Established A Transformation System For Hairy Roots And Functional Analysis Of Aphid Alarm Pheromone Related Genes From Chamomile (Matricaria Chamomilla L.)

German chamomile(Matricaria chamomilla L.),terpenoids from their flowers have important medicinal value.It has medicinal uses,including anti-inflammatory properties and is effective against bacteriostasis,anxiety.We established a transformation system for chamomile hairy roots and cloned three sesquiterpene synthase genes of aphid alarm pheromone,Mc GDS1,Mc GDS2 and Mc GDS3,and performed functional analysis.Mc GDS3 promoter and transcription factor interaction was verified by double luciferase assay.Transformation of chamomile hairy roots and the activity of three aphid alarm pheromones here form the molecular basis for the study of the biosynthesis and regulation of terpenes.The main results were as follows:1.A transgenic system for the hairy root of chamomile was established.The strain suitable for the induction of the hairy root of chamomile was screened,and the conditions suitable for the induction of the hairy root of chamomile with different explants,preculture time,infection time and co-culture time were studied.The results showed that the induction rate was the highest(97%)when the whole rootless of 1-month seedlings were used as explants and infected with 15834 agrobacterium rhizoides for 5min and cultured for 2 days to induce hairy roots.2.Candidate genes encoding germacrene D synthase(Mc GDS)were obtained by transcriptome analysis and were named Mc GDS1,Mc GDS2 and Mc GDS3.The The coding sequences and open reading frames of the Mc GDSs genes were cloned.Comparative analysis of the c DNA and DNA sequences of Mc GDS1,Mc GDS2 and Mc GDS3 transcript region revealed the presence of six introns and seven exons,which represent a common structural feature of angiosperm sesquiterpene synthase genes.Sequence alignments,protein structure and phylogenetic analysis of Mc GDSs were analyzed by bioinformatics analysis.3.The open reading frames encoding Mc GDSs was cloned into the expression vector p ET32a(+)to obtain recombinant plasmids.The recombinant proteins were expressed after induction with isopropyl β-D-thiogalactoside(IPTG).Then the cell extracts was performed SDS-PAGE,Western blotting,and soluble proteins were purified.Mc GDS1,Mc GDS2 and Mc GDS3 were confirmed to be(E)-farnesene synthase,germacrene D synthase,and germacrene A synthase,respectively.Overexpression of Mc GDSs resulted in γ-muurolene accumulation of hairy roots.4.The expression of Mc GDSs was analyzed using q PCR in different organs and at different timepoints during flowering,i.e.,R,S,L,FB,F1,F2,3D,3R,4D,4R,5D and 5R.The expression level of the three Mc GDSs was higher in young tissue than older tissue and was higher in disk florets than in ray florets.The expression level of Mc GDS1,Mc GDS2 and Mc GDS3 was highly correlated with amounts of the end-product essential oils((E)-β-farnesene,germacrene D and β-elemene),with coefficients of 0.76,0.83 and 0.68,respectively.5.The recombinant Mc GDS-GFP vector was constructed and then transient expression in tobacco,subcellular localization revealed diffuse GFP reporter-gene signals in the cytoplasm and nucleus.6.The Mc GDS3 promoter was cloned by referenceing the sunflower genome,The coding sequences of Mc GDS3 promoter was 1,500 bp.Five candidate transcription factors were obtained from M.chamomilla by transcriptome analysis and were named MYB1,MYB2,MYB3,AP2 and b Zip.The interaction between promoter and transcription factors was detected by using double luciferase assay.Results of Luci showed that MYB1,MYB2,AP2 and b Zip all combined with the promoter,while MYB3 did not.