CN101981191A

CN101981191A - Nucleotide sequences and corresponding polypeptides conferring modulated growth rate and biomass in plants grown in saline and oxidative conditions

Info

Publication number: CN101981191A
Application number: CN2007801016166A
Authority: CN
Inventors: F·周; K·费尔德曼; J·索萨
Original assignee: Ceres Inc
Current assignee: Ceres Inc
Priority date: 2007-09-19
Filing date: 2007-09-19
Publication date: 2011-02-23
Also published as: WO2009038581A2; WO2009038581A8

Abstract

The invention relates to separated nucleic acid molecules and corresponding polypeptides coded thereby, which endow following characters in saline and/or oxidative conditions, such as improved plant size, nutrition growth, growth speed, seedling activity and/or biomass. And the invention also relates to applications of transgenic plants, plant cells, plant materials or plant seeds with improved plant size, nutrition growth, growth speed, seedling activity and/or biomass in saline and/or oxidative conditions, compared to wild type plants prepared in similar conditions.

Description

Give the growth velocity of growing plants adjusting under salt and oxidizing condition and the nucleotide sequence and the corresponding polypeptide of biomass

Technical field

The present invention relates to the corresponding polypeptide of isolated nucleic acid molecule and coding thereof, described nucleic acid molecule and polypeptide can strengthen plant-growth under salt and/or oxidative stress condition.The invention still further relates to preparation of nucleic acid molecule and polypeptide and simulated condition down the wild-type plant of growth compare, under salt and/or oxidative stress condition, have improved growth velocity, nourish and grow, the purposes of transgenic plant, vegetable cell, vegetable material or the plant seed of seedling vigor and/or biomass.

Background technology

Can use that molecular engineering obtains to be used for agricultural, gardening, biomass transforms and the clear and definite improved plant of other industry (for example paper industry, plant is as the production plant of protein or other compounds).For example, under salt and/or oxidative stress condition, strengthen plant-growth and can obtain huge agronomical value.

Salinity

Multiple plant species with agriculture importance shows the remarkable susceptibility to salt and/or oxidative stress condition.After salt concn surpasses low relatively threshold value, the growth that many plants are downgraded, necrosis and dead, this causes the overall outward appearance of downgrading and the reduction of vegetable material, seed, fruit and other valuable product output.With regard to physiology, the destruction that the plant of attacking with salinity experiences ion and water body homeostasis, the infringement of metabolic inhibition and cell membrane, this causes growth to stop and necrocytosis (Huh etc. (2002) Plant J, 29 (5): 649-59)

In most important in the world many production agricultures zone, rural activity self causes the salinity water and the soil salinity that improve, and this threatens their lasting productivity.An example is the crop irrigation that has in the arid region that enriches sunlight.Behind farmland application irrigation water, described irrigation water is removed by evaporation and rising process.Although these processes are removed water from soil, they stay the dissolved salt that has in the irrigation water.Therefore, soil and underground water salt concn increase in time, make soil and shallow table underground water become salty, and then the infringement crop.

Except people's activity, natural geology process produced a large amount of when non-salt the unusual salt pan of high yield.Amount to about 20% by the field irrigated be subjected to salinity negative impact (Yamaguchi and Blumwald, 2005, Trends in Plant Science, 10:615-620).Owing to these reasons and other, has huge interest and importance so identify following gene, described gene is given improved salt patience characteristic, thereby makes it possible to be created in the transgenic plant (for example crop plants) that have enhanced growth and/or productivity feature under the salt condition.

Although have this progress, still be starved of following blanket method at present, described method promotes forest or agricultural plant growth, to adapt to the concrete needs that depend on certain environmental conditions.For this reason, the present invention relates to advantageously plant modification, thereby make the interests maximization of various crop, and feature of the present invention is the dna molecular of express recombinant in plant according to the interests of being searched to the patience of salt.These molecules can be from plant self, and simply with higher or lower horizontal expression, perhaps described molecule can be from different plant species.

Oxidative stress

Plant carries out the mode of life of set, therefore annotates the place of the seed germination that moves in them usually.Therefore, they can be exposed in the unsuitable environmental condition that causes owing to weather, pollution and zone.Stress conditions is for example ozone and SO of extreme temperature, arid and drying, salinity, soil nutrient content, heavy metal, UV radiation, pollutent for example ₂, machinery coerces, high light and pathogenic agent are attacked that plant-growth and growth are had tremendous influence.The exposure of coercing of these types induces toxic oxygen thing class (toxic oxygenspecies) to form, and described toxic oxygen thing class produces in all aerobic cells, and is accompanied by the oxidative damage of cell levels.Some nearest disclosed reports have characterized that toxic oxygen thing class that abiotic stress causes produces and oxidative damage subsequently (is seen Larkindale and Knight (2002); Borsani etc. (2001); Lee etc. (2004); Aroca etc. (2005); Luna etc. (2005); (2002) such as and Noctor.

Toxic oxygen thing class is known as active oxygen species (ROS), active oxygen intermediate product (ROI) or activatory oxygen thing class (AOS), and partial reduction or activatory oxygen derivative when being.ROS/ROI/AOS comprises with oxygen being the super-oxide (O at center ₂) and hydroxyl (OH) free radical, and hydrogen peroxide (H ₂O ₂), nitrogen protoxide (NO) and O ₂ ¹These oxygen thing classes are as the byproduct of reaction of photosynthesis, respiration and photorespiration and produce, and mainly form in chloroplast(id), plastosome, endoplasmic reticulum, microbody (for example peroxysome and glyoxysome), plasma membrane and cell walls.Although O ₂ ^-And H ₂O ₂The toxicity of self is low relatively, but the metal dependent form of their high toxicity OH transforms the major cause that is considered to cause the most of biological damage relevant with these molecules.

Oxidative stress damaging cells structure also influences the metabolism and the katabolism of cell.Film fat is oxidized by ROS/ROI/AOS, causes high-molecular weight, crosslinked lipid acid and the accumulation of phosphatide.Proteinic oxidation attacked cause that locus specificity is amino acid modified, the fragmentation of peptide chain, crosslinked reaction product accumulation, the electric charge that changes and the proteolysis susceptibility of raising, these all usually cause the elimination of enzymic activity.The ROS/ROI/AOS (for example ionization radiation) that produces oxyradical also induces a large amount of damages in the sugar moieties of DNA and base portion, described damage causes disappearance, sudden change and other lethal hereditations, for example base degraded, single-strand break and and protein cross.From morphology, the detrimentally affect of high-level ROS cumulative turns out to be the growth and the downright bad infringement of dwarfing.

Although can induce damage, ROS/ROI/AOS also is the crucial setter of metabolism and defence path, the effect of its performance signal granting (signaling) or second messenger molecule.For example, it is crucial that the ROS/ROI/AOS of pathogen-inducible is created in the disease resistance, wherein relates to these molecules on three different levels: penetrate resistance, high sensitive and reply the resistance (Levine etc. (1994) that (HR) and systematicness obtain; Lamb and Dixon (1997); Zhou etc. (2000); Aviv etc. (2002)).In penetrating resistance, ROS/ROI/AOS brings into play function by the crosslinked reinforcement cell walls of Polyphenols.High sensitive is replied H ₂O ₂Be that active signal is provided molecule, its effect is a dose dependent.Under high dosage, H ₂O ₂Cause the high sensitive necrocytosis, and therefore pathogenic agent being limited to local infection site (Lamb and Dixon (1997)), low dosage is then blocked cell cycle progression (Reichheld etc. (1999)) and signal transduction secondary wall differentiation (Potikha etc. (1999)).At last, the ROS/ROI/AOS molecule is by causing small-HR and working in wide spectrum systematicness erworbene Krankenheit resistance in systematicness after the pathogenic agent inoculation first time.

In the signal transduction cascade that causes oxidative stress is amplified, Whitfield's ointment (SA) is accredited as under multiple stress conditions mediation ROS/ROI/AOS cumulative signal of interest and provides molecule, and described stress conditions for example salt and osmotic pressure is coerced (Borsani etc. (2001)), arid (Senaratna etc. (2000)), heat (Dat etc. (1998)), cold (Scott etc. (2004)), UV-light (Surplus etc. (1998)), Paraquat (Kim etc. (2003)) and at the disease resistance (Zhou etc. (2004)) of different pathogens.High-caliber SA induces H ₂O ₂Produce and necrocytosis.

The ROS/ROI/AOS accumulation of SA-mediation and some signal transduction assemblies that genetic expression needs characterize.For example, SA inductive PR genetic expression and disease resistance need NPR1 (Cao etc. (1994)).The signal granting of the sudden change blocking-up SA-mediation among eds1 and the eds5 also strengthens disease susceptibility (Rusterucci etc. (2001)).In the various plants species NahG cross to express also prevent the SA-inductive to abiotic stress and biological coerce reply (Delaney etc. (1994)).Recently, Scott and colleague (2004) have reported freezing treatment is induced SA in Arabidopis thaliana (Arabidopsis) accumulation, express degraded SA by crossing of NahG and have strengthened the cold patience in the transgenic plant.

SA also promotes early flowering (Martinez etc. (2004)) as plant hormone.The SA of multiple level can play a part different in plant-growth and stress response.Yet, seemingly useful in the most of the time to the patience of the raising of high-level SA, because it has reduced the side effect of SA cumulative, stimulate the stress response of SA mediation simultaneously.

Similarly, NO can produce ROS/ROI/AOS, and is that a kind of plant signal is provided molecule, and it relates to seed germination, stomatal closure (Mata and Lamattina (2001); Desikan etc. (2002)), the anti-oxidant reaction (Beligni etc. (2002)) of flowering time (He etc. (2004)), inhibitory cell death and to the patience (Mata and Lamattina (2001)) of biological and abiotic stress condition.Although can be by using nitre Pu Na (sodium nitroprusside, SNP) effect of simulation NO, but the activity that the endogenous NO in the plant produces by nitricoxide synthase causes that described nitricoxide synthase uses the reaction (Desikan etc. (2002)) of L-arginine (Guo etc. (2003)) and nitrate reductase mediation.NO can with the redox central reaction in protein and the film, thereby cause cell injury and inducing cell death.

In order to control the double properties of ROS/ROI/AOS molecule, development of plants goes out the complicated adjusting system, and described regulation system relates to generation and the removing of ROS/ROI/AOS in the cell.During normal g and D, the ROS/ROI/AOS level that this path monitoring metabolism produces, and control ROS/ROI/AOS removes the expression and the activity of path.Main ROS/ROI/AOS purge mechanism comprise superoxide dismutase (SOD), ascorbate peroxidase enzyme (ascorbate perioxidase, APX) and catalase (CAT) and for example effect of xitix, alpha-tocopherol and gsh of non-enzyme assembly.

Believe that oxidation resistant enzyme is the critical components of prevention oxidative stress, part is because a kind ofly coerce form pre-treatment plant and can improve the patience (cross tolerance) (Allen (1995)) that difference is coerced with what induce these expression of enzymes.In addition, at having one or more these antioxidase levels of raising usually, and also show cross tolerance (Gressel and Galun (1994)) by the selected plant lines of resistance of inducing the weedicide that ROS/ROI/AOS plays a role.

Development of plants and output depend on that plant is by the signal granting or remove the ability that path is controlled oxidative stress.Therefore, the ability of improving the ability of plant opposing oxidative stress or obtain the higher degree cross tolerance behind the experience oxidative stress has significant agronomical value.Sequence of the present invention and method provide following means, can or remove the patience of path improvement to oxidative stress by the signal granting by described means.

People and domestic animal have had the high-priority level to the operability and the sustainability of food and feed stream in human civilization history, and represent the origin in agricultural.The expert of agronomy science, agricultural, crop science, Horticulture and forest scientific domain and researchist even still strive for seeking and producing the plant of the growth potential with raising constantly in today are to feed increasing world population and to guarantee to provide reproducible starting material.Powerful research level shows that in the world the leader of every kind of geographical environment and weather is to providing the suitable attention level that food, feed and energy sources applied in these scientific domains.

The control of crop performance has been carried out several centuries routinely by selection and plant breeding.Yet breeding process is not only consuming time but also effort.In addition, must specifically design the suitable procedure of breeding for each relevant plant species.

On the other hand, using molecular genetic method controlling plant to obtain significant progress to provide in the better crop.Nucleic acid molecule by introducing and express recombinant in plant, the researchist all set offers social following plant species at present, even described plant species is transformed into and also can more effectively grows under inferior optimum geography and/or climatope and produce more product.These new methods have advantage (Zhang etc. (2004) the Plant Physiol.135:615 that is not limited to a plant species but can be applicable to a plurality of different plant species; Zhang etc. (2001) Proc.Natl.Acad.Sci.USA 98:12832).

Summary of the invention

The invention provides method and the material relevant with following plant, described plant has the patience level of adjusting to salinity and/or oxidative stress.For example, the invention provides transgenic plant and vegetable cell that salinity and/or oxidative stress are had the patience level of raising, be used to produce salinity and/or oxidative stress had the nucleic acid of the plant of patience level of raising and vegetable cell and be used to prepare salinity and/or oxidative stress are had the plant of patience level of raising and the method for vegetable cell.This class plant and vegetable cell provide under salt and/or oxidative stress condition and to have produced crop or plant and do not have stunted growth and reduce the chance of productive rate.The patience level of the raising of salinity and/or oxidative stress is used in produces the biomass that can be converted into liquid fuel or other chemical on the soil that low productivity is arranged at present and/or produce food and feed, the overall expansion that causes to plough the soil.

This paper provides the method that produces plant and/or plant tissue.In one aspect, method comprises cultivates the vegetable cell that comprises exogenous nucleic acid.Exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of coded polypeptide.During HMM that use is produced by aminoacid sequence shown in one of Fig. 1-6, (Hidden Markov Model, HMM) binary value (bit score) is greater than about 30 for the hidden Markov model of polypeptid acid sequence.Plant and/or plant tissue are compared the corresponding patience level of salinity and/or oxidative stress with the control plant that does not comprise exogenous nucleic acid, and be variant to the patience level of salinity and/or oxidative stress.In some embodiments, when using the HMM that aminoacid sequence obtains shown in Fig. 1, amino acid sequence of polypeptide has the HMM binary value greater than about 400.In some embodiments, when using the HMM that aminoacid sequence obtains shown in Fig. 2, amino acid sequence of polypeptide has the HMM binary value greater than about 30.In some embodiments, when using the HMM that aminoacid sequence obtains shown in Fig. 3, amino acid sequence of polypeptide has the HMM binary value greater than about 120.In some embodiments, when using the HMM that aminoacid sequence obtains shown in Fig. 4, amino acid sequence of polypeptide has the HMM binary value greater than about 150.In some embodiments, when using the HMM that aminoacid sequence obtains shown in Fig. 5, amino acid sequence of polypeptide has the HMM binary value greater than about 425.In some embodiments, when using the HMM that aminoacid sequence obtains shown in Fig. 6, amino acid sequence of polypeptide has the HMM binary value greater than about 550.

In another aspect, method comprises that cultivation comprises the vegetable cell of exogenous nucleic acid.Described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of the following polypeptide of coding, described polypeptide and SEQID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the amino acid equivalent (coordinate) 1 to 135 of SEQ ID NO:140 shown in aminoacid sequence have 85% or higher sequence identity.Compare with the corresponding patience level in the control plant that does not comprise exogenous nucleic acid by the plant that described vegetable cell produces, variant to the patience level of salinity or oxidative stress.

In another aspect, described method comprises that cultivation comprises the vegetable cell of exogenous nucleic acid.Described exogenous nucleic acid comprises the regulatory region that effectively is connected with following nucleotide sequence, described nucleotide sequence at least with SEQ ID NO.1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155,157,159, the fragment of the nucleotide sequence shown in 161 and 164 and with code sequence tabulation in the nucleotide sequence of disclosed any aminoacid sequence have 85% or higher sequence identity.Compare with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid by plant and/or plant tissue that described vegetable cell produces, variant to the patience level of salinity and/or oxidative stress.

This paper provides the method for regulation and control plant to salinity patience and/or oxidative stress patience level.In one aspect, method comprises introduces exogenous nucleic acid in vegetable cell, and described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of coded polypeptide.During HMM that use is produced by aminoacid sequence shown in Fig. 1-6, the HMM binary value of described amino acid sequence of polypeptide is greater than about 30.Compare with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid by plant and/or plant tissue that described vegetable cell produces, variant to the patience level of salinity or oxidative stress.

In one aspect, method comprises introduces exogenous nucleic acid in vegetable cell, described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of the following polypeptide of coding, wherein said polypeptide and SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the amino acid equivalent 1 to 135 of SEQ ID NO:140 shown in aminoacid sequence have 85% or higher sequence identity.Compare with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid by plant and/or plant tissue that described vegetable cell produces, variant to the patience level of salinity or oxidative stress.

In some embodiments, method comprises in vegetable cell the exogenous nucleic acid of introducing the following polypeptide of coding, and described polypeptide is selected from SEQ ID NO:43,44,45,86,140,141,142,143,144 and the amino acid equivalent 1 to 135 of SEQ ID NO:140.Compare with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid by plant and/or plant tissue that described vegetable cell produces, variant to the patience level of salinity.In some embodiments, method comprises introduces the exogenous nucleic acid that coding is selected from the polypeptide of SEQ ID NO:136 and 141 in vegetable cell, and compare with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid by plant and/or plant tissue that described vegetable cell produces, variant to the patience level of oxidative stress.

In another aspect, method comprises introduces exogenous nucleic acid in vegetable cell, described exogenous nucleic acid comprises the regulatory region that effectively is connected with following nucleotide sequence, wherein said nucleotide sequence and SEQ IDNO:1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155,157,159, nucleotide sequence shown in 161 and 164 and with code sequence tabulation in the nucleotide sequence of disclosed any aminoacid sequence have 85% or higher sequence identity.Compare with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid by plant and/or plant tissue that described vegetable cell produces, variant to the patience level of salinity or oxidative stress.

This paper provides the vegetable cell that comprises exogenous nucleic acid.In one aspect, exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of coded polypeptide.Use is during based on the HMM of aminoacid sequence shown in one of Fig. 1-6, and the HMM binary value of described amino acid sequence of polypeptide is greater than about 30.Described plant and/or plant tissue are compared with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid, and be variant to the patience level of salinity or oxidative stress.In another aspect, the regulatory region that exogenous nucleic acid comprises and the nucleotide sequence of the following polypeptide of coding effectively is connected, described polypeptide and be selected from SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the aminoacid sequence of the amino acid equivalent 1 to 135 of SEQ ID NO:140 have 85% or higher sequence identity.Compare with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid by plant and/or plant tissue that described vegetable cell produces, variant to the patience level of salinity or oxidative stress.In one aspect, exogenous nucleic acid comprises the regulatory region that effectively is connected with following nucleotide sequence, described nucleotide sequence at least be selected from SEQ ID NO.1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155,157,159, the nucleotide sequence of disclosed any aminoacid sequence has 85% or higher sequence identity in the tabulation of the fragment of 161 and 164 nucleotide sequence or code sequence.Compare with the corresponding patience level of the control plant that does not comprise exogenous nucleic acid by plant and/or plant tissue that described vegetable cell produces, variant to the patience level of salinity or oxidative stress.The transgenic plant that comprise this class vegetable cell also are provided.In some embodiments, transgenic plant are to be selected from following species member: switchgrass (Panicumvirgatum), dichromatism chinese sorghum (Sorghum bicolor) (Chinese sorghum, Schrock), huge Chinese silvergrass (Miscanthus giganteus) (awns genus), saccharum species (Saccharum sp.) (energy sugarcane), baisam poplqr (Populus balsamifera) (white poplar), Zea mays (Zea mays) (corn), soybean (Glycine max) (soya bean), colea (Brassica napus) (canola oil dish), common wheat (Triticum aestivum) (wheat), upland cotton (Gossypium hirsutum) (cotton), rice (Oryza sativa) (paddy rice), Sunflower Receptacle (Helianthus annuus), alfalfa (Medicagosativa) (clover), beet (Beta vulgaris) or cattailmillet (Pennisetum glaucum) (pearl millet).Some embodiments relate to and comprising from seed of above-mentioned transgenic plant or the goods of nutritive issue (product).Some embodiments relate to from food of above-mentioned transgenic plant or feed goods.

In one aspect of the method, isolating nucleic acid comprises the nucleotide sequence of coded polypeptide, and the aminoacid sequence shown in described polypeptide and the SEQ ID NO.2,4,6,22,27,29,49,52,54,56,60,62,68,76,83,88,90,96,98,104,106,112,114,132,134,149,151 or 160 has 80% or higher sequence identity.

The method of the evaluation genetic polymorphism relevant with salinity and/or the variation of oxidative stress patience level is provided in another aspect.Described method comprises provides plant population, and measure one or more genetic polymorphisms in the plant population whether with the locus genetic correlation connection of following polypeptide, described polypeptide is selected from polypeptide and functional homologue thereof shown in Fig. 1-6.Related in the variation of measuring salinity patience in population plant and/or the plant tissue and/or oxidative stress patience level and the population plant between the existence of one or more polymorphisms, thus allow to identify whether one or more polymorphisms make a variation relevant with this class.

In another aspect, provide the method for preparing plant lines.Described method comprises whether one or more genetic polymorphisms are relevant with the locus of following polypeptide in the mensuration plant population, described polypeptide is selected from polypeptide and functional homologue thereof shown in Fig. 1-6, identify one or more plants in the population, wherein the existence of one or more polymorphisms is relevant with the variation of salt patience or oxidative stress patience, each of the plant of one or more evaluations is produced seed with himself or different plant hybridization, will be with at least a progeny plants of described seed culture and himself or different plant hybridizations, hybridization step is repeated 0-5 generation again with the preparation plant lines.Described at least a allelotrope can be present in the plant lines.The method for preparing plant lines can be applicable to for example switchgrass plant population.

Unless otherwise defined, all technology used herein and scientific terminology have the common identical meanings of understanding of those skilled in the art.Although can use or the method and the material that are equal to similar with material to methods described herein, suitable method and material are still described hereinafter.All publications, patent application, patent and other reference mentioned in this article integral body are by reference incorporated this paper into.Under the situation of conflict, be as the criterion with this specification sheets (comprising definition).In addition, material, method and example only are used for setting forth but not are intended to restriction.

The details of one or more embodiments of the present invention is disclosed in accompanying drawing and explanation hereinafter.From specification sheets and accompanying drawing and claims, can understand other features of the present invention, purpose and advantage.

Summary of drawings

Fig. 1 is (ME08768; The aminoacid sequence comparison of homologue SEQ ID NO:86).In all comparison charts shown in this article, the dotted line indication notch in the aligned sequences promptly lacks amino acid on this position.Amino acid or conserved amino acid replacement identical between aligned sequences are annotated with collimation mark.Fig. 1 provided herein and other comparison charts use 3.52 editions MUSCLE programs to obtain.

Fig. 2 is the aminoacid sequence comparison of the homologue of ME06748 (SEQ ID NO:41).

Fig. 3 is the aminoacid sequence comparison of the homologue of ME19173 (SEQ ID NO:109).

Fig. 4 is the aminoacid sequence comparison of the homologue of ME02064C (SEQ ID NO:140).

Fig. 5 is the aminoacid sequence comparison of the homologue of Ceres clone ID No.1792354 (SEQ ID NO:2).

Fig. 6 is the aminoacid sequence comparison of the homologue of Ceres clone ID No.56784328 (SEQ ID NO:35).

Detailed Description Of The Invention

The present invention relates to and regulate and control the method and the material of salt patience in plant and/or the plant tissue and/or oxidative stress patience level.In some embodiments, plant also can have the biomass and/or the output of raising.This method can comprise that wherein said polypeptide expression causes modulated salinity patience and/or oxidative stress patience with the nucleic acid transformed plant cell of coding salinity and/or oxidative stress patience regulation and control polypeptide.The vegetable cell that uses these class methods to produce can be cultivated produces following plant, and described plant is compared salinity patience, oxidative stress patience and/or the biomass with raising with the wild-type plant that the same terms is grown down.The seed of this class plant and this class plant can be used for producing output and/or the biomass that for example is used for biofuel production, and described biofuel is such as but not limited to ethanol and butanols.

I. definition

" amino acid " is meant one of 20 kinds of amino acid that biologically exist and synthesizing amino acid, comprises the D/L optical isomer.

" the preferential promotor of cell type " or " the preferential promotor of tissue " is meant following promotor, and it preferentially drives in target cell type or tissue respectively and expresses, but causes in other cell types or tissue that too some transcribe.

" control plant " is meant and do not contain the exogenous nucleic acid that exists in the purpose transgenic plant, still have the plant with the same or analogous genetic background of this transgenic plant in other respects.Suitable control plant can be not genetically modified wild-type plant, from the non-transgenic chorista of transformation experiment, or contain the transgenic plant of the exogenous nucleic acid except that the purpose exogenous nucleic acid.

" structural domain " is basic successive amino acid group in the polypeptide, and it can be used in profiling protein matter family and/or protein portion.This class formation territory has " fingerprint " or " mark ", and described fingerprint or mark can comprise conservative primary sequence, secondary structure and/or three-dimensional conformation.Usually structural domain is relevant with specific external and/or activity in vivo.Structural domain can have 10 amino acid to 400 amino acid, for example 10 to 50 amino acid or 25 to 100 amino acid or 35 to 65 amino acid or 35 to 55 amino acid or 45 to 60 amino acid or 200 to 300 amino acid or 300 to 400 amino acid whose length.

" downward modulation " is meant with respect to substrate or native mode and reduces the adjusting that expression product (mRNA, polypeptide or the two) produces.

" external source " about nucleic acid is meant that nucleic acid is the part of recombinant nucleic acid construct, perhaps is not in its natural surroundings.For example, exogenous nucleic acid can be from a kind of species, be introduced into the sequence of another species, i.e. heterologous nucleic acids.Usually, such exogenous nucleic acid is introduced in other species by the recombinant nucleic acid construct.Exogenous nucleic acid also can be biological intrinsic and introduced sequence in this biomass cells again.The exogenous nucleic acid that comprises intrinsic sequence can be distinguished by the existence and the naturally occurring sequence of the non-natural sequence that is connected with exogenous nucleic acid usually, and described exogenous nucleic acid is for example regulated sequence for the extrinsic of intrinsic sequence side joint side in the reorganization nucleic acid construct.In addition, the exogenous nucleic acid of stable conversion is incorporated on other positions except that intrinsic sequence location usually.Should be understood that and in progenitor cell, to introduce exogenous nucleic acid, rather than in the cell of considering, introduce.For example, the transgenic plant that contain exogenous nucleic acid can be the offsprings of hybridizing between the plant of stable conversion and the non-transgenic plant.Such offspring is considered to contain exogenous nucleic acid.

" expression " is meant that the genetic information by transcribing polynucleotide is converted into RNA, and is converted into protein by the translation of mRNA on rrna, described transcribing by enzyme (RNA polymerase) catalysis.

Use " heterologous polypeptide " to be meant the natural polypeptide that has polypeptide in the non-plant cell in this article, described vegetable cell is for example for using the transgenosis switchgrass plant that transforms and express described encoding sequence from the encoding sequence of the nitrogen translocator polypeptide of Zea mays plant.

Use " isolating nucleic acid " to comprise that naturally occurring nucleic acid, condition are that sequence of the next-door neighbour of nucleic acid both sides described in its naturally occurring genome one or both of is removed or does not exist in this article.Therefore, isolating nucleic acid includes but not limited to, as the molecule existence of purifying or the nucleic acid that exists as the nucleic acid molecule that mixes in carrier or the virus.Be present in for example cDNA library, genomic library or contain and hundreds ofly in the gel slice of genomic dna restrictive diges-tion product do not think isolating nucleic acid to millions of other nucleic acid intermediary nucleic acid.

" regulation and control (modulation) " of compound or composition level be meant owing to external source expression of nucleic acids in the vegetable cell or transcribe observe shown in the level change of compound or component.The change of level is measured with respect to the level relatively in the control plant.

" nucleic acid " and " polynucleotide " is used interchangeably in this article, and expression RNA and DNA, comprises cDNA, genomic dna, synthetic DNA and contains the DNA or the RNA of nucleic acid analog.Polynucleotide can have any three-dimensional structure.Nucleic acid can be two strands or strand (being sense strand or antisense strand).The limiting examples of polynucleotide comprises gene, gene fragment, exon, intron, messenger RNA(mRNA) (mRNA), transfer RNA, ribosome-RNA(rRNA), siRNA, small-RNA, ribozyme, cDNA, recombination of polynucleotide, ramose polynucleotide, nucleic acid probe and nucleic acid primer.Polynucleotide can contain unconventional or modified Nucleotide.

" effectively connection " is meant and arranges regulatory region and sequence to be transcribed in nucleic acid, make regulatory region can effectively regulate transcribing or translating of described sequence.For example, for encoding sequence effectively is connected with regulatory region, the translation initiation site with the translation reading frame of encoding sequence places between regulatory region downstream 1 and about 50 Nucleotide usually.Yet regulatory region can be placed in the translation initiation site upstream up to about 5,000 Nucleotide places, or about 2,000 Nucleotide places, transcription initiation site upstream.

Use " polypeptide " to be meant the amino acid whose compound of two or more subunits, amino acid analogue or other peptide mimicses herein, for example phosphorylation or glycosylation are irrelevant with posttranslational modification.Subunit can be by peptide bond or for example ester bond or the ehter bond connection of other keys.This definition comprises polypeptide, point mutation body, insertion mutant, splice variant, chimeric protein and the fragment thereof of full-length polypeptide, brachymemma.

" offspring " comprises the descendants of concrete plant or plant lines.The offspring of (instant) plant comprises F when existing ₁, F ₂, F ₃, F ₄, F ₅, F ₆The seed that forms on the generation plant, or BC subsequently ₁, BC ₂, BC ₃The seed that forms on the generation plant, or F subsequently ₁BC ₁, F ₁BC ₂, F ₁BC ₃The seed that forms on the plant from generation to generation subsequently.Title F ₁Be meant the offspring of hybridizing between two upward different parents of heredity.Title F ₂, F ₃, F ₄, F ₅And F ₆Be meant F ₁Plant selfed progeny or close relative's edge powder offspring's generation subsequently.

" regulatory region " is meant the nucleic acid with following nucleotide sequence, and the influence of described nucleotide sequence is transcribed or translation initiation and speed and transcribe or the stability and/or the mobility of translation product.Regulatory region includes but not limited to promoter sequence, enhancer sequence, response element, protein identification site, can induce element, protein bound sequence, 5 ' and 3 ' non-translational region (UTR), transcription initiation site, terminator sequence, polyadenylation sequence, intron and combination thereof.Regulatory region comprises at least one core (basis) promotor usually.Regulatory region also can comprise at least one controlling elements, for example enhancer sequence, upstream element or active region, downstream (UAR).For example, suitable enhanser is the cis regulatory elements (212 to-154) from octopine synthase (ocs) upstream region of gene district.Fromm etc., The Plant Cell, 1:977-984 (1989).

" rise " is meant the adjusting that improves expression product (mRNA, polypeptide or the two) level with respect to basis or native mode.

" carrier " can be to wherein inserting the replicon that section that another DNA section causes inserting duplicates, for example plasmid, phage or glutinous grain thereby be meant.Usually, carrier can duplicate when correct controlling elements combines.Term " carrier " comprises clone and expression vector, and virus vector and integrative vector." expression vector " is the carrier that comprises regulatory region.

Oxidative stress: the ability of plant species tolerance ROS/ROI/AOS is different." oxidative stress " can be defined as the set of following envrionment conditions, the influence of the ROS/ROI/AOS concentration that plant can begin to be improved under described envrionment conditions, for example reduction of enzymic activity, dna break, DNA-protein cross, necrosis and the growth of downgrading.Because these reasons, so the plant of experience oxidative stress shows that usually significant biomass and/or output reduce.

The oxidative stress that improves can be caused by natural, geological process and mankind's activity (for example polluting).Because the ability of plant species tolerance oxidative stress is different, cause the accurate envrionment conditions of coercing so can not summarize.Yet under the oxidative stress condition, oxidative stress patience plant is compared the higher biomass of generation, output and survival degree with non-oxide stress tolerance plant.Quantitatively physiology outward appearance, the rate of recovery and output.

Photosynthetic efficiency: by the relation between maximum fluorescence signal Fm and variable fluorescence Fv assessment photosynthetic efficiency or the electronics transportation by photosystem II.The reduction of optimal dose suboutput (Fv/Fm) shows coerces, and can be used for monitoring the performance of comparing transgenic plant under salt or the oxidative stress condition with non-transgenic plant.

Whitfield's ointment growth index (SAGI): photosynthetic efficiency x seedling area.

Salt growth index (SGI): photosynthetic efficiency x seedling area (under condition of salt stress).

Salinity: the ability of plant species tolerance salinity is different." salinity " can be defined as the set of following envrionment conditions, plant begins the influence of the salt concn that stands to improve under the described envrionment conditions, and for example example is unbalance, the pore conductivity that reduces, the photosynthesis of reduction, the growth velocity of reduction, necrocytosis, forfeiture turgescence (wilting) or the ovule abortion of raising.Owing to these reasons, so the plant that the experience salinity is coerced shows the remarkable reduction of biomass and/or output usually.

The salinity that improves can be caused by natural, geography process and mankind's activity (for example polluting).Because the ability of plant species tolerance salinity is different, cause the accurate envrionment conditions of coercing so can not summarize.Yet under the salt condition, salinity patience plant produces higher biomass, output and survival rate than non-salt patience plant.The quantitative difference of physical appearance, the rate of recovery and output.

The salinity that improves can be caused by natural, geography process and mankind's activity (for example irrigating).Because the ability of plant species tolerance olighydria is different, so can not summarize the accurate environment salt condition of coercing that causes.Yet under the salt condition, salt patience plant produces higher biomass, output and survival rate than non-salt patience plant.Can be quantitatively and use the difference of known measurement and analytical procedure statistical analysis physical appearance, the rate of recovery and output.

II. polypeptide

Polypeptide described herein comprises salinity patience and/or oxidative stress patience regulation and control polypeptide.When expressing, salinity patience and/or oxidative stress patience regulation and control polypeptide can effectively regulate and control salinity patience and/or oxidative stress patience level in plant or vegetable cell.This class polypeptide contains the structural domain of at least one salinity patience and/or oxidative stress patience regulation and control peptide characteristics (indicative) usually, as described in more detail.Salinity patience and/or oxidative stress patience regulation and control polypeptide have the HMM binary value greater than 30 usually, as described in more detail.In some embodiments, salinity patience and/or oxidative stress patience regulation and control polypeptide and SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the identity that has greater than 85% of the amino acid equivalent 1 to 135 of SEQ ID NO:140, as described in more detail.

A. salinity patience and/or oxidative stress patience are regulated and control the characteristic structural domain of polypeptide

Salinity patience and/or oxidative stress patience regulation and control polypeptide can contain IQ calmodulin binding motif structural domain, and described structural domain is predicted to be salinity patience and/or oxidative stress patience regulation and control peptide characteristic.Calmodulin (CaM) is considered to main calcium sensor, and by itself and on the same group interaction management (orchestrator) adjusting incident of cell protein not.For the conjugated protein existence three classes identification of many known CaM motif; As non-Ca ₂ ⁺-dependent form is in conjunction with the IQ motif of consensus sequence and be used for Ca ₂ ⁺Two kinds of relevant motifs of-dependent form bonded, its position with conservative hydrophobic residue PUBMED:9141499 is that the basis is called 18-14 and 1-5-10.

For example, the regulatory region of scallop myosin is three chain protein mixtures, and it replys Ca2 ⁺In conjunction with and on this motor switch.Side chain interacts two light chains and the adjacent sections that has the heavy chain of IQ-sequence motifs is connected in series.Ca2 ⁺-binding site is an EF-hand motif new on the main light chain, and stabilized with being connected of two light chains by relating to heavy chain, is responsible for Ca2 among the complete mhc molecule PUBMED:8127365 ⁺In conjunction with and regulate requirement to all three chains.

For example, SEQ ID NO:86 discloses the Arabidopis thaliana clone's who is accredited as Ceres SEEDLINE IDno.ME08768 herein aminoacid sequence, and it is predicted to be coding and contains polypeptide from the IQ calmodulin binding motif structural domain of residue 116-136.

In some embodiments, salinity patience and/or oxidative stress patience regulation and control polypeptide at the aminoterminal of naturally occurring polypeptide or carboxyl terminal by brachymemma.The polypeptide of brachymemma can keep some structural domain of naturally occurring polypeptide, lacks other structural domains simultaneously.Therefore, short at the most or long 5 amino acid whose length variants show the salinity patience and/or the oxidative stress patience regulation activity of brachymemma polypeptide usually.In some embodiments, the polypeptide of brachymemma is the dominant negative polypeptide.SEQ ID NO:138 discloses for naturally occurring polypeptide and has been regulated and control amino acid sequence of polypeptide at 5 ' end by the salinity patience of brachymemma and/or oxidative stress patience.Compare with the respective horizontal in the control plant that does not comprise brachymemma and/or its tissue, the difference of salinity patience in plant and/or the plant tissue and/or oxidative stress patience level is given in this class brachymemma polypeptide expression in plant.

B. pass through the functional homologue that Reciprocal BLAST identifies

In some embodiments, be suitable as salinity patience and/or oxidative stress patience regulation and control polypeptide by the reference salinity patience of one or more pfam description definition of above pointing out and/or one or more functional homologue of oxidative stress patience regulation and control polypeptide.Functional homologue be with the reference polypeptide have sequence similarity and the performance with reference to one or more biochemical functions of polypeptide or the polypeptide of physiological function.Functional homologue and can be naturally occurring polypeptide with reference to polypeptide, and sequence similarity is attributable to advolution or divergent evolution incident.Equally, functional homologue is known as homologue or lineal homologue or collateral line homologue sometimes in the literature.The variant of naturally occurring functional homologue (for example wild-type sequence encoding mutant body encoded polypeptide) self can be a functional homologue.Functional homologue also can produce by the site-directed mutagenesis of salinity patience and/or oxidative stress patience regulation and control polypeptid coding sequence, or by regulating and control the structural domain combination results (" structural domain exchange ") of the encoding sequence of polypeptide from the naturally occurring salinity patience of difference and/or oxidative stress patience.Term " functional homologue " is applicable to the nucleic acid of encoding function homology polypeptide sometimes.

Can compare by analysis of nucleotide and peptide sequence and identify functional homologue.For example, the database of Nucleotide or peptide sequence is inquired about the homologue that can identify salinity patience and/or oxidative stress patience regulation and control polypeptide.Sequential analysis can relate to uses salinity patience and/or oxidative stress patience regulation and control polypeptid acid sequence as canonical sequence, and nonredundant database is carried out BLAST, Reciprocal BLAST or PSI-BLAST analysis.In some cases, aminoacid sequence is deduced from nucleotide sequence.These polypeptide that have in the database greater than 40% sequence identity are adaptive candidates of further estimating as salinity patience and/or oxidative stress patience regulation and control polypeptide.Amino acid similarity allows conservative amino acid to replace, and for example replaces another with a hydrophobic residue, or replaces another with a polar residues.Can carry out the hand inspection of this class candidate when needing, remain the quantity of the candidate further estimated thereby dwindle.Can be by selecting to show that having the structural domain (for example Bao Shou functional domain) that exists in salinity patience and/or the oxidative stress patience regulation and control polypeptide carries out the hand inspection.

Can identify conservative region by following zone, location in the one-level aminoacid sequence of salinity patience and/or oxidative stress patience regulation and control polypeptide, described zone is a tumor-necrosis factor glycoproteins, form some secondary structures (for example spiral and β lamella), set up positively charged or electronegative structural domain, or represent protein motif or structural domain.See that for example World Wide Web sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/ go up the Pfam website of the consensus sequence of describing multiple proteins motif and structural domain.The declarative description of the information that comprises in the Pfam database is in Sonnhammer etc., Nucl.AcidsRes., 26:320-322 (1998); Sonnhammer etc., Proteins, 28:405-420 (1997); With Bateman etc., Nucl.Acids Res. is among the 27:260-262 (1999).Can also measure conserved regions by the sequence of comparison from the identical or related polypeptide of closely related species.Closely related species are preferably from identical section.In some embodiments, just enough from the sequence alignment of two kinds of different plant species.

Usually, show that the polypeptide at least about 40% amino acid sequence identity can be used for identifying conserved regions.The conserved regions of related polypeptide shows at least 45% amino acid sequence identity (for example at least 50%, at least 60%, at least 70%, at least 80% or at least 90% amino acid sequence identity).In some embodiments, conserved regions shows at least 92%, 94%, 96%, 98% or 99% amino acid sequence identity.

The aminoacid sequence of the functional homologue of the polypeptide shown in the SEQ ID NO:86 is provided in Fig. 1 and the sequence table.This class functional homologue comprises (SEQ ID NO:88,90,91,93,94,96,98,100,101,102,104,106 and 107).In some cases, the functional homologue of SEQ IDNO:86 has following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ IDNO:86 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

The aminoacid sequence of the functional homologue of the polypeptide shown in the SEQ ID NO:41 is provided among Fig. 2.This class functional homologue comprises (SEQ ID NO:42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83 and 84).In some cases, the functional homologue of SEQ ID NO:41 has following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:41 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

The aminoacid sequence of the functional homologue of the polypeptide shown in the SEQ ID NO:109 is provided among Fig. 3.This class functional homologue comprises (SEQ ID NO:110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132 and 134).In some cases, the functional homologue of SEQ ID NO:109 has following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:109 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

The aminoacid sequence of the functional homologue of the polypeptide shown in the SEQ ID NO:140 is provided among Fig. 4.This class functional homologue comprises (SEQ ID NO:136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167 and 168).In some cases, the functional homologue of SEQ ID NO:140 has following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:140 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

The aminoacid sequence of the functional homologue of the polypeptide shown in the SEQ ID NO:2 is provided among Fig. 5.This class functional homologue comprises (SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31 and 33).In some cases, the functional homologue of SEQ ID NO:2 has following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:2 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

The aminoacid sequence of the functional homologue of the polypeptide shown in the SEQ ID NO:35 is provided among Fig. 6.This class functional homologue comprises (SEQ ID NO:35,36,37,38 and 39).In some cases, the functional homologue of SEQ ID NO:35 has following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:35 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

The evaluation of conserved regions helps the generation of salinity patience and/or oxidative stress patience regulation and control polypeptide variants in salinity patience and/or the oxidative stress patience regulation and control polypeptide.Salinity patience and/or oxidative stress patience regulation and control variant polypeptides have 10 or the replacement of conserved amino acid still less usually in the one-level aminoacid sequence, for example 7 or the replacement of conserved amino acid still less, 5 or the replacement of conserved amino acid still less, or the conserved amino acid between 1 and 5 is replaced.Can be built with the variant polypeptide of usefulness based on Fig. 1 to one of comparison shown in Fig. 6.This class polypeptide comprises conserved regions, and described conserved regions is with the series arrangement from the aminoterminal to the carboxyl terminal shown in scheming.This class polypeptide also can comprise zero, one or more than one amino acid in the position of with dashed lines mark.When not having amino acid on the position of dashed lines labeled, the length of this class polypeptide is the summation of amino-acid residue in all conserved regions.When all having amino acid in all positions of with dashed lines mark, the length of this class polypeptide is the summation of amino-acid residue in all conserved regions and all dotted lines.

C. the functional homologue of identifying by HMM

In some embodiments, useful salinity and/or oxidative stress patience regulation and control polypeptide comprises the polypeptide of the hidden Markov model that is fit to based on Fig. 1-6 polypeptide shown in arbitrary.Hidden Markov model (HMM) is the statistical models of the consensus sequence of one group of functional homologue.Consult Durbin etc., Biological Sequence Analysis:Probabilistic Models of Proteinsand Nucleic Acids, Cambridge University Press, Cambridge, UK (1998).The sequence of functions of use homologue group is as input, by program HMMER 2.3.2 default program parameter generating HMM.Use one group of default parameters by 1.11 editions ProbCons (15 (2): 330-40 (2005)) produce the multisequencing comparison, described parameter is for Do etc., GenomeRes. :-c,--consistence REPS is 2;-ir,--iteration-segmentation REPS is 100;-pre,--pre-training REPS is 0.ProbCons is the open structural domain software program that is provided by Stanford University.

The default parameters that makes up HMM (hmmbuild) is as follows: the acquiescence " system structure priority " that the MAP system structure is used is 0.85 (archpri), and the acquiescence cutoff threshold (idlevel) that is used to measure the ordered sequence number is 0.62.HMMER 2.3.2 permitted in the general open license of GNU on October 3rd, 2003, and can obtain at the place, a plurality of source on the World Wide Web.Hmmbuild is as the text output model.

Can use the HMM of one group of functional homologue to measure candidate's salinity patience and/or oxidative stress patience regulation and control peptide sequence and be fit to the possibility of concrete HMM better than following invalid HMM, described invalid HMM uses non-structure or one group of relevant sequence of function to produce.Tried peptide sequence and represented by the HMM binary value, the numeral that described HMM binary value is to use HMMER hmmsearch program that the candidate sequence match is produced during to the HMM collection of illustrative plates than the possibility that invalid HMM is fit to HMM better.Use following default parameters during operation hmmsearch: acquiescence E-value is 10.0 by (E), the acquiescence binary value is a minus infinity by (T), the sequence number of giving tacit consent in the database (Z) is the true sequence number in the database, the acquiescence E value of per-structural domain fractionated hit tabulation is infinitely great by (domE), and the acquiescence binary value of per-structural domain fractionated hit tabulation is a minus infinity by (domT).High HMM binary value represents to be tried sequence, and to be used to produce the possibility of one or more biochemical functions of polypeptide of HMM or physiologic function bigger.High HMM binary value is at least 20, and is higher usually.

It will be appreciated by those skilled in the art that the HMM score that provides in the sequence table only is used for for example.Because multisequencing alignment algorithm for example ProbCons only can produce near optimal results, so because the slight variation of following factor possibility generation model, described factor is the processed order that is used to compare of sequence for example.Yet HMM score variability is less, so the HMM score in the sequence table has been represented the model with each sequence preparation.

HMM shown in hereinafter salinity of Tao Luning and/or oxidative stress regulation and control polypeptide is fit to the HMM binary value greater than 20 (for example greater than 20,30,40,50,60,70,80,90,100,200,300,400 or 500).In some embodiments, 50%, 60%, 70%, 80%, 90% or 95% of the functional homologue HMM binary value that provides in the sequence table is provided the HMM binary value of salinity of hereinafter discussing and/or oxidative stress regulation and control polypeptide.In some embodiments, hereinafter salinity of Tao Luning and/or oxidative stress regulation and control polypeptide is with HMM shown in being fit to greater than 20 HMM binary value, and has the characteristic structural domain of salinity and/or oxidative stress regulation and control polypeptide.In some embodiments, hereinafter salinity of Tao Luning and/or oxidative stress regulation and control polypeptide is with HMM shown in being fit to greater than 20 HMM binary value, and with aminoacid sequence shown in Fig. 1 to 6 is arbitrary or sequence table in Fig. 1 to 6 in arbitrary relevant aminoacid sequence have 85% or higher sequence identity (75%, 80%, 85%, 90%, 95% or 100% sequence identity).

When the HMM match that produces with aminoacid sequence shown in Fig. 1 is provided in the sequence table, has polypeptide greater than 400,450,500,550,600,650,700,750,800,850,900,950,1000,1050 or 1100 HMM binary value.This class polypeptide comprises CeresSEEDLINE ID no.ME08768, Ceres clone ID no.1943807, Ceres ANNOT IDno.1471392, Public GI ID no.6715635, Ceres clone ID no.910109, Public GIID no.115474509, Ceres clone ID no.1780908, Ceres ANNOT ID no.1520883, Ceres clone ID no.148018, Public GI ID no.18378797, Public GI IDno.21553500, Ceres ANNOT ID no.1444522, Ceres ANNOT ID no.146751 and Public GI ID no.125559938 (SEQ ID NO:86,88,90,91,93,94,96,98,100,101,102,104,106 and 107).

When being provided in the sequence table, the HMM match that produces with aminoacid sequence shown in Fig. 2 has polypeptide greater than 30,50,100,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950 or 1000 HMM binary value.This class polypeptide comprises Ceres SEEDLINE ID no.ME06748, Ceres SEEDLINE IDno.ME20711, Ceres SEEDLINE ID no.ME18973, Ceres SEEDLINE IDno.ME08732, Ceres SEEDLINE ID no.ME19657, Ceres clone ID no.835818, Ceres clone ID no.1796745, Public GI ID no.125543896, Ceres ANNOT IDno.1483984, Ceres clone ID no.1924654, Ceres ANNOT ID no.1468861, Ceres clone ID no.1641776, Ceres ANNOT ID no.1438750, Ceres ANNOT IDno.1447395, Public GI ID no.79482785, Public GI ID no.3292832, Ceres clone ID no.1559074, Ceres clone ID no.1726548, Public GI IDno.115459996, Ceres clone ID no.697034, Ceres clone ID no.353438, PublicGI ID no.125593074, Ceres clone ID no.1920115, Ceres clone ID no.21821, Ceres clone ID no.560066, Public GI ID no.115453071, Ceres clone IDno.1968211 and Public GI ID no.116310011_ (SEQ ID NO:42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83 and 84).

When being provided in the sequence table, the HMM match that produces with aminoacid sequence shown in Fig. 3 has polypeptide greater than 120,150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150 or 1200 HMM binary value.This class polypeptide comprises Ceres SEEDLINE ID no.ME19173, PublicGI ID no.115435054, Ceres clone ID no.1847857, Ceres ANNOT IDno.1455219, Ceres clone ID no.352452, Ceres clone ID no.787908, CeresLOCUS ID no.Os01m00929_AP002743, Ceres clone ID no.246398, PublicGI ID no.125527441, Public GI ID no.125595056, Ceres clone ID no.236071, Public GI ID no.125524760, Public GI ID no.125569365, Public GI IDno.115439499, Public GI ID no.15225258, Public GI ID no.115465173, Ceres ANNOT ID no.1477059 and Ceres ANNOT ID no.1530547 (SEQ IDNO:110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132 and 134).

When being provided in the sequence table, the HMM match that produces with aminoacid sequence shown in Fig. 4 has polypeptide greater than 150,200,250,300,350,400,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200,1250,1300 or 1350 HMM binary value.This class polypeptide comprises Ceres SEEDLINE IDno.ME24091, Ceres clone ID no.375578, Ceres clone ID no.375578, CeresSEEDLINE ID no.ME10681, Ceres SEEDLINE ID no.ME03140, CeresSEEDLINE ID no.ME24076, Ceres SEEDLINE ID no.ME24217, Public GIID no.115440873, Ceres clone ID no.826796, Ceres ANNOT ID no.1465047, Ceres clone ID no.1919901, Ceres clone ID no.520008, Public GI IDno.7413581, Ceres clone ID no.228069, Ceres clone ID no.467508, Ceres clone ID no.1829581, Ceres clone ID no.229668, Public GI ID no.125550655, Ceres clone ID no.106263, Public GI ID no.15231175, Public GI IDno.145357576 and Public GI ID no.125528277 (SEQ ID NO:136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167 and 168).

When being provided in the sequence table, the HMM match that produces with aminoacid sequence shown in Fig. 5 has polypeptide greater than 425,450,500,550,600,650,700,750,800,850,900,950,1000,1050,1100,1150,1200,1250,1300,1350,1400,1450,1500 or 1550 HMM binary value.This class polypeptide comprises Ceres clone IDno.1792354, Ceres clone ID no.1925477, Ceres ANNOT ID no.1521592, Ceres clone ID no.463594, Public GI ID no.22330633, Ceres clones IDno.345954, Ceres LOCUS ID no.Os01m05025_AP003288, GI ID no.56784330, Public GI ID no.125527495, Public GI ID no.125553119, Ceres clone ID no.236431, Ceres clone ID no.908518, Public GI ID no.115465121, Ceres clone ID no.1791910, Public GI ID no.125595019, Public GI IDno.42568886, Public GI ID no.2947062, Ceres ANNOT ID no.1468228, Ceres clone ID no.1942388, Public GI ID no.12324824, Public GI IDno.5882749 and Ceres clone ID no.325403 (SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31 and 33).

When being provided in the sequence table, the HMM match that produces with aminoacid sequence shown in Fig. 6 has polypeptide greater than 550,600,650 or 700 HMM binary value.This class polypeptide comprises CeresGI ID no.56784328, Public GI ID no.56784330, Public GI IDno.125528718, Public GI ID no.125572975 and Public GI ID no.125528716 (SEQ ID NO:35,36,37,38 and 39).

D. identity per-cent

In some embodiments, salinity and/or oxidative stress patience regulation and control polypeptide have the IDNO:2 with SEQ, 4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the amino acid equivalent 1 to 135 of SEQ ID NO:140 shown in the aminoacid sequence of aminoacid sequence with sequence identity of at least 50%, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, the aminoacid sequence of 98% or 99% sequence identity.Polypeptide with this class sequence identity per-cent has the characteristic structural domain of salinity and/or oxidative stress regulation and control polypeptide usually and/or has HMM binary value greater than 20, and is as indicated above.The NO:2 with SEQ ID is provided among Fig. 1-6,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the amino acid equivalent 1 to 135 of SEQ ID NO:140 shown in one of aminoacid sequence example with salinity and/or oxidative stress patience regulation and control amino acid sequence of polypeptide of at least 85% sequence identity.

" sequence identity per-cent " is meant any given canonical sequence and candidate's salinity and/or the sequence identity degree between the oxidative stress regulating and controlling sequence, and described canonical sequence for example is SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the amino acid equivalent 1 to 135 of SEQ ID NO:140.Candidate sequence has the length of canonical sequence length 80% to 200% usually, and for example 82% of canonical sequence length, 85%, 87%, 89%, 90%, 93%, 95%, 97%, 99%, 100%, 105%, 110%, 115%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190% or 200%.Can any candidate nucleic acid of following mensuration or polypeptide with respect to the identity per-cent of reference nucleic acid or polypeptide.The program that uses a computer ClustalW (1.83 editions, default parameters) with reference to sequence (for example nucleotide sequence or aminoacid sequence) and one or more candidate sequence comparisons, described computer program ClustalW allows to compare (overall comparison) on the whole length of nucleic acid or peptide sequence.Chenna etc., Nucleic Acids Res., 31 (13): 3497-500 (2003).

ClustalW calculates the optimum matching between canonical sequence and the one or more candidate sequence, and they are compared, thereby can measure identity, similarity and difference.Can insert the breach of one or more residues to canonical sequence, candidate sequence or this in the two, with the maximization sequence alignment.For the quick pairing comparison of nucleotide sequence, use following default parameters: field length (wordsize): 2; Window size: 4; Scoring method: per-cent; Top diagonal lines number (number of topdiagonal): 4; With the breach point penalty: 5.For the multiple ratio of nucleotide sequence for, use following parameter: the open point penalty of breach: 10.0; Breach extends point penalty: 5.0; Weight conversion: be.For the quick pairing comparison of protein sequence, use following parameter: field length: 1; Window size: 5; Scoring method: per-cent; Top diagonal lines number: 5; With the breach point penalty: 3.For the multiple ratio of protein sequence for, use following parameter: weight matrix (weight matrix): blosum; The open point penalty of breach: 10.0; Breach extends point penalty: 0.05; Hydrophilic breach: open; Hydrophilic residue Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg and Lys; The breach point penalty that residue is special: open.What ClustalW exported is the sequence alignment of mutual relationship between the reflection sequence.ClustalW can go up and upward operation of EuropeanBioinformatics Institute webpage (ebi.ac.uk/clustalw) by for example Baylor College of Medicine Search Launcher webpage (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) on the World Wide Web.

In order to measure the identity per-cent of candidate nucleic acid or aminoacid sequence and canonical sequence, use the ClustalW aligned sequences, use the length of the quantity of identical match in the comparison, and the result be multiply by 100 divided by canonical sequence.Attention can be immediate 1/10th with the valuation of identity percentages.For example, can be 78.1 with 78.11,78.12,78.13 and 78.14 valuations, be 78.2 with 78.15,78.16,78.17,78.18 and 78.19 valuations.

In some cases, salinity and/or oxidative stress patience regulation and control polypeptide have following aminoacid sequence, one or more aminoacid sequences shown in described aminoacid sequence and the SEQ ID NO:86 have at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.The amino acid sequence of polypeptide that has height sequence identity with the polypeptide shown in the SEQ ID NO:86 is provided in the sequence table.This class polypeptide comprises SEQ ID NO:88,90,91,93,94,96,98,100,101,102,104,106 and 107.

In some cases, salinity and/or oxidative stress patience regulation and control polypeptide have following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:41 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.The amino acid sequence of polypeptide that has height sequence identity with the polypeptide shown in the SEQ ID NO:41 is provided in the sequence table.This class polypeptide comprises SEQ ID NO:42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83 and 84.

In some cases, salinity and/or oxidative stress regulation and control polypeptide have following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:109 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.The amino acid sequence of polypeptide that has height sequence identity with the polypeptide shown in the SEQ ID NO:109 is provided in the sequence table.This class polypeptide comprises SEQ ID NO:110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132 and 134.

In some cases, salinity and/or oxidative stress regulation and control polypeptide have following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:140 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.The amino acid sequence of polypeptide that has height sequence identity with the polypeptide shown in the SEQ ID NO:140 is provided in the sequence table.This class polypeptide comprises SEQ ID NO:136,138,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167 and 168.

In some cases, salinity and/or oxidative stress regulation and control polypeptide have following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:2 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.The amino acid sequence of polypeptide that has height sequence identity with the polypeptide shown in the SEQ ID NO:2 is provided in the sequence table.This class polypeptide comprises SEQ ID NO:4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31 and 33.

In some cases, salinity and/or oxidative stress regulation and control polypeptide have following aminoacid sequence, aminoacid sequence shown in described aminoacid sequence and the SEQ ID NO:35 has at least 50% sequence identity, for example 50%, 52%, 56%, 59%, 61%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.The amino acid sequence of polypeptide that has height sequence identity with the polypeptide shown in the SEQ ID NO:35 is provided in the sequence table.This class polypeptide comprises SEQ ID NO:36,37,38 and 39.

E. other sequences

Should be understood that salinity and/or oxidative stress patience regulation and control polypeptide can comprise other amino acid that do not relate to salinity and/or the regulation and control of oxidative stress patience, so this class polypeptide may be than longer under other situations.For example, salinity and/or oxidative stress patience regulation and control polypeptide can comprise the purification tag that is added on aminoterminal or carboxyl terminal, chloroplast transit peptides, amyloplast transit peptides, mitochondrial transport peptide or leader sequence.In some embodiments, salinity and/or oxidative stress patience regulation and control polypeptide comprise the aminoacid sequence of performance reporter gene function, for example green fluorescent protein or yellow fluorescence protein.

III. nucleic acid

Nucleic acid as herein described is included in the nucleic acid of effectively regulating and control salinity and/or oxidative stress patience when transcribing in plant or the vegetable cell.This class nucleic acid include but not limited to encode salinity and/or oxidative stress patience regulation and control polypeptide nucleic acid and can be used in the nucleic acid that suppresses salinity patience and/or oxidative stress patience regulation and control expression of polypeptides by method based on nucleic acid.

The nucleic acid of the salinity of A. encoding patience and/or oxidative stress patience regulation and control polypeptide

This paper has described the nucleic acid of coding salinity patience and/or oxidative stress patience regulation and control polypeptide.This class nucleic acid comprises SEQ ID NO:1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155,157,159,161 and 164, as hereinafter in greater detail.

Salinity patience and/or oxidative stress patience regulation and control nucleic acid can comprise the nucleotide sequence shown in the SEQ ID NO:85.Perhaps, salinity patience and/or oxidative stress patience regulation and control nucleic acid can be the nucleic acid variant with the nucleotide sequence shown in the SEQ ID NO:85.For example, salinity patience and/or oxidative stress patience regulation and control nucleic acid can have following nucleotide sequence, nucleotide sequence shown in described nucleotide sequence and the SEQ IDNO:85,87,89,92,95,97,99,103 and 105 has at least 80% sequence identity, for example 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

Salinity patience and/or oxidative stress patience regulation and control nucleic acid can comprise the nucleotide sequence shown in the SEQ ID NO:40.Perhaps, salinity patience and/or oxidative stress patience regulation and control nucleic acid can be the nucleic acid variant with the nucleotide sequence shown in the SEQ ID NO:40.For example, salinity patience and/or oxidative stress patience regulation and control nucleic acid can have following nucleotide sequence, nucleotide sequence shown in described nucleotide sequence and the SEQ IDNO:40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79 and 82 has at least 80% sequence identity, for example 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

Salinity patience and/or oxidative stress patience regulation and control nucleic acid can comprise the nucleotide sequence shown in the SEQ ID NO:108.Perhaps, salinity patience and/or oxidative stress patience regulation and control nucleic acid can be the nucleic acid variant with the nucleotide sequence shown in the SEQ ID NO:108.For example, salinity patience and/or oxidative stress patience regulation and control nucleic acid can have following nucleotide sequence, nucleotide sequence shown in described nucleotide sequence and the SEQID NO:108,111,113,115,117,120,124,131 and 133 has at least 80% sequence identity, for example 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

Salinity patience and/or oxidative stress patience regulation and control nucleic acid can comprise the nucleotide sequence shown in the SEQ ID NO:139.Perhaps, salinity patience and/or oxidative stress patience regulation and control nucleic acid can be the nucleic acid variant with the nucleotide sequence shown in the SEQ ID NO:139.For example, salinity patience and/or oxidative stress patience regulation and control nucleic acid can have following nucleotide sequence, nucleotide sequence shown in described nucleotide sequence and the SEQID NO:135,137,139,146,148,150,152,155,157,159,161 and 164 has at least 80% sequence identity, for example 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

Salinity patience and/or oxidative stress patience regulation and control nucleic acid can comprise the nucleotide sequence shown in the SEQ ID NO:1.Perhaps, salinity patience and/or oxidative stress patience regulation and control nucleic acid can be the nucleic acid variant with the nucleotide sequence shown in the SEQID NO:1.For example, salinity patience and/or oxidative stress patience regulation and control nucleic acid can have following nucleotide sequence, nucleotide sequence shown in described nucleotide sequence and the SEQ ID NO:1,3,5,7,10,12,16,18,21,26,28 and 32 has at least 80% sequence identity, for example 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

Salinity patience and/or oxidative stress patience regulation and control nucleic acid can comprise the nucleotide sequence shown in the SEQ ID NO:34.Perhaps, salinity patience and/or oxidative stress patience regulation and control nucleic acid can be the nucleic acid variant with the nucleotide sequence shown in the SEQ ID NO:34.For example, salinity patience and/or oxidative stress patience regulation and control nucleic acid can have following nucleotide sequence, nucleotide sequence shown in described nucleotide sequence and the SEQ IDNO:34 has at least 80% sequence identity, for example 81%, 85%, 90%, 95%, 97%, 98% or 99% sequence identity.

Can use standard technique to produce isolated nucleic acid molecule.For example, can use polymerase chain reaction (PCR) technology to obtain to contain the isolating nucleic acid of nucleotide sequence described herein.Can use PCR specific sequence of amplification from DNA and RNA (comprising sequence) from total genomic dna or total cell RNA.Multiple PCR method for example is described in PCR Primer:A Laboratory Manual, Dieffenbach and Dveksler compiles, and Cold Spring Harbor LaboratoryPress is in 1995.Usually, use from the purpose zone terminal or outside sequence information design oligonucleotides primer, described primer is same or similar with the relative chain of template to be amplified on sequence.Can also obtain multiple PCR strategy, the locus specificity nucleotide sequence can be modified by described strategy and introduce in the template nucleic acid.Can also the isolating nucleic acid of chemosynthesis, for example as single nucleic acid molecule synthetic (for example use the phosphoramidite technology, 3 ' use on 5 ' direction the automatization DNA synthetic) or synthetic as a series of oligonucleotide.For example, can synthesize the one or more pairs of long oligonucleotides (for example＞100 Nucleotide) that contain expected sequence, each forms duplex to containing short complementary section (for example about 15 Nucleotide) when making oligonucleotide to annealing.Use archaeal dna polymerase to extend oligonucleotide, each oligonucleotide is to obtaining single, double-stranded nucleic acid molecule, and described nucleic acid molecule can be connected with carrier subsequently.Also can obtain isolating nucleic acid of the present invention by for example naturally occurring DNA of mutagenesis.

B. nucleic acid is regulated and control the purposes of expression of polypeptides

I. salinity patience and/or oxidative stress patience are regulated and control polypeptide expression

Polypeptide

Usually pass through with following nucleic acid transformed plant cell, with nucleic acid express polypeptide in the purpose plant species of coding salinity patience described herein and/or oxidative stress patience regulation and control polypeptide, described nucleic acid has the sequence of the coded polypeptide that effectively is connected with sense orientation and one or more regulatory regions.Should be understood that the degeneracy owing to genetic codon, a large amount of specific salinity patience of nucleic acid codified and/or oxidative stress patience regulation and control polypeptide promptly for many amino acid, exist more than a kind of sub nucleotide triplet that acts on of this amino acid code of bringing into play.Therefore, can use suitable codon preference table, modify the codon in the encoding sequence of given salinity patience and/or oxidative stress patience regulation and control polypeptide, thereby obtain optimal expression in the described plant species at concrete plant species.

In some cases, salinity patience and/or oxidative stress patience regulation and control polypeptide expression suppresses one or more functions of endogenous polypeptide.For example, can use the TAP29 matter function of coding dominance negative polypeptide.Dominance negative polypeptide is sudden change or brachymemma usually for endogenous wild type peptide, and its existence in cell suppresses one or more functions of wild type peptide in cell, and promptly dominance negative polypeptide is hereditary dominance, and give the forfeiture of function.The mechanism that dominance negative polypeptide is given this phenotype can change, but usually relates to protein-protein interaction or protein-DNA interaction.For example, dominance negative polypeptide can be with respect to intrinsic wild-type enzyme by the enzyme of brachymemma, thereby but the polypeptide of brachymemma keeps and to relate to the structural domain of first protein bound lack the structural domain that relates to second protein bound.Therefore, the polypeptide of brachymemma can not correctly be regulated and control second activity of proteins.See for example US 2007/0056058.As another example, cause the point mutation of non-conserved amino acid alternate can cause dominance negative polypeptide in the catalyst structure domain.See for example US 2005/032221.As another example, dominance negative polypeptide can be with respect to inherent wild-type transcription factor by the transcription factor of brachymemma, thereby but the polypeptide of brachymemma keeps one or more DNA binding domainss lacks one or more activation domains.The polypeptide of this class brachymemma can suppress the wild-type transcription factor and combine with DNA, thereby suppresses transcription activating.

Ii. suppress salinity patience and/or oxidative stress patience regulation and control polypeptide expression

Polynucleotide as herein described and recombinant precursor can be used for suppressing salinity patience and/or oxidative stress patience regulation and control polypeptide expression in the purpose plant species.See Matzke and Birchler on the nature.com/reviews/focus/mai for example, Nature Reviews Genetics6:24-35 (2005); Akashi etc., Nature Reviews Mol.Cell Biology6:413-422 (2005); Mittal, Nature Reviews Genetics5:355-365 (2004); Dorsett and Tuschl, Nature Reviews Drug Discovery3:318-329 (2004); With Nature Reviews RNA interference collection, in October, 2005.Known in a large number based on the genetic expression in the nucleic acid inhibition plant, described method comprises the method for sense-rna, the directed RNA cutting of ribozyme, PTGS (PTGS), and for example RNA disturbs (RNAi) and transcriptional gene silencing (TGS).Antisense technology is a kind of known method.In this method, will effectively be connected with transcription termination sequence from the nucleic acid segment clone of the gene that will prevent and with regulatory region, make that the antisense strand of RNA is transcribed.Then recombinant precursor is transformed in the plant, as described herein, and produce the antisense strand of RNA.Nucleic acid segment needs not be the whole sequence of the gene that will prevent, but usually should be complementary substantially with at least a portion of the gene sense strand that will prevent.Usually, can use higher homology to come the use of the short sequence of compensate for slower.Usually use the sequence of at least 30 Nucleotide, for example at least 40,50,80,100,200,500 Nucleotide or more.

In another approach, can be with transcribed nucleic acid for influencing ribozyme or the catalytic RNA that mRNA expresses.See U.S. Patent No. 6,423,885.In fact ribozyme can be designed as and matches with any target RNA-specific, and cuts phosphodiester backbone on the specificity position, thereby makes the functional inactivation of target RNA.Heterologous nucleic acids can be encoded and is designed to cut the ribozyme of concrete mRNA transcript, thus the prevention polypeptide expression.Hammerhead ribozyme can be used for destroying concrete mRNA, although can use the multiple ribozyme of cutting mRNA on the specific recognition sequence of site.Hammerhead ribozyme is at following flanking region appointed positions cutting mRNA, and described flanking region and said target mrna form the complementary base pair.Unique requirement is that target RNA contains 5 '-UG-3 ' nucleotide sequence.The structure of hammerhead ribozyme and generation are known in the art.See U.S. Patent No. 5,254,678 and WO 02/46449 for example and the reference of wherein quoting.The hammerhead ribozyme sequence can implanted stable RNA for example in the transfer RNA (tRNA), to improve cutting efficiency in the body.Perriman etc., Proc.Natl.Acad.Sci.USA, 92 (13): 6175-6179 (1995); De Feyter and Gaudron, Methods inMolecular Biology, the 74th volume, the 43rd chapter, " Expressing Ribozymes in Plants ", Turner, P.C. write, Humana Press Inc., the RNA endoribonuclease that Totowa, NJ. have described for example among the tetrahymena thermophila (Tetrahymena thermophila) naturally occurring RNA endoribonuclease can be useful.See for example U.S. Patent No. 4,987,071 and 6,423,885.

PTGS for example RNAi also can be used for the expression of suppressor gene.For example, can prepare the construct that comprises following sequence, described sequence be transcribed into can self-annealing RNA, for example have the double-stranded RNA of stem-ring structure.In some embodiments, a chain of double-stranded RNA stem portion comprises following sequence, described sequence and salinity patience and/or oxidative stress patience regulation and control polypeptide similar or the identical and length of adopted encoding sequence is arranged is that about 10 Nucleotide are to about 2,500 Nucleotide.The length of the sequence similar or identical with adopted encoding sequence is arranged can be from 10 Nucleotide to 500 Nucleotide, from 15 Nucleotide to 300 Nucleotide, and from 20 Nucleotide to 100 Nucleotide, or from 25 Nucleotide to 100 Nucleotide.Another chain of double-stranded RNA stem portion comprises following sequence, and the antisense strand of described sequence and salinity patience and/or oxidative stress patience regulation and control peptide coding chain is similar or identical and can have than shorter, the identical or longer length of adopted sequence respective length is arranged.In some cases, a chain of double-stranded RNA stem portion comprises the similar or identical sequence of 3 ' or 5 ' non-translational region with the mRNA of coding salinity patience and/or oxidative stress patience regulation and control polypeptide, and another chain of double-stranded RNA stem portion comprises respectively and coding salinity patience and/or the mRNA 3 ' of oxidative stress patience regulation and control polypeptide or the similar or identical sequence of complementary sequence of 5 ' non-translational region.In other embodiments, a chain of double-stranded RNA stem portion comprises the similar or identical sequence of intron sequences among preceding-mRNA with coding salinity patience and/or oxidative stress patience regulation and control polypeptide, and another chain of stem portion comprise with preceding-mRNA in the similar or identical sequence of complementary sequence of intron sequences.The loop section of double-stranded RNA can be 3 Nucleotide to 5,000 Nucleotide, for example 3 Nucleotide to 25 Nucleotide, 15 Nucleotide to 1,000 Nucleotide, 20 Nucleotide to 500 Nucleotide or 25 Nucleotide to 200 Nucleotide.The loop section of RNA can comprise intron.Double-stranded RNA can have 0,1,2,3,4,5,6,7,8,9,10 or more a plurality of stem-ring structure.The construct that will comprise following sequence transforms in the plant into as herein described, and described sequence effectively is connected with transcription termination sequence with regulatory region and is transcribed into the RNA that can form double-stranded RNA.It is well known by persons skilled in the art using the method for RNAi inhibition of gene expression.See for example United States Patent (USP) 5,034,323; 6,326,527; 6,452,067; 6,573,099; 6,753,139; With 6,777,588.Also see WO 97/01952; WO 98/53083; WO 99/32619; WO 98/36083; With U.S. Patent Publication 20030175965,20030175783,20040214330 and 20030180945.

Also can use the expression of following construct suppressor gene, described construct contains the regulatory region that effectively is connected with sense orientation and nucleic acid molecule.Transcription product can with salinity patience and/or oxidative stress patience regulation and control polypeptide have adopted encoding sequence similar or identical.Transcription product also can be not polyadenylation, lack 5 ' cap structure or contain can not montage intron.It is known in the art using the method for full-length cDNA and Partial cDNA Sequence inhibition of gene expression.See for example U.S. Patent No. 5,231,020.

In some embodiments, with the expression of the construct suppressor gene that contains following nucleic acid, it is the template that justice and antisense sequences are arranged complimentary to one another that described nucleic acid has at least one chain.It can be the part of large nucleic acids molecule more that justice and antisense sequences are arranged, and perhaps can be to have the not part of the independent nucleic acid molecule of complementary sequence.It can be to regulate and control the mRNA sequence of polypeptide, 3 ' or 5 ' non-translational region or the identical or complementary sequence of the intron among preceding-mRNA of mRNA with coding salinity patience and/or oxidative stress patience that justice or antisense sequences are arranged.In some embodiments, have justice or antisense sequences identical or complementary with the sequence of following regulatory region, described regulatory region drives the gene transcription of coding salinity patience and/or oxidative stress patience regulation and control polypeptide.In each case, there is adopted sequence to be and antisense sequences complementary sequence.

It can be any length greater than about 12 Nucleotide (for example 13,14,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30 or more a plurality of Nucleotide) that justice and antisense sequences are arranged.For example, antisense sequences length can be 21 or 22 Nucleotide.Usually, it is from about 15 Nucleotide to about 30 Nucleotide that the justice and the length range of antisense sequences are arranged, for example from about 18 Nucleotide to about 28 Nucleotide, or from about 21 Nucleotide to about 25 Nucleotide.

In some embodiments, antisense sequences is a mRNA sequence complementary sequence of regulating and control polypeptide with coding salinity patience described herein and/or oxidative stress patience.With the antisense sequences complementary adopted sequence being arranged can be the sequence that is present among the mRNA of salinity patience and/or oxidative stress patience regulation and control polypeptide.Usually, there are justice and antisense sequences to be designed to sequence, make the level reduction of said target mrna corresponding to 15-30 Nucleotide of said target mrna.

In some embodiments, can use the expression of the construct suppressor gene that contains following nucleic acid, it is the template that adopted sequence (for example 2,3,4,5,6,7,8,9,10 or adopted sequence arranged) is arranged more more than that described nucleic acid has at least one chain.Similarly, can use the expression of the construct suppressor gene that contains following nucleic acid, it is template more than an antisense sequences (for example 2,3,4,5,6,7,8,9,10 or more antisense sequences) that described nucleic acid has at least one chain.For example, construct can contain following nucleic acid, and it is two templates that adopted sequence and two antisense sequences are arranged that described nucleic acid has at least one chain.Many have the adopted sequence can be identical or different, and many antisense sequences can be identical or different.For example, construct can have following nucleic acid, described nucleic acid have a chain be two identical have adopted sequence with described two identical templates that two same anti-sense sequences of adopted sequence complementary are arranged.Perhaps, it is following template that isolating nucleic acid can have a chain: (1) length be two of 20 Nucleotide identical adopted sequence arranged, (2) length is the antisense sequences of adopted sequence complementary that have 20 Nucleotide, identical with two, (3) length is the adopted sequence of having of 30 Nucleotide, with (4) length be 30 Nucleotide, with three identical antisense sequences of adopted sequence complementary are arranged.Construct provided herein can be designed as has any arrangement that justice and antisense sequences are arranged.For example, two identical adopted sequences that have can be closelyed follow two identical antisense sequences, perhaps can be placed between two identical antisense sequences.

Having at least one chain and be one or more has the nucleic acid of justice and/or antisense sequences template effectively to be connected with regulatory region, contains one or more the transcribing of RNA molecule that justice and/or antisense sequences arranged with driving.In addition, such nucleic acid can with the Transcription Termination subsequence for example the terminator of nopaline synthase (no) gene effectively be connected.In some cases, two regulatory regions can instruct transcribing of two transcripts: one comes from the top chain, and one comes from the bottom chain.See for example Yan etc., Plant Physiol., 141:1508-1518 (2006).Two regulatory regions can be identical or different.Two transcripts can form the double stranded rna molecule of inducing target RNA degraded.In some cases, nucleic acid can place in T-DNA or the transfering DNA (P-DNA) from plant, makes the left side of P-DNA and right side T-DNA border sequence or left side and right side boundary sample sequence both sides or the either side at nucleic acid.See US2006/0265788.Nucleotide sequence length between two regulatory regions can from about 15 to about 30 Nucleotide.In some embodiments, nucleotide sequence length between two regulatory regions from about 15 to about 200 Nucleotide, length from about 15 to about 100 Nucleotide, length from about 15 to about 50 Nucleotide, length from about 18 to about 50 Nucleotide, length is from about 18 to about 40 Nucleotide, and length is from about 18 to about 30 Nucleotide, or length from about 18 to about 25 Nucleotide.

Suppress in some methods based on nucleic acid of plant genetic expression being used for, suitable nucleic acid can be nucleic acid analog.Nucleic acid analog can be modified on base portion, sugar moieties or phosphate backbone, to improve for example stability, hybridization or the solubleness of nucleic acid.Modification on the base portion comprises deoxyuridine replacement deoxythymidine and 5-methyl-2 '-Deoxyribose cytidine and 5-bromo-2 '-Deoxyribose cytidine replacement Deoxyribose cytidine.The modification of sugar moieties comprises that the 2 ' hydroxyl of modifying ribose forms 2 '-O-methyl or 2 '-O-allyl group sugar.Can modify the deoxyribose phosphate main chain and produce morpholino nucleic acid, wherein each base portion is connected with hexa-atomic morpholino ring, or produces peptide nucleic acid(PNA), wherein substitutes the deoxidation phosphate backbone and keeps four bases with false peptide main chain.See for example Summerton and Weller, 1997, Antisense Nucleic AcidDrug Dev., 7:187-195; Hyrup etc., Bioorgan.Med.Chem., 4:5-23 (1996).In addition, the deoxidation phosphate backbone can be replaced with for example thiophosphatephosphorothioate or phosphorodithioate main chain, phosphoramidate (phosphoroamidite) or alkyl phosphotriester main chain.

C. construct/carrier

Can use recombinant precursor provided herein to transform plant or vegetable cell, thus regulation and control salinity patience and/or oxidative stress patience level.The nucleic acid construct of reorganization can comprise the nucleic acid of coding salinity patience described herein and/or oxidative stress patience regulation and control polypeptide, its be adapted at plant or cell in express salinity patience and/or oxidative stress patience and regulate and control the regulatory region of polypeptide and effectively be connected.Therefore, nucleic acid can comprise the encoding sequence of any salinity patience shown in the coding SEQ ID NO:1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155,157,159,161 and 164 and/or oxidative stress patience regulation and control polypeptide.The example of the nucleic acid of coding salinity patience and/or oxidative stress patience regulation and control polypeptide is shown in SEQID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the amino acid equivalent 1 to 135 of SEQ ID NO:140 in.The salinity patience of recombinant nucleic acid coding and/or oxidative stress patience regulation and control polypeptide can be inherent salinity patience and/or oxidative stress patience regulation and control polypeptide, can pair cell be allogenic perhaps.In some cases, recombinant precursor contains the nucleic acid that suppresses salinity patience and/or oxidative stress patience regulation and control expression of polypeptides, and it effectively is connected with regulatory region.The example of suitable regulatory region is described in title in the part of " regulatory region ".

The carrier that contains recombinant nucleic acid construct (recombinant nucleic acid construct as described herein) also is provided.The suitable carriers main chain comprises for example conventional carrier main chain that uses in this area, for example plasmid, virus, artificial chromosome, BAC, YAC or PAC.Suitable expression vector includes but not limited to, from the plasmid and the virus vector of for example phage, baculovirus and retrovirus.A large amount of carriers and expression system for example can commerce derive from Novagen (Madison, WI), Clontech (Palo Alto, CA), Stratagene (La Jolla, CA) and Invitrogen/Life Technologies (Carlsbad, company CA).

Carrier provided herein can comprise for example replication orgin, scaffold attached region (SAR) and/or marker.Marker gene can give vegetable cell selectable phenotype.For example, marker can be given the biocide resistance, for example to the resistance of microbiotic (for example kantlex, G418, bleomycin or Totomycin), or to the resistance of weedicide (for example glyphosate, chlorine sulphur grand (chlorsulfuron) or glufosinates (phosphinothricin)).In addition, expression vector can comprise the sequence label that is designed to be convenient to operation or detects (for example purifying or location) expressed polypeptide.Sequence label is luciferase, β-glucuronidase (GUS), green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidyl, c-myc, hemagglutinin or Flag for example ^TM(CT) sequence is expressed as the fusions with encoded polypeptide to label usually for Kodak, New Haven.This class label can be inserted in the polypeptide Anywhere, comprises carboxyl or amino arbitrary end.

D. regulatory region

The selection of waiting to be included in the regulatory region in the recombinant precursor depends on a number of factors, and includes but not limited to that the expression level of efficient, selectivity, inducibility, expectation and cell or tissue preferentially express.Expression by suitably selecting to regulate with the placement regulatory region relevant with encoding sequence encoding sequence is general issues to those skilled in the art.Can regulate and control transcribing of nucleic acid by similar mode.

Some suitable promotors only or main initial transcribing in some cell type.The selection of waiting to be included in the regulatory region in the recombinant precursor depends on a number of factors, and includes but not limited to that the expression level of efficient, selectivity, inducibility, expectation and cell or tissue preferentially express.Expression by suitably selecting to regulate with the placement regulatory region relevant with encoding sequence encoding sequence is general issues to those skilled in the art.Can regulate and control transcribing of nucleic acid by similar mode.

Some suitable regulatory regions only or main initial transcribing in some cell type.The method that is used for identifying and characterize at plant genome DNA regulatory region is known, for example comprises below with reference to the method for describing in the document: Jordano etc. Plant Cell, 1:855-866 (1989); Bustos etc., Plant Cell, 1:839-854 (1989); Green etc., EMBO J., 7:4035-4044 (1988); Meier etc., Plant Cell, 3:309-316 (1991); With Zhang etc., Plant Physiology, 110:1069-1079 (1996).

Multiple regulatory region examples of types is described hereinafter.Some regulatory regions of hereinafter pointing out and other regulatory regions are in U.S. Patent Application Serial Number 60/505,689; 60/518,075; 60/544,771; 60/558,869; 60/583,691; 60/619,181; 60/637,140; 60/757,544; 60/776,307; 10/957,569; 11/058,689; 11/172,703; 11/208,308; 11/274,890; 60/583,609; 60/612,891; 11/097,589; 11/233,726; 11/408,791; 11/414,142; 10/950,321; 11/360,017; PCT/US05/011105; PCT/US05/23639; PCT/US05/034308; PCT/US05/034343; And PCT/US06/038236; PCT/US06/040572; With description in more detail among the PCT/US07/62762.

For example, regulatory region p326, YP0144, YP0190, p13879, YP0050, p32449,21876, YP0158, YP0214, YP0380, PT0848, PT0633, YP0128, YP0275, PT0660, PT0683, PT0758, PT0613, PT0672, PT0688, PT0837, YP0092, PT0676, PT0708, YP0396, YP0007, YP0111, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115, YP0119, YP0120, YP0374, YP0101, YP0102, YP0110, YP0117, YP0137, YP0285, YP0212, YP0097, YP0107, YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879, PT0740, PT0535, PT0668, PT0886, PT0585, YP0381, YP0337, PT0710, YP0356, YP0385, YP0384, YP0286, YP0377, PD1367, PT0863, PT0829, PT0665, PT0678, YP0086, YP0188, YP0263, the sequence of PT0743 and YP0096 is shown in the sequence table of PCT/US06/040572; The sequence of regulatory region PT0625 is shown in the sequence table of PCT/US05/034343; The sequence of regulatory region PT0623, YP0388, YP0087, YP0093, YP0108, YP0022 and YP0080 is disclosed in the sequence table of U.S. Patent Application Serial Number 11/172,703; The sequence of regulatory region PR0924 is disclosed in the sequence table of PCT/US07/62762; The sequence of regulatory region p530c10, pOsFIE2-2, pOsMEA, pOsYp102 and pOsYp285 is shown in the sequence table of PCT/US06/038236.

Be understood that regulatory region can satisfy a kind of criteria for classification based on its activity in a kind of plant species, satisfies different criteria for classifications but be based on its activity in another kind of plant species.

I. the promotor of wide expression

When promotor promoted to transcribe in many (but must not be whole) plant tissue, it can b referred to as " wide expression ".For example, the promotor of wide expression can promote the sequence that effectively is connected transcribe in one or more branches, an end (top) and leaf, but promotes a little less than in as the tissue of root or stem or do not promote fully to transcribe.Another example is, the promotor of wide expression can promote the sequence that effectively is connected transcribe in one or more stems, branch, branch end (top) and leaf, but as flower and produce in the germinal tissue of seed can a little less than promotion or do not promote fully to transcribe.The limiting examples that can be included in the wide expression promotor in the nucleic acid construct provided herein comprises p326, YP0144, YP0190, p13879, YP0050, p32449,21876, YP0158, YP0214, YP0380, PT0848 and PT0633 promotor.Other example comprises cauliflower mosaic virus (CaMV) 35S promoter, mannopine synthase (MAS) promotor, 1 ' or 2 ' promotor from agrobacterium tumefaciens (Agrobacteriumtumefaciens) T-DNA, radix scrophulariae (figmort) mosaic virus 34S promotor, such as the actin promoter of rice actin promoter, and such as the ubiquitin promoter of corn ubiquitin-1 promotor.In some cases, the CaMV 35S promoter is excluded from the promotor category of wide expression.

Ii. root promotor

The active promotor of root is given in root tissue (for example root endodermis, epiblem or root vascular tissue) and being transcribed.In some embodiments, the active promotor of root is the preferential promotor of root, promptly only or mainly in root tissue, give and transcribing.The preferential promotor of root comprises YP0128, YP0275, PT0625, PT0660, PT0683 and PT0758 promotor.The preferential promotor of other roots comprises PT0613), PT0672, PT0688 and PT0837 promotor, it mainly drives in root tissue and transcribes, and drives than low degree ground in ovule and/or seed and transcribes.Other examples of the preferential promotor of root comprise the different subdomain of the Gent of CaMV 35S promoter (people such as Lam, Proc.Natl.Acad.Sci. U.S. 86:7890-7894 (1989)), people such as Conkling, the root cells specific promoter and the tobacco RD2 promotor of Plant Physiol.93:1203-1211 (1990) report.

Iii. ripe endosperm promotor

In some embodiments, it can be useful driving the promotor of transcribing in the endosperm in maturation.Begin from transcribing usually after fertilization of ripe endosperm promotor, and mainly betide in the endosperm tissue during the seed development, and the highest in the cell phase (cellularization phase) usually.Only is promoters active in the main endosperm in maturation, although can use in its hetero-organization also promoters active sometimes.The limiting examples that can be included in endosperm promotor in the maturation in the nucleic acid construct provided herein comprises: the rapeseed protein promotor, the Arcelin-5 promotor, phaseolin promoter (people Plant Cell 1 (9): 839-853 (1989) such as Bustos), Trypsin inhibitor SBTI promotor (people Plant Cell 1 (6): 609-621 (1989) such as Riggs), ACP promotor (people Plant Mol Biol such as Baerson, 22 (2): 255-267 (1993)), stearyl-ACP desaturase promotor (people Plant Physiol 104 (4): 167-176 (1994) such as Slocombe), soybean α ' subunit the promotor of beta-conglycinin (conglycinin) (people Proc.Natl.Acad.Sci. U.S. 83:8560-8564 (1986) such as Chen), oleosin promotor (people Plant MolBiol 34 (3): 549-555 (1997) such as Hong) and zein promotor are (as 15kD zein promotor, 16kD zein promotor, 19kD zein promotor, 22kD zein promotor and 27kD zein promotor).Also suitable is from the paddy protein-1 gene Osgt-1 promotor (people Mol.Cell Biol.13:5829-5842 (1993) such as Zheng), beta-amylase gene promoter and hordein gene promotor.The endosperm promotor comprises YP0092, PT0676 and PT0708 promotor in other maturations.

Iv. ovary is organized promotor

The ovary tissue as ovule wall and mesocarp in promoters active also can be useful, for example polygalacturonic acid anhydrides enzyme (polygalacturonidase) promotor, banana TRX promotor, melon actin promoter, YP0396 and PT0623.Mainly the example of promoters active is YP0007, YP0111, YP0092, YP0103, YP0028, YP0121, YP0008, YP0039, YP0115, YP0119, YP0120 and YP0374 in ovule.

V. blastular/early stage endosperm promotor

In order to realize expressing at blastular/early embryo Ruzhong, can use following regulatory region, described regulatory region or has activity in the polar core precursor in polar core (polar nuclei) and/or centrocyte, but in ovum or ovum precursor activity is not arranged.Only be only or mainly at polar core or its precursor, and/or drive expression promoter in the centrocyte.Use blastular/early stage preferential promotor of endosperm also can find to enter the transcriptional profile of early stage endosperm development, although during the cell phase and transcribe afterwards late and significantly reduce usually in the endosperm development from the polar core expansion.Expression in the zygote or the embryo of growing does not exist with blastular/early stage endosperm promotor usually.

Promotor applicatory comprises the promotor from following gene: Arabidopis thaliana viviparous-1 (seeing GenBank U93215); Arabidopis thaliana atmycl (sees Urao (1996) Plant Mol.Biol., 32:571-57; Conceicao (1994) Plant, 5:493-505); Arabidopis thaliana FIE (GenBank AF129516); Arabidopis thaliana MEA; Arabidopis thaliana FIS2 (GenBank AF096096) and FIE 1.1 (United States Patent (USP) 6,906,244).Other promotors applicatory can comprise the promotor from following gene: corn MAC1 (sees Sheridan (1996) Genetics, 142:1009-1020); Corn C at3 (sees GenBank L05934; Abler (1993) Plant Mol.Biol., 22:10131-1038).Other promotors comprise following arabidopsis thaliana promoter: YP0039, YP0101, YP0102, YP0110, YP0117, YP0119, YP0137, DME, YP0285 and YP0212.Other promotors applicatory comprise following rice promoters: p530c10, pOsFIE2-2, pOsMEA, pOsYp102 and pOsYp285.

Vi. embryo promotor

The preferential regulatory region of transcribing that drives in the zygote cell of after fertilization can provide embryo preferential expression.Only is to drive the promotor of transcribing in the preferential embryo in early days before the heart-shaped stage, but the expression in late period and the mature embryo also is suitable.The preferential promotor of embryo comprises barley lipid transfer protein (Ltp1) promotor (Plant Cell Rep (2001) 20:647-654), YP0097, YP0107, YP0088, YP0143, YP0156, PT0650, PT0695, PT0723, PT0838, PT0879 and PT0740.

Vii. photosynthetic tissue's promotor

Promoters active is given in chlorenchyma (as leaf and stem) and being transcribed in photosynthetic tissue.Only be only or mainly in this class tissue, drive expression promoter.The example of this class promotor comprises ribulose-1,5-bisphosphate, 5-bisphosphate carboxylase (RbcS) promotor is as the RbcS promotor from American Larch (Larixlaricina), pine tree cab6 promotor (people Plant CellPhysiol.35:773-778 (1994) such as Yamamoto), Cab-1 gene promoter (people Plant Mol.Biol.15:921-932 (1990) such as Fejes) from wheat, CAB-1 promotor (people Plant Physiol.104:997-1006 (1994) such as Lubberstedt) from spinach, cab1R promotor (people Plant Cell 4:971-981 (1992) such as Luan) from rice, pyruvate orthophosphate dikinase (PPDK) promotor (people Proc Natl Acad.Sci U.S. 90:9586-9590 (1993) such as Matsuoka) from corn, tobacco Lhcb1*2 promotor (people Plant Mol.Biol.33:245-255 (1997) such as Cerdan), Arabidopis thaliana SUC2 sucrose-H+ symport body promotor (people Planta 196:564-570 (1995) such as Truernit) and from the quasi-sac film protein matter promotor (psaD of spinach, psaF, psaE, PC, FNR, atpC, atpD, cab, rbcS).Other photosynthetic tissue's promotors are PT0535, PT0668, PT0886, PR0924, YP0144, YP0380 and PT0585.

Viii. vascular tissue promotor

The example that has high reactivity or preferential active promotor in vascular bundle comprises YP0087, YP0093, YP0108, YP0022 and YP0080.The preferential promotor of other vascular tissues comprises cell wall protein GRP 1.8 promotors (Keller and the Baumgartner that is rich in glycine, PlantCell, 1051-1061 (1991)), Herba Commelinae belongs to (Commelina) macula lutea virus (CoYMV) promotor (Medberry etc. 3 (10):, Plant Cell, 185-192 (1992)) and rice Dong Gelu baculovirus (tungro bacilliform virus 4 (2):, RTBV)) promotor (Dai etc., Proc.Natl.Acad.Sci.USA, 101 (2): 687-692 (2004)).

Ix. inducible promoter

Inducible promoter responds external stimulus (as chemical substance or environmental stimulus) and gives and transcribing.For example, inducible promoter can respond hormone (as Plant hormones regulators,gibberellins or ethene) or response light photograph or arid and give and transcribing.Drought-inducible promoter comprises YP0380, PT0848, YP0381, YP0337, YP0337, PT0633, YP0374, PT0710, YP0356, YP0385, YP0396, YP0388, YP0384, PT0688, YP0286, YP0377, PD1367 and PD0901.The example of nitrogen inducible promoter comprises PT0863, PT0829, PT0665 and PT0886.The example of dark inducible promoter comprises PR0924 and PT0678.An example of salt inductive promotor is rd29A (Kasuga etc. (1999) Nature Biotech 17:287-291).

X. basic promotor

The basis promotor is the required essential minmal sequence of transcription complex assembling of transcription initiation.The basis promotor usually comprise can be in the transcription initiation site upstream about 15 " TATA box " elements between about 35 Nucleotide.The basis promotor also can comprise " CCAAT box " element (being generally sequence C CAAT) and/or GGGCG sequence, and it can be generally upstream about 60 between about 120 Nucleotide between transcriptional start point upstream about 40 and about 200 Nucleotide.

Xi. other promotors

The promotor of other kinds includes but are not limited to that branch is preferential, callus is preferential, the algal filament cell is preferential, the guard cell preferential as PT0678, stem tuber is preferential, parenchymatous cell is preferential and old and feeble preferential promotor.Described in above-mentioned referenced patent application, the promotor that is named as YP0086, YP0188, YP0263, PT0758, PT0743, PT0829, YP0119 and YP0096 is also useful.

Xii. other regulatory regions

5 ' non-translational region (UTR) can be included in the nucleic acid construct as herein described.5 ' UTR is transcribed, but is not translated, and between the starting point and transcription initiation codon of transcript, and can comprise+1 Nucleotide.3 ' UTR can be between Transcription Termination codon and transcript end.UTR can have as improving mRNA stability or weakening the concrete function of translation.The example of 3 ' UTR includes but not limited to polyadenylation signal and transcription termination sequence, for example nopaline synthase terminator sequence.

Be to be understood that more than a kind of regulatory region can be present in the recombination of polynucleotide, for example intron, enhanser, active region, upstream, transcription terminator and induction type element.Therefore, for example can effectively be connected with the polynucleotide sequence of coding salt and/or oxidative stress patience regulation and control polypeptide more than a kind of regulatory region.

Regulatory region (for example promotor of native gene) can obtain by chemosynthesis, or obtains by subclone from the genomic dna that comprises this type of regulatory region.The nucleic acid that comprises this class regulatory region also can comprise the flanking sequence that contains restriction enzyme site, and described restriction enzyme site helps operation subsequently.

Perhaps, can use two component system to finish unconventionality expression, wherein first component is made up of transgenic plant, described transgenic plant comprise the activating transcription factor that effectively is connected with promotor, second component is made up of following transgenic plant, and described transgenic plant comprise the nucleic acid molecule of the present invention that effectively is connected with the target binding sequence/district of activating transcription factor.With two kinds of transgenic plant hybridization, and nucleic acid molecule of the present invention is expressed in described plant offspring.In another alternate embodiment of the present invention, can transform by sequence in the into same transgenic plant strain and finish unconventionality expression two component system.

IV. transgenic plant and vegetable cell

A. transform

The invention still further relates to the transgenic plant cells and the plant that comprise at least a recombinant nucleic acid construct described herein.Plant or vegetable cell can transform by construct is integrated in its genome, promptly can be stabilized conversion.The cell of stable conversion keeps the nucleic acid of introducing usually when each cell fission.Plant or vegetable cell also can be made the construct unconformability advance in its genome by instantaneous conversion.The cell of instantaneous conversion is lost the nucleic acid construct that all or part is introduced usually when each cell fission, make the nucleic acid of introducing can not detect in daughter cell after the cell fission of capacity.The transgenic plant of instantaneous conversion and stable conversion and vegetable cell but can be used in the method as herein described.

The transgenic plant of using in the methods described herein can be formed the part or all of of whole strain plant.This class plant can be in incubator, in the greenhouse or big Tanaka, grow in the mode that is fit to described species.Transgenic plant can be realized concrete purpose according to the expectation breeding, for example recombinant nucleic acid are introduced in other strains, and recombinant nucleic acid is transferred to other species, or the proterties of further selecting other to want.Perhaps, for being fit to the species of this class technology, transgenic plant can nourish and generate.When using in this article, transgenic plant also represent to have genetically modified initial transgenic plant offspring.Can cultivate the seed that transgenic plant produce,, obtain the seed that nucleic acid construct is isozygotied then with its selfing (or outcross and selfing).

Can in suspension culture or tissue or organ cultures, cultivate transgenic plant.With regard to purpose of the present invention, can use solid and/or liquid tissue culture technique.When using solid medium, transgenic plant cells directly can be placed on the substratum, or place on the filter, then this filter be contacted placement with substratum.When using liquid nutrient medium, transgenic plant cells can be placed on the floatation device (for example porous-film of contact liq substratum).Solid medium can be Murashige and Skoog (MS) substratum for example, its growth hormone that contains agar and suitable concn for example 2, the 4-dichlorphenoxyacetic acid (2,4-D) and the phytokinin of suitable concn (cytokinin) phytokinin (kinetin) for example.

When using the vegetable cell of instantaneous conversion, can comprise the reporter gene sequence that coding has the reporter gene polypeptide of reporter gene activity in the step of converting, and can be after conversion the suitable time carry out reporter gene activity or express and measure.The suitable time of measuring is generally and transforms back about 1-21 days, for example about 1-14 days, about 1-7 days or about 1-3 days.The use of instantaneous measurement is especially convenient for the real-time analysis in the different plant species, is used for perhaps confirming that concrete recipient cell did not before confirm the allos salinity patience and/or the oxidative stress patience regulation and control polypeptide expression of its expression.

The technology that nucleic acid is introduced in monocotyledons and the dicotyledons is known in the art, and includes but not limited to that conversion, electroporation and the particle gun of agriculture bacillus mediated conversion, virus vector mediation transform, and for example United States Patent (USP) 5,538,880; 5,204,253; 6,329,571 and 6,013,863.If the tissue that uses cell or cultivation can be by technology well known by persons skilled in the art aftergrowth from the culture that transforms when then needing as the receptor tissue that transforms.

B. screening/selection

Can screen and/or select following kind of group members in the transgenic plant population, it has proterties or phenotype that genetically modified expression is given.For example, can screen following plant from the offspring population of single transformation event, described plant has salinity patience and/or the oxidative stress patience regulation and control polypeptide or the expression of nucleic acids level of expectation.Can use physics and biochemical method to identify expression level.These methods comprise Southern analysis or the pcr amplification that is used to detect polynucleotide; Be used to detect Northern trace, S1 RNase protection, primer extension or the RT-PCR amplification of rna transcription thing; Be used to detect the enzyme of polypeptide and polynucleotide or the enzymatic determination of ribozyme activity; With the gel electrophoresis of protein that is used to detect polypeptide, Western trace, immunoprecipitation and enzyme-linked immunoassay.Also can use for example in situ hybridization of other technologies, enzyme dyeing and immunostaining to detect the existence or the expression of polypeptide and/or polynucleotide.The method of carrying out the technology of all references is known.As a kind of alternatives, can in comprising the plant population of independent transformation event, screen the plant with anticipant character, the salinity patience of for example being regulated and/or oxidative stress patience level.Can select in a generation or in many generations and/or in and/or screen more than a geographical position.In some cases, can grow and select transgenic plant, described condition to induce under the following conditions expects phenotype or produces the necessary mode of expectation phenotype with other in transgenic plant.In addition, can application choice and/or screening during the concrete etap, expectation shows described phenotype by plant in the described etap.Can select and/or screen and select the transgenic plant that for lacking genetically modified control plant, in salinity patience and/or oxidative stress patience level, have statistically-significant difference.The transgenic plant of selecting or screening are compared the phenotype with change with corresponding control plant, described in this paper " transgenic plant phenotype " part.

Can screen and/or select following kind of group members in the transgenic plant population, it has proterties or phenotype that genetically modified expression is given.For example, can screen following plant from the offspring population of single transformation event, described plant has salt and/or the oxidative stress patience regulation and control polypeptide and/or the expression of nucleic acids level of expectation.Can use physics and biochemical method to identify expression level.These methods comprise Southern analysis or the pcr amplification that is used to detect polynucleotide; Be used to detect Northern trace, S1 RNase protection, primer extension or the RT-PCR amplification of rna transcription thing; Be used to detect the enzyme of polypeptide and polynucleotide or the enzymatic determination of ribozyme activity; With the gel electrophoresis of protein that is used to detect polypeptide, Western trace, immunoprecipitation and enzyme-linked immunoassay.Also can use for example in situ hybridization of other technologies, enzyme dyeing and immunostaining to detect the existence or the expression of polypeptide and/or polynucleotide.The method of carrying out the technology of all references is known.As a kind of alternatives, can in comprising the plant population of independent transformation event, screen the plant with anticipant character, the salinity patience of for example being regulated and/or oxidative stress patience level.Can select in a generation or in many generations and/or in and/or screen more than a geographical position.In some cases, can grow and select transgenic plant, described condition to induce the expectation phenotype under the following conditions or otherwise be that to produce the expectation phenotype in the transgenic plant necessary.In addition, can application choice and/or screening during the concrete etap, expectation shows described phenotype by plant in the described etap.Can select and/or screen and select for lacking genetically modified control plant, the transgenic plant that in salinity patience and/or oxidative stress patience level, have statistically-significant difference.The transgenic plant of selecting or screening are compared the phenotype with change with corresponding control plant, described in this paper " transgenic plant phenotype " part.

C. plant species

Can use polynucleotide described herein and carrier to transform a large amount of monocotyledonss and dicotyledons and vegetable cell system, comprise species: Acanthaceae (Acanthaceae) from one of following section, green onion section (Alliaceae), six go out Hua Ke (Alstroemeriaceae), Amaryllidaceae (Amaryllidaceae), Apocynaceae (Apocynaceae), Palmae (Arecaceae), composite family (Asteraceae), Berberidaceae (Berberidaceae), Bixaceae (Bixaceae), Cruciferae (Brassicaceae), Bromelia family (Bromeliaceae), Cannabaceae (Cannabaceae), Caryophyllaceae (Caryophyllaceae), cephalotaxaceae (Cephalotaxaceae), Chenopodiaceae (Chenopodiaceae), Colchicaceae (Colchicaceae), Curcurbitaceae (Cucurbitaceae), Dioscoreaceae (Dioscoreaceae), Ephedraceae (Ephedraceae), Erythroxylaceae (Erythroxylaceae), Euphorbiaceae (Euphorbiaceae), pulse family (Fabaceae), Labiatae (Lamiaceae), flax family (Linaceae) (Linaceae), Lycopodiaceae (Lycopodiaceae), Malvaceae (Malvaceae), Melanthiaceae (Melanthiaceae), Musaceae (Musaceae), Myrtaceae (Myrtaceae), Nyssaceae (Nyssaceae), papaveracease (Papaveraceae), Pinaceae (Pinaceae), Plantaginaceae (Plantaginaceae), Gramineae (Poaceae), the Rosaceae (Rosaceae), Rubiaceae (Rubiaceae), Salicaceae (Salicaceae), Sapindaceae (Sapindaceae), Solanaceae (Solanaceae), taxaceae (Taxaceae), Theaceae (Theaceae) or Vitaceae (Vitaceae).

Suitable species can comprise the member with the subordinate: Abelmoschus (Abelmoschus), Abies (Abies), Acer (Acer), Agrostis (Agrostis), allium (Allium), six go out Pittosporum (Alstroemeria), pine apple nanas belongs to (Ananas), punching Nelumbo (Andrographis), Andropogon (Andropogon), artemisia (Artemisia), giantreed belongs to (Arundo), Atropa (Atropa), Berberis (Berberis), Beta (Beta), Bixa (Bixa), Btassica (Brassica), calendulin (Calendula), Camellia (Camellia), camplotheca acuminata belongs to (Camptotheca), Cannabis (Cannabis), Capsicum (Capsicum), red blue Pittosporum (Carthamus), Vinca (Catharanthus), Cephalotaxus (Cephalotaxus), crowndaisy chrysanthemum belongs to (Chrysanthemum), quinine belongs to (Cinchona), Citrullus (Citrullus), Coffea (Coffea), Colchicum (Colchicum), Coleus (Coleus), Cucumis (Cucumis), Cucurbita (Cucurbita), Cynodon (Cynodon), Datura (Datura), Carnation (Dianthus), Digitalis (Digitalis), Wild yam (Dioscorea), oil palm belongs to (Elaeis), Ephedra (Ephedra), Plumegrass (Erianthus), coca belongs to (Erythroxylum), eucalyptus belongs to (Eucalyptus), festuca (Festuca), Fragaria (Fragaria), snowdrop (Galanthus), Glycine (Glycine), cotton belongs to (Gossypium), Helianthus (Helianthus), Hevea (Hevea), Hordeum (Hordeum), poison tobacco (Hyoscyamus), Jatropha (Jatropha), Lactuca (Lactuca), linum (Linum), lolium (Lolium), lupinus (Lupinus), tomato belongs to (Lycopersicon), Lycopodium (Lycopodium), cassava (Manihot), Medicago (Medicago), Mentha (Mentha), awns belongs to (Miscanthus), Musa (Musa), Nicotiana (Nicotiana), Oryza (Oryza), Panicum (Panicum), papaver (Papaver), elargol Chrysanthemum (Parthenium), Pennisetum (Pennisetum), Petunia (Petunia), reed canary grass belongs to (Phalaris), ladder forage spp (Phleum), Pinus (Pinus), annual bluegrass belongs to (Poa), Euphorbia (Poinsettia), Populus (Populus), Rauwolfia (Rauwolfia), Ricinus (Ricinus), rose (Rosa), saccharum (Saccharum), willow belongs to (Salix), tetterwort (Sanguinaria), Root of Common Scopolia belongs to (Scopolia), Secale (Secale), Solanum (Solanum), jowar (sorghum), the rice grass belongs to (Spartina), spinach belongs to (Spinacea), chrysanthemum artemisia (Tanacetum), Taxus (Taxus), Theobroma (Theobroma), triticale belongs to (Triticosecale), Triticum (Triticum), Uniola, Veratrum (Veratrum), Vinca (Vinca), Vitis (Vitis) and Zea (Zea).

Suitable species comprise the Panicum species, the sorghum species, the awns species, the saccharum species, the Plumegrass species, the Populus species, big bluestem grass (Andropogon gerardii), napier grass (Pennisetumpurpureum), silk ribbon grass (Phalaris arundinacea) (reed canary grass), Bermuda grass (Cynodondactylon) (Herba cynodontis), Festuca Arundinacea (Festuca arundinacea), grassland net thatch (Spartinapectinata), alfalfa (Medicago sativa), giantreed (Arundo donax), rye (Secalecereale), willow species (willow), eucalyptus species (eucalyptus globolus tree), triticale belongs to (Triticum-wheat X rye) and bamboo.

Suitable species also comprise Sunflower Receptacle (Helianthus annuus), safflower (Carthamustinctorius), manioca (Jatropha curcas), castor-oil plant (Ricinus communis), oil palm (Elaeis guineensis), flax (Linum usitatissimum) and leaf mustard (Brassicajuncea).

Suitable species also comprise beet (Beta vulgaris) and cassava (Manihot esculenta).

Suitable species also comprise tomato (Lycopersicon esculentum), lettuce (Lactucasativa), powder bajiao banana (Musa paradisiacal) (banana), potato (Solanum tuberosum) (potato), wild cabbage (Brassica oleracea) (asparagus broccoli, Cauliflower, brussels sprouts), tea tree (Camellia sinensis) (tea), strawberry (Fragaria ananassa), cocoa (Theobromacacao), fruitlet coffee (Coffea arabica) (coffee), grape (Vitis vinifera), pineapple (Ananas comosus) (pineapple), capsicum (Capsicum annum) (capsicum and pimento), onion (Allium cepa), muskmelon (Cucumis melo) (melon), cucumber (Cucumis sativus), winter squash (Cucurbita maxima) (pumpkin), pumpkin (Cucurbita moschata) (pumpkin), spinach (Spinacea oleracea), watermelon (Citrullus lanatus), coffee ambrette (Abelmoschusesculentus) (swordweed) and eggplant (Solanum melongena).

Suitable species also comprise opium poppy (Papaver somniferum), east opium poppy (Papaverorientale), european yew (Taxus baccata), yewtree (Taxus brevifolia), sweet wormwood (Artemisia annua), hemp (Cannabis sativa), camplotheca acuminata (Camptothecaacuminate), Vinca (Catharanthus roseus), Vinca (Vinca rosea), Peruvian bark (Cinchona officinalis), Colchicum autumnale (Colchicum autumnale), Veratrumcalifornica., narrow leaf foxglove (Digitalis lanata), foxglove (Digitalis purpurea), the Wild yam species, Herba Andrographis (Andrographis paniculata), belladonna (Atropabelladonna), Datura stomonium, the Berberis species, the Cephalotaxus species, ephedra sinica (Ephedra sinica), the Ephedra species, coca (Erythroxylum coca), Galanthuswornorii, the Root of Common Scopolia species, Herba Lycopodii serrati (Lycopodium serratum=Huperziaserrata), the Lycopodium species, snakewood (Rauwolfia serpentina), the Rauwolfia species, bloodroot (Sanguinaria canadensis), the poison tobacco species, Potmarigold Calendula (Calendulaofficinalis), exquisite chrysanthemum (Chrysanthemum parthenium), Coleus forskohlii Briq. (Coleusforskohlii) and feverfew (Tanacetum parthenium).

Suitable species comprise that also guayule (Parthenium argentatum), Hevea species (rubber), spearmint (Mentha spicata) (peppermint), lavender (Mentha piperita) (peppermint), Arnotto (Bixa orellana) and six go out the Pittosporum species.

Suitable species also comprise rose species (rose), carnation (Dianthuscaryophyllus) (carnation), Petunia species (petunia) and painted euphorbia (Poinsettiapulcherrima).

Suitable species also comprise tobacco (Nicotiana tabacum), Lupinus albus (Lupinusalbus) (lupine), Uniola paniculata (oat), bentgrass (Agrostis species), Populus tremuloides (white poplar), Pinus species (pine tree), Abies species (fir), Acer species (maple), barley (Hordeum vulgare), English grass (Poa pratensis) (bluegrass), lolium species (rye grass) and timothy grass (Phleum pratense) (thimothy grass).

Therefore, can in wide in range plant species scope, use described method and composition, comprise from dicot genus Btassica, red blue Pittosporum, Glycine, cotton genus, Helianthus, Jatropha, elargol Chrysanthemum, Populus and Ricinus; Belong to the species of oil palm genus, festuca, Hordeum, lolium, Oryza, Panicum, Pennisetum, ladder forage spp, annual bluegrass genus, saccharum, Secale, Solanum, triticale genus, Triticum and Zea with unifacial leaf.In some embodiments, plant is the member of switchgrass, dichromatism chinese sorghum (Chinese sorghum, Schrock), huge Chinese silvergrass (awns genus), saccharum species (energy sugarcane), baisam poplqr (white poplar), Zea mays (corn), soybean (soya bean), colea (canola oil dish), common wheat (wheat), upland cotton (cotton), rice (paddy rice), Sunflower Receptacle, alfalfa (clover), beet or cattailmillet (pearl millet) species.

In certain embodiments, can use polynucleotide described herein and carrier to transform a large amount of unifacial leaves and dicotyledons and vegetable cell system, wherein this class plant is the hybrid of different plant species or species mutation (for example saccharum species X awns species).

D. transgenic plant phenotype

In some embodiments, wherein salinity and/or the oxidative stress regulation and control polypeptide expression plant that obtains regulating and control can have the salinity and/or the oxidative stress patience level of raising.For example, can in transgenic plant, express the regulation and control of salinity as herein described and/or oxidative stress polypeptide, the salinity that is improved and/or oxidative stress patience level.Compare with the level in the corresponding control plant of express transgenic not, salinity and/or oxidative stress patience level can improve at least 2%, and for example 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60% or greater than 60%.

Nucleic acid molecule of the present invention and polypeptide are interested, because when nucleic acid molecule (is promptly expressed in the non-natural site by unconventionality expression, or reach with the scale that is enhanced or reduce with respect to wild-type) time, they produce following plant, described plant is compared with wild-type plant, show the salt patience and/or the oxidative stress patience that improve, as hereinafter disclosed a plurality of result of experiment part confirmation.Particularly, can have a large amount of modified features of comparing with wild-type plant with nucleic acid molecule of the present invention and polypeptide plant transformed.The example of modified feature comprises photosynthetic efficiency, seedling area and the biomass that can pass through plant height, leaf or lotus throne area or dry-matter measurement.Can for example observe and measure modified feature in seed, seedling, bolting, aging or the like in the different development of plants stages.Usually, salt or oxidation patience can be expressed as the ratio or the combination of measurement, for example salt growth index value or Whitfield's ointment growth index value.For example, with sequence plant transformed of the present invention can show at least 5%, at least 10%, at least 25%, at least 50%, at least 75%, at least 100%, at least 200%, at least 300%, at least 400% or even at least 500% SGI, seedling area and/or SAGI value improve.These proterties can be used to exploitation or maximization plant prod.For example, nucleic acid molecule of the present invention and polypeptide are used to improve expression of gene, and this causes that plant comparing biomass, growth velocity and/or the seedling vigor with raising with the wild-type plant under the same terms under salt and/or the oxidizing condition.

Because disclosed sequence and method improve nourishing and growing and growth velocity under salt and/or the oxidative stress condition,, disclosed method is increased in the plant-growth in the growing plants under salt and/or the oxidizing condition so can be used in.For example, plant of the present invention is compared the light compositing efficient of demonstration raising and the seedling area of raising with following plant under salt and/or oxidizing condition, and described plant is that same species is still not genetically modified for mainly nourishing and growing.The example that biomass produce to improve comprise with the same terms under the biomass of wild-type plant of same species produce and compare, at least 5%, at least 20% or even at least 50% raising.

Usually, when using suitable parameter or nonparametric statistics (for example χ side's check, student t-check, Mann-Whitney check or F-check), transgenic plant or cell o'clock are considered to statistics to the difference of salinity and/or oxidative stress patience amount in p≤0.05 for control plant or cell significant.In some embodiments, the difference of salinity and/or oxidative stress patience amount in p＜0.01, p＜0.005 or p＜0.001 o'clock is that statistics is significant.

Estimate the phenotype of transgenic plant with respect to control plant.The polypeptide amount that shows when plant or the mRNA amount of coded polypeptide are less than 10% of purpose plant demonstration amount, for example be less than 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.01% or at 0.001% o'clock, plant is known as " not expressing " described polypeptide.Can use following method evaluation expression, described method comprises that for example RT-PCR, Northern trace, S1 RNase protection, primer extension, Western trace, gel electrophoresis of protein, immunoprecipitation, enzyme-linked immunoassay, chip are measured and mass spectroscopy.Should be noted that if polypeptide is preferential at tissue or the control of wide expression promotor under express, then can be in the tissue of whole strain plant or selection evaluation expression.Similarly, if polypeptide specified time (for example grow or induce after specified time) express, then can be in the time period of expectation evaluation expression optionally.

V. plant breeding

Genetic polymorphism is an allelic sequence variation discrete in the population.Usually, with 1% or the allelotrope that exists more be considered to genetic polymorphism.The discovery that polypeptide disclosed herein can be regulated and control salinity patience and/or oxidative stress patience content can be used for plant breeding, because the locus of this class polypeptide is shown that to a certain degree chain genetic polymorphism more may be relevant with the variation of salinity patience and/or oxidative stress patience proterties.For example, the genetic polymorphism related with this class polypeptide gene seat more may be used for the auxiliary procedure of breeding of marker, is created in the strain that has the expectation regulation and control in salinity patience and/or the oxidative stress patience proterties.

Therefore, one aspect of the present invention comprise identify one or more genetic polymorphisms whether with the relevant method of variation of salinity patience and/or oxidative stress patience proterties.Whether these class methods relate to the genetic polymorphism of measuring in the given population chain with the locus demonstration of one of polypeptide shown in Fig. 1 to 6 and/or its functional homologue (such as but not limited to functional homologue shown in the sequence table).Association between the existence of one or more genetic polymorphisms in the variation of salinity patience proterties and/or oxidative stress patience proterties and the population plant in the measurement plant population, thus identify whether one or more genetic polymorphisms are relevant with the variation of proterties.If concrete allelicly have statistics the expectation regulation and control with salinity patience and/or oxidative stress patience proterties are relevant significantly, then allelotrope is relevant with the variation of described one or both proterties, and can be used as the marker of one or more described proterties.On the other hand, if concrete allelic existence not with the expectation the regulation and control significant correlation, then the variation with one or more described proterties is not relevant for allelotrope, and the unavailable thing of marking.

Certain methods can be used for following population, and described population contains the exogenous nucleic acid of naturally occurring endogenous polypeptide rather than coding said polypeptide, and promptly described population is not genetically modified for exogenous nucleic acid.Yet, should be appreciated that the population that is applicable to described method can contain the transgenosis at another various trait (for example Herbicid resistant).

Be applicable to that the genetic polymorphism in these class methods comprises that simple sequence repeats the fragment length polymorphism (AFLP) and the restriction fragment length polymorphism (RFLP) of the rapid amplifying (RAPD) of (SSR, or little satellite), polymorphic DNA, single nucleotide polymorphism (SNP), amplification.For example can following evaluation SSR polymorphism: preparation sequence-specific probe, and by the individual amplification template DNA of PCR from the purpose population.If the both sides of probe SSR in population then can produce different big or small PCR products.See for example United States Patent (USP) 5,766,847.Perhaps, can identify the SSR polymorphism at Southern trace by using one or more PCR products as probe from Different Individual in the population.See U.H.Refseth etc., (1997) Electrophoresis 18:1519.The evaluation of RFLP is for example at (Methods in Molecular Biology, the 82nd volume, " ArabidopsisProtocols " such as Alonso-Blanco, the 137-146 page or leaf, J.M.Martinez-Zapater and J.Salinas compile, c.1998, Humana Press, Totowa, NJ); Burr (" Mapping Genes with RecombinantInbreds ", 249-254 page or leaf, Freeling, M. and V.Walbot (volume), The MaizeHandbook, c.1994, Springer-Verlag New York, Inc.:New York, NY, USA; Berlin Germany; Genetics such as Burr (1998) 118:519; And Gardiner, J. etc., (1993) Genetics 134:917) the middle discussion.The evaluation of AFLP is discussed in for example EP 0 534 858 and United States Patent (USP) 5,878,215.

In some embodiments, this method relates to the plant lines breeding.These class methods are used the genetic polymorphism of evaluation as indicated above so that develop following strain in the auxiliary procedure of breeding of marker, described product tie up to the change that has expectation in one or more salinity patience and/or the oxidative stress patience proterties.In case it is relevant with the variation of proterties that suitable genetic polymorphism is accredited as, then identify a relevant polymorphic allelic strain or the many strains bion of variation that has with expectation.In the procedure of breeding, use these plants then, polymorphic allelotrope is made up with a large amount of other allelotrope on other locus relevant with the expectation variation.Be applicable to that the technology in the plant breeding program is known in the art, and include but not limited to, backcross, mix selection (mass selection), pedigree breeding, mix selection (bulkselection), hybridize and recurrent selections with other populations.These technology can be used separately or be used in combination with one or more other technologies in the procedure of breeding.Therefore, make plant selfing that every strain identifies or produce seed, make described seed germination form progeny plants subsequently with different plant hybridization.Make this class progeny plants selfing of at least one strain then or form follow-up offspring (subsequent progeny generation) from generation to generation with different plant hybridization.The procedure of breeding can suitably repeat 0 to 5 generation with selfing or outcross again, thereby realizes the homogeneity and the stability of expectation in the plant lines that obtains, and the described plant lines that obtains keeps polymorphic allelotrope.In most of procedure of breeding, should carry out concrete polymorphic allelotrope analysis from generation to generation at each, although can analyze from generation to generation at alternative when needing.

In some cases, also carry out other useful proterties are arrived selection, for example select fungus resistant or bacterial resistance.At the selection of these other proterties of class can be before evaluation has the polymorphic allelic bion of expectation, during or carry out afterwards.

VI. make article (article of manufacture)

Transgenic plant provided herein serve many purposes in agricultural and Energy production industry.For example, transgenic plant described herein can be used for making animal-feed and food.Yet this class plant especially can be used as the raw material of Energy production usually.

Transgenic plant described herein are for the control plant that lacks exogenous nucleic acid, and per hectare produces higher cereal and/or biomass yield usually.In some embodiments, when growing under the condition of the input (for example fertilizer and/or water) that reduces, these class transgenic plant per hectare for control plant provides and equates or even the cereal and/or the biomass yield that improve.Therefore, can use these class transgenic plant for example to provide yielding stability under the arid in the condition of lower input cost and/or environment-stress.In some embodiments, plant described herein has following composition, and described composition allows more effectively to be processed into free sugar and is processed into alcohol subsequently, is used for Energy production.In some embodiments, this class plant every kilogram of vegetable material for control plant provides the output of higher ethanol, butanols, other biological fuel molecule and/or sugared deutero-by product.By equate or even the production cost that reduces under the output higher with respect to contrast is provided, transgenic plant as herein described have been improved earning rate to peasant and processor, and the human consumer has been reduced cost.

Can seed from transgenic plant described herein be maintained a good state and pack in wrapping material by means known in the art, form and make article.Wrapping material for example paper and cloth are well known in the art.The packing of seed can have describes the wherein mark of seed speciality, for example is sewn on label or tag on the wrapping material, is printed on the mark on the wrapping material or inserts mark in the packing.

Enhanced salt and/or oxidative stress patience provide following chance: cultivation of crops under salt or oxidative stress condition and do not have the destruction of salt inductive ion imbalance, water homeostasis, metabolic inhibition, stunted growth that the infringement and/or the necrocytosis of film caused and the output of minimizing.Under salt or oxidative stress condition the ability of culturing plants can cause can plough ground comprehensive expansion and at present because salinity that improves or oxidative stress condition have the output of the raising in the soil that hangs down productivity.

Seed or seedling vigor are significantly to influence the key character that plant (for example crop plants) success is grown.Hostile environment condition for example salt and/or oxidizing condition can influence plant-growth cycle, seed germination and seedling vigor (promptly vitality and intensity can be variant between the plant-growth of success under this class condition and failure).The seedling vigor is generally defined as and comprises following seed characteristics, and described seed characteristics is determined " under field condition on a large scale normal seedling fast, the homogeneous potential that takes place and grow ".Therefore, it is favourable developing following plant seed, and described plant seed has the vigor of raising, especially under salinity that improves and/or oxidative stress condition.

For example, for example for the generation of rice, corn, wheat or the like, the seed vitality of raising can be favourable for the cereal plant.For these crops, salinization and/or oxidation can slow down or stop to sprout and growth usually.Therefore the gene that the seed vitality that improves under searching and salt and/or the oxidative stress condition is relevant produces improved botanical variety (Walia etc. (2005) Plant Physiology 139:822-835).

The present invention will further describe in following examples, and described embodiment does not limit scope of the present invention described in claims.

VII. embodiment

Embodiment 1: agriculture bacillus mediated Arabidopis thaliana transforms

Host plant and transgenosis: independently transform wild-type Arabidopis thaliana Wassilewskija (WS) plant with the Ti-plasmids that contains following clone, described clones coding SEQ ID NO:43,44,45,86,136,138,140,141,142,143,144 and the polypeptide of the amino acid equivalent 1 to 135 of SEQ ID NO:140.Example comprises Ceres clone ID no.1792354, Ceres SEEDLINE IDno.ME06748, Ceres SEEDLINE ID no.ME08768, Ceres SEEDLINE IDno.ME19173 and Ceres clone ID no.375578.Except as otherwise noted, each Ceres clone and/or be in respect to arbitrary 35S promoter in the Ti-plasmids and on the direction of Yan Youyi from clone's seedling.But the Ti-plasmids support C RS 338 that is applicable to this class construct contains the plant selectable marker gene phosphinothricin acetyl transferase (PAT) that Ceres-makes up, and it is given by the plant transformed Herbicid resistant.

The preparation of soil mixture: with 24L SunshineMix#5 soil (Sun Gro Horticulture, Ltd., Bellevue, WA) with 16L Therm-O-Rock vermiculite (Therm-O-Rock West, Inc., Chandler AZ) mixes in cement mixing machine (cement mixer), prepares 60: 40 soil mixture.In this soil mixture, add 2 Tbsp Marathon, 1% particle (Hummert, EarthCity, MO), 3Tbsp OSMOCOTE

14-14-14 (Hummert, Earth City, MO) and 1Tbsp Peters fertilizer 20-20-20 (J.R.Peters, Inc., Allentown, PA), these compositions that are added at first are added in 3 gallons waters, add then and advance in the soil and thorough mixing.Generally fill the flowerpot of 4-inch diameter with soil mixture.Nylon membrane square with the 8-inch covers flowerpot then.

Plantation: the seed mixture that uses 60mL syringe pump 35mL.In every basin, add 25.Place transparent breeding lid (propagation dome) at the flowerpot top, then basin is placed the cloth of 55% shade to carry out sub-irrigation (subirrigated) down and by adding 1 inch water.

Plant culturing: plantation back 3 to 4 days, remove lid and shade cloth.Water a plant as required.After 7-10 days, use tweezers with the flowerpot thinning to every basin 20 strain plants.After 2 weeks, with the ratio of per gallon water 1Tsp all plants are carried out sub-irrigation with Peters fertilizer.When the about 5-10 cm long of bud (bolt), it is pruned to induce secondary bud (secondary bolt) between first segment and stem bottom.Prune the back and soaked infiltration (Dipping infiltration) in 6 to 7 days.

The preparation of Agrobacterium: Gepcillin, spectinomycin and the Rifampin (respectively being the storage concentration of 100mg/ml) that in the fresh YEB of 150mL, add each 0.1mL.The acquisition Agrobacterium plays daughter board (starter block) and (contains and grow to OD ₆₀₀The 96-orifice plate of about 1.0 Agrobacterium culture) and by from the appropriate well of daughter board, shifting 1mL inoculate culturing bottle of every kind of construct.Then with culture 27 ℃ of shaking culture.Reach about 1.0 OD ₆₀₀After (about 24 hours) that culture is centrifugal.In resuspended Agrobacterium throw out, add 200mL and soak into substratum.Soak into substratum by in 900mL water, adding 2.2g MS salt, 50g sucrose and the preparation of 5 μ l 2mg/ml benzyladenines.

Soak to soak into: flowerpot is inverted and was immersed in the water 5 minutes, thereby the over-ground part of plant is placed in the agrobacterium suspension.Allow plant normal growth and collect seed.

Embodiment 2: the salt conditional filtering

The salt conditional filtering: screening measures by high salt agar plate routinely and high salt soil mensuration is carried out.The proterties of estimating under high salt condition comprises: seedling area, photosynthetic efficiency, salt growth index and regenerative power.

The seedling area: about 2 the week age young plant total leaf area.

Photosynthetic efficiency (Fv/Fm): by assessed in relation seedling photosynthetic efficiency between maximum fluorescence signal Fm and the variable fluorescence Fv or the electron transport by photosystem II.Herein, the reduction of the sub-productive rate of optimal dose (Fv/Fm) is represented to coerce, and therefore can be used to monitor the performance of comparing transgenic plant under the condition of salt stress with non-transgenic plant.

The salt growth index=seedling area x photosynthetic efficiency (Fv/Fm).

Regenerative power: with stem excision back plant vimineous again ability in salt soil, soil is irrigated with 200mM NaCl solution.

Transformant is identified: use PCR amplification cDNA in a T2 plant of selecting at random to insert fragment.This PCR product that checks order then confirms sequence in the plant.

Identify salt stress patience plant: screening shows the transgenic plant that strengthen patience to SA (as hereinafter describing in detail) and high salt in the super pond of seed (superpool).To three independently candidate plant order-checkings, and transgenic sequence and ME02064 coupling.

Evaluation is to the patience of salt stress: select usually four to ten independent plant transformed strains and qualitative evaluation they at T ₁From generation to generation to the patience of salt stress.Select two or three at T ₁The conversion strain that shows the strongest salt stress patience from generation to generation qualitatively is at T ₂And T ₃Further estimate from generation to generation.This evaluation relate to from the planting seed of selected conversion plant lines on the MS agar plate that contains 100mM or 150mM NaCl, and seed hatched 5 to 14 days, allow its sprouting and growth.For example, for ME02064, on the salt flat board, compare the salt stress of five T2 incidents and wild-type Ws.Ws compares with contrast, selects three incident ME02064-01 ,-03 and-04 based on the measurement to each incident 36 strain plant seedlings area.Use T ₂And T ₃In ME02064-01 ,-03 and-04, carry out from generation to generation the further evaluation of salt patience.

Calculate SGI: after sprouting and growing, measure the seedling area and the photosynthetic efficiency that transform strain and wild-type contrast.From these are measured, calculate salt growth index (SGI), and between the seedling of wild-type and conversion, compare.Multiply by photosynthetic efficiency measurement (result) by the duplicate seedling area that obtains of 36 strain seedling that transforms strain and wild-type contrast with each and calculate SGI, and carry out the t-check.

Measure the transgenosis copy number: at BASTA ^TMTest T on the flat board ₂From generation to generation transform plant, thereby measure the transgenosis copy number that each transforms strain.15: 1 BASTA ^TMResistance: BASTA ^TMThe responsive separation than the transgenosis that shows two copies usually, this class of 3: 1 is separated than the transgenosis that shows a copy usually.

Embodiment 3: the oxidative stress conditional filtering

Under normal growth conditions, the Arabidopis thaliana lotus throne comprises the free SA of about 0.5 μ g/g fresh weight.When replying the attack of stress conditions or pathogenic agent, free SA level can reach up to 10 μ g/g fresh weights, approximates 60 μ M greatly.Can induce strong defence to reply and not cause tangible downright bad infringement by spraying exogenous application 100-500 μ M SA on the Arabidopis thaliana leaf and form.In case SA concentration is increased to 5mM or higher, the necrocytosis meeting of downright bad infringement form appears on the leaf of being sprayed.If use SA by growth medium, then Arabidopis thaliana is responsive more to SA-inductive oxidative stress, may be owing to the absorption that continues.Adding 100-150 μ M SA in growth medium is enough to reduce plant-growth, but does not kill the plant among the wild-type Arabidopis thaliana Ws.Therefore, we use the SA screening enhanced oxidative stress patience of this scope.

Whitfield's ointment screening: routinely, use 100 μ M or 150 μ M allogenic water poplar acid sodiums, measure by agar plate and screen.Substratum contains 1/2X MS (Sigma), 150 μ M sodium salicylates (Sigma), 0.5g MES hydrate (Sigma) and 0.7%phytagar (EM Science), uses 10N KOH to be adjusted to pH 5.7.

In order to screen super pond,, repeat flushing with sterilized water then with seed surface sterilization 5 minutes in 30% liquid lime chloride.With the density of each dull and stereotyped 850 seed, about 2500 seed individual layers are sowed on culture medium flat plate.On suitable flat board, cultivate wild-type and positive control.Twine flat board with zone of aeration, and place its stratification of three angels in the dark at 4 ℃.When finishing during this period of time, flat board is transferred in the Conviron incubator, described incubator is set at 22 ℃, 16: 8 hours light: dark circulation, 70% humidity, the incandescent light with emission～100 μ einstein light intensity and the combination of silver-colored light modulation.

Screened seedling since the 6th day every day.Selection is compared with the WS control plant and is grown greatlyyer and keep greener seedling as positive candidate, and is transferred to and recovers in the soil and set seeds.

By placing together on the identical sodium salicylate flat board with the WS contrast, the candidate plant is screened again from 36 seeds of each candidate.Processing plant as indicated above, and in the screening of sprouting beginning in back 4 days seedling.Results leaf texture is used for DNA extraction from confirm indefatigable candidate, and by the pcr amplification transgenosis.

Perhaps, super pond is directly sowed on soil also with 10mM SA spraying.Results leaf texture is used for the genetically modified DNA of pcr amplification with separation from patience candidate plant, subsequently the PCR product is checked order.

The proterties of estimating under the sodium salicylate condition comprises: seedling area, photosynthetic efficiency, Whitfield's ointment growth index (SAG) and regenerative power.

The seedling area: total leaf area of the young plant in about 2 ages in week.

Photosynthetic efficiency (Fv/Fm): by assessed in relation seedling photosynthetic efficiency between maximum fluorescence signal Fm and the variable fluorescence Fv or the electron transport by photosystem II.Herein, the reduction of the sub-productive rate of optimal dose (Fv/Fm) is represented to coerce, and therefore can be used to monitor the performance of comparing transgenic plant under the oxidative stress condition with non-transgenic plant.

The Whitfield's ointment growth(SAG) Index=seedling area (cm ²) x photosynthetic efficiency (Fv/Fm).

The T that selects at random in a strain ₂Use pcr amplification cDNA to insert fragment in the plant.Then this PCR product is checked order with the sequence in the checking plant.

Evaluation is to the patience of oxidative stress: they contrast the patience that compares oxidative stress with wild-type at all obtainable independent T2 plant lines qualitative evaluations that transform at first.Select qualitative demonstration to the oxidative stress the most positive transgenic strain of strong patience, be used to use inner non-transgenic chorista in contrast at T ₂And T ₃Further estimate from generation to generation.This evaluation relate to from the planting seed of selected conversion plant lines on the MS agar plate that contains 100 μ M or 150 μ M sodium salicylates, and seed hatched 4 days at least, allow sprouting and growth and transgenosis state analysis.

Calculate SAG: after sprouting and the growth, measure the seedling area and the photosynthetic efficiency that transform strain and wild-type contrast.From these measuring results, calculate Whitfield's ointment growth index (SAG) and comparison between wild-type and conversion seedling.Multiply by the photosynthetic efficiency measuring result by the duplicate seedling area that obtains of 36 strain seedling that transforms strain and wild-type contrast with each and calculate SAG, and carry out the t-check.

In some cases, use the substratum that has also replenished 100 μ M SNP to verify.

Embodiment 4:ME02064 (Ceres clone 375578; SEQ ID No.138)

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has 35S promoter and Ceres clone 375578.In the pre-authentication experiment, three transform strain ME02064-01 and ME02064-03, the ME02064-04 demonstration qualitative patience (table 4-1) the strongest to salt stress.In two generation confirmatory experiments, further estimate their patience to 150mM NaCl.Separation is than (BASTA ^TMResistance: BASTA ^TMResponsive) show that ME02064-01 and ME02064-03, ME02064-04 transform the transgenosis that strain respectively carries a copy.

Table 4-1: compare the pre-authentication experiment of ME02064 salt patience with wild-type Ws

	The Ws wild-type	ME02064 -01	ME02064 -02	ME02064 -03	ME02064 -04	ME020 64-05
							Average ^*	0.0359	0.0435	0.0346	0.0441	0.0438	0.0305
Standard error	0.0016	0.0048	0.004	0.0041	0.0035	0.0019

^*At the average seedling area that contains 14 days 36 strain plants of growth on the MS agar plate of 150mM NaCl

When growing on containing the MS agar plate of 150mM NaCl, ME02064-01 and ME02064-03, ME02064-04 transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As show as shown in the 4-2, when comparing with non-transgenic contrast seedling, the T2-of ME02064-01 seedling SGI value from generation to generation improves 110%, and the ME02064-03 seedling improves 131%, and the ME02064-04 seedling improves 72%.At T ₃From generation to generation, the SGI of ME02064-01 improves 43%, and ME02064-03 improves 47%, and ME02064-04 improves 64%.Difference between transgenosis and the non-transgenic seedling is that statistics is significant, and proves that clearly enhanced in the ME02064 transformant strain is the result of Ceres clone 375578 ectopic expression to the patience of salt stress.

Table 4-2: ME02064 is to the confirmatory experiment of salt stress patience in two generations

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in the patience of the ectopic expression enhancing of the Ceres clone 375578 under the 35S promoter control to salt stress, described salt stress causes downright bad infringement and the growth of downgrading in wild-type Ws seedling.

Embodiment 5:ME03140; Clone 375578; SEQ ID No.142

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres clone 375578 (SEQ ID NO:142), and study five transgenic strain ME03140-01, ME03140-02, ME03140-03, ME03140-04 and ME03140-05 patience salt stress.When on containing the MS agar plate of 150mM NaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, these transgenic strains show the patience to salt stress that improves.

When growing on containing the MS agar plate of 150mM NaCl, ME03140-01, ME03140-02, ME03140-03, ME03140-04 and ME03140-05 transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As shown in table 5, when comparing with non-transgenic contrast seedling, the T2-of ME03140-01 seedling SGI value from generation to generation improves 102.18%, the ME03140-02 seedling improves 60.78%, the ME03140-03 seedling improves 120.32%, the ME03140-04 seedling improves 45.07%, and the ME03140-05 seedling improves 90.53%.The difference of SGI value has the significant P value of statistics to all transgenic strains between transgenosis and the non-transgenic seedling, and these quantitative experiments prove that clearly Ceres clone 375578 ectopic expression gives in the transgenosis seedling enhanced to the patience of salt stress.

Table 5: the confirmatory experiment of ME03140 salt stress patience in the generation

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Embodiment 6:ME08732; Clone 560066; SEQ ID No.44

With the Ti-plasmids conversion wild-type Arabidopis thaliana Wassilewskija that has the 35S promoter that effectively is connected with Ceres clone 560066 (SEQ ID NO:44), and study three transgenic strain ME08732-01, ME08732-02 and ME08732-03 patience to salt stress.When on containing the MS agar plate of 150mM NaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, these transgenic strains show the patience to salt stress that improves.

When growing on containing the MS agar plate of 150mM NaCl, ME08732-01, ME08732-02 and ME08732-03 transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As shown in table 6, when comparing with non-transgenic contrast seedling, the T2-of ME08732-01 seedling SGI value from generation to generation improves 88.35%, and the ME08732-02 seedling improves 41.72%, and the ME08732-03 seedling improves 26.23%.The difference of SGI value has the significant P value of statistics to ME08732-01 and ME08732-02 transgenic strain between transgenosis and the non-transgenic seedling, and these quantitative experiments prove that clearly Ceres clone 560066 ectopic expression gives in the transgenosis seedling enhanced to the patience of salt stress.

Table 6: the checking of ME08732 salt stress patience in the generation

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in the patience of the ectopic expression enhancing of the Ceres clone 560066 under the 35S promoter control to salt stress, described salt stress causes downright bad infringement and the growth of downgrading in wild-type Ws seedling.

Embodiment 7:ME08768; Clone 539458; SEQ ID No.86

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres clone 539458 (SEQ ID NO:86), and study five transgenic strain ME08768-01, ME08768-02, ME08768-03, ME08768-04 and ME08768-05 patience salt stress.When on containing the MS agar plate of 150mM NaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, these transgenic strains show the patience to salt stress that improves.

When growing on containing the MS agar plate of 150mM NaCl, ME08768-01, ME08768-02, ME08768-03, ME08768-04 and ME08768-05 transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As shown in table 7, when comparing with non-transgenic contrast seedling, the T2 of ME08768-01 seedling improves 80.04% for the SGI value, the ME008768-02 seedling improves 111.63%, the ME008768-03 seedling improves 22.62%, the ME008768-04 seedling improves 115.40%, and the ME008768-05 seedling improves 74.41%.The difference of SGI value has the significant P value of statistics to ME08768-01, ME08768-02, ME08768-04 and ME08768-05 transgenic strain between transgenosis and the non-transgenic seedling, and these quantitative experiments prove that clearly Ceres clone 539458 ectopic expression causes in the transgenosis seedling enhanced to the patience of salt stress.

Table 7: the confirmatory experiment of ME08768 salt stress patience in the generation

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in the patience of the ectopic expression enhancing of the Ceres clone 539458 under the 35S promoter control to salt stress, described salt stress causes downright bad infringement and the growth of downgrading in wild-type Ws seedling.

Embodiment 8:ME10681; Clone 335348; SEQ ID No.141

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres clone 335348 (SEQ ID NO:141), and study six transgenic strain ME10681-01-T ₂, ME10681-01-T3, ME10681-02-T ₂, ME10681-02-T3, ME10681-04-T ₂And ME10681-05-T ₂Patience to salt stress.When on containing the MS agar plate of 150mM NaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, these transgenic strains show the patience to salt stress that improves.

When on containing the MS agar plate of 150mM NaCl, growing, ME10681-01-T ₂, ME10681-01-T ₃, ME10681-02-T ₂, ME10681-02-T ₃, ME10681-04-T ₂And ME10681-05-T ₂Transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As shown in table 8, when comparing with non-transgenic contrast seedling, ME010681-01-T ₂The T2-of seedling SGI value from generation to generation improves 39.17%, ME010681-01-T ₃Seedling improves 19.77%, ME10681-02-T ₂Seedling improves 119.17%, ME10681-02-T ₃Seedling improves 6.21%, ME010681-04-T ₂Seedling raising 113.51% and ME010681-05-T ₂Seedling improves 103.98%.The difference of SGI value is to ME10681-01-T between transgenosis and the non-transgenic seedling ₃, ME10681-02-T ₂, ME10681-04-T ₂And ME10681-05-T ₂Transgenic strain has the significant P value of statistics, and these quantitative experiments prove that clearly Ceres clone 335348 ectopic expression causes in the transgenosis seedling enhanced to the patience of salt stress.

Table 8: the confirmatory experiment of ME10681 salt stress patience in two generations

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in the patience of the ectopic expression enhancing of the Ceres clone 335348 under the 35S promoter control to salt stress, described salt stress causes downright bad infringement and the growth of downgrading in wild-type Ws seedling.

Embodiment 9:ME18973; Ceres cDNA ID 23457556; SEQ ID No.43

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres cDNA ID 23457556 (SEQ ID NO:43), and study six transgenic strain ME18973-01-T ₂, ME18973-02-T ₂, ME18973-02-01-T ₃, ME18973-03-T ₂, ME18973-05-T ₂And ME18973-05-03-T ₃Patience to salt stress.When on containing the MS agar plate of 150mMNaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, these transgenic strains show the patience to salt stress that improves.

When on containing the MS agar plate of 150mM NaCl, growing, ME18973-01, ME18973-02-T ₂, ME18973-02-01-T ₃, ME18973-03-T ₂, ME18973-05-T ₂And ME18973-05-03-T ₃Transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As shown in table 9, when comparing with non-transgenic contrast seedling, ME018973-01-T ₂The T2﹠amp of seedling; T3-SGI value from generation to generation improves 230.01%, ME18973-02-T ₂Seedling improves 22.44%, ME18973-02-01-T ₃Seedling improves 14.96%, ME18973-05-T ₂Seedling improves 16.12%, ME18973-05-03-T ₃Seedling improves 13.97%.The difference of SGI value has the significant P value of statistics to the ME18973 transgenic strain between transgenosis and the non-transgenic seedling, and these quantitative experiments prove that clearly the ectopic expression of Ceres cDNA ID23457556 causes in the transgenosis seedling enhanced to the patience of salt stress.

Table 9: the confirmatory experiment of ME18973 salt stress patience in two generations

ME18973-02-01-T ₃	4.82	0.205971746	44	4.19	0.3832982	25	7.71E-02	14.96％
									ME18973-05-T ₂	4.74	0	1	4.09	0.503725	26	0.160517	16.12％
ME18973-05-03-T ₃	4.38	0.233610226	32	3.84	0.503725	37	6.89E-02	13.97％

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in 35S promoter control Ceres cDNA ID 23457556 ectopic expressions down and strengthen patience to salt stress, described salt stress causes the growth of downright bad infringement and dwarfing in wild-type Ws seedling.

Embodiment 10:ME19657; CDNA ID 23621377; SEQ ID No.45

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres cDNA ID 23621377 (SEQ ID NO:45), and study two transgenic strain ME19657-01-T ₂, ME19657-01-05-T ₃, ME19657-01-08-T ₃, ME19657-02-T ₂, ME19657-03-T ₂, ME19657-04-T ₂And ME19657-04-01-T ₃Patience to salt stress.When on containing the MS agar plate of 150mM NaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, these transgenic strains show the patience to salt stress that improves.

When on containing the MS agar plate of 150mM NaCl, growing, ME19657-01-T ₂, ME19657-01-05-T ₃, ME19657-01-08-T ₃, ME19657-02-T ₂, ME19657-03-T ₂, ME19657-04-T ₂And ME19657-04-01-T ₃Transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As shown in table 10, when comparing with non-transgenic contrast seedling, ME19657-01-T ₂The T2﹠amp of seedling; T3-SGI value from generation to generation improves 82.29%, ME19657-01-05-T ₃Seedling improves 82.29%, ME19657-01-08-T ₃Seedling improves 21.90%, ME19657-02-T ₂Seedling improves 39.50%, ME19657-03-T ₂Seedling improves 98.28%, ME19657-04-T ₂Seedling improves 4.38%, ME19657-04-01-T ₂Seedling improves 7.44%.The difference of SGI value is to ME19657-01-T between transgenosis and the non-transgenic seedling ₂, ME19657-01-05-T ₃, ME19657-01-08-T ₃, ME19657-02-T ₂And ME19657-03-T ₂Transgenic strain has the significant P value of statistics, and these quantitative experiments prove that clearly the ectopic expression of Ceres cDNA ID 23621377 causes in the transgenosis seedling enhanced to the patience of salt stress.

Table 10: the confirmatory experiment of ME19657 salt stress patience in two generations

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in the patience of the ectopic expression enhancing of the Ceres cDNA ID 23621377 under the 35S promoter control to salt stress, described salt stress causes downright bad infringement and the growth of downgrading in wild-type Ws seedling.

Embodiment 11:ME24076; Clone 229668; SEQ ID No.143

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres Clone:229668 (SEQ ID NO:143), and study two transgenic strain ME24076-01 and ME24076-02 patience salt stress.When on containing the MS agar plate of 150mM NaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, these transgenic strains show the patience to salt stress that improves.

When on containing the MS agar plate of 150mM NaCl, growing, ME024076-01-T only ₂Show significantly bigger seedling area and SGI with transgenic plant with respect to non-transgenic plant.As shown in table 11, when comparing with non-transgenic contrast seedling, ME24076-01-T ₂The T2-of seedling SGI value from generation to generation improves 65.57%, ME24076-02-T ₂Seedling reduces by 1.12%.The difference of SGI value has the significant P value of statistics to transgenic strain ME24076-01 between transgenosis and the non-transgenic seedling, and these quantitative experiments prove that clearly Ceres clone 229668 ectopic expression causes in the transgenosis seedling enhanced to the patience of salt stress.

Table 11: the confirmatory experiment of ME24076 salt stress patience in the generation

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in the patience of the ectopic expression enhancing of the Ceres clone 229668 under the 35S promoter control to salt stress, described salt stress causes downright bad infringement and the growth of downgrading in wild-type Ws seedling.

Embodiment 12:ME24217; Clone 375578; SEQ ID No.144

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres clone 375578 (SEQ ID NO:144), and study two transgenic strain ME24217-07-T ₂And ME24217-09-T ₂Patience to salt stress.When on containing the MS agar plate of 150mM NaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, these transgenic strains show the patience to salt stress that improves.

When on containing the MS agar plate of 150mM NaCl, growing, ME24217-07-T ₂And ME24217-09-T ₂Transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As shown in Table 12, when comparing with non-transgenic contrast seedling, the T2-of ME24217-07 seedling SGI value from generation to generation improves 30.41%, and the ME24217-09 seedling improves 134.46%.The difference of SGI value is to ME24217-07-T between transgenosis and the non-transgenic seedling ₂And ME24217-09-T ₂Transgenic strain has the significant P value of statistics, and these quantitative experiments prove that clearly Ceres clone 375578 ectopic expression causes in the transgenosis seedling enhanced to the patience of salt stress.

Table 12: the confirmatory experiment of ME24217 salt stress patience in the generation

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Embodiment 13:ME02064C; Clone 375578C; SEQ ID No.140

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres clone 375578 (SEQ ID NO:140), and study six transgenic strain ME02064C-01-T ₂, ME02064C-02-T ₂, ME02064C-03-T ₂, ME02064C-04-T ₂, ME02064C-05-T ₂And ME02064C-06-T ₂Patience to salt stress.When on containing the MS agar plate of 150mMNaCl, growing, with quantitative experiment that non-transgenic contrast seedling is compared in, most of these transgenic strains do not show the patience to salt stress.

Table 13 is presented at when growing on the MS agar plate that contains 100mM NaCl, compares ME02064C-01-T with non-transgenic contrast seedling ₂The T2-of seedling SGI value from generation to generation is 0.55%; Compare ME02064C-02-T with non-transgenic contrast seedling ₂Seedling is 1.31%; Compare ME02064C-03-T with non-transgenic contrast seedling ₂Seedling is 9.67%; Compare ME02064C-04-T with non-transgenic contrast seedling ₂Seedling is-7.78%; Compare ME02064C-05-T with non-transgenic contrast seedling ₂Seedling is-15.77%; Compare ME02064C-06-T with non-transgenic contrast seedling ₂Seedling is 17.78%.

Table 13: the confirmatory experiment of ME02064C salt stress patience in the generation

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

The ectopic expression that is in 35S promoter control Ceres clone 375578 down may not promote the enhancing to salt stress patience, and described salt stress causes the growth of downright bad infringement and dwarfing in wild-type Ws seedling.

Embodiment 14:ME02064P1; The amino acid/11 of clone 375578P1-SEQ ID No.140 To 135

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with the nucleic acid of coding Ceres clone 375578P1 (amino acid/11 to 135 of SEQ ID NO:140), described nucleic acid is that above Ceres described in the embodiment 1 clones 3 ' truncated variant of 375578.Study five transgenic strain ME02064P1-03-T ₂, ME02064P1-07-T ₂, ME02064P1-09-T ₂, ME02064P1-10-T ₂And ME02064P1-15-T ₂Patience to salt stress.With quantitative experiment that non-transgenic contrast seedling is compared in, all these five transgenic strains show the patience to salt stress.As shown in table 10, compare with not genetically modified contrast seedling, the T2-of ME02064P1 seedling SGI from generation to generation improves 32.57%, 89.52%, 66.84%, 25.43%, 36.95%.

When growing on containing the MS agar plate of 150mM NaCl, ME02064P1-03, ME02064P1-07, ME02064P1-09, ME02064P1-10 and ME02064P1-15 transgenic plant show significantly bigger seedling area and SGI with respect to non-transgenic plant.As shown in table 14, when comparing with non-transgenic contrast seedling, the T2-of ME02064P1-03 seedling SGI value from generation to generation improves 32.57%, the ME02064P1-07 seedling improves 89.52%, the ME02064P1-09 seedling improves 66.84%, the ME02064P1-10 seedling improves 25.43%, and the ME02064P1-15 seedling improves 36.95%.The difference of SGI value is to transgenic strain ME02064P1-03-T between transgenosis and the non-transgenic seedling ₂, ME02064P1-07-T ₂, ME02064P1-09-T ₂, ME02064P1-10-T ₂And ME02064P1-15-T ₂Have the significant P value of statistics, and these quantitative experiments prove that clearly the ectopic expression of Ceres clone 37558P1 causes in the transgenosis seedling enhanced to the patience of salt stress.

Table 14: the confirmatory experiment of ME02064P1 salt stress patience in the generation

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in the patience of the ectopic expression enhancing of the clone 375587P1 under the 35S promoter control to salt stress.

Embodiment 15:ME02064P2; The amino acid of clone 375578P2-SEQ ID No.140 188 to 498

Ti-plasmids with the nucleic acid that has 35S promoter and coding Ceres clone 375578P2 (amino acid/11 88 to 498 of SEQ ID NO:140) transforms wild-type Arabidopis thaliana Wassilewskija, and described nucleic acid is 5 ' truncated variant of the clone of Ceres described in the embodiment 375578 above.Study the patience of eight ME02064P2 transgenic strains to salt.With quantitative experiment that non-transgenic contrast seedling is compared in, compare four transgenic strain ME02064P2-01-T with non-transgenic contrast seedling ₂, ME02064CP2-04-T ₂, ME02064P2-05-T ₂, ME02064P2-06-T ₂, ME02064P2-07-T ₂, ME02064P2-T ₂-08 and ME02064P2-09-T ₂Do not show the significant salt patience of statistics; A transgenic strain ME02064P2-10-T ₂The statistics that shows salt patience significantly reduces.

Table 15 is presented at when growing on the MS agar plate that contains 100mM NaCl, compares ME02064P2-01-T with not genetically modified contrast seedling ₂The T2-of seedling SGI value from generation to generation is 1.62%; Compare ME02064P2-04-T with not genetically modified contrast seedling ₂Seedling is 20.31%; Compare ME02064P2-05-T with not genetically modified contrast seedling ₂Seedling is 31.24%; Compare ME02064P2-06-T with not genetically modified contrast seedling ₂Seedling is 41.14%; Compare ME02064P2-07-T with not genetically modified contrast seedling ₂Seedling is 15.91%; Compare ME02064P2-08-T with not genetically modified contrast seedling ₂Seedling is 40.82%; Compare ME02064P2-09-T with not genetically modified contrast seedling ₂Seedling is 135.79%; Compare ME02064P2-10-T with not genetically modified contrast seedling ₂Be-12.36%.

When on containing the MS agar plate of 100mM NaCl, growing, for non-transgenic plant, ME02064P2-01-T ₂, ME02064P2-04-T ₂, ME02064P2-05-T ₂, ME02064P2-06-T ₂, ME02064P2-07-T ₂, ME02064P2-08-T ₂And ME02064P2-09-T ₂Transgenic plant show significantly bigger seedling area and SGI.Yet as shown in table 3, ME02064P2-10-T ₂The T2-of seedling SGI value from generation to generation compares the reduction that shows SGI with non-transgenic contrast seedling.

Table 15: ME02064P2 is to the confirmatory experiment of salt patience in the generation

^*SGI (salt growth index)=seedling area xFv/Fm (photosynthetic efficiency)

The result sums up:

Be in the patience of the ectopic expression enhancing of the clone 375587P2 under the 35S promoter control to salt stress.

Ceres clone 375578P2 keeps the Ceres clone's 375578 who is positioned at SEQ ID NO:140 amino-acid residue 137-157 alpha-beta structural domain, but does not keep б-г structural domain of the Ceres clone 375578 of SEQ ID NO:140.

Embodiment 16:ME10681; Clone 335348; SEQ ID No.141

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres cDNA 335348 (SEQ ID NO:141).When growing on the flat board that contains 100 or 150 μ M SA, wild-type Ws seedling shows downright bad infringement and the growth of downgrading, and transgenic plant show significantly better growth.

By the growth on the MS agar plate that contains 100 μ M SA, quantitative examination three transgenic strain ME10681-01, ME10681-02 and ME10681-05.After 14 days, use the scanning of EPSON color scanner or fluorescent scanning instrument dull and stereotyped, and calculate SAGI at every strain plant.Data are summarized in the table 16.

When on the MS agar plate that contains 100 μ M SA, growing, ME10681-02-T ₂And ME10681-05-T ₂Seedling area and SAGI that transgenic plant significantly improve with respect to non-transgenic plant.Yet, ME10681-01-T ₂Show that with respect to non-transgenic plant slight SAGI reduces.As shown in Table 12, ME10681-01-T ₂, ME10681-02-T ₂And ME10681-05-T ₂The T of seedling ₂Be respectively-3.29%, 17.65% and 51.84% for the SAGI value.Difference between transgenosis and the non-transgenic seedling is to strain ME10681-02-T ₂And ME10681-05-T ₂Have the significant P value of statistics, and prove that clearly enhanced is the result of Ceres cDNA 36505846 ectopic expressions in the ME10681 transformant strain to the patience of oxidative stress.

Table 16: the Whitfield's ointment confirmatory experiment of ME10681 in the generation

The result sums up:

In a word, the ectopic expression that is in the Ceres clone 335348 under the 35S promoter control strengthens oxidative stress patience, and described oxidative stress patience causes downright bad infringement and the growth of downgrading in wild-type Ws seedling.

Embodiment 17:ME24091; Clone 106263; SEQ ID No.136

Transform wild-type Arabidopis thaliana Wassilewskija with the Ti-plasmids that has the 35S promoter that effectively is connected with Ceres cDNA 016263 (SEQ ID NO:135).When growing on the flat board that contains 100 or 150 μ M SA, wild-type Ws seedling shows downright bad infringement and the growth of downgrading, and transgenic plant show significantly better growth.

By the growth on the MS agar plate that contains 100 μ M SA, ten transgenic strain ME24091-01-T of quantitative examination ₂, ME24091-02-T ₂, ME24091-03-T ₂, ME24091-04-T ₂, ME24091-05-T ₂, ME24091-06-T ₂, ME24091-07-T ₂, ME24091-08-T ₂, ME24091-09-T ₂And ME24091-10-T ₂After 14 days, use the scanning of EPSON color scanner or fluorescent scanning instrument dull and stereotyped, and calculate SAGI at every strain plant.

When on the MS agar plate that contains 100 μ M SA, growing, ME24091-01-T ₂, ME24091-02-T ₂, ME24091-03-T ₂, ME24091-04-01-T ₃, ME24091-04-T ₂, ME24091-05-01-T ₃, ME24091-05-T ₂, ME24091-06-01, ME24091-06, ME24091-07-01, ME24091-07, ME24091-08-01, ME24091-08, ME24091-09-01, ME24091-09, ME24091-10-01 and ME24091-10 transgenic plant the seedling area and the SAGI that significantly improve with respect to non-transgenic plant.As shown in Table 17, the T of ME24091-01, ME24091-02, ME24091-03, ME24091-04, ME24091-05, ME24091-06, ME24091-07, ME24091-08, ME24091-09 and ME24091-10 seedling ₂Improve 119.47%, 198.00% and 133.67%, 241.50%, 143.70% and 248.12%, 186.59%, 188.86%, 285.42% and 180.46% respectively for the SAGI value.Difference between transgenosis and the non-transgenic seedling has the significant P value of statistics to strain ME24091-01, ME24091-02, ME24091-03, ME24091-04-01, ME24091-04, ME24091-05-01, ME24091-05, ME24091-06-01, ME24091-06, ME24091-07-01, ME24091-07, ME24091-08, ME24091-09-01, ME24091-09 and ME24091-10, and proves that clearly enhanced is that Ceres clones the result of 106263 ectopic expressions in the ME24091 transformant strain to the patience of oxidative stress.

Table 17: the Whitfield's ointment confirmatory experiment of ME24091 in two generations

ME24091-04 -T ₂	0.54	0.050859903	45	0.22	0.077668008	19	0.0006 34704	141.50％
									ME24091-04 -01-T ₃	0.39	0.07715765	20	0.24	0.07271465	20	0.0819 50663	61.93％
ME24091-05 -T ₂	0.55	0.048581793	42	0.38	0.072915009	27	0.0298 49118	43.70％
									ME24091-05 -01-T ₃	0.38	0.068463201	21	0.15	0.05109963	30	0.0059 58129	144.90％
ME24091-06 -T ₂	0.71	0.049360913	39	0.29	0.063969074	23	1.1383 1E-06	148.12％
									ME24091-06 -01-T ₂	0.49	0.073404661	19	0.22	0.063271768	22	0.0046 91952	118.19％
ME24091-07 -T ₂	0.69	0.054095931	37	0.37	0.07390372	25	0.0004 14138	86.59％
									ME24091-07 -01-T ₃	0.49	0.052850446	33	0.19	0.049649799	22	5.3153 E-05	162.61％
ME24091-08 -T ₂	0.64	0.059981819	24	0.34	0.071776729	23	0.0011 1815	88.86％
									ME24091-08 -01-T ₃	0.44	0.050181996	27	0.40	0.074557785	26	0.3068 77156	11.48％
ME24091-09 -T ₂	0.81	0.056031311	38	0.29	0.067403065	22	5.8868 5E-08	185.42％

ME24091-09 -01-T ₃	0.45	0.055439617	36	0.28	0.05131548	31	0.0116 714	62.95％
									ME24091-10 -T ₂	0.56	0.048643058	39	0.31	0.062146975	29	0.0012 40527	80.46％
ME24091-10 -01-T ₃	0.36	0.051198395	31	0.26	0.066281225	22	0.1144 18402	39.44％

The result sums up:

In a word, the ectopic expression that is in the Ceres cDNA clone 106263 under the 35S promoter control strengthens oxidative stress patience, and described oxidative stress patience causes downright bad infringement and the growth of downgrading in wild-type Ws seedling.

Embodiment 18: determine functional homologue by Reciprocal BLAST

If candidate and canonical sequence coding have identity function and/or active protein, think that then candidate sequence is the functional homologue of canonical sequence.Use is known as Reciprocal BLAST (Rivera etc., Proc.Natl.Acad.Sci.USA, 95:6239-6244 (1998)) method is identified possible function homologous sequence from database, described database is made up of all obtainable open and patent peptide sequences, comprises from the NR of NCBI with from the peptide that Ceres clones and translating.

Before the beginning Reciprocal BLAST method, use BLAST specific for polypeptide at search for this from specific all peptides with reference to polypeptide source species, thereby identify following polypeptide, described polypeptide with on 85% comparison length, have 80% or bigger BLAST sequence identity with reference to polypeptide, perhaps in comparison, have bigger BLAST sequence identity on the shorter sequence.Be called bunch with reference to the polypeptide of polypeptide and any above-mentioned evaluation.

Use is from Washington University at Saint Louis, Missouri, the 2.0 editions BLASTP program determination BLAST sequence identity of USA and E-value.2.0 version BLASTP program comprises following parameter: a) the E-value is by being 1.0e-5; 2) word length is 5; With 3)-the postsw option.Based on a BLAST HSP (High-scoring SegmentPairs) and specific comparison of the potential function homologue sequence identified, calculating BLAST sequence identity with reference to polypeptide.Divided by HSP length, multiply by 100 with the residue number that mates fully in the BLASTHSP comparison then, obtain BLAST sequence identity.HSP length comprises the breach in the comparison usually, but gets rid of breach in some cases.

Main Reciprocal BLAST method comprises the two-wheeled blast search: forward lookup and reverse search.In the forward lookup step, at from all proteins sequence B LAST of purpose species from source species SA with reference to peptide sequence " polypeptide A ".Use 10 ^-5The E-value by and 35% sequence identity by determining that climax hits (top hit).In climax hit, the sequence with minimum E-value is called as the best hit, and was considered to potential functional homologue or lineal homologue.Hit with the best or with original with reference to polypeptide have 80% or any other climax of bigger sequence identity hit functional homologue or the lineal homologue that is considered to possible equally.Repeat this method at all purpose species.

In the reverse search wheel, hit at the climax of identifying in the forward lookup of all proteins sequence B LAST from source species SA from all species.Following climax from forward lookup hits the functional homologue that also is considered to possible, and described climax hits the polypeptide that returns from above-mentioned bunch and hits as its best.

Functional homologue is identified in hand inspection by possible functional homologue sequence.Showed SEQ ID NO.2,35,41,43,44,45,86,109,135,136,138,140,141,142,143 and the representative functions homologue of the amino acid X-Y of the amino acid X-Y of SEQ ID NO:140 and SEQ ID NO:140 in Fig. 1-6 and the sequence table.

Embodiment 19: determine functional homologue by hidden Markov model

Produce hidden Markov model (HMM) by program HMMER 2.3.2.In order to produce each HMM, use acquiescence HMMER 2.3.2 program parameter, be used for the conFigured of overall comparison.

Use sequence shown in Fig. 1 as input generation HMM.In these sequence input models, the HMM binary value of each sequence is shown in the sequence table.In extra sequence input model, showed the HMM binary value of additional sequences in the sequence table.The result shows the functional homologue that these extra sequences are SEQ ID NO:86.

Also use sequence shown in Fig. 2-6 to produce HMM as input.These sequences are imported in each model, showed the corresponding HMM binary value of each sequence in the sequence table.In extra sequence input model, showed the HMM binary value of additional sequences in the sequence table.The result shows that these extra sequences are functional homologue of group among Fig. 2-6.

Produce HMM in an alternate embodiment, preceding topic is not use the aminoacid sequence of clearly describing among the PCT/US2007/06544.Particularly, get rid of the following amino acid of showing in the PCT/US2007/06544 sequence table: SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ IDNO:180, SEQ ID NO:252, SEQ ID NO:298, SEQ ID NO:300, SEQ IDNO:301, SEQ ID NO:306 and SEQ ID NO:312.

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Claims

1. produce the method for plant of the oxidative stress patience of salinity patience with raising or raising, described method comprises cultivates the vegetable cell that comprises exogenous nucleic acid, described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of coded polypeptide, and wherein the HMM binary value of the aminoacid sequence of coding said polypeptide is greater than about 30, described HMM is based on the aminoacid sequence shown in one of Fig. 1-6, and wherein said plant is compared the corresponding patience level of salinity or oxidative stress with the control plant that does not comprise described nucleic acid, and is variant to the patience level of salinity or oxidative stress.

2. according to the process of claim 1 wherein the HMM binary value of aminoacid sequence of coding said polypeptide greater than about 400, described HMM is based on aminoacid sequence shown in Fig. 1.

3. according to the process of claim 1 wherein the HMM binary value of aminoacid sequence of coding said polypeptide greater than about 30, described HMM is based on aminoacid sequence shown in Fig. 2.

4. according to the process of claim 1 wherein the HMM binary value of aminoacid sequence of coding said polypeptide greater than about 120, described HMM is based on aminoacid sequence shown in Fig. 3.

5. according to the process of claim 1 wherein the HMM binary value of aminoacid sequence of coding said polypeptide greater than about 150, described HMM is based on aminoacid sequence shown in Fig. 4.

6. according to the process of claim 1 wherein the HMM binary value of aminoacid sequence of coding said polypeptide greater than about 425, described HMM is based on aminoacid sequence shown in Fig. 5.

7. according to the process of claim 1 wherein the HMM binary value of aminoacid sequence of coding said polypeptide greater than about 550, described HMM is based on aminoacid sequence shown in Fig. 6.

8. produce the method for plant of the oxidative stress patience of salinity patience with raising or raising, described method comprises cultivates the vegetable cell that comprises exogenous nucleic acid, described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of the following polypeptide of coding, described polypeptide be selected from SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168, have at least 85% or higher sequence identity with the aminoacid sequence of the amino acid equivalent 1 to 135 of SEQ ID NO:140, wherein the described plant that is produced by described vegetable cell is compared with the corresponding patience level in the control plant that does not comprise described nucleic acid, and is variant to the patience level of salinity or oxidative stress.

9. produce the method for plant, described method comprises cultivates the vegetable cell that comprises exogenous nucleic acid, described exogenous nucleic acid comprises the regulatory region that effectively is connected with following nucleotide sequence, described nucleotide sequence be selected from SEQ ID NO:1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155,157,159,161 and 164 nucleotide sequence has 85% or higher sequence identity, wherein the plant that is produced by described vegetable cell is compared the corresponding patience level of salinity or oxidative stress with the control plant that does not comprise described nucleic acid, and is variant to the patience level of salinity or oxidative stress.

10. the regulation and control plant is to the method for salinity or oxidative stress patience level, described method comprises introduces exogenous nucleic acid in vegetable cell, described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of coded polypeptide, wherein the HMM binary value of the aminoacid sequence of coding said polypeptide is greater than about 30, described HMM is based on the aminoacid sequence shown in one of Fig. 1-6, and wherein the plant that is produced by described vegetable cell is compared the corresponding patience level of salinity or oxidative stress with the control plant that does not comprise described exogenous nucleic acid, and is variant to the patience level of salinity or oxidative stress.

11. the regulation and control plant is to the method for salinity or oxidative stress patience level, described method comprises introduces exogenous nucleic acid in vegetable cell, described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of the following polypeptide of coding, wherein said polypeptide be selected from SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168, have 85% or higher sequence identity with the aminoacid sequence of the amino acid equivalent 1 to 135 of SEQ ID NO:140, wherein the plant that is produced by described vegetable cell is compared the corresponding patience level of salinity or oxidative stress with the control plant that does not comprise described nucleic acid, and is variant to the patience level of salinity or oxidative stress.

12. each method among the claim 1-8,10 or 11, wherein said polypeptide is selected from SEQID NO:43,44,45,86,140,141,142,143,144 and the amino acid equivalent 1 to 135 of SEQ ID NO:140, and described plant is compared the corresponding patience level of salinity with the control plant that does not comprise described nucleic acid, and is variant to the patience level of salinity.

13. each method among the claim 1-8,10 or 11, wherein said polypeptide is selected from SEQID NO:136 and 141, and described plant is compared the corresponding patience level of oxidative stress with the control plant that does not comprise described nucleic acid, and is variant to the patience level of oxidative stress.

14. the regulation and control plant is to the method for salinity or oxidative stress patience level, described method comprises introduces exogenous nucleic acid in vegetable cell, described exogenous nucleic acid comprises the regulatory region that effectively is connected with following nucleotide sequence, wherein said nucleotide sequence be selected from SEQ ID NO:1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155,157,159,161 and 164 nucleotide sequence has 85% or higher sequence identity, wherein the plant that is produced by described vegetable cell is compared the corresponding patience level of salinity or oxidative stress with the control plant that does not comprise described nucleic acid, and is variant to the patience level of salinity or oxidative stress.

15. comprise the vegetable cell of exogenous nucleic acid, described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of coded polypeptide, the HMM binary value of wherein said amino acid sequence of polypeptide is greater than about 30, described HMM is based on the aminoacid sequence shown in one of Fig. 1-6, and wherein said plant is compared the corresponding patience level of salinity or oxidative stress with the control plant that does not comprise described nucleic acid, and is variant to the patience level of salinity or oxidative stress.

16. comprise the vegetable cell of exogenous nucleic acid, described exogenous nucleic acid comprises the regulatory region that effectively is connected with the nucleotide sequence of coded polypeptide, wherein said polypeptide be selected from SEQ ID NO:2,4,6,8,9,11,13,14,15,17,19,20,22,23,24,25,27,29,30,31,33,35,36,37,38,39,41,42,43,44,45,47,49,50,52,54,56,58,60,62,63,64,66,68,69,71,73,74,76,78,80,81,83,84,86,88,90,91,93,94,96,98,100,101,102,104,106,107,109,110,112,114,116,118,119,121,122,123,125,126,127,128,129,130,132,134,136,138,140,141,142,143,144,145,147,149,151,153,154,156,158,160,162,163,165,166,167,168 and the aminoacid sequence of the amino acid equivalent 1 to 135 of SEQ ID NO:140 have at least 85% or higher sequence identity, wherein, variant to the patience level of salinity or oxidative stress by in described vegetable cell plant that produces and the control plant that does not comprise described nucleic acid the corresponding patience level of salinity or oxidative stress being compared.

17. comprise the vegetable cell of exogenous nucleic acid, described exogenous nucleic acid comprises the regulatory region that effectively is connected with following nucleotide sequence, described nucleotide sequence be selected from SEQ ID NO:1,3,5,7,10,12,16,18,21,26,28,32,34,40,46,48,51,53,55,57,59,61,65,67,70,72,75,77,79,82,85,87,89,92,95,97,99,103,105,108,111,113,115,117,120,124,131,133,135,137,139,146,148,150,152,155,157,159,161 and 164 nucleotide sequence has 85% or higher sequence identity, wherein the plant that is produced by described vegetable cell is compared the corresponding patience level of salinity or oxidative stress with the control plant that does not comprise described nucleic acid, and is variant to the patience level of salinity or oxidative stress.

18. comprise the transgenic plant of each vegetable cell among the claim 15-17.

19. the transgenic plant of claim 18, wherein said plant is to be selected from following species member: switchgrass, dichromatism chinese sorghum (Chinese sorghum, Schrock), huge Chinese silvergrass (awns genus), saccharum species (energy sugarcane), baisam poplqr (white poplar), Zea mays (corn), soybean (soya bean), colea (canola oil dish), common wheat (wheat), upland cotton (cotton), rice (paddy rice), Sunflower Receptacle, alfalfa (clover), beet or cattailmillet (pearl millet).

20. comprise from according to the seed of each transgenic plant among the claim 15-17 or the goods of nutritive issue.

21. the goods of claim 20, wherein said goods are food or feed goods.

22. comprise the isolating nucleic acid of the nucleotide sequence of the following polypeptide of encoding, the aminoacid sequence shown in described polypeptide and the SEQID NO:2,4,6,22,27,29,49,52,54,56,60,62,68,76,83,88,90,96,98,104,106,112,114,132,134,149,151 or 160 has 80% or higher sequence identity.

23. identify the method that polymorphism is whether relevant with character variation, described method comprises:

A) whether one or more genetic polymorphisms are relevant with the locus of following polypeptide in the mensuration plant population, and described polypeptide is selected from polypeptide and functional homologue thereof shown in Fig. 1-6; With

B) dependency of the existence of one or more polymorphisms described in the plant of the variation of proterties and described population described in the plant of the described population of measurement, thus identify whether described one or more polymorphisms are relevant with the variation of described proterties.

24. the method for claim 23, wherein said proterties are the patience to salinity that improves, the patience to oxidative stress of raising, or the biomass that improves.

25. prepare the method for plant lines, described method comprises:

A) whether one or more genetic polymorphisms are relevant with the locus of following polypeptide in the mensuration plant population, and described polypeptide is selected from polypeptide and functional homologue thereof shown in Fig. 1-6;

B) identify one or more plants in described population, at least a allelic existence is relevant with the variation of salt patience or oxidative stress patience in wherein said one or more polymorphisms;

C) with each and himself or different plant hybridizations of the plant of described one or more evaluations, produce seed;

D) will be from least one strain progeny plants of described seed growth and himself or different plant hybridizations; With

E) with step c) and d) repeat 0-5 again for preparing described plant lines, wherein said at least a allelotrope is present in the described plant lines.

26. the method for claim 24 or 25, wherein said population are the populations of switchgrass plant.

The polypeptide that 27. each method in the claim 1,8,10 or 11, prerequisite are the described nucleotide sequences of coded polypeptide does not encode and clearly describe in the sequence table of PCT/US2007/06544.

28. each method in the claim 1,8,10 or 11, prerequisite are the described nucleotide sequences of coded polypeptide one of following peptide sequence of PCT/US2007/06544 of not encoding: SEQ IDNO:99, SEQ ID NO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ IDNO:104, SEQ ID NO:180, SEQ ID NO:252, SEQ ID NO:298, SEQ IDNO:300, SEQ ID NO:301, SEQ ID NO:306 and SEQ ID NO:312.

29. the method for claim 9 or 14, prerequisite are in the sequence table of the indeterminate PCT/US2007/06544 of being described in of described nucleotide sequence.

30. the method for claim 9 or 14, prerequisite are described nucleotide sequences is not one of following nucleotide sequence of PCT/US2007/06544: SEQ ID NO:98, SEQ IDNO:101, SEQ ID NO:225 and SEQ ID NO:299.

The polypeptide that 31. the vegetable cell of claim 15 or 16, prerequisite are the described nucleotide sequences of coded polypeptide does not encode and clearly describe in the sequence table of PCT/US2007/06544.

32. the vegetable cell of claim 15 or 16, prerequisite are the nucleotide sequences of coded polypeptide one of following peptide sequence of PCT/US2007/06544 of not encoding: SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ IDNO:180, SEQ ID NO:252, SEQ ID NO:298, SEQ ID NO:300, SEQ IDNO:301, SEQ ID NO:306 and SEQ ID NO:312.

33. the vegetable cell of claim 17, prerequisite are in the sequence table of the indeterminate PCT/US2007/06544 of being described in of described nucleotide sequence.

34. the vegetable cell of claim 17, prerequisite are described nucleotide sequences is not one of following nucleotide sequence of PCT/US2007/06544: SEQ ID NO:98, SEQ IDNO:101, SEQ ID NO:225 and SEQ ID NO:299.

35. the nucleotide sequence of claim 22, prerequisite are in the sequence table of the indeterminate PCT/US2007/06544 of being described in of described nucleotide sequence.

36. the nucleic acid of claim 22, prerequisite are described nucleotide sequences is not one of following nucleotide sequence of PCT/US2007/06544: SEQ ID NO:98, SEQ IDNO:101, SEQ ID NO:225 and SEQ ID NO:299.

37. the method for claim 23 or 25, prerequisite are in the sequence table of the indeterminate PCT/US2007/06544 of being described in of described polypeptide.

38. the method for claim 23 or 25, prerequisite are described polypeptide is not one of following peptide sequence of PCT/US2007/06544: SEQ ID NO:99, SEQ IDNO:100, SEQ ID NO:102, SEQ ID NO:103, SEQ ID NO:104, SEQ IDNO:180, SEQ ID NO:252, SEQ ID NO:298, SEQ ID NO:300, SEQ IDNO:301, SEQ ID NO:306 and SEQ ID NO:312.