Isolation and expression analysis of NtCHS6, a new chalcone synthase gene from Nicotiana tabacum

Chalcone synthases （CHS, EC 2.3.1.74） are key enzymes that catalyze the first committed step in flavonoid biosynthesis. In this study, we isolated a chalcone synthase, named NtCHS6, from Nicotiana tabacum. This synthase shared high homology with the NSCHSL （Y14507） gene and contained most of the conserved active sites that are in CHS proteins. The phylogenetic analysis suggested that NtCHS6 protein shared a large genetic distance with other Solanaceae CHS proteins and was the most closely-related to an uncharacterized CHS from Solanum lycopersicum. The expression analysis indicated that NtCHS6 was abundantly expressed in leaves, especially in mature leaves. By scrutinizing its upstream promoter sequences, multiple cis-regulatory elements involved in light and drought responsive were detected. Furthermore, NtCHS6 expression decreased significantly under dark treatment and increased significantly under drought stress suggested that NtCHS6 expression exhibited both light responsiveness and drought responsiveness, and important roles in ultraviolet protection and drought tolerance. Our results might play

Cloning of a flower-specific expression promoter from Arabidopsis thaliana and its plant expression vector construction

Given the sequence of Chs gene promoter from Arabidopsis thaliana reported in GenBank （AF248988）, a pair of specific PCR primers was designed with the Primer Premier 5.0 software. PCR products of about 0.5 kb were successfully amplified with the genome DNA of A. thaliana as a DNA template and Taq polymerase as DNA polymerase. The purified PCR products were ligated to the pMD18-T vector. The sequencing result showed that the Chs promoter from A. thaliana was 531 bp long. Sequence alignment analysis based on the DNAMAN software revealed that the sequence similarity between the cloned promoter and target promoter （AF248988） was up to 100%. Online PLACE analysis indicated that the Chs promoter contained cis-elements such as TATA-box, CAAT-box, pollen-box, G-box, ACGT-containing element, R response element, Myb recognition element and TACPyAT-box. At the same time, a plant expression vectorpAtChs：：GUS which fused the Chs promoter and the marker gene GUS was successfully constructed.

Effects of Temperature on Seed Germination and Metabolism of Scutellaria baicalensis Georgi

[Objectives]To explore the effects of temperature on the primary and secondary metabolism of Scutellaria baicalensis Georgi during the seed germination.[Methods]The superoxide dismutase(SOD)activity was determined using riboflavin-NBT;peroxidase(POD)activity was determined using guaiacol colorimetric method,catalase(CAT),ascorbate peroxidase(APX),phenylalanine ammonia lyase(PAL)and cinnamic acid-4-hydroxylase(C4H)activity were detected by ultraviolet spectrophotometry,and chalcone synthase(CHS)activity and the content of secondary metabolites were measured by high performance liquid chromatography(HPLC).[Results]The germination rate,germination potential and germination index of S.baicalensis seeds were significantly affected by temperature.The most suitable temperature for the germination of S.baicalensis seeds was 25℃.The activities of SOD,POD and CAT in S.baicalensis seeds treated at low and high temperature were higher than that treated at suitable temperature;the activities of PAL,C4H and CHS of S.baicalensis seeds treated at low and high temperature were lower than that treated at suitable temperature.There was a good positive correlation between flavonoids and soluble sugar,PAL activity and C4H activity,and the correlation coefficients were R=0.894*,R=0.956*and R=0.951*,respectively.[Conclusions]In adverse environment,S.baicalensis seeds have good defense capabilities.During the germination of seeds,the formation of secondary metabolites is significantly correlated to the activity of key enzymes.Therefore,high-quality medicinal materials can be obtained by taking measures to improve the activity of key enzymes.

Cloning,expression and activity analysises of chalcone synthase genes in Carthamus tinctorius

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.

Co-suppression in transgenic Petunia hybrida expressing chalcone synthase A (chsA)

Chalcone synthase A is a key enzyme in the anthocyanin biosynthesis pathway. Expression of chsA gene in transgenic Petunia hybrida resulted in flower color alterations and co-suppression of transgenes and endogenous genes. We fused the β-glucuronidase (uidA) gene to the C-terminal of chsA gene, and transferred the fusion gene into Petunia hybrida via Agrobacterium tumefaciens. GUS histochemical staining analysis showed that co-suppression occurred specifically during the development of flowers and co-suppression required the mutual interaction of endogenous genes and transgenes. RNA in situ hybridization analysis suggested that co-suppression occurred in the entire plant, and RNA degradation occurred in the cytoplasm.

Molecular and structural characterization of a promiscuous chalcone synthase from the fern species Stenoloma chusanum

The key enzymes involved in the flavonoid biosynthesis pathway have been extensively studied in seed plants,but relatively less in ferns.In this study,two 4-Coumarate:coenzyme A ligases(Sc4CL1 and Sc4CL2)and one novel chalcone synthase(ScCHS1)were functionally characterized by mining the Stenoloma chusanum transcriptome database.Recombinant Sc4CLs were able to esterify various hydroxycinnamic acids to corresponding acyl-coenzyme A(CoA).ScCHS1could catalyze p-coumaroyl-CoA,cinnamoyl-CoA,caffeoyl-CoA,and feruloyl-CoA to form naringenin,pinocembrin,eriodictyol,and homoeriodictyol,respectively.Moreover,enzymatic kinetics studies revealed that the optimal substrates of ScCHS1were feruloyl-CoA and caffeoyl-CoA,rather than p-coumaroyl-CoA,which was substantially different from the common CHSs.Crystallographic and sitedirected mutagenesis experiments indicated that the amino acid residues,Leu87,Leu97,Met165,and Ile200,located in the substrate-binding pocket near the B-ring of products,could exert a significant impact on the unique catalytic activity of ScCHS1.Furthermore,overexpression of ScCHS1 in tt4 mutants could partially rescue the mutant phenotypes.Finally,ScCHS1 and Sc4CL1 were used to synthesize flavanones and flavones with multi-substituted hydroxyl and methoxyl B-ring in Escherichia coli,which can effectively eliminate the need for the cytochrome P450 hydroxylation/O-methyltransferase from simple phenylpropanoid acids.In summary,the identification of these important Stenoloma enzymes provides a springboard for the future production of various flavonoids in E.coli.

The chromosome-level genome assembly of Astragalus sinicus and comparative genomic analyses provide new resources and insights for understanding legume-rhizobial interactions

The legume species Astragalus sinicus(Chinese milk vetch[CMV])has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops for improving rice productivity and preventing soil degeneration.In this study,we generated the first chromosome-scale reference genome of CMV by combining PacBio and Illumina sequencing with high-throughput chromatin conformation capture(Hi-C)technology.The CMV genome was 595.52 Mb in length,with a contig N50 size of 1.50 Mb.Long terminal repeats(LTRs)had been amplified and contributed to genome size expansion in CMV.CMV has undergone two whole-genome duplication(WGD)events,and the genes retained after the WGD shared by Papilionoideae species shaped the rhizobial symbiosis and the hormonal regulation of nodulation.The chalcone synthase(CHS)gene family was expanded and was expressed primarily in the roots of CMV.Intriguingly,we found that resistance genes were more highly expressed in roots than in nodules of legume species,suggesting that their expression may be increased to bolster plant immunity in roots to cope with pathogen infection in legumes.Our work sheds light on the genetic basis of nodulation and symbiosis in CMV and provides a benchmark for accelerating genetic research and molecular breeding in the future.