Cloning, Expression And Sequence Analysis Of Bmimd, An Innate Immunity-related Gene In The Silkworm, Bombyx Mori

Silkworm is not only one of the first economic domesticated insects, but also one of the model organisms for classical genetics research. And it has weak immunity against disease, which always threatened the sericulture. So study on immunity in Bombyx mori will not only bring in economic benefit, but also have important scientific value on study of insect immunity. Studies had suggested that insect, when infected by microorganism, can synthesis several antimicrobial peptides which are regulated by two distinct signalling pathways: Toll and Immune deficiency (imd). Imd gene is a key gene of Imd pathway. This gene has low orthology among various species and its function was known little by people, but the advanced technique of modern molecular biology, genetics and others provided us powerful tools to its research. Learning from advanced achievements in Drosophila, this paper researched the imd homolog of Drosophila in Bombyx mori by using silkworm genome. This study will give solid base to improving the anti-diseases ability of silkworm, provide theoretical evidence to study the mechanism of silkworm immunity, and also provide valuable clue to study the molecular mechanism of insect immunity response.The main research contents and results are as follows:1 Cloning and sequence analysis of BmimdWe got a homolog of Drosophila imd from Bombyx mori successfully through silico cloning based on the silkworm EST data, and named it as Bmimd. Analyzed by bioinformation method, the gene is 1092 bp in length, consisting of 4 exons and 3 introns, encoding a protein with 250 amino acids, the protein molecular weight was predicted 28.6 kD, and the isoelectric point is 6.45. Using on-line lookuping in signaIP, we found that the location of l-19aa is the singal peptide of Bmimd. Bmimd contains a death-domain which locates from 152aa to 246aa. Blast anlignment in NCBI and clustering analysis by the program of Clustalx suggested that the death-domain of Bmimd in carboxy-terminal region is similar to receptor interaction protein (RIP) of mammalian. Then, we designed primers according to the predicted sequence, cloned the coding sequence of this gene in vitro, and got a taget fragment of 750bp.2 Prokaryotic expression of BmimdThe complete ORF of bmimd was subcloned into the pET-50b(+) expression vector, and the recombinant plasmid was transformed into Escherichia coli BL21. Then induced imd/PET-50b/BL-21 to express protein by IPTG, we got a recombinant protein with molecular weight about 97kD, including the tag protein (contain two six-His tags and a Nus·Tag) with 68kD and taget protein with about 29kD, which is consistent with prediction. The optimal condition of protein expression is 37℃, 0.4mmol/L IPTG induced 5 hours through optimization. The protein is expressed as inclusion bodies by appraisal.3 Expression feature of BmimdBased on the silkworm microarray database, the expression profile of Bmimd (Dazao, day three of fifth instar) in different tissue is as follows: head is the highest; germen, cuticle and fat body have morsel; and there are nearly no in blood, malpighian tubule and silk gland.Bmimd protein was purified by affinity chromatography and elector-elution, and then used to produce specific antibody. Protein was extracted from Bombyx mori of day four of fifth instar, which subjected to immunoblotting with anti-bmimd antibody. The result is similar to above- mentioned result from microarray database analysis, suggested that the regulation of Bombyx mori imd gene is mainly in transcriptional level.Conclusion: In this work, we got a homology of imd which is the key gene of insect innate immunity from Bombyx mori, and named it Bmimd. Then we cloned and expressed Bmimd in vitro, and got the pure protein of it by purification. At last, we studied the expression feature of Bmimd in Bombyx mori at the end of fifth instar. All the results indicated those conclusions: (1) Bmimd may be a ligand of silkworm Imd pathway, which can pass activation signal of pathogeny. (2) The regulation of Bombyx mori imd gene is mainly in transcriptional level. So once infected, Bombyx mori can express Bmimd immediately, regulate body to generate plenty of resistant effectors, and resist infection as soon as possible. (3) Imd pathway of Bombyx mori may be like Drosophila's, which is not only involved in systemic immunity, but also in local immunity.