Dataset Information

Expression of Cry1Ab and Cry2Ab by a polycistronic transgene with a self-cleavage peptide in rice.

ABSTRACT: Insect resistance to Bacillus thuringiensis (Bt) crystal protein is a major threat to the long-term use of transgenic Bt crops. Gene stacking is a readily deployable strategy to delay the development of insect resistance while it may also broaden insecticidal spectrum. Here, we report the creation of transgenic rice expressing discrete Cry1Ab and Cry2Ab simultaneously from a single expression cassette using 2A self-cleaving peptides, which are autonomous elements from virus guiding the polycistronic viral gene expression in eukaryotes. The synthetic coding sequences of Cry1Ab and Cry2Ab, linked by the coding sequence of a 2A peptide from either foot and mouth disease virus or porcine teschovirus-1, regardless of order, were all expressed as discrete Cry1Ab and Cry2Ab at high levels in the transgenic rice. Insect bioassays demonstrated that the transgenic plants were highly resistant to lepidopteran pests. This study suggested that 2A peptide can be utilized to express multiple Bt genes at high levels in transgenic crops.

SUBMITTER: Zhao Q

PROVIDER: S-EPMC4198207 | biostudies-literature | 2014

REPOSITORIES: biostudies-literature

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Expression of Cry1Ab and Cry2Ab by a polycistronic transgene with a self-cleavage peptide in rice.

Zhao Qichao Q Liu Minghong M Tan Miaomiao M Gao Jianhua J Shen Zhicheng Z

PloS one 20141015 10

Insect resistance to Bacillus thuringiensis (Bt) crystal protein is a major threat to the long-term use of transgenic Bt crops. Gene stacking is a readily deployable strategy to delay the development of insect resistance while it may also broaden insecticidal spectrum. Here, we report the creation of transgenic rice expressing discrete Cry1Ab and Cry2Ab simultaneously from a single expression cassette using 2A self-cleaving peptides, which are autonomous elements from virus guiding the polycistr ...[more]

PMID: 25333312

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Project description:Brazil and South Africa, countries with economies in transition, are characterized by a dual agrarian structure co-occurring, sometimes, alongside in the same region. Large-scale commercial farming produces crops for export to global markets on the one hand, and small-scale farming, on the other hand, provides for subsistence and local markets. In both systems, maize (Zea mays) is a key crop for these two countries. For the commercial system, maize is a commodity crop while for the small-scale system it is the prime staple crop. In commercial systems, farmers predominantly grow genetically modified (GM) hybrid maize. In small-scale systems, farmers rely on open pollinated varieties (OPVs) and/or landraces and are largely dependent on seed saving systems. The aim of this study was to understand the relationship between transgene expression rates, the resulting concentrations of the transgene product (Bt protein) and its bioactivity in insect pests following transgene flow from GM hybrid maize into non-genetically modified, non-GM near-isogenic maize hybrid (ISO) and OPVs. We modeled segregation patterns and measured cry1Ab transgene expression (mRNA quantification), Cry1Ab protein concentration and pest survival. Two groups of F1, F2 crosses and backcrosses with GM, ISO and OPV maize varieties from Brazil and South Africa were used. Bioassays with the larvae of two lepidopteran maize pest species, Helicoverpa armigera and Spodoptera littoralis, were carried out. Overall, the cry1Ab transgene outcrossed effectively into the genetic backgrounds tested. The cry1Ab transgene was stably expressed in both ISO and OPV genetic backgrounds. Transgene introgression led to consistent, though highly variable, concentrations of Cry1Ab toxins that were similar to those observed in GM parental maize. Most crosses, but not all, suggested the expected Mendelian segregation pattern. Transgene expression rates were significantly higher than expected from purely Mendelian segregation in the South African crosses. In South African materials, ISO and OPV crosses produced significantly lower Cry1Ab concentrations compared to the GM parental maize. The Cry1Ab toxins from crosses were bioactive and induced mortality rates of ≥92.19% in H. armigera and ≥40.63% in S. littoralis after a period of only 4 days. However, no correlations were observed between the quantitation of mRNA for cry1Ab and the corresponding Cry1Ab protein concentrations, nor between the Cry1Ab concentrations and insect mortality rates across different genetic backgrounds. We therefore suggest that while transcription of the cry1Ab transgene reliably determines the presence of Cry1Ab protein, mRNA levels do not reflect, by themselves, the end Cry1Ab protein concentrations found in the plant. Because predictably high Cry1Ab concentrations are a key prerequisite for effective insect resistance management (IRM) programs, this observation raises questions about the effectiveness of such programs in scenarios with complex crop genetic backgrounds. On the other hand, confirmed bioactivity in all crosses should be expected to impact small farmer's selection behavior, unknowingly favoring the insecticidal trait. This may lead to a fixation of the trait in the wider population, and may influence the genetic diversity of varieties maintained by small-scale farmers.

Project description:Experimental models that capture the genetic complexity of human disease and allow mechanistic explorations of the underlying cell, tissue, and organ interactions are crucial to furthering our understanding of disease biology. Such models require combinatorial manipulations of multiple genes, often in more than one tissue at once. The ability to perform complex genetic manipulations in vivo is a key strength of Drosophila, where many tools for sophisticated and orthogonal genetic perturbations exist. However, combining the large number of transgenes required to establish more representative disease models and conducting mechanistic studies in these already complex genetic backgrounds is challenging. Here we present a design that pushes the limits of Drosophila genetics by allowing targeted combinatorial ectopic expression and knockdown of multiple genes from a single inducible transgene. The polycistronic transcript encoded by this transgene includes a synthetic short hairpin cluster cloned within an intron placed at the 5' end of the transcript, followed by two protein-coding sequences separated by the T2A sequence that mediates ribosome skipping. This technology is particularly useful for modeling genetically complex diseases like cancer, which typically involve concurrent activation of multiple oncogenes and loss of multiple tumor suppressors. Furthermore, consolidating multiple genetic perturbations into a single transgene further streamlines the ability to perform combinatorial genetic manipulations and makes it readily adaptable to a broad palette of transgenic systems. This flexible design for combinatorial genetic perturbations will also be a valuable tool for functionally exploring multigenic gene signatures identified from omics studies of human disease and creating humanized Drosophila models to characterize disease-associated variants in human genes. It can also be adapted for studying biological processes underlying normal tissue homeostasis and development that require simultaneous manipulation of many genes.

Dataset Information

Expression of Cry1Ab and Cry2Ab by a polycistronic transgene with a self-cleavage peptide in rice.

Publications

Expression of Cry1Ab and Cry2Ab by a polycistronic transgene with a self-cleavage peptide in rice.

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