JP2001500378A - Materials and methods for altering lignin content in plants - Google Patents

Abstract

  (57) [Summary] Novel isolated DNA sequences involved in the lignin biosynthetic pathway are provided, along with DNA constructs containing such sequences. Also disclosed is a method for altering the lignin content in a plant, the method comprising introducing one or more DNA sequences of the invention or a sequence complementary to a DNA sequence of the invention into a kenom of a plant. Including.

Description

DETAILED DESCRIPTION OF THE INVENTION           Materials and methods for altering lignin content in plantsTECHNICAL FIELD OF THE INVENTION   The present invention modifies the lignin content and composition of plants. About the field to be made More specifically, the present invention relates to an enzyme involved in the lignin biosynthesis pathway. And nucleotide sequences encoding such enzymes.Background of the Invention   Lignin is an insoluble polymer that is primarily responsible for plant stem rigidity. Specifically, the rig Nin acts as a matrix around the polysaccharide component of some plant cell walls. Lignin The higher the content of, the greater the rigidity of the plant. For example, Takagi species produce large amounts of lignin Synthesized, lignin makes up 20% to 30% of the dry weight of wood. In addition to providing rigidity Lignin transports water in plants by making cell walls hydrophobic and making water impermeable. Promote shipping. Lignin also inhibits the invasion and growth of virulence factors and It plays a role in disease resistance.   High concentrations of lignin in trees cause serious problems for the paper industry. In the paper industry, Large amounts of resources are used to separate lignin from the cellulose fibers required for paper production. Must be used. The typical method used to remove lignin is extremely energy Energy and chemical intensive, increasing costs and undesired waste levels Result. About 20 million tons of lignin annually from wood in the United States alone Are separated.   Lignin has a significant effect on the digestibility or lack of digestion of feed grains, and lignin in plants A slight increase in nin content results in a relatively large decrease in digestion. For example, Cereals with reduced lignin content are more effective feeds for livestock, And the yield of meat is high compared to the amount of forage grain consumed. Normal growth period of the plant Medium, an increase in dry matter content is accompanied by a corresponding decrease in digestion. Therefore, the highest When deciding on the right time to harvest, growers should consider high yields of indigestible substances and low yields. You must choose from substances that are easily digested.   For some applications, increasing the lignin content of plants increases the mechanical strength of wood However, high lignin content is desirable because it changes its color and increases rot resistance. Mycorrhizal species composition and abundance are favored by altering lignin content and structural composition. Can be operated as desired.   As described in detail below, lignin is an enzyme at each stage of the synthetic pathway. Of at least three different monolignols synthesized in a multi-step route catalyzed by Produced by polymerization. Cinnamyl alcohol dehydrogenase (CAD) and caffeine Gene encoding certain enzymes such as 3-0-methyltransferase (COMT) Manipulating copy number of offspring has been shown to alter lignin production (US National patent no. 5,451,514 and PCT Publication No. WO 94/23044). In addition, Pop Antisense expression of a sequence encoding CAD in a ligase with altered composition It has also been shown to produce nin (Grand (C) et al.Planta(Berl.)163: 232 -237 (1985)).   Encodes several enzymes involved in the lignin biosynthetic pathway DNA sequence isolated in some plant species, but encodes many enzymes in a wide range of plant species The transforming gene has not yet been identified. Therefore, the lignin content and There is still a need in the art for materials useful for altering It is important.Summary of the Invention   Briefly, the present invention encodes enzymes involved in the lignin biosynthetic pathway. Isolated DNA sequences available from eucalyptus and pine, such sequences DNA constructs and methods of using such constructs are provided. Lignin content Also provided are transformed plants having altered compositions.   In a first aspect, the invention relates to the following enzymes, isolated from eucalyptus and pine, In other words, cinnamate 4-hydroxylase (C4H), coumarate 3-hydro Xylase (C3H), phenolase (PNL), 0-methyltransferase (OMT), Nnamyl alcohol dehydrogenase (CAD), cinnamoyl-CoA reductase (CCR), Nylalanine ammonia-lyase (PAL), 4-coumarate: CoA ligase (4C L), coniferol glucosyltransferase (CGT), coniferin β-g With lucosidase (CBG), laccase (LAC), and peroxidase (POX) Simply encodes ferulate 5-hydroxylase (F5H) from eucalyptus Provide the isolated DNA sequence. In one embodiment, the isolated DNA sequence comprises (a) SEQ ID NO: 3,13, Sequences listed in 16-70 and 72-88; (b) listed in SEQ ID NOs: 3,13, 16-70 and 72-88 (C) the reverse complement of the sequences listed in SEQ ID NOs: 3,13,16-70 and 72-88 (D) the reverse of the sequences listed in SEQ ID NOs: 3,13,16-70 and 72-88 Sequence, and (e) the same as sequences (a)-(d) as measured by computer algorithm FASTA. A sequence having a probability of being at least about 99% Contains a reotide sequence.   In another embodiment, the DNA sequence of the invention is used alone, in combination with one or more sequences of the invention. Either in combination or in combination with one or more known DNA sequences. The present invention provides a DNA construct that is contained together with a transformed cell containing such a construct.   In a related aspect, the invention provides a gene promoter sequence in the 5'-3 'direction. At least one of the enzymes encoded by the DNA sequences of the invention or variants thereof. Open reading frame encoding the active region, and a gene termination sequence. A DNA structure. The open reading frame is in the sense direction or It may be oriented in any of the orientation directions. The above DNA sequence or non-coding Gene encoding the enzyme encoded by the nucleotide sequence complementary to the region DNA constructs containing non-coding regions of offspring also contain gene promoter sequences and Provided with child termination sequence. Preferably, the gene promoter and termination sequence Is functional in the host plant. Most preferably, the gene promoter and termination The sequence is native to the enzyme gene, but others commonly used in the art, For example, an enhancer such as a Kozak sequence or an Omega enhancer. Cauliflower mosaic virus (CMV) promoter with or without, andUg Lobacterium tumefaciens (Agrobacterium tumefaciens) Nopalinecin Tase terminators can be used effectively in the present invention. One or more desired A tissue-specific promoter may be used for target expression in a given tissue. Like In a preferred embodiment, the gene promoter sequence results in transcription in xylem. DNA structure May further comprise a marker identifying the transformed cell.   In another embodiment, a transformed plant cell comprising a DNA construct of the present invention is such a plant cell. Provided with plants containing transformed cells, fruits and seeds of such plants .   In yet another aspect, a method is provided for regulating the lignin content and composition of a plant. Such a method can stably integrate the DNA structure of the present invention into the genome of a plant. And In a preferred embodiment, the target plant is woody, preferably eucalyptus. And most preferably from the group consisting of pine seedsEucalyptus grandis (Eucalyptus grandis)andPinus Radiata(Pinus radiataGroup) Selected from In a related aspect, a method of producing a plant having an altered lignin content This method comprises transforming a plant cell with the DNA construct of the present invention. Providing cells and promoting regeneration of transformed cells and growth of mature plants Culturing under the conditions.   In yet another aspect, the invention provides a method of altering the activity of a plant enzyme, comprising the steps of: Stably incorporating the DNA constructs of the invention into the kenom of the plant. Preferred practice In embodiments, the target plant is a woody plant, preferably consisting of eucalyptus and pine seeds From the group, most preferablyEucalyptus grandisandPinus Radio Ta Selected from the group consisting of:   The above and other features of the invention, as well as methods for obtaining them, are described in the appended claims. BRIEF DESCRIPTION OF THE DRAWINGS The invention will become apparent from the following more detailed description, taken in conjunction with the drawings, in which: Seems to be well understood.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 is a schematic diagram of the entire lignin biosynthesis pathway.Detailed description   Lignin is at least three different monolignols, mainly p-coumarilua Produced by polymerization of alcohol, coniferyl alcohol and sinapyl alcohol . Although the three subunits of lignin are well known, It is possible that few variants are involved in the lignin biosynthetic pathway in various plants You. Relative concentrations of these residues in lignin vary between different plant species and within species . In addition, the composition of lignin varies between different tissues within a particular plant. Three kinds of monoliths Gunol is derived from phenylalanine in a multi-step process, It is thought that lignin is polymerized by the mechanism.   Figure 1 illustrates the different steps in the biosynthesis pathway of coniferyl alcohol, Are shown together with the enzymes that catalyze them. p-Cumaryl alcohol and Sinapyral Calls are synthesized by a similar route. First, phenylalanine is replaced by phenylalanine Deamination with ammonia-lyase (PAL) to give cinnamate, which Is hydroxylated with cinnamate 4-hydroxylase (C4H) to give p-bear Produces luic ester. p-coumarate is 3-coumarate It is hydroxylated with a lase to give a caffeic acid ester. Newly added The hydroxyl group is then methylated with 0-methyltransferase (OMT) to produce ferulic acid. Ester, which is co-enzyme by 4-coumarate: CoA ligase (4CL) Joined to A Produces Royl-CoA. Reduction of feruloyl-CoA to coniferaldehyde Catalyzed by namoyl-CoA reductase (CCR). Coniferaldehyde is It is further reduced by the action of cinnamyl alcohol dehydrogenase (CAD) to conif Glyceryl alcohol, which in turn produces coniferol glucosyltransfer Glucosylated for transport from the cytoplasm to the cell wall by the enzyme (CGT) Converted to shape. Following transport, deglycosylated coniferyl alcohol The form is obtained by the action of coniferin β-glucosidase (CBG). Finally, Phenolase (PNL), Lacquer Catalyzed by zea (LAC) and peroxidase (POX).   An additional enzyme, ferulic acid ester-5-hydrogen, is used to produce synapyr alcohol. Xylase (F5H) is involved. For more details on the lignin biosynthesis pathway, see Whetton, R. and Sederoff, R .;The Plant Cell,7: 100 See 1-1013 (1995).   Qualitative and quantitative changes in the lignin content in plants are based on photostimulation, low calcium levels. It is known to be triggered by external factors such as bells and mechanical stimuli. New species Lignin synthesis sometimes does not lignify properly in tissues, but is caused by pathogen infection. Even triggered. In addition to lignin, other types of plant products are Derived from nin. These products include flavonoids, coumarins, and stilbes. Benzoic acid and benzoic acid derivatives, and the initial stages of the synthesis of these compounds are all the same. It is. Therefore, alteration of the action of PAL, C4H and 4CL is not only in addition to lignin, but also in other plants. It may affect the synthesis of the product.   Using the methods and materials of the present invention, enzymes involved in the lignin biosynthetic pathway are By additionally incorporating a copy of the dosing gene into the genome of the target plant , Can increase the lignin content of the plant. Similarly, such a gene Lignin content by transforming the target plant with a copy of the antisense You can do it. In addition, genes encoding different enzymes in the lignin biosynthetic pathway Manipulating the number of child copies to change the relative amount of each synthesized monolignol Can be. This can lead to the production of lignin with a changed composition. Re A change in the composition of gnin would be beneficial, for example, in the treatment of paper timber and spoil the wood It will also be effective to change the fungus to your liking.   In one embodiment, the invention encodes an enzyme involved in the lignin biosynthesis pathway To provide fully or partially isolated DNA sequences that encode or partially encode . Its DNA sequence was obtained from eucalyptus and pine. Specifically, the book The invention isEucalyptus grandisEnzyme CAD (SEQ ID NO: 1, 30), PAL ( (SEQ ID NO: 16), C4H (SEQ ID NO: 17), C3H (SEQ ID NO: 18), F5H (SEQ ID NO: 19-21), OMT ( (SEQ ID NO: 22-25), CCR (SEQ ID NO: 26-29), CGT (SEQ ID NO: 31-33), CBG (SEQ ID NO: 34) , PNL (SEQ ID NOs: 35 and 36), LAC (SEQ ID NOs: 37-41) and POX (SEQ ID NOs: 42-44), ToPinus RadiataEnzymes C4H (SEQ ID NO: 2, 3, 48, 49), C3H (SEQ ID NO: 4, 50-52), PNL (SEQ ID NO: 5, 81), OMT (SEQ ID NO: 6, 53-55), CAD (SEQ ID NO: 7, 71), C CR (SEQ ID NO: 8, 58-70), PAL (SEQ ID NO: 9-11, 45-47), 4CL (SEQ ID NO: 12, 56, 57) , CGT (SEQ ID NO: 72), CBG (SEQ ID NO: 73-80), LAC (SEQ ID NO: 82-84) and POX (SEQ ID NO: 82) Nos. 13, 85-88) are provided. like this The complement of the isolated DNA sequence, the reverse complement of the isolated DNA sequence, and the reverse sequence of the isolated DNA sequence are also included. Provided with sequence variants such as The DNA sequence encompassed by the present invention includes cDNA, Includes genomic DNA, recombinant DNA and wholly or partially chemically synthesized DNA molecules. I will.   As used herein, the terms “complement”, “reverse complement” and “reverse sequence” The definition is best illustrated in the following example. For sequence 5'AGGACC3 ', complement, reverse phase The complement and reverse sequence are as follows.                       Complement 3 'TCCTGG 5'                       Reverse complement 3'GGTCCT 5 '                       Inverse sequence 5 'CCAGGA 3'   As used herein, the term "variant" refers to at least about 50%, preferably at least about 50%, of a sequence of the invention. Any sequence that exhibits at least about 70%, more preferably at least about 90%, homology. Covers columns as well. Most preferably, the "variant" has a probability of being the same as the sequence of the present invention. A sequence that is at least about 99%. DNA sequence probability is a computer algorithm FASTA (version 2.0u4, February 1996; Pearson, W.R.) et al.Pro . Natl . Acad. Sci. ,85: 2444-2448, 1988) and the probability of translated DNA sequence is Using the computer algorithm TBLASTX, the probability of protein sequence Gorism BLASTP (Altschul, SF) et al.J . Mol. Biol.,215: 403-410 , 1990). Thus, the term “variant” happens to be coincidental in the database. The probability of seeing (minimum sum probability) is less than about 1% as measured by any of the above tests Sequences.   Sequence variants isolated from other eucalyptus and pine species have been used in the timber industry. Variants from other commercially important species were considered as well. These include the following gymnosperms as examples. Loblolly pine (Pinus taeda), Elio Pine (Pinus elliotti), Crow pine (Pinus clausa), Rhubarb (Pinus palustrus ), Kimatsu (Pinus echinata), Ponderosa pine (Pinus ponderosa), Jeffrey Pine (Pinus jeffrey), American red pine (Pinus resinosa), Yanima Tsu (Pinus rigida), Banks pine (Pinus banksiana), Pound pine (Pinus seroti na ), Eastern Strobe Pine (Pinus strobus), Western strobe Pine tree(Pinus monticola), Sugar pine (Pinus lambertiana), Virginia pine (Pin us virginiana ), Contorta pine (Pinus contorta), Caribbean pine (Pinus car ibaea , P. pinaster ), Calabrian pine (P . brutia), Afgammatz (P . eldari ca ), Cool Tarmatsu (P . coulteri), European pine (P . nigraandP . sylve stris ), Bay pine (Dowgrass-Phil) (Pseudotsuga menziesii);T suga heterophylla ), Canada Tsuga (Tsuga canadensis), Mountain Tsuga (Tsuga m ertensiana Bleeding moth including spruce; German spruce (Picea abies), Red spruce (Picea rubens ), Canadian spruce (Picea glauca), Black spruce (Picea mariana), Bait Uhi (Picea sitchensis), Engleman spruce (Picea engelemanni), Pungen Spruce (Picea pungens) Containing spruce;Sequoia sempervirens); Lots Key fir (Abies lasiocarpa), European fir (Abies amabilis) Fir (Abies grandis), Noble fir (Abies procera), Bay fir (Abies concolo r ), California red fir (California red fir) (Abies magnifica), Balsa Momi (Abies balsamea) Containing this fir;Thuja plicata), Onihiva (li bocedrus decurrens ),Thuja occidentalis), Nu Mahinoki (Chamaecyparis lawsoniona), Atlantic Numa Hinoki (Chamaecyp aris thyoides ), Alaska Hinoki (Chamaecyparis nootkatensis), Empitsby Kushin (Huniperus virginiana) Containing cedar; Eastern larch (Larix laricin a ), Western larch (Larix occidentalis), European larch (Larix dec idua ), Larch (Larix leptolepis), Siberian larch (Larix siberica) Larch;Taxodium distichum) And Sequoia (Sequoia g igantae ); As well as the following angiosperms:Eucalyptus Aruba (Eucalyptus alba),E. Bancrofty(E.bancroftii) ,E. Botiloid(E . botyroides),E. Brigesiana(E.bridgesiana),E. Caro Huila (E . calophylla),E. Camaldrensis(E . camaldulensis),E. Citrio Dora (E . citriodora),E. Cladocalix(E . cladocalyx),E. Cosifera(E . coccifera ),E. Kurtishii(E . curtisii),E. Darling Pleana(E . dalrymple ana ),E. Degrappa(E . deglupta),E. Delagatensis(E . delagatensis),E. De Iversi color (E . diversicolor),E . Donny(E . dunnii),E. Fisifou rear (E . ficifolia),E. Globras(E . globulus),E. Gonfosefara(E . gom phocephala ),E. Gunny(E . gunnii),E. Henry(E . henryi),E. Lavopine A (E . laevopinea),E. Makarturi(E . macarthurii),E. Macro linker(E . mac rorhyncha ),E. Macrata(E . maculata),E. Marginata(E . marginata),E. Mega Kalpa (E . megacarpa),E. Meliodora(E . melliodora),E. Nikolii(E . nicholi i ),E. Nitens(E . nitens),E. Nova Angelica(E . novaanglica),E. Obli Kuah (E . obliqua),E. Obssiflow La (E . obtusiflora),E. Oleades(E . oreades),E. Porciflora(E . pauciflo ra ),E. Polybractair(E . polybractea),E. Legnance(E . regnans),E. Resi Nifera (E . resinifera),E. Robusta(E . robusta),E. Rudis(E . rudis),E . Saligna (E . saligna),E. Sideroxilone(E . sideroxylon),E. Stuarti Anna (E . stuartiana),E. Teleticolmis(E . tereticornis),E. Toleriana(E . torelliana ),E. Urnigella(E . urnigera),E. Urofira(E . urophylla),E . Vimiralis (E . viminalis),E. Viridis(E . viridis),E. Wandoo(E . wandoo )andE. Yumanni(E.youmanni).   The DNA sequences of the present invention were prepared as described in Examples 1 and 2 below.Eucalyptus Grandis andPinus RadiataCDNA library, as prepared from Can be isolated by high yield sequencing. As another option, SEQ ID NO: 1- Synthesize oligonucleotide probes based on sequences given in 13 and 16-88 Can be made by cDNA or by hybridization or PCReucalyptus Putus grandis andPinus RadiataThe above identified ones Genomic DNA libraries from other gymnosperms and angiosperms, including Can be used to identify positive clones. Probe here May be shorter than the sequence provided in, but the length of nucleotides is at least about 10, Preferably it should be at least about 15, most preferably at least about 20. Hybridization suitable for use with such oligonucleotide probes And PCR technology is a well-known technology. Positive clones are digested with restriction enzymes and DNA It can also be analyzed by column determination etc. You.   Furthermore, the DNA sequences of the present invention can be created by synthetic means using well-known techniques. You. An automated oligonucleotide synthesizer is available from PerkinElmer Applied Bi Osystems Division (Perkin Elmer / Applied Biosystems Division) Commercial products can be obtained from suppliers such as Foster City (CA) and follow the manufacturer's instructions. Can be operated.   In one embodiment, the DNA construct of the invention comprises a nucleotide sequence of the invention or An open library encoding at least a functional part of the enzyme encoded by the variant Includes the loading frame. The “functional part” of the enzyme used here affects the metabolic stage. A moiety containing the active site essential to provide, ie, bind one or more reactants Or the part of the molecule that can improve or control the rate of the reaction. Activity A sex site can be composed of discrete parts present on one or more polypeptide chains. Will generally show high substrate specificity. The term "nucleotide" used here `` Enzyme encoded by a sequence '' includes a nucleoside comprising a partially isolated DNA sequence of the present invention. Includes enzymes encoded by the peptide sequences.   For applications where amplification of lignin synthesis is desired, transformation of target plants with DNA constructs Increase the number of copies of the gene and thus the amount of enzyme. The reading frame is inserted into the DNA construct in the sense orientation. Lignin synthesis If down regulation is desired, the RNA produced by transcription of the DNA sequence is complementary to the endogenous mRNA. As you can see, the open reading frame is inserted into the DNA construct in the antisense orientation. insert. As a result, the number of gene copies decreases and the amount of enzymes decreases right. Alternatively, sequences or subsequences that are appropriate for the ribozyme construct (eg, (DNA or RNA, for example).   In a second embodiment, the DNA construct of the invention is encoded by a DNA sequence of the invention A nucleotide sequence containing the non-coding region of the gene encoding the enzyme, or Includes nucleotide sequences complementary to such non-coding regions. Used here The term "noncoding region" includes both untranslated and translated sequences. Or about 5 'or 3' base pairs of the open reading frame Both sequences are included. Non-code that can be usefully used for the structure of the present invention Examples of coding regions include introns and 5'-noncoding leader sequences It is. Transformation of a target plant with such a DNA construct can be performed, for example, by using Napoli (Plant Cell,Two279-290, 1990) and de Carvalho N. iebel) et al. (Plant Cell,7; 347-358, 1995). The process can also reduce the amount of lignin synthesis by the plant.   In addition, the DNA constructs of the present invention are genetically linked to a transcribed DNA sequence. It contains a gene promoter sequence that controls offspring expression and a gene termination sequence. Gene The promoter sequence is usually located at the 5 'end of the DNA sequence to be transcribed and Used to get started. The gene promoter sequence is usually the 5 'non-coding region of the gene. Introns (Luehrsen, K.R.),Mol . Gen . Genet .225: 81-93, 1991) or in the coding region as in the example of tomato PAL May be present (Bloksberg 1991, "Phenylalanine ann Monialiase biology and plant diseases Studies on the Interaction of the Active Substances "Ph.D. Dissertation, Univ. of California. Davis, U niversity Microfilms International order number 9217564). Structure is openly When the coding frame is included in the sense orientation, the gene promoter sequence is also Begin translation of the reading frame. Open reading of antisense orientation For DNA constructs that contain either a coding frame or non-coding regions, The gene promoter sequence is only from the translation initiation site with the RNA polymerase binding site Become.   Various gene promoter sequences effectively used in the DNA construct of the present invention are well-known. Technology. The sequence of the promoter gene and the termination sequence of the gene are As long as it is functional in the target host, it may be internal or external to the target plant host. No. For example, promoters and termination sequences may be used in other plant species, plant viruses, bacterial plasmids. It may be from a sumid or the like. Preferably, the gene promoter and termination sequence are It is from the sequence of the invention itself.   Factors affecting promoter selection include tissue specificity of the desired construct and And the timing of transcription and translation. For example, 35S California Mosaic Constitutive promoters, such as the virus (CaMV 35S) promoter, are found in all parts of the plant. Affects the activity of the enzyme. The use of tissue-specific promoters is notable Will produce only the desired sense or antisense RNA in certain tissues. U. A DNA construct that uses an inducible gene promoter sequence to bind RNA polymerase and And the rate of onset are controlled by external stimuli such as light, heat, anaerobic stress, and altered nutrient status. It may be clause. Generation of transformed cells using a temporally regulated promoter RNA poly at a specific point in time The regulation of the binding and initiation rate of the merase can be affected. Preferably , The native promoter from the enzyme gene in question, or a form such as eucalyptus or pine A promoter from the specific tissue target gene of the organism to be transformed is used. Departure Another example of a gene promoter that can be used effectively in (Mas), octopine synthase (ocs) and Chua et al. (Science,244: 17 4-181, 1989).   The gene termination sequence is located 3 'to the transcribed DNA sequence and is identical to the promoter sequence. It can be obtained from the same gene or from another gene.Aggro Bacterium tumefaciens nopa Many known sources such as the phosphorus synthase gene Gene termination sequences can be effectively utilized in the present invention. But preferred genes The termination sequence may be from the gene for the native enzyme or from the target species to be transformed.   The DNA structure of the present invention contains a selectable marker effective for plant cells, and the structure of the present invention Enables the detection of transformed cells contained in E. coli. Such a marker is It is well known in the art and confers a unique resistance to one or more toxins. This mer One example of a car is the NPTII gene, whose expression is kanamycin or hygromycin. Resistant to antibiotics that are generally toxic to plant cells at moderate concentrations, such as (Rogers et al., “Methods of plant molecular biology”, Weissbach (A. Weissbach) and H. Weissbach (eds.). Academic Press San Diego, CA (1988)). Alternatively, the desired composition in the transformed cells The presence of the structure is confirmed by a well-known technique, such as Southern Western blotting. can do.   Techniques for operatively linking the components of the DNA structure of the present invention are also well known. Yes, one or more controls, e.g., as described by Maniatis et al. And the use of synthetic linkers containing restriction endonuclease sites (``Molecular black Ning: Laboratory Procedures ”, Cold Spring Harbor Laboratories, Co. Spring Harbor, New York, 1989). The DNA structure of the present invention A vector having at least one replication system, such as E. coli (E . coli) To phosphorus After each operation, the cloning of the resulting structure Can perform sequencing and sequencing, and confirm the accuracy of the operation.   The DNA construct of the present invention may be used for monocotyledonous plants (eg, rice grass, corn, cereals, oats). Wheat, wheat and barley), dicotyledonous plants (eg, Arabidopsis (Arabidopsis) , Tobacco, legumes, green coconut palm, oak, eucalyptus, maple) And gymnosperms (eg, Scots pine (Aronen). Finnish Forest Re s. Papers, vol. 595, 1966), white spruce (Ellis et al.,Biotech nology 11 : 94-92,1993), Larch (Huang et al.,In Vitro Cell 27: 201- 207, 1991)). In a preferred embodiment, The DNA construct of the present invention is a woody plant (here, it survives for several years and adds woody tissue every year). (Defined as trees or shrubs of increasing diameter). Good The preferred target plant is from the group consisting of eucalyptus and pine species, most preferablyYou Calyptus Grandis andPinus RadiataSelected from the group consisting of . As already discussed, the open reading frame is oriented in the sense direction. Open ready encoding an enzyme encoded by the DNA construct of the present invention. Plants with DNA structures containing Transversion increases the lignin content of the plant and, in some cases, cosuppressor Will result in a reduction. Open sense antisense orientation DNs containing coding frames or non-coding (non-translated) regions of genes Transforming plants with A structures reduces the lignin content of the transformed plants Will result.   Techniques for stably integrating a DNA structure into the genome of a target plant are well known,Ug Lobacterium tumefaciens Mediation introduction, electroporation, prototype Includes fusion, reproductive organ insertion, vermilion embryo insertion, rapid firing induction, and the like. Technical Selection will depend on the target plant to be transformed. For example, dicotyledonous plants and Certain monocots and gymnosperms, for example, Bevan (Bevan) (Nucl . Acid Res.,12: 8711-8721, 1984)AgrobacteriumTi plasmid method Can be transformed. Targets for introducing the DNA construct of the present invention include leaf tissue , Seeding cells, protomorphs, seeds, embryos, divisions and other tissues, cotyledons, hypocotyls, etc. . One of the preferred methods of transforming eucalyptus and pine is pollen (e.g., allo Nen, 1996, Finnish Forest Res. Papers vol. 595, 53pp) or easily This is a biolistic method using regenerable embryo tissue. Useful in the method of the present invention Other transformation techniques that can be used include Ellis et al. (Plant Cell Reports,8 : 16-20, 1989), Wilson et al. (Plant Cell Reports,7: 704-707, 198 9) and Tautorus et al. (Theor . Appl. Genet.78: 531-536, 1989) So there is a method taught.   When the cells are transformed, the DNA structure of the present invention integrated into the genome. The cells carrying the construct are identified by a marker, such as the kanamycin resistance marker described above. Selected. The transformed cells are then used to regenerate the whole plant using well-known techniques. It can be cultured in a medium. The cell surface of the prototypical body should be placed under appropriate permeation conditions. Renovate with. For seed or embryo platforms, use appropriate germination or callus media. To use. Use appropriate regeneration media for explants. Plant regeneration is established in many species Have been. For a report on forest tree regeneration, see Dunstan et al. Somatic embryogenesis in books, edited by Thorpe, T.A., 1995: "Recent in agriculture" Plant Science and Biotechnology ”, Chapter 12, pp. In 471-540, " In Vitro Embryogenesis ", Vol. 20. Special professional for spruce regeneration Tocol is considered by Roberts et al. ("Spruce somatic embryogenesis") ,Synseed."Application of synthetic seeds to improve yield”, Redenbaug h, K.) et al., CRP Press, Chapter 23, pp. 427-449, 1993). Transformation obtained Plants are sexually or asexually reproduced. The methods used are well known and The object can be continuously regenerated.   As discussed earlier, RNA production in target cells depends on the choice of promoter sequence. Or the integration site or machinery of the DNA sequence integrated into the genome of the target plant host It is controlled by selecting the number of active copies. The target plant is one or more DNA structures of the present invention. Rigs suitable for the activity of one or more enzymes. Regulates the nin biosynthetic pathway, affects the activity of enzymes in one or more tissues, Or affects the activity of the enzyme during one or more of its expressions. Similarly, DNA structures One or more open readings encoding the enzyme encoded by the DNA sequence of the invention. Frame or such an enzyme Can be constructed to include one or more non-coding regions of the gene to be encoded. it can. The DNA sequence of the present invention encodes an enzyme involved in the lignin biosynthetic pathway It can be used in combination with other known sequences. Rig to non-kimoto in this way The addition of the nin biosynthetic pathway makes it possible to produce new woody plants.   The isolated DNA sequence of the present invention comprises a DNA sequence encoding an enzyme involved in the lignin synthesis pathway. The sequence can also be used as a probe to isolate from other plant species, and the technique used is It is well known to those skilled in the art.   The following examples are provided for illustration and not limitation.                                   Example 1 From Eucalyptus grandis cDNA Clone isolation and characterization   Two of the following (one from various tissue mixtures from a single tree, the leaves of a single tree) One)Eucalyptus grandisBuild cDNA expression library and script Learning.   Chang et al.'S protocol (Plant Molecular Biology Reporter 11： 113-1 16, 1993) with slight modifications to extract mRNA from plant tissues. In detail, the sample CPC-RNAXB (Tris-Cl, (pH 8.0, 100 mM); EDTA (25 mM); NaCl (2.0 M); CTAB2%; PVP2% ; Spermidine^*Dissolved in 3HCl 0.05%), chloroform: isoamyl alcohol ( 24: 1). Precipitate the mRNA with ethanol and transfer the entire RNA preparation to poly (A ) Purified using Quick mRNA Isolation Kit (Stratagene, Radiola, CA) . CDNA from reverse transcriptase synthesis from purified mRNA Build an expression library and use the resulting cDNA clones to synthesize ZAP Express cDNA Lambda ZAP using kit (Stratagene) according to manufacturer's protocol Was inserted. The obtained cDNA was prepared from 5 μl of the ligation mixture using 1 μl of a DNA sample. Package using Gigapack II Packaging Extract (Stratagene) Jing. Using XL1-blue MRF 'cells and XLOLR cells (Stratagene) , X-assist helper phage (Stratagene) Removal. Removed phagemid from NZY broth (Gibco BRL, Geyser) X-gal and isopropylthio-β-galactoside (IPTG ) Was plated on an LB-kanamycin agar plate.   99% of the selected colonies plated and plated in the DNA minipreparation are suitable for sequencing. The input fragment was included. Grow positive colonies in NZY broth containing kanamycin And cDNA is purified by alkaline lysis and polyethylene glycol (PEG) precipitation. Made. Use agarose gel at 1% for screen sequencing template for chromosome contamination Was. Turbo Catalyst 800 Machine (PerkinElmer Applied Biosystem) (Stems, Foster City, CA) and according to the manufacturer's protocol Primer sequences were prepared.   The DNA sequence of the positive clone was Applied Biosystems Prism 377 Obtained using a Kencer. First, cDN from both 5 'ends and, in some cases, 3' end The A clone was sequenced. For some clones, subcloned fragments To obtain the internal sequence. Uses standard methods for restriction mapping and subcloning And subcloned into pBluescript II SK + vector.   FASTA algorithm of February 1996 (version 2.0u4) (Internet site, ftp://ftp.virginia.edu/pub/fasta/) Sequence compared to known sequences in EMBL database (46, released March 1996) . Multiple alignment of overlapping sequences to build reliable consensus sequences Was used. DNA sequence isolated based on similarity to known sequences from other plant species (SEQ ID NO: 1) was identified as the sequence encoding the CAD enzyme.   In a further study, using the above approach,Eucalyptus grandisYeast The cDNA sequence encoding the element was isolated. PAL (SEQ ID NO: 16); C4H (SEQ ID NO: 17); C3H ( (SEQ ID NO: 18); F5H (SEQ ID NO: 19-21); OMT (SEQ ID NO: 22-25); CCR (SEQ ID NO: 26-29); CAD (SEQ ID NO: 30); CGT (SEQ ID NOs: 31-33); CBG (SEQ ID NO: 34); PNL (SEQ ID NOs: 35, 36); LAC ( And POX (SEQ ID NOs: 42-44).                                   Example 2 Isolation and characterization of cDNA from Pinus radiata (A) Isolation of cDNA clones by high yield screening   From KibePinus RadiataA cDNA expression library was constructed and described in Example 1. Was screened as follows. Applied Biosystems Prism 377 Sequence Use forward and reverse primers to obtain the DNA sequence of the positive clone The determined sequence was compared to known sequences in the above database.   Based on similarities to known sequences from other plant species, the isolated DNA Enzyme, C4H (SEQ ID NOs: 2 and 3); C3H (SEQ ID NO: 4); PNL (SEQ ID NO: 5); OMT (SEQ ID NO: 6 ); CAD (SEQ ID NO: 7); CCR (SEQ ID NO: No. 8); identified as encoding PAL (SEQ ID NO: 9-11) and 4CL (SEQ ID NO: 12) did.   In a further study, using the above approach,Pinus RadiataEnzyme, PAL SEQ ID NOs: 45-47); C4H (SEQ ID NOs: 48, 49); C3H (SEQ ID NOs: 50-52); OMT (SEQ ID NOs: 53-55); L (SEQ ID NO: 56, 57); CCR (SEQ ID NO: 58-70); CAD (SEQ ID NO: 71); CGT (SEQ ID NO: 72); CBG ( SEQ ID NOS: 73-80); PNL (SEQ ID NO: 81); LAC (SEQ ID NOs: 82-84); and POX (SEQ ID NOs: 85-8) An additional cDNA sequence encoding 8) was isolated.(b) PCR Of cDNA clones by HPLC   Domain conserved in the following peroxidase sequence previously identified in other species Based on two PCR probes (hereinafter LNB010 and LNB011 (SEQ ID NOs: 14 and 1 respectively) 5)) was designed. That is, bumppox (vanpox), hibupox 6 (h vupox6), taepox, hibpox1 (hvupoxl), osapox (osa pox), notopox2 (ntopox2), notopox1 (ntopox1), respox (le spox), pokpox, luspox, aspox , Hillpox, hrspox, spopox and tvepox (remains) Gene bank access number, D11337, M83671, X56011, X58396, X66125, J02979, D11396, X71593, D11102, L07554, M58381, X57564, Z22920 and Z31011).   RNA was isolated from pine xylem and first strand cDNA was synthesized as described above. LNB010 (4μ M), LNB011 (4 μM), 1 × Kogen's buffer, BSA (0.1 mg / ml), dN TP (200mM), Mg²⁺(2 mM) and PCR using Taq polymerase (Kibco BRL) (0.1 U / μl). Performed with this cDNA. The condition is 2 minutes at 94 ° C, 1 minute at 55 ° C and 1/2 minute at 72 ° C on a Tratagen robocycler 25 cycles of 1 minute at 94 ° C, 1 minute at 55 ° C and 1 minute at 72 ° C; 1 minute at 94 ° C 18 cycles of 1 minute at 55 ° C and 3 minutes at 72 ° C. Regenerate genes in the same way Width. Approximately 200 bp band for Schleicher and Sur Eleuquick (Schleicher & Schull Elu-Quik) TAE agarose skeleton using DNA purification kit And purified by T-tail p blue script vector (Marchuk, D.)N ucleic Acids Res .19: 1154, 1991). Kind to known sequence Based on the similarity, the isolated gene (SEQ ID NO: 13) was converted to pine peroxidase (POX). Was identified as a gene encoding                                   Example 3 For altering lignin biosynthesis O- Use of the methyltransferase (OMT) gene (A) Transformation of tobacco with the Pinus radiata OMT gene Pinus RadiataA sequence containing the coding region of OMT (SEQ ID NO: 53) from The sense and antisense construct comprising the compound can be prepared by published methods (An, G). Ebert (PR), Mitra (A), Ha (SB): "binary vector", Kelvin (Gelvin, SB), Schilperoort (RA) ed .: "Plant molecular biology" Manual ", Kluwer Publishers, Dordrecht (1988))) In conversionAgrobacterium tumefaciensLBA4301 (Doctor Cado (C. Kado ), Donated by the University of California, Davis, CA). Transformation construct Restriction of presence and integration of digestion and DNA Validated by sequencing.   Tobacco leaves (Nicotiana Tabacam(Nicotiana tabacum) cv.Samsung) Hold the section Transformation was performed using the method of Horsch et al. (Science, 227: 1229-1231, 1985). 5 independent lines of transformed plants per sense sense construct and antisense structure of OMT Eight independent transformed plant lines were established for each product. Suitable lignin gene constructs The transformed plants containing were verified by Southern blot experiments. In Table 1 shown below, "+" In the "Southern" column confirms that the transformed plant line is an independent transformation line Indicates that it was done.(B) Expression of Pinus OMT in transgenic plants   Independently transformed plants created with OMT sense and antisense constructs Total RNA was isolated from the product line. RNA samples were analyzed by Northern blotting The transgene expression level in the quality conversion line was determined. "Northern" column in Table 1 The data shown in the figure are for a transformed plant library containing OMT sense and antisense constructs. High levels of expression compared to the Northern blot background Is presented. OMT expression in sense plant line number 2 indicates that RNA samples It was not measured because it showed signs. From a control plant with an empty vector No hybridization could be detected in the RNA samples.(C) Regulation of OMT enzyme activity in transgenic plants   In transgenic tobaccoPinusOMT gene and endogenous tobacco OMT gene Activity of the oxidized OMT enzyme Each of the transformed plant lines created with the antisense construct was analyzed. Crude protein extracts were prepared from each transformed plant and were prepared according to Zhang et al. Law (Plant Physiol.,113: 65-74, 1997)). "Enzyme" column in Table 1 The data shown in the above shows that the transformed plant line containing the OMT sense construct Normally, the activity of the OMT enzyme is increased (up to 199%) as compared to the introduced control plant. Transformed plant lines containing the antisense construct usually have reduced OMT enzyme activity (minimal 35%). Do not evaluate the activity of OMT enzyme of sense plant line number 3 Was.(D) Effect of Pinus OMT on lignin concentration in transgenic plants   The lignin concentration of the transformed tobacco plants was determined using an established method, thioglycol. Cholic acid extraction (Freudenberg et al., "Structure and biosynthesis of lignin. , "Springer-Verlag, Berlin, 1968). Outline And freeze the whole tobacco plant on average age 38 days in liquid nitrogen and pulverize it with a mortar and pestle did. Empty vector transfer control plant line 1, independent transformation containing OMT sense construct Transgenic plant line 5, and an independent transformed plant line containing the OMT antisense construct 8.Extract 100mg of frozen powder from methanol individually with methanol, then with 10% thioglycolic acid And finally dissolved in 1M NaOH. The absorbance of the final extract was assayed at 280 nm. The data in the “TGA” column in Table 1 All transformed plant lines containing the E. coli and antisense OMT gene constructs Lignin levels were significantly reduced compared to Show.Table 1   These data indicate that lignin concentrations were measured by lignin such as OMT, as measured by the TGA assay. Manipulate directly with either sense or antisense expression of biosynthetic genes Show clearly what you can do.                                   Example 4 To alter lignin biosynthesis Four- Coumarate: Use of CoA ligase (4CL) gene (A) Transformation of tobacco plant with Pinus radiata 4CL gene Pinus RadiataA sequence containing the coding region of 4CL (SEQ ID NO: 56) from The comprising sense and antisense constructs are directly transformed as described above.A Globacterium tumefaciens Inserted into LBA4301. Of the transformed structure Presence and integration were verified by restriction digestion and DNA sequencing.   tobacco(Nicotiana Tabacamcv. Samsung) Transform leaf sections as described above. Changed. 5 independent lines of transformed plants and 4CL antisense Eight independent lines of transformed plants were established for each construct. Proper lignin inheritance Transgenic plants containing the offspring were verified by Southern blot experiments. Table shown below 2, “+” in the “Southern” column indicates that the listed transformed plant lines were transformed independently. Indicates that the line was confirmed as a replacement line.(B) Pinus 4CL expression in transformed plants   Independently transformed plants created with 4CL sense and antisense constructs Total RNA was isolated from the product line. RNA samples were analyzed by Northern blotting The expression level of the transgene in the quality conversion line was determined. In Table 2 below, "Northern" Transformed plants containing 4CL sense and antisense constructs All lines have high expression levels compared to the Northern blot background. This indicates that the file was presented. 4CL expression of antisense plant line number 1 As no RNA sample was obtained, no measurement was performed. Vs introducing an empty vector No hybridization could be detected in RNA samples from control plants.(C) Regulation of 4CL enzyme activity in transgenic plants   In transgenic tobacco plantsPinus4CL gene and endogenous tobacco 4CL gene The full activity of the 4CL enzyme encoded by 4CL sense and antisense structures Each transformed plant created with the product was analyzed. Crude protein extract Prepared from each transformed plant, the method of Zhang et al. (Plant Physiol.,11 Three : 65-74, 1997)). The data shown in the "Enzyme" column in Table 2 is 4CL The transformed plant line containing the sense construct is compared to the empty vector transfected control plant. Shows an increase in activity of the 4CL enzyme (up to 258%) but includes the 4CL antisense construct It shows that the activity of 4CL enzyme is reduced (minimum 59%) in the transformed plant line.(D) Effect of Pinus 4CL on lignin concentration in transgenic plants   Lignin concentration in a sample of transformed plant material is determined as described in Example 3. did. The data in the "TGA" column in Table 2 are the sense and antisense 4CL gene constructs All transformed plant lines containing 4 shows that it exhibited a significant decrease in lignin levels. These data are lignin The concentration was determined by the TGA assay, as indicated by the sense expression of lignin biosynthesis genes such as 4CL. Or that it can be directly manipulated with either antisense expression.                                   table2 Example 5 Transformation of tobacco using the lignin biosynthesis gene of the present invention Eucalyptus grandisC3H (SEQ ID NO: 18), F5H (SEQ ID NO: 19), CC R (SEQ ID NO: 25) and CGT (SEQ ID NO: 31),Pinus LachitaFrom PAL (SEQ ID NO: 45,47), C4H (SEQ ID NO: 48,49), PNL (SEQ ID NO: 81) and LAC (SEQ ID NO: 83) Sense and antisense constructs comprising sequences comprising the Sea urchin directly By transformationAgrobacterium tumefaciensInserted into LBA4301. Trait The presence and integration of the converted construct was verified by restriction digestion and DNA sequencing.   tobacco(Nicotiana Tabacamcv.Samsung) Leaf sections were characterized as shown in Example 3. Converted. Up to 12 independent transformed plant lines are listed in the previous paragraph Established for each sense structure and each antisense structure . Transgenic plants containing the appropriate lignin gene constructs are examined by Southern blotting Testified. All of the transformed plant lines analyzed were identified as independent transformation lines. It has been certified.                                   Example 6 Manipulation of lignin content in transgenic plants (A) Confirmation of transgene expression by Northern blotting   All RNA was isolated from each of the independent transformed plant lines shown in Example 5. . RNA samples were analyzed by Northern blotting and transferred to each transformation line. The expression level of the transgene was determined. The "Northern" column in Table 3 shows all plants tested. The transgene expression level for the line was compared to the background of the Northern blot. Shown in comparison. RNA samples from empty vector transfection control plants include hybrid Could not be detected.(B) Determination of lignin concentration in transgenic plants   Empty vector-transferred control plants and the respective sense constructs shown in Example 5 and Independent transformation lines for antisense constructs Lignin concentrations up to 12 were determined as described in Example 3. "TGA" column shown in Table 3 Is thioglycolic acid extractable lignin for all tested plant lines Indicates the average percentage of TGA extractable lignin in the transformed plant relative to the control plant To display. The range of fluctuation is shown in parentheses.                                   Table 3   Transgenic plants containing sense and antisense lignin biosynthetic gene constructs All of the lignin levels were significantly higher than the empty vector transfection control plant line. Showed a decrease. The most dramatic effect on lignin concentration was with F5H antisense plants. In the plant Of lignin in the PAL antisense plant and 37% in the control plant %Met. These data show that lignin concentrations measured by the TGA assay can be The sense method and the sense overexpression using the lignin biosynthesis gene of the present invention It clearly shows that it can also be manipulated directly.                                   Example 7 Regulation of lignin enzyme activity in transgenic plants   The activity and substrate specificity of the selected lignin biosynthetic enzymePinus Radiata PAL (SEQ ID NO: 45), PNL (SEQ ID NO: 81) and LAC (SEQ ID NO: 83),Eucalyptus Tas Grandis Sense and antisense per CGT from SEQ ID NO: 31) Assays were performed on crude extracts from transformed tobacco plants containing the structure.   Enzyme assays were performed as published. That is, for PAL, (Southerton, S.G.) and Deverall, BJ.Plant P ath .39: 223-230,1990), CGT is Vellekoop (P.) et al. (FEBS,330: 36- 40, 1993), and PNL is based on Espin (C.J.) et al.Phytochemistry,44: 17-22, 1997) As for LAC and LAC, Bao et al.'S method (Science,260: 672-674, 1993) You. The data shown in the "Enzyme" column of Table 4 are for all plant lines tested. The average enzyme activity from the replication treatment is shown, and the enzyme activity of an empty vector-transferred control plant %. The values in parentheses indicate the fluctuation range.Table 4   All transformed plant lines are empty except PNL antisense transformed plant lines Of average lignin enzyme activity is significantly lower than that observed in Showed sex. The most dramatic effect on lignin enzyme activity is that all lines A PAL antisense transformed plant line showing reduced PAL activity and an empty vector LAC antisense-transformed plant line showing 14% of the LAC activity of the incoming control plant line Was seen in                                   Example 8 Functional identification of lignin biosynthesis gene Pinus RadiataPAL (SEQ ID NO: 47), OMT (SEQ ID NO: 53), 4CL (SEQ ID NO: 56,57) and POX (SEQ ID NO: 86),Eucalyptus grandisOMT (array from Nos. 23, 24), CCR (SEQ ID NOs: 26-28), CGT (SEQ ID NOs: 31, 33) and POX (SEQ ID NOs: 42, 4) A sense construct comprising a sequence comprising the coding region of 4) is commercially available Natan It was inserted into pProEX-1 (Kibco BRL) which is a protein expression vector. The resulting structure Was transformed into E. coli XL1-Blue (Stratadiene), which was then induced to Was added to produce a recombinant protein.PinusOMT and 4CL structures,Eucalyptus For OMT and POX structures, purify the purified protein Matography (Janknecht, R.)Proc . Natl. Acad. Sci.,88 : 8972-8976, 1991). About each purified protein In the enzyme assay, the expected substrate specificity and activity of the enzyme for the tested gene Was finally demonstrated.   Pinus Radiata4CL gene (SEQ ID NO: 56) andPinus RadiataOMT genetics Data on two representative enzyme assays showing evidence of activity of the offspring (SEQ ID NO: 53). Table 5 shows the results. For 4CL enzymes, one unit is the rate of the substrate at 0.1 absorbance units per minute. Equal to the amount of protein required to convert to product. 1 unit for OMT enzyme Is equal to the amount of protein required to convert 1 pmol substrate to product per minute. New                                   Table 5   The data shown in Table 5 shows that both the purified 4CL enzyme and the purified OMT enzyme were high in the enzyme assay. Activity and confirm the identity of the 4CL and OMT genes described in this application. You. Crude protein preparation from Escherichia coli transformed with empty vector was tested for 4CL enzyme And no activity in any of the OMT enzyme assays.   For a clear understanding of the present invention, the present invention will be described in some detail by way of illustration and example. Although described, changes and modifications may be made without departing from the scope of the invention. It is intended that the invention be limited only by the scope of the appended claims. And intended.

[Procedure for Amendment] [Date of Submission] March 12, 1999 (1999.3.12) [Content of Amendment] (1) The following statement follows “Including Sequence” on page 4, 3 lines of the specification. Subscribe. "In a preferred embodiment, the isolated DNA sequence is: nucleotides 1-535 of SEQ ID NO: 1; nucleotides 46-671 of SEQ ID NO: 2; nucleotides 1-535 of SEQ ID NO: 3; nucleotides 290-949 of SEQ ID NO: 4; 5 nucleotides 15-959; SEQ ID NO: 6 nucleotides 15-1026; SEQ ID NO: 7 nucleotides 15-1454; SEQ ID NO: 8 nucleotides 15-740; SEQ ID NO: 9 nucleotides 108-624; SEQ ID NO: 10 nucleotides 68- 274; nucleotides 1-765 of SEQ ID NO: 11; nucleotides 1-384 of SEQ ID NO: 12; nucleotides 1-278 of SEQ ID NO: 13; nucleotides 14-472 of SEQ ID NO: 16; nucleotides 1-6 72 of SEQ ID NO: 17; SEQ ID NO: 18 nucleotides 15-469; SEQ ID NO: 19 nucleotides 15-469; SEQ ID NO: 20 nucleotides 1-341; SEQ ID NO: 21 nucleotides 15-387; SEQ ID NO: 22 nucleotides 1-443; SEQ ID NO: 23 nucleotides 15- 607; nucleotides 15-421 of SEQ ID NO: 24; SEQ ID NO: SEQ ID NO: 26 nucleotides 1-760; SEQ ID NO: 26 nucleotides 58-469; SEQ ID NO: 27 nucleotides 15-495; SEQ ID NO: 28 nucleotides 15-472; SEQ ID NO: 29 nucleotides 13-396; SEQ ID NO: 30 nucleotide 15 -592; nucleotides 15-468 of SEQ ID NO: 31; nucleotides 1-405 of SEQ ID NO: 32; nucleotides 1-380 of SEQ ID NO: 33; nucleotides 1-305 of SEQ ID NO: 34; nucleotides 15-693 of SEQ ID NO: 35; SEQ ID NO: 36 nucleotides 1-418; SEQ ID NO: 37 nucleotides 15-777; SEQ ID NO: 38 nucleotides 1-344; SEQ ID NO: 39 nucleotides 1-341; SEQ ID NO: 40 nucleotides 15-358; SEQ ID NO: 41 nucleotide 1 -409; nucleotides 1-515 of SEQ ID NO: 42; nucleotides 15-471 of SEQ ID NO: 43; nucleotides 15-487 of SEQ ID NO: 44; nucleotides 108-664 of SEQ ID NO: 45; nucleotides 15-418 of SEQ ID NO: 46; sequence Nucleotides 47-479 of SEQ ID NO: 47; nucleotides 127-1785 of SEQ ID NO: 48; Nucleotides 15-475 of SEQ ID NO: 50; nucleotides 288-801 of SEQ ID NO: 50; nucleotides 51-711 of SEQ ID NO: 51; nucleotides 1-426 of SEQ ID NO: 52; nucleotides 92-562 of SEQ ID NO: 53; nucleotides 1 of SEQ ID NO: 54 -1074; nucleotides 1-1075 of SEQ ID NO: 55; nucleotides 1-11961 of SEQ ID NO: 56; nucleotides 1-1010 of SEQ ID NO: 57; nucleotides 15-741 of SEQ ID NO: 58; nucleotides 1-643 of SEQ ID NO: 59; SEQ ID NO: Nucleotides 15-441 of SEQ ID NO: 61; nucleotides 15-913 of SEQ ID NO: 62; nucleotides 15-680 of SEQ ID NO: 63; nucleotides 15-524 of SEQ ID NO: 64; nucleotides 1-524 of SEQ ID NO: 65 417; nucleotides 1-511 of SEQ ID NO: 66; nucleotides 176-609 of SEQ ID NO: 67; nucleotides 1-474 of SEQ ID NO: 68; nucleotides 1-474 of SEQ ID NO: 69; nucleotides 176-608 of SEQ ID NO: 70; SEQ ID NO: 71 Nucleotides 15-1474 of SEQ ID NO: 72 -1038; nucleotides 1-372 of SEQ ID NO: 73; nucleotides 18-545 of SEQ ID NO: 74; nucleotides 40-463 of SEQ ID NO: 75; nucleotides 32-435 of SEQ ID NO: 76; nucleotides 15-451 of SEQ ID NO: 77; SEQ ID NO: 78 nucleotides 1-374; SEQ ID NO: 79 nucleotides 1-457; SEQ ID NO: 80 nucleotides 1-346; SEQ ID NO: 81 nucleotides 15-957; SEQ ID NO: 82 nucleotides 40-452; SEQ ID NO: 83 nucleotide 15- 471; nucleotides 1-338 of SEQ ID NO: 84; nucleotides 150-1229 of SEQ ID NO: 85; nucleotides 1-1410 of SEQ ID NO: 86; nucleotides 1-687 of SEQ ID NO: 87; and nucleotides 1-688 of SEQ ID NO: 88. A nucleotide sequence selected from the group consisting of: "(2) From page 15, line 15 to the end of the same book," Specifically, the present invention provides ... an isolated DNA sequence. " The "Specifically, the present invention is the enzyme CAD (SEQ ID NO: 1, in particular nucleotides 1-535; and SEQ ID NO: 30, in particular nucleotides 15-592) from Eucalyptus grandis, PAL (SEQ ID NO: 16, in particular nucleotides 14-472 ), C4H (SEQ ID NO: 17, especially nucleotides 1-672), C3H (SEQ ID NO: 18, especially nucleotides 15-469), F5H (SEQ ID NO: 19, especially nucleotides 15-469; SEQ ID NO: 20, especially nucleotides 1-341; And SEQ ID NO: 21, especially nucleotides 15-387), OMT (SEQ ID NO: 22, especially nucleotides 1-443; SEQ ID NO: 23, especially nucleotides 15-607; SEQ ID NO: 24, especially nucleotides 15-421; and SEQ ID NO: 25, especially Nucleotides 1-760), CCR (SEQ ID NO: 26, especially nucleotides 58-469; SEQ ID NO: 27, especially nucleotides 15-495; SEQ ID NO: 28, especially nucleotides 15-472; and SEQ ID NO: 29, especially nucleotides 13-396), CGT (SEQ ID NO: 31, especially nucleotides 15-468; SEQ ID NO: 32, especially nucleotides 1-4) 05; and SEQ ID NO: 33, especially nucleotides 1-380), CBG (SEQ ID NO: 34, especially nucleotides 1-305), PNL (SEQ ID NO: 35, especially nucleotides 15-693; and SEQ ID NO: 36, especially nucleotides 1-418) LAC (SEQ ID NO: 37, especially nucleotides 15-777; SEQ ID NO: 38, especially nucleotides 1-344; SEQ ID NO: 39, especially nucleotides 1-341; SEQ ID NO: 40, especially nucleotides 15-358; and SEQ ID NO: 41, especially nucleotides 1-409) and POX (SEQ ID NO: 42, in particular the nucleotide 1-515; SEQ ID NO: 4 3, particularly nucleotides 15-571; and SEQ ID NO: 44, in particular nucleotides 15-487), as well as enzymes C4H (SEQ ID from Painasu Radjiata 2, especially nucleotides 46-671; SEQ ID NO: 3, especially nucleotides 1-535; SEQ ID NO: 48, especially nucleotides 127-1785; and SEQ ID NO: 49, especially nucleotides 15-475), C3H (SEQ ID NO: 4, especially nucleotides 290- 949; SEQ ID NO: 50, especially nucleotides 288-801; SEQ ID NO: 51, especially nucleotides Ptide (SEQ ID NO: 5, especially nucleotides 15-959; and SEQ ID NO: 81, especially nucleotides 15-957), OMT (SEQ ID NO: 6, especially nucleotide 15). -1026; SEQ ID NO: 53, especially nucleotides 92-562; SEQ ID NO: 54, especially nucleotides 1-1074; and SEQ ID NO: 55, especially nucleotides 1-1075), CAD (SEQ ID NO: 7, especially nucleotides 15-1454; and SEQ ID NO: 71, especially nucleotides 15-1474), CCR (SEQ ID NO: 8, especially nucleotides 15-740; SEQ ID NO: 58, especially nucleotides 15-741; SEQ ID NO: 59, especially nucleotides 1-643; SEQ ID NO: 60, especially nucleotides 15-441 SEQ ID NO: 61, especially nucleotides 15-913; SEQ ID NO: 62, especially nucleotides 15-680; SEQ ID NO: 63, especially nucleotides 15-492; SEQ ID NO: 64, especially nucleotides 15-524; SEQ ID NO: 65, especially nucleotides 1-417 SEQ ID NO: 66, especially nucleotides 1-511; SEQ ID NO: 67, especially nucleotides 176-609; SEQ ID NO: 68, especially nucleotides 1-474; SEQ ID NO: 69, especially nucleotides 1-474; and SEQ ID NO: 70, especially nucleotides 176-608), PAL (SEQ ID NO: 9, especially nucleotides 108-624; SEQ ID NO: 10, especially Nucleotides 68-274; SEQ ID NO: 11, especially nucleotides 1-765; SEQ ID NO: 45, especially nucleotides 108-664; SEQ ID NO: 46, especially nucleotides 15-418; and SEQ ID NO: 47, especially nucleotides 65-479), 4CL (sequence SEQ ID NO: 12, especially nucleotides 1-384; SEQ ID NO: 56, especially nucleotides 1-1196; and SEQ ID NO: 57, especially nucleotides 1-1010), CGT (SEQ ID NO: 72, especially nucleotides 15-1038), CBG (SEQ ID NO: 73, SEQ ID NO: 74, especially nucleotides 40-463; SEQ ID NO: 76, especially nucleotides 32-435; SEQ ID NO: 77, especially nucleotides 15-451; SEQ ID NO: 78; Especially nucleotides 1-374; SEQ ID NO: 79, especially nucleotides 1-457; and SEQ ID NO: 80, particularly Nucleotides 1-346), LAC (SEQ ID NO: 82, especially nucleotides 40-4 52; SEQ ID NO: 83, especially nucleotides 15-471; and SEQ ID NO: 84, especially nucleotides 1-338) and POX (SEQ ID NO: 13, especially nucleotides 1-278; SEQ ID NO: 85, especially nucleotides 150-1229; SEQ ID NO: 86, especially nucleotides 1-1410; SEQ ID NO: 87, especially nucleotides 1-687; and SEQ ID NO: 88, especially nucleotides 1-688). Provide the isolated DNA sequence. "

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (GH, KE, LS, MW, S D, SZ, UG, ZW), EA (AM, AZ, BY, KG) , KZ, MD, RU, TJ, TM), AL, AM, AT , AU, AZ, BA, BB, BG, BR, BY, CA, CH, CN, CU, CZ, DE, DK, EE, ES, F I, GB, GE, GH, HU, ID, IL, IS, JP , KE, KG, KP, KR, KZ, LC, LK, LR, LS, LT, LU, LV, MD, MG, MK, MN, M W, MX, NO, NZ, PL, PT, RO, RU, SD , SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UG, UZ, VN, YU, ZW (72) Inventors Gliason, Alistair Walas             New Zealand Auckland Buck             Lands Beach Medina Place 1             /twenty four (72) Inventors Havkkala, Ilkka Jacco             New Zealand Auckland Missis             Yong Bay Atkin Avenue 3             / 121

Claims (1)

[Claims] 1. (a) the sequences listed in SEQ ID NOs: 3,13,16-70,72-88,   (b) the complement of the sequence listed in SEQ ID NOs: 3,13,16-70,72-88,   (c) reverse complements of the sequences listed in SEQ ID NOs: 3,13,16-70,72-88,   (d) a reverse sequence of the sequence strengthened to SEQ ID NOs: 3,13,16-70,72-88,   (e) Probability of being the same as sequence (a)-(d) measured by computer algorithm FASTA Having at least about 99% of An isolated DNA sequence comprising a nucleotide sequence selected from the group consisting of: 2. A DNA construct comprising the DNA sequence of claim 1. 3. A transformed cell comprising the DNA construct of claim 2. 4. In the 5'-3 'direction,   (a) a gene promoter sequence;   (b) the sequences and computer algorithms listed in SEQ ID NOs: 3,13,16-70,72-88 Have the same probability as the sequence of SEQ ID NO: 3,13,16-70,72-88 as measured by FASTA A nucleotide sequence selected from the group consisting of at least about 99% sequences. Open reading frame that encodes at least the functional part of the read enzyme And   (c) gene termination sequence A DNA structure comprising: 5. The DN of claim 4, wherein the open reading frame is in a sense orientation. A structure. 6. The method of claim 4, wherein the open reading frame is in an antisense orientation. DNA structure shown. 7. The gene promoter sequence and gene termination sequence are functional in a plant host. The DNA construct according to claim 4. 8. The DNA structure of claim 4, wherein the gene promoter sequence results in transcription in xylem. Structure. 9. The DNA structure according to claim 4, further comprising a marker for identifying a transformed cell. 10. In the 5'-3 'direction,   (a) a gene promoter sequence;   (b) the sequences and computer algorithms listed in SEQ ID NOs: 3,13,16-70,72-88 The probability of being the same as the sequence of SEQ ID NO: 3,13,16-70,72-88 At least 99% by nucleotide sequence selected from the group consisting of A non-coding region of the gene encoding the encoded enzyme;   (c) gene termination sequence A DNA structure comprising: 11． 11. The DNA construct according to claim 10, wherein the non-coding region is in a sense orientation. 12． The DNA structure according to claim 10, wherein the non-coding region is in an antisense orientation. Structure. 13. The gene promoter sequence and the gene termination sequence are functional in a plant host. 11. The DNA structure according to claim 10, wherein 14. 11. The DNA structure according to claim 10, wherein the gene promoter sequence results in transcription in xylem. Structure. 15. In the 5'-3 'direction,   (a) a gene promoter sequence;   (b) the sequences and computer algorithms listed in SEQ ID NOs: 3,13,16-70,72-88 The probability of being the same as the sequence of SEQ ID NO: 3,13,16-70,72-88 A nucleotide sequence selected from the details consisting of at least about 99% sequence. Open reading frame that encodes at least the functional part of the read enzyme And   (c) gene termination sequence A transformed plant cell comprising a DNA construct comprising: 16． The shape of claim 15, wherein the open reading frame is in a sense orientation. Transgenic plant cells. 17． The method according to claim 15, wherein the open reading frame is in an antisense orientation. Transformed plant cells as described above. 18． The method according to claim 15, wherein the DNA construct further comprises a marker for identifying a transformed cell. Transformed plant cells as described above. 19. A plant comprising the transformed plant cell of claim 15 or a fruit or seed thereof. . 20. 20. The plant according to claim 19, wherein the plant is a woody plant. twenty one. The method according to claim 20, wherein the plant is selected from the group consisting of eucalyptus and pine species. On the plant. 22.5 In the 5'-3 'direction,   (a) a gene promoter sequence;   (b) the sequences and computer algorithms listed in SEQ ID NOs: 3,13,16-70,72-88 The probability of being the same as the sequence of SEQ ID NO: 3,13,16-70,72-88 A nucleotide sequence selected from the details consisting of at least about 99% sequence. A non-coding region of the gene encoding the encoded enzyme;   (c) gene termination sequence A transformed plant cell comprising a DNA construct comprising: twenty three. The transformed plant of claim 22, wherein the non-coding region is in a sense orientation. cell. twenty four. The transformation of claim 22, wherein the non-coding region is in an antisense orientation. Transgenic plant cells. twenty five. A plant comprising the transformed plant cell of claim 22 or a fruit or seed thereof. . 26. 26. The plant according to claim 25, wherein the plant is a woody plant. 27. The method according to claim 26, wherein the plant is selected from the group consisting of eucalyptus and pine species. On the plant. 28.5 In the 5'-3 'direction,   (a) a gene promoter sequence;   (b) the sequences and computer algorithms listed in SEQ ID NOs: 3,13,16-70,72-88 The probability of being the same as the sequence of SEQ ID NO: 3,13,16-70,72-88 At least 99% by nucleotide sequence selected from the group consisting of Open reading frame that encodes at least the functional part of the loaded enzyme And   (c) gene termination sequence Comprising stably integrating a DNA structure comprising A method for adjusting the lignin content. 29. The method according to claim 28, wherein the plant is selected from the group consisting of eucalyptus and pine species. The method described. 30. 29. The method of claim 28, wherein the open reading frame is in a sense orientation. Law. 31. The method according to claim 28, wherein the open reading frame is in an antisense orientation. The method described. 32.5 In the 3'-3 'direction,   (a) a gene promoter sequence;   (b) Sequences listed in SEQ ID NOs: 3,13,16-70,72-88 and computer alcohol The probability of being the same as the sequence of SEQ ID NO: 3,13,16-70,72-88 At least 99% by nucleotide sequence selected from the group consisting of A non-coding region of the gene encoding the encoded enzyme;   (c) gene termination sequence Comprising stably integrating a DNA structure comprising A method for adjusting the lignin content. 33. 33. The method of claim 32, wherein said non-coding region is in a sense orientation. 34. 33. The method of claim 32, wherein said non-coding region is in an antisense orientation. 35. The method according to claim 32, wherein the plant is a woody plant. 36. 36. The method of claim 35, wherein said plant is selected from the group consisting of eucalyptus and pine species. Law. 37. (a) In the 5'-3 'direction, the gene promoter sequence and SEQ ID NOs: 3,13,16-70,7 The sequences listed in 2-88 and the sequence numbers determined by computer algorithm FASTA No. 3,13,16-70,72-88 from sequences that have at least about 99% probability of being the same as the sequences At least one of the enzymes encoded by the nucleotide sequences selected from the group Reading frame encoding a functional part, and transformed cells Transforming a plant cell with a DNA construct comprising When,   (b) culturing the transformed cells under conditions that promote regeneration and growth of mature plants thing A method for producing a plant having an altered lignin structure comprising: 38. The method of claim 37, wherein the open reading frame is in a sense orientation. Law. 39. The method according to claim 37, wherein the open reading frame is in an antisense orientation. The method described. 40. The method according to claim 37, wherein the plant is a woody plant. 41. The method according to claim 40, wherein the plant is selected from the group consisting of eucalyptus and pine species. The method described. 42. (a) In the 5'-3 'direction, the gene promoter sequence and SEQ ID NOs: 3,13,16-70,7 Sequences listed in 2-88 and sequence numbers measured by computer algorithm FASTA No. 3,13,16-70,72-88 from sequences that have at least about 99% probability of being the same as the sequences Encoding an enzyme encoded by a nucleotide sequence selected from the group consisting of: A non-coding region of the gene, as well as a gene termination sequence that gives the transformed cell Transforming a plant cell with a DNA structure comprising   (b) culturing the transformed cells under conditions that promote regeneration and growth of mature plants thing A method for producing a plant having an altered lignin structure comprising: 43. 43. The method of claim 42, wherein said non-coding region is in a sense orientation. 44. 43.The non-coordinator region is in an antisense orientation. The method described in. 45. 43. The method according to claim 42, wherein the plant is a tree. 46. The method of claim 45, wherein the plant is selected from the group consisting of eucalyptus and pine species. The method described. 47. (a) a gene promoter sequence;   (b) the sequences and computer algorithms listed in SEQ ID NOs: 3,13,16-70,72-88 The probability of being the same as the sequence of SEQ ID NO: 3,13,16-70,72-88 A nucleotide sequence selected from the details consisting of at least about 99% sequence. Open reading frame that encodes at least the functional part of the read enzyme And   (c) gene termination sequence Comprising stably incorporating a DNA structure comprising To change the activity of the enzyme in 48. The method of claim 47, wherein the open reading frame is in a sense orientation. Law. 49. The method of claim 47, wherein the open reading frame is in an antisense orientation. The method described. 50. (a) a gene promoter sequence;   (b) the sequences and computer algorithms listed in SEQ ID NOs: 3,13,16-70,72-88 The probability of being the same as the sequence of SEQ ID NO: 3,13,16-70,72-88 At least 99% by nucleotide sequence selected from the group consisting of A non-coding region of the gene encoding the encoded enzyme;   (c) gene termination sequence And stably incorporating a DNA structure comprising A method for changing the activity of an enzyme in a plant comprising: 51. 51. The method of claim 50, wherein said non-coding region is in a sense orientation. 52. 51. The method of claim 50, wherein said non-coding region is in an antisense orientation. 53. The method according to claim 50, wherein the plant is a woody plant. 54. The method according to claim 53, wherein the plant is selected from the group consisting of eucalyptus and pine species. The method described.