Multicellular living organisms and unmodified parts thereof and – Plant – seedling – plant seed – or plant part – per se – Higher plant – seedling – plant seed – or plant part
Reexamination Certificate
2001-10-05
2004-07-06
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Plant, seedling, plant seed, or plant part, per se
Higher plant, seedling, plant seed, or plant part
C800S274000, C800S287000, C800S288000, C800S306000, C800S314000, C800S320100, C800S320200, C800S320300, C435S199000, C536S023700
Reexamination Certificate
active
06759575
ABSTRACT:
The present invention relates to an improved barstar gene and improved barstar protein that can be used to neutralize the activity of a barnase in eucaryotic cells, particularly in plant cells. Thus the improved barstar gene can be used to produce fertility restorer plants capable of restoring the fertility to a line of male-sterile plants that contain in the nuclear genome of their cells a chimeric gene comprising a stamen-selective promoter and a DNA coding for a barnase. The present invention also relates to the restorer plants that contain in the nuclear genome of their cells the improved barstar gene.
BACKGROUND TO THE INVENTION
In many, if not most plant species, the development of hybrid cultivars is highly desired because of their generally increased productivity due to heterosis: the superiority of performance of hybrid individuals compared with their parents (see e.g. Fehr, 1987, Principles of cultivar development, Volume 1: Theory and Technique, MacMillan Publishing Company, New York; Allard, 1960, Principles of Plant Breeding, John Wiley and Sons, Inc.).
The development of hybrid cultivars of various plant species depends upon the capability to achieve almost complete cross-pollination between parents. This is most simply achieved by rendering one of the parent lines male sterile (i.e. bringing them in a condition so that pollen is absent or nonfunctional) either manually, by removing the anthers, or genetically by using, in the one parent, cytoplasmic or nuclear genes that prevent anther and/or pollen development (for a review of the genetics of male sterility in plants see Kaul, 1988, ‘Male Sterility in Higher Plants’, Springer Veriag).
For hybrid plants where the seed is the harvested product (e.g. corn, oilseed rape) it is in most cases also necessary to ensure that fertility of the hybrid plants is fully restored. In systems in which the male sterility is under genetic control this requires the existence and use of genes that can restore male fertility. The development of hybrid cultivars is mainly dependent on the availability of suitable and effective sterility and restorer genes.
Endogenous nuclear loci are known for most plant species that may contain genotypes which affect male sterility, and generally, such loci need to be homozygous for particular recessive alleles in order to result in a male-sterile phenotype. The presence of a dominant ‘male fertile’ allele at such loci results in male fertility.
Recently it has been shown that male sterility can be induced in a plant by providing the genome of the plant with a chimeric male-sterility gene comprising a DNA sequence (or male-sterility DNA) coding, for example, for a cytotoxic product (such as an RNase) and under the control of a promoter which is predominantly active in selected cells of the male reproductive organs. In this regard stamen-selective promoters, such as the promoter of the TA29 gene of
Nicotiana tabacum
, have been shown to be particularly useful for this purpose (Mariani et al., 1990, Nature 347:737, European patent publication (“EP”) 0,344,029). By providing the nuclear genome of the plant with such a male-sterility gene, an artificial male-sterility locus is created containing the artificial male-sterility genotype that results in a male-sterile plant.
In addition it has been shown that male fertility can be restored to the plant with a chimeric fertility-restorer gene comprising another DNA sequence (or fertility-restorer DNA) that codes, for example, for a protein that inhibits the activity of the cytotoxic product or otherwise prevents the cytotoxic product to be active in the plant cells (European patent publication “EP” 0,412,911). For example the barnase gene of
Bacillus amyloliquefaciens
codes for an RNase, the barnase, which can be inhibited by a protein, the barstar, that is encoded by the barstar gene of
B. amyloliquefaciens
. The barnase gene can be used for the construction of a sterility gene while the barstar gene can be used for the construction of a fertility-restorer gene. Experiments in different plant species, e.g. oilseed rape, have shown that a chimeric barstar gene can fully restore the male fertility of male sterile lines in which the male sterility was due to the presence of a chimeric barnase gene (EP 0,412,911, Mariani et al., 1991, Proceedings of the CCIRC Rapeseed Congress, Jul. 9-11, 1991, Saskatoon, Saskatchewan, Canada; Mariani et al., 1992, Nature 357:384). By coupling a marker gene, such as a dominant herbicide resistance gene (for example the bar gene coding for phosphinothricin acetyl transferase (PAT) that converts the herbicidal phosphinothricin to a non-toxic compound [De Block et al., 1987, EMBO J. 6:2513]), to the chimeric male-sterility and/or fertility-restorer gene, breeding systems can be implemented to select for uniform populations of male sterile plants (EP 0,344,029; EP 0,412,911).
The production of hybrid seed of any particular cultivar of a plant species requires the: 1) maintenance of small quantities of pure seed of each inbred parent, and 2) the preparation of larger quantities of seed of each inbred parent. Such larger quantities of seed would normally be obtained by several (usually two) seed multiplication rounds, starting from a small quantity of pure seed (“basic seed”) and leading, in each multiplication round, to a larger quantity of pure seed of the inbred parent and then finally to a stock of seed of the inbred parent (the “parent seed” or “foundation seed”) which is of sufficient quantity to be planted to produce the desired quantities of hybrid seed. Of course, in each seed multiplication round larger planting areas (fields) are required.
Barnase is the ribonuclease which is secreted by
Bacillus amyloliquefaciens
and barstar is the inhibitor of barnase that is produced by the same microorganism (Hartley, 1988, J. Mol. Biol. 202:913-915). Several mutant barnase and barstar proteins have been described (Hartley, 1993, Biochemistry 32:5978-5984; Schreiber and Fersht, 1993, Biochemistry 32:5145-5150; Guillet et al, 1993, Current Biology 1:165-177; Hartley, 1989, TIBS 14:450-454; Axe et al, 1996, PNAS 93:5590-5594; Serrano, 1993, J. Mol. Biol. 233:305-312).
Some of these mutants were shown to essentially retain the biological activity of the barnase and barstar as produced by
Bacillus amyloliquefaciens
. However, at least two mutant barstars have been described that have no detectable barstar activity (Hartley, 1993, Biochemistry 32:5978-5984; Guillet et al, 1993, Current Biology 1:165-177).
Also other related microorganisms are known to produce proteins that are highly similar to barnase and barstar. Thus
Bacillus intermedius
produces binase and binstar (Schulga et al, 1992, NAR 20:2375; Guillet et al, 1993, supra).
SUMMARY OF THE INVENTION
The present invention provides improved barstar DNAs that encode a barstar with an amino acid sequence that starts with Met-Xaa wherein Xaa is Alanine, Valine, Glycine, Aspartic acid or Glutamic acid. Preferably the barstar DNAs encode barstar having an amino acid sequence which is 1) the amino acid sequence of SEQ ID No 2 in which the second amino acid is not Lysine but is Alanine, Valine, Glycine, Aspartic acid or Glutamic acid; 2) the amino acid sequence of SEQ ID No 4; or the amino acid sequence of SEQ ID No 4 in which the second amino acid is not Alanine, but is Valine, Glycine, Aspartic acid or Glutamic acid.
The present invention further provides improved synthetic barstar DNAs that contain less than 40% A and T nucleotides and/or that have a codon usage that is optimized for oilseed rape, cotton, maize, rice and wheat, preferably for oilseed rape, maize and rice. Preferably the synthetic barstar DNAs contain no more than 7% CG dinucleotides and/or no more than 9.5% of CNG trinucleotides. A preferred synthetic barstar DNA encodes a barstar having the amino acid sequence of SEQ ID No. 4. A particularly preferred synthetic barstar DNA has the nucleotide sequence of SEQ ID No 3.
The present invention also provides: chimeric genes in which the improved barstar DNAs a
Michiels Frank
Williams Mark
Bayer Bioscience N.V.
Fox David T.
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