Expression of an aphid-induced barley methyltransferase in
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Expression of an aphid-induced barley methyltransferase in Escherichia coli , purification and characterisation of the enzyme. by Irene Ingvor Zetterlund. Aim. To test the hypothesis that OMT is involved in gramine biosynthesis

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Expression of an aphid-induced barley methyltransferase in Escherichia coli, purification and characterisation of the enzyme

by Irene Ingvor Zetterlund


Aim

  • To test the hypothesis that OMT is involved in gramine biosynthesis

  • To clone the OMT gene into an expression vector with purpose to synthesize the enzyme in E. coli

  • To characterize the enzyme kinetically


Background barley hordeum vulgare
Background: Barley (Hordeum vulgare)

  • Is an important cereal in Sweden

  • Is cultivated on the large area of arable land, about 400 000 ha

  • Is used in the malting industry and for livestock feed

  • All kind of farm animals can be fed on it


Background bird cherry oat aphid rhopalosiphum padi
Background: Bird cherry-oat aphid (Rhopalosiphum padi)

  • One of the most serious barley pests

  • Transmit virus infections, i. a. barley yellow dwarf virus

  • Phloem-feeding insects, cause little tissue damage

  • Are perceived by plants as pathogens

  • Breed by sexual reproduction and parthenogenesis

  • Overwinter as eggs on its primary host, bird cherry

  • In summer make use of diverse grasses as secondary hosts, among them barley


Background plant defence reactions
Background: Plant defence reactions

  • Alkaloids – a big group of N-containing secondary metabolites, have strong physiological effects in defence against herbivores

  • Aphids induce pathogen-defence response

  • Jasmonic acid signalling pathway induces expression of a wide range of defense genes

  • One of them was identified as an O-methyltransferase gene


Background plant defence reactions gramine
Background: Plant defence reactions - Gramine

  • Gramine - indole proto-alkaloid, secondary metabolite in barley and some other species in the grass family Poaceae

  • Induced in barley upon aphid infestation

  • Found in epidermis and in mesophyll parenchyma

  • Missing in the vascular bundles

  • The higher gramine amount

    the lower vulnerability of

    barley to aphids

  • Synthesized from tryptophan

    via 3-aminomethylindole

  • NMT catalyzes SAM-dependent

    conversion of AMI to MAMI

    and from MAMI to gramine


Background omt
Background: OMT

  • One gene, induced by the aphid, is encoding an O-methyltransferase, OMT

  • It is also induced by the jasmonic acid signalling pathway

  • OMTs generally

    • methylate caffeic acid

    • lead to lignin precursors

    • or various classes of flavonoids

  • But not all of barley cultivars had OMT gene in their genome

  • In the barley varieties missing the gene, gramine was not found either

  • In all gramine-containing lines OMT was present


Hypothesis
Hypothesis

  • The gene, characterized as encoding for an O-methyltransferase acting on caffeic acid,

    • might actually be encoding an N-methyltransferase, involved in gramine biosynthesis


Methods impact cn protein purification system
Methods: IMPACT-CN Protein Purification System

  • IMPACT = intein mediated purification with an affinity chitin-binding tag

  • A target protein is fused to a self-cleavable intein tag

  • A chitin-binding domain in intein tag allows purification of the target protein on the chitin column

  • The intein tag undergoes specific self-cleavage in presence of DTT

  • The target protein releases from the chitin-bound intein tag


Methods
Methods

  • RT-PCR and PCR

  • Cloning of the target gene into the vector

  • Transformation of the competent cells

  • Agarose and SDS-PAGE gel electrophoresis

  • Western blotting

  • Bradford microassay for protein quantification

  • Silica gel thin-layer chromatography (TLC)


Materials growth and treatment of plants
Materials: Growth and treatment of plants

  • H. vulgare, variety Lina, susceptible to the aphids

  • Sown in November 2003

  • Grown in a growth chamber at 26oC, long day, (18 h light/6 h darkness)

  • 5-day-old barley plants were harvested

  • Their green tissue was treated with 45 μM jasmonic acid for 24 hours to induce the OMT-gene


Results synthesis of the coding region of the omt gene
Results: Synthesis of the coding region of the OMT gene

  • The total RNA was isolated from barley green tissue

  • RNA was reverse transcribed into single-stranded cDNA using the First-Strand Synthesis System for RT-PCR

  • To amplify the coding region of the OMT gene by PCR primers OMTcloneF and OMTcloneR2 were used

  • A product of about 1100 bp was visualized by 2% agarose gel electrophoresis


Results cloning of the omt gene into the ptyb 12 vector
Results: Cloning of the OMT gene into the pTYB 12 vector

  • The plasmid pTYB12, chosen as a vector - allows the fusion of the cleavable intein tag to the N-terminus of a target protein

  • The plasmid - digested with

    the restriction nucleases SmaI

    and NdeI

  • The DNA fragment - digested

    with restriction nuclease NdeI

  • The digested DNA - ligated

    into the pTYB12 using the

    BioLabs Quick Ligation Kit


Results transformation of e coli dh5 t1 and screening for recombinants
Results: Transformation of E. coliDH5α-T1 and screening for recombinants

  • Toamplify the OMT sequence E. coliDH5α-T1 were transformed with the new plasmid pTYB12-OMT

  • The recombinant cells were selected on Petri dishes with LB medium containing 100 μg/ml ampicillin

  • 96 randomly chosen colonies were

    inoculated in a microtitre plate in LB/amp

    medium

  • PCR test for inserts using intern primers

    OMT F1 and OMT R1 obtained 3 clones


Results control of the inserts
Results: Control of the inserts

  • To confirm the obtained recombinant clones, digesting reactions with restriction nucleases Kpn I, Nco I, Nde I and Sap I were carried out over night at 37oC

  • The digested DNA was analyzed on 1 % agarose gel

  • Plasmid 1 gave the expected fragment pattern and thus was chosen as the pTYB12-OMT plasmid

  • The digesting reaction with restriction

    nucleases resulted in bands as follow:

    • Kpn I - 6706 and 1801 bp

    • Nco I - 7380, 680 and 447 bp

    • Nde I - 8507 bp

    • Sap I - 7810 and 697 bp


Results control of the insert
Results: Control of the insert

  • The plasmid was controlled for the right insert by PCR with 3 pairs of primers: OMT clone F and OMT clone R2 (1); OMT F1 and OMT R1 (2), and Intein Forward and T7 Terminator Reverse (3)

  • Bands of the correct sizes were visible on 2 % agarose gel, lane 1- 1100 bp, 2 - 348 bp and 3 - 1300 bp

  • To make sure that there was no error in the sequence of the cloned fragment, the plasmid pTYB12-OMT was sequenced at Cybergene

  • The sequence proved to be identical to the

    one published earlier


Results transformation of e coli er2566 and screening for recombinants
Results: Transformation of E. coliER2566 and screening for recombinants

  • The E. coli strain ER2566 was provided by Impact-CN as a host strain for the expression of a target gene cloned in the pTYB12 vector

  • ER2566 have a chromosomal copy of the T7 RNA polymerase gene inserted into the lacZ gene, and therefore under the control of the lacZ promoter

  • Expression of T7 RNA polymerase is suppressed in the absence of IPTG, by the binding of lac I repressor to the lac promoter

  • The transcription of the fusion protein takes place

    when IPTG is accessible

  • Transformed cells ER2566 were selected on Petri

    dishes with LB/amp medium

  • To control the protein induction ER2566 was

    transformed with the pMYB5 vector


Results induction of protein expression
Results: Induction of protein expression

  • Induced with 0,5 mM IPTG at RT O/N

  • SDS-PAGE analysis showed bands 100 kDa

  • 100 kDa = OMT-intein fusion protein

  • Positive control - ER2566 transformed with pMYB5 vector

  • Negative control –

    • uninduced E1

  • E2 and E6 chosen to

    continue the experiment


Results optimizing of the protein induction conditions
Results: Optimizing of the protein induction conditions

  • Different conditions were verified:

    • Induction with 0,5 mM and 1 mM IPTG

    • Temperature and time:

      • 37oC, 4 and 6 h

      • RT, O/N

      • 15oC, O/N

  • SDS-PAGE analysis showed

    the strongest band about

    100 kDa for the induction with

    1 M IPTG at RT O/N


Results western blot
Results: Western Blot

  • Protein bands were transferred onto PVDF membrane by semi-dry transfer apparatus

  • Immunoblotting:

    • primary antibodies - against the chitin binding domain

    • Secondary antibodies - Goat Anti-Rabbit HRP

  • The protein was detected using the ECL Plus Western Blotting kit and chemiluminescence in the CCD-camera

  • The strongest bands of about 100 kDa

    in lanes 4 – 7

  • E6 induced at RT with 1 mM IPTG

    showed the strongest band,

    conditions were the best for the protein

    expression.


Results purification of the target protein
Results: Purification of the target protein

  • Purified using the IMPACT-CN Protein Purification System

  • 1 l cell culture was induced with 1 mM IPTG at RT O/N

  • Cells were broken by sonication

  • Clarified cell extract, obtained by centrifugation, was loaded onto chitin column

  • Cleavage reaction - started by

    adding Cleavage Buffer with DTT

  • The protein was eluted using

    the Column Buffer

  • SDS-PAGE analysis showed band ~

    43 kDa corresponding to the purified

    methyltransferase


Results purification of the target protein conditions
Results: Purification of the target protein - conditions

  • Different conditions for the on column cleavage reaction were tested: at 4oC and RT for 24 and 40 hours

  • Elution with the Column Buffer containing 0,5 M and 1 M NaCl

    • The highest protein concentration – elution with 0,5 M NaCl

  • The protein concentration was measured spectrophotometrically, using Bradford microassay method for protein quantification

Table 1Concentration of the target protein


Results silica gel thin layer chromatography
Results: Silica gel thin-layer chromatography

  • For determination of the kinetic parameters of the methyltransferase were used as substrates:

    • AMI, MAMI and caffeic acid

  • The methylation reactions were

    started and stopped by adding of:

    • Start – SAM+3H-SAM (95+5)

    • Stop - stop buffer

  • The methylation products were

    separated by means of TLC-

    plates standing in TLC solvent

  • The regions with the reactions

    products were scraped from the

    TLC-plates for liquid

    scintillation counting


Results assay of the methyltransferase activity
Results: Assay of the methyltransferase activity

  • Methyl­transferase activity was measured by estimation of the amount of 3H-labelled product produced with methyl-3H-SAM

  • 3H count per minute was calculated into built product per 1 mg protein

Table 2 AMI and MAMI methylation products built per 1 mg protein, pmol/min, development in time

Table 3 AMI and MAMI methylation products built per 1 mg protein, pmol/min, relative to the substrates concentration


Results assay of the methyltransferase activity1
Results: Assay of the methyltransferase activity

  • The reactions with the enzyme extract from barley green tissue did not show any activity

  • The reactions with the methyltransferase purified by IMPACT-CN obtained some built product, but the data are questionable.

Table 5 Caffeic acid methylation products built per 1 mg protein, pmol/min,

development in time


Discussion transformation of e coli dh5 t1
Discussion: Transformation of E. coliDH5α-T1

  • E. coli had difficulties to survive after its uptake of the plasmid with the insert OMT

  • Few recombinant colonies were obtained and the survivors turned out to have mutations in the OMT sequence

  • The third transformation resulted in a frameshift mutation

  • The fourth transformation was succesful


Discussion purification of the target protein
Discussion: Purification of the target protein


Discussion assay of the methyltransferase activity
Discussion: Assay of the methyltransferase activity

  • The methylation of AMI:

    • highest after 30 min incubation, decreasing later

    • that contradicts the kinetic development in time as a logarithmic function

  • The methylation of MAMI:

    • increases in time

    • highest after 60 min incubation

  • An explanation - the scraped samples were contaminated and thus are not trustworthy

  • The methyltransferase activity was analyzed relatively to AMI and MAMI concentration:

    • production of MAMI from AMI is inversely proportional to the substrate concentration


Discussion assay of the methyltransferase activity1
Discussion: Assay of the methyltransferase activity

  • Enzyme activity with caffeic acid as substrate - very little activity.

  • The purified enzyme was going through several freeze-thaw cycles between the first measurement with AMI and MAMI as substrate and those with caffeic acid.

  • This could have resulted in the loss of enzymatic activity.

  • These experiments have to be repeated with freshly purified enzyme.


Summary
Summary

  • The enzyme exhibit little activity with caffeic acid but did methylate AMI and MAMI

  • Thus it might be involved in gramine synthesis by methylating AMI and MAMI rather than acting as caffeic acid OMT

  • Described as an O-methyltransferase, but a sequence similarity with other OMTs is only 40%


Conclusion
Conclusion

  • The enzyme carries out the transfer of a methyl group from S-adenosylmethionine to AMI, methylating it to MAMI and a methyl group from SAM to MAMI, with the formation of gamine, in fact acting as an N-methyltransferase in gramine biosynthesis

  • This work supports the idea that the methyltransferase gene accession number U54767 should be classified as an NMT-gene involved in gramine biosynthesis


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