梨是蔷薇科(Rosaceae)梨亚料(Romaceae)梨属(Pyrus)多年生木本植物，是世界主要的果树作物之一，栽培面积和产量均仅次于苹果。砂梨(Pyrus pyrifolia Nakai)原产于我国中部、南部和西部各省，适于在温暖多雨地区栽培，是原产于中国的四个梨栽培种之一，具有耐热、抗旱、抗火疫病等优良性状。但其果实大多贮藏性差，如何提高果实耐贮性是解决好砂梨产业化的关键问题。目前的研究主要是针对砂梨果实的生理特点，通过改善果实的贮藏条件和采用一些乙烯抑制剂来延长砂梨果实的货架期，不能解决根本问题。本实验中以‘明水’（Pyrus pyrifolia Akemizu）梨为主要试材，通过转基因技术导入DSACO基因，提高该品种的耐储性，提高其市场竞争力。主要研究内容如下： 1、为了将植物表达载体转入模式植物番茄中，本研究初次研究了不同浓度ZT和IAA对‘江疏14号’番茄子叶再生的影响，表明该品种在MS+ZT 1.5mg/L+IAA 0.7mg/L的培养基中再生率为81.3%。建立了DSACO和DSLOX基因转化番茄的遗传转化体系，对获得的抗性植株进行初步检测，结果表明上述2个基因的转化效率低，用于转化后抗性植株假阳性率很高。 2、对原有的植物表达载体pYF028-DSACO和pYL028-DSLOX进行改造。将原有DSACO和DSLOX基因经PCR验证后，提取农杆菌质粒转化大肠杆菌DH5α, 提取大肠杆菌质粒，PCR验证并进行双酶切，将酶切后的目的片段回收，连接与pYH4215空载体上。重组质粒转化农杆菌EHA105感受态。结果表明：pYF028-DSACO改造成功并重新命名为pYH4215-ACOi。 3、ACOi基因转化番茄‘江苏14号’，实验中对不同筛选压浓度进行了梯度实验，确定潮霉素浓度。对抑菌素的种类和浓度进行选择，确定抑制超强活性农杆菌EHA105的方法，最终调整了适合ACOi基因转化番茄的遗传转化体系。获得12个抗性株系，通过GUS染色、PCR和PT-PCR检测，得到了3个转基因植株。结果表明：ACOi用于番茄的转化效率要高于DSACO。 4、以‘明水’梨枝条为外植体，研究了无菌苗的获得，无菌苗的继代培养和增殖培养。以无菌苗的叶片为外植体，研究不同基本培养基，不同细胞分裂素和生长素组合等再生影响因素对叶片不定芽再生的影响。实验表明：以在NN69＋TDZ 1.5 mg/L＋IBA 0.2 mg/L＋AgNO3 1.0 mg/L，暗培养25d，叶片不定芽再生率最高为71.8％。 5、研究了不同浓度筛选压、不同菌液浓度和不同侵染时间、以及不同抑菌素浓度等因素对遗传转化的影响。分别建立了农杆菌介导DSACO基因和ACOi基因转化‘明水’梨的遗传转化体系。实验共获得9个抗性株系，对抗性株系进行了GUS染色，PCR和RT-PCR检测，确定在2个株系的‘明水’梨的基因组中成功整合入外源的DSACO基因并得到表达。

Pear, Rosaceae Romaceae Pyrus, is one of important fruit crops on the word. The culture area and production of pear is only less than that of apple. Sand pear (Pyrus pyrifolia Nakai) originated in central, southern and western of China, and it is suitable for cultivation in the warm and wet area with some advantages such as heat-resisting, combats drought, the fire blight-resistant and so on. It is important for sand pear industrialization to prolong fruit storage duration. At present research has been focus on extending fruit shelf life by improving storage condition and application of ethylene inhibitors. These methods are usable but unresolved totally. My research mainly focuses on improving pear fruits storage characteristics by transform DSACO gene into pear. We choose Pyrus Akemize as materials with good quality and taste but un-storable. 1. In order to transform plant expression vector to model plant tomato, we researched different concentration combinations ZT and IAA on cotyledon regeneration of tomato ‘Jiangsu NO. 14’. The result showed this type of tomato regeneration ratio was 81.3% on the medium MS+ZT 1.5mg/L+IAA 0.7mg/L. The transformation system of DSACO and DSLOX were adjusted, the resistance plants were detected and the result shows false positive ratio was very high. 2. Plant expression vector pYF028-DSACO and pYL028-DSLOX were reconstructed. Agrobacterium plasmids were transformed into DH5α. After plasmid enzyme cutting, we linked pYH4215 vector with functional fragment .We transformed linkage production into EHA105. The result shows the reconstructed vector is working, named pYH4215-ACOi. 3. We transformed ACOi into tomato ‘Jiangsu 14’. We screened different concentration of Hyg and built new transgenic system. We got 3 transgenic plants. The transformation ratio of ACOi is higher than that of DSACO. 4. We obtained aseptic seeding and a lot of sterile plants. We made research about the effect of medium, cytokine and growth hormone on regeneration of pear leaves. The result shows that leaves regeneration ratio is 71.8％ after culturing leaves with NN69＋TDZ 1.5 mg/L＋IBA 0.2 mg/L＋AgNO3 1.0 mg/L, dark, 25d. 5. We researched the effect of bacteria concentration and dipping time on transformation on the basis of tomato regenerating system construction. We also optimized transformation system of Agrobacterium mediating DSACO gene and ACOi. We obtained 9 transgenic plants. Transgenic plants were identified by GUS stain and PCR and RT-PCR test. The result showed 2 of them have integrated DSACO gene successfully.