Studies On Interaction And Fruit Peel Coloration Of Its Regulation Mechanism MYB, BHLH And WD40Transcription Factor Of Yunnan Red Pear

Yunnan Red Pears are popular with consumers because of their external beauty and health value. There are many red pear germplasms in Yunnan province owing to its unique geological and climatic conditions. Yunnan Red Pear belongs to sand pear, skin coloring large area, and this trait can be inherited stably. At present, the early maturing95-2, mid-maturing32and35and late maturing’Yunhong-1’four varieties are large-scale cultivated of Yunnan Red Pear in Yunnan Province. The skin color is an important genetic trait of fruits, and determines the value of goods. Red skins pear whose colors are mainly due to the accumulation of anthocyanins. The present study showed that anthocyanin biosynthesis were controlled by MYB, bHLH and WD40three of transcription factor formed MYB-bHLH-WD40complex (MBW complex), and this complex could activate the expression of structural genes by binding to the promoters of structural genes. It has important practical significance and theoretical foundation for solve the Yunnan Red Pear coloring and breeding of quality new varieties to elucidate the molecular mechanism fruit coloration. Therefore, In this study, using Yunhongli-1cultivars of Yunnan Red pear, studying of the interaction among of PyMYB, PybHLH and PyWD40three transcription factors and its regulation mechanism for fruit coloration. The main research results are as follows:1. Western blotting analysis the expression levels of PyMYB, PybHLH and PyWD40transcription factor in Yunhongli-1’s peels. The results suggested that the expression level of PyMYB, PybHLH and PyWD40proteins showed up-regulated, and these results agree with that of anthocyanin accumulation in "Yunhongli-1"skin. Co-immunoprecipitation analysis the PyMYB, PybHLH and PyWD40proteins interaction in vivo, the results showed that immunoprecipitation of PybHLH co-precipitated PyMYB and PyWD40proteins and also the immunoprecipitation of PyMYB co-precipitated PyWD40protein in ’Yunhongli-1’red skin. However, neither PyMYB nor PyWD40was detected in the absence of the polyclonal antibody or when exposure0d skin total protein was used as a negative control. These finding suggested that PybHLH interacts with PyMYB or PyWD40and PyMYB interacts with PyWD40in vivo. Bimolecular fluorescence complementation (BIFC) analysis showed that YFP fluorescence was predominantly detected in the nuclei of the onion cells when co-transformed PyWD40-NYFP and PyMYB-CYFP, PyWD40-NYFP and PybHLH-CYFP, PyMYB-NYFP and PybHLH-CYFP. However, YFP fluorescence not detected when PybHLH-CYFP and pNYFP, PyMYB-CYFP and pNYFP co-transformedinto respectively onion epidermal cells. Therefore, these finding also certificated that the PybHLH, PyMYB and PyWD40each others interacts in vivo in onion epidermal cells, which located in the nucleus.2. In order to study the interaction of PyMYB, PybHLH and PyWD40in vitro, the PyMYB, PybHLH and PyWD40prokaryotic expression vectors were constructed and obtained PyMYB, PybHLH and PyWD40of recombinant proteins. GST-pull down analysis showed that PyWD40and PybHLH proteins were pulled down by PyMYB, and PyWD40protein was pulled down by PybHLH, the results show that the PybHLH, PyMYB and PyWD40each others interacts in vitro. Far Western blotting analysis results show that the PyMYB protein combination with PybHLH and PyWD40, and PybHLH combination with PyMYB, therefore, the PybHLH, PyMYB and PyWD40each others interacts.3.In order to study the regulation mechanism of fruit coloration from Yunnan Red Pear, the PyDFR, PyANS, PyUFGT and PyF3H structural genes promoter region was amplified by Genome Walking kit from Yunhongli-1’peel. The promoter software sequence analysis showed that this promoter sequences not only contain TATA-box, CAAT-box elements but also contain many other promoter elements, such as MYB transcription factor binding element (MBS and MRE), G-box, I-box light-responsive element. Chromatin co-immunoprecipitation (CHIP) and electrophoretic mobility shift (EMS) in vitro and in vivo assays that PyMYB protein combination to the promoter region of PyDFR, PyANS and PyUFGT genes. Therefore, PyMYB transcription factor could activate the expression of structural genes PyANS, PyDFR and PyUFGT by binding to the promoters of structural genes which regulation of anthocyanin biosynthesis in Yunnan Red Pear.In summary, the PyMYB interacts with PybHLH or PyWD40and PyWD40interacts with PybHLH formed protein complexes, the complexes binding to PyANS, PyDFR and PyUFGT the promoters of structural genes, which activate the expression of structural genes, to promote anthocyanin biosynthesis and accumulation. This study clarifies of the anthocyanin biosynthesis regulatory mechanism of Yunhongli-1from Yunnan Red Pear will make foundation for the breeding quality red pear resources.