Multicellular living organisms and unmodified parts thereof and – Method of introducing a polynucleotide molecule into or... – The polynucleotide alters pigment production in the plant
Reexamination Certificate
2001-10-29
2004-07-20
Fox, David T. (Department: 1638)
Multicellular living organisms and unmodified parts thereof and
Method of introducing a polynucleotide molecule into or...
The polynucleotide alters pigment production in the plant
C800S278000, C800S292000, C800S293000, C800S294000, C800S298000, C536S023100, C536S023200, C536S023600
Reexamination Certificate
active
06765129
ABSTRACT:
The invention relates to a DNA encoding a polypeptide with 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXPRI) activity which originates from plants. In addition, the invention relates to the use of DNA sequences encoding a polypeptide with DXPRI activity which originates from plants for the generation of plants with an elevated tocopherol, carotenoid, vitamin K, chlorophyll and polyterpene content, specifically to the use of the DNA sequence SEQ ID No. 1 or of DNA sequences hybridizing herewith, to a method for the generation of plants with an elevated tocopherol, carotenoid, vitamin K, chlorophyll and polyterpene content, and to the resulting plant itself.
The generation of plants with an elevated sugar, enzyme and amino acid content has hitherto been an important objective in plant molecular genetics. The development of plants with an elevated vitamin content, such as, for example, an elevated tocopherol content, is, however, also of economic interest.
The naturally occurring eight compounds with vitamin E activity are derivatives of 6-chromanol (Ullmann's Encyclopedia of Industrial Chemistry, Vol. A 27 (1996), VCH Verlagsgesellschaft, Chapter 4., 478-488, Vitamin E). The first group (1a-d) is derived from tocopherol, while the second group is composed of tocotrienol derivatives (2a-d):
1a, &agr;-tocopherol: R
1
═R
2
═R
3
=CH
3
1b, &bgr;-tocopherol [148-03-8]: R
1
═R
3
=CH
3
, R
2
=H
1c, &ggr;-tocopherol [54-28-4]: R
1
=H, R
2
═R
3
=CH
3
1d, &dgr;-tocopherol [119-13-1]: R
1
═R
2
=H, R
3
=CH
3
2a, &agr;-tocotrienol [1721-51-3]: R
1
═R
2
═R
3
=CH
3
2b, &bgr;-tocotrienol [490-23-3]: R
1
═R
3
=CH
3
, R
2
=H
2c, &ggr;-tocotrienol [14101-61-2]: R
1
=H, R
2
═R
3
=CH
3
2d, &dgr;-tocotrienol [25612-59-3]: R
1
═R
2
=H, R
3
=CH
3
&agr;-Tocopherol has great economic importance.
The development of crop plants with an elevated tocopherol, vitamin K, carotenoid, chlorophyll and polyterpene content by means of tissue culture or seed mutagenesis and natural selection is set a limit. Thus, on the one hand, it must be possible to appraise, for example, the tocopherol content, or content of the desired catabolite, as early as in the tissue culture stage, and, on the other hand, only those plants whose regeneration from cell cultures to entire plants is successful can be manipulated by tissue culture techniques. Also, crop plants may show undesirable characteristics after mutagenesis and selection, and these characteristics must be reeliminated by, in some cases repeated, back crosses. Also, for example, the increase in tocopherol content would be restricted to crosses between plants of the same species.
This is why the genetic engineering approach of isolating essential biosynthesis genes which encode, for example, tocopherol synthesis performance and introducing them into crop plants in a directed fashion is superior to the traditional breeding method. Knowledge of the biosynthesis and its regulation, and identification of genes which affect biosynthesis performance, are prerequisites for this method.
Isoprenoids or terpenoids are composed of a variety of classes of lipid-soluble molecules, and they are formed partially or exclusively from C
5
-isoprene units. Pure prenyl lipids (for example carotenoids) are composed of C skeletons based exclusively on isoprene units, while mixed prenyl lipids (for example chlorophylls, tocopherols and vitamin K), have an isoprenoid side chain linked to an aromatic nucleus.
The biosynthesis of prenyl lipids starts with 3×acetyl-CoA units which are converted into the starting isoprene unit (C
5
), namely isopentenyl pyrophosphate (IPP), via &bgr;-hydroxymethylglutaryl-CoA (HMG-CoA) and mevalonate. Recent C
13
in vivo feeding experiments have demonstrated that the IPP formation pathway in various eubacteria, green algae and plant chloroplasts is mevalonate-independent (FIG.
1
). In this pathway, hydroxyethylthiamine, which is formed by decarboxylation of pyruvate, and glycerolaldehyde-3-phosphate (3-GAP) are first converted into 1-deoxy-D-xylulose-5-phosphate in a “transketolase” reaction mediated by 1-deoxy-D-xylulose-5-phosphate synthase (DOXS) (Lange et al., 1998; Schwender et al., 1997; Arigoni et al., 1997; Lichtenthaler et al., 1997; Sprenger et al., 1997). In an intramolecular rearrangement reaction, this 1-deoxy-D-xylulose-5-phosphate is converted by DXPRI into 2-C-methyl-D-erythritol-4-phosphate and then into IPP (Arigoni et al., 1997; Zeidler et al., 1998). Biochemical data suggest that the mevalonate pathway operates in the cytosol and leads to the formation of phytosterols. The antibiotic mevinolin, a specific mevalonate formation inhibitor, only leads to sterol biosynthesis inhibition in the cytoplasma, while prenyl lipid formation in the plastids remains unaffected (Bach and Lichtenthaler, 1993). In contrast, the mevalonate-independent pathway is located in the plastids and leads predominantly to the formation of carotenoids and plastid prenyl lipids (Schwender et al., 1997; Arigoni et al., 1997).
IPP is in equilibrium with its isomer, dimethylallyl pyrophosphate (DMAPP). Condensation of IPP with DMAPP head to tail results in the monoterpene (C
10
) geranylpyrophosphate (GPP). Addition of further IPP units results in the sesquiterpene (C
15
) farnesyl pyrophosphate (FPP), and to the diterpene (C
20
) geranylgeranyl pyrophosphate (GGPP). Bonding between two GGPP molecules results in the formation of the C
40
precursors of carotenoids.
In the case of mixed prenyl lipids, the isoprene side chain, whose length varies, is linked to non-isoprene rings such as, for example, a porphyrine ring in the case of chlorophylls a and b. The chlorophylls and phylloquinones contain a C
20
phytyl chain, in which only the first isoprene unit contains a double bond. GGPP is converted by geranylgeranyl pyrophosphate oxidoreductase (GGPPOR) to give phytyl pyrophosphate (PPP), the starting material for the subsequent formation of tocopherols.
The ring structures of the mixed prenyl lipids which lead to the formation of vitamins E and K are quinones whose starting metabolites are derived from the shikimate pathway. The aromatic amino acids phenylalanine or tyrosine are converted into hydroxyphenyl pyruvate, which is dioxygenated to give homogentisic acid. The chorismate is formed, on the one hand, via erythrose-4-phosphate, 3′-dehydroquinate, 3′-dehydroshikimate, shikimate, shikimate-3-phosphate and 5′-enolpyruvylshikimate-3-phosphate (FIG.
1
). In this process, fructose-6-phosphate and glycerolaldehyde-3-phosphate are reacted to give xylulose-5-phosphate and erythrose-4-phosphate. The above-described homogentisic acid is subsequently bonded to PPP to form the precursor of &agr;-tocopherol and &agr;-tocoquinone, namely 2-methyl-6-phytylquinol. Methylation steps with S-adenosylmethionine as methyl group donor lead first to 2,3-dimethyl-6-phytylquinol, subsequent cyclization leads to &ggr;-tocopherol and further methylation to &agr;-tocopherol (Richter, Biochemie der Pflanzen [Plant biochemistry], Georg Thieme Verlag Stuttgart, 1996).
Examples which demonstrate that manipulation of an enzyme may directionally affect metabolite flow can be found in the literature. A direct effect on the quantities of carotenoids in these transgenic tomato plants was measured in experiments on an altered expression of phytoene synthase, which links two GGPP molecules to give 15-cis-phytoene (Fray and Grierson, Plant Mol. Biol. 22(4), 589-602 (1993); Fray et al., Plant J., 8, 693-701 (1995)). As expected, transgenic tobacco plants which have reduced quantities of phenylalanine-ammonium lyase show reduced quantities of phenylpropanoid. The enzyme phenylalanine-ammonium lyase catalyzes the degradation of phenylalanine and thus withdraws it from phenylpropanoid biosynthesis (Bate et al., Proc. Natl. Acad. Sci USA 91 (16): (1994
Herbers Karin
Lichtenthaler Hartmut
Reindl Andreas
Schwender Jörg
BASF - Aktiengesellschaft
Fox David T.
Kallis Russell
Keil & Weinkauf
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