title: Molecular cloning and characterization of an α-amylase cDNA highly expressed in major feeding stages of the coffee berry borer, Hypothenemus hampei.
abstract: α-Amylases are common enzymes responsible for hydrolyzing starch. Insect-pests, whose larvae develop in seeds, rely obligatorily on α-amylase activity to digest starch, as their major food source. Considering the relevance of insect α-amylases and the natural α-amylase inhibitors present in seeds to protect from insect damage, we report here the molecular cloning and nucleotide sequence of the full-length AmyHha cDNA of the coffee berry borer, Hypothenemus hampei, a major insect-pest of coffee crops. The AmyHha sequence has 1879 bp, containing a 1458 bp open reading frame, which encodes a predicted protein with 485 amino acid residues, with a predicted molecular mass of 51.2 kDa. The deduced protein showed 55-79% identity to other insect α-amylases, including Anthonomus grandis, Ips typographus and Sitophilus oryzae α-amylases. In depth analysis revealed that the highly conserved three amino acid residues (Asp184, Glu220, and Asp285), which compose the catalytic site are also presented in AmyHha amylase. The AmyHha gene seems to be a single copy in the haploid genome and AmyHha transcription levels were found higher in L2 larvae and adult insects, both corresponding to major feeding phases. Modeling of the AmyHha predicted protein uncovered striking structural similarities to the Tenebrio molitor α-amylase also displaying the same amino acid residues involved in enzyme catalysis (Asp184, Glu220 and Asp285). Since AmyHha gene was mostly transcribed in the intestinal tract of H. hampei larvae, the cognate α-amylase could be considered a high valuable target to coffee bean insect control by biotechnological strategies.
authors.affiliation: Catholic University of Brasilia, Brasília, DF, Brazil; Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil.
authors.forename: C A
authors.initials: CA
authors.lastname: Bezerra
authors.affiliation: Catholic University of Brasilia, Brasília, DF, Brazil; Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil.
authors.forename: L L P
authors.initials: LL
authors.lastname: Macedo
authors.affiliation: Federal University of Rio Grande do Norte, Natal, RN, Brazil.
authors.forename: T M L
authors.initials: TM
authors.lastname: Amorim
authors.affiliation: Federal University of Rio Grande do Norte, Natal, RN, Brazil.
authors.forename: V O
authors.initials: VO
authors.lastname: Santos
authors.affiliation: Embrapa Cerrados, Planaltina, DF, Brazil.
authors.forename: R R
authors.initials: RR
authors.lastname: Fragoso
authors.affiliation: Embrapa Cotton, Campina Grande, PB, Brazil.
authors.forename: W A
authors.initials: WA
authors.lastname: Lucena
authors.affiliation: Agronomic Institute of Paraná, Londrina, PR, Brazil.
authors.forename: A M
authors.initials: AM
authors.lastname: Meneguim
authors.affiliation: University of Caldas, Manizales, Colombia.
authors.forename: A
authors.initials: A
authors.lastname: Valencia-Jimenez
authors.affiliation: Institut National de la Recherche Agronomique, Sophia-Antipolis, France.
authors.forename: G
authors.initials: G
authors.lastname: Engler
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil.
authors.forename: M C M
authors.initials: MC
authors.lastname: Silva
authors.affiliation: Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil.
authors.forename: E V S
authors.initials: EV
authors.lastname: Albuquerque
authors.affiliation: Catholic University of Brasilia, Brasília, DF, Brazil; Embrapa Genetic Resources and Biotechnology, Brasília, DF, Brazil. Electronic address: fatima.grossi@embrapa.br.
authors.forename: M F
authors.initials: MF
authors.lastname: Grossi-de-Sa
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D001517:Coleoptera / Q000145:classification / Q000201:enzymology / Q000502:physiology*; D018076:DNA, Complementary / Q000235:genetics*; D005247:Feeding Behavior; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016366:Open Reading Frames; D010802:Phylogeny; D060888:Real-Time Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D000516:alpha-Amylases / Q000737:chemistry / Q000235:genetics*
chemical_list: D018076:DNA, Complementary; D000516:alpha-Amylases
keywords: Curculionidae; Enzyme modeling; Gene expression; Insect pest; Starch hydrolysis