8
M. Galata et al. / Phytochemistry xxx (2014) xxx–xxx
algorithms in the publicly available Velvet software (v.1.0). The
resulting contig libraries became the input for the also publicly
available Oasis transcript assembler with default settings (initial
release). The sequencing and library construction were performed
by service providers at the Plant Biotechnology Laboratory, Univer-
sity of Lethbridge (Lethbridge, AB, CA).
The CsLINS ORF, excluding the signal peptide, was amplified
using the forward (50-ACA CAT ATG ACT AAG GTT CCT GTT CCA
GTA CCA G-30) and reverse (50-ATA AAG CTT GAG AGG AAT GGG
CTC GAC AAG AAG-30) primers in the same Kapa ready-mix. The
Nde I and Hind III restriction enzyme (NEB) sites were designed
into the forward and reverse primers (underlined), respectively.
The PCR program used was 95 °C for 5 min, followed by 35 cycles
of 95 °C for 1 min, 60 °C for 30 s and 72 °C for 2 min, and a final
extension at 72 °C for 5 min. The amplicons were purified with
an E.Z.N.A DNA Purification Kit (Omega Bio-Tek, GA, USA) and each
fused to a C-terminal eight histidine moiety in the pET41b(+) vec-
tor to facilitate downstream protein purification.
4.6. Bioinformatics
Using the BLASTx algorithm, C. sativum transcripts were aligned
against the whole non-redundant database with a cut-off e-value
of <10ꢀ10. All unique transcript sequences were aligned against se-
quences in The Arabidopsis Information Resource (TAIR v.2.2.8)
and the Universal Protein Resource Knowledgebase (UniProtKB)
protein databases via the BLASTx algorithm. All sequences with
blast hits were annotated with Gene Ontology (GO) terms by ser-
vice providers at the National Research Council Plant Biotechnol-
ogy Institute (NRC-PBI, Saskatoon, SK, Canada). Sequences were
also assigned Kyoto Encyclopedia of Genes and Genomes (KEGG)
annotations via the KEGG Automatic Annotation Server (KAAS)
(Moriya et al., 2007).
Differential transcript expression was performed via RSEM
algorithm and expression comparisons were based on log2 ratios
of samples 2 and 3 to sample 1. Raw transcript expression data
was obtained by service providers at the Plant Biotechnology Lab-
oratory (University of Lethbridge) and raw read counts were re-
ceived as a tab delimited file containing gene ID’s, expression
level and log2 ratios. Using GO and KEGG annotations all the genes
involved with isoprenoid biosynthesis (DXP and MVA pathway as
well as terpene synthase/cyclases) were identified and their differ-
ential expression patterns analyzed. The GO and KEGG annotations
were also used to identify all photosynthetic genes (Supplemen-
tary Table S1) as well as fatty acid biosynthetic genes (Supplemen-
tary Table S4).
The CsLINS PCR product was digested with NdeI and HindIII as
per manufacturer’s protocol and the digested DNA was extracted
from an agarose gel via the Omega Gel Extraction Kit (Omega).The
fragment was ligated into pET41b(+) expression vector as was
done with CscTRPS.
4.9. Recombinant protein expression, crude enzyme assay
and purification
The expression constructs were transformed via heat shock into
Escherichia coli Rosetta (DE3) pLysS expression cells (Novagen,
Darmstadt, Germany). Successful transformants were selected on
solid Lauria–Bertani (LB) medium containing 30
lg/ml of kanamy-
cin and 34 g/ml of chloramphenicol. Single colonies were inocu-
l
lated in 5 ml LB media with the same antibiotics and grown at
37 °C and 190 rpm for 14–16 h. The 5 ml cultures were transferred
to 95 ml LB media with antibiotics and incubated at 30 °C and
190 rpm until OD600 of ꢂ0.8 was reached. Protein expression was
induced by the addition of isopropyl-b-D-thiogalactopyranoside
(IPTG) to a final concentration of 0.5 mM, and incubation at 18 °C
and 190 rpm for 14–16 h. Induced cells were split into two 50 ml
portions and pelleted at 4 °C and 4000 rpm for 20 min before stor-
age at ꢀ80 °C to lyse the cells.
For initial activity determination, frozen induced pellets were
resuspended in 1 ml crude assay buffer (25 mM TRIS–Cl, 5% glyc-
erol, 1 mM dithiothreitol (DTT), 10 mM MgCl2 and 1 mM MnCl2
at pH 7.5) (Crowell et al., 2002) supplemented with 1 mM protein-
ase inhibitor, phenylmethanesulfonylfluoride (PMSF). Cells were
sonicated on ice for six 10 s intervals with 1 min cool down periods
using a Sonic Dismembrator Model 100 (Fisher Scientific, Ottawa,
ON, Canada). The lysate was centrifuged at 14,000 rpm and 4 °C
for 11 min to obtain the soluble protein fraction. One hundred
microliters of crude soluble lysate was added to 2.9 ml of crude as-
4.7. Monoterpene synthase candidate selection
Two mTPS candidate genes, CscTRPS and CsLINS, were chosen
based on sequence homology to known mTPS genes, especially
the presence of certain conserved motifs shared by all known
TPS genes, DDXXD, (N,D)D(L,I,V)X(S,T)XXXE and RRX8W, as well
as one partially conserved motif, LQLYEASFLL. Protein sequence
alignments between CscTRPS and CsLINS, and Citrus limon c-terpin-
ene synthase, Lavandula angustifolia linalool synthase, Salvia fruti-
cosa 1,8-cineole synthase, Cannabis sativa limonene synthase, L.
angustifolia b-phellandrene synthase and Salvia officinalis sabinene
synthase were performed with ClustalW2. Those transcripts with
GO annotation ‘‘monoterpene biosynthetic process’’ as well as
transcripts with BLASTx hits to known mTPS genes having an e-va-
lue <10ꢀ60 were selected as coriander mTPS gene candidates.
say buffer with 25 lM geranyl diphosphate (GPP (18)) as substrate.
The assay mixture was overlaid with 1 ml pentane to trap the vol-
atile products and incubated in a 30 °C water bath for 2 h. Follow-
ing incubation, the assay was mixed vigorously and pentane
overlay was removed for product analysis.
For purification, E. coli pellets containing CscTRPS or CsLINS
were resuspended in 5 ml Ni–NTA binding buffer (50 mM NaH2-
PO4, 300 mM NaCl and 10 mM imidazole at pH 8.0) supplemented
with 1 mM PMSF. Cells were sonicated on ice for eight 15 s inter-
vals with 1 min cool down periods in between. The soluble fraction
was collected by centrifugation at 10,500 rpm and 4 °C for 15 min.
The His-tagged protein was then purified from the soluble lysate
by Ni–NTA agarose affinity chromatography (EMD Chemicals, Ger-
many) according to the manufacturer’s protocol. The eluted CsLINS
protein was further treated with an Amicon buffer exchange col-
umn (50 MWCO) to remove salts and dilute with protein storage
buffer (50 mM tris, 1 mM EDTA, 1 mM DTT with 10% glycerol at
pH 7.0).
4.8. Cloning of full length monoterpene synthase
The N-terminal signal peptide sequences of Cs
were predicted using ChloroP (v.1.1). The open reading frame of
Cs TRPS excluding the N-terminal signal peptide was sticky end
cTRPS and CsLINS
c
PCR amplified (Zeng, 1998) by Kapa HiFi DNA polymerase ready-
mix (Kapa Biosystems Inc., Woburn, MA, USA). Sticky end primers
(Supplementary Table S5) were designed to create EcoR1 and Xho1
(New England Biolabs, Whitby, ON, Canada) overhang regions for
use during ligation of CscTRPS into pET41b(+) bacterial expression
vector (EMD Chemicals, Darmstadt, Germany). The PCR program
used was as follows: 95 °C for 5 min, followed by 35 cycles of
98 °Cfor 20 s, 60 °C for 15 s and 72 °C for 1 min, and a 5 min final
extension at 72 °C.
Partially purified recombinant CscTRPS and CsLINS, as well as
soluble and insoluble total protein extracts from induced and
non-induced E. coli cells was resolved with 12% sodium dodecyl
Please cite this article in press as: Galata, M., et al. Transcriptome profiling, and cloning and characterization of the main monoterpene synthases of Cori-
Galata, Mariana
