Biosynthetic Mechanism Of Indole Alkaloids Under Ultraviolet Irradiation And Cloning And Prokaryotic Expression Analysis Of CtIPT In Clematis Terniflora DC.

China is one of the countries that possess abundant resources of medicinal plants which play a prominent role in disease prevention and treatment of human beings.UV-B has enormous influence on the growth,development,and secondary metabolism of medical plants.However,different medical plant responses differently under the exposure to UV-B irradiation.Thus,more mechanisms need to be further explored.The indole alkaloid,which has pharmaceutical potential in a diverse range of applications,is one sort of the secondary metabolite in medicinal plants.It is increased under UV-B irradiation,which not noly increase self-defensive ability of plants to resist adversity stress,but also increase phamaceutical effect of plants with cytotoxic,antiviral,anti-malarial,anti-inflammatory,and anti-cancer activities.Cytokinin is one of the plant hormones,and it is synthesised by adenylate isopentenyltransferases.It is decreased under UV-B irradiation,which not noly control the growth and development for morphology change of plants to resist adversity stress,but also increase phamaceutical effect of plants indirectly by taking part in secondary metabolism(ie.indole alkaloid,coumarin,flavone)biosynthesis based on the control of certain regulatory factors in UVR8 receptor.Clematis terniflora DC.with important pharmaceutical properties belongs to Ranunclaceae.A novel indole alkaloid(6-hydroxyl-1H-indol-3-yl)carboxylic acid methyl ester was previously isolated from C.terniflora,supplying us with the foundation to investigate deeply in this organism.The main contents and results are as follows:(1)Biosynthetic mechanism of indole alkaloids under UV-B in Clematis terniflora DC.Indole alkaloid(6-hydroxyl-lH-indol-3-yl)carboxylic acid methyl ester was identified and quantified using HPLC-TOF-MS/MS.The peak area was evaluated in response to high level of UV-B irradiation followed by the dark,implying that the indole alkaloid biosynthesis pathway may be activated in C.terniflora.Proteins and metabolites related to amino acid metabolism were increased by the binary stress through detections of 2-DE and GC-TOF-MS techniques,demonstrating that amino acid metabolism was increased.The activity of L-serine deaminase,which transforms serine into pyruvate,was increased significantly by the binary stress,implying that the increase in amino acid metabolism contributed to an enhancement of shikimate metabolic pathway.The genes participated in the metabolic process from shikimate to L-tryptophan were concurrently upregulated by the binary stress using qRT-PCR technique.Metabolites involved in indole biosynthesis pathway including anthranilate,indole,and L-tryptophan,increased by the binary stress through HPLC-TOF-MS/MS detection,as well as the enzymatic activity of L-tryptophan synthase through enzyme activity assay.These suggested an enhancement in shikimate metabolic pathway promotes the indole/tryptophan pathway,which laid the foundation for indole alkaloid biosynthesis.(2)Cloning and prokaryotic expression of CtIPT in Clematis terniflora DC.A base sequence with the length of 1374 bp,including the whole ORF with the length of 332 aa,was obtained through EST database and RACE technique.After physicochemical properties predication,sequence alignment,and phylogenetic tree analysis,it was showed that the amino acid sequence encoded by the base sequence might be translated to a hydrophily protein with the molecular weight of 37.2 kDa,that there was a signature sequence region of adenylate isopentenyltransferases(ATP/GTP binding region),and that the amino acid sequence encoded by the base sequence was quite similar to SlIPT3/4 in tomato and AtIPT3/5/7 in Arabidopsis thaliana.The results all above demonstrated that the protein which translated by the base sequence was adenylate isopentenyltransferases,and then the sequence was named CtIPT.The expression of CtIPT was significantly suppressed by the binary stress using qRT-PCR technique,implying that CtIPT might have ability to resist adversity stress.pET-28a(+),pGEX-4T-1,and pMAL-c2X were used to cloning and expresstion of CtIPT in Escherichia coli BL21,respectively.It was showed that the target protein was successfully expressed under the condition of 16?,180 rpm,and 0.5 mM IPTG to incubation for 6 h.However,soluble expression was only occurred in BL21-pMAL-c2X-CtIPT(contains MBP tag).Finally,the target protein of CtIPT was obtained after separation and purfication using affinity column of amylose resin.