Study on cloning, expression and subcellular localization of one RPA43 related gene(BmRPA43_N) in silkworm, Bombyx mori

• 1、Institute of Biochemistry, Zhejiang Sci-tech University

Abstract：A43, an essential subunit of yeast RNA polymerase I (pol I), encoded by RPA43, interacts with Rrn3, a typical general transcription factor required for rDNA transcription, involved in recruiting RNAP I to the pol I promoter. By scanning the cDNA library of silkworm pupae constructed by our laboratory, we identified one cDNA sequence, which contains a 630 bp ORF encoding for 209 amino acid residues. Bioinformatical methods were applied to analyze the obtained sequence and its induced amino acid sequence. The result shows that this gene has low similarity with other species, but it contains a conserved domain RNAP_I_Rpa43_N. Therefore the gene was named BmRPA43_N（Bombyx mori RPA43_N）。BmRPA43_N was inserted into the expression vector pET-28a to construct the recombinant plasmid. The fusion protein was expressed in the E.coli BL21(DE3) induced by IPTG, and then was purified. Polyclonal antibodies were generated by immunization of New Zealand rabbit with the purified fusion protein and the titer was larger than 1:12800, measured by ELISA. We found that the levels of transcription and expression had great difference in various developmental stages of Bombyx mori detected by Western blotting and RT-PCR methods, but the differences were not obvious in various tissues from 5th instar of silkworm larva . The highest level is in egg and blood, and the lowest is in moth and fat body. Meanwhile, The technology of immunofluorescence cytochemistry was applied to identify the subcellular localization of BmRPA43_N in the cytoplasm of Bm1cell. According to the results of our experiment, it was presumed that BmRPA43_N play an important role in the reproductive development process of Bombyx mori. Its specific functions still need further studies.

Keywords： Bombyx mori BmRPA43_N Western blotting RT-PCR subcellular localization