JPH10501978A - Trehalose-producing transgenic plant - Google Patents

Abstract

  (57) [Summary] The present invention relates to transgenic plants that produce trehalose and methods for increasing the trehalose content of plants. According to the present invention, plants of interest are transformed with a sequence encoding trehalose-6-phosphate synthase fused to a non-structural plant promoter to induce temporal, local or stress on gene expression. Control. The present invention can be used to protect specialty crop plants from drought, high salinity or extreme temperatures, and to improve the storability of harvested plants, including green foods, picked fruits and ornamental plants.

Description

DETAILED DESCRIPTION OF THE INVENTION                   Trehalose-producing transgenic plantField of the invention   The present invention relates to genetic engineering of plants for inserting trehalose synthesis ability . In particular, the invention provides plants with increased trehalose content and the production thereof About the method. The present invention also provides a plant withstands stresses such as cold and drought. A method of increasing sexuality and a method of producing trehalose.Background of the Invention   Trehalose (α-glucopyranosyl-α-D-glucopyranose) is It is a dimer of glucose molecules linked via a primary group. Loss of reducing group, slow Compared to other sugars, including the ability to form poor hydrolysis and non-tide soluble glasses Due to a unique combination of chemical properties, proteins, cell membranes and other in vitro organisms Is one of the most effective known preservatives of biological compounds. Also, a large amount of trehalose Living organisms, including insects, are characteristically insects, animals from certain coastal areas and yeasts and fungi. Often exposed to infiltration, dehydration and heat stress like many microorganisms, including fungi Things. The major role of trehalose in baker's yeast is There is circumstantial evidence that it is to confer resistance to the disease (Wiemken [1990] Antoni e van Leeuwenhoek 58, 209-217 abstract). However, trehalose in baker's yeast It has also been suggested that source accumulation does not itself confer stress tolerance (Nwaka et al. [1994] FEBS Letters 344, 225-228; Van Dijk et al [1995] Ap plied Environ. Microbiol. 61, 109-115).   High concentrations of trehalose are important for fern plants that can survive prolonged drought and heat exposure. Occur in so-called resurrection plants (Avigad [1982] Encyclopedia of Plant R) esearch (New Series) 13A, pp217-347). However, vascular plants Most cannot synthesize trehalose. Such plants are often Other "compatible" lysates, including quinone, proline and various polyols, are available Can be synthesized in response to stress, such as that caused by the loss of effective intracellular water ( McCue & Hanson [1990] Trends in Biotechnology 8, 358-362).   There have been few reports of trehalose in angiosperms, and Listed as small amounts that may reflect contamination (eg, Kandler & Se nse [1965] Z. Pflanzenphysiol. 53, 157-161; Oesch & Meier [1967] Phytoch emistry 6, 1147-1148). In fact, trehalose is found in many plant tissues (Vel uthambi et al. [1981] Plant Physiol. 68, 1369-1374), especially trehalase activity For those that have no or very little activity (trehalase (It is an enzyme that turns it into a course). However, few At least one angiosperm, Myrothamnus flabel lifolia) (another "resurrected" plant) has an apparent accumulation of trehalose (Bi anchi et al. [1993] Physiologia Plantarum 87, 223-226), trehalose and This indicates that there is absolutely no compatibility defect between the progeny plants.   The absence and reported toxicity of trehalose in most angiosperms is Insertion of the trehalose synthesis pathway into these plants has, in some cases, had toxic effects. Some suggestions may be made. On the other hand, sufficient training Haloose production can be a substantial advantage. For example, sugar beet, potato Trehalose accumulated in storage organs such as potatoes and onions is commercially extracted and Provides trehalose at a cost comparable to sucrose in use. These uses Trehalose is used for many foods in an economically attractive drying process. And to preserve the condition and, in this respect, better than sucrose, Includes the production of foods (milk and egg powder, soups, fruit purees, etc.) (eg, Rose r [1991] Trends in Food Science & Technology, July, pp.166-169: Ro And the non-sweetness of trehalose is often an additional benefit (soup, egg powder) Does not produce fructose, which is actually perceived as harmful to health. However, Trehalose is expensive and its use in the dry food industry is prohibitively expensive. Second, The production of trehalose in the edible parts of a plant is limited by the accumulation of tomato-like products. The shelf life can be extended. Third, trehalose accumulation in susceptible tissues can be caused by frost, drought Can increase the tolerance of plants to high salinity and similar stresses.Summary of the Invention   Based on the above description, the provision of a plant having a novel trehalose-producing ability It is an object of the invention. In particular, reduce the harmful potential of trehalose in plant growth Meanwhile, the use of trehalose in plant tissues as a commercial source of trehalose Providing controlled accumulation or stress tolerance or both is the purpose of the present invention. is there.   The present invention provides for the production of transgenic plants with increased trehalose content. Genes for trehalose synthesis under control of suitable plant promoters And is based on the idea of transforming the plant of interest. In particular, the invention Of the enzyme that produces trehalose 6-phosphate or trehalose itself The coding sequence for one or more genes encoding the repeptides may be It is expressed in plants under the control of plant promoters.   Thus, the plants of interest may have a complete expression of the gene in mature plants only, Is at least trehalose-, as is done when unique environmental conditions are encountered. Transformation with the coding sequence of the gene for 6-phosphate synthase (Tre6P synthase) Replace. Accordingly, the promoter is preferably non-structural and directs the expression of the gene. Temporal (e.g., daytime), topical (e.g., tissue specific) or stress-activated (Or "stress-induced") Select so that control is possible. Plants also Rehalose-6-phosphatase (Tre6Pase) encoding gene or Tre6P One regulatory polypeptide that interacts with Notase or Tre6Pase or both Or more.   Transformation of plants can be performed using transformed Agrobacter tumefaciens (Agrobacter tumefaciens), microinjection, electroporation Direct insertion of exogenous DNA by application, particle bombardment and direct DNA uptake. And methods available in the art, including. The structural genes are preferably each 56 kDa Tre6P synthase, 102 kDa Tre6Pase and 123 kDa Yeast gene TPS1 encoding yeast trehalose synthase regulatory subunit , TPS2 and TSL1.   Plants of interest are transformed with structural genes for trehalose synthesis, in particular Tre6P synthase. Transformase as described above, with time, local or Under the control of a suitable promoter to allow for stress-induced regulation, Transgenic with increased trehalose content, including the step of expressing the gene It is another object of the present invention to provide a method for producing a plant.   -At least Tre6P synthase to produce transgenic plants; Transform plants with one structural gene,   -Transgenic plants under conditions that induce trehalose synthesis expression in the plant Cultivate, and   -Extract trehalose from plant tissue The production of trehalose, including a step, is a third object of the present invention.   A fourth object of the present invention is to reduce drought, high salinity, extreme temperatures and other stresses. A method for protecting plants against adverse adversity, said method comprising at least Suitable for plant promoters such that complete expression of offspring does not occur until the plant is adversity The coding sequence of the gene for trehalose-6-phosphate synthase fused to And transforming the plant. The plant is optionally treated with trehalose-6-phos. Phosphatase and trehalose-6-phosphate synthase or trehalose Encodes a regulatory protein that interacts with -6-phosphatase or both Can be co-transformed with one or more genes. For example, the present invention Plant promoters to ensure that full expression of offspring does not occur until the plant encounters low temperatures Transformation with a trehalose-6-phosphate gene suitably fused with Protecting plants with berries or other fruits from frost damage to flowering, including Provide the law.   A fifth object of the present invention is to provide a thorough and sophisticated The present invention provides a transgenic ornamental plant that does not require care. Trehal, preferably fused to a plant promoter to include trehalose in the tissue Including transforming with the gene for ribose-6-phosphate synthase.BRIEF DESCRIPTION OF THE FIGURES   FIG. 1 shows the TPS1 yeast gene encoding the Tre6P synthase subunit. A. thaliana Rubico small fused submerged Arabidopsis Knit promoter (pats1A) and Agrobacterium tumefacien nopa Chimeric gene construct containing a transcription termination signal from the phosphorus synthase gene (nos) Is described. Only the chimeric gene and the plasmid pKOH51 are shown. Chimera Shows the unique restriction enzyme cleavage site of the gene construct.   FIG. 2 shows a transgenic tobacco showing the 56 kDa TPS1 product indicated by the arrow. Figure 4 shows the results of Western blot analysis of plants. The protein was constructed as shown in Figure 1. Object (lines 1, 3, 4, 5, 6, 8, 10, 12, 15, 16, and 19) or Cauliflower fused with β-gluconidase gene (UIDA) in the same vector Chimeric gene (GUS) or non-form at the mosaiuku virus 35S promoter Extracted from transgenic tobacco plants (SR1).   FIG. 3 shows the chromatographic identification of trehalose. Water extract of tobacco leaves Samples (20 μl) were analyzed by HPLC as described in General Materials and Methods. Was. Extracts A and B were cultivated in greenhouse before (A) and after (B) trehalose treatment. Transformant 19 raw weight 192mg ml^-1Was included. Extracts C and D are both sterile Plasmid lacking (C) transformant 4 or (D) TPS1 gene grown under conditions 149 mg ml of leaves from a control plant transformed with pDE1001^-1Was included. Trehalo The peak is indicated by T. Two large peaks (peaks 7 and 8) of about 25 minutes Lucose and sucrose.   Fig. 4 TPS1-subunit tissue analyzed for ats1-TPS1 transformation line 4 It is a protein immunoblot showing localization. Wild type tobacco (SR1) leaves vs. negative Tre6P synthase subunit 0 purified as control and from yeast (TPS1) . 1 μg was used as a positive control. Immunize the trehalose content of the same tissue Indicate in mg / g dry weight on the top. nt, not tested. Use the following abbreviations: FT Bud, UL is upper leaf, ML is middle leaf, LL is lower leaf, US is upper trunk, R is root and EC is enzyme control.   Figure 5 Enhanced drought tolerance in trehalose producing plants. in vitro breeding plant Another leaf from the same developmental stage at 6 to 8 weeks of age was exposed to air drying (25% RH). ( A) The leaves are weighed at the indicated times and the results are expressed as the relative fresh weight of the leaves at each time point. The dry weight of each leaf was obtained after drying the leaf at + 60 ° C. for 48 hours. Wild type control tobacco Is indicated by SR1, and the trehalose-producing transgenic lines 1, 4 and 8 are Each line number is shown. (B) Transgenic when selected during stress treatment Appearance of different leaves from Cookline 4 and control plants. Figure 6 Drought survival of control and trehalose producing tobacco plants. (A) 6-8 weeks old in  All plants propagated in vitro were exposed to air drying (D) for the times indicated. 17:00 stress treatment Shortly after, the untransformed control plant (SR1) and the trehalose-producing transgenic Plant lines 4 and 8 were refilled (R) by placing them in water, respectively. (B) Transgenic line 8 and non-transformed (SR1) and vector transformation Exchange (C) Both control plants were exposed to air-drying (D) for 3 weeks of age. Subsequently, the seedlings were re-watered (R) by placing them in water after 7 hours. Figure 7 Mycobacterium smeg eluted from the first heparin-separose column SDS-PA of fraction containing M. smegmatis Tre6P synthase GE analysis. Lanes 1 to 4 are H33 (7.0 mU), H35 (20 mU), H Includes 36 (21 mU) and H37 (12 mU) samples (Table 3). 8% acrylic After SDS-PAGE with mid, the gel is stained with Coomassie Brilliant Blue did. The size of the molecular weight standard in Lane 5 is indicated in kDa. 55kDa Tre6P sinter The position of the protease polypeptide is indicated by an arrow. Figure 8 Mycobacterium smeg eluted from the second heparin-separose column SDS-PAGE analysis of the peak fraction of Matis Tre6P synthase. H Fraction T21 (Table 3) was concentrated in a Centricon 10 tube and then concentrated to three times its half volume. Mixed with compacted SDS-PAGE sample buffer. All concentrations were 6-fold. Tre6 A sample containing 2.9 mU (lane 1) and 8.7 mU (lane 3) of P synthase 8% acrylamide on SDS-PAGE, Coomassie Brilliant Stained with roux. Lane 2 molecular weight standards are 109, 84, 47, 3 from top to bottom. 3, 24 and 16 kDa. Fig. 9: Tre6P synthase purified from Mycobacterium smegmatis This is a Western analysis. 8% acrylamide gel was used for molecular weight markers before staining (lane 1: 200, 117, 80 and 47 kDa; Lane 10: only visible marker 84 kDa), pure Tre6P synthase from fraction T21 (Table 3) (lane 7 And 8: 2.9 mU; lanes 3, 4 and 9: 8.7 mU) and G100 cephe Storage activity fraction from Adex (Table 3) (lane 6: 7.8 μl total protein) g; lanes 2 and 5: 23 μg of total protein). Electrophoresis and nitro After blotting on cellulose, the nitrocellulose membrane was applied to lanes 3 and 4 and 7 And 8 were cut. Lanes 1-3 dyed with Coomassie Brilliant Blue The lanes 4 to 7 are colored and the purified 56 kDa Tre6P synthase subunit derived from yeast is used. And probed with anti-57 serum raised against And probed. The immunoreactive band was analyzed with a goat anti-rabbit IgG algo from Promega. Using a kaliphosphatase conjugate, both at the time of color development, according to the manufacturer's instructions The interval was visualized in 2.8 minutes. The diagonal lines across lanes 4 to 7 are nitrocellulose Sudden membrane fold during transition to. Fig. 10. Eight transgenes of Arabidopsis thalina Nickline Western analysis. Extract plants grown from seeds of primary transformants And a purified 56 kDa Tre6P synthase derived from yeast essentially as described in FIG. Analyzed with antiserum raised against buunit. T purified from yeast (0.1 μg) The re6P synthase subunit was used as a positive control. Untransformed arabid Psys Salina had no signal.Detailed description of the invention   In the following description, the term "structural plant promoter" refers to its associated coding sequence. Causes the continuous and general expression of the sequence, and the product of the sequence Plant promoters are found at all stages of the vesicle and development. In contrast Non-structural promoters form distinct tissues as plants grow and mature. Specific, such as the differentiation of cells to change or changes in the plant environment Activated by various internal and external events. Many types of environmental changes are B It is known to activate motors. Examples are ats1 used in Examples 1-3. Ribulose-1,5-biphosphate carboxylase such as A promoter -Induced activation of small subunits of Krebbers et al. [1988] Plant Mol. Biol. . 11, 745-759) and LTI78 (Nordin et al. [1933] Plant Mol. Biol. 2). 1, 641-653) and RAB18 (Lang & Plava [1992] Plant Mol. Biol. 20, 9). 51-962). But However, our invention is not limited to these examples of non-structural promoters. Rather An important and novel part of the present invention is that the structural synthesis of trehalose by plants is always A concept that is economic and, in some cases, harmful, and therefore described herein. Benefits of trehalose production are best understood by using non-structural promoters Wear. By means of these non-structural promoters, transgenic plants Trehalose biosynthesis is temporally regulated (i.e., occurs only at certain times), Control (ie limited to certain parts of the plant) or both .   As used herein, the names of plant organs are generally used by ordinary fruit and vegetable merchants and consumers. Is what you are doing. Thus, for example, "fruit" refers to those storage groups that contain seeds. Woven is not itself a fruit from a strict botanical sense, but is sold for eating Including apple or strawberry plants.   It has already been stated that the majority of vascular plants seem to lack trehalose synthesis capacity. Therefore, "trehalose-producing" plants do not require a quantitative definition. However A normal plant (other than the so-called resurrection plant) produces trehalose during stress What can be done does not seem to be studied. The present invention relates to trehalose synthesis. For genetic engineering of plants to insert heterologous capabilities for Thus, trehalose synthesis is also referred to as a “new” ability). Grown under similar conditions The required increase in trehalose compared to untransformed plants is Can be usefully extracted from plants or can be advantageously extracted from plants for commercial use. Is one of them.   Many microorganisms have an enzyme that produces trehalose-6-phosphate (Tre6P). System, which is hydrolyzed to trehalose. For example, Saccharomyces Saccharomyces cerevisiae has 56, 102 and 123 kDa. Contains trehalose synthase complex containing subunits (Londesborough & Vuorio [1993] Eur. J. Biochem. 216, 841-848), which are uridine diphospho Glucose (UDPG) and glucose-6-phosphate (Glc6P) were initially Tr e6P (Tre6P synthesis reaction), and then condensed to free trehalose (Tre6Pase reaction). Tre6P synthase and Tre6Pase activities were 56 and 10 respectively. Attributable to the 2 kDa subunit, the 123 kDa subunit imparts regulatory properties on the complex. Give it, it seems to stabilize. Candida utilis ( Soler et al [1989] FEMS Micobiol. Letters 61, 273-278), Escherichia E. coli (Glaever et al [1988] J. Bacteriol. 170, 2841-2849), D. Dictyostelium discoideum (Killick [197 9] Arch. Biochem. Biophys. 196, 121-133) and M. bacterium Others, including Gumatis (Lapp et al [1971] J. Biol. Chem. 246, 4567-4579). The microbial system uses adenine diphosphoglucose (AD) instead of UDPG for the latter two. PG) can be used. Most of the subunit structures of these microbial enzyme systems There is no information. A variety of trehalose producing species including nematodes, insects and resurrected plants Fungi are also thought to contain Tre6P synthase and enzymes with Tre6Pase activity. Can be   In principle, the transfer of these Tre6P synthases to the plant will Gives a new ability to synthesize Plants like tobacco turn Tre6P into trehalose It is described as having an intrinsic ability to transform, and surprisingly, for example, Transformation of Bacco with Tre6P synthase alone results in effective trehalose itself. Guides production. However, the endogenous conversion of Tre6P to trehalose is slow. Or, if missing, may be transformed again with Tre6Pase from a suitable source. Tr Enzymes in which e6P synthase and Tre6Pase are subunits of trehalose synthesis Plants are derived from the same source so that a natural enzyme complex can be formed in a system like the mother enzyme. Preferably co-transformed with Tre6P synthase and the gene for Tre6Pase . Whatever the source of the enzyme, trehalose formed in the transgenic plant , It can then be extracted or impart increased resistance to certain stresses. Ah The production of trehalose in plants transformed in this way with different yeast genes is already Have been described (PCT / FI93 / 00049).   However, in some cases (e.g., when exposed to stress or Medium) and certain tissues (e.g., storage organs or frost-sensitive tissues at appropriate times). Trehalose accumulation is considered beneficial or at least harmless to the plant. Yes, trehalose accumulation at other times or in other tissues is harmful to plants. There are obvious possibilities (Veluthambi et al. [1971] supra). Therefore, the plant Plants are planted until they mature or encounter environmental conditions including drought and cold. Until the benefits of rehalose outweigh its disadvantages, it causes trehalose synthesis A plant promoter that does not completely express a gene (plant promoter Which are part of the gene that promotes transcription of the nucleotide sequence).   Ribulose-1,5-bisphos drives light-induced expression of small subunits of RUBISCO Fate carboxylase (Rubisco promoter) (Krebbers et al. [1988] Pl ant Mol. Biol. 11, 745-759). Are known to those skilled in the art.   A yeast TPS1 fragment fused exactly to the ats1 promoter of the Rubisco small subunit Tobacco and Arabi transformed with the gene coding sequence (open reading frame, ORF) Disclosed are Dopsis plants. TPS1 is 56 kDa of yeast trehalose synthase. Code. Transformed plants are healthy, fertile and have trehalose in their leaves. including. Transformed with untransformed tobacco or a similar vector lacking the TPS1 gene The changed tobacco does not contain trehalose. The disclosed transgenic plant is shaped It was obtained using Agrobacter tumefaciens as a medium for transmutation. Direct DNA injection, including injection, electroporation or particle bombardment Methods feasible in the art, including insertion, may be used (Gasser & Fraley [1989] Scie nce 244, 1293).   One of these transformed tobacco plants (Transformant 4) has Tre6P synthase activity. Is included. The free 56 kDa subunit is yeast intact trehalose. It is known to be unstable when isolated from synthase complexes (Londesbo rough & Vuorio [1993] supra). The method can be carried out by those skilled in the art using plant promoters such as ats Under the control of the 1A promoter or other convenient promoter, which may be structural, The TPS1 gene and other yeast trehalose synthase genes (TPS2 and It has been described that plants can be co-transformed with one or both TSL1). Such co-transformation is based on the subunits encoded by TPS2 and TSL1. To stabilize the 56 kDa subunit, compared to plants containing only TPS1 Can increase the trehalose content of the plant.   A Tre6P synthase gene controlled by an inducible promoter is inserted into a structural promoter. One or more of the motor-controlled Tre6Pases or regulatory proteins Trehalose production in plants co-transformed with the Intase catalyzes the first and unique step in trehalose biosynthesis, which results in an inducible Controlled by a motor.   Trehalose-producing transgenic tobacco plants show enhanced drought tolerance Make it easy. This is shown in both cut leaf and whole plant experiments. Break Improved drought tolerance is shown in mature plants and young seedlings of self-pollinated offspring of the transformants. Is done. In these progeny, drought tolerance was determined by the 52 kDa synthase in the green tissue of the progeny. TPS1 is evident by the presence of the Tase subunit⁺Co-inherit with traits.   The water stress tolerance of the disclosed transgenic plants is a measure of osmotic buffering of cell contents. The amount of trehalose found in the tissue seems to be too low to provide It is surprising. Perhaps trehalose protects specialized sites such as membranes Or perhaps trehalose or its precursor Pre6P It disrupts plant metabolism and causes secondary changes that protect cells from stress.   Most of the primary transformants containing the chimeric TPS1 gene Spear-shaped leaves, reduced apex dominance and reduced, not found in loin producing offspring Note that slight morphological changes, such as height, are seen. These changes are Appears mainly in tissue culture artifacts. However, Treha containing self-pollinated offspring Loin-producing tobacco plants are slow growing and grown under optimal conditions for 8 weeks before Reveal a one to two week delay from the plants. This is a Rehalose synthesis may slow growth even under the control of the ats1A promoter. And suggest. Therefore, LT178 (Nordin et al [1993] Plant Mol. Biol. 21, 641-65). Stress-inducible promoters, such as 3), are more important for maintaining normal growth rates. Trehalose-induced under stress as described in Example 5. Provides protection, but produces under non-stress conditions.   These transgenic plants contain a TPS gene encoding Tre6Pase. But only with the yeast TPS1 gene encoding Tre6P synthase To synthesize trehalose. Thus, some plants have Tre6Ps Has intrinsic ability to convert to loin. In some cases, T encoding Tre6Pase It may also be advantageous to co-convert the plant with a gene such as PS2, Tre6P Increase the rate of conversion of trehalose to trehalose. Furthermore, similar results were obtained with microbes other than yeast. That can be obtained with the gene for Tre6P synthase (and Tre6Pase) It is obvious. Many of these genes are homologous to TPS1 (and TPS2), Immunological or oligonucleotide probes derived from yeast enzymes and genes Can be easily cloned using. For example, Mycobacterium smegmatis Tre6P synthase is a purified 56 kDa Tre of yeast trehalose synthase. Approximately 55 kDa that cross-reacts with antisera raised against the 6P synthase subunit It is disclosed that it is a polypeptide. Mycobacterium smegmatis enzymatic A new tryptic peptide has been disclosed and has close sequence homology to yeast enzymes Sex is obvious. Those skilled in the art will recognize that the Tre6P synthase and the Tre6Pase gene , Can be easily cloned from many organisms using these approaches. Our invention is , Tre6P synthase and the like obtained from a suitable organism and fused to a suitable plant promoter. And transformation of plants with the gene for Tre6Pase.   Transformation with one or more genes for trehalose synthase Plants containing rehalose can be used in several ways. For example, trehalose Can be extracted from plants on a commercial scale. Such trehalose can be used in drying plants. Inexpensive enough to get no bulk use, including scent and condition retention. In this use, Rehalose is preferably potato tuber, sugar beet or turnip root It accumulates in storage organs such as bulbs of onions. The methods are described in these examples and many more Transformation of other crops (Lindsey [1992] J. Biotechnol. 26, 1-28) Although described, our invention is frequently used by molecular biologists as a model. It is not limited to plants that are present. Those skilled in the art will recognize that methods developed for model plants Applicable to plants. Thus, the present invention provides, for example, a transformed sugarcane stem or Also performed on leaves or banana fruits. Specific expression in storage organs Plant promoters that result in (i.e., patatin promoter Data). In one embodiment of the invention, a Tre6P synthase such as TPS1 The TPS1 coding sequence is at such a promoter that the TPS1 coding sequence is at Fused to such a promoter in the same manner as fused to the s1A promoter A suitable plant is transformed with these DNA constructs. Then accumulates in storage organs The extracted trehalose can be extracted.   In another embodiment of the present invention, the trehalose accumulated in the plant tissue is post-harvest. May improve storage characteristics. Therefore, the cut leaves of the transformed tobacco are untransformed. Loses water slowly compared to cigarettes, and does not change color even after losing water (Fig. 5). This embodiment is applicable to both edible and ornamental plants. Edible plant With regard to the variety of lettuce products that occur after harvesting, A significant financial burden, especially at retail stores. Final cost is paid by the consumer . Trehalose producing lettuce provides a cheaper and more attractive lettuce. Similar considerations Eggs, edible plants, especially cabbage, broccoli, spinach, inondo and Is a leaf product like parsley and green like peas and safflower beans Applies to colored vegetables. Many ornaments like roses, tulips, daffodils Plants are transported after cutting. Even expensive transportation (refrigerated, air) can waste a lot of money Occur. Again, the costs are borne by the consumer, who gives them a good moisture content. Cheaper and more comfortable with trehalose-producing ornamental plants that hold and are less sensitive to drying Product will be provided. In this aspect of the invention, Tre6P synth The gene is selected so that trehalose accumulates at the site of the plant to be harvested. Was Fuse with plant promoter. For example, the ats1A promoter is a tobacco leaf, It leads to the expression of Tre6P in the upper stem and bud, and trehalose is in these parts. Accumulates in place However, the inventor has found that plants such as tobacco are Transports trehalose from its synthesis site to tissues that do not synthesize trehalose You. Therefore, expression of Tre6P synthase in roots with the non-structural ats1A promoter But not a small amount of trehalose, these transgenic tobacco It was also found in plant roots.   In a related aspect of the invention, a trait, such as tomatoes, berries and other fruits Trehalose-containing edible sites in transformed plants are also new due to their trehalose content. Produces puree, paste, jelly and jam with a rich and rich aroma Processed for The addition of trehalose to such plants, especially when the treatment is drying In addition, it has been shown to improve the retention of the scent when it is included (WO89 / 00012). Book The present invention provides a plant that already contains trehalose, Avoid the need for additional Primarily directs trehalose synthesis in storage organs of target plants The use of a promoter, such as a patatin promoter, is useful in this aspect of the invention. May be useful.   Trehalose-producing transformed tobacco is disclosed to have increased drought tolerance You. In general, trehalose-containing transformed plants are free from drought, frost, high salinity and other soils. Tresses can be more resistant than untransformed plants. Therefore, drought, frost and All osmotic stress afflicts plants primarily by removing intracellular water, resulting in membranes and And trehalose can reduce it in vitro. (Crowe et al [1992] Annu. Rev. Physiol. 54, 579). The present invention In one embodiment, the plant promoter used is one induced by stress. It is. Such promoters are known in the art, for example, LT178 ( Nordin et al. [1993] Plant Mol. Biol. 21, 641-653) and RAB18 (La ng & Palva [1992] Plant Mol. Biol. 20, 951-962). This is Trehalo Inhibit source synthesis until needed. A plant that contains trehalose but grows well For this aspect of the invention, this can be achieved without the means of a stress-inducible promoter. Come You. However, stress-inducing programs that block trehalose production until needed The use of motors would otherwise convert photosynthetic capacity to trehalose synthesis. Avoid the production disadvantages that result, and evident in tobacco containing pats1A-TPS1 chimeras. It is further advantageous to avoid such possible delays in growth. This embodiment relates to an edible plant It also applies to ornamental plants intended for garden or interior decoration. Transformation Tress-tolerant ornamental plants are more concentrated and specialized than their non-transformed counterparts. Not necessary. At least some trehalose-producing plants found in primary transformants Growth retardation and slight morphological changes in ornamental plants may not be disadvantageous. -Growth retardation and new appearance can be attractive properties, especially for upholstery Can be.   In yet another embodiment of the present invention, the Tre6P synthase has a specific event or condition. (E.g., cold or drought stress) For example, it is preferably fused with LTI78 or RAB18) and is commercially extractable. Trehalose accumulation in plants should occur in mature plants shortly before harvest. Can be pulled, avoiding the harmful effects of trehalose during the early development of certain plants.   Based on the above description, the transgenic plant of the present invention Barley, millet, wheat, rice, barley, sorghum, amaranth, onions, asparagus Or monocotyledonous plants such as sugarcane or alfalfa, soy, petunia, Cotton, sugar beet, sunflower, carrot, celery, cabbage, cucumber, pepper , Tomato, potato, lentils, flax, broccoli, tobacco, beans, lettuce, a Brassica, collard, cauliflower, spinach, brussels sprouts, artichoke Twins like croak, peas, okra, pumpkin, kale, tea or coffee It can be a leaf plant.   Yeast gene TPS1 and its products are compatible with tobacco biochemical tissue It is disclosed that the gene is highly expressed and the 56 kDa subunit Resulted in the expression of loin. However, in the art, plant genes are e.g. It is known to often have a lower A + T ratio compared to microbial genes, Expression levels of heterologous genes in plants indicate codon usage, especially near the start of the coding sequence Can be increased by altering it like that found in plants (Perlak et al. [1 991] 88, 3324-3328). We note that these and similar modifications of the gene are I anticipate that it may be useful.   It is also well known that natural mutations occur in genes. These DNA sequences And artificial changes can alter the amino acid sequence of the encoding polypeptide. In this specification In the use of TPS1 (or TPS2 or TSL1), the yeast trehalose At the 56 kDa (or 102 kDa or 123 kDa) subunit of synthase TPS1 (or TPS2) encoding a polypeptide of desired function or structural properties Or all DNA sequences homologous to TSL1). Similarly, “Tre6P sinter Gene (or Tre6Pase or regulatory polypeptide) Such genes that can be readily isolated (eg, TPS1 or TPS2 or T Alone (using nucleotide probes designed from known genes of SL1) Without the desired function or function of the polypeptide encoded by the originally isolated gene. Also includes natural and artificial variants that encode polypeptides of structural characteristics.Example General materials and methods material   The plant used was Nicotiana tabacum cv. SR1 and Arabidopsis thalian a L. Heyng. It was ecotype C-24.   Copies the 56 kDa Tre6P synthase subunit of trehalose synthase. Yeast gene TPS1 (formerly called TSS1), trehalose synth The yeast gene TPL1, encoding the 123 kDa control subunit of the enzyme, is Vuo Plasmid pAL described in rio et al. ([1993] Eur. J. Biochem. 216, 849-861). Obtained from K752 and pALK754. The yeast gene TPS2 is from the Swiss Bot And obtained from Dr. Andres Wiemken and cloned into the SacI site of plasmid YCplac111. And contained the activated TPS2 gene. This gene (De Virgilio et al. [1993] Eu r. J. Biochem. 212, 315-323) are listed in Table 1 of Vuorio et al. (1993, supra). As shown, it encodes an amino acid sequence derived from 102 kDa. Yeast trehalose Produced for non-nase (anti-TPS / P) and 56 kDa subunit (anti-57K) Antisera and common biochemicals are derived from sources described in Vuorio et al. (1993 supra). Met. Small vacuole trehalase, suc^-gal^-mel^-mal^-Lon from yeast strain (ALKO2967) Part as described in desborough & Varimo ([1984] Biochem. J. 219, 511-518). Purified and does not hydrolyze sucrose, maltose or melibiose Was.DNA manipulation   All DNA manipulations were performed according to established laboratory methods (Samb rook et al. [1989] Molecular Cloning: A Laboratory Manual, Cold Spr. ing Harbor, New York). Escherichia coli strains DH5α and MC1061 Was used for plasmid production. The promoter used to control TPS1 expression was Ats1A of Arabidopsis thaliana, encoding a small subunit of Rubisco It was derived from the gene (Krebbers et al [1988] supra).   To construct the chimeric ats1A-TPS1 gene, the sequence of the transit peptide was deleted. Amplifies the ats1A promoter fragment from plasmid pGSFR401 by PCR did. A synthetic oligonucleotide primer was placed 5 ′ of the amplified fragment with EcoR I site and used to generate an XbaI site at the 3 'end. PCR amplification production The product was digested with suitable restriction enzymes, followed by purification on an agarose gel, EcoRI and It was ligated into the MluI digested pUC19 plasmid. The yeast TPS1 gene Was amplified from plasmid pALK752. The resulting fragment was 5 'MluI and And a 3 'XbaI site. After digestion and purification, the fragment was I ligated behind ats1A. Fragment of promoter-TPS1 construct Is digested with EcoRI and XbaI, then its polyadenylation signal and T- PBI derived from a plasmid containing the 3 'end of the T-DNA gene G7 containing the right end of the DNA uescriptII SK⁺(Stratagene). Finally, the complete chimera gene is As a RI-SacI fragment, the chimeric kanamycin resistance gene pNOS-N Plant transformation vector pDE100 containing EO-3'OCS as selection marker 1 (Denecke et al [1992] EMBO J. 11, 2345-2355). This obtained Rasmid pKOH51 contains the chimeric pats1A-TPS-1-3′G7 gene. The structure is shaped Cloned into Escherichia coli strain DH5α by Poration (Dower, Miller & Ragsdale [1988] Nucl. Acids Res. 16, 612). 7-6145), the non-tumorigenic Ti plasmid pGV2260 (Deblare et al [1995] Nucl. Acids Res. Agrobacterium tumefacien containing 13,2777-2788) (C58C1 rif^R).   Other constructs were produced in a similar manner.Plant growth   For aseptic growth, sterile explants from Nicotiana tabacum (SR1) Solid MS with 2% sucrose (Murashige & Skoog [1962] Physiol. Plant) 15,473-497) Planted in glass jars containing medium (MS-2). These jars The plants are then grown under controlled growth conditions in a culture room where they can grow at 22 ° C. for 16 hours with light hours. Placed. The explants were transferred to a new jar and MS-2 for continuous growth of sterile material. Transferred to medium constantly. Greenhouse plants were grown on soil in flower pots and watered daily. The transformed Arabidopsis thaliana plants were first transformed into tobacco as described above. It was grown aseptically in a baby food shear under your environment, but later used for seed production. The plant was transplanted to the soil of a flowerpot in the soil. Primary transformant seeds are used to produce new seeds 24 well tissue culture plate, plated directly on soil or surface sterilized for molecular analysis Or aseptically grown.Plant transformation   Both tobacco and Arabidopsis thaliana were According to the call, starting materials are tobacco cut leaves and Arabidopsis thaliana The roots were transformed. Transformation and tissue culture are basically performed by Valvekens et al ([1988] Proc. Natl. Acad. Sci. U.S.A. 85, 5536-5540). Yes, with the following modifications: The isolated roots or leaves are incubated with solidified callus induction medium (CIM) for 4 hours. Preincubation for one day, cut the roots into small segments (1-2 mm) Was cut into large pieces (10-20 mm) and transferred to 20 ml liquid CIM. 3 ', 5'-di Methoxy-4'-hydroxyacetophenone (0.2 mg / l) was added to Agrobacterium (C56C1 rif^R) Added before infection. Bacteria used for infection are treated with suitable antibiotics overnight. In a YEB medium (Vervliet et al [1975] J. Gen. Virol. 26, 33-48) containing C., and collected by centrifugation. Bacterial pellet with 10 mM MgSO_FourResuspend in Then, the mixture was added to the plant tissue and gently stirred for about 15 minutes. Run off excess liquid and discard, root Was blotted onto sterile filter paper. After co-culture with solid CIM for 2 days, the plant tissue was The bacteria were washed once by rinsing with liquid CIM and transferred to a selective germination induction medium (SIM). 7th After growth, explants of the differentiated morphological sector were transferred to fresh SIM.Protein extraction for western analysis   100-200 mg fresh weight vegetable sump Using a protein extraction buffer (50 mM Tris / HCl pH 7.2, 250 mM Claus, 5 mM EDTA, 10 mM MgCl_Two, 1 mM CaCl_Two, 10 mM   β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride (PM SF), 30 μM pepstatin, 50 μM leupeptin and 15 μM (Protinin) was homogenized with a glass rod in a 1.5 ml microcentrifuge tube containing 100 μl. Insoluble Sexual matter was removed by two centrifugations (13,000 g, 10 minutes). Supernatant protein concentration The degree of bovine albumin was determined according to Bradford ([1976] Anal. Biochem. 72, 248-254). Was measured as a standard. Equivalent amount of soluble protein is used for immunological studies in SDS -PAGE.SDS-PAGE and immunological techniques   Proteins were separated by SDS-PAGE (Laemmli [1970] Nature 227, 680-685). For protein detection, Lottin was treated with antiserum anti-57 (1/1000 dilution) and anti-rabbit alkaline phospha Performed with a second antibody conjugated to the enzyme. Protein for Western blotting Was electrophoretically transferred to a nitrocellulose filter and 0.5% Poncea in 5% acetic acid. u Staining with Red confirmed successful filling and transfer of the sample in equal volumes. Then Luther was blocked overnight with 5% non-fat dry milk, probed and stained using standard methods.Tre6P synthase assay   Weigh about 500 mg of frozen plant material, then mortar and Using a pestle to fine powder, solid CO_TwoGround on. Add 1 mM PMSF and powder 1 mM benzamine containing 10 μg / ml of either pstatin A or leupeptin Gin, 2 mM MgCl_TwoContains 1 mM EDTA and 1 mM dithiothreitol Transfer to 0.7 ml of 50 mM HEPES / KOH, pH 7.0 (HBMED) and dissolve Was. The obtained homogenate was centrifuged at 17,000 g for 10 minutes, and the supernatant was treated with Tre6P sinter. For Lze et al., Essentially Lapp et al. ([1971] J. Biol. Chem. 246, 4567-4579). Tested according to Samples (10 μl) were prepared at 40 mM HEPES / KOH, pH 7.0, 10mg MgCl_Two, 10 mM glucose 6-phosphate, 5 mM Fructose 6-phosphate, 5 mM uridine diphosphoglucose (UDPG ) And 1 mg / ml bovine albumin containing 90 μl of the reaction mixture and required at 30 ° C. Incubation was carried out for various times. Trehalose and trehalose 6-phosphate Other sugar derivatives (including sucrose formed) were added to 50 μl of 0.6 M HCl 5 minutes at 100 ° C. and then 50 μl of 8% NaOH and 15 minutes at 100 ° C. Destroyed by heating. The remaining carbohydrates (i.e., trehalose and Rehalose 6-phosphate) anthrone test (Trevelyan & Harri) son [1956] Biochem. J. 63, 23-33).Trehalose test   Approximately 500 mg of frozen plant material is quickly weighed into a glass tube. Hot distilled water (1 ml) is added, the mixture is boiled for 20 minutes and the leaf material is beaten with a flat glass rod. Destroyed. The liquid phase is collected with a Pasteur pipette and the solid is re-extracted with 0.5 ml of water. Was. The combined liquid phases were clarified by centrifugation. Combined solid residue until constant weight At 107 ° C. (the dry weight of the leaves is an average of 5.1% of the fresh weight). Supernatant Was analyzed by Dionex DX-300 liquid chromatography with Dionex pulsed electrochemical detector ( Analysis was performed using PED-2). Samples (20 μl: 3 each) were transferred to Carbopac PA -1 (4 x 50 mm) pre-column to Carbopac PA-1 (4 x 250 mm) column Injected and eluted with water at 1 ml / min. The eluate was added to the post-column reagent (0.6 ml / min. .3M NaOH). Trehalose is glucose that elutes in about 20 minutes. And about 3 minutes before the sucrose peak.Embodiment 1 FIG. Tobacco transformed with the yeast TPS1 gene under the ats1A promoter Trehalose production by plants   Tobacco transformants and control plants are grown under both sterile in vitro conditions and greenhouses I let you. Mature transformants are either untransformed controls or vector pDE1001 (TP Lacks any apparent phenotype compared to controls transformed with (lacking S1) Was. Leaves are collected at 0900 hours, frozen prior to analysis, and at or below -70 ° C Stored at   Twenty of the 26 kanamycin resistant transformants tested were yeast trehalose. Purification of sucrose synthase generated against the 56 kDa Tre6P synthase subunit Detectable levels of immunoreactive polyproteins when probed with different antisera It was found to produce peptides. An example is shown in FIG. Table 1 shows leaves 1 summarizes the trehalose content of   These results indicate that the yeast TPS1 gene has its promoter ats1A promoter. And that it is effectively expressed in tobacco when replaced with is there. The specific signal observed in the Western blot has the correct molecular weight You. Strongest signal (eg, from transformant 4 grown in vitro) Is higher than the signal obtained from the yeast in the stationary phase, per unit of protein loaded on the gel. It was only slightly weaker. TPS1 expression was detected in trehalose in leaf tissue. Is associated with the appearance of HPLC, its HPLC behavior and its Identified by both facts that they were degraded by different trehalases (FIG. 3 also reference). Various transformants have been established at various levels, for reasons that have not yet been established. Expresses the TPS1 product and is found in the leaves (with the obvious exception of transformant 8) The amount of trehalose obtained was approximately the same as the intensity of the 56 kDa signal in the western. There was a correlation (compare FIG. 2 with Table 1). These transformants are recombinant Tre6P Did not have the gene encoding the Aase, but the plant had a higher T than trehalose. No evidence was found to accumulate more re6P. Obviously, Bako has a phosphatase capable of converting Tre6P to trehalose, At least in this case, the key to introducing the trehalose synthesis pathway is The enzyme is Tre6P synthase and, although not necessarily optimally, This indicates that the introduction of the enzyme alone is appropriate.   Organization of the 56 kDa Tre6P synthase subunit of transgenic line 4 Measurement of distribution indicates that a substantial amount of this polypeptide is in all but the lower stem of the plant. It shows that it can be in the green area but not in the root. This distribution is ats1A gene This is due to the tissue specificity of the expression (De Almeida et al. [1989] Mol. Gen. Genet. 218, 78). FIG. 4 also shows that tissues expressing Tre6P synthase also contained trehalose. And In addition, lower amounts of trehalose are also found in the roots, Showing that tobacco transports trehalose from its synthesis site to other tissues I have.   This result indicates that TPS1 was expressed under the ats1A promoter and their green color during the day. Tobacco plants that accumulate trehalose in color tissues must be natural and normal in appearance Is revealed. The major transformant containing the chimeric TPS1 gene was spear Has some morphological changes, such as stalks, reduces top dominance and lowers height (See FIGS. 5 and 6), but most of the changes were in self-pollinated TPS1-positive progeny. Not shown, this still produced trehalose (see Figure 6). Therefore, the main form Morphological changes in transgenics are more a consequence of trehalose production than It appears to be an artifact of tissue culture. However, Tre6P synthase High levels of buunit and the presence of trehalose 20-50% reduction in growth rate under normal conditions compared to untransformed control I was sorry. Transgenic plants with these apparently normal phenotypes In contrast to the reported toxicity of exogenously added trehalose to foods (Ve luthambi et al. [1981] cited above). These results indicate that the ats1A promoter Controlled production of trehalose in plants may slightly decrease the growth rate, It reveals no toxicity for tobacco. Specific including drought or cold Trehalose when necessary using an inducible promoter triggered by the Can reduce growth rate by minimizing growth rate You. Trealose content of the best transformants in Table 1 on a protein basis ( For example, in the case of transformant 4>16 mg / g protein) already in yeast A clear increase in heat resistance is at least 20% of the level observed (De Virgili [1990] FEBS Letters 273, 107-110).   Certain TPS1-transformants and controls are assayed for Tre6P synthase activity did. The results shown in Table 2 are the average of two zero and 15 or 30 minute assays. ± Maximum range. In the control, Tre6P synthase activity was different from that at zero minutes. Did not. In transformant 4, acid and alkali stable carbohydrates were UDPG and And accumulated in the presence of Glc6P. This accumulation requires Glc6P, but Toose 6-phosphate (Fru6P; this hexose phosphate is Does not require trehalose synthase complex) Replacement with PG hindered (enzyme purified from yeast also cannot use ADPG) No). The accumulated carbohydrate is probably trehalose or Tre6P. Seems, because other possible products have been destroyed by hydrolysis is there. HPLC analysis indicated that it was not trehalose. Therefore Under these in vitro assay conditions, Tre6P is converted to trehalose. As a result, Tre6P is synthesized by the extract of the transformant 4 earlier. This means , The overall rate of trehalose synthesis in leaves encodes the Tre6P subunit This shows that it can be increased by co-transformation with TPS2.   The Tre6P synthase activity of the yeast extract found by the method used in Table 2 is , Londesborough & Vuorio ([1991] J. Gen. Microbiol. 137, 323-330). Measurements of the appearance of UDP were consistent with those found. In addition, Yeast extract, measured in the presence of Bacillus plant extract, was not inhibited. Therefore The lack of activity of the control plants in Table 2 is due to some factors present in the tobacco extract. Is not the cause.   Tre6P synthase activity found in transformant 4 was unstable. Some , The activity disappeared during storage on ice for several hours. However, Western Certain bands found in the analysis are not visible in extracts stored at room temperature for 24 hours. Still existed near the intensity of Therefore, the conformation of the Tre6P synthase subunit The transformation appears to change during storage of the tobacco extract. These results , Tre6P synthase activity increases tobacco to TPS1 and yeast trehalose. Co-transform with one or more other subunits of the Increase the conformational stability of the Tre6P synthase subunit. And is achieved by:Embodiment 2. FIG. Transgenic Arabidopsis thaliana   An A. thaliana plant containing TPS1 under the ats1A promoter was isolated from the tobacco as described above. It was constructed in the same manner as the transformant. These transformed Arabidopsis plants also It was natural and normal in appearance and produced fertilized seeds. Main Transformant Species Western analysis of plants grown from offspring shows that they are yeast trehalose synthase (Fig. 10). These plants are The expectation that trehalose accumulates in the green tissue of E. coli is clear.Embodiment 3 FIG. Trehalose-Drought tolerance of producing tobacco plants   The amount of trehalose produced in transgenic tobacco depends on their drought tolerance In vitro grown vs. The isolated leaves from the terrestrial and transgenic lines were subjected to air drying (Relative humidity R H25%) (FIG. 5). 3 hours of stress due to rapid water loss of isolated leaves of control plants The leaves of the trehalose-producing plants initially showed clear browning signs, It remained green for up to 24 hours, although showing a clear reduction in water loss. Follow Thus, the protective effect of trehalose seems to be double: first, synthesize trehalose Leaves from transgenic plants that improve water retention in the early stages Met. Only after prolonged drought treatment, the phase of trehalose-producing and control plants The difference is gone. Second, control leaves showed clear signs of aging and browning . In contrast, leaves from trehalose-containing plants appear green for at least 24 hours. Yes, some browning occurred when the drought treatment was extended (several days). Trehalo ー There was a clear difference between the water producing and control plants even at the same water content. Therefore, Trehalose reduces the rate of water loss as well as protects plant tissues from dehydration. Seems to decrease. Browning is at least partially due to the release of the reducing sugar Is the reaction of amino acids to brown amino acids, resulting from the Maillard reaction. possible. Trehalose does not participate in this reaction as a non-reducing sugar, and It even inhibits the reaction with proteins (Roser & Colaco [1993] New Scientist 1873, 24).   Trehalose protects isolated leaves from drought damage by trehalose. Have similar effects. Trehalose thirst for intact plants Plants grown in vitro were emptied to assess whether they would increase water tolerance. Exposure to air drying (RH 30%, FIG. 6A). Within 3-4 hours, the control plant Lost turgor pressure and withered. Conversely, trehalose-producing transgenic plants Only showed signs of turgor loss after prolonged air drying. 17 hours drying After treatment, the plants were rehydrated and the survival of the plants was documented in detail (FIG. 6A). Non-trait Converted control plants showed no recovery even after prolonged rehydration and were resistant to this treatment. Did not. Conversely, trehalose-producing transgenic lines are And lost most of their tissue moisture (down to 30% residual fresh weight) ) Withstood the drying treatment for 17 hours.   High drought viability was also demonstrated in young seedlings (FIG. 6B). Transgenic 3 weeks old seedlings from the cultivar line along with untransformed and vector transformed control seedlings When exposed to air drying (RH 50%), trehalose-producing plants and control plants become thirsty. It showed a clear difference in water tolerance. Transgenic trehalose positive line 8 shows delayed turgor loss and viability against dehydration stress compared to control plants (Figure 6B).Embodiment 4. FIG. A gene encoding Tre6P synthase and Tre6Pase or Co-transformed plants with one or more genes encoding regulatory polypeptides Trehalose production   One skilled in the art will recognize that the yeast genes TPS2 and TSL under the control of the ats1A promoter. A vector containing one coding sequence can be prepared using General Materials and Method and tobacco, Arabidopsis, and other plants by the method described in Example 1. They can be used to transform. TPS1 and other genes, TPS2 and Plants co-transformed with one or both of TSL1 and TSL1 Crossing the body, further transforming one transformed plant with the second gene, or 2 or 3 genes linked to a promoter; for example, TPS1 is non-constitutive ats1 A promoter can be linked to provide a control for trehalose synthesis. And vectors containing other genes driven by constitutive promoters. It can be obtained by transformation.   Two or more subunits of the yeast trehalose synthase complex, only Controlled expression of the gene for at least one of which is a 56 kDa subunit As a result, the 56 kDa subunit is stabilized by the presence of other subunits. Predicted that trehalose accumulation would be increased in green tissue of plants It is. In addition, the introduction of the 102 kDa Tre6Pase subunit is compatible with the Tre6P synthase. Potential storage of Tre6P expected when the stability of the This is beneficial because it reduces the product.   In this type of construction, the TPS1 coding sequence is replaced with other Tre6P synthase structures. Replace the TPS2 sequence with the other Tre6Pase sequence, replacing it with the coding sequence of the gene. Alternatively, the TSL1 sequence is further regulated by Tre6P synthase and Tre6Pase. The TSL1 product may be found to be a subunit of the natural yeast trehalose synthase Encodes a polypeptide that gives it in a manner similar to that imparting such properties to the knit Obviously, it can be replaced by the sequence of another gene.Embodiment 5 FIG. Tre6P synthase gene under the control of stress-inducible promoter Plant transformation   LTI78 (Nordin et al. [1993] Plant Mol. Biol. 21, 641-653) and RAB1 8 (Lang & Palva [1992] Plant Mol. Biol. 20, 951-962) and other plant promoters Is known to be induced in response to drought and cold stress. Taba Mosquitoes, Arabidopsis and other plants under the control of such a stress-inducible promoter , The coding sequence of the Tre6P synthase gene such as TPS1 alone or a simple plant Tre6Pase or a regulatory polypeptide or both under the control of a promoter Transformation with gene coding sequences, such as TPS2 and TSL1 More trehalose accumulation in plant tissues Can occur. The advantage is that some tissues have plants with trehalose levels May be harmful to the plant, may reduce the yield of photosynthetic capacity, and (1) When plants are accidentally exposed to stress, (The protective effect of trehalose overcoming adverse effects) or ( 2) plants to bring about the accumulation of trehalose, which is extracted from subsequently harvested plants That trehalose accumulates only when stressed intentionally It is.Embodiment 6 FIG. Purification, analysis, and cloning of other Tre6P synthases   While there may be other metabolic pathways for trehalose, Cabib & Leloir (1958, supra) It appears that the major synthetic pathway is through Tre6P, as described in . The key enzyme in this pathway is that Tre6P is once Many cells have the ability to dephosphorylate it to free trehalose Therefore, it is Tre6P synthase. Therefore, the main features of the present invention The concept is to introduce Tre6P synthase activity into the target plant. This activity The origin is not so important. We happen to be the yeast trehalose Subunit of the intase complex and contains at least two other subunits. The yeast enzyme used was used. In such a case, the Tre6P synthase subunit For optimal activity of the kit, a 56 kDa Tre6P synthase subunit derived from yeast was used. Is apparently effective without tobacco 102 and 123 kDa subunits in tobacco Function, but may require the presence of one or more other subunits. In some cases.   Enzymes that catalyze the same reaction in a wide variety of different organisms are often cognate. It is well known that once a family member is cloned, , It is easy to clone other members. Yeast Tre6P synthase B (Londesborough & Vuorio, [1992] USPA 07 / 836,021) Native Tre6P synthase, and Methanobacterium thermoautotrophyca Homologous tan containing proteins derived from bacteria (Methanobacterium thermoautotrophicum) It has been revealed that there is a family of proteins (McDougall et al., [1993] FEMS Mi crobiology Letters 107, 25-30). We also believe that other organisms are cognate Tre6P synth It was decided to test for the presence of the enzyme.   Mycobacterium smegmatis contains heparin-activated Tre6P synthase, This was partially purified and studied by the group of Elbein (Liu et al. [1969] J Chem. 44, 3728-3791; Lapp et al. [1971] J. Biol. Chem. 246, 4567-4579; Elbein  & Mitchell [1975] Arch. Biochem. Biophys. 168, 369-377; Pan et al. [1978] Arch. B iochem. Biophys. 186,392-400). The enzyme purified by these researchers was Glc 0.8 U / m 2 at 37 ° C. in the presence of optimal heparin using 6P and UDPG as substrates g protein (the enzyme is a nucleoside diphosphoglucose Derivative spectra can be used). The preparation had a molecular weight of about 4 on SDS-PAGE. It contained two polypeptides of 5 and 90 kDa. We inhibit proteases Factors and other protein protection reagents are included in the buffer used and are non-ionic Add a final chromatography step in the presence of the detergent, Triton X-100. Improved the purification method of the previous researchers. Our final processing is as follows Was: 1) M. smegmatis cells (grown in Luria broth for 3 days and stored frozen , Fresh weight 28 g) in 1 mM benzamidine, 2 mM MgCl_Two, 1 mM EDTA, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride (PM SF) and 50 mM HEPES / KO containing 10 μg pepstatin A / ml H, pH 7.5, dissolved in 40 ml and then broken in a French press. Homo Genate was centrifuged at 28000 g for 20 minutes. (NH_Four)_TwoSO_Four(Every 100ml Was added to the supernatant. The precipitated protein was collected and contained 0.1 mM EDTA. 20 mM HEPES / KOH, pH 7.5 and 1 mM PMSF and 1 0.2 mM dithiothreitol containing 0 μg pepstatin A / ml (HED buffer) Liquid) And dialyzed against HED buffer overnight. 2) When the dialysate is applied to a DEAE-cellulose 3.4 × 25 cm column, the enzyme is Elution was carried out at 20 ml / h with a linear gradient up to 0.9 M NaCl in 1.2 L of HED buffer. The peak enzyme fraction (0.2 M NaCl) was collected (33 ml), 0.5 mM PMSF and Adapted to 5 μg pepstatin A / ml. (NH_Four)_TwoSO_FourThe addition of 9.9 g The protein was precipitated, 50 mM NaCl, 0.1 mM EDTA and 0.2 mM dithio. 50 mM Tris / HCl, pH 7.5 containing threitol (TNED buffer) Was dissolved. 3) Separate the dissolved protein (3.7 ml) with TNED buffer It was passed through 2.8 × 34 cm of Dex G100 at 36 ml / h. Fast peak enzyme fractions The column was applied to a 1.5 × 8.5 cm column of heparin sepharose in TNED buffer. Mosquito The ram was developed with a gradient to 1.0 M NaCl in 100 ml at 5 ml / h. The enzyme is about 0. Elution was with 5M NaCl. 4) Heparin Sepharose eluate sample (fraction H37 in Table 3) was Transfer to TNED / 0.1% (v / v) Triton X100 on PM10 membrane in Hepari equilibrated with TNED containing 0.1% (v / v) Triton X100 Apply to a Sepharose 0.7 × 8 cm column, 1.0 M Na in 50 ml of the same buffer. Elution was performed with a gradient up to Cl.   Specific activity after the first heparin sepharose step is reported by Pan et al. (1978) SDS-PAGE (FIG. 7), although much higher than Minutes are not pure. Surprisingly, the band at about 55 kDa has an intensity It was the only major band that correlated well with enzyme activity. Cut the 55kDa band Take, digest with trypsin in the gel and tryptic peptide by HPLC Separated and sequenced as described (Londesborough and Vuorio, 1993, supra). did. The sequences are shown in Table 4 and the Sequence Listing. Peptide peak 29 giving double sequence For and 31, amino acids were optionally assigned in the same manner as shown in Table 4, Nos. 3 and 4 (from peak 29) and SEQ ID NOs: 6 and 7 (from peak 31) Has given to. The first 9 residues of peptide 13 are 56 kDa Tre6P derived from yeast. Residues 250-258 of synthase (VGAFPIGID; Vuorio et al., 1993, supra) 89% identical to EMBL accession number X67499).   To confirm that this approximately 55 kDa band represents Tre6P synthase Further purification was attempted. Enzyme is applied to UDP-glucuronate agarose column But could not be recovered. Therefore, fraction H37 was Transfer to buffer containing ton X100 (two-thirds of the activity was lost during this transfer) ), Re-chromatography on heparin-Sepharose in the presence of Triton X100 - FIG. 8 shows the removal of two faint bands smaller than cytochrome c. The only approximately 55 kDa band is the only Coomassie blue reactive substance present in the active fraction. Indicates that there is. Clearly, the 90 kDa poly reported by Pan et al. (1978; supra). The peptide is not an essential component of this M. smegmatis-derived Tre6P synthase, Also, the size of the essential polypeptide, about 55 kDa, was reported by these researchers. Significantly larger than the size of the smaller 45 kDa component. FIG. 9 shows this poly. The peptide is a subtype of the 56 kDa Tre6P synthase of yeast trehalose synthase. Unity Recognized by antisera raised against And that Tre6P synthase of M. smegmatis converts antigenic determinant to yeast enzyme. It indicates that you want to share. Other immune reactions present in relatively crude enzyme preparations The nature of the sex bands (see lanes 5 and 6 in FIG. 9) has not been investigated: Not all are large protein-loaded artifacts, but related proteins Represents. Nevertheless, with antisera raised against yeast enzymes, M. Detect positive clones from host cells transformed with smegmatis gene bank. And can be isolated. Similarly, using the amino acid sequence data in Table 4, You can check the array. Immunological and amidate of yeast-derived enzymes with M. smegmatis Noic acid sequence similarity makes nucleotide probes designed from the TPS1 gene successful. Shows that it can also be used to screen for the M. smegmatis gene.   In conclusion, immunological and sequencing studies of the M. smegmatis enzyme have Confirm that body-derived Tre6P synthase is a member of a family It is. Trehalose is synthesized using the genes of these enzymes in the same manner as TPS1. And make transgenic plants with enhanced stress tolerance .

[Procedure of Amendment] Article 184-8 of the Patent Act [Submission date] September 9, 1996 [Correction contents]                                 The scope of the claims 1. Non-structural plants, such that the transformed plant has a novel capacity for trehalose synthesis Trehalose-6-phosphate synthase gene fused to promoter Plant transformed with the coding sequence. 2. 2. The plant of claim 1, wherein the plant promoter is a specific tissue. 3. 2. The plant of claim 1, wherein the plant promoter is photoactive. 4. Plant promoters are activated by stresses such as drought, high salinity or extreme temperatures 2. The plant of claim 1, which can be sexualized. 5. The trehalose-6-phosphate synthase gene is from a microorganism. The plant according to any one of claims 1 to 4. 6. The trehalose-6-phosphate synthase gene is 5% of yeast trehalose. The plant according to claim 5, which is yeast TPS1 encoding 6 kDa. 7. Trehalose-6-phosphatase, or introduced trehalose-6- Interacts with phosphate synthase or trehalose-6-phosphatase Transformed with at least one gene encoding a regulatory polypeptide Item 10. The plant according to any one of Items 1 to 6. 8. The gene encoding trehalose-6-phosphatase is the yeast TPS2 gene Offspring, wherein the gene encoding the regulatory polypeptide is the yeast TSL1 gene, The plant of claim 7. 9. The plant of claim 6, wherein the plant promoter is pats1A. 10. The plant promoter used was a strain such as LTI78 or RAB18. 9. The plant according to any one of claims 1 to 8, wherein the plant is active. 11. 11. The plant according to claim 1, wherein the plant is more stress-tolerant than a non-transformed plant. Any of the plants. 12. Corn, oats, millet, wheat, rice, barley, sorghum, amaranth 2. A monocotyledonous plant such as, onion, asparagus or sugarcane. Any of the plants according to 13. Alfalfa, soy, petunia, cotton, sugar beet, sunflower, d Carrot, celery, cabbage, cucumber, pepper, tomato, potato, lentil, a Ma, broccoli, tobacco, beans, lettuce, rape, collard, cauliflower, Spinach, Brussels sprouts, artichokes, peas, okra, pumpkin 12. A dicotyledonous plant such as kale, tea or coffee. Some plants. 14． Production of trehalose from the plant of any of claims 1 to 13. 15. A seed produced from the plant according to any one of claims 1 to 13. 16. The plant of interest is at least one trehalose-6-phosphate Transformed with the structural gene for intase, and -The gene is controlled in terms of temporal, local or stress-induced Expression under the control of a suitable promoter to allow A method for increasing the trehalose content of a plant, comprising a step. 17． A yeast gene whose gene encodes 56 kDa of yeast trehalose synthase 17. The method of claim 16, which is a child TPS1. 18. Trehalose Synthesis Is Trehalose-6-phosphate Synthase Gene Non-structural promoters such as pats1A, as regulated by a promoter that controls Trehalose-6-phosphate synthase gene under the control of Trehalose-6-phosphatase or trehal under the control of a suitable promoter. Transforming plants with at least one gene of the regulatory subunit of ribose synthase 18. The method of claim 16 or 17, wherein 19. The trehalose-6-phosphatase or regulatory subunit gene is 19. The method of claim 18, wherein the yeast gene is TPS2 or TPS1, respectively. 20. Sequence encoding trehalose synthase gene is specific in storage organs. 20. Any one of claims 16 to 19, which is fused with a plant promoter that causes expression. the method of. 21. Trehalose-6-phosphine to produce transgenic plants A plant is transformed with a structural gene for a synthase (the gene Promoters that allow for temporal, local or stress-induced control over reality Expressed under the control of) -Under conditions that cause expression of trehalose-6-phosphate synthase in plants Grow transgenic plants, -Extract trehalose from plant tissue, A method for producing trehalose, comprising the steps of: 22. 3. The transgenic plant stores trehalose in a storage organ. Method 1. 23. Claims wherein the plant is a root crop such as potato, sugar beet or turnip Item 23. The method according to Item 23. 24. Until plants encounter drought, high salinity or extreme temperatures, the complete gene Trehalose-6-phospho fused to a plant promoter to prevent expression from occurring Transforming a plant with a sequence encoding at least one gene for a synthase Protects specialty crops from drought, high salinity or extreme temperatures, including Method. 25. Until the plants are exposed to low temperatures, the plants should be planted to prevent full expression of the gene. Trehalose-6-phosphate synthase fused with a product promoter A method for controlling flowering, including transforming a plant with a sequence encoding both genes. How to protect edible fruit plants from frost damage. 26. Provide plants whose harvesting part has improved moisture retention, improved dehydration tolerance, or both. Trehalose-6-phosphate fused to a plant promoter to provide Transforming a plant with a sequence encoding at least one gene of an intase Improve the shelf life of harvested plants, including green foods, picked fruits and ornamental plants. How to be good. 27. Provide plants having improved stress tolerance as compared to non-transformed plants Trehalose-6-phosphate synthase fused to a plant promoter Transforming a plant with a sequence encoding at least one gene of A method for facilitating the cultivation and maintenance of ornamental plants. 28. Plant promoters should be used to accumulate trehalose in the edible parts of the plant. At least one residue of trehalose-6-phosphate synthase fused to Edible parts of the plant, including transforming the plant with sequences encoding the gene. Improves the scent and condition of jelly purees, pastes, jellies and jams Method.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁶ Identification code Agency reference number FI C12N 5/10 9735-4B C12N 5/00 C (C12N 9/00 C12R 1:91) (C12N 9/16 C12R 1:91) (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE), OA (BF, BJ , CF, CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, MW, SD, SZ, UG), AM, AT, AU, BB, BG, BR , BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, L U, LV, MD, MG, MN, MW, MX, NO, NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TT, UA, US, UZ, VN (72) Inventor Menturé, Einal Sweden, S-750 07 Uppsala, Box 7010 (No address shown) Swedish University of Agricultural Sciences, Uppsala Genetic Center (72) Inventor Wellin , Bjorn Sweden, S-756 54 Uppsala, Celloben No. 5 (72) Inventor Mandal, Abul Sweden, S-756 54 Uppsala, Diligent Begen 179 (72) Inventor Parva, A Tapio Sweden, S- 753 10 Uppsala, Drottning Gartan No. 5

Claims (1)

[Claims] 1. Non-structural plants, such that the transformed plant has a novel capacity for trehalose synthesis Trehalose-6-phosphate synthase gene fused to promoter Plant transformed with the coding sequence. 2. 2. The plant of claim 1, wherein the plant promoter is a specific tissue. 3. 2. The plant of claim 1, wherein the plant promoter is photoactive. 4. Plant promoters are activated by stresses such as drought, high salinity or extreme temperatures 2. The plant of claim 1, which can be sexualized. 5. The trehalose-6-phosphate synthase gene is from a microorganism. The plant according to any one of claims 1 to 4. 6. The trehalose-6-phosphate synthase gene is 5% of yeast trehalose. The plant according to claim 5, which is yeast TPS1 encoding 6 kDa. 7. Trehalose-6-phosphatase, or introduced trehalose-6- Interacts with phosphate synthase or trehalose-6-phosphatase Transformed with at least one gene encoding a regulatory polypeptide Item 10. The plant according to any one of Items 1 to 6. 8. The gene encoding trehalose-6-phosphatase is the yeast TPS2 gene Offspring, wherein the gene encoding the regulatory polypeptide is the yeast TSL1 gene, The plant of claim 7. 9. The plant of claim 6, wherein the plant promoter is pats1A. 10. The plant promoter used was a strain such as LTI78 or RAB18. 9. The plant according to any one of claims 1 to 8, wherein the plant is active. 11. 11. The plant according to claim 1, wherein the plant is more stress-tolerant than a non-transformed plant. Any of the plants. 12. Corn, oats, millet, wheat, rice, barley, sorghum, amaranth 2. A monocotyledonous plant such as, onion, asparagus or sugarcane. Any of the plants according to 13. Alfalfa, soy, petunia, cotton, sugar beet, sunflower, d Carrot, celery, cabbage, cucumber, pepper, tomato, potato, lentil, a Ma, broccoli, tobacco, beans, lettuce, rape, collard, cauliflower, Spinach, Brussels sprouts, artichokes, peas, okra, pumpkin 12. A dicotyledonous plant such as kale, tea or coffee. Some plants. 14． Production of trehalose from the plant of any of claims 1 to 13. 15. A seed produced from the plant according to any one of claims 1 to 13. 16. The plant of interest is at least one trehalose-6-phosphate Transformed with the structural gene for intase, and -The gene is controlled in terms of temporal, local or stress-induced Expression under the control of a suitable promoter to allow A method for increasing the trehalose content of a plant, comprising a step. 17． A yeast gene whose gene encodes 56 kDa of yeast trehalose synthase 17. The method of claim 16, which is a child TPS1. 18. Trehalose Synthesis Is Trehalose-6-phosphate Synthase Gene Non-structural promoters such as pats1A, as regulated by a promoter that controls Trehalose-6-phosphate synthase gene under the control of Trehalose-6-phosphatase or trehal under the control of a suitable promoter. Transforming plants with at least one gene of the regulatory subunit of ribose synthase 18. The method of claim 16 or 17, wherein 19. The trehalose-6-phosphatase or regulatory subunit gene is 19. The method of claim 18, wherein the yeast gene is TPS2 or TPS1, respectively. 20. Sequence encoding trehalose synthase gene is specific in storage organs. 20. Any one of claims 16 to 19, which is fused with a plant promoter that causes expression. the method of. 21. Trehalose-6-phosphine to produce transgenic plants Transforming a plant with the structural gene for the acid synthase, -Under conditions that cause expression of trehalose-6-phosphate synthase in plants Grow transgenic plants, -Extract trehalose from plant tissue, A method for producing trehalose, comprising the steps of: 22. Genes can be controlled in terms of temporal, local or stress-induced 22. The method of claim 21, wherein the expression is under the control of a promoter that allows control. 23. 3. The transgenic plant stores trehalose in a storage organ. Method 1. 24. Claims wherein the plant is a root crop such as potato, sugar beet or turnip Item 23. The method according to Item 23. 25. Until plants encounter drought, high salinity or extreme temperatures, the complete gene Trehalose-6-phospho fused to a plant promoter to prevent expression from occurring Transforming a plant with a sequence encoding at least one gene for a synthase Protects specialty crops from drought, high salinity or extreme temperatures, including Method. 26. Until the plants are exposed to low temperatures, the plants should be planted to prevent full expression of the gene. Trehalose-6-phosphate synthase fused with a product promoter A method for controlling flowering, including transforming a plant with a sequence encoding both genes. How to protect edible fruit plants from frost damage. 27. Provide plants whose harvesting part has improved moisture retention, improved dehydration tolerance, or both. Trehalose-6-phosphate fused to a plant promoter to provide Transforming a plant with a sequence encoding at least one gene of an intase Improve the shelf life of harvested plants, including green foods, picked fruits and decorative plants How to be good. 28. Provide plants having improved stress tolerance as compared to non-transformed plants Trehalose-6-phosphate synthase fused to a plant promoter Transforming a plant with a sequence encoding at least one gene of A method for facilitating the cultivation and maintenance of ornamental plants. 29. Plant promoters should be used to accumulate trehalose in the edible parts of the plant. At least one residue of trehalose-6-phosphate synthase fused to Edible parts of the plant, including transforming the plant with sequences encoding the gene. Improves the scent and condition of jelly purees, pastes, jellies and jams Method.