| There are various insects in the earth,which are widely distributed.Because of its strong hydrophobicity and water retention that can prevent the invasion of foreign harmful substances and the evaporation of water,thus insects could adapt to the terrestrial life well.The whole life of insects is accompanied by the process of cuticularization and ecdysis,and the structure and content of cuticular lipids is the key factor to determine that if it could survive in the extreme environments such as high temperatures.Besides,the cuticular lipids also contribute to sustaining structural integrity of the cuticle.To find a new target for the pest control,study on the molecular mechanism of lipid synthesis is essential.This paper takes Locusta migratoria as the research object,based on its transcriptome database and other species related genes,we did gene search and splicing.Through RNAi,combined with other detecting method,the expression and functional analysis of some key genes of the lipid synthesis pathway in locust were studied.And the main results are as follows:1.Identification and functional study on fatty acid synthase genes in L.migratoria.Fatty acid synthase(FAS)is a key enzyme of the lipid synthesis pathway,which contains seven functional domains,including acyl carrier protein(PP-binding),malonyl transacylase(acly-transf-1),ketoacyl synthase(KS),β-ketoacyl reductase(PKS-KR),β-hydroxyacyl dehydratase(PS-DH),enoyl reductase(PKS-ER),and thioesterase(TE).Based on the transcriptome and genome databases of locust,three FAS genes(Lm FAS1,Lm FAS2 and Lm FAS3)were identified.The results of RT-q PCR showed that Lm FAS1 and Lm FAS3 were highly expressed in the integument of fifth instar nymphs while Lm FAS2 was mostly expressed in the testis and ovary.Besides,the expression level of Lm FAS1 reached to a peak before and after the ecdysis,and the expression level of Lm FAS3 maintained a relatively high level during the whole fifth instar.RNAi was used to suppress the expression of Lm FASs,and the results showed that about 80% of locusts died during ecdysis and 24 h after ecdysis gradually when treated with ds Lm FAS1 and ds Lm FAS3,but there was no obvious phenotype in the ds Lm FAS2-treated locusts.In addition,a fluorochrome Bodipy505/515 was chosen to stain the neutral lipid of cuticle,and the results showed that compared with the control group treated with ds GFP,there were less lipid droplets and the fluorescence signal became weaker in the locusts with ds Lm FAS1 and ds Lm FAS3 injection,respectively.Meanwhile,the ultrastructure of cuticle was observed,and the result indicated that the material content in pore canal structure decreased obviously after suppressing the expression level of Lm FAS1 and Lm FAS3,respectively.Moreover,the results of GC-MS showed that both cuticular hydrocarbons(CHC)and inner hydrocarbons(IHC)content reduced after treated with ds Lm FAS1 and ds Lm FAS3,respectively.Considering that free fatty acids(FFAs)are the direct product of the FAS,so the content of FFAs was also measured,and the results showed that the content of FFAs significantly decreased after suppressing the expression level of Lm FAS1 and Lm FAS3,respectively.In conclusion,Lm FAS1 and Lm FAS3 are essential for hydrocarbon production in locust.2.Identification and functional study on elongase of very long chain fatty acids in L.migratoria.There are several kinds of enzymes taking part in the lipid synthesis,and the fatty acid elongase catalyzes palmitate-Co A into long chain acetyl Co A thioester.Based on the transcriptome database of L.migratoria,here seven fatty acid elongases genes(Lm ELO1-7)were identified,and all the proteins these genes encoded contained elongase domain and four to seven transmembrane domains.The expression pattern of Lm ELOs is diverse: Lm ELO1 and Lm ELO4 mainly expressed in the hindgut(HG),Lm ELO2 had a high expression level in the foregut(FG)and hindgut(HG),whereas Lm ELO5 was mainly expressed in the wing pads(WP),ovary(OV)and testis(TE),and Lm ELO6 mainly detected in the ovary(OV).Notably,the transcripts of Lm ELO3 and Lm ELO7 were almost specifically expressed in the integument(IN),but little or no detectable in other tested tissues.In addition,the results of developmental expression pattern showed that Lm ELO3 and Lm ELO7 had a high m RNA expression level in the early stage of fifth instar nymphs,but the expression level of Lm ELO3 reached a peak at day 2,whereas Lm ELO7 had the highest expression level at day 4.After the specific double stranded RNA(dsRNA)was injected separately,lethal phenotypes were observed during molting only when injected with ds Lm ELO7,while suppressing the expression of other Lm ELOs did not induce any obvious phenotypes.The results of Bodipy505/515 staining showed that the fluorescence signal became weaker and the epidermis contained less neutral lipids after the locusts treated with ds Lm ELO7.Furthermore,GC-MS analysis indicated that suppression of Lm ELO7 resulted in a decrease in CHC and IHC amounts by 43.3% and 31.8%,respectively,which is mainly due to the reduction of C28,C29 and C30.Consequently,Lm ELO7 plays an important role in the synthesis of cuticular and inner lipids in L.migratoria.3.Effects of Lm FASs and Lm ELO7 on cuticular permeability in L.migratoriaAs we know,the content of the cuticular lipids in insect affect its permeability of cuticle,and the GC-MS analysis showed that the synthesis of lipids was impeded when silencing the expression of Lm FAS1,Lm FAS3 and Lm ELO7.Thus,Eosin Y staining,insecticide test and desiccation experiment were performed to detect the changes of the cuticular permeability.And as the results showed,comparing to the control group treated with ds GFP,when injected with ds Lm FAS1,ds Lm FAS3 and ds Lm ELO7,respectively,the nymphs cuticle was colored deeper stained at the same condition with the control;and it caused a higher mortality when exposure to the insecticides.In addition,the viability and the survival time of the locusts were significantly reduced in dry environment after suppressing the expression of Lm FAS1,Lm FAS3,and Lm ELO7,respectivelyIn summary,in this study,we found that after the expression of Lm FAS1,Lm FAS3 and Lm ELO7 were suppressed,respectively,a high probability of lethal phenotype was observed.Meanwhile,through neutral lipid staining,cuticular ultrastructure observation and GC-MS analysis,it can be seen that the lethal phenotype is probably because of the reduction of the lipid content.In turn,decreased lipid content enhanced the cuticular permeability and weakened the water retention of the cuticle,and thus resulted in death of the locusts in the environmental impact.In the present study,three key genes of the lipid synthesis pathway were identified and their biological functions were preliminarily studied,which will provide a further theoretical basis for pest control. |
