Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters plant part growth
Reexamination Certificate
1999-06-08
2001-05-29
Benzion, Gary (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters plant part growth
C800S295000, C800S278000, C536S023600, C536S024100
Reexamination Certificate
active
06239332
ABSTRACT:
BACKGROUND OF THE INVENTION
All photosynthetic organisms depend on the light-harvesting reactions of photosynthesis for energy to produce important compounds for growth and metabolism. Energy-rich carbohydrates, fatty acids, sugars, essential amino acids, and other compounds synthesized by photosynthetic organisms are the basis of the food chain on which all animal life depends for existence. Photosynthetic organisms are also the major source of oxygen evolution in the atmosphere, recycling carbon dioxide in the process. Thus life on earth is reliant on the productivity of photosynthetic organisms, especially plants.
Plant productivity is limited by the amount of resources available and the ability of plants to harness these resources. The conversion of light to chemical energy requires a complex system which combines the light harvesting apparatus of pigments and proteins. The value of light energy to the plant can only be realized when it is efficiently converted into chemical energy by photosynthesis and fed into various biochemical processes.
The thylakoid protein apparatus responsible for the photosynthetic conversion of light to chemical energy is one of the most complex mechanisms in the chloroplast and remains one of the most difficult biological systems to study. Oxygen-evolving photosynthetic organisms, such as cyanobacteria, algae and plants, possess two photosystems, PSI and PSII, which cooperate in series to acquire electrons from H
2
O and deliver them energetically up a gradient to NADP
+
. The photosynthetic production of NADPH and ATP then, in turn, feeds into all biochemical pathways. The force driving the uphill flow of these electrons comes from the light energy absorbed by the 100-300 chlorophyll molecules associated with the two photosystems. An important pair of chlorophyll a molecules in the center of each photosystem modulates the movement of electrons. The remaining chlorophyll molecules are associated with proteins which in turn are organized into light gathering antennae that surround the reaction centers and transfer the light energy to them (Green et al. (1991)
TIBS
16:181).
The capacity to absorb light, especially in shade, depends largely on the size and organization of the light harvesting complexes (Lhc) in the thylakoid membranes. The LhcII light harvesting complex is the major ensemble of chlorophyll a/b binding protein (Cab) acting as an antenna to photosystem II (PSII) and plays a key role in harvesting light for photosynthesis (Kuhlbrandt, W. (1984)
Nature
307:478). Plants are capable of adjusting the size of the antennae in accordance with the light intensity available for growth. In shade, the allocation of nitrogen is shifted from polypeptides in the stroma, by decreasing ribulose 1,5-bisphosphate carboxylase (Rbc or Rubisco) levels, to the thylakoidal proteins. Nitrogen redistribution is a compensating response to low irradiance, balancing light harvesting and CO
2
fixation (Evans, J. R. (1989)
Oecologia
78:9); Stitt, M. (1991)
Plant, Cell and Environment
14:741).
In addition to the shift in the investment of nitrogen into different proteins, photosynthetic organisms can adapt to low light conditions by molecular reorganization of the light harvesting complexes (Chow et al. (1990)
Proc. Natl. Acad. Sci. USA
87:7502; Horton et al. (1994)
Plant Physiol.
106:415; Melis, (1991)
Biochim. Biophys. Acta.
1058:87). A plant's reorganizational ability to compensate for changes in the characteristics of the light limits its productivity. Although a mechanism is in place to adapt to low light conditions, photosynthesis in plants grown in suboptimal illumination remains significantly lower due to a limited capacity to generate ATP and NADPH via electron transport (Dietz, K. J. and Heber, U. (1984)
Biochim. Biophys. Acta.
767:432; ibid (1986) 848:392). Under such conditions the capacity to generate ATP and NADPH, the assimilatory force, will dictate the capacity to reduce CO
2
. When light is limiting, plants reorganize to maximize their photosynthetic capacity; however, the ability to adapt is limited by molecular parameters ranging from gene expression to complex assembly to substrate and cofactor availability.
If productivity of a plant or other photosynthetic organism is to be increased, methods to enhance the light-gathering capacity without restricting CO
2
fixation must be developed.
SUMMARY OF THE INVENTION
The present invention provides a chimeric gene construct comprising a promoter region, a 5′ untranslated region containing a translational enhancer, DNA encoding a plastid-specific transit peptide which enhances protein import, a gene encoding a plastid protein, and a 3′ untranslated region containing a functional polyadenylation signal. This construct produces a high level of expression and importation of the functional protein to the site of its function.
In one embodiment of the present invention the promoter is a 35S cauliflower mosaic virus (CaMV) promoter. In another embodiment, the translational enhancer is from the 5′ untranslated region of the pea small subunit of ribulose-1,5-bisphosphate carboxylase. In another embodiment, the transit peptide is from the pea small subunit of ribulose-1,5-bisphosphate carboxylase. In a further embodiment, the gene encoding a protein is the pea cab gene, encoding a chlorophyll a/b binding protein. In yet another embodiment, the 3′ untranslated region containing a functional polyadenylation signal is from the pea cab gene.
This invention also provides a method for enhancing the light harvesting capability of a photosynthetic plant or organism comprising: preparing a gene construct comprising a promoter, a 5′ untranslated region containing a translational enhancer, DNA encoding a plastid-specific transit peptide which enhances protein import, DNA encoding a protein, preferably a structural gene encoding a chlorophyll a/b binding protein, and a 3′ untranslated region containing a functional polyadenylation signal; inserting the gene construct into a suitable cloning vector; and transforming a photosynthetic plant or other photosynthetic organism with the recombinant vector. Alternatively, the gene construct is coated directly on biolistic particles with which the cells are bombarded.
This invention provides a DNA construct which can increase the amount of one or more proteins in a plastid, especially a chloroplast, or in the cells of photosynthetic prokaryotes. These constructs can alter the photosynthetic apparatus to increase the ability of the plant to harvest light, especially under conditions of low illumination.
This invention also provides methods of increasing the light-harvesting efficiency of photosynthesis and the yield of photosynthetic products (such as carbohydrates) in plants and other photosynthetic organisms. These methods can be used to increase the commercial value of plants and seeds, and be used to increase the yields of products produced from fermentation and plant tissue culture operations.
This invention also provides a transgenic (TR) plant or photosynthetic organism containing the construct described above. These transgenic plants and photosynthetic organisms have enhanced photosynthetic capacity and enhanced growth capabilities useful for increased yield, tissue culture, fermentation and regeneration purposes. Compared to wild-type (WT) plants, transgenic plants of this invention demonstrate increased yield, enhanced pigmentation, increased carbohydrate content, increased biomass, more uniform growth, larger seeds or fruits, increased stem girth, enhanced photosynthesis, faster germination, and increased ability to withstand transplant shock. Seeds produced from these plants are also provided by this invention, as well as plant parts useful for production of regenerated plants and other derived products.
REFERENCES:
patent: 4400471 (1983-08-01), Johal
patent: 4940835 (1990-07-01), Shah et al.
patent: 5185253 (1993-02-01), Tumer
patent: 5187267 (1993-02-01), Comai et al.
patent: 5254799 (1993-10-01), DeGrev
Granell Antonio
Ko Kenton
Ko Zdenka W.
Labate Carlos A.
Benzion Gary
Miernicki Steeg Carol
Pearlmutter Nina L.
Queen's University at Kingston
Scribner Stephen J.
LandOfFree
Constructs and methods for enhancing protein levels in... does not yet have a rating. At this time, there are no reviews or comments for this patent.
If you have personal experience with Constructs and methods for enhancing protein levels in..., we encourage you to share that experience with our LandOfFree.com community. Your opinion is very important and Constructs and methods for enhancing protein levels in... will most certainly appreciate the feedback.
Profile ID: LFUS-PAI-O-2506713