Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
1998-01-08
2001-10-30
Hutzell, Paula K. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C435S069100, C435S468000, C435S410000, C435S419000, C536S023100, C536S023600, C800S285000, C800S290000, C800S295000, C800S306000, C800S312000, C800S314000, C800S317200, C800S317300, C800S317400, C800S320000, C800S320100, C800S320200, C800S320300, C800S322000
Reexamination Certificate
active
06310271
ABSTRACT:
FIELD OF THE INVENTION
BACKGROUND OF THE INVENTION
While the world population is constantly increasing, the need for food to feed this growing population directly increases as well. One unfortunate side effect correlated with increased populations is the rapid dwindling of available crop producing farm land. For instance, land in certain countries of Africa and Asia once marginally able to support food crops only decades ago is now completely arid and infertile, making crop production impossible. Unless drastic measures are taken, this situation is only going to worsen with growing populations.
In an effort to better current conditions and to stave off any exacerbation of the problem, much interest has been directed toward ways in which to optimize food production in harsh, barren conditions. The principle is that land currently unable to support crops might be utilized for cultivation. Current research involves looking at the defense mechanisms plants naturally use to survive in stressful environmental conditions and to determine whether these natural mechanisms can be exploited artificially, e.g., by cross-breeding or gene transformation, thereby producing more robust crops. One such defense mechanism which displays promise is organic solute accumulation.
When bacteria, marine algae, and many higher plants are exposed to salinity or drought, they accumulate organic solutes. These solutes include polyols, proline, and quaternary ammonium compounds. They are thought to confer stress tolerance to the organism by balancing the osmotic pressures between the outside and the inside of their cells, thus enabling them to maintain turgor and growth. Unsurprisingly, the biosynthetic pathways for these osmoprotective compounds have become targets for metabolic engineering to improve the stress tolerance of a target species. To date, there have been preliminary studies that have shown these pathways can be genetically manipulated in higher plants and that this can improve tolerance to various abiotic stresses (Tarczynski et al., 1993; Kishor et al., 1995; Lilius et al., 1996; Hayashi et al., 1997).
There is evidence to suggest that the quaternary ammonium compound glycine betaine may be a more effective osmoprotectant than polyols or proline (Mackay et al., 1984; Warr et al., 1988). Further, glycine betaine is more attractive as a potential genetic engineering target because, unlike proline or polyols, glycine betaine has no subsequent metabolic fate. Thus, in principle, this makes it simpler to engineer glycine betaine accumulation because its rate of degradation is not a concern.
In plants, as in bacteria, glycine betaine is synthesized by a two-step oxidation of choline. The first step (oxidation of choline to betaine aldehyde) is catalyzed by choline monooxygenase (CMO). The second step (oxidation of betaine aldehyde to glycine betaine) is catalyzed by betaine aldehyde dehydrogenase (BADH).
Certain higher plants, e.g., spinach and sugar beet, accumulate glycine betaine in response to osmotic stress. But many other species including tomato, tobacco, potato, legumes, rice, and some cultivars of corn and sorghum lack an ability to synthesize it. Metabolic engineering of glycine betaine synthesis in these crops could therefore improve their stress tolerance. Although bacterial choline oxidases (Rozwadowski et al., 1991; Hayashi et al., 1997) or dehydrogenases (Lilius et al., 1996) are being explored for this purpose, use of CMO (in conjunction with BADH) is preferable for the following reason. CMO requires for its function reduced ferredoxin from the light reactions of photosynthesis. Thus, CMO links glycine betaine synthesis with the light reactions of photosynthesis. This helps to match the supply of glycine betaine with the demand for osmotic adjustment and osmoprotection, which climbs rapidly after sunrise as the water potential and water content of salt- or drought-stressed leaves start falling (Hanson and Hitz, 1982).
In some circumstances, however, the synthesis of glycine betaine is an unwanted occurrence. For instance, in sugar manufacturing from beet, glycine betaine is one component of sugarbeets which complicates processing of sugar by inhibiting the crystallization of sugar. Hence sugar beet cultivars with no or reduced levels of glycine betaine in the roots will be desirable.
It has been suggested that glycine betaine accumulation may make plants susceptible to insect pests (Corcuera, 1993) or microbial pathogens (Pearce et al., 1976). Hence, under certain circumstances, it may be possible to improve a plant's resistance to pests or pathogens by blocking the synthesis of glycine betaine. The potential for such an application is available for many important crops that naturally accumulate glycine betaine, for example, wheat, barley, corn, sugarcane, sugar beet, spinach, cotton and sunflower.
Blocking CMO in crop species used as animal feed may also improve their nutritional value. Choline is a frequent animal feed supplement, and therefore cells which contain a higher concentration of choline by virtue of blocking its conversion into glycine betaine would be desirable. Accordingly, a process of genetically altering plants to prevent them from producing glycine betaine would be very beneficial to many agriculturally related industries.
To date, the gene coding for the enzyme responsible for oxidizing betaine aldehyde to glycine betaine, BADH, has been cloned and has been successfully expressed in transformed tobacco. In contrast, the gene encoding CMO is to date unknown and, accordingly, there has been no means to genetically engineer plants using the CMO gene. Since BADH and CMO are both required for glycine betaine production, the singular transformation of BADH without CMO is useless for increasing stress resistance, as glycine betaine is not produced. Also, when blocking of glycine betaine synthesis is desired it is more useful to block CMO than BADH. A block at the step catalyzed by BADH can cause accumulation of betaine aldehyde resulting from the oxidation of choline by CMO. This may inhibit plant growth and productivity because betaine aldehyde is a toxic metabolite and a structural analog of amino aldehyde intermediates of polyamine catabolism.
For the foregoing reasons, there is a need for a means to isolate a gene encoding a CMO-resemblant enzyme, and ideally to identify the sequence of a gene encoding a CMO-resemblant enzyme. As a corollary, there is a need for a purified CMO-resemblant enzyme. There is a need for a method to increase or decrease the glycine betaine concentration of plants. Still further, there is a need for a method to genetically engineer organisms to increase their resistance to stressful conditions.
SUMMARY OF THE INVENTION
The present invention is directed to a method of increasing the resistance of plants to harsh environmental conditions. Specifically exemplified is a method which involves the transformation of plants with a gene encoding a protein having choline monooxygenase activity. Although any method of transformation can be used, in a preferred embodiment of such a method, an
Agrobacterium tumefaciens
host cell is transformed with a vector containing a DNA sequence encoding choline monooxygenase and cultured; the cultured
A. tumefaciens
cell is then used to transform a plant cell according to procedures well known in the art. As a specific example to illustrate the teachings herein it is disclosed that tobacco, which does not express a CMO-type enzyme, has been successfully transformed through the procedures of the present invention, to display CMO activity. Transgenic tobacco expressing chimeric genes for BADH and CMO synthesizes glycine betaine. This aspect of the invention increases the transformed plant's resistance to stressful conditions by increasing the concentration of glycine betaine in tissues.
Another aspect of the present invention relates to an isolated DNA sequence encoding an enzyme having choline monooxygenase characteristics. An example of a DNA sequence according to the present invention is displayed
Burnet Michael
Hanson Andrew D.
Rathinasabapathi Bala
Hutzell Paula K.
Saliwanchik Lloyd & Saliwanchik
University of Florida Research Foundation Inc.
Zaghmout Ousama
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