title: Molecular characterization, tissue distribution, subcellular localization and actin-sequestering function of a thymosin protein from silkworm.
abstract: We identified a novel gene encoding a Bombyx mori thymosin (BmTHY) protein from a cDNA library of silkworm pupae, which has an open reading frame (ORF) of 399 bp encoding 132 amino acids. It was found by bioinformatics that BmTHY gene consisted of three exons and two introns and BmTHY was highly homologous to thymosin betas (Tβ). BmTHY has a conserved motif LKHTET with only one amino acid difference from LKKTET, which is involved in Tβ binding to actin. A His-tagged BmTHY fusion protein (rBmTHY) with a molecular weight of approximately 18.4 kDa was expressed and purified to homogeneity. The purified fusion protein was used to produce anti-rBmTHY polyclonal antibodies in a New Zealand rabbit. Subcellular localization revealed that BmTHY can be found in both Bm5 cell (a silkworm ovary cell line) nucleus and cytoplasm but is primarily located in the nucleus. Western blotting and real-time RT-PCR showed that during silkworm developmental stages, BmTHY expression levels are highest in moth, followed by instar larvae, and are lowest in pupa and egg. BmTHY mRNA was universally distributed in most of fifth-instar larvae tissues (except testis). However, BmTHY was expressed in the head, ovary and epidermis during the larvae stage. BmTHY formed complexes with actin monomer, inhibited actin polymerization and cross-linked to actin. All the results indicated BmTHY might be an actin-sequestering protein and participate in silkworm development.
authors.affiliation: Institute of Biochemistry, College of Life Science, Zhejiang Sci-Tech University, Hangzhou, China.
authors.forename: Wenping
authors.initials: W
authors.lastname: Zhang
authors.forename: Changrong
authors.initials: C
authors.lastname: Zhang
authors.forename: Zhengbing
authors.initials: Z
authors.lastname: Lv
authors.forename: Dailing
authors.initials: D
authors.lastname: Fang
authors.forename: Dan
authors.initials: D
authors.lastname: Wang
authors.forename: Zuoming
authors.initials: Z
authors.lastname: Nie
authors.forename: Wei
authors.initials: W
authors.lastname: Yu
authors.forename: Hanglian
authors.initials: H
authors.lastname: Lan
authors.forename: Caiying
authors.initials: C
authors.lastname: Jiang
authors.forename: Yaozhou
authors.initials: Y
authors.lastname: Zhang
mesh_terms: D000199:Actins / Q000737:chemistry*; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D012831:Bombyx / Q000502:physiology; D019295:Computational Biology / Q000379:methods; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D015723:Gene Library; D007438:Introns; D008856:Microscopy, Fluorescence / Q000379:methods; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010957:Plasmids / Q000378:metabolism; D011817:Rabbits; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013947:Thymosin / Q000096:biosynthesis* / Q000235:genetics*; D014018:Tissue Distribution
chemical_list: D000199:Actins; D018076:DNA, Complementary; D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D013947:Thymosin