Sequence analysis of target genes
The full amino acid sequences translated from ORFs (primers are provided
in table S1) were used for multiple sequence alignent performing on
an
ESPript
(http://espript.ibcp.fr/ESPript/ESPript/)
as described previously (Robert et al. 2014). Phylogenetic
trees
were constructed by MEGA7
using
the Neighbor-Joining method, and Interactive Tree of Life (iTOL )
was used for visual editing (http://itol2.embl.de/) (Letunicet al. 2007). Analysis of signal peptides in TPSs, and prediction
localization of candidate proteins were performed on
Phyre2
(http://www.sbg.bio.ic.ac.uk/phyre2)
and signal P (http://www.cbs.dtu.dk/services/SignalP-4.1/) as
described previously (Kelley et al.2015).
Protein secondary structure was predicted using I-TASSER
(http://zhanglab.ccmb.med.umich.edu/I-TASSER).
Determination oftissue-specific
gene expression in A. thaliana
Approximately
1.5 kb of a promoter for each gene was isolated by FPNI-PCR from genomic
DNA using three specific gene primers and three arbitrary primers and
PCR settings described by Wang et al. (2011). Resulting sequences
were analysed on PlantCARE
(bioinformatics.psb.ugent.be/webtools/plantcare/html)
(Rombauts et al. 1999) and then integrated into
pCXGUS-P
vector harbouring a cloning cassette upstream of GUS(uidA ). After the constructs were confirmed via sequencing, they
were introduced into GV3101Agrobacterium ,
which was used to transform A. thaliana using a floral dip method
described previously (Clough et al. 1998). Transformed lines were
selected on MS plates containing 50
µg·mL-1hygromycin, and were then confirmed via PCR. Enzymatic assay using
5-bromo-4-chloro-3-indolyl-β-D-glucuronide (GUS) was
performed according to the method
of Jefferson et al. (1987) to assess candidate genes
tissue-specific expression.
Subcellular
localization of the LaTPS7, LaTPS8, and LaCYP71D582
in N. benthamiana
GV3101 with eGFP expression vectors controlled by a mannopine synthase
promoter (MAS), constructed using a Trelief™ SoSoo Cloning Kit
(TSINGKE,
China), were used for transient transformation of 4-week-oldN.
benthamiana plant as described by Jin et al. (2015). After 3
days of infiltration, N. benthamiana leaves were excised, mounted
on slides, and analyzed using
a
confocal laser-scanning microscope equipped with a standard filter set
(Leica TCS SP5). Images were processed by
Image
J (https://imagej.nih.gov/ij).
Heterologous
expression of TPSs in Escherichia coli and enzymatic assayin vitro
TPS sequences of truncated
predicted signal peptide were inserted into a pDE2 vector with a
recombinant C-terminal and poly-histidine tag using primers shown in
table S1 and a pDE2 Directional Expression Kit Ver.2 (TSINGKE, China).
The final constructs was transformed into E. coli BL21 (DE3);
recombinant proteins were subjected to
isopropyl-β-D-thiogalactopyranoside (IPTG) induction at the
concentration of 1.0 mM until OD600 reached
0.6~0.8, and then to purification using a His-Tagged
Gravity Column (Merck Millipore). The purified proteins were assayed by
sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) to
ensure that the proteins had been extracted successfully.
The in-vitro enzymatic assay for TPS activity was performed
according to the method of Chen et al. (2011) in a final volume
of 500
μL
buffer [25mM HEPES, pH 7.3; 10 mM MgCl2; 10 mM
MgCl2; 10% glycerol; 10 mM DTT], approximately 20 μL
pure protein, and 10 µg of either FPP,
NPP,
or GPP
(Sigma-Aldrich).
The mixtures were vortexed, incubated at 30°C for 2 h, after which 250
μL hexane was added to each mixture and vortexed for 1 min. The upper
layers were centrifuged at 1200 g and 4°C for 30 min, and then
transferred into 2-mL glass vials for analysis via GC-MS.
Heat-inactivated recombinant protein was used as negative control.