Analysis of the copy number of exogenous genes in transgenic cotton using real-time quantitative PCR and the 2-△△CT method

Abstract

Presence of multiple copies of a transgene has been found to associate with gene silencing that may manifest early or only over a period of time. As such, transgenic copy number should be analyzed as soon as possible. The commonly used method to analyze gene copy number, Southern blotting, has been found to be unreliable for determination of gene copy number when rearrangement or tandem repeats integration occurred. In recent years, a powerful real-time fluorescence quantitative real-time PCR (qRT-PCR) method has been used to analyze gene copy number in genetically modified plants, but these methods have also their own application conditions. Thus, based on real-time quantitative PCR technology, the modified 2-△△CT method, which has been used usually in analyzing gene expression, was used for the first time, to analyze transgenic copy numbers in transgenic cotton. In this paper, a single-copy homozygous transgenic plant whose identity was confirmed by Southern blotting using restriction enzymes was used as a calibrator. Genomic DNA that was extracted from the calibrator and six transgenic T0 cotton plants transformed by pollen tube pathway or gene gun bombardment was amplified by quantitative PCR, and the gene copy numbers were estimated by the modified 2-△△CT method to be 6, 3, 2, 2, 3 and 2 copies. In addition, except 6 copies all other identifications (3, 2, 2, 3 and 2) were equally indicated by the corresponding Southern blotting. This study supports the modified 2-△△CT method using quantitative real-time PCR technology to provide a fast, high-throughput method to analyze the copy number of foreign genes in a large transgenic T0 population. Key words: Cotton, transgene copy number, modified 2-△△CT method, Southern blotting.