In order to identify promoter regions involved in the regulation of the birch NR gene expression,
the 1535 bp sequence 5 flanking this gene, and eight sequences obtained by sequential 5 deletion
were fused to the b-glucuronidase GUS reporter gene. The fusion constructs were introduced into
Nicotiana plumbaginifolia via agrobacteria. Trans- formants harbouring a distinct NR promoter GUS
fusion either were grown on a medium containing nitrate, and GUS activity was determined at the
end of both prolonged darkness 3 days and a 3-h light period thereafter, or GUS activity was com-
pared between nitrate-grown and ammonium-fed plants.
2. Materials and methods
2
.
1
. DNA and RNA analysis DNA was sequenced by the dideoxy chain-ter-
mination method. DNA for PCR analysis was isolated from leaves of transgenic N. plumbaginifo-
lia according to Cheung et al. [6]. RNA was isolated from leaves of young birch plants accord-
ing to de Vries et al. [7], and the transcription start of the NR gene was mapped by nuclease SI pro-
tection and primer-extension analysis [8].
2
.
2
. Construction and screening of a birch genomic library
DNA was extracted from leaves of hydroponi- cally grown birch plants according to Murray and
Thompson [9] and partially digested with MboI. Fragments between 9 and 23 kb were ligated into
the BamHI cloning site of the EMBL4 vector, and the DNA was packaged using an in vitro Lambda
Packaging
System Promega.
Recombinant phages were amplified with Escherichia coli strain
LE392. The library was screened for plaques which hybridised to the 5 region of a birch NR
cDNA clone [4]. Phage DNA from a hybridising clone was digested by SalI + EcoRI. A 2.1 kb
SalIEcoRI fragment that hybridised to the NR cDNA was found to contain 1149 bp 5 upstream
the translation start. Additional 499 bp of the NR promoter further upstream could be determined
by primer walking.
2
.
3
. Construction of NR promoter GUS gene fusions
The plasmid pSLJ4D4 was used to fuse NR promoter sequences with the GUS gene. pSLJ4D4
is a modified pUC19 with an insert that contains the 35S-CaMV promoter, the GUS gene and the
polyadenylation signal of the octopine synthase gene OCS in fusion. Binary vectors derived from
pGA482 were used to transform agrobacteria [10].
From the 2.1 kb SalIEcoRI fragment obtained from recombinant phage, a 1.121 kb RsaIEcoRI-
subfragment was excised that represents a 5 flank- ing sequence of the birch NR gene spanning from
+ 85 to − 1036 relative to the transcription start
site. This RsaIEcoRI fragment was inserted into the plasmid pSLJ4D4 adjacent to the GUS gene
thereby displacing the 35S-promoter. In this fu- sion construct, 29 nucleotides of the NR leader
sequence between the translation start site and position + 85 are replaced by the first 51 nucle-
otides of the GUS leader sequence containing a SacI site that was used later on for the cloning of
promoter deletion constructs in the pGA482 bi- nary vector. A fragment comprising the fused
1,121 bp birch sequence and the GUS encoding sequence was isolated from pSLJ4D4, inserted
into the phagemid pBluescript KSII + Strata- gene, La Jolla, USA, and 5 deleted. Unidirec-
tional deletion reactions in cloned inserts in pBluescript II KS + were performed with the
double-stranded Nested Deletion Kit Pharmacia based on a strategy described by Henikoff [11].
This resulted in NR promoter GUS fusions with end points at positions − 820, − 643, − 535, −
408, − 320 and − 237 of the NR promoter se- quence. A longer strech of the NR promoter with
the endpoint at − 1535 could be cloned and fused to the GUS gene in pBluescript KSII + after
having created a NotI site by primer directed mutagenesis of the original recombinant Lambda
phage DNA adjacent to the birch DNA insert.
From recombinant pBluescript KSII + , a se- quence spanning from position − 183 of the NR
promoter to GUS and OCS was isolated by a HindIII digest and inserted into pGA482. The
obtained pGA482 derived vector was used both per se and as a host to conceive the other pro-
moter sequences with the various endpoints de- scribed above. To this end the promoter sequences
upstream the already mentioned SacI site were
isolated from the corresponding recombinant pBluscript KSII + by a SacI + BamHI double
digest and ligated into the recombinant pGA482 cleaved by SacI + BglII. The various NR pro-
moter GUS gene fusion constructs are depicted in Fig. 1. A 35S-CaMV promoter GUS gene con-
struct derived from pBI121 Clontech, Palo Alto, USA was also inserted into pGA482. Verification
of constructs was carried out by restriction enzyme digests,
DNA – DNA hybridisation
and sequencing.
2
.
4
. Transformation of N. plumbaginifolia, growth of transformants, and fluorimetric GUS assay
The pGA482-derived vectors were introduced into A. tumefaciens strain LBA4404 by the direct
transformation method [10]. For selection of transformants, A. tumefaciens cells were grown on
a medium containing streptomycin, tetracycline, and kanamycin. Successful transformation was
demonstrated by restriction analysis of A. tumefa- ciens DNA and Southern hybridisation with a
radioactive labelled birch NR DNA fragment po- sitions + 85 to − 183. The infection of leaf discs
from N. plumbaginifolia with A. tumefaciens, the selection of transformed calli by use of cefotaxime
and kanamycin, and the regeneration of transgenic plants were performed as described [12]. A PCR
approach was employed to verify that regenerated kanamycin resistant N. plumbaginifolia plants con-
tain a NR promoter GUS gene construct. A se- quence between nucleotide positions + 199 and
−
197 relative to the transcription start site of the fusion construct was amplified using 5-TC-
TACAGGACGTAACATAAG-3 as the antisense primer and 5-AAGTGAAAAGCTTGACCGCA-
3 as the sense primer. The PCR-products were identified by hybridisation with a DNA sample
spanning from positions + 105 to − 184 of the fusion construct.
Transgenic plants were further cultivated on Murashige and Skoog MS medium containing
18.8 mM KNO
3
and 20.6 mM NH
4
NO
3
under a 16 h light8 h dark regime at 24°C in a plant
growth chamber. Light was generated by a cool- white fluorescent source with a light intensity at
150 mmol m
− 2
s
− 1
. Extraction of GUS from leaves and roots of
primary transformants and fluorometric assays for GUS activity were performed according to Jeffer-
son et al. [13] with methylumbelliferone MU as a standard. A detectable GUS activity was found in
about 30 of primary transformed plants.
2
.
5
. Statistical analysis Gene expression in populations of first-genera-
tion transgenic plants usually does not follow a normal distribution. The measure most suitable to
describe the location of an unknown distribution probably is the median. An informative descrip-
tion of the shape of such a distribution is the median absolute deviation or MAD, which is
defined as the mean of the absolute differences between each observation and the median of all
observations [14]. As a distribution-free statistical method we employed the nonparametric Mann –
Whitney U-test that does not use the actual mea- surements,
but instead
the ranks
of the
measurements. This method was also used to test proposed hypotheses employing a multiplication
constant [15]. In one set of experiments see Sec- tion 3.3, the obtained data occur in pairs, and the
Wilcoxon paired-sample test was applied [15]. Cal- culated values of the statistical significance level a
are given in Section 3.
3. Results
