1Cooperative Research Centre for Molecular Plant Breeding,, Centre for Plant Conservation Genetics,
Southern Cross University, Lismore-2480, Australia.
2The Department of Biochemistry, The University of Queensland, St Lucia-4072, Australia.
Abstract
Genetic engineering provides an opportunity to express specific trans-genes in barley. Specific promoter regions of genes can be used to control spatial and temporal expression of trans-genes in genetically engineered barley. The development of transgenic barley for commercial use depends on freedom-to-operate (FTO), with both the trans-genes and the promoters used. We are interested in applying genetic engineering to improve barley with respect to disease resistance and quality. Promoters for which we have FTO are the prb-1 (a pathogen-inducible promoter) and the asi (an endosperm-specific promoter). Investigation of both these promoters indicates that they could be used to produce commercial varieties with highly desirable traits. These represent valuable tools for use in the genetic engineering of barley.
Introduction
Conventional breeding has been used modify the genetic makeup of plants for the benefit of mankind and their domesticated animals. However, with advances in gene-transfer technology researchers have applied molecular tools for the expression of novel genes (trans-genes) to modify a range of agronomic or end use characteristics. Genetic engineered plants have been generated for various uses such as increased tolerance to disease or the generation of novel proteins for increased nutritional value (Tabe L. and Higgins T. J. V.1998), or the production of vaccines, pharmaceuticals, plastics, industrial oils etc (Taylor C.B. 1998). However, in the normal process of plant development the expression of many genes is regulated in response to internal and/or external cues. Regulation of gene expression evolved in the plant is crucial for normal metabolism, growth and plant development in general and is controlled at the transcriptional or post-transcriptional level. Transcriptional regulation of genes results from interactions of DNA binding proteins with regions on the 5’-flanking sequences of genes also known as promoter regions. Thus using genetic engineering and the right promoter sequences the activation or suppression or trans-genes can be achieved. However, the development of transgenic barley for commercial use depends on freedom-to-operate (FTO), with both the trans-genes and the promoters used. We are interested in applying genetic engineering to improve barley with respect to disease resistance and quality. Promoters for which we have FTO are the asi (an endosperm-specific promoter) and the prb-1 (a pathogen-inducible promoter).
Studies on the promoter of the asi gene.
The bifunctional alpha-amylase subtilisin inhibitor protein (ASI) is synthesized in developing barley grain and its expression is regulated by the hormone abscisic acid and gibberellic acid (Mundy, Svendsen et al. 1983). ASI-protein inhibits the high-pI group of alpha-amylases in wheat and barley (Battershell and Henry 1990) and could have a role in regulating the action of alpha-amylase (Henry, Battershell et al. 1992; Henry, McKinnon et al. 1994). In addition, ASI-protein could have a defensive role as it inhibits the bacterial protein subtilisin (Leah and Mundy 1989). Using an ELISA-based assay, asi-protein level as compared to total seed protein was found relatively stable under various environmental conditions in Australia. The promoter of the asi gene was isolated by inverse-PCR and using homology-search putative elements for hormonal and endosperm specific expression was identified. The asi-promoter was analyzed for promoter-strength and tissue-specific expression by transient reporter-gene expression assay as well as in transgenic rice plants. Gene constructs were designed to express the green fluorescent protein gene (gfp) under the control of the asi-promoter and its deletions. In barley the isolated asi-promoter confers on gfp, transient aleurone-specific expression (figure 1). Comparison of the asi-promoter to other endosperm-specific cereal promoters and ABA and GA-responsive promoters is being carried out using transgenic plants and transient reporter-gene expression assay. In rice plants transformed with the asi-gfp.nos construct, GFP-protein is expressed in immature leaf tissue, stigma and expression of GFP in developing endosperm is currently being investigated.
Fig 1. Aleurone-specific expression of gfp under the control of the asi-promoter.
Aleurone (b), pericarp (c) and leaf (d) tissues were bombarded with the construct asi.gfp.nos (a) and cells expressing green fluorescence were detected only in the aleurone layers. Green fluorescence of the GFP-protein was observed under blue light (excitation 490 and emission at 510 nm) using a compound microscope.
Studies on the prb-1 promoter from barley
In barley the pathogenesis related genes type 1 (pr1) comprise a gene family of at least six genes and are expressed in response to pathogens and certain chemicals (Davidson, Manners et al. 1987, Muradov, Petrasovits et al. 1993). The prb-1 gene is a member of this pr1-gene family and homologues have been reported to be present in both wheat (Molina A., EMBL: AJ007349) and rice ( Bhargava, T. and Hamer, J. E., EMBL U89895). Using northern analysis transcript homologous to the prb-1 gene was expressed in barley in response to infection by Erysiphe graminis f.sp. hordei (barley powdery mildew fungus, BPMF) and after treatment with chemical such as 2,4-dichloro isonicotinic acid (DCINA) and benzotidiazole (BTH). Similar patterns of expression were obtained in wheat after treatment with DCINA, BTH and after infection with Erysiphe graminis f.sp. tritici (wheat powdery mildew fungus, WPMF). Investigation of the prb-1 promoter and its deletions using a transient reporter-gene expression assay indicated that the promoter is not induced in response to infection with the BPMF, WPMF or treatment with DCINA or BTH. In transgenic rice plants the prb-1 promoter was not induced after treatment with DCINA and BTH. However, in transgenic rice plants the prb-1 promoter confers on gfp gene constitutive expression in the terminal cell of trichomes and induced-expression in leaf tissue around necrotic-like lesions.
Conclusions
Applications of the asi-promoter in barley.
The promoter of the asi gene could be used to express novel proteins in barley for various uses such as to improve nutritional quality of fodder or for end-use applications such as in the beer industry. The promoter could also be used for endosperm specific expression of proteins in other cereals.
Applications of the prb-1 promoter in barley.
Study of plant microbe interaction has contributed immensely in understanding the molecular basis of resistance and susceptibility to a disease. Plants react to pathogen attack by activating an elaborate defence mechanism that acts both locally and systemically. However, resistance or susceptibility to a pathogen depends on the recognition of the pathogen followed by the timely induction of a defence response. Crop protection against pests and disease has been advanced tremendously by resistance breeding, application of pesticides and by a variety of agronomic practices such as crop rotation. Using gene transfer techniques the expression of resistance related trans-gene/s could further contribute to crop improvement by genetically modifying susceptible plants for increased resistance. Control of spatial and temporal expression of trans-gene/s in response to a range of pathogens can be achieved by using pathogen inducible promoters. Our results indicate that the promoter of the prb-1 gene could be used to direct expression of defense genes at the site of infection for increased resistance.
References
1. Battershell, V. G. and R. J. Henry (1990). Journal of Cereal Science 12(1): 73-82.
2. Davidson, A. D., J. M. Manners, et al. (1987. Plant Molecular Biology 8: 77-85.
3. Henry, R. J., V. G. Battershell, et al. (1992). Journal of Science of Food and Agriculture 58(2): 281-284.
4. Henry, R. J., G. McKinnon, et al. (1994). In: Improvement of Cereal Quality by Genetic Engineering. R. J. Henry and J. A. Ronalds. New York, Plenum Press: 129-132.
5. Leah, R. and J. Mundy (1989). Plant Molecular Biology 12(6): 673-682.
6. Mundy, J., I. Svendsen, et al. (1983). Carlsberg Research Communication 48: 81-90.
7. Muradov, A., L. Petrasovits, et al. (1993). Plant Molecular Biology 23: 439-442.
8. Tabe L. and Higgins T.J.V. (1998). Trends in Plant Science 3: 282-286.
9. Taylor C.B. (1998). The Plant Cell 10: 641-644.
