The Discovery Of A New Splicing Isoform Of Bombyx Mori Spatzle-4 Gene And Its Immune Responses In The Integument Against Different Microorganisms

In this study,we cloned the 1567 bp BmSpz4 cDNA which contains a complete 1386 bp open reading frame,encoding 461 amino acids,the ORF of the cloned sequence was 108 bp longer than the predicted ORF of BmSpz4(BGIBMGA008841-TA)in the SilkDB,and the108 bp encodes 36 amino acids at the 5'end,among which the first 19 amino acids were the signal peptide.A signal peptide is a short peptide present at the N-terminus of the majority of newly synthesized proteins that are destined towards the secretory pathway.The BmSpz4 gene is located on Bm_scaf174 form the chromosome 3,with merely 1 copy in the genome.And it contains 6 exons,5 introns and a Spatzle domain.ExPASy predicted that the molecular weight of BmSpz4 protein was 53.19 kDa and the isoelectric point was 5.56.It was showed by the hydrophobicity analysis of the amino acid sequence of ProtScale that the maximum hydrophobicity of BmSpz4 protein was 3.333,the minimum was-3.011,and the hydrophobic and hydrophilic regions of the protein sequence were staggered.The amino acid sequence of BmSpz4 and those of Anopheles gambiae,Apis dorsata,Drosophila melanogaster,Nasonia vitripennis,Tribolium castaneum SPZ have closer relationship than those of mammalian SPZs.Judging from the preliminary results we conclude that there are2 kinds of spliced variants of BmSpz4.It was shown by semi-quantitative RT-PCR that BmSpz4 gene was expressed in the tissues that the integument,head,malpighian tubules,ovary and testis from the fifth instar day-3 silkworm larva,with the highest expression in the head,then the integument.In this study,Phosphate buffer,Bacillus subtilis(gram-positive bacteria),Escherichia coli(gram-negtive bacteria)and Saccharomyces cerevisiae(fungus)were injected into the body cavity of the fifth instar day-3 silkworm larva respectively.24 h after injecting different types of microorganisms to the body cavity,we extracted the total RNA from integumentary tissues for semi-quantitative reverse transcription PCR.And the results showed that the expression of BmSpz4 in the integument was upregulated by Gram-positive bacteria and fungi but not by Gram-negative bacteria.This phenomenon was the same as the result of microbial-induced immune response of the Toll signaling pathway reported in the Drosophila.In addition,the result of double-stranded RNA interference of BmSpz4 also demonstrated that it had a corresponding interference effect on the expression of antimicrobial peptides induced by bacillus and yeast.Thus,as an important part of silkworm innate immunity,BmSpz4 plays an important role against microbial infection in thesilkworm,Bombyx mori.