Analysis Of A HAT Family Transposase Gene In Silkwork, Bombyx Mori

Transposons are specific and independent DNA sequences with translocation features in genome. For a long time, they are thought to be useless part of genetic material. But now it is clear that transposons can cause some beneficial changes to adaptation and biological survival. Analysis of BmhAT will contribute to the functional and characteristical study of BmhAT transposon, and will also facilitate us further understanding the complexity of silkworm genome.In this paper, we first analyzed the structure and properties of BmhAT using bioinformatical method. From the results we found that BmhAT has significant homology with some hAT family transposases in sequence and contains conserved domains of this family transposase, Zf-BED,DUF-659 and hAT dimerization domain. At the same time, we measured the copy numbers of BmhAT in Bombyx mandarin and different varieties of Bombyx mori by Real-time PCR. Results showed that BmhAT has about three to six copies in Bombyx mori genome, highest in FA 403 and lowest in P50. This may be concerned with movable properties of transposons. We analyzed the relative expression of BmhAT at mRNA level in different tissues of Bombyx mori by Real-time RT-PCR. Results indicated that the relative expression of BmhAT to actin 3 reference gene in posterior silk gland and fat body is 0.4 folds and 0.6 folds, respectively. Quantitative analysis of BmhAT gene provides a basis to understand the features and expression of BmhAT protein.Secondly, we expressed BmhAT fusion protein in E.coli and made a preliminary exploration for the conditions of BmhAT protein's expression and purification by Ni+-NTA affinity chromatography etc. As results showed, BmhAT could only bind to Ni+-NTA affinity chromatography column easily. This may be related to its structure and properties. Therefore, we truncated it into BmhAT-1 by the prediction of protein epitopes and antigenic peptides. We used DEAE anion column to purify preliminarily. Then we utilized Ni+-NTA affinity chromatography column to purify again. In the end, we obtained BmhAT-1 protein with higher purity. Through the analysis of BmhAT protein's expression and purification, it will be in favor of preparing antibody to detect the true expression of silkworm.Finally, we expressed BmhAT protein in BmN cultured cells and silkworm by using the BmNPV's Bac-to-Bac recombinant baculovirus expression system. The results showed that BmhAT could express normally, but stayed in the cultured cells or in blood cells of silkworm basically. Little could be secreted into cell culture medium or hemolymph of silkworm, and we analyzed the nuclear localization of BmhAT in cells by the Orange fluorescent protein OFP. Consequently, we saw the obvious fluorescence in the center of cell. It illustrated that BmhAT may be a nucleoprotein. Through the analysis of BmhAT protein's expression in silkworm, it can make for the further study of BmhAT protein's advanced structures.