Characterization of tergal gland-secreted proteins in the German cockroach, Blattella germanica
Abstract
Tergal glands play a key role in German cockroach precopulatory behavior. Tergal gland secretion contains phagostimulatory sugars and phospholipids that encourage female feeding and arrest the female in a position favorable for mating to proceed. SDS-PAGE separation of proteins occurring in tergal secretion revealed four main proteins of approximately 23, 45, 63, and 94 kDA (molecular size). Comparison of these four proteins with ubiquitous surface proteins resulted in identification of a ∼63 kDa putative tergal gland-secreted protein (bla63). Following purification and tryptic digestion, two peptides corresponding to bla63 were sequenced and shared putative amino acid identity with known α-amylases from two bacteria: Halomonas meridiana and Pseudomonas sp. A 2,051 nucleotide cDNA encoding a full-length α-amylase was obtained by RACE PCR. Sequencing of clones revealed a 1,497 nucleotide open reading frame encoding a protein of 498 amino acids (aa) with a predicted isoelectric point of 6.5 and a predicted secretory signal peptide of 18 aa. The primary structure of bla63 shares sequence similarity with known α-amylases in seven conserved protein sequence regions common to all known α-amylases. Alignment of the deduced protein sequence of bla63 with five insect amylases from Apis mellifera mellifera, Diabrotica virgifera virgifera, Diatraea saccharalis, and Tenebrio molitor showed in 58.4–55.1% sequence identity. Sequence identity with human salivary amylase and a bacterial amylase were 50.1 and 42.8%, respectively. Northern hybridization of total RNA from tergal gland tissue with a 32P-labeled bla63 probe resulted in hybridization to an RNA of ∼2000 bases. This confirms the bla63 cDNA size of 2051 nucleotides. Bla63 amylolytic activity was demonstrated using a gel-based assay in which degradation of starch can be visualized. This assay confirmed the presence of an α-amylase in tergal gland secretion ∼63 kDa. The possible role of bla63 in processing phagostimulatory sugars present in B. germanica tergal gland secretion is discussed.
Degree
Ph.D.
Advisors
Bennett, Purdue University.
Subject Area
Entomology
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