Abstract
Manipulation of a single abiotic stress-related gene could improve plant performance under abiotic stress conditions. To simultaneously increase plant tolerance to multiple stresses, it is usually required to overexpress two (or more) genes in transgenic plants. The common strategy is to assemble two or more expression cassettes, where each gene has its own promoter and terminator, within the same T-DNA. Does the arrangement of the two expression cassettes affect expression of the two transgenes? Can we use the Drosophila gypsy insulator sequence to increase the expression of the two transgenes? Answers to these questions would contribute to design better transformation vectors to maximize the effects of multi-gene transformation. Two Arabidopsis genes, PP2A-C5 and AVP1, and the gypsy insulator sequence were used to construct six transformation vectors with or without the gypsy insulator bracketing the two expression cassettes: uni-directional transcription, divergent transcription, and convergent transcription. Total RNAs were isolated for reverse transcription- quantitative real-time polymerase chain reaction (RT-qPCR) assays and a thorough statistical analysis was conducted for the RT-qPCR data. The results showed that the gypsy insulator does promote the expression of two transgenes in transgenic plants. Besides, the plants containing the divergent transcription cassettes tend to have more correlated expression of both genes.
Highlights
Could be stacked in transgenic plants by transforming linked genes, crossing different transgenic plants, or performing sequential or co-transformations[8]
phosphatase 2A (PP2A)-C5 is one of the five genes encoding the catalytic subunit of PP2A in Arabidopsis, and it plays an important role in plant salt resistance
Our results indicate that transgenic plants independently containing uni-directional or convergent transcription cassettes have higher fold of change (FOC) in PP2A-C5 transcript compared with that of Arabidopsis vacuolar H+-pyrophosphatase 1 (AVP1) and transgenic plants independently containing divergent transcription cassettes have a more similar, or ‘balanced’ FOC for AVP1 and PP2A-C5 transcripts (Fig. 3)
Summary
Could be stacked in transgenic plants by transforming linked genes (multiple genes on the same plasmid), crossing different transgenic plants, or performing sequential or co-transformations[8]. When two or more open reading frames (ORFs) are integrated on one plasmid as linked genes, each exogenous gene is transcribed independently because every ORF has its own promoter, enhancer, and terminator This method has a disadvantage that it is very difficult to control the transcript levels of multiple transgenes, because stacking of multiple strong promoters might cause silencing. The transgene’s promoters might compete for RNA polymerase II, resulting in unexpected differential expression levels of two genes[21] This could be the reason why some plants co-overexpressing multiple anti-stress genes don’t perform better than a single gene transgenic plants[22,23]. Our results can be used to guide vector design for less labor intensive and more efficient multiple transgene expression
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