| Drosophila has no acquired immune system as mammals, gut and innate immunity are the major immune defense mechanism against foreign pathogens in Drosophila. Gut immunity including the induction of antimicrobial peptides (AMPs) and the antibacterial role of reactive oxygen species (ROS) against pathogens intrusion from oral ingestion. Drosophila gut immunity is composed of two complementary facets, the clearance of pathogens and the repair of damage from infection. Innate immunity including phagocytosis, encapsulation and melanization as well as the antimicrobial peptides induced by humoral immunity against pathogens intrusion from skin and wounds. The models of Drosophila gut and innate immunity have been stated clearly, however the key genes involved in these immune pathways and how these genes cooperate with the signal pathways should be investigated more deeply.We have found that the rgn low expression mutant flies have a defect in the phagocytosis of spores in our previous study. It has been reported that Rgn protein sequence contains a C-type lectin domain at its N-terminus. C-type lectins are reported to have functions in extracellular matrix formation, endocytosis, complement activation, pathogen recognition and cell-cell interactions. The rgn homologue in mammals is Reg, which has been shown to be involved in the proliferation of stem cells in the intestine of mice. So we speculated rgn gene played a role in gut and innate immunity in Drosophila.In this study, w1118 (WT) and rgn low expression mutant (rgnG4035) were used as research material, Drosophila survival, apoptosis, intestinal stem cells differentiation, blood cell function and antimicrobial peptide expression were studied by using SDS (sodium dodecyl sulfate) and pathogens feeding, morphology, immunostaining, blood cells count, fluorescent pathogens injectioin and RT-qPCR. The main results obtained are as follows:1. The P-element insertion site of rgnG4035 is located at-590 bp upstream of the ATG of rgn gene by PCR; the mRNA level of rgn is significantly decreased in rgnG4035 compared with WT by RT-qPCR analysis.2. The survival of rgnG4035 and rgnRNAiflies is significantly reduced compared with controls following ingestion of SDS and DSS (dextran sulfate sodium).3. The length of fly guts is significantly reduced after feeding SDS; more melanotic mass is observed in rgnG4035 fly guts compared with WT; more necrotic cells appear in rgnG4035 fly guts compared with WT; rgnG4035 and rgnRNAi flies have a defect in intestinal stem cell differentiation.4. The ROS level is significantly elevated in fly guts following SDS ingestion, but no significant difference is observed between them; The expression of AMPs in fly guts is significantly increased following SDS ingestion, though the induction time is delayed in rgnG4035 compared with WT, rgnG4035 mutant flies show no sensitivity to ingested pathogens.5. Hematopoietic function analysis shows that the differentiation of plasmatocytes is abnormal in in rgnG4035 fly; the differentiation of lamellocytes is defective in rgnG4035 fly; no significant difference is observed between WT and rgnG4035 in the number of crystal cells; rgnG4035 mutant has a defect in the phogocytosis of spore-Alexa Flour 488, abnormal blood cell differentiation may affect the cellular immune function in Drosophila.6. The expression of AMPs is significantly increased in both WT and rgnG4035 flies following septic injection of spores, but no significant difference is observed between them.In summary, in terms of gut immunity, rgn gene participates in the process of gut cell damage repair and cell renewal so as to maintain the gut cell homeostasis; in terms of innate immunity, Drosophila rgn gene affects the hematopoietic function as well as the phagocytosis of pathogens by hemocytes. This study provides a good theoretical basis on the reveal of mechanisms of gut and innate immunity in Drosophila and mammals. |
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