Organic compounds -- part of the class 532-570 series – Organic compounds – Carbohydrates or derivatives
Reexamination Certificate
2000-08-22
2004-03-02
Bui, Phuong T. (Department: 1638)
Organic compounds -- part of the class 532-570 series
Organic compounds
Carbohydrates or derivatives
C800S278000, C435S468000
Reexamination Certificate
active
06699984
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to genetic engineering of plants. More particularly, the invention provides DNA sequences and constructs that are useful to control expression of recombinant genes in plants. Specific constructs of the invention use novel regulatory sequences derived from a maize root preferential cationic peroxidase gene.
BACKGROUND OF THE INVENTION
Through the use of recombinant DNA technology and genetic engineering, it has become possible to introduce desired DNA sequences into plant cells to allow for the expression of proteins of interest. However, obtaining desired levels of expression remains a challenge. To express agronomically important genes in crops at desired levels through genetic engineering requires the ability to control the regulatory mechanisms governing expression in plants, and this requires access to suitable regulatory sequences that can be coupled with the genes it is desired to express.
A given project may require use of several different expression elements, for example one set to drive a selectable marker or reporter gene and another to drive the gene of interest. The selectable marker may not require the same expression level or pattern as that required for the gene of interest. Depending upon the particular project, there may be a need for constitutive expression, which directs transcription in most or all tissues at all times, or there may be a need for tissue specific expression. For example, a root specific or root preferential expression in maize would be highly desirable for use in expressing a protein toxic to pests that attack the roots of maize.
Cells use a number of regulatory mechanisms to control which genes are expressed and the level at which they are expressed. Regulation can be transcriptional or post-transcriptional and can include, for example, mechanisms to enhance, limit, or prevent transcription of the DNA, as well as mechanisms that limit the life span of the mRNA after it is produced. The DNA sequences involved in these regulatory processes can be located upstream, downstream or even internally to the structural DNA sequences encoding the protein product of a gene.
Initiation of transcription of a gene is regulated by a sequence, called the promoter, located upstream (5′) of the coding sequence. Eukaryotic promoters generally contain a sequence with homology to the consensus 5′-TATAAT-3′ (TATA box) about 10-35 base pairs (bp) upstream of the transcription start (CAP) site. Most maize genes have a TATA box 29 to 34 base pairs upstream of the CAP site. In most instances the TATA box is required for accurate transcription initiation. Further upstream, often between −80 and −100, there can be a promoter element with homology to the consensus sequence CCAAT. This sequence is not well conserved in many species including maize. However, genes which have this sequence appear to be efficiently expressed. In plants the CCAAT “box” is sometimes replaced by the AGGA “box”. Other sequences conferring tissue specificity, response to environmental signals or maximum efficiency of transcription may be found interspersed with these promoter elements or found further in the 5′ direction from the CAP site. Such sequences are found within 400 bp of the CAP site, but may extend as far as 1000 bp or more.
Promoters can be classified into two general categories. “Constitutive” promoters are expressed in most tissues most of the time. Expression from a constitutive promoter is more or less at a steady state level throughout development. Genes encoding proteins with housekeeping functions are often driven by constitutive promoters. Examples of constitutively expressed genes in maize include actin and ubiquitin. Wilmink et al. (1995). “Regulated” promoters are typically expressed in only certain tissue types (tissue specific promoters) or at certain times during development (temporal promoters). Examples of tissue specific genes in maize include the zeins (Kriz et al., (1987)) which are abundant storage proteins found only in the endosperm of seed. Many genes in maize are regulated by promoters that are both tissue specific and temporal.
It has been demonstrated that promoters can be used to control expression of foreign genes in transgenic plants in a manner similar to the expression pattern of the gene from which the promoter was originally derived. The most thoroughly characterized promoter tested with recombinant genes in plants has been the 35S promoter from the Cauliflower Mosaic Virus (CaMV) and its derivatives. U.S. Pat. No. 5,352,065; Wilmink et al. (1995); Datla et al. (1993). Elegant studies conducted by Benfey et al. (1984) reveal that the CaMV 35S promoter is modular in nature with regards to binding to transcription activators. U.S. Pat. No. 5,097,025; Benfey et al. (1989) and (1990). Two independent domains result in the transcriptional activation that has been described by many as constitutive. The 35S promoter is very efficiently expressed in most dicots and is moderately expressed in monocots. The addition of enhancer elements to this promoter has increased expression levels in maize and other monocots. Constitutive promoters of monocot origin (that are not as well studied) include the polyubiquitin-1 promoter and the rice actin-1 promoter. Wilmink et al. (1995). In addition, a recombinant promoter, Emu, has been constructed and shown to drive expression in monocots in a constitutive manner, Wilmink et al. (1995).
Few tissue specific promoters have been characterized in maize. The promoters from the zein gene and oleosin gene have been found to regulate GUS in a tissue specific manner. Kriz et al. (1987); Lee and Huang (1994). No root specific promoters from maize have been described in the literature. However, promoters of this type have been characterized in other plant species.
Despite both the important role of tissue specific promoters in plant development, and the opportunity that availability of a root preferential promoter would represent for plant biotechnology, relatively little work has yet been done on the regulation of gene expression in roots. Yamamoto reported the expression of
E. coli:
uidA gene, encoding &bgr;-glucuronidase (GUS), under control of the promoter of a tobacco (
N. tabacum
) root-specific gene, TobRB7. Yamamoto et al. (1991), Conkling et al. (1990). Root specific expression of the fusion genes was analyzed in transgenic tobacco. Significant expression was found in the root-tip meristem and vascular bundle. EPO Application Number 452 269 (De Framond) teaches that promoters from metallathionein-like genes are able to function as promoters of tissue-preferential transcription of associated DNA sequences in plants, particularly in the roots. Specifically, a promoter from a metallathionein-like gene was operably linked to a GUS reporter gene and tobacco leaf disks were transformed. The promoter was shown to express in roots, leaves and stems. WO 9113992 (Croy, et al.) teaches that rape (
Brassica napus
L.) extensin gene promoters are capable of directing tissue-preferential transcription of associated DNA sequences in plants, particularly in the roots. Specifically, a rape extensin gene promoter was operably linked to a extA (extensin structural gene) and tobacco leaf disks were transformed. It was reported that northern analysis revealed no hybridization of an extensin probe to leaf RNA from either control or transformed tobacco plants and hybridization of the extensin probe to transgenic root RNA of all transformants tested, although the levels of hybridization varied for the transformants tested. While each of these promoters has shown some level of tissue-preferential gene expression in a dicot model system (tobacco), the specificity of these promoters, and expression patterns and levels resulting from activity of the promoters, has yet to be achieved in monocots, particularly maize.
DNA sequences called enhancer sequences have been identified which have been shown to enhance gene expression when placed proximal to the promoter. Such seq
Ainley Michael
Armstrong Katherine
Belmar Scott
Folkerts Otto
Hopkins Nicole
Baum Stuart F.
Bui Phuong T.
Dow AgroSciences LLC
Kraus Eric J.
Ludwig Kenneth B.
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