The Colorado potato beetle (CPB), Leptinotarsa decemlineata, is a major pest of potato plants, and its digestive system is a promising target for development of pest control strategies. This work focuses on functional proteomic analysis of the digestive proteolytic enzymes expressed in the CPB gut. We identified a set of peptidases using imaging with specific activity-based probes and activity profiling with selective substrates and inhibitors. The secreted luminal peptidases were classified as: (i) endopeptidases of cathepsin D, cathepsin L, and trypsin types and (ii) exopeptidases with aminopeptidase (cathepsin H), carboxypeptidase (serine carboxypeptidase, prolyl carboxypeptidase), and carboxydipeptidase (cathepsin B) activities. The proteolytic arsenal also includes non-luminal peptidases with prolyl oligopeptidase and metalloaminopeptidase activities. Our results indicate that the CPB gut employs a multienzyme network of peptidases with complementary specificities to efficiently degrade ingested proteins. This proteolytic system functions in both CPB larvae and adults and is controlled mainly by cysteine and aspartic peptidases and supported by serine and metallopeptidases. The component enzymes identified here are potential targets for inhibitors with tailored specificities that could be engineered into potato plants to confer resistance to CPB.

• MeSH
• brouci enzymologie   genetika   růst a vývoj   metabolismus   MeSH
• fyziologie výživy zvířat MeSH
• gastrointestinální trakt enzymologie   MeSH
• hmyzí proteiny genetika   metabolismus   MeSH
• larva genetika   růst a vývoj   metabolismus   MeSH
• proteasy genetika   metabolismus   MeSH
• proteolýza MeSH
• proteomika MeSH
• rostlinné proteiny metabolismus   MeSH
• trávení MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

Ips typographus (L.), the eight-spined spruce bark beetle, causes severe damage throughout Eurasian spruce forests and suitable nuclear markers are needed in order to study its population structure on a genetic level. Two closely related genes encoding α-amylase in I. typographus were characterized and named AmyA and AmyB. Both α-amylase paralogs consisted of six exons and five introns. AmyA encodes a polypeptide of 483 amino acids, whereas AmyB has two alternative transcripts encoding polypeptides of 483 and 370 amino acids. The expression levels of both genes were high during larval stage and adulthood. The AmyB transcripts were absent in the pupal stage. A modification of the allozyme staining method allowed us to detect two clusters of bands on the electrophoretic gel that may correspond to the two α-amylase genes. There was a correlation between the lack of AmyB expression in pupa and the absence of the fast migrating isozyme cluster at this stage, suggesting that the faster migrating isoforms are products of the AmyB gene, whereas the slowly migrating bands are derived from the AmyA.

• MeSH
• alfa-amylasy genetika   metabolismus   MeSH
• brouci enzymologie   genetika   MeSH
• ektroforéza na škrobovém gelu MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• fylogeneze MeSH
• hmyzí proteiny genetika   metabolismus   MeSH
• izoenzymy genetika   metabolismus   MeSH
• klonování DNA MeSH
• messenger RNA metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie aminokyselin MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The enzyme glucose-6-phosphate dehydrogenase (G6PD) of the bark beetle Ips typographus is derived from a gene that includes eight exons and spans over 7100 nucleotides (nt). By means of two transcription starts, the gene generates two mRNA isoforms that are present in similar amounts in the larvae, pupae and adults. The A isoform includes exon IA of 115 nt, which is followed by intron 1a extending to position 3457 of the gene. The B mRNA isoform begins with exon IB (100 nt) that occupies positions 3291-3390 within the 1a intron. Exons II to VII are included in both mRNA isoforms. The gene contains 31.6% (36.5% in the translated region) of the GC nucleotides. Two transcription starts and the exon/intron organization distinguish bark beetle G6PD from the homologous genes known in other insects. Two enzyme variants were detected in the protein extracts of individual bark beetles but their relationship to the A and B mRNA isoforms is uncertain.

• MeSH
• brouci enzymologie   genetika   MeSH
• financování organizované MeSH
• genom hmyzu MeSH
• glukosa-6-fosfátdehydrogenasa genetika   MeSH
• hmyzí geny MeSH
• komplementární DNA MeSH
• konzervovaná sekvence MeSH
• messenger RNA MeSH
• molekulární evoluce MeSH
• molekulární sekvence - údaje MeSH
• počátek transkripce MeSH
• regulace genové exprese enzymů MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• Southernův blotting MeSH
• vývojová regulace genové exprese MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• zvířata MeSH