Abstract
The E. coli phosphomannose isomerase (EcPMI) gene is widely used as a selectable marker gene (SMG) in mannose (Man) selection-based plant transformation. Although some plant species exhibit significant PMI activity and active PMIs were even identified in Man-sensitive plants, whether plant PMIs can be used as SMGs remains unclear. In this study, we isolated four novel PMI genes from Chlorella variabilis and Oryza sativa. Their isoenzymatic activities were examined in vitro and compared with that of EcPMI. The active plant PMIs were separately constructed into binary vectors as SMGs and then transformed into rice via Agrobacterium. In both Indica and Japonica subspecies, our results indicated that the plant PMIs could select and produce transgenic plants in a pattern similar to that of EcPMI. The transgenic plants exhibited an accumulation of plant PMI transcripts and enhancement of the in vivo PMI activity. Furthermore, a gene of interest was successfully transformed into rice using the plant PMIs as SMGs. Thus, novel SMGs for Man selection were isolated from plants, and our analysis suggested that PMIs encoding active enzymes might be common in plants and could potentially be used as appropriate genetic elements in cisgenesis engineering.
Highlights
During plant transformation, transformed cells are identified from a matrix consisting primarily of untransformed cells using a selection system and regenerated to transgenic plants
We showed that the utilization of the plant codon-optimized E. coli phosphomannose isomerase (EcPMI) exhibited higher selection efficiency in rice transformation, which suggested that the expression of the original EcPMI may be
The proteins of the transgenic plants expressing AtPMI2, OsPMI1 and EcPMI exhibited the same levels of biochemical activity regarding M-6-P conversion
Summary
During plant transformation, transformed cells are identified from a matrix consisting primarily of untransformed cells using a selection system and regenerated to transgenic plants. The most-used selection systems consist of an antibiotic or herbicide selective agent, which is toxic for the non-transformed cells, and the corresponding selectable marker gene (SMG) confers the tolerance to transformed cells[3]. These systems are stable and efficient in both scientific research and commercial breeding. The active plant PMI genes were expressed in cells to select and regenerate transgenic rice under Man pressure. Our results suggested that plant PMIs could be applied as effective alternative SMGs in the genetic engineering of rice and other plants
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