【作者】 杜灵娟；

【导师】 王跃进； 刘雅莉；

【作者基本信息】 西北农林科技大学 ， 园艺植物种质资源学， 2013， 博士

【摘要】 百合（Lilium spp.）是五大切花花卉之一，属于乙烯跃变型花卉。花卉衰老受乙烯的调控，通过RNAi技术下调或者敲除乙烯生物合成过程中的关键基因（ACC氧化酶基因，ACO），可以延缓花卉衰老，延长切花寿命。ACO基因在花卉衰老过程中呈花组织特异调控模式，选择一个强的花特异表达启动子驱动ACO RNAi载体，可以降低或消除因组成型启动子驱动ACO RNAi载体在植物的各个组织和所有发育阶段表达可能造成的负面影响。课题组前期构建了四个花特异嵌合启动子，转化了模式植物夏堇。本研究分析了四个嵌合启动子在转基因夏堇中的活性，目的是为了筛选出一个增强的、且花特异表达的启动子，用于切花百合抗衰老遗传改良的研究。此外，针对百合再生率及遗传转化率低、嵌合体多的问题，初步建立了百合体细胞胚再生体系，通过农杆菌介导法及基因枪法转化了ACO RNAi载体。本研究取得的主要结果如下：1.在课题组前期研究的基础上，分析了四个嵌合启动子p35S-PCHS-Ω，p35S-LCHS-Ω，pOCS-PCHS-Ω和pOCS-LCHS-Ω的功能。运用GUS组织化学染色、GUS荧光定量和实时定量PCR方法，分析了GUS基因在转基因夏堇中不同的表达水平及组织特异性。结果表明：嵌合启动子p35S-PCHS-Ω和p35S-LCHS-Ω驱动强的、组成型GUS表达，尤其在蓝紫色的花冠（p35S-PCHS-Ω），蓝紫色的花冠和根（p35S-LCHS-Ω）。嵌合启动子pOCS-PCHS-Ω仅在蓝紫色的花冠驱动较强的GUS表达，但表达量低于p35S-PCHS-Ω。嵌合启动子pOCS-LCHS-Ω驱动GUS表达在蓝紫色的花冠和根。其中，pOCS-PCHS-Ω展现出增强的、花特异表达的模式，可以用于取代35S启动子驱动ACO RNAi载体，进行百合抗衰老遗传改良的研究。2.构建了由35S启动子和增强花特异启动子pOCS-PCHS-Ω驱动的ACO RNAi植物表达载体。从东方百合‘索邦’（Orential hybrid lily ‘Sorbonne’）花瓣的cDNA中克隆得到ACO正反义片段ACO-S和ACO-A，将正反义片段分别插入干扰载体pKANNIBAL的相应位置，构建了由35S启动子驱动的含有内含子发夹结构的ACO RNAi载体pK-ACO-S-A；经两步酶切连接最终得到由35S启动子驱动的ACO RNAi植物表达载体：pCAMBIA1301-35S-RNAi-ACO，并导入根癌农杆菌EHA105。为了降低或消除因组成型启动子35S驱动ACO RNAi载体在植物的所有组织和各个发育阶段表达可能所造成的负面影响，用增强的花特异启动子pOCS-PCHS-Ω替换35S启动子，构建了由增强的花特异启动子pOCS-PCHS-Ω驱动的ACO RNAi植物表达载体：pCAMBIA1301-OPCHS-RNAi-ACO。3.初步建立了百合体细胞胚再生体系。选用东方百合‘索邦’（Orential hybrid lily‘Sorbonne’）、‘伯尼尼’（Oriental hybrid lily ‘Bernini’）和OT百合‘罗宾那’（Oriental×Trumpet hybrid lily ‘Robina’）3个百合品种不同花器官（花丝、花梗、花柱、子房和花药）诱导胚性愈伤组织，结果表明花丝的胚性愈伤诱导率最高（‘索邦’22.79%，‘伯尼尼’16.51%，‘罗宾那’17.77%）；取五个不同时期的花丝，幼嫩时期花丝（stageⅠ）胚性愈伤诱导率最高，是stageⅤ的3-4倍；stageⅤ的花丝取上、中、基三部分，仅基部花丝底部切口处诱导出胚性愈伤。对于胚性愈伤的增殖，使用MS培养基添加不同浓度的PIC和蔗糖，筛选最佳增殖培养基，结果显示添加2mg L^-1PIC和60g L^-1蔗糖胚性愈伤的增殖率高，体胚早期萌发现象少。体胚再生植株时，MS培养基添加1g L^-1AC及30g L^-1的蔗糖，再生率最高。组织学及扫描电镜观察显示，百合体胚发生经历了四个典型的胚胎发育时期：球形胚、心形胚、鱼雷形胚和子叶期胚。4.将ACO RNAi载体（pCAMBIA1301-35S-RNAi-ACO）导入东方百合‘索邦’（Oriental hybrid lily ‘Sorbonne’）的基因组中。采用农杆菌介导法和基因枪法转化‘索邦’胚性愈伤，转化时，胚性愈伤的潮霉素抗性浓度确定为25mg L^-1。得到的潮霉素抗性植株经PCR检测、Southern杂交和GUS组织化学染色，表明ACO RNAi载体已导入百合的基因组中，获得了3个百合转基因株系，为进一步研究提供了材料基础。更多还原

【Abstract】 Lily （Lilium spp.）, is one of the five most famous cutting flowers in the world. It is also anethylene-sensitive plant species. Senescence of flowers is stimulated by ethylene in manyornamental plants. Down-regulation or knock-out of the key genes related to ethylenebiosynthesis （ACO gene） by RNA interference （RNAi） technology would delay the flowersenescence and extend the vase life of cut flower. ACO genes are regulated in a tissue-specificmanner during flower senescence. In the previous work, we isolated an enhancedflower-specific promoter to replace of35S promoter to drive ACO RNAi vector with aim tominimize negative effects, we therefore constructed four chimeric promoters （p35S-PCHS-Ω,p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω） fusion vectors and transformed withTorenia fournieri. In the current study, we examined the levels and tissue specificity of thesefour chimeric promoter fusion vectors in transgenic Torenia fournieri. Due to the relativelylow frequencies in stable transformations and many chimeras occurred in the regeneratedplants, we established an efficient regeneration system via somatic embryogenesis in lily.Finally, we introduced ACO RNAi vector into lily by Agrobacterium-mediated transformationand microprojectile bombardment. The main results are described as follows.1. Based on the preliminary studies, we further analysis the roles of four chimericpromoters （p35S-PCHS-Ω, p35S-LCHS-Ω, pOCS-PCHS-Ω and pOCS-LCHS-Ω）. Weexamined the levels and tissue specificity of GUS expression in transgenic Torenia fournieriusing histochemical staining, fluorometric assay and qRT-PCR. The results showed that:p35S-PCHS-Ω and p35S-LCHS-Ω displayed strongly constitutive GUS expression in alltissues, especially in colored corollas （p35S-PCHS-Ω） or in colored corollas and roots（p35S-LCHS-Ω）. pOCS-PCHS-Ω showed stronger GUS expression in colored corollas than inother tissues but expression was weaker than that of p35S-PCHS-Ω. pOCS-LCHS-Ω droveGUS in colored corollas but also in roots. Among the four chimeric promoters,pOCS-PCHS-Ω exhibited enhanced flower-specific expression and can replace35S promoterto drive ACO RNAi vector with minimal negative effects for transgenic enhancement of thevase life of lily.2. We constructed two ACO RNAi vector under the control of35S and the enhanced flower-specific promoter pOCS-PCHS-Ω, respectively. We cloned319bp ACO fragmentisolated from Orential hybrid lily ‘Sorbonne’ into specific sites （Xho I/EcoR I and Xha I/Cla I） of the interference vector pKANNIBAL, to generate pK-ACO-S-A RNAi vector, whichcontained a hairpin structure droved by35S promoter. Subsequently, pK-ACO-S-A subcoledinto the intermediate vector pAR-CAM. Eventually, the binary plant expression vectorpCAMBIA1301-35S-RNAi-ACO was constructed and introduced into Agrobacterium strainEHA105. To minimize potential adverse effects that can be present in transgenic plantstransformed with the35S promoter to control ACO RNAi vector expression, we used theenhanced flower-specific promoter pOCS-PCHS-Ω to replace35S promoter and constructedthe ACO RNAi plant expression vector pCAMBIA1301-OPCHS-RNAi-ACO.3. W established a regeneration system via somatic embryogenesis in lily. Filaments,pedicels, styles, ovaries and anthers of three cultivars: Orential hybrid lily ‘Sorbonne’,Oriental hybrid lily ‘Bernini’, and Oriental×Trumpet hybrid lily ‘Robina’ were used asexplants to identify and optimize the parameters influencing somatic embryogenesis. Theresults showed that the highest numbers of embryogenic cultures were induced from filaments（Sorbonne22.79%, Bernini16.51%, Robina17.77%）. The youngest developmental filaments（stagesⅠ） had the highest embryogenic response in each genotype, showing3–4-foldincreases compared with stageⅤ explants. Of the three regions of the filament used asexplants, only the basal ones produced embryogenic cultures. The highest embryogenic callusproliferation frequency was obtained using MS medium supplemented with2mg L^-1PIC and60.0g L^-1sucrose. For plant regeneration, these cultures produced the highest numbers ofplantlets when transferred to hormone-free MS medium with1g L^-1AC and30g L^-1sucrose.Histological and SEM observations of the various stages of somatic embryo developmentdemonstrated four typical stages: globular stages, heart-shaped stages, torpedo stages andcotyledonary stages.4. We introduced the ACO RNAi vector （pCAMBIA1301-35S-RNAi-ACO） into Orentialhybrid lily ‘Sorbonne’ genome by Agrobacterium-mediated transformation andmicroprojectile bombardment. When transformed, the critical concentration of hygromycinselection was25mg L^-1. Hygromycin-resistant lily plants were analyzed by PCR, Southernblot analysis and histochemical staining assay, which further proved that the ACO RNAivector （pCAMBIA1301-35S-RNAi-ACO） was introduced into Orential hybrid lily‘Sorbonne’ genome. We eventually obtained three transgenic lines of lily.更多还原