Cloning and Functional Characterization of NADPH-Cytochrome P450 Reductases in Aconitum vilmorinianum.

Diterpenoid alkaloids (DAs) are major pharmacologically active ingredients of Aconitum vilmorinianum, an important medicinal plant. Cytochrome P450 monooxygenases (P450s) are involved in the DA biosynthetic pathway, and the electron transfer reaction of NADPH-cytochrome P450 reductase (CPR) with P450 is the rate-limiting step of the P450 redox reaction. Here, we identified and characterized two homologs of CPR from Aconitum vilmorinianum. The open reading frames of AvCPR1 and AvCPR2 were found to be 2103 and 2100 bp, encoding 700 and 699 amino acid residues, respectively. Phylogenetic analysis characterized both AvCPR1 and AvCPR2 as class II CPRs. Cytochrome c and ferricyanide could be reduced with the recombinant proteins of AvCPR1 and AvCPR2. Both AvCPR1 and AvCPR2 were expressed in the roots, stems, leaves, and flowers of A. vilmorinianum. The expression levels of AvCPR1 and AvCPR2 were significantly increased in response to methyl jasmonate (MeJA) treatment. The yeasts co-expressing AvCPR1/AvCPR2/SmCPR1 and CYP76AH1 all produced ferruginol, indicating that AvCPR1 and AvCPR2 can transfer electrons to CYP76AH1 in the same manner as SmCPR1. Docking analysis confirmed the experimentally deduced functional activities of AvCPR1 and AvCPR2 for FMN, FAD, and NADPH. The functional characterization of AvCPRs will be helpful in disclosing molecular mechanisms relating to the biosynthesis of diterpene alkaloids in A. vilmorinianum.

Comparative chloroplast genome analysis of medicinally important Veratrum (Melanthiaceae) in China: Insights into genomic characterization and phylogenetic relationships.

Members of Veratrum are perennial herbs widely used in traditional Chinese medicine to induce vomiting, resolve blood stasis and relieve pain. However, the intrageneric classification and phylogenetic relationships within Veratrum have long been controversial due to the complexity of morphological variations and lack of high-resolution molecular markers. In this study, we reevaluated the infrageneric relationships with the genus Veratrum using complete chloroplast genome sequence data. Herein, the complete cp genomes of ten species of Veratrum were newly sequenced and characterized. The complete cp genomes of ten species of Veratrum had the typical quadripartite structure, ranging from 151,597 bp to 153,711 bp in size and comprising a total of 135 genes. The structure of Veratrum cp genomes (i.e., gene order, content, and genome components) was highly similar across species. The number of simple sequence repeats (SSRs) ranged from 63 to 78, and of long repeats ranged from 31 to 35. Eight highly divergent regions (ndhF, psbC-psbZ, psbK-psbI, rpoB-trnC_GCA, trnK_UUU-trnQ_UUG, trnS_GCU-trnG_UCC, trnT_UGU-trnL_UAA and ycf1) were identified and are potentially useful for the DNA barcoding of Veratrum. Phylogenetic analysis among 29 taxa based on cp genomes, total genes, protein-coding genes and intergenic regions strongly supported the monophyly of Veratrum. The circumscription and relationships of the infrageneric taxa of Veratrum were well-presented with great resolution. These results will facilitate the identification, taxonomy, and utilization of Veratrum plants as well as the evolutionary studies of Melanthiaceae.

[Selection of optimal qRT-PCR reference genes for Aconitum vilmorinianum].

Screening suitable reference genes is the premise of quantitative Real-time PCR(qRT-PCR)for gene expression analysis. To provide stable reference genes for expression analysis of genes in Aconitum vilmorinianum, this study selected 19 candidate re-ference genes(ACT1, ACT2, ACT3, aTUB1, aTUB2, bTUB, 18S rRNA, UBQ, eIF2, eIF3, eIF4, eIF5, CYP, GAPDH1, GAPDH2, PP2A1, PP2A2, ACP, and EF1α) based on the transcriptome data of A. vilmorinianum. qRT-PCR was conducted to profile the expression of these genes in the root, stem, leaf, and flower of A. vilmorinianum. The Ct values showed that 18S rRNA with high expression level and GAPDH2 with large expression difference among organs were not suitable as the reference genes. NormFinder and geNorm showed similar results of the expression stability of the other candidate reference genes and demonstrated PP2A1, EF1α, and CYP as the highly stable ones. However, BestKeeper suggested EF1α, ACT3, and PP2A1 as the top stable genes. In view of the different results from different softwares, the geometric mean method was employed to analyze the expression stability of the candidate re-ference genes, the results of which indicated that PP2A1, EF1α, and ACT3 were the most stable. Based on the comprehensive analysis results of geNorm, NormFinder, BestKeeper, and geometric mean method, PP2A1 and EF1α presented the most stable expression in different organs of A. vilmorinianum. PP2A1 and EF1α were the superior reference genes for gene expression profiling in different organs of A. vilmorinianum.

[Therapeutic mechanism of Psammosilene tunicoides extract on rheumatoid arthritis based on NLRP3 inflammasome].

In this study, we investigated the mechanism of crude extract of Psammosilene tunicoides(CEPT) in the treatment of rheumatoid arthritis(RA) based on the Nod-like receptor protein 3(NLRP3) inflammasome. The collagen-induced arthritis(CIA) mouse model was established. On day 32 after the primary immunization, according to the arthritis score, the mice were randomly divided into model group, positive control(methotrexate) group, low-and high-dose CEPT groups, and normal group, with 10 mice in each group. According to the administration dose of each group, the mice were continuously administered for 21 days. Every four days during the administration, the paw edema degree, arthritis score, and spleen index of the mice were measured; histopathological examination was performed for the ankles of the mice; the contents of IL-1ß and IL-18 in the serum were determined; the protein expression levels of NLRP3, caspase-1, and apoptosis-associated speck-like protein containing a CARD(ASC), as well as the mRNA expression levels of NLRP3 and caspase-1 in the ankle joints of the mice were detected. The results showed that compared with those in the model group, the mice in the positive control group and CEPT groups had significantly decreased the contents of IL-1ß and IL-18 in the serum and spleen index(P<0.01), significantly lowered arthritis score and degree of paw edema(P<0.01), alleviated arthritic infiltration of the knee, and down-regulated protein and mRNA levels of NLRP3, ASC, and caspase-1 in the ankle joint(P<0.01). These results suggest that P. tunicoides may reduce the paw edema and arthritis score and alleviate the inflammatory response in CIA mice by inhibiting the expression of NLRP3. This study provides a basis for the study of immune regulation of P. tunicoides in RA.

De Novo Biosynthesis of the Oleanane-Type Triterpenoids of Tunicosaponins in Yeast.

Tunicosaponins are natural products extracted from Psammosilene tunicoides, which is an important ingredient of Yunnan Baiyao Powder, an ancient and famous Asian herbal medicine. The representative aglycones of tunicosaponins are the oleanane-type triterpenoids of gypsogenin and quillaic acid, which were found to manipulate a broad range of virus-host fusion via wrapping the heptad repeat-2 (HR2) domain prevalent in viral envelopes. However, the unknown biosynthetic pathway and difficulty in chemical synthesis hinder the therapeutic use of tunicosaponins. Here, two novel cytochrome P450-dependent monooxygenases that take part in the biosynthesis of tunicosaponins, CYP716A262 (CYP091) and CYP72A567 (CYP099), were identified from P. tunicoides. In addition, the whole biosynthesis pathway of the tunicosaponin aglycones was reconstituted in yeast by transforming the platform strain BY-bAS with the CYP716A262 and CYP716A567 genes, the resulting strain could produce 146.84 and 314.01 mg/L of gypsogenin and quillaic acid, respectively. This synthetic biology platform for complicated metabolic pathways elucidation and microbial cell factories construction can provide alternative sources of important natural products, helping conserve natural plant resources.

The complete chloroplast genome sequence of Veratrum oxysepalum and phylogenetic analysis.

Veratrum oxysepalum Turcz. is a medicinal plant belonging to Melanthiaceae occurring in Northeast China. However, there are still limited genomic resources available for genus Veratrum. The complete chloroplast (cp) genome of V. oxysepalum was determined and analyzed in this study. The complete cp genome was 153,705 bp. That contains a large single copy (LSC) region of 83,384 bp, a small single copy (SSC) region of 17,607 bp, which were separated by a pair of 26,358 bp inverted repeat regions (IRs). A total of 135 genes were annotated, including 83 protein-coding genes, 38 tRNAs, and eight rRNAs. Phylogenetic analysis using total chloroplast genome sequence of 21 species revealed that V. oxysepalum was closely relates to V. patulum of Veratrum with 100% bootstrap value.

Complete chloroplast genome sequences of Lagotis yunnanensis (Scrophulariaceae): an Endangered species endemic to the Hengduan Mountains region.

Lagotis yunnanensis is a perennial plant in the Scrophulariaceae family with a high value of medicinal in Tibetan medicine. In this study, we assembled and characterized the complete chloroplast genome of L. yunnanensis as a resource for future studies on this species. The chloroplast genome was 152,789 bp in size, with a large single-copy (LSC) region of 83,642 bp, a small single-copy (SSC) region of 17,795 bp, separated by two inverted repeat (IR) regions of 25,676 bp each. A total of 131 genes were predicted. Phylogenetic analysis showed a close relationship between L. yunnanensis and Veronicastrum sibiricum with 100% bootstrap value.

Characterization of the complete chloroplast genome of Amomum longiligulare (Zingiberaceae).

Amomum longiligulare T. L. Wu (Zingiberaceae) is a herbaceous perennial grown in Hainan Province, China, which is an important medicinal plant used for the improvement of gastrointestinal motility. The complete chloroplast genome of A. longiligulare was assembled based on next-generation sequencing. The plastome was a quadripartite circular with 16,3608 bp in length, including two inverted repeat (IR, 22,696 bp) regions, one large single-copy region (LSC) and one small single-copy region (SSC) of 88,680 bp and 29,536 bp, respectively. The chloroplast genome contained 123 genes, including 85 protein-coding genes, 30 tRNA genes, and 8 rRNA genes. The overall GC content of the whole genome is 36.1%. Phylogenetic analysis strongly supported A. longiligulare and its congeneric species, A. kravanh and A. compactum, as sister group with 100% bootstrap value.

The complete plastid genome sequence of Neopicrorhiza scrophulariiflora (Plantaginaceae): an endangered species endemic to The Himalayas regions.

Neopicrorhiza scrophulariiflora (Pennell) Hong, an endangered perennial species, is endemic to the Eastern Himalayas and Hengduan Mountains. In this study, we have sequenced the complete chloroplast genome of N. scrophulariiflora, which is 152,643 bp in length, including two inverted repeat (IR, 25,829 bp) regions, one large single copy region (LSC) and one small single copy region (SSC) of 83,191 bp and 17,794 bp, respectively. The cp genome has 131 annotated genes, including 86 protein-coding genes, 37 tRNA genes, and eight rRNA genes. The overall GC content of it is 38.1%. Phylogenetic analysis using total chloroplast genome DNA sequence of 14 species revealed that N. scrophulariiflora was closely relates to two species of Veronica with 100% bootstrap value.

The complete chloroplast genome sequence of Erigeron breviscapus and Erigeron multiradiatus (Asteraceae).

The complete chloroplast genomes of Erigeron breviscapus (Vant.) Hand-Mazz and Erigeron multiradiatus (Lindl.) Benth were reported in this study. The complete chloroplast genomes were 152,367 bp and 152,281 bp for E. breviscapus and E. multiradiatus, respectively. Erigeron breviscapus including two inverted repeat (IRs, 24,692 bp) regions, one large singe copy region (LSC) and one small singe copy region (SSC) of 84,881 bp and 18,102 bp, whereas E. multiradiatus contained IRs of 24,691 bp, LSC of 84,789 bp and SSC of 18,110 bp. The chloroplast genomes both contained 129 genes, including 84 protein-coding genes, 37 tRNA genes, and 8 rRNA genes.

Complete chloroplast genome sequence of Swertia mileensis (Gentianaceae): a medicinal species endemic to China.

Swertia mileensis is an important medicinal plant endemic to South-east Yunnan, China, which has been widely used to treat icteric hepatitis. The complete chloroplast genome sequence of S. mileensis is presented in this study, the total size is 153,015 bp in length with a typical quadripartite structure including a pair of inverted repeat (IRs, 25,786 bp) regions separated by a large single copy (LSC, 83,048 bp) region and a small single copy (SSC, 18,395 bp) region. The overall GC content of it is 38.2%. The cp genome has 134 annotated genes, including 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Among these genes, nine genes have one intron and two genes contain two introns. The phylogenetic tree based on 16 complete plastomes of support close relationships among two species of Swertia with 100% bootstrap value.

The complete chloroplast genome and phylogenetic analysis of Veratrum mengtzeanum Loes. F. (Liliaceae).

Veratrum mengtzeanum Loes. F. is a medicinal plant belonging to the genus Veratrum (Liliaceae). In the present study, we assembled and characterized the complete chloroplast (cp) genome of this species. The chloroplast genome is 152,051 bp in length, with one large single copy (LSC) region and one small single copy (SSC) region of 82,112 bp and 17,544 bp, respectively; two inverted repeat (IR) regions of 26,198 bp. It contains 131 annotated genes, including 85 protein-coding genes (PCGs), 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. Phylogenetic analysis indicated that V. mengtzeanum was closely related to Veratrum japonicum with 100% bootstrap value.

[Construction of core collection of Psammosilene tunicoides based on polymorphism of ß-AS gene].

Psammosilene tunicoides is one of the main ingredients of the "Yunnan Baiyao". P. tunicoides is an endangered species included in the secondary protection list in China Plant Red Data Book as well as the endemic species in Southwest China. Its natural resources could not meet the needs of pharmaceutical production. Construction of core collection of P. tunicoides will lay the foundation for germplasm improvement and molecular breeding. The sequence variation of the key enzymes gene locus (ß-AS) were carried out to survey the population structure and population history of the species. Among the 11 populations across its geographical range, 36 haplotypes were identified. The levels of haplotype diversity (Hd=0.905) were high, while the levels of population differentiation (GST=0.280) were low. Analysisof molecular variance (AMOVA) indicated that a significantly greater proportion of total genetic variationpartitioned among populations thanwithin populations (values of 77.43% and 22.57%, respectively). These results in combination with the star-like phylogenetic network analysis indicate that Hap1 as an ancestral haplotypewas shared in four populations, Hap2, Hap4, Hap15 and Hap16 are occurred in two populations, the remains as private haplotype only distributed in single population. The strategy of core collection was constructed in order to maximumpreserve genetic diversity of P. tunicoides.

Genotypic and Environmental Effects on the Volatile Chemotype of Valeriana jatamansi Jones.

Valeriana jatamansi Jones is an aromatic medicinal herb and important alternative to V. officinalis, which is utilized for medicinal purposes in China and India and also as spices in India. Bioactive ingredients of V. jatamansi vary in different regions. However, no information is currently available on influence of genotype and environmental factors in the volatile compounds, especially when germplasms and planting locations need to be selected. Based on the results of SNP and volatile constituents from GC-MS analysis, this study found various genotypes and chemotypes of V. jatamansi for wild plants from seven regions in China and common-garden samples; correlations between genotype and chemotype were revealed for the plants. Two distinct populations (PX, FY) were distinguishable from five others (GJ, YL, SY, DD, DY) according to their genotypes and volatile profiles, the consistency of which was observed showing that genotype could significantly influence chemotype. Wild populations and common-garden samples were also separated in their volatile profiles, demonstrating that environmental factors strongly affected their chemotypes. Compounds contributing to the discrimination were identified as discriminatory compounds. This investigation has explored and provided essential information concerning the correlation between genotype and chemotype as well as environmental factors and chemotype of V. jatamansi in some regions of China. Feasible plantation and conservation strategies of V. jatamansi could be further explored based on these results.

Development, characterization, and cross-amplification of microsatellite markers for Psammosilene tunicoides (Caryophyllaceae).

PREMISE OF THE STUDY: Psammosilene tunicoides (Caryophyllaceae) is a narrowly distributed and endemic plant species in southwestern China. The overexploitation of natural P. tunicoides has led to the destruction of many populations. Population and genetic studies will provide crucial data for the protection and management of P. tunicoides. In this study, we develop simple sequence repeat markers of P. tunicoides to analyze population diversity. METHODS AND RESULTS: Microsatellite loci of P. tunicoides were isolated with FIASCO. Eleven polymorphic and 10 monomorphic primers were developed. The 11 polymorphic primers were tested in three P. tunicoides populations, yielding two to nine alleles per locus. Levels of observed heterozygosity varied from 0.000 to 1.000, and levels of expected heterozygosity ranged from 0.000 to 0.615. In addition, three of these loci were successfully amplified, and showed polymorphism, in three Silene species. CONCLUSIONS: These microsatellite markers can be valuable tools to investigate the genetic diversity and population structure of P. tunicoides.

Differentiation in fructification percentage between two morphs of Amomum tsaoko (Zingiberaceae).

Amomum tsaoko is a flexistylous ginger. Flexistyly is a unique floral mechanism promoting outcrossing, which is known only in some species of Zingiberaceae till date. This is a pioneer report on flexistyly in A. tsaoko from the aspect of fructification percentage to clarify its influence on reproduction. We observed in 2007 and 2008 that the fructification percentage of the anaflexistyled and the cataflexistyled inflorescence were 14.89 ± 10.35% and 11.31 ± 7.91% respectively, with significant difference (d.f. = 141.920, t = 2.518, P = 0.013 < 0.05). The greatly significant difference between 2007 and 2008 were present in both the flower number (d.f. = 93, t = -2.819, P = 0.006 < 0.01) and the fructification percentage (d.f. = 93, t = -2.894, P = 0.005 < 0.01) of the cataflexistylous inflorescence. Although the two morphs were similar in morphological characteristics, there was some gender differentiation between them, showing a possibility that the anaflexistylous morph might function more as females and the cataflexistylous morph more as males. Reproduction of the cataflexistylous morph was significantly sensitive to change of environmental factors, in contrast to the anaflexistylous morph, thus the yield varied between the abundant year (2008) and the off year (2007).

[Cloning of ß-actin Gene in Psammosilene tunicoides and Its Role as a Reference Gene].

Objective: To establish a real-time quantitative PCR method to detect Psammosilene tunicoides ß-actin, and to provide a reference gene for the detection of Psammosilene tunicoides genes by q PCR. Methods: Specific primers were designed based on the conserved region of the ß-actin gene( Gen Bank) and were used to amplify ß-actin by PCR. ß-actin was also used as a reference gene in the q PCR analysis of glycosyltransferase gene( UGT) expression in the roots,stems,and leaves of Psammosilene tunicoides. Results: The length of the ß-actin gene amplicon from Psammosilene tunicoides was 153 bp and shared relatively high homology with ß-actin found in Vaccaria segetalis, Myosoton aquaticum and Portulaca oleracea. Furthermore, UGT was revealed to be stably expressed in different Psammosilene tunicoides tissues when ß-actin was employed as the reference gene. Conclusion: ß-actin is a reliable and suitable reference gene for studies on the expression of triterpenoid saponin biosynthesis-related genes in Psammosilene tunicoides.

The Genus Letrouitia (Letrouitiaceae: Lichenized Ascomycota) New to Cambodia.

The genus Letrouitia is newly recorded for Cambodia, including the four species as L. domingensis, L. leprolytoides, L. sayeri, and L. subvulpina. A brief description and illustrations are provided.

Two new chalcones from Shuteria sinensis.

Two new chalcones, 2',3,4,4'-tetrahydroxy-2-prenylchalcone (1) and 3-methoxy-2',4,4'-trihydroxy-2-prenylchalcone (2), together with two known compounds, munsericin (3) and 3,4-dihydroxylonchocarpin (4), were isolated from the ethanol extract of the whole plant of Shuteria sinensis. Their structures were identified by spectroscopic analysis methods, such as 1D and 2D NMR, along with HR-MS data. Glucose metabolism activity of four compounds was tested, compounds 3 and 4 showed effect on the glucose consumption of insulin-resistant HepG2 cells.

[Study on content of volatile oil in Amomum tsao-ko seeds and its influencing factors].

OBJECTIVE: To lay the foundation for quality evaluation of medicinal materials of Amomum tsao-ko and its selection of fine varieties, the variation of percentage of volatile oil in the seeds among populations and individuals and its influencing factors were studied. METHODS: Extracted volatile oil from the seeds according to the Chinese Pharmacopoeia, studied quantitative characters of fruit, and analyzed the data according to SPSS 13.0. RESULTS: The percentage of volatile oil in seeds was greatly significantly different among the 7 populations (P = 0.000 < 0.001), and among different individuals (P = 0.000 < 0.001). A significantly positive correlation was detected between the percentage of volatile oil in seeds and length of fruit (P = 0.024 < 0.05), and significantly negative correlation occurred between the percentage and other quantitative characters or factors, viz. thousand seeds weight (P = 0.031 < 0.05), flower number per inflorescence (P = 0.028 < 0.05), weight of seeds masses per fruit (P = 0.038 < 0.05), altitude (P = 0.014 < 0.05), and latitude (P = 0.000 < 0.001). The regression equation (Y = 1.031 - 1.744 X2 - 1.119 X3 + 2.207 X1) was the optimal regression model of percentage of volatile oil in seeds (X1, X2 and X3 were respectively represented as the length and length-width ratio of fruit,and the weight of seeds masses). CONCLUSION: Environmental factors influence greatly significantly on the percentage of volatile oil in seeds. The production and accumulation of volatile oil would decrease with altitude and latitude increasing. The longer fruit has the higher percentage volatile oil.