Cloning and functional analysis of flavanone 3-hydroxylase gene in Rhododendron hybridum Hort / 生物工程学报

Flavanone 3-hydroxylase (F3H) is a key enzyme in the synthesis of phycocyanidins. In this experiment, the petals of red Rhododendron hybridum Hort. at different developmental stages were used as experimental materials. The R. hybridum flavanone 3-hydroxylase (RhF3H) gene was cloned using reverse transcription PCR (RT-PCR) and rapid-amplification of cDNA ends (RACE) techniques, and bioinformatics analyses were performed. Petal RhF3H gene expression at different developmental stages were analyzed by using quantitative real-time polymerase chain reaction (qRT-PCR). A pET-28a-RhF3H prokaryotic expression vector was constructed for the preparation and purification of RhF3H protein. A pCAMBIA1302-RhF3H overexpression vector was constructed for genetic transformation in Arabidopsis thaliana by Agrobacterium-mediated method. The results showed that the R. hybridum Hort. RhF3H gene is 1 245 bp long, with an open reading frame of 1 092 bp, encoding 363 amino acids. It contains a Fe2+ binding motif and a 2-ketoglutarate binding motif of the dioxygenase superfamily. Phylogenetic analysis showed that the R. hybridum RhF3H protein is most closely related to the Vaccinium corymbosum F3H protein. qRT-PCR analysis showed that the expression level of the red R. hybridum RhF3H gene tended to increase and then decrease in the petals at different developmental stages, with the highest expression at middle opening stage. The results of the prokaryotic expression showed that the size of the induced protein of the constructed prokaryotic expression vector pET-28a-RhF3H was about 40 kDa, which was similar to the theoretical value. Transgenic RhF3H Arabidopsis thaliana plants were successfully obtained, and PCR identification and β-glucuronidase (GUS) staining demonstrated that the RhF3H gene was integrated into the genome of A. thaliana plants. qRT-PCR, total flavonoid and anthocyanin contentanalysis showed that RhF3H was significantly higher expressed in the transgenic A. thaliana relative to that of the wild type, and its total flavonoid and anthocyanin content were significantly increased. This study provides a theoretical basis for investigating the function of RhF3H gene, as well as for studying the molecular mechanism of flower color in R. simsiib Planch.

Identification of flavonoids 3-hydroxylase from Silybum marianum (L.) Gaertn and its application in enhanced production of taxifolin / 生物工程学报

(2S)-taxifolin is an important flavonoid that has anti-inflammatory and anti-oxidation effects. It is widely used in pharmaceutical and nutraceutical industries. Flavone 3-hydroxylase (F3H) can catalyze the synthesis of (2S)-taxifolin and other 3-hydroxylated flavonoids from (2S)-eriodictyol. Due to the low catalytic efficiency of F3H, the titer of many 3-hydroxyflavones, such as taxifolin, synthesized by microbial method is relatively low. In this study, a SmF3H was identified from the transcriptome of Silybum marianum (L.) Gaertn. The results of fermentation showed that SmF3H can catalyze the flavone 3-hydroxylation reaction, and its catalytic efficiency was significantly higher than that of commonly used SlF3H from Solanum lycopersicum. Six promoters with different transcription strength were selected to optimize the synthesis pathway from the flavonoid precursor (2S)-naringenin to (2S)-taxifolin. The results showed that the highest titer of (2S)-taxifolin (695.90 mg/L in shake flask) could be obtained when the P(GAL7) promoter was used to control the expression of SmF3H. The titer of (2S)-taxifolin was further improved to 3.54 g/L in a 5-L fermenter, which is the highest titer according to current available literatures.

Cloning, sequence analysis, and prokaryotic expression of LaF3H gene from Lepidium apetalum / 中草药

Objective In order to study the key genes involved in flavonoid biosynthesis pathway, the flavanone-3-hydroxylase (F3H) gene was isolated from Lepidium apetalum, which is named as LaF3H. Meanwhile, the sequence analysis, prokaryotic expression, and purification were also performed. Methods Specific primers were designed according to LaF3H gene sequences in the transcriptome data of L. apetalum, and the cDNA sequence of LaF3H gene was isolated from L. apetalum. By construction the prokaryotic expression vector pET-32a-LaF3H, the recombinant LaF3H protein was expressed in Escherichia coli BL21 (DE3) cells under IPTG induction. Results The open reading frame (ORF) of LaF3H was 1 080 bp, which encoded a protein of 359 amino acid residues, with a predicted molecular mass of 40 320. Sequence analysis showed that LaF3H contains five conserved motifs of F3H protein. The phylogenetic analysis indicated that LaF3H protein showed the highest homology with F3H protein from cruciferous plants (such as AtF3H from Arabidopsis thaliana). The prokaryotic expression vector pET-32a-LaF3H was constructed and the recombinant LaF3H protein was successfully expressed in E. coli BL21 (DE3) cells. Furthermore, the recombinant LaF3H protein was purified through Ni2+ affinity chromatography. Conclusion The LaF3H gene was isolated from L. apetalum and the recombinant LaF3H protein was obtained. The results of this study provided the foundation for the further preparation of LaF3H antibody and detection of LaF3H enzyme activity, and were helpful for functional characterization of LaF3H gene involved in flavonoid biosynthesis pathway of L. apetalum.

Cloning of flavanone 3-hydroxylase gene from Lithocarpus polystachyus and its sequence analysis / 中草药

Objective: To clone flavanone 3-hydroxylase (F3H) gene from Lithocarpus polystachyus, and to understand its gene characteristics and initially investigate its expression level in different organs. Methods: The total RNA and genomic DNA from blade of L. polystachyus were extracted. Based on the result of RNA-seq, a pair of specific primers were designed. cDNA and DNA sequences of F3H gene from L. polystachyus were amplified by PCR, then bioinformation analysis was performed after sequencing. The expression level of F3H gene in different organs of L. polystachyus was detected by qRT-PCR. Results: The full length of cDNA of F3H gene was 1 340 bp containing a 1 092 bp open reading frame that encoded 393 amino acids, and F3H was located in the cytoplasm. The result of qRT-PCR showed that F3H gene expressed in different organs of L. polystachyus, and the expression levels of F3H gene were significantly different in different organs (P < 0.05). Conclusion: The F3H gene of L. polystachyus was cloned and its bioinformation was analyzed for the first time, proving that the expression level of F3H gene in different organs of L. polystachyus was different. This finding lays a foundation for the studies on secondary metabolism of flavonoids in L. polystachyus.

Bivalent RNA interference to increase isoflavone biosynthesis in soybean (Glycine max)

In this work, a bivalent RNA interference (RNAi) plant-transformation vector was constructed to silence both the flavanone 3-hydroxylase (F3H) gene and the flavone synthase II (GmFNSII) gene in soybean (Glycine max). Two further unit RNAi vectors were constructed for each of these two genes. RNAi-mediated suppression of these genes effectively regulated flavone and isoflavone production in hairy roots that arose from soybean cotyledons transformed with Agrobacterium rhizogenes ATCC15834. Notably, the bivalent RNAi vector had a significantly higher effect for increasing isoflavone production compared with the two unit RNAi vectors. The study highlighted molecular methods that could be used to enhance isoflavone production in soybean and demonstrated the challenges associated with such metabolic engineering for the production of plant natural products.