| Olea europaea, belonging to the Oleaceae family, is an important woody oil species in the world. Obtained by pressing O. europaea fruit, O. europaea oil is the world’s most recognized healthy edible oil, with excellent effects on health and beauty. Therefore, it is known as "liquid gold" and "vegetable oil queen". Flavonoids are a class of polyphenolic compounds in O. europaea, and have important biological functions in plant life activities. Besides, flavonoids also can be used as drugs with a variety of pharmacological actions. So these compounds are with very important development potential and research value.Flavonoids are secondary metabolites of plant phenylpropanoid pathway. Phenylalanine ammonia lyase (PAL), chalcone synthase (CHS) and chalcone isomerase (CHI) are the three key enzymes in plant flavonoids biosynthesis pathway, and they have an important effect on the accumulation of flavonoids in plant. The full-length DNA and cDNA of the three key enzyme genes were isolated from O. europaea by homology cloning, RT-PCR, FPNI-PCR (Fusion Primer and Nested Integrated PCR) and 3’-RACE, and the three genes, analyzed by related to Bioinformatics, were named as OePAL, OeCHS, OeCHI respectively. In addition, the full-length exon of OePAL was expressed in Pichia pastoris strain X33, and carried out the analysis on enzymatic properties of recombinant OePAL. The main research results are as follows:1. Sequencing showed that the full-length DNA of OePAL was 2970 bp with an intron (393-1220 bp). Meanwhile, the full-length cDNA of OePAL was 2142 bp. The open reading frame (ORF) of OePAL encoded 713 amino acid residues, and sequence alignment had suggested that OePAL showed high homology with other botanic PALs. Amino acid sequence analysis showed that OePAL with a molecular formula of C3424H5490N944O1052S25 and an isoelectric point of 5.83, belonging to the stable protein, didn’t contain a signal peptide sequence. The polypeptide chain of OePAL, having 4 deamination residues sites (L203, V204, L253, A254) and 11 catalytic active sites (N257, G258, N379, D380, N381, H393, H4S3, N484, Q485, D486, V487), contained 56.52% alpha helix,5.61% beta turn and 30.86% random coil. The full-length exon of OePAL expression vector pPICZa A-OePAL was constructed and expressed in Pichia pastoris strain X33. SDS-PAGE analysis showed that the molecular weight of recombinant enzyme was approximately 77.5 kD, and the specific activity of purified enzyme was 196.3 U/mg. The analysis on enzymatic properties of recombinant OePAL indicated that the optimum reaction conditions of recombinant enzyme were temperature 40℃ and pH 8.8. The enzyme could keep relative stability at temperature below 50 ℃, pH 7.7-9.2 for a half hour. Ions such as Cu2+and Na+ could enhance the enzyme activity within the tested range. The enzyme had a Km of 4.89x10-4 mol/L for L-phenylalanine at the optimum reaction conditions.2. The full-length DNA of OeCHS contained 3 exons, and the ORF of OeCHS, being 1173 bp, encoded 390 amino acid residues, which showed high similarity with the complete amino acid sequences of CHS from Vitis vinifera and Solarium lycopersicum, about 90%. Amino acid sequence analysis showed that OeCHS with a molecular weight of 43.0445 kD, a molecular formula of C1915H3053N519O568S19 and an isoelectric point of 6.24, belonging to the stable protein, didn’t contain a signal peptide sequence. The active center of OeCHS was composed by the 4 highly conserved amino acid residues (C164, H303, N336, F215). Meanwhile, there were 44.36% alpha helix,6.92% beta turn and 33.08% random coil in the spatial structure of OeCHS.3. The OeCHI DNA sequence had four exons and three introns, and the cDNA sequence had a complete ORF of encoding 249 amino acids. Sequence alignment had suggested that the ORF of OeCHI had high homology with Pyrus communis and Prunus avium, reaching respectively 68.84% and 67.81%, and the amino acid sequence of OeCHI showed the highest homology with Lonicera japonica, only 65.87%. The polypeptide chain of OeCHI had 4 important catalytic sites (T50, Y108, N115, S192) and 7 substrate-binding sites (R38, G39, L40, F49, I52, K111, V112). The OeCHI, belonging to an unstable protein in the cytoplasm, had a calculated molecular weight of 26.64 kD, a theoretical pi value of 6.01, and it’s molecular formula was C1202H1890N306O368S4. The secondary structure of OeCHI contained alpha helix, beta turn and random coils, which accounted for 42.57%,8.03%,32.53% of the full-length sequence, respectively. |
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