Research On Two FKBP12 Genes From Silkworm, Bombyx Mori

FKBPs are cytosolic receptors for the immunosuppressive drug FK506, which participate in a variety of pathways, including protein folding, down-regulation of T-cell activation and inhibition of cell-cycle progression. The 12 kD FKBPs (also known as FKBP12) are the most comprehensively studied proteins of FKBPs,which are highly conserved, abundant in various cells and possess peptidyl-prolyl cis-trans isomerase(PPIase) activity. It is also of note that among the animal taxa, only the vertebrates were found to possess multiple FKBP12 genes. We searched two complete mRNA sequences encoding the 12 kD FKBP gene orthologue (FKBP12) in Bombyx mori by NCBI GenBank database,named Bm-FKBP12A and Bm-FKBP12B, of which the accession number was DQ443197 and DQ443423. A multiple sequence alignment among species from various taxonomic groups shows high similarity among FKBP 12 paralogues and orthologues at the amino acid level. Homogeneous analysis showed that Bm-FKBP12A shared 77% of identity with that in homo-sapiens, and Bm-FKBP12B shared 73% of identify to that in homo-sapiens respectively. Each other's similarity of 83%. According to the sequencing data, the molecular weights, and isoelectric points were estimated as 11.82kD and 7.86 for Bm-FKBP12A, and 11.63kD and 8.79 for Bm-FKBP12B. These characterizations of two FKBP 12 proteins were similar with other FKBP 12 proteins identified.The cDNA sequences of the two genes was blasted with Whole-genome sequence of silkworm database on NCBI, two genes both composed of by three exons and two introns,and Bm-FKBP12A have long-introns than Bm-FKBP12B. We analysis the promoter sequences,and precipite chromosomal localizationof them by online web. They both was located on chromosome 20,have the 1500 kbp away. Reporter gene analyses revealed that the regions of (-361)—(-311)bp of Bm-FKBP12A and (-161)—(-121) bp of Bm-FKBPI2B are important for promoter activities in the 5'flanking region. Tissue specific cis-elements were found in the 5'flanking region of neither the Bm-FKBP12A nor Bm-FKBP12B. These results are consistent with the fact that Bm-FKBP12 mRNAs are expressed ubiquitously in a variety of tissues and cells.The gene of Bm-FKBP12B was cloned into pGEX-4T-3 vector and expressed in Escherichia coli BL21 (E. coli). After the recombinant protein purified by the GST-affinity chromatography, digested by the thrombin and further purified by the Sephadex G-75 chromatography, we attained highly purified Bm-FKBP12B. The purified protein was injected into the New Zealand white rabbit to produce the polyclonal antibody, the titer of the antibodies was determined to be above 1:128000 by ELISA.The purified Bm-FKBP12B show a high activity of peptidyl-prolyl cis-trans isomerase (PPIase). Using similar EST searching, real-time quantitative PCR and Western blotting, the expression of two silkworm FKBP12 proteins in every stage and every tissue was investigated. Bm-FKBP12 localized in almost every organ no matter which development stage the silkworm in was. We found that transcripts of the Bm-FKBP12A gene were more abundant in every development stage than Bm-FKBP12B;And transcripts of Bm-FKBP12B were more abundant in the stage of moth and embryo.According to Western blotting, the Bm-FKBP12 expression levels are higher in silk gland and gut,suggest that Bm-FKBP12 before spinning stage might play important roles in development of silkworm particular the gene expression regulation of silkgland and the formation of silk fiber.There are no interation between FKBP12 and Hmol by GST-Pull down essay. These results laid a good foundation of further studies of Bm-FKBP12.