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.

Cloning and function analysis of chalcone isomerase gene and chalcone synthase gene in Lonicera macranthoides / 中国中药杂志

In order to explore the functions of genes of key rate-limiting enzymes chalcone isomerase(CHI) and chalcone synthase(CHS) in the biosynthesis of flavonoids in Lonicera macranthoides, this study screened and cloned the cDNA sequences of CHI and CHS genes from the transcriptome data of conventional variety and 'Xianglei' of L. macranthoides. Online bioinformatics analysis software was used to analyze the characteristics of the encoded proteins, and quantitative reverse-transcription polymerase chain reaction(qRT-PCR) to detect the expression of CHI and CHS in different parts of the varieties at different flowering stages. The content of luteo-loside was determined by high performance liquid chromatography(HPLC) and the correlation with the expression of the two genes was analyzed. The results showed that the CHI and CHS of the two varieties contained a 627 bp and 1170 bp open reading frame(ORF), respectively, and the CHI protein and CHS protein were stable, hydrophilic, and non-secretory. qRT-PCR results demonstrated that CHI and CHS of the two varieties were differentially expressed in stems and leaves at different flowering stages, particularly the key stages. Based on HPLC data, luteoloside content was in negative correlation with the relative expression of the genes. Thus, CHI and CHS might regulate the accumulation of flavonoids in L. macranthoides, and the specific functions should be further studied. This study cloned CHI and CHS in L. macranthoides and analyzed their expression for the first time, which laid a basis for investigating the molecular mechanism of the differences in flavonoids such as luteoloside in L. macranthoides and variety breeding.

Cloning and expression analysis of chalcone isomerase from Aquilaria sinensis / 药学学报

Chalcone isomerases (CHIs) play an essential role in the biosynthesis of flavonoids important in plant self-defense. Based on the transcriptome data of Aquilaria sinensis Calli, a full-length cDNA sequence of CHI1 (termed as AsCHI1) was cloned by reverse transcription PCR. AsCHI1 contains a complete open frame (ORF) of 654 bp. The deduced protein is composed of 217 amino acids, with a predicted molecular weight of 23.11 kDa. The sequence alignment and phylogenetic analysis revealed that AsCHI1 has conserved most of the active site residues in type I CHIs, indicating a close relationship with the CHI from Gossypium hirsutum. The recombinant AsCHI1 protein was obtained by heterologous expression of AsCHI1 in E. coli BL21(DE3). The purified AsCHI1 protein exhibited CHI activity by catalyzing the production of naringenin from naringenin chalcone. Remarkably, AsCHI1 expression in A. sinensis Calli treated with various abiotic stresses including salt, mannitol, cold, and heavy metals could be markedly increased, and plant hormones such as abscisic acid (ABA), gibberellin (GA3), and salicylic acid (SA) could also increase the expression of AsCHI1, suggesting that AsCHI1 might play an important role in plant self-defense. The results expand our understanding of the biosynthesis of flavonoids in A. sinensis and give further insight into the defensive responses of A. sinensis to abiotic and biotic stresses.

Overexpressing of chalcone isomerase (CHI) gene enhances flavonoid accumulation in Glycyrrhiza uralensis hairy roots / 药学学报

Chalcone isomerase (CHI) is the second rate-limiting enzyme involved in the biosynthetic pathway of flavonoids in Glycyrrhiza uralensis. Based on our previous studies, we selected the specific CHI haplotype (GenBank Accession No. KY115232) to maximize flavonoid accumulation. We constructed a plant binary expression vector for overexpression of this CHI gene by the gene fusion method and transfected the plasmid into Agrobacterium tumefaciens ACCC10060 by electroporation. The recombinant A. tumefaciens ACCC10060 subsequently was used to infect cotyledons and hypocotyls of G. uralensis to obtain transgenic hairy roots. A qRT-PCR method was used to determine the copy number of CHI and a UPLC method was used to assay the content of four flavonoids in different hairy root lines. The qRT-PCR results showed that the copy number of CHI in hairy roots was 1 or 5. UPLC results showed that the content of total flavonoids, liquiritin, liquiritigenin, and isoliquiritigenin in transgenic hairy root samples was significantly higher than that in wild-type samples. This study demonstrates that overexpression of CHI significantly increases the content of flavonoids in hairy roots of G. uralensis. This work provides a theoretical basis for clarifying the function of CHI. Three transgenic hairy root lines of G. uralensis were isolated which can be used to increase the accumulation of licorice flavonoids in vitro.

Cloning,expression and characterization of chalcone isomerase from medicinal plant Chinese sumac (Rhus chinensis) / 中国中药杂志

Flavonoids are a group of secondary metabolites found in plants. They have many pharmacological functions and play an important role in Chinese sumac( Rhus chinensis),which is a well-known traditional Chinese medicinal plant. Chalcone isomerase( CHI,EC 5. 5. 1. 6) is one of the key enzymes in the flavonoids biosynthesis pathway. In this paper,the full-length c DNA sequence encoding the chalcone isomerase from R. chinensis( designated as Rc CHI) was cloned by RT-PCR and rapid-amplification of c DNA Ends( RACE). The Rc CHI c DNA sequence was 1 058 bp and the open reading frame( ORF) was 738 bp. The ORF predicted to encode a 245-amino acid polypeptide. Rc CHI gene contained an intron and two exons. The sequence alignments revealed Rc CHI shared47. 1%-71. 6% identity with the homologues in other plants. Real-time PCR analysis showed that the total flavonoid levels were positively correlated with tissue-specific expressions of Rc CHI mRNA in different tissues. The recombinant protein was successfully expressed in an Escherichia coli strain with the p GEX-6 P-1 vector. In this paper,the CHI gene was cloned and characterized in the family of Anacardiaceae and will help us to obtain better knowledge of the flavonoids biosynthesis of the flavonoid compounds in R. chinensis.

Cloning and prokaryotic expression of CHI in Chrysanthemum morifolium cv.'Hangju' / 中国中药杂志

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 / 中国中药杂志

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.

Biocatalytic access to diverse prenylflavonoids by combining a regiospecific -prenyltransferase and a stereospecific chalcone isomerase

Prenylflavonoids are valuable natural products that have diverse biological properties, and are usually generated biologically by multiple metabolic enzymes in nature. In this study, structurally diverse prenylflavonoids were conveniently synthesized by enzymatic catalysis by combining GuILDT, a regiospecific chalcone prenyltransferase, and GuCHI, a stereospecific chalcone isomerase that has promiscuous activity for both chalcones and prenylchalcones as substrates. Our findings provided a new approach for the synthesis of natural/unnatural bioactive prenylflavonoids, including prenylchalcones and optical prenylflavanones with chalcone origins.

Cloning and bioinformatic analysis of chalcone isomerase gene in Lithocarpus polystachyus / 中草药

Objective: To understand the chalcone isomerase (CHI) gene expression by cloning it in Lithocarpus polystachyus. Methods: A full-length cDNA of CHI gene from Lithocarpus polystachyus (Lpr-CHI) was obtained by PCR cloning technique according transcriptomics sequences infromation, which bioinformatics analysis was carried out. The expression of CHI gene in different organs of Lithocarpus polystachyus was detected by qRT-PCR. Results: Lpr-CHI was 772 bp in full length with an open reading frame (ORF) of 696 bp, which encoded a protein with 231 amino acids. The protein did not contain a transmembrane domain and is localized in the cytoplasm. Lpr-CHI gene expression was found in different parts, and reached the highest in leaf, which was 9.75 times of the least gene expression in root. Conclusion: Lpr-CHI was obtained for the first time, and it was clear that the gene belongs to CHI type II. And the expression of Lpr-CHI in each organ was significantly different.

Construction of CHI gene expression vector of safflower and its transformation in Arabidopsis thaliana / 中草药

Objective: Plant expression vector of chalcone isomerase in safflower was built and its function was verified by over- expression of CHI in Arabidopsis thaliana. Methods: CHI isolated from safflower in our previous study was introduced by BamH I and EcoR I restriction sites and constructed into the over-expression vector pBASTA-CHI containing 35S promoter, transformed into A. thaliana by Flora-dip method. T2 plants of transgenic A. thaliana were detected by PCR and content of total flavones. Results: Plant expression vector containing the safflower CHI gene was built and over expressed in A. thaliana successfully. T2 plants of transgenic A. thaliana were obtained. Conclusion: PCR of transgenic T2 in A. thaliana is detected that CHI gene of safflower has been integrated into the A. thaliana genome, flavone content is determined in leaves, and the results show that the flavone in transgenetic A. thaliana is higher than that in wild type, the highest strain increases by nearly 2.3 times.

Cloning and bioinformatic analysis of chalcone isomerase in Scutellaria baicalensis / 中草药

Objective: To clone chalcone isomerase (sbCHI) gene from the callus of Scutellaria baicalensis and to analyze its bioinformatics. Methods: RNA was obtained from scutellariae callus, cDNA was reversely transcribed, specific primers were designed, and then CHI was cloned. The protein characteristics was analyzed using bioinformatics and the phylogenetic tree of CHI was constructed using MEGA5.1. Results: The 648 bp sbCHI (accession number: KP064512) sequence was obtained, which has a complete open reading frame (ORF), encoding an unstable protein with 215 amino acids. The sbCHI encoding protein has isoelectric point (pI) of 5.09 and a calculated molecular weight about 22 980, without transmembrane regions and signal peptide has a conserved domain of chalcone-flavanone isomerase family. In the secondary structure, the percentage of alpha helix, β-extended, and random coil were 37.21%, 23.25%, and 39.54%, respectively. The homologous analysis indicates the nucleotide sequence 99.69% similarity and the amino acid sequence 99.07% similarity with S. baicalensis (ADQ13184.1), only in 31 and 160 were different. Conclusion: The sbCHI in scutellariae callus is successfully cloned, which provides the foundation for further characterization sbCHI functionality and the synthetic biology research of baicalin.

Gene cloning and expression level of chalcone isomerase during florescence and content of flavonoids in Fagopyrum dibotrys / 中草药

Objective: To clone and analyze the full-length cDNA of chalcone isomerase (CHI) gene from Fagopyrum dibotrys (FdCHI). To analyze the expression of CHI and the total flavonoids content during florescence of F. dibotrys. Methods: The cDNA sequence of CHI was cloned by homology cloning from F. dibotrys. The expression of CHI was analyzed in the different tissues during florescence by semi-quantitative RT-PCR. The content of total flavonoids was measured by AlCl3 method. Results: The open reading frame (ORF) of FdCHI was 750 bp and encoded a protein with 249 amino acids. Bioinforamtion analysis suggested that the amino acid sequence of FdCHI had the high homology with those in other plants. Gene expression analysis showed that FdCHI was highly expressed in the flowers, followed by the roots and leaves, while lower in the stems. The content of total flavonoids was the highest in the flowers then the leaves and stems, and the lowest was in the roots. Conclusion: The cDNA sequence of FdCHI is firstly obtained from F. dibotrys and its coding protein has the typical characteristic of CHI homologous protein. The gene expression of FdCHI shows the same to the total flavonoids content in the stems, leaves, and flowers, but different in the roots of F. dibotrys.

Presencia del elemento genético transportable dTdic1 en Dianthus caryophyllus con flores variegadas y no variegadas / Presence of the transposable genetic element dTdic1 in Dianthus caryophyllus with variegated and no variegated flowers

El objetivo de este trabajo era la búsqueda del EGT (Elemento Genético Transponible) dTdic1 que ha sido asociado con la variegación del color de las flores de clavel y su relación con dos genes que codifican para enzimas involucradas en la biosíntesis de antocianinas, Chalcona isomerasa (CHI) y Dihidroflavonol reductasa (DFR). Su presencia y expresión se evaluó en siete genotipos con flores variegadas (líneas híbridas) y en cuatro genotipos de flores no variegadas (una línea híbrida y tres cultivares comerciales). Un alto número (indefinido) de copias del elemento dTdic1 se detectó en todas las líneas variegadas y no variegadas. En consecuencia, la sola presencia de este EGT no pudo asociarse directamente con la variegación de los pigmentos florales de flores de clavel. Adicionalmente, dTdic1 se encontró interrumpiendo el gen CHI en cuatro genotipos variegados y uno no variegado. No se observó evidencia de inserción de dTdic1 en el gen DFR en ninguno de los genotipos.

The objective of this work was to search for the EGT (Transposable Genetic Element) dTdic1 that has been associated with color variegation of carnation flowers and its relationship with two genes that code for enzymes involved in the synthesis of anthocyanins, Chalcona isomerase (CHI) and Dihidroflavonol reductase (DFR). Its presence and expression was evaluated in seven genotypes of variegated flowers and four no variegated flower genotypes (one hybrid line and three commercial cultivars). A high number of copies (undefined) of copies of the dTdic1 element was detected in variegaated and no variegated lines. Therefore, the main presence of this EGT was not associated directly with variegation of floral pigments. Additionally, dTdic1 was found interrupting the CHI gene in four variegated and one no variegated phenotypes. No evidence was observed of insertion of dTdic1 in the DFR gene in any of the genotypes.

Cloning and expression analysis of peanut (Arachis hypogaea L. ) CHI gene

Chalcone isomerase (CHI) is the key enzyme that catalyzes chalcone into (2S)-flavanol or (2S)-5-desoxidation flavanol. The full length cDNA (1050 bp) of AhCHI (Arachis hypogaea CHI gene) was cloned by large scale EST sequencing using a peanut immature seed cDNA library. Sequence analysis results indicated that it was a type I CHI gene (with the accession number JN660794). The ORF of AhCHI was 768 bp, encoding a peptide of 255 amino acids with a pI of 5.189. Sequence alignment showed that the coding region of AhCHI gene is highly conserved to compare with CHI genes from other plant species. Peanut cDNA microarray and semi-quantitative RT-PCR analysis indicated that AhCHI was highly expressed in pegs. The expression level in flower and root was higher than the expression level in stem and leaf. AhCHI was expressed in a high level in seeds with a purple seed coat, while its expression was low in seed with white seed coat.