Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1999-08-17
2002-04-16
Wang, Andrew (Department: 1635)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S295000, C800S300100, C435S006120, C435S091100, C435S320100, C435S419000, C435S468000, C536S023100
Reexamination Certificate
active
06372961
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to the genetic manipulation of plants, particularly to the expression of hemoglobin genes in transformed plants.
BACKGROUND OF THE INVENTION
Despite the common perception of hemoglobin as a blood protein, the protein is also found in many invertebrates, bacteria, fungi, and in higher plants. In fact, it exists widely in a variety of organisms. Hemoglobin and hemoglobin genes have been discovered in both leguminous and nonleguminous plants. Most notably, it is found in dicots engaged in symbiotic relationships with bacteria.
Hemoglobin is characterized by its conserved structure, high oxygen affinity, and reversible contamination with oxygen in the ferrous state. Its function is normally associated with the facilitation of oxygen diffusion, oxygen storage, and oxygen utilization in organisms.
Leghemoglobins are genetically heterogenous monomeric heme proteins found in root nodules, specialized structures that form after symbiotic infection of leguminous root cells by nitrogen-fixing Rhizobium bacteria. This plant-encoded hemoglobin assists in this symbiotic relationship by binding oxygen and facilitating its diffusion through the root nodule to the respiring bacteroids, whose nitrogen fixation consumes large amounts of energy. In this manner, oxygen for bacterial respiration is provided at a partial pressure low enough to prevent oxidative damage to the nitrogenase complex, which is readily poisoned by oxygen.
Leghemoglobin genes have been characterized in many legumes, particularly soybean. Expression of these genes in root nodules results in abundant leghemoglobin to facilitate oxygen diffusion. The amino acid sequences of leghemoglobins differ from those of vertebrate globin gene products at about 80% of the positions. However, leghemoglobin folds into the same three dimensional structure as the animal globins.
Hemoglobin genes from several nonleguminous plants have also been characterized. The significance of the presence of hemoglobin products in nonnodulating plants is still unresolved. Studies have shown that in such dicots, hemoglobin occurs mainly in the roots at a concentration of approximately 100 nM, which is likely lower than the concentration of predissolved oxygen in the cells.
Plants not only make oxygen during photosynthesis, but they use it for respiration through the electron transfer chain in mitochondria. Studies suggest that they use hemoglobin to bind and transfer that oxygen. The supply of oxygen to plant cells can be an important factor limiting metabolic activity and hence the potential productivity of crop plants. Therefore, it would be beneficial to manipulate oxygen levels in a plant.
SUMMARY OF THE INVENTION
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to maize hemoglobin. It is an object of the present invention to provide a method for enhancing seed germination and seedling growth.
It is a further object of the present invention to provide methods for improving plant response to flood conditions.
It is a further object of the present invention to provide methods for manipulating oxygen concentration in the plant cell and organelles contributing to the overall growth and metabolism of the plant.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of: a) a nucleotide sequence encoding a maize hemoglobin; b) a nucleotide sequence encoding the amino acid sequence of SEQ ID NOs: 2 or 4; c) a nucleotide sequence set forth in SEQ ID NOs: 1 or 3; d) a nucleotide sequence comprising at least 14 or from 15 to 20, or from 21 to 25 contiguous nucleotides of SEQ ID NOs: 1 or 3; e) a polynucleotide having at least 60% sequence identity to SEQ ID NOS: 1, and 3 wherein the % sequence identity is based on the entire sequence and is determined by GAP version 10 using default parameters; f) nucleotide sequences that hybridize to the nucleotide sequences of a), b), c), d) or e) under stringent conditions; and g) a polynucleotide complementary to a polynucleotide of (a) through (f). The isolated nucleic acid can be DNA.
In another aspect, the present invention relates to recombinant expression cassettes, comprising a nucleic acid as described, supra, operably linked to a promoter. In some embodiments, the nucleic acid is operably linked in antisense orientation to the promoter.
In another aspect, the present invention is directed to a host cell transfected with the recombinant expression cassette as described, supra. In some embodiments, the host cell is a corn, soybean, wheat, rice, alfalfa, barley, sunflower, sorghum, canola, rye, safflower or cotton cell.
In a further aspect, the present invention relates to an isolated protein comprising a polypeptide having at least 6 contiguous amino acids encoded by the isolated nucleic acid referred to, supra.
In another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide of at least 14, or from 15 to 20, or from 21 to 25 contiguous nucleotides which selectively hybridizes under stringent conditions to a nucleic acid of the present invention, or a complement thereof. In some embodiments, the isolated nucleic acid is operably linked to a promoter.
In yet another aspect, the present invention relates to an isolated nucleic acid comprising a polynucleotide, the polynucleotide having 80% sequence identity to an identical length of a nucleic acid of the present invention or a complement thereof.
In an additional aspect, the present invention is directed to an isolated nucleic acid comprising a polynucleotide encoding a polypeptide wherein: (a) a polypeptide comprising at least 6, or from 7 to 10, or from 11 to 15 contiguous amino acids of SEQ ID NOS: 2, and 4; (b) a polypeptide which is a maize hemoglobin; (c) a polypeptide comprising at least 60% sequence similarity to SEQ ID NOS: 2, and 4, wherein the % sequence similarity is based on the entire sequence and is determined by GAP analysis using Gap Weight of 12 and Length Weight of 4; (d) a polypeptide encoded by a nucleic acid of claim
1
; and (e) a polypeptide encoded by a nucleic acid of SEQ ID NOS: 1, and 3.
In yet another aspect, the present invention relates to a transgenic plant comprising a recombinant expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention. In some embodiments, the transgenic plant is Zea mays. The present invention also provides transgenic seed from the transgenic plant.
In a further aspect, the present invention relates to a method of modulating expression of the genes encoding the hemoglobin proteins of the present invention in a plant, comprising the steps of (a) transforming a plant cell with a recombinant expression cassette comprising a plant promoter operably linked to any of the isolated nucleic acids of the present invention; (b) growing the plant cell under plant growing conditions to produce a regenerated plant; and (c) expressing the polynucleotide for a time sufficient to modulate hemoglobin in the plant. Expression of the genes encoding the hemoglobin proteins of the present invention can be increased or decreased relative to a non-transformed control plant.
Compositions and methods for expressing hemoglobin genes in plants, plant cells, and plant tissues are provided. The compositions comprise nucleotide sequences encoding monocot, particularly maize, hemoglobin genes. The sequences are useful in transforming plants for tissue-specific or constitutive expression of hemoglobin.
Expression cassettes comprising the hemoglobin sequences of the invention are provided. Additionally provided are transformed plant cells, plant tissues, and plants.
DETAILED DESCRIPTION OF THE INVENTION
The present invention is drawn to compositions and methods for expressing hemoglobin genes in plants, plant cells, and specific plant tissues. Compositions are nucleic acids and proteins relating to hemoglobin or hemoglobin-like genes in plants. More particularly, nucleo
Shen Bo
Tarczynski Mitchell C.
Lacourciere Karen A
Pioneer Hi-Bred International , Inc.
Pioneer Hi-Bred International Inc.
Wang Andrew
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