1. Field of the Invention
The invention relates to nucleic acid molecules isolated from the 5' flanking region of endosperm-specific storage protein genes of monocotyledonous plants, and which possess nuclear matrix binding activity.
2. Description of the Related Art
In the past fifteen years it has become possible to transfer genes from any organism into a wide range of crop plants including the major monocotyledonous cereal crops wheat, rice, barley, oat and maize. However, even though the introduced genes can be expressed in the transformed crops, the level of expression can be very low. Indeed, Peach and Velten (1991) found that the majority of detectable transformants exhibited very low expression. The variation in expression is due to the influence of the surrounding chromatin at the site of insertion of the transgene (position effects). As a result, large numbers of transgenic plants must be produced in order to be sure of producing a single high-expressing plant. This is not trivial in cereal crops where the transformation efficiency is only 1 to 5%. In addition to expression variability, there is also the possibility that transgene silencing will occur in subsequent generations.
For the purposes of crop improvement, it would be highly beneficial to reduce the numbers of transgenic plants which need to be produced to find a high-expressing plant, and also to ensure that there will be no transgene silencing in subsequent generations.
In eukaryotes, the DNA is folded into chromosomes in the form of loops. These loops are anchored to a proteinaceous nucleoskeleton, known as the nuclear matrix or scaffold, by segments of DNA known as matrix attachment regions ("MAR"s). The DNA loop structure, which allows for the unwinding of DNA to permit access by transcriptional regulatory proteins, has important implications for gene regulation and expression. One function of the loop structure and matrix attachment regions is to insulate genes from the effects of surrounding chromatin, thereby allowing copy number dependent, position independent expression of adjacent genes. For this reason, various studies have investigated the hypothesis that MARs may allow position independent expression of any introduced genes in transgenic plants.
In several studies, the hypothesis was validated, and MARs were found to increase transgene expression, decrease silencing, prevent silencing after crossing or backcrossing to non-transformed plants, normalize expression, and to provide copy number independent expression (Allen et al. (1996); Allen et al. (1993); Ulker et al. (1997); Mlynarova et al. (1995); Breyne et al. (1992)). These studies utilized MARs derived from either animal or tobacco sources.
Recently, MARs from other plant species such as maize and rice have been isolated (Avramova and Bennetzen (1993); Nomura et al.(1997)). The maize MAR was derived from the promoter of an alcohol dehydrogenase gene. The rice MARs were isolated on the basis of functional binding to the nuclear matrix, and their association with any transcribed gene is unknown.
All of the MARs isolated to date have similar features in that: (1) they are AT rich (greater than 60%); (2) they contain motifs such as A-boxes (consensus sequence AATAAA(T/C)AAA--SEQ ID NO: 6, T-boxes (consensus sequence TT(T/A)T(T/A)TT(T/A)TT--SEQ ID NO: 7), ATATTT boxes, and boxes showing homology to DNA topoisomerase II consensus cleavage sites (consensus sequence GTN(A/T)A(T/C)ATTNATNN(G/A)--SEQ ID NO: 8); and (3) they usually have a length of about 300 to 500 nucleotides (see e.g. Breyne et al. (1992)). None of the mentioned motifs are universal, however, and different MARs do not have extensive homology, nor are there strictly conserved DNA elements. Specific MAR DNA sequences are therefore species specific and differ in their ability to bind the nuclear matrix. Each putative MAR must therefore be investigated by functional assays.
Given the low transformation efficiency in cereal crop plants, MARs functional in cereal crop plants to increase transgene expression, decrease silencing, prevent silencing after crossing or backcrossing to non-transformed plants, normalize expression, and to provide copy number independent expression, would be very useful. The species-specificity of MARs limits the utility of the known rice and maize MARs for improving transgene expression in monocotyledonous cereal crops such as wheat (Triticum aestivum). Thus, there is an ongoing need for MARs functional in cereal crop plants.