酶标探针在转基因植物检测中应用.pdfVIP

HEREDITAS (Beijing) 2007 12 , 29(12): 1533―1537 ISSN 0253-9772 技术与方法 DOI: 10.1360/yc-007-1533 涂知明, 陈泠, 杨广笑, 何光源 , , 430074 : 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。 : 组织特异性启动子；UidA 基因；杂交；酶标探针 Application of enzyme-labeled probe in testing of transgenic plant TU Zhi-Ming, CHEN Lin, YANG Guang-Xiao, HE Guang-Yuan 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 Abstract: Used alkaline-phosphatase-labeled DNA as a probe to examine the expression of foreign UidA gene in trans- genic 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. Keywords: tissue-specific promoter; UidA gene; hybridization; enzyme-labeled probe tritordeum , ,