Abstract
We present a method for detecting spliced products from transiently expressed genes using an intron construct containing the target intron and neighboring short exons. The downstream exon was fused with theGUS reporter gene and inserted behind the cauliflower mosaic virus 35S promoter. A particle gun was used to introduce the construct directly into leaf tissues. Leaf discs were harvested 3–6 h after bombardment. Total nucleic acids were prepared and treated with RNase-free DNase. The RNA fraction was subjected to reverse transcription-PCR (RT-PCR) analysis using primers for the upstream exon sequence and theGUS gene. Correct splicing was confirmed by sequencing amplified cDNAs. This method allows a quick assessment of the splicing efficiency of target introns.
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Abbreviations
- CaMV:
-
cauliflower mosaic virus
- RLU:
-
relative fluorescence unit
- RT-PCR:
-
reverse transcription-PCR
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Mitsui, M., Murohashi, Y., Asano, Y. et al. Transient assay for in vivo splicing in Tobacco leaf cells by particle bombardment. Plant Mol Biol Rep 21, 21–30 (2003). https://doi.org/10.1007/BF02773393
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DOI: https://doi.org/10.1007/BF02773393