Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato ( Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products or fluorescent signals were obtained with all of them. No amplified products and fluorescent signals were observed when DNA samples from 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thalianawere used as templates. These results demonstrated that the amplified LAT52DNA sequence was specific for tomato. Furthermore, results of Southern blot showed that the LAT52gene was a single-copy gene in the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detection sensitivities were 0.05 and 0.005ng of tomato genomic DNA, respectively. In addition, two real-time assays employing this gene as an endogenous reference gene were established, one for the quantification of processed food samples derived from nontransgenic tomatoes that contained degraded target DNA and the other for the quantification of the junction region of CaMV35spromoter and the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No.1 samples. All of these results indicated that the LAT52gene could be successfully used as a tomato endogenous reference gene in practical qualitative and quantitative detection of transgenic tomatoes, even for some processed foods derived from transgenic and nontransgenic tomatoes.

With the development of transgenic crops, many countries have issued regulations to label the genetically modified organisms (GMOs) and their derived products. Polymerase Chain Reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods generally need to amplify the transgene and compare the amplified result with that of the corresponding reference gene to obtain reliable results. In this article, we reported the development of specific primers and probe for the rice (Oryza sativa) sucrose phosphate synthase (SPS) gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 13 different rice varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other species, such as wheat, maize, barley, tobacco, soybean, rapeseed, tomato, sunflower, carrot, pepper, eggplant, lupine, mung bean, plum, and Arabidopsis thaliana, were used as templates, which demonstrated that this system was specific for rice. In addition, the results of the Southern blot analysis confirmed that the SPS gene was a single copy in the tested rice varieties. In qualitative and quantitative PCR analyses, the detection sensitivities were 0.05 and 0.005ng of rice genomic DNA, respectively. To test the practical use of this SPSgene as an endogenous reference gene, we have also quantified the,-glucuronidase (GUS) gene in transgenic rice using this reference gene. These results indicated that the SPSgene was species specific, had one copy number, and had a low heterogeneity among the tested cultivars. Therefore, this gene could be used as an endogenous reference gene of rice and the optimized PCR systems could be used for practical qualitative and quantitative detection of transgenic rice.

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.

Genetically modified (GM) tomatoes have approved for commercialization in many countries since the first GM tomato FLAVER AVAR was permitted for planting in 1994. In 1994. In China, GM tomato Huafan No1 with a character of long shelf-life was the first GM plant which was approved forcommercialization in 1996. To meet the require ment of the GM tomatoes labeling policy that has been universal elements transformed into tomato, such as cauliflower mosaic virus 35S (CaMV35s) promoter and the nopaline synthase ●(NOS) terminator of Agrobacterium tumefaciens, and the specifically inserted heterologous DNA sequence between CaMV35s promoter and anti-sense ethylene-forming enzyme ●(EFE) gene were set up. To make the detection methods normative, a novel single copy tomato gene LAT52 was also used as an endogenous reference gene in the PCR detection systems. The limit of detection of screening and construct specific detection methods for Huafan No1 was 68 haploid genome copies in conventionl PCR detection, and three copies in TaqMan real-time PCR detection. The limit of quantitation of screening quantitative PCR assays for Huafan No1 was three copies and was 25 copies for construct-specific quantitative PCR. Two samples with know Huafan No1 was three copies and was 25 copies for construct-specific quantitative PCR. Two samples with known Huafan No1 tomato content were detected using the established conventional and real-time PCR systems, and these results also indicted that the established Huafan No1 screening and construct-specific PCR detection systems were reliable, sensitive and accurate.

Transgenic tobacco plants stably expressing recombinant FaeG, which is the major subunit and adhesin of K88ad fimbriae, were obtained. Analysis of sera from immunized mice indicates that in mice, the immunogenicity induced by plant-derived FaeG protein is comparable to that generated with traditional approaches.