ABSTRACT
We cloned flavonol synthase gene (named as CmFLS) by RACE from Chrysanthemum morifolium cv. 'Hangju' based on transcriptome database. Sequencing results showed that 1 235 bp sequence was acquired with the largest open reading frame (ORF) of 1 008 bp, which encoded 335 amino acids. The predicted CmFLS encoded protein had an isoelectric point (pI) of 5.41. The phylogenetic tree analysis indicated that CmFLS was highly homologous to other FLSs, which identified from the species of Compositae. The recombinant fusion protein, with a molecular mass of 43 kDa, was successfully expressed by prokaryotic expression system. Meanwhile, Ni-NTA resin was used to purify the recombinant fusion protein, and the Ni-Native Buffer containing 250 mmol·L⁻¹ imidazole was most favorable for elution. The purified recombinant fusion protein was subjected to in vitro catalytic reaction, and then the products were extracted and analyzed by HPLC. The results showed that the recombinant fusion protein CmFLS was able to catalyze the production of quercetin by dihydroquercetin under specific buffer and reaction conditions, which indicated that the functional protein encoded by CmFLS had dioxygenase activity in the biosynthetic pathway of flavonoids biosynthesis in Ch. morifolium cv. 'Hangju'. The above results laid the foundation for further studying on CmFLS, and provided new ideas for the regulation of flavonoids metabolism from the molecular level and the catalytic synthesis of flavonols in vitro.
ABSTRACT
The flavonoids metabolic pathway plays central roles in floral coloration, in which anthocyanins and flavonols are derived from common precursors, dihydroflavonols. Flavonol synthase (FLS) catalyses dihydroflavonols into flavonols, which presents a key branch of anthocyanins biosynthesis. The yellow flower of Camellia nitidissima Chi. is a unique feature within the genus Camellia, which makes it a precious resource for breeding yellow camellia varieties. In this work, we characterized the secondary metabolites of pigments during floral development of C. nitidissima and revealed that accumulation of flavonols correlates with floral coloration. We first isolated CnFLS1 and showed that it is a FLS of C. nitidissima by gene family analysis. Second, expression analysis during floral development and different floral organs indicated that the expression level of CnFLS1 was regulated by developmental cues, which was in agreement with the accumulating pattern of flavonols. Furthermore, over-expression of CnFLS1 in Nicotiana tabacum altered floral colour into white or light yellow, and metabolic analysis showed significant increasing of flavonols and reducing of anthocyanins in transgenic plants. Our work suggested CnFLS1 plays critical roles in yellow colour pigmentation and is potentially a key point of genetic engineering toward colour modification in Camellia.
ABSTRACT
Objective: To clone the coding sequence of flavonol synthase gene from Fagopyum dibotrys (FdFLS) and to analyze its sequence, prokaryotic expression, and activity. Methods: The cDNA sequence of FdFLS gene was obtained by homology cloning and analyzed by bioinformatic method; The FdFLS prokaryotic expression vector pET-30b (+)-FdFLS was established and induced to express in Escherichia coli BL21 (DE3), as well as the enzymatic activity of the target protein was measured. Results: The full length of open reading frame (ORF) of FdFLS gene was 1008 bp that encoded a protein of 334 amino acids; The recombinant FdFLS protein had a relative molecular mass of 40000. It also has a catalytic activity, which could make dihydroquercetin and dihydrokaempferol into quercetin and kaempferol, respectively. Conclusion: The FdFLS gene is successfully cloned and reported for the first time, which has an active expression in E. coli.