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Soil cadmium (Cd) contamination seriously reduces the production and product quality of Tartary buckwheat (Fagopyrum tataricum), and strategies are urgently needed to mitigate these adverse influences. Herein, we investigated the effect of salicylic acid (SA) on Tartary buckwheat seedlings grown in Cd-contaminated soil in terms of Cd tolerance and accumulation. The results showed that 75-100 µmol L-1 SA treatment enhanced the Cd tolerance of Tartary buckwheat, as reflected by the significant increase in plant height and root and shoot biomass, as well as largely mitigated oxidative stress. Moreover, 100 µmol L-1 SA considerably reduced the stem and leaf Cd concentration by 60% and 47%, respectively, which is a consequence of increased root biomass and root Cd retention with promoted Cd partitioning into cell wall and immobile chemical forms. Transcriptome analysis also revealed the upregulation of the genes responsible for cell wall biosynthesis and antioxidative activities in roots, especially secondary cell wall synthesis. The present study determines that 100 µmol L-1 is the best SA concentration for reducing Cd accumulation and toxicity in Tartary buckwheat and indicates the important role of root in Cd stress in this species.

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Metal tolerance proteins (MTP) as divalent cation transporters are essential for plant metal tolerance and homeostasis. However, the characterization and the definitive phylogeny of the MTP gene family in Fagopyrum tartaricum, and their roles in response to metal stress are still unknown. In the present study, MTP genes in Fagopyrum tartaricum were identified, and their phylogenetic relationships, structural characteristics, physicochemical parameters, as well as expression profiles under five metal stresses including Fe, Mn, Cu, Zn, and Cd were also investigated. Phylogenetic relationship analysis showed that 12 Fagopyrum tartaricum MTP genes were classified into three major clusters and seven groups. All FtMTPs had typical structural features of the MTP gene family and were predicted to be located in the cell vacuole. The upstream region of FtMTPs contained abundant cis-acting elements, implying their functions in development progress and stress response. Tissue-specific expression analysis results indicated the regulation of FtMTPs in the growth and development of Fagopyrum tataricum. Besides, the expression of most FtMTP genes could be induced by multiple metals and showed different expression patterns under at least two metal stresses. These findings provide useful information for the research of the metal tolerance mechanism and genetic improvement of Fagopyrum tataricum.

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Buckwheat is a promising pseudo cereal and its cultivation history can be traced back to thousands of years ago in China. Nowadays, buckwheat is not only an ordinary crop but also a symbol of healthy life because of its rich nutritional and pharmacological properties. In this research, the current suitable areas of 19 wild buckwheat species were analyzed by the MaxEnt model, which proved that southwestern China was the diversity center of buckwheat. Their morphological characteristics and geographical distribution were analyzed for the first time. In addition, it was found that the change of buckwheat cultivation in three periods might be related to the green revolution of main crops and national policies. Meanwhile, the Sustainable Yield Index (SYI) value of buckwheat in China was the lowest from 1959 to 2016. Through the MaxEnt model, the potentially suitable areas of wild buckwheat would contract while cultivated buckwheat would expand under climate change. Accordingly, the diversity of wild buckwheat will decrease. Therefore, it is necessary to protect buckwheat resources as much as possible to strengthen the development and utilization of buckwheat resources. Moreover, the promotion of buckwheat diversity will be an important trade-off between food security, population growth, and land use under climate change.

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Tartary buckwheat tea (TBT) is the most popular and widely consumed buckwheat product in many countries. However, the perfect quality control standards for TBT were still lacking, and the content of heavy metals in TBT and their health risks to consumers were still unknown. In this research, the total phenolic content, total flavonoid content, and antioxidant capacity as well as six metal contents and their health risks in TBT were detected. The results showed that the total phenolic content, total flavonoid content, and antioxidant capacity varied significantly among different types of TBT. Meanwhile, six metal concentrations in TBT leaves and infusions decreased in the order of Zn, Cu, Cr, Ni, Pb, and Cd. Health risk assessment indicated that the heavy metal intake only from TBT would not cause a noncarcinogenic risk to consumers. However, a strong carcinogenic risk of Cr in TBT for consumers should be paid more attention.

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Fagopyrum cymosum has been considered as a traditional medicinal plant that belongs to Fagopyrum, which has exhibited great pharmaceutical potential due to its abundant flavonoid accumulation. The hairy roots induced by Agrobacterium rhizogenes has been utilized to produce valuable specialized metabolites or reveals plant metabolic processes, whereas the underlying regulatory networks of flavonoid biosynthesis in hairy roots of F. cymosum remained unexplored. Here, the regulatory transcription factor TrMYB4 cloned from Trifolium repens with purple striped leaves was considered to investigate the mechanism of flavonoids biosynthesis in hairy roots of F. cymosum. Results showed that the expression of key genes involved in rutin biosynthesis pathway from TrMYB4 hairy roots were significantly up-regulated compared with non-transgenic hairy roots, while the content of total flavonoids and rutin in TrMYB4 hairy roots also increased consistently. It revealed the TrMYB4 transcription factor could regulate the rutin biosynthesis in F. cymosum. Meanwhile, our research provided a theoretical reference for the industrial production of rutin using F. cymosum hairy roots.

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Buckwheat is an important functional food material with high nutritional value. However, it is still a difficult task for the taxonomy studies of wild buckwheat that are only based on morphology. In order to demonstrate the most efficient DNA barcode in the phylogenetic research of buckwheat, promote the investigation of wild buckwheat, and also reveal the phylogenetic relationship between Fagopyrum species, psbE-psbL and ndhA intron were validated here, which previously have been proved to be promising DNA barcode candidates for phylogenetic studies in genera Fagopyrum. Meanwhile, ndhA intron + psbE-psbL and matK + psbE-psbL could distinguish the relationship between species clearly. Combining the results of morphology and molecular markers, we suggested the buckwheat species should be divided into two subgroups, one subgroup consisted of F. tataricum, F. esculentum, F. cymosum and its related wild species, and the other subgroup included other wild buckwheat species. Our results could fulfill molecular markers of taxonomy research in genera Fagopyrum, promote wild buckwheat species identification, and assist in the use of wild buckwheat resources in the future. Additionally, the phylogenetic relationship revealed here could provide valuable information for molecular breeding of buckwheat and provide reference for inter-species hybridization.

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To investigate the characteristics of heavy metals (Cr, Cd, Pb, Zn, Cu and Ni) in household dust in urban household environment of Chengdu, China, 90 household dust samples were collected from 6 districts of the city. The information of houses and residents were also recorded during dust sampling to identify the correlations between heavy metals in household dust and the house attributes. And also the principal component analysis and cluster analysis for sources and impactor factors. The concentrations of Pb (123mg·kg-1), Zn (675mg·kg-1), Cu (190mg·kg-1), Cr (82.7mg·kg-1), Cd (2.37mg·kg-1) and Ni (52.6mg·kg-1) in household dust are in low or moderate levels when compare with that from other counties or areas. The heavy metals of household dust samples from Chengdu is higher concentrations than that in the street dust from Chengdu, except for Cr. Statistical analysis result showed traffic sources and corrosion of alloys are important factors contributing to the rise concentrations of heavy metals in household dust. In addition, there is negative correlation (p<0.05) between the heavy metals concentrations and floor levels. The ingestion is the important pathway for Pb, Zn, Cu and Ni via household dust exposure to the residents, and the dermal contact was identified as a main route for Cr and Cd in household dust exposure to the residents. There are minor non-carcinogenic and carcinogenic risks from the heavy metals in household dust for the residents in Chengdu.

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Buckwheat is a nutritional and economically crop belonging to Polygonaceae, Fagopyrum. To better understand the mutation patterns and evolution trend in the chloroplast (cp) genome of buckwheat, and found sufficient number of variable regions to explore the phylogenetic relationships of this genus, two complete cp genomes of buckwheat including Fagopyrum dibotrys (F. dibotrys) and Fagopyrum luojishanense (F. luojishanense) were sequenced, and other two Fagopyrum cp genomes were used for comparative analysis. After morphological analysis, the main difference among these buckwheat were height, leaf shape, seeds and flower type. F. luojishanense was distinguishable from the cultivated species easily. Although the F. dibotrys and two cultivated species has some similarity, they different in habit and component contents. The cp genome of F. dibotrys was 159,320 bp while the F. luojishanense was 159,265 bp. 48 and 61 SSRs were found in F. dibotrys and F. luojishanense respectively. Meanwhile, 10 highly variable regions among these buckwheat species were located precisely. The phylogenetic relationships among four Fagopyrum species based on complete cp genomes was showed. The results suggested that F. dibotrys is more closely related to Fagopyrum tataricum. These data provided valuable genetic information for Fagopyrum species identification, taxonomy, phylogenetic study and molecular breeding.

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Little is known about the molecular mechanism of the R2R3-MYB transcriptional repressors involved in plant phenylpropanoid metabolism. Here, we describe one R2R3-type MYB repressor, FtMYB11 from Fagopyrum tataricum. It contains the SID-like motif GGDFNFDL and it is regulated by both the importin protein 'Sensitive to ABA and Drought 2' (SAD2) and the jasmonates signalling cascade repressor JAZ protein. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that FtMYB11 interacts with SAD2 and FtJAZ1. Protoplast transactivation assays demonstrated that FtMYB11 acts synergistically with FtSAD2 or FtJAZ1 and directly represses its target genes via the MYB-core element AATAGTT. Changing the Asp122 residue to Asn in the SID-like motif results in cytoplasmic localization of FtMYB11 because of loss of interaction with SAD2, while changing the Asp126 residue to Asn results in the loss of interaction with FtJAZ1. Overexpression of FtMYB11or FtMYB11D126N in F. tataricum hairy roots resulted in reduced accumulation of rutin, while overexpression of FtMYB11D122N in hairy roots did not lead to such a change. The results indicate that FtMYB11 acts as a regulator via interacting with FtSAD2 or FtJAZ1 to repress phenylpropanoid biosynthesis, and this repression depends on two conserved Asp residues of its SID-like motif.

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Urban street dusts were collected in commercial area (CA), traffic area (TA), educational area (EA), residential area (RA) and parks area (PA) of Chengdu, China, to investigate the concentrations of heavy metals (Pb, Zn, Cu, Ni, Cd and Cr), and analyzed to evaluated possible sources and health risk assessment. The average concentrations (mg/kg) of Pb (82.3), Zn (296), Cu (100), Cd (1.66) and Cr (84.3) in urban street dusts were all higher than the local soil background values. The concentrations of heavy metals in each functional area could be classified as follows: CA>TA>RA>EA>PA. Principal component analysis and Cluster analysis showed mainly derived from the mixed sources of nature and traffic (51.7%). The results of health risk assessment showed no non-carcinogenic and carcinogenic risks of the metals for inhabitants. However, higher concentrations and oral bioaccessibility of the heavy metals in the dusts from CA and TA, indicating there was more health risks to the inhabitants in than that in other functional areas.

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Pb hyper-accumulated Carex putuoshan was taken as experimental material and subjected to combined stress of Pb and Zn. The differential expression of proteins in their roots were analyzed by Proteomic Approach. The protein that was directly involved in the cellular defense under the Pb and Zn combined stress was separated, and expression of those genes was analyzed with Carex Evergold as control. The results were obtained by MALDI-TOF/MS analysis. After applying Pb and Zn combined stress, the expression of 9 protein spots (including 7 different proteins, 2 identical proteins, 1 unknown protein) in Carex putuoshan root was found to be significantly up-regulated. Five proteins were obtained from the 9 proteins related to carbohydrate metabolism, including malate dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, frutose-1,6-bisphosphate aldolase, enolase, and 6- phosphogluconate dehydrogenase. Two proteins were related to protein biosynthesis, including isoflavone reductase and phytochelatin synthase (PCS). From these proteins, the most important protein is PCS, which is a key enzyme in the synthesis of phytochelatins (PCs) and plays an important role in chelation. It is directly involved in cellular defense under Pb and Zn stress. After Pb and Zn combined stress, the CpPCS in Carex putuoshan was cloned. The full length of cDNA is 1461 bps, and it encodes 486 amino acids with molecular weight of 53.86 kD and pI value of 6.12. Two typical phytochelatin synthase subfamily domains constitute CpPCS protein, which includes three adjacent Cys-Cys elements in the C-terminal region. Phylogenetic analysis of PCS proteins from different species showed that it had the closest relationship with the Oryza sativa and Triticum aestivum. Real-time quantitative PCR analysis indicated that CpPCS and CePCS (Carex Evergold) genes were expressed in the root. The CpPCS and CePCS genes were up-regulated by Pb and Zn treatments. The expression of CpPCS was higher than that of CePCS under the same condition. The study found that CpPCS expression was increased by Pb and Zn stress in the Carex putuoshan enrichment process of Pb, which lead to high expression of PCS protein. CpPCS improved the accumulation ability and resistance of Carex putuoshan to heavy metals with the expression level of glucose metabolism related proteins increasing after Pb and Zn stress.

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Tartary buckwheat is an ancient annual dicotyledonous herb, which is widely distributed around the world, specifically in the high altitude area of southwestern China and in the hill region of Himalayan. The plantlet regeneration of tartary buckwheat via somatic embryogenesis or multiple shoot induction was investigated in two different tartary buckwheats, Yuanzi and Xichang. The regeneration ability of Yuanzi was better than Xichang tartary buckwheat, and the hypocotyls were better than cotyledons as tartary buckwheat plantlet regeneration explants via somatic embryogenesis. The most suitable medium for callus induction was Murashige and Skoog basal medium added 2 mg/L 2, 4- dichlorophenoxyacetic acid and 1 mg/L Kinetin, which could reach up to 98.96% callus induction percentage. The plantlet regeneration percentage from callus of tartary buckwheat could reach up to 55.77%, which induced on 2.0 mg/L Benzyladenine and 1.0 mg/L KT in MS basal medium. In addition, maximum of multiple shoot induction percentage was 69.05%, which was observed in case of Yuanzi tartary buckwheat in MS basal medium with added 3.0 mg/L 6-BA and 1.0 mg/L Thidiazuron. Roots induction of regenerated plants were achieved on 1/2 MS basal medium with added 1mg/L Indole-3-Butytric acid, which has 75% survival after transferred regenerated plants to soil under field conditions.

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Sub-group 4 R2R3-type MYB transcription factors, including MYB3, MYB4, MYB7 and MYB32, act as repressors in phenylpropanoid metabolism. These proteins contain the conserved MYB domain and the ethylene-responsive element binding factor-associated amphiphilic repression (EAR) repression domain. Additionally, MYB4, MYB7 and MYB32 possess a putative zinc-finger domain and a conserved GY/FDFLGL motif in their C-termini. The protein 'sensitive to ABA and drought 2' (SAD2) recognizes the nuclear pore complex, which then transports the SAD2-MYB4 complex into the nucleus. Here, we show that the conserved GY/FDFLGL motif contributes to the interaction between MYB factors and SAD2. The Asp → Asn mutation in the GY/FDFLGL motif abolishes the interaction between MYB transcription factors and SAD2, and therefore they cannot be transported into the nucleus and cannot repress their target genes. We found that MYB4(D261N) loses the capacity to repress expression of the cinnamate 4-hydroxylase (C4H) gene and biosynthesis of sinapoyl malate. Our results indicate conservation among MYB transcription factors in terms of their interaction with SAD2. Therefore, the Asp → Asn mutation may be used to engineer transcription factors.

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Plant annexins are Ca(2+)- and phospholipid-binding proteins forming an evolutionary conserved multi-gene family. They are implicated in the regulation of plant growth, development, and stress responses. With the availability of the maize genome sequence information, we identified 12 members of the maize annexin genes. Analysis of protein sequence and gene structure of maize annexins led to their classification into five different orthologous groups. Expression analysis by RT-PCR revealed that these genes are responsive to heavy metals (Ni, Zn, and Cd). The maize annexin genes were also found to be regulated by Ustilago maydis and jasmonic acid. Additionally, the promoter of the maize annexin gene was analyzed for the presence of different stress-responsive cis-elements, such as ABRE, W-box, GCC-box, and G-box. RT-PCR and microarray data show that all 12 maize annexin genes present differential, organ-specific expression patterns in the maize developmental steps. These results indicate that maize annexin genes may play important roles in the adaptation of plants to various environmental stresses.

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Nicotianamine (NA) is an important divalent metal chelator and the main precursor of phytosiderophores. NA is synthesized from S-adenosylmethionine in a process catalyzed by nicotianamine synthase (NAS). In this study, a set of structural and phylogenetic analyses have been applied to identify the maize NAS genes based on the maize genome sequence release. Ten maize NAS genes have been mapped; seven of them have not been reported to date. Phylogenetic analysis and expression pattern from microarray data led to their classification into two different orthologous groups. C-terminal fusion of ZmNAS3 with GFP was found in the cytoplasm of Arabidopsis leaf protoplast. Expression analysis by reverse transcription polymerase chain reaction revealed ZmNAS genes are responsive to heavy metal ions (Ni, Fe, Cu, Mn, Zn, and Cd), and all 10 ZmNAS genes were only observed in the root tissue except of ZmNAS6. The promoter of ZmNAS genes was analyzed for the presence of different cis-element response to all kinds of phytohormones and environment stresses. We found that the ZmNAS gene expression of maize seedlings was regulated by jasmonic acid, abscisic acid, and salicylic acid. Microarray data demonstrated that the ZmNAS genes show differential, organ-specific expression patterns in the maize developmental steps. The integrated comparative analysis can improve our current view of ZmNAS genes and facilitate the functional characterization of individual members.

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The rutin degrading enzyme (RDE) was isolated and purified from tartary buckwheat seeds. The RDE was purified about 11.34-fold and its final yield was 3.5%, which was very low, due to our purification strategy of giving priority to purity over yield. The RDE molecular weight was estimated to be about 60 kDa. When rutin was used as substrate, an optimal enzyme activity was seen at around pH 5.0 and 40 °C. Strains isolation strategy characterized by the use of rutin as sole carbon source in enrichment cultures was used to isolate RDE-producing strains. Then the active strains were identified by morphology characterization and 18s rDNA-ITS (Internal Transcribed Spacer) gene sequencing. Three isolates coded as B3, W2, Y2 were successfully isolated from fusty Fagopyrum tataricum flour cultures. Strain B3 possessed the highest unit activity among these three strains, and its total activity reached up to 171.0 Unit. The active isolate (B3) could be assigned to Penicillium farinosum. When the Penicillium farinosum strains were added to tartary buckwheat flour cultures at pH 5.0, 30 °C after 5 days fermentation, the quercetin production raised up to 1.78 mg/l, almost 5.1 times higher than the fermentation without the above active strains. Hence, a new approach was available to utilize microorganism-aided fermentation for effective quercetin extraction from Fagopyrum tataricum seeds.

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To obtain protective human monoclonal antibody from HIV-1 infected person, we adapted a technology for isolating antigen specific monoclonal antibody from human memory B cells through in vitro B cell activation coupled with RT-PCT and expression cloning. Human B cells were purified by negative sorting from PBMCs of HIV-1 infected individuals and memory B cells were further enriched using anti-CD27 microbeads. Two hundred memory B cells per well were cultured in 96-well round-bottom plates Env-specific antibodies in supernatants were with feeder cells in medium containing EBV and CpG. screened by ELISA after 1-2 weeks' culture. Cells from positive wells of Env-specific antibody were harvested and total RNA was isolated. Human VH and Vkappa or Vlambda genes were amplified by RT-PCR and cloned into IgG1 and kappa or lambda expressing vectors. Functional VH and Vkappa or Vlambda were identified by cotransfecting 293T cells with individual heavy chain and light chain clones followed by analysis of culture supernatants by ELISA for Env-specific antibodies. Finally, corresponding mAb was produced by transient transfection of 293T cells with the identified VH and Vkappa/lambda pair and purified by protein A affinity chromatography. Purified monocolonal antibodies were used for HIV-1 specific antibody-dependent cell-mediated cytotoxicity (ADCC) and neutralizing activity assay. Four monocolonal Env-specific antibodies were isolated from one HIV-1 subtype B' infected individual. Two of them showed strong ADCC activity and one showed weak neutralizing activity against HIV-1. Its further studies on their application in therapeutic or prophylactic vaccines against HIV-1 should be grounded.

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Maize (Zea mays ssp. mays L.) is an important model organism for fundamental research in the agro-biotechnology field. Aldehydes were generated in response to a suite of environmental stresses that perturb metabolism including salinity, dehydration, desiccation, and cold and heat shock. Many biologically important aldehydes are metabolized by the superfamily of NAD(P)(+)-dependent aldehyde dehydrogenases. Here, starting from the database of Z. mays, we identified 28 aldehyde dehydrogenase (ALDH) genes and 48 transcripts by the in silico cloning method using the ALDH-conserved domain amino acid sequence of Arabidopsis and rice as a probe. Phylogenetic analysis shows that all 28 members of the ALDH gene families were classified to ten distinct subfamilies. Microarray data and quantitative real-time PCR analysis reveal that ZmALDH9, ZmALDH13, and ZmALDH17 genes involve the function of drought stress, acid tolerance, and pathogens infection. These results suggested that these three ZmALDH genes might be potentially useful in maize genetic improvement.

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The raffinose family oligosaccharides (RFOs), such as raffinose and stachyose, are synthesized by a set of distinct galactosyltransferases, which sequentially add galactose units to sucrose. The accumulation of RFOs in plant cells are closely associated with the responses to environmental factors, such as cold, heat and drought stresses. Systematic analysis of genes involved in the raffinose metabolism has not been reported to date. Searching the recently available working draft of the maize genome, six kinds of enzyme genes were speculated, which should encode all the enzymes involved in the raffinose metabolism in maize. Expression patterns of some related putative genes were analyzed. The conserved domains and phylogenetic relationships among the deduced maize proteins and their homologs isolated from other plant species were revealed. It was discovered that some of the key enzymes, such as galactinol synthase (ZmGolS5, ZmGolS45 and ZmGolS37), raffinose synthase (ZmRS1, ZmRS2, ZmRS3 and ZmRS10), stachyose synthase (ZmRS8) and ß-fructofuranosidase, are encoded by multiple gene members with different expression patterns. These results reveal the complexity of the raffinose metabolism and the existence of metabolic channels for diverse RFOs in maize and provide useful information for improving maize stress tolerance through genetic engineering.

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Evidence from in vitro and in vivo studies indicates that rutin, the main flavonoid in tartary buckwheat ( Fagopyrum tataricum ), may have high value for medicine and health. This paper reports the finding of a flavonol synthase (FLS) gene, cloned and characterized from F. tataricum and designated FtFLS1, that is involved in rutin biosynthesis. The FtFLS1 gene was expressed in Escherichia coli BL21(DE3), and the recombinant soluble FtFLS1 protein had a relative molecular mass of 40 kDa. The purified recombinant protein showed, with dihydroquercetin as substrate, total and specific activities of 36.55 × 10(-3) IU and 18.94 × 10(-3) IU/mg, respectively, whereas the total and specific activities were 10.19 × 10(-3) IU and 5.28 × 10(-3) IU/mg, respectively, with dihydrokaempferol. RT-PCR revealed that during F. tataricum florescence there was an organ-specific expression pattern by the FtFLS1 gene, with similar trends in flavonoid content. These observations suggest that FtFLS1 in F. tataricum encodes a functional protein, which might play a key role in rutin biosynthesis.

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