PNNL-13501 - Pacific Northwest National Laboratory

Discovery, Isolation, and Characterization of Strong Plant Promoters
Study Control Number: PN98024/1270
Brian Hooker, Ziyu Dai
This project was initiated to develop the molecular biology capabilities needed for analysis of higher plant gene
expression under post-harvest and senescence conditions and the subsequent isolation of novel promoters related to these
plant life stages. These new capabilities provide the scientific foundation for engineering senescence and stress-induced
responses in specific crop plants. Potential applications could impact the productivity of plants grown for food, fiber,
energy, or other applications, while reducing the environmental impacts of agricultural production.
Project Description
The focus of this project was to isolate and characterize
novel plant promoters and promoter regulatory elements
that might be applied to improve heterogeneous gene
expression in higher plants. Various regulatory
(promoter) sequences can potentially control gene
expression in transgenic plants to achieve different
expression systems and targets related to temporal or
spatial expression, or create inducible systems that are
responsive to different external factors and chemicals, etc.
Further characterization of novel promoters and their
associated regulatory elements also provides greater
understanding of regulatory mechanisms for gene
expression in higher plants.
Introduction
The introduction of new traits into crop plants through
molecular biology techniques is beginning to play an
important role in maintaining and improving agricultural
productivity. The discovery and development of novel,
efficient promoters and expression systems is very
attractive for the genetic engineering of any organisms. A
promoter is defined as the region of DNA that RNA
polymerase binds to, initiating transcription. In its most
minimal form the promoter typically includes a TATA
box, which is located about 20 to 30 base pairs upstream
of the transcription start site, and a CAAT box. In the
upstream promoter region of the TATA box and CAAT
box, there exist other regions that influence transcription.
These regions are called cis-acting elements, which can
regulate transcription strength as a function of temporal or
spatial conditions and/or in response to external stimuli.
Promoters directly control the initiation of transcription
and the subsequent expression level of the gene of
66 FY 2000 Laboratory Directed Research and Development Annual Report
interest, as well as the plant portion in which the encoded
protein is produced. Promoters may initiate gene
expression throughout the entire plant (the cauliflower
mosaic virus 35S promoter) or only in specific tissues,
such as green portions (the chlorophyll a/b binding
protein promoter). In addition, the activity of some
promoters may be induced by certain stresses or chemical
stimuli (pathogenesis related protein promoters).
Protein synthesis in ribosomes occurs in all known
organisms. Cytoplasmic ribosomal protein genes have
been studied in animals and yeast, but there is little
information on the regulatory elements of these ribosomal
protein genes for plant systems (Dai et al. 1996). Two
cytoplasmic ribosomal protein genes (rpL25 and rpL34)
were previously isolated from tobacco NT1 suspension
cells at the stage of cell rapid division (Gao et al. 1994).
The rpL34 promoter was identified and functional
analysis was completed using the cat reporter gene (Dai et
al. 1996).
Plant senescence-associated genes are important targets
for the development of new plant traits. For example, the
cytokinin biosynthesis gene was linked with an
Arabidopsis senescence-specific promoter, resulting in
senescence-resistant tobacco plants (Gan and Amasino
1995). In addition, senescence-specific promoters may
comprise good targets for “post-harvest” foreign protein
production in plant bioreactors (Cramer et al. 1997). The
senescence of plant tissues may be artificially triggered
through application of chemical stimuli such as abscisic
acid, ethylene, or methyl jasmonate (Park et al. 1998), or
through harvest from intact plants. The carbon and
nitrogen sources of senescence tissues can then be
partitioned into new products (proteins or other
chemicals) in targeted plant portions (non-food portions).
For example, related research at our Laboratory showed