CN1159833A - Transgenic plants producing trehalose - Google Patents

Abstract

The present invention concerns transgenic plants producing trehalose and methods of increasing the trehalose content of plants. According to the invention, the plants of interest are transformed with the coding sequence of a gene for trehalose-6-phosphate synthase fused to a non-constitutive plant promoter, which allows for temporal, topological or stress-induced control over the expression of the gene. The invention can be used for protecting staple crop plants against drought, high salinity or temperature extremes and for improving the storage properties of harvested plants including green food stuffs, picked fruits and ornamental plants.

Description

Produce the transgenic plant of trehalose

FIELD OF THE INVENTION

The present invention relates in plant, import the genetically engineered of trehalose synthesis capability.Specifically, the present invention relates to increase the plant of content of trehalose and produce the methods of these plants.The invention still further relates to and improve the method for plant environmental stresss such as cold-peace dryings, and by the method for plant production trehalose.

The background of invention

Trehalose (α-glucopyranosyl-α-D-pyranoglucose) is the dimer by the glucose molecule of its reduction group connection.Owing to compare with other sugar, it has and lacks reduction group, hydrolysis and reach uncommon character such as can forming not deliquescent vitreum slowly, so it is the most effective known external sanitas of a kind of protein, cytolemma and other biological chemical combination thing, in addition, the live organism that contains a large amount of trehaloses usually is to be feature to be exposed under osmotic pressure, dehydration and the heat stress state, for example insect, some shallow water fauna and comprise yeast and many microorganisms of bacterium.There is circumstantial evidence (see Wiemken[1990] Antonie van Leeuwenhoek 58, the summary of 209-217) to show that the main effect of trehalose in bread yeast provides the resistance of these environmental stresss of antagonism.But also the someone points out (Nwaka et al (1994) FEBS Letters 344,225-228; Van Dijk et al (1995) Applied Environ.Microbiol.61,109-115), trehalose gathering in bread yeast itself is not sufficient to provide the tolerance to environmental stress.

In so-called resurrection plant such as pteridophyte (Selaginella lepidophylla), have high-caliber trehalose, make it can be under dry and thermal environment long-term surviving (referring to Avigad (1982) in Encyclopedia of Plant Research (new series) 13A, the commentary of being done among the pp 217-347).Yet most of vascular plants can not trehalose synthesis.These plants often come environmental stresss such as drying are reacted by the usability that reduces ICW; usually gather some other " compatible " solute; for example glycine front three betaine, proline(Pro) and various polyvalent alcohol are (referring to McCue and Hanson (1990) Trends in Biotechnology 8,358-362).

The report of trehalose is very limited in the relevant angiosperm, and a small amount of trehalose described in the report may reflect and has microbiological contamination (for example referring to Kandler﹠amp; Senser (1965) Z.Pflanzenphysiol.53,157-161; Qesch﹠amp; Meier (1967) Phytochemistry 6,1147-1148).It is virose (Veluthambi et al. to many plant tissues that existing people points out trehalose, (1981) Plant Physiol.68,1369-1374), particularly have only very little or do not have the active plant of trehalase (trehalase is a kind of enzyme that trehalose is changed into glucose) those.But having a kind of angiosperm at least is Myrothamnus flakellifolia.(another kind of " more Soviet Union " plant) (Bianchi et al. (1993) Physiologia Plantarun 87 223-226) shows there is not absolute consistency barrier between trehalose and angiosperm to gather relatively large trehalose.

Do not have trehalose in most of angiosperms, and to report it be virose in certain plants, this shows that importing the trehalose route of synthesis in these plants may bring disadvantageous effect to plant sometimes.On the other hand, in plant, successfully produce trehalose and will have tangible benefit.For example the trehalose that gathers in the storage organ of sugar beet, potato, onion etc. can be had with sucrose to be worth the trehalose that compares favourably after commerce is produced in some applications.These application comprise makes desiccating food products (milk and powdered egg, soup stock and puree etc.), because trehalose can keep the local flavor and the tissue signature of numerous food product raw material by the drying process at commercially attractive, and much more superior than sucrose in this respect (for example referring to Roser (1991) Trends in Food Science﹠amp; Technology, July issue, pp.166-169; Roser﹠amp; Colaco (1993) NewScientist, May issue, pp.25-28).May be because trehalose does not produce discovery to the disadvantageous fructose of health, so (as making soup stock, powdered egg) trehalose has more advantage than sucrose in many cases.But it is too high so that be difficult to bear that the high price of trehalose makes it application cost in desiccating food products industry again.Secondly, in the edible part of certain plants, produce trehalose and can prolong for example shelf-lives of tomato of product.The 3rd, trehalose gathering in sensitive organization can increase the tolerance of plant to frost, drying, high salt and similar pressure.

The summary of invention

Based on the above-mentioned fact, an object of the present invention is to provide plant with new trehalose synthesis ability.Specifically, an object of the present invention is to provide trehalose and gathering, or environmental stress tolerance or both are provided, reduce the disadvantageous effect of trehalose simultaneously to greatest extent plant-growth as the controllability in the plant tissue of the commercial source of trehalose.

The present invention is based on the responsible trehalose synthetic structure gene that is under the suitable plant promoter control and transforms suitable plant, has increased this notion of transgenic plant of content of trehalose with generation.Specifically, promptly according to the present invention under the control of specific plant promoter, in plant, express the encoding sequence of the one or more genes of polypeptide that coding can produce the enzyme of trehalose-6-phosphate or trehalose itself.

Therefore, at least the useful plant of using the encoding sequence of trehalose-6-phosphate synthase (Tre6P synthetic enzyme) gene that suitably merges with plant promoter to transform is only when plant is ripe or could realize expression of gene when it suffers from special environmental conditions.Therefore, promotor is non-composition preferably, and select in the genetic expression process, to be able to sequential (as by carrying out round the clock) control, by the control or pressure activation (or " pressure inducement ") control of local distribution (as tissue-specific).Also can transform plant with one or more encoding trehalose-6-phosphate enzymes (Tre6Pase) or with the Tre6P synthetic enzyme or with interactional regulatory polypeptide of Tre6Pase or the two gene.

Available any method known in the art transforms plant, these methods comprise uses the agrobacterium tumefaciens (Agrobacter tumefaciens) that is transformed to infect, or through micro-injection, electroporation or partickle bombardment, and the dna direct absorption directly imports foreign DNA.Structure gene preferably is selected from 102 KDaTre6Pase of encode respectively 56 KDa Tre6P synthetic enzyme, yeast TreP and yeast genes TPS1, TPS2 and the TSL1 that 123KDa regulates subunit.

Another object of the present invention provides the method for producing the transgenic plant that increased content of trehalose, this method comprises with trehalose synthetic structure gene, the plant that the gene transformation of particularly aforesaid Tre6P synthetic enzyme is useful, and under the control of suitable promotor, express these genes, with allow in the genetic expression process, to carry out sequential, by local distribution or pressure inducement control.

The 3rd purpose of the present invention provides the method for producing trehalose, and this method comprises:

-transform plant with the structure gene of at least one Tre6P synthetic enzyme, with the generation transgenic plant,

-these transgenic plant of cultivation under the condition that can induce trehalose synthesize enzyme gene in this plant, to express, and

-from the tissue of plant, extract trehalose.

The 4th purpose of the present invention provides the method that makes plant avoid bad condition influences such as arid, high salinity, extreme temperature and other pressure, this method comprises that the encoding sequence of the trehalose-6-phosphate synthase gene that suitably merges with at least one and plant promoter transforms plant, has only and just realize giving full expression to of gene when plant suffers from unfavourable condition.Also available one or more encoding trehalose-6-phosphate enzyme and with the adjusting albumen of trehalose-6-phosphate synthase or trehalose-6-phosphate enzyme interacting or both genes cotransfection operationally.For example, the invention provides a kind of method that prevents to avoid in flowering period frost damage with berry or other fruit plants, this method comprises the gene-transformed plant of using the trehalose-6-phosphate enzyme that suitably merges with plant promoter, only just realizes giving full expression to of gene when plant suffers from low temperature.

The 5th purpose of the present invention provides a kind of production and transformed, do not need to resemble not by the method for the ornamental plant that carries out meticulously the plant transformed and ad hoc take care of, this method comprises the gene-transformed plant of using the encoding trehalose-6-phosphate synthetic enzyme that suitably merges with plant promoter, contains trehalose thereby make in some tissue of plant.

Brief description of drawings

Fig. 1 diagram shows to contain has merged A.thaliana 1, the structure of the mosaic gene construct of the transcription termination signal of the female gene of the TPS1 enzyme with the Tre6P synthetase subunit unit of encoding of 5-diphosphoribulose carboxylase small subunit promotor (pats1A) and the rouge alkali synthetase gene (no) of agrobacterium tumefaciens.Wherein only demonstrate the part of the plasmid pKOH51 of band mosaic gene.The restriction enzyme site that has shown the uniqueness that is used for the mosaic gene structure.

Fig. 2 provides the result who rotaring gene tobacco plant is made the Western engram analysis, wherein with arrow indication 56KDa TPS1 product.From containing construct shown in Figure 1 ( swimming lane 1,3,4,5,6,8,10,12,15,16 and 19), or (GUS) another has in same carrier in the tobacco transformed plant with the mosaic gene of the cauliflower mosaic virus 35S promoter of beta-glucuronidase gene (UIDA) fusions, or extraction protein in the tobacco plant that is never transformed (SR1).Each swimming lane adds the protein of equivalent.Employed antiserum(antisera) is the antiserum(antisera) of the 56KDa Tre6P synthetase subunit unit of anti-yeast.

Fig. 3 shows the chromatography qualification result to trehalose.According to general " material and method " the described water extract sample (20 μ l) of analyzing tobacco leaf with the HPLC method.Extract A and B contain the transformant 19ml of 192mg/ml fresh weight greenhouse growth ^-1, be before handling with trehalase (A), (B) be after handling with trehalase.Extract C and D contain 149mg/ml (C) transformant 4 or (D) use the blade of the adjoining tree of the plasmid pDE1001 conversion that lacks the TPS1 gene, and both all grow under the aseptic condition.T indication trehalose peak value.Two big peaks elution time appearance in about 25 minutes are dextrose plus saccharoses.

Fig. 4 shows the protein immunoblotting analytical results that ats1-TPS1 is transformed the tissue positioned that is TPS1 subunit in 4.As negative control, and the Tre6P synthetase subunit unit that uses 0.1 μ g purifying from yeast (TPS1) is as positive control with the leaf of wild-type tobacco (SR1).Above immunoblotting, represent content of trehalose in the same tissue with the mg/g dry weight.The nt representative does not detect.Used following dummy suffix notation: FB represents bud, and UL represents upper leaf, and ML represents middle leaf, and LL represents the bottom leaf, stem in the US representative, and R represents root, and EC represents the enzyme contrast.

Fig. 5 show produce that the trehalose plant improved to the exsiccant tolerance.The excised leaf that 6 to 8 weeks made plant be in the same etap of breeding in vitro is exposed to air dried environment (25%RH).(A) in the described time blade is weighed, and the result is shown among the figure as the relative fresh weight of leaf on each time point.Leaf is obtained the dry weight of each blade after 48 hours in+60 ℃ of dryings.SR1 indication wild-type contrast tobacco produces trehalose transgenic lines 1,4 and 8, is indicated by their number of being respectively.(B) derive from the selected time point during being presented at environmental stress and handling outside the excised leaf of transgenic lines 4 and control plant and see.

Fig. 6 shows control group and the arid viability of producing trehalose group tobacco plant.(A) show the time that 6 to 8 weeks made the whole plant of breeding in vitro expose under dry air (D) condition.Environmental stress was handled after 17 hours, with the adjoining tree (SR1) that do not transformed be that 4 and 8 product trehalose transgenic plant are placed in the water aquation (R) again.(B) made the seedling of (C) adjoining tree of transgenic lines 8 and (SR1) that do not transformed and carrier conversion under dry air (D) condition, expose the specified time in 3 weeks.Environmental stress was handled after 7 hours, seedling is placed on makes it again aquation (R) in the water.

Fig. 7 shows the result that the SDS-PAGE that component is done that contains the M.SmegmatisTre6P synthetic enzyme of wash-out on heparin-Sepharose post first time is analyzed.Swimming lane 1 to 4 contains the sample (table 3) of H33 (7.0mU), H35 (20mU), H36 (21mU) and H37 (12mU) respectively.After carrying out SDS-PAGE on 8% acrylamide gel, dye with Coomassie brilliant blue.The molecular weight size of molecular weight standard product is shown with the KDa number in the swimming lane 5.Position with arrow indication 55KDa Tre6P synthetic enzyme polypeptide.

Fig. 8 shows the result that the SDS-PAGE to the component peaks of the M.SmegmatisTre6P synthetic enzyme of wash-out from second time heparin-Sepharose post analyzes.In Centricon 10 test tubes, concentrate component T21 (table 3), mix with 3 times that partly measure volume spissated SDS-PAGE sample buffers then.Cumulative volume concentrates 6 times.On 8% acrylamide gel, the sample that contains (swimming lane 1) 2.9mU and (swimming lane 3) 8.7mU Tre6P synthetic enzyme is carried out SDS-PAGE, and dye with Coomassie brilliant blue.Molecular weight standard shown in the swimming lane 2 from the top on earth portion be respectively 109,84,47,33,24 and 16KDa.

Fig. 9 shows the result who the Tre6P synthetic enzyme of purifying from M.Smegmatis is made the Western engram analysis.With prestained molecular marker (swimming lane 1:200,117,80 and 47KDa; The molecular weight sign of seeing 84KDa only), pure Tre6P synthetic enzyme (swimming lane 7 and the 8:2.9mU that obtain from component T21 (table 3) swimming lane 10:; Swimming lane 3,4 and 9:8.7mU) and active part (the swimming lane 6:7.8 μ g gross protein that derives from the merging of G100 Sephadex chromatography column (table 3); Swimming lane 2 and 5:23 μ g gross protein).Electrophoresis and transfer printing mark to nitrocellulose filter after, between swimming lane 3 and 4 and swimming lane 7 and 8 between cutting nitrocellulose filter.Dye swimming lane 1 to 3 with Coomassie brilliant blue, detect swimming lane 4 to 7 with anti-57 serum of the yeast 56KDa Tre6P synthetic enzyme subunit of antivenom purification, and detect swimming lane 8 to 10 with pre-immune serum.Use the goat-anti rabbit LgG-alkaline phosphatase enzyme conjugates available from Promega to manifest the immunocompetence band according to the operation instruction that manufacturers provides, developing time is 2.8 minutes in both cases.The oblique line of crossing over swimming lane 4 to 7 is because on transferring to nitrocellulose filter the time due to the unexpected gauffer that forms of film.

Figure 10 shows the Western engram analysis result that 8 transgenic lines of Arabidopsis thaliana are carried out.The plant that extraction becomes from the seed growth of initial conversion body, and analyze it by the antiserum(antisera) of the anti-yeast 56KDa of usefulness shown in Figure 2 Tre6P synthetic enzyme subunit basically.The Tre6P synthetic enzyme subunit (0.1 μ g) of use purifying from yeast is as positive control.The A.thaliana that is not the transformed reflection that is negative.

The detailed description of invention

In describing hereinafter, term " composition plant promoter " refers to cause their correlative coding The continuity of sequence and generality are expressed, thereby in all cells of these plant strains and growth All can find the expression product of these sequences in the stages. On the contrary, non-composition promoter then is Activate by special inside or external procedure, for example along with development of plants and mature cell differentiation Form different tissues, perhaps the environment of plant changes etc. Known many kinds of environment changes all Yes activate specific promoter. Its example comprises photoinduced ribulose-1,5-bisphosphate, 5-diphosphonic acid carboxylase Small subunit promoter (Krebbers et al. (1988) Plant Mol.Biol.11,745-759), The ats1A promoter of for example using among the embodiment 1-3, and can be induced by various environmental pressures Promoter is LTI78 (Nordin et al. (1993) Plant Mol.Biol.21,641-653) for example And RAB18 (Lang﹠PalVa (1992) Plant Mol.Biol.20,951-962). But The present invention is not only limited to these non-composition promoters of giving an example. On the contrary, of the present invention one important The part with novelty be based on such concept, by plant composition trehalose synthesis always namely Uneconomical and sometimes be harmful to, thereby preferably by using non-composition promoter to embody this Shen Please in the benefit of disclosed trehalose production method. By these non-composition promoters, can be right To carry out SECO (be that it is when only occurring in some to the marine alga monose in the genetically modified plants Between), locally distributed feedback control (being its some position that is only limited to plant) or carry out simultaneously this two side The control of face.

The title of the plant organ that uses in this specification all is that common vegetable fruit shop and its client are logical Normal use. For example, " fruit " comprises the edible entity part of apple or strawberry plant, but These contain seed-bearing stored tissue is not fruit itself on the strict botany meaning.

Mentioned the ability that as if nearly all vascular plant all lack trehalose synthesis, so right Term " product trehalose " plant does not need to carry out quantitative definition. Yet, as if also not research Some conventional plant (rather than so-called resurrection plant) produces marine alga during standing environmental pressure The possibility of sugar. The present invention relates to external source importing trehalose synthesis capability (is also referred to as " new herein " the trehalose synthesis capability) genetically engineered plant. With not turned to of growing under the same conditions The plant that changes is compared, and it is anti-that the increase of desired content of trehalose is that it can cause environmental pressure The useful improvement of being subjected to property perhaps can advantageously be extracted it from plant, to be used for commercial object.

Many microorganisms have generation trehalose-6-phosphate (Tre6P) also and then with it to be hydrolyzed into The enzyme system of trehalose. For example, brewer's yeast namely has and comprises 56,102 and the 123KDa subunit Trehalose synthetase compound (Londesborough﹠Vuorio (1993) Eur.J.Biochem. 216,814-848), this multienzyme complex at first with uridine diphosphate glucose (UDPG) and G-6-P (Glc6P) is condensed into Tre6P (reaction of Tre6P synzyme), then Be hydrolyzed into again free trehalose (Tre6Pase reaction). Tre6P synzyme and Tre6Pase activity Be present in respectively 56 and the 102KDa subunit in, it is special that the 123KDa subunit then makes compound have to regulate Property also plays stabilization. Other microfloras comprise candida utili (Soler at al. (1989) FEMS Microbiol Letters 61,273-278), Escherichia coli (Glaever et al. (1988) J.Bacteriol.170,2841-2849), plate-like net handle bacterium (Dictyostelium Discoideum) (Killick (1979) Arch.Biochem.Biophys.196,121-133) And smegma bacillus (Mycobacterium smegmatis) (Lapp et al. (1971) J. Biol.Chem.246,4567-4579) enzyme system, latter two systems can be used adenosine two phosphorus Acid glucose (ADPG) replaces UDPG. The Asia of these microbial enzyme systems that the relevant latter mentions The data of based structures seldom. Also can guess the generation that comprises nematode, insect and resurrection plant The multicellular organisms of trehalose contains the enzyme of poly-Tre6P synzyme and Tre6Pase activity.

In principle, any the transferring in the plant with in these Tre6P synzyme all can obtain The plant that new synthetic Tre6P ability is arranged. Disclosed certain plants, for example tobacco has Tre6P Change into the capability of trehalose, thereby be surprisingly found out that, close with coding Tre6P separately The genetic transformation of one-tenth enzyme for example tobacco can produce trehalose effectively. If but Tre6P is to marine alga The endogenous of sugar transforms very slow or lacks such ability, then also can shift to derive from and anyly suitably come The Tre6Pase in source. For example close using wherein Tre6P synzyme and Tre6Pase to be trehalose When becoming the yeast enzyme system of subunit of enzyme, with the Tre6P synzyme and the Tre6Pase that derive from same source It may be favourable that gene carries out cotransformation, because so just can form the native enzyme compound. No matter The source of enzyme how, all can extract the trehalose that generates in the genetically modified plants, perhaps can give plant With the higher tolerance to some environmental pressure. Described and used by this way some ferment Produce the method (PCT/FI93/00049) of trehalose in the plant of female genetic transformation.

Although can expect trehalose some time (for example plant contact during the environmental pressure or In the ripe plant) or some tissue in (for example in storing organ, or in due course in frost Freeze in the sensitiveness tissue) gather plant usefully, perhaps at least harmless to plant, but also exist Opposite possibility, namely gathering of trehalose may be to plant in other times and its hetero-organization (the Veluthambi et al. (1971), document is the same) that is harmful to. Therefore, use in the plant maturation Or run into planting that arid and the environmental pressures such as low temperature can not cause that the gene of trehalose gives full expression to before Thing promoter (it is as the part of the gene of facilitating coded sequence to transcribe) will be favourable, In this case trehalose to the benefit of plant surpass its may to plant bring unfavorable. This class is non-Some examples of composition plant promoter are well known by persons skilled in the art, comprising driving The small subunit ribulose-1,5-bisphosphate that the photoinduction of RUBISCO small subunit is expressed, 5-diphosphonic acid carboxylase (Rubisco) promoter (Krebbers et al. (1988) Plant Mol.Biol.11,745-759).

Disclose with the yeast TPS1 gene that correctly is fused on the Rubisco small ylidene gene promoter Coded sequence (open reading frame, the tobacco and the Arabidopsis plant that ORF) transform. TPS1 The 56KDa subunit of coding Trehalose in Yeast synzyme. The plant that is converted is healthy and can educates, And contain trehalose at its leaf. The tobacco that is not converted with lacking similar year of TPS1 gene The tobacco that body transforms does not then contain trehalose. Published genetically modified plants are to use agrobacterium tumefaciens to be situated between Lead to transform obtaining, but also can use any method known in the art to transform, wherein Comprise through micro-injection, electroporation or partickle bombardment with direct importing DNA (Gasser﹠Fralev (1989) Science 244,1293).

There is a strain (transformant 4) to demonstrate in these tobacco plants that are converted to contain Tre6P synthetic Enzymatic activity. Know that free 56KDa subunit is compound from the complete trehalose synthetase of yeast That unsettled (Londesborough﹠Vuorio (1993), document goes out when separating in the thing Locate the same). Described the method for the available cotransformation plants of those skilled in the art, namely used Be in plant promoter, under for example the convenient promoter of ats1A promoter or some composition is controlled One of TPS1 gene and other Trehalose in Yeast synthase genes (TPS2 and TSL1) or both advance The row cotransformation. Because the subunit by TPS2 and TSL1 coding can be stablized the 56KDa subunit, so Compare with the plant that only contains TPS1, this cotransformation can increase the content of trehalose of plant.

Be in the Tre6P synthase gene under the inducible promoter control and be in composition in usefulness The Tre6Pase that promoter control is lower or adjusting protein (for example TSL1 product) one or many In the plant of individual gene cotransformation, because Tre6P synzyme catalysis trehalose biosynthetic first Individual unique step will be so wherein the generation of trehalose will be subjected to the adjusting of inducibility promoter.

Disclosing the product trehalose tobacco plant that is converted is proved to be and has the raising drought tolerance. This Click-through is crossed the experiment of carrying out with the blade that exsomatizes and whole plant and is confirmed. Ripe plant and turning to The immature seedling of changing the filial generation of strain self-pollination shows the drought tolerance that is improved. In these filial generations In, as existing in the chlorenchyma according to filial generation as indicated in the 52KDa Tre6P synzyme subunit, Drought tolerance and TPS1+ characteristic be divided into from.

Published genetically modified plants are wonderful to the tolerance of water environment pressure, because exist The amount all too of the trehalose in its tissue is little so that be difficult to provide infiltration buffering to cellular content Effect. May be trehalose by protection specificity position for example film work, perhaps may be Trehalose or its precursor (Tre6P) have been upset the metabolism of plant and have been stoped cell to take place this pressure The secondary change of power.

Disclose to see and contained the slight morphological change in the level transformant at the beginning of the chimeric TPS1 gene, pocket knife shape leaf for example, mainly the reduction that in the filial generation that still produces trehalose, disappears top growth vigor and lowered plant height.As if these changes mainly manually cause owing to tissue culture.But under optimum condition, after 8 weeks of growth, comprise that the product trehalose tobacco plant growth of the filial generation of self-pollination is all slowed down to some extent, lagged for 1 to 2 week than control plant.Even this shows that trehalose synthesis is under the control of ats1A promotor in plant, also can retarding of growing.Therefore; promotor that can the environment for use pressure inducement is gone into LT178 (Nordin et al. (1993) Plant Mol.Biol.21; 641-653) further to help under the non-ambient pressure condition, keeping normal growth speed and the productive rate of plant; but, still can under stress provide trehalose inductive protection mechanism simultaneously as explaining among the embodiment 5.

Though the TPS2 gene-transformed plant of the Tre6Pase that need not encode with the yeast TPS1 gene of coding Tre6P synthetic enzyme only, these transgenic plant are trehalose synthesis still.This shows that certain plants has the endogenous ability that Tre6P is transformed into trehalose.TPS2 gene with the Tre6Pase that for example encodes carries out cotransfection in some cases, also is feasible to improve Tre6P to the speed that trehalose transforms.Moreover, use Tre6P synthetic enzyme (with the Tre6Pase) gene that derives from yeast organism in addition to transform obviously and can obtain similar result.These genes are many all to be and TPS1 (and TPS2) homologous, and can use or be derived from the enzyme of Yeast system and the immunology and the oligonucleotide probe of gene cloned it at an easy rate.For example, reported Tre6P synthetic enzyme from smegma bacillus be a kind of can with about 55KDa polypeptide of the antiserum(antisera) generation cross reaction of the yeast TreP 56KDaTre6P synthetic enzyme subunit of antivenom purification.The pancreatin that discloses smegma bacillus Tre6P enzyme is separated the aminoacid sequence of peptide, discloses the close sequence homology of itself and yeast enzyme.Those skilled in the art can use these means to clone the gene of Tre6P synthetic enzyme and Tre6Pase at an easy rate from many organisms.The present invention includes with derive from any practical organism and be fused to Tre6P synthetic enzyme and Tre6Pase gene-transformed plant on the suitable plant promoter.

Can use by several modes and transform with one or more trehalose synthesize enzyme genes that plants are resulting to contain the trehalose plant.For example, can on commercially producing scale, from plant, extract trehalose.These trehaloses can be with enough cheap price high volume applications, as is used for during drying keeping the local flavor and the structure of food raw material.For this application purpose, trehalose preferably accumulates in and stores in the organ, for example in the bulb of root of the stem tuber of potato, sugar beet or turnip or onion.(Lindsey (1992) J.Biotechnol.26,1-28), but the present invention is not only limited to the plant that these molecular biologists often are used as experimental model to have described the method that transforms these plants of giving an example and many other crop plants.Those skilled in the art can be applied to other plant with the method for progressions model plant.Therefore, also can in the fruit of the cane stalk that is for example transformed or leaf or banana, realize the present invention.Causing plant promoter specific expressed in storage organ is (for example patatin promotor) known in the art.In one aspect of the invention, with the Tre6P synthase gene for example the encoding sequence of TPS1 and transform suitable plant being fused to as the TPS1 encoding sequence on the same manner on ats1A promotor promotor being fused to these DNA construct.Be extracted in the trehalose that gathers in the storage organ then.

In another aspect of the present invention, the trehalose that gathers in plant tissue can improve the storage characteristics after the results.Therefore, slower by the excised leaf moisture loss of transformation of tobacco than the tobacco that is not transformed, even after losing moisture, also can not fade gradually (Fig. 5).The advantage of this aspect is applicable to food plant and ornamental plant.With regard to edible plant, particularly wither and fade and to bring serious economy loss for what retail market extensive stock lettuce class plant occurred in the results back.Loss is added on one's body the human consumer at last.Produce the trehalose romaine lettuce and will provide more cheap and more attracting salad for the human consumer.Similar consideration also relates to some other food plant, particularly leaf with product for example wild cabbage, sprouting broccoli, dill, spinach or Parsley and other green vegetables such as pea and Flos Carthami Kidney bean.Many ornamental plants all are to transport after cutting-out as rose, turmeric, daffodil etc.Both used expensive mode of transport (refrigeration, air transport) also can lose than juicy.Keep its moisture content preferably and then be that the human consumer provides the product of more liking with the lower price to the less product trehalose ornamental plant of the susceptibility of fading.In this aspect of the invention, the Tre6P synthase gene is fused on the selected plant promoter, thereby marine alga is accumulated in the plant part to be gathered in the crops.For example, the ats1A promotor is expressed the Tre6P synthetic enzyme in leaf, top stem and the bud of tobacco, and trehalose is accumulated in these positions.But the inventor discloses, and plants such as tobacco can be transported to trehalose the tissue of trehalose synthesis not from its synthesising part.Therefore, though non-composition ats1A promotor can not make the Tre6P synthetic enzyme express, in the root of these rotaring gene tobacco plants, also can see trehalose in a small amount in root.

In related fields of the present invention, contain edible portion such as tomato, berry and other fruits of the conversion plant of trehalose, owing to contain trehalose, so can be processed to fresher puree, paste, jelly and the jam that more is rich in local flavor.Show that (WO89/00012) adds trehalose and can help preserving its local flavor in these food raw materials, particularly in the man-hour that adds that comprises drying step.The present invention needn't add trehalose again by the plant that contains trehalose is provided.In this aspect of the invention, the instruction trehalose mainly in the storage organ of plant the synthetic promotor for example the patatin promotor may be useful.

The transformation of tobacco of generation trehalose disclosed by the invention has improved the tolerance to arid.In general, containing the plant that transformed of trehalose can be than by plant transformed bigger resistance not being arranged to arid, frost, high salt and other environmental stresss.Therefore, arid, frost and seepage water pressure be basically all because of discharging moisture in cell, damaging cells film and protein and destroy plant, and known trehalose can slow down these infringements (Crowe et al. (1992) Annu.Rev.Physiol.54,579) external.In this aspect of the invention, employed plant promoter can be an environmental stress inductive promotor.Such promotor is as known in the art, for example have LTI78 (Nordin et al. (1993) Plant Mol.Biol.21,641-653) and RAB18 (Lang﹠amp; PalVa (1992) Plant Mol.Biol.20,951-962).Use these promotors will prevent trehalose synthesis when still not needing to synthesize.For well-grown and contain the plant of trehalose, can not need the promotor of pressure inducement to realize this aspect of the present invention.But applying pressure inductive promotor prevent have required before trehalose synthesis also have the other advantage, comprising the production loss of avoiding convert light synthesis capability by other means to come trehalose synthesis to cause, and avoid as carrying the delayed growth that tobacco occurs of pats1A-TPS1 mosaic.The advantage of this aspect not only can be used for food plant, and is used in the ornamental plant of garden or indoor displaying.The force resistance ornamental plant that is transformed only need not transformed less the looking after of plant than corresponding.Cause in the initial conversion strain observed some to produce the slow and very little morphological change of trehalose plant-growth be not the shortcoming of ornamental plant: particularly for decorative indoor plant, the slower and new outward appearance of growing may be attractive feature.

In another aspect of the present invention, the Tre6P synthase gene suitably is fused on the plant promoter (for example LTI78 or RAB18) that is activated when running into particular incident or envrionment conditions (for example arid or the cold pressure that freezes), thereby but the process that can promote plant to gather commercial extracted amount trehalose occurs in the results maturation plant not long ago, to avoid any disadvantageous effect of trehalose to the early development process of certain plants.

Based on disclosing as above; transgenic plant of the present invention can be monocotyledonss such as corn, oat, grain, wheat, rice, barley, Chinese sorghum, three-coloured amaranth, onion, asparagus or sugarcane, or dicotyledons such as clover, soybean, petunia, cotton, beet, Sunflower Receptacle, Radix Dauci Sativae, celery, wild cabbage, cucumber, pepper, tomato, potato, root of Szemao crotalaria, flax, sprouting broccoli, tobacco, Kidney bean, lettuce, rape, Cauliflower, spinach, brussels sprouts, arithoke, pea, gumbo, pumpkin, leafy cabbage, kale, tea or coffee.

Yeast genes TPS1 and its expression product are that the biochemical mechanism of tobacco adapts: gene is able to high expression level and the 56KDa subunit causes manifesting of trehalose.But known in the artly be, compare plant gene with for example microorganism lower A+T ratio is usually arranged, and can use by changing codon, particularly change the codon that begins part near encoding sequence make it to become the codon that sees in the plant improve the expression level of heterologous gene in plant (Perlak et al. (1991) 88,3324-3328).Our imagination to gene carry out these with similar modification applicable to the present invention.

The known natural sudden change of generation in gene.These changes in the dna sequence dna and other people wage reform become can cause coded amino acid sequence of polypeptide change.Term as used herein " TPS1 " (or TPS2 or TSL1) comprises all and TPS1 (or TPS2 or TSL1) homologous dna sequence dna, and latter's coding has yeast TreP 56KDa (or 102KDa or 123KDa) expectation function of subunit or the polypeptide of constitutional features.Equally, term " gene of coding Tre6P synthetic enzyme (or Tre6Pase or regulatory polypeptide) " not only comprises the gene that can be easy to separate (for example using the nucleotide probe according to the known array design of TPS1 or TPS2 or TSL1) from the natural biological body, and comprising natural or artificial variation's body, the polypeptide of these varient encoded polypeptides and original isolating genes encoding has identical function and constitutional features.

General material of embodiment and method

Material.Employed plant is the environmental C-24 of Nicotiana tabacum cv.SR1 and Arabidopsisthaliana L.Heynh..

((1993) Eur.J.Biochem.216,849-861) 123KDa that obtains the yeast genes TPS1 (being called TSS1 in the past) of 56KDa Tre6P synthetic enzyme subunit of encoding trehalose synthetic enzyme and encoding trehalose synthetic enzyme among described plasmid pALK752 and the pALK754 regulates the TSL1 of subunit from people such as Vuorio.Free Drs Claudio De Virgilio and Andres Wiemken (Botaniches Institut der Universit  t Basel, the plasmid that Switzerland) provides and contain the TPS2 gene in the SacI site of being cloned into plasmid YCplac111 are provided yeast genes TPS2.The aminoacid sequence that be derived from 102KDa subunit of this gene (as Virgilio er al. (1993) Eur.J.Biochem.212,315-323 is described) coding shown in the table 1 of people such as Vuorio (1993, ibid for document).Prepared antiserum(antisera) is the antiserum(antisera) (anti-TPS/P) of anti-yeast TreP and the antiserum(antisera) (anti-57K) of anti-56KDa subunit, and the general biological chemical process is referring to people's such as Vuorio above-mentioned document.The vacuole trehalase is according to Londesborough and Varimo ((1984) Biochem.J.219,511-518) described method is partially purified from suc-gal-mel-mal-yeast strain (ALKO2967), and it can not sucrose hydrolysis, maltose and close disaccharides.

The DNA operation.All (Cold Spring Harbor NewYork) carries out for Sambrooket al., Molecular Cloning:A Laboratory Manual according to the laboratory method of having set up in all DNA operations.Use coli strain DH5 α and MC1061 to carry out the plasmid preparation.Be used to control the ats1A gene (Krebbers et al. (1988), document ibid) of TPS1 expression promoter from the small subunit of the coding Rubisco of A.thaliana.

In order to make up ats1A-TPS1 gene mosaic, lack the ats1A promoter fragment of the encoding sequence of transit peptides from plasmid pGSFR401 amplification through PCR.Use the synthetic Oligonucleolide primers to cause an EcoRI site and cause an XbaI site at 3 ' end at the segmental 5 ' end of amplification.Restriction Enzyme digestion pcr amplification product with suitable is connected to behind the purifying on sepharose in the pUC19 plasmid with EcoRI and MluI digestion.Amplification yeast TPS1 gene from above-mentioned plasmid pALK752.Resulting fragment promptly contains 5 ' MluI restriction enzyme site and 3 ' XbaI enzyme cutting site.Behind enzymic digestion and agarose gel purifying, the gained fragment is connected to the back of pats1A among the pUC19.The fragment that has promotor-TPS1 construct with EcoRI and XbaI cutting, be inserted into pBluescript II SK+ (Stratagene) then and derive in the plasmid, wherein this plasmid of deriving carries 3 ' end of the T-DNA gene GF that comprises its polyadenylation signal and T-DNA right side edge.At last, with whole mosaic gene as the EcoRI-SacI fragment be inserted into chimeric selective mark kalamycin resistance gene pNOS-NEO-3 ' OCS carrier pDE1001 (Denecke et al. (1992) EMBO J.11,2345-2355) in.The plasmid pKOH51 that obtains like this carries chimeric pats1A-TPS1-3 ' GF gene.By transforming the gained construct is changed among the coli strain DH5 α, then through electroporation (Dower, Miller﹠amp; Ragsdale (1988) Nucl.Acids Res.16 6127-6145) transfers to it and contains non-carcinogenic Ti-plasmids pGV2260 (Deblare et al. (1995) Nucl.Acids Res.13, agrobacterium tumefaciens (C58C1rif 2777-2788) ^R) in.

Make other constructs with similarity method.

The growth of vegetable material.In order to carry out the cultivation of pure property, will plant the MS (Murashige﹠amp that has added 2% sucrose in containing through the explant of the Nicotiana tabacum (SR1) of sterilization; Skoog (1962) Physiol.Plant 15 is 473-497) in the glass pot of solid medium (SM-2).Then these jars are placed in the culturing room of control culture condition, make plant in 22 ℃ of illumination growths 16 hours.Explant is moved in the new jar that is added with the MS-2 substratum on time and continue to cultivate pure property material.Make hothouse plants grow in the interior soil of box and watering every day.At first the method by above-mentioned cultivation tobacco makes the A.thaliana plant pure property growth in small-sized food pot in check environment that is transformed, and transfers to then in the greenhouse, makes it to produce seed in filling the basin of soil.To directly implant the new seed of generation in the soil by the seed that the initial conversion strain produces, perhaps through making it pure property growth in 24 hole tissue culturing plates behind the surface sterilization, to carry out analysis of molecules.

Plant Transformation.According to root transformation of tobacco and the A.thaliana of following method with cut tobacco leaf and A.thaliana.Basically according to people such as Valvekens ((1988) Proc.Natl.Acad.Sci, USA, 85,5536-5540) described method transforms and tissue culture, but method has been done following change.With isolating or leaf pre-cultivation 4 days on callus induction solid medium (CIM), undercut is become segment (1-2mm) and leaf is cut into bigger fragment (10-20mm), move into then in the 20ml CIM liquid nutrient medium.Add 3 ', 5 '-dimethoxy-4 ' '-carry out Agrobacterium (C58C1 rif behind the glycoloyl benzene (0.2mg/L) ^R) infect.The bacterium that will be used to infect be added in contain suitable antibiotic YE β substratum (Vervliet et al. (1975) J.Gen.Virol.26,33-48) in 28 ℃ of propagation spend the night, and centrifugal collection it.Then the bacterial precipitation thing is suspended in 10mM MgSO again ₄In, add in the plant tissue and also mixed gently about 15 minutes.Outwell excessive liquid and with the root transfer printing on aseptic filter paper.After cultivating 2 days altogether on the solid CIM, with liquid CIM with plant tissue drip washing 3-4 time washing bacterium off, and transfer on the substratum of sprouting (SIM) of selection.Grow after 7 days, the explant that form is taken place in differentiation is partly transferred among the fresh SIM.

Extracting protein analyzes in order to Westem.Containing 100 μ l proteins extraction damping fluid (50mM Tris/HCl pH7.2,250mM sucrose, 5mM EDTA, 10mM MgCl ₂, 1mM CaCl ₂, 10mM beta-mercaptoethanol, 1mM phenylmethylsulfonyl fluoride (PMSF), 30 μ M pepstatins, 50 μ M leupeptins and 15 μ M aprotinins) the 1.5ml Eppendorf tube in, with the plant sample of glass homogenate 100-200mg fresh weight.Remove insoluble material through two times centrifugal (13,000g, 10 minutes).((1976) Anal.Biochem.72, method 248-254) use bovine serum albumin as the protein concn in the standard substance mensuration supernatant liquor according to Bradford.The soluble protein of equivalent is added on carries out immunology research on the SDS-PAGE gel.

SDS-PAGE and immunological technique.With SDS-PAGE method (Laemmli (1970) Nature 227,680-685) isolated protein.In order to detect protein, antiserum(antisera) with anti-57 (1/1000 dilution) and coupling the anti-rabbit second antibody of alkaline phosphatase carry out immunoblotting assay.In order to carry out the reaction of Western trace, protein electrophorese is transferred on the nitrocellulose filter and with 0.5% ponceau (Ponceau Red) that is added in 5% acetate dyeed, to guarantee equal applied sample amount being arranged and shifted success.Then with 5% degreasing dry powder sealing filter membrane, and detect and dye with standard method.

Tre6P synthetic enzyme detection method.Take by weighing about 500mg refrigerated vegetable material, then at solid-state CO ₂Upward be ground into fine powder with mortar and pestle.Powder transfer is contained 1mM benzamidine, 2mM MgCl to 0.7ml ₂, 1mM EDTA and 1mM dithiothreitol (DTT) (wherein also containing 1mM PMSF and each 10 μ g/ml pepstatin A and leupeptin) 50mMHEPES/KOH (pH7.0) (HBMED) in, and make it to dissolve gradually.Also ((1971) J.Biol.Chem.246,4567-4579) described method detected the Tre6P synthetic enzyme in the supernatant liquor according to people such as Lapp basically in centrifugal 10 minutes with the gained homogenate with 17,000 * g.Sample (10 μ l) is added to 90 μ l contains 40mM HEPES/KOH (pH7.0), 10mMMgCl ₂, 10mM G-6-P, 5mM fructose-6-phosphate, 5mM uridine diphosphate glucose (UDPG) and 1mg/ml bovine serum albumin reaction mixture in, and in 30 ℃ of times that insulation is required.Be heated to 100 ℃ of termination reactions during by 2 minutes.Add 50 μ l 0.6M HCl and in 100 ℃ of heating 5 minutes, add 50 μ l 8%NaOH then and in 100 ℃ of heating 15 minutes, to destroy the sugar derivatives (comprising the sucrose that is generated) except that trehalose and trehalose-6-phosphate.Use anthrone detection method (Trevelyan﹠amp then; Harrison (1956) Biochem.J.63 23-33) detects the carbohydrate (being trehalose and trehalose-6-phosphate) that keeps.

The trehalose detection method.The about 500mg refrigerated of weighing vegetable material is added in the glass test tube fast.Add behind the hot distilled water mixture boiled 20 minutes, leaf is smashed to pieces with the round end glass stick.Also extract solid substance once more with suction pipe results liquid phase with 0.5ml water.The liquid phase that centrifugal clarification merges.Make it to reach constant weight (the dry weight average out to fresh weight of leaf 5.1%) in 107 ℃ of dry solid residue that merge.The DionexDX-300 liquid chromatography (LC) instrument that use is furnished with Dionex pulse electrochemical detector (PED-2) is clear liquid analytically.(4 * 50mm) pre-columns are with sample (20 μ l by Carbopac PA-1; In triplicate) be expelled to Carbopac PA-1 (on 4 * 250mm) posts and with 1ml/ minute flow velocity water wash-out.Eluate is mixed with post column reagent (flow velocity is 0.6ml/ minute 0.3M NaOH).When trehalose appears at about 3 minutes, before the dextrose plus saccharose peak that in the time of about 20 minutes, occurs.Embodiment 1: be under the ats1A promotor control yeast TPS1 gene transformation tobacco plant also

By the generation trehalose

Tobacco transformant and control plant are grown under aseptic " in vitro " condition and the greenhouse in.Compare with the control group that transforms with carrier pDE1001 (lacking TPS1) with the control group that is not transformed, sophisticated transformant does not have obvious phenotypes to change.Collected blade, freezing and be stored in when back-up is analysed below-70 ℃ or-70 ℃ and use in 0900 hour.

Tested 26 kalamycin resistance transformants, the result when detecting with the antiserum(antisera) of the yeast TreP 56 KDa Tre6P synthetic enzyme subunits of antivenom purification, but find to have 20 strain generation detection levels that the immunoreactivity polypeptide of expection size is arranged.Example is shown among Fig. 2.Summed up the content of trehalose of blade in the table 1.

The content of trehalose tobacco plant special processing trehalose of the TPS1 transformant of table 1 tobacco

(mg/ fresh leaf) in vitro the SR1-that is not converted of plant≤0.002 pDE1001 contrast-≤0.002 transformant, 1-0.02 transformant, 3-0.009 transformants, 4-0.067 transformants, 8-0.075 transformants, 8 alcohol extracts replaces water extraction to get 0.055 greenhouse plant pDE1001 contrast-≤0.002 transformant, 1-0.16 transformant, 4-0.16 transformants, 4 alkaline phosphatases^a0.13 transformant 5-0.052 transformants 6-0.044 transformants 8-0.039 transformants 8 specificity trehaloses 0.021 transformant 19-0.053 transformants 19 alkaline phosphatases ^a0.060 transformant 19 specificity trehaloses 0.016 transformant 25-0.036 transformants 26-0.11

A make carrier [ ¹⁴C] use the alkaline phosphatase treatment extract under the dephosphorylized condition of trehalose-6-phosphate.

These results disclose, and when the promotor of yeast TPS1 gene was replaced by the ats1A promotor, it can be expressed in tobacco effectively.Observed signal specific has correct molecular weight on the Westem trace.Be added on the gel the proteinic peak signal of per unit (for example deriving from the signal that grows in invisible spectro transformant 4) only than derive from stationary phase the zymic signal slightly a little less than.The expression of TPS1 is followed and is occurred trehalose in the leaf texture, and the latter then can be degraded by the high specific trehalase according to its HPLC behavior and it and assert (also referring to Fig. 3).Based on not clear and definite as yet reason, different transformants is with different horizontal expression TPS1 products, and (with regard to the apparent expression of transformant 8) see in the blade the trehalose amount roughly with the Western trace in the intensity relevant (comparison diagram 2 and table 1) of 56KDa signal.Though these transformants do not carry the gene of coding reorganization Tre6Pase, not evidence suggests and have gathered in the plant than the more TreP of trehalose.Clearly, tobacco has the Phosphoric acid esterase that Tre6P can be transformed into trehalose, show to be that the necessary key enzyme of importing trehalose route of synthesis is the Tre6P synthetic enzyme at least for this kind of plant, although and not necessarily best, it also is enough importing this enzyme separately.

Detecting 56KDa Tre6P synthetic enzyme subunit is that organizing respectively in 4 shows transgenic plant, can see this peptide species (referring to Fig. 4) that a great deal of is arranged in all green portion except that plant bottom stem and root.This distribution is (DeAlmeida et al. (1989) Mol.Gen.Genet.218,78) that are consistent with the tissue specificity of ats1A genetic expression.Fig. 4 also shows in the tissue of expressing the Tre6P synthetic enzyme and also contains trehalose.In addition, as seen trehalose is in a small amount arranged in root, show that transgene tobacco can be from its synthesising part to its hetero-organization conveying trehalose.

The result also discloses, express be in the control of ats1A promotor down TPS1 and the tobacco plant that in its chlorenchyma, gathers trehalose between photoperiod be health and outward appearance be normal.Though contain the initial conversion strain of chimeric TPS1 gene some little morphological change is arranged, for example present pocket knife shape blade, reduced apical dominance and lowered height (seeing Fig. 5 and 6), but the great majority change does not come across in the positive filial generation of the autophilous TPS1 that still produces trehalose.Therefore, the morphological change of initial conversion strain is what cause because of the tissue culture people seemingly, rather than trehalose causes because of producing.But compare with the control group that is not transformed, produce high-level Tre6P synthetic enzyme subunit and exist trehalose not cause obviously (20～50%) reduction of the transgenic plant speed of growth under normal operation.Obviously normal phenotype and the external source of having reported of these transgenic plant add the toxicity opposite (Veluthambi et al. (1981), document ibid) of trehalose to certain plants.These results disclose, though produce trehalose in plant under the control of sts1A promotor the speed of growth of plant are slowed down a bit, and tobacco plant is not had toxicity.Can use the inducibility promotor that excites by particular procedures such as arid or colds, by only causing just that when needed trehalose produces the reduction that reduces the speed of growth as much as possible.

Based on protein content, the content of trehalose of the best transformant shown in the table 1 protein of 16mg/g transformant 4 (for example 〉=) be in yeast observed make contents level that heat tolerance has clearly improvement at least 20% (De Virgilio et al. (1990) FEBS Letters273,107-110).

Detect the Tre6P synthase activity in some TPS1 transformant and the control plant.Result shown in the table 2 detected resulting mean value ± maximum range from duplicate 0 and 15 or 30 minute.With regard to control group, the Tre6P synthase activity is zero no better than.With regard to transformant 4, in the presence of UDPG and G1c6P, gathered bronsted lowry acids and bases bronsted lowry stability carbohydrate.This gathering needs G1c6P, but do not need fructose-1, 6-diphosphate (Fru6P; This phosphohexose activates zymic natural seaweed sugar synthetic enzyme mixture), and when replacing UDPG, can suppress this gathering (enzyme of purifying can not use ADPG from yeast) with ADPG.Because other possible products all are hydrolyzed destruction, infer that the carbohydrate that wherein gathers is trehalose or Tre6P.Therefore, under these vitro detection conditions, synthetic Tre6P is faster than being converted into trehalose by the extract of transformant 4.This shows can increase the total speed of trehalose synthetic in the leaf with the TPS2 cotransformation of coding Tre6Pase subunit.

The Tre6P synthase activity of the yeast extract that predicts with method shown in the table 2 with by Londesborough and Vuorio ((1991) J.Gen.Microbiol.137,323-330) method that occurs of described detection UDP predict active consistent.In addition, the yeast extract that detects in the presence of the tobacco plant extract is not suppressed.Therefore, in the table 2 control plant not record activity be not owing to be subjected to due to the interference of existing some factor in the tobacco plant.

The tobacco leaf that table 2 TPS1 transforms and the Tre6P synthase activity of control group tobacco leaf

(all results are used under the aseptic condition in vitro, and growing plants obtains)

Transformant detects mixture Tre6P synthase activity

(mU/g fresh leaf)

Whole 22 ± 35 7 ± 8 transformants 4 of the whole 3 ± 47 3 ± 21PDE1001 of the control group that 15min 30min is not transformed control group, test 1 whole 259 ± 147 60 ± 6 transformants 4, test 2 whole 128 ± 39 155 ± 22

Less Glc6P 12 ± 19-1 ± 12

Less Fru6P 153 ± 10 144 ± 3

ADPG replaces UDPG 0 ± 52 4 ± 21

The Tre6P synthase activity that sees in the transformant 4 is unsettled.Some extract is deposited the several hrs activity and just can be disappeared on ice.But the specificity band that sees in the Western analysis still is present in the extract that has stored 24 hours under the room temperature with its similar original intensity.Therefore, might be the change that Tre6P synthetic enzyme subunit conformation has taken place between the shelf lives of tobacco extract.These results show, can with the conformational stability of raising Tre6P synthetic enzyme subunit, thereby reach the purpose that improves the Tre6P synthase activity simultaneously with other subunit gene transformation of tobacco of TPS1 and one or more yeast TrePs.Embodiment 2: transgenosis Arabidopsis thaliana

Same quadrat method structure with above-mentioned structure tobacco transformant contains the A.thaliana plant that is in the following TPS1 of ats1A promotor control.The Arabidopsis plant that these are transformed also is healthy and normal outward appearance is arranged, and produced the seed that can educate.The plant that seed from the initial conversion strain is grown carries out Western to be analyzed, and shows that they contain the 56KDa subunit (Figure 10) of yeast TreP.What obviously can omit in advance is that these plants can gather trehalose in its chlorenchyma.Embodiment 3: the arid resistance of producing the trehalose tobacco plant

For the amount that detects the trehalose that is produced in the transgene tobacco whether is enough to improve their drought tolerance, in vitro the control group of propagation and the excised leaf of transgenic lines plant carry out air-dry (25% relative humidity, RH) (Fig. 5).The excised leaf of control plant is lost moisture rapidly and is demonstrated the sign that becomes brown after 3 hours applying environmental stress, and the blade that produces the trehalose plant still is green after reaching 24 hours, obviously reduced loss of moist simultaneously.At first therefore, it seems that the provide protection of trehalose be two aspects: as if at first, the excised leaf of the transgenic plant of trehalose synthesis just has the water retention that has improved.Just just disappear having prolonged this species diversity of producing between trehalose plant and the control group plants after the drying treatment.The second, the control group blade table reveals the sign of clear and definite sign and browning look.On the contrary, the blade that contains the trehalose plant is still to be green after 24 hours, and only after the exposure duration that prolongs under the dry environment (for example several days) sign of browning look just appears.Even between product trehalose plant and the control plant clearly visible difference is being arranged also under the same water-content situation.Therefore, the influence that as if trehalose can stop plant tissue to be dewatered, and can reduce the speed of loss of moist.A part of reason of blade browning look may be because due to the Maillard reaction, and promptly wherein the free amine group of reducing sugar and polypeptide and the amino acid that produces brown color react.Silver non-reducing sugar trehalose does not participate in this reaction, even suppresses the reaction (Roser﹠amp between other sugar and the protein; Colaco (1993), New Sciennist 1973,24).

The cut leaf of trehalose protection is avoided the arid damage and is shown that trehalose had both made on complete plant level and also has similar effect.In order to estimate the drought tolerance whether trehalose can improve complete plant, we make the complete plant that in vitro breeds be exposed to (30%RH, Fig. 6 A) under the air drying condition.Lose full state and wilting occurs 3 to 4 hours internal reference plants.On the contrary, the gene plant that produces trehalose is through the long-time sign that only demonstrates full state forfeiture after air-dry.After the drying treatment 17 hours, make plant aquation and write down the survival rate (Fig. 6 A) of plant again.The control plant that is not transformed had both made and had not still recovered after the aquation again for a long time, thereby failed to survive after this handles.On the contrary, though produce the transgenic plant system of trehalose and obviously wilt and lost most tissues moisture (reduce to fresh weight 30%), still survival after dry 17 hours.

Immature seedling also shows the arid viability (Fig. 6 B) of raising.The seedling that made in 3 weeks that transgenic lines 8 is produced is exposed under the air drying condition (50%RH) together with the contrast seedling with the carrier conversion that is not transformed, and proves to produce between trehalose plant and control plant to have evident difference on drought tolerance.Compare with control plant, the genetically modified trehalose positive be 8 demonstrate delay full loss of state and dehydration under the environmental stress than high survival probability (Fig. 6 B).Embodiment 4: Tre6P synthetic enzyme and one or more coding Tre6Pase or modulability are many by encoding

The plant of the gene cotransformation of peptide produces trehalose

Those skilled in the art can prepare to contain and are in the ats1A promotor control yeast TPS2 down and the carrier of the encoding sequence of TSL1 gene, and by the method described in " generally material and method " and the embodiment 1 with their transformation of tobacco, Arabidopis thaliana and other plants.In order to obtain being one of TPS1 and TSL1 or both plant transformed with TPS1 and other genes simultaneously, can make the mutual cross of indivedual transformant phase, or further transform a strain by plant transformed with second gene, perhaps transform: for example with containing the carrier that is connected to two or three genes on the suitable promotor, TPS1 can be connected on the non-composition ats1A promotor in the trehalose building-up process, providing control action kou, and by other expression of gene of composition promoters driven.

Expect that expression of gene will cause the increase of trehalose accumulated amount in the green plant tissue for two or more subunits (at least one is 56 KDa subunits) of in check yeast TreP mixture, because the existence of other genes will make the 56KDa subunit be able to stabilization.In addition, it will be favourable importing 102KDa Tre6Pase subunit, because when the stability of Tre6P synthetic enzyme subunit increases, be expected the potential accumulated amount that it will reduce Tre6P.

Obviously, for such structure, the encoding sequence that can replace TPS1 with the encoding sequence of some other Tre6P synthetic enzyme structure gene, with the encoding sequence replacement TSL1 sequence that encoding sequence replaces the TPS2 sequence and other codings are given the gene of accommodation property or stability to Tre6P synthetic enzyme and Tre6Pase with some of some other Tre6Pase gene, wherein the mode of being taked is as giving these character by the TSL1 product to the unartificial yeast TreP.Embodiment 5: change with the Tre6P synthase gene that is under the control of environmental stress inductive promotor

Change plant

Known for example LTI78 (Nordin et al. (1993) Plant:Mol.Biol.21,641-653) and RAB18 (Lang﹠amp; Palva (1992) Plant Mol.Biol.20, it is derivative in the reaction to arid and cold pressure 951-962) waiting plant promoter.Separately with Tre6P synthase gene under the promotor control that is in these pressure inducements such as the encoding sequence of TPS1, perhaps, trehalose only gathering in plant tissue when being reacted, these environmental stresss occurred together with being in the Tre6Pase under the control of conventional plant promotor or regulating peptide or both gene transformation of tobacco, Arabidopsis plant or other plant.Like this; advantage is to have only (1) (to provide provide protection by trehalose during in environmental stress when the plant adventitious exposure; overcome any disadvantageous effect then); perhaps (2) just cause gathering of trehalose when being exposed to environmental stress preparatively when plant has in advance; and then from the plant of results, extract trehalose; the disadvantageous effect that the trehaloses that so just may avoid different amounts bring some tissue of certain plants, and photosynthetic synthesis capability reduces direction deflection and possible plant-growth sluggishness to productive rate.Embodiment 6: the purifying of other Tre6P synthetic enzyme, analysis and clone

As if although have other pathways metabolisms that produce trehalose, described as Cabib and Leloir (1958, ibid for document), main route of synthesis still passes through Tre6P.Key enzyme in this approach is the Tre6P synthetic enzyme, because as mentioned above, in case make Tre6P, many cells just can make it dephosphorylation and become free trehalose.Therefore, the key principle among the present invention is to import the Tre6P synthase activity in the target plant.Coming as for this activity then is not most important wherefrom.We have used the yeast enzyme of the subunit (this mixture at least also contains other two subunits) that is yeast TreP mixture by chance.In this case, though evidence suggests from zymic 56KDa Tre6P synthetic enzyme subunit, can do not have 102 and the situation of 123KDa subunit under in tobacco, bring into play function effectively, but the optimal activity of Tre6P synthetic enzyme subunit may need to have one or more other subunits.

The enzyme of same reaction usually is a homologous in the multiple different organisms of known catalysis, thereby in case after having cloned a member of this family, just will help finishing the clone's work to other members.Because cloned yeast Tre6P synthetic enzyme (Londesborough and Vuorio, (1992) USPA07/836,021), so further recognize and exist one to comprise from the Tre6P synthetic enzyme of intestinal bacteria with from protein (the McDougall et al. of hot autotrophic methane bacteria (Methanobacterium thermoautotrophicum), (1993) FEMS Microbiology Letters 107, homologous protein family 25-30).We determine to test whether the other biological body also contains homology Tre6P synthetic enzyme.

Smegma bacillus contains partially purified heparin activated T re6P synthetic enzyme (Liu et al., (1969) J.Biol.Chem.244, the 3728-3791 that is crossed by the group study of Elbein; Lapp et al. (1971) J.Biol.Chem 246,4567-4579; Elbein and Mitchell (1975) Arch.Biochem.Biophys.168,369-377; Pan et al., (1978) Arch.Biochem.Biophys.186,392-400).Enzyme by these investigator's purifying reaches in the presence of the optimal dose heparin at 37 ℃, makes substrate (this enzyme can use the spectrum of nucleoside diphosphate glucose derivative) with Glc6P and UDPG and detects, and shows that it has the proteinic specific activity of 0.8U/mg.Said preparation contains the SDS-PAGE molecular weight and is about 45 and two polypeptide of 90KDa.We change author's purification process, comprising adding proteinase inhibitor and other protein protectants in employed damping fluid, and add a last chromatographic step in the presence of non-ionic detergent Triton X-100.Below be the method for our last use:

1) containing 1mM benzamidine, 2mM MgCl ₂, 1mM EDTA, 1mM dithiothreitol (DTT), 1mM phenylmethylsulfonyl fluoride (DMSF) and 10 μ g pepstatin A/ml 40ml 50mM HEPES/KOH (pH7.5) in cracking smegma bacillus cell (28 weight in wet base cells of growth 3 days and freezing preservation in the Luria nutrient solution), in the Franch homogenizer, destroy it then.With homogenate with 28, centrifugal 20 minutes of 000g.In supernatant liquor, add (NH ₄) ₂SO ₄(every 100ml adds 30g).The protein of collecting precipitation, be dissolved in contain 0.1mMEDTA and be added with 0.1mM PMSF and the 20mM HEPES/KOH (pH7.5) of the 0.2mM dithiothreitol (DTT) of 10 μ g pepstatin A/ml (HED damping fluid) in, and to HED damping fluid dialysed overnight.

2) dialyzate is splined on 3.4 * 25cm DEAE cellulose column also with the linear gradient that reaches 0.9M NaCl that is added in the 1.2 LHED damping fluids, with 20ml/ hour flow velocity wash-out enzyme.The peak value part (when 0.2M NaCl by wash-out) that merges enzyme (33ml) and is adjusted to and is wherein contained 0.5mM PMSF and 5 μ g pepstatin A/ml.Add 9.9g (NH ₄) ₂SO ₄With precipitating proteins, and sedimentary proteolytic enzyme is dissolved among the 50mM Tris/HCl (pH7.5) (TNED damping fluid) that contains 50mM NaCl, 0.1mMEDTA and 0.2mM dithiothreitol (DTT).

3) make dissolved protein (3.7ml) with 36ml/ hour flow velocity by 2.8 * 34cm Sephadex G100 post of crossing with TNED damping fluid balance.Peak value part with enzyme directly is splined on 1.5 * 8.5cm heparin-Sepharose (in the TNED damping fluid) post then.With the flow velocity wash-out post of the gradient that reaches 1.0M NaCl (in the 100ml damping fluid) with 5ml/ hour.Wash-out goes out enzyme when about 0.5M NaCl.

4) with in the sample transfer of heparin-Sepharose eluate (the component H37 in the table 3) TNED/0.1% (V/V) Triton * 100 to the PM10 film that is added in the Amincon Xiao Chi, be splined on then on 0.7 * 8cm heparin-Sepharose post of crossing with the TNED balance that contains 0.1% (V/V) Triton * 100, and with the NaCl gradient elution that reaches 1.0M that is added in the above-mentioned damping fluid of 50ml.

Table 3 purifying Tre6P synthetic enzyme from smegma bacillus is pressed Londesborough and the described method of Vuorio (1993) basically, in 35 ℃ and the following Tre6P synthetic enzyme that detects of 0.25 μ g heparin/ml existence

The active gross activity of component volume ratio

(ml) (mU/mg) (U) 28, (the NH of 000g supernatant liquor 154 9 25.0 dialysis ₄) ₂SO ₄Sediment 52 21 35.2DE52 eluates 33 139 23.4G100 eluates 21 385 14.6 first heparin-Sepharose eluate component H33 2.7 1,880 0.9 component H34 2.7 4,380 2.1 component H35 2.7 6,190 2.7 component H36 2.7 7,890 2.8 component H37 2.7 6,340 1.6 second heparin-Sepharose eluate (only upper sample H37) component T21 1.4 ND 0.08 component T22 1.4 ND 0.14 component T23 1.4 ND 0.06

Though the specific activity after process first heparin-Sepharose chromatographic step is more much higher than people such as Pan (19878) report, SDS-PAGE (Fig. 7) shows that these components are impure.Surprisingly, the band at about 55KDa place is the closely-related master tape of intensity and enzymic activity.Cut away 55KDa band and in gel, use tryptic digestion, then through HPLC isolate pancreatin separate peptide also as stated above (Londesborough and Vuorio, 1993, document is the same) carry out sequential analysis.Sequence is shown in Table 4 and provides in sequence table.For the peptide peak value part 29 and 31 of the bifilar sequence that provides, amino acid optionally is appointed as SEQ ID NO 3 and 4 (from peak 29) and SEQ ID NO 6 and 7 (from peak 31) by the same manner described in the table 4.Preceding 9 residues of peptide 13 have 89% to be entirely identical to the residue 250-258 of the 56KDa Tre6P synthetic enzyme of yeast (VGAFPIGID is referring to people's such as Vuorio above-mentioned document (1993); The EMBL accession number is X67499).

Table 4 derives from from the Tre6P of smegma bacillus purifying

The inside pancreatin of synthetic enzyme is separated the aminoacid sequence of peptide

Peptide peak sequence nucleotide sequence table

13 VGAFPISIDSAEL SEQ?ID?NO：1

21 AT/GFLDALAATGETGDSGVT SEQ?ID?NO：2

29 (bifilar) RVVVNNTSR SEQ ID NO:3

YLEGAR SEQ?ID?NO：4

25 QVLAHDVDR SEQ?ID?NO：5

31 (bifilar) IGGAQPAD SEQ ID NO:6

VGALQVLL SEQ?ID?NO：7

43 GEVQVGFR SEQ?ID?NO：8

Represent the Tre6P synthetic enzyme in order to confirm that further this about 55KDa is with, make further purifying and attempt.This enzyme is attached on the UDP-glucuronic acid agarose column forcefully, but can not be recovered.Therefore, component H37 is transferred in the damping fluid that contains 0.1%Triton * 100 (in this transfer process, have 2/3 activity lose) and in the presence of Triton * 100, cross heparin-Sepharose post once more and carry out chromatography.Fig. 8 shows, except two than the little faint band of cytochrome c, it is the only Coomassie brilliant blue reactive explosive that is present in the active part that about 55KDa is with.Clearly, the 90KDa polypeptide that people such as Pan (1978) are reported not is the basal component from this Tre6P synthetic enzyme of smegma bacillus, and the size of the basic polypeptide of about 55KDa is significantly greater than less 45KDa composition that these authors reported.Fig. 9 shows that this polypeptide is that this Tre6P synthetic enzyme that shows smegma bacillus is shared epitope in the yeast enzyme by the antiserum(antisera) of the 56KDa Tre6P synthetic enzyme subunit of anti-yeast TreP rather than by pre-immune serum identification.Studied the character of other immunoreactivity bands (seeing swimming lane 5 and 6 among Fig. 9) that are present in the thick relatively zymin: all these is not because big protein applied sample amount people causes, and represents some relevant protein.However, the antiserum(antisera) that still can use anti-yeast enzyme is from detecting and isolate positive colony the skin scurf mycobacterium gene pool transformed host cells.Equally, can use the sequence of the separated gene of the amino acid data check shown in the table 4.Immunology and amino acid sequence similarity from yeast and smegma bacillus between the isolating enzyme show, also can successfully use the nucleotide probe screening smegma bacillus gene according to the TPS1 gene design.

In a word, smegma bacillus immunology that enzyme is done and order-checking being studies confirm that, all is the member of same family from the Tre6P synthetic enzyme of different organisms.Can use the gene of these enzymes to make the transgenic plant of can trehalose synthesis and having improved the environmental stress tolerance by the same manner that uses TPS1.

Sequence table (1) general information (i) applicant: Alko Group Ltd and

Londesborough，John

Tunnela，Outi

Holmstrm，Kjell-Ove

Mntyl，Einar

Welin，Bjrn

Mandal，Abul

Palva, E.Tapio be the invention exercise question (ii): the transgenic plant that produce trehalose are sequence number (iii): 8 (iv) addresses:

(A) address: Alko Group Ltd

(B) street: Salmisaarenranta 7

(C) city: Helsinki

(D) state :-

(E) country: Finland

(F) area code: FIN-00180 (v) computer-reader form:

(A) media type: Diskette, 3.5 inch, 720 kb

(B) computer: IBM PC/XT/AT

(C) operating system: PC-DOS

(D) software: WP5.1 file exported as DOS text file (vi) existing application materials:

(A) application number:?

(B) submit to day: 29 June 1995

(C) classification:? (application materials are vii) arranged earlier:

(A) application number: FI 943133

(B) submit to day: the information of 29-JUNE-1994 (2) SEQ ID NO:1: (i) sequence signature:

(A) length: 13 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide is (iii) supposed: do not have (iv) clip types: N-terminal (v) sequence description: SEQ ID NO:1Val Gly Ala Phe Pro Ile Ser Ile Asp Ser Ala Glu Leu

The information of 5 10 (2) SEQ ID NO:2: (i) sequence signature:

(A) length: 19 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide is (iii) supposed: do not have (iv) clip types: N-terminal (v) sequence description: SEQ ID NO:2Ala Xaa Phe Leu Asp Ala Leu Ala Ala Thr Gly Glu Thr Gly Asp

The information of 5 10 15Ser Gly Val Thr (2) SEQ ID NO:3: (i) sequence signature:

(A) length: 9 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide is (iii) supposed: (iv) clip types is arranged: N-terminal (v) sequence description: SEQ ID NO:3Arg Val Val Val Asn Asn Thr Ser Arg

The information of 5 (2) SEQ ID NO:4: (i) sequence signature:

(A) length: 6 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide is (iii) supposed: (iv) clip types is arranged: N-terminal (v) sequence description: SEQ ID NO:4Tyr Leu Glu Gly Ala Arg

The information of 5 (2) SEQ ID NO:5: (i) sequence signature:

(A) length: 9 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide is (iii) supposed: do not have (iv) clip types: N-terminal (v) sequence description: SEQ ID NO:5Gln Val Leu Ala His Asp Val Asp Arg

The information of 5 (2) SEQ ID NO:6: (i) sequence signature:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide is (iii) supposed: (iv) clip types is arranged: N-terminal (v) sequence description: SEQ ID NO:6

Ile Gly?Gly Ala Gln Pro Ala Asp

The information of 5 (2) SEQ ID NO:7: (i) sequence signature:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide is (iii) supposed: (iv) clip types is arranged: N-terminal (v) sequence description: SEQ ID NO:7

Val Gly Ala Leu Gln Val Leu Leu

The information of 5 (2) SEQ ID NO:8: (i) sequence signature:

(A) length: 8 amino acid

(B) type: amino acid

(D) topological framework: linearity is molecule type (ii): peptide is (iii) supposed: do not have (iv) clip types: N-terminal (v) sequence description: SEQ ID NO:8Gly Glu Val Gln Val Gly Phe Arg

5

Claims (29)

1. one kind with the encoding sequence plant transformed that is fused to the trehalose-6-phosphate synthase gene on the non-composition plant promoter, has new trehalose synthesis capability thereby make by plant transformed.

2. the plant of claim 1, wherein plant promoter is tissue-specific.

3. the plant of claim 1, wherein plant promoter is photoactivation.

4. the plant of claim 1, wherein plant promoter is to be activated because of standing environmental stresss such as drying, high salinity or extreme changes of temperature.

5. each plant in the claim 1 to 4, wherein the trehalose-6-phosphate synthase gene is microbe-derived.

6. the plant of claim 5, wherein the trehalose-6-phosphate synthase gene is the yeast genes TPS1 of the 56KDa subunit of coding yeast TreP.

7. each plant in the claim 1 to 6, its for the encoding trehalose-6-phosphate enzyme or encode and at least one gene cotransformation of the regulatory polypeptide of the trehalose-6-phosphate synthase that is imported into or trehalose-6-phosphate enzyme interacting.

8. the plant of claim 7, wherein the gene of encoding trehalose-6-phosphate enzyme is a yeast TPS2 gene, the proteinic gene of coding and regulating is a yeast TSL1 gene.

9. the plant of claim 6, wherein plant promoter is pats1A.

10. each plant in the claim 1 to 8, wherein employed plant promoter is environmental stress activated promotor, for example LTI78 or RAB18.

11. each plant in the claim 1 to 10, wherein said plant is than by plant transformed more overall situation force resistance not being arranged.

12. each plant in the claim 1 to 11, it is for example corn, oat, grain, wheat, rice, barley, Chinese sorghum, three-coloured amaranth, onion, asparagus or a sugarcane of monocotyledons.

13. each plant in the claim 1 to 11, it is for example clover, soybean, petunia, cotton, beet, Sunflower Receptacle, Radix Dauci Sativae, celery, wild cabbage, cucumber, pepper, tomato, potato, root of Szemao crotalaria, flax, sprouting broccoli, tobacco, Kidney bean, lettuce, rape, Cauliflower, spinach, brussels sprouts, arithoke, pea, gumbo, pumpkin, leafy cabbage, kale, a tea or coffee of dicotyledons.

14. the generation of trehalose in each the plant in the Accessory Right requirement 1 to 13.

15. seed by each plant generation in the claim 1 to 13.

16. increase the method for the content of trehalose of plant, this method may further comprise the steps:

-structure gene with trehalose-6-phosphate synthase transforms interested plant at least, and

-express the gene that is under the suitable promotor control, to allow in the genetic expression process, carrying out sequential, local distribution or the control of environmental stress inductive.

17. the method for claim 16, wherein gene is the yeast genes TPS1 of the 56KDa subunit of coding yeast TreP.

18. the method for claim 16 or 17, wherein plant is with the gene that is in the trehalose-6-phosphate synthase under non-composition promotor such as the pats1A control, be in any suitable promotor control trehalose-6-phosphate enzyme or gene cotransformation of the adjusting subunit of TreP down, the adjusting of synthesizing the promotor that promptly is controlled the trehalose-6-phosphate synthase gene of trehalose like this with at least one.

19. the method for claim 18, wherein the gene of trehalose-6-phosphate enzyme or adjusting subunit is respectively yeast genes TPS2 or TSL1.

20. each method in the claim 16 to 19, wherein the encoding sequence of trehalose synthesize enzyme gene is fused on the plant promoter, is provided with causing the expression specifically in storage organ.

21. produce the method for trehalose, it may further comprise the steps:

-transform plant with the structure gene of trehalose-6-phosphate synthase, with the generation transgenic plant,

-under the condition that will induce trehalose-6-phosphate synthase in plant, to express, cultivate transgenic plant, and

-from the tissue of plant, extract trehalose.

22. the method for claim 21, wherein gene is to express under the control of the promotor that allows expression of gene is carried out sequential, local distribution or the control of environmental stress inducibility.

23. the method for claim 21, wherein transgenic plant gather trehalose in its storage organ.

24. the method for claim 23, wherein plant is root crop such as potato, beet or turnip.

25. protection staple crops plant is with the method to drought-resistant, high salinity or extreme changes of temperature; it comprises that the encoding sequence of using the trehalose-6-phosphate synthase gene that merges with plant promoter at least transforms plant, so only could realize giving full expression to of gene when plant suffers from arid, high salinity or extreme temperature conditions.

26. protection have can carpophagous plant in case flower is subjected to the method for frost damage; it comprises that the encoding sequence of using the trehalose-6-phosphate synthase gene that merges with plant promoter at least transforms plant, so only could realize giving full expression to of gene when plant suffers from low temperature.

27. improve the method for the storing property of gathering in the crops plant of the fruit comprise green food raw material, harvesting and ornamental plant, this method comprises uses the trehalose-6-phosphate synthase gene-transformed plant that merges with plant promoter at least, so that provide such plant, part that it has been gathered in the crops to have the water retention capacity of improvement, improve dehydration tolerance or both.

28. help cultivating and safeguarding the method for ornamental plant, it comprises uses the trehalose-6-phosphate synthase gene-transformed plant that merges with plant promoter at least, so that make plant have the environmental stress tolerance of having improved with not compared by plant transformed.

29. improve by the mud of the edible portion preparation of plant, stick with paste, freeze and the taste of sauce and the method for quality, it comprises that the encoding sequence of using the trehalose-6-phosphate synthase gene that merges with plant promoter transforms plant, so that realize trehalose gathering in the plant edible portion.

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