Regulatory Mechanism Of Squalene And Methyl Jasmonate On Azadirachtin A Biosynthesis

Pest management relying heavily on synthetic pesticides has serious drawbacks in terms of insect resistance,toxic residues in/on food,and threat to human health and the environment.Such drawbacks have brought up new challenges for developing safer chemicals for crop protection which besides active against the target pests should be safe to humans,non-target organisms and the environment.The secondary metabolite azadirachtin is a tetranortriterpenoid obtained from the neem tree?Azadirachta indica?.It is a natural insecticide,known to affect feeding,growth,reproduction and metamorphosis of the insect pests.Because of the broad-spectrum control of insects and the relatively low nontarget toxicity it has been widely used in agriculture.However,the supply of plant-derived azadirachtin is unstable,resulting in shortages and price fluctuations.A stable source of affordable azadirachtin is required.It is a highly oxygenated and complex molecule,which makes its chemical synthesis difficult as well as uneconomical.Commercial production of chemically complex molecules like azadirachtin can be made feasible via in vitro cultivations.But the biggest challenge is that the biosynthetic pathway of azadirachtin is poorly understood.Therefore,it is of great scientific significance to reveal the biosynthetic pathway and metabolic regulation of azadirachtin to improve the yield of azadirachtin and achieve the large-scale industrial production of azadirachtin.In this study,a cell-free synthesis system of azadirachtin A?AZ-A?was constructed,and some synthesis precursors of AZ-A were clarified.The biosynthesize pathway of AZ-A from squalene was proposed.The effect of squalene on intracellular synthesis of AZ-A was studied in callus,which verified the feasibility of studying azadirachtin synthesis in non-cellular system.The yield of AZ-A in callus was further increased by binding methyl jasmonate.Transcriptome and metabolome analysis were used to study the effects of methyl jasmonate and squalene on callus metabolism,to explore the regulatory mechanism of AZ-A synthesis and improve the pathway of squalene synthesis of AZ-A.The main results are as follows:?1?A cell-free synthesis system of AZ-A was established.Squalene as substrate,buffer type,concentration,initial p H,extraction time and solid-liquid were optimized of preparation conditions.The Optimal AZ-A cell-free system of synthetic preparation conditions was neem leaves were extracted 1 h in 200 mmol·L^-1 Tris-HCl buffer?p H7.0?,solid-liquid ratio was 1:20,and 4?was extract temperture.Then the reaction termination reagent and stabilizing agent,reaction temperature,reaction time and concentration of substrate and auxiliary factor of synthetic reaction conditions were optimized.The total system was 800?L,it included 300?L neem cell free extracts?approximate 0.015 g neem leaves?,100?L 250?mol·L^-1 substrates?squalene?and 400?L buffer containing 1 mmol·L^-1 Mg²⁺,1 mmol·L^-1 Mn²⁺,0.1 mmol·L^-1 ATP,0.1 mmol·L^-1 NADPH⁺and 5 mmol·L^-1ascorbic acid.The reaction extracted60 min under 30?,and 200?L acetic acid was terminator.Seven kinds of terpenes were used as the substrates to screen the synthesis precursors of AZ-A in the established cell-free synthesis system.The results showed that 2,3-epoxysqualene,butyrospermol,euphol,nimbin,deacylsa-nannin and salannin were the synthetic precursors of AZ-A,and salannin was located in the downstream of AZ-A synthesis pathway.Lanosterol was not the synthetic precursor of azadirachtin A.The biosynthesis of squalene to AZ-A was further proposed.?2?The effects of squalene and methyl jasmonate on the biosynthesis of azadirachtin A were determined.The results showed that squalene and methyl jasmonic acid promoted AZ-A synthesis significantly.Callus were cultured under red and blue light?16/8h L/D?for 24 h.Adding 500mmol·L^-1 squalene and 100 mmol·L^-1 methyl jasmonate increased the yield of azadirachtin A by79.1%and 73.0%respectively compared with the control.The synergistic effect of squalenen and methyl jasmonate was more conducive to the synthesis of azadirachtin A.The yield of AZ-A was increased by 139.0%when 500 mmol·L^-1 squalene and 100 mmol·L^-11 methyl jasmonate were added to the medium and cultured for 24 h by red and blue light?16/8h L/D?irradiation..Four unknown substances also changed significantly after squalene treatment.Combined with mass spectrometry information,three compounds were speculated 1-senecioyl-3-acetylvilasininlactol presumably,1-1-Detigloyl-isobutyroyl-epoxymethacroyl azadirachtin and Nimbosodione.1-senecioyl-3-acetylvilasininlactol presumably may participate in azadirachtin A synthesis.?3?The effects of methyl jasmonate and squalene on the metabolic network of azadiaditin A biosynthesis were preliminarily revealed.767 metabolites were obtained from the callus treated with sterile water,methyl jasmonate,squalene,and methyl jasmonate and squalene combined based on the extensive targeted metabolome technique.Furthersystematic analysis of different metabolites in different treatment groups revealed that methyl jasmonate,squalene and methyl jasmonate combined with squalenen were treated with 132,137 and 132 different metabolites,respectively.The metabolic effects of methyl jasmonate and squalene on organic acids,amino acids and derivatives and phenylpropanes and derivatives were significant.Compared with squalene,methyl jasmonate had an obvious effect on lipid regulation,while squalene effected greater on amine metabolism.This study preliminarily elucidated the mechanism which methyl jasmonate and squalene regulate the synthesis of AZ-A based on the analysis of different metabolites.Methyl jasmonate promoted Entner-Doudoroff pathway reaction,glycolysis and other primary metabolism,and reduced the secondary metabolism of sterols to regulate thesynthesis of AZ-A.Squalene affected Entner-Doudoroff pathway reaction and glycolysis.On the other hand,the tricarboxylicacid cycle,lipid metabolism,and the activity of enzymes involved in structural modification.was also affected the synthesis of AZ-A.The metabolic analysis of Azadirodione,azadirachtin B,azadirachtin D,azadirachtin H,Azadirodione,Nimbin and Salannin was established.The results showed that squalene promoted the synthesis of azadirachtin H,and methyl jasmonate had inhibitory effects on the synthesis of azadirachtin D and azadirachtin H,methyl jasmonate and squalene inhibited the synthesis of azadirachtin B.Nimbin and Salannin were furtherverified to be the synthetic precursors of AZ-A and Azadirodione probably icipated in the biosynthesis of azadirachtin A.?4?The mechanisms by which methyl jasmonate and squalene regulate the biosynthesisof azadirachtin A were preliminarily elucidate.Transcriptome sequencing of azadiabem callus treated with sterile water,methyl jasate,squalene and both obtained 96.88gb Clean Data,and assembled 44313 Unigene,including 25038Unigene in the database annotation information.Methyl jasmonate,squalene and the two treatment groups influenced 261,32 and 316 genes expressed.560 differentially expressed genes were compared with common database and annotated into functional information.Methyl jasmonate has regulatory effects on the growth,metabolism,enzyme activity and hormone signal transduction of neem,and squalene has effects on the metabolism,enzyme catalysis and information transduction.The synergistic effects of the two drugs on the growth,metabolism and catalysis of azadirachtin were more significant and the range of regulation waswider.Combining transcriptome with metabolomics of callus indicated methyl jasmonate regulates the Entner-Doudoroff pathway and glycolysis reaction,promotes the synthesis of acetyl Co A,pyruvate and 3-phosphoglyceraldehyde,and then causes the high expression of HMGR,DXR and SQLE in the synthesis of terpenes,and promotes the synthesis of azadirachtin A.At the same time,methyl jasmonate inhibits the synthesis of sterols and makes more squalene enter the metabolism of azadirachtin A.Being a precursor of AZ-A,squalene also influences the synthesis of AZ-A by regulating the synthesis of acetyl Co A and the activity of enzymes related to structural modification.This study constructed a cell-free synthesis system of AZ-A and identified 2,3-epoxysqualene,Euphol,butyrospermol,salannin,deacelysalannin and nimbin were the synthesis precursors of AZ-A.It proposed the biosynthesis pathway from squalene to AZ-A.Based on metabolomics and transcriptome studies,the effectsof methyl jasmonate and squalene on the biosynthesis of AZ-A in callus and their regulatory mechanisms were preliminarily revealed.The results lay a foundation for elucidation of azadirachtin biosynthesis and allobiosynthesis of azadirachtin.