Studies On The Mechanism Of Red Pigment Development In Red Pear

Pear is one of the most important fruits in China. China ranks first in the world for pear production, accounting for 60% of world production, 17% of world exports. Pear occupies an important place in the national economy. In the situation that common pear supply is over demand in contrast to red pear supply is unable to meet the demand. Besides, red pear has good appearance quantity and high market value. Therefore, red pear varieties have a brighter future. However, mechanism of red pigment development of red pear is lack. In order to study the regulatory mechanism of red pigment development in red pear, in this study, changes in the level of anthocyanin and the activities of enzymes of anthocyanin biosynthesis including plenylalanine ammonia lyase (PAL), chalcone ismoerase (CHI), were studied in the pericarp of Asian pear (Pyrus pyrifolia)'Aoguan'and'Mantianhong'during the pigment formation period; furthermore, a R2R3-MYB transcription factor gene, PyMYB10, which regulated anthocyanin synthesis, was isolated from'Aoguan', the expression pattern and regulatory mechanism of PyMYB10 were also studied. Proteome maps of both bagged and normal P. communis'Placer'fruit skin were obtained by performing two-dimensional electrophoresis analysis and compared to assess the extent to which protein distribution differed in pear skin. The composition of anthocyanin both in bagged and normal P. communis'Placer'fruit skin were measured by HPLC. The main results are as follows:1. Changes in the level of anthocyanin and the activities of enzymes of anthocyanin biosynthesis including plenylalanine ammonia lyase (PAL), chalcone ismoerase (CHI), were studied in the pericarp of'Aoguan'and'Mantianhong'during the pigment formation period. Unlike apple, highest anthocyanin concentration of two varieties appeared in immature fruit, faded towards harvest. Activity of PAL was apparently not limiting to colour development, while CHI activity was closely related to the synthesis of anthocyanin.2. According to the homologous sequences from other plants, degenerate primers were designed to amplify specific DNA fragment using cDNA prepared from'Aoguan'. The 735-bp full-length cDNA of PyMYB10 was obtained using RACE PCR. The predicted protein of PyMYB10 had 245 amino acids. Alignment of the predicted translations of PyMYB10 gene with other MYB TFs at the R2R3 domain indicated a high degree of homology, especially some MYB TFs involved in anthocyanin biosynthesis. The gene contains two distinct MYB domain transcription factors are R2R3MYB. Phylogenetic analysis revealed that PyMYB10 is in the same cluster with apple MdMYB10, which has been proved to regulate anthocyanin synthesis. These results suggested that PyMYB10 might have the same function as MYBs, which play an important role in regulating anthocyanin synthesis.3. The genomic sequence and the promoter region of PyMYB10 were also cloned. Sequence analysis revealed that the genomic fragment of PyMYB10 with molecular size of about 1.7 kb, contains three exons and two introns. Motif analysis of the 0.7-kb upstream region from the start codon, probably corresponding to the promoter, suggested that the 0.7-kb region comprised cis-acting regulatory elements responsed to light, temperature, abscisic acid and methyl jasmonate signals.4. Transcription profiling of PyMYB10 and six anthocyanin biosynthetic genes PyCHS, PyPAL, PyCHI, PyANS, PyDFR and PyF3H were investigated in'Aoguan'skins by qPCR at four developmental stages. Anthocyanin contents of'Aoguan'were determined at the four sampling times accordingly. In'Aoguan', transcription of PyMYB10 was assayed in young leaves, flower buds and fruit skin in which anthocyanins appeared to accumulate by QPCR. The results showed that the link between PyMYB10, PyANS, PyFTH, PyDFR expressions and anthocyanin content was particularly strong.5. The 35S:PyMYB10 expression matrix was constructed by cloning the full-length PyMYB10 cDNA into the binary vector pBI121 constructed plant expression vector 35s:PyMYB10. PyMYB10 cDNA was overexpressed in Arabidopsis. There was expression of PyMYB10 in the transgenic plants with clear ectopic pigmentation in their immature seeds, but no expression was detected in wild-type control plants. These data suggest that PyMYB10 plays a role in anthocyanin biosynthesis.6. Effect of fruit bagging treatment on the synthesis of anthocyanin of red pear were studied in this study. HPLC chromatography of pear skin anthocyanins showed two major peaks, representing cyanidin-3-galactoside with the largest peak found in apple and pear and peonidin 3-galactoside with the smaller peak. The results showed that the synthesis of cyaniding 3-galactoside was inhibited larger by fruit bagging treatment than peonidin 3-galactoside. 7. Proteome maps of both bagged and normal P. communis'Placer'fruit skin were obtained by performing two-dimensional electrophoresis analysis and compared to assess the extent to which protein distribution differed in pear skin. Differentially expressed protein spots were subjected to matrix-assisted laser desorption ionization time of flight (MALDI-TOF) analysis and the data compared to that of known proteins to deduce their possible functions. These identified proteins were mainly involved in photosynthesis, signal transduction, energy pathway, protein folding and assembly, and carbohydrate and acidity metabolisms, and were under-expressed in bagged fruit skins.