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

Purpose

• To isolate gene which involved in Biosynthesis of Benzylisoquinoline alkaloids in Lotus

Content

• Introduction

• Literature Reviews

• Research Methodology

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

The research of Separation and Purification of Alkaloid in Lotus

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

(Chen et al., 2013)

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)

• DNA sequence

• Pharmaceutical source

THANK YOU

Acknowledgement

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