Molecular Cloning And Characterization Of Endodormancy Related Genes IRE1、bZIP60 In Peach Bud

As far as deciduous fruit trees, bud dormancy is a biocompatible alteration for fighting against lower temperature in winter. With the development of facilities fruit trees, the feature of bud dormancy has been one of variouis factors hindering the production of fruit trees. The warming of greenhouse was directly dertermined by the period of dormancy, which was related with the fruit market and production efficiency. Therefore, how to control the dormancy of fruit trees is crucial to the production of fruit trees.As one of the critical factors of influencing fruit trees is dormancy, it is of great significance to study the process of the dormancy of fruit trees, which is helpful to regulation of stage of ripe for the fruit trees.The 5-year-old peach（Prunus persica（L.） Batsch cv. Nectarina Shuguang） was selected in this study to investigate the alteraion of ulturastructure of cells in the buds of flowers by transmission eletron microscopy during the dormancy. Meanwhile, the association of dormancy in both bud and seed with key genes was speculated, and also analysis of the changes of key genes involved in the pathway of endoplasmic reticulum stress in the process of dormancy. In addition, the chilling dependent peaches were selected to check whether chilling played an improtant role in the expression of Pp IRE1 and Ppb ZIP60. We speculated roles of that genes of Pp IRE1 and Ppb ZIP60 played in the dormancy throught construction of superexpression and mutant strains of complementary of Pp IRE1 and Ppb ZIP60, and verified the function of genes of Pp IRE1 and Ppb ZIP60 by an ectopic expression in Arabidopsis.The main results were as follows:1, It was consistent in both bud dormancy and seed dormancy by observing the changes of ultrastructure, including changes from thin to thick in the cytoplasm; increase in volume of nuclear and loose nucleoli gradually; cell membrane, nuclear memebrane and plasma membrane going from incomplete to full in the cell membrane, boundary turning from fuzzy to clear; the change of plasmodesmata being from less, thin to more, thick; the recovery of intercellular channels after release of dormancy, the transfer of nutrients becoming more frequent throught plasmodesmata. The proliferation and increasing in individual of mitochondrial and clearer ridge stuctures made preparation for rapid growth and differentiation after germination. The transport channel was emeraging from shorter and smaller the endoplasmic reticulum arranged surrounding the larger vacuole, the edage of plasma membrane and nuclear memebrane. The saccular sheets of Golgi body became more and more clear and complete. The increase of endoplasmic reticulum and Golgi body where were synthesis of protein showed the increase of metabolic enzymes for protein synthesis. Liposomes, starch granules, and fat granule became increase prior to dormancy, while turned to less after the release of dormancy.2, The accumulation in endoplasmic reticulum Bi P（Bi P1 and Bi P2）demonstrated the occurrence of endoplasmic reticulum stress. The critical genes of Pp IRE1a、Pp IRE1b、Ppb ZIP60、Ppb ZIP28、Pp NF-YA4、Pp NF-YB3 and Pp NF-YC2 being discovered in the pathways of IRE1-b ZIP60,b ZIP28-NF-Y showed the trendline from rising and then falling during the dormancy, revealing an close link between them and further being involved in the regulation of dormancy. The function of genes could be speculated by observation of germination of seeds throught ectopic exression without system of genetic transformation in peach.3, The m RNA expression of Pp IRE1、Ppb ZIP60 in Prunus persica（L.） Batsch cv. Nectarina Super Crimson（the requriement of chilling 800 C.U.） and Prunus persica（L.） Batsch cv. Nectarina May Glo（the requriement of chilling 200 C.U.） reached no significant difference during dormancy.4, The overexpression and analysis of function of genes of Pp IRE1 and Ppb ZIP60 and rate of germination in the day 4d, 8d, 12 d after seeding throught recording the superexpress strains in Arabidosis showed that no significant difference in the seed germination between Arabidospsis and wild types, however, the germination time was obviously in advance. All germinated on the fourth day and the time was shorten half compared with the wild type（8d）.5, The rate of germination reached 98% on the twelvth day after seeding in bzip60-1、ire1-1 by the observation of T- DNA Insertion mutant strains of homozygous lines of Sail₂83B03（bzip60-1）, Salk₀50203（bzip60-2）, Salk₀18112（ire1-1） and Sail₁256F04（ire1-2） failed to reach significant difference compared with that of 100% germination in wild types. The germiantio rate of bzip60-2（20%）、ire1-2（15%） in seeds showed less than that of in wild types. The germination rate was recorded in T3 generation of seeds by contructing complementary function of mutant strains of Pp IRE1、Ppb ZIP60 in mutants of bzip60-2、ire1-2, conluding that the rate of germation in complementary function of mutant strains being higher compared with mutant strains, especially the mutant of ire1 being improved by three fold than that of 35S::Pp IRE1/ire1, while part of phenotypes in complementary strains got recovered; and similar trend in 35S::Pp IRE1/bzip60, of which the rate of germination improved by two fold compared with bzip60 mutants. Therefore, we conclude that break of dormancy in peach was linked to higher expression of Pp IRE1 and Ppb ZIP60 m RNA.The hypotheis of consistency existing in dormancy of both seed and bud was confirmed on the basis of celluar and genes and preliminarlly revealed that the regulation of dormancy was associated with involvement of critical genes in the the pathway of endoplasmic reticulum stress by further verification of Pp IRE1 and pb ZIP60. Therefore, foundation was laid for revealing the mechanism of bud dormancy in peach at molecular level, and also theroritical basis was provide for further investigating regulation of ripen period.