This invention relates in general to plant genetic engineering. In particular, the present invention relates to a particular promoter from a figwort mosaic virus which is useful in the expression of genes in plants.
One of the primary goals of plant genetic engineering is to obtain plants having improved characteristics or traits. The type and number of these characteristics or traits are innumerable, but may include virus resistance, insect resistance, herbicide resistance, enhanced stability or improved nutritional value, to name a few. Recent advances in genetic engineering have enabled researchers in the field to incorporate heterologous genes into plant cells to obtain the desired qualities in the plant of choice. This permits advantageous genes from a source different than the transformed plant to be incorporated into the plant's genome. This new gene can then be expressed in the plant cell to exhibit the new trait or characteristic.
In order for the newly inserted gene to express the protein for which it codes in the plant cell, the proper regulatory signals must be present and in the proper location with respect to the gene. These regulatory signals include a promoter region, a 5' non-translated leader sequence and a 3' polyadenylation sequence. The promoter is a DNA sequence that directs the cellular machinery to produce RNA. The promoter region influences the rate at which the RNA product of the gene and resultant protein product of the gene is made. The 3'-polyadenylation signal is a non-translated region that functions in plant cells to cause the addition of polyadenylate nucleotides to the 3' end of the RNA to stabilize the RNA in the cytoplasm for subsequent translation of the RNA to produce protein.
It has previously been shown that certain promoters are able to direct RNA synthesis at a higher rate than others. These are called strong promoters. Certain promoters have also been shown to direct RNA production at higher levels only in particular types of cells and tissues. Those promoters that direct RNA production in many or all tissues of a plant are called constitutive promoters.
Previous work had shown that the 35S promoter from the cauliflower mosaic virus (CaMV35S) was the strongest constitutive promoter known in plants (Odell et al., 1985; Jensen et al., 1986; Jefferson et al., 1987; Kay et al., 1987; Sanders et al., 1987). This had been shown by demonstrating measurable levels of reporter gene proteins or mRNAs in extracts prepared from the leaves, stems, roots and flowers of transgenic plants. As a result, the CaMV35S promoter has been widely used by scientists in the field of plant genetic engineering.
Although the CaMV35S promoter appeared to be a strong, constitutive promoter in assays involving cell extracts, detailed histological analysis of a reporter gene product that is detectable at the cell and tissue level showed a rather high degree of variability of expression of this gene product. This histological analysis revealed an unknown and unexpected variability in the expression of a gene product driven by the CaMV35S promoter. This variable level and site of expression is believed to have two primary causes. The first is that variability is an intrinsic property of the CaMV35S promoter. The second is that the variability is caused by the position that the CaMV35S promoter driven DNA sequence is integrated into the genome of the transformed plant. When a gene is introduced into a plant cell, the new DNA becomes incorporated at random locations in the plant DNA. This variability in location or insert position leads to a variation in the level of promoter activity and protein production from individual transformants. As a result, a large number of individual transgenic plants must be assayed to find those that produce the highest levels of gene product in most or all of the tissues of the plants. Even the presumed strong, constitutive CaMV35S promoter is subject to the effect of insertion position variability and its use requires that a relatively large number of transformed plants be screened to find ones having appropriate levels of gene expression. Thus, it is clear that a need exists in plant genetic engineering for promoters that express high levels of chimeric gene product, but that is less subject to the wide variation in tissue level expression due to intrinsic properties of the promoter or caused by the effect of insertion position in transgenic plant DNA.
Other caulimoviruses, a group of double-stranded DNA viruses to which the cauliflower mosaic virus belongs, were considered as a potential source for such a promoter. Two caulimoviruses that are distantly related to CaMV have been previously described. The figwort mosaic virus (FMV) was described by Richins et al. (1987) and the carnation etched ring virus (CERV) was described by Hull et al. (1986). The DNA sequence and predicted gene organization of each of these two viruses were similar enough to the CaMV to permit Richins et al. to speculate as to the locations of the FMV and CERV homologues of the CaMV35S promoter. There was, however, little conservation of DNA sequences in these presumptive promoter regions and no confirming RNA transcript analysis had been carried out to provide a demonstration of the exact location of the promoter sequences, much less a showing that a promoter from FMV would provide an increased and more uniform level of expression of a chimeric gene in plants.
It is therefore a primary object of the present invention to provide a promoter for use in transgenic plants that exhibits an increased and more uniform level of expression of a gene product driven by the promoter than that exhibited by previously known plant promoters.
It is another object of the present invention to provide a promoter for use in transgenic plants that is less affected by insertion position effects than previously known and used plant promoters.
It is a further object of the present invention to provide a promoter for use in transgenic plants that exhibits a higher level of expression of a gene product driven by the promoter in many of the tissues and cells of the plant, particularly the floral buds, than that exhibited by previously known plant promoters.
It is yet another object of the present invention to provide such a promoter for the expression of a chimeric gene in plants that is obtained from the full-length transcript of the figwort mosaic virus.
Other and further objects of the invention will be made clear or become apparent from the following description.
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