植酸酶基因定性PCR检测方法及阳性质粒分子的构建

植酸酶基因在农业生产中具有很高的应用价值，正被广泛用于农作物的基因工程研究。为了满足转植酸酶基因作物安全监管的需要，通过优化PCR检测条件，建立了植酸酶基因特异性定性PCR检测方法。该方法具有良好的扩增特异性和检测灵敏度，可以满足我国植酸酶转基因作物监管需要。此外，我们将6大作物(小麦、水稻、棉花、大豆、玉米和油菜)的内标准基因和植酸酶基因克隆到同一载体上，构建成了阳性质粒分子pBS Endogenous-phytase。该质粒分子适用于这6大作物中植酸酶基因的筛查检测。本研究为转植酸酶基因作物的安全监管提供了阳性材料和检测方法。

Establishment of Phytase-Specific Qualitative PCR Detection Method and Construction of a Positive Plasmid Molecule

Phytase gene is valuably applicated in agricultural production, especially in genetic engineering of crops. In order to meet the requirements of safety regulation of transgenic crops, the gene-specific qualitative PCR detection method targeting the fungal-originated phytase gene was developed. A primer pair Phytase-F5/R5 yielding a 389 bp amplicon was selected from 11 primer pairs, then the PCR reaction system was optimized by improving Mg2+ concentration, primer concentration and primer anneal temperature. Twenty transgenic and nontransgenic lines from different crops were used as templates in PCR, showing that the PCR method had good amplification specificity. The results of sensitivity testing indicated that the phytase amplicon was still observed when the template concentration was down to 0.05%, which reaches the national standards for GMO (Genetically Modi-fied Organism) detection method. In addition, we cointegrated the phytase gene and the endogenous reference genes from six major crops of wheat, rice, cotton, soybeans, corn, and rapeseed into a vector, yielding a positive plasmid molecule pBS Endoge-nous-phytase. The positive plasmid molecule was suitable for screening phytase gene in the six crops about wheat, soybeans, corn, cotton, rice, and rape. This study provides positive materials and detection method for safety regulation of genetically modified crops carrying phytase gene.