Validation of a Tomato-Specific Gene, <i>LAT52</i>, Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Transgenic Tomatoes

Yang, Litao; Pan, Aihu; Jia, Junwei; Ding, Jiayu; Chen, Jian-Xiu; Huang, Cheng; Zhang, Chengmei; Zhang, Dabing

doi:10.1021/jf0493730

Cited by 70 publications

(54 citation statements)

References 19 publications

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“…Our lab have validated four novel endogenous reference genes suitable for GMOs qualitative and quantitative PCR detection, i.e., the sucrose phosphate synthase (SPS) gene of rice (Ding et al, 2004), high mobility group protein I/Y (HMG I/Y) gene for rapeseed (Weng et al, 2005), Lat52 gene of tomato (Yang et al, 2005b), and Stearoyl-Acyl Carrier Protein Desaturase (Sad1) gene for cotton (Yang et al, 2005a). Also our lab has successfully used the established SPS and HMG I/Y endogenous PCR systems to estimate the copy number of the introduced target gene in GM rice and rapeseeds (Weng et al, 2004;Yang et al, 2005c).…”

Section: Resultsmentioning

confidence: 99%

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

et al. 2005

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Based on the DNA sequences of the junctions between recombinant and cotton genomic DNA of the two genetically modified (GM) cotton varieties, herbicide-tolerance Mon1445 and insect-resistant Mon531, event-specific primers and probes for qualitative and quantitative PCR detection for both GM cotton varieties were designed, and corresponding detection methods were developed. In qualitative PCR detection, the simplex and multiplex PCR detection systems were established and employed to identify Mon1445 and Mon531 from other GM cottons and crops. The limits of detection (LODs) of the simplex PCR were 0.05% for both Mon1445 and Mon531 using 100 ng DNA templates in one reaction, and the LOD of multiplex PCR analysis was 0.1%. For further quantitative detection using TaqMan real-time PCR systems for Mon1445 and Mon531, one plasmid pMD-ECS, used as reference molecule was constructed, which contained the quantitative amplified fragments of Mon1445, Mon531, and cotton endogenous reference gene. The limits of quantification (LOQs) of Mon1445 and Mon531 event-specific PCR systems using plasmid pMD-ECS as reference molecule were 10 copies, and the quantification range was from 0.03 to 100% in 100 ng of the DNA template for one reaction. Thereafter, five mixed cotton samples containing 0, 0.5, 0.9, 3 and 5% Mon1445 or Mon531 were quantified using established real-time PCR systems to evaluate the accuracy and precision of the developed real-time PCR detection systems. The accuracy expressed as bias varied from 1.33 to 8.89% for tested Mon1445 cotton samples, and from 2.67 to 6.80% for Mon531. The precision expressed as relative standard deviations (RSD) were different from 1.13 to 30.00% for Mon1445 cotton, and from 1.27 to 24.68% for Mon531. The range of RSD was similar to other laboratory results (25%). Concluded from above results, we believed that the established event-specific qualitative and quantitative PCR systems for Mon1445 and Mon531 in this study are acceptable and suitable for GM cotton identification and quantification.

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Section: Resultsmentioning

confidence: 99%

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

et al. 2005

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“…the amount of tomato DNA in each sample was quantified as reported by Le Floch et al (25) and by using specific primers and a probe for the tomato Lat52 gene (65). A MiniOpticon thermocycler (Bio-Rad) was used for PCR; the reaction volume was 15 l and consisted of 7.5 l of 2ϫ Quantitect probe mix (Qiagen) and a 0.4 M concentration of each primer and probe.…”

Section: Vol 75 2009 Influence Of P Oligandrum On Rhizosphere Popumentioning

confidence: 99%

Influence of Pythium oligandrum Biocontrol on Fungal and Oomycete Population Dynamics in the Rhizosphere

Vallance

Floch

Déniel

et al. 2009

Appl Environ Microbiol

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Fungal and oomycete populations and their dynamics were investigated following the introduction of the biocontrol agent Pythium oligandrum into the rhizosphere of tomato plants grown in soilless culture. Three strains of P. oligandrum were selected on the basis of their ability to form oospores (resting structures) and to produce tryptamine (an auxin-like compound) and oligandrin (a glycoprotein elicitor). Real-time PCR and plate counting demonstrated the persistence of large amounts of the antagonistic oomycete in the rhizosphere throughout the cropping season (April to September). Inter-simple-sequence-repeat analysis of the P. oligandrum strains collected from root samples at the end of the cropping season showed that among the three strains used for inoculation, the one producing the smallest amount of oospores was detected at 90%. Single-strand conformational polymorphism analysis revealed increases in the number of members and the complexity of the fungal community over time. There were no significant differences between the microbial ecosystems inoculated with P. oligandrum and those that were not treated, except for a reduction of Pythium dissotocum (ubiquitous tomato root minor pathogen) populations in inoculated systems during the last 3 months of culture. These findings raise interesting issues concerning the use of P. oligandrum strains producing elicitor and auxin molecules for plant protection and the development of biocontrol.

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“…Studies on the expression of housekeeping genes have mainly been performed on human tissues (Thellin et al 1999;Schmittgen and Zakrajsek 2000;Warrington et al 2000;Radonic et al 2004), bacteria, and viruses (Stöcher et al 2002(Stöcher et al , 2003Savli et al 2003). To the best of our knowledge, few studies have been performed on plants (Frost and Nilsen 2003;Ronning et al 2006;Yang et al 2005); some plants used in such studies are barley (Burton et al 2004), rice (Kim et al 2003), and Arabidopsis thaliana (Orsel et al 2002). In plants, the common reference genes used are Actin, GAPDH, ribosomal genes, Cyp, and EF-1-α (Bézier et al 2002;Dean et al 2002;Thomas et al 2003).…”

Section: Introductionmentioning

confidence: 99%

Reference Gene Selection for Real-Time Quantitative Polymerase Chain Reaction of mRNA Transcript Levels in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

et al. 2010

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The Chinese cabbage is a crop belonging to the family Cruciferae; very few studies have focused on the analysis of gene expression in this economically important crop. In this study, we used real-time quantitative polymerase chain reaction to identify the control genes that are the most stably expressed in a given set of tissues and under conditions of drought stress and downy mildew infection. We characterized the transcript stability of nine candidate reference genes, namely, those encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), ubiquitin (Ubc), elongation-factor-1-α (EF-1-α), adenine phosphoribosyltransferase (Apr), clathrin, cyclophilin (Cyp), tubulin (Tub), Actin, and 18s rRNA, using the geNorm and Normfinder software programs. The results of a geNorm analysis indicated that EF-1-α and Apr were the most suitable reference genes among the given set of tissues and that GAPDH and Ubc were the most stable genes under conditions of drought stress and downy mildew infection. Furthermore, the results of an analysis performed using the Normfinder software program indicated EF-1-α to be the best normalization factor in the given set of tissues and under conditions of downy mildew infection and GAPDH to be the best factor under drought stress conditions. The results of the geNorm analysis also helped determine the minimum number of genes required to calculate a reliable normalization factor. A study on the variability of expression of PSY expression showed that the relative quantification of this gene varied depending on the internal control and the number of internal controls used, thus highlighting the importance of the choice of internal controls in such experiments.

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Validation of a Tomato-Specific Gene, LAT52, Used as an Endogenous Reference Gene in Qualitative and Real-Time Quantitative PCR Detection of Transgenic Tomatoes

Cited by 70 publications

References 19 publications

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

Qualitative and Quantitative PCR Methods for Event-specific Detection of Genetically Modified Cotton Mon1445 and Mon531

Influence of Pythium oligandrum Biocontrol on Fungal and Oomycete Population Dynamics in the Rhizosphere

Reference Gene Selection for Real-Time Quantitative Polymerase Chain Reaction of mRNA Transcript Levels in Chinese Cabbage (Brassica rapa L. ssp. pekinensis)

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