Regulation Of Juvenile Hormone Epoxide Hydrolase On The Growth And Development Of Mythimna Separata

Mythimna separata belongs to Lepidoptera Noctuidae,it feeds on more than 104 species of plants in 16 families,such as corn and beans.When the outbreak occurs,it will cause huge economic losses.It is an important national agricultural pest.Due to the increasing drug resistance and the toxic effect of chemical control on the environment,we urgently need to find and develop eco-friendly pesticides with new modes of action.Juvenile hormone（JH）is an important terpenes compound secreted by the corpora allata（CA）of insects.It plays a very important role in the growth and development of insects.Juvenile hormone epoxide hydrolase（JHEH）,as an important degradation enzyme in JH metabolism,may become an insecticide target gene for biological control of pests in the future.Therefore,clarifying the important role and biological function of JHEH in JH biological metabolism pathway can provide a theoretical basis for proposing biotechnology methods to control pests through JH degradation pathway in the future.In this experiment,six MsJHEH genes were cloned,their expression and effects on the growth and development for both M.separata larvae,adult reproduction and the survival rate of offspring were studied.The results are as follows:（1）Six new juvenile hormone epoxide hydrolase gene c DNA sequences containing complete open reading frame（ORF）were obtained from the M.separata transcriptome library.They were named MsJHEH1-6 respectively and were registered on NCBI Gen Bank with the login numbers of MT802191-MT802196.The c DNA sequences of MsJHEH were 1595,1533,1514,1811,1513 and2694 bp,and they contained open reading frames of lengths 1410,1389,1470,1380,1389 and 2064bp,encoding 469,462,489,459,462 and 687 amino acids respectively.The MsJHEH amino acid sequence contained the N-terminal transmembrane motif XWG,a catalytic triad,the conserved motif HGWP and two tyrosines,and all include EHN epoxide hydrolase superfamily domain.（2）Juvenile hormone analogue（JHA）had significant induction effect on MsJHEH expression.Due to the different metabolic ability of different MsJHEH to JH,its response to JHA induction is different.After JHA treatment,the expression of each gene was up-regulated.The expression of genes MsJHEH2,MsJHEH3 and MsJHEH4 was up-regulated most significantly.These three genes were selected for follow-up research.（3）The expression of MsJHEH2,MsJHEH3 and MsJHEH4 had obvious developmental stage and tissue specificity.The expression of MsJHEH2 was the highest in pupal stage and was higher in prepupal stage and 5^thinstar larva stage.The expression of MsJHEH3 was the highest at 5^thinstar larvae.MsJHEH4 had the highest expression in eggs and the lowest in adult stage.MsJHEH was differentially expressed at different development times during the 3^rdinstar larvae.The results showed that the expression of MsJHEH2 increased first and then decreased.The expression of MsJHEH2 was the highest level at 12 h.MsJHEH3 and MsJHEH4 expression showed an upward trend,and reached the highest at 48 h.The expression of MsJHEH2 was the highest in the midguts and the lowest in the salivary glands.The highest expression of MsJHEH3 was in fat bodies and the lowest in hindguts,salivary glands and Malpighian tubules.The expression of MsJHEH4 was the highest in the integuments of M.separata,which was 90.91 times that in salivary glands.It can be seen that each MsJHEH plays a different role in different developmental stages and tissues.（4）Different concentrations of JHA had different effects on MsJHEH2,MsJHEH3 and MsJHEH4 expression.In general,when the concentration of JHA was low（0.3125μg/μL,0.625μg/μL,1.25μg/μL）,there was a significant induction effect on gene expression,when the concentration of JHA was high（2.5μg/μL,5μg/μL）,the induction effect was poor,or no significant difference.The induction of MsJHEH by JHA confirmed that MsJHEH was involved in the metabolic process of JH.（5）RNA interference successfully inhibited the expression of MsJHEH in larvae and the content of JH in M.separata was significantly up-regulated,MsJHEH silencing led to larval weight gain and delayed the emergence of M.separata.The body weight treated with si RNA of MsJHEH2 and MsJHEH3 showed an increasing trend with time after gene silencing,most significantly on the 5th-7th day.After si RNA of MsJHEH4 treatment,the body weight of M.separata showed a temporary upward trend in the first 3 days after gene silencing,but there was no significant difference.After si RNA of MsJHEH treatment,the pupation rate,emergence rate decreased and the emergence time were significantly prolonged.（6）MsJHEH silencing promoted the vitellogenesis and ovarian development,improve oviposition level,and promoted the reproduction of M.separata adults.After si RNA treatment,the content of JH and the expression of vitellogenin were significantly up-regulated.The level of ovarian development was significantly higher than that of the control 72 h after interference.The total number of eggs production and the total number of impregnated eggs by female adults increased significantly,the oviposition period prolonged and the preoviposition period shortened.The female adult longevity was relatively shortened,but not significant.MsJHEH silencing had no significant effect on the survival rate of offspring.MsJHEH is involved in JH metabolism,and through its regulation of JH titer,it can affect the growth,development and reproduction of M.separata.The research provided a theoretical basis for formulating new pest control strategies.