| Tripterygium is a traditional Chinese herbal medicine and insecticide plant which has important medical and agricultural value.However,the resources of wild T.wilfordii are scarcly,slow growth,low contents,severely restricting with its wide application in the field of medicine and pesticide research;therefore,the study of the synthesis pathway and its molecular regulation mechanism is a key biological genetic engineering technology to improve tripterygium active secondary metabolites production.To better understand the analysis and synthesis related gene expression induced by regulation mechanism,this study systematically studied tripterygium active secondary metabolites,from the expression analysis,and induce aspects of the regulation of gene function identification of a series of work,biological and triptolide We achieved the following results:1.A rapid analytical method for the simultaneous determination of triptolide,wilforgine and wilforine was developed using high performance liquid chromatography-electrospray ionization tandem mass spectrometry(HPLC-ESI-MS/MS).The contents of three active substances in different tissues and plant tissue culture of Tripterygium wilfordii were compared.The ultrasonic extraction and reflux extraction technologies were respectively used for extraction of three active substances in T.wilfordii.In the reflux extraction,the analytes was extracted by V(methanol): V(acetonitrile)= 1: 1 solution,cleaned up by Supelclean LC-Si solid phase extraction cartridges.During the ultrasonic extraction,the analytes were extracted by ethanol and purified by OASIS HLB SPE column,and separated on a reversed phase C18 column with gradient elution with acetonitrile and water as the mobile phase.The extract was then determined with high performance liquid chromatography-electrospray ionization tandem mass spectrometry HPLC-ESI-MS / MS).The results showed that the highest contents of triptolide is adventitious roots,followed by hairy root,rarely stems and leaves content.The highest contents of wilforgine is hairy root,followed by the root bark,leaves not detected.Hairy root and root bark of wilforine content fairly.When the addition level ranged from 0.01 to 2 mg/kg,the recoveries of three active substances of T.wilfordii was 74.35-108.71%.The detection limits of the three active substances ranged from 0.08-0.12 ?g/mL.Results indicated that this method is highly efficient,with good sensitivity and accuracy.which can quickly monitor the quality of T.wilfordii extract and its products.2.In this study,ten candidate reference genes(PP2A,TBP,TUB,26 S,SAND,GAPDH,TIP41,eIF-4?,EF-1? and ACTINT7)of T.wilfordii Hook.f.were evaluated in different tissues and under different treatments.Three statistical algorithms,geNorm?NormFinder?BestKeeper??Ct ? RefFinder were used to evaluate the expression level stabilities of candidate reference genes.The five methods use different calculation algorithms and therefore can give different results.TIP41 and ACTINT7 were identified as the most stable reference genes no matter in different tissues or in methyl jasmonate,salicylic acid induced and under salt stress,osmotic stress treatment.The effects of the methyl jasmonate induction treatment,the biosynthesis 23 genes induced found that these genes are subject to varying degrees and synergistically induced upregulation reveal elicitors may be induced by an upstream activity of regulatory genes which regulate the entire metabolic pathways;Comparative analysis of the regulatory effect of methyl jasmonate biosynthetic pathway for triptolide,wilforgine and wilforine effects alkaloid production.Based on our results,TwHMGR1,Tw DXR and TwTPS21 are gradually upregulated by MeJA over the time course experiment until the 6 h time point(when maximum wilfogine accumulated),so probably their expression affects wilfogine biosynthesis.TwFPPS1 and TwCMK expression began to increase at 3 h and gradually increased till 9h(when maximum wilforine and triptolide accumulated),followed by a gradually decrease at 12 and 24 h.Tw6OMT,TwTR-1 and TwFPPS2 in methyl jasmonate during the test process,the wilforine content of their expression and expression of views coincide,may be their expression of alkaloid synthesis.The key role of triptolide,wilforgine and wilforine based on the accumulation,it can be concluded trends TwHMGR1,TwDXR,Tw6 OMT,TwTR-1,Tw TPS21 and TwFPPS2 six basic gene and triptolide consistent with active secondary metabolites content in the product trend,suggesting three habitats accumulated metabolites may modulate the expression of genes related to the above-mentioned biosynthesis.3.Constructed T.wilfordii Hook.f.biosynthetic pathway related genes TwTR-1,Tw6 OMT prokaryotic expression vector,TwTR-1,Tw6 OMT protein was added to the crude extracts of different polarity tripterygium segment of the catalytic reaction,the reaction was subjected to LC-ESI /MS/MS analysis.It was found TwTR-1,Tw6 OMT catalyze methylene chloride and(7: 3,v / v)and methylene chloride and methanol(9: 1,v / v)section extracts a compound of retention time slightly long product.Further attempts to use ESI/MS/MS identification of substrate and product structure,suggesting Tw TR-1,Tw6 OMT may be involved in sesquiterpene backbone carbonyl reduction reaction,and methyl transfer reaction.Follow-up can be improved by this test method,such as by switching to more sophisticated separation system will be able to make it widely used in the identification of unknown function catalytic enzyme gene.This study established HPLC-ESI-MS/MS method for the determination of triptolide,wilforgine and wilforine,and resolved T.wilfordii Hook.f.biosynthesis genes in three secondary metabolites induced component content material relationship with the molecular level operation,for the purposes of cloning and gene function analysis,regulation of the biosynthetic pathway improve plant cells of the active ingredient content,the development of resources to solve the insecticidal plant Tripterygium triptolide plant as well as the limited resources more effectively to lay basis. |
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