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Gene isolation related to biosynthesis of benzylisoquinoline alkaloid in Nelumbo nucifera Gaertn. (Lotus)
Thitirat Meelaph
Department of Biochemistry and Microbiology, Biomedicinalchemistry
Faculty of Pharmaceutical Science
Introduction
KEYWORDS
• Alkaloid : Benzylisoquinoline alkaloid
• Coclaurine N-methyltransferase (CNMT)
• Lotus
• Gene Isolation
Alkaloid
Plant secondary
metabolites
Low-molecular-weightNitrogen-containing compounds
Anticancer agent paclitaxel(Taxus baccata)
Antimalarial quinine (Cinchona officinalis)
Antineoplastic vinblastine(Catharanthus roseus)
(Inui, T., et al., 2007)
(The defense of plants against herbivores and pathogens)
Benzylisoquinoline Alkaloid
• Benzyllisoquinolinealkaloids (BIAs)
A large and structurally diverse group of plant-metabolites derived from aromatic amino acid tryrosinevia a complex array of biosynthetic enzymes. (David K. Liscombe er al., 2009)
Staniek A, et al., Natural product-modifying pathways in plant, Biotech.J 2013;8: 1159-1171-DOI10.1002/biot201300224
Benzylisoquinoline Alkaloid
Analgesics morphine and codeine (Papaver somniferum)
Antibacterial agents sanguinarine (P. somniferum, Eschscholzia Californica)
Berberine (Coptis japonica)
Coclaurine N-methyl transferase
• Coclaurine N-methyltransferase (CNMT)
: S-adenosyl-L-methionine (SAM): CNMT catalyzes the transfer of a methyl group from S-adenosyl-L-methionine to the amino group of the tetrahydrobenzylisoquinoline alkaloid coclaurine. (This is a unique N-methyltransferase in the biosynthesis of benzylisoquinoline alkaloids)
(Inui, T., et al., 2007)
Gene isolation
METABOLIC ENGINEERING APPLICATIONS
Identify&functional
characterization
Transgenic plants
Metabolite profiles,
Alkaloid pathways
Manipulate of Enzyme
(Peter J Facchini,2001)
Isolation of novel N-methyltransferases from three benzylisoquinoline alkaloid-producing species
Plant species Study topic Investigator Year
Coptis japonica Molecular Cloning and Characterization of CoclaurineN-Methyltransferase
Kum-Boo Choi., et al. 2002
Coptis japonica Thalictrum minus
Overexpression of Coptisjaponica Norcoclaurine 6-O-Methyltransferase in Cultured Eschscholzia californica
Inui T., et al. 2007
Eschscholzia californicaPapaver bracteatumThalictrum flavumcell
Metabolite profiling (CNMT genes already isolated)
Liscombe., K.D., et al. 2009
LOTUS
Lotus is perennial aquatic plant that is consumed around
the world (600 cultivars to date)
1. Nelumbo nucifera Gaertn : widely distributed in East Asia, especially in China and India,
2. Nelumbo lutea (Willd.) Pers. : from North America.
Lotus has been cultivated based on its use : - flower production, - seed production, and for - rhizome production (Guo 2009)
Taxonomy Hierarchy
Kingdom PlantaeSubkingdom Viridiplantae
Division Tracheophyta – vascular plantSubdivision Spermatophytina – seed plants
Class MagnoliopsidaOrder Proteales
Family NelumbonaceaeGenus Nelumbo
Bioactive compounds in Lotus
• Lotus plants provide several bioactive compounds
Alkaloids FlavonoidsTherpenes Phenolics
(T C M D, Vo., et al.,2013)(Akinjokunla, OJ., et al.,2010)(Shidhar, KR., et al.,2007)
LC/MS guided isolation of alkaloids from Lotus leaves by pH-Zone Refining counter current-chromatography
Preparative seperation of alkaloids from NelumboNucifera leaves by conventional and pH-zone-refining Counter-Current Chromatography
Determination of Lotus leave alkaloids by solid phase extraction combined with high performance liquid chromatography with diode array and tandem mass spectrometry detection
Bioactive alkaloid in Lotus
Yang G, et al.,Rapidsimuluneous Determination of four alkaloids in Lotus plumule by CZE with Ephedrine Hydrochloride as an internal standard, Chromatographai 2012;75 :1295-1300-DOI/s10337 10.1007012-2312-7
Pharmaceutical Value of Bioactive alkaloids in Lotus plants
Found in Leaf, Seed and EmbryoValue : Inhibit Bacteria growth
and Anti-HIVAkinjokunla, OJ et al. (2010)
Sridhar, K.R. and Bhat, J. (2007)
Yang G, et al., Rapidsimuluneous Determination of four alkaloids in Lotus plumule by CZE with Ephedrine Hydrochloride as an internal standard, Chromatographai 2012;75 :1295-1300-DOI/s10337 10.1007012-2312-7
Pharmaceutical Value of Bioactive alkaloids in Lotus plants
Found in Leaves Embryo and PlumuleValue : Antibacterial and
Anti-HIV activity
Sridhar, K.R. and Bhat, J. (2007)
Yang G, et al.,Rapidsimuluneous Determination of four alkaloids in Lotus plumule by CZE with Ephedrine Hydrochloride as an internal standard, Chromatographai 2012;75 :1295-1300-DOI/s10337 10.1007012-2312-7
Pharmaceutical Value of Bioactive alkaloids in Lotus plants
Found in Leave and SeedValue : Anti-diabetic and HIV activity
K. Hou Nguyen, at al., Nuciferine stimulates insulin secretion from beta cells-An in vitro comparison with glibenclamide,Journal of Ethnopharmacology 2012;142:488-495
T C M V Do, et al., Analysis of Alkaloids in Lotus(Nelumbo Nucifera Gaertn.) leaves by non-aqueous capillaryelec-
trophoresis using altraviolet and mass spectrometric detection, Journal of Chromatography A 2013;174-180
(Shidrha, KR. Bhat,R. (2007)
(Akinjokunla, OJ et all, 2010)
Nuciferine
Research Methodology
Gene isolation and recombinant construction
Alignment
RNA extraction
Primer design
Recombinant DNA (pET28B)
EST database (Liscombe., K.D., et al.,2009)
Lotus transformation- Agrobacterium
Expression of recombinant in E.coli (BL21)
Enzyme activity
Lotus seed germination
Explants
callus induction
Explants
Shoot induction
Subcloning to pCambia1302
Co-transformation with Agrobacterium
Whole plant
transformation with Agrobacterium
Explants sampling and gene profile
BamHI
SalI
Kanamycin
E.Coli BL21
induce protein expression(1 mM IPTG, 50mg/ml Kana 37ᵒC, 4hr) in shaker
Culture in LB broth at 37ᵒC, CO2 incubator
Ni ,Co Affinity resin
6000 rpm, 5 min4ᵒC , lysis pellet
20 mM Tris/HCl, pH 7.5, 0.5 M KCl, 10% glycerol,10 mM b-mercaptoethanol
10 000 g20 min at 4C,
Comassieblue dye
HPLC analysis
80 µM S-adenosyl-L-methionine, 2 µM S-adenosyl-L-[methyl-14C]-methionine, 1–500 µM alkaloid substrate in 100 mM Tris/HCl, pH 7.5, and 12 mMb-mercaptoethanol, at 37C.
(Choi., et al.,2002)
BamHI
SalI
KanamycinBamHI
SalI
BamHI
SalI
pCambia1302
pET28B in BL21
Agrobacterium
Explantsampling
Real-time quantitative PCR analysis of gene expression
Stable transformation
Transient transformation
MS medium + 4 mM2,4-D and 1 mM BA
MS medium + 1 mg/ml Chitosan
(Arunyanart., S. et al.,2005)