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Sezai Ercisli - One of the best experts on this subject based on the ideXlab platform.
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characterization of genetic diversity in turkish common Bean gene pool using phenotypic and whole genome dartseq generated silicodart marker information
PLOS ONE, 2018Co-Authors: Muhammad Azhar Nadeem, Ephrem Habyarimana, Vahdettin Çiftçi, Muhammad Amjad Nawaz, Tolga Karaköy, Gönül Cömertpay, Muhammad Qasim Shahid, Rüştü Hatipoğlu, Mehmet Zahit Yeken, Sezai ErcisliAbstract:Turkey presents a great diversity of common Bean landraces in farmers’ fields. We collected 183 common Bean accessions from 19 different Turkish geographic regions and 5 Scarlet Runner Bean accessions to investigate their genetic diversity and population structure using phenotypic information (growth habit, and seed weight, flower color, bracteole shape and size, pod shape and leaf shape and color), geographic provenance and 12,557 silicoDArT markers. A total of 24.14% markers were found novel. For the entire population (188 accessions), the expected heterozygosity was 0.078 and overall gene diversity, Fst and Fis were 0.14, 0.55 and 1, respectively. Using marker information, model-based structure, principal coordinate analysis (PCoA) and unweighted pair-group method with arithmetic means (UPGMA) algorithms clustered the 188 accessions into two main populations A (predominant) and B, and 5 unclassified genotypes, representing 3 meaningful heterotic groups for breeding purposes. Phenotypic information clearly distinguished these populations; population A and B, respectively, were bigger (>40g/100 seeds) and smaller (<40g/100 seeds) seed-sized. The unclassified population was pure and only contained climbing genotypes with 100 seed weight 2–3 times greater than populations A and B. Clustering was mainly based on A: seed weight, B: growth habit, C: geographical provinces and D: flower color. Mean kinship was generally low, but population B was more diverse than population A. Overall, a useful level of gene and genotypic diversity was observed in this work and can be used by the scientific community in breeding efforts to develop superior common Bean strains.
Robert B. Goldberg - One of the best experts on this subject based on the ideXlab platform.
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Using Giant Scarlet Runner Bean (Phaseolus coccineus) Embryos to Dissect the Early Events in Plant Embryogenesis.
Methods in Molecular Biology, 2020Co-Authors: Min Chen, Anhthu Q. Bui, Robert B. GoldbergAbstract:The giant embryo of the Scarlet Runner Bean (Phaseolus coccineus) has been used historically to investigate the molecular and developmental processes that control the early events of plant embryo development. In more recent years, our laboratory has been using Scarlet Runner Bean embryos to uncover the genes and regulatory events that control embryo proper and suspensor region differentiation shortly after fertilization. In this chapter we describe methods that we have developed to isolate Scarlet Runner Bean embryos at the globular stage of development, and capture embryo proper and suspensor regions by either hand dissection or laser capture microdissection (LCM) for use in downstream genomic analysis. These methods are also applicable for use in investigating the early events of common Bean (Phaseolus vulgaris) embryo development, a close relative of Scarlet Runner Bean, which also has a giant embryo in addition to a sequenced genome.
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A shared cis-regulatory module activates transcription in the suspensor of plant embryos.
Proceedings of the National Academy of Sciences, 2018Co-Authors: Kelli F. Henry, Anhthu Q. Bui, Tomokazu Kawashima, Robert B. GoldbergAbstract:The mechanisms controlling the transcription of gene sets in specific regions of a plant embryo shortly after fertilization remain unknown. Previously, we showed that G564 mRNA, encoding a protein of unknown function, accumulates to high levels in the giant suspensor of both Scarlet Runner Bean (SRB) and Common Bean embryos, and a cis-regulatory module containing three unique DNA sequences, designated as the 10-bp, Region 2, and Fifth motifs, is required for G564 suspensor-specific transcription [Henry KF, et al. (2015) Plant Mol Biol 88:207-217; Kawashima T, et al. (2009) Proc Natl Acad Sci USA 106:3627-3632]. We tested the hypothesis that these motifs are also required for transcription of the SRB GA 20-oxidase gene, which encodes a gibberellic acid hormone biosynthesis enzyme and is coexpressed with G564 at a high level in giant Bean suspensors. We used deletion and gain-of-function experiments in transgenic tobacco embryos to show that two GA 20-oxidase DNA regions are required for suspensor-specific transcription, one in the 5' UTR (+119 to +205) and another in the 5' upstream region (-341 to -316). Mutagenesis of sequences in these two regions determined that the cis-regulatory motifs required for G564 suspensor transcription are also required for GA 20-oxidase transcription within the suspensor, although the motif arrangement differs. Our results demonstrate the flexibility of motif positioning within a cis-regulatory module that activates gene transcription within giant Bean suspensors and suggest that G564 and GA 20-oxidase comprise part of a suspensor gene regulatory network.
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A shared cis-regulatory module activates transcription in the suspensor of plant embryos
2018Co-Authors: Kelli F. Henry, Anhthu Q. Bui, Tomokazu Kawashima, Robert B. GoldbergAbstract:The mechanisms controlling the transcription of gene sets in specific regions of a plant embryo shortly after fertilization remain unknown. Previously, we showed that G564 mRNA, encoding a protein of unknown function, accumulates to high levels in the giant suspensor of both Scarlet Runner Bean (SRB) and Common Bean embryos, and a cis-regulatory module containing three unique DNA sequences, designated as the 10-bp, Region 2, and Fifth motifs, is required for G564 suspensor-specific transcription [Henry, K. F. et al., Plant Mol. Biol. 88(3):207-217 (2015); Kawashima, T. et al., Proc. Natl. Acad. Sci USA 106(9):3627-3632 (2009)]. We tested the hypothesis that these motifs are also required for transcription of the SRB GA 20-oxidase gene, which encodes a gibberellic acid hormone biosynthesis enzyme and is co-expressed with G564 at a high level in giant Bean suspensors. We used deletion and gain-of-function experiments in transgenic tobacco embryos to show that two GA 20-oxidase DNA regions are required for suspensor-specific transcription − one in the 5′ untranslated region (UTR) (+119 to +205) and another in the 5′ upstream region (-341 to -316). Mutagenesis of sequences in these two regions determined that the cis-regulatory motifs required for G564 suspensor transcription are also required for GA 20-oxidase transcription within the suspensor, although the motif arrangement differs. Our results demonstrate the flexibility of motif positioning within a cis-regulatory module that activates gene transcription within giant Bean suspensors, and suggest that G564 and GA 20-oxidase comprise part of a suspensor gene regulatory network.
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A cis-regulatory module activating transcription in the suspensor contains five cis-regulatory elements
Plant Molecular Biology, 2015Co-Authors: Kelli F. Henry, Tomokazu Kawashima, Robert B. GoldbergAbstract:Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean ( Phaseolus coccineus ) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis -regulatory sequences, including the 10-bp motif (5′-GAAAAGCGAA-3′), the 10 bp-like motif (5′-GAAAAACGAA-3′), and Region 2 motif (partial sequence 5′-TTGGT-3′). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis -regulatory elements within the 54-bp cis -regulatory module that are required for G564 suspensor transcription: the Fifth motif (5′-GAGTTA-3′) and a third 10-bp-related sequence (5′-GAAAACCACA-3′). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis -regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis -regulatory module was found upstream of the G564 ortholog in the Common Bean ( Phaseolus vulgaris ), indicating that the regulation of G564 is evolutionarily conserved in closely related Bean species.
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A cis -regulatory module activating transcription in the suspensor contains five cis -regulatory elements
Plant Molecular Biology, 2015Co-Authors: Kelli F. Henry, Tomokazu Kawashima, Robert B. GoldbergAbstract:Little is known about the molecular mechanisms by which the embryo proper and suspensor of plant embryos activate specific gene sets shortly after fertilization. We analyzed the upstream region of the Scarlet Runner Bean (Phaseolus coccineus) G564 gene in order to understand how genes are activated specifically in the suspensor during early embryo development. Previously, we showed that a 54-bp fragment of the G564 upstream region is sufficient for suspensor transcription and contains at least three required cis-regulatory sequences, including the 10-bp motif (5′-GAAAAGCGAA-3′), the 10 bp-like motif (5′-GAAAAACGAA-3′), and Region 2 motif (partial sequence 5′-TTGGT-3′). Here, we use site-directed mutagenesis experiments in transgenic tobacco globular-stage embryos to identify two additional cis-regulatory elements within the 54-bp cis-regulatory module that are required for G564 suspensor transcription: the Fifth motif (5′-GAGTTA-3′) and a third 10-bp-related sequence (5′-GAAAACCACA-3′). Further deletion of the 54-bp fragment revealed that a 47-bp fragment containing the five motifs (the 10-bp, 10-bp-like, 10-bp-related, Region 2 and Fifth motifs) is sufficient for suspensor transcription, and represents a cis-regulatory module. A consensus sequence for each type of motif was determined by comparing motif sequences shown to activate suspensor transcription. Phylogenetic analyses suggest that the regulation of G564 is evolutionarily conserved. A homologous cis-regulatory module was found upstream of the G564 ortholog in the Common Bean (Phaseolus vulgaris), indicating that the regulation of G564 is evolutionarily conserved in closely related Bean species. Electronic supplementary material The online version of this article (doi:10.1007/s11103-015-0308-z) contains supplementary material, which is available to authorized users.
Singh, Shree P. - One of the best experts on this subject based on the ideXlab platform.
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Selecting common Bean with genes of different evolutionary origins for resistance to Xanthomonas campestris pv. phaseoli
'Crop Science Society of America', 2007Co-Authors: Lema Márquez Margarita, Terán Henry, Singh, Shree P.Abstract:Common bacterial blight (CBB) is an important seed-borne disease of common Bean (Phaseolus vulgaris L.). Low levels of resistance occur in the common and Scarlet Runner Bean (Phaseolus coccineus L.), with higher levels available in the tepary Bean (Phaseolus acutifolius A. Gray). Germplasm lines with CBB resistance separately from each of the three Phaseolus species and pyramided resistance are available. The objectives of this study were to: (i) determine the main and interaction effects of two isolates of Xanthomonas campestris pv. phaseoli (Xcp), one from Colorado and one from Wisconsin, and their inoculum densities with CBB resistance from the three species separately and pyramided; and (ii) identify the most useful germplasm for breeding for resistance. Thirty-one genotypes were evaluated at 14 and 21 d after inoculation (DAI) using two inoculum densities in 2005 and three in 2006 of each of the two Xcp isolates. Large differences in response to Colorado and Wisconsin Xcp isolates, densities, and evaluation time were observed. The resistance derived from the three species separately was not effective against the aggressive Wisconsin Xcp isolate at higher densities ≥108, especially at 21 DAI. Resistance pyramided with the tepary Bean was the most effective. No crossover interactions were observed between the 31 common Bean germplasm sources and the two Xcp isolates at any density and evaluation date. Use of only pyramided resistance for breeding is advised.Peer reviewe
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Inheritance of white mold resistance in Phaseolus vulgaris × P. coccineus crosses
'Scientific Societies', 2006Co-Authors: Schwartz, Howard F., Lema Márquez Margarita, Terán Henry, Otto Kristen, Singh, Shree P.Abstract:The fungus Sclerotinia sclerotiorum, cause of white mold, is known to attack >400 plant species. It is a widespread problem in dry Bean (Phaseolus vulgaris) in the United States, causing >30% average yield losses. Low to moderate levels of resistance are found in dry Bean. However, some accessions of P. coccineus (commonly known as Scarlet Runner Bean) possess a relatively higher level of resistance. Our objective was to verify the reaction of 13 known white mold-resistant P. coccineus germ plasms and determine inheritance of resistance in accessions PI 433246 and PI 439534. Pinto Othello was crossed with PI 433246, and the resulting interspecific F1 was back-crossed onto Othello and allowed to produce F2 seed. Similarly, pinto UI 320 was crossed with PI 439534. The F1 was backcrossed onto UI 320 and allowed to produce F2 seed. The two parents, F1, F2, and backcross to dry Bean of each set were evaluated in the greenhouse using the straw test at Fort Collins, CO in 2004. All 13 P. coccineus accessions and the two F2 also were evaluated using the modified petiole test at Kimberly, ID in 2005. All 13 P. coccineus accessions were variable in a 2002 straw test when rated for white mold reaction on a 1-to-9 scale, because the mean disease score ranged from 1.9 for PI 433246 to 4.4 for PI 189023 and 8.8 for the susceptible check Bill Z. For the petiole test, when rated on a 1-to-9 scale, the accessions exhibited an intermediate white mold score of 4 or 5 in 2005. In 2004, the susceptible check Othello exhibited a mean score of 7.9 compared with 3.4, 3.2, and 2.1 for PI 433246, UI 320, and PI 439534, respectively. The white mold reaction of PI 433246 and PI 439534 was dominant in their respective F1. The F2 segregation further indicated that white mold resistance in PI 433246 and PI 439534 was controlled by a single dominant gene. These two and other white mold-resistant P. coccineus accessions and selected breeding lines from the interspecific crosses should be useful for future improvement of white mold resistance of pinto and other market classes of dry and green or snap Bean.Financial support was provided by the Colorado Dry Bean Administrative Committee, Colorado Seed Growers Association, and USDA-ARS National Sclerotinia Initiative Grant No. 58-5442-2-256, entitled “Introgressing White Mold Resistance from the Secondary Gene Pool of Common Bean.”We thank M. Welsh, Phaseolus collection curator, for supplying seed of 13 P. coccineus accessions.Colorado Dry Bean Administrative CommitteeColorado Seed Growers AssociationUSDA-ARS National Sclerotinia InitiativePeer reviewe
Shree P. Singh - One of the best experts on this subject based on the ideXlab platform.
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Selecting Common Bean with Genes of Different Evolutionary Origins for Resistance to Xanthomonas campestris pv. phaseoli
Crop Science, 2007Co-Authors: Margarita Lema Márquez, Henry Terán, Shree P. SinghAbstract:Common bacterial blight (CBB) is an important seed-borne disease of common Bean (Phaseolus vulgaris L.). Low levels of resistance occur in the common and Scarlet Runner Bean (Phaseolus coccineus L.), with higher levels available in the tepary Bean (Phaseolus acutifolius A. Gray). Germplasm lines with CBB resistance separately from each of the three Phaseolus species and pyramided resistance are available. The objectives of this study were to: (i) determine the main and interaction effects of two isolates of Xanthomonas campestris pv. phaseoli (Xcp), one from Colorado and one from Wisconsin, and their inoculum densities with CBB resistance from the three species separately and pyramided; and (ii) identify the most useful germplasm for breeding for resistance. Thirty-one genotypes were evaluated at 14 and 21 d after inoculation (DAI) using two inoculum densities in 2005 and three in 2006 of each of the two Xcp isolates. Large differences in response to Colorado and Wisconsin Xcp isolates, densities, and evaluation time were observed. The resistance derived from the three species separately was not effective against the aggressive Wisconsin Xcp isolate at higher densities ≥10 8 , especially at 21 DAI. Resistance pyramided with the tepary Bean was the most effective. No crossover interactions were observed between the 31 common Bean germplasm sources and the two Xcp isolates at any density and evaluation date. Use of only pyramided resistance for breeding is advised.
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Inheritance of White Mold Resistance in Phaseolus vulgaris × P. coccineus Crosses.
Plant Disease, 2006Co-Authors: Howard F. Schwartz, Henry Terán, Kristen Otto, Margarita Lema, Shree P. SinghAbstract:Schwartz, H. F., Otto, K., Teran, H., Lema, M., and Singh, S. P. 2006. Inheritance of white mold resistance in Phaseolus vulgaris × P. coccineus crosses. Plant Dis. 90:1167-1170. The fungus Sclerotinia sclerotiorum, cause of white mold, is known to attack >400 plant species. It is a widespread problem in dry Bean (Phaseolus vulgaris) in the United States, causing >30% average yield losses. Low to moderate levels of resistance are found in dry Bean. However, some accessions of P. coccineus (commonly known as Scarlet Runner Bean) possess a relatively higher level of resistance. Our objective was to verify the reaction of 13 known white mold-resistant P. coccineus germ plasms and determine inheritance of resistance in accessions PI 433246 and PI 439534. Pinto Othello was crossed with PI 433246, and the resulting interspecific F1 was backcrossed onto Othello and allowed to produce F2 seed. Similarly, pinto UI 320 was crossed with PI 439534. The F1 was backcrossed onto UI 320 and allowed to produce F2 seed. The two parents, F1, F2, and backcross to dry Bean of each set were evaluated in the greenhouse using the straw test at Fort Collins, CO in 2004. All 13 P. coccineus accessions and the two F2 also were evaluated using the modified petiole test at Kimberly, ID in 2005. All 13 P. coccineus accessions were variable in a 2002 straw test when rated for white mold reaction on a 1-to-9 scale, because the mean disease score ranged from 1.9 for PI 433246 to 4.4 for PI 189023 and 8.8 for the susceptible check Bill Z. For the petiole test, when rated on a 1-to-9 scale, the accessions exhibited an intermediate white mold score of 4 or 5 in 2005. In 2004, the susceptible check Othello exhibited a mean score of 7.9 compared with 3.4, 3.2, and 2.1 for PI 433246, UI 320, and PI 439534, respectively. The white mold reaction of PI 433246 and PI 439534 was dominant in their respective F1. The F2 segregation further indicated that white mold resistance in PI 433246 and PI 439534 was controlled by a single dominant gene. These two and other white moldresistant P. coccineus accessions and selected breeding lines from the interspecific crosses should be useful for future improvement of white mold resistance of pinto and other market classes of dry and green or snap Bean.
Jiménez, Arnubio Valencia - One of the best experts on this subject based on the ideXlab platform.
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As enzimas presentes no trato digestivo dos insetos : um alvo susceptível de inibição
2010Co-Authors: Jiménez, Arnubio ValenciaAbstract:Sementes do feijão (Phaseolus coccineus) foram analisadas pela presença e atividade do inibidor de amilase (-AI). Por meio do uso de anticorpos policlonais gerados contra o inibidor -AI-1 do feijão comum (P. vulgaris), foi possível detectar polipeptídios típicos deste inibidor (Mr. 14 18 kDa), além do polipeptídeo de 32 kDa. Os resultados encontrados nos testes de inibição permitem sugerir que o inibidor presente nas sementes de P. coccineus 35590 seria o melhor candidato para a transformação genética do café visando à geração de plantas com resistência a larvas da broca-do-café. A digestão proteolítica in vitro do inibidor puro causou uma perda rápida da sua atividade inibitória, afetando seriamente sua capacidade natural de interagir com as -amilases. Também, neste trabalho foram estudadas as propriedades das -amilases presentes no trato digestivo de larvas de Tecia solanivora, um inseto-praga que gera um dano importante à batata (Solanum tuberosum). O inseto tem no mínimo três isoformas principais de -amilases digestivas, as quais apresentaram pontos isoelétricos de 5.30, 5.70 e 5.98. A atividade da -amilase digestiva tem um pH ótimo entre 7.0 e 10.0, com um pico de atividade em pH 9.0. A enzima foi inibida por inibidores de natureza protéica presentes nas sementes de P. coccineus (70% de inibição) e de P. vulgaris cv Radical (87% de inibição) em pH 6.0. Os resultados mostram também que o inibidor de - amilase de amaranto pode ser o melhor candidato para gerar batatas modificadas geneticamente resistentes a este inseto-praga. Futuras pesquisas precisam ser desenvolvidas para esclarecer se estes inibidores de -amilase são resistentes às proteases presentes no trato digestivo da lagarta de batata, T. solanivora. Em um ensaio subseqüente, foi desenvolvido e testado um novo e sensível método espectrofotométrico para a detecção da atividada leucine aminopeptidase (-aminoacyl-peptide hydrolase, EC 3.4.11.1) proveniente do trato digestivo de larvas de Telchin licus (Lepidoptera: Castniidae), fazendo uso do substrato L-leucyl-2- naphthylamide. _______________________________________________________________________________ ABSTRACTSeeds of Scarlet Runner Bean (Phaseolus coccineus L.) were analyzed for -amylase inhibitor (-AI) activity. By using polyclonal antibodies raised against pure a−AI-1 from common Bean (Phaseolus vulgaris L.) it was possible to detect the typical -AI polypeptides (Mr 14 -18 kDa) as well as a large polypeptide of Mr 32,000 Da. Differential inhibition curves lead us to suggest that the gene encoding one of the inhibitors in P. coccineus (in accession G35590) would be a good candidate for genetic engineering of coffee resistance towards the coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae). An in vitro proteolytic digestion treatment of pure a−AI−1, resulted in a rapid loss of the inhibitory activity, seriously affecting its natural capacity to interact with mammalian -amylases. In addition, the properties of the digestive -amylase from T. solanivora larvae, an important insect pest of potato (Solanum tuberosum), were studied. This insect has three major digestive -amylases with isoelectric points 5.30; 5.70 and 5.98 respectively, which were separated using native and isoelectric focusing gels. The -amylase activity has an optimum pH between 7.0 and 10.0 with a peak at pH 9.0. The enzyme was inhibited by proteinaceous inhibitors from P. coccineus (70% inhibition) and P. vulgaris cv. Radical (87% inhibition) at pH 6.0. The results show that the -amylase inhibitor from Amaranthus may be a better candidate to make genetically modified potatoes resistant to this insect. However, it is necessary to know if these -amylase inhibitors are resistant to the gut proteases from T. solanivora. On the other hand, a simple and sensitive spectrophotometric assay to determine the activity of leucine aminopeptidase (-aminoacyl-peptide hydrolase, EC 3.4.11.1) from insect intestinal tracts of Telchin licus (Lepidoptera: Castniidae), using L-leucyl-2- naphthylamide as substrate, was developed
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As enzimas presentes no trato digestivo dos insetos : um alvo susceptível de inibição
2009Co-Authors: Jiménez, Arnubio ValenciaAbstract:Tese (doutorado)—Universidade de Brasília, Instituto de Ciências Biológicas, Departamento de Biologia Celular, 2009.Sementes do feijão (Phaseolus coccineus) foram analisadas pela presença e atividade do inibidor de amilase (-AI). Por meio do uso de anticorpos policlonais gerados contra o inibidor -AI-1 do feijão comum (P. vulgaris), foi possível detectar polipeptídios típicos deste inibidor (Mr. 14 18 kDa), além do polipeptídeo de 32 kDa. Os resultados encontrados nos testes de inibição permitem sugerir que o inibidor presente nas sementes de P. coccineus 35590 seria o melhor candidato para a transformação genética do café visando à geração de plantas com resistência a larvas da broca-do-café. A digestão proteolítica in vitro do inibidor puro causou uma perda rápida da sua atividade inibitória, afetando seriamente sua capacidade natural de interagir com as -amilases. Também, neste trabalho foram estudadas as propriedades das -amilases presentes no trato digestivo de larvas de Tecia solanivora, um inseto-praga que gera um dano importante à batata (Solanum tuberosum). O inseto tem no mínimo três isoformas principais de -amilases digestivas, as quais apresentaram pontos isoelétricos de 5.30, 5.70 e 5.98. A atividade da -amilase digestiva tem um pH ótimo entre 7.0 e 10.0, com um pico de atividade em pH 9.0. A enzima foi inibida por inibidores de natureza protéica presentes nas sementes de P. coccineus (70% de inibição) e de P. vulgaris cv Radical (87% de inibição) em pH 6.0. Os resultados mostram também que o inibidor de - amilase de amaranto pode ser o melhor candidato para gerar batatas modificadas geneticamente resistentes a este inseto-praga. Futuras pesquisas precisam ser desenvolvidas para esclarecer se estes inibidores de -amilase são resistentes às proteases presentes no trato digestivo da lagarta de batata, T. solanivora. Em um ensaio subseqüente, foi desenvolvido e testado um novo e sensível método espectrofotométrico para a detecção da atividada leucine aminopeptidase (-aminoacyl-peptide hydrolase, EC 3.4.11.1) proveniente do trato digestivo de larvas de Telchin licus (Lepidoptera: Castniidae), fazendo uso do substrato L-leucyl-2- naphthylamide. _______________________________________________________________________________ ABSTRACTSeeds of Scarlet Runner Bean (Phaseolus coccineus L.) were analyzed for -amylase inhibitor (-AI) activity. By using polyclonal antibodies raised against pure a−AI-1 from common Bean (Phaseolus vulgaris L.) it was possible to detect the typical -AI polypeptides (Mr 14 -18 kDa) as well as a large polypeptide of Mr 32,000 Da. Differential inhibition curves lead us to suggest that the gene encoding one of the inhibitors in P. coccineus (in accession G35590) would be a good candidate for genetic engineering of coffee resistance towards the coffee berry borer Hypothenemus hampei Ferrari (Coleoptera: Scolytidae). An in vitro proteolytic digestion treatment of pure a−AI−1, resulted in a rapid loss of the inhibitory activity, seriously affecting its natural capacity to interact with mammalian -amylases. In addition, the properties of the digestive -amylase from T. solanivora larvae, an important insect pest of potato (Solanum tuberosum), were studied. This insect has three major digestive -amylases with isoelectric points 5.30; 5.70 and 5.98 respectively, which were separated using native and isoelectric focusing gels. The -amylase activity has an optimum pH between 7.0 and 10.0 with a peak at pH 9.0. The enzyme was inhibited by proteinaceous inhibitors from P. coccineus (70% inhibition) and P. vulgaris cv. Radical (87% inhibition) at pH 6.0. The results show that the -amylase inhibitor from Amaranthus may be a better candidate to make genetically modified potatoes resistant to this insect. However, it is necessary to know if these -amylase inhibitors are resistant to the gut proteases from T. solanivora. On the other hand, a simple and sensitive spectrophotometric assay to determine the activity of leucine aminopeptidase (-aminoacyl-peptide hydrolase, EC 3.4.11.1) from insect intestinal tracts of Telchin licus (Lepidoptera: Castniidae), using L-leucyl-2- naphthylamide as substrate, was developed
