Molecular Regulation Mechanism Of Toll Pathway Of Neoseiulus Barkeri Against Beauveria Bassiana

Using natural enemies to carry pathogenic fungi for cooperative control is an important mode which promote efficient utilization of biological control products.However,pathogenic fungi that act on target pests can also adversely affect natural enemies.Therefore,it is necessary to analyze and enhance the tolerance of natural enemies to pathogenic fungi and the potential tolerance mechanism.The infection of pathogenic fungi can activate the innate immune response system of insects.The Toll signaling pathway,an important signal transduction pathway in the innate immune system,has attracted much attention for its physiological regulation of antimicrobial factors during insect resistance to exogenous stimuli.However,the response and regulatory mechanism of Toll pathway in predatory mites remains unclear.Therefore,this thesis takes commercial natural enemy Neoseiulus barkeri as the object,and comprehensively uses bioinformatics and molecular biology methods to explore the Toll pathway regulatory network of N.barkeri;define the basic composition of the Toll pathway;analysis the response patterns of Toll pathway related genes after Beauveria bassiana infection;clarify the functional characteristics and regulation patterns of Toll pathway key response genes;verify the function of downstream antimicrobial factors,and construct the regulatory network of Toll pathway on antimicrobial factors.The purpose of this research is to clarify the molecular mechanism of the N.barkeri Toll pathway in response to B.bassiana infection,improve the research basis of the immune response of natural enemy insects(mites),and provide theoretical support for the coordinated control of target pests by N.barkeri carrying B.bassiana.The main research contents and results are as follows:1 Identification and sequence analysis of Toll pathway signaling factors.10 Toll Pathway related genes were screened by transcriptome data.Molecular cloning and sequence analysis showed that the selected genes had their own conserved domains and characteristic sequences,which were consistent with characteristics of Toll pathway signaling factors.This indicates that the Toll pathway of N.barkeri is structurally complete and functionally conserved.Phylogenetic analysis showed that the Toll pathway genes of N.barkeri converged with those of Gallendomus occidentalis with high similarity.Evolutionary selection pressure analysis showed the Toll pathway genes were in negative selection state,eliminating harmful mutations and tending to conserved structure.Using q PCR to analyze the expression patterns of the Toll pathway genes in different developmental stages of N.barkeri,the results showed that Toll pathway genes were generally highly expressed in larva and nymph.This suggests that Toll pathway plays an important regulatory role in the rapid development of N.barkeri.2 Regulation patterns and function analysis of Toll pathway signaling factors.Using q PCR to detect the response patterns of the N.barkeri Toll pathway genes after B.bassiana long-term infection,the results showed that the up-regulated expression of Toll pathway genes was induced by B.bassiana,and the response lasted for a long time and was violent.The expression of Toll pathway genes was down-regulated at 120 h after Beauveria bassiana infection,which was speculated to be related to the immune escape mechanism derived from B.bassiana or inhibition of mite immune system.Using RNAi to knock down the expression of five key genes with significant response(Nb Spz-3,Nb Toll-4,Nb Myd88-1,Nb Cactus and Nb Dorsal),and detect the regulation patterns of Toll pathway related genes.The results showed that the five Toll pathway key genes were involved in signal transduction through linearized regulation mode.At the same time,we measured the tolerance of N.barkeri to B.bassiana infection after the silencing of Toll pathway genes.The results showed that the expression of Nb Spz-3,Nb Myd88-1,Nb Cactus and Nb Dorsal which were knocked down could significantly reduce the survival rate of N.barkeri.After the B.bassiana infection,the Toll pathway key genes of N.barkeri will express rapidly and stably for a long time,and conduct signal transduction in a linearized regulation pattern,which can enhance the tolerance of N.barkeri to B.bassiana.3 Identification and expression patterns analysis of antimicrobial protein genes of N.barkeri.Three c-type lysozyme genes,one i-type lysozyme gene,two new defensin genes and one Reeler domain protein gene were cloned.We analyzed the expression patterns of the 7 antimicrobial protein genes in different developmental stages of N.barkeri,and the results showed that the 7 antimicrobial protein genes were highly expressed in the larva and nymph,and the expression level in the egg stage was low.These were similar to the expression patterns of Toll pathway genes at different developmental stages.In addition,the response patterns of antimicrobial protein genes to B.bassiana showed that the lysozyme genes and Reeler domain protein genes were rapidly up-regulated in a short time and the up-regulated expression lasted for a long time;although the expression of the new defensin genes was also up-regulated,it lasted for a short time and was upregulated again at the late stage of infection.These results indicate that the lysozyme and Reeler protein families play a major role in the process of resisting B.bassiana infection,while the new defensin family plays a complementary role.4 Functional analysis of antimicrobial factors and regulation patterns of Toll pathway key genes on antimicrobial factors in N.barkeri.The effect of four lysozyme genes with significant response on the tolerance of N.barkeri to B.bassiana was verified using RNAi.The results showed that inhibition of the four genes expression significantly reduced the survival rate of N.barkeri.This indicated that the expression of the selected antimicrobial protein genes enhanced the tolerance of N.barkeri to B.bassiana.In addition,after knocking down the expression of Toll pathway genes,we measured the change of antimicrobial protein genes expression.The results showed that Nb Spz-3 and Nb Dorsal had a powerful regulatory effect on all 7antimicrobial factors,while Nb Toll-4,Nb Cactus and Nb Myd88-1 had complementary regulatory roles with different emphasis.In conclusion,we analyzed the basic composition of the Toll signal pathway of N.barkeri.We also elucidated the molecular mechanism that the key genes in the Toll pathway regulated the downstream antimicrobial factors to resist the B.bassiana infection.