Terminal Synthesis Of R-JH Ⅲ Optical Enantiomers In The Honeybee And The Mechanisms

Insect Juvenile hormone(JH)is a sesquiterpenoids synthesized and secreted primarily by the corpora allata(CA)cells of insects.It is mainly involved in regulating a series of important physiological and biochemical processes such as body appearance,molting,diapause and so on.At present,a total of 8 JH species have been identified,of which 7 JH species have stereochiral configurations,and most insects can specifically synthesize stereochiral R-JH Ⅲ.At present,it is still unclear how insects specifically regulate the biosynthesis of stereoconformation R-JH Ⅲ from geometrical precursors.Honeybee is an extremely important ecological resource insect and plays an irreplaceable role in global ecosystem services.In this study,Apis mellifera was selected as the research object,and corresponding research methods were established to determine the types and titers of juvenile hormone in bees;identify the terminal synthesis pathway of JH Ⅲ in bees,and verify the main regulatory genes of JH Ⅲ biosynthesis in bees;the effect of related genes on JH Ⅲ chiral enantiomers of honeybee was determined.The main results obtained are as follows:(1)The results of separation of racemes of JH Ⅲ by CHIRALPAK AY-RH showed that only R-JH Ⅲ and no S-JH Ⅲ were detected in bees.R-JH Ⅲ decreased first and then increased at different growth stages,and finally tended to be stable.The distribution of R-JH Ⅲ content in different parts of bees was as follows: head > chest > abdomen.The results of "epoxidation" and "methylation" blocking tests showed that the final synthesis path of R-JH Ⅲ in honeybees was “methylation first,epoxidation later”,and there was no supplementary pathway of “epoxidation first,methylation later”.(2)After RNAi,the expression of Am CYP15A1 gene was significantly reduced,and the content of JH Ⅲ in bees was also significantly reduced.The verification results confirmed that CYP15A1 enzyme of bees was the main ratelimiting enzyme for the synthesis of JH Ⅲ in bees.The exogenous induced expression of purified Am-CYP15A1 enzyme can convert excess MF into S-JH Ⅲ: R-JH Ⅲ≈1:1.(3)The epoxidization of substrates(2E,6E)-MF,(2E,6Z)-MF and(2Z,6Z)-MF with concentrations of 100 μM in honeybee pharyngeal body(CA)showed that high concentrations of MF with different cis-trans configurations can produce S/R-JH Ⅲ under the action of CYP15A1 enzyme.The results of bioepoxidation of different concentrations of(2E,6E)-MF in vitro and by microinjection in bees showed that bees could specifically synthesize R-JH Ⅲ from low concentrations of(2E,6E)-MF in vivo.High concentrations of(2E,6E)-MF can be converted into S-JH Ⅲ and R-JH Ⅲ in equal proportions in bees.(4)Compared with CK,the expression level of JHEH gene in bees treated with microinjection of exogenous JH Ⅲ was significantly increased,while the expression level of JHE gene was almost unchanged.Therefore,Am-JHEH enzyme was the main metabolic enzyme of JH Ⅲ in bees.The expression levels of JHBP and JHEH gene in bees treated with single injection of exogenous RJH Ⅲ and S-JH Ⅲ,as well as the enzymatic reaction of S/R-JH Ⅲ mediated in vitro,showed that Am-JHBP enzyme could bind S-JH Ⅲ and R-JH Ⅲ without difference and protect them from hydrolysis.Am-JHEH also showed no difference in metabolism of S-JH Ⅲ and R-JH Ⅲ,and the two chiral enantiomers of S-JH Ⅲ and R-JH Ⅲ did not convert to each other in bees.In summary,this study identified a “esterify then epoxidize” sequence of R-JH Ⅲ terminal synthesis pathway for honeybee specific synthesis via FA to MF and then R-JH Ⅲ.The stereospecific regulatory mechanism by which A.mellifera can precisely regulate the synthesis of R-JH Ⅲ enantiomeric in vivo by specifically binding(2E,6E)-MF and tightly controlling its titer was also revealed in detail.The results provide strong data support for exploring the social behavior and population evolution of honey bees,provide a basis for engineering and guiding the selective synthesis of chiral enantiomers at the molecular level,and open a new perspective for revealing the complex mechanism of juvenile hormone synthesis in different insect species.At the same time,this study provides a new application thinking for the protection and utilization of honey bee populations and the safe use of pesticides.