal endophytes PHY-24, PHY-38, PHY-40, PHY-51, PHY-53, which belonged to genus Chaetosphaeronema, inhibited strand transfer reaction catalyzed by HIV-1 integrase with IC50 values, of 6.60, 5.20, 2.86, 7.86, 4.47, 4.56 and 3.23 microg x mL(-1) respectively. In conclusion, the endophytic fungi of Phlomis younghusbandii Mukerjee are valuable for further screening anti-HIV-1 integrase agents.

Fluid suspension co-culture of Dendrobium officinale protocorm and living fungus / 中国中药杂志

<p><b>OBJECTIVE</b>To set up a system for fluid suspension co-culture of Dendrobium officinale protocorm and living fungus MF24, so as to provide certain scientific evidence for industrial production of protocorm.</p><p><b>METHOD</b>Whether the protocorm culture system was suitable for the normal growth of MF24 fungus were studied, the growth of protocorm cultured alone and co-cultured with the fungus were researched under light and dark culture conditions, the biomass and proliferation times were determined, and HPLC method was used to analyze and compare the changes of 11 characteristic peak areas in D. officinale protocorm.</p><p><b>RESULT</b>The MF24 fungus could grow normally in the 6,7-V liquid medium used to culture the protocorm, and when it was cultured by 8-10 hours per day under 1 500 lx, the growth rate of the fungus was slowed. Protocorm could grow normally in light and dark culture conditions, and add the MF24 fungus in the early cultivation stages of protocorm, both inhibit the growth of each other. In the protocorm for the growth stability to add 5 diameter 9 mm fungi block, the protocorm growth and chemical composition type had no significant effect. However, under illumination, co-cultured for 5 days protocorm of which 10 compounds content decreased 13.64% to 138.47%, in dark conditions, co-cultured for 5 days protocorm of which 7 compounds increased by 0.71% to 12.82%, and 4 compounds slightly reduced by 3.03% to 14. 14% compared with the control.</p><p><b>CONCLUSION</b>Under the appropriate condition, living fungus MF24 could co-culture with the D. officinale protocorm, and affected the latter's secondary metabolite levels.</p>

Sesquiterpenes and monoterpene from Aquilaria sinensis / 中国中药杂志

The column chromatography on silica gel, semi-preparative HPLC were used to separate and purify the compounds from the petroleum ether and ethanol extract of Aquilaria sinensis. Nine compounds were isolated. On the basis of their spectroscopic data, the structures were identified as 3, 3, 7-trimethyltricycloundecan-8-one (1), longifolene (2), norlongilactone (3), caryophyllenol-II (4), humulene diepoxide A (5), kobusone (6), (-)-bornyl ferulate (7), (24R) -24-ethylcholesta-4, 22-dien-3-one (8), (24R)-24-3-ono-4-en-sitosterone (9). Compounds 2-9 were isolated from this plant for the first time. compounds 1-6 are sesquiterpenes, compound 7 is a monoterpene derivative, compound 8 and 9 are steroids.

Antimicrobial activity of endophytic fungi isolated from Dendrobium species in southwestern China / 中国中药杂志

<p><b>OBJECTIVE</b>To isolate and characterize endophytic fungi from seven Dendrobium species, and detect their antimicrobial activities.</p><p><b>METHOD</b>Fungal endophytes were isolated by strictly sterile sample preparation and fungal identification methods were based on their ITS ribosomal DNA (ITS rDNA gene) sequences. The agar well diffusion method was then employed to evaluate the antimicrobial activity against six pathogenic organisms and the phylogenetic tree of active isolates was constructed by the MEGA.</p><p><b>RESULT</b>Ninety-eight endophytic fungi obtained from seven Dendrobium spp., and among them twenty-four isolates, representing 11 genera and 14 species, displayed anti-microbial activities. The phylogenetic assay based on ITS-rDNA showed that 24 active isolates were sorted to 7 taxonomic orders: Hypocreales, Sordariales, Capnodiales, Eurotiales, Botryosphaeriales, Xylariales and Mucorales. The results of antimicrobial activity assay revealed that 1.02%, 10.2%, 18.4%, 1.02%, 1.02% and 10.2% of fermentation broths of 98 isolates displayed significant antimicrobial activities against E. coli, B. subtilis, S. aureus, C. albicans, C. neoformans and A. fumigatus, respectively. Four strains DL-R-3, DL-S-6, DG-R-10 and DN-S-1 displayed strong and broad antimicrobial spectrum.</p><p><b>CONCLUSION</b>Endophytic fungi associated with Dendrobium species have fungal diversity, and possess diverse antimicrobial activity.</p>

Molecular characterization of a mitogen-activated protein kinase gene DoMPK1 in Dendrobium officinale / 药学学报

The mitogen-activated protein kinase (MAPK) cascade, composed of MAPK kinase kinase (MAP3K), MAPK kinase (MAP2K), and MAPK, is abundantly conserved in all eukaryotes. MAPK along with MAPK cascade plays a vital regulatory role in the plant-arbuscular mycorrhiza/rhizobium nodule symbioses. However, the biological function of MAPK in orchid mycorrhiza (OM) symbiosis remains elusive. In the present study, a MAPK gene, designated as DoMPK1 (GenBank accession No. JX297594), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of DoMPK1 was 1 263 bp and encoded a 372 aa protein with a molecular weight of 42.61 kD and an isoelectric point (pI) of 6.07. The deduced DoMPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain (39-325) and MAP kinase signature (77-177). Multiple sequence alignment and phylogenetic analysis demonstrated that DoMPK1 was highly homologous (71%-85%) to MAPK genes from various plant species and was closely related to those from monocots. Real time quantitative PCR (qPCR) analysis revealed that DoMPK1 was constitutively expressed in leaves, stems, roots and seeds, and the transcript abundance was not significantly different in the four included tissues. Furthermore, DoMPK1 transcript was markedly induced in roots at 30 d after fungal infection, with 7.91 fold compared to that of the mock inoculated roots, suggesting implication of DoMPK1 in the early D. officinale and Mycena sp. interaction and an essential role in the symbiosis. Our study characterized a MAPK gene associated with OM symbiosis for the first time, and will be helpful for further functional elucidation of DoMPK1 involving in D. officinale and Mycena sp. symbiotic interaction.

Cloning and expression analysis of a calcium-dependent protein kinase gene in Dendrobium officinale in response to mycorrhizal fungal infection / 药学学报

Calcium-dependent protein kinases (CDPKs) play an important regulatory role in the plantarbuscular mycorrhiza/rhizobium nodule symbiosis. However, the biological action of CDPKs in orchid mycorrhiza (OM) symbiosis remains unclear. In the present study, a CDPK encoding gene, designated as DoCPK1 (GenBank accession No. JX193703), was identified from D. officinale roots infected by an OM fungus-Mycena sp. using the reverse transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) methods, for the first time. The full length cDNA of DoCPK1 was 2137 bp in length and encoded a 534 aa protein with a molecular weight of 59.61 kD and an isoelectric point (pI) of 6.03. The deduced DoCPK1 protein contained the conserved serine/threonine-protein kinase catalytic domain and four Ca2+ binding EF hand motifs. Multiple sequence alignment demonstrated that DoCPK1 was highly homologous (85%) to the Panax ginseng PgCPK1 (ACY78680), followed by CDPKs genes from wheat, rice, and Arabidopsis (ABD98803, ADM14342, Q9ZSA2, respectively). Phylogenetic analysis showed that DoCPK1 was closely related to CDPKs genes from monocots, such as wheat, maize and rice. Real time quantitative PCR (qPCR) analysis revealed that DoCPK1 was constitutively expressed in the included tissues and the transcript levels were in the order of roots > stems > seeds > leaves. Furthermore, DoCPK1 transcripts were significantly accumulated in roots 30 d after fungal infection, with 5.16 fold compared to that of the mock roots, indicating involvement of DoCPK1 during the early interaction between D. officinale and Mycena sp., and a possible role in the symbiosis process. This study firstly provided important clues of a CDPK gene associated with OM symbiosis, and will be useful for further functional determination of the gene involving in D. officinale and Mycena sp. symbiosis.

Mould and mycotoxin contamination of medicinal materials / 中国中药杂志

Medicinal materials may be contaminated with a broad variety of fungi, which are represented by Aspergillus spp, Penlicillium spp, Fusarium spp, Rhizopus spp, Mucor spp et al. This fact limits the utilization of medicinal materials, besides, medicinal materials may also be contaminated with mycotoxins produced by these fungi, and bring harm to human health. Several mycotoxins have been detected in medicinal materials, such as AFTs, OTA, FBs, et al. The contamination may originate from the conditions in which the medicinal plants are cultivated, stored and in the finished product manufacturing stages. Some methods have been used for detoxifcation and disinfection for medicinal materials, but they have limited effects. Taking into consideration the background situation, it is important for medicinal materials to be protected from contamination of fungi at every stage of production. The present study intends to give a review of contamination of medicinal materials by moulds and mycotoxins and discuss the factors influencing this situation, expecting to contribute to the knowledge for reducing the contamination.

Diterpenoids from Chinese eaglewood / 中国中药杂志

Column chromatography on silica gel, Sephadex LH-20, semi-preparative HPLC were used to separate and purify the compounds from the petroleum ether and ethanol extract of Chinese eaglewood. Nine compounds were isolated. On the basis of their spectroscopic data, their structures were identified as dehydroabietic acid (1), methyl dehydroabietate (2), methyl 7-oxodehydroabietate (3), 7alpha, 15-dihydroxydehydroabietic acid (4), 7alpha-hydroxypodocarpen-8(14)-en-13-on-18-oic acid (5), pimaric acid (6), pimarol (7), 18-norpimara-8 (14), and 15-dien-4alpha-ol (8), 18-norisopimara-8 (14), 15-dien-4beta-ol (9). All of the compounds were isolated from this plant for the first time, and compounds 5, 8 and 9 are norditerpenoids.

Chemical constituents of Dendrobium candidum / 中国中药杂志

The column chromatography on silica gel, sephadex LH-20 preparative HPLC were used to separate and purify the compounds from the stems of Dendrobium candidum. Twenty compounds were isolated and identified as 3,4'-dihydroxy-5-methoxybibenzyl(1), dihydroresveratrol(2), dendromoniliside E(3), denbinobin(4),2,4,7-trihydroxy-9, 10-dihydrophenanthrene(5), aduncin(6), (-)-loliolide(7), adenosine(8), uridine(9), guanosine(10), sucrose(11), 5-hydroxymethyl-furaldehyde(12), n-octacostyl ferulate(13), defuscin(14), n-triacontyl cis-p-coumarate(15), daucosterol(16), beta-sitosterol(17), hexadecanoic acid(18), hentriacontane(19), and heptadecanoic acid(20). Their structures were elucidated on the basis of spectroscopic data and physicochemical properties. All of the compounds were isolated from this plant for the first time.

Comparison of rutin and syringin content between tissue culturing seedlings and botanical drug of Saussurea involucrata / 中国中药杂志

<p><b>OBJECTIVE</b>To compare the rutin and syringin content in tissue culturing seedlings and in botanical drug of Saussurea involucrata.</p><p><b>METHOD</b>The HPLC with Hydro-RP C18 (4.6 mm x 250 mm, 5 microm) column was used, a mixture of acetonitrile-water (5:95) was used as a mobile phase, with flow rate of 1 mL x min(-1), column temperature at 25 degrees C and detection wavelength at 220 nm.</p><p><b>RESULT</b>The effective constituents of tissue culturing seedlings were almost similar to the botanical drug. And syringin in tissue culturing seedlings was increased 4.35 times.</p><p><b>CONCLUSION</b>It has a good prospect to acquire high-quality S. involucrata by tissue culturing seedlings.</p>

Application of SPSS orthogonal design in tissue culture of Anoectochilus roxburghii / 中国中药杂志

<p><b>OBJECTIVE</b>To study the effect of the different constitutions of plant hormone on the development of Anoectochilus roxburghii.</p><p><b>METHOD</b>A. roxburghii were harvested after having been cultured for 60 days. An orthogonal design was used to study the effect of NAA and 6-BA on the leaf number, eustipe number, lateral branch number of the stem tip and stem section, and the height of the stem tips. All of the data were processed by SPSS.</p><p><b>RESULT AND CONCLUSION</b>It is reported for the first time that NAA could make different development of A. roxburghii at low concentration ( < 1 mg L(-1)) and high concentration ( > 1 mg L(-1)). The optimum constitution of MS medium was NAA 0.5 mg L(-1) + 6-BA 1 mg L(-1) for the growth of the stem tip of A. roxburghii, and NAA 1 mg L(-1) + 6-BA 2 mg L(-1) for the differentiation of bud and the formation of lateral branch of the stem section. The different concentrations of NAA and 6-BA had different effects on the growth and differentiation of the stem tip and the stem section of A. roxburghii.</p>

Study on the protoplast preparation and regeneration of Gliocladium sp. producing antiinflammation compound 6,22-diene-5,8-epidioxy ergosta-3-hydroxy / 中国生化药物杂志

Purpose　The aim is to study the conditions of preparation and regeneration of Gliocladium sp. (F) protoplast. Methods　 Different enzyme systems, enzymolysis time, osmotic pressure stabilizers were studied to investigate their influence on the productivity and regenerating rate of Gliocladiumsp. F protoplasts. The HPLC method was used to determine EP (6,22-diene-5,8-epidioxy ergosta-3-hydroxy) content.Results　The higher productivity of protoplasts was obtained when mycelia of strain F growing for 60 hours was digested at 28℃ for 4 hours by solution containing 2% cellulase and 2% helicase dissolved in 0.5 mol/L mannitol and the medium containing 0.5mol/L mannitol as osmotic pressure stabilizer would be suitable for protoplast regeneration. According to the EP productivity detected by HPLC, high positive rate of the regenerated strains growing in the medium containing 0.5mol/L mannitol could be got. Conclusion　The results will promote the research of strain F mutantgenesis and will be helpful for obtaining the strain more effectively biosythesizing compound EP.

STUDIES ON PHARMACOLOGICAL ACTIVITY OF MYCENA DENDROBII / 微生物学通报

The pharmacological activity of Mycena dendrobii Fan et Guo, a new species of endophytic fungus was studied. It was revealed that the mycelia methanol extracts and the fermentation liquid ethanol extracts of Mycena dendrobii showed anglgesic effect to mice, which have the correlations to that of the traditional Chinese medicine ‘shihu’. The fermentation liquid ethanol extracts of Mycena dendrobii showed excitation effect to central nervous system of mice. Then the effective parts of anglgesic effect was determined.

Suspension culture of protocorm in Dendrobium candidum / 中草药

Object To study the feasibility of suspension culture of protocorm in Dendrobium candidum Wall ex Lindl and effect of inoculum and medium volume on the growth of protocorm Methods Effect of four basic media MS, 1/2 MS, 67 V, and B 5, inoculum and medium volume on the growth of protocorm were studied by completely random experimental design and orthogonal test design Results The growth of D candidum protocorms in liquid medium was markedly better than that in solid medium (P0 05), B 5 was much better than 1/2 MS (P