A chromosome-level genome assembly for Dracaena cochinchinensis reveals the molecular basis of its longevity and formation of dragon's blood.

Dracaena, a remarkably long-lived and slowly maturing species of plant, is world famous for its ability to produce dragon's blood, a precious traditional medicine used by different cultures since ancient times. However, there is no detailed and high-quality genome available for this species at present; thus, the molecular mechanisms that underlie its important traits are largely unknown. These factors seriously limit the protection and regeneration of this rare and endangered plant resource. Here, we sequenced and assembled the genome of Dracaena cochinchinensis at the chromosome level. The D. cochinchinensis genome covers 1.21 Gb with a scaffold N50 of 50.06 Mb and encodes 31 619 predicted protein-coding genes. Analysis showed that D. cochinchinensis has undergone two whole-genome duplications and two bursts of long terminal repeat insertions. The expansion of two gene classes, cis-zeatin O-glucosyltransferase and small auxin upregulated RNA, were found to account for its longevity and slow growth. Two transcription factors (bHLH and MYB) were found to be core regulators of the flavonoid biosynthesis pathway, and reactive oxygen species were identified as the specific signaling molecules responsible for the injury-induced formation of dragon's blood. Our study provides high-quality genomic information relating to D. cochinchinensis and significant insight into the molecular mechanisms responsible for its longevity and formation of dragon's blood. These findings will facilitate resource protection and sustainable utilization of Dracaena.

Genome-wide investigation and expression analysis of the AP2/ERF family for selection of agarwood-related genes in Aquilaria sinensis (Lour.) Gilg.

Aquilaria sinensis is an important non-timber tree species for producing high-value agarwood, which is widely used as a traditional medicine and incense. Agarwood is the product of Aquilaria trees in response to injury and fungal infection. The APETALA2/ethylene responsive factor (AP2/ERF) transcription factors (TFs) play important roles in plant stress responses and metabolite biosynthesis. In this study, 119 AsAP2/ERF genes were identified from the A. sinensis genome and divided into ERF, AP2, RAV, and Soloist subfamilies. Their conserved motif, gene structure, chromosomal localization, and subcellular localization were characterized. A stress/defense-related ERF-associated amphiphilic repression (EAR) motif and an EDLL motif were identified. Moreover, 11 genes that were highly expressed in the agarwood layer in response to whole-tree agarwood induction technique (Agar-Wit) treatment were chosen, and their expression levels in response to methyl jasmonate (MeJA), salicylic acid (SA), or salt treatment were further analyzed using the quantitative real time PCR (qRT-PCR). Among the 11 genes, eight belonged to subgroup B-3. All 11 genes were significantly upregulated under salt treatment, while eight genes were significantly induced by both MeJA and SA. In addition, the gene clusters containing these upregulated genes on chromosomes were observed. The results obtained from this research not only provide useful information for understanding the functions of AP2/ERF genes in A. sinensis but also identify candidate genes and gene clusters to dissect their regulatory roles in agarwood formation for future research.

[Panax notoginseng: a review on chemical components, chromatographic analysis, P. notoginseng extracts, and pharmacology in recent five years].

As a famous and precious Chinese medicinal material, Panax notoginseng(PN) has been commonly used for a long history in China. As reported, PN exhibits significant pharmacological actions in protecting cardiocerebral vascular system and nervous system and suppressing tumors. In recent years, with the innovation in ideas, as well as the development of methods and equipment, PN has been extensively investigated, and notable progress has been made. This paper reviewed the advancements of PN in recent five years from chemical components, chromatographic analysis, P. notoginseng extracts, and pharmacology, in which the application of PN extracts in quality control was first summarized. The present study aims to provide a theoretical basis for quality control, product development, and rational medication of PN.

The complete chloroplast genome sequence of the medicinal plant Persicaria filiformis.

Persicaria filiformis, known as a traditional Chinese herbal medicine, is a perennial plant of Polygonaceae wildly distributed in China. The complete chloroplast (cp) genome of P. filiformis was assembled and analyzed in this study. The length of the circular genome is 159,741 bp, with a rich GC content of 41.3%. The cp genome structure consists of a large single-copy region (LSC 84,432 bp), a small single-copy region (SSC 13,073 bp) and a pair of inverted repeat regions (IR 31,118 bp). The complete genome encodes 130 genes, including 85 protein-coding genes, 37 tRNA genes and 8 rRNA genes. Phylogenetic analysis indicated that P. filiformis is most related to P. japonica.

The Separation of Antler Polypeptide and Its Effects on the Proliferation and Osteogenetic Differentiation of Bone Marrow Mesenchymal Stem Cells.

BACKGROUND: Colla Cornus Cervi (CCC) has been used as a traditional Chinese medicine in the treatment of osteoporosis and osteonecrosis of the femoral head. However, the bioavailability of CCC is seriously limited owing to its large molecular weight and complex ingredients. In the present study, antler polypeptide was separated from CCC, and the effects of antler polypeptide on rat bone marrow mesenchymal stem cells (BMSCs) were investigated. METHODS: Antler polypeptide was separated from Colla Cornus Cervi by ultrafiltration into different samples according to the molecular weight. The total peptide content of these samples was determined by the biuret method. The content of antler polypeptide in different samples was quantified by high-performance liquid chromatography (HPLC). The effects of antler polypeptide at different concentrations on the proliferation, cell cycle, alkaline phosphatase activity, and BMP7 expression of BMSCs were investigated. RESULTS: Antler polypeptide was separated by ultrafiltration into different samples: A (molecular weight <800 Da), B (molecular weight 800-1500 Da), and C (molecular weight >1500 Da). The total peptide contents of A, B, and C were 0.602 mg/mL, 8.976 mg/mL, and 38.88 mg/mL. Antler polypeptide B eluted at 14.279∼15.351 min showed that the content of antler polypeptide was significantly higher than that of A and C with a peak area of 933.80927. The BMSCs proliferation rate (84.66%) of polypeptide B was the highest at the concentration of 1.578 × 10-2 g/mL. Antler polypeptide B significantly promoted the proliferation of BMSCs with a proliferation index of 38.68%, which was significantly higher than that of the other groups. Antler polypeptide B significantly enhanced the activity of alkaline phosphatase in BMSCs compared to that of the blank group (P < 0.001). Antler polypeptide B increased the BMP7 protein expression in BMSCs. CONCLUSIONS: Results suggested that antler polypeptide may promote the proliferation and osteogenic differentiation of BMSCs. Our study lays an experimental foundation for the further development and application of antler polypeptide in medicine.

[Characterization of AOX family members from Aquilaria sinensis and their responses to wounding].

Aquilaria sinensis is a typical inducible medicinal plant, that can produce agarwood only after it is wounded by external stimuli. Alternative oxidase(AOX) is one of the terminal oxidases of the plant mitochondrial electron transport, which plays an important role in plants' response to environmental stress. In order to reveal the physiological function of AOX gene in the process of agarwood formation from A.sinensis induced by wounding, AOX gene was cloned based on the transcriptome database and then identified by the bioinformatics analysis, and their expression pattern in different tissues and under wounding stress were detected by qRT-PCR. The results as follows. Three AOX genes were cloned from A.sinensis for the first time. They were named AsAOX1a, AsAOX1d and AsAOX2, respectively. The tissue expression shown that AsAOX1a is mainly expressed in the stem and the seed, and the AsAOX1d and AsAOX2 genes are mainly expressed in the pulp and the stem. AsAOX1a and AsAOX1d genes are highly responsive to wounding stress, and their response time was different. In addition, the expression of AsAOX1a and AsAOX2 induced by wounding are reduced by H_2O_2 treatment, but promoted by AsA treatment. The cloning, bioinformatics analysis and expression characteristics of AOX genes from A.sinensis provided basic information for further study the function of AOX genes in the development of A.sinensis, especially in the process of agarwood formation of A. sinensis induced by wounding.

Molecular Mechanism Underlying Mechanical Wounding-Induced Flavonoid Accumulation in Dalbergia odorifera T. Chen, an Endangered Tree That Produces Chinese Rosewood.

Dalbergia odorifera, a critically endangered tree species, produces heartwood containing a vast variety of flavonoids. This heartwood, also known as Chinese rosewood, has high economic and medicinal value, but its formation takes several decades. In this study, we showed that discolored wood induced by pruning displays similar color, structure, and flavonoids content to those of natural heartwood, suggesting that wounding is an efficient method for inducing flavonoid production in D. odorifera. Transcriptome analysis was performed to investigate the mechanism underlying wounding-induced flavonoids production in D. odorifera heartwood. Wounding upregulated the expression of 90 unigenes, which covered 19 gene families of the phenylpropanoid and flavonoid pathways, including PAL, C4H, 4CL, CHS, CHI, 6DCS, F3'5'H, F3H, FMO, GT, PMAT, CHOMT, IFS, HI4'OMT, HID, IOMT, I2'H, IFR, and I3'H. Furthermore, 47 upregulated unigenes were mapped to the biosynthesis pathways for five signal molecules (ET, JA, ABA, ROS, and SA). Exogenous application of these signal molecules resulted in the accumulation of flavonoids in cell suspensions of D. odorifera, supporting their role in wounding-induced flavonoid production. Insights from this study will help develop new methods for rapidly inducing the formation of heartwood with enhanced medicinal value.

WRKY44 represses expression of the wound-induced sesquiterpene biosynthetic gene ASS1 in Aquilaria sinensis.

Agarwood is derived from wounds in Aquilaria trees and is widely used in traditional medicine, incense, and perfume. Sesquiterpenes are one of the main active components in agarwood and are known to be induced by wounding or injury; However, the molecular mechanisms by which wounding leads to sesquiterpene formation remain largely unknown. Agarwood sesquiterpene synthase 1 (ASS1) is one of key enzymes responsible for the biosynthesis of sesquiterpenes and is a crucial jasmonate (JA)-responsive wound-inducible synthase. However, it is not known why ASS1 is not expressed in healthy trees and how its expression is induced as a result of wounding. Here, we report that ASS1 is a wound-induced gene with a promoter in which a 242-bp region (-973 to -731bp) is identified as the core sequence for responding to wound signals. AsWRKY44 binds directly to this region and represses ASS1 promoter activity. Down-regulation or disruption of AsWRKY44 can relieve the inhibition and activate ASS1 expression. In addition, AsWRKY44 is degraded and the expression of ASS1 is significantly up-regulated in response to exogenous application of methyl jasmonate. Thus, AsWRKY44 is a crucial negative regulator of wound-induced ASS1 transcription, and is central to the mechanism of sesquiterpene biosynthesis in agarwood.

Treatment with metformin and sorafenib alleviates endometrial hyperplasia in polycystic ovary syndrome by promoting apoptosis via synergically regulating autophagy.

This study aimed to investigate the molecular mechanisms underlying the roles of metformin (MET) and Sorafenib (SOR) in the treatment of endometrial hyperplasia (EH) in polycystic ovary syndrome (PCOS). Effects of MET and SOR on the area of endometrium and myometrium were detected. Western blot analysis and immunohistochemistry assays were carried out to detect the levels of mammalian target of rapamycin complex 1 (mTORC1), mTORC2, LC3-II, P62, and Caspase-3 in rats and cultured cells. Furthermore, cell counting kit-8 assay and flow cytometry analysis was carried out to determine the apoptotic profiles of treated cells. MET and SOR could apparently decrease the areas of endometrium and myometrium in PCOS. MET notably enhanced the expression of LC3-II and Caspase-3 in PCOS while substantially reducing the level of mTORC1 and P62. Similarly, SOR also enhanced the expression of LC3-II and Caspase-3 in PCOS while substantially reducing the level of mTORC2 and P62. Treatment with MET and SOR significantly inhibited the proliferation of HCC-94 and HEC-1-A cells while promoting their apoptosis by upregulating the expression of Caspase-3. In cells treated with MET, the expression of mTORC1 and LC3-II was upregulated while the expression of P62 was downregulated. Similarly, in cells treated with SOR, the expression of mTORC2 and LC3-II was also upregulated while the expression of P62 was also downregulated. Furthermore, MET showed no effect on mTORC2 expression, while SOR showed no effect on mTORC1 expression. In this study, we suggested that MET and SOR alleviated the risk of EH in PCOS via the mTORC1/autophagy/apoptosis axis and mTORC2/autophagy/apoptosis axis, respectively.

Selection and Validation of Reference Genes for Gene Expression Studies by RT-PCR in Dalbergia odorifera.

Perennial tree Dalbergia odorifera T. Chen could form the precious heartwood used to produce chinese traditional medicine, rosewood furniture and fragrances. However the formation of heartwood is time-consuming and low efficient, leading to the severe destruction of its wild resources. Thus, it is urgent to study the molecular mechanism of heartwood formation in D. odorifera. But till now, there is no report about the reference gene selection in this species. In this study, the expression stability of nine candidate reference genes were evaluated across different tissues and stems treated by wound and chemical stimulators. Four algorithms were applied to obtain the robust genes. The results support HIS2, GAPDH, and CYP to be the most stable reference genes in samples under different wound treatments while DNAj was the least stable. In different tissues, HIS2, UBQ, and RPL were the most stable reference genes while DNAj was the least stable. The selected reference genes were validated through the normalization of the qRT-PCR data of six heartwood related genes in terpene biosynthesis pathway and ethylene signal pathway. The results showed that their expression levels were accurate when they were normalized by the most stable reference gene HIS2, or by the combination of the two or three most stable reference genes. These results demonstrated that these selected reference genes are reliable.

[Advances in study of plant type â ¢ polyketide synthases].

Polyketides are a large class of natural products with notable structural diversity and different biological activities. They have essential pharmacological value for human health. In plants, the enzymes responsible for the formation of phenolic metabolites backbone structures are collectively known as type Ⅲ polyketide synthases (PKSs), which are the key enzymes for the polyketides biosynthesis. The PKSs catalyze a series of condensation reactions of two-carbon acetate units with an acyl starter. A brief overview of this group of enzymes, including their reaction mechanisms, function modification, expression regulation, molecular evolution, and recent interesting findings are presented here.

[Cloning and gene expression of acetyl-CoA C-acetyl transferase gene (AsAACT) from Aquilaria sinensis].

OBJECTIVE: This study aimed to clone the acetyl-CoA C-acetyl transferase (AACT) gene from Aquilaria sinensis and analyze the bioinformatics and expression of the gene. METHOD: One unique sequence containing partly AACT gene sequence was discovered in our previous transcriptome dataset of A. sinensis. AACT gene was cloned by RT-PCR and RACE strategy with the template of RNA extracted from A. sinensis stem. The bioinformatic analysis of this gene and its corresponding protein was performed. The AsAACT expression in calli was analyzed with GADPH gene as an internal control gene in wounded condition by qRT-PCR technique. RESULT: One unique sequence of AACT, named as AsAACT, was cloned from A. sinensis. The full length of AsAACT cDNA was containing a 1 236 bp ORF that encoded 411 amino acids. The result of qRT-PCR displayed that the highest expression level was at 4 h. which indicated that it was possibly involved in early-stage response to wound. CONCLUSION: Cloning and analyzing AsAACT gene from A. sinensis provided basic information for study the function and expression regulation of AsAACT in terpenoid biosynthesis.

[Study of production of sesquiterpenes of Aquilaria senensis stimulated by Lasiodiplodia theobromae].

To investigate the mechanism of agarwood formation in Aquilaria sinensis induced by Lasiodiplodia theobromae, the fermentation liquor of L. theobromae was analyzed qualitatively and quantitatively by gas chromatography-mass spectrometry (GC-MS). JAs were detected in the fermentation liquor. The effect of the fermentation liquor on the abundance of sesquiterpenes in the callus of A. sinensis was analyzed by solid phase microextraction-gas chromatography-mass spectrometry (SPME-GC-MS). And the fermentation liquor stimulated alpha-guaiene, alpha-humulene and delta-guaiene biosynthesis in calli. It was inferred that L. theobromae produced JAs, which resulted in a significant increase of sesquiterpenes in A. sinensis.

Whole-tree agarwood-inducing technique: an efficient novel technique for producing high-quality agarwood in cultivated Aquilaria sinensis trees.

Agarwood is the fragrant resin-infused wood derived from the wounded trees of Aquilaria species. It is a valuable non-timber forest product used in fragrances and as medicine. Reforestation for Aquilaria trees in combination with artificial agarwood-inducing methods serves as a way to supply agarwood and conserve of wild Aquilaria stock. However, the existing agarwood-inducing methods produce poor-quality agarwood at low yield. Our study evaluated a novel technique for producing agarwood in cultivated Aquilaria trees, called the whole-tree agarwood-inducing technique (Agar-Wit). Ten different agarwood inducers were used for comparison of Agar-Wit with three existing agarwood-inducing methods. For Aquilaria trees treated with these ten inducers, agarwood formed and spread throughout the entire tree from the transfusion point in the trunk to the roots and branches of the whole tree. Agarwood yield per tree reached 2,444.83 to 5,860.74 g, which is 4 to 28 times higher than that by the existing agarwood-inducing methods. Furthermore, this agarwood derived from Agar-Wit induction was found to have a higher quality compared with the existing methods, and similar to that of wild agarwood. This indicates Agar-Wit may have commercial potential. Induction of cultivated agarwood using this method could satisfy the significant demand for agarwood, while conserving and protecting the remaining wild Aquilaria trees.

[The mechanical wound transcriptome of three-year-old Aquilaria sinensis].

Chinese agarwood is formed in the aromatic resinous wood formed in Aquilaria sinensis (Lour.) Gilg (botanical family: Thymelaeaceae). Only when suffering stress of wound, etc, can A. sinensis produce sesquiterpenes etc. compounds of agarwood around wounds. However, little is known about how wound induced the biosynthesis pathway of sesquiterpenes. To reveal the molecular mechanism of wound-induced agarwood formation, RNA sequencing (RNA-seq) technology was used to investigate the profile of gene expression in A. sinensis treated by mechanical wounding and elucidate its functional gene. A total of 40,295 ESTs with an average read length of 305 bp were generated and 22 095 unigenes were formed by initial gene splicing. 61.6% of these unigenes (13 611) were annotated using BLAST searches against the SwissProt, KEGG, Nr and Nt databases. Twenty-six unigenes (encoding 7 enzymes) were found to be involved in sesquiterpene of agarwood biosynthesis by bioinformatic tools of Gene Ontology and KEGG. Novel genes that are potentially involved in sesquiterpenes biosynthesis were identified in A. sinensis, providing data for further sesquiterpenes biosynthesis pathway by molecular methods and the EST data establish a foundation for future studies in the molecular mechanisms of wound-induce agarwood formation in A. sinensis.

Transcriptome analysis of Bupleurum chinense focusing on genes involved in the biosynthesis of saikosaponins.

BACKGROUND: Bupleurum chinense DC. is a widely used traditional Chinese medicinal plant. Saikosaponins are the major bioactive constituents of B. chinense, but relatively little is known about saikosaponin biosynthesis. The 454 pyrosequencing technology provides a promising opportunity for finding novel genes that participate in plant metabolism. Consequently, this technology may help to identify the candidate genes involved in the saikosaponin biosynthetic pathway. RESULTS: One-quarter of the 454 pyrosequencing runs produced a total of 195, 088 high-quality reads, with an average read length of 356 bases (NCBI SRA accession SRA039388). A de novo assembly generated 24, 037 unique sequences (22, 748 contigs and 1, 289 singletons), 12, 649 (52.6%) of which were annotated against three public protein databases using a basic local alignment search tool (E-value ≤1e-10). All unique sequences were compared with NCBI expressed sequence tags (ESTs) (237) and encoding sequences (44) from the Bupleurum genus, and with a Sanger-sequenced EST dataset (3, 111). The 23, 173 (96.4%) unique sequences obtained in the present study represent novel Bupleurum genes. The ESTs of genes related to saikosaponin biosynthesis were found to encode known enzymes that catalyze the formation of the saikosaponin backbone; 246 cytochrome P450 (P450s) and 102 glycosyltransferases (GTs) unique sequences were also found in the 454 dataset. Full length cDNAs of 7 P450s and 7 uridine diphosphate GTs (UGTs) were verified by reverse transcriptase polymerase chain reaction or by cloning using 5' and/or 3' rapid amplification of cDNA ends. Two P450s and three UGTs were identified as the most likely candidates involved in saikosaponin biosynthesis. This finding was based on the coordinate up-regulation of their expression with ß-AS in methyl jasmonate-treated adventitious roots and on their similar expression patterns with ß-AS in various B. chinense tissues. CONCLUSIONS: A collection of high-quality ESTs for B. chinense obtained by 454 pyrosequencing is provided here for the first time. These data should aid further research on the functional genomics of B. chinense and other Bupleurum species. The candidate genes for enzymes involved in saikosaponin biosynthesis, especially the P450s and UGTs, that were revealed provide a substantial foundation for follow-up research on the metabolism and regulation of the saikosaponins.

Combined effect of total alkaloids from Feculae Bombycis and natural flavonoids on diabetes.

Both total alkaloids from Feculae Bombycis (TAFB) and natural flavonoids can inhibit alpha-glucosidase activity to depress the glucose level in blood. To investigate the cooperative effect of TAFB and flavonoids on blood glucose, we have studied their combined function compared with individual ingredients on enzymology, in-vitro and in-vivo. In the enzymological assay, the combination of TAFB and flavonoids showed more effective inhibition, compared with either TAFB or flavonoids alone, to alpha-glucosidase activity. In the everted intestine model in-vitro, the combined inhibition of starch hydrolysation and glucose transference to blood was much stronger than with separate components. In short-term studies with normal and experimentally-induced diabetic mice in-vivo, the combination of TAFB and flavonoids also had a stronger suppressive effect on the postprandial elevation in blood glucose after oral administration. In long-term treatment to diabetic mice in-vivo, the compound prescription could depress not only the fasting blood glucose, but also the fasting blood total cholesterol. These results demonstrated that TAFB and flavonoids could inhibit alpha-glucosidase activity cooperatively, which would successfully depress blood glucose level in the therapy of diabetes.