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中国农学通报

中国农学通报 ›› 2014, Vol. 30 ›› Issue (27): 252-257.doi: 10.11924/j.issn.1000-6850.2014-0463

所属专题： 生物技术； 马铃薯

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利用改良RACE方法克隆木薯淀粉合成关键酶AGPase小亚基基因的研究

郭雅静,罗兴录,陈会鲜   

  1. 广西大学,广西大学,广西大学农学院
  • 收稿日期:2014-02-26 修回日期:2014-02-26 接受日期:2014-05-19 出版日期:2014-10-15 发布日期:2014-10-15
  • 通讯作者: 罗兴录
  • 基金资助:
    国家“973”计划课题“木薯淀粉高效积累途径及关键基因”(2010CB126601)；广西自然科学基金重点项目“木薯种质资源创新与高产高粉耐旱耐寒品种选育”(2010GXNSFD013025)

The Study on Cloning of Starch Synthesis Key Enzyme AGPase Small Subunit Gene of Cassava with Improved RACE Method

罗兴录 and   

  • Received:2014-02-26 Revised:2014-02-26 Accepted:2014-05-19 Online:2014-10-15 Published:2014-10-15

摘要： 首次从木薯中分离克隆出AGPase 小亚基的基因5’端cDNA序列，最终为进一步成功获得木薯淀粉合成关键酶AGPase小亚基基因的cDNA全长序列提供依据。本研究以木薯作为试验材料，设计锚定引物，并根据木薯AGPase 小亚基基因已知序列设计PCR特异引物，利用逆转录引物AUP1 反转录总RNA，然后分离纯化反转录产物，通过对三大酶促反应（逆转录反应，TdT 加尾，巢式及降落PCR技术）的具体改良，并将其与传统方法进行比较。利用改良后方法，首次成功获得木薯淀粉合成关键酶AGPase 小亚基基因5’端cDNA序列。同时，本研究通过对RACE技术的改良与传统方法相比，在操作上更加简单、快速；在序列获得上更加高效；在试验成本上更加低廉。本研究可以高效且快速的成功获得木薯淀粉关键酶AGPase小亚基基因5’端序列，具有一定的实用性及简便性。

关键词: 形状, 形状

Abstract: The research, for the first time, isolated cloned AGPase small subunits cDNA sequence of gene 5' end from cassava, and successfully obtained cassava AGPase small subunit gene key enzymes of starch synthesis in full length cDNA sequence. The study used cassava as test materials, set up anchor primers, and designed specific PCR primers according to the known sequences of cassava AGPase small subunit gene. Then, the study used reverse transcription primers AUP1 retroviruses total RNA, separated and purified the reverse transcription product, and based on the improvement of three enzymatic reaction (the reverse transcription reaction, the TdT and tail, nested and touchdown PCR technology), to carry out the comparison of the new method with the traditional one. Using the modified method, the cassava AGPase key enzymes of starch synthesis in the small subunit gene 5' end cDNA sequence was obtained successfully for the first time. Meanwhile, compared with the traditional method, the improved RACE technology was much simple and fast in operation，more efficient in obtaining sequence and cheaper in test cost. Therefore, this study can be efficient and quick to obtain cassava starch key enzyme AGPase small subunit gene 5' end sequences, and had certain practicability and simplicity.