The Lethal Effects Of Beauveria Bassiana On Ectropis Obliqua And Transcriptome Analysis Of Its Immune-related Genes

Ectropis obliqua is one of the most common and most harmful chewing pests in tea gardens in our country.At present,chemical pest control is still the mainstay.Beauveria bassiana,as a widely used fungal insecticide,is limited in its development and utilization in the field of E.obliqua control due to its relatively slow growth rate and long insecticidal cycle.In order to fully understand the natural immune mechanism of the E.obliqua,in this study,we established an axenic E.obliqua larva model to compare the effects of different infection methods of B.bassiana spore suspension on the lethality of the E.obliqua larvae,constructed 4 c DNA libraries of the hemocytes and fat bodies from infected larva and healthy larvae,screened out a series of differential genes related to the immune response of the looper at the transcriptome level.The main results of the full text are as follows:（1）Construction of a relatively sterile E.obliqua larva model in the laboratoryThe orthogonal experiment was designed to investigate the effects of egg age,different disinfectant concentration and treatment time on the removal of microorganisms on the surface of E.obliqua eggs.Tissue culture tea seedlings were used to feed the larvae.The guts homogenates of the 4^th instar larvae were spread on LB plate and the 16S r DNA-PCR method was used to further verify the axenic situation.This feeding method supplied a stable research system for the subsequent interaction research between insect and symbionts.（2）The effect of different infection methods of B.bassiana on the mortality rate of E.obliquaWe set up 4 groups of experiments:nonaxenic+Bb,axenic+Bb,nonaxenic+Tween 80,axenic+Tween 80.The B.bassiana spore suspension with 1×10⁷spores/m L concentration was used to infect the larvae by immersion and injection respectively.Through analysis,it was found that the larvae infected by B.bassiana by immersion had a long death cycle and low mortality.On the 10th day after infection,the mortality rate of the nonaxenic group was51.7%and that of the axenic group was 45%,which showed no significant difference in mortality between the two groups（P>0.05）.However,the death rate of injection infection is fast,and the mortality rate is high.After 48 hours,the cumulative mortality of nonaxenic and axenic after being infected by B.bassiana was 41.8%and 24.3%,respectively,and a large number of deaths occurred thereafter.By 60 hours after vaccination,the cumulative mortality rate of the two groups reached 96.4%and 94.3%,and severe acute immune reactions occurred,but there was also no significant difference between the two groups（P>0.05）.（3）RNA-seq analysis of B.bassiana infected with E.obliquaFrom the perspective of insect innate immunity,the dynamic changes of gene expression in hemocytes and fat body tissues of the larvae infected by B.bassiana were analyzed using RNA-seq.It was found that a total of 5,877 differential genes were co-expressed in hemocytes and fat bodies,indicating that the E.obliqua needed a large number of genes to regulate and complete the process of immune response in the process of fighting with fungal infections.There were 5,826 differential genes up-regulated in hemocytes.51 were down-regulated;5,784 were up-regulated in the fat body,and 93 were down-regulated,indicating that fungal infection caused the general up-regulation of genes in the fat body and hemocytes of E.obliqua.Through GO annotation analysis of the differential genes of hemocytes and fat bodies,most genes were regarded to be used in the catalytic activity,binding,cellular process,metabolic process,and cell Part,membrane part,organelle and protein-containing complex account for a relatively high proportion.KEGG functional classification of 5,877genes co-expressed in hemocytes and fat bodies showed most of the up-regulated genes are mainly involved in immune response,signal transduction,amino acid metabolism,carbohydrate metabolism,lipid metabolism,Folding,sorting and degradation,transcription,and so on.It is speculated that the infection of B.bassiana may activate a series of cellular and molecular responses of E.obliqua,and at the same time lots of energy and carbohydrates in the body needed to be consumed.（4）Identification and q RT-PCR analysis of genes related to the immunity of the E.obliquaWe screened and identified 249 immune-related genes,including 53 pattern recognition protein genes:17 CTL,9 PGRP,5 GNBP,11 SR-B,3 TEP,4 galectin,1 FREP,1draper,1DSCAM,1 hemolin;67 signal modulation genes:14 c SP,11 serpin,42 SPH;89 signal transduction genes:44 IMD signaling pathway genes,31 Toll signaling pathway genes,9JAK/STAT pathway genes and 5 JNK pathway genes;15 immune effector genes,and 25other immune-related genes.q RT-PCR method was used to verify the expression changes of10 immune-related genes.CTL-10 was significantly up-regulated in hemocytes and fat bodies,PGRP-5 and PGRP-6 were down-regulated in immune organs,and GNBP-2 is down-regulated in hemocytes and up-regulated in fat body;serpin-7 and SPH-5 are both significantly up-regulated;SPZ-2 in the Toll signaling pathway is up-regulated in hemocytes and down-regulated in fat body;immune effect factors ATT,PPO-1 are inhibited,showing a downward trend in hemocytes and fat bodies.This study initially understood that B.bassiana did not interact with guts microorganisms during infection,and provided a large number of E.obliqua sequence resources through RNA-seq technology for the first time to study the molecule of the E.obliqua against B.bassiana.