| Eurydema gebleri a common pest on cruciferous vegetables,is widely distributed in China.E.gebleri sucks the host juice through its piercing and sucking mouthparts to provide it with the nutrients needed for growth and development,which can make the host unable to set pods or empty seeds.In severe cases,it can lead to the host wilt or even die.Beauveria bassiana is one of the entomogenous fungi widely used in biological control.At present,there are increasing research reports on the use of B.bassiana to infect insects,but there are no reports about the infection of B.bassiana on E.gebleri.Therefore,this study used the spot method to infect the E.gebleri with B.bassiana,and observed the changes of the protective enzymes(SOD,POD,CAT)and detoxification enzymes(Car E,GST)in E.gebleri after the infection.The RNA-seq technology was used to sequence E.gebleri infected with B.bassiana,and the genes related to antioxidant and detoxification metabolism were analyzed.The results of this study can provide a theoretical basis for studying pathogenic mechanism of entomogenous fungal to E.gebleri.The research results are mainly shown in the following four aspects:1.After infection with different concentrations of B.bassiana strain FDB,the activities of protective enzymes(SOD,POD,CAT)in E.gebleri were increased first and then decreased.After treated with B.bassiana(1×106 conidia/ml,1×107 conidia/ml and 1×108 conidia/ml),The SOD activity in E.gebleri had the peak value on the 6d,4d and 4d respectively,which were significantly higher than those of the control group(P<0.05).The POD activity peaked on the 6d,6d,and 4d respectively,which were significantly higher than those of the control group(P<0.05).The CAT activity peaked on the 6d,6d,and 4d respectively,which were significantly higher than those of the control group(P<0.05).After infected with B.bassiana strain FDB,the activity of Car E in B.bassiana showed a trend of increasing-decreasing-increasing.The Car E activity in E.gebleri had the peak value on the 4d,2d and 2d respectively,of which 1×107 conidia/ml and 1×108 conidia/ml treatment group was significantly higher than the control group(P<0.05);after that,the Car E activity in E.gebleri infected with B.bassiana reached the lowest value on the 6th day,Among them,the 1×108 conidia/ml treatment group was significantly lower than the control group(P<0.05).However,the GST activity in E.gebleri significantly decreased,and reached the lowest value on the 6,8 and 2 days respectively,which were significantly higher than those of the control group(P<0.05).2.Using RNA-Seq technology,we sequenced the adult E.gebleri 96 h after infected with B.bassiana.A total of 2.52 ×108 Clean reads were obtained,and a total of 117,851 Transcript and 47345 Unigene were obtained by assembly and splicing.Annotate Unigene with the databases NR,GO,KEGG,egg NOG,Swiss-Prot,and Pfam;the annotation rates were 37.10%,7.88%,15.21%,30.58%,22.51%,and 20.97%respectively.Through annotation with the NR database,the homology between the E.gebleri and Halyomorpha halys was the highest.Through annotation with the GO database,Unigene was classified into20 subclasses of biological processes,17 subclasses of Cellular components,and 13 subclasses of molecular functions.Through annotation with the KEGG database,Unigene was classified into 12 subclasses of metabolism,4 subclasses of genetic information processing,3 subclasses of environmental information processing,4 subclasses of cellular processes,and 10 subclasses of organic systems.According to the egg NOG annotation results,Unigene was divided into 26 categories.The number of Unigenes from high to low was general function prediction only,function unknown and signal transduction mechanism,etc.3.The number of differentially expressed genes,GO enrichment and KEGG Pathway enrichment of E.gebleri after 96 h infected with B.bassiana were statistically analyzed.A total of 834 differentially expressed genes were screened.There were 402 up-regulated genes and 432 down-regulated genes.The differentially expressed genes were enriched into 64 GO terms of cell components,119 GO terms of molecular functions and 276 GO terms of biological processes.Among them,24 GO terms had more than10 differentially expressed genes.The differentially expressed genes were enriched by KEGG,and the differentially expressed genes were annotated into 138 pathways,which were divided into 6 categories and37 subcategories according to their levels.And 11 of them had the number of differentially expressed genes greater than 4.In addition,5 up-regulated and down-regulated genes were screened out for q RT-PCR verification,which finally showed that the sequencing results are reliable.4.Antioxidant and detoxification genes were identified and analyzed based on transcriptome data.A total of 2 SOD,3 POD,1 CAT,10 Car E and 14 GST genes were identified with complete open reading frame(ORF).Among them,there were 1 CAT(up-regulated),1 POD(up-regulated),2 Car E(down-regulated)and 1 GST(down-regulated)differentially expressed genes.POD differentially expressed genes failed to be successfully annotated into the pathway,there were 8 pathways involved in the differentially expressed genes of CAT,1 pathway involved in the differentially expressed genes of Car E,and 9 pathways involved in the differentially expressed genes of GST.Construct a phylogenetic tree of antioxidant and detoxification metabolic genes.SOD was divided into 3 categories.One of the SOD genes of E.gebleri belongs to Mn and one belongs to copper chaperone.POD was divided into 4 categories.One of the POD genes belonged to Chorion.Car E was divided into 3 categories.One of the Car E genes belonged to juvenile hormone esterase.GST was divided into 7 categories.One of the GST genes of E.gebleri belonged to delta,one gene belonged to theta,one gene belonged to microsomal and two genes belonged to sigma.In summary,this dissertation conducted a systematic study on the antioxidant and detoxification metabolic reactions in the body of E.gebleri after infection with B.bassiana.Macroscopically,we observed the dynamic changes of protective enzymes and detoxification enzymes in the body of E.gebleri.At the micro level,sequencing,biological information analysis,and differentially expressed gene analysis of E.gebleri,on this basis,the genes and differentially expressed genes related to antioxidant and detoxification metabolic reactions were screened for analysis. |
PDF Full Text Request