Cloning and expression analysis of superoxide dismutase SmMSD2 gene from Salvia miltiorrhiza / 药学学报

Superoxide dismutase (SOD) is a key enzyme that scavenge superoxide anion free radical (O2·-) in vivo, and plays an important role in plant growth and development and stress. In this study, according to the genome and transcriptome data of Salvia miltiorrhizae, 9 SOD genes were identified and the expression patterns of SOD family genes were further analyzed, including 5 Cu/Zn-SOD, 2 Fe-SOD and 2 Mn-SOD. On the basis of proteomic analysis, combined with transcriptome data, one full-length cDNA of Mn-SOD gene, namely SmMSD2 was cloned from Salvia miltiorrhizae. The results of amino acid sequence alignment and phylogenetic analysis showed that SmMSD2 protein belongs to the manganese superoxide dismutase (Mn-SOD) subfamily, and SmMSD2 protein shares high sequence identity with the Mn-SOD proteins of various plants that all contain a C-terminal conserved metal-binding domain "DVWEHAYY". The prokaryotic expression vector pMAL-c2X-SmMSD2 was constructed and transformed into E. coli BL21 expressing strain, and the target recombinant protein was successfully induced and its enzymatic properties were analyzed. Spatiotemporal expression analysis showed that SmMSD2 gene was expressed in all tissues, indicating that SmMSD2 gene was constitutively expressed at a stable level. Real-time quantitative PCR indicated that drought (15% PEG6000), abscisic acid (ABA) and indole-3-acetic acid (IAA) could induce the expression of SmMSD2 gene, suggesting that SmMSD2 may be involved in the response of Salvia miltiorrhizae to abiotic stress such as drought, as well as the signaling pathways of phytohormone ABA and IAA. These results lay the foundation for further elucidating the involvement of superoxide dismutase in the stress response and accumulation of active components of Salvia miltiorrhiza.

The UGT74L2 of Andrographis paniculata catalyzes phloretin to produce trilobatin and its enzymatic study / 药学学报

The last essential enzyme in the biosynthetic pathway of trilobatin, phloretin-4'-O glycosyltransferase (P4'-OGT), catalyzes the conversion of trilobatin to phloretin in vitro. However, only a few P4'-OGTs have been found in plants. This study used Malus domestica phloretin-4'-O glycosyltransferase (MdPh-4'-OGT) as a query to identify and clone two UDP-glucuronosyltransferase (UGT) genes, designated UGT74L2 and UGT74L3, from the transcriptome of Andrographis paniculata. According to a phylogenetic tree analysis, UGT74L2 and UGT74L3 belonged to the UGT74 family, which has been linked to several activities in other species. The in vitro enzymatic reaction demonstrated that UGT74L2 could particularly catalyze the formation of trilobatin from phloretin, but UGT74L3 had no effects. By using Ni-NTA affinity chromatography to extract the soluble UGT74L2 recombinant protein, the enzymatic kinetics of the activity was investigated using phloretin as the substrate. The results showed that the optimal temperature and pH for UGT74L2 enzymatic reaction were 40 ℃ and 8.0 (Tris-HCl system), respectively. Three metal ions (Ca2+, Mn2+ and Co2+) showed inhibitory effect on the activity of UGT74L2, while Mg2+ could improve the activity of UGT74L2. Other tested metal ions have no significant effect on UGT74L2. The results of enzymatic kinetic parameters that the Km value was 29.84 μmol·L-1, the kcat was 0.02 s-1, and the kcat·Km-1 was 572.6 mol-1·s-1. By homology modeling, molecular docking and mutation experiments, we found that multiple amino acids residues around the substrate binding pocket play quite an important role during catalytic process, In summary, we identified a novel P4'-OGT gene from medicinal plant Andrographis paniculata and provided a new efficient catalyst to synthesize trilobatin. Meanwhile, this study provides a reference for mining new efficient glycosylation modules from plants.

Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province / 中国中药杂志

This study investigated the choroplast genome sequence of wild Atractylodes lancea from Yuexi in Anhui province by high-throughput sequencing, followed by characterization of the genome structure, which laid a foundation for the species identification, analysis of genetic diversity, and resource conservation of A. lancea. To be specific, the total genomic DNA was extracted from the leaves of A. lancea with the improved CTAB method. The chloroplast genome of A. lancea was sequenced by the high-throughput sequencing technology, followed by assembling by metaSPAdes and annotation by CPGAVAS2. Bioiformatics methods were employed for the analysis of simple sequence repeats(SSRs), inverted repeat(IR) border, codon bias, and phylogeny. The results showed that the whole chloroplast genome of A. lancea was 153 178 bp, with an 84 226 bp large single copy(LSC) and a 18 658 bp small single copy(SSC) separated by a pair of IRs(25 147 bp). The genome had the GC content of 37.7% and 124 genes: 87 protein-coding genes, 8 rRNA genes, and 29 tRNA genes. It had 26 287 codons and encoded 20 amino acids. Phylogenetic analysis showed that Atractylodes species clustered into one clade and that A. lancea had close genetic relationship with A. koreana. This study established a method for sequencing the chloroplast genome of A. lancea and enriched the genetic resources of Compositae. The findings are expected to lay a foundation for species identification, analysis of genetic diversity, and resource conservation of A. lancea.

Prokaryotic expression, subcellular localization and enzymatic activity analysis of DXS gene from Platycodon grandiflorum / 药学学报

1-Deoxy-D-xylulose-5-phosphate synthase (DXS), the first key enzyme in 2-methyl-D-erythritol-4-phosphate (MEP) pathway, catalyzes the condensation of glyceraldehyde-3-phosphate with pyruvate to 1-deoxy-xylose-5-phosphate (DXP). In this study, PgDXS1, PgDXS2, and PgDXS3 genes were cloned from the root of Platycodon grandiflorum (P. grandiflorum). The open reading frame (ORF) of PgDXS1, PgDXS2, and PgDXS3 were 2 160, 2 208, and 2 151 bp in full length, encoding 719, 735, and 716 amino acids, respectively. Homologous alignment results showed a high identity of PgDXSs with DXS in Hevea brasiliensis, Datura stramonium and Stevia rebaudiana. The recombinant expression plasmids of pET-28a-PgDXSs were constructed and transformed into Escherichia coli (E. coli) BL21 (DE3) cells, and the induced proteins were successfully expressed. Subcellular localization results showed that PgDXS1 and PgDXS2 were mainly located in chloroplasts, and PgDXS3 was located in chloroplasts, nucleus and cytoplasm. The expression of three DXS genes in different tissues of two producing areas of P. grandiflorum were assayed via real-time fluorescence quantitative PCR, and the results showed that all of them were highly expressed in leaves of P. grandiflorum from Taihe. Under methyl jasmonate (MeJA) treatment, the expression levels of three PgDXS genes showed a trend of first decreasing and then increasing at different time points (3 - 48 h), and the activity of DXS showed a trend of first increasing and then decreasing in three tissues of P. grandiflorum. This study provides a reference for further elucidating the biological function of PgDXS in terpenoid synthesis pathway in P. grandiflorum.

Cloning and prokaryotic expression analysis of AlCMK from Atractylodes lancea / 药学学报

4-(Cytidine 5′-diphospho)-2-C-methyl-D-erythritol kinase (CMK) was one of the key enzymes in the methylerythritol-4-phosphate (MEP) pathway to generate terpenoids. In this study, based on the transcriptome data of Atractylodes lancea, the sequence of the CMK gene was cloned, named AlCMK (GenBank accession number OM283293). The results showed that AlCMK contains a 1 230 bp open reading frame (ORF) encoding 409 amino acids. The deduced protein had a theoretical molecular weight of 44 752.53 and an isoelectric point of 6.67. Transmembrane structure analysis showed that there was no transmembrane structure, and the secondary structure of AlCMK was predicted to be mainly composed of random coil. Homologous alignment revealed that AlCMK shared high sequence identity with the CMK proteins of Tanacetum cinerariifolium, Osmanthus fragrans, Eucommia ulmoides, Lonicera japonica and Salvia miltiorrhiza. Phylogenetic analysis indicated that AlCMK protein had the higher homology with CMK protein of Compositae. The pET-32a-AlCMK prokaryotic expression vector was constructed and a fusion protein with molecular mass of about 65 kDa was expressed in the E. coli BL21 (DE3). The qRT-PCR was used to analyze the expression pattern of AlCMK gene in different tissues and after MeJA treatment. Meanwhile, the enzyme activity was determined by ELISA kit. The results showed that AlCMK gene was tissue-expressed in different origins and its expression was induced by MeJA, and the results of the enzyme activity assay showed that the AlCMK enzyme activity in different regions was higher in the leaves. The subcellular localization showed that AlCMK was located in the chloroplast. This study provides a reference for further elucidating the biological function of AlCMK gene in terpenoid synthesis pathway in Atractylodes lancea.

Clinical characteristics and genetic analysis of a neonate with Smith-Magenis syndrome / 中华医学遗传学杂志

OBJECTIVE@#To explore the clinical features and genetic etiology for a neonate with Smith-Magenis syndrome (SMS).@*METHODS@#Copy number variation sequencing (CNV-seq) was applied to the neonate and his parents, and the genotype-phenotype correlation was analyzed.@*RESULTS@#On the second day after birth, the neonate had presented with pathological jaundice and immunodeficiency. Cranial MRI revealed ventricular enlargement and enlargement of cisterna magna. At 3 months, the infant has presented with square face, prominent forehead, deep-set eyes, hypertelorism, palpebral fissure upward and button noses. Genetic testing showed that he had carried a 2.9 Mb deletion in 17p11.2 region, seq[GRCh37] del(17)(p11.2)(chr17:16 836 379-19 880 992). The same deletion was not found in either parent.@*CONCLUSION@#SMS is mostly diagnosed in child and adulthood, but rarely in neonates. For neonates with SMS, the neurological and behavioral abnormalities have not been shown, but pathological jaundice, CNS abnormalities and immune deficiency may be the characteristics, which require attention of neonatal physicians.

Molecular cloning and functional characterization of an isoflavone glucosyltransferase from Pueraria thomsonii / 中国天然药物

Pueraria thomsonii has long been used in traditional Chinese medicine. Isoflavonoids are the principle pharmacologically active components, which are primarily observed as glycosyl-conjugates and accumulate in P. thomsonii roots. However, the molecular mechanisms underlying the glycosylation processes in (iso)flavonoid biosynthesis have not been thoroughly elucidated. In the current study, an O-glucosyltransferase (PtUGT8) was identified in the medicinal plant P. thomsonii from RNA-seq database. Biochemical assays of the recombinant PtUGT8 showed that it was able to glycosylate chalcone (isoliquiritigenin) at the 4-OH position and glycosylate isoflavones (daidzein, formononetin, and genistein) at the 7-OH or 4'-OH position, exhibiting no enzyme activity to flavonones (liquiritigenin and narigenin) in vitro. The identification of PtUGT8 may provide a useful enzyme catalyst for efficient biotransformation of isoflavones and other natural products for food or pharmacological applications.

Different development phase of transcriptome analysis from Crataegus pinnatifida Bge. and cloning, structure and function prediction of squalene epoxidase involved in triterpenic acid biosynthesis / 药学学报

italic>Crataegus pinnatifida is a traditional Chinese medicine, which contains organic acids, triterpenoid acids and other active components, has important medicinal and edible value. In order to study the difference of gene expression level in different developmental stages of hawthorn and explore the genes of active ingredient biosynthesis in Crataegus pinnatifida, high-throughput Illumina HiSeq 2000 technology were used to conduct transcriptome sequencing and bioinformatics analysis on Crataegus pinnatifida fruits from the same origin at different developmental stages. 78 496 Unigenes with an average length of 941 nt were obtained by Trinity software. Among them, 58 395 Unigenes can be annotated by NR, NT, Swiss prot, KEGG, COG, GO and other public databases. KEGG pathway analysis showed that 52 Unigenes encoding 15 key enzymes involved in the citric acid cycle. There are 62 Unigenes were involved in the triterpene biosynthesis pathway of Crataegus pinnatifida. Two key enzymes SQE of triterpenoid metabolism pathway in Crataegus pinnatifida were cloned and performed bioinformatic analysis. The results showed that ORF of CpSQE1 and CpSQE2 were 1 594 bp and 1 597 bp, respectively, encoding 530 and 531 amino acids. The molecular weight of proteins was 57.6 kDa and 57.5 kDa. Bioinformatics analysis showed that both CpSQE1 and CpSQE2 proteins have a PLN02985 superfamily conserved domain, belonging to the squalene monooxygenase superfamily. The phylogenetic tree shows that CpSQE1 and CpSQE2 are clustered together with SQE with squalene epoxidase function in other plants. This study provides a research basis for further exploring the key genes in the biosynthesis process of hawthorn active ingredients and analyzing the regulation pathway of its active ingredient biosynthesis.

Cloning and prokaryotic expression of 3-ketoacyl-CoA thiolase gene AIKAT from Atractylodes lancea / 中国中药杂志

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid β-oxidation pathway of Atractylodes lancea was cloned. Meanwhile, bioinformatics analysis, prokaryotic expression and gene expression analysis were carried out, which laid a foundation for the study of fatty acid β-oxidation mechanism of A. lancea. The full-length sequence of the gene was cloned by RT-PCR with the specific primers designed according to the sequence information of KAT gene in the transcriptomic data of A. lancea and designated as AIKAT(GenBank accession number MW665111). The results showed that the open reading frame(ORF) of AIKAT was 1 323 bp, encoding 440 amino acid. The deduced protein had a theoretical molecular weight of 46 344.36 and an isoelectric point of 8.92. AIKAT was predicted to be a stable alkaline protein without transmembrane segment. The secondary structure of AIKAT was predicted to be mainly composed of α-helix. The tertiary structure of AIKAT protein was predicted by homology modeling method. Homologous alignment revealed that AIKAT shared high sequence identity with the KAT proteins(AaKAT2, CcKAT2, RgKAT and AtKAT, respectively) of Artemisia annua, Cynara cardunculus var. scolymus, Rehmannia glutinosa and Arabidopsis thaliana. The phylogenetic analysis showed that AIKAT clustered with CcKAT2, confirming the homology of 3-ketoacyl-CoA thiolase genes in Compositae. The prokaryotic expression vector pET-32 a-AIKAT was constructed and transformed into Escherichia coli BL21(DE3) for protein expression. The target protein was successfully expressed as a soluble protein of about 64 kDa. A real-time quantitative PCR analysis was performed to profile the AIKAT expression in different tissues of A. lancea. The results demonstrated that the expression level of AIKAT was the highest in rhizome, followed by that in leaves and stems. In this study, the full-length cDNA of AIKAT was cloned and expressed in E. coli BL21(DE3), and qRT-PCR showed the differential expression of this gene in different tissues, which laid a foundation for further research on the molecular mechanism of fatty acid β-oxidation in A. lancea.

Identification of Corydalis yanhusuo,C. turtschaninovii,C. decumbens by allele-specific PCR / 中国中药杂志

To establish a DNA molecular markers method for identification of Corydalis yanhusuo,C. turtschaninovii and C. decumbens,the mat K,trn G and psb A-trn H sequences of 56 samples from 14 species of C. yanhusuo,C. turtschaninovii,C. decumbens and their related species were obtained by sequencing. The SNP loci were obtained by Bio Edit 7. 2. 2 software. The primers for AS-PCR identification were designed based on the mutation sites,and the conditions of PCR were optimized to identify C. yanhusuo,C. turtschaninovii,and C. decumbens according to the specific bands. The results showed that the amount of template( 0. 6-1 200 ng)and annealing temperature( 42-60 ℃) had little influence on the amplification results,and the number of cycles had much influence on the amplification results. When the number of cycles was 20,the specific bands of 297 bp( mat K),353 bp( trn G) and 544 bp( mat K) were amplified from C. yanhusuo,C. turtschaninovii and C. decumbens,respectively. The method established in this study had a minimum detection limit of 6 ng for C. yanhusuo,60 ng for C. decumbens and less than 0. 6 ng for C. turtschaninovii. Thus,the allelespecific PCR method established in the research can specifically identify C. yanhusuo,C. turtschaninovii,and C. decumbens.

HBsAg loss with Pegylated-interferon alfa-2a in hepatitis B patients with partial response to nucleos(t)-ide analog: new switch study / 中华肝脏病杂志

Objective@#Hepatitis B surface antigen (HBsAg) loss is seldom achieved with nucleos(t)ide analog (NA) therapy in chronic hepatitis B patients but may be enhanced by switching to finite pegylated-interferon (Peg-IFN) alfa-2a. We assessed HBsAg loss with 48- and 96-week Peg-IFN alfa-2a in chronic hepatitis B patients with partial response to a previous NA.@*Methods@#Hepatitis B e antigen (HBeAg)-positive patients who achieved HBeAg loss and hepatitis B virus DNA < 200 IU/mL with previous adefovir, lamivudine or entecavir treatment were randomized 1:1 to receive Peg-IFN alfa-2a for 48 (n = 153) or 96 weeks (n = 150). The primary endpoint of this study was HBsAg loss at end of treatment. The ClinicalTrials.gov identifier is NCT01464281.@*Results@#At the end of 48 and 96 weeks' treatment, 14.4% (22/153) and 20.7% (31/150) of patients, respectively, who switched from NA to Peg-IFN alfa-2a cleared HBsAg. Rates were similar irrespective of prior NA or baseline HBeAg seroconversion. Among those who cleared HBsAg by the end of 48 and 96 weeks' treatment, 77.8% (14/18) and 71.4% (20/28), respectively, sustained HBsAg loss for a further 48 weeks. Baseline HBsAg < 1 500 IU/mL and week 24 HBsAg < 200 IU/mL were associated with the highest rates of HBsAg loss at the end of both 48- and 96-week treatment (51.4% and 58.7%, respectively). Importantly, extending treatment from 48 to 96 weeks enabled 48.3% (14/29) more patients to achieve HBsAg loss.@*Conclusion@#Patients on long-term NA who are unlikely to meet therapeutic goals can achieve high rates of HBsAg loss by switching to Peg-IFN alfa-2a. HBsAg loss rates may be improved for some patients by extending treatment from 48 to 96 weeks, although the differences in our study cohort were not statistically significant. Baseline and on-treatment HBsAg may predict HBsAg loss with Peg-IFN alfa-2a.

Species and medical history of "Xishuang" medicinal materials / 中国中药杂志

"Xishuang" is a special phenomenon that chemical composition of medicinal materials crystallize on the surface exposed to air for a long time. We summarized Herbal textual research of "Xishuang" phenomenon of six herbs, such as Schisandrae Chinensis Fructus, Moutan Cortex, Atractylodis Rhizoma, Magnoliae Officinalis Cortex, dried persimmon frost and watermelon frost. From historical perspective, cream of Schisandrae Chinensis Fructus was firstly discovered in Lei Gong's Moxibustion Theory. Thereafter, dried persimmon frost was found in Song Dynasty, which was named "white persimmon" in Ben Cao Tu Jing and had become an independent medicine in Compendium of Materia Medica. Then, watermelon frost was found in Yang Yi Da Quan of the Qing Dynasty, and Moutan Cortex's "sand star" was recorded in Zeng Ding Wei Yao Tiao Bian of the Republic of China. After that, "Xishuang" phenomenon of Atractylodis Rhizomaand Magnoliae Officinalis Cortex were reported in 1950s and 1960s in succession. The pattern of "Xishuang" is divided into different type, natural "Xishuang" includes Schisandrae Chinensis Fructus, Moutan Cortex, Atractylodis Rhizoma and Magnoliae Officinalis Cortex, artificial "Xishuang" includes watermelon frost, and dried persimmon frost formed crystals by using artificial intervention. The above 6 kinds of herbs have different crystal structure and chemical composition. Therefore, according to traditional identification experience, "Xishuang" phenomenon is related to varieties and quality of medicinal herbs. These research provide herbalism basis for the modern study of "Xishuang" medicinal materials.

Study on identification of Dendrobium officinale and related species by bidirectional PCR amplification of mismatched and specific alleles / 中国中药杂志

Based on rDNA ITS sequences of Dendrobium officinale and the other 69 species of Dendrobium, a pair of dismatched allele-specific diagnostic primers, TPSH-AS1F and TPSH-AS1R were designed to authenticate D. officinale from the other species. Thebidirectional PCR amplification were performed using the diagnostic primers with the total DNAs of the original plants or processing products as a template. When the annealing temperature was raised to 60 ℃, only the template DNA of D. officinale could be amplified whereas the diagnostic PCRs of the other Dendrobium species were all negative. Compared with the other authentification methods, the bidirectional PCR amplifications is not only simpler and time-saving but practical and effective.

Growth rings in roots of seven Sect. Paeonia species and its application on identification of Paeoniae Radix Rubra / 中国中药杂志

The growth years of medicinal materials are closely related to their quality, and "Herb-chronology" has been used to determine the growth years of perennial dicotyledonous plants in recent years. On the basis of conventional paraffin section and freehand section, the anatomical study on roots of seven Sect. Paeonia species and main roots of cultivated Paeonia lactiflora was conducted in this paper. The results showed that, there existed some differences in microstructure of the seven species such as P. lactiflora, P. obovata, P. veitchii, P. mairei, P. anomala, P. sinjiangensis and P. anomala var. intermedia, and this could be used to distinguish different species. In the roots of seven Sect. Paeonia species, distinct growth rings were formed because that the different diameters or density of xylem vessels in the secondary xylem formed clusters and arranged interrupted rings in tangential direction. There were growth rings in the main roots of P. lactiflora cultivated 1-4 years in Siping, Jilin, which were all consistent with their growth years. Due to the similar growth characteristics between wild Sect. Paeonia species and cultivated P. lactiflora, the growth rings can provide a basis for the age identification and lay the foundation for the quality evaluation of Paeoniae Radix Rubra.

Variety and distribution of Anhui native medicinal materials in ancient Chinese materia medica / 中国中药杂志

Anhui is located in the middle and lower reaches of the Yangtze River Plain, its across warm temperate zone and subtropics. The mountain and water next to each other, which leads to Chinese medicine resources ranked first in East China. The utilization of traditional Chinese medicine resources in Anhui has a long history, which could date back to the publishing time of Ming Yi Bie Lu (Appendant Records of Famous Physicians). And the kinds of traditional Chinese medicine in Song Dynasty ups to 80. There are also some differences in the distribution of various geographical units in terms of the types: Jianghuai hilly region's ups to 64, 25 in Wannan mountainous area, the species in Dabie Mountains and Huaibei plain are 16 and 14 respectively. In addition, the Jianghuai hilly region's and Wannan mountainous area have a long history among of them, which have been reached a peak in the Song Dynasty. The history of native medicinal materials in Anhui recorded in different periods, though combing herbal books. And the results showed that the vast majority of varieties in ancient are the same as modern ones, which provide the historical basis for the rich bulk medicinal materials in Anhui. The distinctions in natural and social environment of different geographical units have effects on the history of the usage of Chinese medicine resources in respective regions. Thus, the variety and distribution of native medicinal materials in Anhui among the Bencao works of different period provides herbalism basis for the protection and utilization of Chinese medicine resources currently.

Evolution and characteristics of system,assessing quality by distinguishing features of traditional Chinese medicinal materials, of Dao-di herbs of Astragali Radix / 中国中药杂志

"Assessing the quality by distinguishing features of traditional Chinese medicinal materials" is a characteristic quality evaluation system of traditional Chinese medicine, and it is also the basis of "Rating according to characters and setting the price by the grade" on the market. Astragali Radix was regarded as a famous traditional Chinese medicine (TCM), and this paper has carried out herbal textual research on the development and formation of the concept, "assessing the quality by distinguishing features of traditional Chinese medicinal materials", of Astragali Radix. The authentic medicine producing areas of Astragalus in China have experienced a great change, Gansu , Sichuan and adjacent areas before the Tang Dynasty; Shanxi during the Tang and Song Dynasty. The concept, "assessing the quality by distinguishing features of traditional Chinese medicinal materials", of Astragali Radix was formed in the Song and Ming Dynasty and still used today, which described as that the shape is "straight as an arrow"; the texture is "soft as cotton"; the section looks like" gold well and jade hurdle"; it was sweet in taste and has beany flavor. The system, "assessing the quality by distinguishing features of traditional Chinese medicinal materials", of Astragali Radix has undergone the adjustments from "true or false" to "good or bad", advance with the times, pick out the advantages from others and absorb the experience of traditional identification actively. Besides, it always returns to laconism from erudition and was summarized highly. Assessing the quality by distinguishing features of traditional Chinese medicinal materials and commodity specifications have the same root, so the former has reference meaning to revise the latter.

Screening target genes for bimolecular marking methods of Magnolia quality / 中国中药杂志

The study used use bimolecular marking methods to evaluate the lignans of Magnolia officinalis and M. officinalis var. biloba. First, we compare the chemical constituents between M. officinalis and M. officinalis var.biloba. There were significant differences in concentration of magnolignan I between leaves of these two varieties. Then we further select the p-hydroxyphenyl lignin to mining the key enzyme genes of biosynthesis from Magnolia transcriptome, and screened an encoding cinnamyl alcohol dehydrogease gene as the candidate marker of bimolecular marking methods of Magnolia quality by comparing of the expression level and structure variation in homologous gene between M. officinalis and M. officinalis var.biloba. The established method provides the technical support for bimolecular marking methods of Magnolia quality evaluation.

Cloning and prokaryotic expression analysis of DNA methyltransferas 1(MET1) in Ganoderma lucidum / 中国中药杂志

DNA methyltransferase is the key enzyme in the process of DNA methylation, playing an important role in regulation of gene expression in vivo. According to the Ganoderma lucidum transcriptome data, a full-length cDNA sequence of MET1 from G. lucidum was cloned for the first time, the GenBank registration number is KU239998, and we conducted a comprehensive bioinformatics analysis of the genetic characteristics and spatial structure. The prokaryotic expression analysis showed that E.coli[pET28a(+)-GlMET1] in BL21(DE3) could induce objective protein, shaking the culture at 16 ℃ until the host bacterium(A₆₀₀) was approximately 0.8, and added IPTG to finally concentration of 0.2 mmol•L⁻¹, and then the optimal expression of GlMET1 recombinant protein was accumulated for the induction time of 20 h. The real-time PCR results showed that the expression levels of GlMET1 had obvious differences among varieties of G. lucidum. During the maturity stage, the expression levels of GlMET1 were lower than that in juvenile stage, the results showed that with the growth of G. lucidum, the expression levels of GlMET1 were on the decline. The research provided an important basis for studying the mechanism of DNA methyltransferase thoroughly.

Research of regulating synthesis of luteolin and luteoloside of Lonicera japonica by LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1 treated with 5-azaC / 中国中药杂志

This study is aimed to explore the mechanism of catalyzing the synthesis of luteolin and luteoloside by LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1.The leaves of Lonicera japonica were treated with different concentrations of 5-azaC(20,40,60,80,100 μmol•L-1) for three periods（1,2,3 d）. Firstly, we cloned LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1. Secondly, we analyzed the expression levels of LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1 by Real-Time PCR and the contents of luteolin and luteoloside determined by UPLC-MS/MS. The results explained the expression levels of LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1 consistent with the content variation of luteolin in general, but there was no significant correlation with the contents of luteoloside. And we found the expression levels of LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1 were slightly different. The research indicated that the contents of luteolin and luteoloside got higher by improving the expression levels of LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1. This will provide technical support and lay a theoretical foundation for regulating the synthesis of luteolin and luteoloside by LjFNS Ⅱ 1.1 and LjFNS Ⅱ 2.1.

Identification of 1-deoxy-D-xylulose-5-phosphate synthase and analysis on essential oil in Magnolia officinalis / 中草药

Objective: Based on the data of transcriptome sequencing of Magnolia officinalis, MoDXS1 and MoDXS2 genes were completed in detail by using bioinformatics methods. Methods: MoDXS1 and MoDXS2 genes were analyzed and predicted by the tools of bioinformatics in the following aspects: physical and chemical characteristics of amino acid sequences, function domain, hyophobicity or hydrophilicity, secondary structure and tertiary structure of protein, molecular phylogenetic evolution, and so on; The expression levels of MoDXS1 and MoDXS2 were identified by real-time PCR. Results: ORF Finder indicated that MoDXS1 and MoDXS2 genes were full-length, and they all were unstable hydrophobic proteins; Structural domain of MoDXS1 and MoDXS2 showed high homology with other plants; The secondary structures all were hybrid architecture, and alpha helixes were the major motifs, tertiary structure of protein was predicted by Homology modeling; Sequence alignment that MoDXS family had relative close relationship to the DXS of Nicotiana tabacum, Salvia miltiorrhiza, and Arabidopsis thaliana; The results of evolutionary relationship analysis showed that MoDXS1 had relative close relationship to angiosperm, but MoDXS2 was clustered in a clade solely; The expression levels of DXS1 in M. officinalis and M. officinalis var. biloba were not significantly different, but the expression level of DXS2 in M. officinalis var. biloba was higher than that in M. officinalis. By applying the technique of GC-MS, the contents of major volatile components β-caryophyllene, β-caryophyllene oxide, and β-eudesmol in M. officinalis var. biloba are higher than those in M. officinalis. Conclusion: The results provide theoretical reference for studies on secondary metabolic regulation in terpenoid of M. officinalis.