Functional characterization of the bHLH family transcription factors from the red flour beetle, Tribolium castaneum

The basic helix-loop-helix transcription factors are present in animals, plants and fungi and play important roles in controlling cellular proliferation, tissue differentiation, development and detoxification. RNAi has become a widely used tool to knock-down and analyze the function of genes. We searched the red flour beetle genome sequence deposited into National Center for Biotechnology information and Human genome sequencing center databases and identified 53 bHLH genes. TcNeuroD is a unique gene identified in red flour beetle and is absent in all the six insect genome sequences available and is present in vertebrates. Phylogenetic analyses using Mr. Bayes program has classified these 53 genes into ten families as defined by bHLH-PAS, HES, Myc/USF, Hand, Mesp, Shout, p48, NeuroD/Neurogenin, Atonal and AS-C. As RNAi works well in Tribolium castaneum, double-stranded RNA's of all 53 bHLH genes were injected into larvae and the effects of RNAi-mediated knock-down in expression of these genes were monitored throughout development. Out of 53 genes tested, 18 of them affected the growth and development of T.castaneum. The phenotypic effects of bHLH gene knock-down include block in growth, larval-pupal, pupal to adult metamorphosis and impaired wing development during pupal and adult stages. This first attempt at functional characterization of all the 53 bHLH genes identified in T.castaneum showed the power of RNAi to study the functions of families of genes in this model insect. Supported by NSF grant, IBN-0421856.