Identification and functional characterization of two trans-isopentenyl diphosphate synthases and one squalene synthase involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Two trans-isopentenyl diphosphate synthase and one squalene synthase genes were identified and proved to be involved in the triterpenoid biosynthesis in Platycodon grandiflorus. Platycodon grandiflorus is a commonly used traditional Chinese medicine. The main bioactive compounds of P. grandiflorus are triterpenoid saponins. The biosynthetic pathway of triterpenoid saponins in P. grandiflorus has been preliminarily explored. However, limited functional information on related genes has been reported. A total of three trans-isopentenyl diphosphate synthases (trans-IDSs) genes (PgFPPS, PgGGPPS1 and PgGGPPS2) and one squalene synthase (SQS) gene (PgSQS) in P. grandiflorus were screened and identified from transcriptome dataset. Subcellular localization of the proteins was defined based on the analysis of GFP-tagged. The activity of genes was verified in Escherichia coli, demonstrating that recombinant PgFPPS catalysed the production of farnesyl diphosphate. PgGGPPS1 produced geranylgeranyl diphosphate, whereas PgGGPPS2 did not exhibit catalytic activity. By structural identification of encoding genes, a transmembrane region was found at the C-terminus of the PgSQS gene, which produced an insoluble protein when expressed in E. coli but showed no apparent effect on the enzyme function. Furthermore, some triterpenoid saponin synthesis-related genes were discovered by combining the component content and the gene expression assays at the five growth stages of P. grandiflorus seedlings. The accumulation of active components in P. grandiflorus was closely associated with the expression level of genes related to the synthesis pathway.

Isolation, characterisation, and expression profiling of DXS and DXR genes in Atractylodes lancea.

1-Deoxy-d-xylulose-5-phosphate synthase and 1-deoxy-d-xylulose-5-phosphate reductoismerase are considered two key enzymes in the 2-C-methyl-d-erythritol-4-phosphate pathway of terpenoid biosynthesis and are related to the synthesis and accumulation of sesquiterpenoids. We cloned two DXS and DXR genes from Atractylodes lancea and analysed their expression in different tissues and in response to methyl jasmonate (MeJA). Subcellular localisation analysis revealed that the AlDXS and AlDXR1 proteins are located in the chloroplasts and cytoplasm, whereas AlDXR2 is only located in the chloroplasts. pET-AlDXS-28a and pGEX-AlDXR-4T-1 were expressed in Escherichia coli BL21(DE3) and BL21, respectively. Based on the abiotic stress analysis, the growth rate of the recombinant pGEX-AlDXR-4T-1 was higher than that of the control in HCl and NaOH. AlDXS exhibited the highest expression level in rhizomes of A. lancea from Hubei but was highest in leaves from Henan. In contrast, AlDXR showed maximum expression in the leaves of A. lancea from Hubei and Henan. Moreover, DXS and DXR gene expression, enzyme activities, and antioxidant enzyme activities oscillated in response to MeJA, with expression peaks appearing at different time points. Our findings indicated that the characterisation and function of AlDXS and AlDXR could be useful for further elucidating the functions of DXR and DXR genes in A. lancea.

[Complete chloroplast genome sequencing and phylogeny of wild Atractylodes lancea from Yuexi, Anhui province].

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.

Comparative analysis in different organs and tissue-specific metabolite profiling of Atractylodes lancea from four regions by GC-MS and laser microdissection.

Traditional Chinese medicine is made from the rhizome of Atractylodes lancea (Thunb.) DC. (Compositae), known as Cangzhu. In this study, gas chromatography-mass spectrometry was used to identify and quantify the volatile oils of different organs of A. lancea from four regions of China: Jiangsu, Anhui, Henan, and Hubei provinces. The volatile oils of A. lancea were qualitatively and quantitatively characterized using gas chromatography-mass spectrometry combined with laser microdissection. The results identified 21 components in A. lancea, the majority of the components were found in the rhizomes, followed by the fibrous roots, flowers, leaves, and stems. According to the contents of volatile oils in A. lancea, it was divided into Dabieshan (mainly includes hinesol and ß-eudesmol) and Maoshan types (mainly includes atractylon and atractylodin), and the ratios of hinesol:ß-eudesmol:atractylon:atractylodin were 17.06:4.55:0:1, 12.66:11.71:0.99:1, 7.43:6.23:0:1, and 0.13:0.16:1.52:1 in A. lancea from AH, HN, HB, and JS, respectively. Tissue-specific study indicated that Dabieshan type mainly includes elemol, hinesol, and ß-eudesmol in the periderm and secretory cavities of A. lancea, whereas Maoshan type mainly includes atractylon, atractylodin, little hinesol, and ß-eudesmol in the secretory cavities. Conversely, no volatile oils were detected in the cortex, phloem, xylem, vascular ray, or pith. This study provides a foundation for further evaluation and utilization of A. lancea.

Transcriptome analysis identifies putative genes involved in triterpenoid biosynthesis in Platycodon grandiflorus.

MAIN CONCLUSION: Comprehensive transcriptome analysis of different Platycodon grandiflorus tissues discovered genes related to triterpenoid saponin biosynthesis. Platycodon grandiflorus (Jacq.) A. DC. (P. grandiflorus), a traditional Chinese medicine, contains considerable triterpenoid saponins with broad pharmacological activities. Triterpenoid saponins are the major components of P. grandiflorus. Here, single-molecule real-time and next-generation sequencing technologies were combined to comprehensively analyse the transcriptome and identify genes involved in triterpenoid saponin biosynthesis in P. grandiflorus. We quantified four saponins in P. grandiflorus and found that their total content was highest in the roots and lowest in the stems and leaves. A total of 173,354 non-redundant transcripts were generated from the PacBio platform, and three full-length transcripts of ß-amyrin synthase, the key synthase of ß-amyrin, were identified. A total of 132,610 clean reads obtained from the DNBSEQ platform were utilised to explore key genes related to the triterpenoid saponin biosynthetic pathway in P. grandiflorus, and 96 differentially expressed genes were selected as candidates. The expression levels of these genes were verified by quantitative real-time PCR. Our reliable transcriptome data provide valuable information on the related biosynthesis pathway and may provide insights into the molecular mechanisms of triterpenoid saponin biosynthesis in P. grandiflorus.

Molecular cloning and functional characterization of two squalene synthase genes in Atractylodes lancea.

MAIN CONCLUSION: Two squalene synthase genes AlSQS1 and AlSQS2 were isolated from Atractylodes lancea and functionally characterized using in vitro enzymatic reactions. Atractylodes lancea is a traditional herb used for the treatment of rheumatic diseases, gastric disorders, and influenza. Its major active ingredients include sesquiterpenoids and triterpenes. Squalene synthase (SQS; EC 2.5.1.21) catalyzes the first enzymatic step in the central isoprenoid pathway towards sterol and triterpenoid biosynthesis. In this study, we aimed to investigate two SQSs from A. lancea using cloning and in vitro enzymatic characterization. Bioinformatics and phylogenetic analyses revealed that the AlSQSs exhibited high homology with other plant SQSs. Furthermore, AlSQS1 was observed to be localized in both the nucleus and cytoplasm, whereas AlSQS2 was localized in the cytoplasm and endoplasmic reticulum. To obtain soluble recombinant enzymes, AlSQS1 and AlSQS2 were successfully expressed as glutathione S-transferase (GST)-tagged fusion proteins in Escherichia coli Transetta (DE3). Approximately 68 kDa recombinant proteins were obtained using GST-tag affinity chromatography and Western blot analysis. Results of the in vitro enzymatic reactions established that both AlSQS1 and AlSQS2 were functional, which verifies their catalytic ability in converting two farnesyl pyrophosphates to squalene. The expression patterns of AlSQS and selected terpenoid genes were also investigated in two A. lancea chemotypes using available RNA sequencing data. AlSQS1 and AlSQS2, which showed relatively similar expression in the three tissues, were more highly expressed in the stems than in the leaves and rhizomes. Methyl jasmonate (MeJA) was used as an elicitor to analyze the expression profiles of AlSQSs. The results of qRT-PCR analysis revealed that the gene expression of AlSQS1 and AlSQS2 plummeted at lowest value at 12 h and reached its peak at 24 h. This study is the first report on the cloning, characterization, and expression of SQSs in A. lancea. Therefore, our findings contribute novel insights that may be useful for future studies regarding terpenoid biosynthesis in A. lancea.

[Cloning and prokaryotic expression of 3-ketoacyl-CoA thiolase gene AIKAT from Atractylodes lancea].

In this study, the gene encoding the key enzyme 3-ketoacyl-CoA thiolase(KAT) in the fatty acid ß-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 ß-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 ß-oxidation in A. lancea.

Ginsenosides regulate adventitious root formation in Panax ginseng via a CLE45-WOX11 regulatory module.

Adventitious root branching is vital to plant growth and regeneration, but the regulation of this process remains unclear. We therefore investigated how ginsenosides regulate adventitious root branching in Panax ginseng. Cell proliferation and adventitious root branching were decreased in the presence of ginsenoside Rb1 and a high concentration of ginsenoside Re, but increased when treating with a low concentration of Re. Moreover, the exogenous application of a synthetic dodeca-amino acid peptide that has a CLAVATA3/EMBRYO SURROUNDING REGION-related (CLE) motif corresponding to PgCLE45 retarded root growth in both ginseng and Arabidopsis. The root Re levels and the expression of the DDS, CYP716A47, and CYP716A53 genes that encode enzymes involved in ginsenoside synthesis were decreased in the presence of PgCLE45. The expression profiles of PgWOX and PgCLE genes were determined to further investigate the CLE-WOX signaling pathway. The levels of PgWOX11 transcripts showed an inverse pattern to PgCLE45 transcripts. Using yeast one-hybrid assay, EMSA, and ChIP assay, we showed that PgWOX11 bound to the PgCLE45 promoter, which contained the HD motif. Transient expression assay showed that PgWOX11 induced the expression of PgCLE45 in adventitious roots, while PgCLE45 suppressed the expression of PgWOX11. These results suggest that there is a negative feedback regulation between PgCLE45 and PgWOX11. Taken together, these data show that ginsenosides regulate adventitious root branching via a novel PgCLE45-PgWOX11 regulatory loop, providing a potential mechanism for the regulation of adventitious root branching.

[Cloning and prokaryotic expression analysis of squalene synthase CpSQS1 and CpSQS2 from Crataegus pinnatifida].

In order to understand the structural characteristics of squalene synthase genes in the triterpenoids biosynthesis pathway of Crataegus pinnatifida, the squalene synthase genes of C. pinnatifida was cloned and analyzed by bioinformatics and prokaryotic expression. Two squalene synthase genes CpSQS1 and CpSQS2 were cloned from C. pinnatifida fruit by RT-PCR. The ORF length of CpSQS1 and CpSQS2 were 1 239 bp and 1 233 bp respectively, encoding 412 aa and 410 aa respectively. CpSQS1 and CpSQS2 were predicted to be stable acidic proteins by online tools. The secondary structure was mainly composed of α-helix structure, and the tertiary structure was predicted by homology modeling. Structural functional domain analysis showed that 35-367 aa of CpSQS1 and CpSQS2 cDNA containing conserved trans-isoprenyl pyrophosphate synthase domains. Transmembrane domain analysis predicted that two transmembrane domains were founded in CpSQS1 and CpSQS2. The squalene synthase amino sequence of C. pinnatifida had higher homology with the known SQS of Salvia miltiorrhiza and Glycyrrhiza glabra. Phylogenetic tree analysis showed that CpSQS1 and CpSQS2 were clustered into one branch of MdSQS1 and MdSQS2, which were consistent with the phylogenetic rule. Prokaryotic expression vector pGEX-4 T-1-CpSQS1 and pGEX-4 T-1-CpSQS2 were transformed into Escherichia coli Transetta(DE3) for induction, and the target protein was successfully expressed at 65 kDa. The expression levels of CpSQS2 were significantly higher than that of CpSQS1 in three different developmental stages of C. pinnatifida. In this study, the full-length cDNA sequences of C. pinnatifida SQS1 and SQS2 were cloned and analyzed for the first time, which provided the foundation for further study on the metabolic pathway of C. pinnatifida triterpenoids.

[Comparative study on appearance characters and internal structure of cultivated and wild Ganoderma lucidum in Huoshan].

Through the comparative study on the appearance characters and internal structure of cultivated and wild Ganoderma lucidum in Huoshan,this paper provides a reference for the further study of G. lucidum. In this study,the similarities and differences between cultivated G. lucidum " Huozhi No. 1" and wild G. lucidum in Huoshan were compared by means of character observation,optical microscopy and scanning electron microscope( SEM). The results showed that the pileus color of " Huozhi No. 1" was yellowish brown and thicker,while that of wild G. lucidum was mainly reddish brown,the context was thinner,and there were gravel and rotten wood at the bottom of the stipe. A clear skeletal hyphae and binding hyphae were observed in cultivated and wild G. lucidum,but there was no significant difference. The shell layer,context layer,mediostratum layer and spores of cultivated and wild G. lucidum were observed by SEM,and the results showed that there was no significant difference. It was found that the mediostratum of " Huozhi No. 1" was thin and irregular,while the mediostratum of wild G. lucidum was neat and compact. There were two types of spores in wild G. lucidum,one of which retained the outer wall of spore type Ⅰ,with tiny pores on the surface. The other is type Ⅱ spores with many spinous processes on the surface,which may be formed by type Ⅰ spores falling off the outwall. In this study,the appearance characters and internal structure of cultivated and wild G. lucidum in Huoshan were systematically observed and compared,which provided theoretical basis and reference for the identification and quality evaluation of cultivated and wild G. lucidum.

[Identification of Corydalis yanhusuo,C. turtschaninovii,C. decumbens by allele-specific PCR].

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.

Molecular Identification and Taxonomic Implication of Herbal Species in Genus Corydalis (Papaveraceae).

Many species of Corydalis (Papaveraceae) have been used as medicinal plants in East Asia, and the most well-known species are Corydalis yanhusuo and C. decumbens in the Pharmacopoeia of China. However, authentication of these species remains problematic because of their high morphological variation. Here, we selected 14 closely related species and five genomic regions (chloroplast: matK, trnG, rbcL, psbA-trnH; nuclear: ITS) to explore the utility of DNA barcoding for authenticating these herbs. In addition, the Poisson tree process (PTP) and automatic barcode gap discovery (ABGD) were also used and compared with DNA barcoding. Our results showed that the ITS region was not suitable for molecular analysis because of its heterogeneous nature in Corydalis. In contrast, matK was an ideal region for species identification because all species could be resolved when matK was used along with the other three chloroplast regions. We found that at least five traditional identified species were lumped into one genetic species by ABGD and PTP methods; thus, highlighting the overestimation of species diversity using the morphological approach. In conclusion, our first attempt of molecular analysis of Corydalis herbs presented here successfully resolved the identification of medicinal species and encouraged their taxonomic re-assessment.

[Species and medical history of "Xishuang" medicinal materials].

"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.

[Variety and distribution of Anhui native medicinal materials in ancient Chinese materia medica].

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.

[Growth rings in roots of seven Sect. Paeonia species and its application on identification of Paeoniae Radix Rubra].

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.

[Evolution and characteristics of system,assessing quality by distinguishing features of traditional Chinese medicinal materials, of Dao-di herbs of Astragali Radix].

"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.

[Study on identification of Dendrobium officinale and related species by bidirectional PCR amplification of mismatched and specific alleles].

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.

Cloning, prokaryotic expression and functional analysis of squalene synthase (SQS) in Magnolia officinalis.

Magnolia officinalis Rehder et Wilson is a traditional Chinese herbal medicine that is used to treat various diseases such as neurosis, anxiety, and stroke. The main secondary metabolites in magnolia bark are phenolic compounds and terpenoids. Squalene synthase plays a significant role in catalyzing two farnesyl diphosphate molecules to form squalene, the first precursor of triterpenoid, phytosterol, and cholesterol biosynthesis. In this study, a full-length cDNA of squalene synthase was cloned from M. officinalis and designated MoSQS (GenBank accession no. KT223496). The gene contains a 1240-bp open reading frame and it encodes a protein with 409 amino acids. Bioinformatic and phylogenetic analysis clearly suggested that MoSQS shared high similarity with squalene synthases among other plants. Prokaryotic expression showed that a transmembrane domain-deleted (385-409 aa) MoSQS mutant (MoSQSΔTM) could be expressed in its soluble form in Escherichia coli Transetta (DE3). GC-MS analysis showed that squalene was detected in an in vitro reaction mixture. These results indicated that MoSQSΔTM was functional, thereby establishing an important foundation for the study of triterpenoid biosynthesis in M. officinalis.

[Research of regulating synthesis of luteolin and luteoloside of Lonicera japonica by LjFNS â ¡ 1.1 and LjFNS â ¡ 2.1 treated with 5-azaC].

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.