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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 Gen├ęticos e Biotecnologia, Bras├şlia-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

PCRs of positive C. arabica plants, showing nptII and ╬▒-AI1 amplifications PCRs from T0 (A and B) and T1 plants (C and D). A, 204-bp band from the ╬▒-AI1 gene of T0 plants. B, 411-bp band from the nptII gene of T0 plants. M, 1 kb plus leader; 1 - 6, T0 transformed coffee plants; 7, non-transgenic coffee plant; 8 positive control (pBIN19╬▒-AI1 vector). C, 204-bp band from the ╬▒-AI1 gene of T1 plants. D, 411-bp band from the nptII gene of T1 plants. M, 1 kb plus leader; 1 - 11, positive T1 plants; 12, positive control (pBIN19╬▒-AI1 vector); 13 non-transgenic coffee plant.
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Figure 3: PCRs of positive C. arabica plants, showing nptII and ╬▒-AI1 amplifications PCRs from T0 (A and B) and T1 plants (C and D). A, 204-bp band from the ╬▒-AI1 gene of T0 plants. B, 411-bp band from the nptII gene of T0 plants. M, 1 kb plus leader; 1 - 6, T0 transformed coffee plants; 7, non-transgenic coffee plant; 8 positive control (pBIN19╬▒-AI1 vector). C, 204-bp band from the ╬▒-AI1 gene of T1 plants. D, 411-bp band from the nptII gene of T1 plants. M, 1 kb plus leader; 1 - 11, positive T1 plants; 12, positive control (pBIN19╬▒-AI1 vector); 13 non-transgenic coffee plant.

Mentions: Six PCR positive coffee plants (T0) were obtained after biolistic transformation, showing the presence of both nptII and ╬▒-AI1 genes (Figures 3A and 3B). The transformation efficiency obtained in this work was 23.1% (6 positive: 20 negative plants). Although generation of spontaneous kanamycin resistance is very well documented [27], the low efficiency may also be a consequence of unstable genome insertion.


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)

PCRs of positive C. arabica plants, showing nptII and ╬▒-AI1 amplifications PCRs from T0 (A and B) and T1 plants (C and D). A, 204-bp band from the ╬▒-AI1 gene of T0 plants. B, 411-bp band from the nptII gene of T0 plants. M, 1 kb plus leader; 1 - 6, T0 transformed coffee plants; 7, non-transgenic coffee plant; 8 positive control (pBIN19╬▒-AI1 vector). C, 204-bp band from the ╬▒-AI1 gene of T1 plants. D, 411-bp band from the nptII gene of T1 plants. M, 1 kb plus leader; 1 - 11, positive T1 plants; 12, positive control (pBIN19╬▒-AI1 vector); 13 non-transgenic coffee plant.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 3: PCRs of positive C. arabica plants, showing nptII and ╬▒-AI1 amplifications PCRs from T0 (A and B) and T1 plants (C and D). A, 204-bp band from the ╬▒-AI1 gene of T0 plants. B, 411-bp band from the nptII gene of T0 plants. M, 1 kb plus leader; 1 - 6, T0 transformed coffee plants; 7, non-transgenic coffee plant; 8 positive control (pBIN19╬▒-AI1 vector). C, 204-bp band from the ╬▒-AI1 gene of T1 plants. D, 411-bp band from the nptII gene of T1 plants. M, 1 kb plus leader; 1 - 11, positive T1 plants; 12, positive control (pBIN19╬▒-AI1 vector); 13 non-transgenic coffee plant.
Mentions: Six PCR positive coffee plants (T0) were obtained after biolistic transformation, showing the presence of both nptII and ╬▒-AI1 genes (Figures 3A and 3B). The transformation efficiency obtained in this work was 23.1% (6 positive: 20 negative plants). Although generation of spontaneous kanamycin resistance is very well documented [27], the low efficiency may also be a consequence of unstable genome insertion.

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 Gen├ęticos e Biotecnologia, Bras├şlia-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