Studies On The Expression And Function Of Insulin-Like Signalling Key Genes In Leptinotarsa Decemlineata(Say)

In this study,the key components of the Leptinotarsa decemlineata insulin signaling pathway,akt(protein kinase B)and tsc1 and tsc2 genes were cloned,multiple sequence alignment and phylogenetic analysis were performed,and the spatial and temporal expression of several genes were analyzed by qPCR technology.The function of several key genes in the growth and development of larvae and their effects on the metabolism of glucose and lipids is studied,as well as the relationship between insulin-like hormones and juvenile hormones and ecdysone.The relationship between the function and the regulation of hormone interaction is described.AKT is recommended as a new target for pest control,considering the stable transmission of AKT gene functions during evolution,and the significantly reduced pupation and eclosion rate of larvae.This article mainly obtained the following results1.Sequence alignment and phylogenetic analysis of key genes of the potato beetle insulin pathwayThe amino acid sequence of the Ldakt gene translation was similarly searched and compared by NCBI online BLAST.After comparing the amino acid sequences of 5 insects,it was found that the sequence identity between LdAKT and AKT of other species is higher,which indicates that the LdAKT gene is It has better conservation during evolution.The NJ phylogenetic method was used to find that the potato beetle was closely related to Anoplophora glabripennis and Agrilus planipennis.Online-BLAST performed a similarity search comparison of the amino acid sequence of the Ldtsc1/2gene translation.After comparing the amino acid sequences with orthologous codes of insects of different orders,it can be seen that the tsc1 and tsc2 genes encode hamartin and tuberin proteins,respectively.LdTSC1 a nd the coleopteran corn root DvTSC1 are aggregated into one branch,and the self-consistent consistency is 100%;the potato beetle LdTSC2 is aggregated with the DvTSC2,self-expansion consistency is 100%;TSC2 of other insects clustered into small groups,indicating that LdTSC2 has better conservation than LdTSC1 during evolution.2.Spatiotemporal expression profile of key genes of potato insulin-like pathway(1)Age expression was analyzed by qPCR.When the expression of potato beetles at differentdevelopmental ages was different,Ldakt expression was relatively low in eggs and first instar larvae,and then in the third instar Gradually increased,4th instar larvae expressed the highest.it was found that Ldtsc1 expression was relatively low in eggs and first instar larvae,and then gradually increased in the second and fourth instar larval stages.The expression of Ldtsc2 was relatively low in eggs and 1st instar larvae,followed by higher expression at the end of 1st,early 2nd and 4th instars.(2)Tissue expression The relative expression levels of the tissues measured on the six tissues of the third instar larva of the potato beetle showed that the gene did not have a consistent expression pattern in each tissue,and the expression of each gene was different.Ldakt gene expression is relatively high in brain and hemolymph.The expression of Ldtsc1 gene was found to be relatively high in brain and hemolymph when the expression of different tissues of potato beetle was different.However,the expression of Ldtsc2 gene was relatively high in the brain and midgut.3.Effects of knockdown of key genes of insulin pathway by RNAi on the growth and development of potato beetlesAfter feeding the second-instar larvae to knock down the target genes Ldakt and Ldtsc1/2,it was detected that the expression of tissues in the test worms decreased,weight loss,fat body loss,and delayed development occurred.At the same time,the larvae had a certain degree of mortality.It has a significant effect on the pupation rate and emergence rate.Most test insects appear delayed in soil and cannot pupate normally,and it is easy to form black pupae,which causes most pupae to fail to emerge normally.4.Effects of key genes of Leptinotarsa decemlineata insulin pathway on carbohydrate and lipid metabolism in larvaeStudies have shown that after silencing Ldakt and Ldtsc1/2,the expression levels of target genes are both down-regulated,and their glucose and lipid metabolism is also blocked.Studies have found that after the glucose ingested by the test insect enters the hemolymph,the absorption and utilization of glucose are reduced;while trehalose is enriched in the test tissue,the decomposition rate of trehalose in the fat body is reduced,resulting in a reduction in the amount of glycogen synthesis;The synthesis of qualitative triglycerides and total cholesterol were reduced in tissue.Glycolipid metabolism disorders of test insects seriously and downregulation of key insulin-like gene expression leads to reduced anabolism that affectedthe development of larvae.5.Cross-talk between key genes of Leptinotarsa decemlineata insulin pathway and juvenile hormone and ecdysoneThis study further tested the transcription levels of juvenile hormone synthesis genes Ldjhamt,juvenile hormone resting hormone Ldas-c or juvenile hormone analog Ldkr-h1,ecdysone synthesis-related genes Ldptth,Ldshd,and Ldftz-f1 in vivo.All of them decreased in different degrees,and it was found that insulinoids may positively regulate ecdysone and juvenile hormone.