Cytochrome P450 (CYP450) is a key element in the Ganoderma triterpenoid biosynthetic pathway. The catalytic reaction process for CYP450 requires NADPH / NADH for electron transfer. After searching the genome dataset of Ganoderma lucidum, the unique sequence encoding CYP450 and NADPH were discovered, separately. The open reading frames of GLCYP450 and GLNADPH were cloned separately using RT-PCR strategy from G lucidum. The appropriate restriction enzyme cutting sites were introduced at the 5' and 3' ends of gene sequence. The genes of GLCYP450 and GLNADPH were recombined into the yeast expression vector pESC-URA, leading to the formation of the yeast expression plasmid pESC-GLNADPH-GLCYP450. This study provides a foundation for researching Ganoderma triterpene biosynthesis using the approach of synthetic biology.

Object To develop a new method of discriminating the principle components of Radix Paeoniae Rubra (RPR) from different areas, and to find the reason for forming high-quality of RPR in the place of the genuine. Methods Using Foruier transform infrared (FTIR) absorption spectrometry, the characteristic peaks of fingerprint infrared spectra of RPR samples from 18 habitats were recognized and compared. Results Frequency, intensity and shape of infrared absorption spectra were obviously different between wild and cultivated groups of RPR. The infrared absorption peaks of RPR in Duolun, a famous Chinese orthodox drug, were of distinct characteristics. Conclusion FTIR technique is first applied to rapid analysis of principle component of RPR from different areas. So an operable method in the quality control and discrimination of RPR in the place of the genuine is provided.

Codon usage bias is an important characteristic of genetic information transfer in organisms. Analysis of codon usage bias of different species is important for understanding the rules on genetic information transfer. The previous method for analysis of codon usage bias is mainly based on genomic data. However, this method is greatly limited, because the genome sequences of higher organisms are still not available up to now. In this study, we found that we could obtain the same optimal codons of Ganoderma lucidum (Curtis: Fr.) P. Karst based on its whole genomic data or large-scale transcriptomic data from its liquid-cultured hyphae, primordium and fruiting body, separately. This result indicated the feasibility to understand the codon usage bias based on the large-scale transcriptomic data. By calculating the proportion of rare codons of Escherichia coli and Saccharomyces cerevisiae in 26 terpene synthases (TS) of G. lucidum, we found that the rare codons of S. cerevisiae have a higher proportion in TS genes, while the rare codons of E. coli have relatively lower, suggesting that the TS genes of G. lucidum are possibly more difficult to be expressed in S. cerevisiae than in E. coli. Chemical synthesis of TS genes according to the yeast optimal codons will be an effective way to solve the problem on the mismatch of gene codon bias between the foreign genes and the host strain.

Synthetic biology of traditional Chinese medicine (TCM) is a new and developing subject based on the research of secondary metabolite biosynthesis for nature products. The early development of synthetic biology focused on the screening and modification of parts or devices, and establishment of standardized device libraries. Panax notoginseng (Burk.) F.H.Chen is one of the most famous medicinal plants in Panax species. Triterpene saponins have important pharmacological activities in P. notoginseng. Squalene epoxidase (SE) has been considered as a key rate-limiting enzyme in biosynthetic pathways of triterpene saponins and phytosterols. SE acts as one of necessary devices for biosynthesis of triterpene saponins and phytosterols in vitro via synthetic biology approach. Here we cloned two genes encoding squalene epoxidase (PnSE1 and PnSE2) and analyzed the predict amino acid sequences by bioinformatic analysis. Further, we detected the gene expression profiling in different organs and the expression level of SEs in leaves elicited by methyl jasmonate (MeJA) treatment in 4-year-old P notoginseng using real-time quantitative PCR (real-time PCR). The study will provide a foundation for discovery and modification of devices in previous research by TCM synthetic biology. PnSE1 and PnSE2 encoded predicted proteins of 537 and 545 amino acids, respectively. Two amino acid sequences predicted from PnSEs shared strong similarity (79%), but were highly divergent in N-terminal regions (the first 70 amino acids). The genes expression profiling detected by real-time PCR, PnSE1 mRNA abundantly accumulated in all organs, especially in flower. PnSE2 was only weakly expressed and preferentially in flower. MeJA treatment enhanced the accumulation of PnSEI mRNA expression level in leaves, while there is no obvious enhancement of PnSE2 in same condition. Results indicated that the gene expressions of PnSE1 and PnSE2 were differently transcribed in four organs, and two PnSEs differently responded to MeJA stimuli. It was strongly suggested that PnSEs play different roles in secondary metabolite biosynthesis in P. notoginseng. PnSE1 might be involved in triterpenoid biosynthesis and PnSE2 might be involved in phytosterol biosynthesis.

3-Hydroxy-3-methylglutaryl coenzyme-A reductase (HMGR), the first enzyme of mavalonic acid pathway, is one of the key devices involved in ginsenoside biosynthesis based on synthetic biology approach. The open reading frame of a novel HMGR gene from Panax ginseng (PgHMGR2) was cloned and analyzed in this study. PgHMGR2-encoding protein showed 71.6% sequence similarity to a P. ginseng HMGR in GenBank. The full-length cDNA sequence of PgHMGR2 containing 1 770 bp, which encodes 589 amino acids, was cloned by RT-PCR strategy from P. ginseng. The bioinformatic analysis showed that PgHMGR2-encoding protein contained two transmembrane regions and the HMG_CoA_reductase domain, without signal peptide. The protein sequence of PgHMGR2 had the highest sequence similarity (99%) with Panax quinquefolius HMGR (GenBank accession No. ACV65036). The expression level of PgHMGR2 was the highest in flower based on a real-time PCR analysis, followed by leaf and root, and the lowest was in stem. The result will provide a foundation for exploring the molecular function of PgHMGR2 involved in ginsenoside biosynthesis based on synthetic biology approach in P. ginseng plants.

Research on medicinal model organism is one of the core technologies to promote the modernization of traditional Chinese medicine (TCM). The research progress of Salvia miltiorrhiza as medicinal model plant is summarized in this paper. The genome of S. miltiorrhiza is small and its life cycle is short, as well as this plant can be stably genetically transformed. Because S. miltiorrhiza possesses the important medicinal and economic values, recently the transcriptome and genome of S. miltiorrhiza have been significantly recovered. The research prospect of S. miltiorrhiza as medicinal model plant in TCM was discussed, including biosynthesis of active components and their genetic regulation, relationship between quality of TCM and ecological environments, and selective breeding of good quality lines. Furthermore, as medicinal model plant, the construction of mutant library for S. miltiorrhiza, the genome map with high quality, and the functional genome should be investigated. Accompanying modern investigation of life sciences, the platform for medicinal model plant, S. miltiorrhiza, will be promoted to be established. It is important to develop the ethnopharmacology and new drugs around the world.

Objective To evaluate the efficacy of double filtration plasmapheresis (DFPP) combined with immunosuppressive agents (leflunomide plus methotrexate) on synovitis in magnetic resonance imaging (MRI) in patients with high active rheumatoid arthritis (RA). Methods Fifty eight patients with RA (disease duration 6 months to 12 years) were randomly divided. Thirty-one were randomized to the treatment group and 27 were randomized to the control group. All patients received leflunomide 10 mg, two times daily; plus methotrexate 15 mg orally once weekly. DFPP was performed in the treatment group once 1-2 weeks for 3-4 sessions. Control patients did not receive DFPP. All patients underwent contrast-enhanced MRI of the right wrist at the baseline and 6 months, 1 month in the treatment group. The signs including synovitis pannus, bone marrow edema and effusion were observed on MRI. The scoring of synovial hypertrophy, pannus, bone marrow edema were measured according to the outcome measures in RA MRI scoring system. Comparisons between groups were performed with paired-samples t test and independent-sample t test. Results The MRI synovitis score, MRI pannus score and MRI bone marrow edema in the treatment group was (1.4±1.6), (0.13± 0.35) and (5±4) respectively,so was significantly lower than that of the control group [respectively for (7.9± 1.3), (2.76±0.43), (16±12),P<0.01]. 53% of the treatment group satisfied both the disease activity score 28-joint assessment and MRI synovitis assessment (no enhancement of synovium or pannus, no effusion), but none in the control group (P<0.01). Significant changes at 1 month was observed in DAS28 and HAQ scores (P<0.01), but not in the MRI synovitis score, MRI pannus score, MRI bone marrow edema score and effusion in the treatment group (P>0.05). Conclusion DFPP combined with immunosuppressive agents can significantly improve synovitis in MRI in patients with high active RA. Improvement of the signs of MRI is later than that in the clinic. So imaging assessment may be necessary for accurate evaluation of disease status and selection of therapy.

Objective Based on the principles of laser radiation protection, medical metroiogy and photoelectron technology, an automatic verification device and testing technology to provide verification and performance evaluation for laser protective spectacles and equipments which have the various functions in laser protection have been developed and established. Methods The current system comprises laser source, laser measuring instruments, software of computer detection and control and modules of optics and mechanics used in auxiliary equipment. By use of the verification device and the special test-recording method to be designed and studied by us, the measured data can be automatically acquired, processed, recorded, saved and printed and, furthermore, many parameters on protective performance of the measured laser protective spectacles can be tested and given. Results Laser wavelength of the verification apparatus are 1 064 nm and 532 nm, response range of wavelength is from 0.4 m to 1.1 μm, measuring range of laser energy is from 10-8 J to 0.3 J, measuring range of optical density for laser protective spectacles is from 0.1 to 8.0, stability is 0.21%, measuring uncertainty is 5%(k=2). Conclusion The automatic verification device is steady and reliable. The achieved performance indexes accord with the requirement of national standard on laser protection.

ERF family transcription factor (TF) represented ethylene-responsive protein which harbored a conserved AP2 domain. After searching the plant transcription factor database, a total of 75 unigenes was found which contained AP2 domain from the transcriptome dataset of Panax quinquefolius L. One unique sequence of ERF transcript, named as PqERF1, was cloned with entire open reading frame of 933 base pairs (bp). Protein prediction result indicated that the gene was localized in nucleus and had a conserved AP2 domain. PqERF1 gene could be induced by methyl jasmonate (MeJA) which was consistent to the inducing profile of triterpene ginsenosides. InterproScan prediction indicated that PqERF1 was probably a pathogenesis-related gene. Sequence alignment and phylogenetic analysis demonstrated PqERF1 was with high identity and had relative close relationship to the NtERF4 (Nicotiana tabacum), PhERF12 (Petunia x hybrida) and DcERF1 (Daucus carota) which was related to plant defense, regulation of secondary metabolism and the flower senescence respectively. Therefore, the gene was likely involved in regulation of secondary metabolism, plant defense and physical processes which would provide gene resource for further study on secondary metabolite synthesis and molecular breeding of P. quinquefolius.

To investigate the profile of gene expression in Salvia miltiorrhiza and elucidate its functional gene, 454 GS FLX platform and Titanium regent were used to produce a substantial expressed sequence tags (ESTs) dataset from the root of S. miltiorrhiza. A total of 46 722 ESTs with an average read length of 414 bp were generated. 454 ESTs were combined with the S. miltiorrhiza ESTs from GenBank. These ESTs were assembled into 18 235 unigenes. Of these unigenes, 454 sequencing identified 13 980 novel unigenes. 73% of these unigenes (13 308) were annotated using BLAST searches (E-value < or = 1e-5) against the SwissProt, KEGG TAIR, Nr and Nt databases. Twenty-seven unigenes (encoding 15 enzymes) were found to be involved in tanshinones biosynthesis, and 29 unigenes (encoding 11 enzymes) involved in phenolic acids biosynthesis. Seventy putative genes were found to encode cytochromes P450 and 577 putative transcription factor genes. Data presented in this study will constitute an important resource for the scientific community that is interested in the molecular genetics and functional genomics of S. miltiorrhiza.