| Locusta migratoria is the major agricultural pest all over the world.At present,the use of chemical pesticides is still the main method to control locusts.Although chemical pesticides are high-effective,long-term use of chemical pesticides can cause environmental pollution and damage to the ecological environment.Therefore,it is especially important to develop green pest control methods.Biological control is the main method for green pest control.However,the innate immune system in insects has greatly reduced the efficiency of biological control.In recent years,many studies have shown that ecdysone plays an important role in regulating insect immune response.Entomogenous fungi are important biocontrol factors,but the mechanism by which ecdysone regulates insect antifungal immune response is still unclear.In this paper,i TRAQ technology was used to screen for differential fat body proteins with ecdysone treatment in the locusta migratoria.The molecular and expression characteristics of immulectin-1 were studied.The function of the recombinant protein r Lm IML-1 was analyzed and the immune function of immulectin-1 was studied by using RNAi technology.The regulation mechanism of ecdysone on immulectin-1 mediated antifungal immune response was further explored.The main results are as follows:1.Using i TRAQ technology to compare the differentially expressed protein profiles of the fat body after treating with ecdysone.The results showed that 134 differentially expressed proteins were successfully identified in total protein of fat body compared with the control group,among which 118 were up-regulated and 16 were down-regulated,and the immune-related protein C-type lectin was identified that responded to ecdysone.2.The C-type lectin in response to ecdysone was cloned,and the full-length c DNA of the gene was obtained.The gene was successfullyIV uploaded into the Gen Bank database with the accession number was MK250966.It contains two typical carbohydrate recognition domains and was designated as immulectin-1(IML-1).The results showed that Lm IML-1was highly expressed in fat body and hemolymph,and the expression level of Lm IML-1 increased gradually in the first 5 days of 5th instar,and decreased sharply before molting.3.The prokaryotic expression vector was constructed and the immunological activity of the recombinant protein r Lm IML-1 was analyzed.The results showed that the agglutination of E.coli by r Lm IML-1 was in Ca2+-dependent manner,and r Lm IML-1 promotes the encapsulation of hemocytes to beads,which indicates that Lm IML-1 plays an important role in cellular immune response.After RNA interference of Lm IML-1,the encapsulation rate of hemocytes to beads was decreased significantly.After the injection of ds Lm IML-1,the antifungal activity of the plasma of nymphs significantly decreased.Silencing the Lm IML-1 and further treating the nymphs with the spores of Metarhizium anisopliae,the mortality of nymphs was significantly increased compared to the control.The above results demonstrated that Lm IML-1 plays an important role in the antifungal invasion process of locusts.4.The ecdysone induction experiment proved that both endogenous and exogenous ecdysone can induce the expression of Lm IML-1,and the expression level of Lm IML-1 was significantly down-regulated after RNAi of the ecdysone receptor Lm Ec R.After ecdysone treatment,the encapsulation of hemocytes to fungal spores and the antifungal activity of plasma were increased.After RNAi of Lm IML-1,ecdysone neither enhanced the cellular immune response of hemocytes to fungal spores,nor induced the increase of plasma antifungal activity.The above results revealed that ecdysone activates Lm IML-1 to regulate the antifungal cellular and humoral immune response by binding to its receptor complex.This study reveals the mechanism of ecdysone regulates the anti-fungal immune response in Locusta migratoria,and has important scientificsignificance for improving the efficiency of biological control,exploring new targets for pest control,and exploring the green control strategy based on hormone system and immune system. |
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