农杆菌介导miR396基因对大麦愈伤组织的遗传转化

发布时间：2018-11-28 20:30

【摘要】：miRNAs是植物体内一类长度在21个核苷酸左右、非编码具有调控功能的小RNA分子,它们通过参与基因的转录后调控,在植物生长发育和抗病抗逆方面发挥着至关重要的作用。前人研究表明,miR396广泛参与到植物的细胞分裂、叶片性状形成、花器官变异和耐旱性等过程。为有效利用该优良基因资源,本研究使用农杆菌介导的遗传转化技术,将来源于水稻的miR396基因导入大麦中过量表达,以期获得性状变异的优良转基因植株,尤其是在耐旱性方面表现突出的材料。文中选取了6个大麦品种的幼胚愈伤组织和3个品种的成熟胚愈伤组织进行遗传转化,以抗潮霉素hptⅡ基因为筛选标记,成功获得了部分含有目的基因Os-miR396的不同大麦品种的转基因阳性株,同时对大麦转基因过程的影响因素进行了分析和优化,主要结果如下:(1)基因型的差异依然是影响幼胚和成熟胚愈伤诱导率的主要因素;(2)当成熟胚采用只接种盾片诱导时,不需要添加ABA便可有效减少发芽率,添加ABA反而会抑制愈伤生长发育;(3)相同潮霉素浓度对不同品种幼胚愈伤的分化筛选效果存在差异,25mg/L的潮霉素剂量对美里黄金、华大麦4号和华大麦7号而言筛选压可能过高,对愈伤的生长及增殖可能有着不良影响;(4)不同浓度的Timentin抑菌效果不同,当侵染浓度高时,需要提高Timentin的浓度才能很好的抑制农杆菌的污染;(5)农杆菌介导法对幼胚愈伤转化实验中,得到17株抗性大麦植株,经PCR检测:miR396基因阳性株为5株,分别为干木二条1株、苏啤1号2株和浙农大三号2株。(6)农杆菌介导法对成熟胚愈伤转化实验中,得到13株再生大麦植株,PCR检测表明:miR396基因阳性株为4株,均来自于大麦品种干木二条。
[Abstract]:MiRNAs is a kind of small RNA molecules which are about 21 nucleotides in length and are not encoded in plants. They play an important role in plant growth and development and resistance to disease and stress by participating in the post-transcriptional regulation of genes. Previous studies have shown that miR396 is widely involved in plant cell division, leaf character formation, floral organ variation and drought tolerance. In order to effectively utilize this excellent gene resource, the miR396 gene derived from rice was introduced into barley by Agrobacterium tumefaciens-mediated genetic transformation in order to obtain excellent transgenic plants with variant characters. Especially in the field of drought resistance to outstanding materials. In this paper, the immature embryogenic callus of 6 barley varieties and the mature embryo callus of 3 varieties were selected for genetic transformation. The hygromycin resistant hpt 鈪，

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