MXPA00012340A - Enzyme - Google Patents
EnzymeInfo
- Publication number
- MXPA00012340A MXPA00012340A MXPA/A/2000/012340A MXPA00012340A MXPA00012340A MX PA00012340 A MXPA00012340 A MX PA00012340A MX PA00012340 A MXPA00012340 A MX PA00012340A MX PA00012340 A MXPA00012340 A MX PA00012340A
- Authority
- MX
- Mexico
- Prior art keywords
- nucleic acid
- acid sequence
- gibberellin
- leu
- oxidase
- Prior art date
Links
- 108090000790 Enzymes Proteins 0.000 title claims description 57
- 102000004190 Enzymes Human genes 0.000 title claims description 57
- 229920001850 Nucleic acid sequence Polymers 0.000 claims abstract description 72
- 150000007523 nucleic acids Chemical group 0.000 claims abstract description 64
- 101710044974 GA2OX1 Proteins 0.000 claims abstract description 60
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- 239000003448 gibberellin Substances 0.000 claims abstract description 16
- 238000007254 oxidation reaction Methods 0.000 claims abstract description 11
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims abstract description 10
- 230000003647 oxidation Effects 0.000 claims abstract description 7
- 238000002360 preparation method Methods 0.000 claims abstract description 7
- 241000196324 Embryophyta Species 0.000 claims description 138
- 230000014509 gene expression Effects 0.000 claims description 39
- 229920003013 deoxyribonucleic acid Polymers 0.000 claims description 34
- 240000005799 Phaseolus coccineus Species 0.000 claims description 27
- 230000000694 effects Effects 0.000 claims description 24
- 150000001413 amino acids Chemical class 0.000 claims description 17
- 239000000463 material Substances 0.000 claims description 15
- 235000010632 Phaseolus coccineus Nutrition 0.000 claims description 11
- 229920001405 Coding region Polymers 0.000 claims description 10
- 230000000295 complement Effects 0.000 claims description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N acetone Chemical class CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 8
- 108020004707 nucleic acids Proteins 0.000 claims description 8
- 230000002829 reduced Effects 0.000 claims description 6
- 229920001184 polypeptide Polymers 0.000 claims description 4
- 125000003275 alpha amino acid group Chemical group 0.000 claims 4
- 229910052709 silver Inorganic materials 0.000 claims 1
- 239000004332 silver Substances 0.000 claims 1
- 150000002576 ketones Chemical class 0.000 abstract 1
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- JKHXYJKMNSSFFL-IUCAKERBSA-N Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(O)=O)CCCCN JKHXYJKMNSSFFL-IUCAKERBSA-N 0.000 description 7
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- YSWHPLCDIMUKFE-QWRGUYRKSA-N Glu-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 YSWHPLCDIMUKFE-QWRGUYRKSA-N 0.000 description 6
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- RDIKFPRVLJLMER-BQBZGAKWSA-N Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)N RDIKFPRVLJLMER-BQBZGAKWSA-N 0.000 description 5
- HXWUJJADFMXNKA-UHFFFAOYSA-N Asparaginyl-Leucine Chemical compound CC(C)CC(C(O)=O)NC(=O)C(N)CC(N)=O HXWUJJADFMXNKA-UHFFFAOYSA-N 0.000 description 5
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- SENJXOPIZNYLHU-IUCAKERBSA-N Leu-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-IUCAKERBSA-N 0.000 description 5
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Abstract
A nucleic acid sequence is provided which encodes a gibberellin 2-oxidase gene which catalyses the 2&bgr;-oxidation of a gibberellin molecule to introduce a hydroxyl group at C-2 and further catalyses the oxidation of the hydroxyl group introduced at C-2 to yield the ketone derivative. Such sequences can find application in the preparation of transgenic plants with altered levels of gebberellin 2-oxidase.
Description
ENZIMA The present invention relates to a novel enzyme comprised in the control of plant growth, coding of DNA sequences for the enzyme and uses of encoding nucleotide sequences for the enzyme in the production of transgenic plants with improved growth characteristics. or altered. Gibberes (GAs) are a large group of diterpenoid carboxylic acids that occur in all higher plants and some fungi. Certain members of the group function as plant hormones and are understood in various experimental processes, including seed germination, stem extension, leaf enlargement, development and initiation of flowering, and growth of seeds and fruit. The biologically active GAs are usually C? 9 compounds which contain a 1-9-1 0 lactone, a C-7 carboxylic acid and a 3? -hydroxyl group. The delayed periods of its biosynthesis comprise the oxidative removal of C-20 and hydroxylation at C-3. Hydroxylation at the 2-β position results in the production of biologically inactive products. This reaction is the most important path for the metabolism of GA in plants and ensures that active hormones do not accumulate in plant tissues. The enzymes 7-oxidase, 20-oxidase, 3-β-hydroxylase and biosynthetic 2ß-hydroxylase of GA, are all dioxygenases dependent on 2-oxoglutarate. These are a large group of enzymes for which 2-oxoglutarate is a co-substrate that is decarboxylated for succinate as part of the reaction (see review by Hedden, P. and Kamiya, Y., in Annu, Rev. Plant Physiol. Plant Mol. Biol. 48 431-460 (1 997)). The chemical regulators of plant growth have been used in horticulture and agriculture for many years. Many of these compounds work by changing the concentration of GA in plant tissues. For example, growth retardants inhibit the activity of the enzymes comprised in GA biosynthesis and thereby reduce the GA content. Such chemicals are commonly used, for example, to prevent lodging in cereals and to control the growth of ornamental and horticultural plants. Conversely, GAs can be applied to plants, such as in the application of GA3 to seedless grapes to improve the size and shape of the strawberry, and to barley wheat to improve the production of fermented barley. The mixtures of GA4 and GA7 are applied to apples to improve fruit quality and certain conifers to stimulate pineapple production. There are several problems associated with the use of growth regulators. Some of the growth retardants are highly persistent in the field, making it difficult to develop other crops after a treated crop. Others require repeated applications to maintain the required effect. It is difficult to restrict the application to target plant organs without spreading to other organs or plants and having undesirable effects. The precise choice of objectives of the application of growth regulator can vary the intensive work. A non-chemical option to control the morphology of the plant is, in this way, highly desirable. Development seeds often contain high concentrations of GAs and relatively large amounts of GA-biosynthetic enzymes. The mature seeds of the tendril bean (Phaseolus coccineus) contain extremely high concentrations of 2ß-hydroxy GA, GA8, as well as their glucoside, indicating that high levels of 2ß-hydroxylase activity should be present. This has been confirmed by the related species Phaseolus vulgaris in which there is a rapid increase in GA 2ß-hydroxylase activity shortly before the seeds reach full maturity (Albone er al., Planta 177 1 08-1 1 5 ( 1 989)). The 2 ß-hydroxylases have been partially purified from the cotyledons of Pisum sativum (Smith, V. A. and MacMillan, J. , Plant 167 9-18 (1983)) and Phaseolus vulgaris (Griggs er al. Phytochemistry 30 2507-2512 (1 991) and Smith, VA and MacMillan, J., J. Plant Growth Regul. 2 251-264 (1984) ). These studies show that there is evidence that, for both sources, at least two enzymes with different substrate specificities are presented. Two cotyledon activities of absorbed P. vulgaris were separated by chromatography of cation exchange and gel filtration. The main activity corresponds to an enzyme of Mr 26,000 by size exclusion by HPLC, GAT and GA4 hydroxylated, in preference to GA9 and GA20, while GA9 was the preferred substrate for the second enzyme (Mr 42,000). However, attempts to purify the enzyme activity to obtain N-terminal information for the amino acid sequence have proved impossible because of the low abundance of the enzyme in plant tissues relative to other proteins and the co-purification of a lectin. contaminant with enzyme activity yielding N-terminal amino acid sequence impossible. The regulation of gibberellin deactivation has been examined in Pisum sativum (orchard granite) using the no (slender) mutation as reported in Ross et al (The Plant Journal 7 (3) 51 3-523 (1995)). The mutation does not block the deactivation of GA20, which is the precursor of the GA .. bioactive. The results of these studies indicate that the gene without can be a regulatory gene that controls the expression of two separate structural genes involved in the deactivation of GA, especially the oxidation of GA20 to GA29 by 2ß-hydroxylation to C-2 followed by the further oxidation of the hydroxyl group to an acetone (GA29 to GA29-catabolite). The conversion of GA29 to GA29-catabolh or in granule seeds was inhibited by prohexadione-calcium, an inhibitor of 2-oxoglutarate-dependent dioxygenases (Nakayama et al Plant Cell Physiol. 31 1 1 83-1 1 90 (1990)) , indicating that the reaction was catalyzed by an enzyme of this type. In spite of the slender (without) mutation in granules it was found to block both, the conversion of GA20 to GA29 and from GA2g to GA29-catabolite in seeds, the inability of unlabeled GA20 to inhibit the oxidation of radiolabeled GA29, and vice versa, He indicated that the stages were catalyzed by separate enzymes. Furthermore, in sperm tissues, the slender mutation inhibits the 2ß-hydroxylation of GA20, but not the formation of GA29-catabolite. These observations lead to the theory that there were two separate enzymes comprised in the metabolic pathway controlling the deactivation of GA in plants (Hedden, P. and Kamiya, Y., in Annu, Rev. Plant Physiol. Plant Mol. Biol. 48 431 -460 (1 997)). However, it has now been surprisingly found that a simple enzyme can, in fact, catabolize these different reactions. The present invention represents the first reported cloning of a cDNA encoding a GA 2ß-hydroxylase that acts on C? 9-GAs and for which, 2? -hydroxylation has only its hydroxylase activity. A cDNA clone of pumpkin seed encodes an enzyme that has both 2β- and 3β-hydroxylase activities (Lange et al., Plant Cell 9 1459-1467 (1 997)), but its main activity is 3β-hydroxylation and acts as a 2-hydroxylase only with tricarboxylic acid substrates (C20); no 2β-hydroxylate C1 9-GAs. Since the new enzyme of the present invention catalyzes both the β-hydroxylation and the further oxidation of the hydroxyl group substituted to an acetone group at C-2, the enzyme has been named a "GA 2-oxidase". According to a first aspect of the present invention there is provided an isolated, purified or recombinant nucleic acid sequence encoding a gibberellin 2-oxidase enzyme comprising a nucleic acid sequence as shown in Figure 1 or a functional derivative of it, or its complementary filament or a sequence homologous to it. A naming system for GA-biosynthesis genes has now been presented (Coles er to The Plant Journal 17 (5) 547-556 (1999). References in the present application to the gibberellin 2-oxidase gene of Phaseolus coccineus should The references in the present application to the gibberellin 2-oxidase genes of Arabidopsis thaliana such as at-2bt3, at-2bt24 and T31 E 1 0.1 1 should also be understood as referring to AtGA2ox1, AtGA2ox2 and AtGA2ox3 respectively The nucleic acid sequences of the present invention, which encode a gibberellin 2-oxidase (GA 2-oxidase), are 2-oxoglutarate-dependent dioxygenases that introduce a hydroxyl group to C-2β in GAs, particularly C? 9-GAs, including bioactive GAs such as GAT and GA4.They also oxidize the 2ß-hydroxylated GAs further to give GA-catabolites, which have a function of acetone at C-2 The lactone bridge of these catabolites can also be of opening to produce a C-1 9 carboxylic acid and a double bond to C-1 0. The activity of the 2-oxidases results in the inactivation of bioactive GAs or in the conversion of active GAs biosynthetic precursors to products that do not they can become bioactive forms. A preferred nucleic acid sequence of the present invention, therefore, encodes a gibberellin 2-oxidase enzyme capable of oxidizing C1-gibberellin compounds by the introduction of a hydroxyl group into C-2β. The enzyme can oxidize the 2ß-hydroxyl group to an acetone group. The preferred gibberellin 2-oxidase substrates of the present invention are GA9, GA4. In the context of the present invention, the degree of identity between the amino acid sequences can be at least 40%, suitably 50% or higher, for example 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95%. At a nucleotide level, the degree of identity can be at least 50%, suitably 60% or higher, for example 65%, 70%, 75%, 80%, 85%, 90% or 95%. A homologous sequence according to the present invention may therefore have a sequence identity as described above. Sequence homology can be determined using any conveniently available protocol, for example, using Clustal X ™ from the University of Strasbourg and identity tables produced using Genedoc ™ (Karl B. Nicholas). Also included within the scope of the present invention are nucleic acid sequences, which hybridize to a sequence according to the first aspect of the invention under stringent conditions, or a nucleic acid sequence, which is homologous to or hybridized under stringent conditions to such a sequence but for the degeneration of the genetic code, or an oligonucleotide sequence specific for any sequence. The stringent conditions of hybridization can be characterized by low salt concentrations or high temperature conditions. For example, highly stringent conditions can be defined as being hybridization to DNA binding to a solid support in 0.5M NaHPO4, 7% sodium dodecyl sulfate (SDS), 1 mm EDTA at 65 ° C, and washing in 0.1 xSSC /0.1% SDS at 68 ° C (Ausubel et al., "Current Protocols in Molecular Biology" 1, page 2.1 0.3, published by Green Publishing Associates, Inc. and John Wiiey & amp;; Sons, Inc., New York, (1989)). In some circumstances, less stringent conditions may be required. As used in the present application, moderately stringent conditions can be defined as comprising washing in 0.2xSSC / 0. 1% SDS at 42 ° C (Ausubel et al (1988) supra). Hybridization can also be made more stringent by the addition of increased amounts of formamide to destabilize the double nucleic acid hybrid. In this way, the particular conditions of hybridization can be easily handled, and will generally be selected according to the desired results. In general, suitable hybridization temperatures in the presence of 50% formamide are 42 ° C for a probe, which is 95 to 1 00% homologous to the target DNA, 37 ° C for 90 to 95% homology, and 32 ° C for 70 to 90% homology. An example of a preferred nucleic acid sequence of the present invention is one that encodes an enzyme, which has the activity of a gibberellin 2-oxidase enzyme from Phaseolus coccineus (for example PcGA 2ox1) or an equivalent protein from another member of the Fabaceae family. A nucleic acid sequence of the present invention can also encode a gibberellin 2-oxidase enzyme from Phaseolus vulgaris or from Arabidopsis thaliana (for example AtGA2ox1, AtGA2ox2 or AtGAox3). Other nucleic acid sequences according to this aspect of the present invention can also comprise a nucleic acid sequence as previously defined, in which the coding sequence is operatively linked to a promoter. The promoter can be constitutive and / or specific for expression in a particular plant cell or tissue, for example in roots using tobacco RB7 (Yamamoto eí al Plant Cell 3 371-382 (1991)); in green tissues using rbcS-3A tomato (Ueda e al Plant Cell 1 217-227 (1989)); dividing cells using corn histone H3 (Brignon et al Plant Mol. Biol. 22 1007-1015 (1993)), or Arabidopsis CYC07 (Ito e al Plant Mol. Biol. 24863-878 (1994)); in vegetable meristem using Arabidopsis KNAT1 (Lincoln eí to Plant Cell 6 1859-1876 (1994)); in vascular tissue using GRP1.8 bean (Keller, B., &Heierli, D., Plant Mol. Biol. 26 747-756 (1994)); in flower using Arabidopsis ACT11 (Huang ei al Plant Mol. Biol. 33 125-139 (1997)) or chalcone synthase of perunia (Vandermeer ei al Plant Mol. Biol. 15 95-109 (1990)); in pistil using potato SK2 (Ficker eí al Plant Mol. Biol. 35 425-431 (1997)); in anther using Brassica TA29 (Deblock, M., &Debrouwer, D., Plant 189 218-225 (1993)); in fruit using tomato polygalacturonase (Bird eí al Plant Mol. Biol. 11 651-662 (1988)). Alternative promoters can be derived from the viruses of
. A ~ &gw - - plant, for example the 35S promoter Cauliflower mosaic virus (CaMV). Suitable promoter sequences can include promoter sequences from plant species, for example from the family Brassicaceae. The present invention therefore also extends to a recombinant, purified, or isolated nucleic acid sequence comprising a promoter, which naturally reduces the expression of a gene encoding a gibberellin 2-oxidase enzyme comprising a sequence of nucleic acid as shown in Figure 1 or a functional derivative thereof, or its complementary filament, or a homologous sequence thereof. The gibberellin 2-oxidase enzyme can be from Phaseolus coccineus (for example PcGA2ox1) or an equivalent protein from another member of the Fabaceae family. Such nucleic acid sequences can also encode a gibberellin 2-oxidase enzyme from P. vulgaris or A. Thaliana (for example AtGA2ox1, AtGA2ox2 or AtGA2ox3). Preferably, the nucleic acid sequence comprises a promoter, which drives the expression of a gibberellin 2-oxidase enzyme from P. coccineus, P. vulgaris or A. Thaliana. Such promoter sequences include promoters, which occur naturally 5 'to the coding sequence of the sequence shown in Figure 1. The promoters can also be selected for constitutively expressing nucleic acid coding for the gibberellin 2-oxidase gene. Promoters that are induced by internal or external factors, such as chemicals, plant hormones, light or tension could be used. Examples are genes related to pathogenesis capable of being induced by salicylic acid, copper controllable gene expression (Mett ei al Proc. Nat'l Acad. Sci USA 90 4567-4571 (1 993)) and gene expression. regulated by tetracycline (Gatz eí al Plant Journal 2 397-404 (1992)). Examples of genes capable of being induced by gibberellin are? -TIP (Phillips, AL, & Useful, AK, Plant Mol. Biol. 24 603-61 5 (1 994)) and GAST (Jacobsen, S. E., &Olszewski, N. E., Plant 198 78-86 (1 996)). The gibberellin 20-oxidase genes are down-regulated by GA (Phillips et al Plant Physiol. 108 1 049-1 057 (1 995)) and their promoter coupled to the GA 2-oxidase ORF can also find application in this aspect of the invention The gibberellin 2-oxidase enzymes encoded for the nucleic acid sequences of the present invention can suitably act to catalyze the 2β-oxidation of a Cig-gibberellin molecule to introduce a hydroxyl group into C-2 followed by further oxidation to produce the derivative of acetone. The nucleic acid sequences of the present invention can also encode RNA, which is antisensitive to the RNA normally found in a plant cell or can encode RNA, which is capable of cleaving RNA normally found in a plant cell. In accordance with the above, the present invention also provides a nucleic acid sequence encoding a ribozyme capable of specific cleavage of RNA encoded by a gibberellin 2-oxaddase gene. Such DNA encoding the ribozyme would generally be useful in inhibiting the deactivation of gibberellins, particularly C? 9-GAs. The nucleic acid sequences according to the present invention can further comprise 5 'signal sequences to direct expression of the expressed protein product. Such signal sequences can also include the target sequences of proteins, which can direct an expressed protein to a particular location inside or outside a host cell expressing such a nucleic acid sequence. Alternatively, the nucleic acid sequence 10 may also comprise a 3 'signal such as a polyadenylation signal or other regulatory signal. The present invention therefore offers significant advantages for agriculture in the provision of nucleic acid sequences to regulate the metabolism of gibberellin plant hormones. The regulation can be either to inhibit plant growth by promoting the action of gibberellin 2-oxidase or to promote plant growth by preventing the inactivation of gibberellin by gibberellin 2-oxidase. For example, in 1 997, there was lodging in approximately 1 5% of the wheat and 30% of the 20 harvest of barley in the UK with an estimated cost for the growths of £ 1 00m. The availability of host-resistant cereals with shorter stems, stronger as a result of the reduced GA content, can be of considerable economic benefit. According to another aspect of the present invention there is provided an antisensitive nucleic acid sequence, which
^ MMtói - ^^ í ^ ^ i ^^^^^^^^^^^^^^^^^^^ j ^^^^^^^^^^^^^^^^^ ^? ¡^ Y¡¡ ^^ yj¿j¿ < Does it include a filament that can be transcribed from DNA complementary to at least part of the strand of DNA that is naturally transcribed in a gene encoding a gibberellin 2-oxidase enzyme, such as the gibberellin 2-oxidase enzymes of P. coccineus, P vulgaris or A. thaliana. Preferred genes according to the present invention include PcGA2ox1, AtGA2ox1, AtGA2ox2 and AtGA2ox3. The antisensitive nucleic acid and ribozyme-encoding nucleic acid described above are examples of a more general principle: according to a further aspect of the invention DNA is provided, which causes (eg by its expression) the selective dissolution of the expression own gibberellin 2-oxidase genes, or in preferred embodiments the gene P. coccineus PcGA2ox1. According to another aspect of the present invention that provides an isolated, purified or recombinant polypeptide comprising a gibberellin 2-oxidase enzyme having the amino acid sequence as shown in Figure 2. The recombinant DNA according to the invention can be in the form of a vector. The vector can, for example, be a plasmid, cosmid, bacteriophage, or artificial chromosome. The vectors will frequently include one or more selected markers to allow the selection of transferred (or transformed: the terms are used interchangeably in this specification) with them and, preferably, to allow the selection of refuge vector cells incorporating heterologous DNA. The appropriate "start" and "stop" signs will usually be presented.
Additionally, if the vector is attempted for expression, regulatory sequences sufficient to direct expression will be presented; however, the DNA according to the invention will generally be expressed in plant cells, and such microbial host expression will not be among the main objects of the invention, although it is not discarded (such as, for example, in host cells of the invention). yeast or bacterial). Vectors that do not include regulatory sequences are useful as cloning vectors. Cloning vectors can be presented inside E. coli or another suitable host, which facilitates their management. According to another aspect of the invention, there is thus provided a host cell transferred or transformed with a nucleic acid sequence as described above. A further embodiment of the invention is the provision of enzymes by expression of GA 2-oxidase cDNAs in heterologous hosts, such as Escherichia coli, yeasts including strains of Saccharomyces cerevisiae, or insect cells infected with a baculovirus containing recombinant DNA. The enzymes could be used for the production of 2ß-hydroxylated GAs and GA-catabolites or for the preparation of raised antibodies against GA 2 -oxidases. The host cell may also be suitable for a plant cell in a plant cell culture or as part of a callus. The nucleic acid sequences according to this invention can be prepared by any convenient method comprising coupling together successive nucleotides, and / or ligating oligo- and / or polynucleotides, including cell-free in vitro processes, but recombinant DNA technology. form the method of choice. Ultimately, the nucleic acid sequences according to the present invention will be introduced into plant cells by any suitable means. According to still a further aspect of the invention, there is provided a plant cell that includes a nucleic acid sequence according to the invention as described above. Preferably, the nucleic acid sequences of the present invention are presented within the plant cells by transformation using the binary vector pLARS 1 20, a modified version of pGPTV-Kan (Becker ei al Plant Mol. Biol. 20 1 1 95-1 1 97 (1 992)), in which the ß-glucuronidase reporter gene is replaced by the cauliflower 35S mosaic virus pBI220 promoter (Jefferson, R.A., Plant Mol. Biol. Rep. 5 387-405 (1987)). Such plasmids can then be introduced into Agrobacterium numefaciens by electroporation and can then be transferred into the host through a vacuum filtration process. Alternatively, the transformation can be achieved using a tiplasmid vector disarmed and carried by Agrobacterium by methods known in the art, for example as described in E P-A-01 1671 8 and EP-A-0270822. Where the Agrobacterium is ineffective, foreign DNA could be introduced directly into the plant s using only an electric discharge apparatus, such as for example in the transformation of monocotyledons. Any other method that provides stable incorporation of the nucleic acid sequence within the Nuclear DNA or mitochondrial DNA from any plant could also be adequate. This includes plant species, which are not yet capable of genetic transformation. Preferably, the nucleic acid sequences according to the invention for introduction into host s also contain a second chimeric gene (or "marker" gene) that allows a transformed plant containing the foreign DNA to be easily distinguished from other plants that do not contain the foreign DNA. Examples of such a marker gene include antibiotic resistance (Herrera-Estrella ei to EMBO J. 2 987-995 (1 983)), herbicidal resistance (EP-A-0242246) and expression (EP-A-0344029) of glucuronidase (GUS). ). The expression of the marker gene is preferably controlled by a second promoter, which allows expression in s at all periods of development such that the presence of the marker gene can be determined in all periods of regeneration of the plant. A whole plant can be regenerated from a single transformed plant , and the invention therefore provides transgenic plants (or parts thereof, such as propagation material, ie proplasts, s, cali, tissues, organs, seeds, embryos, ovules, zygotes, tubers, roots, etc.) which include nucleic acid sequences according to the invention as described above. In the context of the present invention, it should be noted that the term "transgenic" should not be taken to be limited in relation to an organism as defined above that contains in its germline one or more genes of other species, despite the fact that many organisms will contain such a gene or genes. Still, the term refers more clearly to any organism, of which its germline has been the subject of technical intervention by recombinant DNA technology. Therefore, for example, an organism in whose germ line an endogenous gene has been deleted, duplicated, activated or modified is a transgenic organism for the purposes of this invention as well as an organism in whose germ line a sequence has been added. of exogenous DNA. Preferred plant species include but are not limited to monocotyledons which include seed and offspring or propagules thereof, for example, Lolium, Zea, Triticum, Sorghum, Triticale, Saccharum, Bromus, Oryzae, Avena, Hordeum, Sécale and Setaria Especially the useful transgenic plants are corn, wheat, barley plants and seed thereof. Dicotyledonous plants are also within the scope of the present invention and preferred transgenic plants include but are not limited to the species Fabaceae, Solanum, Brassicaceae, especially potatoes, beans, cabbages, forest trees, roses, clematis, seed oil rapeseed. , sunflower, chrysanthemum, poinsettia and antirino (calf grass). The selection of plant s, tissue and plants for the presence of specific DNA sequences can be performed by Southern analysis as described in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Second edition (1989)). This selection can also be executed using the Polymerase Chain Reaction (PCR) by techniques well known in the art. The transformation of plant cells includes the separation of transformed cells from those that have not been transformed. A convenient method for such preparation or selection is to incorporate within the material to be inserted into the transformed cell a gene for a selection marker. As a result only those cells, which have been successfully transformed, will contain the marker gene. The translation product of the marker gene is then conferred to a phenotypic trait that will make selection possible. Typically, the phenotypic trait is the ability to survive in the presence of some chemical agent, such as an antibiotic, for example kanamycin, G41 8, paromomycin, etc. , which is placed in a selection medium. Some examples of genes conferring antibiotic resistance include, for example, those that are encoded for kanamycin resistance of neomycin phosphotransferase (Velten ei to EMBO J. 3 2723-2730 (1988)), hygromycin resistance (van den Elzen ei to Plant Mol. Biol. 5 299-392 (1985)), the kanamycin resistance gene (NPT II) derived from Tn5 (Bevan eí al Nature 304 1 84-1 87 (1 983); McBride went to the Plant Mol. Biol. 14 (1 990)) and chloramphenicol acetyltransferase. The PAT gene described in Thompson et al (EMBO J. 6 251 9-2523 (1987)) can be used to confer herbicidal resistance.
An example of a gene primarily useful as a selectable marker in tissue culture for identification of plant cells containing genetically technical vectors is a gene that encodes an enzyme that produces a chromogenic product. An example is the gene that is encoded for the production of β-glucuronidase (GUS). This enzyme is widely used and its preparation and use is described in Jefferson (Plant Mol. Biol. Repórter 5 387-405 (1988)). Once the transformed plant cells have been cultured in the selection medium, the surviving cells are selected for further study and management. Selection methods and materials are well known to those skilled in the art, allowing one to choose the surviving cells with a high degree of predictability that the chosen cells will have to be successfully transformed with exogenous DNA. After the transformation of the plant cell or plant using, for example, the Agrobacterium Ti-plasmid, those plant cells or plants were transformed by the Ti-plasmid in such a way that the enzyme is expressed, it can be selected by an appropriate phenotypic marker. These phenotypic markers include, but are not limited to, antibiotic resistance. Other phenotypic markers are known in the art and can be used in this invention. Positive clones are regenerated following procedures well known in the art. Subsequently
^ j ^ transformed plants are evaluated for the presence of the desired properties and / or extension, at which the desired properties are expressed. A first evaluation may include, for example, the level of bacterial / fungal resistance of the transformed plants, the stable inheritance of the desired properties, field tests and the like. By way of illustration and summary, the following scheme indicates a typical process by which, the transgenic plant material, including complete plants, can be prepared. The process can be considered as understanding five steps: (1) first isolate from a suitable source or synthesize by means of known processes a DNA sequence encoding a protein exhibiting GA 2-oxidase activity; (2) operably linking said DNA sequence in a 5 'to 3' direction for plant expression sequences as defined above; (3) transforming the construction of stage (2) into plant material by means of known processes and expressing it therein. (4) Select the plant material treated according to step (3) by the presence of a DNA sequence that encodes a protein that exhibits gibberellin 2-oxidase activity: and
. & & - ^ (5) Optionally regenerate the transformed plant material according to step (3) to a complete plant. The present invention thus also comprises transgenic plants and the sexual and / or asexual offspring thereof, which have been transformed with a recombinant DNA sequence according to the invention. The regeneration of the plant can proceed by any known convenient method of the suitable propagation material either prepared as described above or derived from such material. The expression "asexual or sexual offspring of transgenic plants" includes by definition according to the invention, all mutants and variants obtainable by means of known process, such as, for example, cell fusion or mutant selection and, which still exhibits the characteristic properties of the initial transformed plant, together with all the cross products and fusion of the transformed plant material. Another object of the invention concerns the proliferation material of transgenic plants. The proliferation material of transgenic plants is defined in relation to the invention as any plant material that can be propagated sexually in vivo or in vitro. Particularly preferred within the scope of the present invention are the protoplasts, cells, cali, tissues, organs, seeds, embryos, egg cells, zygotes, along with any other propagation material obtained from transgenic plants. A further aspect of the invention is the provision of an antibody raised against at least a part of the amino acid sequence of gibberellin 2-oxidase. Such an antibody is useful in the selection of a cDNA library in suitable vectors derived from the RNA of the plant tissue. The gibberellin 2-oxidase gene according to the invention is useful in the modification of growth and experimental processes in transgenic plants. Another important aspect of the present invention is, therefore, its use in the preparation of transgenic plants or seeds, in which the gibereli na 2-oxidase is constitutively over-expressed to reduce the concentration of bioactive gibberellins (GAs) in plants or semi llas. Preferred gibberellin 2-oxidase genes include PcGA2ox1, AtGA2ox1, AtGA2ox2 and AtGA2ox3. Such transgenic plants that over-express GA 2-oxidase would resemble plants that have been treated with growth retardants. The invention could therefore be used to reduce vegetative growth as in, for example, the prevention of lodging in cereals, including rice, and the improvement in grain production, the prevention of lodging in seed oil rape and the improvement of foliage structure, the improvement in seedling quality for transplantation, the reduction in growth of pleasant grasses, the reduction in growth of scion in orchid and ornamental trees, the production of ornamental plants with habit of more compact growth, the improvement in
^^^^^^^ & g ^ H ^ * tolerance for cold, extraction and infection, the increase in productions for fun of assimilated from vegetative to reproductive organs, the prevention of curl in rosette plants, such as sugar beet , lettuce, brassica and spinach. The invention can also be used to induce male and / or female sterility by expression in floral organs, to prevent pre-harvest sprouting in cereals, to reduce the growth of stem in guard plants, to inversely inhibit seed development or germination and to reduce the growth of offspring of commercial timber species. Overexpression of the nucleic acid sequences encoding giberilin 2-oxidase can be achieved using DNA constructs comprising constitutive promoters and nucleic acid coding sequences in transgenic plants prepared by recombinant DNA technology. Alternatively, overexpression can be achieved by using the homologous recombination technique to insert into the nucleus of a cell a constitutive promoter upstream of the normally silent copy of the nucleic acid sequence of the present invention. The present invention also provides in a further aspect the use of a nucleic acid sequence as previously defined in the preparation of transgenic plants and / or seeds, in which the expression of endogenous GA 2-oxidase genes in transgenic plants is reduces (ie, silences), by, for example, the expression of antisensitive copies of GA DNA sequences -
Endogenous 2-oxidase, the expression of truncated sensitive copies of the endogenous gene (co-suppression) or the use of synthetic ribozymes targeted to endogenous transcripts. Preferred giberilin 2-oxidase genes according to this aspect of the invention include PcGA2ox1, AtGA2ox1, AtGA2ox2 and AtGA2ox3. This would result in plants with reduced change, and therefore increased concentrations, of bioactive GAs. In this form, the invention could be used, for example, to improve the growth and establishment of fruits in seedless grapes, citron and pear, improve the texture of the skin and the shape of the fruit in apple, increase the length of the stem and therefore, produce in sugarcane, increase the product and precocity in celery and rhubarb, improve malt products and quality in cereals, particularly barley. It could also be used to increase growth in wood species. The preferred features of the second and subsequent aspects of the invention are as for the first aspect mutatis mutandis. The invention will now be described by reference to the following examples and drawings that are provided for the purpose of explanation only and should not be construed as limiting the present invention. In the examples, reference is made to a number of drawings in which: Figure 1 shows the nucleotide sequence of the cDNA 2-oxidase clone of P. coccineus pc-2boh.dna (PcGA2ox1) with the coding region in the residues 68-1 063nt (332 amino acids). Figure 2 shows the deduced amino acid sequence for the nucleotide sequence of P. coccineus (PcGA2ox1) shown in Figure 1. Figure 3 shows the DNA probe sequence for
A. thaliana probe T3 (Figure 3a) and probe T24 (Figure 3b). Figure 4 shows the two main trajectories of gibberellin biosynthesis (GA). Figure 5 shows the partial nucleotide sequence for the clone of A. thaliana at-2bt3 (AtGA2ox1) with the coding region at residues 41-1 027nt (329 amino acids). Figure 6 shows the deduced amino acid sequence for the clone of A. thaliana at-2bt3 (AtGA2ox1). Figure 7 shows the partial nucleotide sequence for the clone of A. thaliana at-2bt24 (AtGA2ox2), with the coding region at residues 1 09-1 1 31 nt (341 amino acids). Figure 8 shows the deduced amino acid sequence for the clone of A. thaliana at-2bt24 (AtGA2ox2). Figure 9 shows the nucleotide sequence for the genomic clone of A. thaliana T31 E 10.1 1 (AtGA2ox3). Figure 10 shows the deduced amino acid sequence for the genomic clone T31 E 1 0. 1 1 (AtGA2ox3). Figure 1 1 shows a photograph of plants
Transformed Arabidopsis (Columbia ecotype) expressing GA 2-oxidase cDNA of P. coccineus under control of the CaMV 35S promoter, - including a transformed plant that does not show phenotype (far right). Example 1. Isolation of cDNA clone encoding GA 2ß-hydroxylase from Phaseolus coccineus A cDNA clone encoding a GA 2ß-hydroxylase was isolated from Phaseolus coccineus embryos by screening a cDNA library for functional enzyme expression as follows . RNA was extracted from the cotyledons of mature Phaseolus coccineus seeds according to Dekker et al. (1989). Poly (A) + mRNA was purified by chromatography on oligo (dT) cellulose. cDNA was synthesized from 5 μg of poly (A) + mRNA using a directional cDNA synthesis kit (? -ZAP II cDNA synthesis kit, Stratagene). The cDNA was ligated into the arms of? -ZAP II, packed using Gigapack Gold III (Stratagene) and 1 x 1 06 recombinant clones were amplified according to the manufacturer's instructions. A phagemid graft was prepared from the cDNA library of Phaseolus coccineus (1 x 1 09 pfu) according to the manufacturer's in vivo excision procedure (Stratagene). For the primary selection, SOLR E. coli were infected with the phagemid graft according to the manufacturer's instructions (Stratagene), resulting in approxima 1 000 units of colony formation (cfu). These were subdivided into 48 cavities (6x8 set) of a micro-main plate (cavity volume = 3.5 ml) and amplified by overnight growth at 37 ° C with shaking, in 0.5 ml of 2xYT broth supplemented with 50 μg / ml of kanamycin and 100 μg / ml of carbenicillin. The aliquots (20 μl) of the six cavities in each row, and from the eight cavities in each column were combined to make 14 groups and each was added to 10 ml of 2YT broth, supplemented with 50 μg / ml kanamycin and 100 μg / ml carbenicillin, and grown at 37 ° C with shaking spent an OD of 600 nm of 0.2-0.5. The cultures were then transferred to a shaking incubator at 30 ° C and the production of recombinant fusion protein was induced by the addition of 1 mM IPTG. The cultures were induced for 1 6 hours. The bacteria were pelleted by centrifugation (3000g x 10 min) and resuspended in 750 μl of lysis buffer (1 00 mM Tris HCl pH 7.5, 5 mM DTT). The bacteria were used by sonication (3 x 10 s) and the cell debris was pelleted by centrifugation for 10 min in a microfuge. The supernatants were analyzed for GA 2ß-hydroxylase activity as described below. The cell lysates of bacteria in group 6 (R6) colu mna 1 (C 1) were able to catalyze the release of 3H 2 O from [1, 2-3H2] GA4 and [2, 3-3H2] GA9. For the second selection, the bacteria of the R6C1 cavity were anodized on 2YT agar plates, supplemented with 1 00 μg / ml of carbenicillin and 50 μg / ml of kanamycin and grown for 16 hours at 37 ° C. One hundred single colonies were randomly collected and transferred to 5 ml of 2xYT broth containing 1 00 mg / ml carbenicillin and 50 mg / ml kanamycin and grown with shaking for 1 6 hours at 37 ° C. The cultures were installed on a 10 x 10 grid and the groups of each row and column were induced and tested for GA 2ß-hydroxylase activity as described above. Rows 2 and 9 and columns 7 and 1 0 were able to catalyze the release of 3H2O from [1, 2-3H2] GA4 and [2, 3-3H2] GA9. It was shown that crops 27 and 29 are responsible for this activity. The putative GA 2ß-hydroxylase clone was designated 2B27. Plasmid DNA, isolated from clone 2B27 using the Promega SVA miniprep kit, was sequenced using the Taq cycle sequencing kit from Amersham with the inverse and universal sequencing primaries M 1 3 (-20). The chain termination products produced from the sequencing reactions were analyzed using an Applied Biosystems 373A automatic sequencer. The sequence analysis was executed using the Sequencer 3.0 program of Gene Codes Corporation. Additional protein and nucleotide sequence analyzes were carried out using the Genetics Computer Group program series from the University of Wisconsin. Example 2. Analysis of GA 2ß-hydroxylase activity The activity of GA 2ß-hydroxylase was determined by measuring the 3H2O lineage of a tritiated 2β GA substrate., as described by Smith and MacMillan (Smith, V.A. and MacMillan, J. in J. Plant Growth Regulation 2 251-264 (1988)). Bacterial lysate (90 μl) was incubated with [1, 2-3H2] GA4 and [2, 3-3H2] GA9 (ca. 50000 dpm), in the presence of 4 mM 2-oxoglutarate, 0 5 mM Fe (11) SO4, 4 mM ascorbate, 4 mM DTTm 1 mg / ml catalase, 2 mg / ml BSA, in a final reaction volume of 100 μl. The mixture was incubated at 30 ° C for 60 min. The tritiated GAs were removed by the addition of 1 ml of activated charcoal (5% w / v) and subsequent centrifugation for 5 min in a microfuge. The aliquots (0.5 ml) of the supernatant were mixed with 2 ml of scintillation fluid and the radioactivity was determined by scintillation counter. In order to confirm the function of the cDNA expression products, the bacterial lysate was incubated with [1-7C14] GAs in the presence of cofactors, as described above. After incubation, acetic acid (10 μl) and water at pH3 (140 μl) were added, and the mixture was centrifuged at 3,000 rpm for 10 min. The supernatant was analyzed by HPLC with on-line radiomonitoring and the products identified by GC-MS, as previously described (MacMillan et al Plant Physiol. 1 1 3 1 369--1 377 (1 997)). Example 3. Cloning of cDNAs encoding GA 2-oxidase from Arabidopsis thaliana The predicted protein sequence of clone 2B27 was used to search the Genome Survival Sequence database at the National Biological Information Center (ncbi.nlm.nih. gov) using the TblastN program. Two genomic sequences of Arabidopsis, T3M9-Sp6 and T24E24TF, demonstrated high amino acid sequence identity with the sequence 2B27. Oligonucleotide primaries were designed based on the T3M9-Sp6 genomic sequences.
^^^^ j ^^^^^^^ ^^^ j «g§ fetf ^ 5'-TAATCACTATCCACCATGTC-3 '(sensitive), 5'-TGGAGAGAGTCACCCACGTT (antisensitive), and sequences T24E24TF: 5'-GGTTATGACTAACGGGAGGT-3 '(sensitive), 5'-CTTGTAAGCAGAAGATTTGT-3' (antisensitive), and used in PCR reactions with Arabidopsis genomic DNA as a template. PCR reactions consisted of 200 mg of genomic DNA, 1 x PCR of regulator, 1.5 mM MgCl2, 200 μM deoxinucleoside trisphosphates, 1 μM of each primary and 2 units of Taq DNA polymerase (Promega). Reactions were heated at 94 ° C for 3 min then 35 cycles of amplification were carried out (94 ° C for 30 seconds, 55 ° C for 30 seconds and 72 ° C for 30 seconds), followed by a final incubation of 1 0 min at 72 ° C. The resulting PCR products were cloned directly into the vector pCR2.1 using the TA cloning kit (Invitrogen) and sequenced as described above. The clones were designated as AtT3 and AtT24. The siliques, flowers, upper stems (maximum 2 cm of stem), lower stems, leaves (caulina and rosette) and roots of the Columbia ecotype were harvested and frozen in liquid N2. Poly (A) + mRNA mRNA was extracted as described above Northern blots were prepared by electrophoresis of 5 μg of poly (A) + mRNA samples through agarose gels containing formaldehyde and subsequent transfer to nictrocellulose (Sambrook and Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Plainview, New York (1989)). Probes labeled 32P random barley were generated for AtT3 and AtT4 using Ready to Go labeling beads (Pharmacia). Figure 3 shows the DNA probe sequences for A. thaliana probe T3 (Figure 3a) and probe T24 (Figure 3b). Hybridizations were carried out in the presence of 50% formamide at 42 ° C for 16 hours (hybridization buffer: 5xSSPE, 2x Denhardts, 0.5% (w / v) SDS, 1 00 μg / ml salmon sperm DNA denatured sonicate, 1.0% Dextran sulphate). The spots were rinsed twice for 1 0 min in 1 x SSC / 0.5% SDS at 20 ° C. Additional 2 x 10 min rinsings were carried out in 0. 1 x SSC / 0.5% SDS at 60 ° C. the spots were exposed to Kodak MS film at -80 ° C with MS intensification screens: the highest expression of both genes was detected in the inflorescence. A cDNA library constructed using 5 μg of poly (A) + mRNA influencing as described above. A total of 5 x 1 05 recombinant phage in E. coli XL1-Blue MRF 'were anodized in five square plates of 24 cm x 24 cm. The plates grew to confluence (8-10 h), then duplicate elevations were taken on nitrocellulose filters supported 20 x 22 cm (Nitropure, MSI) and processed as described by Sambrook et al (Molecular Cloning: A Laboratory Manual. Spring Harbor Laboratory Press, Plainview, New York (1989)). Hybridization of labeled 32T AtT3 and AtT24 probes was carried out as described above. Positive plaques were identified by autoradiography and recessed from the plates in 750 μl of SM buffer (50 mM Tris HCl pH 7.5, 1 00 mM NaCl, 1.0 mM MgSO4, 0.5% gelatin) and reselected until the Plaque pure clones were isolated. Plasmid rescue was carried out using Stratagene Rapid Expression Kit. The cDNA clones were sequenced and the recombinant protein expressed in E. coli and tested for GA 2-oxidase activity as described above. The amino acid and partial nucleotide sequences for all clones are shown in Figures 5, 6, 7 and 8. A third genomic sequence of Arabidopsis T3E 10. 1 1 (AtGa2ox3), with a high amino acid identity with GA 2- P. coccineus oxidase (PcGA2ox1) was also detected in the database of the Gene Bank. Its derived amino acid sequence has 53%, 49% and 67% identity (67%, 67% and 84% similarity) with GA 2-oxidase from P. coccineus (PcGA2ox1), T3 (PcGA2ox1), and T24 (PcGA2ox2) ), respectively. The nucleotide sequence of T31 is shown in Figure 9 and the deduced amino acid sequence is shown in Figure 1 0. Example 4. Transformation of Arabidopsis with sensitive and antisensible GA 2-oxidase cDNA constructs. The predicted coding region of 2B27 was amplified by PCR using the primers of oligonucleotides: 5, -TGAGCTTCAACCATGGTTGTTCTGTCTCAGC-3 '(sensitive); and 5'-TGAGCTCTTAATCAGCAGCAGATTTCTGG-3 '(antisensitive), each of which had a Sacl restriction site incorporated at its 5' end. The PCR product was sub-cloned into pCR2 1 to facilitate DNA sequencing as previously described The 2B27 coding region was digested with Sacl and sub-cloned into the Sacl site of the binary vector pLARS120, a modified version of pGPTV-Kan (Becker et al., Plant Mol Biol. 20 1 195-1 197 (1992)) in which the ß-glucuronidase reporter gene is replaced by the 35S cauliflower mosaic virus promoter from pBI220 (Jefferson, RA, Plant Mol Biol Rep 5 387-405 (1987)). The DNA was inserted into the sensitive orientation under the control of the 35S promoter. The plasmid was introduced into Agrobacterium tumefaciens by electroporation and then transferred into Arabidopsis cv. Columbia through a vacuum infiltration method (Bechtold et al., Compt Rend. Acad. Sci. Series iii-Sciences of Vie-Life Science 31 6 1 1 94-1 1 99 (1 993)). Similarly, the Sacl fragments of AtT3 and AtT24 were subcloned into pLARS 1 20, except in these two cases the DNA was inserted in the antisensitive orientation under the control of the 35S promoter. Aarabidopsis was transformed with those two antisensitive constructions as described above. Example 5 (a). Altered expression of GA 2-oxidase in transgenic plants. The 2-oxidase cDNA of P. coccineus in sensitive orientation (PcGA2ox1) and the 2-oxidase cDNA of A. thaliana in antisensitive orientations were inserted between the 35S CaMV promoter and nos terminator in the vector pLARS 120. The vector pLARS 1 20 is a binary vector for plant transformation mediated by Agrobacterium: the T-DNA contains, in addition to the 35S CaMV promoter and terminator nos, a selectable marker nptll under the promoter nos The vector is derived from pGPTV-Kan (Becker et al. Mol Biol. 20 1 195-1 197 (1992)), the uidA reporter gene in pGPTV-Kan replaced by the 35S promoter. The binary expression constructs were introduced into GV31 01 strain of Agrobacterium tumefaciens which carries the plasmid pAD1289 which confers WrG overexpression by electroporation. These were introduced into Arabidopsis by the vacuum infiltration method (Bechtold ei al Compt. Rend. Acad. Sci. Series iii-Sciences of Vie-Life Science 31 6 1 1 94-1 1 99 (1 993)). To identify transgenic plants, the seeds of infiltrated plants were grown on MS plates supplemented with kanamycin (50 μg / ml) for approximately 14 days and the resistant plants were transferred to the compost. T-DNA containing the construction of 2-oxidase of P. coccineus was also introduced into Nicotiana sylvestris by infection of leaf discs with transformed Agrobacterium tumefaciens Transformation of Arabidopsis plants with GA 2-oxidase cDNA of P. coccineus under the control of the 35S CaMV promoter produced the following results. More than half of the transformants examined showed some degree of dwarfism, many were squashed severely and some did not manage to roll up. Figure 11 shows a photograph of a selection of dwarf transformed plants compared to a transformed plant that shows no phenotype (Columbia ecotype). The transformants responded to treatment with GA3 with increased stem lengthening and normal flower development so that it was possible to obtain seeds. Overexpression of GA 2- cDNA oxidase from P. coccineus in Nicotiana sylvestris resulted in plants with reduced stem height. A transformant did not curl or produce flowers, although untransformed plants have already flowered. Example 5 (b). Altered expression of GA 2-oxidase in transgenic plants Five lines of Arabidopsis that are homozygous for the 35S-PcGA2ox1 transgene were obtained. One line was wound at the same time as the wild type plants (Columbia) but had reduced stem height, while the other four lines remained as rosettes and did not roll when grown in long (16 hours) or short (10 hours) photoperiods. ). A severely stunted line failed to produce homozygous plants, even when the 15 seeds were germinated in the presence of GA3, indicating that seed development deteriorated in this line when two copies of the gene were presented. The severely stubby lines have dark, small leaves that remain closed at spiral level. They produced flower buds when they grew 20 long photoperiods although the flowers did not develop normally and were infertile. No flower bud was obtained when the plants grew in short photoperiods. The treatment of the transgenic lines with 10 μM of GA3 allowed them to roll up and produce normal flowers that establish viable seeds. 25 The metabolism of C19-Gas were compared in a line
^^^^^ J ^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^^ - l? s 35S-PcGA2ox1 severely scrubbed with that in wild-type plants. At the HPLC base, the squat line converted GA ^ GA4, GA9 and GA30 into 2-oxidized products to a much greater extent than did the wild type. The squat line did not metabolize GA3, confirming the results with recombinant enzyme indicating that GA3 is not a substrate for GA 2 -oxidases. Therefore, this GA can be used, when required, to reverse GA deficiency resulting from overexpressing GA 2-oxidase genes. Northern blot analysis of the 35S-PcGA2ox1 lines confirmed high levels of transgene expression. The abundance of transcription for GA 2-oxidase (AtGA2ox1) and 3β-hydroxylase (AtGA3ox1) was elevated in rosettes of the 35S-PcGA2ox1 lines compared to wild-type plants, while the transcription level of GA2-oxidase (AtGA2ox2 ) native was reduced, as a consequence of the control mechanisms for GA homeostasis. Example 6. Patterns of expression of GA 2-oxidase genes of Arabidopsis T3 and T24 (AtGA2ox1 and AtGA2ox2), respectively. The gene expression patterns of GA 2-oxidase Arabidopsis T3 and T24 were examined by testing Northern blots of RNA extracted from different tissues with full-length cDNAs. The genes showed similar patterns of expression, with transcription for both genes present in the leaves, lower stems, upper stems, flowers and siliques. The highest levels of expression were in the flowers, siliques and upper stems in descending order of abundance of transcription, T24 but not T3, also expressed in roots. The abundance of transcription for both T3 and T24 in buttons of immature flowers and pedicels of the GA deficient Arabidopsis mutant, gal-2, increased after treatment with GA3, indicating that the expression of these 2-oxidase genes is supraregulated by GA. This contrasts with the expression of the 3β-hydroxylase and GA 20-oxidase genes, which are down-regulated by GA. The abundance of transcription for T31 was much lower in all tissues than for T3 or T24. Transcription of T31 was detected by RT-PCR in flowers, upper stems and leaves but not in roots or siliques. Example 7. Function of recombinant GA 2-oxidases from Phaseolus coccineus and Arabidopsis thaliana. The catalytic properties of the recombinant proteins obtained by expressing the cDNAs of P. coccineus (PcGA2ox1) and A. thaliana (AtGA2ox1, AtGA2ox2 and AtGA2ox3) in E. coli were examined by incubation in the presence of dioxygenase cofactors with a range of GA substrates marked 14C, consisting of GA ,, GA4, GA9 and GA20 of C19-GAs and C19-GAs, GA12 and GA? 5- The latter compound was incubated in both its open and closed lactone form. No conversion of GA12 was obtained with any of the enzymes, whereas GA1 5 was converted into a single product by PcGA2ox1 and AtGA2ox2. The open lactone form of GA15 (20-hydroxy GA12) was converted to the same product by AtGA2ox2, but less efficiently than was the
t. i 'lactone form, while there was no conversion of open lactone from GA15 to PcGA2ox1. The mass spectrum of the product of GA15 is consistent with it being 2β-hydroxy GA1 5, although, due to the authentic compound is not available for comparison, the identity of this product is tentative. Comparison of the substrate specificities of the recombinant enzyme for C19-GAs (Table 1) indicated that GA9 was the preferred substrate for PcGA2ox1, AtGA2ox1 and AtGA2ox2. The recombinant enzymes differed in some way in their substrate specificities, with GA, becoming as effectively as GA9 by PcGA2ox1 and AtGA2ox3, but a relatively poor substrate for AtGA2ox1 and AtGA2ox2. Although GA20 was 2β-hydroxylated more efficiently than GA by AtGA2ox1 and AtGA2ox2, no GA29 catabolite was detected after incubation with GA20, while low GA34 catabolite products were obtained when GA4 was incubated with PcGA2ox1, AtGA2ox2 and AtGA2ox3 . The activities of recombinant PcGA2ox1, AtGA2ox2 and AtGA2ox3 for 2β-hydroxylation of GA9 varied little between pH 6.5 and 8, and that of AtGA2ox1 with maximum at pH 7 with undetectable activity at pH <; _5.9 and > 8. 1. The results indicate that non-3β-hydroxy C19-GAs, which are immediate precursors of the biologically active compounds, are better substrates for the GA 2-oxidase than are the Gases per se. Therefore, overexpression of GA 2-oxidase genes would result in very little active GA occurring.
& Specificity of recombinant GA 2-oxidase for substrates C19-GAs
Values are% product by HPLC radio monitoring of products after incubation of the E. coli cell lysates expressing the cDNA with 1 C labeled GA substrates and cofactors for 2.5 hours. The products and substrate were E. coli cell expressing the cDNA with labeled GA substrates 14C and cofactors for 2.5 hours. The products and substrate were separated by HPLC and the products were identified by GC-MS. Where product productions were combined < 1 00%, the rest is substrate without converting. Gibberellin Biosynthesis (GA) Figure 4 shows the two main trajectories of gibberellin (GA) biosynthesis, from GA? 2 to GA4 and from GA53 to GA,. GA, and GA4 are biologically active GAs. The conversion of GA12 to GAg and from GA53 to GA20 are catalyzed by GA 20-oxidase. The conversion of GAg to GA4, and from GA20 to GA, are catalyzed by GA 3ß-hydroxylase. GA9, GA4, GA20 and GA, are all substrates for the 2β-hydroxylase activity of GA 2-oxidase, becoming GA51, GA34, GA29 and GA8, respectively. These 2ß-hydroxylated GAs can also be oxidized to the corresponding catabolites. The present invention shows that the enzyme of P. coccineus and the two enzymes of Arabidopsis thaliana catalyze the 2β-hydroxylation of each substrate. In addition, the present invention shows that the enzyme P. coccineus and one of the enzymes of A. thaliana form GA51-catabolite and GA34-catabolite when incubated with GA9 and GA4, respectively
Sequence Listing
< 110 > University of Bristol Thomas, Stephen G Hed on, Peter Phillips, An re L
< 120 > Enzyme
< 141 > 1999-06-11
< 150 > GB 9812821.8 < 151 > 1998-06-12
< 150 > GB 9815404.0 < 151 > 1998-07-15
< 160 > 16
< 170 > PatentIn Ver. 2.1
< 210 > 1 < 211 > 1318 < 212 > DNA < 213 > Phaseolus coccineus
< 400 > 1 gtttctcttc cttaccctgt tctgcttctc tttttcatag taacaatcga caacaacaac 60 aacaaccatg gttgttctgt ctcagccagc attgaascag tttttccttc tgaaaccatt 120 cccttgttca caagtccacg cggggattcc tgtggtcgac ctcacgcacc ccgatgccaa 180 gaatctcata gtgaacgcct gtagggactt cggcttcttc aagcttgtga accatggtgt 240 ttaatggcca tccattggag atttagaaaa aggttcttta cgaggccctc aaaaatctca 300 gtccgagaaa gacagagctg gtccccccga ccctttcggc tatggtagca agaggattgg 360 cccaaacggt gatgtcggtt gggtcgaata cctcctcctc aacaccaacc ctgatgttat 420 ctcacccaaa ttttccgaga tcactttgca aaatcctcat catttcaggg cggtjgtgga 480 gaactacatt acagcagt? to agaacatgtg ctatgcggtg ttggaattga tggcggaggg 540 aggcagagga gttggggata atacgttaag caggttgctg aaggatgaga aaagt.gattc 600 gtgcttcagg ttgaaccact acccgccttg ccctgaggtg caagcactga accgcfaattt 660 ggggagcaca ggttgggttt cagacccaca gataatttct gtcttaagat ctaacagcac 720 atctggcttg caaatctgtc tcacagatgg cacttgggtt tcagtcccac ctgat.cagac 780 ttcctttttc atcaatgttg gtgacgctct acaggtaatg actaatggga ggttt.aaaag 840 tgtaaagcat aggg ttttgg ctgacacaac gaagtcaagg ttatcaatga TCTA tttgg 900 aggaccagcg ttgagtgaaa atatagcacc tttaccttca gtgatgttaa aaggeigagga 960 aaagagttca gtgtttgtac catggtgtga atacaagaag gctgcgtaca cttcciaggct 1020 agctgataat aggcttgccc ctttccagaa atctgctgct gattaaccaa acacaccctt 1080 caaattccac tcattttacg cacgtgttat taccccaatt ttstttcctt tttct.tttcc 1140 taggtttcaa tgtgtctgtc acagttgact ctacttgaca ATGA 1200 tatatagaaa taggt taagatgttt atcattttct ttttcttgtt tcatctaagt gtaacagttg gtctcaactt 1260 ccctttcctc aattgtcaat ggaacgcaac tctagttaca aaaaaaaaaa aaaaaaaa 1318
< 210 > 2 < 211 > 331 < 212 > PRT < 213 > Phaeeolus coccineus
< 400 > 2 Met Val Val Leu Ser Gln Pro Ala Leu Asn Gln Phe Phe Leu Leu Lys 1 5 10 15
Pro Phe Lys Ser Thr Pro Leu Phe Thr Gly lie Pro Val Val Asp Leu 20 25 30
Thr His Pro Asp Ala Lys Asn Leu lie Val Asn Ala Cys Arg Asp Phe 35 40 45
Gly Phe Phe Lys Leu Val Asn His Gly Val Pro Leu Glu Leu Met Wing 50 55 60
Asn Leu Glu Asn Glu Ala Leu Arg Phe Phe Lys Lys Ser Gln Ser Glu 65 70 75 80
Sgg Lys Asp Arg Wing Gly Pro Pro Asp Pro Phe Gly Tyr Gly Ser Lys Arg 85 90 95
lie Gly Pro Asn Gly Asp Val Gly Trp Val Glu Tyr Leu Leu Leu Asn 100 105 110
Thr Asn Pro Asp Val lie Ser Pro Lys Ser Leu Cys lie Phe Arg Glu 115 120 125
Asn Pro His His Phe Arg Ala Val Val Glu Asn Tyr lie Thr Ala Val 130 135 140
Lys Asn Met Cys Tyr Wing Val Leu Glu Leu Met Wing Glu Gly Leu Gly 145 150 155 160
lie Arg Gln Arg Asn Thr Leu Ser Arg Leu Leu Lys Asp Glu Lys Ser 165 170 175
Asp Ser Cys Phe Arg Leu Asn His Tyr Pro Pro Cys Pro Glu Val Gln 180 185 190
Ala Leu Asn Arg Asn Leu Val Gly Phe Gly Glu His Thr Asp Pro Gln 195 200 205
lie lie Ser Val Leu Arg Ser Asn Ser Thr Ser Gly Leu Gln lie Cys 210 215 220
Leu Thr Asp Gly Thr Trp Val Ser Val Pro Pro Asp Gln Thr Ser Phe 225 230 235 240
Phe Lie Asn Val Gly Asp Ala Leu Gln Val Met Thr Asn Gly Arg Phe 245 250 255
Lys Ser Val Lys His Arg Val Leu Wing Asp Thr Thr Lys Ser Arg Leu 260 265 270
Ser Met lie Tyr Phe Gly Gly Pro Wing Leu Ser Glu Asn lie Wing Pro 275 280 285 Leu Pro Ser Val Met Leu Lys Gly Glu Glu Cys Leu Tyr Lys Glu Phe 290 295 300
Thr Trp Cys Glu Tyr Lys Lys Wing Wing Tyr Thr Ser Arg Leu Wing Asp 305 310 315 320
Asn Arg Leu Ala Pro Phe Gln Lys Ser Ala Ala 325 330
< 210 > 3 < 211 > 210 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of Artificial Sequence: Probe
< 400 > 3 taatcactat ecaccatgtc ctcttagcaa taagaaaacc aatggtggta agaatgtgat 60 tggttttggt gaacacacag atcctcaaat catctctgtc ttaagatcta acaacacttc 120 tggtctccaa attaatctaa atgatggctc atggatctct gtccctcccg atcacacttc 180 cttcttctte aacgtgggtg actctctcca 210
< 210 > 4 < 211 > 199 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of Artificial Sequence Probe
< 400 > 4 ggttatgact aacgggaggt tcaagagtgt taaacacagg gtcttagccg atacaaggag 60 atcgaggatt tcaatgatat atttcggcgg accgccattg agccagaaga tcgcaccatt 120 gccatgcctt gtccctgagc aagatgattg gctttacaaa gaattcactt ggtctcaata 180 caaatcttct gcttacaag 199
< 210 > 5 < 211: > 1318 < 212 > DNA < 213 > Arabidopsis thaliana
< 220 > < 221 > mise-feature < 222 > (1243, 1265) < 223 > unidentified waste
< 400 > 5 aaaaattcta tcaaaatcaa tcaaacaagg aaatatatca atggcggtat tgtctaaacc 60 ggtagcaata ccaaaatccg ggttctctct aatcccggtt atagatatgt ctgacccaga 120 gccctcgtga atecaaacat aagcatgcga agacttcggc ttcttcaagg tgatcaacca 180 tggcgtttcc gcagagctag tctctgtttt agaacaegag accgtcgatt tcttctcgtt 240 gcccaagtca gagaaaaccc aagtcgcagg ttatcccttc ggatacggga acagtaagat 300 tggtcggaat ggtgacgtgg gttgggttga gtacttgttg ateatgatte atgaacgcta 360 cggttcgggt ccactatttc caagtcttct caaaagcccg ggaactttca gaaacgcatt 420 ggaagagtac acaacatcag tgagaaaaat gacattegat gttttggaga agatcacaga 480 tgggctaggg atcaaaccga ggaacacact tagcaagctt gtgtctgacc aaaaca gga 540 etegatattg agaettaate actatccacc atgtcctctt ageaataaga aaaccaatgg 600 tggtaagaat gtgattggtt ttggtgaaca cacagatcct caaatcat t ctgtcttaag 660 atetaacaac acttctggtc t caaattaa tctaaatgat ggctcatgga tctctgtccc 720 tcccgatcac acttccttct tcttcaacgt tggtgactct ctccaggtga tgacaaatgg 780 gaggttcaag agcgtgaggc atagggtttt agctaactgt aaaaaat ta gggtttctat 840 gatttaette gctggacc tt cattga tca gagaateget ccgttgacat gtttgataga 900 caatgaggac gagaggttgt acgaggagtt tacttggtct? aatacaaaa actctaccta 960 ttgtctgata caactctaga ataggcttca acaattcgaa aggaagacta taaaaaatct 1020 cctaaattga tgtgatatat ctatttaatc tataagtgtg tgetacatac agacaatgea 1080 tctgtatatt ttgaagtata atgttatttg ttaatccaat aactgtaaaa acatgcaaga 1140 gtgtgtttgt ttgtttcgta atatcaacat cgctcccatc ttttatggat aaaaaaaaaa 1200 aaaaaaaaaa cactgttttg atgtaagcta cattttactt tangtgtaca tcttattgtg 1260 ttaantaaat tatttcaaaa taaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaa 1318 < 210 > 6 < 211 > 329 < 212 > PRT < 213 > Arabidopsis thaliana
< 400 > 6 Met Wing Val Leu Ser Lys Pro Val Wing Pro Lys Ser Gly Phe Ser 1 5 10 15
Leu He Pro Val He Asp Met Ser Asp Pro Glu Ser Lys His Ala Leu 20 25 30
Val Lys Ala Cys Glu Asp Phe Gly Phe Phe Lys Val He Asn His Cly 35 40 45
Val Ser Wing Glu Leu Val Ser Val Leu Glu His Glu Thr Val Asp E'he 50 55 60
Phe Ser Leu Pro Lys Ser Glu Lys Thr Gln Val Wing Gly Tyr Pro Phe 65 70 75 80
Gly Tyr Gly ñsn Ser Lys He Gly Arg Asn Gly Asp Val Gly Trp Val 85 90 95
Glu Tyr Leu Leu Met Asn Wing Asn His Asp Being Gly Ser Gly Pro Leu 100 105 110
Phe Pro Ser Leu Leu Lys Ser Pro Gly Thr Phe Arg Asn Ala Leu Glu 115 120 125
Glu Tyr Thr Thr Ser Val Arg Lys Met Thr Phe Asp Val Leu Glu Lys 130 135 140
He Thr Asp Gly Leu Gly He Lys Pro Arg Asn Thr Leu Ser Lys Leu 145 150 155 160
Val Ser Asp Gln Asn Thr Asp Ser He Leu Arg Leu Asn His Tyr Pro 165 170 175
i cagaa »• -, ..-« ...
Pro Cys Pro Leu Ser Asn Lys Lys Thr Asn Gly Gly Lys Asn Val He 180 185 190
Gly Phe Gly Glu His Thr Asp Pro Gln He He Ser Val Leu Arg Ser 195 200 205
Asn Asn Thr Ser Gly Leu Gln He Asn Leu Asn Asp Gly Ser Trp He 210 215 220
Ser Val Pro Pro Asp His Thr Ser Phe Phe Phe Asn Val Gly Asp Ser 225 230 235 240
Leu Gln Val Met Thr Asn Gly Arg Phe Lys Ser Val Arg His Arg Val 245 250 255
Leu Wing Asn Cys Lys Lys Ser Arg Val Being Met He Tyr Phe Wing Gly 260 265 270
Pro Ser Leu Thr Gln Arg He Wing Pro Leu Thr Cys Leu He Asp Asn 275 280 285
Glu Asp Glu Arg Leu Tyr Glu Glu Phe Thr Trp Ser Glu Tyr Lys Asn 290 295 300
Ser Thr Tyr Asn Ser Arg Leu Ser Asp Asn Arg Leu Gln Gln Phe Glu
305 310 315 320
Arg Lys Thr He Lys Asn Leu Leu Asn 325
< 210 > 7 < 211 > 1237 < 212 > DNA < 213 > Arabidopsis thaliana
< 400 > 7 gaattcggca cgagtttcct tcttcttcct caacctttgc ttcaatcttc aacaactttc 60 gattttgcaa tttttataaa gttaagtgta aacctacaaa aaccaaacat ggtggttttg 120 ccacagccag tcactttaga taaccacatc tccctaatcc ccacatacaa accggttccg 180 gttctcactt cccattcaat ccccgtcgtc aacctagccg atccggaagc gaaaacccga 240 atcgtaaaag cctgcgagga gttcgggttc ttcaaggtcg taaaccacgg agtccgaccc 300 ctcggttaga gaactcatga gcaggaggct attggcttct tcggcttgcc tcagtctctt 360 aaaaaccggg ccggtccacc tgaaccgtac ggttatggta ataaacggat tggaccaaac 420 ggtgacgttg gttggattga gtatctcctc ctcaatgcta atcctcagct ctcctctcct 480 aaaacctccg ccgttttccg tcaaacccct caaattttcc gtgagtcggt ggaggagtac 540 atgaaggaga ttaaggaagt gtcgtacaag gtgttggaga tggttgccga agaactaggg 600 atagagccaa gggacactct gagtaaaatg ctgagagatg agaagagtga ctcgtgcctg 660 agactaaacc attatccggc ggcggaggaa gaggcggaga agatggtgaa ggtggggttt 720 cagacccaca ggggaacaca gataatctca gtgctaagat ctaataacac ggcgggtctt 780 caaatctgtg tgaaagatgg aagttgggts gctgtccctc ctgatcactc ttctttcttc 840 attaatgttg gagatgct ct tcaggttatg actaacggga ggttcaagag tgttaaacac 900 agggtcttag ccgatacaag gagatcgagg atttcaatga tatatttcgg cggaccgcca 960 ttgagccaga agatcgcacc attgccatgc cttgtccctg agcaagatga ttggctttac 1020 cttggtctca aaagaattca atacaaatct tctgcttaca agtctaagct tggtgattat 1080 agacttggtc tctttgagaa acaacctctt ctcaatcata aaacccttgt atgagagtag 1140 tcatgatgat ctttatcatc tagaaagtca ctttgtacga taatcacaaa aagaaggaaa aaaaaaaaaa aaaaaaa tggatagtgt tttggattaa 1200 1237
< 210 > 8 < 211 > 341 < 212 > PRT < 213 > Arabidopsis thaliana
< 400 > 8 Met Val Val Leu Pro Gln Pro Val Thr Leu Asp Asn His He Ser Leu
1 5 10 15 He Pro Thr Tyr Lys Pro Val Pro Val Leu Thr Ser His Ser He Pro 20 25 30
Val Val Asn Leu Wing Asp Pro Glu Wing Lys Thr Arg He Val Lys Wing 35 40 45
Cys Glu Glu Phe Gly Phe Phe Lys Val Val Asn His Gly Val Arg Pro 50 55 60
Glu Leu Met Thr Arg Leu Glu Gln Glu Wing He Gly Phe Phe Gly Leu 65 70 75 80
Pro Gln Ser Leu Lys Asn Arg Wing Gly Pro Pro Glu Pro Tyr Gly Tyr 85 90 95
Gly Asn Lys Arg He Gly Pro Asn Gly Asp Val Gly Trp He Glu Tyr 100 105 110
Leu Leu Leu Asn Ala Asn Pro Gln Leu Ser Ser Pro Lys Thr Ser Ala 115 120 125
Val Phe Arg Gln Thr Pro Gln lie Phe Arg Glu Ser Val Glu Glu Tyr 130 135 140
Met Lys Glu He Lys Glu Val Ser Tyr Lys Val Leu Glu Met Val Wing 145 150 155 160
Glu Glu Leu Gly He Glu Pro Arg Asp Thr Leu Ser Lys Met Leu Arg 165 170 175
Asp Glu Lys Ser Asp Ser Cys Leu Arg Leu Asn His Tyr Pro Ala Wing 180 185 190
Glu Glu Glu Ala Glu Lys Met Val Val Lys Val Gly Phe Gly Glu His Thr 195 200 205
Asp Pro Gln He He Ser Val Leu Arg Ser Asn Asn Thr Wing Gly Leu 210 215 220 Gln He Cys Val Lys Asp Gly Ser Trp Val Wing Val Pro Pro Asp His
225 230 235 240
Be Ser Phe Phe He Asn Val Gly Asp Ala Leu Gln Val Met Thr Asn 245 250 255
Gly Arg Phe Lys Ser Val Lys His Arg Val Leu Wing Asp Thr Arg Arg 260 265 270
Ser Arg He Ser Met He Tyr Phe Gly Gly Pro Pro Leu Ser Gln Lys 275 280 285
He Ala Pro Leu Pro Cys Leu Val Pro Glu Gln Asp Asp Trp Leu Tyr 290 295 300
Lys Glu Phe Thr Trp Ser Gln Tyr Lys Ser Ser Ala Tyr Lys Ser Lys 305 310 315 320
Leu Gly Asp Tyr Arg Leu Gly Leu Phe Glu Lys Gln Pro Leu Leu Asn 325 330 335
His Lys Thr Leu Val 340
< 210 > 9 < 211 > 1008 < 212 > DNA < 213 > Arabidopsis thaliana
< 400 > 9 atggtaattg tgttacagcc agccagtttt gatagcaacc tctatgttaa tccaaaatgc 60 aaaccgcgtc cggttttaat ccctgttata gacttaaccg actcagatgc caaaacccaa 120 atcgtcaagg catgtgaaga gtttgggttc ttcaaagtca tcaaccatgg ggtccgaccc 180 gatcttttga ctcagttgga gcaagaagcc atcaacttct ttgctttgca tcactctctc 240 aaagacaaag cgggtccacc tgacccgttt ggttacggta ctaaaaggat tggacccaat 300 ggtgaccttg gctggcttga gtacattctc cttaatgcta atctttgcct tgagtctcac 360 aaaaccaccg ccattttccg gcacacccct gcaattttca gagaggcagt ggciagagtac 420 attaaagaga tgaagagaat gtcgagcaaa tttctggaaa tggtagagga agcigctaaag 480 atagagccaa aggagaagct gagccgtttg gtgaaagtga aagaaagtga ttcgtgcctg 540 agaatgaacc attacccgga gaaggaagag actccggtca aggaagagat tgc.gttcggt 600 gagcacactg atccacagtt gatatcactg ctcagatcaa acgacacaga gg? ttttgcaa 660 atctgtgtca aagatggaac atgggttgat gttacacctg atcactcctc ttt.cttcgtt 720 cttgtcggag atactcttca ggtgatgaca aacggaagat tcaagagtgt gaaacataga 780 gtggtgacaa atacaaagag gtcaaggata tcgatgatct acttcgcagg tcctcctttg 840 agcgagaaga ttg caccatt atcatgcctt gtgccaaagc aagatgattg cct.ttataat 900 gagtttactt ggtctcaata caagttatct gcttacaaaa ctaagcttgg tgaictatagg 960 cttggtctct ttgagaaacg acctccattt tctctatcca atgtttga 1008
< 210 > 10 < 211 > 335 < 212 > PRT < 213 > Arabidopsis thaliana
< 400 > 10 Met Val He Val Leu Gln Pro Ala Ser Phe Asp Ser Asn Leu Tyr Val 1 5 10 15
Asn Pro Lys Cys Lys Pro Arg Pro Val Leu He Pro Val He Asp Leu 20 25 30
Thr Asp Ser Asp Ala Lys Thr Gln He Val Lys Ala Cys Glu Glu Phe 35 40 45
Gly Phe Phe Lys Val He Asn His Gly Val Arg Pro Asp Leu Leu Thr 50 55 60
Gln Leu Glu Gln Glu Wing He Asn Phe Phe Wing Leu His His Ser Leu 65 70 75 80
Lys Asp Lys Ala Gly Pro Pro Asp Pro Phe Gly Tyr Gly Thr Lys Arg 85 90 95 He Gly Pro Asn Gly Aep Leu Gly Trp Leu Glu Tyr He Leu Leu Asn 100 105 110
Wing Asn Leu Cys Leu Glu Ser His Lys Thr Thr Wing He Phe Arg His 115 120 125
Thr Pro Wing He Phe Arg Glu Wing Val Glu Glu Tyr He Lys Glu Met 130 135 140
Lys Arg Met Being Ser Lys Phe Leu Glu Met Val Glu Glu Glu Leu Lys 145 150 155 160
He Glu Pro Lys Glu Lye Leu Ser Arg Leu Val Lys Val Lys Glu Ser 165 170 175
Asp Ser Cys Leu Arg Met Asn His Tyr Pro Glu Lys Glu Glu Thr Pro 180 185 190
Val Lys Glu Glu He Gly Phe Gly Glu His Thr Asp Pro Gln Leu He 195 200 205
Being Leu Leu Arg Being Asn Asp Thr Glu Gly Leu Gln He Cys Val Lys 210 215 220
Asp Gly Thr Trp Val Asp Val Thr Pro Asp His Ser Ser Phß Phe Val
225 230 235 240
Leu Val Gly Asp Thr Leu G n Val Met Thr Asn Gly Arg Phe Lys Ser 245 250 255
Val Lys His Arg Val Val Thr Asn Thr Lys Arg Ser Arg He Ser Met 260 265 270
He Tyr Phe Wing Gly Pro Pro Leu Ser Glu Lys He Wing Pro Leu Ser 275 280 285
Cys Leu Val Pro Lys Gln Asp Asp Cys Leu Tyr Asn Glu Phe Thr Trp 290 295 300 Ser Gln Tyr Lys Leu Ser Wing Tyr LyS Thr Lys Leu Gly Asp Tyr Arg
305 310 315 320
Leu Gly Leu Phe Glu Lys Arg Pro Pro Phe Ser Leu Ser Asn Val 325 330 335
< 210 > 11 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of Artificial Sequence: Primary
< 400 > 11 taatcactat ccaccatgtc 20
< 210 > 12 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence I
< 220 > < 223 > Description of Artificial Sequence: Primary
< 400 > 12 tggagagagt cacccacgtt 20
< 210 > 13 < 211 > 20 < 212 > DNA < 213 > Artificial sequence
< 220 > < 223 > Description of Artificial Sequence: Primary
< 400 > 13 ggttatgact aacgggaggt 20
< 210 > 14 < 211 > 20 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of Artificial Sequence: Primary
< 400 > 14 cttgtaagca gaagatttgt 20
< 210 > 15 < 211 > 30 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of Artificial Sequence: Primary
< 400 > 15 tgagctcaac catggttgtt ctgtctcagc 30 < 210 > 16 < 211 > 29 < 212 > DNA < 213 > Artificial Sequence
< 220 > < 223 > Description of Artificial Sequence: Primary
< 400 > 16 tgagctctta atcagcagca gatttctgg 29
Claims (22)
- CLAIMS 1. An isolated, purified or recombinant nucleic acid sequence encoding a gibberellin 2-oxidase enzyme wherein the nucleic acid sequence: a) comprises the nucleic acid sequence as shown in Figure 1; or b) comprises a nucleic acid sequence having at least 75% nucleic acid sequence identity to the nucleic acid sequence shown in Figure 1 or a functional derivative thereof or its complementary filament.
- 2. A nucleic acid sequence according to claim 1, characterized in that it has at least 80%, 85%, 90% or 95% of nucleic acid sequence identity to the nucleic acid sequence shown in Figure 1 or a functional derivative of it or its complementary filament.
- 3. A nucleic acid sequence according to claim 1 or claim 2, characterized in that it encodes an enzyme having the activity of a gibberellin 2-oxidase enzyme from Phaseolus coccineus.
- 4. A nucleic acid sequence according to claim 1 or claim 2, characterized in that it encodes a gibberellin 2-oxidase enzyme from P. coccineus or Arabidposis thaliana.
- 5. A nucleic acid sequence according to any of claims 1 to 4, characterized in that the coding sequence is operably linked to a promoter.
- 6. A nucleic acid sequence according to claim 5, characterized in that the promoter is a constitutive promoter.
- 7. A nucleic acid sequence according to claim 5, characterized in that the promoter is specific for expression in a particular plant cell.
- 8. An isolated, purified or recombinant nucleic acid sequence encoding a gibberellin 2-oxidase enzyme comprising a nucleic acid sequence as shown in Figure 1 or a functional derivative thereof, or its complementary filament, or a sequence homologous thereto, wherein the coding sequence is operably linked to a promoter that is specific for expression in a particular plant cell.
- 9. A nucleic acid sequence according to any of claims 1 to 8, characterized in that gibberellin 2-oxidase catalyses the 2β-oxidation of a C-9-gibberellin molecule to introduce a C-2 hydroxyl group. 1.
- An isolated, purified or recombinant nucleic acid sequence encoding a gibberellin 2-oxidase enzyme comprising a nucleic acid sequence as shown in Figure 1 or a functional derivative thereof, or its complementary filament, or a sequence homologous to it, where gibberellin 2-oxasease catalyses the 2ß-oxidation of a molecule of C19- The gibberellin to introduce a hydroxy group into C-2. eleven .
- A nucleic acid sequence according to claim 9 or claim 10, characterized in that the gibberellin 2-oxidase further catalyzes the oxidation of the hydroxyl group introduced in C-2 to produce the acetone derivative.
- 12. A nucleic acid sequence that includes a transcribable DNA strand complementary to at least part of the DNA strand that is naturally transcribed in a gene encoding a gibberellin 2-oxidase enzyme.
- 1 3. An isolated, purified or recombinant polypeptide comprising a gibberellin 2-oxidase enzyme wherein the polypeptide a) has the amino acid sequence as shown in Figure 2; or b) has at least 75% amino acid identity to the amino acid sequence as shown in Figure 2.
- 14. A polypeptide according to claim 13, characterized in that it has at least 80%, 85%, 90% or 95% of amino acid sequence identity to the amino acid sequence shown in Figure 2.
- 1 5. A vector comprising a nucleic acid section according to any of claims 1 to 12.
- 16. A host cell transfected or transformed with a sequence of nucleic acid according to any of claims 1 to 1 2
- 1 7 A plant cell according to claim 16.
- 18 A silver or a part of a plant I minus some of those cells is according to claim 17.
- 19. Propagate material from a plant according to claim 18.
- 20. The use of an isolated nucleic acid sequence, purified or recombinant that encodes a gibberellin 2-oxidase enzyme comprising a nucleic acid sequence as shown in Figure 1, or a functional derivative thereof, or its complementary filament, or a sequence homologous thereto, in the preparation of a plant. twenty-one .
- The use according to claim 20, characterized in that the gibberellin 2-oxidase is constitutively overexpressed in the plant to reduce the concentration of active gibberellins (G! As) in the plant.
- 22. The use according to claim 20, characterized in that the expression of endogenous GA2-oxidase genes in transgenic plants is reduced.
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
GB9812821.8 | 1998-06-12 | ||
GB9815404.0 | 1998-07-15 |
Publications (1)
Publication Number | Publication Date |
---|---|
MXPA00012340A true MXPA00012340A (en) | 2002-07-25 |
Family
ID=
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