Site-directed mutagenesis enhances the activity of benzylidene acetone synthase of polyketide synthase from Polygonum cuspidatum / 生物工程学报

Polygonum cuspidatum polyketide synthase 1 (PcPKS1) has the catalytic activity of chalcone synthase (CHS) and benzylidene acetone synthase (BAS), which can catalyze the production of polyketides naringenin chalcone and benzylidene acetone, and then catalyze the synthesis of flavonoids or benzylidene acetone. In this study, three amino acid sites (Thr133, Ser134, Ser33) that may affect the function of PcPKS1 were identified by analyzing the sequences of PcPKS1, the BAS from Rheum palmatum and the CHS from Arabidopsis thaliana, as well as the conformation of the catalytic site of the enzyme. Molecular modification of PcPKS1 was carried out by site-directed mutagenesis, and two mutants were successfully obtained. The in vitro enzymatic reactions were carried out, and the differences in activity were detected by high performance liquid chromatography (HPLC). Finally, mutants T133LS134A and S339V with bifunctional activity were obtained. In addition to bifunctional activities of BAS and CHS, the modified PcPKS1 had much higher BAS activity than that of the wild type PcPKS1 under the conditions of pH 7.0 and pH 9.0, respectively. It provides a theoretical basis for future use of PcPKS1 in genetic engineering to regulate the biosynthesis of flavonoids and raspberry ketones.

Cloning, expression and activity analysises of chalcone synthase genes in Carthamus tinctorius / 中草药·英文版

OBJECTIVE@#Flavonoids are the bioactive compounds in safflower (Carthamus tinctorius), in which chalcone synthase (CHS) is the first limiting enzyme. However, it is unclear that which chalcone synthase genes (CHSs) are participated in flavonoids biosynthesis in C. tinctorius. In this study, the CHSs in the molecular characterization and enzyme activities were investigated.@*METHODS@#Putative chalcone biosynthase genes were screened by the full-length transcriptome sequences data in C. tinctorius. Chalcone biosynthase genes in C. tinctorius (CtCHSs) were cloned from cDNA of flowers of C. tinctorius. The cloned gene sequences were analyzed by bioinformatics, and their expression patterns were analyzed by real-time PCR (RT-PCR). The protein of CtCHS in the development of flowers was detected by polyclonal antibody Western blot. A recombinant vector of CtCHS was constructed. The CtCHS recombinant protein was induced and purified to detect the enzyme reaction (catalyzing the reaction of p-coumaryl-CoA and malonyl-CoA to produce naringin chalcone). The reaction product was detected by HPLC and LC-MS.@*RESULTS@#Two full-length CtCHS genes were successfully cloned from the flowers of safflower (CtCHS1 and CtCHS3), with gene lengths of 1525 bp and 1358 bp, respectively. RT-PCR analysis showed that both genes were highly expressed in the flowers, but the expression of CtCHS1 was higher than that of CtCHS3 at each developmental stage of the flowers. WB analysis showed that only CtCHS1 protein could be detected at each developmental stage of the flowers. HPLC and LC-MS analyses showed that CtCHS1 could catalyze the conversion of p-coumaryl-CoA and malonyl-CoA substrates to naringin chalcone.@*CONCLUSION@#CtCHS1 is involved in the biosynthesis of naringin chalcone in safflower.

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 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.

Cloning and expression pattern analysis of chalcone synthase gene in Microcos paniculata / 中草药

Objective To clone the full-length cDNA of MpCHS and analyze its expression pattern in different parts of Microcos paniculata and different growth periods of M. paniculata leaves. Methods Based on the transcriptome data of M. paniculata, we designed specific primers for MpCHS gene. The full-length cDNA of MpCHS was amplified by PCR and the positive clones were then sequenced, analyzed, and constructed prokaryotic expression vector. The bioinformatics analysis of MpCHS was also performed. Meanwhile, the mRNA expression of MpCHS was detected using real-time quantitative PCR. Results The relative molecular mass was 42 700, and its theoretical isoelectric point was 6.11, with three conserved functional active sites (165 C, 304 H, and 337 N) of the CHS family proteins and the tag sequence of RLMMYQQGCFAGGTVLR and GVLFGFGPGL. Phylogenetic tree analysis showed that MpCHS had close relationship with woody plants such as cocoa and upland cotton. We successfully cloned the full-length cDNA of MpCHS (GenBank: KY472608). It had an ORF of 1 176 bp which encoded a protein of 391 amino acid residues. RT-qPCR results showed that MpCHS was expressed in all parts of M. paniculata and its expression in leaves was gradually decreased along with its development. Conclusion MpCHS is cloned from M. paniculate for the first time, and the gene expression pattern of MpCHS in different parts of M. paniculata and different growth periods of M. paniculata leaves was analyzed. This study facilitates the further purification and functional validation of MpCHS protein and provides reference for further analysis of flavonoids biosynthesis pathway in M. paniculata.

Analysis on the correlation between chalcone synthase gene polymorphism and content of liquiritin in licorice / 药学学报

Chinese pharmacopoeia stipulates that the content of liquiritin in licorice slices should be no less than 0.5%. However, there are lots of unqualified licorice slices in the herbal medicine markets. Due to the important role of functional gene polymorphism in secondary metabolism, this study attempts to analyze the influence of chalcone synthase (CHS) gene polymorphism on liquiritin biosynthesis and find out the unique haplotypes in licorice samples with high or low content of liquiritin, and to provide a basis for further analysis of molecular mechanism in flavonoid biosynthetic pathway. The contents of the 4 main flavonoids (liquiritin, isoliquiritin, liquiritigenin, isoliquiritigenin) in 60 licorice samples were assayed by HPLC and the results were analyzed by Spearman and χ2 tests. The contents of the 4 main flavonoids were related to each other and obviously different in different original plants. They were highest in Glycyrrhiza uralensis samples and lowest in Glycyrriza inflate samples. Five G. uralensis samples with the highest liquiritin contents and five G. inflate samples with the lowest liquiritin contents were selected to clone the CHS cDNA sequences. 336 CHS cDNA sequences with a full length of 1 175 bp were obtained, 249 variable sites (141 missense mutation sites) were found, and 137 haplotypes were determined. 130 variable sites were found in the 336 CHS amino acid sequences and 102 types were determined. AA-3 is the major type of CHS in licorice, AA-35 is the special major type of CHS in the group with high flavonoids contents and AA-36 is the special major type of CHS in the group with low flavonoids contents. The mutation sites between AA-35 and AA-36 are I/V at 193 and V/T at 229. Discovery Studio 2.5 analysis of the three-dimensional structure of the CHS protein shows that the valine at site 229 of AA-35 is combined with malonyl-CoA. Homology analysis indicates that the homology of CHS among different species is low. This study is significant for identification of the unique haplotypes in licorices with high or low content of liquiritin and guiding the further molecular breeding of high-quantity licorice.

In Silico Cloning and Bioinformatics Analysis of Chalcone Synthase Gene from Ribes Americanum / 中国药师

Objective:To study the in silico cloning and bioinformatics of chalcone synthase gene from Ribes americanum. Meth-ods:Ribes nigrum chalcone synthase sequence and Ribes americanum were cloned by retrieving the EST database as the querying probe, which were assembled by CAP3 sequence assembly program, and the bioinformatics database and related software were used to predict the structure and perform the function analysis. Results:Bioinformatical analysis showed chalcone synthase gene encoded 1423 bp and contained a 1173bp ORF, the protein was with 390 amino acid, which was a hydrophilic protein located in cytoplasm including three transmembrane regions with the secondary structure composed of alpha helix. Conclusion:The study is helpful to the further ex-planation for the molecular function mechanism of chalcone synthase gene from Ribes americanum.

Construction of RNA interference vector of Polygonum cuspidatum chalcone synthase gene and its genetic transformation / 中草药

Objective: To construct the RNAi expression vector of Polygonum cuspidatum chalcone synthase (PcCHS1) gene, and to obtain the transgenic plants in which PcCHS1 expression was down-regulated. Methods: According to known sequence (EF090604) of PcCHS1 gene in GenBank, right primers were designed and the conserved sequence was cloned. The conserved fragment (574 bp) targeting at PcCHS1 gene was inserted into the expression vector pYLRNAi in both forward and reverse directions, and RNA interference (RNAi) expression vector pYLRNAi-PcCHS1 was constructed. Using the method of Agrobacterium-mediated transformation, the expression vector was used to transform the shoot tips of P. cuspidatum, and transgenic plants were obtained. The expression of PcCHS1 was confirmed by Northern blotting and the accumulation of polydatin was detected by HPLC. Results: RNAi expression vector of PcCHS1gene was constructed successfully, and five transgenic plants were obtained. Northern blotting analyses indicated that the expression levels of PcCHS1 were significantly down-regulated in the transgenic plants. Polydatin concentration in the transgenic plants was up to 3.8 times higher than that in non-transformed control plants. Conclusion: Transgenic P. cuspidatum plants with down-regulated expression of PcCHS1 gene were obtained successfully. The content of polydatin in the transgenic P. cuspidatum was significantly increased by RNAi against PcCHS1. This work might establish an experimental basis for the effective application of PcCHS1 in improving polydatin accumulation in P. cuspidatum.

Cloning, bioinformatic analysis, and expression of chalcone synthase gene in Carthamus tinctorius / 中草药

Objective: To clone the chalcone synthase (CHS) gene in Carthamus tinctorius, to analyze the bioinformation of CHS, to compare the expression of CHS during the florescence, and to provide the foundation for composition and regulation mechanism of the active ingredients in C. tinctorius. Methods: RNA was obtained from fresh safflower corolla, cDNA was reversely transcriped, specific primers were designed, and then CHS was cloned. The protein characteristics was analyzed using bioinformatics, the phylogenetic tree of CHS was constructed using MEGA5.1, the expression of CHS during the florescence was analyzed using real time-PCR. Results: The 1 149 bp CHS sequence in C. tinctorius was obtained, which has a 1 041 bp ORF, encoding 346 amino acids. This protein belongs to the CHS family according to Blastp in NCBI. The CHS in safflower was similar to that in above 100 plants, and the similarities to Silybum marianum, Callistephus chinensis, Chrysanthemum x morifolium, and Gynura bicolor were respectively reaching 95%, 95%, 94%, and 94%. It has the closest relationship to S. marianum according to the phylogenetic tree using MEGA5.1. The CHS formula was C1678H2693N451O493S20 and the molecular weight was 37 700, with the isoelectric point of 6.10. The number of negatively charged amino acid residues (Asp + Glu) was 42, and the number of positively charged amino acid residues (Arg + Lys) was 38. The gene expression analysis showed that the highest expression of CHS in safflower was on day 3 of florescence, much higher than that on the other days. Conclusion: The CHS in safflower is successfully cloned, analyzed, and expressed, which provides the foundation for composite and regulation mechanism of the active ingredients in C. tinctorius.

Cloning and differential expression of chalcone synthase gene 1 in Houttuynia cordata and its protein sequence analysis / 中草药

Objective: To clone and sequence the chalcone synthase1 (CHS1) gene from the leaves of Houttuynia cordata. Methods: The cloning primers were designed on the basis of the conserved sequences of the cloned CHS gene in other plants. The total RNA was extracted from the leaves of H. cordata. The sequence of CHS gene was cloned by reverse transcription polymerase chain reaction (RT-PCR) and SON-PCR techiniques, and then the gene was ligated with pMD18-T Simple vector. The positive clone was sequenced after the identification of clone by PCR. Results: We cloned a fragment of 1 188 bp. The analysis of sequencing indicated that the fragment encoded 395 amino acids and shared the sequence homology of more than 62.3% with CHS gene sequences from other higher plants. Bioinformatic analysis indicated that the CHS amino acids had no signal peptide sequences, but possessed the CHS family's characteristic sequences, RLMMYQQGCFAGGTVLR and GVLFGFGPGL. Relative real-time PCR analysis indicated that CHS1 showed the highest transcript abundance in the flowers, moderate levels in the stems, lower levels in the rhizomes, and the lowest levels in the leaves. Conclusion: It is the first report that a novel CHS gene is cloned from H. cordata, which lays a foundation for the effective use of CHS1 gene.

Molecular cloning and expression profiling of a chalcone synthase gene from hairy root cultures of Scutellaria viscidula Bunge

A cDNA encoding chalcone synthase (CHS), the key enzyme in flavonoid biosynthesis, was isolated from hairy root cultures of Scutellaria viscidula Bunge by rapid amplification of cDNA ends (RACE). The full-length cDNA of S. viscidula CHS, designated as Svchs (GenBank accession no. EU386767), was 1649 bp with a 1170 bp open reading frame (ORF) that corresponded to a deduced protein of 390 amino acid residues, a calculated molecular mass of 42.56 kDa and a theoretical isoelectric point (pI) of 5.79. Multiple sequence alignments showed that SvCHS shared high homology with CHS from other plants. Functional analysis in silico indicated that SvCHS was a hydrophilic protein most likely associated with intermediate metabolism. The active sites of the malonyl-CoA binding motif, coumaroyl pocket and cyclization pocket in CHS of Medicago sativa were also found in SvCHS. Molecular modeling indicated that the secondary structure of SvCHS contained mainly α-helixes and random coils. Phylogenetic analysis showed that SvCHS was most closely related to CHS from Scutellaria baicalensis. In agreement with its function as an elicitor-responsive gene, the expression of Svchs was induced and coordinated by methyl jasmonate. To our knowledge, this is the first report to describe the isolation and expression of a gene from S. viscidula.

Comparative study of Chalcone synthase promoters across plant families / Estudio Comparativo de Promotores de la Chalcón Sintasa en Diferentes Familias de Plantas

In the post – genomic era the understanding of gene regulation has become a challenge and a research priority. In this research, we performed a comparative study of the regulator sequences of the chalcone synthase gene across plant families. Twenty-two sequences of chalcone synthase promoters were compared considering three regulator Cis elements: G-Box, H-Box and TATA Box. Our results show that these Cis elements are conserved among species and even at the family level. However, in some species all of the Cis elements were not found, showing that the expression and regulation of these promoters via the Cis elements can be variable. Additionally, a comparison between promoters from a species with a chalcone synthase multigene family showed that the duplicate genes are variable in the composition of the Cis elements, suggesting that these genes could be expressing in different ways.