The regulation of triacylglycerol biosynthesis in cocoa ..
by hydrolysis of triacylglycerols
The gene sequence of PAP2 listed in the JGI database (JGI Protein ID: 343983) was used for the design of primers for the amplification of the gene. A full-length CrPAP2 gene was amplified by PCR. A DNA fragment of about 1 000 bp was inserted into the pMD-18T vector and sequenced. Compared with the 343983 sequence in the JGI C. reinhardtii v4.0 database, the cloned CrPAP2 CDS had a sequence with 978 bp and showed 100% consistency. This cloned fragment was named CrPAP2.
processing one acetate unit at a time
The method of Ullah et al. () was used to measure the CrPAP2 activities. The amount of Pi in nmoles released by PAP from the substrate dioleoyl-phosphatidate (1,2-dioleoyl-sn-glycero-3-phosphate, sodium salt) was determined. The reaction was stopped by the addition of 2 ml AMA reagent (acetone: 10 mmol/L ammonium molybdate:5 mol/L sulfuric acid mixture, 2:1:1 (v/v/v)), followed by 100 ml of 1 mol/L citrate solution. The resulting turbidity was removed using centrifugation at 12 000×g for 6 min at room temperature. The absorbance of the resulting yellow color was read at 355 nm using a spectrophotometer. The enzyme unit, kat, was defined as the moles of the substrate converted per second (Heinonen and Lahti, ; Ullah et al., ).