Molecular Cloning And Function Of New Immune-related Gene Spatzle In The Silkworm And The Infectious Research Of Fluorescence Pseudomonas

The silkworm is a model organism in the study of Lepidopteran. It is also an economicallyimportant insect, being a primary producer of silk. Sericulture has a history of more than5,000years. Sericulture production is a traditional industry in China. However, silkworm diseaseshave caused a great loss in economy. Due to the needs of industrial development, immunedefense mechanism of the silkworm is a long-term focus of attention. Studies on thesilkworm immune-related genes establish a good basis in the immune response againstpathogen. This not only supplies a theory for disease-resistant abilities of silkworm variety,but also has significance in the development of new environmental pesticides and a newpathway for pest control.A new immune-related gene BmSpz4was cloned from the epidermis of silkworm,Bombyx mori in this study. The evolutionary relationship of homologous genes of differentspecies was analyzed. The microbe-induced expression profile of BmSpz4and its functionwas also studied and possible molecular mechanism of silkworm immune response wasexpored. The main research is as follows:1. Cloning of silkworm spatzle4geneA DNA fragment of about600bp was cloned and sequenced when we were trying toclone ATP-binding cassette genes from silkworm, Bombyx mori. By Blast search using thesilkDB database, we found it is part of predicted spatzle-like gene in silkworm.To clone thecomplete ORF of the gene, RNA was extracted from epidermal tissue of fifth instar larvaeBombyx mori, then cDNA was synthesized by reverse transcription with RNA as a template.The complete open reading frame was cloned and sequenced. We also cloned andsequenced its3’end untranslated region using3’-RACE technology.2. Structure and molecular evolution analysis of the new cloned silkworm spatzle geneThe BmSpz4is located in the chromosome3scaffold2927, found only1copy in thegenome. The gene has four exons and three introns, encoding425amino acid residues. Itsmolecular mass is49.36kD, isoelectric point5.31. It has no predicted N-terminal signalpeptide. BmSPZ hydrophobicity is maximum1.733, minimum value is-3.011.Thehydrophobic and hydrophilic regions of the protein amino acid sequence are staggered. SPZhas no transmembrane domains. The amino acid sequence of BmSPZ and those of Drosophilamelanogaster, Nasonia vitripennis, Gambia mosquitoes, Anopheles gambiae, Tribolium castaneum, Acyrthosiphon pisum SPZ have closer relationship than those of mammalian SPZs. Bysequence alignment and evolution analysis, it is shown that the newly cloned spatzle gene ofsilkworm is most closely related to the spatzle4gene in Drosophila melanogaster, so it isnamed as BmSpz4.3. Tissue expression profile and microbe-induced expression analysisTo examine mRNA levels of BmSpz4in different tissues, total RNA samples wereisolated from dissected fat body, midgut, silk gland, head, integument, ovary, and testis offifth instar silkworm larvae as previously described. By Semi-quantitative RT-PCR analysis,spatzle4gene expression was analyzed in different tissues on the third day of the fifth instarlarvae, and the result shows that it is expressed differently in tissues and is detected thehighest expression level in head. Followed by a small amount of expression in the epidermisand testis.And there is no expression of this gene in the rest of the tissues.To test the hypothesis that BmSpz4participates in immune responses, the levels ofBmSpz4mRNA was compared in integument after the silkworm larvae were injected withwater and different microorganisms. There was a significant increase at24h after theimmune challenge of Bacillus laterosporus and Saccharomyces cerevisia, and there was noobvious increase in the gene’s transcription when injected with Escherichia coli. As we allknow that Bacillus laterosporus and Saccharomyces cerevisia belong to Gram-positivebacteria and fungi, and Escherichia coli belong to Gram-negative bacteria. Our results areconsistent with those obtained using Drosophila melanogaster as an organism inmicrobe-induced response experiments.4. Molecular Cloning of Gene Pf-1in the P. fluorescens and its infection to silkwormIn the process of cloning genes in the silkworm, a gene fragment was amplified byRT-PCR and sequenced. After blastn in the NCBI, we found that this sequence has95%homology with Pseudomonas flusorescens gene. It suggested that the silkworm used in theexperiment might be infected with Pseudomonas fluorescens. Te test this hypothesis, weinject Pseudomonas fluorescens underneath the skin of silkworm. The infection experimentdid show that Pseudomonas flusorescens is a pathogen to the silkworm, Bombyx mori.