【作者】 余义勋；

【导师】 包满珠；

【作者基本信息】 华中农业大学 ， 植物学， 2004， 博士

【摘要】 香石竹（Dianthus caryophyllus L.）是世界四大切花之一，在花卉市场中占有重要地位。延长瓶插寿命对延长观赏期和调节流通有重要作用。传统的保鲜方法多采用乙烯抑制剂或乙烯拮抗剂的保鲜剂，但这些化学物质多会造成环境污染，且成本较高。本研究通过克隆香石竹ACC氧化酶（ACO）基因，将其构建成不同T-DNA结构的植物表达载体，并导入香石竹不同品种，分析转基因香石竹切花的瓶插寿命。 1 ACO基因的克隆及其植物表达载体的构建 根据已发表的香石竹ACO基因序列，设计一对特异引物，以香石竹品种‘America’基因组DNA为模板，扩增出香石竹ACO基因，将其连接到pMD18-T质粒载体上，对序列测定结果与已发表的ACO基因cDNA序列进行对比分析显示，该克隆片段含3个外显子，2个内含子，外显子序列与已发表的ACO基因cDNA序列完全一致。通过中间载体pBluescript SK和pIC19R，经PCR分析及酶切检测，成功地构建了ACO基因正义、反义、正义重复和反义重复等4种不同T-DNA结构的植物表达载体，并用冻融法将该4种植物表达载体导入了农杆菌LBA4404菌株。 2 香石竹5个品种再生体系的建立 采用香石竹无菌苗叶片为外植体，建立了香石竹5个品种‘Aicardi’、‘Master’、‘Yellow Star’、‘Opera’和‘Mabel’的再生体系。幼叶比老叶分化率高，叶片在含BA和TDZ两种细胞分裂素中都可以分化，但在含TDZ培养基中，不定芽玻璃化程度较高，且发现AgNO₃（0-25 mmol／L）对不定芽分化起抑制作用，因此选用MS+NAA 0.3mg／L+BA 1.0mg／L为分化培养基，5个品种的再生率在38.6％与61.8％之间。无菌苗茎段在R1（MS+NAA 0.1mg／L）、R2（MS+IBA 0.1mg／L）和R3（MS+IAA 0.1mg／L）三种培养基中均能生根，但以R1培养基生根率最高（100％）。无菌苗生根后经炼苗移栽至河沙、珍珠岩和腐叶土的3种基质中，结果表明在珍珠岩中的成活率（78.7％）明显高于河沙和腐叶土。 3 香石竹5个品种的遗传转化 用农杆菌介导法对上述5个品种进行了遗传转化。实验表明：香石竹对潮霉素比较敏感，叶片分化选择压力为4-5mg／L。在转化过程中对预培养时间、根癌农杆菌菌液浓度、侵染时间、乙酰丁香酮浓度以及共培养时间等转化条件进行了摸索。将香石竹叶片在分化培养基上预培养2天，于根癌农杆菌菌液(OD₆₀₀值为0.6-0.9)侵染8-12min,在含100mmol／L的乙酰丁香酮的培养基上共培养3d，转移到选择培养基（MS+NAA 0.3mg／L+BA 1.0mg／L+Hyg 4mg／L+Cef 400mg／L）中培养，剔除 华中农业大学博士学位毕业论文选择培养前两周分化出的腋芽，每3周换一次培养基。在此期间，抗性芽长至1 cm时切下转移到选择生根培养基（MS+NAA 0.1 mg/L十Hyg 4.0 mg几）中生根培养。不同品种抗性芽分化率在4.3%和11 .2%之间，4转基因植株的获得及检测 比较四种提取香石竹基因组DNA的方法，其中改良SDS法所提DNA较纯，且拖带较少。抗性不定芽在选择生根培养基上生根率为48.3%，PCR扩增HPT基因表明，24.5%的抗性生根植株为阳性。Southem杂交证实，14.2%的抗性生根植株外源基因已整合到植物基因组中。共得到33株转化株系，其中30株为单拷贝，3株为两个拷贝。5不同T一DNA结构导入后对香石竹瓶插寿命及乙烯释放量的影响 分析了4种T一DNA结构导入‘Master’、‘Mabel’和‘Yellow Star’3个香石竹品种17个转化株系瓶插寿命及部分株系乙烯释放量的测定结果。 在转化‘Master’品种正义ACO基因的3株转化株系中，有l株瓶插寿命显著延长;在转化反义ACO基因的4株转化株系中，3株瓶插寿命显著延长;在转化正义重复ACO基因的2株转化株系瓶插寿命显著延长，抑制乙烯生成达90%以上;在转化反义重复ACO基因的3株转化株系中，瓶插寿命都显著延长，延长的时间较长 （达12d以上），而且抑制乙烯生成达100%。 以上结果表明，4种结构的T一DNA均能在一定程度上抑制内源基因的表达，相比之下，反义强于正义，重复基因结构强于单一基因结构。6转化同一T一DNA结构不同品种间的瓶插寿命比较。 在转化‘Master’品种的n株系中有8株瓶插寿命得以显著延长，在转化Mabel’品种的3株系中有l株瓶插寿命得以显著延长，在转化‘Yelfow Star’只有1株，其瓶插寿命得以显著延长。 转化同一T-DNA结构不同品种间的瓶插寿命有一定差异。在转化正义ACO基因的‘Master’品种3株系中有1株系瓶插寿命得以显著延长，‘Mabel’品种2株系瓶插寿命都未延长;在转化反义ACO基因的‘Master’品种的4株系中有3株系延长，‘Yellowst盯’品种1转化株系瓶插寿命延长;在转化正义重复ACO基因的 ‘Master’品种的2株系瓶插寿命均达12d左右，‘Mabel’品种2株系有1株系瓶插寿命延长。 另在同一株系的不同花朵之间瓶插寿命也存在着差异，可能是转化嵌合体所致。更多还原

【Abstract】 As one of the major contributors and a commercial leader to the cut-flower market, carnation (Dianthus caryophyllus L.) is an important target for the breeding of new cultivars with novel characteristics. In conventional method of prolonging vase life of carnation cut flower, the substance containing Ag+ was used and this led to pollution of environment. In this study, ACC oxidase (AGO) gene genetic DNA was cloned from carnation cultivar ’America’ genome. Four T-DNA structures of plant expression vectors of AGO gene were constructed and integrated into carnation cultivars ’Master’, ’Mabel’, ’Yellow Star’, ’Opera’ and ’Aicardi’. The vase lifeof transgenic cut flower was prolonged.1 Cloning of ACC oxidase gene of carnation and four constructions of its plant expression vectorsA pair of primers was designed according to the 1-aminocyclopropone-l-carboxylic acid (ACC) oxidase gene sequence of carnation, and the primers were used to amplify the genomic DNA fragment of 1.2 kb by polymerase chain reaction (PCR) by taking genome DNA from carnation cutivar ’America’ leaves as template. The PCR product was cloned into pMD18-T vector. Sequencing indicated that the cloned ACC oxidase gene consisted of 3 exons interrupted by 2 introns. Totally four T-DNA structures of plant expression binary vectors were constructed. Those were sense, antisense, direct repeated of sense and antisense.2 Establishment of efficient regeneration system from leaf of 5 carnation cultivarsThe regeneration system of carnation was established with the method of direct adventitious shoots inducing. The adventitious shoots were induced from leaf explants based on MS basal medium supplemented with 1 .0mg/L BA and 0.3mg/L NAA and its differentiation frequencies were between 38.6% and 61.8%. The age of leaf affects its differentiation frequency and AgNO3 in adventitious medium inhibits adventitious shoot inducing. The regenerated plants were rooted on MS medium containing 0.1mg/L NAA.3 Genetic transformation of carnationTaken the young leaf as explants, the Agrobacterium rwme/ac/ens-mediated genetic transformation system of carnation was established. A. tumefaciens strain LBA4404 harboring pMOGMON containing four T-DNA structures of ACO gene were used totransform 5 carnation cultivars. Leaf explants were pre-cultured on shoot-inducing medium for 2 days, then immersed in Agrobacteriwn suspension for 8-12 min. The co-cultivation was carried out on medium containing 1 00 mol/L acetosyringone (AS) for3 days. After that the transformants were obtained by transferring explants to selection medium supplemented with 4-5 mg/L hygromycin (Hyg) and 400mg/L cefotaxime (Cef). Some factors that affect the transformation frequency were compared. The regeneration frequencies on selection medium were between 4.3% and 11.2%.4 Identification of transgenic plantsIn four methods of DNA extraction, the modified SDS method was the best. 48.3 percent transformed plants rooted on rooting medium containing 4mg/L Hyg. And 24.5 percent of the PCR amplification of HPT gene was positive. Southern blot analysis demonstrated that 33 transformed plants, which was 14.2 percent of resistant rooting plants, were positive, and foreign gene were integrated into plant genome. 3 of the 33 transformed plants have two copies of foreign gene and the rest of transformed plants have single copy of foreign gene.5 Comparison of vase life and ethylene production of transgenic plant cut flowers with different T-DNA structuresVase life and ethylene production of 17 transgenic lines of 3 cultivars were analyzed. Vase life and ethylene production were different in transgenic lines with different T-DNA structures.Vase life of 5 direct repeat transgenic lines of ’Master’ cultivar was all prolonged by about 6 days, and antisense direct repeat transgene produced only a negligible amount of ethylene during cut flower natural senescence. 3 lines vase life of 7 single transgenic lines of ’Master’ cultivar did not change compared with control. This suggested that direct repeat transgene ind更多还原