Validation of a Tomato-Specific Gene, LAT52, Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Transgenic Tomatoes
详细信息 查看全文
- 作者：Litao Yang ; Aihu Pan ; Junwei Jia ; Jiayu Ding ; Jianxiu Chen ; Huang Cheng ; Chengmei Zhang ; and Dabing Zhang
- 刊名：Journal of Agricultural and Food Chemistry
- 出版年：2005
- 出版时间：January 26, 2005
- 年：2005
- 卷：53
- 期：2
- 页码：183 - 190
- 全文大小：295K
- 年卷期：v.53,no.2(January 26, 2005)
- ISSN：1520-5118
文摘
Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR)detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specificgene, LAT52, was selected and validated as suitable for using as an endogenous reference gene intransgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with16 different tomato varieties, and identical amplified products or fluorescent signals were obtainedwith all of them. No amplified products and fluorescent signals were observed when DNA samplesfrom 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thaliana were usedas templates. These results demonstrated that the amplified LAT52 DNA sequence was specific fortomato. Furthermore, results of Southern blot showed that the LAT52 gene was a single-copy genein the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detectionsensitivities were 0.05 and 0.005 ng of tomato genomic DNA, respectively. In addition, two real-timeassays employing this gene as an endogenous reference gene were established, one for thequantification of processed food samples derived from nontransgenic tomatoes that containeddegraded target DNA and the other for the quantification of the junction region of CaMV35s promoterand the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No. 1 samples.All of these results indicated that the LAT52 gene could be successfully used as a tomato endogenousreference gene in practical qualitative and quantitative detection of transgenic tomatoes, even forsome processed foods derived from transgenic and nontransgenic tomatoes.Keywords: Lycopersicon esculentum; LAT52; endogenous reference gene; GMO detection; qualitativeand quantitative PCR