Cloning and catalytic analysis of Isatis indigotica chalcone isomerase in vitro / 中国中药杂志

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.

Genome-wide analysis of AP2/ERF superfamily in Isatis indigotica / 中西医结合学报

OBJECTIVE@#AP2/ERF (APETALA2/ethylene-responsive factor) superfamily is one of the largest gene families in plants and has been reported to participate in various biological processes, such as the regulation of biosynthesis of active lignan. However, few studies have investigated the genome-wide role of the AP2/ERF superfamily in Isatis indigotica. This study establishes a complete picture of the AP2/ERF superfamily in I. indigotica and contributes valuable information for further functional characterization of IiAP2/ERF genes and supports further metabolic engineering.@*METHODS@#To identify the IiAP2/ERF superfamily genes, the AP2/ERF sequences from Arabidopsis thaliana and Brassica rapa were used as query sequences in the basic local alignment search tool. Bioinformatic analyses were conducted to investigate the protein structure, motif composition, chromosome location, phylogenetic relationship, and interaction network of the IiAP2/ERF superfamily genes. The accuracy of omics data was verified by quantitative polymerase chain reaction and heatmap analyses.@*RESULTS@#One hundred and twenty-six putative IiAP2/ERF genes in total were identified from the I. indigotica genome database in this study. By sequence alignment and phylogenetic analysis, the IiAP2/ERF genes were classified into 5 groups including AP2, ERF, DREB (dehydration-responsive element-binding factor), Soloist and RAV (related to abscisic acid insensitive 3/viviparous 1) subfamilies. Among which, 122 members were unevenly distributed across seven chromosomes. Sequence alignment showed that I. indigotica and A. thaliana had 30 pairs of orthologous genes, and we constructed their interaction network. The comprehensive analysis of gene expression pattern in different tissues suggested that these genes may play a significant role in organ growth and development of I. indigotica. Members that may regulate lignan biosynthesis in roots were also preliminarily identified. Ribonucleic acid sequencing analysis revealed that the expression of 76 IiAP2/ERF genes were up- or down-regulated under salt or drought treatment, among which, 33 IiAP2/ERF genes were regulated by both stresses.@*CONCLUSION@#This study undertook a genome-wide characterization of the AP2/ERF superfamily in I. indigotica, providing valuable information for further functional characterization of IiAP2/ERF genes and discovery of genetic targets for metabolic engineering.

Genetic Diversity Analysis of Isatis indigotica Germplasm Materials by Chloroplast DNA Variation and ISSR Molecular Marker / 中国实验方剂学杂志

ObjectiveTo analyze the sequence variation and genetic diversity of 47 Isatis indigotica germplasm materials， and carry out the study on the genetic differentiation and structure. MethodGenomic DNA of 47 I. indigotica germplasm materials were extracted by kit extraction method. Two chloroplast DNA （cp DNA） sequences and five inter-simple sequence repeat （ISSR） primers were used for amplification and sequencing. Chromas， Mega 7.0， DanSP5， and GenALEx were used to calibrate， splice， and analyze the sequence characteristics. PERMUT and PopGen 1.31 were used to analyze the genetic diversity parameters and genetic structure， and NTSYS was used to obtain the unweighted pair-group method with arithmetic means（UPGMA） clustering tree plot of 47 I. indigotica germplasm materials. ResultA total of 129 samples from 47 I. indigotica germplasm materials were successfully amplified and sequenced. The length of 2 cp DNA sequences after spliced was 1 412 bp， and there were 377 polymorphic variation loci， and 36 haplotypes. Fu and Li's D* test was significant （P<0.01）. The values of Pi， HS， and HT based on cp DNA were 0.119 89， 0.787， and 0.891， respectively. The genetic differentiation coefficients of gene differentiation coefficient（Gst）， nucleotide differentiation coefficient（Nst）， and fixation index（Fst） were 0.117， 0.468， and 0.488， respectively， and the gene flow （Nm） was 0.615. The mean values of PPB， Shannon information diversity index（I）， Nei's genetic diversity index（H）， and Gst based on ISSR were 78.85%， 0.334 8， 0.218 6， and 0.754 4， respectively， and the Nm value was 0.162 8. ConclusionI. indigotica has high genetic diversity and abundant haplotypes at the species level， with abundant haplotypes. Genetic differentiation among different germplasm materials is obvious， and gene exchange is not frequent. Genetic variation mainly exists among populations. The population has accumulated various low-frequency gene mutations recently， suggesting that it has experienced significant regional expansion in the history.

Genetic Diversity Analysis of Isatis indigotica Germplasm Materials by Chloroplast DNA Variation and ISSR Molecular Marker / 中国实验方剂学杂志

ObjectiveTo analyze the sequence variation and genetic diversity of 47 Isatis indigotica germplasm materials， and carry out the study on the genetic differentiation and structure. MethodGenomic DNA of 47 I. indigotica germplasm materials were extracted by kit extraction method. Two chloroplast DNA （cp DNA） sequences and five inter-simple sequence repeat （ISSR） primers were used for amplification and sequencing. Chromas， Mega 7.0， DanSP5， and GenALEx were used to calibrate， splice， and analyze the sequence characteristics. PERMUT and PopGen 1.31 were used to analyze the genetic diversity parameters and genetic structure， and NTSYS was used to obtain the unweighted pair-group method with arithmetic means（UPGMA） clustering tree plot of 47 I. indigotica germplasm materials. ResultA total of 129 samples from 47 I. indigotica germplasm materials were successfully amplified and sequenced. The length of 2 cp DNA sequences after spliced was 1 412 bp， and there were 377 polymorphic variation loci， and 36 haplotypes. Fu and Li's D* test was significant （P<0.01）. The values of Pi， HS， and HT based on cp DNA were 0.119 89， 0.787， and 0.891， respectively. The genetic differentiation coefficients of gene differentiation coefficient（Gst）， nucleotide differentiation coefficient（Nst）， and fixation index（Fst） were 0.117， 0.468， and 0.488， respectively， and the gene flow （Nm） was 0.615. The mean values of PPB， Shannon information diversity index（I）， Nei's genetic diversity index（H）， and Gst based on ISSR were 78.85%， 0.334 8， 0.218 6， and 0.754 4， respectively， and the Nm value was 0.162 8. ConclusionI. indigotica has high genetic diversity and abundant haplotypes at the species level， with abundant haplotypes. Genetic differentiation among different germplasm materials is obvious， and gene exchange is not frequent. Genetic variation mainly exists among populations. The population has accumulated various low-frequency gene mutations recently， suggesting that it has experienced significant regional expansion in the history.

Discovery of alkaloids from the leaves of Isatis indigotica Fortune with neuroprotective activity / 中国天然药物

Seven alkaloids including five undescribed ones (1a/1b, 2, 3 and 5) were obtained from the leaves of Isatis indigotica Fortune. Their structures were established by extensive spectroscopic analyses. The absolute configurations of compounds 1a, 1b, 3 and 5 were determined by comparison of the experimental and calculated electronic circular dichroism (ECD) spectra. Subsequently, the neuroprotective effects of all the isolates against H

Erucic acid from Isatis indigotica Fort. suppresses influenza A virus replication and inflammation in vitro and in vivo through modulation of NF-kB and p38 MAPK pathway / 药物分析学报

Isatis indigotica Fort. (Ban-Lan-Gen) is an herbal medicine prescribed for influenza treatment. However, its active components and mode of action remain mostly unknown. In the present study, erucic acid was isolated from Isatis indigotica Fort., and subsequently its underlying mechanism against influenza A virus (IAV) infection was investigated in vitro and in vivo. Our results demonstrated that erucic acid exhibited broad-spectrum antiviral activity against IAV resulting from reduction of viral polymerase transcription activity. Erucic acid was found to exert inhibitory effects on IAV or viral (v) RNA-induced pro-inflam-matory mediators as well as interferons (IFNs). The molecular mechanism by which erucic acid with antiviral and anti-inflammatory properties was attributed to inactivation of NF-kB and p38 MAPK signaling. Furthermore, the NF-kB and p38 MAPK inhibitory effect of erucic acid led to diminishing the transcriptional activity of interferon-stimulated gene factor 3 (ISGF-3), and thereby reducing IAV-triggered pro-inflammatory response amplification in IFN-β-sensitized cells. Additionally, IAV- or vRNA-triggered apoptosis of alveolar epithelial A549 cells was prevented by erucic acid. In vivo, erucic acid administration consistently displayed decreased lung viral load and viral antigens expression. Meanwhile, erucic acid markedly reduced CD8+cytotoxic T lymphocyte (CTL) recruitment, pro-apoptotic signaling, hyperactivity of multiple signaling pathways, and exacerbated immune inflammation in the lung, which resulted in decreased lung injury and mortality in mice with a mouse-adapted A/FM/1/47-MA(H1N1) strain infection. Our findings provided a mechanistic basis for the action of erucic acid against IAV-mediated inflammation and injury, suggesting that erucic acid may have a therapeutic potential in the treatment of influenza.

Indole alkaloid glycosides with a 1'-(phenyl)ethyl unit from leaves

Seven indole alkaloid glycosides containing a 1'-(4″-hydroxy-3″,5″-dimethoxyphenyl)ethyl unit (-) were isolated from an aqueous extract of leaves (da qing ye). Their structures were determined by spectroscopic data analysis combined with enzymatic hydrolysis as well as comparison of their experimental CD (circular dichroism) and calculated ECD (electrostatic circular dichroism) spectra. Based on analysis of and/or Cotton effect (CE) data of -, two simple roles to assign location and/or configuration of -glycopyranosyloxy and 1'-(phenyl)ethyl units in the indole alkaloid glycosides are proposed. Stereoselectivity in plausible biosynthetic pathways of - is discussed. Compounds and and their mixture in a 3:2 ratio showed activity against KCNQ2 in CHO cells. The mixture of and (3:2) exhibited antiviral activity against influenza virus H1N1 PR8 with IC 64.7 μmol/L (ribavirin, IC 54.3 μmol/L), however, the individual or was inactive. Preliminary structure-activity relationships were observed.

Textual research on medicinal blue herbs / 中国中药杂志

In this paper, through the collection and collation of ancient herbs, medical books and prescriptions, combined with modern literature, the historical changes of the name, origin, position, medicinal parts, collection, processing and processing of bluegrass were systematically combed and verified.It can be seen from the research that bluegrass was first used as medicine by the fruit, namely blueberry, which was originally Polygonum tinctorium. Since the Ming and Qing Dynasties, blueberry was rarely used, and it has been no longer used medicinally. In the Wei and Jin Dynasties, the medicinal parts extended to the stems and leaves, and most of them used juice as medicine.Since the Tang Dynasty, origin has been extended to Isatis indigotica, Baphicacanthus cusia, Indigofera tinctoria, Compositae plant Wulan, etc. In the Song Dynasty, the medicinal parts extended to the roots, and the "Banlangen" began to appear, and gradually became the main medicinal parts of blue medicinal materials, the main base of which was B. cusia. Since the Qing Dynasty, I. indigotica, a Cruciferae, has gradually become a genuine indigo root, while B. cusia has become a southern indigo root. It was the first mineral dye imported from abroad for thrush, and then used as medicine, also known as clam powder. Because it was found that it had the same effect with the extract of bluegrass, it was also named indigo naturalis in China, which has lasted till now. The main stream of Isatidis Folium in the past dynasties is the dry stem and leaf of Clerodendrum cyrtophylum. Since the Qing Dynasty, the stem and leaf of Isatis indigotica, P. tinctorium and other blue grasses have been gradually mixed as substitutes and gradually become the mainstream.

Glycosides from roots of Isatis indigotica / 中草药

Objective: To study the glycosides from the roots of Isatis indigotica. Methods: The chemical components were isolated and purified by silica gel, Sephadex LH-20, ODS C18 gel, and semi preparative-HPLC chromatographic techniques while the structures were deduced by NMR, MS and IR spectral data analysis. Results: A total of 12 glycosides were obtained and elucidated as isatindigoside C (1), epiprogoitrin (2), progoitrin (3), o-aminobenzoic acid 7-O-β-D-glucopyranosyl ester (4), 2’-O-methyladenosine (5), 2-methoxyphenyl β-D-glucopyranoside (6), coniferin (7), syringin (8), cis-coniferin (9), cis-syringin (10), corchoionoside C (11), and cannabiside D (12). Conclusion: Compound 1 is a new indole alkaloid glycoside which named as isatindigoside C, while compounds 5-7 and 9-12 are isolated from the plant for the first time.

Improvement of Identification Method （3）of Isatis indigotica from 2015 Edition of Chinese Pharmacopeia （PartⅠ） / 中国药房

OBJECTIVE： To investigate the rationality of TLC identification method （3） of （R，S）-epigoitrin in Isatis indigotica stated in 2015 edition of Chinese Pharmacopeia （partⅠ） （later abbreviated as pharmacopeia）， and make some improvements. METHODS： Three batches I. indigotica were collected and prepared into decoction pieces according to the processing method of I. indigotica in pharmacopoeia. TLC identification of （R，S）-goitrin in I. indigotica decoction piece and medicinal material were conducted according to identification method （3） in pharmacopeia （80％ ethanol as solvent for sample treatment， ultrasound extraction）； the rationality of pharmacopoeia method was investigated. Then the method was improved by changing the extraction solvent and pretreatment method （method one： using water as solvent， ultrasound extraction； method two： soaking in water for 1 h， then adding into methanol， ultrasound extraction； method three： the sample was wetted and then dried， using 80％ methanol as solvent， ultrasound extraction） of samples， and the optimal method was verified. According to the optimal method， the TLC identification of （R，S）-goitrin was detected by using chromatographic plates from different manufacturers， under the conditions of low temperature and low humidity （7 ℃， relative humidity 48％） and high temperature and high humidity （35 ℃， relative humidity 75％） respectively，to investigate the durability of the method. RESULTS： According to the method of pharmacopeia， in the chromatograms of decoction pieces， the same color spots appeared at the corresponding chromatographic positions of reference substance， but no corresponding spots appeared in the medicinal material chromatograms. After the samples were treated by three improvement methods， in medicinal material chromatograms， the same color spots appeared in the corresponding chromatographic positions of reference substances. There were single chromatographic spot after medicinal materials were treated with method one， and there were more spots after medicinal materials were treated with method two and three， and method two consumed less time than method three. The results of validation tests and method durability tests  showed that after the treatment of I. indigotica and its decoction pieces according to method two， the same color spots appeared in the corresponding positions of the decoction pieces and the medicinal materials chromatograms as those of the control. CONCLUSIONS： The improved TLC identification method is effective， the chromatographic spots are clear， and the repeatability is good.

Chemical constituents from roots of Isatis indigotica / 中国中药杂志

Nineteen compounds were isolated from the roots of Isatis indigotica through silica gel, Sephadex LH-20, ODS and pre-HPLC chromatography technique. Their structures were elucidated by the MS and NMR spectra as 7-hydroxydeoxyvasicinone (1), (1H-indol-3-yl) oxoacetamide (2), 1-methoxy-1H-indole-3-acetonitrile (3), arvelexin (4), 1H-indole-3-acetonitrile (5), 1H-indole-3-aldehyde (6), 1H-indole-3-acetic acid (7), 2,3-dihydro-4-hydroxy-2-oxo-indole-3-acetonitrile (8), deoxyvasicinone (9), indigotiisocoumarin A (10), cycloanthranilylproline (11), quinazoline-2,4(1H,3H)-diones (12), indirubin(13), (+)-pinoresinol (14), (+)-epipinoresinol (15), burselignan (16), (+)-isolariciresinol (17),vanillic acid (18) and 5-hydroxymaltol (19). Among them, compound 1 is a new natural product, and compound 2, 14, 15, 18 and 19 were isolated from the title plant for the first time.

Effects of nitrogen and sulfur combined application on nutritional components and active components of Isatis indigotica at seedling stage / 中国中药杂志

Using split plot design, a pot experiment with sand culture was conducted to investigate the effects ofnitrogen and sulfur combined application on nutritional components and active component of Isatis indigotica at seedling stage under different N (5，15，25 mmol·L⁻¹)and S(0.00，1.25，2.50，5.00，7.50 mmol·L⁻¹) levels. The results showed thatthe two elements had obvious effects and the leaf and root dry weights of I. indigotica seedlings increased greatly at N₂ level. Under the same nitrogen concentration, the leaf and root dry weights increased firstly and decreased with the rising of sulfur concentrations in which S₂ was conducive to the growth and biomass accumulation. Soluble sugar, soluble protein, soluble amino acids contents were the highest in N₁, N₂ and N₃ treatments, respectively. The influence of sulfur concentrations on nutritional components was same as biomass, but the peak of different nutritional components was diversity in different nitrogen levels. The effects on secondary metabolites (total flavones, indigo, indriubin, epigotrin contents) were not obvious significantly, in which these indexes by N₁S₃，N₁S₂，N₃S₀，N₃S₁were the highest, respectively. In conclusion, the combination of nitrogen and sulfur of N₂S₂(N 15 mmol·L⁻¹ and S 2.5 mmol·L⁻¹) was beneficial to the growth and secondary metabolites accumulation of I. indigotica. These results could provide a theoretical basis for rational fertilization and cultivation of I. indigotica seedling.

Sulfur-enriched alkaloids from the root of

Five new sulfur-enriched alkaloids isatithioetherins A-E (-), and two pairs of scalemic enantiomers (+)- and (-)-isatithiopyrin B ( and ) and isoepigoitrin and isogoitrin and ), along with the known scalemic enantiomers epigoitrin and goitrin ( and ), were isolated and characterized from an aqueous extract of the roots. Their structures were determined by extensive spectroscopic data analysis, including 2D NMR and theoretical calculations of electronic circular dichroism (ECD) spectra based on the quantum-mechanical time-dependent density functional theory (TDDFT). Compounds - represent a novel group of sulfur-enriched alkaloids, biogenetically originating from stereoselective assemblies of epigoitrin-derived units. Isolation and structure characterization of and support the postulated biosynthetic pathways for the diastereomers and a rare thio-Diels-Alder reaction. Compounds and showed antiviral activity against the influenza virus A/Hanfang/359/95 (H3N2, IC 0.60 and 1.92 μmol/L) and the herpes simplex virus 1 (HSV-1, IC 3.70 and 2.87 μmol/L), and also inhibited Coxsackie virus B3 (IC 0.71 μmol/L).

Bioinformatics analysis of laccases involved in active lignans′ biosynthesis in Isatis indigotica / 药学实践杂志

Objective Identifying laccases, as one of the key synthetases in the lariciresinol biosynthetic pathway, by analyzing the transcriptome sequencing results in Isatis indigotica would provide a dependable foundation for later functional study of Isatis indigotica′s laccases. Methods Bioinformatical softwares and kinds of analytical methods online were used to find out the characteristics of the laccases from I. indigotica, including physical and chemical properties, homology, and the properties after induction of MeJA. Results The transcriptional results showed that Iilac3 and Iilac5 from I. indigotica were corresponded ith the accumulation of the effective metabolites, making them the potential functional genes participated in lariciresinol synthesis. Conclusion Through the detailed bioinformatical analysis of Iilacs,which laid a solid foundation for the further study of the physiological and biochemical mechanisms and structural characteristics of the functional proteins.

Aromatic compounds from an aqueous extract of "ban lan gen" and their antiviral activities

A pair of new diphenyl glycerol ether enantiomers (-)-and (+)-and two new methyl benzamidobenzoatesand, named (-)-()- and (+)-()-isatindigotrioic acid [(-)-and (+)-] and isatindigoticamides A () and B (), respectively, were isolated from an aqueous decoction of the roots of(ban lan gen). Their structures were elucidated by spectroscopic data analysis including 2D NMR experiments. The absolute configurations of (-)-and (+)-were assigned based on the CD exciton chirality method. Compoundsandexhibited antiviral activities against HSV-1 with ICvalues of 4.87 and 25.87 μmol/L, respectively. Compoundwas also found active against Coxsackie virus B3 and LPS-induced NO production.

Indole alkaloid sulfonic acids from an aqueous extract ofroots and their antiviral activity

Six new indole alkaloid sulfonic acids (-), together with two analogues (and) that were previously reported as synthetic products, were isolated from an aqueous extract of theroot. Their structures including the absolute configurations were determined by spectroscopic data analysis, combined with enzyme hydrolysis and comparison of experimental circular dichroism and calculated electronic circular dichroism spectra. In the preliminary assay, compoundsandshowed antiviral activity against Coxsackie virus B3 and influenza virus A/Hanfang/359/95 (H3N2), respectively.

8,4'-Oxyneolignane glucosides from an aqueous extract of "ban lan gen" (root) and their absolute configurations

Three pairs of glycosidic 8,4'-oxyneolignane diastereoisomers, named isatioxyneolignosides A-F (-), were isolated from an aqueous extract ofroots. Their structures and absolute configurations were elucidated by comprehensive spectroscopic data analysis and enzyme hydrolysis. The validity of Δvalues to distinguishandaryl glycerol units and Cotton effects at 235±5 nm to determine absolute configurations at C-8 in-and their aglycones (-) are discussed.

Advances in Biosynthesis of Antiviral Active Components of Isatidis Radix / 世界科学技术-中医药现代化

Isatidis Radix,the root of Isatis indigotica Fort.(Cruciferae),is a representitive herb widely used for clearing away heat-toxin in traditional Chinese medicine (TCM).Lariciresinol is a representitive component of lignans and an important efficacious substance with the antiviral effect.This review elucidated the progress on its biosynthetic pathways,the screening of key regulatory genes and metabolic engineering of lignans components in Isatidis Radix,providing a favorable reference for the full understanding of biosynthesis of antiviral active components,the quality improvement of Isatidis Radix and the sustainable utilization of TCM resources.

Computational identification and systematic classification of novel GRAS genes in Isatis indigotica / 中国天然药物

Isatis indigotica Fort., belonging to Cruciferae, is one of the most commonly used plants in traditional Chinese medicine. The accumulation of the effective components of I. indigotica is related with its growth conditions. The GRAS genes are members of a multigene family of transcriptional regulators that play a crucial role in plant growth. Although the activities of many GRAS genes have long been recognized, only in recent years were some of them identified and functionally characterized in detail. In the present study, 41 GRAS genes were identified from I. indigotica through bioinformatics methods for the first time. They were classified into ten groups according to the classification of Arabidopsis and rice. The characterization, gene structure, conserved motifs, disordered N-terminal domains, and phylogenetic reconstruction of these GRASs were analyzed. Forty-three orthologous gene pairs were shared by I. indigotica and Arabidopsis, and interaction networks of these orthologous genes were constructed. Furthermore, gene expression patterns were investigated by analysis in methyl jasmonate (MeJA)-treated I. indigotica hairy roots based on RNA-seq data. In conclusion, this comprehensive analysis would provide rich resources for further studies of GRAS protein functions in this plant.

The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis / 中国天然药物

Phenolic compounds, metabolites of the phenylpropanoid pathway, play an important role in the growth and environmental adaptation of many plants. Phenylalanine ammonia-lyase (PAL) is the first key enzyme of the phenylpropanoid pathway. The present study was designed to investigate whether there is a multi-gene family in I. Indigotic and, if so, to characterize their properties. We conducted a comprehensive survey on the transcription profiling database by using tBLASTn analysis. Several bioinformatics methods were employed to perform the prediction of composition and physicochemical characters. The expression levels of IiPAL genes in various tissues of I. indigotica with stress treatment were examined by quantitative real-time PCR. Protoplast transient transformation was used to observe the locations of IiPALs. IiPALs were functionally characterized by expression with pET-32a vector in Escherichia colis strain BL21 (DE3). Integration of transcripts and metabolite accumulations was used to reveal the relation between IiPALs and target compounds. An new gene (IiPAL2) was identified and both IiPALs had the conserved enzymatic active site Ala-Ser-Gly and were classified as members of dicotyledon. IiPAL1 and IiPAL2 were expressed in roots, stems, leaves, and flowers, with the highest expression levels of IiPAL1 and IiPAL2 being observed in stems and roots, respectively. The two genes responded to the exogenous elicitor in different manners. Subcellular localization experiment showed that both IiPALs were localized in the cytosol. The recombinant proteins were shown to catalyze the conversion of L-Phe to trans-cinnamic acid. Correlation analysis indicated that IiPAL1 was more close to the biosynthesis of secondary metabolites than IiPAL2. In conclusion, the present study provides a basis for the elucidation of the role of IiPALs genes in the biosynthesis of phenolic compounds, which will help further metabolic engineering to improve the accumulation of bioactive components in I. indigotica.