Studies On The Function Of Bmtbx20 And Bmtbx1 In Bombyx Mori

T-box genes are important transformation factors related to various development progresses in creatures from lower animals to higher animals and they are conserved during evolution. Tbx20 and tbx1 are members of the Tbx1 subfamily. Tbx20 plays an important role in a variety of physiological processes, such as cardiogenesis, motor neuron cell migration, development of limbs and maintenance of segment polarity. Tbx1 has an important significance in the normal development of ossature, the proliferation and differentiation of thymic epithelial cell and the endodermis cells of brain, the neural crest cells migration, the maintenance of second heart field and angiocellular morphous. Although functions of tbx20 and tbx1 have been studied a lot in mouse and human, the investigations in invertebrate,especially the insects, are rare.The silkworm Bombyx mori has great research value served as a lepidopteran model animal with many experimental distinguishing features, such as ease of rearing, Simple structure for dissection, a short lifecycle, a longer history of research, a large body size. Furthermore, the complication of silkworm genome sequencing is much helpful to the study. As there is no report of tbx20 and tbx1 in silkworm,we studied the function of tbx20 and tbx1 in silkworm.In this study, the T-box genes, tbx20 and tbx1 from the silkworm, bombyx mori, were cloned successfully and designated Bmtbx20 and Bmtbx1. Tissue immunofluorescence staining and evolutionary analysis were used to the preliminary investigation of BmTbx20 and BmTbx1 in the silkworm. The research will provide a basis for the functional investigations of Bmtbx20 and Bmtbx1 in the future as well as treatment of human neurological diseases.The main results are as follows:(1) Using cDNA of the newly-hatched silkworm as a template, the fragments of Bmtbx20 and Bmtbx1 were amplificated via primer STAR kit with the primers designed according to the sequences predict on NCBI database. Bmtbx20 was located on chromosome 4 of B. mori according to the silkworm genome database, and contained 3 exons and 2 introns. The full length of Bmtbx20 CDS sequence consisted of 1497 bp, encoding 498 amino acid residues with a calculated molecular weight of 55.77 kDa, a theoretical isoelectric point of 7.27. Multiple sequence alignments and domain analysis showed that BmTbx20 contained a T-box DNA binding domain and five low complexity regions(LCRs), lacking one low complexity region comparing with its homologue in Drosophila, and two more low complexity regions than the homologue in human. The low complexity regions have a relationship with the evolution. Bmtbx1 was located on chromosome 16 of B. mori according to the silkworm genome database. The length of partial sequence we cloned consisted of 915 bp, encoding 305 amino acid residues with a calculated molecular weight of 34.13 KDa, a theoretical isoelectric point of 8.29, containing a T-box domain.(2) BmTbx20 and BmTbx1 were selected as the antigen for antibody preparation. The results of SDS-PAGE indicated that the recombinant protein Bm Tbx20 was expressed in inclusion and BmTbx1 in soluble protein. The protein was purified by His Trap HP column, and was injected into 2 New Zealand rabbits and 5 mice respectively to generate polyclonal antibodies. The results of ELISA showed that the prepared antibody displayed the antigen-specific immune responses.(3) The antibody staining of embryos showed that BmTbx20 expressed in dorsal blood vessel, ventral nerve cord and brain, while BmTbx1 expressed in the nerves of foot and brain. The result presented that Bmtbx20 and Bmtbx1 play an important role in the development of brain and nerves. Furthermore, Bmtbx20 has a relationship with the development of dorsal blood vessel.(4) The antibody staining of larvae of silkworm showed that BmTbx20 and BmTbx1 both expressed in the cell nucleus of trachea and fat body. The colocalization of BmTbx20 and BmTbx1 in trachea and fat body indicates that Bmtbx20 and Bmtbx1 related with the development of trachea and fat body.(5) The antibody staining of larvae from newly-hatched silkworm to fifth instar larvae showed that BmTbx20 was localized to the cytoplasm of middle silk gland in 1-3 instar larvaes and the cell nucleus in 4-5 instar larvaes. Furthermore BmTbx20 was localized to the cytoplasm of posterior silk gland in 3-4 instar larvaes and the cell nucleus in 1-2 instar larvaes and 5 instar larvaes. BmTbx1 was localized to the cytoplasm of middle silk gland and posterior silk gland in 1-3 instar larvaes and 5 instar larvaes. Furthermore BmTbx1 was localized to the cell nucleus in 4 instar larvaes.(6) Sequences of Tbx20 and Tbx1 from other species collected from the NCBI and Ensemble databases were used to construct the phylogenetic tree with MEGA 5. Phylogenetic analysis showed that Bmtbx20 and Bmtbx1 were conservative during evolution. BmTbx20 and BmTbx1 stayed together with the sequences of insect, and then with the ones of the other invertebrates, finally with the sequences of vertebrate. The evolution of tbx20 and tbx1 was consistent with the evolution of species.