Chalcone Isomerase Gene Research Articles

Identification and Characterization of Chalcone Isomerase Genes Involved in Flavonoid Production in Dracaena cambodiana.

Dragon’s blood is a traditional medicine in which flavonoids are the main bioactive compounds; however, the underlying formation mechanism of dragon’s blood remains largely poorly understood. Chalcone isomerase (CHI) is the key enzyme in the flavonoid biosynthesis pathway. However, CHI family genes are not well understood in Dracaena cambodiana Pierre ex Gagnep, an important source plant of dragon’s blood. In this study, 11 CHI family genes were identified from D. cambodiana, and they were classified into three types. Evolutionary and transcriptional profiling analysis revealed that DcCHI1 and DcCHI4 might be involved in flavonoid production. Both DcCHI1 and DcCHI4 displayed low expression levels in stem under normal growth conditions and were induced by methyl jasmonate (MeJA), 6-benzyl aminopurine (6-BA, synthetic cytokinin), ultraviolet-B (UV-B), and wounding. The recombinant proteins DcCHI1 and DcCHI4 were expressed in Escherichia coli and purified by His-Bind resin chromatography. Enzyme activity assay indicated that DcCHI1 catalyzed the formation of naringenin from naringenin chalcone, while DcCHI4 lacked this catalytic activity. Overexpression of DcCHI1 or DcCHI4 enhanced the flavonoid production in D. cambodiana and tobacco. These findings implied that DcCHI1 and DcCHI4 play important roles in flavonoid production. Thus, our study will not only contribute to better understand the function and expression regulation of CHI family genes involved in flavonoid production in D. cambodiana but also lay the foundation for developing the effective inducer of dragon’s blood.

Correlation between DNA methylation of the chalcone isomerase gene in Lithocarpus polystachyus Rehd and phlorizin accumulation

Correlation between DNA methylation of the chalcone isomerase gene in Lithocarpus polystachyus Rehd and phlorizin accumulation

Cloning and Functional Characterization of Chalcone Isomerase Genes Involved in Anthocyanin Biosynthesis in <i>Clivia miniata</i>

Chalcone isomerase (CHI), catalyzing isomerization of chalcones, is a crucial enzyme in flavonoid biosynthesis. Three <i>CHI</i> genes were isolated from <i>Clivia miniata</i> and designated as <i>CmCHI1, CmCHI2</i> and <i>CmCHI3</i>, respectively. Multiple sequence alignments and phylogenetic analysis showed that CmCHI1 and CmCHI2 were members of type I CHI proteins, whereas CmCHI3 belonged to type IV CHI proteins. Subcellular localization analysis found that all three CmCHIs had diffused distribution in the cytoplasm similar to green fluorescent protein (GFP). Anthocyanin biosynthesis and gene expression analysis demonstrated that <i>CmCHIs</i> were highly expressed in anthocyanin accumulated tissues. To further functionally characterize the role of CmCHIs, an <i>in vitro</i> enzymatic activity assay was carried out using the purified recombinant proteins. Results showed that CmCHI1 and CmCHI2 could completely convert the substrate naringenin chalcone (NC) into the product naringenin (NA), whereas CmCHI3 seemed nonfunctional as no increment of NA was detected. Further genetic transformation of <i>Arabidopsis tt5-1</i> mutant validated that CmCHI1 and CmCHI2 rather than CmCHI3 could complement the <i>chi</i> deficient phenotypes. In summary, <i>CmCHI1</i> and <i>CmCHI2</i> are the real active <i>CHI</i> genes in <i>Clivia miniata</i>. The results not only broaden our knowledge on flavonoid biosynthesis in <i>C. miniata</i> but also lay a new foundation for further flavonoid modification in <i>C. miniata</i>.

Adaptive Evolution of Chalcone Isomerase Superfamily in Fagaceae.

Chalcone Isomerase (CHI) catalyzes the biosynthesis of flavonoids and secondary metabolism in plants. Currently, there is no systematic analysis of CHIs gene family in Fagaceae which is available. In this study, twenty-two CHI proteins were identified in five species of the Fagaceae family. The CHI superfamily in Fagaceae can be classified into three subfamilies and five groups using phylogenetic analysis, analysis of physicochemical properties, and structural prediction. Results indicated that serine (Ser) and isoleucine (Ile) residues determine the substrate preferred by active Type I Fagaceae CHI, and the chalcone isomerase-like (CHIL) of Fagaceae had active site residues. Adaptive analysis of CHIs showed that CHIs are subject to selection pressure. The active CHI gene of Fagaceae was located in the cytoplasm, and it had the typical gene structure of CHI and contains four exons. All the twenty-two identified CHIs had the conserved domain motif 3, and the different groups had their own structural characteristics. In the process of fatty acid binding protein (FAP) evolution to CHIL and CHI, the physical and chemical properties of proteins also had significant differences in addition to changes in protein functions.

Gene polymorphism of chalcone isomerase influence the accumulation of flavonoids in licorice (Glycyrrhiza spp.)

Licorice has been used in traditional Chinese medicine for more than 2000 years. Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in licorice. The purpose of this study is to analyze the CHI gene polymorphism and identify CHI haplotypes associated with high/low flavonoid contents. Five Glycyrrhiza uralensis samples with the high flavonoids contents and five Glycyrrhiza inflate samples with low flavonoids contents were selected to clone the CHI cDNA sequences. The CHI haplotypes related to the accumulation of flavonoids were determined and induced to express in Pichia pastoris GS115. Enzymatic reactions and HPLC method were used to verify the functions of the specific CHI haplotypes. 100 CHI cDNA sequences with a full length of 690 bp were obtained and 48 haplotypes were determined. Haplotype 1 and 31 are the major haplotypes in groups with high/low flavonoids contents, respectively. CHI-1 and CHI-31 were successfully expressed and the HPLC results indicated that the catalytical activities of CHI-1 were higher than that of CHI-31. This study analyzed the gene polymorphism of CHI and determined that CHI-1 was conducive to flavonoid biosynthesis. The results of this study will provide a basis for clarifying the function of CHI and further molecular breeding of the high-quality licorice.

Molecular cloning and enzymatic characteristics of chalcone isomerase from Rheum palmatum

Rheum palmatum has been widely developed in traditional Chinese medicine. One of the major active compounds in R. palmatum is flavonoids. Until now, few genes involved in flavonoid biosynthesis have been isolated from this medicinal plant. In this study, a new chalcone isomerase gene RpCHI was cloned from an RNA-seq library of R. palmatum. The 714bp RpCHI cDNA was cloned and sequenced. The RpCHI recombinant protein was successfully overexpressed in a prokaryotic expression system and purified to homogeneity by a nickel-nitrilotriacetic acid Ni-NTA prepacked column, and the enzymatic activity was investigated. The results have clarified that RpCHI was assembled into the type I CHI group based on phylogenetic analysis. Activity examination revealed that RpCHI exhibits high substrate affinity for naringenin chalcone, and it can also use isoliquiritigenin in relatively low activity. Optimum pH and temperature for enzymatic reaction was determined to be 7.0 and 40 °C, respectively. RpCHI was successfully cloned in the experiment, and the enzymatic activity revealed RpCHI uses various substrates to produce antioxidant flavonoids.

Isolation and Characterization of Chalcone Isomerase (CHI) Gene from Boesenbergia rotunda

Chalcone isomerase (CHI; E.C. 5.5.1.6) is an enzyme that plays critical roles in the flavonoid biosynthesis pathway and plant secondary metabolism. Although CHI genes have been widely studied in various higher plants, to date there are no studies about CHI genes in Boesenbergia rotunda. The CHI gene (747 bp) was isolated from different parts of the plant, including rhizomes, leaves and flowers of B. rotunda. The isolated B. rotunda CHI, designated BrCHI, contained one intron and two exons and encoded a peptide of 248 amino acid with a predicted molecular mass of 26.2 kDa and pI of 5.03. The deduced amino acid sequence of BrCHI shares 63–65% identity with CHI from other plants within the order of Zingiberales such as banana and canna lily. Sequence alignment showed that the coding region of the BrCHI gene is highly similar to CHI genes from other plant species and has four conserved amino acid residues (Thr53, Tyr112, Asn119, and Ser191) which are essential for CHI function. Phylogenetic analysis has clustered BrCHI together with other CHIs belonging to CHI type I. Recombinant BrCHI protein was successfully expressed in E. coli using the pMAL-c4X vector cloning system. In vitro enzyme activity, assayed by HPLC, indicated that recombinant BrCHI could catalyse the formation of naringenin from naringenin chalcone. This study demonstrates the ability of bacterial host to overexpress BrCHI gene isolated from B. rotunda to produce functional BrCHI that successfully converts naringenin chalcone to naringenin

A high-quality chromosomal genome assembly of Diospyros oleiferaCheng.

Background Diospyros oleifera Cheng, of the family Ebenaceae, is an economically important tree. Phylogenetic analyses indicate that D. oleifera is closely related to Diospyros kaki Thunb. and could be used as a model plant for studies of D. kaki. Therefore, development of genomic resources of D. oleifera will facilitate auxiliary assembly of the hexaploid persimmon genome and elucidate the molecular mechanisms of important traits.FindingsThe D. oleifera genome was assembled with 443.6 Gb of raw reads using the Pacific Bioscience Sequel and Illumina HiSeq X Ten platforms. The final draft genome was ∼812.3 Mb and had a high level of continuity with N50 of 3.36 Mb. Fifteen scaffolds corresponding to the 15 chromosomes were assembled to a final size of 721.5 Mb using 332 scaffolds, accounting for 88.81% of the genome. Repeat sequences accounted for 54.8% of the genome. By de novo sequencing and analysis of homology with other plant species, 30,530 protein-coding genes with an average transcript size of 7,105.40 bp were annotated; of these, 28,580 protein-coding genes (93.61%) had conserved functional motifs or terms. In addition, 171 candidate genes involved in tannin synthesis and deastringency in persimmon were identified; of these chalcone synthase (CHS) genes were expanded in the D. oleifera genome compared with Diospyros lotus, Camellia sinensis, and Vitis vinifera. Moreover, 186 positively selected genes were identified, including chalcone isomerase (CHI) gene, a key enzyme in the flavonoid-anthocyanin pathway. Phylogenetic tree analysis indicated that the split of D. oleifera and D. lotus likely occurred 9.0 million years ago. In addition to the ancient γ event, a second whole-genome duplication event occurred in D. oleifera and D. lotus.ConclusionsWe generated a high-quality chromosome-level draft genome for D. oleifera, which will facilitate assembly of the hexaploid persimmon genome and further studies of major economic traits in the genus Diospyros.

The Chalcone Isomerase Family in Cotton: Whole-Genome Bioinformatic and Expression Analyses of the Gossypium barbadense L. Response to Fusarium Wilt Infection.

Chalcone isomerase (CHI) is a key component of phenylalanine metabolism that can produce a variety of flavonoids. However, little information and no systematic analysis of CHI genes is available for cotton. Here, we identified 33 CHI genes in the complete genome sequences of four cotton species (Gossypium arboretum L., Gossypium raimondii L., Gossypium hirsutum L., and Gossypium barbadense L.). Cotton CHI proteins were classified into two main groups, and whole-genome/segmental and dispersed duplication events were important in CHI gene family expansion. qRT-PCR and semiquantitative RT-PCR results suggest that CHI genes exhibit temporal and spatial variation and respond to infection with Fusarium wilt race 7. A preliminary model of CHI gene involvement in cotton evolution was established. Pairwise comparison revealed that seven CHI genes showed higher expression in cultivar 06-146 than in cultivar Xinhai 14. Overall, this whole-genome identification unlocks a new approach to the comprehensive functional analysis of the CHI gene family, which may be involved in adaptation to plant pathogen stress.

Dissection of Chemical Composition and Associated Gene Expression in the Pigment-Deficient Tea Cultivar 'Xiaoxueya' Reveals an Albino Phenotype and Metabolite Formation.

The tea cultivar ‘Xiaoxueya’, a temperature-sensitive albino mutant, is a rare tea germplasm because of its highly enriched amino acid content and brisk flavour. In comparison with green leaf tissues of ‘Xiaoxueya’, albino leaves show significant deficiency in chlorophylls and carotenoids and severely disrupted chloroplasts. Furthermore, the accumulation of quality-related secondary metabolites is altered in ‘Xiaoxueya’ albino leaf, with significantly increased contents of total amino acids, theanine, and glutamic acid and significantly decreased contents of alkaloids, catechins, and polyphenols. To uncover the molecular mechanisms underlying albinism and quality-related constituent variation in ‘Xiaoxueya’ leaves, expression profiles of pivotal genes involved in the biosynthetic pathways of pigments, caffeine, theanine, and catechins were investigated by quantitative real-time PCR technology. The results revealed that suppressed expression of the chloroplast-localized 1-deoxy-D-xylulose-5-phosphate synthase genes DXS1 and DXS2 involved in the 2-C-methyl-D-erythritol-4-phosphate (MEP) pathway and protochlorophyllide oxidoreductase genes POR1 and POR2 involved in the chlorophyll biosynthetic pathway is responsible for the pigment deficiency in ‘Xiaoxueya’ albino leaf. Additionally, the low expression of the tea caffeine synthase gene (TCS) involved in caffeine biosynthesis and the chalcone synthase genes CHS1, CHS2, and CHS3, the chalcone isomerase gene CHI, the flavonoid 3’,5’-hydroxylase genes F3’5’H1 and F3’5’H2, and the anthocyanidin reductase genes ANR1 and ANR2 involved in the flavonoid pathway is related to the reduction in alkaloid and catechin levels in ‘Xiaoxueya’ albino leaves.

Functional and genomic characterization of a wound- and methyl jasmonate-inducible chalcone isomerase in Eremochloa ophiuroides [Munro] Hack

Eremochloa ophiuroides, a perennial warm-season lawn grass, has a characteristic phenotype of red pigmentation in tissues during maturation. The putative gene families associated with the red coloration were previously identified in E. ophiuroides. These genes encode chalcone synthases, flavonol 3-hydroxylases, and flavonol 3′-hydroxylases, acting on the early flavonoid-biosynthesis pathway. Here, a type-I chalcone isomerase (CHI) gene was isolated from E. ophiuroides based on leaf-transcriptome data, and the corresponding enzyme was functionally characterized in vitro and in planta. Complementation of Arabidopsis tt5 mutants by overexpressing EoCHI recapitulated the wild-type seed coat color. Wounding and methyl jasmonate treatments significantly elevated the transcript level of EoCHI and total anthocyanin content in shoots. Confocal microscopy indicated the localization of EoCHI to the endoplasmic reticulum. The genomic EoCHI sequence contained two introns with a novel pattern of exon‒intron organization. Further examinations on genomic structures of CHI family from ancient to advanced plant lineages should be of interests to decipher evolutionary pathways of extant plant CHI genes.

Integrating molecular characterization and metabolites profile revealed CtCHI1\u2019s significant role in Carthamus tinctorius L.

BackgroundAs a traditional Chinese herb, safflower (Carthamus tinctorius L.) is valued for its florets to prevent cardiovascular and cerebrovascular diseases. Basing on previous chemical analysis, the main active compounds are flavonoids in its florets. Although flavonoid biosynthetic pathway has been well-documented in many model species, unique biosynthetic pathway remains to be explored in safflower. Of note, as an important class of transitional enzymes, chalcone isomerase (CHI) has not been characterized in safflower.ResultsAccording to our previous research, CHIs were identified in a safflower transcriptome library built by our lab. To characterize CHI in safflower, a CHI gene named CtCHI1 was identified. A multiple sequences alignment and phylogenetic tree demonstrate that CtCHI1 shares 92% amino acid identity and close relationship with CHI to Saussurea medusa. Additionally, subcellular localization analysis indicated CtCHI1-GFP fusion protein was mainly in the cell nucleus. Further, we purified CtCHI1 protein from E. coli which can effectively catalyze isomerization of 2′,4′,4,6′-tetrahydroxychalcone into naringenin in vitro. Via genetic engineer technology, we successfully obtained transgenic tobacco and safflower lines. In transgenic tobacco, overexpression of CtCHI1 significantly inhibited main secondary metabolites accumulation, including quercetin (~ 79.63% for ovx-5 line) and anthocyanins (~ 64.55% for ovx-15 line). As shown in transgenic safflower, overexpression of CtCHI1 resulted in upstream genes CtPAL3 and CtC4H1 increasing dramatically (up to ~ 3.9fold) while Ct4CL3, CtF3H and CtDFR2 were inhibited. Also, comparing the whole metabolomics database by PCA and PLS-DA between transgenic and control group, 788 potential differential metabolites were marked and most of them displayed up-regulated trends. In parallel, some isolated secondary metabolites, such as hydroxysafflor yellow A (HSYA), rutin, kaempferol-3-O-β-rutinoside and dihydrokaempferol, accumulated in transgenic safflower plants.ConclusionsIn this study, we found that CtCHI1 is an active, functional, catalytic protein. Moreover, CtCHI1 can negatively and competitively regulate anthocyanins and quercetin pathway branches in tobacco. By contrast, CtCHI1 can positively regulate flavonol and chalcone metabolic flow in safflower. This research provides some clues to understand CHI’s differential biochemical functional characterization involving in flavonoid pathway. More molecular mechanisms of CHI remain to be explored in the near future.

Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.'Hangju'

Three Chrysanthemum-chalcone-isomerase genes( CmCHI) were successfully cloned by PCR from the database of Chrysanthemum transcriptome and named CmCHI1,CmCHI2 and CmCHI3,respectively. Bioinformatics analysis showed that the base numbers of CmCHI1-3 open reading frame were 708,633 and 681 bp,encoding 235,210 and 226 amino acids,respectively. Three fusion proteins of about 30 kDa were successfully induced by prokaryotic expression technology,and the corresponding recombinant fusion proteins were isolated and purified by Ni-NTA resin column. Clustering analysis showed that the 3 CmCHI were homologous with Compositae plants,and CmCHI1 and CmCHI3 belonged to type Ⅰ CHI. CmCHI2 belongs to type Ⅳ CHI. Using β-actin as an internal reference gene,RT-qPCR was used to detect and analyze the expression of CmCHI1-3 genes in Hangju. The results showed that the expression levels of CmCHI1 and CmCHI3 were higher,while the expression levels of CmCHI2 were lower. It was concluded that CmCHI1 and CmCHI3 were the main chalcone isomerase genes involved in the synthesis of flavonoids in Hangju,and CmCHI2 was a helper gene. Flooding treatment significantly promoted the expression of CmCHI1 and CmCHI3 genes,but had no regulatory effect on CmCHI2. The above results provided a basis for further study of the molecular regulation mechanism of CHI gene in the metabolism of flavonoids in Hangju,which laid a foundation for improving the content of flavonoids in Hangju and finally improving the medicinal quality of Hangju.

Cloning and characterization of chalcone synthase and chalcone isomerase genes in Arisaema heterophyllum

Chalcone synthase( CHS) and chalcone isomerase( CHI) are key enzymes in the biosynthesis pathway of flavonoids. In this study,unigenes for CHS and CHI were screened from the transcriptome database of Arisaema heterophyllum. The open reading frame( ORFs) of chalcone synthase( Ah CHS) and chalcone isomerase( Ah CHI) were cloned from the plant by RT-PCR. The physicochemical properties,expression and structure characteristics of the encoded proteins Ah CHS and Ah CHI were analyzed. The ORFs of Ah CHS and Ah CHI were 1 176,630 bp in length and encoded 392,209 amino acids,respectively. Ah CHS functioned as a symmetric homodimer. The N-terminal helix of one monomer entwined with the corresponding helix of another monomer. Each CHS monomer consisted of two structural domains. In particular,four conserved residues define the active site. The tertiary structure of Ah CHI revealed a novel open-faced β-sandwich fold. A large β-sheet( β4-β11) and a layer of α-helices( α1-α7) comprised the core structure. The residues spanning β4,β5,α4,and α6 in the three-dimensional structure were conserved among CHIs from different species. Notably,these structural elements formed the active site on the protein surface,and the topology of the active-site cleft defined the stereochemistry of the cyclization reaction. The homology comparison showed that Ah CHS had the highest similarity to the CHS of Anthurium andraeanum,while Ah CHI had the highest similarity to the CHI of Paeonia delavayi. This study provided the basis for the functional study of Ah CHS and Ah CHI and the further study on plant flavonoid biosynthesis pathway.

Cloning and expression analysis of a new chalcone isomerase gene duringflowering in safflower

Cloning and expression analysis of a new chalcone isomerase gene duringflowering in safflower

Pruning induced yield and quality variations and the correlated gene expression and phytohormone changes in Lonicera japonica

Lonicera japonica Thunb. is widely used in China to make teas, aromatic oils or medicines. The demand for L. japonica is large in China. Pruning measures are used to increase the yield of L. japonica in practice. However, there remains a question whether the quality of L. japonica is increased as well. Our study focused on the accumulations of the primary bioactive components and their molecular mechanisms, as well as the roles of endogenous plant hormones on L. japonica after pruning. To achieve this aim, the flower buds of L. japonica were randomly collected and divided into four groups for agronomic traits investigation, bioactive compound changes detection by liquid chromatography coupled with tandem mass spectrometry (LCMS/MS), the related gene expression levels quantification by quantitative real-time reverse transcription-PCR (qRT-PCR), and endogenous phytohormone changes determination by indirect enzyme-linked immunosorbent assay (ELISA), respectively. Results showed that pruning produced superior flower bud yield but inferior quality in L. japonica, especially at the first harvest time. More specifically, pruning significantly increased the yield (1.4-fold) but significantly decreased contents of chlorogenic acid (1.5-fold), isochlorogenic acid A (1.3-fold), luteoloside (1.4-fold), and isoquercetrin (1.3-fold) at the first harvest time. The decreased hydroxycinnamoyl-CoA quinate hydroxycinnamoyl transferase (HQT) and chalcone isomerase (CHI) gene expression levels were probably the main reasons for L. japonica quality decrease. Pruning also induced flexible phytohormone changes in L. japonica. Indole-3-acetic acid (IAA), cytokines (CKs), jasmonic acid (JA), and abscisic acid (ABA) were the main hormones involving in building shoot architecture, flower development, and plant secondary metabolism. In conclusion, we think the pruning technique should be used carefully or with other agronomic practices to obtain the well balance between yield and quality in L. japonica.

Cloning and analyzing of chalcone isomerase gene (AaCHI) from Artemisia annua

Artemisinin, isolated from Chinese medical herbal plant Artemisia annua L., was reported to be the main compound of anti-malaria drugs. However, the artemisinin content was very low in A. annua. Great efforts have been made to increase the artemisinin in A. annua. Chalcone isomerase (CHI) was a rate-limiting enzyme in flavonoids metabolic pathways. Terpenoids and flavonoids are from different biosynthetic pathways, but there is a synergistic action between them on a variety of biological processes in plants. Here, the full-length cDNA of CHI was isolated from A. annua. The AaCHI gene, contained a 690 bp open reading frame, which encoded a protein with 229-amino acids. An analysis of AaCHI transcript levels in multifarious tissues of A. annua showed that flower and bud had the highest transcription levels. Unexpectedly, the artemisinin biosynthetic genes and artemisinin content showed an increase in the AaCHI overexpression transgenic A. annua plants. The results indicated that overexpression of AaCHI gene was an effective method to improve the artemisinin content in A. annua. Taken together, our findings showed that AaCHI is involved in artemisinin production in A. annua and revealed a link between flavonoids and terpenoid production.

The Research Progress of Chalcone Isomerase (CHI) in Plants.

Chalcone isomerase (CHI) is the second rate-limiting and the first reported enzyme involved in the biosynthetic pathway of flavonoids. It catalyzes the intramolecular cyclization reaction, converting the bicyclic chalcone into tricyclic (2S)-flavanone. In this paper, we obtained and analyzed 916 DNA sequences, 1310 mRNA sequences, and 2403 amino acid sequences of CHI registered in NCBI by Jan 2018. The full length of CHI DNA sequences ranges from 218 to 3758bp, CHI mRNA sequences ranges from 265 to 1436bp, and CHI amino acid sequences ranges from 35 to 465 amino acid residues. Forty representative species were selected from each family to construct the maximum likelihood tree and analyze the evolutionary relationship. According to the medicinal and agricultural use, 13 specific species were selected, and their physicochemical properties were analyzed. The molecular weight of CHI ranges from 23 to 26kD, and the isoelectric point of CHI ranges from 4.93 to 5.85. All the half-life periods of CHI are 30h in mammalian reticulocytes in vitro, 20h in yeast, and 10h in E. coli in vivo, theoretically. The consistency of the 13 CHI amino acid sequences is 63.55%. According to the similarity between each sequence, we selected four CHI sequences of Paeonia suffruticosa, Paeonia lactiflora, Taxus wallichiana, and Tradescantia hirsutiflora for secondary structure, three-dimensional protein models, conserved domains, transmembrane structure, and signal peptide prediction analysis. It was found that CHI sequences of Paeonia suffruticosa and Paeonia lactiflora owned a higher similarity; they both share the template 4doi.1.A. The four CHI all have no signal peptides, and they exert their activities in cytoplasm. Then, PubMed, Web of Science, Science Direct, and Research Gate were used as information sources through the search terms 'chalcone isomerase', 'biosynthesis', 'expression', and their combinations to get the latest and comprehensive information of CHI, mainly from the year 2010 to 2018. More than 300 papers were searched and 116 papers were reviewed in the present work. We summarized the classification of CHI, catalytic reaction mechanism of CHI, and progress of genetic engineering regarding CHI clone, expression, and exogenous stimulator regulation. This paper will lay a foundation for further studies of CHI and other functional genes involved in flavonoids biosynthetic pathway.

Overexpression of the Glycine max chalcone isomerase ( GmCHI ) gene in transgenic Talinum paniculatum plants

Talinum paniculatum plants contain flavonoids and saponins, which have strong antioxidant properties and are used in the treatment of numerous diseases, such as inflammation, allergies, and gastric ulcers. Currently, there is no published research on the flavonoid content of T. paniculatum plants; however, it has been determined that the species in the genus Talinum have very low flavonoid content, and therefore the effective approach to enhance the flavonoid content in T. paniculatum plants is overexpression of the Glycine max chalcone isomerase (GmCHI) gene. In this study, we analyzed GmCHI gene overexpression, which has increased total flavonoid content in transgenic T. paniculatum lines. We have successfully transformed T. paniculatum plants with the pCB301-GmCHI vector and created 2 lines in generation T1 (T1-2.2 and T1-10) with a transgenic frequency of 0.27%. Recombinant CHI protein expressed in transgenic lines has a molecular weight of approximately 25 kDa, and the concentration of this protein in the two transgenic lines T1-2.2 and T1-10 was 4.29 ?g mg?1 and 6.14 ?g mg?1, respectively. Total flavonoid contents of the two transgenic lines T1-2.2 and T1-10 were 4.24 mg g?1 and 2.74 mg g?1, respectively, which showed increases of 7.4-fold and 4.8-fold compared to that of the nontransgenic plants, respectively. Thus, overexpression of the GmCHI gene enabled T. paniculatum plants to improve their total flavonoid content. This work also confirmed the effectiveness of Agrobacterium-mediated plant transformation in enhancement of flavonoid accumulation in T. paniculatum plants.

Age-associated methylation change of CHI promoter in herbaceous peony (Paeonia lactiflora Pall).

Chalcone isomerase gene (CHI) is a key gene that regulates the formation of yellow traits in petals. To reveal transcriptional regulatory mechanisms of CHI gene in petals of Paeonia lactiflora, we investigated the CHI expression using qPCR, the pigment content by HPLC, and methylation levels using BSP+Miseq sequencing in ‘Huangjinlun’ variety during different developmental stages including flower-bud stage (S1), initiating bloom (S2), bloom stage (S3), and withering stage (S4). Results showed that the expression level of CHI gene at S2 stage was significantly higher than that at other stages (P<0.05), and at S4 stage was extremely significantly lower than other stages (P<0.01). Besides, total anthocyanin, anthoxanthin, and flavonoid contents in petals presented a similar trend with CHI expression during developmental stages. A total of 16 CpG sites varying methylation levels were detected in CHI gene core promoter region, of which the methylation levels at mC-4 and mC-16 sites were extremely significantly negatively correlated with CHI mRNA expression (P<0.01). mC-16 site is located in the binding region of C/EBPα transcription factor, suggesting that methylation at the mC-16 site may inhibit the binding of C/EBPα to CHI promoter DNA, thereby regulating the tissue-specific expression of CHI gene. Our study revealed the expression pattern of CHI gene in petal tissues of P. lactiflora at different developmental stages, which is related to promoter methylation. Moreover, the important transcription regulation element–C/EBPα was identified, providing theoretical reference for in-depth study on the function of CHI gene in P. lactiflora.