【作者】 张亚林；

【导师】 李唯；

【作者基本信息】 甘肃农业大学 ， 作物生态生理， 2010， 硕士

【摘要】 桃(Prunus persica(L. ) Batsch)是世界性重要果树,我国是桃主产国,产量居世界之首,甘肃省是桃的原产地和最适栽培区之一。桃是典型的呼吸跃变型果实,成熟果实采后很快出现呼吸高峰,果肉迅速变软,给贮藏运输带来极大不便,这是长期制约桃生产的主要问题之一。而通常采用的低温冷藏等方法易造成桃果冷害,结果十分不理想。因此,通过植物基因工程技术控制果实软化,选育出耐贮运的桃新品种是从根本上解决桃贮运难题的有效途径之一。本研究通过反义基因技术特异性抑制果实成熟后PG的表达,有望延迟桃果实的软化,获得耐贮性硬溶质桃新种质。本研究以甘肃省常栽品种白粉桃为试材,建立了桃遗传转化再生体系,并以白粉桃茎段为材料,在优化桃遗传转化再生体系的基础上,通过根癌农杆菌介导反义PG基因转化,得到了转化植株并用PCR进行了检测。研究主要取得如下结果:1.以白粉桃幼茎和花后不同时间成熟的果实种胚为外植体,进行桃遗传转化再生体系优化。其幼茎最佳诱导培养基为MS+6-BA 1.0mg/L+ NAA 0.5mg/L,诱导率可达93.5%。开花75d后的种胚能在MS+6-BA 0.5mg/L+NAA 0.05mg/L上直接诱导分化成苗,诱导分化率可达91.5%。将不定芽用100mg/L IBA浸蘸其基部转移到不含激素的1/2MS培养基中诱导生根,生根率达到78%。2.用携带有PG基因反义片段和35S启动子的表达载体pCAMBIA2300,转化白粉桃茎段。受体材料预培养3d后,经根癌农杆菌菌液(OD600值0.4)浸染10min后共培养60h,转移至分化培养基MS+6-BA 1.0mg/L +NAA 0.5 mg/L+kan 50mg/L+Cef 250mg/L上诱导产生不定芽,将抗kan的不定芽先后置于含有kan 50mg/L的生长、增殖和生根培养基上继续选择,获得完整植株。经PCR检测初步证明,反义PG基因已导入桃中。更多还原

【Abstract】 Peach〔Prunuspersica (L.) Batsch.〕is an important fruit in the worldwide. China is a major producing country of peach, whose output ranks first in the world, and Gansu Province is the original and the most appropriate cultivation area of peach. Peach is a tipical respiratory climacteric fruit. Soon ripe fruits picked appeared the respiratory peak and flesh became soft rapid, which caused great inconvenience in transport and storage and it is a major problem of restraining the production in peach .While it is unsatisfactory that the common method such as cold storage usually leads to chilling injury of peach. Therefore, using plant genetic engineering technology can control fruit softening, and changing fundamentally storage and transport properties in peach has an important significance.In this study, Baifen peach often planted in Gansu Province was used as test materials, and then established a peach regeneration system. with Baifen peach stem segments as materials, based on the optimization genetic transformation system of peach, taking advantage of Agrobacterium mediated transformed into antisense PG gene and obtained transformed plants, and then tested by PCR. The main results obtained are as follows:1. With tender stem and matural fruit in different time of Baifen after flowering as explants, optimize the genetic transformation system. The best medium for induction was MS+1.0mg/L6-BA+0.5mg/LNAA, and induction frequency was up to 93.5%. After flowering 75d embryo can directly differentiate into seedlings, induction frequency was up to 91.5%. The shoots dipped its base with 100mg/L1IBA were shifted to 1/2MS medium without hormone to induce roots whose rate was up to 78%.2. Transform into the stem of Baifen peach by expressive vector pCAMBIA2300 carrying the antisense PG gene fragments and 35S promoter. Receptor materials precultivated for 3 days, were co-cultured for 60h by Agrobacterium bacteria (OD600 value of 0.4) after being disseminated, transferred into the differentiative medium MS +1.0 mg/L6-BA +0.5 mg / LNAA +50 mg / L km +250 mg / LCef to induce adventitious buds, and anti-km adventitious buds were placed in the 50mg/Lkm mediums of growth, proliferation and rooting. Ultimately the whole plants were obtained by The PCR test which showed that the antisense PG gene has been introduced into the plants.更多还原