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Abstract

The gene encoding green fluorescent protein (GFP) from Aequorea victoria was resynthesized to adapt its codon usage for expression in plants by increasing the frequency of codons with a C or a G in the third position from 32 to 60%. The strategy for constructing the synthetic gfp gene was based on the overlap extension PCR method using 12 long oligonucleotides as the starting material and as primers. The new gene contains 101 silent nucleotide changes compared to its wild-type counterpart used in this study. Several transgenic tobacco lines containing the wild-type gfp gene contained minute amounts of a smaller protein cross-reacting with GFP antiserum, whereas only one protein of the expected size was found in transgenics with the synthetic gfp gene. The smaller protein was probably encoded by a truncated gfp mRNA created by splicing of a 84 bp cryptic intron as detected by a reverse transcription-PCR technique. A comparison of GFP production in transgenics with the wild-type and the synthetic gfp gene under the control of the enhanced CaMV 35S promoter showed that the large-scale alterations in the gfp gene increased the frequency of high expressors in the transgenic population but hardly changed the maximum GFP concentrations. The latter phenomenon may be attributed to a reduced regeneration capacity of transformed cells with higher GFP concentrations.

Complete Chloroplast Genome Sequence of Poisonous and Medicinal Plant Datura stramonium: Organizations and Implications for Genetic Engineering

“…Since all cp genomes have high AT content, AT biased mutational pressure is believed to be the factor responsible for codon usage bias. Previous studies demonstrated that there existed a significant relationship between codon usage bias and gene expression level [57], [58], which suggested stronger natural selection constraints on highly expressed genes to optimize translation efficiency using major codons [59]. Information about the rare and preferred codons can be effectively used for enhancing gene expression by optimizing synonymous codons, which may provide us a further understanding of synthesis and metabolism of secondary metabolites in D. stramonium .…”

Complete Chloroplast Genome of the Wollemi Pine (Wollemia nobilis): Structure and Evolution

“…[45,46]) and may also be associated with the codon bias of chloroplast protein-coding genes and hence the regulation of gene expression (e.g. [45,46]).…”

“…[45,46]) and may also be associated with the codon bias of chloroplast protein-coding genes and hence the regulation of gene expression (e.g. [45,46]). AT-rich regions in the Wollemia chloroplast genome include intergenic (67.88%), protein-coding (62.41%) and intronic (62.39%) regions, while rRNAs (45.75%) and tRNAs (46.47%) have a much lower AT content.…”

Seeing the Wood through the Trees: A Review of Techniques for Distinguishing Green Fluorescent Protein from Endogenous Autofluorescence

“…For example, Liu et al (113) demonstrated the induction of apoptosis (programmed cell death) by three mutant GFPs in four mammalian cell lines. GFP toxicity has also been noted in plants (100,108), particularly A. thaliana (107), although Niwa and co-workers (79) have since demonstrated nontoxic high-level expression of a synthetic, codon-optimized GFP in Arabidopsis.…”