Engineered GFP as a vital reporter in plants

Chiu et al. (1996). Curr Biol DOI: 10.1016/s0960-9822(02)00483-9 Citations: 1388

Native jellyfish GFP gave weak fluorescence in plants, limiting reporter use. This paper engineered synthetic GFP with optimized codons and an S65T mutation, sGFP(S65T). The variant was 100-fold brighter and revealed drought-inducible promoter activity in tobacco.

Parts used

SV40 NLS

Key findings

  • sGFP with optimized human codons achieved 20-fold higher expression than native jellyfish GFP in maize cells. Combined with S65T mutation, sGFP(S65T) produced >100-fold brighter fluorescence under 490nm excitation and enabled detection within 3-4 hours versus 15-16 hours for native GFP.

  • sGFP(S65T) detected weak Arabidopsis CAB2 promoter activity in maize cells (previously undetectable with native GFP) and enabled light-responsive expression. The construct worked across multiple plant systems including tobacco protoplasts (>80% transfection efficiency), Arabidopsis tissues, and onion epidermal cells.

  • Transgenic tobacco plants expressing 35SC4PPDK-sGFP(S65T) showed clear yellow fluorescence in all cell types, enabling rapid visual identification without drug selection or PCR. Nuclear and plastid targeting via NLS and transit peptide fusions demonstrated precise subcellular localization in living cells.

1 3

Abstract

The green-fluorescent protein (GFP) of the jellyfish Aequorea victoria has recently been used as a universal reporter in a broad range of heterologous living cells and organisms. Although successful in some plant transient expression assays based on strong promoters or high copy number viral vectors, further improvement of expression efficiency and fluorescent intensity are required for GFP to be useful as a marker in intact plants. Here, we report that an extensively modified GFP is a versatile and sensitive reporter in a variety of living plant cells and in transgenic plants.