CN102361988A

CN102361988A - Transgenic plants with altered redox mechanisms and increased yield

Info

Publication number: CN102361988A
Application number: CN2010800132513A
Authority: CN
Inventors: B·麦克尔西
Original assignee: BASF Plant Science Co GmbH
Current assignee: BASF Plant Science Co GmbH
Priority date: 2009-03-23
Filing date: 2010-03-17
Publication date: 2012-02-22
Also published as: AU2010227625A1; AR075918A1; EP2411524A1; MX2011009550A; DE112010001241T5; WO2010108836A1; BRPI1006259A2; AU2010227625A2; CA2754916A1

Abstract

Polynucleotides are disclosed which are capable of enhancing yield of a plant transformed to contain such polynucleotides. Also provided are methods of using such polynucleotides, and transgenic plants and agricultural products, including seeds, containing such polynucleotides as transgenes.

Description

Transgenic plant with redox mechanism and the output that improves of change

Invention field

This application requires the benefit of priority of the U.S. Provisional Patent Application series number 61/162,427 of submission on March 23rd, 2009, at this its full content is incorporated herein by reference.

Background of invention

Population increases and climate change makes the possibility of global food, feed and fuel crunch become sharp-pointed focus point in recent years.Agricultural consumes 70% human used water, and many local rainfall amounts descend in the world simultaneously.In addition, to the change of land use, the arable land of less hectare can be used for planting farm crop along with change urban district and outskirts of a town into from the farmland.Agricultural biotechnologies attempt changing to satisfy the human demand that increases through the plant genetic that can increase crop yield, for example, and through giving better to the tolerance of abiotic stress or through improving living weight.

Crop yield is defined as the bushelage of the relevant agricultural prods (like seed, army provisions or seed) of every acre of results at this.Crop yield receives the influence of abiotic stress, like arid, heat, salinity and cold coercing, and receives the influence of plant size (living weight).Traditional plant breeding strategy is slow relatively, and in giving the abiotic stress tolerance of raising, is unsuccessful usually.Grain output raising through conventional breeding in the corn has reached a plateau recently.The harvest index of corn, that is, output living weight and total ratio of accumulating living weight during results, the centuries is in the selection breeding process of grain output in the past, remains unchanged basically.Therefore, the output that in corn, takes place recently improves the result of the total biomass generation that is the raising of per unit cultivated area.Through improving the total biomass that planting density has obtained this raising, this has caused adaptive phenotype to change, and like reducing of leaf angle, this can reduce the size of blocking of bottom leaf and male flower fringe, and this can improve harvest index.

If when soil moisture exhausts, perhaps can not obtain water during a drought, crop yield is restricted.If the transpiration of leaf surpasses the water supply from root, will produce plant moisture and lack.The available water supply relates to the water yield and the plant that keep in the soil and uses its root system to obtain the ability of water.Water is relevant with the photosynthetic carbon dioxide fixation that passes through pore from the transpiration of leaf.These two process positive correlations make to flow into the loss of moist through transpiration closely related through photosynthetic high carbon dioxide.Along with water comes out from the leaf transpiration, the potential energy of Ye Shui reduces, and it is closed that pore is tending towards in the waterpower process, limits photosynthetic amount.Because the carbon dioxide fixation in the photosynthesis is depended in crop yield, so water absorbs and transpiration is the acting factor of crop yield.Can use less water to fix the carbonic acid gas of same amount or have and carry out more how photosynthetic potential, and so in many agrosystems, have and produce the more potential of multi-biomass and economic yield the plant that low water potential energy can normally act on down.

Agricultural biotechnologies have been used test in the greenhouse of model plant system, crop research and field test, demonstrate the transgenic plant of improving output to research and develop as possible, through abiotic stress tolerance raising or the living weight through improving.For example, water use efficiency (WUE) is usually relevant with drought tolerance parameter.Also utilized plant that tolerance or the resistance of plant to abiotic stress confirmed in the research of replying of drying, osmotic shock and temperature extremal.

The increase of the living weight under the low water operability possibly be because the growth efficiency that improves relatively or the water consumption of reduction cause.When selecting to be used to improve the proterties of crop, water uses and reduces, and has special value and grow to change to import in the high Irrigation farming system of cost at water.Do not need corresponding water to use the growth of rise to improve and have suitability for all agrosystems.In the unrestricted agrosystem of many water supplies, growth improves, and has also improved output even increase to cost with the water use.

Agricultural biotechnologies are also utilized the measurement of expression transgenic to other parameters of crop yield potential impact.For fodder crop, like clover, silage corn and hay, phytomass is relevant with ultimate production.Yet; For cereal crop; Used other parameters to estimate output; Like the plant size, as measured through total plant dry weight, over-ground part dry weight, over-ground part fresh weight, leaf area, the stem volume of timber, plant height, lotus throne (rosette) diameter, leaf length, root length, root amount, tiller number and the number of sheets.The plant size of early development stage is relevant with metacyclic plant size usually.The common smaller plant of big plant with big leaf area can absorb more light and carbonic acid gas, therefore will in the identical time period, obtain higher weight probably.For plant size and growth velocity, have strong genetic constitution, therefore for many different gene types, the plant size under envrionment conditions probably with another envrionment conditions under size relevant.Like this, the different and dynamic environment that uses standard environment to come the crop in the simulated farmland to run in different positions and time.

Therefore harvest index is metastable under many envrionment conditionss, and the firm dependency between plant size and the grain yield is possible.The plant size is relevant inherently with grain yield, because most of cereal living weight depends on the leaf of plant and stem is current or the photosynthesis yield-power of storage.The same as abiotic stress tolerance, under the normalization condition in growth room or greenhouse, measure the size of the plant of early development, be the standard practices method of measuring the potential production advantage of giving by genetically modified existence.

Plant can not be moved the searching energy derive or avoided preying on or coercing.Therefore, plant its environment of multiple bio-chemical pathway and network response of having evolved out is supplied with the energy of developmental plant under the varying environment condition, to keep.At these bad conditions, for example arid, extreme temperature and be exposed under the heavy metal a major challenge to plant be some meta-bolites be have highly toxic.Under the situation of oxidative stress, these toxin comprise the high activity oxygen classification (ROS) of super-oxide, superoxide, hydroxyl radical free radical, and organic derivative.ROS is to for example unsaturation lipid of organic molecule, and nucleic acid and protein have highly reactive.ROS extracts hydrogen from these organic molecules, cause the formation of reduced form oxygen (water or reduced form organic product) and second kind of organic ROS, thereby this destruction that makes chain reaction continue to cause the pair cell composition to continue is eliminated up to ROS.The removing of ROS relates to the formation of the non-activity end product that does not belong to the ROS classification.Known multiple hydrogen donor is brought into play function as the ROS scavenging agent in vegetable cell, comprise Viteolin, ascorbate salt, gsh and Trx.These different ROS scavenging agents all have 2 common characteristics; Their oxidised form can be by the reduction form of the reduction of the metabolic reaction in the cell with the scavenging agent of in circulating reaction, living again, through directly or indirectly obtaining reducing equivalent from NAD (P) H to other organic cpds anergies and said oxidised form.

Under unsuitable environmental condition, when the generation of the ROS that forms as metabolic by-prods surpasses oxidative stress appears when the plant scavenge system purifies the ability for stable end product with ROS.In order to tackle oxidative stress, vegetable cell must contain q.s can inactivation ROS scavenging agent or enzyme.In addition, cell also needs the supply of the reducing equivalent of enough NAD (P) H forms, with the scavenging agent of the activated form of living again.If one of this two deficiency, then the titre of ROS increases, and cell suffers lipid, nucleic acid or proteinic oxidative damage.Under serious situation, damage will cause necrocytosis, downright bad with lose yield-power.

In nearly all vegetable cell compartment, all detect gsh, for example tenuigenin, chloroplast(id), endoplasmic reticulum, vacuole and plastosome.Gsh is the main source of nonprotein mercaptan in the vegetable cell; The chemical reactivity of thiol group makes gsh participate in multiple biochemical function just.Gsh is water-soluble and stable, and except making the ROS detoxification, it also protects antagonism, and other coerce for example heavy metal, organic chemicals and pathogenic agent.Lyoenzyme, " classics " Selenoperoxidase is with reduced form monomer gsh and H ₂O ₂Be converted into its oxidised form, glutathione disulfide (GSSG) and H ₂O.The cellular oxidation reduction balance of cell has been indicated the GSH/GSSG ratio, there are some researches show that it relates to ROS perception and signal conduction.Second kind of form phospholipid hydroperoxide glutathione peroxidase (PHGPx) of Selenoperoxidase maybe be relevant for film.PHGPx is relevant with different functions, and for example signal conducts and cytodifferentiation, and maybe be relevant with the Trx approach.PHGPx also reduces the lipid hydroperoxide of esterification on film.Therefore, PHGPx is relevant with the snperoxiaized reparation of membrane lipid.

Gsh is also participated in glutathione (glutathionylation), and glutathione comes modifying protein through protecting specific cysteine residues to avoid irreversible oxidation, regulates some activity of proteins thus.Through glutathione isocitrate lyase is deactivated.Isocitrate lyase catalysis is from the formation of isocitric acid to succsinic acid and oxoethanoic acid, and this is the part of glyoxylate cycle, and wherein the acetyl-CoA with 2 molecules is converted into 1 succinic acid molecules.

Gsh also can be degraded through the effect of γ gamma glutamyl transpeptidase, the transfer of gamma-glutamyl part to the acceptor of gamma glutamyl transpeptidase catalysis gsh, and said receptor can be amino acid, peptide or water.Based on the homology of animal GGT, in Arabidopis thaliana, found 4 gene: GGT1, GGT2, GGT3 and GGT4.GGT1 is responsible for the activity of 80-99%, and except in seed, GGT2 is responsible for 50% activity in seed.Knocking out of GGT2 and GGT4 do not show obvious phenotypes, and after blooming, to occur too early lotus throne soon old and feeble but GGT1 knocks out.The silique number that knocks out the demonstration reduction of GGT3 and the seed production of reduction.

When atom passes through to take place when the electron-transfer reaction experience changes oxidation-reduction (redox) reaction in its oxidation state.Oxidation has been described through losing hydrogen or obtaining oxygen and has been obtained oxidation state.Reduction has been described through obtaining hydrogen or lose oxygen to lose oxidation state.Biologically, many important store energy or delivery pathways all relate to redox reaction.Cellular respiration is CO with glucose oxidase ₂, and with O ₂Be reduced to water.In photosynthesis, CO in photosystem II ₂Be reduced to sugar, H ₂O is oxidized to O ₂In photosystem I, electronic gradient is reduced to NADH with cofactor NAD+.Proton gradient produces, and drives the synthetic of ATP, and like what in respiratory chain, taken place, it pumps H+; H+ transhipment atp synthase and H+ picked-up coupling are with synthetic ATP.At non-photosynthesis biology for example in the intestinal bacteria, thus the commutative electronics of redoxomorphism and utilize hydrogen to allow anaerobic growth as energy derive, and this needs the effect of hydrogenase.

The redox state of cell mainly is reflected as the ratio of NAD+/NADH or NADP+/NADPH.This balance is reflected in metabolite for example in the amount of pyruvic acid and lactic acid.Plant-growth needs the supply of carbon, ATP, NADH and NADPH.These need be met through glycolysis-and phosphopentose pathway, and above-mentioned approach provides the oxidative pathway of the NADPH that lives again and the hexose that runs into from metabolism to produce the non-oxide approach of ribose and other pentoses.Transaldolase is the enzyme in non-oxide phosphopentose pathway, but its catalysis three carbon keto-alcohol unit are from the countertransference of sedoheptulose-7-phosphoric acid to glyceraldehyde-3-phosphate with formation E4P and fructose-6-phosphate.Transaldolase and transketolase provide the connection between glycolysis-and the phosphopentose pathway together.

The semi-lactosi metabolism has constituted the part in the cellular metabolism, and it provides glucose for fructose and seminose metabolism, nucleotide sugar metabolism and glycolysis-.The conversion of semi-lactosi to Cori ester needs the effect of 3 kinds of enzymes through the Leloir approach: galactokinase, galactose-1-phosphate uridyl transferring enzyme and UDP-semi-lactosi 4-epimerase.In the first step of approach, galactokinase uses ATP specificity phosphorylation semi-lactosi to form galactose-1-phosphate.

Relate to stress response although in plant, characterized some, the gene of water conservancy usefulness and/or living weight, up to now, the achievement of cultivating the genetically modified crops with raising output is still limited, and such plant does not have commercialization.Therefore need to identify other genes with the ability that improves crop yield.

Summary of the invention

Contriver of the present invention has found to change genetic expression relevant with the ROS scavenge system in the plant can improve plant biomass.When like target described herein, polynucleotide and polypeptide listed in the table 1 can improve the output of transgenic plant.

Table 1

In one embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The isolating polynucleotide of coding chloroplast transit peptides; Polynucleotide with separated coding total length galactokinase enzyme polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding total length transaldolase A polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of the auxiliary polypeptide of separated coding total length hydrogenase-2; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively, said promotor can improve the genetic expression in the leaf; The polynucleotide of separated coding mitochondrial transport peptide; Polynucleotide with separated coding total length isocitrate lyase polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding chloroplast transit peptides; The polynucleotide of separated coding total length phospholipid hydroperoxide glutathione peroxidase polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding total length gamma glutamyl transpeptidase polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding mitochondrial transport peptide; The polynucleotide of separated coding total length atp synthase subunit B ' polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding chloroplast transit peptides; The polynucleotide of separated coding total length C-22 sterol desaturase polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.

In further embodiment, the invention provides the seed that produces by transgenic plant of the present invention, wherein seed isozygotys for the transgenic that comprises above-mentioned expression vector.The plant that is derived from seed of the present invention has shown under normal and/or stress conditions, compares the tolerance to environment-stress of raising, and/or the plant-growth that improves, and/or the output that improves with the wild-type kind of plant.

In one aspect of the method, the present invention relates to through transgenic plant of the present invention, their plant part, their seed produce or from its product, like food, feed, food supplement, feed additive, fiber, makeup or medicine.

The present invention further provides in the table 1 the specific isolated polypeptide of identifying in the specific isolating polynucleotide identified and the table 1.The present invention also comprises the recombinant vectors that comprises the isolating polynucleotide of the present invention.

Again in another embodiment; The present invention relates to produce the method for above-mentioned transgenic plant; Wherein this method comprises with the expression vector transformed plant cells that comprises isolating polynucleotide of the present invention, and produces the transgenic plant of expressing by the polypeptide of polynucleotide encoding from vegetable cell.Polypeptide expression causes under normal and/or stress conditions in the plant, compares the tolerance to environment-stress of raising, and/or growth, and/or output with the wild-type kind of plant.

In another embodiment, the invention provides and improve the tolerance of plant again environment-stress, and/or growth, and/or the method for output.This method may further comprise the steps: with the expression cassette transformed plant cells that comprises separation polynucleotide of the present invention, and from vegetable cell generation transgenic plant, wherein these transgenic plant comprise said polynucleotide.

The accompanying drawing summary

Fig. 1 has shown b0757 by name (SEQ ID NO:2), GM59594085 (SEQ ID NO:4), the comparison of the aminoacid sequence of the galactokinase of GM59708137 (SEQ ID NO:6) and ZMBFb0152K10 (SEQ ID NO:8).Use the Align X of Vector NTI to generate comparison.

Fig. 2 has shown b2464 by name (SEQ ID NO:10), the comparison of the proteic aminoacid sequence of transaldolase A of BN43182918 (SEQ ID NO:12) and GM48926546 (SEQ ID NO:14).Use the Align X of Vector NTI to generate comparison.

Fig. 3 has shown YIR037W by name (SEQ ID NO:20); BN42261838 (SEQ ID NO:22), BN43722096 (SEQ ID NO:24), BN51407729 (SEQ ID NO:26); GM50585691 (SEQ ID NO:28); GMsa56c07 (SEQ ID NO:30), GMsp82f11 (SEQ ID NO:32), GMs s66f03 (SEQ ID NO:34); HA03MC1446 (SEQ ID NO:36); HV03MC9784 (SEQ ID NO:38), OS34914218 (SEQ ID NO:40), the comparison of the aminoacid sequence of the phospholipid hydroperoxide glutathione peroxidase of ZM61990487 (SEQ ID NO:42) and ZM68466470.r01 (SEQ ID NO:44).Use the Align X of Vector NTI to generate comparison.

Fig. 4 has shown the comparison of the proteinic aminoacid sequence of atp synthase subunit B ' of SLL1323 by name (SEQ ID NO:48) and Gmsb 38b04 (SEQ ID NO:50).Use the Align X of Vector NTI to generate comparison.

Fig. 5 has shown the comparison of aminoacid sequence of the C-22 sterol desaturase of YMR015C by name (SEQ ID NO:52) and GMso65h07 (SEQ ID NO:54).Use the AlignX of Vector NTI to generate comparison.

Description of Preferred Embodiments

Run through this application, with reference to various publications.The disclosure of those reference of quoting in all these publications and these publications is all introduced among the application as a reference at this, more fully to describe the situation in field according to the invention.Just in order to describe the purpose of particular, and plan restriction at this used term.As used at this, " one (a) " or " one (an) " can represent one or more, and this will depend on the content of using it.Therefore, for example, mention that " cell (a cell) " can represent to use at least one cell.

In one embodiment, the invention provides the transgenic plant that cross the isolating polynucleotide shown in the expression table 1 in said subcellular compartment and the tissue.Transgenic plant of the present invention have shown the output of comparing raising with the wild-type kind of plant.As used at this, term " output of the raising " meaning is any raising of plant prod (like seed, fruit or the fiber) output of any measurement.According to the present invention, the variation of different phenotypic characters can improve output.For example (but and unrestricted); Parameter tolerance, leaf one-tenth, phototaxis, apical dominance and the fruit development of coercing like development of floral organs, root of hair, root living weight, seed number, seed weight, harvest index, to abiotic environment all is the appropriate measurements that improves output.Any raising of output is the output according to raising of the present invention.For example, the raising of output can comprise 0.1%, 0.5%, 1%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or higher increase of any measuring parameter.For example; With compare from the bushel/acre of be untreated soybean or corn of cultivation under the same conditions; Being derived from the increase of the bushel/acre of the genetically modified plant of soybean comprise its crop that is to(for) the Nucleotide and the polypeptide of table 1 or corn, is the output according to raising of the present invention.

As in this qualification, " transgenic plant " thus be to use recombinant DNA technology to change to contain otherwise be not present in the plant of the isolating nucleic acid in the plant.As used at this, term " plant " comprises complete plant, vegetable cell and plant part.Plant part includes, but not limited to stem, root, ovule, stamen, leaf, embryo, meristem zone, callus, gametophyte, sporophyte, pollen, sporule etc.Transgenic plant of the present invention can be male sterile or male-fertile, and may further include those transgenics that other are not included in these described isolating polynucleotide.

As used at this, term " kind " refers to one group of plant of enjoying common trait in the species, these common traits with they with canonical form and these species in other possible kinds separate.Although have at least a difference proterties, the characteristic of kind is that also between the intravarietal individuality some change, and this Mendelian who mainly is based on proterties among the offspring that success goes down to posterity separates.If the homozygote of proterties to following degree in the heredity: when the kind of isozygotying when body is pollinated; Do not observe the independent separate (independent segregation) of this proterties significant amounts among the offspring, think that then kind is " isozygotying " for this specific proterties.In the present invention, proterties originates from the transgene expression of the one or more separation polynucleotide in the introduced plant kind.As still in this use; Term " wild-type kind " refers to one group of plant analyzing in order to compare purpose as control plant; Wherein wild-type article kind of plant is identical with transgenic plant (using according to isolating polynucleotide plant transformed of the present invention), except wild-type article kind of plant isolating polynucleotide of the present invention of no use transform.Do not use isolating polynucleotide according to the present invention altered vegetable cell, seed, plant constituent, plant tissue, plant organ or complete plant in heredity as referring at this used term " wild-type ".

Like the purpose of enhanced phenotype in this used term " control plant " refers to for the plant of identifying transgenic or hereditary change or required proterties, be used for vegetable cell, explant, seed, plant constituent, plant tissue, plant organ or complete plant that the plant of relative transgenic or hereditary change compares.In some cases, " control plant " can be the transgenic plant system that comprises empty carrier or marker gene, but do not contain the target recombination of polynucleotide in the plant that is present in transgenic to be evaluated or hereditary change.Control plant can be the plant of the kind identical with the plant of transgenic to be detected or hereditary change system or kind, maybe can be that another kind is or kind, like known plant with particular phenotype, characteristic or known type.Suitable control plant will comprise the hereditary unaltered or not genetically modified plant of the parental line of the transgenic plant that are used to be created in this.

As in this qualification, term " nucleic acid " and " polynucleotide " can exchange, and refer to straight or branched, strand or double-stranded RNA or DNA, or its hybrid.Term also comprises the RNA/DNA hybrid." isolating " nucleic acid molecule is other nucleic acid molecule of existing in the natural origin with the nucleic acid sequence of other polypeptide (that is, encode) isolated nucleic acid molecule basically.For example, think that the nucleic acid of cloning is isolating.If change, or place locus or the position that is not natural site, or be introduced in the cell, think that also nucleic acid is isolating through conversion through human intervention.In addition, isolated nucleic acid molecule (like the cDNA molecule) can not have natural other cell materials that link to each other of some and its, or the substratum when producing through recombinant technology, or precursor or other chemical during chemosynthesis.Although can choose the untranslated sequence that comprises 3 ' and the 5 ' end that is positioned at gene coding region wantonly, preferably remove the native sequences of both sides, coding region in the replicon of natural generation.

As used at this, term " environment-stress " refers to and salinity, arid, nitrogen, temperature, metal, chemical, morbific or oxidative stress, or the relevant non-top condition of its arbitrary combination.As used at this, term " arid " refers to and can be used for wherein supporting that the water yield of plant-growth or growth is lower than the envrionment conditions of optimum water.As used at this, term " fresh weight " refers to all materials in the plant, comprises water.As used at this, term " dry weight " refers to the material of all except water in the plant, and comprises, for example, and glucide, protein, oil and mineral nutrient.

Any plant species can transform formation according to transgenic plant of the present invention.Transgenic plant of the present invention can be dicotyledons or monocotyledons.For example but and unrestricted, transgenic plant of the present invention can be derived from any of following dicotyledons section: pulse family comprises the plant as pea, clover and soybean; Umbelliferae comprises the plant as Radix Dauci Sativae and celery; Solanaceae comprises the plant as tomato, yam, eggplant, tobacco and pepper; Cruciferae, particularly Btassica, it comprises the plant as rape, beet, Caulis et Folium Brassicae capitatae, Cauliflower and sprouting broccoli; And Arabidopis thaliana; Composite family, it comprises the plant as lettuce; Malvaceae, it comprises cotton; Pulse family, it comprises such plants such as peanut.Transgenic plant of the present invention can be derived from monocotyledons, as, for example, wheat, barley, Chinese sorghum, broomcorn millet, rye, triticale (triticale), corn, rice, oat and sugarcane.Transgenic plant of the present invention also are embodied in tree; Like apple, pears, Wen Bai (quince), Lee, cherry, peach, nectarine, apricot, papaya, mango and other woody kinds; Comprise coniferals and fallen leaves trees, like poplar, pine, Chinese larch, cdear, Oak Tree etc.Especially preferred is that Arabidopis thaliana (A.thaliana), tobacco (Nicotiana tabacum), rice, rape, Kano draw (canola), soybean, corn (corn) (corn (maize)), cotton and wheat.

In one embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding chloroplast transit peptides; Polynucleotide with separated coding total length galactokinase enzyme polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.Like what in following examples 2, prove, the transgenic arabidopsis plant that contains the bacillus coli gene b0757 (SEQ ID NO:1) of target chloroplast(id) is compared the output that has proved raising with the contrast arabidopsis thaliana.B0757 genes encoding galactokinase and be Partial Feature to have characteristic sequence GHMP_ kinases _ C (Pfam:PF08544) and GHMP_ kinases _ N (PF00288).In Fig. 1 listed galactokinase zymoprotein illustrated such characteristic sequence.

The transgenic plant of this embodiment possibly comprise any polynucleotide of coding galactokinase enzyme polypeptide.Preferably; The transgenic plant of this embodiment comprise the polynucleotide that coding has the full-length polypeptide of galactokinase activity; Wherein said polypeptide comprises and is selected from GHMP_ kinases _ C and GHMP_ kinases _ N characteristic sequence at least one characteristic sequence in the two, and wherein GHMP_ kinases _ C characteristic sequence is selected from the 278th to 362 amino acids of SEQ ID NO:2; The the 378th to 426 amino acids of SEQ ID NO:4; The the 326th to 404 amino acids of SEQ ID NO:6; The the 391st to 473 amino acids with SEQ ID NO:8; Wherein GHMP_ kinases _ N characteristic sequence is selected from the 114th to 182 amino acids of SEQ ID NO:2; The the 152nd to 219 amino acids of SEQ ID NO:4; The the 138th to 205 amino acids of SEQ ID NO:6; The the 159th to 226 amino acids with SEQ ID NO:8.Preferably said polypeptide comprise GHMP_ kinases _ C characteristic sequence and GHMP_ kinases _ N characteristic sequence the two.Most preferably, the transgenic plant of this embodiment comprise coding and have the 1st to 382 amino acids that is selected from SEQ ID NO:2; The the 1st to 460 amino acids of SEQ ID NO:4; The the 1st to 431 amino acids of SEQ ID NO:6; Polynucleotide with the galactokinase enzyme polypeptide of the sequence of the 1st to 504 amino acids of SEQ ID NO:8.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding total length transaldolase A polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.Like proof in following examples 2, the transgenic arabidopsis plant and the transgenic plant of this embodiment that contain the bacillus coli gene b2464 (SEQ ID NO:9) of coding transaldolase A polypeptide are compared the output that has proved raising with the contrast arabidopsis thaliana.Transaldolase A polypeptide is a Partial Feature there to be transaldolase (PF00923) characteristic sequence.In Fig. 2 listed transaldolase A protein illustrated such characteristic sequence.

The transgenic plant of this embodiment possibly comprise the proteinic any polynucleotide of coding transaldolase A.Preferably, the transgenic plant of this embodiment comprise the polynucleotide that coding has the active full-length polypeptide of transaldolase A, and wherein said polypeptide comprises the 12nd to 312 amino acids that is selected from SEQ ID NO:10; The the 1st to 275 amino acids of SEQ ID NO:12; Transaldolase characteristic sequence with the 1st to 277 amino acids of SEQ ID NO:14.Most preferably, the transgenic plant of this embodiment comprise coding and have the 1st to 316 amino acids that is selected from SEQ ID NO:10; The the 1st to 284 amino acids of SEQ ID NO:12; Polynucleotide with the transaldolase A polypeptide of the sequence of the 1st to 283 amino acids of SEQ ID NO:14.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of the auxiliary polypeptide of separated coding total length hydrogenase-2; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.Like what in following examples 2, prove, the transgenic arabidopsis plant that contains bacillus coli gene b2990 (SEQ ID NO:15) is compared the output that has proved raising with the contrast arabidopsis thaliana.B2990 genes encoding hydrogenase-2 accessory protein.Under anaerobic, this protein is chaperone appearance protein in intestinal bacteria, and it is that to produce active hydrogenase 2 necessary, and hydrogenase 2 is [NiFe] picked-up hydrogenases, its with hydrogenase 1 with H ₂Oxidation and the coupling mutually of reduction fumaric acid.Hydrogenase-2 accessory protein is a Partial Feature there to be HupF-HypC (PF01455) characteristic sequence.

The transgenic plant of this embodiment can comprise any polynucleotide of coding hydrogenase-2 accessory protein.Preferably, the transgenic plant of this embodiment comprise the polynucleotide that coding has the active full-length polypeptide of hydrogenase assembling chaperone, and wherein said polypeptide comprises the 1st to 79 the amino acid whose HupF_HypC characteristic sequence of SEQ ID NO:16.Most preferably, the transgenic plant of this embodiment comprise the polynucleotide that coding has hydrogenase-2 accessory protein of the 1st to 82 the amino acid whose sequence that comprises SEQ ID NO:16.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively, said promotor can improve the genetic expression in the leaf; The polynucleotide of separated coding mitochondrial transport peptide; Polynucleotide with separated coding total length isocitrate lyase polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.Like what in following examples 2, prove, to compare with the contrast arabidopsis thaliana, the transgenic arabidopsis plant that contains the genes of brewing yeast YER065C (SEQ ID NO:17) of the mitochondrial coding isocitrate lyase of target has proved the output that improves.Isocitrate lyase is a Partial Feature there to be ICL (PF00463) characteristic sequence.

The transgenic plant of this embodiment possibly comprise any polynucleotide of the isocitrate lyase of encoding.Preferably, the transgenic plant of this embodiment comprise the polynucleotide that coding has the active full-length polypeptide of isocitrate lyase, and wherein said polypeptide comprises the ICL characteristic sequence of the 22nd to 550 amino acids that contains SEQ ID NO:18.Most preferably, the transgenic plant of this embodiment comprise the polynucleotide of isocitrate lyase that coding has the sequence of the 1st to 557 amino acids that comprises SEQ ID NO:18.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding chloroplast transit peptides; The polynucleotide of separated coding total length phospholipid hydroperoxide glutathione peroxidase polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.Like what in following examples 2, prove, the transgenic arabidopsis plant that contains the genes of brewing yeast YIR037W (SEQ ID NO:19) of target chloroplast(id) is compared the output that has proved raising with the contrast arabidopsis thaliana.YIR037W genes encoding phospholipid hydroperoxide glutathione peroxidase albumen, it serves as transmitter and the redox signal of the cell endoperoxide level transducer to transcription factor Yap1, and Yap1 regulates the peroxide level in the yeast saccharomyces cerevisiae.Phospholipid hydroperoxide glutathione peroxidase is a Partial Feature there to be GSHPx (PF00255) characteristic sequence of representing the Selenoperoxidase family gene.In Fig. 3 listed phospholipid hydroperoxide glutathione peroxidase illustrated such characteristic sequence.

The transgenic plant of this embodiment possibly comprise any polynucleotide of the phospholipid hydroperoxide glutathione peroxidase of encoding.Preferably, the transgenic plant of this embodiment comprise the polynucleotide that coding has the active full-length polypeptide of phospholipid hydroperoxide glutathione peroxidase, and wherein said polypeptide comprises the 4th to 111 amino acids that is selected from SEQ ID NO:20; The the 10th to 118 amino acids of SEQ ID NO:22; The the 37th to 145 amino acids of SEQ ID NO:24; The the 9th to 117 amino acids of SEQ ID NO:26; The the 9th to 117 amino acids of SEQ ID NO:28; The the 9th to 117 amino acids of SEQ ID NO:30; The the 12nd to 120 amino acids of SEQ ID NO:32; The the 12nd to 120 amino acids of SEQ ID NO:34; The the 11st to 119 amino acids of SEQ ID NO:36; The the 12nd to 120 amino acids of SEQ ID NO:38; The the 9th to 117 amino acids of SEQ ID NO:40; The the 12nd to 120 amino acids of SEQ ID NO:42; GSHPx characteristic sequence with the 24th to 132 amino acids of SEQ ID NO:44.Most preferably, the transgenic plant of this embodiment comprise coding and have the 1st to 163 amino acids that is selected from SEQ ID NO:20; The the 1st to 169 amino acids of SEQ ID NO:22; The the 1st to 201 amino acids of SEQ ID NO:24; The the 1st to 169 amino acids of SEQ ID NO:26; The the 1st to 166 amino acids of SEQ ID NO:28; The the 1st to 166 amino acids 0 of SEQ ID NO:3; The the 1st to 170 amino acids of SEQ ID NO:32; The the 1st to 170 amino acids of SEQ ID NO:34; The the 1st to 185 amino acids of SEQ ID NO:36; The the 1st to 176 amino acids of SEQ ID NO:38; The the 1st to 166 amino acids of SEQ ID NO:40; The the 1st to 170 amino acids of SEQ ID NO:42; Polynucleotide with the phospholipid hydroperoxide glutathione peroxidase of the sequence of the 1st to 182 amino acids of SEQ ID NO:44.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding total length gamma glutamyl transpeptidase polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.Randomly, said expression cassette also comprises the polynucleotide of separated coding chloroplast transit peptides, and it effectively links to each other with the polynucleotide of separated coding promotor and the polynucleotide of separated coding total length gamma glutamyl transpeptidase polypeptide.Like what in following examples 2, prove, the transgenic arabidopsis plant that contains the synechocystis gene slr1269 (SEQ ID NO:45) of coding gamma glutamyl transpeptidase polypeptide is compared the output that has proved raising with the contrast arabidopsis thaliana.Gamma glutamyl transpeptidase is a Partial Feature there to be G_glu_transpept (PF01019) characteristic sequence.

The transgenic plant of this embodiment possibly comprise any polynucleotide of the gamma glutamyl transpeptidase of encoding.Preferably; The transgenic plant of this embodiment comprise the polynucleotide that coding has the active full-length polypeptide of gamma glutamyl transpeptidase, and wherein said polypeptide comprises the G_glu_transpept characteristic sequence of the 21st to 511 amino acids that contains SEQ ID NO:46.Most preferably, the transgenic plant of this embodiment comprise the polynucleotide of gamma glutamyl transpeptidase that coding has the sequence of the 1st to 518 amino acids that comprises SEQ ID NO:46.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding mitochondrial transport peptide; The polynucleotide of separated coding total length atp synthase subunit B ' polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.Like what in following examples 2, prove, the transgenic arabidopsis plant that contains the mitochondrial synechocystis gene of target SLL1323 (SEQ ID NO:47) is compared the output that has proved raising with the contrast arabidopsis thaliana.SLL1323 genes encoding atp synthase subunit B ' albumen.Subunit B and B ' come in the comfortable chloroplast(id) and the F0 mixture in the F-ATP enzyme of in the bacterium plasma membrane, finding, and form the part of the peripheral shaft (peripheral stalk) that F1 and F0 mixture are linked together.Atp synthase subunit B ' albumen is Partial Feature there to be ATP-synt_B (PF00430) characteristic sequence of representing atp synthase B/B ' CF (0) family gene.Atp synthase subunit B ' the albumen example of listing at Fig. 4 such characteristic sequence.

The transgenic plant of this embodiment possibly comprise the proteic any polynucleotide of coding atp synthase subunit B '.Preferably; The transgenic plant of this embodiment comprise coding and have the polynucleotide of the active full-length polypeptide of atp synthase subunit B ', and wherein said polypeptide comprises the ATP-synt_B characteristic sequence of the 82nd to 213 amino acids of the 7th to 138 amino acids that is selected from SEQ ID NO:48 and SEQ ID NO:50.Most preferably, the transgenic plant of this embodiment comprise the proteic polynucleotide of atp synthase subunit B ' of sequence that coding has the 1st to 215 amino acids of the 1st to 143 amino acids that comprises SEQ ID NO:48 and SEQ ID NO:50.

In another embodiment, the invention provides the polynucleotide of using expression cassette transgenic plant transformed, this expression cassette to comprise the separated coding promotor that links to each other effectively; The polynucleotide of separated coding chloroplast transit peptides; The polynucleotide of separated coding total length C-22 sterol desaturase polypeptide; Wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and have shown the output that improves.Gene YMR015C (SEQ ID NO:51) coding C-22 sterol desaturase, this is a kind of cytochrome P 450 enzymes (ERG5), the formation of the two keys of C-22 (23) in the sterol side chain in the biosynthesizing of catalysis ergosterol in yeast.C-22 sterol desaturase is with near the K-spiral motif (xExxR) around the protoporphyrin IX heme halfcystine part of C-end, PERF consensus sequence (PxRx) and FGRCG motif exist for Partial Feature.The C-22 sterol desaturase polypeptide illustrated of in Fig. 5, listing so conservative motif.

The transgenic plant of this embodiment possibly comprise any polynucleotide of coding C-22 sterol desaturase.Preferably; The transgenic plant of this embodiment comprise the polynucleotide that coding has the active full-length polypeptide of C-22 sterol desaturase; Wherein said polypeptide comprises the structural domain that contains K-spiral motif, PERF consensus sequence and FGRCG motif, and wherein K-spiral motif has the sequence of the 365th to 368 amino acids of the 395th to 398 amino acids that is selected from SEQ ID NO:52 and SEQ ID NO:54; The PERF motif has the sequence of the 418th to 421 amino acids of the 450th to 453 amino acids that is selected from SEQ ID NO:52 and SEQ ID NO:54; Has the sequence of the 438th to 447 amino acids of the 469th to 478 amino acids that is selected from SEQ ID NO:52 and SEQ ID NO:54 with the FGRCG motif.More preferably, said polynucleotide encoding has the active full-length polypeptide of C-22 sterol desaturase, and wherein said polypeptide comprises the structural domain of the 27th to 498 amino acids of the 61st to 529 amino acids that is selected from SEQ ID NO:52 and SEQ ID NO:54.Most preferably, the transgenic plant of this embodiment comprise the polynucleotide of coding C-22 sterol desaturase, and said C-22 sterol desaturase comprises the 1st to 538 amino acids of SEQ ID NO:52 and the 1st to 513 amino acids of SEQ ID NO:54.

It is the seed that isozygotys that the present invention further provides for said expression cassette (being also referred to as " transgenic " at this), and the transgenic plant that wherein obtain from said seed plantation are compared with the plant of wild-type kind and shown the output that improves.The present invention also provides the product that is produced by the transgenic plant of expressing polynucleotide, its plant part or its seed or produce from transgenic plant, its plant part or its seed of expressing polynucleotide.Can use the whole bag of tricks well known in the art to obtain product.As used at this, word " product " includes, but not limited to food, feed, food supplement, feed additive, fiber, makeup or medicine.Food thought to be used to the compsn that nutrition is provided or is used to supplement the nutrients.Especially, animal-feed and animal-feed supplement are thought food.The present invention further provides the agricultural-food that produce through any transgenic plant, plant part and plant seed.Agricultural-food include, but not limited to plant milk extract, protein, amino acid, glucide, fat, oil, polymkeric substance, VITAMINs etc.

The present invention also provides isolating polynucleotide, and it has the SEQ of being selected from ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:49; Sequence with SEQ ID NO:53.The isolating polynucleotide of the present invention also comprise the separation polynucleotide of coded polypeptide, and polypeptide has the SEQ of being selected from ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:50; Aminoacid sequence with SEQ ID NO:54.Can use the standard molecule biotechnology and, for example, use the robotization dna synthesizer, separate polynucleotide of the present invention at this sequence information that provides.

Isolating polynucleotide of the present invention comprise the homologue of the polynucleotide of table 1." homologue " is defined as two nucleic acid or the polypeptide that has similar or substantially the same Nucleotide or aminoacid sequence separately at this.Homologue comprises allele variant, analogue or directly to homologue, as with delimit.As used at this, term " analogue " refers to two nucleic acid with same or similar function, but in incoherent organism, separately evolves.As used at this, term " directly to homologue " refers to two nucleic acid from different plant species, evolves but formed from total ancestral gene through species.The term homologue further comprise be different from the table 1 nucleotide sequence that shows once because genetic code degeneracy and the nucleic acid molecule of the phase homopolypeptide of therefore encoding.

In order (for example to measure two aminoacid sequences; One of nucleotide sequence of table 1 and homologue thereof) per-cent sequence identity; For the best compares purpose than right sequence (for example, can breach be introduced in the sequence of a polypeptide, be used for the best comparison with other polypeptide or nucleic acid).The amino-acid residue of more corresponding amino acid position then.When the position in the sequence by another sequence in the same amino acid of corresponding position when occupying, then molecule is identical in this position.Can between two nucleotide sequences, carry out the comparison of same type.

Preferably, isolating amino acid homologue, analogue and the straight whole piece aminoacid sequence of in homologue and table 1, identifying have at least about 50-60%, preferably at least about 60-70%; More preferably at least about 70-75%, 75-80%, 80-85%; 85-90% or 90-95% are most preferably at least about 96%, 97%; 98%, 99% or higher identity.In a further preferred embodiment, isolating nucleic acid homologue of the present invention comprise with table 1 in the nucleotide sequence that shows have at least about 40-60%, preferably at least about 60-70%; More preferably at least about 70-75%, 75-80%, 80-85%; 85-90% or 90-95%, even more preferably at least about 95%, 96%; 97%, 98%, 99% or the nucleotide sequence of higher identity.

For the object of the invention; Use Align 2.0 (Myer s and Miller; CABIOS (1989) 4:11-17) all parameter settings of the default situation of use, or Vector NTI 9.0 (PC) software package (Introgen, 1600 Faraday Ave.; Carl sbad CA92008) has measured per-cent sequence identity between two nucleic acid or the peptide sequence.For the per-cent identity of calculating with Vector NTI, the breach that the breach of use 15 is opened punishment and 6.66 extends punishment, the per-cent identity of two nucleic acid of mensuration.The breach that the breach of use 10 is opened punishment and 0.1 extends punishment, is used to measure the per-cent identity of two polypeptide.Every other parameter setting is a default settings.For the purpose (Clustal W algorithm) of a plurality of comparisons, the open punishment of breach is 10, and breach extends to punish to be 0.05, use blosum62 matrix.Should understand when dna sequence dna is compared with the RNA sequence, in order to measure the purpose of sequence identity, thymidylic acid and uridylate are of equal value.

Can be based on the identity of they and said polypeptide; Use encode polypeptide separately or based on its polynucleotide of primer; As according to the hybridization probe of standard hybridization technique under rigorous hybridization conditions, separate homologue, analogue and straight nucleic acid molecule to homologue corresponding to polypeptide listed in the table 1.As used at this, for the hybridization of DNA and southern blotting technique, term " rigorous condition " refers to the solution at 10 * Denhart ' s, and 6 * SSC is hybridized in 0.5%SDS and the 100 μ g/ml denatured salmon sperm dnas and spent the night.Trace was washed 30 minutes down at 62 ℃ according to the order of sequence, in 3 * SSC/0.1%SDS, follow 1 * SSC/0.1%SDS at every turn, and last 0.1 * SSC/0.1%SDS.In preferred embodiments, like what also use at this, phrase " rigorous condition " refers to is hybridizing in 6 * SSC solution under 65 ℃.In another embodiment, " high rigorous condition " refers to the solution at 10 * Denhart ' s, and 6 * SSC is hybridized in 0.5%SDS and the 100 μ g/ml denatured salmon sperm dnas and spent the night.Trace was washed 30 minutes down at 65 ℃ according to the order of sequence, in 3 * SSC/0.1%SDS, follow 1 * SSC/0.1%SDS at every turn, and last 0.1 * SSC/0.1%SDS.The method that is used to carry out nucleic acid hybridization is well known in the art.

Can optimize isolating polynucleotide used among the present invention, that is, genetically engineeredly improve it in given plant or the expression in the animal.Nucleic acid in order to provide plant to optimize can change over the dna sequence dna of gene: 1) comprise by the preferred codon of the plant gene of high expression level; 2) comprise the A+T content that the nucleotide base in plant, found is basically formed; 3) form the plant homing sequence; 4) elimination causes RNA instability, unsuitable polyadenylation, degraded and terminated sequence, or forms the sequence of secondary hairpin structure or RNA splice site; Or 5) eliminate the antisense ORFs.Can use distribution frequency to realize the expression of nucleic acid that improves in the plant through utilizing the codon in general plant or the specified plant.The method of optimizing the expression of plant amplifying nucleic acid can be at EPA0359472; EPA0385962; PCT applies for No.WO91/16432; U.S. patent No.5,380,831; U.S. patent No.5,436,391; Perlack etc., 1991, Proc.Natl.Acad.Sci.; USA88:3324-3328; With Murray etc., 1989, Nucleic Acids Res.17:477-498.

The present invention also provides recombinant expression vector, and it comprises and is selected from following expression cassette: a) expression cassette, and it comprises the polynucleotide of the separated coding promotor that links to each other effectively; The polynucleotide of separated coding chloroplast transit peptides; Polynucleotide with separated coding total length galactokinase enzyme polypeptide; B) expression cassette, it comprises the polynucleotide of the separated coding promotor that links to each other effectively; Polynucleotide with separated coding total length transaldolase A polypeptide; C) expression cassette, it comprises the polynucleotide of the separated coding promotor that links to each other effectively; Polynucleotide with separated coding total length hydrogenase 2 auxiliary polypeptide; D) expression cassette, it comprises the polynucleotide of the separated coding promotor that links to each other effectively; The polynucleotide of separated coding mitochondrial transport peptide; Polynucleotide with separated coding total length isocitrate lyase polypeptide; E) expression cassette, it comprises the polynucleotide of the separated coding promotor that links to each other effectively; The polynucleotide of separated coding chloroplast transit peptides; Polynucleotide with separated coding total length phospholipid hydroperoxide glutathione peroxidase polypeptide; F) expression cassette, it comprises the polynucleotide of the separated coding promotor that links to each other effectively; Polynucleotide with separated coding total length gamma glutamyl transpeptidase polypeptide; G) expression cassette, it comprises the polynucleotide of the separated coding promotor that links to each other effectively; The polynucleotide of separated coding mitochondrial transport peptide; Polynucleotide with separated coding total length atp synthase subunit B ' polypeptide; And h) expression cassette, it comprises the polynucleotide of the separated coding promotor that links to each other effectively; The polynucleotide of separated coding chloroplast transit peptides; Polynucleotide with separated coding total length C-22 sterol desaturase polypeptide.

In another embodiment, recombinant expression vector of the present invention comprises isolating polynucleotide, and it has the SEQ of being selected from ID NO:3; SEQ ID NO:5; SEQ ID NO:7; SEQ ID NO:11; SEQ ID NO:13; SEQ ID NO:21; SEQ ID NO:23; SEQ ID NO:25; SEQ ID NO:27; SEQ ID NO:29; SEQ ID NO:31; SEQ ID NO:33; SEQ ID NO:35; SEQ ID NO:37; SEQ ID NO:39; SEQ ID NO:41; SEQ ID NO:43; SEQ ID NO:49; Sequence with SEQ ID NO:53.In addition, recombinant expression vector of the present invention comprises isolating polynucleotide, and its coding has the SEQ of being selected from ID NO:4; SEQ ID NO:6; SEQ ID NO:8; SEQ ID NO:12; SEQ ID NO:14; SEQ ID NO:22; SEQ ID NO:24; SEQ ID NO:26; SEQ ID NO:28; SEQ ID NO:30; SEQ ID NO:32; SEQ ID NO:34; SEQ ID NO:36; SEQ ID NO:38; SEQ ID NO:40; SEQ ID NO:42; SEQ ID NO:44; SEQ ID NO:50; Polypeptide with the aminoacid sequence of SEQ ID NO:54.

Recombinant expression vector of the present invention also comprises one or more regulating and controlling sequences, selects based on the host cell that is used to express, and it links to each other with polynucleotide isolating to be expressed effectively.As used at this; About recombinant expression vector; " effectively connect " or " linking to each other effectively " meaning is that the mode that herbicide-tolerant polynucleotide allows this herbicide-tolerant polynucleotide to express when introducing host cell (for example, bacterium or plant host cell) with carrier links to each other with regulating and controlling sequence.Term " regulating and controlling sequence " is used for comprising promotor, enhanser and other expression controlling elementss (for example, polyadenylation signal).

As implied above, particular of the present invention is used the promotor that can strengthen the genetic expression in the leaf.In some embodiments, promotor is the leaf specificity promoter.In these embodiments of the present invention, can use any leaf specificity promoter.Many such promotors are known, for example, and from the USP promotor (Baeumlein etc. of broad bean (Vicia faba); (1991), Mol.Gen.Genet.225,459-67); The promotor (rbcS promotor) of photoinduction type gene such as ribulose-1,5-bisphosphate .5-di-phosphate carboxylase, coding chlorophyll a/b-conjugated protein (Cab), ribulose activating enzyme, from promotor (Kwon etc., (1994) of the gene of the B-subunit of the chloroplast(id) Glycerose 3-phosphate dehydrogenase of Arabidopis thaliana; Plant Physiol.105 is 357-67) with other leaf specificity promoters, like Aleman; I. those that identify in, (2001), Isolation and characterization of leaf-specific promoters from alfalfa (Medicago sativa); Master thesis; New Mexico State University, Los Cruces, NM.

In other embodiments of the present invention, root or seedling specificity promoter have been used.For example, super promotor provides the high level expression in root and the seedling (Plant J.7:661-676 for Ni etc., (1995)).Other root-specific promoters include, but not limited to the TobRB7 promotor (Yamamoto etc., (1991), Plant Cell 3,371-382), the rolD promotor (Leach etc., (1991), Plant Science 79,69-76); (Science 244,174-181) etc. for Benfey etc., (1989) for CaMV 35S structural domain A.

In other embodiments, used constitutive promoter.Constitutive promoter is activated under most of condition.The instance that is applicable to the constitutive promoter in these embodiments comprises the parsley ubiquitin promoter described in the WO2003/102198, CaMV 19S and 35S promoter, sX CaMV35S promotor, Sep1 promotor, rice actin promoter, Arabidopis thaliana actin promoter, corn ubiquitin promoter, pEmu, radix scrophulariae mosaic virus 35 S promoter, Smas promotor, super promotor (U.S. patent No.5; 955; 646), GRP1-8 promotor, cinnamyl-alcohol dehydrogenase promotor (U.S. patent No.5; 683; 439), from the promotor of the T-DNA of Agrobacterium; Like mannopine synthase, nopaline synthase and octopine synthase, the small subunit of diphosphoribulose carboxylase (ssuRUBISCO) promotor etc.

According to the present invention, chloroplast transit sequence refer to the to encode nucleotide sequence of chloroplast transit peptides.The instance of chloroplast transit peptides comprises the conjugated protein transit peptides of chlorophyll a/b, carboxydismutase transit peptides small subunit, EPSPS transit peptides and dihydrodipicolinate synthase's transit peptides.As definition here, the mitochondrial transport sequence refers to coding line plastochondria presequence and pilot protein matter to mitochondrial nucleotide sequence.The instance of plastosome presequence comprises the ATP enzyme subunit, atp synthase subunit, Rieske-FeS albumen; Hsp60, MDH, Oxalacetic transacetase; Aconitase, isocitric enzyme, pyruvic oxidase; Malic enzyme, glycine decarboxylase, serine hydroxymethylase and superoxide-dismutase.

Such transit peptides is known in the art.See, for example people (1991) Plant Mol.Biol.Rep.9:104-126 such as Von Heijne; People such as Clark (1989) J.Biol.Chem.264:17544-17550; People such as Della-Cioppa (1987) Plant Physiol.84:965-968; People such as Romer (1993) Biochem.Biophys.Res.Commun.196:1414-1421; With people (1986) Science 233:478-481 such as Shah.Chloroplast targeted sequence is known in the art and comprises ribulose-1,5-bisphosphate, the chloroplast(id) small subunit of 5-di-phosphate carboxylase (Rubisco) (people (1996) Plant Mol.Biol.30:769-780 such as de Castro Silva Filho; People such as Schnell (1991) J.Biol.Chem.266 (5): 3335-3342); 5-enol acetone shikimic acid-3-phosphate synthase (5-(enolpyruvyl) shikimate-3-phosphate synthase, EPSPS) (people (1990) J.Bioenerg.Biomemb.22 (6) such as Archer: 789-810); Tryptophan synthetase (people (1995) J.Biol.Chem.270 (11) such as Zhao: 6081-6087); Plastocyanin (people (1997) J.Biol.Chem.272 (33) such as Lawrence: 20357-20363); Chorismate synthase (people (1993) J.Biol.Chem.268 (36) such as Schmidt: 27447-27457); With light harvesting chlorophyll a/b conjugated protein (LHBP) (people (1988) J.Biol.Chem.263:14996-14999 such as Lamppa).See people (1991) Plant Mol.Biol.Rep.9:104-126 such as Von Heijne again; People such as Clark (1989) J.Biol.Chem.264:17544-17550; People such as Della-Cioppa (1987) Plant Physiol.84:965-968; People such as Romer (1993) Biochem.Biophys.Res.Commun.196:1414-1421; With people (1986) Science 233:478-481 such as Shah.

In a preferred embodiment of the invention, listed polynucleotide are expressed in the vegetable cell from higher plant (for example, spermatophyte is like crop) in the table 1.Any way be can pass through, transfection, conversion or transduction, electroporation, partickle bombardment, agroinfection etc. comprised, in polynucleotide " introducing " vegetable cell.For example, use U.S. patent No.4,945,050; 5,036,006; 5,100,792; 5,302,523; 5,464,765; 5,120,657; Listed partickle bombardment in 6,084,154 grades discloses the appropriate method of conversion or transfection of plant cells.More preferably, according to U.S. patent No.5,591,616; 5,731,179; 5,981,840; 5,990,387; 6,162,965; 6,420,630, described in U.S. patent application publication number 2002/0104132 grade, use edaphic bacillus to transform and formed transgenic corn seed of the present invention.Can use for example European patent No.EP0424047, U.S. patent No.5,322,783, European patent No.EP0397687, U.S. patent No.5, any technology of describing in 376,543 or U.S. patent No.5,169,770 is carried out the conversion of soybean.The particular instance that wheat transforms can apply for finding among the No.WO93/07256 at PCT.Can use U.S. patent No.5,004,863; 5,159,135; Disclosed method is come converting cotton in 5,846,797 grades.Can use U.S. patent No.4,666,844; 5,350,688; 6,153,813; 6,333,449; 6,288,312; 6,365,807; Disclosed method transforms rice in 6,329,571 grades.For example, use like U.S. patent No.5,188,958; 5,463,174; 5,750,871; EP1566443; Middle those disclosed method such as WO02/00900 transforms the Kano and draws.The other plant method for transformation is disclosed in, for example, and U.S. patent No.5,932,782; 6,153,811; 6,140,553; 5,969,213; 6,020,539 etc.Can use according to the present invention and to be applicable to any methods for plant transformation that transgenic is inserted specified plant.

According to the present invention,, then can the polynucleotide of introducing stably be maintained in the vegetable cell if incorporate non-chromosome self-replicating into or be integrated in the plant chromosome.Perhaps, the polynucleotide of introducing may reside on the extrachromosomal non-replicating vector and can transient expressions or of short duration activity arranged.

The present invention also is presented as the method for producing the transgenic plant that comprise at least one listed in the table 1 polynucleotide; Wherein the expression of polynucleotide causes comparing with the wild-type kind of plant in the plant; The growth that plant is improved under normal or lack of water condition and/or the tolerance to environment-stress of output and/or raising; The method comprising the steps of: (a) with in the above-described expression cassette introduced plant cell, (b) from plant transformed cell regeneration transgenic plant; And from the regenerated vegetable cell, select the higher plant of output.Vegetable cell can be, but be not limited to the cell of protoplastis, gamete generation cell and the complete plant of regeneration.As used at this, term " transgenic " refers to any plant, vegetable cell, callus, plant tissue or the plant part that contains above-mentioned expression cassette.According to the present invention, expression cassette stably is integrated in the karyomit(e) or in the stable extra-chromosomal element, it can be successfully gone down to posterity.

Can pass through normally and/or not too planting the plant of modifying under the appropriate condition, and the effect of genetic modification to plant-growth and/or output and/or stress tolerance assessed in growth characteristics and/or the metabolism of analysis plant.Such analytical technology is well known to a person skilled in the art, and comprise that dry weight, weight in wet base, seed weight, seed amount, polypeptide are synthetic, glucide is synthetic, lipid is synthetic, evapotranspiration rate, general plant and/or crop yield, bloom, breed, set seeds, the measurement of root growth, respiration rate, photosynthetic rate, metabolite composition etc.

Further specify the present invention through following examples, these embodiment are not interpreted as by any way dielectric imposed limits in scope of the present invention.

Embodiment 1

The sign of gene

Use standard recombinant technology clone takes the lead (lead) gene b0757 (SEQ ID NO:1); B2464 (SEQ ID NO:9), b2990 (SEQ ID NO:15), SLL1323 (SEQ ID NO:47); Slr1269 (SEQ ID NO:45); YER065C (SEQ ID NO:17), YIR037W (SEQ ID NO:19), and YMR015C (SEQ ID NO:51).Each takes the lead the function of gene aminoacid sequence prediction through icp gene and other known function coded by said gene.Use currently known methods from the proprietary library of species separately, to separate homologue cDNA.Use bioinformatic analysis to handle and the note sequence.

From colibacillary b0757 gene (SEQ ID NO:1) coding galactokinase.With the full length amino acid sequence (SEQ ID NO:2) of b0757 to proprietary cDNA DB with e ^-10The e value carry out BLAST comparison people such as (, on seeing) Altschul.Identify from 2 homologues of soybean with from a homologue of corn.Indicated the amino acid dependency of these sequences in the comparison that in Fig. 1, shows.

From colibacillary b2464 gene (SEQ ID NO:9) coding transaldolase A.With the full length amino acid sequence (SEQ ID NO:10) of b2464 to proprietary cDNA DB with e ^-10The e value carry out BLAST comparison people such as (, on seeing) Altschul.Identify 1 homologue drawing from the Kano and from the homologue of soybean.Indicated the amino acid dependency of these sequences in the comparison that in Fig. 2, shows.

YIR037W gene (SEQ ID NO:19) coding phospholipid hydroperoxide glutathione peroxidase from yeast saccharomyces cerevisiae.With the full length amino acid sequence (SEQ ID NO:20) of YIR037W to proprietary cDNA DB with e ^-10The e value carry out BLAST comparison people such as (, on seeing) Altschul.Identify 3 homologues that draw from the Kano, from 4 homologues of soybean, from 1 homologue of Sunflower Receptacle, from 1 homologue of barley, from 1 homologue of rice with from 2 homologues of corn.Indicated the amino acid dependency of these sequences in the comparison that in Fig. 3, shows.

SLL1323 gene (SEQ ID NO:47) coding atp synthase subunit B ' from synechocystis.With the full length amino acid sequence (SEQ ID NO:48) of SLL1323 to proprietary cDNA DB with e ^-10The e value carry out BLAST comparison people such as (, on seeing) Altschul.Identify 1 homologue from soybean.Indicated the amino acid dependency of these sequences in the comparison that in Fig. 4, shows.

YMR015C gene (SEQ ID NO:51) coding C-22 sterol desaturase from yeast saccharomyces cerevisiae.With the full length amino acid sequence (SEQ ID NO:52) of YMR015C to proprietary cDNA DB with e ^-10The e value carry out BLAST comparison people such as (, on seeing) Altschul.Identify 1 homologue from soybean.Indicated the amino acid dependency of these sequences in the comparison that in Fig. 5, shows.

Embodiment 2

Take the lead gene cross expressing in plant

Use currently known methods that the polynucleotide of table 1 are connected to expression cassette.3 kinds of different promotor control transgenic expression in Arabidopis thaliana have been used: from the USP promotor (" USP ") (SEQ ID NO:61 or SEQ ID NO:62) of broad bean (Vicia faba); Super promotor (" super "; SEQ ID NO:63); With parsley ubiquitin promoter (" PCUbi "; SEQ ID NO:64).For targeted expression, use mitochondrial transport peptide (SEQ ID NO:56 or SEQ ID NO:58; In table 2-9, be called " plastosome ") or chloroplast transit peptides (SEQ ID NO:60; In table 2-10, be called " plastid ").

Use currently known methods with comprising the environmental C24 of construct arabidopsis thaliana transformation that takes the lead gene that describes among the embodiment 1.Merge the seed that T2 transforms plant based on driving expression promoter, gene source species and target type (chloroplast(id), plastosome or do not have target).The living weight that seed bank is used under that fully irrigate and the growth conditions water restriction screens for the first time.From seed bank, be chosen in hitting in the first screening, carry out analysis of molecules and collect seed.Then the seed of collecting is used for the analysis at programmed screening, has wherein analyzed more a plurality of bodies of each transgenic event.If in programmed screening, be accredited as with contrasting from the plant of construct and compare the living weight with raising, then it gets into screening for the third time.In this screening, fully irrigate with the growth conditions of arid measure down from all transgenic events of this construct above 100 strain plants.Using canonical statistics to learn program will compare with the wild-type arabidopsis thaliana or from the transgenic arabidopsis seed bank growing plants of selecting at random from the data of transgenic plant.

The plant of under abundant irrigation conditions, cultivating 2 acceptance is weekly irrigated to soil saturation.Use commercial imaging system to obtain the image of transgenic plant at the 17th and 21 day.Perhaps, culturing plants under the limited growth conditions of water, through seldom irrigating to soil saturation, this makes soil irrigate the drying that becomes between the processing.In these experiments, irrigated at after planting the 0th, 8 and 19 day.Use commercial imaging system to obtain the image of transgenic plant at the 20th and 27 day.

Use image analysis software to come the transgenic plant that comparison grows and the image of control plant in identical experiment.Use relative size or the living weight of the pixel mensuration plant of image, use the color of deep green than the ratio measuring plant of the total area.Therefore the ratio in back is called as health index, and it is the tolerance of chlorophyll relative quantity in the leaf, and is the tolerance of the relative quantity of leaf aging or yellow, and record in the time of the 27th day only.In the transgenic plant that contain the different band tau gene, exist to change, this is because the different loci that DNA inserts influences the factor of gene expression dose and pattern with other.The positive of relevant proterties and the number of heliophobous plant have been indicated in order to show this effect data table.

Table 2 to 9 shown the arabidopsis thaliana observed value comparison." CD " plant indicator grows under the cycle drought condition; The condition that " WW " indication is fully irrigated.Numeral indication repeatedly independent experiment under the same conditions after the abbreviation.The percentage difference indication is represented with the per-cent of contrast non-transgenic plant with respect to the genetically modified measurement of control plant; The p-value is the significance,statistical of difference between transgenic and the control plant, and based on the T-of all independent eventss check relatively, wherein NS is illustrated under 5% the probability level, does not have significance.The independent transgenic event sum that the event number indication detects in experiment; Positive events number indication comparison in experiment is total according to big independent transgenic event; Negative event number indication comparison in experiment is total according to little independent transgenic event.

A. galactokinase

In Arabidopis thaliana, express and be under the super promotor control and the galactokinase gene b0757 (SEQ ID NO:1) of target chloroplast(id).Table 2 has been listed and has fully been irrigated and the cycle drought condition living weight and the health index that obtain of the arabidopsis thaliana of these constructs conversions of usefulness of detection down.

Table 2

Table 2 shows that the arabidopsis thaliana of the b0757 gene of expressing the target chloroplast(id) causes plant bigger under the water confined condition, but is not fully causing under the irrigation conditions.In these experiments, to express all independent transgenic events of b0757 gene and all compare according to big, indication is to coercing the better adaptation of environment.

B. transaldolase A

In Arabidopis thaliana, express the transaldolase A gene b2464 (SEQ ID NO:9) that does not have the ubcellular target that is under USP or the control of super promotor.Table 3 has been listed and has fully been irrigated and the cycle drought condition living weight and the health index that obtain of the arabidopsis thaliana of these constructs conversions of usefulness of detection down.

Table 3

Table 3 is presented under the water confined condition, and the arabidopsis thaliana that expression is in the b2464 gene under the super promotor control is bigger.Between the transgenic plant that contain the b2464 gene, exist really because the difference that the different loci that DNA inserts and other factors that influence gene expression dose or pattern cause.In these experiments, most of independent transgenic event comparison of expressing the b2464 gene is according to big, and indication is to coercing the better adaptation of environment.In addition, the b2464 expression of gene that is under the control of USP promotor causes bigger plant under abundant irrigation conditions.In these experiments, express all transgenic events of b2464 gene and all compare according to big.

C. hydrogenase-2 accessory protein

In Arabidopis thaliana, express the hydrogenase that the does not have the ubcellular target-2 accessory protein b2990 (SEQ ID NO:15) that is under the super promotor control.Table 4 has been listed and has fully been irrigated and the cycle drought condition living weight and the health index that obtain of the arabidopsis thaliana of these constructs conversions of usefulness of detection down.

Table 4

Table 4 shows that the arabidopsis thaliana of expressing the b2990 gene is all bigger under abundant irrigation and water confined condition.Between the transgenic plant that contain the b2990 gene, exist really because the difference that the different loci that DNA inserts and other factors that influence gene expression dose or pattern cause.In these experiments, most of independent transgenic event comparison of expressing the b2990 gene is according to big, and indication is to coercing the better adaptation of environment.Under abundant irrigation conditions, the plant that the b2990 expression of gene causes health index to reduce; This effect is not found under the water confined condition.

D. isocitrate lyase

In Arabidopis thaliana, express and be under the control of USP promotor and the mitochondrial isocitrate lyase gene of target YER065C (SEQ ID NO:17).Table 5 has been listed living weight and the health index that the arabidopsis thaliana of these constructs conversions of usefulness that detect down at abundant irrigation conditions obtains.

Table 5

Table 5 shows that the arabidopsis thaliana of expressing the YER065C gene is bigger under abundant irrigation conditions.Between the transgenic plant that contain the YER065C gene, exist really because the difference that the different loci that DNA inserts and other factors that influence gene expression dose or pattern cause.In these experiments, most of independent transgenic event comparison of expressing the YER065C gene is according to big.

E. lipid hydroperoxide Selenoperoxidase

In Arabidopis thaliana, express and be under USP or the control of PCUbi promotor and target chloroplast(id) or mitochondrial lipid hydroperoxide glutathione peroxidase gene YIR037W (SEQ ID NO:19).Table 6 has been listed and has fully been irrigated and the water confined condition living weight and the health index that obtain of the arabidopsis thaliana of these constructs conversions of usefulness of detection down.

Table 6

Table 6 shows that expressing the arabidopsis thaliana that is in the YIR037W gene under the control of PCUbi promotor compares according to big under the water confined condition when the target chloroplast(id), indication is to coercing the better adaptation of environment.In addition, fully irrigate the transgenic plant of expressing YIR037W down with the water confined condition on color, compare shine green get darker, such as the health index of raising demonstration.This prompting YIR037W transgenic plant are compared with control plant and produce more chlorophyll or have less chlorophyll degradation.

When the YIR037W expression of gene received control of USP promotor and target chloroplast(id), the YIR037W transgenic plant were all compared according to plant little under abundant irrigation and water confined condition.In addition, under abundant irrigation conditions the comparison of YIR037W transgenic plant shallowly green according to plant, such as the health index that reduces demonstration.This prompting has the YIR037W transgenic plant of this particular build body and compares the less chlorophyll of generation with control plant or have more chlorophyll degradation.If with YIR037W gene target plastosome and controlled by the USP promotor, then when relatively YIR037W transgenic and control plant, do not observe the significant difference in living weight or health index.

H. gamma glutamyl transpeptidase

In Arabidopis thaliana, express and be in the control of PCUbi promotor also target chloroplast(id), plastosome or the not subcellular gamma glutamyl transpeptidase gene of target slr1269 (SEQ ID NO:45) down.Table 7 has been listed and has fully been irrigated and the cycle drought condition living weight and the health index that obtain of the arabidopsis thaliana of these constructs conversions of usefulness of detection down.

Table 7

Table 7 demonstration is expressed the arabidopsis thaliana of the mitochondrial slr1269 gene of target and under the water confined condition, is compared according to little.In addition, fully irrigate with the water confined condition under the slr1269 transgenic plant all compare according to plant shallowly green, such as the health index of reduction demonstration.The mitochondrial slr1269 transgenic plant of this prompting target are compared with control plant and produce less chlorophyll or have more chlorophyll degradation.When slr1269 expression of gene target chloroplast(id), observe similar result.Under the water confined condition, the comparison of slr1269 transgenic plant is according to little.Under abundant irrigation conditions, the comparison of slr1269 transgenic plant is according to shallowly green, such as the health index that reduces demonstration.

When the slr1269 expression of gene did not have the ubcellular target, the slr1269 transgenic plant were compared according to plant big under the water confined condition, and indication is to coercing the better adaptation of environment.In addition, express the transgenic plant of slr1269 and under the water confined condition, on color, compare, show like the health index that improves according to green deeplyer.The mitochondrial slr1269 transgenic plant of this prompting target are compared with control plant and produce more chlorophyll or have less chlorophyll degradation.

G.ATP synthase subunit B '

In Arabidopis thaliana, express and be under the control of PCUbi promotor and the mitochondrial atp synthase subunit of target B ' gene SLL1323 (SEQ ID NO:47).Table 8 has been listed and has fully been irrigated and the cycle drought condition living weight and the health index that obtain of the arabidopsis thaliana of these constructs conversions of usefulness of detection down.

Table 8

The arabidopsis thaliana that table 8 show to be expressed the SLL1323 gene cause fully irrigate and the water confined condition under plant all bigger.Between the transgenic plant that contain the SLL1323 gene, exist really because the difference that the different loci that DNA inserts and other factors that influence gene expression dose or pattern cause.In these experiments, most of independent transgenic event comparison of expressing the SLL1323 gene is according to big, and indication is to coercing the better adaptation of environment.In addition, express the transgenic plant of SLL1323 and under the water confined condition, on color, compare, show like the health index that improves according to green deeplyer.This points out said plant to compare with control plant and produces more chlorophyll or have less chlorophyll degradation.

H.C-22 sterol desaturase

In Arabidopis thaliana, express the YMR015C gene (SEQ ID NO:51) and the target chloroplast(id) of coding C-22 sterol desaturase, use 3 kinds of constructs.In a kind of construct, transcribe and controlled by the PCUbi promotor.In another kind, transcribe and controlled by super promotor.Transcribing of YMR015C controlled by the USP promotor in the third construct.Table 9 has been listed and has fully been irrigated and the water confined condition living weight and the health index that obtain of the arabidopsis thaliana of these constructs conversions of usefulness of detection down.

Table 9

Table 9 demonstration has the Arabidopis thaliana of the YMR015C expression of PCUbi promotor control and when protein also target chloroplast(id), compares according to plant big significantly.In addition, these transgenic plant impinge upon on the color green deeplyer with the transgenic plant comparison that the YMR015C with super promotor control expresses.These data are indicated said plant in coercing, to compare according to plant and are produced more chlorophyll or have chlorophyll degradation still less.Table 9 shows that also most of independent transgenic event comparison is according to big.

Table 9 shows that Arabidopis thaliana that YMR015C with PCUbi promotor or the control of super promotor expresses compares down at abundant irrigation conditions when protein also target chloroplast(id) that to shine plant significantly little.Table 9 shows that also most of independent transgenic event comparison is according to little.In addition, these 2 kinds of constructs all significantly reduce the amount of green color of plant when growth under abundant irrigation conditions.

Claims

1. use the expression cassette transgenic plant transformed, said expression cassette comprises effectively continuous

A) polynucleotide of separated coding promotor, said promotor can improve the genetic expression in the leaf;

B) polynucleotide of separated coding mitochondrial transport peptide; With

C) separated coding comprises the polynucleotide of total length isocitrate lyase polypeptide of the 22nd to 550 amino acids of SEQ ID NO:18, and wherein transgenic plant are compared with the wild-type plant that does not contain expression cassette of same breed and shown the output that improves.

2. be purebred seed to transgenic, said transgenic comprises effectively continuous

B) polynucleotide of separated coding mitochondrial transport peptide; With

C) separated coding comprises the polynucleotide of total length isocitrate lyase polypeptide of the 22nd to 550 amino acids of SEQ ID NO:18, the transgenic plant that wherein obtain by said seed growth with not containing of same breed said genetically modified wild-type plant compare and shown the output that improves.

3. improve the method for plant biomass, said method comprising the steps of:

A) use the expression cassette transformed plant cells, said expression cassette comprises effectively continuous

I) polynucleotide of separated coding promotor, said promotor can improve the genetic expression in the leaf;

The ii) polynucleotide of separated coding mitochondrial transport peptide; With

Iii) separated coding comprises the polynucleotide of total length isocitrate lyase polypeptide of the 22nd to 550 amino acids of SEQ ID NO:18;

B) from plant transformed cell regeneration transgenic plant; With

C) transgenic plant that shown the output that improves are compared in selection with the wild-type plant that does not contain said expression cassette of same breed.