Cloning And Analysis Of UFGT Structure Gene And Transcription Factor In Pyruspyrifolia Nakai Cv.Huobali From Yunnan

The ‘Pyrus pyrifolia Nakai cv．Huobali’ is local variety native in Yunnan. The all redpear collectively referred to as the‘Pyrus pyrifolia Nakai cv．Huobali’ in Yunnan. The redpericarp phenotype can stable genetic. The most critical factor is anthocyanin synthesis of fruitcoloring of pear. Anthocyanins are the colored end products of the flavonoid pathway andcomposed of anthocyanin and sugar, And a class of important secondary metabolites in plants,And is a water-soluble pigment widely exists in the angiosperm epidermal cell vacuole.Anthocyanins make flowers, leaves, fruits, pericarp and other organs showing different color. Redpears have high commercial value if they coloring good. So it is also very significant toresearch the mechanism of anthocyanin biosynthesis and regulation from the molecular biologyfield. This study clone the cDNA sequence of the structural genes UDP-glucose:flavonoid3-O-glucosyl transferase （UFGT）, and transcription factor MYB（Family of transcriptionfactors with Trp cluster motif）, WD40（WD element，Trp-ASP） and bHLH from the Pyruspyrifolia Nakai cv．Huobali and ‘Wenshan’ red pear. use pear Actin gene as an internal control,and expression analysis of MYB gene were carried out by qT-PCR, in young leaves, flowers,pericarp and seeds.1. The fragment cDNA of UFGT gene was obtained by homologous cloning from Pyruspyrifolia Nakai cv．Huobali. Bioinformatics analysis of UFGT gene showed that the1465bpcDNA and encoding479amino acids. The homology on amino acid level of UFGT gene withUFGT of Malus domestica （BAI44431）, UFGT of Citrus clementina （XP₀06443177），UFGTof Vitis vinifera （XP₀02270947） was99%,97%,96%respectively. The protein predicationof UFGT showed that there was a function domains GT1_Gtf_Like and a GlycosylTransferase_GTB_type super gene family. Furthermore, the investigation also found thatUFGT gene was non trans-membrane protein without leader peptides and signal peptide inN-terminal, UFGT Belongs to the non secreted protein, and subcellular localization was incytosol. The main motifs of predicted secondary structure of UFGT were alpha helix andrandom coil. The Ramachandran map based on the Swiss Model display the protein spatialstructure is stable, and the spacesmaller obstacles.2. The fragment cDNA of WD40gene was obtained by homologous cloning from Pyruspyrifolia Nakai cv．Huobali. Bioinformatics analysis of WD40gene showed that the1126bpcDNA and encoding371amino acids. The homology on amino acid level of WD40with WD40of Prunus persica（XP₀07215608）, WD40of Fragaria vesca subsp. Vesca （XP₀04304579）,WD40of Populus trichocarpa（XP₀02321066）, WD40of Vitis vinifera （NP₀01268006） was 94%,93%,92%,91%, respectively. WD40proteins belong to the hydrophilic protein, located incytoplasmic matrix, belongs to the non secretory proteins. Contains a signaling WD40domain function,analysis showed that the protein tertiary structure prediction evaluation space conformation stability.3. The fragment cDNA of bHLH gene was obtained by homologous cloning from Pyruspyrifolia Nakai cv．Huobali. Bioinformatics analysis of bHLH gene showed that the1947bpcDNA and encoding581amino acids. The homology on amino acid level of bHLH with bHLHof Malus domestica（ABB84474）, bHLH of Prunus persica（XP₀07220209）, bHLH ofFragaria vesca subsp. Vesca（XP₀04308377） was95%、95%、78%, respectively. The bHLHbelongs to the unstable protein, the polypeptide chain is hydrophilic, belongs to the hydrophilic protein.Contains two typical curly spiral structure, in which the confidence value of HR2is very high, the analysisis seven peptide repeat area. The protein three-dimensional structure conformation stability, contains abHLH-MYC-N super family and a bHLH gene families.4.The full-length cDNA of MYB gene was obtained by homologous cloning from‘Wenshan’ red pear （Pyrus pyrifolia Naki）. The nucleotide sequence had been deposited in theGenBank under accession KF601227. Bioinformatics analysis of MYB gene showed that the1551bp cDNA and encoding244amino acids. The homology on amino acid level of MYB withMYB10（ADN26574） and R2R3MYB （ABX71487） was100%,98%, respectively. Thehomology on amino acid level of MYB with MYB of Pyrus bretschneideri x（EU153577）,MYB of Malus domestica（AB744001）, MYB of Mespilus germanica（EU153574）, MYB ofRoyal Gala（EU518249） was97%,95%,95%,93%, respectively. This protein has two DNAbinding sites, and two SNAT gene families. Expression analysis of MYB gene of ‘Wenshan’ redpear （Pyrus pyrifolia Naki） showed that MYB gene all expressed in young leaves, flower,pericarp and seeds.