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.

Objective To study the chemical constituents in the leaves of Lindera aggregate.MethodsVarious column chromatographic techniques were used to separate and purify the chemical constituents and their structures were elucidated by spectral analyses.Results Nine compounds were isolated and identified as quercetin(Ⅰ),and kaempferol(Ⅱ),avicularin(Ⅲ),afzelin(Ⅳ),dihydrokaempferol(Ⅴ),astragaline(Ⅵ),kaempferol-3-O-?-D-xylopyranoside(Ⅶ),juglalin(Ⅷ),and kaempferol-3-O-(2″-O-?-D-glucopyranosyl)-?-L-rhamnopyranoside(Ⅸ).Conclusion Compounds Ⅲ—ⅠⅩ are isolated from the title plant for the first time.Compounds Ⅴ—Ⅸ are isolated from the plants of Lindera Thunb.for the first time.

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.

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.

DNA barcoding is a technique in which species identification and discovery are performed by using short and standard fragments of DNA sequences. In this study, eleven species of Paris, including seven varieties, were sampled. Five chloroplast sequences, psbA-trnH, rpoB, rpoC1, rbcL, matK, and one nuclear marker, the second internal transcribed spacer (ITS2) of ribosomal DNA, were amplified and sequenced. The PCR amplification and sequencing efficiency, intra- and inter-specific divergence and barcoding gap were used to evaluate different loci, and the identification efficiency was assessed using BLAST1 and Nearest Distance methods. The ITS2 sequences in the studied samples of Paris were amplified and sequenced successfully using primers designed by our group, while matK showed low level in the amplification and psbA-trnH was difficult for sequencing because of over 800 bp and poly (A) structure. Analysis of the intra- and inter-specific divergence and barcoding gap showed ITS2 was superior to other loci. The ITS2 showed a much higher percentage of success (100%) in identification than other five loci, none of which indicated more than 50% except matK (52.9%). The 2-locus combination of rbcL+matK didn't improve ability of authentication. In addition, the rate of successful identification with ITS2 kept 100% when the samples were expanded to 67 samples of 29 species. In conclusion, ITS2 can be used to correctly identify medicinal plants of Paris, and it will be a potential DNA barcode for identifying medicinal plants of other taxa.

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.

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 identify the original plant of Daturae Flos from its adulterants by DNA barcoding, the sequences of ITS2, psbA-trnH, matK, rbcL of four species including Datura metel, Darura innoxia, Darura stramonium and Brugmansia arborea were compared and analyzed. The PCR and sequencing success rate of the four regions (ITS2, psbA-trnH, matK, rbcL) was 100%, 90%, 100% and 85%, respectively. Sequences were assembled with CodonCode Aligner. K2P distances were calculated and NJ tree was performed by MEGA 4.1. Thirty SNPs were found among ITS2 sequences, and 33 insert/deletes were found among psbA-trnH intergenic regions. The interspecific K2P distance of ITS2 and psbA-trnH was obviously higher than that of the intraspecific one. As to matK and rbcL, there was no "Barcoding Gap" existing between inter- and intra-specific distances. The NJ trees of the four regions/combinations were built separately. Samples of Brugmansia arborea were clustered into one clade, and the other species of Datura L. formed another clade. The results showed that either ITS2 or psbA-trnH was useful to identify Daturae Flos from its adulterants.

ObjectiveTo investigate the clinical features of the abnormal chromosome karyotypes in twin pregnancies complicated with fetal malformations. Methods Totally 181 twin pregnancies (362 fetuses) in which one or two fetuses had abnormalities diagnosed by ultrasound were referred to the First Affiliated Hospital of Sun Yat-Sen University from January, 2000 to September, 2010. They were divided into different groups according to ( 1 ) maternal age: the cases with maternal age ≥35 were divided into advanced pregnancy group ( 105 fetuses) , and those with maternal age ＜35 were divided into young pregnancy group (203 fetuses) ; ( 2 ) conceived method : those conceived by assisted reproductive technology were divided into assisted reproductive group (81 fetuses), and the natural conception pregnancies were divided into natural conception group (227 fetuses) ; ( 3 ) chorionicity: the monochorionic twin (MCT) pregnancies were divided into MCT group( 123 fetuses), and the dichorionic twin (DCT) pregnancies were divided into DCT group( 185 fetuses); (4) structural abnormalities: 205 fetuses with structural abnormalities were divided into the abnormal fetal group, and 103 fetuses without structural abnormalities were divided into the normal fetal group. All fetuses were examined by the ultrasound and chromosomes were examined in 308 fetuses. Results( 1 ) The karyotype of fetuses: among 181 twin pregnancies, 23 cases had chromosomal abnormalities in 1 or 2 fetuses ( 12. 7％ ,23/181 ), and chromosomes were exarmined in both fetuses in 20 of 23 cases. Twenty-six of 308 fetuses were found with abnormal chromosomes ( 8.4％, 26/308 ) , and the aneuploid was the most common type of abnormal karyotypes ( 53.8％ , 14/26 ). Twenty-one of 205 fetuses with malformations were found with abnormal karyotypes (10. 2％, 21/205 ). (2) Seven of 123 fetuses in MCT group were with abnormal karyotypes (5.7％, 7/123), and 19 of 185 fetuses in DCT group were with abnormal karyotypes ( 10. 3％, 19/185 ). There was no statistical difference of abnormal chromosome incidence between the two groups. There were 14 fetuses with aneuploid in DCT group ( 7.6％, 14/185 ) ;but there was no fetus with aneuploid in MCT group. There was statistical difference between these two groups. In two cases of DCT group, only one fetus with malformation received chromosome examination because another fetus was dead, and the karyotypes were trisomy 21 and trisomy 18 respectively. Both fetuses of the rest 17 cases received chromosome examination, and the chromosomes of both fetuses in each pregnancy were different. Fifteen of 19 fetuses with abnormal chromosomes in DCT group were complicated with structural abnormalities, and 7 fetuses of 4 twin pregnancies in MCT group were with chromosomal abnormalities. (3) The comparison of the abnormal karyotype incidence between the advanced pregnancy group and young pregnancy group: the abnormal karyotype incidence of the advanced pregnancy group was 7. 6％ (8/105), and that was 8.9％ (18/203) in young pregnancy group. There was no statistical difference between the two groups ( P ＞ 0. 05 ). Six of 105 fetuses in advanced pregnancy group were aneuploids (5. 7％, 6/105), and 8 of 203 fetuses in young pregnancy group were aneuploids (3.9％, 8/203). The aneuploid incidence in advanced pregnancy group was significantly higher than that in young pregnancy group ( P ＜ 0. 05 ). (4) The comparison of the abnormal karyotype incidence between the assisted reproductive group and the natural conception group: 11 of 81 fetuses were with the abnormal karyotypes in assisted reproductive group ( 13. 6％, 11/81 ), and 15 of 227 fetuses were with the abnormal karyotypes in assisted reproductive group (6. 6％, 15/227). There was statistical difference between the two groups ( P ＜0. 05). There were 7 fetuses with the aneuploid in assisted reproductive group ( 8. 6％, 7/81 ) and 7 fetuses with the aneuploid in natural conception group ( 3. 1％, 7/227 ), which showed no statistical difference ( P ＞0. 05 ).(5) The comparison of the abnormal karyotype incidence between the abnormal fetal group and normal fetal group: 21 of 205 fetuses in abnormal fetal group were with abnormal karyotypes (10. 2％, 21/205), and 5 of 103 fetuses in normal fetal group were with abnormal karyotypes ( 4. 9％, 5/103 ) . There was no statistical difference (P ＞ 0. 05 ). 13 fetuses in abnormal fetal group were with the aneuploid (6. 3％, 1 3/205), and only one fetus in normal fetal group was aneuploid (1.0％, 1/103 ). There was statistical difference between the two groups ( P ＜ 0. 05 ). ConclusionsAneuploid is the most common abnormal karyotype in twin pregnancy complicated with fetal abnormalities, especially trisomy 21. Aneuploid mainly occurs in only one fetus of DCT, and chromosomal discordance is usually found in DCT. While in MCT, the twin fetuses with the same abnormal karyotype may have different pbenotypes. The results suggest that it is necessary to analyze both karyotypes of twins even if only one fetus is complicated with structural abnormalities.

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.