Combined analysis of Polygonum cuspidatum transcriptome and metabolome revealed that PcMYB62, a transcription factor, responds to methyl jasmonate and inhibits resveratrol biosynthesis.

A comparative transcriptomic and metabolomic analysis of Polygonum cuspidatum leaves treated with MeJA was carried out to investigate the regulatory mechanisms of its active compounds. A total of 692 metabolites and 77,198 unigenes were obtained, including 200 differentially accumulated metabolites and 6819 differentially expressed genes. We screened potential regulatory transcription factors involved in resveratrol and flavonoids biosynthesis, and successfully identified an MYB transcription factor, PcMYB62, which could significantly decrease the resveratrol content in P. cuspidatum leaves when over-expressed. PcMYB62 could directly bind to the MBS motifs in the promoter region of stilbene synthase (PcSTS) gene and repress its expression. Besides, PcMYB62 could also repress PcSTS expression and resveratrol biosynthesis in transgenic Arabidopsis thaliana. Our results provide abundant candidate genes for further investigation, and the new finding of the inhibitory role of PcMYB62 on the resveratrol biosynthesis could also potentially be used in metabolic engineering of resveratrol in P. cuspidatum.

[Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum].

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Investigation of the Potential Key Genes and the Multitarget Mechanisms of Polygonum cuspidatum against Heart Failure Based on Network Pharmacology and Experimental Validation.

In this study, systematic pharmacology and bioinformatic approaches were employed to identify the potential targets of Polygonum cuspidatum (PC) for treating heart failure (HF). The active ingredients of PC were screened by using the TCMSP database, and HF-related genes were identified in the GEO database. Then, the herb-HF targeted-gene networks were constructed using Cytoscape software. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional analyses were performed to obtain the enriched molecular pathways associated with the pathogenesis of HF. Finally, in vitro experiment was performed to evidence network pharmacology analysis. 170 intersection genes were obtained, and key genes (FOXO3, NFKB1, and TNF) were identified. Besides, GO and KEGG findings indicated that PC treatment of HF was achieved via regulating apoptosis, IL-17 signaling pathway, TNF signaling pathway, response to oxidative stress, and response to reactive oxygen species. And cell experiment revealed that PC could decrease the expression of NFKB1 and TNF and increase the expression of FOXO3, SOD1, and GPX1 in H9C2 cells. These findings showed that the therapeutic mechanism of PC in the treatment of HF may be associated with the regulation of inflammation-related and oxidative stress-related genes.

PcWRKY11, an II-d WRKY Transcription Factor from Polygonum cuspidatum, Enhances Salt Tolerance in Transgenic Arabidopsis thaliana.

Being an invasive plant, Polygonum cuspidatum is highly resilient and can survive in unfavorable environments for long periods; however, its molecular mechanisms associated with such environmental resistance are largely unknown. In this study, a WRKY transcription factor (TF) gene, PcWRKY11, was identified from P. cuspidatum by analyzing methyl jasmonate (MeJA)-treated transcriptome data. It showed a high degree of homology with WRKY11 from Arabidopsis thaliana, containing a WRKY domain and a zinc finger structure and II-d WRKY characteristic domains of HARF, a calmodulin-binding domain (C-motif), and a putative nuclear localization signal (NLS) through sequence alignment and functional element mining. qPCR analysis showed that the expression of PcWRKY11 can be induced by NaCl, osmotic stress, and UV-C. In this study, we also found that overexpression of PcWRKY11 in A. thaliana could significantly increase salt tolerance. To explore its possible molecular mechanism, further investigations showed that compared with the wild type (WT), under salt stress, the transgenic plants showed a lower malondialdehyde (MDA) content, higher expression of ascorbate peroxidase (APX) and superoxide dismutase (SOD), and higher enzyme activity of peroxidase (POD), superoxide dismutase (SOD), and catalase (CAT). Moreover, the transgenic plants also showed higher expression of Δ1-pyrroline-5-carboxylate synthase (AtP5CS), and higher contents of proline and soluble sugar. Taken together, these results indicate that PcWRKY11 may have a positive role in plants' adaptation to salinity conditions by reducing reactive oxygen species (ROS) levels and increasing osmosis substance synthesis.

Expression, purification and structural characterization of resveratrol synthase from Polygonum cuspidatum.

Polygonum cuspidatum, an important medicinal plant in China, is a rich source of resveratrol compounds, and its synthesis related resveratrol synthase (RS) gene is highly expressed in stems. The sequence of the resveratrol synthase was amplified with specific primers. Sequence comparison showed that it was highly homologous to the STSs. The RS gene of Polygonum cuspidatum encodes 389 amino acids and has a theoretical molecular weight of 42.4 kDa, which is called PcRS1. To reveal the molecular basis of the synthesized resveratrol activity of PcRS1, we expressed the recombinant protein of full-length PcRS1 in Escherichia coli, and soluble protein products were produced. The collected products were purified by Ni-NTA chelation chromatography and appeared as a single band on SDS-PAGE. In order to obtain higher purity PcRS1, SEC was used to purify the protein and sharp single peak, and DLS detected that the aggregation state of protein molecules was homogeneous and stable. In order to verify the enzyme activity of the high-purity PcRS1, the reaction product was detected at 303 nm. By predicting the structural information of monomer PcRS1 and PcRS1 ligand complexes, we analyzed the ligand binding pocket and protein surface electrostatic potential of the complex, and compared it with the highly homologous STSs protein structures of the iso-ligand. New structural features of protein evolution are proposed. PcRS1 obtained a more complete configuration and the optimal orientation of the active site residues, thus improving its catalytic capacity in resveratrol synthesis.

Regional differences in clonal Japanese knotweed revealed by chemometrics-linked attenuated total reflection Fourier-transform infrared spectroscopy.

BACKGROUND: Japanese knotweed (R. japonica var japonica) is one of the world's 100 worst invasive species, causing crop losses, damage to infrastructure, and erosion of ecosystem services. In the UK, this species is an all-female clone, which spreads by vegetative reproduction. Despite this genetic continuity, Japanese knotweed can colonise a wide variety of environmental habitats. However, little is known about the phenotypic plasticity responsible for the ability of Japanese knotweed to invade and thrive in such diverse habitats. We have used attenuated total reflection Fourier-transform infrared (ATR-FTIR) spectroscopy, in which the spectral fingerprint generated allows subtle differences in composition to be clearly visualized, to examine regional differences in clonal Japanese knotweed. RESULTS: We have shown distinct differences in the spectral fingerprint region (1800-900 cm- 1) of Japanese knotweed from three different regions in the UK that were sufficient to successfully identify plants from different geographical regions with high accuracy using support vector machine (SVM) chemometrics. CONCLUSIONS: These differences were not correlated with environmental variations between regions, raising the possibility that epigenetic modifications may contribute to the phenotypic plasticity responsible for the ability of R. japonica to invade and thrive in such diverse habitats.

De Novo Biosynthesis of Polydatin in Saccharomyces cerevisiae.

Polydatin, with better structural stability and biological activities than resveratrol, is mainly extracted from the traditional Chinese medicinal plant Polygonum cuspidatum. In this study, based on the transcriptome analysis of P. cuspidatum, we identified the key glycosyltransferase of resveratrol and achieved the biosynthesis of polydatin from glucose by incorporation with the resveratrol biosynthesis module, UDP-glucose supply module, and glycosyltransferase expression module. Through metabolic engineering and fermentation optimization, the production of polydatin reached 545 mg/L, and the dry cell weight was 27.83 mg/g DCW, which was about twice that of extracted from the P. cuspidatum root (11.404 mg/g DCW). Therefore, it is possible to replace the production mode of polydatin from plant extraction to microbial chassis in the future.

Tissue-specific transcriptome analyses reveal candidate genes for stilbene, flavonoid and anthraquinone biosynthesis in the medicinal plant Polygonum cuspidatum.

BACKGROUND: Polygonum cuspidatum Sieb. et Zucc. is a well-known medicinal plant whose pharmacological effects derive mainly from its stilbenes, anthraquinones, and flavonoids. These compounds accumulate differentially in the root, stem, and leaf; however, the molecular basis of such tissue-specific accumulation remains poorly understood. Because tissue-specific accumulation of compounds is usually associated with tissue-specific expression of the related biosynthetic enzyme genes and regulators, we aimed to clarify and compare the transcripts expressed in different tissues of P. cuspidatum in this study. RESULTS: High-throughput RNA sequencing was performed using three different tissues (the leaf, stem, and root) of P. cuspidatum. In total, 80,981 unigenes were obtained, of which 40,729 were annotated, and 21,235 differentially expressed genes were identified. Fifty-four candidate synthetase genes and 12 transcription factors associated with stilbene, flavonoid, and anthraquinone biosynthetic pathways were identified, and their expression levels in the three different tissues were analyzed. Phylogenetic analysis of polyketide synthase gene families revealed two novel CHS genes in P. cuspidatum. Most phenylpropanoid pathway genes were predominantly expressed in the root and stem, while methylerythritol 4-phosphate and isochorismate pathways for anthraquinone biosynthesis were dominant in the leaf. The expression patterns of synthase genes were almost in accordance with metabolite profiling in different tissues of P. cuspidatum as measured by high-performance liquid chromatography or ultraviolet spectrophotometry. All predicted transcription factors associated with regulation of the phenylpropanoid pathway were expressed at lower levels in the stem than in the leaf and root, but no consistent trend in their expression was observed between the leaf and the root. CONCLUSIONS: The molecular knowledge of key genes involved in the biosynthesis of P. cuspidatum stilbenes, flavonoids, and anthraquinones is poor. This study offers some novel insights into the biosynthetic regulation of bioactive compounds in different P. cuspidatum tissues and provides valuable resources for the potential metabolic engineering of this important medicinal plant.

Octaketide Synthase from Polygonum cuspidatum Implements Emodin Biosynthesis in Arabidopsis thaliana.

Plant anthranoids are medicinally used for their purgative properties. Their scaffold was believed to be formed by octaketide synthase (OKS), a member of the superfamily of type III polyketide synthase (PKS) enzymes. Here, a cDNA encoding OKS of Polygonum cuspidatum was isolated using a homology-based cloning strategy. When produced in Escherichia coli, P. cuspidatum octaketide synthase (PcOKS) catalyzed the condensation of eight molecules of malonyl-CoA to yield a mixture of unphysiologically folded aromatic octaketides. However, when the ORF for PcOKS was expressed in Arabidopsis thaliana, the anthranoid emodin was detected in the roots of transgenic lines. No emodin was found in the roots of wild-type A. thaliana. This result indicated that OKS is the key enzyme of plant anthranoids biosynthesis. In addition, the root growth of the transgenic A. thaliana lines was inhibited to an extent that resembled the inhibitory effect of exogenous emodin on the root growth of wild-type A. thaliana. Immunochemical studies of P. cuspidatum plants detected PcOKS mainly in roots and rhizome, in which anthranoids accumulate. Co-incubation of E. coli - produced PcOKS and cell-free extract of wild-type A. thaliana roots did not form a new product, suggesting an alternative, physiological folding of PcOKS and its possible interaction with additional factors needed for anthranoids assembling in transgenic A. thaliana. Thus, transgenic A. thaliana plants producing PcOKS provide an interesting system for elucidating the route of plant anthranoid biosynthesis.

Identification and evaluation of reference genes for quantitative real-time PCR analysis in Polygonum cuspidatum based on transcriptome data.

BACKGROUND: Polygonum cuspidatum of the Polygonaceae family is a traditional medicinal plant with many bioactive compounds that play important roles in human health and stress responses. Research has attempted to identify biosynthesis genes and metabolic pathways in this species, and quantitative real-time PCR (RT-qPCR) has commonly been used to detect gene expression because of its speed, sensitivity, and specificity. However, no P. cuspidatum reference genes have been identified, which hinders gene expression studies. Here, we aimed to identify suitable reference genes for accurate and reliable normalization of P. cuspidatum RT-qPCR data. RESULTS: Twelve candidate reference genes, including nine common (ACT, TUA, TUB, GAPDH, EF-1γ, UBQ, UBC, 60SrRNA, and eIF6A) and three novel (SKD1, YLS8, and NDUFA13), were analyzed in different tissues (root, stem, and leaf) without treatment and in leaves under abiotic stresses (salt, ultraviolet [UV], cold, heat, and drought) and hormone stimuli (abscisic acid [ABA], ethylene [ETH], gibberellin [GA3], methyl jasmonate [MeJA], and salicylic acid [SA]). Expression stability in 65 samples was calculated using the △CT method, geNorm, NormFinder, BestKeeper, and RefFinder. Two reference genes (NDUFA13 and EF-1γ) were sufficient to normalize gene expression across all sample sets. They were also the two most stable genes for abiotic stresses and different tissues, whereas NDUFA13 and SKD1 were the top two choices for hormone stimuli. Considering individual experimental sets, GAPDH was the top-ranked gene under ABA, ETH, and GA3 treatments, while 60SrRNA showed good stability under MeJA and cold treatments. ACT, UBC, and TUB were suitable genes for drought, UV, and ABA treatments, respectively. TUA was not suitable because of its considerable variation in expression under different conditions. The expression patterns of PcPAL, PcSTS, and PcMYB4 under UV and SA treatments and in different tissues normalized by stable and unstable reference genes demonstrated the suitability of the optimal reference genes. CONCLUSIONS: We propose NDUFA13 and EF-1γ as reference genes to normalize P. cuspidatum expression data. To our knowledge, this is the first systematic study of reference genes in P. cuspidatum which could help advance molecular biology research in P. cuspidatum and allied species.

A WRKY transcription factor, PcWRKY33, from Polygonum cuspidatum reduces salt tolerance in transgenic Arabidopsis thaliana.

KEY MESSAGE: PcWRKY33 is a transcription factor which can reduce salt tolerance by decreasing the expression of stress-related genes and increasing the cellular levels of reactive oxygen species (ROS). WRKY transcription factors play important roles in the regulation of biotic and abiotic stresses. Here, we report a group I WRKY gene from Polygonum cuspidatum, PcWRKY33, that encodes a nucleoprotein, which specifically binds to the W-box in the promoter of target genes to regulate their expression. The results from qPCR and promoter analysis show that expression of PcWRKY33 can be induced by various abiotic stresses, including NaCl and plant hormones. Overexpression of PcWRKY33 in Arabidopsis thaliana reduced tolerance to salt stress. More specifically, several physiological parameters (such as root length, seed germination rate, seedling survival rate, and chlorophyll concentration) of the transgenic lines were significantly lower than those of the wild type under salt stress. In addition, following exposure to salt stress, transgenic plants showed decreased expression of stress-related genes, a weakened ability to maintain Na+/K+ homeostasis, decreased activities of reactive oxygen species- (ROS-) scavenging enzymes, and increased accumulation of ROS. Taken together, these results suggest that PcWRKY33 negatively regulates the salt tolerance in at least two ways: by down-regulating the induction of stress-related genes and by increasing the level of cellular ROS. In sum, our results indicate that PcWRKY33 is a group I WRKY transcription factor involved in abiotic stress regulation.

Protein preparation, crystallization and preliminary X-ray analysis of Polygonum cuspidatum bifunctional chalcone synthase/benzalacetone synthase.

The chalcone synthase (CHS) superfamily of type III polyketide synthases (PKSs) generate the backbones of a variety of plant secondary metabolites. An active bifunctional chalcone synthase/benzalacetone synthase (CHS/BAS) from Polygonum cuspidatum was overexpressed in Escherichia coli as a C-terminally polyhistidine-tagged fusion protein, purified to homogeneity and crystallized using polyethylene glycol 4000 as a precipitant. The production of well shaped crystals of the complex between PcPKS1 and benzalacetone was dependent on the presence of sorbitol and barium chloride as additives. The crystals belonged to the orthorhombic space group P212121, with unit-cell parameters a = 80.23, b = 81.01, c = 122.89 Å, and diffracted X-rays to at least 2.0 Å resolution.

The Japanese knotweed invasion viewed as a vast unintentional hybridisation experiment.

Chromosome counts of plants grown from open-pollinated seed from Japanese knotweed around the world have revealed the presence of extensive hybridisation with both native and other introduced taxa. These hybrids fit into three categories: inter- and intraspecific hybrids involving the taxa of Fallopia section Reynoutria (giant knotweeds), hybrids between Japanese knotweed and F. baldschuanica (Regel) Holub and hybrids between Japanese knotweed and the Australasian endemics of the genus Muehlenbeckia. In this minireview, the viability of the different classes of hybrid and the potential threats they pose are discussed in the context of recent examples of allopolyploid speciation, which generally involve hybridisation between a native and an alien species. Such wide hybridisations also challenge accepted taxonomic classifications. Japanese knotweed s.l. provides a fascinating example of the interplay between ploidy level, hybridisation and alien plant invasion. The octoploid (2n=88) Fallopia japonica var. japonica (Houtt.) Ronse Decraene is a single female clone throughout much of its adventive range, and provides an ideal system for investigating the potential for wide hybridisation.

Invasion of diverse habitats by few Japanese knotweed genotypes is correlated with epigenetic differentiation.

The expansion of invasive species challenges our understanding of the process of adaptation. Given that the invasion process often entails population bottlenecks, it is surprising that many invasives appear to thrive even with low levels of sequence-based genetic variation. Using Amplified Fragment Length Polymorphism (AFLP) and methylation sensitive-AFLP (MS-AFLP) markers, we tested the hypothesis that differentiation of invasive Japanese knotweed in response to new habitats is more correlated with epigenetic variation than DNA sequence variation. We found that the relatively little genetic variation present was differentiated among species, with less differentiation among sites within species. In contrast, we found a great deal of epigenetic differentiation among sites within each species and evidence that some epigenetic loci may respond to local microhabitat conditions. Our findings indicate that epigenetic effects could contribute to phenotypic variation in genetically depauperate invasive populations. Deciphering whether differences in methylation patterns are the cause or effect of habitat differentiation will require manipulative studies.

De novo characterization of the root transcriptome of a traditional Chinese medicinal plant Polygonum cuspidatum.

Various active components have been extracted from the root of Polygonum cuspidatum. However, the genetic basis for their activity is virtually unknown. In this study, 25600002 short reads (2.3 Gb) of P. cuspidatum root transcriptome were obtained via Illumina HiSeq 2000 sequencing. A total of 86418 unigenes were assembled de novo and annotated. Twelve, 18, 60 and 54 unigenes were respectively mapped to the mevalonic acid (MVA), methyl-D-erythritol 4-phosphate (MEP), shikimate and resveratrol biosynthesis pathways, suggesting that they are involved in the biosynthesis of pharmaceutically important anthraquinone and resveratrol. Eighteen potential UDP-glycosyltransferase unigenes were identified as the candidates most likely to be involved in the biosynthesis of glycosides of secondary metabolites. Identification of relevant genes could be important in eventually increasing the yields of the medicinally useful constituents of the P. cuspidatum root. From the previously published transcriptome data of 19 non-model plant taxa, 1127 shared orthologs were identified and characterized. This information will be very useful for future functional, phylogenetic and evolutionary studies of these plants.

Overexpression of a resveratrol synthase gene (PcRS) from Polygonum cuspidatum in transgenic Arabidopsis causes the accumulation of trans-piceid with antifungal activity.

Although resveratrol-forming stilbene synthase (STS) genes have been well characterized in many plant species, there are only a few descriptions about STS genes from Polygonum cuspidatum Sieb. et Zucc, an important medicinal crop in Asian countries. To evaluate the biological functions of a Polygonum cuspidatum resveratrol synthase gene (PcRS), the PcRS gene was expressed in Arabidopsis under the control of Cauliflower mosaic virus (CaMV) 35S promoter. Integration and expression of transgene in the plant genome of Arabidopsis was confirmed by Southern blot and Northern blot analyses. Transgenic plants accumulated a new compound in both the leaves and seeds, which was identified as trans-piceid by high-pressure liquid chromatography (HPLC) and electrospray mass spectrometry (HPLC-ESI-MS). Overexpression of PcRS in transgenic Arabidopsis caused restriction of Colletotrichum higginsianum colonization by inhibition of spore production, resulting in enhanced resistance against C. higginsianum. So, the PcRS gene could be deployed in other crop plants to significantly enhance resistance to fungal pathogens and improve the nutritional quality. In addition, altered seed coat pigmentation and significant reduction in anthocyanin levels were observed in transgenic Arabidopsis, while the expression of endogenous chalcone synthase (CHS) gene was not down-regulated. These results suggest that additional STS activities cause a lack of precursors for CHS which leads to the disturbance of the subsequent flavonoid biosynthesis steps in Arabidopsis.

Isolation of the 5'-end of plant genes from genomic DNA by TATA-box-based degenerate primers.

We report a rapid and simple method for isolating the 5'-end of plant genes from genomic DNA by polymerase chain reaction (PCR) with TATA-box-based degenerate primers (TDPs). The TDPs were specially designed according to the TATA box, which is conserved in the promoter region of most plant genes. The unknown 5'-ends of several genes in different plants were isolated by PCR with gene-specific primers of the known core fragment and the TDPs. Our method does not require the arduous RNA manipulations and expensive enzyme treatments of the popular rapid amplification of cDNA ends (RACE) and its variants, and so could be a cheap practical alternative.

Identification of a Polygonum cuspidatum three-intron gene encoding a type III polyketide synthase producing both naringenin and p-hydroxybenzalacetone.

Benzalacetone synthase (BAS) is a member of the plant-specific type III PKS superfamily that catalyzes a one-step decarboxylative condensation of 4-coumaroyl-CoA with malonyl-CoA to produce p-hydroxybenzalacetone. In our recent work (Ma et al. in Planta 229(3):457-469, 2008), a three-intron type III PKS gene (PcPKS2) was isolated from Polygonum cuspidatum Sieb. et Zucc. Phylogenetic and functional analyses revealed this recombinant PcPKS2 to be a BAS. In this study, another three-intron type III PKS gene (PcPKS1) and its corresponding cDNA were isolated from P. cuspidatum. Sequence and phylogenetic analyses demonstrated that PcPKS1 is a chalcone sythase (CHS). However, functional and enzymatic analyses showed that recombinant PcPKS1 is a bifunctional enzyme with both, CHS and BAS activity. DNA gel blot analysis indicated that there are two to four CHS copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS1 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS1 transcripts in leaves were inducible by pathogen infection and wounding. BAS is thought to play a crucial role in the construction of the C(6)-C(4) moiety found in a variety of phenylbutanoids, yet so far phenylbutanoids have not been isolated from P. cuspidatum. However, since PcPKS1 and PcPKS2 (Ma et al. in Planta 229(3):457-469, 2008) have been identified in P. cuspidatum, it is possible that such compounds are also produced in that plant, albeit in low concentrations.

A novel type III polyketide synthase encoded by a three-intron gene from Polygonum cuspidatum.

A type III polyketide synthase cDNA and the corresponding gene (PcPKS2) were cloned from Polygonum cuspidatum Sieb. et Zucc. Sequencing results showed that the ORF of PcPKS2 was interrupted by three introns, which was an unexpected finding because all type III PKS genes studied so far contained only one intron at a conserved site in flowering plants, except for an Antirrhinum majus chalcone synthase gene. Besides the unusual gene structure, PcPKS2 showed some interesting characteristics: (1) the CHS "gatekeepers" Phe215 and Phe265 are uniquely replaced by Leu and Cys, respectively; (2) recombinant PcPKS2 overexpressed in Escherichia coli efficiently afforded 4-coumaroyltriacetic acid lactone (CTAL) as a major product along with bis-noryangonin (BNY) and p-hydroxybenzalacetone at low pH; however, it effectively yielded p-hydroxybenzalacetone as a dominant product along with CTAL and BNY at high pH. Beside p-hydroxybenzalacetone, CTAL and BNY, a trace amount of naringenin chalcone could be detected in assays at different pH. Furthermore, 4-coumaroyl-CoA and feruloyl-CoA were the only cinnamoyl-CoA derivatives accepted as starter substrates. PcPKS2 did not accept isobutyryl-CoA, isovaleryl-CoA or acetyl-CoA as substrate. DNA gel blot analysis indicated that there are two to four PcPKS2 copies in the P. cuspidatum genome. RNA gel blot analysis revealed that PcPKS2 is highly expressed in the rhizomes and in young leaves, but not in the roots of the plant. PcPKS2 transcripts in leaves were induced by pathogen infection, but not by wounding.

[Increasing the content of active constituents in Polygonum cuspidatum hairy root by gene transformation technology].

To increase the content of active constituent--RE and PD of Polygonum cuspidatum hairy root, through Ri-mediated gene transformation technology, modified high salt low pH method was used to distill genome DNA of grapevine (Vitis raparia). Primer was designed according to sequence of Genebank (AF128861). Through PCR amplification obtain RS gene sequence was obtained. Binary vector pCAMBIA1300-35S-RS was constructed. Frost thawing method was used to transform Agrobacterium rhizogenes ATCC11325. Scratched aseptic seedling leaf of Polygonum cuspidatum was contaminated subsequently. DNA conformity and mRNA expression of RS gene were investigated by PCR and RT-PCR respectively. RE and PD in transgenic hairy root were determined by HPLC. For the first time successfully inducement acquires transformed RS gene hairy root of Polygonum cuspidatum. Content of active constituents--RE and PD were 17 - 187 microg x g(-1) DW and 836 - 1 970 microg x g(-1) DW, respectively, the non-transgenic hairy root was 0 - 130 microg x g(-1) DW and 190 - 320 microg x g(-1) DW. In the different root selected, the content of PD was much higher than that in non-transformed hairy roots of Polygonum cuspidatum, the highest content is 5 times, but the content of RE has not increased apparently.