Cloning And Characterization Of Genes Involved In Juvenile Hormone Degradation Of Lymantria Dispar

Gypsy moth(Lymantria dispar Linnaeus)is a member of the bombinidae family,which is widely distributed around the world.At present,the control of gypsy moth mainly relies on chemical insecticides,and long-term use can lead to the formation of insect resistance,high residue,serious pollution and other problems.Therefore,it has become an urgent problem to find new prevention methods.With the rapid development of molecular biology,finding and using molecular targets to control the growth and development of pests can be a new strategy to control pests.An ideal target gene is a key regulatory gene involved in the growth and development of insects.By changing the transcription level of this gene,the activity of the corresponding substance of the target gene can be regulated,so as to affect the physiological mechanism of pests and eventually lead to their death.Juvenile hormone(JH)in insects plays an important regulatory role in the growth and development of insects,and jheh is a metabolic gene in the metabolic pathway of JH.The exploration of the function of jheh gene and the relationship between related genes is of great significance for the clarification of insect physiology and the search for insecticidal targets.Therefore,based on the gypsy moth transcriptome database,three JH degrading enzyme genes.jhe,jheh and jhdk,were cloned from gypsy moth and annotated.QPCR was used to detect the time expression patterns of jhe,jheh and jhdk in different development stages of gypsy moth,and ELISA method was used to detect the change of JHIII titer in different development stages of gypsy moth.Finally,RNAi experiment of jheh gene was conducted on gypsy moth sixth-instar larvae by injection,and its effect on jhe,jhdk and JH signaling pathway gene kr-hl gene was verified,and JHIII titer was detected again,and pupation and subsequent physiological changes of insects were recorded.The main research results are as follows:1.Cloning and analysis of JH degradation enzyme gene of gypsy mothBased on gypsy moth transcriptome database,using rt-pcr cloning technology for JH metabolic pathway in three degradation enzyme gene jhe,jheh and jhdk ORF,deduce the amino acid sequence of its encoding,and then use the ClustalW2,ProtParam,ProtScale and TMHMM raw software analyzes the three letter JH protein degradation enzyme gene conserved sequence,the structure of the core fields,across the membrane structure and other potential physiological function.The reading frame(ORF)of jhe,jheh and jhdk genes was 552～1743 bp,encoding 183～601 amino acids.The molecular weight of protein was 21.3～65.0kDa and pI was 6～6.87.A11 are acidic proteins.The prediction results of transmembrane regions of jhe,jheh and jhdk proteins showed that jhe and jhdk had no transmembrane region,while jheh contained a transmembrane region.2.Expression patterns of JH degradation enzyme gene at different developmental stagesResults show that jhe in gypsy moth in the age of three to five instars expression quantity showed a trend of increase after the fi&st cut,expressed in each instar larvae of early and late quantity is higher,this is consistent with the JHIII drop degree change trend,but the expression of JHIII time lag,the jhe in gypsy moth larvae of JHIII has certain regulation effect.jhdk and jheh genes were up-regulated and down-regulated in each phase,while the titer of JHIII decreased first and then increased in the third to fifth instar larvae.Both jhe and jhdk expressions were very low at the pupal stage,which was consistent with the low titer of JHIII at the pupal stage.The results suggested that JH signal negatively regulated the expressions of jheh and jhdk.3.Effects of jheh gene silencing on the development of sixth instar larvae of gypsy mothThe results showed that RNAi mediated jheh gene silencing significantly reduced the expression of jheh gene,resulting in up-regulation of jhe gene at 24h and down-regulation of jhdk gene at 36h.Degradation due to jheh gypsy moth for juvenile hormone plays an important role,and the gene silencing jheh gypsy moth larvae in the body after the increase of the content of JH which jhe indicates that the experiments in jheh silence after high expression may in order to degrade the body drops of JH,while the lower jhdk may be due to jheh silence which causes the lack of degradation of the substrate JHd.Subsequently,it was found that the pupal stage of the larvae was prolonged by 1.09d,but the mortality and pupation rate did not change significantly,indicating that jheh gene silencing affected the metabolic process of JH.These results are helpful to elucidate the role of jheh in JH degradation of gypsy moth and its metabolic regulation mechanism in the JH signaling pathway,and provide a potentially valuable new target for the prevention and treatment of gypsy moth.