Key message
The novel AP2/ERF transcription factor SmERF128 positively regulates diterpenoid tanshinone biosynthesis by activating the expression of
SmCPS1, SmKSL1
, and
SmCYP76AH1
in
Salvia ...miltiorrhiza
.
Certain members of the APETALA2/ethylene-responsive factor (AP2/ERF) family regulate plant secondary metabolism. Although it is clearly documented that AP2/ERF transcription factors (TFs) are involved in sesquiterpenoid biosynthesis, the regulation of diterpenoid biosynthesis by AP2/ERF TFs remains elusive. Here, we report that the novel AP2/ERF TF SmERF128 positively regulates diterpenoid tanshinone biosynthesis in
Salvia miltiorrhiza
. Overexpression of
SmERF128
increased the expression levels of copalyl diphosphate synthase 1 (
SmCPS1
), kaurene synthase-like 1 (
SmKSL1
) and cytochrome P450 monooxygenase 76AH1 (
SmCYP76AH1
), whereas their expression levels were decreased when
SmERF128
was silenced. Accordingly, the content of tanshinone was reduced in
SmERF128
RNA interference (RNAi) hairy roots and dramatically increased in
SmERF128
overexpression hairy roots, as demonstrated through Ultra Performance Liquid Chromatography (UPLC) and Liquid Chromatography-Tandem Mass Spectrometry (LC-MS/MS) analysis. Furthermore, SmERF128 activated the expression of
SmCPS1, SmKSL1
, and
SmCYP76AH1
by binding to the GCC box, and to the CRTDREHVCBF2 (CBF2) and RAV1AAT (RAA) motifs within their promoters during in vivo and in vitro assays. Our findings not only reveal the molecular basis of how the AP2/ERF transcription factor SmERF128 regulates diterpenoid biosynthesis, but also provide useful information for improving tanshinone production through genetic engineering.
Numerous adverse reactions have arisen following the use of inaccurately identified medicinal plant ingredients, resulting in conditions such as aristolochic acid nephropathy and herb-induced ...poisoning. This problem has prompted increased global concern over the safety of herbal medicines. DNA barcoding, a technique aiming at detecting species-specific differences in a short region of DNA, provides a powerful new tool for addressing this problem. A preliminary system for DNA barcoding herbal materials has been established based on a two-locus combination of ITS2+psbA–trnH barcodes. There are 78,847 sequences belonging to 23,262 species in the system, which include more than 95% of crude herbal drugs in pharmacopeia, such as those of China, Japan, Korea, India, USA, and Europe. The system has been widely used in traditional herbal medicine enterprises. This review summarizes recent key advances in the DNA barcoding of medicinal plant ingredients (herbal materia medica) as a contribution towards safe and efficacious herbal medicines.
Adulterant herbal materials are a threat to consumer safety. In this study, we used DNA barcoding to investigate the proportions and varieties of adulterant species in traditional Chinese medicine ...(TCM) markets. We used a DNA barcode database of TCM (TCMD) that was established by our group to investigate 1436 samples representing 295 medicinal species from 7 primary TCM markets in China. The results indicate that ITS2 barcodes could be generated for most of the samples (87.7%) using a standard protocol. Of the 1260 samples, approximately 4.2% were identified as adulterants. The adulterant focused on medicinal species such as Ginseng Radix et Rhizoma (Renshen), Radix Rubi Parvifolii (Maomeigen), Dalbergiae odoriferae Lignum (Jiangxiang), Acori Tatarinowii Rhizoma (Shichangpu), Inulae Flos (Xuanfuhua), Lonicerae Japonicae Flos (Jinyinhua), Acanthopanacis Cortex (Wujiapi) and Bupleuri Radix (Chaihu). The survey revealed that adulterant species are present in the Chinese market, and these adulterants pose a risk to consumer health. Thus, regulatory measures should be adopted immediately. We suggest that a traceable platform based on DNA barcode sequences be established for TCM market supervision.
The presence of duplicates introduced by PCR amplification is a major issue in paired short reads from next-generation sequencing platforms. These duplicates might have a serious impact on research ...applications, such as scaffolding in whole-genome sequencing and discovering large-scale genome variations, and are usually removed. We present FastUniq as a fast de novo tool for removal of duplicates in paired short reads. FastUniq identifies duplicates by comparing sequences between read pairs and does not require complete genome sequences as prerequisites. FastUniq is capable of simultaneously handling reads with different lengths and results in highly efficient running time, which increases linearly at an average speed of 87 million reads per 10 minutes. FastUniq is freely available at http://sourceforge.net/projects/fastuniq/.
Summary
Danshen, Salvia miltiorrhiza Bunge, is one of the most widely used herbs in traditional Chinese medicine, wherein its rhizome/roots are particularly valued. The corresponding bioactive ...components include the tanshinone diterpenoids, the biosynthesis of which is a subject of considerable interest. Previous investigations of the S. miltiorrhiza transcriptome have relied on short‐read next‐generation sequencing (NGS) technology, and the vast majority of the resulting isotigs do not represent full‐length cDNA sequences. Moreover, these efforts have been targeted at either whole plants or hairy root cultures. Here, we demonstrate that the tanshinone pigments are produced and accumulate in the root periderm, and apply a combination of NGS and single‐molecule real‐time (SMRT) sequencing to various root tissues, particularly including the periderm, to provide a more complete view of the S. miltiorrhiza transcriptome, with further insight into tanshinone biosynthesis as well. In addition, the use of SMRT long‐read sequencing offered the ability to examine alternative splicing, which was found to occur in approximately 40% of the detected gene loci, including several involved in isoprenoid/terpenoid metabolism.
Significance Statement
Here, we combined NGS and SMRT sequencing to generate a more complete/full‐length S. miltiorrhiza transcriptome. Our study provides a general model for studying the full‐length transcriptome, alternative splicing, and the biosynthesis of active constituents in medicinal plants, as well as important information about the important Danshen constituent of traditional Chinese medicine more specifically.
There is a long history of traditional medicine use. However, little genetic information is available for the plants used in traditional medicine, which limits the exploitation of these natural ...resources. Third-generation sequencing (TGS) techniques have made it possible to gather invaluable genetic information and develop herbal genomics. In this review, we introduce two main TGS techniques, PacBio SMRT technology and Oxford Nanopore technology, and compare the two techniques against Illumina, the predominant next-generation sequencing technique. In addition, we summarize the nuclear and organelle genome assemblies of commonly used medicinal plants, choose several examples from genomics, transcriptomics, and molecular identification studies to dissect the specific processes and summarize the advantages and disadvantages of the two TGS techniques when applied to medicinal organisms. Finally, we describe how we expect that TGS techniques will be widely utilized to assemble telomere-to-telomere (T2T) genomes and in epigenomics research involving medicinal plants.
Background: The plant working group of the Consortium for the Barcode of Life recommended the two-locus combination of rbcL + matK as the plant barcode, yet the combination was shown to successfully ...discriminate among 907 samples from 550 species at the species level with a probability of 72%. The group admits that the two-locus barcode is far from perfect due to the low identification rate, and the search is not over. Methodology/Principal Findings: Here, we compared seven candidate DNA barcodes (psbA-trnH, matK, rbcL, rpoC1, ycf5, ITS2, and ITS) from medicinal plant species. Our ranking criteria included PCR amplification efficiency, differential intra- and inter-specific divergences, and the DNA barcoding gap. Our data suggest that the second internal transcribed spacer (ITS2) of nuclear ribosomal DNA represents the most suitable region for DNA barcoding applications. Furthermore, we tested the discrimination ability of ITS2 in more than 6600 plant samples belonging to 4800 species from 753 distinct genera and found that the rate of successful identification with the ITS2 was 92.7% at the species level. Conclusions: The ITS2 region can be potentially used as a standard DNA barcode to identify medicinal plants and their closely related species. We also propose that ITS2 can serve as a novel universal barcode for the identification of a broader range of plant taxa.
American ginseng (Panax quinquefolius L.) is one of the most widely used herbal remedies in the world. Its major bioactive constituents are the triterpene saponins known as ginsenosides. However, ...little is known about ginsenoside biosynthesis in American ginseng, especially the late steps of the pathway.
In this study, a one-quarter 454 sequencing run produced 209,747 high-quality reads with an average sequence length of 427 bases. De novo assembly generated 31,088 unique sequences containing 16,592 contigs and 14,496 singletons. About 93.1% of the high-quality reads were assembled into contigs with an average 8-fold coverage. A total of 21,684 (69.8%) unique sequences were annotated by a BLAST similarity search against four public sequence databases, and 4,097 of the unique sequences were assigned to specific metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes. Based on the bioinformatic analysis described above, we found all of the known enzymes involved in ginsenoside backbone synthesis, starting from acetyl-CoA via the isoprenoid pathway. Additionally, a total of 150 cytochrome P450 (CYP450) and 235 glycosyltransferase unique sequences were found in the 454 cDNA library, some of which encode enzymes responsible for the conversion of the ginsenoside backbone into the various ginsenosides. Finally, one CYP450 and four UDP-glycosyltransferases were selected as the candidates most likely to be involved in ginsenoside biosynthesis through a methyl jasmonate (MeJA) inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay.
We demonstrated, with the assistance of the MeJA inducibility experiment and tissue-specific expression pattern analysis, that transcriptome analysis based on 454 pyrosequencing is a powerful tool for determining the genes encoding enzymes responsible for the biosynthesis of secondary metabolites in non-model plants. Additionally, the expressed sequence tags (ESTs) and unique sequences from this study provide an important resource for the scientific community that is interested in the molecular genetics and functional genomics of American ginseng.
Angiogenesis, in which vascular endothelial growth factor receptor (VEGFR) 2 plays an essential role, is associated with a variety of human diseases including proliferative diabetic retinopathy and ...wet age-related macular degeneration. Here we report that a system of adeno-associated virus (AAV)-mediated clustered regularly interspaced short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) is used to deplete VEGFR2 in vascular endothelial cells (ECs), whereby the expression of SpCas9 is driven by an endothelial-specific promoter of intercellular adhesion molecule 2. We further show that recombinant AAV serotype 1 (rAAV1) transduces ECs of pathologic vessels, and that editing of genomic VEGFR2 locus using rAAV1-mediated CRISPR/Cas9 abrogates angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization. This work establishes a strong foundation for genome editing as a strategy to treat angiogenesis-associated diseases.Abnormal angiogenesis causes many ocular diseases. Here the authors employ CRISPR/Cas9 gene editing technology to silence VEGFR2, a major regulator of angiogenesis, in retinal endothelium and abrogate angiogenesis in the mouse models of oxygen-induced retinopathy and laser-induced choroid neovascularization.
Five DNA regions, namely, rbcL, matK, ITS, ITS2, and psbA-trnH, have been recommended as primary DNA barcodes for plants. Studies evaluating these regions for species identification in the large ...plant taxon, which includes a large number of closely related species, have rarely been reported.
The feasibility of using the five proposed DNA regions was tested for discriminating plant species within Asteraceae, the largest family of flowering plants. Among these markers, ITS2 was the most useful in terms of universality, sequence variation, and identification capability in the Asteraceae family. The species discriminating power of ITS2 was also explored in a large pool of 3,490 Asteraceae sequences that represent 2,315 species belonging to 494 different genera. The result shows that ITS2 correctly identified 76.4% and 97.4% of plant samples at the species and genus levels, respectively. In addition, ITS2 displayed a variable ability to discriminate related species within different genera.
ITS2 is the best DNA barcode for the Asteraceae family. This approach significantly broadens the application of DNA barcoding to resolve classification problems in the family Asteraceae at the genera and species levels.
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