Functional Study Of Genes On Regulating Cuticular Hydrocarbon Production And Transport In Acyrthosiphon Pisum

Insect cuticular hydrocarbons(CHCs)serve as the first barrier to protecting insects in many ways.Most importantly,they prevent terrestrial insects from losing water via cuticle evaporation in arid environment and blocking the invasion of insects by external harmful microorganisms including bacteria,fungi,and viruses.In addition,insect CHCs serve as the material basis of insect chemical communication,and are very important in the social behaviors of insects for species and sex identification and mating.Therefore,this thesis mainly focused on the three aspects that affect CHC production,transport and deposition,through molecular identification,characterization and functional study of NADPH-cytochrome P450 reductase(CPR),apolipoprotein D(ApoD)and lipophorin receptor(LpR)which are involved in the HCs biosynthesis,transport,and deposition in Acyrthosiphon pisum.It was found that three genes of CPR,ApoD,and LpR all play a regulatory role in CHC accumulation and desiccation tolerance of A.pisum,and ApoD and LpR had an important influence on A.pisum fecundity.The main findings are as follows:1.Study of A.pisum CPR(ApCPR)gene on regulating CHC production:Firstly,molecular characteristics and protein functional domains of ApCPR gene were characterized and its evolutionary relationship was clarified by constructing a phylogenetic tree.Secondly,after desiccant stress treatment,the expression of ApCPR gene was significantly up-regulated compared with the control.Next,on the basis of determining the optimal dsRNA injection dose(1.82?g)of ApCPR gene and duration(5 days)of ApCPR silencing effect at this dose,the contents of CHC and internal hydrocarbon(IHC)in A.pisum were determined by GC-MS at the 3rd,4th,and 5th day after dsRNA injection.It was found that ApCPR silencing led to a significant reduction in the CHC and IHC content of A.pisum.At the same time,the components of CHC and IHC were both qualified and quantified,and results showed that the A.pisum CHC was composed of 9 C₂₅-C₃₃ n-alkanes,but IHC contained a large number of HCs with methyl branches in addition to n-alkanes,as well as ApCPR silencing had different effects on different components of A.pisum CHC and IHC.Then,after ApCPR silencing it has resulted in a significant decrease in CHC content.A flat and clean abdominal body surface of aphids covered with a small amount of lipids by scanning electron microscope from the treatment group was observed,as well as the significantly shortened survival time in arid environment was compared with the control.Finally,we did the insecticidal bioassay on the basis of LC₅₀ values of three insecticides including chlorpyrifos,imidacloprid,and beta-cypermethrin,with applying 0.3?L of insecticide solution to the abdominal cuticle of A.pisum when ApCPR expression was suppressed.The results showed that ApCPR silencing increased the A.pisum susceptibility to three insecticides,and the mortality rate was significantly higher than control.Therefore,ApCPR has potential to become a new target for aphid control.2.Study of A.pisum ApoD(ApApoD)gene on regulating CHC transport:Firstly,molecular characteristics and protein functional domains of ApApoD gene were characterized,and its phylogenetic relationship was analyzed by comparing protein structure with those from other representative insects and constructing a phylogenetic tree.Secondly,under desiccant stress conditions,the expression of ApApoD was significantly up-regulated.Next,RNAi parameters of ApApoD gene were optimized.Based on the optimal dsRNA injection dose of 1.21?g and silence effect duration of 5 days,pea aphids on the 3rd day after dsRNA injection were collected for CHC and IHC content determination by GC-MS and results showed that the contents of both reduced significantly,and it had different effects on different components in CHC and IHC.ApApoD silencing significantly shortened survival time of A.pisum in arid environment and reduced desiccation tolerance.Finally,ApApoD silencing led to a significant decrease in adult fecundity of A.pisum which was manifested in delayed newborn aphid production and lower cumulative average reproduction,as well as reduced embryo number and stunted embryos compared with the control.Therefore,ApApoD gene may become a new target based onRNAi technology for aphid control.3.Study of A.pisum LpR(ApLpR)gene on regulating CHC deposition:Firstly,the protein functional domain of ApLpR gene was compared with LpR of other representative insects,and evolutionary relationship was analyzed by constructing a phylogenetic tree.Secondly,under desiccant stress,the abundance of ApLpR mRNA was significantly up-regulated.Next,the optimal dsRNA injection dose of ApLpR was determined to be 1.21?g,and at this dose,ApLpR silencing effect could be maintained for 4 days.On this basis,CHC and IHC contents of A.pisum on the 3rd day after dsRNA injection were determined by GC-MS,and results showed that the CHC and IHC contents were significantly reduced,as well as the components of CHC and IHC of A.pisum were identified and quantified,and results showed that ApLpR silencing had different effects on different components.Then,under desiccant stress,ApLpR silencing led to a significant decrease in A.pisum desiccation tolerance.Finally,ApLpR silencing also resulted in a significant reduction in adult fecundity of A.pisum.Therefore,ApLpR gene may become another newRNAi target for aphid control.To sum up,this work systematically studied the molecular and functional characteristics of three genes,ApCPR,ApApoD and ApLpR,which regulate CHC synthesis and transport in A.pisum,and revealed their important regulatory role in CHC content accumulation and desiccation tolerance of A.pisum,as well as the important effects of ApApoD and ApLpR on adult fecundity of A.pisum.These results not only provide a basis for further study on their regulation mechanisms in CHC production,transport and deposition,as well as A.pisum reproduction,but also provide potential targets for aphid control based onRNAi technology.