A study published in BMC Plant Biology finds that CitF3H (Flavanone 3-hydroxylase) is a key gene involved in anthocyanin biosynthesis in citrus fruit. This will help develop new strategies to improve the nutritional and commercial values of citrus fruit.
as a query, which has been reported to encode a functional flavanone 3-hydroxylase inisolated from Ponkan mandarin was cloned into the pCold GST vector . The recombinant plasmid was transformed into XL1-BlueCitF3H
was inoculated in a 200 mL of 2×YT medium with carbenicillin . Cultures were grown at 37 ℃ until an optical density at 600 nm of 0.8 was reached. The culture solution was maintained at 15 ℃ for 30 min. The expression of proteins was induced by the addition of IPTG , and the cultures were grown at 15 ℃ for additional 24 h. After centrifugation, the cells were harvested and frozen in liquid nitrogen, and then resuspended in extraction buffer .
To investigate the enzymatic activity of CitF3H protein, the purified recombinant protein was reacted with flavanones , flavones , flavanols . Reaction mixtures consisted of 100 mM Tris-HCl buffer , 250 mM 2-Oxo-glutaric acid, 30 mM sodium ascorbate, 50 µM FeSO4, 10% glycerol, 0.05% Triton X-100, 500 µM substrates, and 40 µg of purified protein in a total volume of 100 µl. Assays were incubated at 37 ℃ with shaking for 2 h.
Anthocyanins were extracted from the freeze-dried juice sacs using a HCl: methanol solution. After homogenization, ultrasonication and centrifugation, the supernatant was filtered through a TORAST disc syringe filter . Then, the samples were analyzed using a HPLC system fitted with a YMC-UltraHT Pro C18 column at a flow rate of 1.0 mL min. The eluent was monitored at 650 nm using a MD4010 PDA detector.
The gene expression was conducted by real-time quantitative PCR according to the method of Ma et al. []. As an endogenous control, the TaqMan Ribosomal RNA Control Reagents VIC Probe was used. The real-time PCR was performed using TaqMan Universal PCR Master Mix on a StepOnePlus™ system . Each reaction contained template cDNA, 900 nM primers, and a 250 nM probe . The thermal cycling conditions were 95 °C for 10 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 1 min.
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