Delivery of foreign proteins into insects

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1. Selective absorption of mulberry urease to the hemolymph of the silkworm : When urease extracted from mulberry roots was applied to the 6^<th> -the 8^<th> day larvae of the 5^<th> instar stage, more than 8% of total injection amount were uptaked into larvae The uptake efficiency was very low in other ureases derived from different organisms. Immunocytological observation shown that urease entry take place via microvilli surface of midgut cells.2. Identification of mulberry urease gene : Full-length urease cDNA was isolated and the complete nucleotide sequence was determined, and partially defective urease as well. Deducted amino acid sequence (837 residues) from full-length cDNA had a high similarity with those of other plant ureases. The ure G gene coding urease accessory protein G was also isolated and sequenced partially.3. Gene expression of urease in mulberry seedlings : High urease activity was detected in roots, whereas cotyledons and hypocotyls had no or little activity of urease. In contract, the expression of urease gene was detected in all of the organs. This suggests that additional gene products including ure G protein are involved in maturation of urease protein.4. Comprehensive detection of protein species uptaked into silkworms : Biotinylated mulberry leaf proteins were applied to the silkworm larvae. The profile by two dimensional electrophoresis shown that 15 biotinylated proteins were uptaked to the larval hemolymph.5. Experimental system for generating transgenic mulberry plants : A vector plasmid encoding GUS and HTP genes was constructed to transform mulberry plants by a chimeric gene composed of urease gene and foreign gene.