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Alpha-amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits alpha-amylases from the coffee berry borer pest.

Barbosa AE, Albuquerque EV, Silva MC, Souza DS, Oliveira-Neto OB, Valencia A, Rocha TL, Grossi-de-Sa MF - BMC Biotechnol. (2010)

Bottom Line: The presence of the alpha-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting.Immunoblotting and ELISA experiments using antibodies against alpha-AI1 inhibitor showed a maximum alpha-AI1 concentration of 0.29% in crude seed extracts.Inhibitory in vitro assays of the alpha-AI1 protein against H. hampei alpha-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: EMBRAPA Recursos Genéticos e Biotecnologia, Brasília-DF, Brazil.

ABSTRACT

Background: Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an alpha-amylase inhibitor gene (alpha-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.

Results: We transformed C. arabica with the alpha-amylase inhibitor-1 gene (alpha-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the alpha-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against alpha-AI1 inhibitor showed a maximum alpha-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the alpha-AI1 protein against H. hampei alpha-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity.

Conclusions: This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.

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Related in: MedlinePlus

Comparison between the amount of α-AI1 in coffee transgenic plants and the inhibition rate of H. hampei α-amylases measured by DNS assay. In the DNS assay, 30 μg of the total seed protein extract was used, and the α-AI1 quantity in these extracts was measured using ELISA.
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Figure 6: Comparison between the amount of α-AI1 in coffee transgenic plants and the inhibition rate of H. hampei α-amylases measured by DNS assay. In the DNS assay, 30 μg of the total seed protein extract was used, and the α-AI1 quantity in these extracts was measured using ELISA.

Mentions: In vivo insect assays described in other works exhibited mortality ranging from 89.8% to 100% [17,20], depending on the inhibitor concentration in each tested plant. The high levels of amylase inhibitor may be related to the fact that in some studies [17], seeds from homozygous plants were used. However, the coffee plants used here were hemizygous. Valencia et al. (2000) showed that an inhibitor from common bean inhibits the H. hampei gut amylases up to 95%. We tested the effect of extracts from transformed seeds, and the level of inhibition of amylase activity correlates with the level of inhibitor in the common bean (P. vulgaris) seeds (Table 1). The plant with the highest amount of α-AI1 inhibitor (Plant 2) exhibited the strongest H. hampei α-amylases inhibition, and the inhibition levels reached by each transgenic plant were directly related to the α-AI1 concentration (Figure 6).


Alpha-amylase inhibitor-1 gene from Phaseolus vulgaris expressed in Coffea arabica plants inhibits alpha-amylases from the coffee berry borer pest.

Barbosa AE, Albuquerque EV, Silva MC, Souza DS, Oliveira-Neto OB, Valencia A, Rocha TL, Grossi-de-Sa MF - BMC Biotechnol. (2010)

Comparison between the amount of α-AI1 in coffee transgenic plants and the inhibition rate of H. hampei α-amylases measured by DNS assay. In the DNS assay, 30 μg of the total seed protein extract was used, and the α-AI1 quantity in these extracts was measured using ELISA.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2914071&req=5

Figure 6: Comparison between the amount of α-AI1 in coffee transgenic plants and the inhibition rate of H. hampei α-amylases measured by DNS assay. In the DNS assay, 30 μg of the total seed protein extract was used, and the α-AI1 quantity in these extracts was measured using ELISA.
Mentions: In vivo insect assays described in other works exhibited mortality ranging from 89.8% to 100% [17,20], depending on the inhibitor concentration in each tested plant. The high levels of amylase inhibitor may be related to the fact that in some studies [17], seeds from homozygous plants were used. However, the coffee plants used here were hemizygous. Valencia et al. (2000) showed that an inhibitor from common bean inhibits the H. hampei gut amylases up to 95%. We tested the effect of extracts from transformed seeds, and the level of inhibition of amylase activity correlates with the level of inhibitor in the common bean (P. vulgaris) seeds (Table 1). The plant with the highest amount of α-AI1 inhibitor (Plant 2) exhibited the strongest H. hampei α-amylases inhibition, and the inhibition levels reached by each transgenic plant were directly related to the α-AI1 concentration (Figure 6).

Bottom Line: The presence of the alpha-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting.Immunoblotting and ELISA experiments using antibodies against alpha-AI1 inhibitor showed a maximum alpha-AI1 concentration of 0.29% in crude seed extracts.Inhibitory in vitro assays of the alpha-AI1 protein against H. hampei alpha-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity.

View Article: PubMed Central - HTML - PubMed

Affiliation: EMBRAPA Recursos Genéticos e Biotecnologia, Brasília-DF, Brazil.

ABSTRACT

Background: Coffee is an important crop and is crucial to the economy of many developing countries, generating around US$70 billion per year. There are 115 species in the Coffea genus, but only two, C. arabica and C. canephora, are commercially cultivated. Coffee plants are attacked by many pathogens and insect-pests, which affect not only the production of coffee but also its grain quality, reducing the commercial value of the product. The main insect-pest, the coffee berry borer (Hypotheneumus hampei), is responsible for worldwide annual losses of around US$500 million. The coffee berry borer exclusively damages the coffee berries, and it is mainly controlled by organochlorine insecticides that are both toxic and carcinogenic. Unfortunately, natural resistance in the genus Coffea to H. hampei has not been documented. To overcome these problems, biotechnological strategies can be used to introduce an alpha-amylase inhibitor gene (alpha-AI1), which confers resistance against the coffee berry borer insect-pest, into C. arabica plants.

Results: We transformed C. arabica with the alpha-amylase inhibitor-1 gene (alpha-AI1) from the common bean, Phaseolus vulgaris, under control of the seed-specific phytohemagglutinin promoter (PHA-L). The presence of the alpha-AI1 gene in six regenerated transgenic T1 coffee plants was identified by PCR and Southern blotting. Immunoblotting and ELISA experiments using antibodies against alpha-AI1 inhibitor showed a maximum alpha-AI1 concentration of 0.29% in crude seed extracts. Inhibitory in vitro assays of the alpha-AI1 protein against H. hampei alpha-amylases in transgenic seed extracts showed up to 88% inhibition of enzyme activity.

Conclusions: This is the first report showing the production of transgenic coffee plants with the biotechnological potential to control the coffee berry borer, the most important insect-pest of crop coffee.

Show MeSH
Related in: MedlinePlus