Abstract
BACKGROUND: The aim of this study was to develop a genetic transformation system to construct an overexpression vector for the mitochondrial gene atp6 in tobacco, thereby providing a foundation to investigate the functional roles of mitochondrial genes in this species. METHODS: A full-length coding sequence (CDS) of the atp6 gene from a sterile line was cloned, along with the mitochondrial leader peptide sequence of atp2-1 from tobacco, using cDNA from kenaf UG93A anthers as a template. An overexpression vector for plants was constructed by employing In-Fusion technology, and wild-type tobacco plants were transformed via Agrobacterium-mediated transformation. Transgenic tobacco plants were then subjected to resistance screening and PCR validation. RESULTS: The overexpression vector PBI121-atp2-1-atp6-EGFP, which includeds the mitochondrial leader peptide sequence, was successfully constructed. PCR validation using two pairs of primers targeting different sites on the overexpression vector confirmed the stable expression of the target gene in six transgenic tobacco plants (H1, H3, H4, H5, H7, and H8) via both primer pairs. A phenotypic analysis and iodine-potassium iodide (I(2)-KI) staining of pollen grains from transgenic tobacco plants revealed the presence of shriveled and malformed pollen grains with reduced viability. These findings suggest that the atp6A gene, including the mitochondrial signal peptide, induces pollen abortion in tobacco. CONCLUSIONS: The genetic transformation system developed for the vector overexpressing the atp6 mitochondrial gene from kenaf provides a valuable framework to investigate the molecular regulatory mechanisms underlying the role of the atp6 gene in kenaf cytoplasmic male sterility (CMS).