DING, X.; LI, S.; BORST, D. W.; Illinois State University, Normal: Identification and cDNA cloning of transferrin, a major fat body protein in the lubber grasshopper

The fat body in many insects contains abundant storage proteins that are used as nutritional reserves. In the lubber grasshopper (Romalea microptera), the fat body of the adult female also contains an abundant protein (MW = ~84 kDa) which changes in abundance during the oviposition cycle. We isolated this protein using anion exchange chromatography and SDS-PAGE. The amino acid composition and the protein sequence of trypsin fragments (by LC-MS) implied that this protein was related to the insect transferrins. To confirm this, we used the sequences of the trypsin fragments to design two degenerated primers. These primers were used to amplify a 1.6-kb cDNA fragment by PCR. 5�-RACE and 3�-RACE were used to recover the entire cDNA (2528-bp). The 2211-bp open reading frame encodes a 736-amino acid protein (MW 80.9 kDa). The alignment of the deduced amino acid sequence of this protein revealed a close relationship (between 38-58% identity and 58-71% similarity) to the sequences of other insect transferrins, identifying this protein as the lubber transferrin (Rom-transferrin). Northern blotting analysis demonstrated the presence of two transferrin transcripts. The more abundant 2.5-kb transcript matches the size of the Rom-transferrin cDNA, while the less abundant 6.2-kb transcript may be an immature Rom-transferrin mRNA. Rom-transferrin transcripts were detected in all regions of fat body but not in gut, ovary, and muscle tissue. The isolation of this cDNA will allow us to investigate the expression of this protein during the oviposition cycle, and how expression is affected by juvenile hormone and by the nutritional status of the animal. (Supported by NSF grant DBI – 9978810 to DWB).