Functional Characterization Of Axon Guidance Molecule Robo In Silkworm Bombyx Mori

The formation of neural network is the basis of the nervous system development, and axon guidance molecules paly a very important role during this course. Four families of axon guidance molecules have been found so far, these are Ephrins family,Netrins family,Semaphorins family and Slits family. Members in these families can express in the surface of cells to play a repulsive or attractive role in short distance, or spread to other parts to play a repulsive or attractive role. As the receptor of the axon guidance molecule Slit, Roundabout(Robo) was identified for the first time in the Drosophila by a genetic screen for mutations affecting the commissure axons cross the midline in the nervous system. Robo plays a repulsive role during the process of axon guidance near the midline of the nervous system, meanwhile, it also can promote the process of axonal growth, dendrite branching as well as mediated neuronal migration process. It is a latest hotspot to research the axon guidance mechanism and molecular mechanism in other tissues of Robo molecule.As a model insect of Lepidoptera, silkworm, Bombyx mori, has a higher degree of differentiation and larger size than Drosophila. It provides an operational research model for the research of biological and molecular mechanisms. Moreover, the nervous system of silkworm is very simple, but it can produce a rich behaviors, moreover, the clear genetic background and rich resources of gene mutations make the silkworm become an ideal animal carrier to research the development of nervous system. At present, researches of axon guidance molecules in silkworm are still in the initial stage. In this study, we identified and cloned three robo genes, Bmrobo1 a, Bmrobo1 b and Bmrobo2/3 in the silkworm for the first time, then analyzed the evolutionary relationship of robo genes in silkworm and other species. Moreover, we studied their functions by immunofluorescence staining and RNAi technique. This study is of great significance to understand the unknown molecular mechanism of neural development in the silkworm, and will also provide theoretical basis for the prevention and treatment of tumors as well as nervous system diseases. The main results of this research were listed as follows.(1) We cloned three robo genes, Bmrobo1 a, Bmrobo1 b and Bmrobo2/3, in the silkworm. Bmrobo1 a and Bmrobo1 b are located in the chromosome 6, and Bmrobo2/3 in the chromosome 13. Bmrobo1 a harbors 15 exons and 14 introns, the full length of CDS is 2931 bp, and encodes 976 amino acids. Bmrobo1 b harbors 17 exons and 16 introns, the full length of CDS is 3018 bp, and encodes 1005 amino acids. Bmrobo2/3 harbors 9 exons and 8 introns, the full length of CDS is 3624 bp, and encodes 1207 amino acids.(2) We collected Robo sequences in databases and built a phylogenetic tree by MEGA6, then found robo gene was originated in the coelenterate animal, Hydra, and eventually emerged 4 members, robo1, robo2, robo3 and robo4, after a number of replications in the evolutionary process.(3) We produced polyclonal antibodies of BmRobo1 a, BmRobo1 b and BmRobo2/3. After the construction of recombinant plasmid pET-28 a as well as prokaryotic expression in Escherichia coli BL21, we purified the recombinant protein, then prepared the rabbit polyclonal antibody. Western blot and ELISA analysis proved that the antibodies of good quality and can produce specific immune response against antigen.(4) Antibody staining showed that BmRobo1 a, BmRobo1 b and BmRobo2/3 were expressed in the axons close to the neural midline, brain and ganglion of embryos as well as ganglion of 3th day 5th instar larvae, which suggests their significant roles during the neural development. Moreover, Robo proteins were colocalized with their ligand, BmSlit, in the nervous system, which indicted Robo exerted its function by binding to its Slit ligand in the silkworm.(5) By RNAi, we clarified that Bmrobo1 a, Bmrobo1 b and Bmrobo2/3 played important role in the repulsive processes of neural midline in the silkworm. After the Synthesis of Bmrobo1 a, Bmrobo1 b and Bmrobo2/3 dsRNA respectively, we found axon bundles were closer to the midline after injected dsRNA to the silkworm Embryo, which suggest functions of Bmrobo1 a, Bmrobo1 b and Bmrobo2/3 in the repulsive processes of neural midline.