| Brown planthopper(Nilaparvata lugens St?l)is a monophagous pest of rice.Owing to its characteristics of migration and strong reproductive capacity,N.lugens often causes huge losses to rice production.In the study of interaction between N.lugens and the resistant rice,it was found that some mitochondria related genes and pathways showed differential expression patterns.Mitochondria play an important role in many life activities,such as ATP synthesis,oxidative stress,calcium signal transmission and cell apoptosis.Mitophagy is a process that cells selectively degrade mitochondria through autophagy,which can remove dysfunctional mitochondria.Mitophagy is a homeostasis regulation and adaptation mechanism of cells.However,the relationship between mitophagy and the adaptation of N.lugens to resistant rice has not been reported.Therefore,it is necessary to characterize some mitophagy-related genes in N.lugens and explore their expression patterns and functions,which is helpful to further reveal the role of mitophagy in the process of N.lugens adapting to resistant rice.5 genes of N.lugens,among which 2 genes encode the alpha subunit and beta subunit of mitochondrial ATP synthase(NlATPSɑand NlATPSβ),and 3autophagy-related genes(NlATG3,NlATG4 and NlATG8)in the ATG8 ubiquitin binding pathway were cloned in this study.The expression patterns of these genes in different developmental stages and different sexes of N.lugens were detected by real-time quantitative PCR.In addition,the expression patterns in different tissues of three autophagy-related genes(NlATG3,NlATG4 and NlATG8)were also detected.The functions of 4 genes(NlATPSɑ,NlATPSβ,NlATG3 and NlATG4)were detected by RNA interference(RNAi).The results of the study are as follows:1.Cloning and functional analysis of NlATPSɑand NlATPSβin N.lugensThe full-length cDNA of NlATPSɑis 2131 bp,with an open reading frame of1656 bp,encoding 551 amino acids.The open reading frame of NlATPSβis 1578 bp,encoding 525 amino acids.The qPCR showed that the expression patterns of NlATPSɑand NlATPSβwere similar,both were expressed in all developmental stages,and the expression levels of nymph were higher than those of the adult.In addition,the expression level of NlATPSɑ,as well as NlATPSβ,between male and female was significant different(P<0.01).RNAi of NlATPSɑand NlATPSβwas conducted using injection method,and the results showed that the expression levels of these genes were drastically declined compared with the control,and showed an extremely significant(P<0.01)at 4~thh day after RNAi.In addition,RNAi can reduce the contents of ATP in N.lugens.RNAi can also cause death of N.lugens,and the survival rate decreased to 45%and 40%at 7~thh day,respectively,compared with the control.2.Cloning and functional analysis of NlATG3,NlATG4 and NlATG8 in N.lugensThe open reading frame of NlATG3 is 990 bp,encoding 329 amino acids;and the open reading frame of NlATG4 is 1140 bp,encoding 379 amino acids;and the open reading frame of NlATG8 is 360 bp,encoding 119 amino acids.The qPCR showed that the expression patterns of NlATG3,NlATG4 and NlATG8 were similar,3 genes were expressed in all developmental stages,and the expression levels of nymph were higher than those of the adult.In addition,the expression levels of NlATG3 and NlATG8 between male and female were significant different(P<0.01),and the expression level of NlATG4 was different(P<0.05).However,the expression patterns of NlATG3,NlATG4 and NlATG8 in defferent tissues were different:NlATG3 were expressed in all tissues and the expression level was the lowest in the head,NlATG4were expressed in all tissues and the expression level in the thorax was the highest,when the expression level of NlATG8 in the thorax was near to 0.RNAi of NlATG3and NlATG4 was conducted using injection method,and the results showed that the expression levels of these genes were drastically declined compared with the control,and showed an extremely significant(P<0.01)at 4~thh day after RNAi.In addition,RNAi can reduce the contents of ATP in N.lugens.Injection of ds NlATG4 can change the cytomorphology of N.lugens.The vacuolization of midgut cells appeared obvious and the morphology and structure of mitochondria near the intestinal villi became very vague compared with the control group.The integrity of fat tissue was destroyed,and the boundaries of their bacteria were not clear.Injection of dsNlATG3 can cause severe death and the survival rate decreased to less than 10%at 7~thh day compared with the control,when the survival rate of injection of dsNlATG4 was decreased to 50%at7~thh day compared with the control.In conclusion,some autophagy-related genes had been cloned and identified,and the expression patterns of these genes had been identified,and the function of these genes were preliminarily studied in this study.The results laid the foundation for further revealing the function of mitophagy in N.lugens,and provided new ideas and potential targets for the prevention and control of N.lugens. |
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