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.

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.

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.

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.

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.

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.

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.

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.

To observe the clinical therapeutic effect of diabetic peripheral neuropathy (DPN) treated by needling combined with drug, 104 patients with DPN were randomly divided into acupuncture plus drug group and control group, and each group had 52 patients. After treatment of two months, the clinical effective rate in acupuncture plus drug group was 51.9%, and the total effective rate was 88.5%, both of them were better than those in control group (P＜0.05). The needling method of nourishing the kidney and dredging the meridian combined with drug had good clinic effect in the treatment of DPN.