1. Validation of a carnation-specific gene, ANS, used as an endogenous reference gene in qualitative and real-time quantitative PCR for carnations
- Author
-
Xiao Wu, Aihu Pan, Jia Junwei, Lingxi Jiang, Heyan Lin, Kai Zhao, Hong Zhu, Furong Tan, Jinbin Wang, Shiru Tao, Xueming Tang, and Peng Li
- Subjects
DNA, Plant ,Carnation ,Biology ,Genes, Plant ,Polymerase Chain Reaction ,Sensitivity and Specificity ,Analytical Chemistry ,Anthocyanins ,chemistry.chemical_compound ,Species Specificity ,Dianthus ,Gene Expression Regulation, Plant ,Environmental Chemistry ,Gene ,Southern blot ,Pharmacology ,Genetics ,Regulation of gene expression ,Reproducibility of Results ,biology.organism_classification ,Molecular biology ,Genetically modified organism ,Real-time polymerase chain reaction ,chemistry ,Low copy number ,Agronomy and Crop Science ,DNA ,Food Science - Abstract
The validation of the anthocyanin synthase (ANS) gene as a carnation endogenous reference gene applicable both in classical and real-time PCR methods is a prerequisite for the development of PCR assays for genetically modifed (GM) carnation detection. This is important due to the fact that GM carnation lines, developed by Florigene Pty Ltd, have been approved for commercialization. In this study, both methods were tested on 14 different carnation cultivars, and identical amplifcation products were obtained with all of them. No amplifcation products were observed with samples from 14 other plant species, which demonstrated that the system was specifc to carnation. The results of Southern blot analysis confrmed that the ANS gene had a low copy number in the 10 tested carnation varieties. In qualitative and real-time PCR assays, the LOD values of 0.05 and 0.005 ng carnation DNA, respectively, were validated. Moreover, the real-time PCR system was validated with high PCR effciency and linearity. Thus, the ANS gene had species specifcity, low heterogeneity, and low copy number among the tested cultivars. These results provide evidence that the gene can be used as an endogenous reference gene of carnation, as well as in qualitative and quantitative PCR systems.
- Published
- 2011