Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly

Abstract

The green f luorescent protein (GFP) from the jellyfish Aequorea victoria is finding wide use as a genetic marker that can be directly visualized in the living cells of many heterologous organisms. We have sought to express GFP in the model plant Arabidopsis thaliana, but have found that proper expression of GFP is curtailed due to aberrant mRNA processing. An 84-nt cryptic intron is efficiently recognized and excised from transcripts of the GFP coding sequence. The cryptic intron contains sequences similar to those required for recognition of normal plant introns. We have modified the codon usage of the gfp gene to mutate the intron and to restore proper expression in Arabidopsis. GFP is mainly localized within the nucleoplasm and cytoplasm of transformed Arabidopsis cells and can give rise to high levels of f luorescence, but it proved difficult to efficiently regenerate transgenic plants from such highly f luorescent cells. However, when GFP is targeted to the endoplasmic reticulum, transformed cells regenerate routinely to give highly f luorescent plants. These modified forms of the gfp gene are useful for directly monitoring gene expression and protein localization and dynamics at high resolution, and as a simply scored genetic marker in living plants.

Localization and in vitro binding studies suggest that the cytoplasmic/nuclear tobacco lectin can interact in situ with high‐mannose and complex N‐glycans

“…Due to its small size, stable and high fluorescence, negligible impact on cell viability and versatility, GFP is widely used in localization studies in living cells [18]. Variants of GFP with improved expression and stability in mammalian and plant cells have been developed.…”

Protein Disulfide Isomerase-2 of Arabidopsis Mediates Protein Folding and Localizes to Both the Secretory Pathway and Nucleus, Where It Interacts with Maternal Effect Embryo Arrest Factor

“…locus At5g60640) was cloned into pSK (Bluescript, Stratagene, USA) and pET25b(+) vectors (Novagen, USA). The mGFP4 gene was obtained from the commercial vector pBIN-gfp4 (Haseloff et al, 1997) by restriction digestion. A 1398 bp PDI2 promoter region was amplified by PCR.…”

Expression analysis and subcellular localization of the Arabidopsis thaliana G-protein β-subunit AGB1

“…In the absence of a targeting signal, mGFP5 localises to the cytoplasm and also diffuses into the nucleus (Haseloff et al 1997). The AGB1-GFP fluorescent pattern is clearly distinguishable from the pattern observed in non-targeted mGFP5 plants, being thinner and different in shape (Fig.…”

“…5c, d). The AGB1-GFP fusion fluorescent signal is also distinct from endoplasmic reticulum (ER)-targeted mGFP5 (Haseloff et al 1997;Haseloff 1999), which lines the periphery of the cytoplasm but also forms a reticulate pattern within it and surrounds the nucleus ( Fig. 5e; Haseloff et al 1997).…”