| In the nature, pathogenic microorganisms infected host mainly by food. Gut epithelia is the first line to defense host from pathogens after bacterial infection via feeding. Gut immunity combat with microorganisms by generation ROS(Reactive oxygen species) and local AMPs(Antimicrobial peptides). Silkworm as the important model insect, also have great value on economic. Study on the mechanisms of immune and disease resistance will provide great significance on mode construction of pathogenic-microorganisms and silkworm disease prevention.In this study, silkworms were infected by bacteria feeding and injection. The bacteria we used belong to gram-positive(Staphylococcus aureus, S.a;Bacillus bombyseptieus, B.b;Micrococcus luteus, M.l and Streptococcus pneumonia, S.p) and Gram-negative(Pseudomonas aeruginosa, P.a;Serratia marcescens, S.m and Yersinia pseudotuberculosis, YPIII). After bacterial infection, the mortality was recorded and the H2O2 level changes in the gut and plasma were measured. Semiquantitative PCR and Quantitative real-time PCR were used to analysis the transcription levels of ROS related genes and AMP genes. Afterwards, recombinant protein of Bm Prx5 was obtain by prokaryotic expression, followed a measurement on activity of Bm Prx5 in vitro. The expression levels of Bm Prx5 in the midgut, fat body and hemocyte after bacterial infection were analyzed by real-time PCR. The main results are as follows:The results after bacteria injection demonstrate the average lethality by Gram-negative bacterium(S.m and P.a) is higher than that by Gram-positive bacterium(S.a and M.l). ROS related genes were significantly up-regulated by S.m oral infection. The fold change on ROS-generation genes is lower than it on ROS-degradation genes which lead intestinal H2O2 level always lower than control after S.m infection.Survival curves showed that B. bombysepticus is deadly when larval silkworms are infected orally. Bacterial infection caused intestinal H2O2 and NO levels to increase significantly by 8 and 16 hpi, respectively. Real-time quantitative PCR(q PCR) analysis showed that the transcription levels of Duox and catalase were highly up-regulated by P. aeruginosa infection at 8 hpi. P. aeruginosa infection induced nitric oxide synthase 2(NOS2) expression at 16 hpi, which contributes to the generation of NO. m RNA levels of AMP genes, specifically Glovorin 2 and Glovorin 3, were obviously increased during the early infection stage. These results indicate that invading bacteria elevate intestinal ROS and NO levels and induce AMP gene transcription, which synergistically contribute to intestinal immune defense.We cloned a c DNA of Bombyx mori peroxiredoxin 5(Bm Prx5), which contained a 565 bp open reading frame for a 188-residue protein. Sequence analysis indicated Bm Prx5 belongs to the atypical 2-Cys peroxiredoxin family. Recombinant Bm Prx5 purified from Escherichia coli showed antioxidant activity that removes H2O2 and protects DNA from oxidative damage. Quantitative real-time PCR showed the Bm Prx5 m RNA level in hemocytes increased early and decreased by 24 h after injection of H2O2 whereas, in the fat body, the transcript level decreased at 6 h and increased at 12 h. Pseudomonas aeruginosa and Staphylococcus aureus infection resulted in higher levels of H2O2 in the hemolymph and Bm Prx5 m RNA in hemocytes at 8 h post infection. These data suggested that Bm Prx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. Further study is needed to elucidate the exact role of Bm Prx5 in the silkworm immune system.The conclusions are as follows: Bacillus bombyseptieus and Serratia marcescens are the most fatal bacteria to the silkworms; Generation of ROS, NO and AMPs induced by bacterial infection play synergistic defense roles in intestinal immunity of Bombyx; Bm Prx5 acts as an antioxidant enzyme to protect the silkworm from oxidative damage induced by bacterial infection. |
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