Study On Cloning, Expression And Localization Of One RPA43 Related Gene (BmRPA43 N) In Silkworm, Bombyx Mori

A43, an essential subunit of yeast RNA polymerase I (pol I), is encoded by RPA43, interacts with Rrn3, and is a typical general transcription factor required for rDNA transcription, which is involved in recruiting RNAP I to the pol I promoter. During this process, the pol I subunit A43 is a major determination for the formation and stabilization of the pol I-Rrn3 complex. It has two domains, N-terminal RNP domain (RNAP_I_Rpa43_N) and C-terminal oligonucleotide- binding (OB) domain. A protein only containing the N-terminal RNP domain, named TWISTNB, was discovered in mammals, avian and amphibian. Patients with the deletions in this coding gene of TWISTNB have significant learning difficulties, which suggested that such mutations define a new microdeletion syndrome. As yet, this protein was not reported in insects.By scanning the cDNA library of silkworm pupae constructed by our laboratory, we identified one cDNA sequence, which contains a 630 bp ORF encoding 209 amino acid residues. Bioinformatics methods were applied to analyze the obtained sequence and its induced amino acid sequence. The result showed that the protein encoded by this gene has low similarity with the related protein in other species, but it contains the conserved domain RNAP_I_Rpa43_N. Therefore the gene was named BmRPA43_N(Bombyx mori RPA43_N)(GenBank accession number is GU288816). We cloned the ORF fragement of BmRPA43_N into pET-28a vector. The fusion protein was expressed successfully in E.coli by adding IPTG for induction, and after Ni2+ affinity chromatography, the His-BmRPA43_N was highly purified. FPLC analysis showed the purity of His-BmRPA43_N was higher than 90%; the molecular weight of the fusion protein was also confirmed by mass spectrometry, which matched the theoretical value very well. In order to prepare the polyclonal antibody of BmRPA43_N, we used this purified fusion protein to immunize a male New Zealand rabbit. The obtained polyclonal antibody had a titer larger than 1:12800 at the concentration of 10μg/ mL, and the specific reaction responsed very well detected in Western blotting analysis. It was detected that the levels of transcription and expression had great differences in various developmental stages of Bombyx mori 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 of expression is in egg and blood, and the lowest is in moth and fat body, respectively. Meanwhile, the technology of immunofluorescence cytochemistry was applied, and subcellular localization of BmRPA43_N was identified in the cytoplasm of BmN cell. According to the results of our experiments, it was speculated that BmRPA43_N play an important role in the reproductive development process of Bombyx mori. Further studies are still needed on its specific functions.