草地学报

草地学报 ›› 2016, Vol. 24 ›› Issue (5): 1146-1149.DOI: 10.11733/j.issn.1007-0435.2016.05.034

• 研究专报 • 上一篇    下一篇

山黧豆CASase基因的克隆及RNAi载体的构建

张明科1, 刘凤娟2, 陶英杰2, 胡鑫2, 李荣硕1, 徐全乐2   

  1. 1. 西北农林科技大学医院, 陕西 杨凌 712100;
    2. 西北农林科技大学生命科学学院, 陕西 杨凌 712100
  • 收稿日期:2015-02-02 修回日期:2015-10-15 出版日期:2016-10-15 发布日期:2017-01-13
  • 通讯作者: 徐全乐
  • 作者简介:a同等贡献作者,张明科(1971-),男,陕西扶风人,硕士,副主任医师,主要从事临床病理研究,E-mail:zmkyl@163.com;刘凤娟(1990-),河南商丘人,硕士研究生,研究方向为生物化学与分子生物学,E-mail:liufengjuan2015@163.com
  • 基金资助:

    中央高校基本科研业务费（2014YB040，2452015401）；国家自然科学基金（31401910）；中国博士后科学基金（2016M590975）；陕西省博士后科研项目（2016BSHEDZZ119）资助

Cloning of CASase Gene from Lathyrus sativus and Construction of its RNAi Vector

ZHANG Ming-ke1, LIU Feng-juan2, TAO Ying-jie2, HU Xin2, LI Rong-shuo1, XU Quan-le2   

  1. 1. College Hospital, Northwest A & F University, Yangling, Shaanxi Province 712100, China;
    2. College of Life Sciences, Northwest A & F University, Yangling, Shaanxi Province 712100, China
  • Received:2015-02-02 Revised:2015-10-15 Online:2016-10-15 Published:2017-01-13

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摘要：

β-腈基丙氨酸合成酶（CASase）是调控山黧豆（Lathyrus sativus）内源毒素β-ODAP合成的关键酶。本研究以山黧豆幼根RNA为模板，利用RT-PCR扩增了505 bp的山黧豆CASase基因序列；通过Gateway BP反应将扩增片段连接到入门载体构建pENTR-CASase。经测序验证后，将目的片段插入到植物表达载体构建pSGRNAi-CASase，并将其转化到农杆菌GV3101中；为进一步的遗传转化奠定了基础。

关键词: 山黧豆, &beta, -腈基丙氨酸合成酶基因, Gateway, RNAi载体构建

Abstract:

CASase is thought to be the first key enzyme in β-ODAP biosynthetic pathways of Lathyrus sativus. To investigate the function of CASase gene and breed novel lines with low β-ODAP and high sulfur-containing amino acids, a 505 bp fragment of CASase gene was cloned from radicle RNA of L. sativus through RT-PCR. And then, an entry clone vector pENTR-CASase was constructed through Gateway BP cloning. An RNAi transformation vector pSGRNAi-CASase was constructed. Finally, the RNAi vector was transformed into Agrobacterium tumefaciens strain GV3101, which laid a foundation for obtaining transgenic plants with CASase gene silenced.

Key words: L. sativus, CASase gene, Gateway, RNAi

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