Cloning And Expression Characteristics Of Genes Fragments Related To Key Fruit-coloring Enzymes In Bagging Pingguoli

Pingguoli fruit was used as material in the experiment,cloned the key colouring gens(PAL、CHI、UFGT) by RT-PCR and analysised express characteristics by Semi-Quantitative RT-PCR. This test have made the foundation to the study of Pingguoli fructification colouring.The results show as below:1. Three bands of28S,18S and5S were identified distinctly in the amplifications of total RNA of Pingguoli peel using the improved CTAB method and the brightness of band28S was twice that of18S. The OD260/OD280values of extracted RNA ranged from1.8to2.0. We considered the improved CTAB method as the perfect way to extract the total RNA of Pingguoli peel.2. A pair of primers were designed according to the conserved sequences of PAL, CHI and UFGT genes. Taking the cDNAs of Pingguoli fruits as templates, the fragments of PAL(407bp), CFI(444bp) and UFGT(422bp) genes were amplified, of which the accession numbers are JN120854, JN120855and JN120856, and which might code128,148and131amino acids.3. The homologous analysis indicated that, the homologies of PAL, CHI and UFGT genes sequences between Pingguoli and most logged horticultural plants were around90%:the homology of PAL genes between Pingguoli and Pyrus bretschneideri Rehd was97%; that of CHI genes between Pingguoli and P. Pyrifolia Nakai was95%; and that of UFGT genes between Pingguoli and Pyrus communis was99%. The percentages of both same amino acids and similar acids were around90%, and the high similarities suggested that the PAL, CHI and UFGT genes of Pingguoli fruit were cloned successfully.4. The phylogenetic evolution analysis showed a closer relationship in PAL genes of Pinggguoli,Pyrus communis and Pyrus bretschneideri Rehd,and CHI gene of Pingguoli had a closer relationship with those of Pyrus communis and Pyrus bretschneideri Rehd, and the closest relationship of UFGT appeared between Pingguoli and Pyrus communis.5. The results of semi-quantitative PCR demonstrated, expression levels of PAL and CHI genes in both bagging and normal fruits were improving along with the ripening process, but it was clearly high in bagging fruits than in normal ones during at134、143days after full bloom and at147days after full bloom. The expressions of UFGT gene were not stable in the whole ripening process, but the expression level in bagging fruits was much higher than that in normal ones. So bagging inhibited the expressions of PAL, CHI and UFGT genes, but the expression levels increased significantly after abandoning the bags.