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Acta Agron Sin ›› 2012, Vol. 38 ›› Issue (04): 639-647.doi: 10.3724/SP.J.1006.2012.00639

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

LI Jun1,2,LIU Xin3,CAO Ying-Long2,WU Yu-Hua2,LI Jian-Meng3,WU Gang2,ZHANG Li2,LU Chang-Ming2,*   

  1. 1 School of Life Science, South-Central University for Nationalities, Wuhan 430074, China; 2 Oil Crops Research Institute, Chinese Academy of Agriculture Sciences, Wuhan 430062 China; 3 Development Center of Science and Technology, Ministry of Agriculture, Beijing 100026 China
  • Received:2011-09-14 Revised:2011-12-19 Online:2012-04-12 Published:2012-02-13
  • Contact: 卢长明, E-mail: cmlu@oilcrops.cn, Tel: 027-86728186

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. And 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 Endogenous-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.

Key words: GMO detection, Phytase gene, Qualitative PCR, Positive plasmid molecule

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