Analysis of ecdysone-pulse responsive region of BMWCP2 in wing disc of Bombyx mori.
The present study was undertaken to clarify the regulation of cuticle-protein gene expression. Bombyx BAC library was screened to obtain the sequences of regulatory regions of cuticle protein genes isolated from wing discs of Bombyx mori. Two kb upstream of BMWCP2 was cloned into a reporter plasmid, and a reporter assay was operated. Plasmids were introduced into wing discs and wing tissues using a gene gun. DNA introduction into wing discs was confirmed with plasmid pA3GFP. The upstream region of BMWCP2 showed stage-specific activity: strongest at P0. EMSA analysis indicated the binding of BmbetaFTZ-F1. Ecdysone pulse-responsive sequences were examined in vitro. A luciferase assay was performed using reporter plasmids that contained different length upstream-regions of BMWCP2. With this method, we identified the ecdysone-responsive region. With deletion of the BMWCP2 upstream region, mutagenesis of the BmbetaFTZ-F1 binding site and EMSA analysis, it was confirmed that the BMWCP2 expression was regulated by BmbetaFTZ-F1 through the ecdysone pulse. This is first to apply the introduction of reporter plasmids into small organs to examine the developmental and hormonal regulation of the cuticle protein gene expression. We demonstrated that the binding of BmbetaFTZ-F1 facilitated the promoter activity of the BMWCP2 cuticle protein gene in vitro.
Nita
Masahiro
M
Faculty of Agriculture, Utsunomiya University, 350 Mine, Utsunomiya, Tochigi 321-8505, Japan.
Wang
Hua-Bing
HB
Zhong
Yang-Sheng
YS
Mita
Kazuei
K
Iwanaga
Masashi
M
Kawasaki
Hideki
H
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D015153:Blotting, Western; D012831:Bombyx / Q000235:genetics* / Q000254:growth & development; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004440:Ecdysone / Q000494:pharmacology; D024202:Electrophoretic Mobility Shift Assay; D015870:Gene Expression / Q000187:drug effects; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D015698:Genomic Library; D019476:Insect Proteins / Q000235:genetics* / Q000378:metabolism; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D011485:Protein Binding; D020218:Response Elements / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D014921:Wings, Animal / Q000254:growth & development / Q000378:metabolism*
D004268:DNA-Binding Proteins; D019476:Insect Proteins; D014157:Transcription Factors; C047663:cuticle proteins, insects; C499713:nuclear hormone receptor FTZ-F1, Bombyx mori; D004440:Ecdysone; D008156:Luciferases