转植酸酶基因玉米phyA2基因标准质粒分子构建及其检测应用

质粒分子以其良好的适应性被较多地用作转基因标准材料的替代品。本文根据转植酸酶基因玉米phyA2基因设计引物与探针，并构建了一种包含phyA2基因片段(243 bp)和内标基因片段(178 bp)的质粒分子pPZ。选取不同转基因模板DNA作用于phyA2特异性引物，PCR结果显示只有转植酸酶基因玉米出现131 bp的目的条带；选取不同转基因作物检测体系作用于质粒分子pPZ，只有以pPZ DNA为模板时出现目的条带(131 bp和88 bp)。同时采用pPZ质粒分子和转基因玉米阳性基因组DNA为标准对4个转植酸酶基因玉米混合样品(3.0%、1.0%、0.5%和0.1%)进行定量检测，t检验表明，2种标准产生的斜率、线性相关系数和定值结果没有显著差异。这些结果表明，pPZ质粒分子可以作为转植酸酶基因玉米替代品用于定性和定量检测。

Construction and Application of a Reference Plasmid Molecule Suitable for phyA2 of Phytase Transgenic Maize

Plasmid molecule based reference material has been shown to be a good alternative as the calibrator for genetically modified organisms (GMOs) identification and quantification. In this study, primers and probe for qualitative and quantitative PCR detection for phytase transgenic maize were designed, and a flexible plasmid pPZ containing phyA2 (243 bp) and zSSIIb (178 bp) sequences, was developed. Primers and plasmid were specifically tested through qualitative PCRs. Results showed that the primers and pPZ plasmid were very specific for phytase transgenic maize since there were no other PCR products amplified using different GMOs and primers except the specific main bands. Four different levels of genetically modified (GM) matrix samples (3.0%, 1.0%, 0.5%, and 0.1%) were detected by real-time PCR when plasmid pPZ and phytase transgenic maize genomic DNA were used as quantification standards, respectively. t-test showed the slop, linearity (R²) and quantification of the two standards for phyA2 and zSSIIb were not significantly different. The results indicated that the pPZ plasmid can be used as standard for phytase transgenic maize detection.