Efficient seed-specifically regulated autoexcision of marker gene (nptII) with inducible expression of interest gene in transgenic Nicotiana tabacum

Abstract

In this study, we developed the seed-specifically regulated autoexcision system based on Cre/loxP site-specific recombination for coelimination of nptII and cre genes from transgenic plants. To accomplish this, a seed-specific gene promoter, BcNA1, from Brassica napus was used to drive conditional expression of recombinase. NptII and recombinase cassettes were placed between two directly repeated loxP sites. The loxP-flanked DNA was located between the SP-DDEE synthetic pathogen inducible promoter and promoterless β-glucuronidase (Gus) gene, as a model gene of interest. Upon seed-specific expression of cre recombinase, the excision event would eliminate loxP-flanked DNA and bring the gus reporter gene under the control of the inducible promoter. This Cre/loxP system was transformed into Nicotiana tabacum via Agrobacterium-mediated transformation. The regenerated plants were obtained by selection on kanamycin medium and PCR screening. Based on seed germination assay on kanamycin-containing medium, the transgenic lines were subdivided into homogeneous and heterogeneous categories. Phenotypic and molecular analysis of T1 progeny plants indicated that completely NptII-free plants were obtained in homogeneous transgenic lines. Coelimination of NptII and recombinase cassettes were verified with PCR, sequencing, and histochemical Gus staining assay. Full excision efficiency (100%) demonstrates the effectiveness of this autoexcision system for the production of marker gene-free transgenic plants.

Engineered Expression of Vip3A in Green Tissues as a Feasible Approach for the Control of Insect Pests in Maize

“…Boszorádová et al combined the Cre/loxP and the embryo-specific CRUC promoter to remove hazardous transgenes from the genome in commercial oilseed rape [28]. For this comprehensive strategy, diverse promoters are crucial regulatory elements and have an efficient impact on the expression of transgenes in defined tissues [36][37][38]. In the current study, the green-tissue-specific promoter Zm1rbcS with a moderate drive capability was chosen to induce the expression of the simple gene Cre in the KEY plants.…”

Transgene Bioconfinement: Don’t Flow There

“…The Cre-loxP recombinase system has been studied in Arabidopsis (Arabidopsis thaliana L.) [67] and tobacco [67][68][69][70] studies, where pollen was targeted for transgene excision to prevent pollenmediated gene flow [67,70]. In Arabidopsis and tobacco greenhouse experiments, the microsporespecific promoter NTM19 (derived from tobacco) was used to drive Cre expression and produce transgene-free pollen [67].…”

“…Additionally, all transgenic lines were normal in regard to vegetative and reproductive morphology. The expression of the Cre/loxP recombinase system was also studied in tobacco (Nicotiana tabacum L.) under the control of the seed-specific promoter BcNA1 (derived from canola) to remove the SMG [68]. Eight transgenic lines were obtained and self-pollinated, and progeny were subjected to a germination assay on selection media to determine whether excision had occurred.…”

Green tissue-targeted expression of the Cry1Ab/c gene in transgenic maize using the Cre/loxP system as an alternative strategy against lepidopteran pests

“…Therefore, as a critically manipulated elicitor, the specific promoter controls the expression tissues of the Cre gene and guides the excision reaction occurring in defined tissues, thereby enabling the expression of transgenes in particular tissues. To date, several tissue-specific promoters have been widely used in genetic engineering, such as green tissue-specific promoters, endosperm-specific promoters, and root-specific expression promoters, which have been described with specific induction in transgenic plants (Mlynarova et al 2006, Li et al 2007, Bai et al 2008, Hamzeh et al 2016). This promising Cre/ lox P-mediated system has previously been applied in transgenic tobacco (Moravcikova et al 2008), rice (Chen et al 2017), and oilseed rape (Boszorádová et al 2019) for manipulating transgene expression in desired tissues.…”