Establishment Of Peach Genetic Transformation Regeneration System And Promoter Cloning Of Fruit-specific Gene E8

Peach〔Prunuspersica (L.) Batsch.〕is one of the most important commercial stone fruit species and a famous crop in Gausu province of China. The annual yield is over 13 million tons all over the world. Peach is a favorite and famous fruit, because of its good taste, excellent quality, attractive color and marvelous fragrance, well-received in the market. However, Peach is tipical respiratory climacteric fruits, its unique and maturity characteristic resulted in bad preservation and freshness. The production was seriously affectted by its bad preservation during storing and transportation.In order to reduce the losses caused by post-harvest fruit rottern, it is necessary to control fruit soften during the ripening and senescence of fruit. However, it has not much improvement in dealing with this problem by using traditional methods.It has important significance to solve this problem by biotechnology.This study described establishment of peach genetic transformation regeneration system and promoter cloning of fruit-specific gene E8. Plants were collected explants of peach'Baifen','Yuhualu','Guangshanbai'and of tomato'Maofen 802'. It laid down a solid foundation and provided beneficial information for a prolonged shelf life using genetic engineering technology. The results were as fallow.1. Peach genetic transformation regeneration system was established. Tender leave, leaf stalk, tender shoot and immature embryos collected 50 days and long time after bloom were cultured in vitro. The results showed that: (1) The different explants of peach could induce callus, but only the callus derived from embryos collected 50 days and long time after bloom succeeded in producing adventitious buds formation. Embryos inoculated on MS medium supplemented with 0.25 mg·L -1 6-BA and 1.0 mg·L -1 2,4-D , gave highest callus induction rate (99.8%) of three variety of peach. (2) The callus was granulometric with good potency of differentiation on MS medium supplemented with 0.1 mg·L -16-BA and 1.0 mg·L -1NAA. The fold of multiplication was 10.16 when the source concentration was 20g·L-1. (3) When the callus was cultured on MS medium containing 1.0 mg·L-1 6-BA and 0.1mg·L-1 NAA, the callus became to embryogenitic callus and the differentiation rate of callus was 37.5%. (4) The optimum condition of the roots from shoots was that soaking the explant in 100mg/L IBA solution and inoculated on 1/2MS medium without hormone, the rooting rate was 82%. (5)The embryos collected 75 days after bloom succeeded in producing adventitious buds formation and roots straightly on medium MS media supplied with 0.5mg·L -1 6-BA and 0.05mg·L -1 NAA.The hightest differentiation rate of'Baifen'peach collected 75d after bloom was 96.1% more than two other variety of peach.2.Using modified CTAB method, high purity and quality genomic DNA solution was acquired from leaf tissue, containing less polysaccharides, without browning and fit for further molecular operation. Two specific primers were designed which were added to restriction enzyme sites HindⅢand BamHⅠ. Grading-up PCR reaction system and a DNA fragment (1.1kb) of an E8 Promoter was amplified by polymerase chain reaction (PCR) and cloned into a pMD18-T Simple Vector. The plasmid DNA was isolated and identified by plasmid PCR and restriction enzyme analysis. After recombinant plasmid was identified, the results proved the correct target fragments inserted into the pMD18-T Simple Vector. Named the recombinant plasmid with pMD-E8.