[Isolation and functional characterization of (iso)eugenol O-methyltransferase (IEMT) gene in Asarum sieboldii].

Methyleugenol is one of the main active constituents in the volatile oil of the traditional Chinese medicine Asari Radix et Rhizoma. It possesses various pharmacological effects such as analgesic, anesthetic, and anti-inflammatory properties. In biosynthesis, the initial precursor phenylalanine is finally converted into methyleugenol through a series of intermediate compounds including coniferyl acid, courmaryl acid, caffeic acid, ferulic acid/ferulic-CoA, coniferyl aldehyde, conferyl alcohol, cnfiferyl acetate, and eugenol/isoeugenol, which are produced through catalysis of a large number of enzymes. Eugenol O-methyltransferase(EOMT) is one of the key enzymes in the biosynthesis pathway, capable of methylating eugenol on the para-site hydroxyl group of the benzene ring, thereby generating methyleugenol. Here, an(iso)eugenol O-methyltransferase(IEMT) gene was cloned for the first time from Asarum siebo-ldii, holding an open reading frame that consisted of 1 113 bp and encoded a protein containing 370 amino acid residues. Bioinformatics analysis results showed that this protein was equipped with the characteristic structural domains of methyltransferases such as S-adenosylmethionine(SAM) binding sites and dimerization domains. The prokaryotic expression recombinant plasmid pET28a(+)-AsIEMT was constructed, and the candidate protein was induced and purified. In vitro enzyme assays confirmed that AsIEMT had dual functions. The enzyme could catalyze the production either of methyleugenol from eugenol or of methylisoeugenol from isoeugenol, although the latter was more prevalent. When isoeugenol was used as the substrate, the kinetics parameters K_m and V_(max) of catalytic reaction were(0.90±0.06) mmol·L~(-1) and(1.32±0.04)nmol·s~(-1)·mg~(-1), respectively. This study expanded our understandings of critical enzyme genes involved in phenylpropanoid metabolic pathways, and would facilitate the elucidation of quality formation mechanisms of the TCM Asari Radix et Rhizoma.

[Discussion on hepatic damage mechanism of Asari Radix et Rhizoma based on network pharmacology and untargeted metabolomics].

Asari Radix et Rhizoma is a common drug for relieving exterior syndrome in clinics, but its toxicity limits its use. In this study, the mechanism of hepatic damage of Asari Radix et Rhizoma was studied by network pharmacology and metabolomics. The hepatic damage-related dataset, namely GSE54257 was downloaded from the GEO database. The Limma package was used to analyze the differentially expressed genes in the dataset GSE54257. Toxic components and target genes of Asari Radix et Rhizoma were screened by TCMSP, ECTM, and TOXNET. The hepatic damage target genes of Asari Radix et Rhizoma were obtained by mapping with the differentially expressed gene of GSE54257, and a PPI network was constructed. GO and KEGG enrichment analysis of target genes were performed, and a "miRNA-target gene-signal pathway" network was drawn with upstream miRNA information. Thirty rats were divided into a blank group, a high-dose Asari Radix et Rhizoma group, and a low-dose Asari Radix et Rhizoma group, which were administered once a day. After continuous administration for 28 days, liver function indexes and liver pathological changes were detected. Five liver tissue samples were randomly collected from the blank group and high-dose Asari Radix et Rhizoma group, and small molecule metabolites were analyzed by ultra-high performance liquid chromatography-mass spectrometry(UHPLC-MS). The orthogonal partial least squares-discriminant analysis(OPLS-DA) method was used to screen differential metabolites, and enrichment analysis, correlation analysis, and cluster analysis were conducted for differential metabolites. Finally, the MetaboAnalyst platform was used to conduct pathway enrichment analysis for differential metabolites. It was found that there were 14 toxic components in Asari Radix et Rhizoma, corresponding to 37 target genes, and 12 genes related to liver toxicity of Asari Radix et Rhizoma were obtained by mapping to differentially expressed genes of GSE54257. The animal test results showed that Asari Radix et Rhizoma could significantly increase the liver function index, reduce the activity of the free radical scavenging enzyme, change the liver oxidative stress level, and induce lipid peroxidation damage in rats. The results of untargeted metabolomics analysis showed that compared with the blank group, nine metabolites were up-regulated, and 16 metabolites were down-regulated in the liver tissue of the Asari Radix et Rhizoma group. These 25 metabolites had strong correlations and good clustering. Pathway enrichment analysis showed that these differential metabolites and the 12 hepatotoxic target genes of Asari Radix et Rhizoma were mainly involved in purine metabolism, as well as the biosynthesis and metabolism of valine, leucine, glycine, serine, and threonine. The study confirmed that the hepatica damage effect of Asari Radix et Rhizoma was the result of multi-component, multi-target, and multi-signaling pathways, and its mechanism may be related to inhibiting nucleotide synthesis and affecting protein metabolism.

Full-length transcriptome analysis and identification of genes involved in asarinin and aristolochic acid biosynthesis in medicinal plant Asarum sieboldii.

Asarum sieboldii, a well-known traditional Chinese medicinal herb, is used for curing inflammation and ache. It contains both the bioactive ingredient asarinin and the toxic compound aristolochic acid. To address further breeding demand, genes involved in the biosynthetic pathways of asarinin and aristolochic acid should be explored. Therefore, the full-length transcriptome of A. sieboldii was sequenced using PacBio Iso-Seq to determine the candidate transcripts that encode the biosynthetic enzymes of asarinin and aristolochic acid. In this study, 63 023 full-length transcripts were generated with an average length of 1371 bp from roots, stems, and leaves, of which 49 593 transcripts (78.69%) were annotated against public databases. Furthermore, 555 alternative splicing (AS) events, 10 869 long noncoding RNAs (lncRNAs) as well as their 11 291 target genes, and 17 909 simple sequence repeats (SSRs) were identified. The data also revealed 97 candidate transcripts related to asarinin metabolism, of which six novel genes that encoded enzymes involved in asarinin biosynthesis were initially reported. In addition, 56 transcripts related to aristolochic acid biosynthesis were also identified, including CYP81B. In summary, these transcriptome data provide a useful resource to study gene function and genetic engineering in A. sieboldii.

Physiological, Biochemical and Transcriptomic Analysis of the Aerial Parts (Leaf-Blade and Petiole) of Asarum sieboldii Responding to Drought Stress.

Asarum sieboldii Miq. is a leading economic crop and a traditional medicinal herb in China. Leaf-blade and petiole are the only aerial tissues of A. sieboldii during the vegetative growth, playing a vital role in the accumulation and transportation of biomass energy. They also act as critical indicators of drought in agricultural management, especially for crops having underground stems. During drought, variations in the morphology and gene expression of the leaves and petioles are used to control agricultural irrigation and production. Besides, such stress can also alter the differential gene expression in these tissues. However, little is known about the drought-tolerant character of the aerial parts of A. sieboldii. In this study, we examined the physiological, biochemical and transcriptomic responses to the drought stress in the leaf blades and petioles of A. sieboldii. The molecular mechanism, involving in drought stress response, was elucidated by constructing the cDNA libraries and performing transcriptomic sequencing. Under drought stress, a total of 2912 and 2887 unigenes were differentially expressed in the leaf blade and petiole, respectively. The detection of many transcription factors and functional genes demonstrated that multiple regulatory pathways were involved in drought tolerance. In response to drought, the leaf blade and petiole displayed a general physiological character, a higher SOD and POD activity, a higher MDA content and lower chlorophyll content. Three unigenes encoding POD were up-regulated, which can improve POD activity. Essential oil in petiole was extracted. The relative contents of methyleugenol and safrole in essential oil were increased from 0.01% to 0.05%, and 3.89% to 16.97%, respectively, while myristicin slightly reduced from 24.87% to 21.52%. Additionally, an IGS unigene, involved in eugenol biobiosynthesis, was found up-regulated under drought stress, which was predicated to be responsible for the accumulation of methyleugenol and safrole. Simple sequence repeats (SSRs) were characterized in of A. sieboldii, and a total of 5466 SSRs were identified. Among them, mono-nucleotides were the most abundant repeat units, accounting for 44.09% followed by tri-, tetra-, penta and hexa-nucleotide repeats. Overall, the present work provides a valuable resource for the population genetics studies of A. sieboldii. Besides, it provides much genomic information for the functional dissection of the drought-resistance in A. sieboldii, which will be useful to understand the bio-regulatory mechanisms linked with drought-tolerance to enhance its yield.

Radiation history of Asian Asarum (sect. Heterotropa, Aristolochiaceae) resolved using a phylogenomic approach based on double-digested RAD-seq data.

BACKGROUND AND AIMS: The genus Asarum sect. Heterotropa (Aristolochiaceae) probably experienced rapid diversification into 62 species centred on the Japanese Archipelago and Taiwan, providing an ideal model for studying island adaptive radiation. However, resolving the phylogeny of this plant group using Sanger sequencing-based approaches has been challenging. To uncover the radiation history of Heterotropa, we employed a phylogenomic approach using double-digested RAD-seq (ddRAD-seq) to yield a sufficient number of phylogenetic signals and compared its utility with that of the Sanger sequencing-based approach. METHODS: We first compared the performance of phylogenetic analysis based on the plastid matK and trnL-F regions and nuclear ribosomal internal transcribed spacer (nrITS), and phylogenomic analysis based on ddRAD-seq using a reduced set of the plant materials (83 plant accessions consisting of 50 species, one subspecies and six varieties). We also conducted more thorough phylogenomic analyses including the reconstruction of biogeographic history using comprehensive samples of 135 plant accessions consisting of 54 species, one subspecies, nine varieties of Heterotropa and six outgroup species. KEY RESULTS: Phylogenomic analyses of Heterotropa based on ddRAD-seq were superior to Sanger sequencing-based approaches and resulted in a fully resolved phylogenetic tree with strong support for 72.0-84.8 % (depending on the tree reconstruction methods) of the branches. We clarified the history of Heterotropa radiation and found that A. forbesii, the only deciduous Heterotropa species native to mainland China, is sister to the evergreen species (core Heterotropa) mostly distributed across the Japanese Archipelago and Taiwan. CONCLUSIONS: The core Heterotropa group was divided into nine subclades, each of which had a narrow geographic distribution. Moreover, most estimated dispersal events (22 out of 24) were between adjacent areas, indicating that the range expansion has been geographically restricted throughout the radiation history. The findings enhance our understanding of the remarkable diversification of plant lineages in the Japanese Archipelago and Taiwan.

Genetic data reveals a complex history of multiple admixture events in presently allopatric wild gingers (Asarum spp.) showing intertaxonomic clinal variation in calyx lobe length.

Clinal variation is a major pattern of observed phenotypic diversity and identifying underlying demographic processes is a necessary step to understand the establishment of clinal variation. The wild ginger series Sakawanum (genus Asarum) comprises four taxa, which exhibit intertaxonomic clinal variation in calyx lobe length across two continental islands isolated by a sea strait. To test alternative hypotheses of the evolutionary history and to determine the implications for the formation of clinal variation, we conducted approximate Bayesian computation (ABC) analysis and ecological niche modeling (ENM). ABC analysis indicated that the scenario assuming multiple admixture events was strongly supported. This scenario assumed two admixture events occurred between morphologically distinct taxa, likely leading to the generation of intermediate taxa. One of the admixture events was estimated to have occurred during the last glacial maximum (LGM), during which the taxa were estimated to have formed a common refugia in southern areas by ENM analysis. Although four taxa are currently distributed allopatrically on different islands and trans-oceanic dispersal appears unlikely, the formation of a land bridge and the geographic range shift to refugia would have allowed secondary contact between previously isolated taxa. This study suggests that clinal variation can be shaped by demographic history including multiple admixtures due to climatic oscillations.

Total duplication of the small single copy region in the angiosperm plastome: Rearrangement and inverted repeat instability in Asarum.

PREMISE OF THE STUDY: As more plastomes are assembled, it is evident that rearrangements, losses, intergenic spacer expansion and contraction, and syntenic breaks within otherwise functioning plastids are more common than was thought previously, and such changes have developed independently in disparate lineages. However, to date, the magnoliids remain characterized by their highly conserved plastid genomes (plastomes). METHODS: Illumina HiSeq and MiSeq platforms were used to sequence the plastomes of Saruma henryi and those of representative species from each of the six taxonomic sections of Asarum. Sequenced plastomes were compared in a phylogenetic context provided by maximum likelihood and parsimony inferences made using an additional 18 publicly available plastomes from early-diverging angiosperm lineages. KEY RESULTS: In contrast to previously published magnoliid plastomes and the newly sequenced Saruma henryi plastome published here, Asarum plastomes have undergone extensive disruption and contain extremely lengthy AT-repeat regions. The entirety of the small single copy region (SSC) of A. canadense and A. sieboldii var. sieboldii has been incorporated into the inverted repeat regions (IR), and the SSC of A. delavayi is only 14 bp long. All sampled Asarum plastomes share an inversion of a large portion of the large single copy region (LSC) such that trnE-UUC is adjacent to the LSC-IR boundary. CONCLUSIONS: Plastome divergence in Asarum appears to be consistent with trends seen in highly rearranged plastomes of the monocots and eudicots. We propose that plastome instability in Asarum is due to repetitive motifs that serve as recombinatory substrates and reduce genome stability.

Comparative Transcriptomics Analysis for Gene Mining and Identification of a Cinnamyl Alcohol Dehydrogenase Involved in Methyleugenol Biosynthesis from Asarum sieboldii Miq.

Asarum sieboldii Miq., one of the three original plants of TCM ASARI RADIX ET RHIZOMA, is a perennial herb distributed in central and eastern China, the Korean Peninsula, and Japan. Methyleugenol has been considered as the most important constituent of Asarum volatile oil, meanwhile asarinin is also employed as the quality control standard of ASARI RADIX ET RHIZOMA in Chinese Pharmacopeia. They both have shown wide range of biological activities. However, little was known about genes involved in biosynthesis pathways of either methyleugenol or asarinin in Asarum plants. In the present study, we performed de novo transcriptome analysis of plant tissues (e.g., roots, rhizomes, and leaves) at different developmental stages. The sequence assembly resulted in 311,597 transcripts from these plant materials, among which 925 transcripts participated in 'secondary metabolism' with particularly up to 20.22% of them falling into phenylpropanoid biosynthesis pathway. The corresponding enzymes belong to seven families potentially encoding phenylalanine ammonia-lyase (PAL), trans-cinnamate 4-monooxygenase (C4H), p-coumarate 3-hydroxylase (C3H), caffeoyl-CoA O-methyltransferase (CCoAOMT), cinnamoyl-CoA reductase (CCR), cinnamyl alcohol dehydrogenase (CAD), and eugenol synthase (EGS). Moreover, 5 unigenes of DIR (dirigent protein) and 11 unigenes of CYP719A (719A subfamily of cytochrome P450 oxygenases) were speculated to be involved in asarinin pathway. Of the 15 candidate CADs, four unigenes that possessed high FPKM (fragments per transcript kilobase per million fragments mapped) value in roots were cloned and characterized. Only the recombinant AsCAD5 protein efficiently converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates employed not only in biosynthesis of lignin but also in that of methyleugenol and asarinin. qRT-PCR revealed that AsCAD5 had a high expression level in roots at three developmental stages. Our study will provide insight into the potential application of molecular breeding and metabolic engineering for improving the quality of TCM ASARI RADIX ET RHIZOMA.

Putative floral brood-site mimicry, loss of autonomous selfing, and reduced vegetative growth are significantly correlated with increased diversification in Asarum (Aristolochiaceae).

The drivers of angiosperm diversity have long been sought and the flower-arthropod association has often been invoked as the most powerful driver of the angiosperm radiation. We now know that features that influence arthropod interactions cannot only affect the diversification of lineages, but also expedite or constrain their rate of extinction, which can equally influence the observed asymmetric richness of extant angiosperm lineages. The genus Asarum (Aristolochiaceae; ∼100 species) is widely distributed in north temperate forests, with substantial vegetative and floral divergence between its three major clades, Euasarum, Geotaenium, and Heterotropa. We used Binary-State Speciation and Extinction Model (BiSSE) Net Diversification tests of character state distributions on a Maximum Likelihood phylogram and a Coalescent Bayesian species tree, inferred from seven chloroplast markers and nuclear rDNA, to test for signal of asymmetric diversification, character state transition, and extinction rates of floral and vegetative characters. We found that reduction in vegetative growth, loss of autonomous self-pollination, and the presence of putative fungal-mimicking floral structures are significantly correlated with increased diversification in Asarum. No significant difference in model likelihood was identified between symmetric and asymmetric rates of character state transitions or extinction. We conclude that the flowers of the Heterotropa clade may have converged on some aspects of basidiomycete sporocarp morphology and that brood-site mimicry, coupled with a reduction in vegetative growth and the loss of autonomous self-pollination, may have driven diversification within Asarum.

Phylogenetic relationships in Asarum: Effect of data partitioning and a revised classification.

PREMISE OF THE STUDY: Generic boundaries and infrageneric relationships among the charismatic temperate magnoliid Asarum sensu lato (Aristolochiaceae) have long been uncertain. Previous molecular phylogenetic analyses used either plastid or nuclear loci alone and varied greatly in their taxonomic implications for the genus. We analyzed additional molecular markers from the nuclear and plastid genomes, reevaluated the possibility of a derived loss of autonomous self-pollination, and investigated the topological effects of matrix-partitioning-scheme choice. METHODS: We sequenced seven plastid regions and the nuclear ITS1-ITS2 region of 58 individuals representing all previously recognized Asarum s.l. segregate genera and the monotypic genus Saruma. Matrices were partitioned using common a priori partitioning schemes and PartitionFinder. KEY RESULTS: Topologies that were recovered using a priori partitioning of matrices differed from those recovered using a PartitionFinder-selected scheme, and by analysis method. We recovered six monophyletic groups that we circumscribed into three subgenera and six sections. Putative fungal mimic characters served as synapomorphies only for subgenus Heterotropa. Subgenus Geotaenium, a new subgenus, was recovered as sister to the remainder of Asarum by ML analyses of highly partitioned datasets. Section Longistylis, also newly named, is sister to section Hexastylis. CONCLUSIONS: Our analyses do not unambiguously support a single origin for all fungal-mimicry characters. Topologies recovered through the analysis of PartitionFinder-optimized matrices can differ drastically from those inferred from a priori partitioned matrices, and by analytical method. We recommend that investigators evaluate the topological effects of matrix partitioning using multiple methods of phylogenetic reconstruction.

Deep sequencing of plant and animal DNA contained within traditional Chinese medicines reveals legality issues and health safety concerns.

Traditional Chinese medicine (TCM) has been practiced for thousands of years, but only within the last few decades has its use become more widespread outside of Asia. Concerns continue to be raised about the efficacy, legality, and safety of many popular complementary alternative medicines, including TCMs. Ingredients of some TCMs are known to include derivatives of endangered, trade-restricted species of plants and animals, and therefore contravene the Convention on International Trade in Endangered Species (CITES) legislation. Chromatographic studies have detected the presence of heavy metals and plant toxins within some TCMs, and there are numerous cases of adverse reactions. It is in the interests of both biodiversity conservation and public safety that techniques are developed to screen medicinals like TCMs. Targeting both the p-loop region of the plastid trnL gene and the mitochondrial 16S ribosomal RNA gene, over 49,000 amplicon sequence reads were generated from 15 TCM samples presented in the form of powders, tablets, capsules, bile flakes, and herbal teas. Here we show that second-generation, high-throughput sequencing (HTS) of DNA represents an effective means to genetically audit organic ingredients within complex TCMs. Comparison of DNA sequence data to reference databases revealed the presence of 68 different plant families and included genera, such as Ephedra and Asarum, that are potentially toxic. Similarly, animal families were identified that include genera that are classified as vulnerable, endangered, or critically endangered, including Asiatic black bear (Ursus thibetanus) and Saiga antelope (Saiga tatarica). Bovidae, Cervidae, and Bufonidae DNA were also detected in many of the TCM samples and were rarely declared on the product packaging. This study demonstrates that deep sequencing via HTS is an efficient and cost-effective way to audit highly processed TCM products and will assist in monitoring their legality and safety especially when plant reference databases become better established.

The expression and phylogenetic analysis of four AP3-like paralogs in the stamens, carpels, and single-whorl perianth of the paleoherb Asarum caudigerum.

The paleoherb species Asarum caudigerum (Aristolochiaceae) is important for research into the origin and evolution of angiosperm flowers due to its basal position in the angiosperm phylogeny. In this study, four MADS-box-containing transcripts were isolated from A. caudigerum by rapid amplification of cDNA ends (RACE). Sequence comparisons and phylogenetic analyses indicated that they possess high homology to AP3 subfamily genes, which have been shown previously to be involved in petal and stamen development in eudicots. Reverse-transcription quantitative PCR (RT-qPCR) and in situ hybridization analyses showed AcAP3-A expression mainly in the second whorl (stamens) and AcAP3-B expression in whorls 1 and 3 (perianth and carpels). Compared with eudicot AP3 homologs, premature translation termination codons were caused by an insertion in the K1 domain of AcAP3-C, and by a deletion in the 7th exon of AcAP3-D. Sequence analyses suggested that the A. caudigerum AP3 lineage had undergone gene duplication and subfunctionalization, diverging in expression patterns during perianth, stamen, and carpel development. Based on comparative genomic and phylogenetic analyses, we concluded that subfunctionalization has likely contributed to the persistence of two functional AP3 paralogs, that two other copies may have become pseudogenes, and that these AP3 duplication and subfunctionalization events may have contributed to the evolution of the unusual floral morphology of A. caudigerum.

[Genetic diversity and volatile components of Asarum sieboldii in Qin-ba region].

OBJECTIVE: To analyze the genetic diversity and the volatile components of Asarum sieboldii from seven habitats in Qin-ba region. METHODS: The genetic diversity of the herb was analyzed by ISSR (inter simple sequence repeat) markers; The relative content volatile components of the herb were dectected by head space solid-phase microextraction gas chromatogrphy-mass spectrometry (HS-SPME-GC-MS). The contents of the 3 main components were analyzed by steam distillation gas chromatography-mass spectrometry (GC-MS). RESULTS: 57 bands were amplified from 7 populations by 6 reliable primers, 51 of which were polymorphic (89.47% of the total). The cluster analysis presented that these resources were divided into two main groups. There were differences in the chemical components and the contents of Asarum sieboldii from the 6 wild habitats. Except for some same components, many unique components were identified in them respectively. In addition, some components could be detected only in some populations which had smaller genetic distance. CONCLUSION: Cluster analysis shows no direct correlation between genetic distance and geographic distance of Asarum sieboldii in Qin-ba region. The accumulations of some volatile components of Asarum sieboldii are possibly related to genetic diversity.

Comparative Transcriptomic Analysis Reveals the Effects of Drought on the Biosynthesis of Methyleugenol in Asarum sieboldii Miq.

Asarum sieboldii Miq., a perennial herb in the family Aristolochiaceae, is widely used to treat colds, fever, headache and toothache in China. However, little is known about the drought-tolerance characteristics of A. sieboldii. In this study, to elucidate the molecular-genetic mechanisms of drought-stress tolerance of A. sieboldii, RNA-seq was conducted. In total, 53,344 unigenes were assembled, and 28,715 unigenes were annotated. A total of 6444 differential-expression unigenes (DEGs) were found, which were mainly enriched in phenylpropanoid, starch and sucrose metabolic pathways. Drought stress revealed significant up-regulation of the unigenes encoding PAL, C4H, HCT, C3H, CCR and IGS in the methyleugenol-biosynthesis pathway. Under the condition of maintaining drought for 15 days and 30 days, drought stress reduced the biosynthesis of volatile oil by 24% and 38%, respectively, while the production of key medicinal ingredients (such as methyl eugenol) was increased. These results provide valuable information about the diverse mechanisms of drought resistance in the A. sieboldii, and the changes in the expression of the genes involved in methyleugenol biosynthesis in response to drought stress.

Extended expression of B-class MADS-box genes in the paleoherb Asarum caudigerum.

Asarum caudigerum (Aristolochiaceae) is a paleoherb species that is important for research in origin and evolution of angiosperm flowers due to its basal position in the angiosperm phylogeny. In this study, a subtracted floral cDNA library from floral buds of A. caudigerum was constructed and cDNA arrays by suppression subtractive hybridization were generated. cDNAs of floral buds at different stages before flower opening and of leaves at the seedling stage were used. The macroarray analyses of expression profiles of isolated floral genes showed that 157 genes out of the 612 unique ESTs tested revealed higher transcript abundance in the floral buds and uppermost leaves. Among them, 78 genes were determined to be differentially expressed in the perianth, 62 in the stamens, and 100 genes in the carpels. Quantitative real-time PCR of selected genes validated the macroarray results. Remarkably, APETALA3 (AP3) B-class genes isolated from A. caudigerum were upregulated in the perianth, stamens and carpels, implying that the expression domain of B-class genes in this basal angiosperm was broader than those in their eudicot counterparts.

Deep sequencing and transcriptome analysis to identify genes related to biosynthesis of aristolochic acid in Asarum heterotropoides.

Asarum spp. are important medicinal plants that have the potential for use in treating various types of fevers. Aristolochic acid is one of the main toxic compounds present in these plants. To improve our understanding of the biosynthetic pathway of aristolochic acid, we sequenced the transcriptome of the root and leaf tissues of Asarum heterotropoides and performed de novo sequence assembly. The data were stitched together to produce 468,357 transcripts with an N50 of 611 bp. The data were annotated with various databases (RefSeq non-redundant proteins [Nr], Swiss-Prot, Kyoto Encyclopaedia of Genes and Genomes [KEGG], Clusters of Orthologous Groups/EuKaryotic Orthologous Groups [COG/KOG], and Gene Ontology [GO]) and were annotated. There were 205,165 transcripts (43.81%) of differentially expressed genes in the roots and leaves, which were shown to be involved in biosynthesis, transport, and catabolism, and 100 genes in defence mechanisms. Three candidate transcripts (TyrDC1, TyrDC2, and TyrDC3) were discovered in these differential genes. TyrDC may be a key enzyme in the biosynthesis pathway of aristolochic acid identified using quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and high-performance liquid chromatography (HPLC). The transcriptome data and analysis presented here lay the foundation for further research into these important medicinal plants.

[Studies on the botanical sources and DNA molecular identification of Herba Asari based on ITS sequence].

OBJECTIVE: To explore the genetic basis of using three species of Asarum as Herba Asari to determine the taxonomic positions of Asarum heterotropoides and A. siebodii; and to apply DNA molecular analysis as a tool for identification of Herba Asari. METHOD: PCR, purification, sequence analysis were prerformed. RESULT: MS sequences of the three Asarum species were obtained. 3 botanical sources of Herba Asari are closely clustered together on the topology tree; one inner branch is composed of A. heterotropoides and A. sieboldii, whereas another branch contains A. sieboldii. Their ITS sequences are different. CONCLUSION: Three plant species of Herba Asari are closely related, and there are genetic reasons that they are used as the sources of the same medicine. The classification placement of A. sieboldii is not certain. The differences of ITS sequences of the botanical sources of Herba Asari can be used as a means of identification.

[Problems and solutions in the use of RAPD to the identification of the Chinese drugs "xi-xin" (Herba asari) and its substitutes].

The use of random amplified polymorphic DNAs (RAPD) on the identification of crude drugs has a few special problems, e.g., the influence of the degree of degradation of DNA templates on the amplified products, the reliability of results by one or two primers and the application range of RAPD, etc., so the problems were discussed through researches on Herba Asari. In this experiment, the DNA of twenty-one samples from different locations and collected at different time were extracted. The suitable concentrations of DNA templates and the suitable primers were selected. After this, the DNA of these samples were amplified by RAPD. The results showed that the concentration and the degree of degradation of DNA templates, and the different sources of crude drugs affected the result of RAPD assay. Some suggestions were made to solve these problems, such as the selection of DNA templates concentration, the screening of suitable primers, and the use of contrast groups and cluster analysis. Furthermore, the application range of RAPD to the identification of crude drugs was discussed. All of these will provide the guide to the use of RAPD for the identification of crude drugs.

Expressed sequence tags (ESTs) and phylogenetic analysis of floral genes from a paleoherb species, Asarum caudigerum.

BACKGROUND AND AIMS: Asarum caudigerum (Aristolochiaceae) is an important species of paleoherb in relation to understanding the origin and evolution of angiosperm flowers, due to its basal position in the angiosperms. The aim of this study was to isolate floral-related genes from A. caudigerum, and to infer evolutionary relationships among florally expression-related genes, to further illustrate the origin and diversification of flowers in angiosperms. METHODS: A subtracted floral cDNA library was constructed from floral buds using suppression subtractive hybridization (SSH). The cDNA of floral buds and leaves at the seedling stage were used as a tester and a driver, respectively. To further identify the function of putative MADS-box transcription factors, phylogenetic trees were reconstructed in order to infer evolutionary relationships within the MADS-box gene family. KEY RESULTS: In the forward-subtracted floral cDNA library, 1920 clones were randomly sequenced, from which 567 unique expressed sequence tags (ESTs) were obtained. Among them, 127 genes failed to show significant similarity to any published sequences in GenBank and thus are putatively novel genes. CONCLUSIONS: Phylogenetic analysis indicated that a total of 29 MADS-box transcription factors were members of the APETALA3(AP3) subfamily, while nine others were putative MADS-box transcription factors that formed a cluster with MADS-box genes isolated from Amborella, the basal-most angiosperm, and those from the gymnosperms. This suggests that the origin of A. caudigerum is intermediate between the angiosperms and gymnosperms.