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中国农学通报

中国农学通报 ›› 2011, Vol. 27 ›› Issue (7): 50-54.

所属专题： 生物技术； 小麦

• 农学 农业基础科学 • 上一篇    下一篇

小麦SSⅡ-A基因片段的克隆及其RNAi表达载体的构建

张付芸 陈伟霞 刘子渲 李保云 梁荣奇   

  • 收稿日期:2010-09-15 修回日期:2010-10-13 出版日期:2011-04-05 发布日期:2011-04-05
  • 基金资助:

    转基因生物新品种培育重大专项“改良淀粉和蛋白品质以及面粉色泽转基因小麦新种质培育”

Cloning of SSⅡ-A Partial Sequence and its Construction of RNAi Expression Vector

  • Received:2010-09-15 Revised:2010-10-13 Online:2011-04-05 Published:2011-04-05

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摘要：

可溶性淀粉合成酶Ⅱ（SSⅡ）是小麦籽粒支链淀粉合成的主要角色，但是其三个部分同源基因（SSⅡ-A、SSⅡ-B和SSⅡ-D）对支链淀粉合成的贡献大小目前还未见报道，探究它们对支链淀粉合成的作用，对小麦淀粉合成的遗传操纵和淀粉品质改良具有重要意义。此文通过RT-PCR方法克隆了小麦SSⅡ-A基因cDNA的部分序列（长度为539 bp），经序列比对发现，它与AB201445.1（Triticum aestivum wSSII-A gene for starch synthase II-A, complete cds）的同源性为100%。以H质粒为中间载体，构建了SSⅡ-A基因片段的发夹结构，并将之连接到pBAC47P上高分子量谷蛋白亚基（HMW-GS）1Dx5启动子的下游，得到了高效特异的SSⅡ-A RNAi表达载体。这些工作为下一步遗传转化获得转基因植株以深入探究SSⅡ-A基因对支链淀粉合成的贡献大小奠定了基础。

关键词: 烟草, 烟草, 钾离子通道, 转基因, 抗逆性

Abstract:

Soluble starch synthaseⅡ (SSⅡ) is the main character of the enzymes that catalyse amylopectin synthesis, the exploration of its three homoeologous genes’ (SSⅡ-A, SSⅡ-B and SSⅡ-D) contribution to amylopectin synthesis is very important to the genetic manipulation of starch synthesis and the improvement of starch quality in wheat. Here we cloned partial sequence (539 bp) of wheat SSⅡ-A cDNA using RT-PCR technology, which completely aligns to AB201445.1 (Triticum aestivum wSSII-A gene for starch synthase II-A, complete cds). The hairpin structure of the sequence was constructed based on H plasmid, and then it was ligated to the downstream of HMW-GS 1Dx5 promoter in pBAC47P. Therefore, the specific and effective RNAi expression vector was formed, which would lay a foundation to further explore SSⅡ-A’s contribution to amylopectin synthesis.