| Research background and question:Free fatty acids(FFA)are important metabolites in animals,involved in oxidative energy supply,lipid synthesis and signal transduction.Free fatty acids in animals are mainly derived from food,or are synthesized from their own excess sugars.When there are insufficient carbohydrates in animals,triglycerides are broken down into FFA and glycerol to maintain the homeostasis of FFA in the body.Lipases in the digestive tract are responsible for decomposition of TAG in food into FFA and glycerol,while lipases in animal tissues break down TAG stored in the body into FFA.However,excessive FFA in the body may also lead to diseases such as hyperlipidemia and angiemphraxis.Therefore,the content of FFA in animals needs to be regulated at an appropriate level(homeostasis).The molecular mechanism of regulating the expression of different lipases to maintain the dynamic balance of FFA in animals is unclear.Complete metamorphosis insects obtain nutrients such as fatty acids by eating and digesting at feeding stage,while they rely on decomposing their own stored fats to provide energy substances at metamorphosis stage.It is known that insulin and juvenile hormone(JH)maintain the growth and development of larvae,while 20-hydroxyecdysone(20E)antagonizes insulin and juvenile hormone to promote metamorphosis.Therefore,complete metamorphosis insects provide a good model for studying the regulation mechanism of FFA metabolism in different developmental and physiological states of animals.Taking the lepidopteran insect Helicoverpa armigera,cotton bollworm,as a model,this thesis reveals the molecular mechanism that JH and 20E differentially upregulate the expression of different lipases via different transcription factors to maintain the dynamic balance of free fatty acids in hemolymph at feeding and metamorphosis stage,and thus maintaining ontogeny.Research results:The phylogenetic tree of lipases in H.armigera was constructed based on the lipases in human genome,and the results showed that the lipases in H.armigera genome were divided into three categories:one was closely related to pancreatic lipase(PTL)in human intestine,and one was related to lipases in human adipocytes,and the other was closely related to human acid lipases.Based on the analysis of the transcriptome data,the expression of lipases in H.armigera showed stage specificity,and they were divided into two categories:one was mainly highly expressed in midgut at feeding stage,and the other was mainly highly expressed in fat body at metamorphosis stage.Lipase member H-A-like(Lha-like),which was highly expressed in midgut at feeding stage and closely related to human pancreatic lipase,and monoacylglycerol lipase ABHD 12-like(Abhd12-like),which was highly expressed in fat body at metamorphosis stage and closely related to lipases in human adipocytes,were selected for study.It was found that JH Ⅲ induced the expression of Lha-like in midgut at feeding satge,however,20E upregulated the expression of Abhd12-like in fat body at metamorphosis stage.In addition,The expression of Lha-like was also upregulated by insulin but not 20E,while the expression of Abhd12-like was not upregulated by insulin and JH Ⅲ.It was found that the 5’ upsream sequence of Lha-like had highly conserved sequences compared with E-box(5’-CACGTG-3’)of JH response element after analysis.Knockdown of methoprene-tolerant 1(Met1)inhibited the expression of Lha-like.The pLha-LUCI-GFP-His luciferase reporter gene plasmid and the MET1-RFP-His overexpression plasmid were co-expressed on HaEpi.Dual-luciferase reporter assay(with the addition of pRL-TK plasmid)showed that JH Ⅲ upregulated transcription of Lha-like via MET1.It was found that 5’upstream sequence of Abhd12-like contained the classical FOXO-binding element(FOXOBE,5’-TTGTTTAC-3’)of forkhead box transcription factor O(FOXO)and the highly conserved sequence compared with edysone response element(EcRE,5’-TCAATG-3’).The results after cotransfection showed that 20E directly upregulated the transcription of Abhd12-like via FOXO,while EcR indirectly activated the transcriptional activity of Abhd12-like.The expression of Lha-like was knocked down by injecting dsRNA into the insect.It was found that the content of free fatty acids in larval hemolymph decreased,the content of triglycerides in fat body decreased,and the larvae showed the phenotype of delayed pupation and small pupation.The expression of Abhd12-like was knocked down by injecting dsRNA into the insect.It was found that the content of free fatty acids in hemolymph also decreased,while the content of triglycerides in fat body increased,and the larvae showed the phenotype of delayed pupation.These results indicated that the two lipases maintained the dynamic balance of free fatty acids in hemolymph at different stages.Conclusion and significance:JH Ⅲ upregulated the transcription of Lha-like via MET1.LHA-like released free fatty acids by decomposing triglycerides in food,thus promoting the synthesis of TAG in fat body and storing energy materials for larval metamorphosis.20E upregulated the transcription of Abhd12-like via FOXO.However,ABHD12-like released free fatty acids by decomposing triglycerides in larval fat body at metamorphosis stage to maintain the energy required for metamorphosis after cessation of feeding.Different hormones regulated the expression of different lipases at different developmental stages through different transcription factors to maintain the dynamic balance of FFA in the body,and provided energy materials for insects at different developmental stages and physiological conditions.By exploring the molecular mechanism of the differential expression of Lha-like highly expressed in larval midgut at feeding stage and Abhd12-like highly expressed in larval fat body at metamorphosis stage,providing a new idea for pest control. |
PDF Full Text Request