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V. Herl et al. / Phytochemistry 67 (2006) 225–231
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5a-POR (Kreis et al., 1998), several conserved functional
protein domains (LEGFXAF, LIHX, IPFV) were described
by Thigpen and Russell (1992) and Bhattacharyya et al.
(1999). None of these domains could be found in the 5b-
PORs isolated so far. On the other hand, the two peptide
fragments sequenced by Ga¨rtner et al. (1994) were identified
in all 5b-POR products sequenced so far (Roca-Perez et al.,
2004; AY585865-68, AY750898, AY574950, AY738710-12).
2.3. Southern blot analysis
The molecular organization of the 5b-POR from D.
lanata was determined by Southern blot analysis of geno-
mic DNA (Sambrook et al., 1989), digested with the
restriction endonucleases EcoR1, BamH1 and HindIII
(Fig. 2B). The full-length cDNA clone of 1170 bp was
32P-dGTP labelled and used as a probe for hybridization.
Under high stringency conditions two bands were detected
for cuts by EcoR1 and BamH1 restrictases, while three
bands were found for HindIII, as outlined in Fig. 2B. These
results indicate that 5b-POR from D. lanata consists of
almost 1–2 genes, as the size of the genomic clone of the
5b-POR is known (1250 bp, AY585867).
2.4. Over-expression
Over-expression of 5b-POR was achieved in Escherichia
coli after IPTG (0.1 mM) induction at low temperature
(4 ꢂC). The enzyme was purified by Ni-NTA batch fraction-
ation under native conditions and analysed by SDS–PAGE
(Fig. 4). As a control, the bacterial host cells were trans-
formed with an empty pQEX vector. The Ni-NTA-purified
r5b-POR was analysed by SDS–PAGE where a strong band
at 40 kDa indicated that the recombinant protein has the
same size as the 5b-POR isolated from D. purpurea leaves
by Ga¨rtner et al. (1994). Traces of other proteins (less than
3% of overall protein) could also be detected in the SDS–
PAGE but these were also found in the control extracts of
the bacteria containing the pQEX vector only. Since Jan-
knecht et al. (1991) stated that proteins containing neigh-
bouring histidins are not common in bacteria we suggest
that these ‘‘contaminating’’ proteins remained bound to
the column material due to the low stringency condition
(20 mM imidazole) chosen in the washing step.
Fig. 2. (A) PCR amplification of 5b-POR from D. lanata; (Lane 1) DNA
fragment of genomic DNA from D. lanata; (Lane 2) cDNA fragment from
RNA of D. obscura after RT-PCR; (Lane 3) cDNA fragment from RNA
of D. purpurea after RT-PCR; (Lane 4) cDNA fragment from RNA of D.
lanata after RT-PCR; (M) DNA-Marker SmartLadder (200–10,000 bp).
(B) Southern blot of D. lanata DNA digested with EcoR1 (E), HindIII (H)
and BamH1 (B) hybridized with 32P-dGTP labelled 5b-POR cDNA.
(AY574950), D. purpurea (AY585868) and the Dop5br gene
of D. obscura (AJ555127). The sequences of the deduced 5b-
POR gene products are 95–99% identical. A high degree of
homology was also seen when the nucleotide sequence of
the cDNA was analysed in silico and compared with the
reports for Dop5br of D. obscura (Roca-Perez et al., 2004)
and Dop5br of D. purpurea (AJ310673) (Fig. 3). Hence, it
seems as if the 5b-POR genes are highly conserved within
the genus Digitalis. Recently, Bra¨uchler et al. (2004) pro-
posed a phylogenetic cladogramme of the genus Digitalis
on the basis of ITS- and trnL-F sequences and found D.
lanata and D. obscura more closely related to each other
than to D. purpurea which is supported by our data.
2.5. Function of r5b-POR
The 5b-POR gene over-expressed in E. coli yielded an
enzymatically active protein. The Ni-NTA-purified protein
was checked for 5b-POR activity using the assay described
earlier (Stuhlemmer and Kreis, 1996). A typical experiment
is shown in Fig. 5A. TLC analysis revealed the formation of
one single product, namely 5b-pregnane-3,20-dione, when
progesterone was used as the substrate (Fig. 5, Lane 3).
5a-Pregnane-3,20-dione, the 5a-isomer of the pregnane-
3,20-dione was not formed. This fact is supported by the
different Rf-values and the different colours of the two
The deduced 5b-POR protein sequences were found
similar to those of Oryza sativa (about 58%), Populus tremu-
loides (about 64%) and to the Arabidopsis thaliana wound-
induced gene AWI (Yang et al., 1997). For the competing