AN EFFICIENT BINARY SYSTEMFOR GENE EXPRESSION IN THESILKWORM, Bombyx mori,USING GAL4 VARIANTS

摘要

A binary gene expression system using the yeast GAL4 DNA-bindingprotein and the upstream activating sequence (UAS) of galactose-drivenyeast genes is an established and powerful tool for the analysis of gene function. However, in the domesticated silkworm, Bombyx mori, thissystem has been limited in its utility by the relatively low transcriptionalactivation activity of GAL4 and by its toxicity. In this study, weinvestigated the potential of several established GAL4 variants (GAL4D,GAL4VP16, GAL4VPmad2, GAL4VPmad3, and GAL4NFkB) and oftwo new GAL4 variants, GAL4Rel and GAL4Relish, which contain thetranscription-activating regions of the BmRel and BmRelish genes,respectively, to improve the utility of the GAL4/UAS system in B. mori.We generated constructs containing these GAL4 variants under thecontrol of constitutive or inducible promoters and investigated theirtranscription-activating activity in cultured B. mori cells and embryosand in transgenic silkworms. GAL4VP16 and GAL4NFkB exhibitedhigh transactivation activity but appeared to be toxic when used astransgenes under the control of a constitutive promoter. Similarly,GAL4VPmad2 and GAL4VPmad3 exhibited higher transactivationactivity than GAL4, combined with strong toxicity. The transcriptionactivating activity of GAL4D was about twice that of GAL4. The two new GAL4 variants, GAL4Rel and GAL4Relish, were less active thanGAL4. Using GAL4VP16 and GAL4NFkB constructs, we havedeveloped a very efficient GAL4/UAS binary gene expression system foruse in cultured B. mori cells and embryos and in transgenic silkworms.