作物学报2012，Vol.38Issue(4)：639-647,9.DOI:10.3724/SP.J.1006.2012.00639
植酸酶基因定性PCR检测方法及阳性质粒分子的构建
Establishment of Phytase-Specific Qualitative PCR Detection Method and Construction of a Positive Plasmid Molecule
摘要
Abstract
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 Modified 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.关键词
转基因检测/植酸酶基因/定性PCR/阳性质粒分子Key words
GMO detection/Phytase gene/Qualitative PCR/Positive plasmid molecule引用本文复制引用
李俊,刘信,曹应龙,武玉花,厉建萌,吴刚,张丽,卢长明..植酸酶基因定性PCR检测方法及阳性质粒分子的构建[J].作物学报,2012,38(4):639-647,9.基金项目
本研究由国家转基因生物新品种培育重大专项(2009ZX08012-009B)资助. （2009ZX08012-009B）
