ABSTRACT
Erigeron breviscapus (Vant.) Hand.-Mazz. is a famous traditional Chinese medicine that has positive effects on the treatment of cardiovascular and cerebrovascular diseases. With the increase of market demand (RMB 500 million per year) and the sharp decrease of wild resources, it is an urgent task to cultivate high-quality and high-yield varieties of E. breviscapus. However, it is difficult to obtain homozygous lines in breeding due to the self-incompatibility (SI) of E. breviscapus. Here, we first proved that E. breviscapus has sporophyte SI (SSI) characteristics. Characterization of the ARC1 gene in E. breviscapus showed that EbARC1 is a constitutive expression gene located in the nucleus. Overexpression of EbARC1 in Arabidopsis thaliana L. (Col-0) could cause transformation of transgenic lines from self-compatibility (SC) into SI. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays indicated that EbARC1 and EbExo70A1 interact with each other in the nucleus, and the EbARC1-ubox domain and EbExo70A1-N are the key interaction regions, suggesting that EbARC1 may ubiquitinate EbExo70A to regulate SI response. This study of the SSI mechanism in E. breviscapus has laid the foundation for further understanding SSI in Asteraceae and breeding E. breviscapus varieties.
Subject(s)
Arabidopsis , Erigeron/genetics , Plant Proteins , Plants, Genetically Modified , Ubiquitin-Protein Ligases , Arabidopsis/enzymology , Arabidopsis/genetics , Erigeron/enzymology , Plant Proteins/biosynthesis , Plant Proteins/genetics , Plants, Genetically Modified/enzymology , Plants, Genetically Modified/genetics , Ubiquitin-Protein Ligases/biosynthesis , Ubiquitin-Protein Ligases/geneticsABSTRACT
Two versatile UDP-glucosyltransferases, UGT75L25 and UGT75X1, were isolated from Erigeron breviscapus. The enzymes display high sequence identity to flavonoid 7- O-glucosyltransferase from Malus species and cluster to the phylogenetic group L of plant glucosyltransferases, also involved in the formation of hydroxycinnamoyl glucose esters, which are used as bifunctional donors in the glucosylation or acylation of anthocyanins. The enzymes, functionally expressed in Escherichia coli, exhibit broad substrate specificity toward 21 structurally diverse types of phenolic acids, including (hydroxy)cinnamates, vanillic acid, 3-hydroxycoumarin, and 7-hydroxyflavonoids. The catalytic characteristics of UGT75L25 and UGT75X1 were exploited to generate the corresponding acyl-glucose-esters or glucosides with high efficiency. These findings demonstrate the significant potential of acyl-glucose-esters in the further enzymatic synthesis of bioactive anthocyanins.
Subject(s)
Erigeron/enzymology , Glucosyltransferases/metabolism , Plant Proteins/metabolism , Amino Acid Sequence , Erigeron/chemistry , Erigeron/genetics , Esters/chemistry , Esters/metabolism , Glucose/chemistry , Glucose/metabolism , Glucosyltransferases/chemistry , Glucosyltransferases/genetics , Molecular Sequence Data , Phylogeny , Plant Proteins/chemistry , Plant Proteins/genetics , Sequence Alignment , Substrate SpecificityABSTRACT
OBJECTIVE: Erigeron breviscapus is a medicinal plant with the most developmental potential in Yunnan province, which is belongs to Erigeron genus of Compositae family. Scutellarin, the main active component of Erigeron breviscapus is one of flavone 7-O-glucuronide derivatives, its biosynthesis pathway is still not clear. METHOD: Full length cDNA encoding flavone syhthase II gene in E. breviscapus was cloned in this study using R-PCR, 3'-RACE and 5'-RACE. RESULT: The opening reading frame of FS II cDNA of E. breviscapus is 1 557 bp long and encoding 518 amino acids, designed as EbFS II, which is highly homologous with FS II of Compositae species, like Callistephus chinensis, Cynara cardunculus var. scolymus, Gerbera hybrida, Dahlia pinnata and Lobelia erinus. CONCLUSION: Phylogenetic analysis showed that EbFS II might has the function of directly converting flavanone to flavone.
Subject(s)
Cytochrome P-450 Enzyme System/genetics , Cytochrome P-450 Enzyme System/metabolism , Erigeron/enzymology , Erigeron/genetics , Genes, Plant , Amino Acid Sequence , Cloning, Molecular/methods , Computational Biology/methods , Molecular Sequence Data , Plants, Medicinal/enzymology , Plants, Medicinal/genetics , Sequence AlignmentABSTRACT
OBJECTIVE: Scutellarin from Erigeron breviscapus is a flavonoid with remarkable pharmacological activity, whose route of biosynthesis is still fully clear. Chalcone synthase (CHS) is the key enzyme regulating flavonoids biosynthesis, and the aim of this study is to explain the relationship between patterns of the gene expression and scutellarin content through studying CHS gene expression patterns combined with scutellarin content in various parts of E. breviscapus. METHOD: Through RT-PCR and RACE, the full length of CHS was cloned and analyzed by fluorescent quantitative PCR. The scutellarin content in tissues was analyzed by HPLC. RESULT: The full-length gene sequence was 1 270 bp, encoding 405 amino acids. Software analysis found that the DNA sequence was 80% similarity with Compositae plant homeo-box gene. Fluorescence quantitative analysis showed that CHS had the highest expression level in leaves, far higher than that in root, stem and flower. HPLC analysis showed that the scutellarin was the highest in leaves, followed by the flowers and stems, scutellarin was not detected in root. CONCLUSION: Correlation analysis showed that CHS expression amount and scutellarin content in different parts of E. breviscapus is positive correlation (r = 0.761, P < 0.05), it suggests that CHS gene expression level has important effect on biosynthesis of scutellarin.
