Manganese(II)-Enhanced Graphene Oxide Fluorescence Switch-Based Hybridization Chain Reaction for the Enzyme-Free and Sensitive Determination of Bacillus thuringiensis (Bt) in Transgenic Crops
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Bacillus thuringiensis, as a special DNA sequence, has been introduced into crops for insect control. At the same time, the safety risk of transgenic crops has received wide concern. Herein, a simple, enzyme-free and sensitive novel nanosensor for the determination of Bacillus thuringiensis by manganese(II)-enhanced graphene oxide (GO) fluorescence switch (Mn(II)-GO-FS)-based hybridization chain reaction (HCR) signal amplification was developed. The high affinity of Mn(II) to oxygen-containing groups in double-stranded DNA and on the surface of GO was able to overcome the steric effect of rigid double chains and drive the efficient adsorption of HCR hairpin probes with sticky ends on GO. The efficient quenching of 6-carboxyfluorescein (FAM) labeled on the HCR hairpin probes guaranteed a low background. The Bacillus thuringiensis acted as a trigger to initiate a cascade of hybridization events between the two metastable HCR hairpin probes (H1 and H2) to polymerize into a nicked long double-helix structure (dsDNA). The products desorbed from the surface of GO and produced strong fluorescence. The results included a wide linear range (0.1 pM-50 nM) and a low detection limit (0.05 pM). This nanosensor was successfully applied to determine Bacillus thuringiensis in maize seeds with spiked recoveries between 97.5% and 103%. The Mn(II)-GO-FS had a simple design and was conveniently used for the determination of other special genes by changing the corresponding HCR hairpin probe sequence. Moreover, this novel nanosensor did not require harsh experimental conditions, operating in a neutral environment.Keywords:
Bacillus thuringiensis
An enzyme-linked immunosorbent assay(ELISA) method was used to detect and quantitate the expression of Bacillus thuringiensis(Bt) insecticidal protein in different growth periods and different tissues of the Bt transgenic insect-resistant corn.From the results,we can see that the expressions of Bacillus thuringiensis(Bt) insecticidal protein in leaves are the highest,while the lowest in pollen.With the growth of the plant,the expression of Bacillus thuringiensis(Bt) insecticidal protein in leaves,stalk and ear continuously increased.The smallest concentration of Bt protein for detection is 0.5 ng/mL.
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Genetically modified maize
Bt Cotton
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Bacillus thuringiensis
Cry1Ac
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Quantitative assays were developed to analyze gene products produced by transgenic plants that express Bacillus thuringiensis insect control proteins. Initially B. thuringiensis proteins were produced in plants at approximately 10,000 fold lower specific activities than the same proteins produced by microbial fermentations. Consequently, accurate and selective assays were developed to quantitate B. thuringiensis specific DNA (gene copy), mRNA and protein produced by insect tolerant transgenic plants. The 100 fold increase in B. thuringiensis protein expression achieved in plants, based on these analysis, enabled the production of transgenic plants that are protected against agronomically important insect pests.
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Bacillus thuringiensis
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Bacillus thuringiensis (Bt) crops are plants genetically engineered (modified) to contain the endospore (or crystal) toxins of the bacterium, Bt to be resistant to certain insect pests. In 1995, the Environmental Protection Agency (EPA) in USA approved the commercial production and distribution of the Bt crops: corn, cotton, potato, and tobacco. Currently, the most common Bt crops are corn and cotton. The crystal, referred to as Cry toxins, is proteins formed during sporulation of some Bt strains and aggregate to form crystals. Such Cry toxins are toxic to specific species of insects belongs to orders: Lepidoptera, Coleoptera, Hymenoptera, Diptera, and Nematoda. In 2016, the total world area cultivated with genetically modified crops (GM crops) reached about 185 million ha. This review shows that there is a worldwide controversy about the safety of Bt crops to the environment and mammals. Some researchers support the cultivation of Bt crops depending upon the results of their laboratory and field studies on the safety of such crops. Others, however, are against Bt crops as they may cause risk to human.
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The insecticidal activity of Bacillus thuringiensis (Bt) is due mainly to the presence of the insecticidal crystal proteins (Cry proteins) and vegetative insecticidal proteins (Vips). The evolution of resistance in insect populations represents a serious threat to the continued success of Bt crops. Resistance management strategies attempt to prevent or diminish the selection of the rare individuals carrying resistance genes and hence to keep the frequency of resistance genes below levels that would result in inefficient insect control. Bt sprayable biopesticides are generally regarded as safe for use as biological control agents and are promoted in both organic and integrated pest management (IPM) systems. In view of the specificity of Cry proteins, the effect of Bt crops on most nontarget insects can be expected to be minimal, especially when compared with the effects of broad-spectrum insecticides.
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Presently, a number of approaches to pest control via genetic engineering have been developed and genetically engineered crops expressing insecticidal characteristics are under cultivation for the last 15 years. Use of Bacillus thuringiensis genes encoding o endotoxins with insecticidal characteristics is the major approach and a number of such B. thuringiensis genes have been expressed in crops with variable level of efficiency. It is very crucial to achieve adequate level of B. thuringiensis gene expression to have durable resistance against target insect pests. As with many aspects of genetic engineering, politics can impact on the success of a project involving the development of B. thuringiensis transgenic crops, irrespective of its apparent social, economic or environmental benefits. Public education will be essential to ensure the widespread adoption of genetic adoption technologies in agriculture, and scientists will have to play an active role in this process.
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This chapter contains sections titled: Introduction Plant Engineering Insecticidal Crystal Proteins from B. thuringiensis 1030 Bt Plants Insect Resistance to Bt Resistance Management with Bt Plants Safety of Bt Plants Conclusions References
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