Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or...
Reexamination Certificate
2000-04-28
2003-09-23
McElwain, Elizabeth F. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
C800S298000, C800S306000, C800S312000, C800S317200, C800S317400, C800S320000, C800S320100, C800S320200, C800S320300, C435S419000
Reexamination Certificate
active
06624342
ABSTRACT:
The present invention relates to novel nucleic acid sequences, which code for a geranylgeranyl reductase, a method for producing novel plants, which contain a novel nucleic acid sequence and the tocopherol and/or chlorophyll content of which is altered in comparison with wild-type plants, these novel plants, parts and products thereof and plant cells as well as the use of the nucleic acid sequences for the manipulation of tocopherol, chlorophyll and/or vitamin K
1
content in transgenic plants, parts and products thereof and plant cells.
The diterpene geranylgeranyl pyrophosphate (GGPP) is formed as C
20
-intermediate in the plant isoprenoid metabolism. It results from the addition of one unit isopentenyl pyrophosphate (IPP) to farnesyl pyrophosphate, a C
15
-sesquiterpene. GGPP enters several synthesis pathways of plant secondary metabolism. For example, two molecules of GGPP can be joined “tail to tail” to give C
40
-bodies, the tetraterpenes, generally called carotenoides and to which, for example, the &bgr;-carotene belongs. By the addition of further molecules of IPP, GGPP furthermore enters the biosynthesis of polyterpenes, such as rubber and guttapercha.
Further, GGPP can be transformed into other diterpenes, such as phytyl pyrophosphate (PPP). The C
20
-body phytol is an obligatory intermediate in the biosynthesis of the tocopherols (Soll and Schulz (1981) Biochem. Biophys. Res. Commun. 99, 907-912) as well as the synthesis of the chlorophylls (Beale and Weinstein (1990) in: Biosynthesis of Heme and Chlorophyll (Daily H. A., ed.) McGraw Hill, N.Y., 287-391). While the basic structure of all chlorophylls (chlorophyll a, b, c, etc.) is a porphyrin system consisting of four pyrrole rings, to which system the phytol is bound by an ester-like bond through the pyrrole ring IV, the tocopherols are characterised by a structure consisting of homogentisate and a phytol tail.
The group of tocopherols, commonly described as vitamin E, comprises several structurally closely related lipophilic vitamins, viz &agr;-, &bgr;-, &ggr;-, &dgr;- und &egr;-tocopherol, &agr;-tocopherol being the most important in biological terms. The tocopherols can be found in many plant oils, specially rich in tocopherols are the seed oils of soybean, wheat, maize, rice, cotton, lucerne and nuts. Also fruits and vegetables, e.g. raspberries, beans, peas, fennel, pepper etc. contain tocophrols. As far as presently known, tocopherols are exclusively synthesized in plants and photosynthetically active organisms.
Due to their redox potential tocopherols contribute to avoid oxidation of unsaturated fatty acids by air oxygen; &agr;-tocopherol is the most important lipophilic antioxidant in human. It is assumed that due to their function as antioxidative agents the tocopherols contribute to the stabilisation of biological membranes, because the fluidity of the membranes is maintained by the protection of the unsaturated fatty acids of the membrane lipids. Moreover, according to recent observations, regular uptake of relatively high tocopherol doses can counteract the development of arteriosclerosis. Further positive physiological properties and influences of tocopherols have been described, such as delay of late damages associated with diabetes, reduction of the risk of cataract development, reduction of oxidative stress in smokers, anticarcinogenic effects, protective effects against skin damages such as erythremes and skin aging etc.
Due to their oxidation inhibiting properties the tocopherols are not only used in food technology applications, but also employed in paintings based on natural oils, in deodorants and other cosmetics, such as sun protection agents, skin care agents, lip sticks etc. In such applications tocopherol compounds like tocopheryl acetate and succinate are usual application forms for the use as vitamin E, in circulation promoting and lipid reducing agents and as food additive in veterinary applications.
In the biosynthesis of tocopherols, in particular in the biosynthesis of &agr;-tocopherol, phytyl pyrophosphate is believed to be the limiting factor. Previous studies indicate that PPP is formed from GGPP by sequential hydrogenation of the isoprenoid group, during which reaction dihydro-GGPP and tetrahydro-GGPP are formed as intermediates (GGPP->dihydro-GGPP->tetrahydro-GGPP->PPP; cf., for example, Bollivar et al. (1994) Biochemistry 33, 12763-12768).
The stepwise hydrogenation of GGPP to PPP is, as presently assumed, catalyzed by the enzyme geranylgeranyl reductase (GGPP reductase, also called geranylgeranyl pyrophosphate hydrogenase and GGPP hydrogenase), which is coded in plants in the gene Chl P. The enzyme geranylgeranyl reductase belongs to the isoprenoid metabolism and functions for two metabolic pathways: the tocoperol biosynthesis and the chlorophyll biosynthesis.
The essential role of this enzyme has been shown for the first time for the biosynthesis of chlorophyll (Benz et al. (1980) Plant Sci. Lett. 19, 225-230; Soll and Schultz (1981) Biochem. Biophys. Res. Commun. 99, 907-912; Schoch et al. (1977) Z. Pflanzenphysiol. 83, 427-436). The final step in chlorophyll biosynthesis is the esterification of chlorophyllide, which may take place with phytyl pyrophosphate as well as with geranylgeranyl pyrophosphate. In systematic studies of
Rhodobacter capsulatus
mutants it could be demonstrated that bacteriochlorophyllide is esterified with GGPP in a first step and that subsequently esterified chlorophyll-GG is hydrogenated (Katz et al. (1972) J. Am. Chem. Soc. 94, 7938-7939). In higher plants, phytyl chlorophyll (chlorophyll-P) can be found for the most part (Rüdiger and Schoch (1991) In: Chlorophylls (Scheer, H., Ed.) pp. 451-464, CRC Press, Boca Raton, Fla., USA). So far it has not been elucidated yet, which substrates are involved in the reductase reaction in plants. Presently, it is assumed that the plant enyzme geranylgeranyl reductase is able to transform chlorophyll-GG into chlorophyll-P (Schoch et al. (1978) Z. Pflanzenphysiol. 83, 427-436) as well as to hydrogenate GGPP to PPP, which is then subsequently joined to chlorophyllide (Soll et al. (1983) Plant Physiol. 71, 849-854).
GGPP serves as the substrate for the synthesis pathways of tocopherol and phyllochinone in the chloroplast outer membranes and for chlorophyll synthesis in the thylakoid membranes. The reduction of GGPP to PPP has been described for the first time 1983 by Soll et al. (Plant. Physiol. (1983) 71, 849-854). However, until now the isolation and characterisation of nuclic acid sequences, which code for the plant enzyme and which can be used for the manipulation of tocopherol content in transgenic plants, was unsuccessful.
The essential role of geranylgeranyl reductase in tocopherol and chlorophyll metabolism makes this enzyme a particular valuable instrument for molecular biotechnology. By means of molecular biological techniques such as the transfer of DNA sequences coding for geranylgeranyl reductase, it should be possible to achieve alterations in tocopherol and/or chlorophyll biosynthesis performance in plants. By this way it would, for example, become possible to produce transgenic plants having an increased or reduced tocopherol content. Such transgenic plants and parts, cells and/or products thereof could subsequently be used as food and feed and in general as production center for tocopherol, for use in chemical, pharmaceutical and cosmetic industrial applications.
Further, there is reason to expect that plants which exhibit an increased content of antioxidative tocopherols, in comparison with wild-type plants, also show increased tolerance against stress conditions, in particular against oxidative stress.
It is therefore an object of the invention to provide new nucleic acid sequences, with the help of which the content of tocopherol can be manipulated in plants, plant cells, plant parts and/or plant products.
Further it is an important object of the invention to provide transgenic plants, plant cells, plant products and plant parts having an altered tocopherol content compared to wild-typ
Grimm Bernhard
Tanaka Ryouichi
Collins Cynthia
Institut fur Pflanzengenetik und Kulturpflanzenforschung
McElwain Elizabeth F.
Schwegman Lundberg Woessner & Kluth P.A.
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