Preliminary Function Analysis Of The Immune-related Gene BmMyD88, Bombyx Mori

Silkworm has been domesticated and used in China for over thousands years. In the long history, Bombyx mori not just as a economic species for silk secretion, more importantly, it is a cultural symbol of the Chinese civilization. However, the harm caused by silkworm diseases has been affecting the entire industry, about nearly billion of annual losses caused by variety of silkworm diseases in China. Recent studies show that the silkworm express a variety of anti-microbial peptides while it was infected by bicrobial, and this process requires a series of complex signaling pathways to complete. The Toll and Imd signal pathways are two important regulatory pathways for signal transduction and antimicrobial peptide expression. In this study, we analysis the key gene of Toll signal pathway:myeloid differentiation factors 88, to explore the humoral immune regulatory mechanisms of signal transduction in silkworm, the main results are as follows:1. Gene clone and expression analysis of BmMyD88Based on the genome database of Bombyx mori, we cloned the cDNA gene sequence of BmMyD88 in silkworm. The cDNA full-length of BmMyD88 is 1188bp, encoding 395 amino acids, molecular weight is 45kDa, and isoelectric point is 6.54. The structure of the protein contains two functional areas:N terminal death domain and C terminal Toll/IL-IR domain, TIR. Homolgy comparison showed that the BmMyD88 has high similarity compared with the Drosophila melanogaster, Aedes aegypti, Homo sapiens, etc, especially in the two functions areas, the homology is higher. Whole genome gene chip showed that, the expression levels of BmMyD88 in various tissues is very low, microbial infection gene chip also showed that after the invasion by the pathogen, expression of BmMyD88 remains low. Using the RT-PCR and quantitative PCR we further validate the chips data.2. Prokaryotic expression and preparation of anti-BmMyD88 antibodyAccording to the gene cDNA sequence, primers were designed to amplify full-length and TIR domain of BmMyD88. and these were cloned into pEGX 4T-1 and pET-28a expression vector. The recombinant plasmids were transformed into Rosetta strain. after the IPTG induction, full-length protein expressed in the supernatant, TIR domain protein expressed in the form of inclusion bodies. We used affinity chromatography to obtain the purified TIR domain protein, and then, prepared the anti-BmMyD88 antibody.3. Western blotting analysis of the protein expression of BmMyD88We use the Western blotting to detect the protein expression of BmMyD88 in fat body and midgut, which were infected by microbial after 24h. The results showed that the BmMyD88 protein can be detected in the fat body and midgut, however, protein expression levels did not change significantly in the induced state compared with the normal state, except midgut sample which was infected by B. bombysepticus, its expression of BmMyD88 was reduced.