Cloning And Expression Analysis Of PpCHS, PpDFR And PpPAL Genes In Red Pear (pyruspyrifolia Naki) Origin From Yunnan

Anthocyanins are a class of important secondary metabolites in plants, and accumulated in vacuole, decided to flower and fruit color. The fruit-skin’s color is an important appearance quality. Red pears have high commercial value if they coloring good. So it is also very significant to research the mechanism of anthocyanin biosynthesis and regulation from the molecular biology field. This study cloned the cDNA sequence of the Chalcone synthase (CHS) gene, Dihydroflavonol4-reductase (DFR) gene and Phenylalanine ammonia lyase (PAL) gene, which related to anthocyanin biosynthesis from the’Yanshan’red pear and’Weishan’red pear (Pyrus pyrifolia Naki). Using pear actin gene as an internal control, and expression analysis of PpCHS and PpDFR gene were carried out by semi-quantity RT-PCR, in young leaves, flowers, pericarp and seeds.1. The full-length cDNA of CHS gene was obtained by homologous cloning from ’Yanshan’red pear(Pyrus pyrifolia Naki). It was named PpCHS, the nucleotide sequence had been deposited in the GenBank under accession JQ060997. Bioinformatics analysis of PpCHS gene showed that the1203bp cDNA contained a complete opening-reading frame (ORF)1170bp encoding389amino acids. The homology on the nucleotide level of CHS gene with CHS of Pyrus communis (DQ901397),(AY786998), CHS of Malus x domestica (AB074485),(EU872158),(DQ026297),(AY786996) and CHS of Sorbus aucuparia (DQ286037) was97%,96%,95%,95%,95%,95%and96%, respectively. Furthermore, the investigation also found that CHS gene was non trans-membrane protein without leader peptides and signal peptide in N-terminal, CHS protein subcellular localization was in cytoplasm, and the polypeptide chain presented hydrophilic. The main motifs of predicted secondary structure of CHS were alpha helix and random coil, beta turn and extended strand were spread in the whole secondary structure. The protein predication of PpCHS showed that there were many function domains such as CHS-like, KAS-Ⅲ, BcsA and so on. Expression analysis of PpCHS gene of’Yanshan’red pear (Pyrus pyrifolia Naki) showed that PpCHS gene all expressed in young leaves, flower, pericarp and seeds. But PpCHS gene expressed stronger in young leaves and flowers and weaker in pericarp and seeds. 2. The full-length cDNA of DFR gene was obtained by homologous cloning from’Yanshan’red pear(Pyrus pyrifolia Naki). It was named PpDFR, the nucleotide sequence had been deposited in the GenBank under accession JQ749637. Bioinformatics analysis of PpDFR gene showed that the1160bp cDNA contained a complete opening-reading frame (ORF)1044bp encoding347amino acids. The homology on the nucleotide level of DFR gene with DFR of Pyrus communis (AY227731),(AY227732),(AY227730), DFR of Malus x domestica (AF117268),(AY227729) and DFR of Crataegus monogyna was99%,99%,99%,98%,98%and98%, respectively. Amino acid sequence alignment found that DFR protein in N-terminal exist a NADPH binding sites is’VTGASGFIGSWLVMRLLEHGY’ and exist a substrate specificity of amino acids with decision sequence ’TVNVEEHQKPVYDESNWSDVEFCRSV’. Furthermore, the investigation also found that DFR gene was non trans-membrane protein without leader peptides and signal peptide in N-terminal, DFR protein subcellular localization was in cytoplasm, and the polypeptide chain presented hydrophilic. The protein predication of PpDFR showed that there was a function domains NADB-Rossmann supeffamily, combined NADPH through the Rossmann folding. Expression analysis of PpCHS gene of ’Yanshan’red pear(Pyrus pyrifoliaNaki) showed that PpDFR gene all expressed in young leaves, flower, pericarp and seeds.3. The fragment cDNA of PAL gene was obtained by homologous cloning from ’Weishan’red pear(Pyrus pyrifolia Naki). It was named PpPAL, the nucleotide sequence has been deposited in the GenBank under accession JQ749640. Bioinformatics analysis of PpPAL gene showed that the1919bp cDNA and encoding389amino acids. The homology on the nucleotide level of PAL gene with PAL of Pyrus communis(DQ230992), PAL of Pyrus bretschneideri (GU906268),(JQ247318), PAL of Primus avium (AF036948) and PAL of Rubus idaeus (AF237955) was98%,95%,95%,88%and83%, respectively. Amino acid sequence alignment found that residues of the deamination site, including the sites of L180, V181, L230, A231were found in PpPAL. The catalytic active sites were found at N234, G235, N356, D357. N358. H370. HNQDV(460-464). Furthermore, the investigation also found that PAL gene was non trans-membrane protein without leader peptides and signal peptide in N-terminal, PAL protein subcellular localization was in cytoplasm, and the polypeptide chain presented hydrophilic. The main motifs of predicted secondary structure of PAL were alpha helix and random coil, beta turn and extended strand were spread in the whole secondary structure. The protein predication of PpPAL showed that there were two function domains such as PAL-HAL and phe_am_lyase.