Identification And Characterization Of Some Atp-binding Cassette Transporters In Silkworm, Bombyx Mori

ATP-binding cassette (ABC) transporter proteins constitute one of the largest protein superfamilies and are present in all organisms from bacteria to human. These proteins utilize the energy derived from ATP binding hydrolysis to drive substrate translocation across the membrane. ABC transporters have been subdivided into either full or half transporters in all organisms. Full transporters contain two nucleotide binding domains (NBD, also called ATP-binding cassettes) and two six-transmembrane helices, referred to as the"transmembrane domain"(TMD), on a single polypeptide. As their name suggests, half transporters contain one ABC domain and one TMD on a single polypeptide. Half transporters are dependent upon the formation of hetero- or homo-dimers. The silkworm genome sequence has been analyzed to find ATP-binding cassette transport protein genes based on the knowledge of bioinformatics. We identified 47 ABC transport proteins in the silkworm genome sequences, which possesses members of all current ABC subfamilies A to H. The largest silkworm is the ABCC genes which have 15 members, and the second largest silkworm group is the ABCB subgroup with 9 members. Silkworm showed 5 proteins homologous to MDR (multidrug resistance) P-glycoproteins (ABCB subfamily) and 4 proteins homologous to MRPs (multidrug resistance-associated proteins) (ABCCsubfamily). At present, ABC transport proteins were studied very limited in silkworm. The present study provides a useful foundation for studying the function of ABC transport proteins in silkworm.ATP-binding cassette transporter isoform B6 (ABCB6) has been considered to be a very important half-transporter. In mammals, ABCB6 was shown to be functionally active in cellular e?ux of certain porphyrins from cells and to be involved in regulation of iron homeostasis. The nucleic acid sequence of reported human ABCB6 was used to TBLASTN search against the silkworm EST database. The ESTs with high score were clustered and assembled into a consensus sequence. Based on the consensus sequence, the open reading frame (ORF) encoding BmAbcb6 was amplified by reverse transcription-polymerase chain reaction (RT-PCR) and sequenced, which is the first member of Abcb subfamily in silkworm, termed as BmAbcb6. Further bioinformatic analysis shows that the BmAbcb6 cDNA has 16 exons and 15 introns, encoding 850 amino acid residues with pI 8.23 and a predicted molecular mass of 96.35 kD, respectively. The BmAbcb6 protein contains a cytosolic nucleotide binding domain and one transmembrance domain that consists of 10α-helices, it belongs to the Abcb subfamily. By Semi-quantitative RT-PCR analysis, BmAbcb6 gene expression was analyzed in different tissues on the third day of the fifth instar larvae, and the result shows that BmAbcb6 is expressed in every tissue and is detected the highest expression level in testis. This result provides a foundation for studying the function of BmAbcb6 gene in silkworm.The open reading frame (ORF) encoding Bmwh2 gene of Bombyx mori was amplified by reverse transcription-polymerase chain reaction and sequenced. Further bioinformation analysis showed that the Bmwh2 cDNA has 14 exons and 13 introns, which codes for 689 amino acid residues with pI 8.42 and a predicted molecular mass of 77.38 kD. Bmwh2 protein is a half-transporter with one cytosolic nucleotide binding domain and one transmembrance domain that consists of 6α-helices. It belongs to subfamily G of the Bombyx mori ABC transporter superfamily. Semi-quantitative RT-PCR analysis to the expression of Bmwh2 gene in different tissues of the 3rd day larvae of the 5th instar indicated that the highest expression level was in testis. A number of white eggs and chimeric RNAi (RNA interference) phenotypes were obtained by microinjecting synthesized siRNA corresponding to the Bmwh2 gene into Bombyx mori eggs at embryonic development stage. Base on the above results, it is suggested that Bmwh2 may be involved in transportation of the precursors of the serosal pigments in silkworm egg.