JPH09502601A - Putrescine N-methyltransferase, recombinant DNA molecules encoding putrescine N-methyltransferase, and transgenic tobacco plants with reduced alkaloid content - Google Patents

Abstract

  (57) [Summary] This invention provides a highly purified tobacco putrescine N-methyltransferase, a method for its purification, and a method for producing a PMT DNA sequence. The purification method includes a step of adding a tobacco root extract to an anion exchange medium and specifically eluting putrescine N-methyltransferase with an elution buffer containing putrescine.

Description

Detailed Description of the Invention     Putrescine N-methyl transferase,     Coat tresin N-methyltransferase     Containing recombinant DNA molecules and alkaloids     Transgenic tobacco plants with reduced abundance                            TECHNICAL FIELD OF THE INVENTION   This invention provides a highly purified putrescine N-methyltransferase, The novel purification method, and the antisense gene and the sense gene You. In particular, the present invention provides transgenics with genetically altered nicotine levels. Sense and antisense putrescine N-meth for creating tobacco plants The use of the le transferase gene. Transgenic plant like this The article is useful for producing dried tobacco leaves for use in the tobacco industry.                               Background of the Invention   Various methods have been adopted to remove nicotine from tobacco. But these Most of these methods are not sufficiently selective for nicotine. These methods are Removing other ingredients from Bako adversely affects the flavor and aroma of tobacco. So Moreover, such methods are generally complex and expensive.   Compounds that are biochemically synthesized with nicotine are generally produced by a series of biochemical reactions. Formed, each reaction is catalyzed by a different enzyme. A feature that yields a given compound A constant reaction sequence is known as one route. Path activation and its final One way to inhibit product production is to reduce the amount of enzyme required in the pathway . Several degrees of that enzyme, compared to other enzymes in the pathway, usually result in the If the enzyme's abundance is low enough to make the final product low enough to make the element rate limiting Production will be low. If the relative abundance of enzymes is not usually rate-limiting, That few degrees is necessary to make the enzyme rate limiting in order to reduce pathway output. It must be reduced to a minute. Similarly, if the relative abundance of enzymes is rate limiting, then An increase in the number of C.sub.i increases the yield of the end product of the pathway.   Nicotine is first formed in the roots of tobacco plants, then transported to the leaves and stored in the leaves. (Tso, Physiology and Biochemistry of Tobacco Plants, 233-234 P., Dowden Hutchinson & Ross, Stroudsburg, PA, USA , 1972). The nicotine molecule consists of two heterocycles, a pyridine moiety and a pyrrolidinium. And each of which is derived from a distinct biochemical pathway. Nicotine pie The lysine moiety is derived from nicotinic acid. The pyrrolidine moiety of nicotine is From succinate to N-methylputrescine and then to N-methylpyrroline Offered by the road. The essential step during nicotine biosynthesis is from putrescine to N -A process that produces methylputrescine (Goodwin and Mercer, Introduction  to Plant Biochemistry, pp. 488-491, pergamon press, Newyo, USA (1983)).   The conversion reaction of putrescine to N-methylputrescine is , S-adenosylmethine, which acts as a methyl group donor, is an enzyme Catalyzed by putrescine N-methyltransferase (“PMT”) . PMT is a pathway that supplies N-methylpyrroline for nicotine synthesis in tobacco And it seems to be a rate-limiting enzyme (Feth et al., “Regulation in Tobacco Callus of Enzyme  Activities of the Nicotine Pathway ", planta, 168, 402-407 P., 1986; Wagner et al., "The Regulation of Enzyme Activities of the N". icotine Pathway in Tobacco ", Physiol, Plant, 68, 667-672, 1986).   Limited characterization of relatively crude PMT formulations (30x purification) (Mizusaki et al., “Phytochemical Studies on Tobacco Alkaloids XIV . The Occurrence and Properties of Putrescine N-Methyltransferase in Tob acco Plants ", Plant cell Physiol., 12: 633-640, 1971. Year). The purification process leading to the drug product is the ammonium sulfate precipitation method from the first extract. And limited to gel filtration chromatography.   Significantly lower levels of enzymes (or other tags) than normally obtained by plants. A plant characterized by You. Normally, transcription of the gene encoding the target enzyme produces single-stranded mRNA. , The RNA is then translated by the ribosome to yield the target enzyme. Anti A sense RNA molecule has its nucleotide sequence paired with a portion of the target mRNA molecule. And is a complementary RNA molecule. Therefore, antisense RN The A molecule undergoes complementary base pairing with the target mRNA molecule (hybridization, hybridization or high hybridization). Bridation), making the target mRNA molecule unavailable for translation Make it easier to decompose, or do both. Therefore, the target mRNA encodes The ability of cells to produce specific enzymes is inhibited.   The antisense method has been adopted in several studies and the specific enzyme is Transgenic plants characterized by low levels have been created. For example, Plants containing Bell chalcone synthase, an enzyme in the flower pigment biosynthesis pathway, Inserting chalcone synthase antisense gene into the tobacco and petunia genomes Is produced by doing. These transgenic tobacco plants and plants Transgenic petunia plants produce lighter-than-normal color flowers ( Van der Krol et al., “An Anti-Sense Chalcone Synthase Gene in Transgenic Pla nts Inhibits Flower Pigmentation ", Nature, 333, 866-869. 1988). In addition, the antisense RNA method is applied to tomatoes and polygalacturoners. It has been successfully adopted to inhibit the production of zymase (Smith et al., “Ant isense RNA Inhibition of Polygalacturonase Gene Expression in Transgenic  Tomatoes ", Nature, 334, 724-726, 1988; Sheehy et al. “Reduction of Polygalacturonase Activity in Tomato Fruit by Antisense R NA ", Proc. Natl. Acad. Sci. USA, 85, 8805-8809, 1988. Years), and tobacco, a small support for the ribulose bisphosphate carboxylase enzyme. Unit Have been successful in inhibiting production (Rodermel et al., “Nuclear-organelle In teractions: Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carbox ylase Enzyme Levels in Transformed Tobacco Plants ”, Cell, 55, 67 3 to 681, 1988). Alternatively, it may be more than the usual amount of a given enzyme. The transgenic plant is characterized by its enzyme in the sense (ie, normal) orientation. It can be created by transforming a plant with the gene.   Increasing Nicotine Levels in Tobacco Plants by Changing PMT Levels Genetic engineering of the tobacco plants to be let down is not yet possible. The reason is PM There are known methods for cloning the PMT gene without prior purification of the T enzyme. And the method for purifying the PMT enzyme was not known before this invention. is there.                            Brief description of the drawings   Figure 1 shows the protein patterns obtained during successive steps of the tobacco PMT purification process. Is a photocopy of a 12.5% SDS-polyacrylamide gel stained with silver. You. Lanes 1 and 6: molecular weight reference protein (phosphorylase B, 95.5 KD; Glutamate dehydrogenase, 55.0 KD; Ovalbumin, 43.0 KD Lactate dehydrogenase, 36.0 KD; carbonic anhydrase, 29. 0KD; lactoglobulin, 18.4KD; cytochrome C, 12.4KD). Les Zone 2: 40-65% saturation ammonium sulfate fraction. Lane 3: Hydrophobic interaction PM from column T activity peak fraction. Lane 4: Elution with putrescine from anion exchange column Concentrated substance. Lane 5: Free flow of concentrated material derived from anion exchange column, etc. PMT activity peak fraction obtained by electrofocusing electrophoresis.   Figure 2 shows a 3mm thick continuous sliver from a 12.5% non-denaturing polyacrylamide gel. FIG. 3 is a graph showing the PMT activity of the column, and the gel of The concentrated material was loaded by elution with trescine. The enzymatic activity of PMT is the product Was recovered as¹⁴Expressed as C break variable / min (on background), It is named "sex".   FIG. 3 shows a band of PMT activity of a non-denaturing electrophoresis gel and a thickness of 3 mm before and after the band. Silver stain 12 serial slices (Figure 2) were analyzed for purity and apparent molecular weight. . It is a photocopy of a 5% SDS-polyacrylamide gel. Named "Sm" The lanes shown contain the starting material (ie the material added to the non-denaturing gel) ing. The lane named “Std” is a molecular weight standard protein (phosphorylase). ZeB, 95.5 KD; glutamate dehydrogenase, 55.0 KD; ovoal Bumin, 43.0 KD; lactate dehydrogenase, 36.0 KD; carbonic acid Nhydrase, 29.0 KD; lactoglobulin, 18.4 KD; cytochrome C , 12.4KD).   Figure 4 shows ammonium sulfate fractionation, hydrophobic interaction chromatography, and Concentrating the sample following elution with putrescine from the anion exchange column By subjecting the tobacco PMT purified by It is a graph which draws the relative PMT activity and pH of the obtained fraction. Enzyme activity of PMT Sex recovered as product¹⁴Expressed as C break variable / min (on background) And is designated as "relative activity".   Figure 5 shows excision (after electroblotting on an inert membrane) and determination of amino acid sequence. Silver stain showing the PMT protein bands ("a1" and "a2") that were determined. It is a photocopy of a 5% SDS-polyacrylamide gel. Loaded in gel The sample is isoelectrically focused on the substance eluted with putrescine from the anion exchange column. It is a part of the fraction obtained by electrophoresis. The actual bands used for sequence analysis are , Loaded with a portion of the same material analyzed in the gel of FIG. ) It is derived from polyacrylamide gel.                               Summary of the invention   The present invention is firstly directed to highly purified putrescine N-methyl transferase. And a novel method for purifying this enzyme.   The purification method of the present invention comprises the steps of adding a tobacco plant extract to an anion exchange medium. The addition temperature and the pH and composition of the extract depend on the PMT being anion exchange medium. It is a condition that is maintained. Next, the PMT contains an effective amount of polyamine Elute from the anion exchange medium with elution buffer. Elution temperature and elution buffer The pH and chemical composition of PMT, in the absence of polyamines, are It is a condition that is maintained.   In a preferred embodiment, the eluate of the anion exchange medium is dissolved in the anion exchange medium. Directly onto chromatographic media with an affinity for PMT in the presence of dissociation buffer Add and then elute the bound material. Most preferably, anion exchange color The outlet from the column is connected to the inlet of the ω-aminohexyl agarose column, Dilute PMT from the anion exchange column was collected in the column and then more concentrated PMT is eluted in a different form.   The PMT of the present invention has a molecular weight of about 55 to 65 kilodaltons and an unmodified isoelectric point. PH of about 5.0-6.0, Km value for putrescine of about 300 μM-50 0 μM and a Km value for S-adenosylmethionine of about 100 μM to 1 50 μM. In a preferred embodiment, PMT has the sequence listing SEQ ID NO: 1, SEQ ID NO: 2 and 17 amino acids selected from the amino acid sequence defined as SEQ ID NO: 3. It has an array of mino acids.   The present invention also provides a cell encoding putrescine N-methyltransferase. Recombinant DNA molecule, antisense recombinant DNA molecule, and these recombinant DNA molecules Vectors containing molecules and transformation with these DNA molecules or vectors A transgenic tobacco cell and a transgenic tobacco plant Things. Transgenic tobacco cells and transgenics of this invention Tobacco plants have higher nicotine content than untransformed control tobacco cells and tobacco plants. Characterized by low or high.                            Detailed description of the invention Purification of PMT   The starting material used for PMT purification consists of tobacco roots. The root is hydroponics It is preferable to harvest from cultivated tobacco plants. Hydroponics is highly controlled It is easy to grow plants under controlled and reproducible conditions and is clean and intact. In this state, the spread filamentous root system can be efficiently harvested.   Tobacco seeds are germinated on or near the surface of a moist plant potting mixture. Can be made. The most preferred conditions are a temperature of about 80 ° F and a relative humidity of 60%. is there. Approximately 2 weeks after the seeds have germinated, the seedlings are thinned (removed) and the residual seedlings are removed. The seedlings are about 6 inches high and grow unhindered until about 6 leaves are formed. Leave enough blank space for. When the seedlings reach a height of about 6 inches, generally, The root system and the pellets of potting material are left in place, and the appropriate nutrient solution is added. The solution is transplanted to a hydroponic device equipped with an aeration (oxidation) means. Also this hydroponic cultivation equipment The equipment must be equipped to supplement the dissolved nutrients, and It must be large enough to accommodate a grown tobacco plant.   Head flower removal (thinning) is a standard practice in commercial tobacco cultivation, but It is known in the art to increase growth and increase leaf nicotine content. You. Therefore, the plants used as starting materials for the purification of PMT usually grow Flowers are picked at the appropriate stage. Suitable for planting and thinning Different intervals include plant varieties, light intensity, photoperiod, soil and air temperature, soil water, among others. Determined by a number of factors including minute and mineral nutrition. However, the root is generally plucked Harvested 3 to 7 days after flowering. Where the plant is cultivated under the specified combination of growth conditions The optimum time for picking a given tobacco variety can be easily determined by a person skilled in the art. Can be   Harvested roots are preferably washed with cold water, then residual water is removed with a Buchner funnel. Aspirate and remove. The washed roots can then be harvested and used immediately or- Freeze to 80 ° C. Frozen roots are stored at about -80 ° C until use.   In the general PMT purification method, about 400 to 600 g of frozen roots per 11 extraction buffers are used. Homogenize the tissue with a high speed mixer. The extraction buffer is generally one or more buffers. Agents, one or more reducing agents, one or more heavy metal chelating agents, one or more water-active modifications And an effective amount of each of the one or more protease inhibitors. Ma The extraction buffer contains an effective amount of one or more phenolic compound adsorbents. Is preferred. Effective amounts of these drugs depend on the particular drug used . However, the amount used is generally the amount commonly used during plant protein purification. Selected from Therefore, the choice of drug and its effective dose will depend on the skill of the ordinary investigator. Within range.   The pH of the extraction buffer should be in the range of about 7.2-8.3, but is preferred. It is about 7.5. The dissociation constant (pKa) that gives an effective pH buffering capacity at the desired pH. ) Having water Soluble compounds are used. Also preferred buffers are virtually transparent to UV radiation. Appropriate Buffers include tris (hydroxymethyl) aminomethane (“Tris”) and imida Sol, phosphate, N-morpholinopropanesulfonic acid (“MOPS”), N- Tris (hydroxymethyl) methyl-2-aminoethanesulfonic acid (“TES” ), Triethanolamine, and N-tris (hydroxymethyl) -methyl- Glycine (“Tricine”) is included. Tris buffer is preferred.   Possible oxidation reactions of sulfhydryl groups of proteins and phenolic compounds of plants In order to inhibit the possible oxidation reaction of substances and to inhibit the formation of reactive free radicals , A reducing agent is added to the extraction buffer. Both of these reactions inactivate PMT Sometimes. Suitable reducing agents include dithiothreitol ("DTT"), dithioethol. Litolitol, 2-mercaptoethanol, thioglycolate, glutathione , Cysteine, and ascorbate. DTT and ascorbate Is preferred.   Heavy metal chelators activate proteases and interact directly with heavy metals. By action or by oxidation of phenolic compounds to PMT inactivating species In order to prevent PMT inactivation that may occur due to the acceleration of the reaction by heavy metals, Added to extraction buffer. As a preferable heavy metal chelating agent, ethylenedia There is mintetraacetic acid (“EDTA”), but other common chelating agents such as ethylene Glycol bis (β-aminoethyl ether) N, N. N ', N'tetraacetic acid ("EGTA") and citrate can also be used.   Water-activated denaturants deactivate PMTs and may cause denaturation and other minor changes. It is added to the extraction buffer to stabilize the PMT against various conformational changes. This Water-active modifiers such as are non-ionic hydrophilic compounds and these are added Decrease the water activity of the aqueous solution. Glycerin, ethylene glycol and small molecules An amount of polyethylene glycol (eg "PEG400") is preferred, but Sucrose, sucrose, fructose and sorbitol are water-active modifiers It is an example of another compound useful.   Protease inhibitors are proteolytic enzymes that are released during tissue homogenization. In order to prevent possible inactivation of PMT due to protein cleavage, Is added to the output buffer. A useful protease inhibitor is phenylmethylsulfone. Nilfluoride (“PMSF”), leupeptin, aprotinin, chymostatin And pepstatin are included. PMSF and leupeptin are preferred.   The presence of phenolic adsorbents, when present, causes acid when plant cells are destroyed. Excludes phenolic plant compounds that are denatured to inactivate or precipitate PMT Therefore, it is preferable to add it to the extraction buffer. In general, phenolic compounds Insoluble polyvinylpyrrolidone (“PVPP”) and amber for adsorption Ito XAD-4 is suspended in extraction buffer. Significant to PMT enzyme activity Or remove phenolic compounds without harm Other substances that inactivate are PVPP or Amberlite XAD-4 Something that can be used instead of these things is included.   Before adding root tissue, the extraction buffer is cooled to about -15 ° C to -20 ° C and frozen. Generate a slurry. During the homogenization process, the temperature of the homogenate is about 3-5 ° C or higher. Should not be raised to.   As is known to one of ordinary skill in the art, the method used above The amount of root tissue used can vary, but the approximate weight of tissue used is measured. The amounts of the other ingredients used are adjusted accordingly.   After homogenization treatment, insoluble material (PVP containing adsorbed phenolic compound (Including P) is preferably removed from the homogenate. This removal is preferred Is a refrigerating centrifuge set to about 10,000 to 20,000 xg and about 30 at about 4 ° C. This is done by allowing to settle for ~ 90 minutes. Soluble after sedimentation of insoluble material The sex extract (ie the supernatant) is decanted. Final protein in soluble extract The quality concentration is generally about 2.5-3.5 mg / ml.   Fractionate soluble extract with ammonium sulphate and use standard methods (Scopes, Protein Pu rification Principles and Practice, pages 48-52, Springer-Verlag, USA , New York, 1982) 40% to 65% ammonium sulfate. Fractions (precipitation) are collected from the soluble extract. Then, the fraction is equivalent to 1 g of root weight. About 0.1-0.4 ml Dissolve in lysis buffer.   A preferred buffer for dissolving the 40% to 65% ammonium sulphate fraction is Efficient Buffers, Heavy Metal Chelating Agents, Reducing Agents, Water Active Modifiers and Protears Ze inhibitors. The most preferred lysis buffer is Tris buffer (pH about 7.5) (about 10-20 mM), EDTA (about 1-10 mM), glycerin (about 10-30%), DTT (about 1-10 mM), PMSF (about 0.2-10.0 m) g / 1), and leupeptin (about 0.2-10.0 mg / l) . These buffer components have the same purpose as described above for similar components in the extraction buffer. It is included. Those skilled in the art will appreciate that these ingredients have similar functions. You will know that you can replace it with something else.   The ammonium sulphate fraction is then subjected to standard methods such as dialysis or sieving. Chromatographic desalting and then the desalted fractions are annealed as described below. Subject directly to on-exchange chromatography. However, in a preferred embodiment, ammonium sulfate The nium fraction is first subjected to hydrophobic interaction chromatography.   The dissolved ammonium sulfate fraction is subjected to hydrophobic interaction chromatography Before adding salt to ensure that PMT binds to the hydrophobic interaction medium. It gives a sufficiently high salt concentration. The preferred concentration of salt added is 1.5N. Added The preferred salt shown is NaCl. Another useful salt is ammonium sulfate.   Preferred hydrophobic interaction media are sized for chromatography and covalently bound. Composed of cross-linked agarose gels with nearly spherical particles, containing phenyl groups . Such phenyl agarose is referred to as "phenyl-Sepharose CL-4B". Commercially available (Pharmacia-LKB, Inc., Pis, NJ, USA) Cataway, catalog number: 17-0810-01).   The hydrated phenyl agarose is then applied to a suitable chromatographic column using standard methods. High salt equilibration buffer filled in ram and having a pH of about 7.2-8.3, preferably about 7.5. Equilibrate at about 4-8 ° C. A preferred high salt equilibration buffer is an effective amount of buffer Agents, heavy metal chelating agents, water-active modifiers and reducing agents, and about 1.5-2. It contains salt at a concentration of 0M. The most preferred high salt equilibration buffer solution is about 10 m M Tris (pH is about 7.5), about 1.5 M NaCl, about 1 mM EDTA, about 2 It contains mM DTT and about 20% (V / V) glycerin.   Sample of soluble extract adjusted with salt (volume of packed column of phenylagarose 1 Approximately 0.5-2.0 ml of extract per ml) is equilibrated with phenyl agarose Inject it into the column until the eluate is free of proteinaceous substances. Wash the membrane with equilibration buffer. If the buffer is transparent to UV light then It is determined by measuring the absorbance of UV light at a wavelength of about 280 nm. General First, wash the phenyl agarose column with about 5 to 7 column volumes of equilibration buffer. I do. PMT is a hydrophobic interaction medium Combine and remain.   Proteins still adsorbed on the phenyl agarose matrix (P (Including MT) at 4-8 ° C. and then loaded salt concentration (preferably about 1.5 M). From about 4 to 6 times the volume of the column containing a linear salt gradient decreasing from about 0.0M to about 0.0M. Elute with elution buffer, then 2-3 column volumes of additional salt-free elution buffer Elute with liquid. Preferably, the elution buffer is Tris (about 10 mM) (pH is about 7). . 5), DTT (about 2 mM) and EDTA (about 1 mM) and glycerin (about 20) % V / V).   Fractions of about 0.01 to 0.03 column volume were collected and PMT activity was performed as follows. The sex was assayed for absorbance at 280 nm wavelength light. Eluate fractions commonly collected Has a column volume of about 1 to 2 times and a protein concentration of about 0.4 to 2.5 mg / ml. I have   It is understood that salts other than the preferred NaCl can also be used in the above buffers. Will Such salts include potassium chloride and ammonium sulfate. .   An important step of the purification method of the present invention is the novel aniline carried out as follows. This is a step of on-exchange chromatography. To perform this step, add it to the column. The added tobacco plant extract (eg, preferably phenyl agarose eluate) is , The pH and chemical composition must be such that the PMT in the extract will bind to the anion exchange medium. I have to. That is, the extract has a pH of about 7.2 to 8.3 and about 0.0 to 1 Must contain 0 mM salt, preferably That is, about 5-10 mM buffer, about 1-10 mM reducing agent, about 10 30% (V / V) water active modifier and about 1-5 mM heavy metal chelator Should be included. Most preferably, the tobacco plant extract is 10 mM Tris / HCl (pH 7.5), 2 mM DTT, 1 mM EDTA and 20% (V / V ) Glycerin is contained.   Those skilled in the art will, of course, adjust the pH and salt concentration of the tobacco plant extract to the above values. The PMT is still bound to the anion exchange medium by changing the value of It will be clear that it can be loaded. Especially when the salt concentration increases Generally, the binding of proteins to anion exchange media is reduced and the pH is increased. Increasing the protein generally increases the binding of the protein to the anion exchange medium. Therefore, those skilled in the art will appreciate that the PMT binds to the anion exchange medium, Various combinations of salt concentration and pH other than could be readily determined. Yes The only constraint on the effective pH / salt combination is that pH denatures and inactivates PMT. It is not expensive enough. In general, PMT is significant at pH above about 9. Should not be exposed for a long time.   From the above, the extract of tobacco root added to the anion exchange medium is sulfated. Fractionation with ammonium or the steps of hydrophobic interaction chromatography described above In the case of the eluate from E. coli, the extract must be desalted in an appropriate buffer. Is clear. This desalting is accomplished by standard methods. For example, gel filtration black Matography (eg Sef Adex G-25) or dialysis can be used using known methods. Such desalting is preferably performed by dialysis.   In the preferred method, the solution collected from the hydrophobic interaction chromatography steps described above is used. Effluent (or other hot tobacco root extract) is 1 ml of collected eluate or extract For about 15 to 25 ml of dialysis buffer per about 8 to 20 hours at about 4 to 8 ° C, Dialyzed. It is preferable to stir the dialysis buffer. 10000KD cut-off Dialysis membranes are preferred. The chemical composition and pH of the dialysis buffer should be such that the PMT in the dialysis fraction It is selected to be retained by the anion exchange medium as described.   Anion exchange media have one or more functional anion exchange moieties and With relatively rigid particles (eg cross-linked agarose) of suitable size for the graph Must be configured. Such anion exchange moieties are diethylamino Ethyl, polyethyleneimine, tertiary amino, quaternary amino, p-aminobenzyl And diethyl- (2-hydroxypropyl) aminoethyl. Selected. Such media are commercially available. Diethylaminoethyl (“DEA Anion exchange media having an E ″) moiety are preferred. DEAE-agarose ( "DEAE-Sepharose, Fast Flow", Pharmacia-LKB, Inc., New Jersey, USA State of Piscataway, Cat. No. 17-0709-01) is the most preferable. .   The anion exchange medium should be adjusted to pH and salt conditions of the equilibration buffer by standard methods. Be balanced.   The equilibrated anion exchange medium then has a means for retaining the medium at the bottom (eg, Standard for columns with a semi-molten glass disc) and outlet tube (ie hollow tube) Fill with the method. Then cap the top of the column and connect to the inlet tube. Then Equilibration buffer is preferably passed through the column and the pH and conductivity of the flow-through is monitored. , It must be ensured that the medium is properly balanced.   The column is used when all the proteins in the added tobacco plant extract are bound. Contain enough anion exchange medium to make up only about 50% of the volume of the medium I have to For example, the added tobacco plant extract is For phenyl agarose eluate, the column contains dialyzed phenyl agarose. About 0.04-0.10 ml (packed column volume) of DEAE- Must contain agarose.   Preferably the same as when filling the column with medium and adding the tobacco plant extract Equilibrate the medium at temperature. The temperature of the column depends on the temperature at which the tobacco plant extract is added. When washing or eluting at a warmer temperature, it contains an anion exchange matrix. The slurry is degassed before it is packed in the column.   As mentioned above for the tobacco plant extract added to the anion exchange medium, The equilibration buffer of the anion exchange medium should be such that the PMT is retained by the medium. Must have a chemical composition with H. Similarly, those skilled in the art are suitable. Easy determination of sensitive pH / chemical composition combinations Can be. The preferred equilibration buffer has no added salt at all and has a pH of It is about 7.2 to 8.3, and most preferably 7.5. More favorable equilibration The buffer solution contains an effective amount of a buffer, a heavy metal chelating agent, a reducing agent and a water activity modifier. Contains. The most preferred equilibration buffer is 10 mM Tris / HCl (pH 7). . 5) 1 mM EDTA, 2 mM DTT and 20% (V / V) glycerin Contains   Tobacco plant extract, equilibration buffer and anion exchange media are all equilibration, Prior to and during loading and washing of the column, the temperature is preferably about 2-10 ° C. Most preferably about 4-8 ° C.   The tobacco plant extract is added at a flow rate of about 0.1 to 0.3 times the volume / minute. Taba The fluid that passed through with the addition of the co-plant extract practically does not contain PMT, and is therefore discarded. Be abandoned. The column is then allowed to elute the proteinaceous material until it stabilizes at a low level. Wash with equilibration buffer. The equilibration buffer contains a buffer that absorbs at 280 nm. If not, the column should have a low level of absorption of UV light at 280 nm. Wash with elution buffer until stabilized. Generally, the anion exchange medium is 5-10. Wash with 2 volumes of equilibration buffer containing 10 mM NaCl, then add another 3-10 volumes Wash the product with equilibration buffer without NaCl. PMT is an anion exchange during the cleaning process. It is held on a replacement medium.   After washing, the PMT is an elution buffer containing an effective amount of polyamine and anion exchange. Elution from the exchange medium, but elution temperature , And the pH and chemical composition of the elution buffer are similar to those of PMT without polyamine. It is a condition to be held in the exchange medium.   Polyamines in the elution buffer are putrescine, N-methylputrescine, spermine. A group consisting of Lumin, spermidine, agmatine, cadaverine and mixtures thereof Selected from Putrescine is the preferred polyamine. Polyamine elutes About 0.5-50 mM, preferably 1-10 mM, most preferably about 5 in buffer. Must be present at a concentration of mM.   The elution buffer further comprises an effective amount of buffer, heavy metal chelating agent, reducing agent and It preferably contains a water activity modifier. These components are And is the same as described above. Effective amounts of these ingredients are determined by those skilled in the art. Can be measured by one without extra testing. The pH of the elution buffer is It should be about 7.2-8.3, preferably about 7.5. Anion exchange medium Replenishing the equilibration buffer of 1. with the polyamine will result in the appropriate elution buffer. Proper melting Isolation buffer is 10 mM Tris / HCl (pH 7.5), 1 mM EDTA, 20 % (V / V) glycerin, 2 mM DTT, and 5 mM putrescine (1 , 4-diaminobutane) (Sigma Chemical Co., St. Louis, Missouri, USA) , Catalog number: P7505).   Elution of PMT from the anion exchange column was about 18-26 ° C (ie room temperature) It is preferable to carry out. With elution buffer The anion exchange column must be at the selected elution temperature before the elution begins. I won't.   To elute the PMT from the column, elute the elution buffer about 0.02-0.10 times. Column volume / minute flow rate is added and fractions are collected from the bottom of the column. The eluate is Collected in a single eluate pool. In the most preferred elution method, about 1 volume of elution is used. Dissociation buffer is added to the column and then the flow is stopped. The anion exchange medium Leave in contact with a portion of the release buffer for about 1 to 6 hours, preferably about 1 hour. Then the addition of elution buffer is restarted.   PMT elutes very slowly from the anion exchange medium. In general, anio The exchange medium is eluted with about 40-70 volumes of elution buffer, most preferably Elute with at least 50 volumes of elution buffer. Monitor the elution state of PMT Therefore, the PMT activity of the eluted fractions is assayed as follows.   Since PMT is recovered in a relatively dilute state and in a relatively large volume, it can be dissolved in anion exchange solution. It is desirable to concentrate the exudate. The eluate is, for example, an anion exchange medium Added to a chromatographic medium that has an affinity for PMT in the presence of a buffer. From the chromatographic medium, the bound material was relatively concentrated in good yield. It can be eluted in the form. Alternatively, the PMT may be precipitated. The invention In the preferred method, during the elution, the outlet of the anion exchange column is connected to the inlet of the concentration column. It is connected. In this way, the eluted PMT is removed from the anion exchange column. Spill out and concentrate directly It enters the shrink column and is adsorbed there. Elution from the anion exchange column of PMT After completion, remove the outlet of the column from the concentration column and put the PMT on the concentration column. Elute from   A preferred concentration column is ω-aminohexyl agarose (“ω-aminohexyl. Le-Sepharose 4B ", Sigma Chemical Co., St. Louis, Missouri, USA , Catalog No .: A8884) (“AHS”) on an anion exchange column. Utilize ~ 30% column volume. PMT is a relatively high concentration of salt, preferably The column is eluted with an elution buffer containing 1.5 M NaCl. Elution buffer The liquid preferably further comprises an effective amount of a buffering agent, a heavy metal chelating agent, a reducing agent, And a water-active modifier. The most preferred elution buffer is 1.5M Na Cl, 10 mM Tris / HCl (pH 7.5), 1 mM EDTA, 20% (V / V) glycerin and 2 mM DTT. Concentration column is 4-8 It is preferably loaded and eluted at ° C.   The first 4-8 volumes of eluate from the concentration column contained most of the activity of PMT. Have. The fraction is preferably an ultrafiltration device (Amicon Corp. USA, Masachi). Further available with the "Centricon 30" available from Danvers, S. Concentrated. After performing ultrafiltration, the sample is generally about 0.04 to 0.70 mg / m 2. It has a protein concentration of 1 l. Generally from several concentration columns Fractions containing PMT are collected and concentrated further.   The PMT obtained after the steps of anion exchange and sample concentration is It is then purified by preparative scale isoelectric focusing. Isoelectric focusing The method involves placing a mixture of samples in a stabilized pH gradient and then Apply voltage. Each protein species has a pH gradient where the net charge of the protein species is zero. It moves electrophoretically toward the point of distribution. If the net charge of the protein is zero The pH of the protein is called the isoelectric point of the protein.   Stabilization of various pH gradient solutions including sucrose solutions and polyacrylamide gels Media can be used. Similarly, fractionate the pH gradient solution and focus on the isoelectric focus. Various methods for recovering the protein after the electrophoresis method can be adopted. pH gradient Distributing fractionation method must be selected to be compatible with the gradient stabilizing medium .   A preferred pH gradient liquid stabilizing medium is a sucrose solution (density gradient) placed in a glass tube. Liquid distribution). Most preferably, the sucrose density gradient solution has a pH of about 5 to about 6. It contains a range of pH gradients. The preferred gradient liquid fractionation method is through a stopcock. The liquid flow rate is accurately controlled. The collected fractions were tested for pH and PMT activity. Be tested. Isoelectric focusing instruments, chemicals and protocols Available from a number of commercial companies.   The PMT isolated by the method of this invention is substantially free of other tobacco proteins. Nonetheless, PMT is the predominant protein in the formulation. Few contaminated tobacco strains in the formulation The protein is sodium dodecyl sulfate polyacrylamide gel electrophoresis (“S Standard method by DS-PAGE ") Be separated. In this way, PMT that is pure enough for amino acid sequence analysis is obtained. can get.Characterization of PMT   Tobacco PMT is about 55-65 kilodalts when measured by SDS-PAGE. It has a molecular weight of about 1.0 to about 5.0 and is measured by isoelectric focusing method. It is characterized by having a native isoelectric point of 6.0.   In addition, tobacco PMT has a methyl group on S-adenosylmethionine. The ability to catalyze the reaction of transferring benzene to the δ-amino group and to putrescine It is characterized by high substrate specificity.   The Michaelis-Menten constant (Km) shows that the initial reaction rate is 1 of the maximum reaction rate. It is defined as the substrate concentration when it equals / 2. Km values catalyze the same reaction There is wide variation in the distinct enzyme species involved. Therefore, the measured value of Km is It is a useful “identification marker”. Lightly purified tobacco PMT It is characterized by a Km value of about 300-500 μM for succinate. The highly purified tobacco PMT of the present invention has been tested for S-adenosylmethionine. The Km value is about 100 to 150 μM.Determination of partial amino acid sequence of PMT   In preparation for amino acid sequence analysis, standard SDS-PAGE methods were used to Remains after the steps of on-exchange, sample concentration and isoelectric focusing Separation of PMT from a small number of contaminating proteins. For SDS-PAGE method For a detailed protocol on this, see Laemmli, “Cleavage of Structural Protein. s During the Assembly of the Head of Bacteriophage T4 ", Nature, 22 Vol. 7, pp. 680-685, 1970, and from the manufacturer of the electrophoresis apparatus. Seen in the provided manual. Contains individual proteins by known methods Electrophoretically transferred the band to a thin sheet or membrane (electroblotting Let's hold it and visualize it. In one known method, protein bands To a hydrophobic polymer such as polyiodide (4-vinyl-N-methylpyridinium). Electroblot to a sheet of glass microfiber coated with thione, then fluor Visualize with a non-anionic drug such as resamine. Another method is protein Quality onto a polyvinylidene difluoride membrane (“Immobilon-P”, Millipore, Electroblot, Amidobra, Bedford, Mass., USA This is a method of visualizing bands with an anionic dye such as Beck (Bauw et al., “Alte rations in the Phenotype of Plant Cells Studied by NH₂-Terminal Amino Ac id Sequence Analysis of Proteins Electroblotted from Two-Dimensional Gel -Separated Total Extracts ", Proc. Nat. Acad. Sci. USA, 84, 4806 ~ 4810, 1987; A Practical Guide to Protein and Peptide Purif ication for Microse-quencing, Paul T. Edited by Matsudaira, Academic Press, US Country, New York, 1989).   Transferring the isolated protein from the electrophoretic gel, The above methods of visualizing transferred proteins are preferred. However, by electrophoresis Changes in materials and methods used to prepare isolated proteins for sequence analysis Is not excluded from the invention, as would be apparent to one of ordinary skill in the art. It will be easy.   The purified PMT that composes the band has the same apparent molecular weight (that is, about 60 KD). Is determined. The protein band was transferred from the electrophoretic gel to the membrane and transferred to a membrane. After visualization of the bands, the membrane strips with individual bands of purified PMT were Cut accurately to avoid contamination by a puffy band.   Purified tobacco PM constituting a protein band (isolated as described above) For T, the amino terminal sequence is analyzed by standard automated methods. Cigarette PM The T protein is an amino acid selected from SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 3. It contains a no acid sequence.   SEQ ID NO: 1 is the sequence from the “a1” band (FIG. 5) and SEQ ID NO: 2 is “a2”. "Is the sequence from the band (FIG. 5). SEQ ID NO: 3 is the sequence of SEQ ID NO: 1 and SEQ ID NO: 2. It is a common array.   The high homology of the sequences from the bands of the purified protein with which they are closely related is due to the It is suggested that a shaped tobacco PMT protein is present. like this The complex form of tobacco PMT protein consists of post-translational modifications of a single gene product. Or, it is generated from the gene of complex PMT.Cloning of PMT DNA sequence   A partial amino acid sequence of the PMT protein of the present invention (SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3) was used to design a set of oligonucleotides such as It is. In other words, one or more of the It hybridizes selectively with the PMT sequences in the rally. This selective hybridization leads to P A cDNA clone containing a sequence encoding part or all of the MT protein It is used to identify Oligonucleotide probe from amino acid sequence For a description of the design of Sambrook et al., Molecular Cloning-A Laboratory Manua. I, Cold Spring Harbor Press, 1989, Chapter 11.   The synthesis of such oligonucleotide probes is routinely performed on commercially available automated equipment. Have been.   Construction of a cDNA library is currently a routine task at a molecular biology laboratory. (See Chapter 8 of Sambrook et al., Supra). Similarly, the clone containing the sequence of interest. CDNA library with an oligonucleotide probe to identify It is well known in the art to screen Lee and one skilled in the art It is fully included in the ability of. For screening a cDNA library For a description of using oligonucleotides, see Sambrook et al., Supra. It is described in chapter 11 of the laboratory manual of. With oligonucleotide probe The cDNA clones selected on the basis of the hybridization reaction of Characterized by presence and nucleotide sequence. Such a DNA analysis method is based on Sambro ok, etc., and Ausube l et al., Short Protocols in Molecular Biology, Green Publishing Associates a nd Wiley Interscience, New York, USA, fully described in 1989 I have. Any PMT cDNA clone thus obtained was , And can be used as a probe to identify other PMT cDNA clones.   Tobacco [Nikotiana Tabakum L. var. (Nicotiana tabacum L. var.) N K326] genomic library is commercially available (Clonetech Laboratories, Inc., Palo Alto, CA, USA). The above genomic library is Tobacco PMT screened according to the vendor's provided protocol A chromosomal gene encoding is obtained.   In addition to the methods described above, one of skill in the art can obtain the desired PMT cDNA clone. The replication chain reaction (“PCR”) method can be advantageously used. Basics of PCR The principle is the rapid amplification of a portion of DNA between two regions of known sequence. various PCR technology was developed for use in the discovery, analysis and construction of unique DNA molecules. It has come. Equipment, chemicals and protocols for numerous uses of PCR technology Commercially available. For a general discussion of PCR, see Sambrook et al. (Supra ), Chapter 14.   A preferred PCR method for practicing the present invention is known as RNA PCR. RNA PCR has two basic steps:   (1) RNA as a template in the presence of reverse primer Reverse transcriptase synthesis of the first cDNA strand using, and (2) After polymerase synthesis of complementary cDNA strands, forward and reverse Taq Polymerase amplification of both strands in the presence of both primers.   The PMT cDNA molecule is an oligonucleotide based on a known PMT partial amino acid sequence. Tobacco root poly (A⁺ ) Using RNA And may be obtained by applying the RNA PCR method.   Therefore, the present invention provides a recombinant DNA molecule encoding a tobacco PMT protein. provide.Transfectants stably transformed with PMT DNA sequences in sense or antisense orientation. Production of transgenic tobacco cells and plants   This invention encodes a tobacco PMT protein and contains a PMT antisense RNA component. Stable with recombinant DNA molecules operably linked to a regular sequence encoding the offspring Provided are transgenic transgenic tobacco cells and plants.   Taba with lower nicotine content than untransformed control tobacco plants To produce a co-plant, tobacco cells are operably linked in a regular array. A partial PMT cDNA sequence cloned in antisense orientation in a row, Relength PMT cDNA sequence, partial PMT chromosomal sequence, or fullerene Transformation with Artificial PMT Antisense Transcription Unit Containing Guts PMT Chromosome Sequence Is done. Suitable regular sequences include transcription initiation sequences ("promoters") and Includes rearadenylation / transcription termination sequences .   Expression of antisense sequences in transgenic tobacco plants is generally associated with Uses the Yasai Mosaic Virus (CaMV) 35S promoter (eg, Co “Both RNA Level and Translation Efficiency are Reduced by rnelissen et al. Anti-Sense RNA in Transgenic Tobacco ”, Nucleic Acids Res., 17:83 Pp.3-843, 1989; Rezaian et al., "Anti-Sense RNAs of Cucumber Mosai. c Virus in Transgenic Plants Assessed for Contol of the Virus ”, Plant M olecular Biology, Vol. 11, pp. 463-471, 1988; Rodermel et al., "Nu. clear-organelle Interactions: Nuclear Antisense Gene Inhibits Ribulose B isphosphate Carboxylase Enzyme Levels in Transformed Tobacco Plants ”, C ell, 55, pp. 673-681, 1988; Smith et al., "Antisense RNA Inh". ibition of Polygalacturonase Gene Expression in Transgenic Tomatoes ”, N ature, 334, 724-726, 1988; Van der Krol et al., "An Ant. i-Sense Chalcone Synthase Gene in Transgenic Plants Inhibits Flower Pigm entation ", Nature, 333, 866-869, 1988). CaMV35 was used to express PMT in transformed tobacco cells and plants of the invention. It is preferable to use the S promoter. Other recombinant genes in tobacco roots The use of the CaMV promoter to express E. coli has been well documented (Lam , “Site-Specific Mutations Alter In Vitro Factor Binding and Change Promoter Expression Pattern in Transgenic Plants ", Proc. Nat. Acad. Sc i. USA, Vol. 86, pp. 7890-7894, 1989; Poulsen et al., "Dissecti. on of 5'Upstream Sequences for selective Expression of the Nicotiana p lumbaginifolia rbcs-8B Gene ", Mol. Gen. Genet., Volume 214, 16-2 Page 3, 1988).   Although it is preferable to use the CaMV35S promoter, other promoters Has been successfully used to express heterologous genes in tobacco plants. It is also possible to use a promoter other than the CaMV35S promoter of the present invention. Included in the range.   Various transcription termination sequences are known. The origin and identity of transcription termination sequences are mainly This is a matter of convenience. For example, nopaline synthase (“NOS”), octopine Synthase (“OCS”) and CaMV polyadenylation / transcription termination sequences are Used to express heterologous genes in transgenic tobacco plants and used in PMT Useful for expression of sequences (eg Rezian et al., Supra and Rodermel et al., Supra). reference).   Next, restriction mapping, Southern blot hybridization, and nucleotide sequences. Using standard methods, such as analytical methods, and having PMT sequences in the antisense orientation, Operable on node sequences (ie promoter and polyadenylation / transcription termination sequences) Identify clones that are ligated to.   To produce transgenic cells stably transformed with exogenous DNA Exogenous D in the tobacco cell genome There are well-developed methods that can be applied to introduce NA. Many known tobacco Any of the cell transformation methods can be used to practice this invention. Tobacco cells Whether the transformation method of A. coli uses components of the Agrobacterium system It is easily classified based on crab.   Agrobacterium tumefasciens is Has a nucleotide sequence called "T-DNA" (as transferred DNA) Efficient in the chromosome of natural dicotyledonous plants (including tobacco) It is a Gram-negative bacterium that has been metastasized to and integrated in and grows tumors on infected plants. Difference Naturally-occurring vector systems for integrating seed DNA into plant chromosomes include plant genes. In engineering, it has been extensively studied, modified, and interspersed (Deblaere et al., “Efficient Octopine Ti Plasmid-Derived Vectors for Agrobacterium-Mediated Gene Tran sfer to Plants ”, Nucleic Acids Research, 13: 4777-4788, 1985). Operable in sense or antisense orientation relative to regulatory sequences A naked recombinant DNA molecule containing a PMT DNA sequence linked to Connect to the DNA sequence by conventional methods. These are polyethylene glycol Method into the tobacco protoplasts by electroporation or electroporation (both standard methods). Be entered. Alternatively, it may contain a recombinant DNA molecule encoding PMT. Is introduced into live Agrobacterium cells. This cell is then the DNA To tobacco plant cells (Rogers et al., “Gene Transfer in Plants: Prod auction of Tr ansformed Plants Using Ti Plasmid Vectors ”, Methods in Enzymology, 1 18: 627-640, 1986).   Although the Agrobacterium method is widely used in the art, It is not a necessary element in the method. Naked DNA without T-DNA vector sequence Transformation by fusing tobacco protoplasts with DNA-containing liposomes or Can be done by electroporation (Shillito et al., “Direct Gene Transfer  to Protoplasts of Dicotyledonous and Monocotyledonous Plants by a Numbe r of Methods, Including Electroporation ", Methods in Enzymology, 15 3, 313-316, 1987). Does not have T-DNA vector sequence Inert high-speed microprojectiles [BIOLISTIC (registered trademark)] Particle Delivery System, Dupont, Wilmington, Delaware, USA) Thus, it can be used to transform tobacco cells.   Used to make transformed tobacco cells and tobacco plants of this invention The PMT recombinant DNA molecule and vector also contain a dominant selectable marker gene. It is preferable to have. Suitable dominant selectable markers for use in tobacco include neo Mycin phosphotransferase, hygromycin phosphotransferase Antibiotics Encoding Zeze and Chloramphenicol Acetyltransferase A quality resistance gene is included. Other well-known prioritized selections suitable for use in tobacco The selection marker is methotrexate resistant di- A mutant dihydrofolate reductase gene encoding hydrofolate reductase. (Deblaere et al., Supra). DNA containing an appropriate antibiotic resistance gene Vectors and corresponding antibiotics are commercially available.   The transformed tobacco cells are the product of the selected dominant selectable marker gene. Antibiotics (or other compounds that are usually toxic to tobacco cells) that confer resistance to Untransformed cells by adding a mixed population of cells to medium containing the appropriate concentration. Selected from the population surrounding the transformed cells. Therefore, it is a transformed tobacco cell Injuries survive and multiply.   The transformed cells may be cultured according to any method known in the art of culturing tobacco cells and tissues. By applying, regenerates an induced, intact and fruitful tobacco plant. plant The regeneration method is selected to be consistent with the transformation method. Estimated transgenesis PMT sequences are stably present and oriented in the genome of the tobacco plant The test for this was performed by Mendelian inheritance of the PMT sequence and controlled hybridization. Revealed by standard DNA analysis performed on offspring resulting from Become.   Transgenic tobacco plants are regenerated from transformed cells and then transformed into The inserted PMT sequence can be transformed into other tobacco varieties without undue experimentation using conventional plant propagation methods. Easily transferred.   Decreased levels of nicotine in PMT antisense transgenic tobacco plants Is tested by the standard nicotine assay.   Full length PMT cDNA molecule or full length The appropriate operably linked regulatory sequences using the PMT chromosomal gene of Transgenic tobacco cells and transgenic tobacco linked in orientation It will be appreciated that plants can be constructed (and those skilled in the art should Appropriate regulatory sequences that express the gene in the mouse orientation are provided in Including any one of the known eukaryotic translation initiation sequences in addition to the transcription / transcription termination sequence. You will see). Such a transformed tobacco plant has PMT The level of protein is increased and, therefore, is higher than that of untransformed control tobacco plants. It is characterized by high content of cotin.   Therefore, the PMT DNA sequence was used to reduce the level of PMT protein. Increase or decrease to reduce or increase the content of nicotine in tobacco plants. It should be understood that addition is included within the scope of this invention.   The following examples are provided for better understanding of the invention. These examples are examples It is for the purpose of showing only and is not intended to limit the scope of the invention. ExampleBuffer solution composition Buffer A   50 mM Tris / HCl, pH 7.5   5 mM EDTA (free acid)   20% (V / V) glycerin   2 mM DTT   0.5% (W / V) sodium ascorbate   2% (W / V) PEG400   0.4 mg / l PMSF (from 1 mg / ml stock solution)   0.4 mg / l leupeptin (from 1 mg / ml stock solution)   100g / l PVPP   40g / l Amberlite XAD-4Buffer B   10 mM Tris / HCl, pH 7.5   1 mM EDTA (free acid)   20% (V / V) glycerin   2 mM DTT   0.4 mg / l PMSF (from 1 mg / ml stock solution)   0.4 mg / l leupeptin (from 1 mg / ml stock solution)Buffer C   10 mM Tris / HCl, pH 7.5   1 mM EDTA (free acid)   20% (V / V) glycerin   2 mM DTTStock solution of protease inhibitor   Dissolve PMSF (1 mg / ml) in dimethylformamide and add 2.1 ml each. Was stored at -20 ° C until use. Leupeptin (1mg / ml) distilled water Dissolved in 2 . 1 ml aliquots were stored at -20 ° C until use.Production of crude extract   Tobacco grown by hydroponics (Nicotiana tabacum L. var. Burley 21) About 1 kg of plant roots was harvested 3 days after flowering. Wash the harvested roots with cold water Placed in a clean Buchner funnel and aspirated to remove water. Frozen washed roots at -80 ° C And stored. Pre-chill the frozen roots in a 1 gallon Waring blender Added to 2.51 buffer that had been slurried. Buffer the roots with a large spoon Mix with liquid slurry. Start the blender at a low speed setting and then the medium speed setting. Homogenization was performed according to the standard. If the homogenate temperature is higher than 3-5 ° C I was careful not to.   Put the resulting extract in a centrifuge bottle and centrifuge at 13680 xg for 70 minutes at 4 ° C. Separated and insoluble debris was pelletized.   The supernatant was decanted and its volume was 2.371. About 0.77g of D TT was added to the extract.Fractionation with ammonium sulfate   Crystals of ammonium sulfate were added to the extract in an amount of 22.6 g per 100 ml of the extract. It was added slowly and the extract was brought to 40% saturation with ammonium sulfate. Obtained sulfuric acid The ammonium-containing extract was stirred at 4 ° C for 2 hours.   Precipitation with ammonium sulphate at 40% saturation was carried out at 27500 xg for 30 minutes 4 It was recovered by centrifugation at ° C. An additional 0.33 g of extract 11 Add DTT did. Extraction of ammonium sulfate crystals with an amount of 15.3 g per 100 ml of extract. Gradually add ammonium sulphate to the solution to adjust the concentration of ammonium sulfate from 40% to 65% saturation. Up to. The extract containing 65% ammonium sulfate was stirred overnight at 4 ° C. Stirred. Fraction with 40-65% saturation ammonium sulphate, 27500 xg Pellet by centrifuging for 70 minutes at 4 ° C and discard the supernatant. Was.   Precipitation with 40-65% saturation ammonium sulfate was dissolved in buffer B to obtain a total volume. The volume is made up to 200 ml, then 17.53 g of NaCl are added and dissolved by stirring on ice. I let you. The solution of the 40-65% fraction containing NaCl was added at 47800 × g for 30 minutes, 4 Centrifuged at 0 ° C and the resulting pellet was discarded.   The production of the crude extract and the fractionation with ammonium sulphate were carried out essentially as described above. The same procedure was repeated three more times to obtain four 40-65% saturated ammonium sulfate solutions. The monium fractions (200 ml each) were pooled. 800 manufactured in this way The ml pool corresponded to a total of 5.239 Kg of root tissue.Hydrophobic interaction chromatography   Phenyl-Sepharose CL4B (Pharmacia Inc., New Jersey, USA State, Piscataway, Catalog Number: 17-0810-01) Hydrophobic interaction Rams (5 cm x 20 cm) were equilibrated with buffer C supplemented with 1.5 M NaCl. . Clear 40-6 of 800 ml pool corresponding to 5.239 Kg of root tissue. The 5% saturated ammonium sulphate fraction was converted to equilibrated phenyl-sepharose as described above. Kara Injected into Until a stable baseline of absorbance of 280 nm wavelength light is obtained. Column (indicating that all unbound protein was removed), the column was  It was washed with buffer C supplemented with NaCl. Then 1.5M to 0.0M in buffer C The PMT was eluted with a linear gradient solution 21 of NaCL that had a decreasing concentration. Additional buffer C The column was further washed with 11. Fractions of 12 ml each were collected and the fractions (P At the peak of MT activity and before and after it, every 3 fractions, and the other fractions of the gradient solution (Every 10 fractions), PMT activity was assayed continuously as follows. Hydrophobic mutual Working chromatography was carried out at 4 ° C. with a flow rate of 4.7 ml / min.   Pool Phenol-Sepharose Fractions # 86- # 116 with PMT activity And continuously agitate the resulting pool for about 18 hours against buffer C 91. It was dialyzed. The dialyzed sample is divided into four 100 ml portions and cooled to -80 ° C. I rejected it.Assay of PMT activity   The following was put in each reaction tube.   12.5 nmol Tris / HCl 8.3   0.25 nmol EDTA   1.25 nmol 2-mercaptoethanol   0.9 nmol putrescine   0.15 nmol unlabeled S-adenosylmethionine   0.18 nmol [¹⁴C-methyl] S-adenosylme                             Thionine                             (57nCi / nmol)                             Enzyme sample                             Total volume = 0.25 ml   The reaction was initiated by adding the enzyme sample and was performed for 30 minutes at 30 ° C. . By adding 0.5 ml of 10% (W / V) NaOH solution saturated with NaCl The reaction was stopped.   Radioactive product, N- [¹⁴C-methyl] putrescine is a solvent to chloroform Separated from the substrate by extraction. The reaction was stopped by adding 1 ml of chlorophosphine to the mixture. After swirling with Rum for 90 seconds, centrifuge at 1600 x gav for 5 minutes The organic and aqueous phases were separated. Then 0.5 ml of the organic phase was assayed. 0.5 ml Organic phase of 9.5 ml of liquid scintillation cocktail (Beckman Instruments , USA, Columbia, USA) to add radioactivity to liquid scintillation Counter using a standard method.   One unit of PMT activity is equivalent to 1 nmole of the product produced in 30 minutes at 30 ° C. Define.   Negative controls are included in all tests. Negative control is reaction mixture without enzyme , Or a mixture quenched with NaOH at time zero.Anion exchange chromatography   Thaw two 100 ml aliquots of phenyl-Sepharose and thaw DEAE-Sepharose "Fast Flow" column equilibrated with buffer C at 4 ° C. Lamb (Pharmacia-LKB, Piscataway, NJ, USA) , Catalog number: 17-0709-01, Lot number: OB-05854, 1c m × 14.5 cm) at 4 ° C. at a flow rate of 1.5 ml / min.   The DEAE-Sepharose column is then placed on a stable 280 nm baseline. To obtain 1.5 ml of buffer C containing 10 mM NaCl. It was washed at a flow rate of / min. The column is then loaded with 50 ml of buffer C without NaCl. Equilibrated again. Next, raise the temperature of the column to room temperature (24 ° C) and The void volume was adjusted to 5 mM putrescine (Sigma Chemical Co., USA, Missouri, USA). Into Louis, catalog number: P7505, lot number: 39F0039) The buffer solution C was replaced. Hold the column at 24 ° C for approximately 1 hour without passing it through, then At 0.7 ml / min (1 ml with 5 mM putrescine-containing buffer C632 ml). PMT was eluted at 24 ° C. with a flow rate of 5 hours).Concentration of PMT by adsorption   The PMT eluted from the DEAE-Sepharose column was treated with ω-aminohexyl. Sepharose 4B ("AHS") (Sigma Chemical Co., Missouri, USA, St. Louis, Catalog No .: A8894) column (1 cm x 3 cm) (4 ° C Directly stored in). PMT was removed from the above AHS column using 1.5 M NaCl. Was eluted at a flow rate of 1.6 ml / min with buffer C containing 12-15 Four fractions of ml were collected and assayed for PMT activity as described above. the first (14.7 ml) of the total PMT activity recovered from the AHS concentration column. Contains 0% or more I wasUltrafiltration   For further concentration, 13.7 ml of the first AHS fraction was divided into 6 parts. "Centricon 30" (Amicon, Denver, Mass., USA) It was placed in an ultrafiltration device and concentrated about 25 times. Pool the concentrates from the six above devices Diluted with buffer C containing no salt, and diluted with a single "Centricon 3". A second ultrafiltration was performed with 0 "until the total volume was approximately 150 μl. The resulting 150 μl concentrate was stored at −80 ° C.Preparative isoelectric focusing   PMT purified in the DEAE / AHS step (including concentration by ultrafiltration), It was further purified by isoelectric focusing. Isoelectric focusing of preparative separation method In a sucrose density gradient liquid (1.6 cm × 21 cm), a commercially available ampholyte (Ph armacia-LKB, Piscataway, NJ, USA) . The pH gradient solution should be prepared according to the ampholyte manufacturer's instructions, and the pH range should be adjusted. It was set to about 5.3 to about 6.3. Apply a voltage of 1000 to 4000 volts (1 to Focusing was performed for about 3 hours at a power of 4 watts. After focusing, Fractions were collected and the pH and PMT activity of each fraction were measured. Focusing and fraction collection 4 C. was performed.   Figure 4 shows the relative activity of PMT and pH vs. fraction number after isoelectric focusing. Is a dual graph of the number (ie, position of sucrose density gradient). Experiment shown in Figure 4 The data is It shows that the isoelectric point of Baco PMT is about 5.7. Other isoelectric focusing In the migration experiment, the PI of tobacco PMT was as low as 5.0 and as high as 5.8. It seems to indicate a high value. In fact, many factors affect the apparent PI, One of ordinary skill in the art will recognize that the measured value of will usually vary somewhat. There will be.   The relative purity of PMT at successive stages of the purification process was evaluated by measuring the specific activity. (Table 1). The purity values (doubled) shown in Table 1 are actually from the crude extract of tobacco roots. Is an underestimated value of the degree of purification. Because 40-65% saturation ammonium sulfate This is because the fraction according to 1. is set to 100% when the activity yield is calculated.   Also, the PMT at successive stages of the method of the present invention Relative purity was measured by standard SDS-PAGE method. Figure 1 shows the PMT purification process. SDS-PAGE protein bandpass that the sample shows (during silver staining) at each step Indicates a turn. The samples on the gel are as follows: Lanes 1 and 6: molecular weight standard Quality (listed in the column of brief description of the drawings below); Lane 2: 40-65% Fractions with ammonium sulfate saturation; Lane 3: Phenyl-Sepharose column. Fraction with peak PMT activity derived from lane 4; lane 4; enrichment from DEAE / AHS stage Material; Lane 5: Isoelectric focusing of concentrated material from DEAE / AHS stage The fraction with the peak PMT activity. Substances purified by DEAE / AHS ( The predominant PMT band in lane 4) (indicated by the arrow) shows the above-mentioned hydrophobic interaction. It can be seen that it is hardly visible in the material from the stage (lane 3).Tobacco PMT molecular weight   The apparent molecular weight of tobacco PMT was calculated using ammonium sulfate, phenyl-cephalon. And PMT material that has passed the DEAE-AHS / ultrafiltration purification step. It was measured in an experiment in which PMT was isolated on a non-denaturing electrophoretic gel. For non-denaturing concentration The gel buffer is 0.27M Tris / HCl (pH 6.8), 10% (V / V) It contained lyserine and 20 mM 2-mercaptoethanol. Non-denaturing 1 Gel buffer for separation of 2.5% polyacrylamide is 0.38M Tris / HCl (pH 8.8) 10% (V / V) glycerin, and 12 mM 2-mercaptoetha It contained a nol.   Cut a single lane from a non-denaturing gel, cut in half along the length, then It was cut into 3 mm thick slices. 1/2 of each gel slice is standard PMT assay Place directly in the mixture and subject the corresponding 1/2 of each gel slice to SDS-PAGE. Was.   The non-denaturing gel slices that showed the highest PMT activity (Fig. 2) were especially apparent. It contained a single protein with an abundance of approximately 60 KD (Fig. 3).Enzyme activity of PMT   Substrate specificity tests were performed on the highly purified tobacco PMTs of this invention. 1,3-diaminopropane and 1,5-diaminopentane (both putrescine , A chemical analog of phosphatidylethanolamine (a methyl group acceptor) and N-methylputrescine (a normal product of PMT) as a substrate for PMT The working performance was compared with putrescine (1,4-diaminobutane). 1, 3-diaminopropane, 1,5-diaminopentane and phosphatidylethano In the standard PMT assay (assay above) instead of putrescine When used, no detectable amount of radioactive product was produced. To test PMT, When N-methylputrescine is used instead of putrescine, radioactive products Production was less than 6% as observed with putrescine.   A look at the substrates of two PMTs, putrescine and S-adenosylmethionine. Multiplying the Km value with other substrates in excess, while controlling various rate-determining concentrations of one substrate In (from above Thus, it was determined by measuring PMT activity. Lightly purified tobacco The Km for putrescine of PMT was about 400 μM. Highly purified The Km for S-adenosylmethionine of tobacco PMT was about 125 μM. Was. K found for putrescine by lightly purified tobacco PMT m-value and S-adenosylmethionine by highly purified tobacco PMT. The Km value found for the PMT is in good agreement with that published for PMT (Mi zusaki et al., supra; Feth et al., "Determination of Putrescine N-methyltransf. erase By High Performance Liquid Chromatography ", Phytochemistry, 24 Vol., 921-923, 1985).Analysis of amino terminal amino acid sequence   Tobacco PMT for sequence analysis is fractionated by ammonium sulphate, Phenyl-Cef Chromatography, DEAE Sepharose chromatography eluting with putrescine Matography (followed by concentration with AHS and ultrafiltration), free flow, etc. The substance that has been subjected to the purification step of the electrofocusing electrophoresis is subjected to the SDS-PAGE method. By isolation. After subjecting the highly purified PMT to SDS-PAGE, The resulting protein band was labeled with polyvinylidene difluoride membrane (“Immobilon”, Millipore, Bedford, Mass., USA) And then visualized by standard methods with Amido Black. "A1" The membrane pieces with the sliver (see FIG. 5) are highly purified showing the molecular weight characteristics of tobacco PMT. Only two bands of formulated product (See Fig. 3), but cut it apart from the adjacent "a2" band. Was. The method recommended by the manufacturer was used for the PMT isolated in this way. Applie equipped with an online 120A analyzer (pulse liquid phase sequencer) Amino terminal amino acid sequence analysis was performed using d Biosystems Model 477A.   Of the first 17 amino acids at the amino terminus of the "a1" band of tobacco PMT The sequence is (SEQ ID NO: 1): LeuSer Xaa Asn Phe Leu P. he Gly Thr Ala Ser Ser Xaa Tyr Gln T It was found to be yr Glu.   The "a2" band (see Figure 5) is the only two bands showing the molecular weight of tobacco PMT. It was the second one (see Figure 3). Create "a2" band (Fig. 5) When analyzed in the same manner as the "band," the "a2" band shows the following partial amino acids: Sequence (sequence number 2): Leu Ser Ser Asn Phe Leu Ph e Gly Thr Ala Ala Pro Tyr Tyr Gln Ty rGlu was generated.Confirmation of amino-terminal sequence of 60kD PMT protein   Additional amount of 60kD tobacco PMT protein was further prepared for amino acid sequence analysis did. Based on this further analysis, the first 29 amino acid sequences of the "a1" band were Column (SEQ ID NO: 4): Leu Ser Ser Asn Phe Leu Ph e Gly Thr Ala Ser Ser Tyr Tyr Gln Ty r Glu Gly Ala Phe Leu Ser Asp Gly Va l Gly Leu Ser Asn.Partial amino acid sequence of CNBr fragment   To obtain a partial internal (as opposed to amino-terminal) PMT amino acid sequence, we Isolated the tobacco PMT electrophoretic band as described above and brominated the purified PMT. It was subjected to cyanine (“CNBr”) decomposition. (CNBr is a methionine residue and is specifically a peptide Disconnect. ) We analyzed the CNBR reaction products by SDS-PAGE, Found a CNBR fragment with an apparent molecular weight of about 15, 6.2 and 3 kD did. We electrophoresed from a polyacrylamide gel containing CNBr fragments. The active band, electroblot the protein from that band, and (see above). Sea urchin) The fragment was subjected to amino acid sequence analysis.   The sequence of the first 13 amino acids of the 15 kD CNBR fragment of PMT is (Column number 5): Phe Ile Thr Glu Asn Gly Phe Al a Gly Arg Ser Gly Arg was found.   The first of the 6.2 kD CNBR fragments of PMT The sequence of 5 amino acids is (SEQ ID NO: 6): Asn Glu Pro Xaa Phe.   Val Ala Ila Ser Gly Tyr Arg Asp Xaa   It was found to be Thr.   The sequence of the first 20 amino acids of the 3 kD CNBR fragment of PMT is (sequence No. 7): Ala Asp Ile Glu His Tyr Ser Lys   Leu Ile Asp Ala Leu Xaa Ile Lys Gly   It was found to be Ile Gln Phe.Isolation and characterization of PMT cDNA clones   Uses common RNA PCR method to isolate PMT cDNA clones did. The template in the RNA PCR method is poly (A⁺ ) R NA. poly (A⁺ The starting material for RNA production is grown in hydroponics Of tobacco plants (N. tabacum, Var. Burly 21). poly (A⁺ ) The first step in the production of RNA was the production of total RNA. Whole tobacco root RNA isolation is accomplished using commercially available kits (Stratagene , LaJolla, CA, Cat. Performed using the knowledge and reagents from No. 200345) . poly (A⁺ ) RNA is poly (A⁺ ) Commercially available for the production of RNA Noh Kit (Pharmacia LKB, Piscataway, NJ, Cat. No. 27-9258- 01) was used to isolate from total RNA production. Oligonucleotide PCR plastic Immersed with partial amino acid originally obtained from purified PMT protein Designed according to standard principles using acid sequence data. In later experiments, the primer Was designed from the sequence of a previously isolated cDNA clone.PCR method   The RNA PCR method was performed using a commercially available kit (“GeneAmp RNA PCR Kit ”, Perkin Elmer Cetus, Norwalk, CT). Essentially followed the advice of the R kit seller. The seller's protocol is modified as follows. Corrected:   Reverse transcriptase reaction (1) Reverse primer concentration was 2.5 μM; (2) poly (A⁺ ) RNA amount was approximately 1 μg for the reverse transcriptase reaction; (3) Thermal cycle program is 42 ℃ 60 minutes, 99 ℃ 10 minutes, 4 ℃ 10 minutes 1 cycle I did a kuru and connected to a soaker file at 4 ℃;   Polymerase chain reaction (1) Primer concentration was 2.5 μM each; (2) Add Taq polymerase enzyme to the final reaction after 98 ° C cDNA denaturation step. Added; (3) Thermal cycle program is step cycle file 98 ℃ 1 minute, 60 ℃ 10 minutes 1 cycle, 94 ° C for 1 minute, 50 ° C for 2 minutes, 72 ° C for 3 minutes for 30 cycles Step cycle file 72 ℃ for 10 minutes, soaker file 4 ℃ Was;     20% of each PCR sample after standard chloroform extraction step (Vol.) On a 2% agarose gel for electrophoretic analysis of PCR products did.Cloning procedure   A PCR product selected for further analysis is provided with a commercially available vector ー pBluescript II (SK⁺ ) (Stratagene). PCR product An aliquot of was treated with Klenow enzyme to generate blunt ends on the DNA. Klen The ow reaction mixture is as follows: about 1 μg PCR-generated DNA, 80 μM 4 deoxynucleotide triphosphates for each final concentration, 2.5 μl of 1OX universal buffer (Stratagene), 5 units of Klenow enzyme (Stratagene), and water to obtain a total volume of 25 μl. Klenow reaction at room temperature 3 Continued for 0 minutes and stopped by heat inactivation at 75 ° C. for 10 minutes.   The PCR-generated DNA fragment was then digested with pBl digested with the restriction enzyme SmaI. The uescript II (SK +) was ligated. The ligation reaction mixture is As: about 1 μg of blunt ended DNA, about 0.2 μg of SmaI digested pBl uescript II, 3 μg 1OX ligation reaction buffer (Stratagene), 12 units T 4 DNA ligase, and water to obtain a total volume of 30 μl. We'll be at 4 ° C overnight A gauze reaction was carried out, and the reaction was stopped by thermal activation at 75 ° C. for 10 minutes.   After each ligation reaction, the following bacterial transformation method was performed. Commercially available competent E. Coli cells (“XL1-Blue” cells, Stratagene) suspension on ice Cooled on . About 30 for a sample of DNA that has been chilled on ice (ligated as described above). μl of suspension was added. Competent cells in the presence of ligated DNA , Put on ice for 30 minutes. Then, using a 42 ° C water bath, incubate the cells for 2 minutes with DNA. In the presence of. After the heat shock, the cells and DNA were returned to the ice bath for 5 minutes. 1 After addition of ml of LB medium, the cells were vigorously shaken and incubated at 37 ° C for 1 hour. I had a cube. The cells were then placed in selective medium. After overnight incubation The plates were examined for colonies of transformed cells. Selection of transformed cells For selection, LB medium containing ampicillin at 100 mg / liter was used. Was.   Selected transformants were produced from cultures of individual transformant colonies Screened by PVuII restriction analysis of DNA. This selection method is the desired insertion Colonies with PCR product are larger than PCR product, about 0.5 kb PVu Based on the fact that it produces II. This is the pBluescript II multiple cloning part Position adjacent to the position (MCS) into which the PCR product is cloned This is because there are PVuII restriction sites at reotide positions 529 and 977. (Seller See the map of the plasmid pBluescript II in the product literature. ) PVuI Clones selected in I-screen were further analyzed by DNA sequence analysis.Clone PMT1.2   Clone PMT1.2 (about 1.2 kb) was cloned into RNA PCR as described above. And obtained by cloning. The mold is Tobacco root poly (A⁺ ) RNA and forward and reverse primers They were designated as P-20 (SEQ ID NO: 8) and P-22 (SEQ ID NO: 9), respectively. Primer The sequence design of P-20 was based on amino acids 13-20 of the 60 kD PMT protein. . The sequence design of primer P22 was based on the 15kD CNBr fragment of PMT protein. Based on mino acids 1-7.   Preliminary DNA sequence analysis was performed on clone PMT1.2. PMT1 . The deduced amino acid sequence encoded by one of the two open reading frames is 60 kD. It showed strong homology (94%) to the N-terminal region of the PMT protein. PM The deduced amino acid sequence encoded by T1.2 is a 60 kD PMT protein. 3kD CNBr Fragment (as described above) representing the internal area of white matter It showed strong homology (95%).Clone PMT-26   Clone PMT-26 (about 1.2 kb) was subjected to RNA PCR as described above. And obtained by cloning. The template is tobacco root poly (A⁺ ) RNA and The reverse and reverse primers are P-24 (SEQ ID NO: 10) and P-, respectively. 21 (SEQ ID NO: 11). The sequence of primer P-24 is intact 60 kD P Based on amino acids 4-10 of MT protein, contains a BamHI site at its 5'end I was out. The sequence of primer P-21 is 15 kD CNBr of 60 kD PMT protein. Based on amino acids 1-6 and the N-terminal methionine of the fragment. In addition, Limer P-21 contained an EcoRI site at its 5'end. Therefore, The test was conducted from amino acid # 4 near the N-terminus of the 60 kD PMT protein to the protein. CDNA encoding the region up to amino acid # 6 of the 15 kD CNBr fragment of Escherichia coli Planned to get a clone.   Preliminary DNA sequence analysis was performed on cDNA clone PMT-26. Clone PMT-26 encoded approximately two-thirds of the 60 kD PMT protein. N- of the deduced amino acid sequence encoded by one of the PMT-26 open reading frames The terminal region showed strong homology to the N-terminal region of the 60 kD protein. So The deduced amino acid sequence C-terminal region is the internal region of the 60 kD PMT protein. A strong homology to the 3 kD CNBr fragment (as described above) Indicated.Clone Q7   Clone Q7 (approximately 0.4 kb) was cloned into RNA PCR and clones as described above. It was obtained by PCR template is tobacco root poly (A⁺ ) RNA and Fowa And PCR primers were P-18 (SEQ ID NO: 12) and P, respectively. -23 (SEQ ID NO: 13). The sequence of primer P-18 was purified as above. N-terminal fragment of the 15 kD cyanogen bromide fragment generated from the isolated 60 kD protein. Based on thionine plus amino acids 1-7. Reverse primer P-23 Sequence of poly (dt) region (20 deoxythymidine), EcoRI site, and And 3 additional deoxythymidines at the 5'end.Clone PMT2-5   Is the clone PMT2-5 (about 1.5 kb) a partial clone PMT-26? RNA PCR and clones in combination with their sequence information (essentially as described above). It was obtained by using the technology. (In RNA PCR method, primer concentration Was 1.0 μM rather than 2.5 μM. Furthermore, the first step cycle f ile specified at 50 ° C rather than 55 ° C, last step cycle 10 min Rather, I specified 30 minutes. ) The template is tobacco root poly (A⁺ ) RNA The word and reverse primers are P-38 (SEQ ID NO: 14) and P-3, respectively. 6 (SEQ ID NO: 15). The sequence of primer P-38 is 60 kD PMT protein. Encodes amino acids 4-11 of the quality (SEQ ID NO: 4) N-terminal region (as described above). Based on the DNA sequence from the 51 region of clone PMT-26. Primer -P-36 was designed from the 3'region of DNA clone Q. Primer P-38 and And P-36 both showed 100% homology to their target sequences.   DNA sequence analysis was performed on the cloned PMT2-5 DNA insert. P The MT2-5 DNA sequence maps to PMT1.2 and Q7 DNA as expected. I did. From this result, clones PMT1.2 and Q7 displayed a single transcribed region. It was confirmed that I encode a single protein region. Clone P MT2-5 is an N-terminal methionine and amino acids 1-3 (ie leu-ser-ser). ) But encoded the complete 60 kD PMT protein. In addition to PMT2-5 , Clone PMT2-5 or Q7 Other clones with partial homology were obtained (data not shown).Clone PMT14-3   The forward primer, P-37, was added to the 5'end of clone PMT2-5. Added codons for PMT amino acids 1-3, alanine and N-terminal methionine It was synthesized for. The sequence of primer P-37 was determined by standard DNA PCR techniques. Were incorporated into PMT2-5 DNA. PMT2-5 DNA is a polymerase chain reaction And the forward and reverse primers are each It was set to -37 (sequence number 16) and P-36 (sequence number 15). D from PCR The NA product was cloned as above. This is the clone PMT14-3 Obtained by law. Clone PMT14-3 immediately after the N-terminal methionine Encodes a complete 60 kD PMT protein with additional alanine residues inserted It was determined to do. This homologous structure of clone PMT14-3 has a known sequence. Confirmed by analytical method. The inserted alanine residue is the N-terminal methionine residue The effect of PMT14-3D on post-translational processing of Recombination produced in a host cell transformed with an expression vector having an NA insert It can be determined by sequence analysis of the PMT protein.   Examples of recombinant DNA sequences prepared by the methods described herein include rice American Type Culture Collection in Rockville, Maryland The culture that was deposited with the company. E. coli PMT14-3 Named after, this culture was deposited on March 11, 1993 and was deposited with the ATCC. The number 69253 is attached. Nicotiana tabacum ) Leaves are sequenced in either the 3'to 5'or the 5'to 3'direction as follows: Agrobacterium tumefaciens containing the DNA sequence of No. 17 (Agroba cterium tumefaciens):       pBI121: control plasmid, Kan '(commercially available)   1.2A3-3B: PMT gene in the antisense direction                     Transformation with 1.2 kb fragment                     Plant   1.2A2-40: PMT gene in the antisense direction                     Transformation with 1.2 kb fragment                     Plant   1.2S3-15: 1.2 of PMT gene in the sense direction                     transformed with the kb fragment                     plant   1.2S4-16: 1.2 of PMT gene in sense orientation                     transformed with the kb fragment                     plant   Callus grew into a plant and took leaf samples before and after the plant was cut . Alkaloids in leaf samples were treated with methanol potassium hydroxide containing an internal standard. And extracted from transformed leaves and control leaves. The supernatant is filtered and turned into nicotine Gas chromatography analysis using a nitrogen-phosphorus detector for analysis. I served. I got the following results Was:   As expected, the amount of nicotine after the plant was cut was clearer than before it was cut. There are many crabs. However, the increase brought about by the pruning was Significantly reduced in plants grown from materials treated according to the invention as compared to I was

[Procedure of Amendment] Article 184-8 of the Patent Act [Submission date] May 31, 1995 [Correction contents]                         The scope of the claims   1. S-adenosylmethionine methyl group to putrescine δ-amino group Coat tobacco proteins characterized by their ability to catalyze a transfer reaction. Recombinant DNA molecule.   2. Nucleotide sequence of SEQ ID NO: 17 or a cross with said SEQ ID NO: 17 (hybridization , A nucleotide sequence to be hybridized or said nucleotide sequence The recombinant according to claim 1, which contains a degenerate nucleotide sequence in any of the following. E DNA molecule.   3. Performing transcription of mRNA encoded by the separated DNA molecule The DNA of claim 1 or 2 operably linked to a sequence capable of A vector containing the molecule.   4. Complementary linked to a sequence capable of transcription of antisense mRNA Claims characterized by a DNA sequence encoding a specific antisense mRNA The vector according to claim 3.   5. A culture stably transformed with the vector according to claim 3 or 4. Transgenic tobacco cells.   6. A plant stably transformed with the vector according to claim 3 or 4. Lancegenic tobacco plants.   7. Contains the nucleotide sequence of SEQ ID NO: 17 and contains putrescine N-methyltra An isolated DNA molecule encoding a transferase.

Claims (1)

[Claims]   1. S-adenosylmethionine methyl group to putrescine δ-amino group Coat tobacco proteins characterized by their ability to catalyze a transfer reaction. Recombinant DNA molecule.   2. Putre having the amino acid sequence of SEQ ID NO: 1, SEQ ID NO: 2 or SEQ ID NO: 3 The recombinant according to claim 1, which encodes succin N-methyltransferase. E DNA molecule.   3. Contains the nucleotide sequence of the cDNA insert of plasmid PMT14-3 Or a nucleotide sequence that hybridizes to the DNA insert or the nucleotide sequence The set of claim 1 containing a degenerate nucleotide sequence in any of the columns. Altered DNA molecule.   4. Nucleotide sequence of SEQ ID NO: 17 or a hybridization (miscellaneous) to such nucleotide sequence. A contract containing a DNA sequence that can be seeded (hybridized) A recombinant DNA molecule according to claim 1.   5. Performing transcription of mRNA encoded by the separated DNA molecule Claim 3, Claim 3, or Claim 4 operably linked to the array A vector containing the DNA molecule of.   6. Complementation linked to a sequence capable of transcription of antisense mRNA Claims characterized by a DNA sequence encoding a specific antisense mRNA The vector according to claim 5.   7. A culture stably transformed with the vector according to claim 5 or 6. Transgenic tobacco cells.   8. A plant stably transformed with the vector according to claim 5 or 6. Lancegenic tobacco plants.   9. Putrescine N-methyl transfer encoded by SEQ ID NO: 17 Rahse.   10. The protocol encoded by SEQ ID NO: 1, SEQ ID NO: 2, or SEQ ID NO: 3. Syn N-methyl transferase.