Functional Research Of PybHLH Transcription Factor And PyMT1Metallothionein Gene From Yunnan Red Pear (Pyrus Pyrifolia)

Basic helix-loop-helix (bHLH) family transcription factors are common in eukaryotes,and they form a large family.They play an important role in growth regulation, anthcyanin biosynthesis, secondary metabolism and abiotic stress. Metallothionein is a kind of ubiquitous and multifunctional protein in organism. Plant metallothionein mainly involved in the storage, transportation and metabolism of trace element as well as heavy metal detoxification, free radicals and antagonist ionizing radiation effect in body. Yunnan Red Pear belongs to sand pear, pear genera, pear subfamilies, rosaceae. At present, four kinds of Yunnan Red Pear contaning early maturing variety ’Zaobaimi’(95-2), mid-maturing Meirensu’(35) and ’Mantianhong’(32) and late maturing ’Yunhong-1’ are widely painted. Yunnan red pear has been planted in large scale and has been created bulk economic benefits in Yunnan province. Due to the differences of pigmentation and inner quality, it is necessary to perform the researches both on transcription factor bHLH, a regulator of anthcyanin biosynthesis and abiotic stress-related, and metallothionein gene PyMT1, a fruit quality related gene. These results will provide a foundation for improving Yunnan red pear fruit coloration and internal qualities. So far, bHLH transcription factor of Yunnan Red Pear has not been studied. In this research, PybHLH transcription factor and PyMT1metallothionein Gene gene was cloned from Yunnan Red Pear, and their functions were verified by transgenic Arabidopsis and transgenic Ecoli,separately. The main results of my research are as follows:The PybHLH gene was cloned from red peel of ’Mantianhong’, and its functions were predicted by BLASTn and alignment with other bHLHs in plant.The paint overexpression vector pK2-p35S-PybHLH was constructed by Gateway technology. The PybHLH transgenic Arabidopsis was obtained by transforming the vector pK2-p35S-PybHLH into Arabidopsis Col-0by floral-dip method. The transgenic strains were confirmed by RT-PCR and western blot with a specific PybHLH antibody.6lines of PybHLH transgenic Arabidopsis were obtained, and there were no observable differences between transgenic ones and the wild type. The differences of phenotype and physiology between PybHLH transgenic Arabidopsis and the wild type were explored before and after treatment with200mM of NaCl. The results showed that distinct differences were observed before and after treatment with200mM of NaCl, however, there is no obvious difference in anthocyanin content between the transgenic and non-transgenic ones. The physiological changes of transgenic Arabidopsis and wild-type were detected after treatment with200mM NaCl. The results showed that although the content of total soluble sugar increased in both transgenic ones and non-transgenic ones after treatment, while the content of total soluble protein in both decreased, the differences between transgenic one and nontransgenic ones were the contents of both total soluble sugar and total soluble protein in transgenic ones were far more than that of the non-transgenic ones. The content of both MDA and H2O2increased in both transgenic ones and non-transgenic ones after treatment, however, the former increased far less than the latter. These results suggested that overexpression of PybHLH in Arabidopsis confered its resistance to NaCl.Results showed that PybHLH transcription factors have no apparent effect in the regulation of anthocyanin biosynthesis in Arabidopsis, however, it showed NaCl resistance in Arabidopsis.PyMT (Pyrus pyrifolia metallothionein) genomic sequences were amplified according to the EST sequence of the red pear ’Yunhong-l’s light-specific supression substration hybridization library from four cultivars of Yunan red pear and these sequences were analyzed. The PyMT cDN A sequence of ’Yunhong-1’ was amplified by RT-PCR and was designated PyMT1(Pyrus pyrifolia metallothionein1). Prokaryotic expression vector pGEX-4T-1-PyMT1was constructed and was transformed into E. coli BL21for expression. The growth curves of E. coli. transformed with pGEX-4T-1-PyMTl and the control were obtained by growing them in liquid medium with different concentration of various metal ions.Sequence analysis showed that PyMT1belongs to typical the Metallothion2. Results of phylogenic analysis showed that PyMTl was at the same evolutionary branch with MdMT. The SDS-PAGE results display that the expressed proteins is consistent with the anticipated size. Results of the growth curve show that PyMT1has a certain tolerance to heavy mental ions such as Zn, Cu and Cd in vivo. The results will provide a foundation for further purification, structure and function research of PyMT1.