PNNL-13501 - Pacific Northwest National Laboratory

accumulation of protein products, including CBHI and E1
cellulases, under senescence conditions in tobacco leaves
(Dai et al. 2000).
Approach
Research focused on following areas: 1) isolating and
characterizing senescence and abscission related
promoters in potato and corn, 2) characterizing cis-acting
regulatory elements of ribosomal protein gene L34
(rpL34) promoter, and 3) isolating ribosomal protein gene
L25 (rpL25) promoter and testing its function. Two
different approaches were used for isolation of
senescence-associated genes in potato and corn.
For potato, the novel cDNA clones were isolated via the
differential hybridization screening method. A cDNA
library was constructed from poly (A) + RNA isolated
from the dark-treated, excised potato leaf tissues with
ZAP cDNA synthesis kit (Stratagene). About 60,000
plaques from the cDNA library were plated, transferred
onto duplicate nitrocellulose filters, hybridized with
radio-labeled cDNA probes synthesized independently
from 1 µg poly (A) + RNA of healthy untreated leaf tissue
and dark-treated tissues. The plaques that displayed
different signal intensity between the two probes were
collected, re-plated, and screened again using newly
synthesized cDNA probes. A single, pure plaque from
each of these clones still demonstrating the differential
hybridization signal intensity was collected, and the
pBluescript phagemid containing the cDNA insert was
excised from the UniZAP vector as the described by the
manufacturer (Stratagene, California, USA). The
nucleotide sequences were determined by automated
sequencing and the searches of the Genbank databases
were performed with BLASTN.
For corn, the novel cDNA clones were isolated with
suppressive subtractive hybridization methods as
described by the manufacturer (CLONTECH, California,
USA). A series of novel cDNA clones that carry partial
cDNA fragments were isolated. The 5’-end and 3’-end
cDNA fragments were further isolated with a Marathon
cDNA amplification kit (CLONTECH, California, USA)
and the cDNA clones were confirmed by DNA
sequencing. The DNA sequences were analyzed via
BLASTN data searches.
The cis-acting elements of rpL-34 promoter were
identified and characterized by the loss-of-function
(internal deletion) and the gain-of-function (multimers of
cis-acting elements with mini-promoter). The effects of
these cis-acting elements on the promoter activity were
determined by GUS reporter gene (its activity and
histochemical staining in different tissues and
developmental stages) in transgenic plants. The rpL25
promoter was isolated with the inverse PCR method.
Briefly, the genomic DNA fragments were first circled by
DNA ligation. Then two DNA primers, which were
complementary to the 3’-end and 5’-end of DNA
sequences of rpL25 cDNA clone, were used for genomic
PCR. The PCR products were confirmed by DNA
sequence. The functions of promoter were determined
with GUS reporter gene by transient and Agrobacteriummediated
stable transformation.
Results and Accomplishments
The function of two important regulatory elements within
the rpL34 promoter region was determined by gain-offunction
analysis using tandem repeats of each element in
a variety of arrangements. Screening and characterization
of these elements in transient assay has been completed.
In addition, stably transformed tobacco lines have been
produced for further characterization of gain-of-function.
In addition, hybrid L34/35S promoters have been tested in
transient assays.
The rpL25 promoter was isolated and tested in transient
assay as well as in stable transformants. The expression
pattern of the L34 and L25 promoters are compared in
young seedlings in Figure 1. Although both promoters
appear to express preferentially in meristematic tissues,
the L25 shows strong expression only during seed
germination and early seedling growth stages.
Figure 1. Transgenic seedlings bearing a B-glucoronidase
(GUS) gene flanking either the L25 (Panels A-E) and L34
(Panels F-J) promoters. Seedlings were stained for GUS
detection at increasing age (A and F, 0 days post-imbibition;
B and G, 3 days; C and H, 6 days; D and I, 9 days; E and J,
16 days).
Senescence cDNA libraries were developed for both
potato (Solanum tuberosum) and corn (Zea mays). When
these libraries were screened with a differential
hybridization method, over 50 different clones, which are
Biosciences and Biotechnology 67