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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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α-AI1 expression in transgenic coffee beans from T0 plants. A. SDS-Polyacrylamide gel (15%) stained with Coomassie Brilliant Blue. M, Benchmark™ prestained protein ladder (Invitrogen); P, purified α-AI1 from Phaseolus vulgaris seeds; 1-3, total seed protein extract from transgenic coffee plants. The arrows indicate possible expressed α-AI1; 4, total seed protein extract of non-transgenic coffee plant. B. Western blot. M, Benchmark™ prestained protein ladder (Invitrogen); P, purified α-AI1 from Phaseolus vulgaris seeds; 1-3, total seed protein extract from transgenic coffee plants; 4, total seed protein extract of the non-transgenic coffee plant.
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Figure 5: α-AI1 expression in transgenic coffee beans from T0 plants. A. SDS-Polyacrylamide gel (15%) stained with Coomassie Brilliant Blue. M, Benchmark™ prestained protein ladder (Invitrogen); P, purified α-AI1 from Phaseolus vulgaris seeds; 1-3, total seed protein extract from transgenic coffee plants. The arrows indicate possible expressed α-AI1; 4, total seed protein extract of non-transgenic coffee plant. B. Western blot. M, Benchmark™ prestained protein ladder (Invitrogen); P, purified α-AI1 from Phaseolus vulgaris seeds; 1-3, total seed protein extract from transgenic coffee plants; 4, total seed protein extract of the non-transgenic coffee plant.

Mentions: To examine α-AI1 protein expression in coffee beans of T0 plants, crude extracts from transformed seeds were analysed using SDS-PAGE and immunoblots (Figures 5A and 3B, respectively). The level of expression was evaluated by ELISA assay (Table 1). The immunoblot showed three positive bands of 19, 17 and 16 kDa that were not present in the extracts of untransformed plants and corresponded to the protein found in P. vulgaris seed extracts (Figure 5B lanes 1, 2, 3 and 4). In the common bean, amylase inhibitor-1 is synthesised as a preproprotein, and in its mature form, it consists of two polypeptides of 19 and 14 kDa [29]. Differential processing of the polypeptide and glycan portions of the protein results in a series of bands on immunoblots, especially in transgenic seeds of different species where processing events may be different. For example, as many as 11 different immunoreactive polypeptides were found in tobacco seeds [30]. In transgenic peas expressing α-AI1, three polypeptides were visualised on immunoblots [17]. The altered molecular mass of α-AI1 in the different transgenic plants may have occured due to a variation in the extent of glycosylation or the processing of the glycans [31]. Even in P. vulgaris seeds, the subunits α and β of α-AI1 are not always glycosylated in the same way, generating polypeptide heterogeneity [32]. Unexpectedly, differences in glycosylation between the inhibitor isolated from the common bean and from transgenic peas expressing α-AI1 resulted in differences in immunogenic properties between the two proteins. The protein made in peas caused immunological responses and inflammation in mice [33]. Nevertheless, no difference in immunogenicity was observed in mice fed with transgenic chickpeas expressing α-AI1 [33]. As a consequence, it is difficult to predict if similar immunogenic alterations will occur in an inhibitor made by transgenic coffee plants.


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)

α-AI1 expression in transgenic coffee beans from T0 plants. A. SDS-Polyacrylamide gel (15%) stained with Coomassie Brilliant Blue. M, Benchmark™ prestained protein ladder (Invitrogen); P, purified α-AI1 from Phaseolus vulgaris seeds; 1-3, total seed protein extract from transgenic coffee plants. The arrows indicate possible expressed α-AI1; 4, total seed protein extract of non-transgenic coffee plant. B. Western blot. M, Benchmark™ prestained protein ladder (Invitrogen); P, purified α-AI1 from Phaseolus vulgaris seeds; 1-3, total seed protein extract from transgenic coffee plants; 4, total seed protein extract of the non-transgenic coffee plant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: α-AI1 expression in transgenic coffee beans from T0 plants. A. SDS-Polyacrylamide gel (15%) stained with Coomassie Brilliant Blue. M, Benchmark™ prestained protein ladder (Invitrogen); P, purified α-AI1 from Phaseolus vulgaris seeds; 1-3, total seed protein extract from transgenic coffee plants. The arrows indicate possible expressed α-AI1; 4, total seed protein extract of non-transgenic coffee plant. B. Western blot. M, Benchmark™ prestained protein ladder (Invitrogen); P, purified α-AI1 from Phaseolus vulgaris seeds; 1-3, total seed protein extract from transgenic coffee plants; 4, total seed protein extract of the non-transgenic coffee plant.
Mentions: To examine α-AI1 protein expression in coffee beans of T0 plants, crude extracts from transformed seeds were analysed using SDS-PAGE and immunoblots (Figures 5A and 3B, respectively). The level of expression was evaluated by ELISA assay (Table 1). The immunoblot showed three positive bands of 19, 17 and 16 kDa that were not present in the extracts of untransformed plants and corresponded to the protein found in P. vulgaris seed extracts (Figure 5B lanes 1, 2, 3 and 4). In the common bean, amylase inhibitor-1 is synthesised as a preproprotein, and in its mature form, it consists of two polypeptides of 19 and 14 kDa [29]. Differential processing of the polypeptide and glycan portions of the protein results in a series of bands on immunoblots, especially in transgenic seeds of different species where processing events may be different. For example, as many as 11 different immunoreactive polypeptides were found in tobacco seeds [30]. In transgenic peas expressing α-AI1, three polypeptides were visualised on immunoblots [17]. The altered molecular mass of α-AI1 in the different transgenic plants may have occured due to a variation in the extent of glycosylation or the processing of the glycans [31]. Even in P. vulgaris seeds, the subunits α and β of α-AI1 are not always glycosylated in the same way, generating polypeptide heterogeneity [32]. Unexpectedly, differences in glycosylation between the inhibitor isolated from the common bean and from transgenic peas expressing α-AI1 resulted in differences in immunogenic properties between the two proteins. The protein made in peas caused immunological responses and inflammation in mice [33]. Nevertheless, no difference in immunogenicity was observed in mice fed with transgenic chickpeas expressing α-AI1 [33]. As a consequence, it is difficult to predict if similar immunogenic alterations will occur in an inhibitor made by transgenic coffee plants.

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