Promoters for gene expression in caryopses of plants

Details Promoters for gene expression in caryopses of plants Promoters for gene expression in caryopses of plants

2001-07-05

2004-09-21

Mehta, Ashwin D. (Department: 1638)

Multicellular living organisms and unmodified parts thereof and

Method of introducing a polynucleotide molecule into or...

The polynucleotide contains a tissue, organ, or cell...

C800S278000, C800S298000, C435S252300, C435S254110, C435S320100, C435S419000, C536S023100, C536S024100

Reexamination Certificate

active

06794559

ABSTRACT:

The present invention relates to promoters which permit a caryopsis-specific expression or suppression of genes in genetically modified plants, to methods for the tissue-specific gene expression or gene suppression in plants, expression cassettes, recombinant vectors and host cells containing such promoters, to transgenic plant cells and plants transformed with said promoters, and to methods for generating such plant cells and plants.
Prior-art documents whose disclosure is herewith incorporated into the present application by reference are cited hereinbelow.
The application of plants whose genetic material has been modified with the aid of genetic engineering methods has proved advantageous in many fields of agriculture in order to transfer certain characteristics to crop plants. The predominant aims are firstly crop protection, and secondly improved quality and yield of the plants or products which can be harvested.
A large number of methods for genetically modifying dicotyledonous and monocotyledonous plants are known (cf., inter alia, Gasser and Fraley, Science 244 (1989), 1293-1299; Potrykus, Ann. Rev. Plant Mol. Biol. Plant Physiol. 42 (1991), 205-225; Newell, Mol. Biotechnol. 16(1), (2000), 53-65). They are frequently based on the transfer of gene constructs which, in most cases, constitute combinations of specific coding regions of structural genes with promoter regions and transcription terminators of the same or other (for example heterologous) structural genes.
In connection with the expression of structural genes, providing promoters is of great importance for generating transgenic plants, since the specificity of a promoter is decisive for the point in time at which, the tissue types in which, the physiological conditions under which and the intensity with which a transferred gene is expressed in the modified plant.
To succeed with these various approaches for the genetic manipulation of plants, it is therefore necessary to place genes to be regulated differently under the control of suitable promoters.
Transcriptional initiation and regulation is subject to the DNA segment of a gene termed promoter. As a rule, promoter sequences are in the 5′-flanking region of a transcribed gene. Individual elements of a promoter (for example transcriptional enhancers) can also be located in the 3′-flanking region or within intron sequences (Kuhlemeier, Plant Mol. Biol. 19 (1992): 1-14; Luehrsen, The Maize Handbook, 636-638) (1994).
The controlled expression of transgenes is very useful, for example for introducing resistance properties or the modification of metabolic processes in plants. If a transgene or its gene product is to engage into defined metabolic pathways of a plant, for example if it is to produce a new constituent or to protect from attack by pathogens, its spatially and/or temporarily controlled expression is only possible when an inducible and/or tissue- and/or development-specific promoter is used. Only this makes possible the specific production of desired constituents at a defined developmental stage or within a certain tissue of the plant. For example, when applying antisense technology, where the expression of plant-homologous genes is to be prevented, the use of tissue- and/or development-specific promoters is advantageous over a tissue- and/or developmental-independent expression when, for example, the antisense effect occurs precisely at the developmental stage, or precisely in the tissue, of the plant at which, or in which, the plant-homologous gene is also expressed.
A large number of promoters capable of governing the expression of transferred genes or structural genes in plants is already known. The most frequently used promoter is the 35S CaMV promoter (Franck et al., Cell 1 (1980), 285-294), which leads to constitutive expression of the gene introduced.
Frequently, inducible promoters are also employed, for example for wound induction (DE-A-3843628), chemical induction (Ward et al., Plant Molec. Biol. 22 (1993), 361-366) or light induction (Fluhr et al., Science 232 (1986), 1106-1112).
Under certain circumstances, the use of the frequently described constitutive promoters (e.g. 35 S) entails certain disadvantages. Promoters which bring about a constitutive expression of the genes controlled by them can be employed, for example, for generating herbicide-tolerant and pathogen-resistant plants, but have the disadvantage that the products of the genes controlled by them are present in all parts of the plant, which may be undesirable, for example when the plants are intended for consumption. A negative aspect of tissue- and/or development-independent expression of a transgene can also be an undesired effect on plant development. The use of inducible promoters likewise entails disadvantages, since the induction conditions are typically difficult to control in the open in the case of agricultural plants.
The use of cell- and tissue-specific promoters has also been described: stomata-specific gene expression (DE-A-4207358), seed-, tuber- and fruit-specific gene expression (reviewed in Edwards and Coruzzi, Annu. Rev. Genet. 24 (1990), 275-303; DE-A-3843627), phloem-specific gene expression (Schmülling et al., Plant Cell 1 (1989), 665-670), root-nodule-specific gene expression (DE-A-3702497) or meristem-specific gene expression (Ito et al., Plant Mol. Biol. 24 (1994), 863-878).
A limited number of promoters which regulate gene expression in the caryopsis are known as yet. The management of certain approaches in the genetic modification of plants therefore requires the provision of alternative promoter systems for gene expression in the caryopsis whose regulation differs from that of the known systems.
Starch biosynthesis genes whose gene products are expressed specifically in the storage tissue of the caryopsis, but not in vegetative tissues, have been isolated from various plant species, for example the relevant genes or cDNA clones of GBSS I. They include the waxy locus from maize (Klögen et al. (1986) Mol. Gen. Genet. 203: 237-244), and barley (Rohde et al. (1988) Nucleic Acid Research 16, 14: 7185-7186), rice (Wang et al. (1990) Nucleic Acid Research 18: 5898), potato (van der Leij et al. (1991) Mol. Gen. Genet. 228: 240-248), pea (Dry et al. (1992) Plant J. 2: 193-202), cassava (Salehuzzaman et al. (1993) Plant Mol. Biol. 20: 947-962), millet (Hsingh et al. (1995) EMBL Database Acc.No. U23954) and sugar beet (Schneider et al. (1999) Mol. Gen. Genet. 262: 515-524).
The gene products which are expressed specifically in the caryopsis also include type II starch synthase (SSII). Corresponding genes were isolated from maize (zSSIIa and zSSIIb; Ham et al. (1998) Plant Mol. Biol. 37: 639-649), pea (Dry et al. (1992) Plant J. 2: 193-202), potato (Edwards et al. (1995) Plant J. 8: 283-294) and sweet potato (Ham et al. (1998) Acc. Nr. AF068834).
The situation for wheat is as follows: cDNA clones both for the waxy gene and for the SSII gee were isolated and sequenced. In total, 3 different GBSSI cDNA clones were isolated from wheat (Clark et al. (1991) Plant Mol. Biol. 16: 1099-1101; Ainsworth et al. (1993) Plant Mol. Biol. 22: 67-82 (Block (1997) “Isolierung, Charakterisierung und Expressionsanalysen von Stärkesynthase-Genen aus Weizen” [Isolation, characterization and expression analyses of wheat starch synthase genes] (
Triticum aestivum
L.), PhD thesis, University of Hamburg).
In addition, coding sequences of a type II starch synthase (SSII) have also been isolated from a caryopsis-specific cDNA library, and their caryopsis-specific expression has been detected. Northern analyses have demonstrated that the transcripts of GBSS I (Block (1997), PhD thesis, University of Hamburg, School of Biology) and of SSII (Walter (2000), PhD thesis, University of Hamburg, School of Biology), WO 97/45545, EMBL Database Acc. No. U66377) are found during early developmental stages of the caryopsis, but not in assimilating leaf tissue. In addition, transcripts were also demonstrated in the endosperm and the pericarp for SSII.
Three cDNA sequences of the w

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