Auxin independent1 (Axi1) as an endogenous gene for copy number determination in transgenic tobacco
Simara da Silva Lopes, Jéssica Fabiane da Veiga Costa, Beatriz de Almeida Barros, Andrea Almeida Carneiro and Sylvia Morais de Sousa
Abstract: Determining the copy number is important because it can greatly influence the expression level and genetic stability of transgenes. This study aimed to establish a methodology that can estimate the transgene copy number in tobacco (Nicotiana tabacum) using the Auxin independent1 (Axi1) gene as an endogenous control. Tobacco c.v. Petit Havana plants were transformed via Agrobacterium tumefaciens with a pMCG1005 vector that contained Bar as a selective gene. Oligonucleotide efficiency was determined and a qPCR using the 2-ΔCt and 2-ΔΔCt methods was performed. Bar was the target gene and Axi1 was the endogenous control in five transgenic tobacco events. The results showed that the Axi1 gene was donated by the maternal parent N. sylvestris when interspecific hybridization occurred between N. sylvestris and N. tomentosiformis. The copy number results agreed with the segregation ratios for the Bar gene in T1 plants, which confirmed that Axi1 is a single copy gene that can be used as an endogenous control.