Validation of a cotton-specific gene, Sad1, used as an endogenous reference gene in qualitative and real-time quantitative PCR detection of transgenic cottons

文献类型： 外文期刊

第一作者： Yang, LT

作者： Yang, LT;Chen, JX;Huang, C;Liu, YH;Jia, SR;Pan, LW;Zhang, DB

作者机构：

关键词： Gossypium hirsutum;stearoyl-acyl carrier protein desaturase gene;endogenous reference gene;genetically modified organism;conventional and real-time PCR

期刊名称：PLANT CELL REPORTS （ 影响因子：4.57； 五年影响因子：4.463 ）

ISSN： 0721-7714

年卷期： 2005 年 24 卷 4 期

页码：

收录情况： SCI

摘要： Genetically modified (GM) cotton lines have been approved for commercialization and widely cultivated in many countries, especially in China. As a step towards the development of reliable qualitative and quantitative PCR methods for detecting GM cottons, we report here the validation of the cotton (Gossypium hirsutum) endogenous reference control gene, Sad1, using conventional and real-time (RT)-PCR methods. Both methods were tested on 15 different G. hirsutum cultivars, and identical amplicons were obtained with all of them. No amplicons were observed when DNA samples from three species of genus Gossypium, Arabidopsis thaliana, maize, and soybean and others were used as amplified templates, demonstrating that these two systems are specific for the identification and quantification of G. hirsutum. The results of Southern blot analysis also showed that the Sad1 gene was two copies in these 15 different G. hirsutum cultivars. Furthermore, one multiplex RT-quantitative PCR employing this gene as an endogenous reference gene was designed to quantify the Cry1A(c) gene modified from Bacillus thuringiensis (Bt) in the insect-resistant cottons, such as Mon531 and GK19. The quantification detection limit of the Cry1A(c) and Sad1 genes was as low as 10 pg of genomic DNA. These results indicat that the Sad1 gene can be used as an endogenous reference gene for both qualitative and quantitative PCR detection of GM cottons.

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