Abstract
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.
Highlights
Genetic engineering incorporates new genes into transgenic plants from completely unrelated species, which can be transmitted to their descendants (Low et al 2018, Nazir et al 2019)
The results from this study showed that Auxin independent1 (Axi1) is a single copy gene and that it came from the maternal donor N. sylvestris
Tobacco is a model plant that is often used in plant-microorganism interaction and gene function studies (Clarkson et al 2017, Chen et al 2018, Bai et al 2019)
Summary
Genetic engineering incorporates new genes into transgenic plants from completely unrelated species, which can be transmitted to their descendants (Low et al 2018, Nazir et al 2019). In 1983, the first transgenic tobacco (Nicotiana tabacum) plants containing a gene coding for resistance against the antibiotic kanamycin were generated (Fraley et al 1983). Several proteins have been expressed in transgenic tobacco and other plants (Jube and Borthakur 2007, Burnett and Burnett 2019, Moon et al 2020). It is considered to be a model plant for scientific research because of its well-established regeneration and transformation methodologies, and the existence of a sequenced genome (Edwards et al 2017). The genetic transformation of model plants is key to validating gene function. Molecular characterization is necessary after transformation because copy number and the insertion site can influence gene stability and the expression levels of the ORCID: 0000-0002-8030-1385
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