Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide confers resistance to heat or cold
Reexamination Certificate
1999-11-08
2002-12-31
Bui, Phuong T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide confers resistance to heat or cold
C800S278000, C800S298000, C800S295000, C536S023100, C536S024100, C536S023600, C435S069100, C435S320100, C435S419000, C435S468000
Reexamination Certificate
active
06501006
ABSTRACT:
FIELD OF THE INVENTION
This invention relates to plant genetic engineering and plant breeding. In particular, it relates to methods of modulating chilling tolerance in plants
BACKGROUND OF THE INVENTION
The prior art lacks means for enhancing seed emergence in cool soils by expressing of genes associated with endomembrane integrity. The present invention addresses these and other needs.
Tropical crops such as cotton, maize, and cowpea, are sensitive to chilling soil temperatures often encountered during early sowing in subtropical regions in spring. Early spring sowing can be beneficial in the subtropics because it results in a longer growing season and higher yields. However, early sown seeds that are subjected to chilling temperatures suffer reductions in rate of emergence and maximal emergence. Variation in chilling sensitivity at germination has been found among genotypes of warm season annuals such as cowpea (Ismail et al.,
Crop Sci
. 37:1270-1277 (1997)), soybean (Ismail et al.
Crop Sci
. 37:1270-1277 (1997)), cotton (Christiansen, M. N. & Lewis, C. F.,
Crop Sci
. 13:210-212 (1973)) and maize (Cal, J. P. & Obendorf, R. L.,
Crop Sci
. 12:369-373 (1972)). Numerous studies have suggested positive associations between the extent of electrolyte leakage from seeds and pre-emergence mortality of embryos at chilling temperature for chilling sensitive crops (Bramlage et al.,
Plant Physiol
. 61:525-529 (1978); Leopold, A. C.,
Plant Physiol
. 65:1096-1098 (1980)).
Soluble sugars (Koster, K. L., & Leopold, A. C.,
Plant Physiol
. 88:829-832 (1988); Chen, Y., & Burris, J. S.,
Crop Sci
. 30:971-975 (1990)) and proteins, typically LEA (late embryogenesis-abundant) (Blackman et al.,
Physiol. Plant
. 93:630-638 (1995); Close, T. J.,
Physiol. Plant
. 97:795-803 (1996); Close, T. J.,
Physiol. Plant
. 100:291-296 (1997); Ingram, J. & Bartels, D.,
Annu. Rev. Plant Physiol. Plant Mol. Biol
. 47:377-403 (1996)) are known to accumulate during seed development and are thought to play a role in protecting the embryo during desiccation. Studies with soybean indicated that accumulation of LEA proteins during embryogenesis might reduce the extent of desiccation-induced electrolyte leakage in immature seeds suggesting a role in membrane protection (Blackman et al.,
Physiol. Plant
. 93:630-638 (1995)). Dehydrins (DHN, LEA D11 family) are among the most commonly observed proteins induced by environmental stress associated with dehydration or low temperature (Close, T. J.,
Physiol. Plant
. 97:795-803 (1996); Close, T. J.,
Physiol. Plant
. 100:291-296 (1997)). Distinct subclasses of DHNs have been noted (Close, T. J.,
Physiol. Plant
. 100:291-296 (1997)). Several lines of evidence suggested a role of DHNs in membrane interactions and/or protein stabilization (Ismail et al.,
Crop Sci
. 37:1270-1277 (1997); Close, T. J.,
Physiol. Plant
. 100:291-296 (1997); Egerton-Warburton et al.,
Physiol. Plant
. 101:545-555 (1997); Danyluk et al.,
Plant Cell
. 10:623-638 (1998)).
In cowpea, two closely related lines (F
6
siblings) were found to vary in maximal emergence under chilling field conditions (Ismail et al.,
Crop Sci
. 37:1270-1277 (1997)), but also in other characters (Ismail, A. M. & Hall, A. E.,
Crop Sci
. 38:381-390 (1998)). Dry, mature seeds of the chilling-tolerant line, 1393-2-11, were found to contain a substantial quantity (estimated to be about 1% of total soluble protein) of a ~35-kDa DHN protein that was not detectable in the seeds of the genetically similar line, 1393-2-1. Also the chilling-tolerant line had slower electrolyte leakage from its seeds during imbibition at low temperature. Based on studies with reciprocal hybrids, the chilling tolerance of 1393-2-11 was hypothesized to be due to additive and independent effects of the DHN under dominant nuclear inheritance and a maternal effect associated with slower electrolyte leakage from seeds during imbibition compared with line 1393-2-1 (Ismail et al.,
Crop Sci
. 37:1270-1277 (1997)).
Despite these advances, the prior does not provide nucleic acids useful for conferring chilling tolerance on seedlings during and after emergence in soil. The present invention addresses these and other needs.
SUMMARY OF THE INVENTION
The present invention provides isolated nucleic acid molecules comprising a polynucleotide sequence encoding a DHN polypeptide that enhances chilling tolerance in plants. The proteins comprise an amino acid sequence as shown in SEQ ID NO:2. The nucleic acid encoding the protein preferably has a polynucleotide sequence that specifically hybridizes to SEQ ID NO: 1. The invention also provides recombinant expression cassettes comprising the polynucleotide sequences of the invention. The expression cassettes typically comprise a seed-specific promoter or promoter from the allele described here.
The invention also provides transgenic plants comprising a recombinant expression cassette comprising a promoter operably linked to the polynucleotide sequence. The transgenic plants of the invention can be, for example, cowpea. In addition, marker assisted selection as described here can be used to identify related genes and alleles in other plants. Using these markers one of skill can use conventional breeding techniques to confer chilling tolerance to a variety of plant species. Such methods are particularly useful in other members of the legume family, such as soybean.
DEFINITIONS
The term “DHN polypeptide” refers to polypeptides having at least substantial identity to SEQ ID NO: 2 and that confer chilling tolerance on plant seedlings. As discussed in more detail below, DHN polypeptides of the invention, and the genes that encode them, are distinct from other allelic variants in a number of ways. Among the differences is the presence of one additional &PHgr;-segment (&PHgr;
7
). The general structure of these proteins, and in particular &PHgr;-segments, is discussed in detail in Close, T. J.,
Physiol. Plant
. 100:291-296 (1997).
The phrase “nucleic acid sequence” refers to a single or double-stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5′ to the 3′ end. It includes chromosomal DNA, self-replicating plasmids, infectious polymers of DNA or RNA and DNA or RNA that performs a primarily structural role.
The term “promoter” refers to regions or sequence located upstream and/or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells. Such a promoter can be derived from plant genes or from other organisms, such as viruses capable of infecting plant cells.
The term “plant” includes whole plants, shoot vegetative organs/structures (e.g. leaves, stems and tubers), roots, flowers and floral organs/structures (e.g. bracts, sepals, petals, stamens, carpels, anthers and ovules), seed (including embryo, endosperm, and seed coat) and fruit (the mature ovary), plant tissue (e.g. vascular tissue, ground tissue, and the like) and cells (e.g. guard cells, egg cells, trichomes and the like), and progeny of same. The class of plants that can be used in the method of the invention is generally as broad as the class of higher and lower plants amenable to transformation techniques, including angiosperms (monocotyledonous and dicotyledonous plants), gymnosperms, ferns, and multicellular algae. It includes plants of a variety of ploidy levels, including aneuploid, polyploid, diploid, haploid and hemizygous.
A polynucleotide sequence is “heterologous to” an organism or a second polynucleotide sequence if it originates from a foreign species, or, if from the same species, is modified from its original form. For example, a promoter operably linked to a heterologous coding sequence refers to a coding sequence from a species different from that from which the promoter was derived, or, if from the same species, a coding sequence which is not naturally associated with the promoter (e.g. a genetically engineered coding sequence
Close Timothy J.
Hall Anthony E.
Ismail Abdelbagi M.
Bui Phuong T.
Ibrahim Medina A..
The Regents of the University of California
Townsend & Townsend & Crew LLP
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