CN1330720A - Thioredoxin and grain processing - Google Patents

Thioredoxin and grain processing Download PDF

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CN1330720A
CN1330720A CN99814535A CN99814535A CN1330720A CN 1330720 A CN1330720 A CN 1330720A CN 99814535 A CN99814535 A CN 99814535A CN 99814535 A CN99814535 A CN 99814535A CN 1330720 A CN1330720 A CN 1330720A
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M·B·拉纳汉
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Syngenta Participations AG
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Abstract

The invention provides methods of processing grain, particularly corn and soybeans, utilizing thioredoxin and/or thioredoxin reductase to enhance extractability and recovery of starch and protein. The invention further provides transgenic plants expressing thermostable thioredoxin and/or thioredoxin reductase.

Description

Trx and cereal processing
The present invention relates to be used for cereal and process particularly corn wet milling and soyabean processing, with the novel method of enhancing protein and recovery of starch, and the new transgenic plant that is used for this method.
Trx (TRX) and thioredoxin reductase (TR) are to utilize the also enzyme of disulfide linkage in the crude protein of NADPH.Protein disulfide plays an important role in cereal working (machining) efficiency and the aspects such as quality that reclaim product from cereal processing.The effective ways of the amount of protein disulfide will increase working (machining) efficiency greatly in exploitation removal or the reduction cereal.In addition, the cereal performance in the animal feed also is subjected to the influence of intramolecularly and intermolecular disulfide bond: cereal digestibility, nutrition availability and antinutritional factor (as, proteolytic enzyme, amylase inhibitor etc.) neutralization all can increase by the degree that reduces disulfide linkage.
The expression in corn and soybean of transgenosis Trx and/or thioredoxin reductase, and the purposes of Trx in cereal processing (as wet-milling) have novelty, and the another kind of method that reduces disulfide linkage in the seed protein in the industrial processes process is provided.Therefore the present invention provides the cereal of the storage protein quality that has change, and (following all be called " processing ") processing characteristics is different from the cereal of normal cereal qualitatively in industrial processes or animal digestion process.
The delivering method of Trx and/or thioredoxin reductase has been avoided the Trx that work in-process is formed for adding and/or the external source needs of thioredoxin reductase.Providing second advantage of Trx and/or thioredoxin reductase by cereal is in first being processed or as extra procedure of processing, needn't physical property destroy seed integrity so that enzyme contact with the storage or the stroma protein of seed.
Three kinds of patterns utilizing Trx in cereal processing are provided:
1) in the seed development process, expresses and does composition and quality in order to the cereal that changes results;
2) in the seed development process, express (but non-activity) to change through adding the product quality in man-hour;
3) generation of Trx and/or thioredoxin reductase is used in the course of processing by adding the quality that changes other grain productss in the cereal.
Therefore the present invention provides:
A kind of method that increases starch and protein separation efficient in the cereal milling method is included in the Trx added and/or thioredoxin reductase and exists flood cereal down in high temperature, with separate cereal in protein and the step of starch component.
A kind of as above-mentioned method, wherein cereal comprises the cereal from transgenic plant, wherein transgene expression Trx and/or thioredoxin reductase, particularly heat-staple Trx and/or thioredoxin reductase.
A kind of as above-mentioned method, wherein plant is selected from dicotyledons or monocotyledons, particularly from cereal, especially particularly from corn (Zea mays) and soybean.
The present invention also provides transgenic plant.
Particularly, the invention provides:
A kind of plant that comprises the allogeneic dna sequence of coding Trx and/or thioredoxin reductase, described dna sequence dna stably is integrated into nuclear or the plastid DNA of plant.
A kind of as above-mentioned plant, wherein Trx and/or thioredoxin reductase are heat-staple.
A kind of as above-mentioned plant, wherein Trx and/or thioredoxin reductase are selected from SEQID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, SEQ ID NO:6 and SEQ ID NO:7.
A kind of as above-mentioned plant, wherein plant is selected from corn and soybean.
The present invention also provides:
The effable expression cassette of one kind of plant, it contains Trx and/or the thioredoxin reductase coding region that effectively is connected with promotor that function is arranged and terminator sequence in plant.
A kind of as the above-mentioned effable expression cassette of plant, wherein Trx and/or thioredoxin reductase are heat-staple.
A kind of as the above-mentioned effable expression cassette of plant, wherein Trx and/or thioredoxin reductase are selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
The present invention also provides:
A kind of preparation comprises the method for the cereal of high-caliber Trx and/or thioredoxin reductase, comprises with transforming plant as above-mentioned expression cassette.
A kind of preparation comprises the method for the cereal of high-caliber Trx and/or thioredoxin reductase, comprise to first kind of plant that contains the heterogenous expression box and be used for pollinating from the pollen of second kind of plant that contains the heterogenous expression box, contain the promotor that trans-activator is regulated in the expression cassette of wherein said first kind of plant, its adjusting and effectively be connected with the dna sequence dna of coding Trx and/or thioredoxin reductase, contain the promotor that effectively is connected with the dna sequence dna of coding trans-activator in the expression cassette of second kind of plant, described trans-activator can be regulated the promotor that this trans-activator is regulated, and also comprises from the plant of pollination like this in this method and reclaims cereal.
In addition, the invention provides according to plant of the present invention or vegetable material purposes as animal-feed.
The present invention described herein is applicable to all bread crops, particularly corn, soybean, wheat and barley, more particularly corn and soybean, especially corn.The expression in cereal of transgenosis Trx and/or thioredoxin reductase is to change raw material (seed) quality for the treatment of cereal processing, change is from the raw materials quality of cereal processing, work in-process maximization specific seed composition output (raising the efficiency) changes working method and produces the fraction of seed source or become the method for the new purposes of branch from the powder process logistics.Wet milling process
Wet milling process is the method for starch, protein and the oil component of a kind of separation cereal (more commonly being cereal, for example corn).They are different with the dry grinding method of only simply cereal being pulverized herein.The first step is normally flooded in the wet milling process, and wherein cereal is soaked in the water under the condition of careful control, with softening grain, and the separation of promotion composition.The butyraceous embryo swims in the surface of aqueous solution, and is moved out of, by adding the method for water, dehydration, grinding, screening, centrifugal and washing, with starch and protein separation and be purified.Difficult point is starch granules is unclamped from the complex matrices of the protein that constitutes the cereal endosperm and cell walls.It is believed that an existence that reason is intramolecularly or intermolecular disulfide bond of this difficulty, it makes protein be difficult for dissolving, and insensitive to proteolytic enzyme, suppresses to discharge in the proteic matrix of starch granules from cereal.At present, the main method that is used to reduce these keys makes floods cereal in the presence of sulfurous gas, but this method cost height, hostile environment and very ineffective.
The useful effect (powdery and opaque) of proteic matrix of corn grain endosperm has been given in some sudden changes, but has destroyed whole-grain.The transgenosis Trx is expressed some such advantages is provided, and does not produce and the relevant whole-grain problem of this class sudden change.
After the results of Trx or processing dependency activity have same beneficial effect.For example, in one embodiment, the Trx enzyme is directed in the cellular compartment and accumulation therein.The protein reduction takes place behind the seed physical damage.In another embodiment, the endosperm Trx through the dipping dormancy is activated.In a preferred embodiment, the invention provides a kind of plant of expressing heat-staple transgenosis Trx and thioredoxin reductase, as from thermophilic Trx of height and thioredoxin reductase, unless for example at high temperature (as, be higher than 50 ℃) otherwise do not have obvious active Trx and thioredoxin reductase.In one embodiment, the saccharification of heat-staple Trx and thioredoxin reductase and the expression by other thermophilic enzymes is collaborative in endosperm.Feed applications
The expression in cereal of transgenosis Trx and/or thioredoxin reductase also can be used for improving especially the cereal characteristic relevant with digestibility in the animal-feed.The susceptibility of feedstuff protein confrontation proteolytic enzyme is the function of time and protein configuration.The grain fragmentation usually is used for the feed preparation as steam film-making (steamflaking).These methods all are designed for the digestibility that is of value to grain.The softer grain of its integrity of destructible is expected in the animal stomach.Between protein the constraint of conformation and crosslinked be the main determining factor of proteolytic enzyme susceptibility.Express these keys of modification by the Trx that increases and to help digestion.Corn dry mill method/corn grit
Protein content and quality are important determinatives in granulate production and corn grit production.The minimizing of disulfide linkage has changed the character of Semen Maydis powder, makes it be suitable for being used as the wheat substitute, especially the Semen Maydis powder that is prepared by the mutation of high protein white maize.The soybean fragmentation
U.S. soybean crop more than half is broken or grinds, and the protein quality in gained low fat soyflour or the defatted soyflour (or coarse grain) is extremely important for processing subsequently.Very important on the protein output of soyabean processing logistics and the quality economy, and depend primarily on protein conformation.Increase the Trx activity by the expression that in the water soluble protein fraction, increases transgenosis Trx and/or thioredoxin reductase and increased protein solubility, increase protein output thus.Extracting promotion by the water-based of defatted soyflour under alkaline condition reclaims.Expression by transgenosis Trx and/or thioredoxin reductase increases the Trx activity, also reduced the pH required to effective extraction, the calcium hydroxide or the sodium hydroxide that add have been reduced thus, and reduced and be used for the acid that Acid precipitation adds subsequently, realize no alkaline destructive protein efficient recovery, reduced the consumption of water and the waste discharge of processing factory (containing main biological oxygen demand (BOD)).
Proteinic redox situation influences functional that soy-protein provides, as solvability, water absorption, viscosity, cohesion/adhesion, gelling and elasticity.By as above-mentioned increase Trx increased activity the fiber removal in soy protein concentrate production and soy-protein isolate protease hydrolysis process.Similarly, as above described about corn, expression by transgenosis Trx and/or thioredoxin reductase increases the Trx activity, improved the functional of enzyme activation soyflour in the animal-feed, the solubility of soyabeen grists fraction steam powder process fraction.
Although preferred target in cellular compartment and be heat-staple transgenosis Trx and/or thioredoxin reductase, as above-mentioned, provide in the seed development process and the method for protein modification in the course of processing, to avoid running into to significant adverse effect as the storage protein accumulation of the active result of Trx in the seed development process.In addition, can add Trx, because (opposite with the corn wet milling method) before the cereal destroy integrity (broken and oil extracts) do not need to destroy disulfide linkage as processive enzyme.Be used for the selection of the Trx and the thioredoxin reductase of heterogenous expression:
Trx, thioredoxin reductase and protein disulfide isomerase (PDI) gene sees eukaryote, eubacterium and archeobacteria, comprise stenothermophiles, as Methanococcus jannaschii (Methanococcus iannaschii) and flicker ancient green-ball bacterium (Archaeoglobusfulgidus).The selection of specific gene depends in part on its intended purposes.For method of the present invention, preferred Trx has following characteristic: 1. thermostability
From the Trx of stenothermophiles and related protein at high temperature (＞50 ℃) find to have the stability of increase, and low activity relatively at room temperature.From stenothermophiles for example from archeobacteria (as, Methanococcus jannaschii and the ancient green-ball bacterium of flicker) or other stenothermophiless in express TRX and/or TR, be preferably used for expressing in the seed development process, making when cereal is impregnated or at high temperature adding man-hour Trx activity just obviously increases.Most of corn processing methods comprise or are suitable for high-temperature step.So preferred heat-staple Trx and thioredoxin reductase.Heat-staple being meant, enzyme advantage activation at high temperature for example, is higher than 40 ℃, most preferably is higher than 50 ℃, is 45-60 ℃ for wet milling process for example, even higher, as 45-95 ℃.2. substrate susceptibility
Also can reduce the effect of not expecting in the seed development process by selecting to act preferentially on some protein (as the structural protein in the matrix) and the metabolic enzyme of key being had SA Trx.Multiple TRX show with control in redox under the different reactivity of enzyme.Therefore, can select mainly to act on the target of expectation, minimize the side effect of overexpression.
Suitable heat-staple Trx and thioredoxin reductase comprise as follows:
Trx sequence (SEQ ID NO:1 from Methanococcus jannaschii; Gi|1591029) MSKVKIELFTSPMCPHCPAAKRVVEEVANEMPDAVEVEYINVMENPQKAMEYGIMA VPTIVINGDVEFIGAPTKEALVEAIKKRL
Trx sequence (SEQ ID NO:2 from the ancient green-ball bacterium of flicker; Gi|2649903) (trx-1) MPMVRKAAFYAIAVISGVLAAVVGNALYHNFNSDLGAQAKIYFFYSDSCPHCREVK PYVEEFAKTHNLTWCNVAEMDANCSKIAQEFGIKYVPTLVIMDEEAHVFVGSDEVR TAIEGMK
Trx sequence (SEQ ID NO:3 from the ancient green-ball bacterium of flicker; Gi|2649838) (trx-2) MVFTSKYCPYCRAFEKVVERLMGELNGTVEFEVVDVDEKRELAEKYEVLMLPTLVL ADGDEVLGGFMGFADYKTAREAILEQISAFLKPDYKN
Trx sequence (SEQ ID NO:4 from the ancient green-ball bacterium of flicker; Gi|2649295) (trx-3) MDELELIRQKKLKEMMQKMSGEEKARKVLDSPVKLNSSNFDETLKNNENVVVDFWA EWCMPCKMIAPVIEELAKEYAGKVVFGKLNTDENPTIAARYGISAIPTLIFFKKGK PVDQLVGAMPKSELKRWVQRNL
Trx sequence (SEQ ID NO:5 from the ancient green-ball bacterium of flicker; Gi|2648389) (trx-4) MERLNSERFREVIQSDKLVVVDFYADWCMPCRYISPILEKLSKEYNGEVEFYKLNV DENQDVAFEYGIASIPTVLFFRNGKVVGGFIGAMPESAVRAEIEKALGA
Thioredoxin (trxB) sequence (SEQ ID NO:6:gi|1592167) MIHDTIIIGAGP GGLTAGIYAMRGKLNALCIE KENAGGRIAEAGIVENYPGFEEIRGYELAEKFKNHAEKFKLPIIYDEVIKIETKER PFKVITKNSEYLTKTIVIATGTKPKKLGLNEDKFIGRGISYCTMCDAFFYLNKEVI VIGRDTPAIMSAINLKDIAKKVIVITDKSELKAAESIMLDKLKEANNVEIIYNAKP LEIVGEERAEGVKISVNGKEEIIKADGIFISLGHVPNTEFLKDSGIELDKKGFIKT DENCRTNIDGIYAVGDVRGGVMQVAKAVGDGCVAMANIIKYLQKL from Methanococcus jannaschii
Trx sequence (SEQ ID NO:7 from the ancient green-ball bacterium of flicker; gi|2649006) (trxB)MYDVAIIGGGPAGLTAALYSARYGLKTVFFETVDPVSQLSLAAKIENYPGFEGSGMELLEKMKEQAVKAGAEWKLEKVERVERNGETFTVIAEGGEYEAKAIIVATGGKHKEAGIEGESAFIGRGVSYCATCDGNFFRGKKVIVYGSGKEAIEDAIYLHDIGCEVTIVSRTPSFRAEKALVEEVEKRGIPVHYSTTIRKIIGSGKVEKVVAYNREKKEEFEIEADGIFVAIGMRPATDVVAELGVERDSMGYIKVDKEQRTNVEGVFAAGDCCDNPLKQVVTACGDGAVAAYSAYKYLTS。
The gene that coding is used for protein of the present invention preferably designs by the codon decompiling that utilizes plant optimization, to increase G-C content and to remove harmful sequence, as detailed below.Activity of proteins can be reset or the additive method enhancing by DNA, as following.Therefore, the present invention comprises from these proteinic protein, especially to kept the active protein of Trx or thioredoxin reductase and gone up similar protein substantially.
Be used in the developmental activity of cereal in order in seed, to make up the Trx expression, preferably instruct the promotor of the seed-specific expression of TRX and TR, its target is in storing thing, makes enzyme to storage protein the effect of expectation be arranged, and this expects in some applications.But, in the present invention, be desirably in the expression that makes up Trx and/or thioredoxin reductase in the seed usually, be used for accumulation and non-activity in the cereal forming process.Utilize several strategies, normal seed development has not been had the seed of obvious damage to produce express transgenic Trx and/or thioredoxin reductase.As:
(i) make active Trx or thioredoxin reductase compartmentation, make it obviously not interact, for example by being directed at amyloplast or in amyloplast, expressing with target protein.Adopt the sequence-directed accumulation in amyloplast of plastid target.Perhaps, utilize secretion signal that Trx and/or thioredoxin reductase are directed in born of the same parents' external position of cell walls.Or final, in monocotyledons, utilize and in the seed development process, in cell type (as aleuron), express remaining stores the composition separation in Trx and/or thioredoxin reductase and the endosperm.
(ii) from thermophile, make up the expression of Trx and/or thioredoxin reductase.At room temperature (being up to 38-39 ℃ in the field) in growth course does not have or does not almost have active enzyme not too can cause problem.Therefore preferably, enzyme mainly at high temperature has activity, for example, is higher than 40 ℃, is most preferably 45-60 ℃ for wet milling process, even higher, for example, and 45-90 ℃.
(iii) Trx and/or thioredoxin reductase are placed under the inducible promoter control, chemical inducible promoter for example, the promotor of wound-induced type promotor or trans-activator-adjusting is activated behind its plant pollination through expressing trans-activator.
(iv) utilize to have the Trx that specific requirements is arranged for specific thioredoxin reductase, make Trx or thioredoxin reductase activity respectively by the acquisition of suitable Trx or thioredoxin reductase by suitable adjusting.For example, in different plants, express Trx and thioredoxin reductase, active combination only can be got in the seed behind the plant pollination through expressing complementarity (complimentary) enzyme.Perhaps, Trx or thioredoxin reductase are confined in the cell, for example in plastid, vacuole or apoplast,, make when cereal is processed, just can get as above-mentioned.Corn processing method
Therefore the present invention provides a kind of Trx and/or thioredoxin reductase of utilizing to strengthen starch isolating novel method from proteic matrix.In first embodiment, find that Trx is useful in multiple seed processed and applied, comprising wet milling process, dry grinding method, oil grain processing, soyabean processing, wheat processing and flour/dough/pasta quality, most preferably is the wet-milling of cereal, especially corn.
Therefore, the invention provides a kind of method
With improvement wet-milling efficient or increase powder process output,
With increase starch and proteinic separation efficiency,
Increasing the starch from cereal and the output of soluble protein, or
Increasing protein in water or the solvability in other solvents,
This method is included in the existence of adding Trx and/or thioredoxin reductase and floods cereal down, and separates starch and protein component in the cereal.
Usually, carry out before being immersed in powder process, but can carry out thereafter, powder process or impregnation steps more than having once in process for extracting increase the protein output from seed in steeping process or in the process behind the dipping.The Trx and/or the thioredoxin reductase of complementarity preferably, can be provided by express transgenic in by the plant of results cereal on it.
The present invention further provides:
Trx or thioredoxin reductase purposes in one approach, described method are used for improving milling efficiency or increasing starch or proteinic powder process output in for example above-mentioned arbitrary method
Steep water wherein comprises by the Trx of significant quantity and/or thioredoxin reductase to promote starch and protein separation in the cereal
Cereal, its Trx that has contacted significant quantity is to promote that starch separates with proteinic; With
Starch or protein, it is produced by aforesaid method.
Can strengthen the activity of Trx in the aforesaid method by in steep water, adding thioredoxin reductase and/or NADPH.Usually be present in other compositions that are used for wet milling process in the steep water and also can exist, as producing the bacterium of lactic acid.Preferably, be immersed under about 52 ℃ of temperature and carried out 22-50 hour, expect that therefore Trx is stable under these conditions.Cereal can be the dicotyledons seed, and for example, oil grain as soybean, Sunflower Receptacle or canola, is preferably soybean; Perhaps cereal can be the monocotyledons seed, for example corn, wheat, oat, barley, rye or rice, most preferably corn.
Trx can be the arbitrary protein that has thiol group, and it can be formed disulfide linkage by oxidation reversibly, and is reduced by NADPH in the presence of thioredoxin reductase.Preferably, Trx is derived from thermophile, as mentioned above.Being used for Trx of the present invention and/or thioredoxin reductase stably produces in a kind of microorganism through through engineering approaches (as yeast or Aspergillus (Aspergillus)), perhaps in the plant that can carry out high expression (as barley) of through engineering approaches, expressing, under for example promoters active (as high pI αDian Fenmei promotor or other Plant hormones regulators,gibberellins dependency promotors) is controlled in saccharifying.Then with Trx (be excretory or extraction form or with produce biological or its part combines) add in the steep water.
As add Trx in steep water alternative method or replenish, can in treating the seed of powder process, directly express enzyme.Preferably, in kernel maturity or controlled process process, express enzyme.
Therefore, in another embodiment, the invention provides:
It is a kind of that (as plant, the such as grain class is as barley or corn at genetically modified organism; Or in the microorganism, for example yeast or Aspergillus) in the method for industrial-scale production Trx, for example, a kind of like this method, comprise cultivating having the genetically modified organism of expressing the Trx mosaic gene, and randomly separate or extract the step of Trx.
A kind of method of utilizing transgenic plant, described transgenic plant are at the Trx of seed maturity or sprouting stage generation increasing amount, making in the seed development process or in impregnation technology influences proteinic quality in this seed by endogenous synthetic Trx, eliminates or be reduced in the needs of handling with exogenous chemical agent or enzyme before the powder process thus.
A kind of method for preparing transgenic plant, described transgenic plant are at the Trx of seed maturity or sprouting stage generation increasing amount, making in the seed development process or in impregnation technology influences proteinic quality in this seed by endogenous synthetic Trx, eliminates or be reduced in the needs of handling with exogenous chemical agent or enzyme before the powder process thus.
A kind of method that is used for cereal powder process, its utilization contains the transgenic seed of Trx, obtains the output of high starch and soluble protein.The expression in transgenic plant of Trx and thioredoxin reductase
The present invention also comprises a kind of genetically modified organism, and described biology has a kind of chimeric expression box in its genome, and described expression cassette comprises a kind of coding region of encode heat-staple Trx or thioredoxin reductase under promotor is operably controlled.
Preferably, genetically modified organism is a kind of plant, formal representation Trx and/or thioredoxin reductase that it exists with non-natural in this kind of plant, or its high level expression Trx that exists with this kind of plant non-natural.Preferably, in the seed development process, in seed, express Trx, and therefore preferred under the control of seed specific promoters.Randomly, the expression of Trx places under the control of derivable or the promotor that trans-activator is regulated, and when making expectation, can activate expression by chemical induction or with trans-activator hybridization.By with vacuole target sequence merge with Trx suitably target in the vacuole of plant.
In the present invention, Trx sequence and promoters active in plant are merged, and be transformed into nuclear gene group or plastom.Promotor is preferably the promotor of seed-specific, as γ-zein promotor.Promotor can be the promotor of chemical induction in addition, as tobacco PR-la promotor, maybe can be the promotor of the trans-activator adjusting of chemical induction, and wherein trans-activator is under the promotor control of chemical induction; But in some situation, constitutive promoter such as CaMV 35S or Gelvin promotor also can be used.The promotor that has chemical induction, the expression that is transformed into the thioredoxin gene of plant can be in due course and activate by the foliage applying chemical inducer.
Perhaps, the Trx encoding sequence and by hybridizing with this sequence plant transformed and second kind of plant of expressing trans-activator, can be realized expression under the promotor control that trans-activator is regulated.In the preferred form of present method, first kind of plant that contains the Trx encoding sequence is seed parent generation, and is male sterile, and second kind of plant of expression trans-activator is the pollina.The expression of Trx is by with first kind of plant and the second kind of plant intercropping in seed, for example make the kind of plant of winning by second kind of pollination, and, in the seed of first kind of plant, express Trx by using trans-activator to activate the promotor that trans-activator is regulated by the trans-activator genetic expression of second kind of parental generation.
Therefore the present invention provides a kind of plant of expressing Trx and/or Trx in the plant of for example Trx and/or the expression of Trx non-natural, for example, a kind of plant that contains the allogeneic dna sequence of the Trx of encoding, described dna sequence dna stably is integrated into its nuclear or plastid DNA, but preferably under a kind of inducible promoter control, for example, the chemistry inducible promoter, for example effectively be connected with inducible promoter, perhaps under the promotor control that trans-activator is regulated, wherein, corresponding trans-activator is under inducible promoter control, or in second kind of plant, express, make by hybridizing with second kind of plant, can activate promotor; Wherein Trx or thioredoxin reductase are preferably heat-staple; This kind of plant also comprises its seed, and wherein seed randomly treated (as primed or dressing) and/or packaged for example, places the bag with operation instruction, comprises the therefrom seed of results, for example is used for above-mentioned milling method.
Transgenic plant of the present invention can randomly comprise the gene that is used to strengthen thioredoxin reductase and/or NADPH generation.
The present invention also provides:
A kind of method of producing Trx comprises and cultivating as above-mentioned Trx expression plant
A kind of production pyrenoid and/or method of protein, comprise from as the seed of above-mentioned results extract starch or protein; And
A kind of wet milling process comprises the seed of expressing the plant of Trx as above-mentioned dipping, and therefrom extracts starch and/or protein.
The present invention also provides:
A kind of expression of plants box, it comprises the zone of coding Trx or thioredoxin reductase, preferably Trx is from thermophile, for example from, for example as above-mentioned from the Methanococcus jannaschii or the ancient green-ball bacterium of glimmering, wherein the coding region is preferably through optimizing to contain the codon of plant preference, described coding region is connected with terminator sequence with the promotor that function is arranged in plant, wherein promotor is preferably seed specific promoters or inducible promoter, chemical inducible promoter or the trans-activator promotor of regulating for example, it (is for example regulated by the nuclear trans-activator, be the T7 promotor when trans-activator is the T7 RNA polymerase, it is expressed randomly under inducible promoter control).
A kind of carrier, it contains effable expression cassette in this kind of plant;
This carrier plant transformed of a kind of usefulness; Or
A kind of transgenic plant for example comprise the effable expression cassette of this kind of plant in its nuclear or the plastom at its genome.
The present invention also comprises the method that a kind of production contains the cereal of high-level Trx or thioredoxin reductase, comprise to first kind of plant that contains the heterogenous expression box and pollinating with second kind of plant that contains the heterogenous expression box, expression cassette in wherein said first kind of plant contains the promotor that trans-activator is regulated, its adjusting and effectively be connected with the dna sequence dna of coding Trx and/or thioredoxin reductase, expression cassette in second kind of plant contains the promotor that effectively is connected with the dna sequence dna of coding trans-activator, and described trans-activator can be regulated the promotor that this trans-activator is regulated; This method also comprises from the plant of pollination like this and reclaims cereal.Definition
For guaranteeing the clear and consistent understanding of specification sheets and claims, provide as giving a definition:
As used herein " expression cassette " meaning be can be in vegetable cell the guiding gene dna sequence dna of expressing, comprise the promotor that is connected with the purpose coding region effectively, this coding region is connected with the terminator effectively.Encode usually target protein but also codified purpose functional r NA is for example having justice or antisense orientation to suppress the sense-rna or the untranslatable rna of the expression of specific gene (as sense-rna) of coding region.Gene can be chimeric, and the meaning is that at least a composition of gene is allogenic for the another kind of at least composition of gene.Gene is natural generation also, but obtains with recombinant forms, can be used for the gene transformation of plant.Yet generally speaking expression cassette is allogenic for the host, i.e. not natural generation in host cell of the specific dna sequence of expression cassette, and must be introduced into the ancestors of host cell or host cell by transformation event.
" nuclear expression box " is to be integrated into the expression cassette that the host examines DNA.
" plastid expression cassette " is the expression cassette that is integrated into host's plastid DNA.Plastid expression cassette described here can randomly comprise a polycistronic operon, this operon comprises the two or more purpose cistron encoding sequences under the control that is positioned at single promotor (as the promotor of trans-activator mediation), for example, one of them purpose encoding sequence coding suppresses the antisense mRNA of clpP or the expression of other plastid proteolytic enzyme, strengthens the accumulation of other encoding sequence or aim sequence expressed protein with this.
" allogenic " meaning is " being different from natural origin " as used herein.For example, if use from other biology especially from the biologically-derived gene-transformed plant of another kind, this gene is allogenic for this plant, and also is allogenic for the offspring who carries this gene of this plant.
" homoplasmon " refers to that on the wherein all plastid inheritances of a kind of plant, plant tissue or vegetable cell be identical.When plastid is transformed, when sudden change or other gene alteration, this is standard state in plant.In the different tissues or the different steps of growth, plastid can have different forms, as chloroplast(id), proplastid, etioplast, amyloplast, chromoplast or the like.
" inducible promoter " is initial promotor of transcribing just when some particular outer stimulator of plant contact only, and this is different from constitutive promoter or to particular organization or organ or special promotor of etap.Particularly preferably be chemical inducible promoter and wound-induced type promotor for the present invention.Chemical inducible promoter comprises the promotor of plant derivation, as the promotor in the tolerance approach of system's acquisition, PR promotor for example, as PR-1, PR-2, PR-3, PR-4 and PR-5 promotor, especially tobacco PR-1a promotor and Arabidopis thaliana (Arabidopsis) PR-1 promotor is when plant contact BTH their initial transcribing during with relevant chemicals.See United States Patent (USP) 5,614,395, be hereby incorporated by, and WO98/03536, be hereby incorporated by.The promotor of chemical induction type also comprises receptor-mediated system, as by other biologically-derived system that comes, as the genetic expression of steroid dependence, the genetic expression that copper relies on, the genetic expression that tsiklomitsin relies on, and the expression system of especially using fruit bat (Drisophila) usp receptor of protecting young tethelin and antagonists to mediate thereof, this has description in PCT/EP96/04224, be hereby incorporated by, and unite the system that uses acceptor, as describing among the PCT/EP96/00686, be hereby incorporated by.Wound-induced type promotor comprises the promotor of proteinase inhibitor, as the proteinase inhibitor II promotor of potato, and the promotor of the plant derivation of other participation wound response pathway, as the promotor of polyphenoloxidase, LAP and TD.Total see C.Gatz, " chemical control of genetic expression ", plant physiology molecular biology of plants annual report, (Annu.Rev.Plant Physiol.PlantMol.Biol.) (1997) 48:89-108, its content is hereby incorporated by.The promotor that trans-activator is regulated describes in detail below, and can be by a kind of plant and second kind of plant hybridization of expressing trans-activator are induced, and described plant contains the thioredoxin gene under the promotor control that trans-activator is regulated.
" isolated DNA molecule " is to exist owing to artificial participation is separated nucleotide sequence and its natural surroundings, and so non-natural product.Isolating nucleotide sequence can be pure form exist or be present in the non-natural environment, in genetically modified host cell.
" protein " is the whole protein by the corresponding nucleotide sequence encoding as described here, or by the part of a part of encoded protein matter of corresponding nucleotide sequence.
" isolating protein " is by isolating nucleotide sequence coded protein, thereby the non-natural product.Isolating protein can be pure form exist or be present in the non-natural environment, as genetically modified host cell, wherein protein is not expressed for normal time in waiting gene transgenic host cell, or reaches with multi-form or different scales.
" plant " is meant arbitrary plant, or the plant part in arbitrary etap, is plant and the vegetable material that is intended to especially contain by pulverizing, destroying or put to death, and the plant of survival, plant slitting, cell or tissue culture and seed.
DNA reorganization: DNA reorganization is meant in dna molecular and imports (preferably importing at random) sudden change or reset, or at two or be listed as between many dna moleculars the method that produces the exchange of (preferably producing at random) dna sequence dna.The dna molecular that DNA reorganization is obtained is the reorganization dna molecular that derives from the non-natural existence of a template DNA molecule at least.The enzyme of template DNA coding is reorganized the enzyme that dna molecule encode is modified, and is preferably had the biologic activity of change relatively.
The most broadly, when when this uses for nucleotide sequence, term " substantially similar " meaning is a kind of nucleotide sequence corresponding to the reference nucleotide sequence, the polypeptide that wherein corresponding sequence encoding is a kind of and reference is nucleotide sequence coded has the polypeptide of substantially the same 26S Proteasome Structure and Function, the amino acid change of polypeptide function for example, only takes place not influence.Wish ground, the similar basically nucleotide sequence coded polypeptide of nucleotide sequence coded reference.Substantially similar nucleotide sequence and be at least 80% with reference to the percentage ratio of identity between the nucleotide sequence more is at least 85% with wishing with wishing, preferably at least 90%.Use Smith-Waterman sequence contrast algorithm carry out the sequence contrast (see, for example, Waterman, M.S. " calculation biology introduction: collection of illustrative plates, sequence and genome ".Chapman﹠amp; Hall. London: 1995.ISBN 0-412-99391-0, or http://www-hto.usc.edu/software/seqaln/indes.html).LocalS program 1.16 editions is used following parameters: join in the district: 1, penalize mispairing: and 0.33, the open interval (open-gap) 2 of penalizing, interval (extended-gap) 2 is penalized in expansion.
With reference nucleotide sequence " substantially similar " nucleotides sequence be listed under the following condition can with the reference nucleotide sequence hybridization: 7% sodium lauryl sulphate (SDS), 0.5M NaPO 4, 1mMEDTA, 50 ℃, 50 ℃ of down washings, that more wish is 7% sodium lauryl sulphate (SDS), 0.5M NaPO with 2 * SSC, 0.1%SDSD 4, 1mM EDTA, 50 ℃, 50 ℃ of down washings, that more wish is 7% sodium lauryl sulphate (SDS), 0.5MNaPO with 1 * SSC, 0.1%SDSD 4, 1mM EDTA, 50 ℃, with 0.5 * SSC, 0.1%SDSD at 50 ℃ of down washing, preferably 7% sodium lauryl sulphate (SDS), 0.5M NaPO 4, 1mM EDTA, 50 ℃, with 0.1 * SSC, 0.1%SDSD at 50 ℃ of down washing, more preferably 7% sodium lauryl sulphate (SDS), 0.5M NaPO 4, 1mM EDTA, 50 ℃, with 0.1 * SSC, 0.1%SDSD 65 ℃ of down washings.
When this uses for protein, term " substantially similar " meaning is a kind of corresponding to the proteinic protein of reference, wherein this protein with substantially the same 26S Proteasome Structure and Function is arranged with reference to protein, for example, the amino acid that the polypeptide function only takes place not influence changes.When being used for protein or aminoacid sequence, the identity percentage ratio between substantially similar and reference protein or the aminoacid sequence is at least 80% with wishing.More wish ground 85%, preferably at least 90%, more preferably at least 95%, more more preferably at least 90%.
" trans-activator " is a kind of protein, by itself or with one or more other proteinic associating, transcribe the coding region that can cause being positioned under the promotor control of corresponding trans-activator mediation.The example of trans-activator system comprises phage t7 gene 10 promotors, and its activation of transcribing depends on special RNA polymerase such as phage t7 RNA polymerase.Trans-activator generally is that RNA polymerase or DNA are conjugated protein, and it can interact with specific promotor and transcribe with initial, and method is directly to activate this promotor or deactivation repressor gene, as suppressing the expression or the accumulation of aporepressor.DNA is conjugated protein can be the chimeric protein that comprises a land (as the GAL4 land) that is connected with suitable transcription activating protein structural domain.Some trans-activator system can have multiple trans-activator, for example not only needs the promotor of polysaccharase but also the specific subunit of needs (the sigma factor) for Promoter Recognition, DNA combination or transcriptional activation.Trans-activator is allogenic for plant preferably. the modification of microbial gene is to optimize the nuclear expression in plant
If wish, clone's thioredoxin gene of describing among the application can be modified being used for and be expressed the transgenic plant host.For example, derived from the transgene expression of microbe-derived gene in plant, need be modified to realize and to optimize its expression in plant these genes.Particularly, the enzyme that coding is separated but its bacterium open reading frame of in natural microbial, being encoded by same transcript, it can be expressed on the transcript of separating in plant best.For reaching this point, each microorganism open reading frame (ORF) of individual separation, and in the expression cassette of the plant transcription terminator of the plant promoter sequence that open reading frame 5 ' end is provided and open reading frame 3 ' end, clone.Isolating open reading frame sequence preferably includes initial ATG codon and stops the STOP codon, but can comprise other sequence except that initial ATG and STOP codon.In addition, open reading frame can be by brachymemma, but still keeps the activity that needs; To long especially open reading frame, the clipped form of retentive activity can be preferably used for expressing in genetically modified organism.
" plant promoter " and " plant transcription terminator " meaning is effective promotor and a transcription terminator in vegetable cell.This comprises can be from the non-plant source as virus (example comprises the octopine synthase gene of cauliflower mosaic virus or Agrobacterium Ti/Ri plasmid) deutero-promotor and transcription terminator.
In some cases, the modification of open reading frame encoding sequence and flanking sequence is unwanted, in these situations, is enough to separate fragment that comprises the purpose open reading frame and the downstream that is inserted into plant promoter.Yet preferred, should leave or remove the adjacent microorganism sequence that is connected in ATG upstream and STOP codon downstream as few as possible.In the reality, this structure depends on the operability of restriction site.
In other situation, can cause problem in the expression derived from microbe-derived expression of gene.These problems characterize in the art well, and are particularly common to particular source such as genus bacillus (Bacillus) deutero-gene.The available technology well known in the art of the modification of these genes is carried out.Following problem is the typical case that possible run into: 1. codon uses
Preferred codon uses and is different from preferred codon use in the specified microorganisms in the plant.Codon use in contrast clone's the microorganism open reading frame and the use (the especially gene of target plant) in the plant gene can be identified the codon that the open reading frame planted agent preferably changes.It is the strong preference of Nucleotide C and G that general plant evolution trends towards monocotyledonous the 3rd base position, and dicotyledons is often used Nucleotide A or T in this position.The preferred codon of particular target genetically modified organism is used to introduce by modifying factor, can overcome the problem of many GC/AT content described below and unconventional montage.2.GC/AT content
Plant gene generally has the GC content more than 35%.The open reading frame sequence that is rich in A and T Nucleotide can cause some problems in plant.At first, the primitive of ATTTA is considered to cause the destabilization of information, and is found at 3 ' end of many short-lived mRNA.Next thinks that the appearance of the interior inappropriate position polyadenylation signal AATAAA of information causes the brachymemma in advance of transcribing.In addition, monocotyledons can discern be rich in AT sequence as splice site (as follows).3. the sequence adjacent with initial methionine
Plant is different from information that the microorganism part is them does not have the ribosome bind site determined.More properly, it is believed that rrna is attached to 5 ' end of information, and scan first available ATG, begin translation at this.Yet, it is believed that the certain Nucleotide adjacent with ATG has preference, and can be by comprise the expression that total translation initiation of eucaryon strengthens microbial gene at the ATG place.Ciontech (1993/1994 catalogue, 210 pages) proposed sequence GTCGACCATGGTC (SEQ IDNo:8) are as total translation initiation of expressing intestinal bacteria uidA gene in plant.In addition, Joshi (NAR 15:6643-6653 (1987)) has contrasted many plant sequences adjacent with ATG, suggestion consensus sequence TAAACAATGGCT (SEQ ID No:9).In plant, express under the situation of meeting difficulty in the microorganism open reading frame, comprise one of these sequences at initial ATG place and can improve translation.In these situations, last three Nucleotide of consensus sequence may be not suitable for being included in because in the sequence of the modified that the modification of second AA residue causes.The preferred sequence adjacent with initial methionine can be different between different plant speciess.Investigation to 14 corn genes being arranged in the GenBank database provides following result:
Initial ATG site before in 14 corn genes
-10 -9 -8 -7 -6 -5 -4 -3 -2 -1
C 3 8 4 6 2 5 6 0 10 7
T 3 0 3 4 3 2 1 1 1 0
A 2 3 1 4 3 2 3 7 2 3
G 6 3 6 0 6 5 4 6 1 5
Can carry out this analysis to the plant species of introducing thioredoxin gene of waiting of hope, and the modification sequence adjacent with ATG is to introduce preferred Nucleotide.4. the removal of improper splice site
From non-plant source cloned genes with also may not comprise and be identified as 5 in the plant ' or the primitive of 3 ' splice site, and be cut, therefore produce information brachymemma or that lack plant, expressing the gene that is optimized.The removal in these sites can be used the technology of describing among the application WO97/02352, is hereby incorporated by.
The modification technique of encoding sequence and flanking sequence is that this area is known.If the initial expression of microorganism open reading frame is very low and think and be fit to carry out modification to above-mentioned sequence, can finish the structure of synthetic gene according to method well known in the art.For example, be described in the patent disclosure EP0 385 962 (Monsanto) that delivers, EP 0 359 472 (Lubrizol) and WO93/07278 (Ciba-Geigy).In most of examples, preferably use instantaneous measurement scheme (being well known in the art) and measure the expression of gene construct before being transferred to transgenic plant.
The major advantage that plastid transforms is that plastid generally can be expressed literalness substantially bacterial gene.Do not need as described above codon adaptability reform etc., and plastid can be expressed in a plurality of open reading frames under the single promotor control.The structure of plant conversion carrier and selected marker
Multiple conversion carrier can be used for Plant Transformation, and gene of the present invention can be used for being connected with in these carriers any.Preferred transformation technology and the target species that are used to transform are depended in the selection of used carrier.To some target species, can preferably different microbiotic or herbicide screenings.The selected marker that is generally used for transforming comprises the nptII gene, and it gives the resistance (Viera﹠amp to kantlex and associated antibiotic; Messing, gene 19:259-268, (1982); Bevan etc., nature, 304:184-187 (1983)), the bar gene, it is given the resistance of weedicide phosphinothricin (White etc., nucleic acids research 18:1062 (1990), Spencer etc., applied genetics theory (Theor.Appl.Genet.) 79:625-631 (1990)), the hpt gene, it gives the resistance (Blochinger﹠amp to antibiotic hygromycin; D:ggelmam, molecular cytobiology 4:2929-2931), the dhfr gene, it gives the resistance (Bourouis etc. to methotrexate, EMBOJ.2 (7): 1099-1104 (1983)), and the mannose-6-phosphate isomerase gene, it gives the ability of metabolism seminose, as described in US 5767378.Make up the requirement of expression of plants box
The gene order that preparation is expressed in transgenic plant at first is assemblied in the expression cassette that is arranged in after the suitable promotor and is positioned at suitable transcription terminator upstream.These expression cassettes can easily be transferred in the above-mentioned plant conversion carrier then.
1. promotor is selected
The selection of the promotor of using in expression cassette will determine genetically modified room and time expression pattern in the transgenic plant.The promotor of selecting will specific cellular type (as leaf epidermal cell, mesophyll cell, root chrotoplast) or in particular organization or organ (as root, leaf or flower) express transgenic, this selection will reflect the location of the biosynthetic hope of Trx.Among the present invention, the preferred seed specificity promoter.In addition, selected promotor can drive expression of gene under photoinduced or other instantaneous adjusting promotor.Further selecting is that selected promotor can be by the outside stimulus thing, as uses special chemical inducer or wound-induced.This will provide the possibility of only just inducing Trx to transcribe when wishing.
2. transcription terminator
Multiple transcription terminator all can use in expression cassette.They are responsible for the Transcription Termination outside transgenosis and its correct polyadenylation.Suitable comprises CaMV 35S terminator, tml terminator, nopaline synthase terminator, pea rbcS E9 terminator with the known transcription terminator that works in plant.These can be used for monocotyledons and dicotyledons.
3. strengthen or regulate the sequence of expression
Found that many sequences can strengthen the genetic expression in the transcription unit, these sequences can be used in combination with gene of the present invention to strengthen its expression in transgenic plant.
Found that multiple intron sequences can strengthen expression, especially in monocot plant cell.For example, find when introducing maize cell that the intron of corn Adhl gene significantly strengthens the expression that is positioned at the wild type gene under the homologous promoter.Find that introne 1 is effective especially, and strengthen with the fusion constructs of chloramphenicol acetyl transferasegene in expression people such as (, gene development (GenesDevelep) 1:1183-1200 (1987)) Callis.In identical experimental system, the intron of corn bronzel gene has similar effect on enhancing is expressed.Intron sequences is by conventional introduced plant conversion carrier, generally in untranslated leader.
Known some amount also can strengthen expression by viral deutero-untranslated leader, and it is especially effective in the dicotyledons cell.Particularly, tobacco mosaic virus (TMV) (TMV, " Ω-sequence "), the leader sequence of corn yellows mottle virus (MCMV) and alfalfa mosaic virus (AMV) express and proved effectively strengthening (as people such as Gallie, nucleic acids research 15:8693-8711 (1987); People such as Skuzeski, molecular biology of plants 15:65-79 (1990)).
4. the orientation of gene product in the cell
The number of mechanisms that has the directed gene product in the known plants has also characterized some details of the sequence of controlling these machining functions.The N-terminal sequence is responsible for being oriented to endoplasmic reticulum, apoplast and from aleurone cell's exocytosis (Koehler and Ho, vegetable cell 2:769-783 (1990)).In addition, amino terminal sequence associating carboxyl terminal sequence is responsible for vacuole guiding people such as (, molecular biology of plants 14:357-368 (1990)) Shinshi of gene product.By suitable targeting sequence described above and purpose transgenic sequence are merged, may guide transgene product to any organoid or cellular compartment.Selected signal sequence should comprise known cleavage site, and the syzygy of structure should consider to cut required cleavage site any amino acid afterwards.In some cases, can satisfy this needs by some amino acid that between cleavage site and transgenosis ATG, adds a spot of amino acid or replace in the transgenic sequence.
The mechanism of cell directional described above not only can be used in combination with its homologous promoter, and can be used in combination with allogeneic promoter, thereby realize the special cell directional purpose under the promoter transcription adjusting, this promotor has the expression pattern different with the promotor of the directional sign of deriving.The embodiment that expression cassette makes up
The present invention includes the expression of any thioredoxin gene under effable promotor is regulated in plant, no matter and the source of promotor how.
The present invention includes the effable promotor of any plant and any thioredoxin gene in addition and express the use of uniting of required or other sequence of selecting.These sequences include but not limited to, transcription terminator, the outside sequence (as intron [as the Adh introne 1], virus sequence [as TMV-Ω]) that strengthens expression and the sequence that is intended to gene product is oriented to specific cells device and cellular compartment.
Can use the number of chemical conditioning agent in plant transformed, to express Trx or thioredoxin reductase encoding sequence according to the present invention to induce.In the context of present disclosure, " chemical regulator " comprises known is the chemical reagent (being set forth in US 5,614,395) of the inductor of PR-1a promotor in plant, or its relevant derivative.Be used for one group of preferred conditioning agent that chemistry of the present invention can induce thioredoxin gene based on phendioxin, 2,3-thiadiazoles (BTH) structure, include but not limited to the compound of following type: phendioxin, 2,3-thiadiazoles carboxylic acid, phendioxin, 2,3-thiadiazoles thiocarboxylic acid, the cyanogen phendioxin, 2, the 3-thiadiazoles, phendioxin, 2,3-carboxylic acid amine, phendioxin, 2,3-thiadiazoles carboxylic acid hydrazides, phendioxin, 2,3-thiadiazoles-7-carboxylic acid, phendioxin, 2,3-thiadiazoles-7-thiocarboxylic acid, 7-benzonitrile-1,2, the 3-thiadiazoles, phendioxin, 2,3-thiadiazoles-7-carboxylic acid amine, phendioxin, 2,3-thiadiazoles-7-carboxylic acid hydrazides, phendioxin, 2,3-thiadiazoles alkyl carboxylates, wherein alkyl comprises one to six carbon atom, phendioxin, 2,3-thiadiazoles-7-carboxylate methyl ester, phendioxin, 2,3-thiadiazoles-7-carboxylic acid n-propyl ester, phendioxin, 2,3-thiadiazoles-7-carboxylic acid benzyl ester, phendioxin, 2,3-thiadiazoles-7-carboxylic acid sec-butyl hydrazides, and suitable derivative.Other chemical induction type thing comprises, for example, and the derivative of phenylformic acid, Whitfield's ointment (SA), polyacrylic acid and replacement thereof; Suitable substituents comprises low alkyl group, lower alkoxy, rudimentary alkylthio and halogen.But another group conditioning agent that is used for chemistry inducing DNA sequence of the present invention is based on the pyridine carboxylic acid structure, as the Yi Yansuan structure, and halo Yi Yansuan structure preferably.Preferably dichloro-isonicotinic acid and derivative thereof are as lower alkyl esters.The suitable conditioning agent of this compounds be as, 2,6-dichloro-isonicotinic acid (INA) and lower alkyl esters, especially methyl esters.
Constitutive expression: actin promoter
Several isoforms of known Actin muscle are expressed in the most cells type, so actin promoter is suitably to select as constitutive promoter.Especially, the promotor of rice ActI gene is cloned and is characterized (people such as McElroy, vegetable cell 2:163-171 (1990)).The 1.3kb fragment of finding this promotor comprises all required regulatory elements of expression in the rice protoplast.In addition, made up and manyly be used for monocotyledonous expression vector (people such as McElroy, molecular gene genetics (Mol.Gen.Genet.) 231:150-160 (1991)) specifically based on the Actl promotor.They have introduced the sequence of Actl-introne 1, Adhl 5 ' flanking sequence and (the corn alcohol dehydrogenase gene) Adhl-introne 1 and CaMV35S promotor.The carrier that shows high expression level is that 35S and Actl intron or Actl 5 ' flanking sequence and Actl include the syzygy of giving.The optimization of sequence also strengthens expression near (GUS accuses gene) initial ATG.The promoter expression cassettes that people such as McElroy (molecular gene genetics 231:150-160 (1991)) describe can be modified easily being used for the expression of thioredoxin gene, and is particularly useful among the monocotyledons host.For example, comprising this segmental promotor can remove from the McElroy structure, is used for replacing the two 35S promoters among the pCGN1761ENX, and this carrier can be used for the insertion of special gene sequence then.The fusion gene that makes up can be transferred to suitable conversion carrier then.In independent report, the paddy rice Actl promotor of also finding to have first intron has guided high expression level in the barley cell of cultivating people such as (, vegetable cell report (Plant CellRep.) 12:506-509 (1993)) Chibbar.
Constitutive expression: ubiquitin promotor
Ubiquitin is the known another kind of gene product that accumulates in many cellular types, its promotor is from several species clonings, in transgenic plant, use (as people such as Sunflower Receptacle-Binet, plant science 79:87-94 (1991), people such as corn-Christensen, molecular biology of plants 12:619-632 (1989)).The corn ubiquitin is studied in transgenosis unifacial leaf system, and its sequence and the carrier that transform to make up for monocotyledons are disclosed among the patent disclosure text EP 0 342 926.Further, people such as Taylor (vegetable cell report 12:491-495 (1993)) have described a kind of carrier (pAHC25), it comprises corn ubiquitin promotor and first intron, and when microparticle bombardment is introduced its high reactivity in many monocot plant cell suspensions.The ubiquitin promotor is suitable for expressing thioredoxin gene in transgenic plant especially monocotyledons.Suitable carriers be describe among the derivative of pAHC25 or the application pass through to introduce suitable ubiquitin promotor and/or the adorned any conversion carrier of intron sequences.
The expression that Gent is different
Another pattern of expression Trx of the present invention and thioredoxin reductase is that root is expressed, and for example, increases from root crop such as beet and Ma Lingzhu and extracts starch and sugar.A kind of suitable root promotor is described (FEBS 290:103-106 (1991)) by de Framond, also describes in disclosed patent application EP 0 452 269.This promotor is transferred to suitable carriers such as pCGN1761ENX, to be used to insert Trx or thioredoxin reductase gene and subsequently complete promotor-gene-terminator box to be transferred to the purpose conversion carrier.
Wound-induced type promotor
Wound-induced type promotor also is applicable to the expression of thioredoxin gene.Many this promotors are described (as people such as Xu, molecular biology of plants 22:573-588 (1993), people such as Logemann, vegetable cell 1:151-158 (1989), Rohrmeier and Lehle, molecular biology of plants 22:783-792 (1993), people such as Firek, molecular biology of plants 22:129-142 (1993), people such as Warner, plant magazine (Plant J.) 3:191-201 (1993)), and all be applicable to the present invention.People such as Logemann have described 5 ' upstream sequence of dicotyledonous potato Wunl gene.People such as Xu show that the wound-induced type promotor (pin2) of dicotyledons potato has activity in monocotyledon rice.Further, Rohrmeier and Lehle the wound-induced type described and can be used for using standard technique to separate the clone of the corn Wipl cDNA of homologous promoter.Similarly, people such as people such as Firek and Warner have described the wound-induced type gene of the medicinal asparagus of monocotyledons (Asparagus officinalis), and it is invaded the position in local wound and pathogenic agent and expresses.Use clone technology well-known in the art, these promotors can be transferred to suitable carriers, merge with thioredoxin gene of the present invention, and be used for expressing these genes in the plant wound site.
The expression of chloroplast(id) orientation
Chen and Jagend open reading frame (journal of biological chemistry (J.Biol.Chem.) 268:2363-2367 (1993)) have been described the successful Application chloroplast transit peptides and have been imported heterologous transgene.Employed peptide is the transit peptides (people such as Poulsen, molecular gene genetics 205:193-200 (1986)) of the rbcS gene of tobacco Nicotiana plumbaginifolia.Use restriction enzyme DraI and SphI, or Tsp509I and SphI, the dna sequence dna of this transit peptides of encoding can be downcut from plasmid prbcS-8B and through operating to use with any structure as described above.The DraI-SphI fragment is from extending to respect to-58 of initial rbcS ATG, and comprise, be right after first amino acid (also being methionine(Met)) of input cleavage site mature peptide afterwards, and the Tsp509I-SphI fragment is from extending to respect to-8 of initial rbcS ATG, and comprise first amino acid of mature peptide.Therefore, these fragments suitably can be inserted the polylinker of any selected expression cassette, generation and selected promotor are (as 35S, PR-1a, Actin muscle, ubiquitin etc.) untranslated leader merge transcribe fusion gene, and can make Trx or thioredoxin reductase gene insert the correct fusion gene in transit peptides downstream.The conversion of dicotyledons
The transformation technology that is used for dicotyledons is well known in the art, and comprises based on the technology of Agrobacterium and does not need the technology of Agrobacterium.The technology of non-Agrobacterium comprises that protoplastis or cell directly absorb exogenous genetic material.This can finish by the picked-up of PEG or electroporation mediation, the transportation or the microinjection of particle bombardment mediation.The case description of these technology is in people such as Paszkowski, the Europe molecular biosciences magazine EMBO J 3:2717-2722 of association (1984), people such as Potrykus, molecular gene genetics 199:169-177 (1985), people such as Reich, biotechnology 4:1001-1004 (1986), and people such as Klein, natural 327:70-73 (1987).In each example, use standard technique as known in the art cell transformed is regenerated as whole plants.
With its high transformation efficiency with to many widespread uses not of the same race, agriculture bacillus mediated conversion is the preferred technology that is used to transform dicotyledons.Comprise tobacco, tomato, Sunflower Receptacle, cotton, oilseed rape, grape, potato, soybean, clover and white poplar (EP 0 317 511 (cotton [1313]) with the conventional many crop varieties that transform of Agrobacterium, EP 0 249 432 (tomato), WO87/07299 (Brassica), US 4,795,855 (white poplars)).Agrobacterium-mediated Transformation generally comprises the binary vector (as pCIB200 or pCIB2001) that will carry the purpose foreign DNA and is transferred to suitable agrobacterium strains, this depend on the symbiosis Ti-plasmids or the integrity of the vir gene that carries by host's agrobacterium strains on the karyomit(e) (as be used for the CIB542 strain (people such as Uknes, vegetable cell 5:159-169 (1993)) of pCIB200 and pCIB2001.The reorganization binary vector is finished by three parent's mating methods to the transfer of Agrobacterium, uses the intestinal bacteria carry the reorganization binary vector, carries plasmid such as pRK2013 and the reorganization binary vector can be transferred to the auxiliary coli strain of target Agrobacterium strain.In addition, can transform the binary vector of to recombinate by DNA and be transferred to Agrobacterium (Hofgen and Willmitzer, nucleic acids research 16:9877 (1988)).
Generally include the common cultivation of Agrobacterium and plant explants by reorganization Agrobacterium-mediated Transformation target plant species, and according in scheme well-known in the art.Transform be organized in to have on the selection substratum of microbiotic that exists between the binary plasmid T-DNA border or Herbicid resistant mark regenerated.Also can utilize particle bombardment (biolistics) to carry out the dicotyledons conversion.Transform preferable methods for soybean and see US 5024944.Monocotyledonous conversion
The conversion of most of monocotyledons kinds has now also become routine.Preferred technology comprise use PEG or electroporation technology directly with transgenosis to protoplastis, and make alpha bombardment be transferred to callus.Available single kind DNA or multiple DNA (being cotransformation) transform, and these methods all are applicable to the present invention.The advantage of cotransformation is a vector construction of having avoided complicated, and the transgenic plant that produce the unlinked genes seat with goal gene and selected marker, can remove selected marker in filial generation subsequently if wish.Yet a shortcoming using cotransformation is that DNA kind separately is integrated into genomic frequency and is lower than 100% people such as (, biotechnology 4:1093-1096 (1986)) Schocher.
Patent application EP 0 292 435 ([1280/1281], Ciba-Geigy), EP 0 392 225 (CibaGeigy) and WO 93/07278 (Ciba-Geigy) described from a good inbred lines of corn and prepared that callus and protoplastis, use PEG or electroporation transform protoplastis and from the technology of the protoplast regeneration maize plant that transforms.People (biotechnology 8:833-839 (1990)) such as people such as Gordon-Kamm (vegetable cell 2:603-618 (1990)) and Fromm have delivered the technology that makes alpha bombardment transform A188-deutero-corn plants system.In addition, people (biotechnology 11:194-200 (1993)) such as application WO 93/07278 (Ciba-Geigy) and Koziel have described the technology through the good inbred lines of particle bombardment maize transformation.This technology is used the long immature maize of 1.5-2.5mm, and downcut from mealie in 14-15 days its pollination back, and uses PDS-1000He Biolistics device to be used for bombardment.
Carry out the conversion of paddy rice and also can use the direct gene transfer techniques of using protoplastis and particle bombardment.The existing description of conversion (people such as Zhang, the vegetable cell report 7:379-384 (1988) that are used for the protoplastis mediation of Japonica-type and lndica-type; People such as Shimamoto, natural 338:274-277 (1989); People such as Datta, biotechnology 8:736-740 (1990)).Make alpha bombardment all can conventional transform these two types people such as (, biotechnology 9:957-962 (1991)) Christou.
Patent application EP 0 332 581 (Ciba-Geigy) has described generation, conversion and the regenerating technique of Pooideae protoplastis.These technology allow the conversion of orchardgrass (Dactylis) and wheat.And, people such as Vasil (biotechnology 10:667-674 (1992)) have described the wheat of answering alpha bombardment to change the long-term renewable callus cell of C type over to and have transformed, and people (plant physiology 102:1077-1084 (1993)) such as people such as Vasil (biotechnology 11:1553-1558 (1993)) and Weeks have also described the wheat of answering alpha bombardment immature embryo and immature embryo deutero-callus and transformed.Yet one preferably is used for the wheat transformed technology, comprises by particle bombardment immature embryo transformed wheat, and comprises high-sucrose or the high malt sugar step that gene transport is preceding.Before the bombardment, any amount of embryo (long 0.75-1mm) places and contains 3% sucrose and 3mg/12, on the MS substratum of 4-D (Murashiga and Skoog, Physiologia Plantarum 15:473-497 (1962)), be used for inducing of the somatic embryo that can in the dark carry out.In selected bombardment day, shift out embryo from inducing culture, be placed on (promptly the concentration with hope generally is 15%, has added the inducing culture of sucrose or maltose) on the osmoticum.The idioplasm wall was separated 2-3 hour, then bombardment.Generally be dull and stereotyped 20 embryos of each target, although be not crucial.Precipitate the suitable plasmid that carries gene (as pCIB3064 or pSG35) on the gold particle of micron-scale with standard method.Each embryo is dull and stereotyped with device bombardment of DuPont Biolistics helium and with the explosion pressure of about 1000psi, 80 purpose sieves of use standard.After the bombardment, embryo is put back to the dark place reply about 24 hours (still on osmoticum).After 24 hours, from osmoticum, shift out embryo, it is put back to inducing culture, before regeneration, placed about one month.After about one month, the embryo explants that will contain the embryo generation callus of growth is transferred to regeneration culture medium (MS+1mg/lNAA, 5mg/lGA), it comprises appropriate selection reagent (being 10mg/lbasta, is the 2mg/l methotrexate) in addition in the situation of pSOG35 in the situation of pCIB3064.After about one month, the seedling of growing is transferred in bigger being called as " GA7 " sterile chamber, it contains the selective reagents of MS, 2% sucrose and the same concentrations of half intensity.Patent application WO94/13822 has described the method that wheat transforms, and is hereby incorporated by.Plastid transforms
The plastid transformation technology is at United States Patent (USP) 5,451, has widely in 513,5,545,817 and 5,545,818 and describes, and they all are incorporated herein by reference thus clearly comprehensively; Apply in WO95/16783 number description being arranged at PCT, be incorporated herein by reference thus comprehensively; And in people such as McBride (1994) the journal 91:7301-7305 of NAS, description is arranged, also be incorporated herein by reference at this comprehensively.It is that the clone's of selected marker plastid DNA district introduces suitable target tissue with goal gene that the basic fundamental that chloroplast(id) transforms comprises flank, as using biolistics or protoplast transformation (as the conversion of calcium chloride or PEG mediation).1 to the flanking region of 1.5kb, is called targeting sequence, has made things convenient for the homologous recombination with plastom, and therefore allows the replacement or the modification of plastom given zone.At first, point mutation in the rps12 gene of chloroplast(id) 16S rRNA and the resistance of giving spectinomycin and/or Streptomycin sulphate is used as the selected marker (Svab of conversion, second, Hajdukiewicz, P., and Maliga, P. (1990) the journal 87:8526-8530 of NAS is hereby incorporated by; Staub, J.M. and Maliga, P. (1992) vegetable cell 4,39-45 is hereby incorporated by).This has caused stable same kytoplasm transformant, and its frequency is that per 100 bombardment target leaves approximately produce a transformant.The existence of cloning site allows to be used to introduce the generation (the European molecular biosciences of P. (1993) association magazine 12,601-606 is hereby incorporated by for Staub, J.M. and Maliga) of the directed carrier of foreign gene plastid between these marks.The method that obtains the essence increase of transformation frequency is that recessive rRNA or r-albumen antibiotics resistance gene are replaced with dominant selectable marker, the coding spectinomycin separate toxenzyme aminoglycoside-3 '-the bacterium aadA gene (Svab of adenosyl transferase, Z. and Maliga, P. (1993) NAS journal 90,913-917 is hereby incorporated by).In the past, this mark successfully was used for the high frequency conversion (4083-4089 is hereby incorporated by for Goldschmidt-Clermont, M. (1991) nucleic acids research 19) of green alga Chlamydomonas reinhardtii chlamydomonas plastom.Other selected marker that is used for the plastid conversion is known in the art, and is included in the scope of the present invention.Generally, need about 15-20 cell division cycle to reach the homoplasmon state after the conversion.
By homologous recombination, the plastid that gene is inserted in all several thousand copies of the annular plastom that each vegetable cell exists is expressed, and utilizes many copy numbers of the gene that is better than nuclear expression to make expression level can surpass 10% of total solvable vegetable-protein easily.Yet this high expression level can cause potential survival problem, and is especially early stage in plant-growth and growth.Expression also causes similar problem to highly deleterious bioactive enzyme of the existence of transgenic plant or some protein, because if composing type ground is expressed and may be made it can't successful introduced plant genome.Therefore, an aspect, (US 5 the tobacco PR-1a promotor of chemical induction type in the present invention, 614,395 are incorporated herein by reference) control down in the nuclear gene group expression of the directed phage t7 RNA polymerase of chloroplast(id) report that with the chloroplast(id) that T7 gene 10 promotors/terminator sequence is regulated transgenosis combines.For example, when in nucleus, being expressed the strain system pollination of T7 polysaccharase with the plastid transformant of kytoplasm, obtain to carry two transgenic constructs but the F1 plant of not expressing gus protein for the uidA gene of coding beta-Glucuronidase (GUS) reporter gene of maternal inheritance.Only after leaf was used PR-1a inducing compounds benzo (1,2,3) thiadiazoles-7-thiocarboxylic acid S-methyl esters (BTH), the GUS's of ability a large amount of enzyme activations of initiation in the plastid of these plants was synthetic.The concise and to the point description of the sequence in the sequence table
SEQ ID NO:1 is from the protein sequence of the Trx of Methanococcus jannaschii.
SEQ ID NO:2 is from the protein sequence of the Trx of the ancient green-ball bacterium of flicker (trx-1).
SEQ ID NO:3 is from the protein sequence of the Trx of the ancient green-ball bacterium of flicker (trx-2).
SEQ ID NO:4 is from the protein sequence of the Trx of the ancient green-ball bacterium of flicker (trx-3).
SEQ ID NO:5 is from the protein sequence of the Trx of the ancient green-ball bacterium of flicker (trx-4).
SEQ ID NO:6 is from the protein sequence of the Trx of Methanococcus jannaschii (trxB).
SEQ ID NO:7 is from the protein sequence of the Trx of the ancient green-ball bacterium of flicker (trxB).
SEQ ID NO:8 Clontech sequence
SEQ ID NO:9 Joshi sequence
Embodiment
The present invention further further specifies by the embodiment of following detailed description.These embodiment only are used to the purpose illustrated, are not intended to restriction unless otherwise indicated.
Standard recombinant dna of Li Yonging and molecule clone technology are known for this area herein, for example, and people such as Sambrook, people such as (1989) molecular cloning and Ausubel, (1994) contemporary molecular biology method.Embodiment 1: with heat-staple Trx maize transformation
Expression has following sequence from the heat-staple thioredoxin gene of Methanococcus jannaschii: MSKVKIELFTSPMCPHCPAAKRVVEEVANEMPDAVEVEYINVMENPQKAMEYGIMA VPTIVINGDVEFIGAPTKEALVEAIKKRL (SEQ ID NO:1)
It is by utilize as United States Patent (USP) 5625136 described corn preference codons under seed-specific γ-zein promotor control, and utilize the expression cassette between the T-DNA border that is incorporated in plasmid pGIGUP to prepare.The T-DNA border of this plasmid is contained by the effable bar gene of the plant of ubiqutin promoters driven so that the resistance to phosphinothricin (people such as Christensen, molecular biology of plants magazine, 18:875-689,1992) to be provided.It also contains the gus gene beta-Glucuronidase that has intron in encoding sequence N-termination codon, described encoding sequence is by driving from the chimeric promoters (SMAS) of octopine and mannopine synthase gene (at the tripolymer of the octopine promotor of the bioactive sequence upstream that has mannopine synthase gene structure territory, people such as Ni, the plant magazine, 7:661-676,1995).This intron-gus gene is expressed the GUS activity in vegetable cell, but does not express in Agrobacterium (Agrobacterium).In addition, will be cloned into plasmid pNOV117 from the heat-staple Trx of Methanococcus jannaschii, it contains by the effable pmi gene of the plant of corn ubiqutin promoters driven, be used on seminose screening (people such as Christensen, 1992, people such as Joersbo, 1998).
These the experiment in utilized agrobacterium tumefaciens (A.Tumefaciens) LBA4404 (pAL4404, pSB1).PAIA404 is a kind of first (disarmed) helper plasmid that unloads.PSB1 is a kind of plasmid of wide host range, and it contains and pGIGUP and pNOV117 homologous zone and the 15.2kb KpnI fragment from the intrusion district of pTiBo542 (people such as Ishida, 1996; The efficient conversion (Zea mays L.) of the corn of agrobacterium tumefaciens mediation, Nature Journal biotechnology fascicle, 14,745-750).(pAL4404 pSB1), obtains the common integration of pGIGUP or pNOV117 and pSB1 to utilize electroporation that plasmid pGIGUP or pNOV117 are imported LBA4404.The T-DNA of pNOV117 contains by mannose-6-phosphate isomerase gene of ubiqutin promoters driven (ability of metabolism seminose is provided) and above-mentioned thioredoxin gene.
Agrobacterium on the YP substratum of having added 50mg/l spectinomycin and 10mg/l tsiklomitsin, grow 3 days (the 5g/l yeast extract, the 10g/l peptone, 5g/lNaCl, 15g/l agar, pH6.8).Collect bacterium with transfering loop, and with density range 10 9To 510 9Individual cell/ml is suspended in the N6 liquid nutrient medium.Also can from the overnight culture of YP substratum, collect agrobatcerium cell, and be suspended in the N6 liquid nutrient medium.For 1 liter of substratum, add: 4g powdery N6 salt (Sigma, St.Louis, MO), 30g sucrose, the 100mg inositol, 2mg glycine, 1mg VitB1,0.5mg pyrodoxine HCL, 0.5mg nicotinic acid, 2mg 2,4-D (from mother liquor [1mg/mL], dissolve 2 in rare KOH, 4-D makes).Be adjusted to pH6.0 with lMKOH, add 3gGelrite, steam sterilizing.
Obtained immature maize after the self-pollination in about 10-14 days.The amount of immature zygotic embryo with about 25 immature embryos of every plate is assigned in the different plates, contains in the plate and can induce and support to give birth to the substratum that the embryo callus forms.
With immature embryonic breeding with contain on the Agrobacterium kind entering plate of the Ti-plasmids that comprises a kind of selected marker thing gene, or inoculation is gone in the liquid.Before the inoculation Agrobacterium or after the inoculation, immature embryo is plated in the callus initial medium that contains Silver Nitrate (10mg/l) immediately.Place 25 immature embryos on every plate approximately.Inoculation back 16-72 hour is transferred to immature embryo in the callus initial medium that contains Silver Nitrate and cefotaxime.The following screening of carrying out transformant:
Utilize the external selection transformant of seminose.Screening can be used and is low to moderate 1g/L, after inoculation 2-20 days and keep 2-12 week altogether.So the living embryo callus that obtains is regenerated on the standard regeneration culture medium being with or without in the presence of the seminose.All plants are all used the tolerance of dichlorophenol sulfonphthalein (CR) test to seminose.This test has utilized pH sensitive indicators dyestuff to be presented at the seminose existence cell of growth down.The cell of growth produces pH change in substratum, its with indicator dichlorophenol sulfonphthalein (CR) by the red stain Huang.Expression can be differentiated in this test easily to the plant of seminose tolerance.CR test male plant by PCR identify mannose group because of existence whether.For PCR test male plant utilization Southern engram analysis.Analyze the expression of Trx in the aftergrowth.Described development of plants is normal.In small-scale wet milling process processing Analysis and Identification from the progeny plant corn cereal of high expression level plant, compare with the corn that does not have the genetically modified homologous genes type of Trx, measured the starch extractibility.The corn of expressing thioredoxin gene has shown than the obvious starch availability of higher wet milling process processing of the non-maize transformation of isotype.Heat-staple Trx of embodiment 2 usefulness and thioredoxin reductase maize transformation
Utilize the step described in the embodiment 1, corn is used as above-mentioned Trx and thioredoxin reductase cotransformation from M.jannaschii.Two genes are all under the control of the γ of seed-specific zein promotor.Two genes link to each other and between the left side and right margin of pGIGUP or pNOV117, mix possibility in the Plant Genome to strengthen two genes as single insertion fragment.
Identify whether the regenerated plant expresses Trx and thioredoxin reductase.Described development of plants is normal.In small-scale wet milling process processing Analysis and Identification from the progeny plant corn cereal of high expression level plant, compare with the corn that does not have the genetically modified homologous genes type of Trx, measured the starch extractibility.The corn of expressing thioredoxin gene has shown than the obvious starch availability of higher wet milling process processing of the non-maize transformation of isotype.
Sequence table＜110〉Novartis AG＜120〉thioredoxin and cereal processing Ver.2.2＜210 in＜130〉S-30758/A＜140〉09/213.208＜141〉1998 on December 17,＜160〉9＜170〉PatentIn〉1＜211〉85＜212〉PRT＜213〉Methanococcus jannaschii＜400〉1Met Ser Lys Val Lys Ile Glu Leu Phe Thr Ser Pro Met Cys Pro His, 15 10 15Cys Pro Ala Ala Lys Arg Val Val Glu Glu Val Ala Asn Glu Met Pro
20 25 30Asp?Ala?Val?Glu?Val?Glu?Tyr?Ile?Asn?Val?Met?Glu?Asn?Pro?Gln?Lys
35 40 45Ala?Met?Glu?Tyr?Gly?Ile?Met?Ala?Val?Pro?Thr?Ile?Val?Ile?Asn?Gly
50 55 60Asp?Val?Glu?Phe?Ile?Gly?Ala?Pro?Thr?Lys?Glu?Ala?Leu?Val?Glu?Ala?65 70 75 80Ile?Lys?Lys?Arg?Leu
85＜210〉2＜211〉119＜212〉PRT＜213〉flicker ancient green-ball bacterium＜400〉2Met Pro Met Val Arg Lys Ala Ala Phe Tyr Ala Ile Ala Val Ile Ser, 15 10 15Gly Val Leu Ala Ala Val Val Gly Asn Ala Leu Tyr His Asn Phe Asn
20 25 30Ser?Asp?Leu?Gly?Ala?Gln?Ala?Lys?Ile?Tyr?Phe?Phe?Tyr?Ser?Asp?Ser
35 40 45Cys?Pro?His?Cys?Arg?Glu?Val?Lys?Pro?Tyr?Val?Glu?Glu?Phe?Ala?Lys
50 55 60Thr?His?Asn?Leu?Thr?Trp?Cys?Asn?Val?Ala?Glu?Met?Asp?Ala?Asn?Cys?65 70 75 80Ser?Lys?Ile?Ala?Gln?Glu?Phe?Gly?Ile?Lys?Tyr?Val?Pro?Thr?Leu?Val
85 90 95Ile?Met?Asp?Glu?Glu?Ala?His?Val?Phe?Val?Gly?Ser?Asp?Glu?Val?Arg
100 105 110Thr?Ala?Ile?Glu?Gly?Met?Lys
115＜210〉3＜211〉93＜212〉PRT＜213〉flicker ancient green-ball bacterium＜400〉3Met Val Phe Thr Ser Lys Tyr Cys Pro Tyr Cys Arg Ala Phe Glu Lys, 15 10 15Val Val Glu Arg Leu Met Gly Glu Leu Asn Gly Thr Val Glu Phe Glu
20 25 30Val?Val?Asp?Val?Asp?Glu?Lys?Arg?Glu?Leu?Ala?Glu?Lys?Tyr?Glu?Val
35 40 45Leu?Met?Leu?Pro?Thr?Leu?Val?Leu?Ala?Asp?Gly?Asp?Glu?Val?Leu?Gly
50 55 60Gly?Phe?Met?Gly?Phe?Ala?Asp?Tyr?Lys?Thr?Ala?Arg?Glu?Ala?Ile?Leu?65 70 75 80Glu?Gln?Ile?Set?Ala?Phe?Leu?Lys?Pro?Asp?Tyr?Lys?Asn
85 90＜210〉4＜211〉134＜212〉PRT＜213〉flicker ancient green-ball bacterium＜400〉4Met Asp Glu Leu Glu Leu IleArg Gln Lys Lys Leu Lys Glu Met Met, 15 10 15Gln Lys Met Ser Gly Glu Glu Lys Ala Arg Lys Val Leu Asp Ser Pro
20 25 30Val?Lys?Leu?Asn?Ser?Ser?Asn?Phe?Asp?Glu?Thr?Leu?Lys?Asn?Asn?Glu
35 40 45Asn?Val?Val?Val?Asp?Phe?Trp?Ala?Glu?Trp?Cys?Met?Pro?Cys?Lys?Met
50 55 60Ile?Ala?Pro?Val?Ile?Glu?Glu?Leu?Ala?Lys?Glu?Tyr?Ala?Gly?Lys?Val?65 70 75 80Val?Phe?Gly?Lys?Leu?Asn?Thr?Asp?Glu?Asn?Pro?Thr?Ile?Ala?Ala?Arg
85 90 95Tyr?Gly?Ile?Ser?Ala?Ile?Pro?Thr?Leu?Ile?Phe?Phe?Lys?Lys?Gly?Lys
100 105 110 Pro?Val?Asp?Gln?Leu?Val?Gly?Ala?Met?Pro?Lys?Ser?Glu?Leu?Lys?Arg
115 120 125Trp?Val?Gln?Arg?Asn?Leu
130＜210〉5＜211〉105＜212〉PRT＜213〉flicker ancient green-ball bacterium＜400〉5Met Glu Arg Leu Asn Ser Glu Arg Phe Arg Glu Val Ile Gln Ser Asp, 15 10 15Lys Leu Val Val Val Asp Phe Tyr Ala Asp Trp Cys Met Pro Cys Arg
20 25 30Tyr?Ile?Ser?Pro?Ile?Leu?Glu?Lys?Leu?Ser?Lys?Glu?Tyr?Asn?Gly?Glu
35 40 45Val?Glu?Phe?Tyr?Lys?Leu?Asn?Val?Asp?Glu?Asn?Gln?Asp?Val?Ala?Phe
50 55 60Glu?Tyr?Gly?Ile?Ala?Ser?Ile?Pro?Thr?Val?Leu?Phe?Phe?Arg?Asn?Gly?65 70 75 80Lys?Val?Val?Gly?Gly?Phe?Ile?Gly?Ala?Met?Pro?Glu?Ser?Ala?Val?Arg
85 90 95Ala?Glu?Ile?Glu?Lys?Ala?Leu?Gly?Ala
100 105＜210〉6＜211〉301＜212〉PRT＜213〉Methanococcus jannaschii＜400〉6Met Ile His Asp Ihr Ile Ile Ile Gly Ala Gly Pro Gly Gly Leu Thr, 15 10 15Ala Gly Ile Tyr Ala Met Arg Gly Lys Leu Asn Ala Leu Cys Ile Glu
20 25 30Lys?Glu?Asn?Ala?Gly?Gly?Arg?Ile?Ala?Glu?Ala?Gly?Ile?Val?Glu?Asn
35 40 45Tyr?Pro?Gly?Phe?Glu?Glu?Ile?Arg?Gly?Tyr?Glu?Leu?Ala?Glu?Lys?Phe
50 55 60Lys?Asn?His?Ala?Glu?Lys?Phe?Lys?Leu?Pro?Ile?Ile?Tyr?Asp?Glu?Val?65 70 75 80Ile?Lys?Ile?Glu?Thr?Lys?Glu?Arg?Pro?Phe?Lys?Val?Ile?Thr?Lys?Asn
85 90 95Ser?Glu?Tyr?Leu?Thr?Lys?Thr?Ile?Val?Ile?Ala?Thr?Gly?Thr?Lys?Pro
100 105 110Lys?Lys?Leu?Gly?Leu?Asn?Glu?Asp?Lys?Phe?Ile?Gly?Arg?Gly?Ile?Ser
115 120 125Tyr?Cys?Thr?Met?Cys?Asp?Ala?Phe?Phe?Tyr?Leu?Asn?Lys?Glu?Val?Ile
130 135 140Val?Ile?Gly?Arg?Asp?Thr?Pro?Ala?Ile?Met?Ser?Ala?Ile?Asn?Leu?Lys145 150 155 160Asp?Ile?Ala?Lys?Lys?Val?Ile?Val?Ile?Thr?Asp?Lys?Ser?Glu?Leu?Lys
165 170 175Ala?Ala?Glu?Ser?Ile?Met?Leu?Asp?Lys?Leu?Lys?Glu?Ala?Asn?Asn?Val
180 185 190Glu?Ile?Ile?Tyr?Asn?Ala?Lys?Pro?Leu?Glu?Ile?Val?Gly?Glu?Glu?Arg
195 200 205Ala?Glu?Gly?Val?Lys?Ile?Ser?Val?Asn?Gly?Lys?Glu?Glu?Ile?Ile?Lys
210 215 220Ala?Asp?Gly?Ile?Phe?Ile?Ser?Leu?Gly?His?Val?Pro?Asn?Thr?Glu?Phe225 230 235 240Leu?Lys?Asp?Ser?Gly?Ile?Glu?Leu?Asp?Lys?Lys?Gly?Phe?Ile?Lys?Thr
245 250 255Asp?Glu?Asn?Cys?Arg?Thr?Asn?Ile?Asp?Gly?Ile?Tyr?Ala?Val?Gly?Asp
260 265 270Val?Arg?Gly?Gly?Val?Met?Gln?Val?Ala?Lys?Ala?Val?Gly?Asp?Gly?Cys
275 280 285Val?Ala?Met?Ala?Asn?Ile?Ile?Lys?Tyr?Leu?Gln?Lys?Leu
290 295 300＜210〉7＜211〉300＜212〉PRT＜213〉flicker ancient green-ball bacterium＜400〉7Met Tyr Asp Val Ala Ile Ile Gly Gly Gly Pro Ala Gly Leu Thr Ala, 15 10 15Ala Leu Tyr Ser Ala Arg Tyr Gly Leu Lys Thr Val Phe Phe Glu Thr
20 25 30Val?Asp?Pro?Val?Ser?Gln?Leu?Ser?Leu?Ala?Ala?Lys?Ile?Glu?Asn?Tyr
35 40 45Pro?Gly?Phe?Glu?Gly?Ser?Gly?Met?Glu?Leu?Leu?Glu?Lys?Met?Lys?Glu
50 55 60Gln?Ala?Val?Lys?Ala?Gly?Ala?Glu?Trp?Lys?Leu?Glu?Lys?Val?Glu?Arg?65 70 75 80Val?Glu?Arg?Asn?Gly?Glu?Thr?Phe?Thr?Val?Ile?Ala?Glu?Gly?Gly?Glu
85 90 95Tyr?Glu?Ala?Lys?Ala?Ile?Ile?Val?Ala?Thr?Gly?Gly?Lys?His?Lys?Glu
100 105 110Ala?Gly?Ile?Glu?Gly?Glu?Ser?Ala?Phe?Ile?Gly?Arg?Gly?Val?Ser?Tyr
115 120 125Cys?Ala?Thr?Cys?Asp?Gly?Asn?Phe?Phe?Arg?Gly?Lys?Lys?Val?Ile?Val
130 135 140Tyr?Gly?Ser?Gly?Lys?Glu?Ala?Ile?Glu?Asp?Ala?Ile?Tyr?Leu?His?Asp145 150 155 160Ile?Gly?Cys?Glu?Val?Thr?Ile?Val?Ser?Arg?Thr?Pro?Ser?Phe?Arg?Ala
165 170 175Glu?Lys?Ala?Leu?Val?Glu?Glu?Val?Glu?Lys?Arg?Gly?Ile?Pro?Val?His
180 185 190Tyr?Ser?Thr?Thr?Ile?Arg?Lys?Ile?Ile?Gly?Ser?Gly?Lys?Val?Glu?Lys
195 200 205Val?Val?Ala?Tyr?Asn?Arg?Glu?Lys?Lys?Glu?Glu?Phe?Glu?Ile?Glu?Ala
210 215 220Asp?Gly?Ile?Phe?Val?Ala?Ile?Gly?Met?Arg?Pro?Ala?Thr?Asp?Val?Val225 230 235 240Ala?Glu?Leu?Gly?Val?Glu?Arg?Asp?Ser?Met?Gly?Tyr?Ile?Lys?Val?Asp
245 250 255Lys?Glu?Gln?Arg?Thr?Asn?Val?Glu?Gly?Val?Phe?Ala?Ala?Gly?Asp?Cys
260 265 270Cys?Asp?Asn?Pro?Leu?Lys?Gln?Val?Val?Thr?Ala?Cys?Gly?Asp?Gly?Ala
275 280 285Val?Ala?Ala?Tyr?Ser?Ala?Tyr?Lys?Tyr?Leu?Thr?Ser
290 295 300＜210〉8＜211〉13＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: oligonucleotides＜400〉8gtcgaccatg gtc 13＜210〉9＜211〉12＜212〉DNA＜213〉artificial sequence＜220〉＜223〉artificial sequence description: oligonucleotides＜400〉9taaacaatgg ct 12

Claims (13)

1. a method that increases starch and protein separation efficient in the cereal milling method is included in Trx and/or the thioredoxin reductase existence added and floods cereal down in high temperature, and separates the protein of cereal and the step of starch component.
2. the process of claim 1 wherein that cereal comprises the cereal from transgenic plant, wherein transgene expression Trx and/or thioredoxin reductase.
3. the method for claim 2, wherein plant is selected from corn (Zea mays) and soybean.
4. plant that comprises the allogeneic dna sequence of coding Trx and/or thioredoxin reductase, described dna sequence dna stably is integrated into nuclear or the plastid DNA of plant.
5. the plant of claim 4, wherein Trx and/or thioredoxin reductase are heat-staple.
6. the plant of claim 5, wherein Trx and/or thioredoxin reductase are selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
7. each plant among the claim 4-6, wherein plant is selected from corn and soybean.
8. the effable expression cassette of a kind of plant, it contains Trx and/or the thioredoxin reductase coding region that effectively is connected with promotor that function is arranged and terminator sequence in plant.
9. the effable expression cassette of the plant of claim 8, wherein Trx and/or thioredoxin reductase are heat-staple.
10. the effable expression cassette of the plant of claim 9, wherein Trx and/or thioredoxin reductase are selected from SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6 and SEQ ID NO:7.
11. a method for preparing the cereal that comprises high-caliber Trx and/or thioredoxin reductase comprises with the expression cassette among the claim 8-10 transforming plant.
12. method for preparing the cereal that comprises high-caliber Trx and/or thioredoxin reductase, comprise to first kind of plant that contains the heterogenous expression box and pollinating with the pollen that contains the heterogenous expression box from second kind of plant, expression cassette in wherein said first kind of plant contains the promotor that trans-activator is regulated, its adjusting and effectively be connected with the dna sequence dna of coding Trx and/or thioredoxin reductase, expression cassette in second kind of plant contains the promotor that effectively is connected with the dna sequence dna of coding trans-activator, and described trans-activator can be regulated the promotor that this trans-activator is regulated; This method also comprises from the plant of pollination like this and reclaims cereal.
13. according among the claim 4-7 each plant or vegetable material as the purposes of animal-feed.
CN99814535A 1998-12-17 1999-12-15 Thioredoxin and grain processing Pending CN1330720A (en)

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US6750046B2 (en) 1991-02-22 2004-06-15 Sembiosys Genetics, Inc. Preparation of thioredoxin and thioredoxin reductase proteins on oil bodies
US7585645B2 (en) 1997-05-27 2009-09-08 Sembiosys Genetics Inc. Thioredoxin and thioredoxin reductase containing oil body based products
US6531648B1 (en) * 1998-12-17 2003-03-11 Syngenta Participations Ag Grain processing method and transgenic plants useful therein
US7179964B2 (en) 1999-03-29 2007-02-20 The Regents Of The University Of California Transgenic plants with elevated thioredoxin levels
CA2368854A1 (en) 1999-03-31 2000-10-05 Regents Of The University Of California Barley gene for thioredoxin and nadp-thioredoxin reductase
US7009087B1 (en) 2000-12-01 2006-03-07 Pioneer Hi-Bred International, Inc. Compositions and methods for altering the disulfide status of proteins
EP2278018A3 (en) * 2002-08-07 2012-01-04 BASF Plant Science GmbH Nucleic acid sequences encoding proteins associated with abiotic stress response
KR100806950B1 (en) * 2006-07-25 2008-02-22 경상대학교산학협력단 Thioredoxin reductase isotype C complex with activity of chaperone and reductase and uses thereof
EP4263850A1 (en) * 2020-12-17 2023-10-25 Novozymes A/S Improved fiber-wash in corn wet-milling

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US5792506A (en) * 1991-10-12 1998-08-11 The Regents Of The University Of California Neutralization of food allergens by thioredoxin
FR2723097B1 (en) * 1994-07-26 1996-10-18 Agronomique Inst Nat Rech THIOREDOXINS H OF SOFT WHEAT AND DURUM WHEAT AND SIMILARITY PROTEINS, ADDCODING FRAGMENTS THEREOF AND PROCESSES FOR OBTAINING SAME
AU6028099A (en) * 1998-09-08 2000-03-27 E.I. Du Pont De Nemours And Company Thioredoxin h homologs

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