[Identification and expression analysis of whole gene family of Isatis indigotica 4-coumarate: CoA ligase].

The 4-coumarate: CoA ligase(4CL) is a key enzyme in the upstream pathway of phenylpropanoids such as flavonoids, soluble phenolic esters, lignans, and lignins in plants. In this study, 13 4CL family members of Arabidopsis thaliana were used as reference sequences to identify the 4CL gene family candidate members of Isatis indigotica from the reported I. indigotica genome. Further bioinformatics analysis and analysis of the expression pattern of 4CL genes and the accumulation pattern of flavonoids were carried out. Thirteen 4CL genes were obtained, named Ii4CL1-Ii4CL13, which were distributed on chromosomes 1, 2, 3, 4, and 6. The analysis of the gene structure and conserved structural domains revealed the intron number of I. indigotica 4CL genes was between 1 and 12 and the protein structural domains were highly conserved. Cis-acting element analysis showed that there were multiple response elements in the promoter sequence of I. indigotica 4CL gene family, and jasmonic acid had the largest number of reaction elements. The collinearity analysis showed that there was a close relationship between the 4CL gene family members of I. indigotica and A. thaliana. As revealed by qPCR results, the expression analysis of the 4CL gene family showed that 10 4CL genes had higher expression levels in the aboveground part of I. indigotica. The content assay of flavonoids in different parts of I. indigotica showed that flavonoids were mainly accumulated in the aboveground part of plants. This study provides a basis for further investigating the roles of the 4CL gene family involved in the biosynthesis of flavonoids in I. indigotica.

Classification of Isatis indigotica Fortune and Isatis tinctoria Linnaeus via comparative analysis of chloroplast genomes.

BACKGROUND: Isatis tinctoria Linnaeus and Isatis indigotica Fortune are very inconsistent in their morphological characteristics, but the Flora of China treats them as the same species. In this work, a new technology that differs from conventional barcodes is developed to prove that they are different species and to clarify their classification. RESULTS AND METHODS: I. indigotica was indistinguishable from I. tinctoria when using ITS2. CPGAVAS2 was used to construct the chloroplast genomes. MAFFT and DnaSP were used to calculate nucleotide polymorphism, the chloroplast genomes of the two have high diversity in the rpl32 ~ trnL-UAG short region. When using this region as a mini barcode, it was found that there are obvious differences in the base numbers of I. tinctoria and different ploidy I. indigotica were found, but diploid and tetraploid I. indigotica had the same number of bases. Moreover, the reconstruction of the maximum likelihood (ML) tree, utilizing the mini-barcode, demonstrated that I. tinctoria and both diploid and tetraploid I. indigotica are located on distinct branches. The genome size of tetraploid I. indigotica was approximately 643.773 MB, the heterozygosity rate was approximately 0.98%, and the repeat sequence content was approximately 90.43%. This species has a highly heterozygous, extremely repetitive genome. CONCLUSION: A new method was established to differentiate between I. indigotica and I. tinctoria. Furthermore, this approach provides a reference and basis for the directional breeding of Isatis.

IiSVP of Isatis indigotica can reduce the size and repress the development of floral organs.

KEY MESSAGE: IiSVP of Isatis indigotica was cloned and its expression pattern was analyzed. Ectopic expression of IiSVP in Arabidopsis could delay the flowering time and reduce the size of the floral organs. SVP (SHORT VEGETATIVE PHASE) can negatively regulate the flowering time of Arabidopsis. In the present work, the cDNA of IiSVP, an orthologous gene of AtSVP in I. indigotica, was cloned. IiSVP was highly expressed in rosette leaves, inflorescences and petals, but weakly expressed in sepals, pistils and young silicles. The results of subcellular localization showed that IiSVP was localized in nucleus. Bioinformatics analysis indicated that this protein was a MADS-box transcription factor. Constitutive expression of IiSVP in Arabidopsis thaliana resulted in decrease of the number of petals and stamens, and curly sepals were formed. In IiSVP transgenic Arabidopsis plants, obvious phenotypic variations in flowers could be observed, especially the size of the floral organs. In comparison with the wild-type plants, the size of petals, stamens and pistil in IiSVP transgenic Arabidopsis plants was decreased significantly. In some transgenic plants, the petals were wrapped by the sepals. Yeast two-hybrid experiments showed that IiSVP could form higher-order complexes with other MADS proteins, including IiSEP1, IiSEP3, IiAP1 and IiSEP4, but could not interact with IiSEP2. In this work, it was proved that the flowering process and the floral development in Arabidopsis could be affected by IiSVP from I. indigotica Fortune.

[Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro].

Chalcone isomerase is a key rate-limiting enzyme in the biosynthesis of flavonoids in higher plants, which determines the production of flavonoids in plants. In this study, RNA was extracted from different parts of Isatis indigotica and reverse-transcribed into cDNA. Specific primers with enzyme restriction sites were designed, and a chalcone isomerase gene was cloned from I. indigotica, named IiCHI. IiCHI was 756 bp in length, containing a complete open reading frame and encoding 251 amino acids. Homology analysis showed that IiCHI was closely related to CHI protein of Arabidopsis thaliana and had typical active sites of chalcone isomerase. Phylogenetic tree analysis showed that IiCHI was classified into type Ⅰ CHI clade. Recombinant prokaryotic expression vector pET28a-IiCHI was constructed and purified to obtain IiCHI recombinant protein. In vitro enzymatic analysis showed that the IiCHI protein could convert naringenin chalcone into naringenin, but could not catalyze the production of liquiritigenin by isoliquiritigenin. The results of real-time quantitative polymerase chain reaction(qPCR) showed that the expression level of IiCHI in the aboveground parts was higher than that in the underground parts and the expression level was the highest in the flowers of the aboveground parts, followed by leaves and stems, and no expression was observed in the roots and rhizomes of the underground parts. This study has confirmed the function of chalcone isomerase in I. indigotica and provided references for the biosynthesis of flavonoid components.

Functional identification of the different regions in B-class floral homeotic MADS-box proteins IiAP3 and IiPI from Isatis indigotica.

APETALA3 (AP3) and PISTILLATA (PI) are B-class MADS-box floral homeotic genes of Arabidopsis and are involved in specifying the identity of petals and stamens. In the present work, IiAP3 and IiPI, the respective orthologous genes of AP3 and PI, were cloned from Isatis indigotica. By expressing in ap3-6 and pi-1 homozygous mutant and in wild-type Arabidopsis under the control of AP3 promoter or CaMV 35S promoter, we demonstrated that IiAP3 and IiPI were functionally equivalent to AP3 and PI of Arabidopsis. Referring to previous reports and the research results in the present work, expression patterns of AP3 and PI homologs are not the same in different angiosperms possessing diverse floral structures. It suggests that the alterations in expression may contribute to the changing morphology of flowers. To further determine the relationship between IiAP3 and IiPI, the coding sequences of the different structural regions in these two proteins were swapped with each other, and the data collected from transgenic Arabidopsis plants of the chimeric constructs suggested that MADS domain was irreplaceable for the function of IiAP3, K domain of IiAP3 was involved in specifying the identity of stamens, K domain of IiPI was mainly related to the formation of petals, and C-terminal region of IiPI was involved in characterization of stamens. In addition, a complete KC region of these two proteins was more effective in phenotypic complementation of the mutants.

(-)-Lariciresinol Isolated from the Roots of Isatis indigotica Fortune ex Lindl. Inhibits Hepatitis B Virus by Regulating Viral Transcription.

Chronic hepatitis induced by hepatitis B virus (HBV) infection is a serious public health problem, leading to hepatic cirrhosis and liver cancer. Although the currently approved medications can reliably decrease the virus load and prevent the development of hepatic diseases, they fail to induce durable off-drug control of HBV replication in the majority of patients. The roots of Isatis indigotica Fortune ex Lindl., a traditional Chinese medicine, were frequently used for the prevention of viral disease in China. In the present study, (-)-lariciresinol ((-)-LRSL), isolated from the roots of Isatis indigotica Fortune ex Lindl., was found to inhibit HBV DNA replication of both wild-type and nucleos(t)ide analogues (NUCs)-resistant strains in vitro. Mechanism studies revealed that (-)-LRSL could block RNA production after treatment, followed by viral proteins, and then viral particles and DNA. Promoter reporter assays and RNA decaying dynamic experiments indicated that (-)-LRSL mediated HBV RNA reduction was mainly due to transcriptional inhibition rather than degradation. Moreover, (-)-LRSL in a dose-dependent manner also inhibited other animal hepadnaviruses, including woodchuck hepatitis virus (WHV) and duck hepatitis B virus (DHBV). Combining the analysis of RNA-seq, we further found that the decrease in HBV transcriptional activity by (-)-LRSL may be related to hepatocyte nuclear factor 1α (HNF1α). Taken together, (-)-LRSL represents a novel chemical entity that inhibits HBV replication by regulating HNF1α mediated HBV transcription, which may provide a new perspective for HBV therapeutics.

[Cloning and functional characterization of lignan glycosyltransferase gene IiUGT349 in Isatis indigotica].

Based on the transcriptome data of Isatis indigotica, a total of 110 putative glycosytransferases were identified. Through prokaryotic expression and enzymic activity assay in vitro, a novel lignan glycosyltransferase gene was screened out and named IiUGT349, which catalyzed lariciresinol into lariciresinol-4-O-ß-D-glucoside and lariciresinol-4'-O-ß-D-glucoside. Bioinformatics analysis suggested that IiUGT349 contained an open reading frame(ORF) of 1 401 bp encoding a protein of 467 amino acids. A protein analysis indicated that IiUGT349 have a predecited molecular weight of 52.77 kDa and pI of 5.96. Phylogenetic analysis showed that IiUGT349 belonging to UGT90 family shared low amino acid sequence identity with the reported lignan glycosyltransferases, which may represent a novel type of lignan glycosyltransferases. Quantitative real-time PCR(qRT-PCR) analysis showed that IiUGT349 was expressed in roots, stems, young leaves and leaves, with the highest expression level in stems. Further biochemical analysis showed that the optimal reaction time of IiUGT349 recombinant protein was 12 h and the optimal temperature was 45 ℃. Subcellular localization demonstrated that IiUGT349 was located in the cytoplasm and nucleus of plants. In this study, a new glucosyltransferase gene IiUGT349 from I. indigotica belonging to the UGT90 family was cloned, which laid a foundation to further investigate its' function and elucidate the lignan glycosides biosynthesis pathway and plays an important role for great significance for the synthetic biology of active lignan glycosides.

Integrative analyses of targeted metabolome and transcriptome of Isatidis Radix autotetraploids highlighted key polyploidization-responsive regulators.

BACKGROUND: Isatidis Radix, the root of Isatis indigotica Fort. (Chinese woad) can produce a variety of efficacious compound with medicinal properties. The tetraploid I. indigotica plants exhibit superior phenotypic traits, such as greater yield, higher bioactive compounds accumulation and enhanced stress tolerance. In this study, a comparative transcriptomic and metabolomic study on Isatidis Radix autotetraploid and its progenitor was performed. RESULTS: Through the targeted metabolic profiling, 283 metabolites were identified in Isatidis Radix, and 70 polyploidization-altered metabolites were obtained. Moreover, the production of lignans was significantly increased post polyploidization, which implied that polyploidization-modulated changes in lignan biosynthesis. Regarding the transcriptomic shift, 2065 differentially expressed genes (DEGs) were identified as being polyploidy-responsive genes, and the polyploidization-altered DEGs were enriched in phenylpropanoid biosynthesis and plant hormone signal transduction. The further integrative analysis of polyploidy-responsive metabolome and transcriptome showed that 1584 DEGs were highly correlated with the 70 polyploidization-altered metabolites, and the transcriptional factors TFs-lignans network highlighted 10 polyploidy-altered TFs and 17 fluctuated phenylpropanoid pathway compounds. CONCLUSIONS: These results collectively indicated that polyploidization contributed to the high content of active compounds in autotetraploid roots, and the gene-lignan pathway network analysis highlighted polyploidy-responsive key functional genes and regulators.

Genome-Wide Identification and Analysis on YUCCA Gene Family in Isatis indigotica Fort. and IiYUCCA6-1 Functional Exploration.

Auxin is one of the most critical hormones in plants. YUCCA (Tryptophan aminotransferase of Arabidopsis (TAA)/YUCCA) enzymes catalyze the key rate-limiting step of the tryptophan-dependent auxin biosynthesis pathway, from IPA (Indole-3-pyruvateacid) to IAA (Indole-3-acetic acid). Here, 13 YUCCA family genes were identified from Isatis indigotica, which were divided into four categories, distributing randomly on chromosomes (2n = 14). The typical and conservative motifs, including the flavin adenine dinucleotide (FAD)-binding motif and flavin-containing monooxygenases (FMO)-identifying sequence, existed in the gene structures. IiYUCCA genes were expressed differently in different organs (roots, stems, leaves, buds, flowers, and siliques) and developmental periods (7, 21, 60, and 150 days after germination). Taking IiYUCCA6-1 as an example, the YUCCA genes functions were discussed. The results showed that IiYUCCA6-1 was sensitive to PEG (polyethylene glycol), cold, wounding, and NaCl treatments. The over-expressed tobacco plants exhibited high auxin performances, and some early auxin response genes (NbIAA8, NbIAA16, NbGH3.1, and NbGH3.6) were upregulated with increased IAA content. In the dark, the contents of total chlorophyll and hydrogen peroxide in the transgenic lines were significantly lower than in the control group, with NbSAG12 downregulated and some delayed leaf senescence characteristics, which delayed the senescence process to a certain extent. The findings provide comprehensive insight into the phylogenetic relationships, chromosomal distributions, and expression patterns and functions of the YUCCA gene family in I. indigotica.

Selection of reference genes for the quantitative real-time PCR normalization of gene expression in Isatis indigotica fortune.

BACKGROUND: Isatis indigotica, a traditional Chinese medicine, produces a variety of active ingredients. However, little is known about the key genes and corresponding expression profiling involved in the biosynthesis pathways of these ingredients. Quantitative real-time polymerase chain reaction (qRT-PCR) is a powerful, commonly-used method for gene expression analysis, but the accuracy of the quantitative data produced depends on the appropriate selection of reference genes. RESULTS: In this study, the systematic analysis of the reference genes was performed for quantitative real-Time PCR normalization in I. indigotica. We selected nine candidate reference genes, including six traditional housekeeping genes (ACT, α-TUB, ß-TUB, UBC, CYP, and EF1-α), and three newly stable internal control genes (MUB, TIP41, and RPL) from a transcriptome dataset of I. indigotica, and evaluated their expression stabilities in different tissues (root, stem, leaf, and petiole) and leaves exposed to three abiotic treatments (low-nitrogen, ABA, and MeJA) using geNorm, NormFinder, BestKeeper, and comprehensive RefFind algorithms. The results demonstrated that MUB and EF1-α were the two most stable reference genes for all samples. TIP41 as the optimal reference gene for low-nitrogen stress and MeJA treatment, while ACT had the highest ranking for ABA treatment and CYP was the most suitable for different tissues. CONCLUSIONS: The results revealed that the selection and validation of appropriate reference genes for normalizing data is mandatory to acquire accurate quantification results. The necessity of specific internal control for specific conditions was also emphasized. Furthermore, this work will provide valuable information to enhance further research in gene function and molecular biology on I. indigotica and other related species.

Combined transcriptome and metabolite profiling reveals that IiPLR1 plays an important role in lariciresinol accumulation in Isatis indigotica.

A lignan, lariciresinol, is an important efficacious compound for the antiviral effect of Isatis indigotica, a widely used herb for the treatment of colds, fever, and influenza. Although some rate-limiting steps of the lariciresinol biosynthetic pathway are well known, the specific roles of gene family members in I. indigotica in regulating lariciresinol production are poorly understood. In the present study, a correlation analysis between the RNA sequencing (RNA-Seq) expression profile and lignan content by using I. indigotica hairy roots treated with methyl jamonate (0.5 µM) at different time points as a source implicated that I. indigotica pinoresinol/lariciresinol reductase 1 (IiPLR1), but not IiPLR2 or IiPLR3, contributed greatly to lariciresinol accumulation. Gene silencing by RNA interference (RNAi) demonstrated that IiPLR1 indeed influenced lariciresinol biosynthesis, whereas suppression of IiPLR2 or IiPLR3 did not change lariciresinol abundance significantly. IiPLR1 was thus further characterized; IiPLR1 was constitutively expressed in roots, stems, leaves, and flowers of I. indigotica, with the highest expression in roots, and it responds to different stress treatments to various degrees. Recombinant IiPLR1 reduces both (±)-pinoresinol and (±)-lariciresinol efficiently, with comparative K cat/K m values. Furthermore, overexpression of IiPLR1 significantly enhanced lariciresinol accumulation in I. indigotica hairy roots, and the best line (ovx-2) produced 353.9 µg g(-1) lariciresinol, which was ~6.3-fold more than the wild type. This study sheds light on how to increase desired metabolites effectively by more accurate or appropriate genetic engineering strategies, and also provides an effective approach for the large-scale commercial production of pharmaceutically valuable lariciresinol by using hairy root culture systems as bioreactors.

[Cloning and expression analysis of a hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyl transferasegene(IiHCT) from Isatis indigotica].

Based on the transcriptome data, we cloned the open reading frame of IiHCT gene from Isatis indigotica, and then performed bioinformatic analysis of the sequence. Further, we detected expression pattern in specific organs and hairy roots treated methyl jasmonate( MeJA) by RT-PCR. The IiHCT gene contains a 1 290 bp open reading frame( ORF) encoding a polypeptide of 430 amino acids. The predicted isoelectric point( pI) was 5.7, a calculated molecular weight was about 47.68 kDa. IiHCT was mainly expressed in stem and undetectable in young root, leaf and flower bud. After the treatment of MeJA, the relative expression level of IiHCT increased rapidly. The expression level of IiHCT was the highest at 4 h and maintained two fold to control during 24 h. In this study, cloning of IiHCT laid the foundation for illustrating the biosynthesis mechanism of phenylpropanoids in I. indigotica.

The dirigent multigene family in Isatis indigotica: gene discovery and differential transcript abundance.

BACKGROUND: Isatis indigotica Fort. is one of the most commonly used traditional Chinese medicines. Its antiviral compound is a kind of lignan, which is formed with the action of dirigent proteins (DIR). DIR proteins are members of a large family of proteins which impart stereoselectivity on the phenoxy radical-coupling reaction, yielding optically active lignans from two molecules of E-coniferyl alcohol. They exist in almost every vascular plant. However, the DIR and DIR-like protein gene family in I. indigotica has not been analyzed in detail yet. This study focuses on discovery and analysis of this protein gene family in I. indigotica for the first time. RESULTS: Analysis of transcription profiling database from I. indigotica revealed a family of 19 full-length unique DIR and DIR-like proteins. Sequence analysis found that I. indigotica DIR and DIR-like proteins (IiDIR) were all-beta strand proteins, with a signal peptide at the N-terminus. Phylogenetic analysis of the 19 proteins indicated that the IiDIR genes cluster into three distinct subfamilies, DIR-a, DIR-b/d, and DIR-e, of a larger plant DIR and DIR-like gene family. Gene-specific primers were designed for 19 unique IiDIRs and were used to evaluate patterns of constitutive expression in different organs. It showed that most IiDIR genes were expressed comparatively higher in roots and flowers than stems and leaves. CONCLUSIONS: New DIR and DIR-like proteins were discovered from the transcription profiling database of I. indigotica through bioinformatics methods for the first time. Sequence characteristics and transcript abundance of these new genes were analyzed. This study will provide basic data necessary for further studies.

Development of a complete set of monosomic alien addition lines between Brassica napus and Isatis indigotica (Chinese woad).

KEY MESSAGE: A complete set of monosomic alien addition lines of Brassica napus with one of the seven chromosomes of Isatis indigotica and the recombinant mitochondria was developed and characterized. Monosomic alien addition lines (MAALs) are valuable for elucidating the genome structure and transferring the useful genes and traits in plant breeding. Isatis indigotica (Chinese woad, 2n = 14, II) in Isatideae tribe of Brassicaceae family has been widely cultivated as a medicinal and dye plant in China. Herein, the intertribal somatic hybrid (2n = 52, AACCII) between B. napus cv. Huashuang 3 (2n = 38, AACC) and I. indigotica produced previously was backcrossed recurrently to parental B. napus, and 32 MAAL plants were isolated. Based on their phenotype, 5S and 45S rDNA loci and chromosome-specific SSR markers, these MAALs were classified into seven groups corresponding to potential seven types of MAALs carrying one of the seven I. indigotica chromosomes. One of the MAALs could be distinguishable by expressing the brown anthers of I. indigotica, other two hosted the chromosome with 5S or 45S rDNA locus, but the remaining four were identifiable by SSR markers. The simultaneous detection of the same SSR maker and gene locus in different MAALs revealed the paralogs on the chromosomes involved. The recombinant mitochondrial genome in MAALs was likely related with their male sterility with carpellody stamens, while the MAAL with normal brown anthers probably carried the restoring gene for the male sterility. The complete set of MAALs should be useful for exploiting the I. indigotica genome and for promoting the introgression of valuable genes to B. napus.

[Identification of cellular morphology and flow cytometry on different tetraploid Isatis indigotica strains].

OBJECTIVE: To identify tetraploid Isatis indigotica strains through morphology and flow cytometry. METHODS: The tissue culture seedlings of tetraploid Isatis indigotica were root-tip squashed and chromosome counted before rooted climatized and transplanted in field. The plants in field were taken as experimental materials. Macroscopic observation was applied to identify by form and structure; Free-hand section was used to observe the length, width and density of stomas; And flow cytometry was applied to identify the ploidy. RESULTS: Compared with diploid plants, tetraploid plants had obvious changes in form and structure. The stomas from the tetraploid were notably longer, and the number of guard cells in chloroplasts was remarkably larger. The experiment materials were proved to be tetraploid by flow cytometry. CONCLUSION: The materials are tetraploid plants. Macroscopic observation, the length of stoma and the number of guard cells in chloroplasts can be taken as aided identification for ploidy of mutagenesis materials. Meanwhile, flow cytometry can be applied to identify the ploidy of Isatis indigotica.

The potential mechanism of response to light intensity in energy metabolism mediated by miRNA in Isatis indigotica.

Light is the main source of energy for plant growth. Studies have shown that I. indigotica is a light-demanding plant and its yield and various active components are positively correlated with light intensity, but no studies of light intensity affecting energy metabolism in I. indigotica have been reported. Mitochondria are the main site of energy metabolism, and miRNAs are important factors in regulating gene expression, this experiment attempts to study the effects of different light intensities on energy metabolism from the perspective of mitochondria and miRNAs. The results show that the biomass、mitochondrial structural integrity and energy metabolism in I. indigotica were found to be positively correlated with light intensity. Small RNA and transcriptome sequencing identified 241 miRNAs and 36,372 mRNAs, and degradomic technology identified 72 miRNAs targeting 106 mRNAs, among which 12 pairs of miRNA-mRNAs were annotated on mitochondria. Combined with RT-qPCR validation, it was concluded that miR167a-5p positively regulates LETM1 and affects mitochondrial structure, miR400-5p and mIR169m-p3_1ss15CT negatively regulate GRXS15 and CMC4, respectively, affecting SDH and CCO activities, and miR395a-APS4 may affect the utilization of ATP and sulfate assimilation. In summary, the results of this study complement and enrich knowledge of light effects on mitochondria from the perspective of miRNA, while providing guidance for the cultivation of I. indigotica.

IiAGL6 participates in the regulation of stamen development and pollen formation in Isatis indigotica.

The AGL6 (AGMOUSE LIKE 6) gene is a member of the SEP subfamily and functions as an E-class floral homeotic gene in the development of floral organs. In this study, we cloned IiAGL6, the orthologous gene of AGL6 in Isatis indigotica. The constitutive expression of IiAGL6 in Arabidopsis thaliana resulted in a late-flowering phenotype and the development of curly leaves during the vegetative growth period. Abnormal changes in floral organ development were observed during the reproductive stage. In woad plants, suppression of IiAGL6 using TRV-VIGS (tobacco rattle virus-mediated virus-induced gene silencing) decreased the number of stamens and led to the formation of aberrant anthers. Similar changes in stamen development were also observed in miRNA-AGL6 transgenic Arabidopsis plants. Yeast two-hybrid and BiFC tests showed that IiAGL6 can interact with other MADS-box proteins in woad; thus, playing a key role in defining the identities of floral organs, particularly during stamen formation. These findings might provide novel insights and help investigate the biological roles of MADS transcription factors in I. indigotica.

[Cloning and functional characterization of the pinoresinol-lariciresinol reductase gene IiPLR2 in Isatis indigotica].

The pinoresinol-lariciresinol reductase (PLR), a crucial enzyme in the biosynthesis of lignans in plants, catalyzes a two-step reaction to produce lariciresinol and secoisolariciresinol. Lignans such as lariciresinol are the effective components of traditional Chinese medicine Radix Isatidis in exerting antiviral activity. In order to study the function of the key enzyme PLR in the biosynthesis of lariciresinol in Isatis indigotica, the original plant of Radix Isatidis, IiPLR2 was cloned from I. indigotica, with a full length of 954 bp, encoding 317 amino acids. Multiple sequence alignment showed that IiPLR2 contained a conserved nicotinamide adenine dinucleotide phosphate (NADPH)-binding motif. The phylogenetic tree showcased that IiPLR2 shared the same clade with AtPrR1 from Arabidopsis thaliana. The prokaryotic expression vector pET32a-IiPLR2 was constructed and then transformed into Escherichia coli BL21(DE3) competent cells for protein expression. The purified enzyme IiPLR2 could catalyze the conversion of pinoresinol to lariciresinol and the conversion of lariciresinol to secoisolariciresinol. The cloning, sequencing, and catalytic functional analysis of IiPLR2 in this study enrich the understanding of this kind of functional proteins in I. indigotica and supplement the biosynthesis pathways of lignans. Moreover, this study provides a functional module for further research on metabolic regulation and synthetic biology and lays a foundation for comprehensively revealing the relationship between the spatial structures and catalytic functions of such proteins.

Biosynthesis of the active compounds of Isatis indigotica based on transcriptome sequencing and metabolites profiling.

BACKGROUND: Isatis indigotica is a widely used herb for the clinical treatment of colds, fever, and influenza in Traditional Chinese Medicine (TCM). Various structural classes of compounds have been identified as effective ingredients. However, little is known at genetics level about these active metabolites. In the present study, we performed de novo transcriptome sequencing for the first time to produce a comprehensive dataset of I. indigotica. RESULTS: A database of 36,367 unigenes (average length = 1,115.67 bases) was generated by performing transcriptome sequencing. Based on the gene annotation of the transcriptome, 104 unigenes were identified covering most of the catalytic steps in the general biosynthetic pathways of indole, terpenoid, and phenylpropanoid. Subsequently, the organ-specific expression patterns of the genes involved in these pathways, and their responses to methyl jasmonate (MeJA) induction, were investigated. Metabolites profile of effective phenylpropanoid showed accumulation pattern of secondary metabolites were mostly correlated with the transcription of their biosynthetic genes. According to the analysis of UDP-dependent glycosyltransferases (UGT) family, several flavonoids were indicated to exist in I. indigotica and further identified by metabolic profile using UPLC/Q-TOF. Moreover, applying transcriptome co-expression analysis, nine new, putative UGTs were suggested as flavonol glycosyltransferases and lignan glycosyltransferases. CONCLUSIONS: This database provides a pool of candidate genes involved in biosynthesis of effective metabolites in I. indigotica. Furthermore, the comprehensive analysis and characterization of the significant pathways are expected to give a better insight regarding the diversity of chemical composition, synthetic characteristics, and the regulatory mechanism which operate in this medical herb.

[Discussion on the botanical origin of Isatidis radix and Isatidis folium based on DNA barcoding].

This paper aimed to investigate the botanical origins of Isatidis Radix and Isatidis Folium, and clarify the confusion of its classification. The second internal transcribed spacer (ITS2) of ribosomal DNA, the chloroplast matK gene of 22 samples from some major production areas were amplified and sequenced. Sequence assembly and consensus sequence generation were performed using the CodonCode Aligner. Phylogenetic study was performed using MEGA 4.0 software in accordance with the Kimura 2-Parameter (K2P) model, and the phylogenetic tree was constructed using the neighbor-joining methods. The results showed that the length of ITS2 sequence of the botanical origins of Isatidis Radix and Isatidis Folium was 191 bp. The sequence showed that some samples had several SNP sites, and some samples had heterozygosis sites. In the NJ tree, based on ITS2 sequence, the studied samples were separated into two groups, and one of them was gathered with Isatis tinctoria L. The studied samples also were divided into two groups obviously based on the chloroplast matK gene. In conclusion, our results support that the botanical origins of Isatidis Radix and Isatidis Folium are Isatis indigotica Fortune, and Isatis indigotica and Isatis tinctoria are two distinct species. This study doesn't support the opinion about the combination of these two species in Flora of China.