Subcellular localization of the LaTPS7, LaTPS8,
and LaCYP71D582 via transient expression in tobacco
When La TPS7 and La TPS8 were predicted by Signal P, the
signal peptide was not detected in the plastid. However, the signal was
detected when we used Phyre2, and La CYP71D582 was predicted to
localize on the membranes by both Signal P and Phyre2.
Hence,
experimental verification is particularly important when prediction of
TPSs subcellular localization is conflicted. Thus, full-ORF fusion
vectors enhanced with green fluorescent protein (eGFP) were constructed
for each candidate gene, and injected into N. benthaiana leaves
via A. tumefaciens -mediated transfection. Analysis of whole
leaves via confocal microscopy showed that both La TPS7 andLa TPS8 were associated with chloroplasts (Figure 3), whereasLa CYP71D582
was located in the endoplasmic reticulum (ER) (Figure 3).
Isolation andin-vitro enzymatic
characteristic of TPSs
To determine the function of each protein, six His-tagged TPS
recombinant proteins,
with
truncated putative signal peptides in their amino acid
sequences,
were expressed and extracted from E. coli BL21 (DE3). These
recombinant proteins were then tested with either GPP, NPP, or FPP (10
μg for each) used as substrate. Our
results show that La TPS7 catalysed GPP to produce seven
monoterpenes corresponding to α -pinene, camphene, myrcene,
limonene, terpinolene, linalool, and terpineol, and catalysed NPP toα -pinene, camphene, limonene, terpinolene, terpineol, and
nerolidol. La TPS8 produced α -pinene, β -pinene,
sylvestrene, linalool, fenchol, and geraniol from GPP, andα -pinene, limonene, terpinolene, terpineol, and nerolidol from
NPP. However, no products were detected when FPP was used as substrate
for both genes (Figure 4). These results indicate that these enzymes
differ in their substrate recognition and product output.