n of endogenous Bm-X protein. Immunostaining showed that Bm-X protein localized in both the cytoplasm and nucleus. But the fluorescence localization signals were stronger in cytoplasm than in nucleus, even in the dividing cells (Figure 7).

4. DISCUSSION

We described the cloning, expression, and subcellular localization of a novel gene Bm-X in the study. We have also obtained and purified an anti-Bm-X serum for further studies. As far as we know, our laboratory is the first to report tissue-specific expression and subcellular localization of this species-specific gene in Bombyx mori.

By bioinformatics tools, we confirmed a novel gene Bm-X of Bombyx mori from our silkworm pupae cDNA library. Bm-X gene belongs to silkworm genome, but lacks any significant homology with other species. It is a species-specific gene of Bombyx mori without any previous knowledge. We predicted the molecular characteristics of Bm-X protein by bioinformatics. Bioinformatics analysis can be informative for the design of experiments to study unknown genes, for example the predicted hydrophilicity was beneficial to protein purification and predicted functional sites were useful for functional studies.

In this study, we obtained polyclonal antibodies specific for the Bm-X protein. In order to purify the natural Bm-X from the fifth instar larvae, we extracted total proteins of this larval instar and salted out with 100% saturated ammonium sulfate. These proteins were then used in the Western blot analysis. The results showed that the antibody has high degree of specificity with Bm-X protein. The Western blot analysis revealed that a protein band over 19.96-kDa appeared in the silkworm protein extracts (Figure 4), indicating that the molecular weight of endogenous Bm-X was approximate 20 kDa. However, there was a significant difference between the molecular weight of the endogenous Bm-X protein and that expressed from bacteria. We believe that the reason for such a difference lies in post-translational modification of natural Bm-X protein. This modification may be related to the two predicted glycosylation sites of natural

Figure 6. Expression level of Bm-X in different tissues of 5th instar larva of Bombyx mori by Western blot analysis. Protein extracts from the head (H), midgut (Md), Genital (G), silk gland (S), epidermis (E), fatty body (F), stigma (T), and Malpighian tubule (M) of 5th instar larva were subjected to immunoblot analysis. Here we use rBm-X as a positive control.

Figure 7. The subcellular localization of Bm-X protein. 1: Visible light images; 2: Cy3 fluorescence images; 3: DAPI fluorescence images; 4: Mix images control. Negative control; A: Positive result by 40× amplified; B: Positive result by 40 × 2.5 amplified.

Bm-X protein.

During the fifth instar larva, the expression of Bm-X is distributed broadly in some tissues, including the silk gland, midgut and epidermis as confirmed by our Western blot analysis. The tissue distributions differed greatly during pupation of Bombyx mori. In addition, we did not detect obvious signals of Bm-X protein in the pupae, suggesting that Bm-X may play a role in molecular mechanisms in Bombyx mori larvae pupation. Our results were reproducible in repeated experiments, and we observed that the sensitivity of detection from fluorescence western blot was 20 times higher than western blot with DAB dried, suggesting that the expression level of Bm-X in these three tissues may be low.

The subcellular localization analysis of Bm-X protein showed that Bm-X protein localizes in both the cytoplasm and nucleus. The fluorescence localization signals were stronger in cytoplasm than in nucleus, even there is no change in the dividing cells. The scanning laser tomography also showed that Bm-X protein is uniformly distributed in BmN cells.

We have identified a novel Bm-X gene which is is specific to Bombyx mori. We immunized New Zealand rabbits with the recombinant Bm-X to obtain the antiBm-X polyclonal antibodies which showed high degree of specificity against rBm-X. We have found that Bm-X is expressed in the epidermis, silk gland, and midgut of the silkworm. However, the specific function of Bm-X in various periods of development in silkworm requires further research. One possible approach to determine these functions would be RNA interference with dsRNA designed to inhibit the expression of Bm-X.

5. ACKNOWLEDGEMENTS

This work was supported by financial grants from the National High Technology Research and Development Program (No. 2011AA100603), Zhejiang Natural Science Foundation (No. Y3090304, Y3090339, Y31- 10051, Y3110354) and Zhejiang Educational Foundation (Y200909740, Y201019098).

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NOTES

*Corresponding author.

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