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作物学报 ›› 2012, Vol. 38 ›› Issue (04): 639-647.doi: 10.3724/SP.J.1006.2012.00639

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

植酸酶基因定性PCR检测方法及阳性质粒分子的构建

李俊1,2，刘信3，曹应龙2，武玉花2，厉建萌3，吴刚2，张丽2，卢长明2,*   

  1. 1 中南民族大学生命科学学院, 湖北武汉430074; 2中国农业科学院油料作物研究所, 湖北武汉430062; 3 农业部科技发展中心, 北京100026
  • 收稿日期:2011-09-14 修回日期:2011-12-19 出版日期:2012-04-12 网络出版日期:2012-02-13
  • 通讯作者: 卢长明, E-mail: cmlu@oilcrops.cn, Tel: 027-86728186
  • 基金资助:

    本研究由国家转基因生物新品种培育重大专项(2009ZX08012-009B)资助。

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 Published:2012-04-12 Published online:2012-02-13
  • Contact: 卢长明, E-mail: cmlu@oilcrops.cn, Tel: 027-86728186

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1019

被引次数

8

摘要： 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。

关键词: 转基因检测, 植酸酶基因定性PCR, 阳性质粒分子

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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