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中国农学通报

中国农学通报 ›› 2010, Vol. 26 ›› Issue (24): 133-136.

所属专题： 生物技术； 园艺

• 林学 园艺 园林 • 上一篇    下一篇

油茶SAD基因原核表达载体构建

彭邵锋 陈永忠 陈隆升 陆 佳   

  • 收稿日期:2010-10-26 修回日期:2010-11-17 出版日期:2010-12-20 发布日期:2010-12-20
  • 基金资助:

    华中丘陵区油茶高产良种应用集成示范;油茶高产优质培育技术研究;湖南省高产优质油茶新品种选育;油茶“两系”杂交种子园营建及其高产培育技术研究;油茶高产优质新种质创制

Construction of Prokaryotic Expression Vector of SAD Gene of Camellia Oleifera

  • Received:2010-10-26 Revised:2010-11-17 Online:2010-12-20 Published:2010-12-20

PDF (PC)

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摘要：

为研究油茶种子SAD基因的预期功能，以报道的油茶SAD的CDS为模板设计引物，RT-PCR及TA克隆获得含有酶切位点的CDS片段。经过PCR及测序验证序列的正确性后，将PCR扩增产物双酶切后与同样经双酶切的原核表达载体pET30a连接，转化BL21（DE3）菌株。菌液PCR所得条带与预期一致，对于重组质粒的双酶切后两类产物电泳的条带分布符合经验分布规律。多种鉴定结果表明已成功构建适宜于原核表达用的重组载体。

关键词: 稳定性, 稳定性

Abstract:

To investigate the expected functions of the SAD from Camellia oleifera seeds, a pair of primer integrated with two restriction sites was designed according to the reported coding sequence (CDS) of the SAD, thereafter the segment of CDS, which contained double digestion sites, was obtained by RT-PCR and TA cloning method. After confirmation of the CDS by PCR and sequencing respectively, the PCR products of the CDS was double digested and ligated to pET30a which was aslo double digested using the same restriction enzymes, followed by transformation of recombinant pET-CoSAD into BL21 (DE3). The length of colony PCR products was the same as the expected, and the distribution of double digested products via electrophoresis was also in accordance with experiential distribution. In summary, a recombinant pET-CoSAD which could be used to analyze the function of CoSAD in pET system had been constructed successfully.