Molecular cloning of alpha-amylases from cotton boll weevil, Anthonomus grandis and structural relations to plant inhibitors: an approach to insect resistance.
Anthonomus grandis, the cotton boll weevil, causes severe cotton crop losses in North and South America. Here we demonstrate the presence of starch in the cotton pollen grains and young ovules that are the main A. grandis food source. We further demonstrate the presence of alpha-amylase activity, an essential enzyme of carbohydrate metabolism for many crop pests, in A. grandis midgut. Two alpha-amylase cDNAs from A. grandis larvae were isolated using RT-PCR followed by 5' and 3' RACE techniques. These encode proteins with predicted molecular masses of 50.8 and 52.7kDa, respectively, which share 58% amino acid identity. Expression of both genes is induced upon feeding and concentrated in the midgut of adult insects. Several alpha-amylase inhibitors from plants were assayed against A. grandis alpha-amylases but, unexpectedly, only the BIII inhibitor from rye kernels proved highly effective, with inhibitors generally active against other insect amylases lacking effect. Structural modeling of Amylag1 and Amylag2 showed that different factors seem to be responsible for the lack of effect of 0.19 and alpha-AI1 inhibitors on A. grandis alpha-amylase activity. This work suggests that genetic engineering of cotton to express alpha-amylase inhibitors may offer a novel route to A. grandis resistance.
Embrapa Recursos Genéticos e Biotecnologia, Brasilia-DF, Brazil.
Osmundo B
OB
Oliveira-Neto
João A N
JA
Batista
Daniel J
DJ
Rigden
Octávio L
OL
Franco
Rosana
R
Falcão
Rodrigo R
RR
Fragoso
Luciane V
LV
Mello
Roseane C
RC
dos Santos
Maria F
MF
Grossi-de-Sá
D000595:Amino Acid Sequence; D000818:Animals; D003001:Cloning, Molecular; D001517:Coleoptera / Q000187:drug effects / Q000201:enzymology*; D018076:DNA, Complementary / Q000032:analysis; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D007305:Insecticide Resistance; D007814:Larva / Q000187:drug effects; D008969:Molecular Sequence Data; D010940:Plant Proteins / Q000737:chemistry* / Q000302:isolation & purification / Q000494:pharmacology; D012434:Secale / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000737:chemistry*; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000302:isolation & purification
D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D010940:Plant Proteins; D014361:Trypsin Inhibitors; D000516:alpha-Amylases