【摘 要】
：
植物核雄性不育现象广泛存在于自然界中。由于用其育成的核不育两用系,既可作为不育系又是保持系,因而具有重要的育种应用价值和理论研究价值。西瓜是重要的园艺作物,在世界
【出 处】
：
西北农林科技大学 西北农林科技大学
论文部分内容阅读
植物核雄性不育现象广泛存在于自然界中。由于用其育成的核不育两用系,既可作为不育系又是保持系,因而具有重要的育种应用价值和理论研究价值。西瓜是重要的园艺作物,在世界十大果品中居第五位。我国年西瓜种植面积约为147万hm2,为世界上第一西瓜生产和消费大国,其产量和消费量占世界总产量和消费量的55%以上。杂种优势利用在西瓜生产上发挥了巨大作用,提高杂交制种效率和杂交种种子纯度,降低杂交种种子生产成本,是杂种优势利用的重要课题。雄性不育系的发现、研究和利用是解决上述问题的有效途径,细胞核雄性不育是西瓜作物中不育材料的主要形式,也是西瓜杂种优势利用的重要途径之一,而核不育源的发掘及其研究是这一领域的重要基础工作。Se18是西北农林科技大学园艺学院西瓜课题组发现的一种西瓜不育源,属一对隐性基因控制的核雄性不育,与G17AB由同一等位基因控制。目前已对其遗传特性、不育性细胞生物基础、生理生化和不育基因RAPD标记等方面进行了系统研究。本文以Se18西瓜核雄性不育两用系花蕾为试才,应用mRNA差异显示技术(mRNA differential display PCR, DDRT-PCR)寻找和筛选与西瓜核雄性不育相关的基因,主要研究结果如下:(1)通过系统的梯度实验,建立了西瓜核雄性不育相关基因DDRT-PCR体系。筛选了DDRT-PCR体系中各种反应因素的浓度,优化后25μL体系中各组分浓度为:cDNA第一链反应产物2μL(RT反应体系中总RNA 2μg,锚定引物0.5μg),2.5mmol/L dNTP 0.75μL,25mmol/L MgCl2 2μL,锚定引物T12M 100ng,差异显示随机引物50ng,Taq酶1.25U,10×Taq Buffer 2.5μL, ddH2O补齐。扩增程序:94℃3min;94℃25s,42℃30s,72℃1min,40个循环,72℃延伸5min,4℃保存。该反应体系的产物经测序胶电泳和银染,可以获得数量适宜、带型清晰、重复性好的cDNA片段。(2)应用mRNA差异显示研究Se18西瓜核雄性不育材料不育株和可育株雄花花蕾中基因表达的差异,获得了西瓜细胞核雄性不育系不育相关特异cDNA片段6个:T12C/B0315S-359、T12A/B0315S-382、T12A/B0318S-506、T12C/B0305S-262、T12C/B0306S-153和T12C/B0309S-439,分别由359bp、382bp、506bp、262bp、153bp和439bp的碱基序列组成。获得了西瓜细胞核雄性不育系育性相关特异cDNA片段4个:T12C/B0315F-175、T12G/B0318F-260、T12A/B0315F-355和T12A/B0315F-253,分别由175bp
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