Effects of egt gene transfer on the development of Bombyx mori |
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Authors: | Zhang Xing Xue Renyu Cao Guangli Hu Xiaolong Wang Xiaojuan Pan Zhonghua Xie Min Yu Xiaohua Gong Chengliang |
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Institution: | a School of Biology & Basic Medical Science, Soochow University, Suzhou 215123, Chinab National Engineering Laboratory for Modern Silk, Soochow University, Suzhou 215123, China |
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Abstract: | This study investigated the effects of gain of ecdysteroid UDP-glucosyltransferase (EGT) gene function mutation on the development of the silkworm, Bombyx mori. A novel piggyBac-derived plasmid containing the egt gene from B. mori nucleopolyhedrovirus (BmNPV) driven by a heat-shock protein (hsp) 23.7 promoter, with a neomycin-resistance gene (neo) controlled by the BmNPV ie-1 promoter and a green fluorescent protein gene (gfp) under the control of the B. mori actin 3 (A3) promoter was constructed. 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, dot-blot hybridization and western blotting. The hatching rate of G1 generation silkworm eggs was about 60% lower than that of normal silkworm eggs. The duration of the G1 generation larval period was extended, and the G2 generation pupal stage lasted four days longer than that in non-transgenic silkworms. The ecdysone blood level in G2 silkworms in the third instar molting stage was reduced by up to 90%. These results show that EGT suppressed transgenic silkworm molting, and that egt expression in egt-transgenic silkworms resulted in arrest of metamorphosis from pupae to moths. |
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Keywords: | egt ecdysteroid glucosyltransferase B mori Bombyx mori BmNPV B mori nucleopolyhedrovirus hsp heat-shock protein gfp green fluorescent protein ie-1 immediate early-stage gene actin 3 A3 neo neomycin-resistance gene AcMNPV Autographa californica nucleopolyhedrovirus polyA egt gene poly(A) signal sequence SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis DsRed Red Fluorescence Protein SIT sterile insect technique ITR inverted terminal repeats of piggyBac SV40-PA SV40 poly(A) signal sequence |
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