【作者】 陆艳；

【导师】 徐刚标；

【作者基本信息】 中南林业科技大学 ， 林木遗传育种， 2006， 硕士

【摘要】 生长素影响植物生长发育的每个阶段。生长素早期或原初的反应基因的启动子通常包括最小的生长素反应元件TGTCTC序列。生长素反应因子(ARF)与这类生长素反应元件特异性地结合后调节着生长素反应基因的表达。已有报道说ARF基因的表达都不受生长素浓度的影响。本实验试图将ARF基因引入烟草植株，使转基因烟草成为该基因表达蛋白的生物反应器。 本研究以烟草无菌苗叶片为外植体，利用叶盘法，通过含生长素应答基因的根癌农杆菌LBA4404携带双元质粒载体(pBin438)介导进行转化，开展ARF基因转化烟草的研究。其主要结果如下： 1．烟草高频再生体系的建立 采用无菌苗烟草叶片为外植体，以MS为基本培养基，筛选出MS+0.8mg／L 6-BA+0.05mg／L NAA以诱导不定芽的分化，并于1／2MS+0.02mg／LIBA+0.02mg／L NAA的生根培养基上生根，获得完整再生植株。其叶片分化率达96.9％，生根率达96.7％。 2．转化体筛选及植株再生 将预培养3d的外植体与农杆菌菌液浸染3-5min后，共培养2-3d，然后转化到含Km75mg／L的选择培养基上进行分化筛选，再转入Km为75mg／L，100mg／L的培养基上进行生根筛选，生根率为66.1％。与对照相比转基因烟草在形态上发生了明显的改变：叶片颜色变深绿；叶片增厚：主叶脉变粗壮等。 3．转基因PCR检测 PCR扩增，获得40株阳性转基因苗，证明ARF基因已导入烟草中，转化率为32.8％。更多还原

【Abstract】 Auxin effects all the growth development periods of plants. The promoters of auxin early or primitive reaction gene generally include the minimal auxin reaction element sequence (TGTCTC). Auxin responsive factor (ARF) combines with this kind of auxin reaction elements uniquely and control the expression of auxin reaction gene. It has been reported that ARF gene expression was not effected by auxin concentration. We expect that the transgenic tobacco plant become a bioreactor and develop a new way to produce auxin protein.In this paper, tobacco aseptic seedling leaf were used as explants, and the gene was transferred into tobacco by Agrobacterium LBA4404 mediation which has been inserted with the plant expression vector pBin438 that contain ARF promoter and laid a foundation for scale production of ARF from transgenic plant. The main results are as follows:1. Regeneration system of plants.The regeneration system of tobacco was established. The adventitious shoots were induced from leaf explants of tobacco based on MS basal medium supplemented with 0.8 mg/L 6-BA and 0.05mg/L NAA. Then the regenerated plants were rooted on 1/2MS medium containing 0.02 mg/L NAA and 0.02 mg/L IBA. And the frequency was about 96.9%, 96.7% respectively.2. Transformation and selection of plants.Leaf explants had been pre-cultured for 2 days, then immersed in Agrobacterium suspension for 5-6minutes. The co-cultivation had been carried out for 2-3 days. After that, the transformants were obtained by transferring explants to selection medium containing 75mg/L kanamycin and rooting medium containing 75mg/L、 100mg/L kanamycin. And its rooted frequency was 66.1%.In comparison with CK, obvious morphological variation was observed in the transgenic tobaccos. Their leaves became greener and thicker, chief veins turned bolder and thicker than those of CK.3. Detection of transgenic plants.The PCR assay of plants showed that the target gene had been integrated更多还原