Effects Of BmKIT₃^R Gene Transfer On Pupal Development Of Bombyx Mori Linnaeus Using A Gal4/UAS Binary Transgenic System

The pupal stage of the silkworm Bombyx mori Linnaeus lasts for approximately2weeks. Prolongation of pupal duration, however, would reduce the labor required to process and dry fresh cocoons. This study investigated the effects of BmKIT3R gene (from the Chinese scorpion Buthus martensii Karsch) transfer on the pupal development of B. mori using a Gal4/UAS binary transgenic system. Gal4driven by a pupa-specific promoter BmWCP4(from a B. mori wing-cuticle protein gene) or PDP (from a B. mori cocoonase gene), and BmKIT3R driven by a UAS cis-acting element were used to construct novel piggyBac-derived plasmids containing a neomycin-resistance gene (neo) controlled by the Bombyx mori nucleopolyhedrovirus (BmNPV) ie-1(immediate-early gene) promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin3(A3) promoter. The vector was transferred into silkworm eggs by sperm-mediated gene transfer. Transgenic silkworms were produced after screening for neo and gfp genes and gene transfer was verified by polymerase chain reaction. The larval development of the hybrid progeny of Gal4-and UAS-ransgenic silkworms was similar to that of normal silkworms, but some pupae failed to metamorphose into moths and the development of surviving pupae was arrested as a result of BmKIT3R expression. Moreover, Gal4driven by the BmWCP4promoter delayed pupal development more effectively than that driven by the PDP promoter in the Gal4/UAS binary transgenic system. Pupal durations of hybrid transgenic silkworm progeny with BmWCP4and PDP promoters were approximately5,2and4days longer, respectively, compared to corresponding normal silkworms, BmWCP4-Gal4, and UAS-BmKIT3R transgenic silkworms, respectively. These results suggest new avenues of research for prolonging the pupal duration of silkworms.