Conclusions The selleck Ruxolitinib sequenced gene, CYP75A31, encodes a flavonoid 35 hydroxylase which accepts luteolin, naringenin, erio dictyol, dihydrokaempferol, dihydroquercetin, kaemp ferol, quercetin and liquiritigenin as substrates. The ability to do 3 and especially 5 hydroxylation of inter mediates in the flavonoid pathway places CYP75A31 at an important branch point in the regulation between flavonol and anthocyanin synthesis. Expression of the CYP75A31 gene increased in response to nitrogen depri vation, in accordance with other genes in the phenylpro panoid pathway, which is an expected response to abiotic stress in plants. Methods Plant Material Suzanne F1 seeds were sown on rock wool and given Hoagland nutrient solution containing 15 mM NO3.
RNA and DNA used to identify coding sequence and introns of the F35H gene was isolated Inhibitors,Modulators,Libraries from plants grown in a 12 h light dark regimen. Expression and metabolite analysis Inhibitors,Modulators,Libraries were performed on plants grown in continuous light, and given complete Hoagland solution before shifted to a nitrogen deprived regimen where KNO3 was replaced by KCl and Ca 2 4H2O was replaced by CaCl2. Identifying the F35H gene RNA was isolated from leaves of the cherry tomato Suzanne F1 using the RNeasy Plant Mini Kit. To identify the 3end of the F35H gene the Gen eRacer Kit was used. The gene speci fic left primer used for the 3 end had the sequence and was based on a F35H sequence for Solanum tuberosum. The cDNA amplified was sequenced, and a nucleotide BLAST against the Gene Bank showed close similarity to other F35H sequences.
An EST sequence was found in the TIGR database which was assumed to be the 5 end of the gene. Based Inhibitors,Modulators,Libraries on the obtained sequences for 3 and 5 ends, new primers cov ering the entire gene were made. The 3 sequence was used to make the primer 75ALerevECO with an additional EcoRI site for the 3 end of the gene. The 5 end primer, 75ALedirBAM, includes an additional BamHI site. cDNA for cloning was made using the SuperScript III First Strand Synthesis SuperMix for qRT PCR. The ORF of CYP75A31 was amplified by PCR introducing BamHI EcoRI rectriction sites upstream of the start ATG and downstream to the stop codon TGA using Inhibitors,Modulators,Libraries Platinum Taq DNA Polymerase High Fidelity. PCR program was as follows 95 C for 5 min, followed by 5 cycles of 95 C for 1 min, 40 C for 1 min and 72 C for 1. 5 min. Then 35 cycles of 95 C for 30 sec, 55 C for 30 sec and 72 C for 1.
5 min. At the end there was an extra 5 min elongation at 72 C before cool ing to 4 C. The product was ligated into a TOPO vector using Inhibitors,Modulators,Libraries the pCR 8 GW TOPO TA Cloning Kit as recommended. The ligated vector was trans formed into OneShot Chemically Competent E. coli and grown on LB media containing specti nomycin. Several individual colonies were picked and grown selleck chem to amplify and isolate the plasmids for sequen cing.