遗传 ›› 2007, Vol. 29 ›› Issue (12): 1533-1537.doi: 10.1360/yc-007-1533

• 技术与方法 • 上一篇    

酶标探针在转基因植物检测中的应用

涂知明; 陈泠; 杨广笑; 何光源   

  1. 华中科技大学生命科学与技术学院中英联合实验室, 分子生物物理学教育部重点实验室, 武汉 430074

  • 收稿日期:2007-04-26 修回日期:2007-07-08 出版日期:2007-12-10 发布日期:2007-12-10
  • 通讯作者: 何光源

Application of enzyme-labeled probe in testing of transgenic plant

TU Zhi-Ming; CHEN Lin; YANG Guang-Xiao; HE Guang-Yuan

  

  1. Key Laboratory of Molecular Biophysics of the Ministry of Education, China-UK Joint Lab, College of life Science &
    Technology, Huazhong University of Science & Technology Wuhan
    , 430074, China
  • Received:2007-04-26 Revised:2007-07-08 Online:2007-12-10 Published:2007-12-10
  • Contact: HE Guang-yuan

摘要:

采用碱性磷酸酶标记DNA制备分子探针, 并首次在植物中应用。酶在苯醌作用下与单链DNA联结, 形成DNA和酶的共价复合物即酶标探针。此探针通过分子杂交与待测DNA结合, 再与酶的底物作用显色, 3~6h 内可观察结果。用此探针检测转基因植物中的UidA基因, 点杂交和Southern杂交结果表明, 所合成的酶标探针具有快速、准确、安全而经济的优点。点杂交证明外源UidA基因被成功转化到受体植物中, Southern 杂交对转基因的材料检测的结果证明, 该材料包含多个外源UidA基因拷贝, 初步确定其外源UidA基因拷贝数在5个以上。

关键词: 组织特异性启动子, UidA基因, 杂交, 酶标探针

Abstract:

Used alkaline-phosphatase-labeled DNA as a probe to examine the expression of foreign UidA gene in transgenic plants. Alkaline phosphatase coupled with polyethyleneimine (PEI) using P-benzoquine as the cross-linking reagent was covalently linked to single-stranded DNA via glutraldehyde. Such DNA-enzyme complexes were used as a probe for dot hybridization and Southern blot. After hybridization and incubation with a substrate solution, results can be observed directly in three to six hours and the results showed that it was a sensitive, specific, rapid, safe and economical probe. Dot hybridization analysis showed that the UidA gene was transformed into the target plants and southern blot showed that there were at least 5 copies of UidA gene in transgenic plants.