| The brassica leaf beetle(Phaedon brassicae Baly)is an important pest on cruciferous vegetables,which causes serious damage.At present,the control of this pest still mainly depends on chemical control.Over-reliance on chemical pesticides will lead to environmental pollution and pest resistance.Entomopathogenic fungi can be used for the control of pesticide-resistant pests.However,the immunity of pests against fungi is one of the reasons for the slow infection and the unsatisfactory control effect of entomopathogenic fungi.Elucidating the immune mechanism of pests against entomopathogenic fungi can provide theoretical basis and insecticidal target for better use of fungi to control pests.Based on this,the susceptibility of P.brassicae to Paecilomyces cicadae was detected in this thesis.Transcriptome of P.brassicae larvae infected by P.cicadae was sequenced,and the immune-related genes were screened and identified.The functional domains and phylogeny of the immune genes were analyzed by bioinformatical methods,and the expression patterns of the selected immune genes of P.brassicae infected by P.cicadae were characterized.For the scavenger receptor family genes(SR)of pattern recognition receptors,the expression levels of them after P.cicadae infection were detected by RT-q PCR,and the immune function of the SR-B4 gene with the highest expression level was further investigated.The main results are as follows:1.Toxicity of P.cicadae against P.brassicae,sequencing and analysis of the transcriptome of P.brassicae infected by P.cicadaeThe results of toxicity assay showed that P.cicadae can efficiently infect the larvae of P.brassicae,and all the larvae died after treatment with 1.0×10~7spores/m L of P.cicadae for9 d.Transcriptomes of P.brassicae larvae non-infected and infected by P.cicadae were sequenced,160164 unigenes were obtained after assembling.Among them,4272 genes were significantly up-regulated and 10399 genes were significantly down-regulated after P.cicadae infection.KEGG enrichment analysis showed that the differential genes were mainly enriched in bile secretion,lysosome,Toll and IMD signaling pathways,receptor interaction,tyrosine metabolism process and cell apoptosis,etc.These pathways are related to insect immunity,which play important roles in insect response to pathogen invasion.2.Screening,bioinformatics and expression pattern analysis of immune genesA total of 214 immune-related genes were screened from the transcriptome of P.brassicae,including 47 pattern recognition receptor genes,62 signaling pathway genes,and 105 immune effector and other effector genes.Among the screened immune-related genes,95 were significantly different expressed,of which 64 were up-regulated and 31were down-regulated.RT-q PCR was used to detect the expression levels of selected immune genes of P.brassicae,and it was found that they were differentially expressed after P.cicadae infection,which were consistent with the results of transcriptome analysis.3.Study on the immune function of scavenger receptor gene SR-B4A total of 20 SR genes were identified in P.brassicae transcriptome,including 3 SR-As,16 SR-Bs and 1 SR-C.The expression patterns of 18 SR genes of P.brassicae were characterized by RT-q PCR after infection by P.cicadae.The results showed that 11 of them were significantly up-regulated compared with the control,and the SR-B4 gene showed the highest expression level,which was 16.16 times higher than that of the control.The ORF of SR-B4 gene is 1515 bp in length,encoding 504 aa with a conserved CD36functional domain.The results of RNAi showed that the larvae injected with ds SR-B4 were died at 5 d after infection with P.cicadae,while 44.44%of the larvae injected with ds GFP still survived,indicating that the SR-B4 gene has the immune function.The conserved CD36 functional domain of the SR-B4 protein was recombinantly expressed in Escherichia coli BL21 cells.Results of microbial aggregation showed that CD36 protein has the obvious ability to aggregate the cells of E.coli and Staphylococcus aureus,and P.cicadae spores,and may be involved in the cellular immunity.Finally,CD36 could significantly clear P.cicadae,E.coli and S.aureus in P.brassicae.These results indicated that SR-B4 was involved in the immunity and played a key role in the immune response of P.brassicae against P.cicadae infection.This study is helpful to clarify the immune mechanism of pests to entomopathogenic fungi,and provides a theoretical basis and insecticidal targets for better use of fungi to control pests. |
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