The BmSpatzle5 Gene Is Involved In The Toll-signaling Pathway Against Fungus And Gram-positive Bacteria Invasion In The Integument Of Silkworm, Bombyx Mori

Insect is the most diverse group of animals on Earth. Contrary to the vertebrates, insects don’t have the acquired immunity, therefore, in order to resist the invasion of external microbes, they can only rely on their own innate immunity. Innate immunity is the first line of defense in organisms. When microbia invade, a group of germline-encoded pattern recognition receptors can recognize the pathogen-associated molecular pattern, and then activates multiple signaling pathways to induce the expression of antimicrobial peptides(AMPs). Toll signaling pathway is the most studied signaling pathway. Spatzle(spz) plays an important role in the Toll signaling pathway of insects. In this study, by means of the experiments including Bombyx mori Spatzle5(Bmspz5) gene cloning, bioinformatics analysis, tissue expression profile analysis, microbial induced expression and RNA interference, We characterized the Bmspz5 and analyzed the signaling pathway in which the gene is involved. The main result is as follow:In this study, the open reading frame(ORF) of Bmspz5 gene and its 3 ’end non-coding region were cloned from Bombyx mori(B. mori). The Bmspz5 gene was located in chromosome 7 with only one copy in the genome, and a total of six exons and five introns, exon /intron boundary, were in line with GT-AG rules. The gene encodes a protein of 405 amino acids, of which 56 amino acids with the C-terminal fragment were shown to be different from the corresponding predicted ORF in silkworm genome database. The deduced amino acid sequence of the Bmspz5 gene correspond to the protein with molecular masses of 45.93 kD and isoelectric point of 8.96. Bioinformatics analysis showed that Bmspz5 was Phylogenetically closest to Drosophila spz5 and Manduca sexta spz5 and has a highly conserved C-terminal seven-cystein domains.Expression of Bmspz5 in different tissues was analyzed by semi-quantitative RT-PCR. The results showed that Bmspz5 gene expression was different in various tissues of the fifth-instar day 3 larvae. The highest expression was found in the head followed by the integument, there was virtually no expression found in other tissues. Microbial induced experimental results show that Bmspz5 was upregulated in the integument 24 h after yeast and Bacillus subtilis injection but presented no changes after the injection of Escherichia coli. In the head, however, the injection of yeast, Bacillus subtilis and Escherichia coli did not lead to the upregulation of Bmspz5 transcription. The results suggest that Bmspz5 participated in the immune response against fungi and gram-positive bacteria invasion in the integument.RNA interference experimental results showed that, by injection of yeast after silencing of Bmspz5 expression, the expression of the transcription factor Cactus, Rel, and AMPs Attain, Gloverin and Moricin in the integument is inhibited compared to injection of PBS after silencing of Bmspz5 expression as a control. A similar result was obtained after the injection of bacillus, but the knockdown efficiency is relatively low. Hence, silencing Bmspz5 transcription could inhibit the transcription of AMPs Attain, Gloverin and Moricin and related transcription factor Cactus and Rel in the Toll signal pathway instead of the transcription factor Relish in the Imd signal pathway. This study demonstrates that Bmspz5 is involved in the Toll signaling pathway in the Silkworm Integument.