【作者】 王涛；

【导师】 朱宝成； 李潞滨；

【作者基本信息】 河北农业大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 兰科(Orchidaceae)是有花植物中最大的一个科,具极高的观赏价值。在全世界花卉业中,兰花具有重要的经济价值。兰花是整个兰科植物的总称,主要分布于热带、亚热带,性喜温暖潮湿,耐寒性、抗旱性能力较差。近年来兰花广泛受到建兰花叶病毒(Cymbidium Mosaic Virus ,CyMV)等病毒的侵染,导致观赏价值下降。目前国际上还未有防治植物病毒病的有效药剂,而随着世界上兰花贸易的不断扩大,兰花病毒病也开始在不同植株中蔓延,严重影响多种兰花的栽培和生产。因此,培育耐寒、抗旱、抗病毒的兰花新品种已成为兰花新品种培育的主要方向。本研究以8个种类具有重要价值的兰花(大花蕙兰、中国兰属附生种类等)为材料,应用农杆菌和基因枪两种转化方法进行了转基因研究,将美花兰抗寒基因CiDREB1和毛竹抗旱基因PeDREB2分别转化大花蕙兰、中国兰花等种类兰花;采用实时荧光定量PCR方法对转CyMV-CP基因兰花进行表达量的检测。本研究旨在利用基因工程手段进行兰花抗逆性品质改良,为最终培育耐寒、抗旱、抗病毒兰花新品种奠定理论和实践基础。1.研究确定了2类兰花的最佳抗生素筛选浓度。表达载体中有新霉素磷酸转移酶基因(NptⅡ)作为筛选标记基因,利用卡那霉素对转化体进行筛选。美花兰( Cymbidium insigne ) MH的卡那霉素最佳筛选浓度为50 mg/L ;树兰(Epidendrum)SL的卡那霉素最佳筛选浓度为15 mg/L。2.采用农杆菌侵染和基因枪介导的方法,以美花兰抗寒基因CiDREB1基因和毛竹抗旱基因PeDREB2基因为外源目的基因,对美花兰MH,大花蕙兰(Cymbidium)PB、PC,兰属(Cymbidium)兰花F5、F7、C2,树兰SL,卡特兰(Cattleya)K1 8种兰花进行遗传转化,初步得到了抗生素水平的阳性苗,并对转化率分别进行了统计。结果表明,农杆菌侵染法出苗率处于3.13-15.6%之间;基因枪介导的转化出苗率处于1.7-92.2%之间。3.基因克隆获得了兰花PB、PC肌动蛋白Actin基因部分序列,设计合成了适合于实时荧光定量PCR试验进行基因相对表达量分析的特异性引物(Actin-S/A, CyMV-S/A)。溶解曲线和标准曲线分析结果表明,Actin-S/A引物扩增Actin基因标准曲线相关系数为r2 = 0.994,线性方程为y =-3.271X+19.008,扩增效率E=102.2%;CyMV-S/A引物扩增CyMV-CP基因标准曲线相关系数为r2 = 0.997,线性方程为y =-3.731X+11.993,扩增效率E=85.4%。表明所设计的CyMV-CP基因引物能够用于目的基因相对表达量的检测。4.利用实时荧光定量PCR方法,以两个种类转CyMV-CP基因大花蕙兰PB、PC PCR检测阳性植株为材料,对CyMV-CP基因在转基因兰花植株体内的表达量进行分析。结果表明,外源基因CyMV-CP在转基因大花蕙兰PB、PC植株体内得到较高水平的表达,表达量明显高于未转基因兰花植株,相对表达量分别是对照植株的14.8倍、100倍。更多还原

【Abstract】 Orchidaceae is one of the largest families in the flowering plants with high ornamental value. As one of important ornamental plants, orchids have an important commercial importance in world flower industry, and distribute mainly in the tropical, subtropical, liking warm and humid, with poor ability to cold and drought tolerance. In recent years, orchids were seriously infected by Cymbidium Mosaic Virus, leadingto declining of ornamental value. Simultaneously, with the world trade widely expanding, kinds of virus diseases spread in plants, and seriously affect the cultivation and production of orchids.Therefore, antiviral, drought and cold resistant new orchid varieties have become the main direction of orchids breeding.In this research, Kanamycin was the antibiotic selection of orchids. Eight species of orchids (Cymbidium hybridium, Cattleya, Chinese Cymbidium, etc.) were transformed by bombardment or Agrobacterium mediated transformation, and protocorm-like bodies (PLBs) were used as target explants, with CiDREB1 gene from Cymbidium insigne and PeDREB2 gene from Phyllostachys edulis. Results showed that the germinat rates of transgenic PLBs were between 1.7-92.2% under the antibiotic selection in the MS culture medium with appropriate Kanamycin concentration.The technique of SYBR Green-based quantitative real-time reverse transcription polymerase chain reaction (real-time RT-PCR) was applied to quantitative detect transgenic Cymbidium with CyMV-CP gene. By using RT-PCR method, partial sequence of Actin gene was cloned from Cymbidium. On the base of Actin gene sequence, the primers of Actin-S/A and CyMV-S/A were synthesized, which were suitable for quantitative real-time fluorescent PCR. The plasmid containing the target sequence was constructed to prepare for the standard curve and detect the sensitivity.The melt curve and standard curve analysis results showed that the regression equation of Actin-S/A primers was y = - 3.271X+19.008, and related coefficient: R2= 0.994, ampliative efficiency E=102.2%; and the regression equation of CyMV-S/A primers was y = -3.731X+11.993,and related coefficient: R2= 0.997, ampliative efficiency E=85.4%.It indicated that the primers can be used for detecting the expression of CyMV-CP gene in the transgenic Cymbidium.The assay showed that the exogenous CyMV-CP genes in transgenic Cymbidium PB, PC plants had higher levels of expression, and the expression level obviously higher than control plants, and the separate relatively expression were 14.8 times and 100 times comparing to the negative Cymbidium.更多还原