A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest-resistant transgenic cotton plants.
Fourteen different cDNA fragments encoding serine proteinases were isolated by reverse transcription-PCR from cotton boll weevil (Anthonomus grandis) larvae. A large diversity between the sequences was observed, with a mean pairwise identity of 22% in the amino acid sequence. The cDNAs encompassed 11 trypsin-like sequences classifiable into three families and three chymotrypsin-like sequences belonging to a single family. Using a combination of 5' and 3' RACE, the full-length sequence was obtained for five of the cDNAs, named Agser2, Agser5, Agser6, Agser10 and Agser21. The encoded proteins included amino acid sequence motifs of serine proteinase active sites, conserved cysteine residues, and both zymogen activation and signal peptides. Southern blotting analysis suggested that one or two copies of these serine proteinase genes exist in the A. grandis genome. Northern blotting analysis of Agser2 and Agser5 showed that for both genes, expression is induced upon feeding and is concentrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two trypsin-like and two chymotrypsin-like were expressed at detectable levels. Under the effect of the serine proteinase inhibitors soybean Kunitz trypsin inhibitor and black-eyed pea trypsin/chymotrypsin inhibitor, expression of one of the trypsin-like sequences was upregulated while expression of the two chymotrypsin-like sequences was downregulated.
Oliveira-Neto
Osmundo B
OB
Embrapa Recursos Genéticos e Biotecnologia, S.A.I.N. Parque Estação Biológica, Final W3, Asa Norte, Brasilia, DF 70770-900, Brazil.
Batista
João A N
JA
Rigden
Daniel J
DJ
Fragoso
Rodrigo R
RR
Silva
Rodrigo O
RO
Gomes
Eliane A
EA
Franco
Octávio L
OL
Dias
Simoni C
SC
Cordeiro
Célia M T
CM
Monnerat
Rose G
RG
Grossi-De-Sá
Maria F
MF
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000032:analysis; D003368:Gossypium / Q000235:genetics; D007814:Larva / Q000201:enzymology / Q000235:genetics; D008969:Molecular Sequence Data; D005810:Multigene Family; D010572:Pest Control, Biological / Q000379:methods; D030821:Plants, Genetically Modified; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012697:Serine Endopeptidases / Q000096:biosynthesis* / Q000235:genetics*; D014361:Trypsin Inhibitors / Q000494:pharmacology; D048889:Weevils / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development
D018076:DNA, Complementary; D014361:Trypsin Inhibitors; D012697:Serine Endopeptidases