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Gene characterization of two digestive serine proteases in Sitodiplosis mosellana: implications for alternative control strategies

  • Lourdes D. Arrueta (a1), Richard H. Shukle (a2), Ian L. Wise (a3) and Omprakash Mittapalli (a1)

Abstract

Two full-length cDNA sequences encoding digestive serine proteases (designated as SmPROT-1 and SmPROT-2) were recovered from the midgut of the orange wheat blossom midge, Sitodiplosis mosellana (Géhin) (Diptera: Cecidomyiidae), in an ongoing expressed sequence tag project. The deduced amino acid sequences shared homology with digestive serine proteases from insect and non-insect species, including conserved regions such as the catalytic triad, active pocket, and conserved structural motifs. Secretory signal peptides in both proteases at the N-terminals indicate that these proteins could function as midgut digestive serine proteases. A phylogenetic analysis grouped SmPROT-1 and SmPROT-2 with trypsin-like and chymotrysin-like serine proteases, respectively. Quantitative real-time PCR analysis showed that SmPROT-1 and SmPROT-2 were expressed predominantly in the midgut rather than in other tissues (fat body and salivary glands). Expression analyses revealed high mRNA levels for the feeding instars (1st- and 2nd-instar larvae) compared with other stages (neonate, 3rd instar, pupa, and adult). These results provide new insights into the biology of S. mosellana and are discussed in the context of developing alternative control strategies.

Nous avons récupéré deux séquences complètes d'ADN complémentaire qui codent pour les sérines protéases digestives (désignées SmPROT-1 et SmPROT-2) dans le tube digestif moyen de la cécidomyie orangée du blé, Sitodiplosis mosellana (Géhin) (Dipera: Cecidomyiidae), dans le cadre d'une étude en cours sur les marqueurs de séquences exprimées. Les séquences d'acides aminés déduites partagent des homologies avec les sérines protéases digestives d'espèces d'insectes et de non insectes, incluant les régions conservées, telles que la triade catalytique, la poche d'interaction et les motifs structuraux conservés. Des peptides de signal de sécrétion dans les deux protéases aux terminaux N indiquent que ces protéines pourraient servir de sérines protéases digestives dans le tube digestif moyen. Une analyse phylogénétique regroupe SmPROT-1 et SmPROT-2 respectivement avec les sérines protéases de type trypsine et chymotrysine. Une analyse d'amplification en chaîne par polymérase (PCR) quantitative en temps réel montre que SmPROT-1 et SmPROT-2 sont exprimées plus dans le tube digestif moyen par comparaison aux autres tissus (corps gras et glandes salivaires). Des analyses d'expression génique montrent des concentrations élevées d'ARNm chez les stades qui s’alimentent (larves de 1er et 2e stades) par rapport aux autres stades (néonates, larves de 3e stade, nymphes et adultes). Nos résultats ouvrent de nouvelles perspectives sur la biologie de S. mosellana; nous en discutons dans le contexte de la mise au point de stratégies de contrôle de rechange.

[Traduit par la Rédaction]

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Corresponding author

1 Corresponding author (e-mail: mittapalli.1@osu.edu; omittapalli@gmail.com).

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Gene characterization of two digestive serine proteases in Sitodiplosis mosellana: implications for alternative control strategies

  • Lourdes D. Arrueta (a1), Richard H. Shukle (a2), Ian L. Wise (a3) and Omprakash Mittapalli (a1)

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