Insect Biochemistry and Molecular Biology 34 (2004) 903–918 www.elsevier.com/locate/ibmb A diverse family of serine proteinase genes expressed in cotton boll weevil (Anthonomus grandis): implications for the design of pest- resistant transgenic cotton plants Osmundo B. Oliveira-Neto a,b , Joa ˜o A.N. Batista a , Daniel J. Rigden a , Rodrigo R. Fragoso a,b , Rodrigo O. Silva a,b , Eliane A. Gomes c , Octa ´vio L. Franco a,d , Simoni C. Dias a,b , Ce ´lia M.T. Cordeiro a , Rose G. Monnerat a , Maria F. Grossi-de-Sa ´ a, a Embrapa Recursos Gene ´ticos e Biotecnologia, S.A.I.N. Parque Estac ¸a ˜ o Biolo ´ gica, Final W3, Asa Norte, Brasilia, DF 70770-900, Brazil b Universidade de Brası ´lia, Brasilia, DF 70910-900, Brazil c Embrapa Milho e Sorgo, Sete Lagoas, MG 35701-970, Brazil d Po ´ s-Graduac ¸a ˜o em Cie ˆncias Geno ˆ micas e Biotecnologia, Universidade Catolica de Brasilia, 916N Asa Norte, Brasilia, DF 70770-900, Brazil Received 22 May 2004; accepted 1 June 2004 Abstract 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 chymo- trypsin-like sequences belonging to a single family. Using a combination of 5 0 and 3 0 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 con- centrated in the gut of larvae and adult insects. Reverse northern analysis of the 14 cDNA fragments showed that only two tryp- sin-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. # 2004 Elsevier Ltd. All rights reserved. Keywords: Anthonomus grandis; Serine proteinases; cDNA cloning; Multigene family 1. Introduction Serine proteinases are one of a diverse group of enzymes capable of cleaving peptide bonds, and are involved in various essential processes such as intra- and extra-cellular protein metabolism, blood coagu- lation, immune response, fertilization and developmen- tal regulation, digestion, among others (Elvin et al., 1994; Barrett and Rawlings, 1995; Rao et al., 1998; Nagano et al., 2003). These enzymes have been detected in several insect orders including Coleoptera (Zhu and Baker, 1999), Diptera (Jiang et al., 1997) and Lepidoptera (Gate- Abbreviations: Agser, Anthonomus grandis serine proteinase; ARE, AU-rich element; BTCI, black-eyed pea trypsin/chymotrypsin inhibitor; CUB domain, complement-Uegf-BMP-1 domain; CI 0.95 , 95% confidence interval; RT-PCR, reverse transcription-polymerase chain reaction; SKTI, soybean Kunitz trypsin inhibitor; UTR, untranslated region  Corresponding author. Cenargen/Embrapa, S.A.I.N. Parque Rural, Final W5, Asa Norte, 70770-990, Brasilia, DF, Brazil. Tel.: +55-61-448-4705; fax: +55-61-340-3624. E-mail address: fatimasa@cenargen.embrapa.br (M.F. Grossi-de- Sa ´). 0965-1748/$ - see front matter # 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.ibmb.2004.06.001