Identification Of Differentially Expressed Genes In The Silkworm, Bombyx Mori Midgut During Bacterial Infection

Silkworm has great economic value as an important economical insect, but because ofsilkworm diseases, sericulture has been difficult to develop all the time. So the further studyof silkworm immune mechanism has been really imperative. When infect the silkworm bytwo different ways-feeding them and hemocoel injection, the results were totally different.The disease that silkworms were infected by eating developed more slowly and caused lowermortality, which indicated that as the first line of defense during pathogens infection,intestinal tract plays a crucial role in insect immune responses.In order to further explore the immune mechanism of silkworm midgut, annealingcontrol primer (ACP) PCR strategy was used to screen expressed genes related to intestinalimmune system after feeding silkworms Pseudomonas aeruginosa and Staphylococcus aureus.126DNA bands in total were obtained using annealing control primer (ACP) based on reversetranscriptional PCR technique, and25DNA bands with significant differences betweeninfected and uninfected groups were obtained. Cloning and sequencing studies were carriedout. Through Bioinformatics technology,21differentially genes were identified, among whichA102c had no homolog in the databases, A11, A12and A107b encoded genes with unknownfunctions. Among these genes we found several genes involved in insect immune, such asPGRP-L1(AK378516), a member of PGRPs (Peptidoglycan Recognition Proteins) familywhich related to the recognition of peptidoglycan;30kP protease A precursor(BGIBMGA012788) and serine protease precursor (BGIBMGA014427), which weremembers of the serine protease family involved in the Toll immune signal transductionpathways and melanization.Immune related genes (PGRP-L1、Serine protease precursor、30kP ptorease A precursor)and differentially gene BGIBMGA004115in semi-quantitative PCR were further tested byquantitative real-time PCR to detect the transcriptional level based on the time of1h,2h,8h,16h,24h after bacterial infection. In the meantime, changes of these immune-related geneexpression induced by bacteria infection were observed. The result showed that peptidoglycanrecognition protein-L1(PRRP-L1) was activated by Gram-negative bacteria (Pa) on some level, which did not occur when infected by Gram-positive bacteria (Sa). The Expressinglevel of serine protease precursor decreased to1/10of control level at16h after bacterialinfection. On the contrary, the transcriptional level of30kP ptorease A precursor, which is alsoa member of serine protease family, was increased after2h of bacterial infection, and thehighest peak of expression level (10fold of the control group) emerged at8h after bacterialinfection, then return to normal levels after16h of infection. This result indicated that differentserine protease precursor play different roles in the immune system of silkworm. Moreover,BGIBMGA004115gene expressing level declined sharply to1/1000of the control groupinfected by Gram-positive in only2hours, which indicated that bacterial infection inhibitedBGIBMGA004115gene transcription, even induced gene silencing. Even though there is nodetailed functional annotation of this gene in database, it may play an important role in theinteraction between silkworm and pathogen. In summary, using ACP technology we screenedand identified a number of immune-related genes, which laid the foundation for the followingstudy of gene function and silkworm immune mechanism.