Identification and expression analysis of ras gene in silkworm, Bombyx mori

Abstract

Ras proteins play important roles in development especially for cell proliferation and differentiation in various organisms. However, their functions in the most insect species are still not clear. We identified three ras cDNAs from the silk worm, Bombyx mori. These sequences corresponded to three Ras of Drosophila melanogaster, but not to three mammalian Ras (H-Ras, K-Ras, N-Ras). Subsequently, the expression profiles of ras were investigated by quantitative real-time PCR using whole body of individuals from the embryonic to adult stages, and various tissues of 4th and 5th instar larvae. Each of three Bombyx ras showed different expression patterns. We also showed membrane localization of their products. These results indicate that the three Bombyx Ras are functional and have different roles.

Figure 1. cDNA and putative amino acid sequences of B. mori Ras.

(A) Ras1 (accession number: AB3892674), (B) Ras2 (AB3892674) and (C) Ras3 (AB3892674). Amino acids which are important for GTP/Mg²⁺ binding are boxed. The C terminal isoprenylation site is underlined. Glycine residues substituted by valine in BmRas-GFP fusion proteins are indicated by arrowheads.

Figure 2. Alignment of amino acid sequences of Ras superfamily members.

Bm, Ce, Dm and Mm means B. mori, C. elegans, D. melanogaster and M. musculus, respectively. The accession number of each sequence is described in Results. Identical and homologous amino acid residues are highlighted and shaded, respectively. Amino acids which are important for GTP/Mg²⁺ binding are boxed. Amino acids of the GEF interaction site and the effector binding site are shown by arrowheads and asterisks, respectively. The C terminal isoprenylation site is indicated with dots.

Figure 3. Phylogenic tree constructed using primary sequences of Ras superfamily members.

An unrooted UPGMA tree was prepared using CLC FREE WORKBENCH VER. 4.01 (CLC Bio A/S, Aarhus, Denmark). References for sequences are shown in Results. A bootstrap value is attached to each node, and the value is a measure of the confidence in the branch. The number of replicates in bootstrap analysis is adjusted to 100.

Figure 4. Changes in the mRNA expression levels of Bmras1, Bmras2 and Bmras3 during development.

mRNA samples were harvested from various organs of various developmental stages from the embryo to adult of B. mori at 24-hours interval. Transcripts of Bmras in these samples were quantified by qRT-PCR. Relative expression levels in whole body (A) and tissues (B) of Bmras1, whole body (C) and tissues (D) of Bmras2, whole body (E) and tissues (F) of Bmras3 against Bmrp49 are shown. Expression levels in whole body samples are indicated by solid squares. Changes in organs, namely, the epidermis, fat body, silk gland, muscle, Malpighian tubles and gut are shown by solid circles, solid triangles, open circles, solid diamonds, open squares and open triangles, respectively.

Figure 5. Localization of Ras-GFP fusion proteins in Sf-9 cells.

GFP (A), Ras1-GFP (B), Ras2-GFP (C) and Ras3-GFP (D) proteins were translated in Sf-9 cultured cells, and the localization of these proteins was observed with their GFP signals by confocal microscopy (left pannels). Right panels show Nomarsky microscope images of left panels. GFP luminescence is localized in nuclei in the case of non-fused GFP (A), but on the plasma membrane, and weakly in the cytoplasm, in cases of fusion proteins (B, C and D).