| Tetranychus urticae（Koch）is a global agricultural and horticultural pest,it is distributed in the main production areas of buckwheat in Guizhou province,causing flower and fruit dropping and affecting the yield of buckwheat in severe cases.The extensive use of chemical acaricides for a long time has brought a series of problems such as enhanced pest resistance,serious pesticide residues,ecological and environmental pollution,etc.Therefore,it is urgent to develop and find new acarid biocontrol agents with high efficiency and low toxicity.Cordyceps cateniannula（also known as Isaria cateniannula）is one of the most potential new biological control agents and has been found to have a good control effect on T.urticae.The mortality of C.cateniannula to T.urticae can reach 100% under laboratory conditions,and is more than 80% after 10 days of field application.However,the immune mechanism of T.urticae in vivo during the infection process by C.cateniannula is still unclear and needs to be explored urgently.In this study,the activity changes of antioxidant enzymes and detoxification enzymes in T.urticae during the infection process by C.cateniannula were measured,and the expression specificities of CAT,GST genes,chitinase genes,and Toll pathway-related genes were analyzed and verified,which will clarify the immune mechanism of T.urticae to C.cateniannula infection and provide a scientific basis for the development and use of C.cateniannula as a biological agent of T.urticae.The main research results are as follows:1、Effect of the antioxidant enzymes and detoxification enzymes activities of T.urticaeAfter the female adult mites of T.urticae were infected by C.cateniannula,the antioxidant enzyme and detoxification enzyme systems in the mites showed a trend of increasing and then decreasing.The activities of POD and MFO reached the maximum of 89.02 U/g,2.65 nmo L/min/g at 4 h respectively.CAT enzyme activity reached a maximum of 4820.00 nmo L/min/g at 12 h and GST enzyme activity reached a maximum of 1238.70 nmo L/min/g at 36 h.These results indicated that T.urticae was under stress after being infected by C.cateniannula,the ROS was increased,and the immune systems such as antioxidant enzymes and detoxification enzymes were activated to cope with the infection by C.cateniannula.2、Effect of the CAT,GST,Chitinase,and Toll pathway of T.urticaeAfter T.urticae is infected by C.cateniannula,the expression levels of different Tu CATs showed a trend of increasing and then decreasing.The expression levels of Tu CAT1,Tu CAT2,and Tu CAT4 reached the maximum and were 2.63 times,3.26 times and 2.19 times that of the control group at 4h.The expression levels of Tu CAT3 reached the maximum and were 1.30 times that of the control group at 36 h.The expression of Tu GSTs showed a trend of decreasing and then increasing.The expression capacity of Tu GST2 and Tu GST3 were all down-regulated at 4 h.At 36 h,Tu GST2 and Tu GST3 expressions reached the maximum and were 1.50 times and 4.23 times that of the control group.Tu GST1 and Tu GST4 expressions reached the maximum and were 1.29 times and 1.72 times that of the control group respectively,at 48 h.The expressions of different chitinase genes（Tu CHTs）all showed a decrease,but the different types of Tu CHTs showed different response patterns,which affected by the response speed,the intensity and the different periods.The expression trends of the same type of Tu CHTs were similar.Tu CHT7,Tu CHT8 and Tu CHT9 were type I chitinase,while Tu CHT11 and Tu CHT12 were type II chitinase.Studies showed that the functions of both types I and type II chitinase were related to the molting process of insects.While Tu CHT10 was a type VII chitinase,which had certain fungistasis effect.The genes related to different Toll pathways all could be expressed,eg.Tu Dorsal expression was up-regulated at 4 h,indicating that the response of Toll pathway in vivo was elicited.The up-regulated expression of other genes except for Tu Toll4 and Tu Toll8 might be closely related to activate of the Toll pathway at 12 h.Tu Myd88 played an important role in the response process.3、Clone and bioinformatics analysis of Chitinase gene and Toll pathway-related gene of T.urticaeThe sequences of Tu CHT12 and Tu Myd88 genes were screened out from the genomic data of T.urticae.The c DNA sequences were cloned and obtained by RT-PCR technology.The physicochemical properties,sequence analysis,and phylogenetic tree of the protein encoded by Tu CHT12 and Tu Myd88 genes were analyzed by bioinformatics.The open reading frame（ORF）of Tu CHT12 was found to be 1020 bp,encoding 339 aa.The predicted isoelectric point and molecular weight of CHT12 were 6.50 and 48.69 k Da,respectively.CHT12 protein contained a signal peptide and forecasted to be a a secreted protein.The results of phylogenetic tree analysis by amino acid sequence alignment showed that Tu CHT12 was clustered on one branch with Drosophila melanogaster CHT10 and forecasted to be a type II chitinase because of Dm CHT10,which might be related to the molting process of mites.Tu Myd88 has an open reading frame of（ORF）1350 bp that encodes 449 amino acids.The predicted isoelectric point and molecular weight of My D88 were 5.83 and 51.25 k Da,respectively.The Myd88 was clustered on the branch of Sarcoptes scabiei and Dermatophagoides pteronyssinus with a similarity of 80%,both of which belonged to Acariforms.4、Functional verification of the Tu CHT12 and Tu Myd88 genes of T.urticaeWhen the Tu CHT12 gene was silenced,the expression of Tu CHT11 would be decreased,which might be because both CHT11 and CHT12 were type II chitinase.When the expression of Tu CHT12 was inhibited,the expression of similar chitinase genes would be affected,which could lead to the molting process of T.urticae affected.Tu Myd88 gene silencing resulted in decreasing expression of Tu Toll8,Tu Pelle,and Tu Dorsal,indicating that Myd88 affected the Toll pathway in the immune response of T.urticae.The Tu CHT12 and Tu Myd88 genes were both silenced,the cumulative mortality of the female T.urticae infected by ds Myd88+XS08-1 in 4thd was 100%,more 1.18 times than the control group;and the number of eggs laid was significantly reduced to 58,less 0.42 times than the control,indicating that C.cateniannula and ds Myd88 could be used simultaneously to control the female T.urticae. |
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