CN1469705A - Enhanced meristematic activity and competence by overexpression of tonoplast pyrophosphatase - Google Patents

Abstract

A transgenic plant having increased meristematic activity and competence effectuating in larger leave, stem, flower, fruit and root structures comprising a polynucleotide sequence causing upregulated expression of vacuolar pyrophosphatase.

Description

Strengthen branch vitality and competence by overexpression of tonoplast pyrophosphatase

Related application

Under the countries concerned's law (relevant) with this PCT application, within the bounds of possibility, the application benefits from: the U.S. Provisional Application the 60/164th of the Roberto Gaxiola that on November 10th, 1999 submitted to, No. 808, exercise question is " proton transport protein and application in plant " (" Proton Transporters and Uses in Plants "); No. the 09/644th, 039, the U.S. Patent application of the Roberto Gaxiola that on August 22nd, 2000 submitted to, exercise question is " proton transport protein and application in plant " (" ProtonTransporters and Uses in Plants "); No. the 60/226223rd, the U.S. Provisional Application of the Roberto Gaxiola that on August 18th, 2000 submitted to, exercise question is " drought-resistant/the freeze injury genetically modified plants " (" Drought/Freeze Resistant TransgenicPlants "); And the PCT that on November 10th, 2000 submitted to applies for PCT/US00/30955 number, exercise question be " can in salinized soil, grow anti-stress super-huge genetically modified plants " (" Stress-Resistant Oversized TransgenicPlant Capable of Growing in Salinized Soil "), the content of each application instruction is hereby expressly incorporated by reference in the scope that suitably foreign law allows.Government supports

The invention that the application describes in whole or in part, obtains following support: from the graduate subsidy of national health GM52414, DK54214, DK43495, DK51509, DK34854 and GM35010; Subsidy MCB9317175 from National Science Foundation.There is certain right in government to the present invention.Background technology related to the present invention

1. the technical field of the invention

The present invention relates to the plant of genetic modification, strongr with respect to these plants of environmental stress, as arid and/or freeze injury; These plants of structure with respect to nutrition organs and/or reproductive organs excessive (comparing) with the homologue of their normal phenotype; And these plants can grow in hypersaline environment.This class plant also shows the higher branch liveliness proof and the cell division activity of increase.

2. background technology related to the present invention

When we entered the new millennium, the prospect that the mankind obtain food was very severe.Because world population is growing, increasing crop yield still is a main target of agricultural research.A main target of horticulture research also is to cultivate more healthy and strong non-crop plants, and is useful or make the joyful plant of people to the mankind as ornamental plants, flowers and plants, shrub and other.

Up to now, the improvement of crops and gardening plant depends on the plant that selection breeding has desired proterties.Yet these selection breeding technology are often not fully up to expectations, because many plants are carried the heterozygosity genetic material, can not have fully with the identical gratifying proterties of their parental generation.

Molecular biological progress makes the mankind to operate the idioplasm of animal and plant.Plant genetic engineering need separate and handle genetic material (generally being the form with DNA or RNA), then this genetic material is imported plant.Use this class technology to cause the generation of multiple merit plant, the plant, the plant that can express medicine or other chemical composition and other plant of expressing useful quality that strengthen as anti-insect power.The advantage of this class plant is not only to contain genes of interest, and can educate.

Cultivating the plant with high resistance to cold and diseases in the recent period is an interesting field of plant science.Generally speaking, plant has and keeps adaptation mechanism, thereby guarantees that they survive in the hostile environment condition.Freeze injury and arid be two classes that usually run into of plant stress, both of these case is all relevant with cell dehydration.Known certain plants has some gene, and these genes are being in cold or are opening under the lack of water situation for a long time, its coded product is directly or indirectly corresponding be used to provide numerous similar stronger with this class plant to arid and/or cold tolerance than it.Much stress relevant gene be determined with high temperature and water (referring to, as: United States Patent (USP) the 5th, 837,545,5,071,962,4,707, No. 359).A lot of people believe that these genes produce some albumen, for example " water stress protein ", and these albumen are believed to be helpful in plant life.For example some plant that is exposed to stressed condition produces a kind of hormone that is called abscisic acid (ABA), and it helps plant to close its stroma, thus alleviate stress severity.Yet unfortunately, known abscisic acid can suppress the generation of young leaves, causes coming off of flower and fruit, thereby causes the underproduction.

It is believed that most of tropical plants do not evolve out withstand prolonged arid and/or cold ability; On the contrary, known a lot of temperate plant some ability of having evolved out at least and having tolerated this class situation.Under drying condition and after freeze injury, the output of plant variant has very big difference.For example the tender leaf of tobacco (Nicotiana spp.) generation is very responsive to arid, and can not commercially produce at insufficient water and the high area of evaporativity.With water stress be opposite, people know little about it to the gene and the albumen that participate in the freeze injury tolerance.Yet, have the main component of theoretical hypothesis freeze injury tolerance may relate to tolerance to dehydration (referring to,, L.Guy (1989) plant physiology (PlantPhysiol.) 89:444-451) as G.C.Yelenosky.

Cultivation has a growing ability of improvement in salinized soil plant is that recent another makes the interested field of people.When the water of pouring contained soluble-salt, the salinization of soil of soil then can take place.After the water evaporation, salt can accumulate in soil gradually.The salinization of soil gradually of irrigateing land makes the agriculture dim future (R.Serrano etc., plant science comment (Crit.Rev.Plant Sci.), 13:121-138 (1994)) in a lot of arable lands on our this celestial body.For example, the arid area can provide desirable light application time and temperature condition to the growth of most of crops, but rainfall is not ideal enough.Man-made irrigation only can address this problem in short-term, because have been found that soil in this class environment is through the rapid salinization of soil of regular meeting.For growing in the environment of salinization of soil, sodium/potassium ion ratio that plant must remain in its intracytoplasmic sodium/potassium ion ratio soil is much lower, and the growth that this stops a lot of plants comprises food crop.

The eliminating of salt in the Physiologic Studies prompting root, and/or leaf cell isolates from vacuole with salt, is the key factor (M.Kirsch etc., molecular biology of plants (Plant Mol.Biol.), 32:543-547 (1996)) of decision salt tolerance.The toxic concentration of sodium chloride at first reaches in the leaf that launches fully, and wherein sodium chloride is separated in the vacuole.Have only when surpassing the bearing capacity of vacuole, can make that just the concentration in cytosol and the non-protogenous matter (apoplasmic) reaches poisonous level, finally cause the forfeiture of turgescence, so plant death.Existing people proposes that the excessive acidifying in the vacuole chamber (by the V-ATP enzyme) can provide sodium/hydrogen ion exchange activity needed extra proton, this exchange activity makes cytosol detoxifcation (M.S.Tsiantis, Deng, plant magazine (Plant J.), 9:729-736 (1996)).Known salts stress increase hydrogen ion transhipment that ATP relies on and that pyrophosphoric acid (PPi) relies in the tonoplast vesicle in the sunflower seedling root for example.Salt is handled and is also brought out the responsive sodium hydrogen ion exchange activity (E.Ballesteros etc., plant physiology (Physiologia Plantarum), 99:328-334 (1997)) of amiloride (amiloride).In halophytes mesembrianthemum (Mesembryanthemum crystallinum), high sodium chloride can stimulate vacuole H in the leaf cell ⁺The activity of-ATP enzyme (V-ATP enzyme) and vacuole sodium/hydrogen ion antiport carrier.

And the aesthetic property of the output of raising crops and some ornamental plants of increase is another point of interest in the agronomy research.Have the plant of bigger trophism and/or genitality structure than wild-type plant and, can improve output of crops and the sight of some ornamental plants of enhancing by plantation by increasing the growth rate of plant.

In the prior art, chemical compound lot is speed that can improve plant growing and the output that improves the biomass in the plant useful component by flatter.For example, declare to be applied to the cyclodextrin in the tissue culture medium (TCM), by comprising the fissional mechanism of increase, can strengthen the cellular tissure incubation growth speed (referring to, as United States Patent (USP) the 6th, 087, No. 176).Also known certain plants somatotropin, for example auximone is (except its many positions, also promote root growth), the basic element of cell division, gibberellic acid (except other many positions, also promoting the stem growth), when being used for plant tissue, can promote the cell division that increases.Also known in the present technique field, some growth factor can be used for increasing the size of plant and/or plant flowers.Unfortunately, these somatotropin and the factor separates and uses not only costliness but also time-consuming.

United States Patent (USP) the 5th, 859 has disclosed for No. 338: the modification of the CLAVATA1 gene of arabidopsis (Arabidopsisthaliana) is caused the normal control of cell division forfeiture in tip of a root meristematic tissue and the floral meristem.It is said that the forfeiture of above-mentioned arbitrary situation division control all can cause merismatic increase.In spending, this increase allegedly can cause the increase of floral organ number, comprises the increase of carpel (carpel) number, and it can increase the size of fruit and the number of seed.United States Patent (USP) the 5th, 859 provides clavatal nucleic acid and albumen No. 338, and the clavatal nucleic acid and the albumen of modified, with mutagenic meristematic tissue phenotype.

United States Patent (USP) the 5th, 750 has disclosed a kind of method of controlling plant cell growth for No. 862, and this method comprises the level and/or the catalytic activity of regulating cell cycle regulating protein in the plant.Especially, this Patent publish, by improving p34 ^Cdc2The level of albumen can promote by individual cells or in groups cytothesis be plant.By regulation and control indirect regulation p34 ^Cdc2The regulating element of protein active also can be realized the regulation and control to regeneration.

Thereby we need such plant: to arid and/or freeze injury have improvement anti-stress, have bigger dimensional characteristic (with respect to wild type to response body) and more can tolerate salinity in the soil of their growths, and can produce the biomass of more useful component.

The invention summary

The present invention discloses to have and raise the genetically modified plants that the vacuole pyrophosphatase is expressed.Discover, show this active plant of raising, usually bigger than its wild type counterparts, show stronger opposing arid and/or freeze injury stress ability, the salinity in their somatomedins is tolerated and shows stronger branch liveliness proof (cell division) more, thereby cause at the biomass of certain plants part higher than wild-type plant.

According to the present invention, anyly can change suitable exogenous nucleic acid molecule that vacuole pyrophosphatase in the plant expresses and can be used for transforming genetically modified plants.This exogenous nucleic acid can comprise the nucleic acid of coding vacuole pyrophosphatase albumen (external source vacuole pyrophosphatase), as AVP1, its funtion part (peptide, polypeptide) or its homologue, and/or the nucleic acid of the endogenous vacuole pyrophosphatase expression of change plant (exogenous nucleic acid imports wherein)." exogenous nucleic acid " refers to and derives from exogenous nucleic acid and import nucleic acid outside wherein the plant cell, or imports after certain part of plant or plant in order to the nucleic acid outside the preparation genetically modified plants.Exogenous nucleic acid in order to conversion can be RNA (ribonucleic acid) or DNA (DNA (deoxyribonucleic acid)), (for example cDNA (being complementary DNA), genomic DNA).In addition, exogenous nucleic acid can be ring-type or straight chain, two strands or single chain molecule.Single-chain nucleic acid can be positive-sense strand or antisense strand." funtion part " of the nucleic acid that vacuole pyrophosphatase albumen is encoded refers to the part nucleic acid that albumen or polypeptide (it keeps the functional characteristic of vacuole pyrophosphatase albumen) are encoded.In certain specific embodiments, this nucleic acid coding AVP1, funtion part or its homologue.This AVP1 nucleic acid can be from as arabidopsis or any other plant or synthetic and obtain.

The nucleic acid that changes the endogenous vacuole pyrophosphatase expression of plant (exogenous nucleic acid imports wherein) is included in regulating and controlling sequence (adjusting sequence for example induction type, composing type) and the antisensenucleic acids that function is arranged in the plant.The example of regulating and controlling sequence comprises promotor, enhancer.This nucleic acid can also comprise, for example, polyadenylation site, reporter gene and/or intron sequences or the like, the existence of these sequences may not be the function of this nucleic acid or expresses necessary, but can be by influence, for example, transcribe and/or stability (for example, mRNA's transcribes and/or stability) and nucleic acid is expressed better and/or strengthen its function.This class component can be included in the nucleic acid molecules to obtain the optimum utility of nucleic acid.

Use known method, the nucleic acid that uses among the present invention can obtain from various sources.The nucleic acid of the coding vacuole pyrophosphatase (as AVP1) that for example adopts among the present invention can obtain from natural source, as tobacco, bacterium, tomato or corn.In a specific embodiment, the corresponding vacuole pyrophosphatase of the wild type of nucleic acid coding and genetically modified plants.In another specific embodiment, the not corresponding vacuole pyrophosphatase of the wild type of this nucleic acid coding and these genetically modified plants.Changing nucleic acid (as regulating and controlling sequence) that the endogenous vacuole pyrophosphatase of plant (exogenous nucleic acid imports wherein) expresses also can chemosynthesis, reorganization generation and/or obtain from commercial source.

The whole bag of tricks that nucleic acid of the present invention imports in the plant is known for a person skilled in the art.For example, can adopt Agrobacterium (Agrobacterium) mediation Plant Transformation, particle bombardment, (for example United States Patent (USP) the 4th, 945, No. 050 in the particulate bombardment; United States Patent (USP) the 5th, 100, No. 792), protoplast transformation, pollen transgenosis, reproductive organs injection and prematurity embryo injection.This exogenous nucleic acid be directed in any suitable cell (or various kinds of cell (group)) of plant, for example the root cells of plant (group), stalk cell (group) and/or leaf cell (group).

Any suitable plant comprises that angiosperm, monocotyledon and dicotyledon, gymnosperm and algae can be used for producing genetically modified plants of the present invention, tissue culture or cell culture.For example, tomato, corn, tobacco, paddy, Chinese sorghum, cucumber, lettuce, turfgrass, view and admire (as bigger flower, bigger leaf) and pulses leguminous plants can as described hereinly transform, to produce genetically modified plants of the present invention.In addition, genetically modified plants of the present invention can grow in the matrix of any support plant growing, as soil or water (aquatic cultivation).

Genetically modified plants of the present invention preferably tolerate the high salt concentration in the soil.Term " salt " comprises all salt, promptly refer to when hydrogen in the acid is substituted by metal or metal equivalents and the compound that generates, and include but not limited to: comprise unit price and poisonous cationic salt, sodium chloride, potassium chloride, calcium chloride, magnesium chloride, caddy, zinc chloride and sulphurizing salt divalence.

Can the salt tolerance be introduced in the plant of the present invention by the exogenous nucleic acid transformed plant cells, this exogenous nucleic acid can change the expression of vacuole pyrophosphatase in the plant, so that its up-regulated.Any suitable vacuole pyrophosphatase wherein has and is severally cloned, all can be used for the compositions and methods of the invention (for example, Z.Sarasian etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 89:1775-1779 (1992); Jenslerchl etc., molecular biology (Molec.Biol.), 29:833-840 (1995); Y.Kim etc., plant physiology (Plant Physiol.), 106:375-382 (1994)).In particular specific embodiment, the present invention relates to the genetically modified plants of salt tolerant, be included as the exogenous nucleic acid assembly (construct) (Z.Sarasian etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 89:1775-1779 (1992)) of expressing AVP1 and designing.The conversion of plant cell can be carried out in whole strain plant, seed, leaf, root or any other plant part.They preferably, these genetically modified plants are changed, to such an extent as to can be grown in the salinity that suppresses corresponding non-transgenic plant growth.The transgenic progeny of genetically modified plants, the seed that is produced by genetically modified plants and the genetically modified plants offspring (it also is a theme of the present invention) who is generated by transgenic seed advantageously carry these salt tolerance attributes.Plant can be screened to obtain the salt tolerance of certain level it from cell transformed regeneration to produce genetically modified plants.In a preferred embodiment, exogenous nucleic acid coding AVP1 or its homologue.Preferably, a certain degree is brought up in the expression of the vacuole pyrophosphatase in the plant, thus when NaCl concentration be during from about 0.2M to about 0.3M, genetically modified plants have tolerance to sodium chloride (NaCl).The genetically modified plants that can be grown in the salt solution also can be obtained by following method: will import one or more plant cells by the nucleic acid of up-regulated expression vacuole pyrophosphatase in plant, to produce transformant.As used herein, " salt solution " comprise containing the water that salt is feature, and preferably wherein the concentration of salt in water be to about 0.4M from about 0.2M.In a specific embodiment, salt solution refers to seawater.

Genetically modified plants of the present invention also can be used to produce the dual genetically modified plants of salt tolerant (approximately 0.2M is to the salinity of about 0.4M).In a specific embodiment, the present invention relates to the dual genetically modified plants of salt tolerant, comprise one or more exogenous nucleic acid plant transformed cells of using, this exogenous nucleic acid can change vacuole pyrophosphatase and Na in the plant ⁺/ H ⁺The expression of antiport carrier.Vacuole pyrophosphatase in a favourable assembly is AVP1 or its homologue, and Na ⁺/ H ⁺The antiport carrier is AtNHX1 or its homologue.The present invention also comprises the transgenic progeny of dual genetically modified plants and seed that is produced by genetically modified plants and the genetically modified plants offspring who is generated by this seed.

Also can be by usefulness exogenous nucleic acid transformed plant cells with arid and/or freeze injury tolerance introduced plant, wherein exogenous nucleic acid can change the expression of vacuole pyrophosphatase in the plant, thereby raises this expression.In a preferred embodiment, provide the genetically modified plants of drought-resistant and/or freeze injury on the certain degree, it comprises genome, and this genome contains the vacuole H that one or more external sources are introduced ⁺Transposition pump gene.The particularly preferred external source chimeric DNA assembly that genetically modified plants (bring out arid and/or freeze injury tolerance, and in the geophilous ability of salt marsh) comprise separation, this assembly coding vacuole H of educating ⁺The transposition pump, and preferably can be operatively connected in promotor, as 35-S promotor or any other strong promoter, include but not limited to tissue-specific promoter.These genetically modified plants can comprise polynucleotide sequence, and this sequence comprises the external source tonoplast pyrophosphoric acid proton pump gene that can be operatively connected in promotor.In the genetically modified plants (can grow in saline soil) of another particularly preferred drought-resistant and/or freeze injury, this polynucleotide sequence comprises the external source tonoplast pyrophosphoric acid proton pump gene of the two-in-series enhancer (double tandem enhancer) that can be operatively connected in 35S promoter.Particularly preferred tonoplast pyrophosphoric acid proton pump gene is the AVP1 gene.

The vacuole pyrophosphatase up-regulated expression that carries out with said method also can be used to provide the plant that has bigger nutrition and/or genitality organ than the corresponding plant of its wild type.That is to say, the invention provides the method that increases plant products, comprise that can change the nucleic acid that the vacuole pyrophosphatase is expressed in the plant introduces one or more plant cells, thereby produce transformant, increase the output of plant thus.This method can further comprise from the transformant regeneration plant, with the generation transfer-gen plant, and selects the transfer-gen plant bigger than its corresponding wild-type plant, and then produces the genetically modified plants bigger than its corresponding wild-type plant.The present invention also comprise obtain than its corresponding wild-type plant have the colored size of increase genetically modified plants (as, ornamental plants) method, comprise that can change the nucleic acid that the vacuole pyrophosphatase is expressed in the plant introduces one or more plant cells, thereby produce transformant.

The vacuole pyrophosphatase up-regulated expression that carries out with said method also can be used to produce the plant that has stronger minute vitality/cell division speed than the corresponding plant of its wild type.As what those skilled in the art understood, meristematic tissue is the center that higher plant grows, because nearly all back embryo (post-embryonic) organ comprises root, leaf, flower, armpit meristematic tissue and cambium, all started by bud or root meristematic tissue.The branch liveliness proof that increases causes higher biomass aspect plant structure one or more, as by plant structure, root architecture and stem structure dry weight proved.The branch liveliness proof that increases is envisioned for the speed of sprouting that causes regenerating in root, leaf, hypocotyl and cotyledon explant increase, and the increase that causes whole plant strain growth speed.

The inventor finds that proton (hydrogen ion) pump that pyrophosphoric acid drives crossing in vacuole expressed and caused bigger proton pump sexuality, bigger proton pump sexuality then to cause the more ability of good general's ion absorption vacuole, and this has reduced the osmotic potential of cell; Proton (hydrogen ion) pump that pyrophosphoric acid drives crossing in vacuole expressed the increase that also causes plant cell division and multiplication capacity.As what those skilled in the art understood, this discovery can have huge commercial value, for example, transforms by pair cell, expresses this type of pump to cross, and reduces the production time of timber, cereal etc. thus; Increase the regeneration capacity of bud, as woody plant, crop (as corn) and ornamental plants (as orchid) with bad or low regeneration capacity plant.Causing the mistake of cell division that this class increases and propagation to express available arbitrary assembly described herein realizes.Though many inducible promoters and tissue-specific promoter can be used for triggering crossing of this gene and/or its homologue and express, can be operatively connected the proton pump gene (AVP-1) that drives in the tonoplast pyrophosphoric acid of chimeric promoters (as the two-in-series enhancer of 35S promoter) but preferred assembly comprises, wherein chimeric promoters is that design was used for expression AVP-1.

The present invention has also disclosed novel box gene, comprises containing the box that can be operatively connected the proton pump gene that drives in the tonoplast pyrophosphoric acid of chimeric promoters.Novel box gene comprises can be operatively connected the proton pump gene that drives in the external source tonoplast pyrophosphoric acid of promotor, and novel coded sequence comprises and can be operatively connected the proton pump gene that drives in the external source tonoplast pyrophosphoric acid of the two-in-series enhancer of 35S promoter.Preferably, this coded sequence is that design was used for expression AVP1.

The present invention has also disclosed novel expression vector, comprise: contain the expression vector of polynucleotide sequence, it comprises and can be operatively connected in the two-in-series enhancer of 35S promoter and further be operatively connected the proton pump gene that drives in the external source tonoplast pyrophosphoric acid of multiple clone site; And the expression vector that contains polynucleotide sequence, it comprises and can be operatively connected in the two-in-series enhancer of 35S promoter and further be operatively connected the proton pump gene that drives in the external source tonoplast pyrophosphoric acid of heterologous coded sequence.

The inventor thinks, the invention of this disclosure can be applicable to any plant, include but not limited to: it is useful or make the joyful plant of people to the mankind to make rerum natura plant, ornamental plants, grass, shrub or any other, comprises being applied to monocotyledon, dicotyledon, angiosperm, gymnosperm and algae.

The accompanying drawing summary

With reference to following detailed Description Of The Invention and accompanying drawing, can understand above description and further purpose of the present invention, characteristics and advantage more completely, wherein:

Figure 1A is the independently vertical view of transgenic lines (1 ' and 2 ') of typical (every kind is come from 10 strain plants) wild type (WT) and two, they 10 hours light and shade in the cycle hydroponics growing 7 weeks;

Figure 1B (1), Figure 1B (2) and Figure 1B (3) be typical 5 days seedling (old seedling) root and the microphotograph of root hair, these seedlings are from the typical wild type, 1 ' and 2 ' among Figure 1A, its parallel surface that grows in the vertical plant nutrient agar panel;

Fig. 1 C is a western blot figure of independently crossing the membrane portions that the transgenic lines (1 ' and 2 ') of expressing AVP-1 separates from wild type (WT) and two;

Fig. 2 be exposed to lack of water stress be after 7 days, typical wild type (WT) plant is to the typical vertical view of crossing the genetically modified plants of expression AVP-1 (1 ' and 2 ');

Fig. 3 is grown in wild type (WT) plant that contains in the high soil of the salinity perspective view to the genetically modified plants of representational overexpression AVP-1 (1 ' and 2 ');

Fig. 4 A is the working model sketch of the related transport protein in vacuole chamber before sodium is isolated from yeast; Nhx1 (Na ⁺/ H ⁺The antiport carrier), Vma1 (tonoplast H ⁺-ATP enzyme), Gef1 (yeast CLC chloride channel), Ena1 (cell membrane Na ⁺-ATP enzyme);

Fig. 4 B is the working model sketch that is shown in the related transport protein in vacuole chamber before sodium is isolated from yeast among Fig. 4 A, and it also comprises Avp1 (proton pump that A.thaliana vacuole pyrophosphoric acid drives);

Fig. 5 A and Fig. 5 B are that expression wild-type yeast system and mutant yeast are Na in the cell ⁺And K ⁺The histogram of content, the multiple sudden change that influences the sodium tolerance is carried by wherein mutant yeast system, and the value among the figure is the mean value of twice determination data and the stub line is represented standard deviation;

Fig. 6 crosses the root of expression (1 ' and 2 ') Arabidopsis (arabidopsis) plant and the vertical view of the bud that cotyledon explant is regenerated from wild type and AVP-1, and these plants are incubated in the SIM medium of Figure 13;

Fig. 7 is wild-type plant (WT) is expressed the genetically modified plants (1 ' and 2 ') of AVP-1 to typical mistake Na ⁺And K ⁺The histogram of content, these plant plantings are in containing the high soil of salinity;

Fig. 8 is that calcium is taken among Fig. 32 ' 35SAVP-1 transgenosis tonoplast vesicle (square) is taken in the vesicle of wild type (WT) among Fig. 3 to calcium curve map;

Fig. 9 A and Fig. 9 B illustrate the 35SAVP-1 postulated mechanism, drive function by proton, can make higher solid volume progression go into vacuole with respect to the wild type vacuole;

Figure 10 is the vertical view that wild type contrast transgenosis (1 ' and 2 ') AVP-1 crosses the leaf of expression Arabidopsis (arabidopsis) plant, and leaf is to place by the length of time;

Figure 11 A and 11B are with the wild type of water (distilled water) pouring and the vertical view of the leaf of crossing transgenosis (1 ' and 2 ') Arabidopsis (arabidopsis) plant of expressing AVP-1, and the growth of root architecture is described;

Figure 12 be typical wild type petunia leaf transplant (petunia leave cuttings) (WT) contrast the vertical view of the shoot regeneration that the typical transgenic petunia leaves of plants of crossing expression AVP-1 (35-S AVP-1) transplants;

Figure 13 be from wild type (WT) and AVP-1 cross that expression (1 ' and 2 ') Arabidopsis (arabidopsis) plant obtains and be placed on the SIM inducing culture 5 day age cotyledon the vertical view of shoot regeneration.

Figure 14 is the histogram that two kinds of AVP-1 from wild type and Arabidopsis (arabidopsis) plant cross the osmotic potential in the leaf that the abundance of expressing system's (1 ' and 2 ') feeds water.

Figure 15 is the vertical view that is incubated at the petunia explant in the MS medium, and the cultivation that was illustrated in for 6 weeks is inducing from the callus of this explant afterwards.

Detailed Description Of The Invention

Because Vacuoles of Plants occupies the 40-99% of cumulative volume in the maturation plant cell born of the same parents, thereby the change of vacuole size has tremendous influence (R.G.Zhen, E.J. Kim, P.A.Rea to cell size, in " Vacuoles of Plants " (The Plant Vacuole) book, (academic publishing Co., Ltd, 1997), the 25th volume, the 298-337 page or leaf). The volume of vacuole is controlled by the flux of ion and water, and the latter two are mediated by various pumps and transport protein. In plant, the motive force that triggers ion, solute and water transmembrane transport is proton gradient. The activity of vacuole proton pump causes acidifying in the chamber, and sets up the hydrogen ion electro-chemical potential gradient of crossing over this tonoplast, and this gradient provides energy for inorganic ions, sugar and organic acid secondary active transport albumen. The activity of these transport proteins can be regulated acid-base value and gas ions homeostasis in the born of the same parents, and the accumulation that causes producing the required solute of osmotic potential, this osmotic potential promotes vacuole expansion (H.Sze, X.Li, M.G.Palmgren, plant cell (The Plant Cell) 11,677-689 (1999)).

The pump that 3 kinds of different produced electrochemical proton gradients are arranged. A kind of cell membrane that is positioned at, it discharges (PM H with hydrogen ion in cell⁺-ATP enzyme), two kinds are positioned at tonoplast or other inner membrance chamber, and it can make acidifying (vacuole type H in the chamber at their places⁺-ATP enzyme and H⁺-PP enzyme) (R.A.Leigh, in " Vacuoles of Plants " (The Plant Vacuole) book that L.a.Sanders edits, (academic press, Santiago, California, 1997), the 25th volume, 171-194 page or leaf).

Work in the past shows, utilizes the antisense assembly to reduce carrot vacuolus H⁺The A subunit of-ATP enzyme (A subunit) level can cause the stretching, extension reduction of plant and the change (J.P.Gogarten etc., plant cell (The Plant Cell) 4,851-864 (1992)) of leaf attitude. The present inventor supposes that the hydrogen ion that enters in the vacuole of increasing supply can accelerate the cell stretching, extension. Recently, be beneficial to the correlation theory of ion accumulation based on vacuolar proton, same inventor supposes that the accumulation of solids in the vacuole may be conducive to protective plant tolerance arid and the plant of more anti-freeze injury is provided.

The present inventor recognizes, plant has a lot of vacuole hydrogen ion transhipment pumps, by activity, the expression that increases them of raising them, the copy number of transcribing and/or translate or increase them that raises them, then can increase the accumulation of solids in the vacuole, this is owing to obtainable proton in the vacuole increases. The present inventor verifies this hypothesis (R.G.Zhen, E.J.Kim, P.A.Rea by the copy number that increases the transhipment of vacuole hydrogen ion pump, inorganic pyrophosphatase or V-PP enzyme (pyrophosphatase) (it is comprised of single polypeptide), in " Vacuoles of Plants " (The Plant Vacuole) book, (academic publishing Co., Ltd, 1997), the 25th volume, the 298-337 page or leaf). In arabidopsis, can produce hydrogen ion gradient by tonoplast by the V-PP enzyme of AVP-1 gene code, itself and vacuole H⁺Hydrogen ion gradient magnitude close (similar in magnitude) (V. Sarafian, Y.Kim, R.J.Poole, P.A.Rea that-ATP enzyme produces, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 89,1775-1779 (1992)). Understand that such as those skilled in the art the similar gene in other plant will work in a similar fashion.

H in the known tissue growing⁺-PP enzyme (hydrogen ion pyrophosphatase) is the main proton pump of tonoplast. This may be because the following fact: in the tissue of growth, in order to make up new cell, nucleic acid, DNA, various RNA, protein and cellulose etc. are actively synthesized, and therefore, pyrophosphoric acid can generate in a large number as the accessory substance of these metabolic processes. The energy that is stored in the pyrophosphoric acid molecule can change into the different energy, namely strides the hydrogen ion gradient of tonoplast. This hydrogen ion gradient consists of vacuole and gathers the motive force of solute, thereby it is poor to produce enough infiltrations that makes plant cell begin to grow. Growth effects for the increase of having seen, although the present invention also is limited to any specific hypothesis never in any form, but present inventor's hypothesis: excessively expressing in the genetically modified plants of AVP-1, more the hydrogen ion pyrophosphatase of big figure has positive-effect to the speed that produces the hydrogen ion gradient, and this causes minute liveliness proof that more enlivens.

In a specific embodiment, produce genetically modified plants of the present invention with assembly, this assembly comprises the vacuole pyrophosphatase gene that is operatively connected in promoter, wherein promoter is that design was used for expression vacuole pyrophosphatase (for example, expression cassette). As employed in this article, term " is crossed and is expressed " and refers to have larger expression/activity than the expression that occurs in the situation without this assembly. In a particular specific embodiment, produce genetically modified plants of the present invention with assembly, this assembly comprises and is operatively connected the gene in the AVP1 of chimeric promoters, wherein chimeric promoters is that design was used for expression AVP1. More particularly, the present invention relates to assembly, wherein the AVP1 gene is the two-in-series enhancer that is operatively connected in 35S promoter.

Genetically modified plants of the present invention except edibility or ornamental value, industrial value (such as timber production), can have other purposes. For example, genetically modified plants of the present invention can be taken in different or more ion than its wild type counterparts. As discussed below, mutant yeast system (enal) be studies confirm that the Proton Transport pump in the vacuole plays a very important role in higher organism (plant component that relates to is by replenishing budding yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) salt-sensitive mutant is identified) in the cation detoxication in the intracellular cation detoxification system. Genetically modified plants and/or its offspring can be used to bioanalysis and recover remediation of soils and growth medium, and these genetically modified plants and/or its offspring comprise exogenous nucleic acid, and exogenous nucleic acid (according to this type of research) can change the expression of vacuole pyrophosphatase in plant. Such plant can be used to from the culture medium that can support plant growth (for example soil, water) except decationizing (as, unit price and/or bivalent cation). For example, conversion of plant of the present invention can be used for removing sodium (Na), plumbous (Pb), manganese (Mn) and/or calcium (Ca) ion from the culture medium of supporting plant growth.

In order to prove that the hydrogen ion that increases in the vacuole supplies with arid and/or freeze injury tolerance, the tolerance of salt growth and the impact of plant volume on plant, the inventor has made the genetically modified plants of the vacuolar proton pump AVP-1 that contains additional copy.

With assembly arabidopsis thaliana transformation (Arabidopsis thaliana (ecotype the columbia)) plant that contains the AVP-1 gene. Then separate the transgenic line that contains this gene additional copy. AVP-1, open reading frame is cloned into Xma1 position [R.Toper, V.Matzeit, B.Gronenbom, J.Shell and the H.H. Steinbiss of modified pTR103, nucleic acids research (Nucleic acid Research), 15,5890 (1987)]. This carrier comprises that the series connection of 35-S promoter repeats. Comprise that the Hind III fragment of 35-S Gene expression, AVP-1 open reading frame (ORF) and polyadenylation signal is subcloned into Hind III site (P.Hajdukiewicz, Z.Svab and the P.Maliga of pPZP212 carrier, molecular biology of plants (Plant Molecular Biology), 25,989-994 (1994)). Carry out agrobacterium-mediated conversion by arabidopsis (Arabidopsis thaliana (ecotype columbia)) the vacuum infiltration of blooming. By the seed of the plant through transforming is planted screening transgenic plant on the plant nutrient agar plate, wherein the plant nutrient agar plate replenishes the kanamycins with 25 mg/litre. Plant continues to select to cultivate for two generations, with the genetically modified plants of evaluation with this gene pure.

Figure 1A be typical (every kind is come from 10 strain plants) wild type (WT) and two independently transgenosis be the top view of (1 ' and 2 '), they 10 hours light and shade in the cycle hydroponics growing 7 weeks. As can seeing from Figure 1A, it is 1 ' relevant with the size of plant with the quantity of the visual comparative descriptions AVP-1 of wild type (WT) that transgenosis is that 2 ' (it is at higher horizontal expression AVP-1 albumen), transgenosis are. Research finds that the quality of genetically modified plants is greater than the quality of wild-type plant. Transgenosis is than the dry weight of wild type (WT) large 1.5 and 3 times respectively of the dry weights (sample number is 4) (measuring after processing 24 hours at 75 ℃) of 1 ' and 2 ' whole genetically modified plants.

Figure 1B (1), Figure 1B (2) and Figure 1B (3) are that (magnifying power is 40 times for the root of typical 5 days seedlings and the microphotograph of root hair, spillikin length is 2 millimeters on the photo), these seedlings are from the typical wild type among Figure 1A, 1 ' transgenosis system and 2 ' transgenosis system, and its Parallel Growth is in the surface of vertical plant nutrient agar panel. Transgenosis is that the average length of 1 ' and 2 ' shoot root hair (is measured the root staple length along whole root, chosen 5 seedlings for every group than wild type (WT) root staple length 40% and 70% (Figure 1B). Average 80 root hairs are measured in every strain). The length of root hair is relevant with the size of vacuole, so the increase of root hair size may be due to the increase of vacuole volume. This situation and Arabidopsis Mutants rdh3 are compared, it is reported that this mutant has the vacuole volume that reduces, a kind of short and small plant (M.E.Galway, J.W.J. Heckman, J.W.Schiefelbein with unusual short and small hair, plant (Planta) 201,209-218 (1997)). The root architecture that has recognized that increase to soil weather, the fixed nitrogen of bean has active influence, and helps plant to the absorption of water and nutrient.

Fig. 1 C is the western blot figure of independently crossing the membrane portions that the transgenosis system (1 ' and 2 ') of expressing AVP-1 separates from wild type (WT) and two. In the water ballast culture medium, after 8 weeks of growth, separate total membrane portions from 8 all old wild types (WT) and the bud (shoots) of AVP-1 genetically modified plants (1 ' and 2 '). The homogenate of plant sprout under 8000 rev/mins and 100,000 rev/mins of conditions sequentially centrifugal 15 and 30 minutes respectively. 100 milligrams film sediments are resuspended to 10mM trishydroxymethylaminomethane (Tris), pH7.5,150mM NaCl, 1mM ethylenediamine tetra-acetic acid (EDTA), 10% glycerine, then 1mM phenylmethylsulfonyl fluoride (PMSF) albumen (10 microgram) separates at 10% SDS-polyacrylamide gel electrophoresis (PAGE), carry out electroblotting and immunostaining with antibody, the antibody that adopts is from the synthetic peptide of (raised against) KLH-conjugation (conjugated), this peptide is corresponding to the 4th hydrophilic loop (V.Sarafian, Y.Kim, R.J.Poole, the P.A.Rea of the supposition of AVP-1 albumen, newspaper (Proc.Natl.Acad.Sci.USA) 89 of institute of NAS, 1778-1779 (1992)). Pyrophosphatase detects by chemiluminescence. Fig. 1 C explanation transgenosis system (1 ' and 2 ') is the horizontal expression AVP-1 albumen higher than wild type (WT) (that is, with respect to WT, 1 ' increases by 1.6 times, and 2 ' increases by 2.4 times).

Because potassium concentration (100mM to 300mM) in the increase cell, the wheat of lack of water has higher drought tolerance (S.Gupta, G.Berkowitz, P.Pier, plant physiology (Plant Physiol.) 89,1358-1365 (1989)), therefore the drought tolerance increase of hypothesis (but the invention is not restricted to this theory) AVP-1 genetically modified plants may be the result of potassium concn in its higher vacuole, and the interior potassium concn of higher vacuole causes the increase of water reserve capability. As if laboratory test support this viewpoint.

Fig. 2 be exposed to lack of water stress be after 7 days, typical wild-type plant (WT) is to the typical top view of crossing the genetically modified plants of expression AVP-1 (1 ' and 2 '). Wild type and the genetically modified plants (Fig. 3 A) of excessively expressing AVP-1 have been carried out drought-enduring test (24 ℃). 7 days lacks of water stress after, wild-type plant is withered, from the then swelling and living of the plant of 35S AVP-1 transgenosis system (1 ' and 2 '). In addition, when water was stood the plant of drought stress, genetically modified plants were proceeded normal growth, bolting (bolted) and set seeds, and wild-type plant is then dead. Relative water content in wild type and the 35S AVP-1 genetically modified plants leaf lack of water stress during test show that transgenosis system has the ability of stronger reservation moisture than wild-type plant.

Figure 14 is the column diagram that two kinds of AVP-1 from wild type and arabidopsis thaliana cross the osmotic potential in the leaf that the abundance of expressing system's (1 ' and 2 ') feeds water. The osmotic potential of the reduction in the leaf of the genetically modified plants that record under constant water content and AVP-1 cross to express and cause the argument of the Solute accumulation that increases consistent, thereby have increased the ability that keeps water.

Although in the accompanying drawings not explanation, concerning many plant species, stood 24 hours or the freeze injury pressure (challenge) (＜0 ℃) of longer time after, also obtained similar result. Although be not restricted to such hypothesis, believed that the genetically modified plants of expression AVP-1 (1 ' and 2 ') provided stronger frost damage prevention ability than wild-type plant, this is owing to have the cation of greater number in vacuole. The cation of greater number can provide higher osmotic pressure, it causes the ability of stronger reservation water, and the ability of stronger reservation water is not only given the ability of the lower soil water potential of Plant Tolerance (Soil water potential), and giving the ability of the stronger opposing freeze injury of plant, freeze injury can cause the plant serious dehydration.

Fig. 3 be grown in contain in the high soil of salinity wild-type plant (WT) to the representational perspective view of crossing the genetically modified plants of expression AVP-1 (1 ' and 2 ').Five strain wild-type plants (WT) and two AVP-1 of five strains cross express transgenic system (1 ' and 2 ') and are planted in the soil, and are in 10 hours light and shade cycle.Plant irrigated for 6 weeks with the nutrient solution (1/8MS salt) of dilution, then with the nutrition liquid irrigation that replenishes with the dilution of NaCl.Originally the concentration of NaCl is 100mM, per then 4 days increase 50mM.Diagram among Fig. 3 is corresponding to the typical plant that existed under the 300mM NaCl condition at the 10th day.Fig. 3 explanation, two kinds of AVP-1 vegetation types (type) (1 ' and 2 ') are obviously more healthy and stronger than wild-type plant in containing the high soil of salinity.The following fact, promptly cross expression AVP-1 (the tonoplast proton pump that pyrophosphoric acid drives, this work) or AtNHX1 (sodium ion/sodium/protein countertransporter, (M.Apse etc., science (Science), 285:1256-1258 (1999)) and this work) genetic engineering modified arabidopsis (Arabidopsis thaliana) plant can in the presence of high concentration NaCl, grow the strategy of supporting this paper strongly and being set forth.Can expect that dual genetically modified plants can show the salt tolerance phenotype of further enhancing.These a little arabidopsiss (Arabidopsis thaliana). transport protein or their homologue can be finished similar function in important agricultural crops.The volume of the increase of 35S AVP-1 arabidopsis genetically modified plants also helps genetic engineering modified crop can increase output potentially.

The working model of plant cell device middle-jiao yang, function of the spleen and stomach gas ions homeostasis

Though the present invention is not limited to any specific hypothesis, the present inventor has designed the working model at plant cell device middle-jiao yang, function of the spleen and stomach gas ions homeostasis, the result of the unanticipated of being found in its can herein interpreted disclosed genetically modified plants.

In plant, the energy of most transport process comes from the elementary transhipment of proton.Hydrogen ion transhipment pump is positioned at cell membrane and tonoplast, forward hydrogen ion to extracellular and vacuole chamber (P.A.Rea etc., " tonoplast ATP and pyrophosphatase " (Tonoplast Adenosine Triphosphate and inorganicPyrophosphatase) from cytosol respectively.In " plant biochemistry method " (Methods Plant Biochem.) book, PP.385-405, academic publishing Co., Ltd, London (1990)).This vegetation water vacuolar membrane comprises two kinds of hydrogen ion transhipment pumps: V-ATP enzyme and inorganic pyrophosphatase or title V-PP enzyme.Their effect makes in the chamber acidifying and set up proton electro-chemical potential gradient (J.M.Davies etc., " bioenergetics of vacuole proton pump " (the The Bioenergetics of Vacular H that strides tonoplast ⁺Pumps), in " plant vacuole " (Plant Vacuole) book that R.A.Leigh, D.Saners edited, pp.340-363, academic press, Santiago (1997)).This tonoplast is relevant with a lot of physiology courses, comprises the storage and the compensation (retrieval) of isolation, turgescence adjusting and the nutrition of cytosolic pH static balancing (cytosolic pH stasis), modulability calcium ion subregion, poisonous ion such as sodium ion.Vacuole occupies the long-pending 40-99% of total endosome of maturation plant cell.The vacuolar proton pump pyrophosphatase is general and a large amount of compositions that exist of vegetation water vacuolar membrane, it can produce the proton electrochemical potentials of striding tonoplast of stable state, its proton electrochemical potentials (P.A.Rea etc., " tonoplast ATP and pyrophosphatase " (Tonoplast Adenosine Triphosphate andInorganic Pyrophosphatase) similar or that produce greater than the V-ATP enzyme.In " plant biochemistry method " (Methods PlantBiochem.) book, pp.385-405, academic publishing Co., Ltd, London (1990)).In various biosynthesis pathways, pyrophosphoric acid (PPi) is the accessory substance in activation or the multimerization process, it also in plant as the alternative energy donor of ATP: be used for sucrose by sucrose synthase and shift; Pass through pyrophosphoric acid: the fructose-6-phosphate phosphotransferase is used for glycolysis; And provide energy (M.Stitt, Botany Gazette (Bot.Acta) 111:167-175 (1998)) for tonoplast by the vacuolar proton pump pyrophosphatase.

The most cells inner cell organ comprises clathrin coated vesicl, endosome, Golgi membrane and vacuole, all has acid inside (X.S.Xie etc., journal of biological chemistry (J.Biol.Chem.), 264:18870-18873 (1989)).This acidifying is mediated by proton transhipment electrogenesis ATP enzyme, and proton pump V-PP enzyme (J.M.Davies etc., " bioenergetics of vacuole proton pump " (The Bioenergetics of Vacular H of in the plant vacuole, also driving by pyrophosphoric acid ⁺Pumps), in " plant vacuole " (Plant Vacuole) book that R.A.Leigh, D.Saners edited, pp.340-363, academic press, Santiago (1997); R.G.Zhen etc., " molecule and the biochemical basis of the proton transhipment that drives at the tonoplast pyrophosphoric acid " (TheMolecular and Biochemical Basis of Pyrophosphate-EnergizedProton Translocation at the Vacuolar Membrane), academic publishing Co., Ltd (1997)).Need anion transport to keep clean electroneutral (A.al-Awquti, cell biology viewpoint (Curr.Opin.Cell.Biol.) in the present age, 7:504-508 (1995)).

Fig. 4 A is the working model sketch of the related transport protein in vacuole chamber before sodium is isolated from yeast; Nhx1 (Na ⁺/ H ⁺The antiport carrier), Vma1 (tonoplast H ⁺-ATP enzyme), Gef1 (yeast CLC chloride channel), Ena1 (cell membrane Na ⁺-ATP enzyme).The yeast member Gef1 of the valtage-gated chloride channel superfamily of GLC, be in yeast, copper changed over to late period the Golgi body vesicle and cation be isolated from before the vacuole chamber necessary.(R.A.Gaxiola etc., newspaper (Proc.Natl.Acad.Sci.USA) 95:4046-4050 (1998) of institute of NAS; R.A.Gaxiola etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 96:480-1485 (1999); Embodiment 1).And, show, the defective of gef1 mutant, can be suppressed by following method: import phenotype member, the Torpedo marmorata CLC-0 of the super family of CLC or import arabidopsis (Arabidobsis thaliana) CLC-c and CLC-d chloride ion channel (M.Hechenberger etc., journal of biological chemistry (J.Biol.Chem.), 271:33662-33638 (1996); R.A.Gaxiola etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 95:4046-4050 (1998)).Fig. 4 B is the working model sketch that is shown in the related transport protein in vacuole chamber before sodium is isolated from yeast among Fig. 4 A, and it also comprises Avp1 (proton pump that A.thaliana vacuole pyrophosphoric acid drives).

Though without wanting to be limited by theory, two observed results that Fig. 4 a and 4b show cause proposing sodium ion and are isolated from model in the yeast.At first, the gef1 mutant is to the high salt concentration sensitivity.Secondly, sodium ion/proton exchange body Nhx1 is vacuole chamber (R.Nass etc., journal of biological chemistry (J.Biol.Chem.), 273:21054-21060 (1998)) before being positioned.The model assumption that proposes in Fig. 4 A and 4B, the sodium ion by Nhx1 are isolated and are depended on vacuole H ⁺-ATP enzyme and chloride channel Gef1.Stream allows by vacuole H in the anion of Gef1 mediation ⁺-ATP enzyme is set up enough proton gradients of size, with going up to accumulation (uphillaccumulation) by sodium ion/proton exchange driving sodium ion.

Based on this model, suppose: the inner coelom that flows to supposition in the increase proton can improve by the sodium ion of Nhx1 permutoid isolates.In order to increase hydrionic availability, expressed function acquisition type (gain-of-function) the mutant gene AVP1-D of A.thaliana, proton pump coding (Fig. 3 B) (R.G.Zhen etc. that this mutant gene drives for the vacuole pyrophosphoric acid, journal of biological chemistry (J.Biol.Chem.), 272:22340-22348 (1997)).Be expressed in the sodium ion tolerance that this proton pump in the yeast can recover to test strain, contain NHX1 and the GEF1 gene that function is arranged but prerequisite is this test strain.In addition, Gef1p and Nhx1p are positioned in the same organelle altogether, promptly preceding vacuole indoor (R.A.Gaxiola etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 96:1480-1485 (1999)).These results effectively support the model among Fig. 4 A and the 4B, and the vacuole chamber can be used to identify the uncertain plant transport protein that participates in sodium detoxifcation in the cell before showing yeast.

The physiological data of vacuole role in the cation detoxication is in full accord in Fig. 4 A and 4B institute's representation model and the higher plant.Yeast and plant cell are shared from approach and signal (J.M.Neuhaus etc., molecular biology of plants (Plant Mol.Biol.), the 38:127-144 (1998) of golgi's network to the vacuole transitional vesicle; (N.Paris etc., plant physiology (Plant Physiol.), 115:29-39 (1997); M.H. etc., journal of biological chemistry (J.Biol.Chem.), 272:24530-24535 (1997); A.V.Vitale etc., plant science trend (Trends Plant Sci.), 4:148-154 (1999)).Therefore, in yeast, study, to identify in higher plant the effect of vacuole in the cation detoxifcation.

In yeast middle-jiao yang, function of the spleen and stomach ionic isolation Study on Mechanism

Embodiment 1: the function of AtNhx1 and Avp1 in yeast system

For the separation model that proposes among proof diagram 4A and the 4B, made up the mutant yeast system (ena1) of disappearance cell membrane sodium outflow pump, this mutant yeast is thereby must depends on inner detoxification system, to grow in hypersaline environment.Isolation model shown in Fig. 4 A and the 4B (R.Nass and R.Rao, journal of biological chemistry (J.Biol.Chem.), 273:21054-21060 (1998), and R.A.Gaxiola etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 95:4046-4050 (1998)) estimates: if can strengthen the availability of proton in the endosome chamber of supposition, ena1 system becomes salt tolerant so, promptly along with flowing in the proton that increases, the cytoplasm sodium ion can be isolated by the NHX permutoid.

Yeast vacuole ATP enzyme is a multimeric protein, and therefore being difficult to increases its activity by crossing arbitrary subunit of expressing wherein.But (instead), increase in the proton and flow by in yeast, expressing A.thaliana AVP1 gene, thereby obtain identical effect.This gene code single chain polypeptide, when expressing in yeast, it can pump into proton the tube chamber (E.J.Kim etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 91:6128-6132 (1994)) of vacuole.For guaranteeing the high activity of this proton pump, the E229D gain-of-function mutation body (R.G.Zhen etc. of the AVP1 gene (AVP1-D) of proton pump sexuality have been expressed with enhancing, journal of biological chemistry (J.Biol.Chem.), 272:22340-22348 (1997)).Saltant and interior sodium and the potassium content of wild-type cell after being grown on SD-uracil (SD-ura) medium that contains high concentration NaCl have been detected.Material and method

Yeast strain and plasmid

(ura3-1 can1-100 leu2-3,112trp1-1 his3-11, (R.A.Gaxiola etc., EMBO magazine (EMBO J.), 11:3157-3164 (1992)) isogenic system have been adopted with W303.In order to make up GEF1 and NHX1 gene delection, plasmid pRG52 (gef1 ∷ HIS3) (R.A.Gaxiola etc. have been adopted, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 95:4046-4050) (1998) and pRG197 (nhx1 ∷ HIS3), thus prepared RGY85 and RGY296 system respectively.Ena1 ∷ HIS3 mutant available from Fink laboratory collection (collection) (L5709).

Method for transformation

Carried out the conversion of yeast cells (D.Gietz etc., nucleic acids research (Nucleic Acids Res.), 20:1425 (1992)) by utilizing the lithium acetate method.Double-mutant RGY324 (gef1 ∷ HIS3 ena1 ∷ HIS3), RGY326 (nhx1 ∷ HIS3 ena1 ∷ HIS3) and RGY343 (gef1 ∷ HIS3 nhx1 ∷ HIS3) obtain through the hybridization of single mutation system.By being given a mark, the phenotype relevant with each single mutant identify the double-mutant among the mitogenetic offspring.Sprout (sporulation), the marking of tetrad division (dissection) and mating type states document described (C.Guthrie and G.R.Fink as follows, " yeast genetics and molecular biology guide " (Guide to Yeast Genetics and MolecularBiology), the academic press, Santiago, (1991)).Cell is at YPD (1% yeast/2% peptone/2% dextrose, Difco company), YPGAL (1% yeast/2% peptone/2% galactose, Difco company), SD (Difco company, add the synthetic medium of 2% dextrose), or APG (APG is synthetic minimal medium, comprise the 10mM arginine, 8mM phosphoric acid, 2% glucose, 2mM magnesium sulfate, 1mM potassium chloride, 0.2mM calcium chloride, and trace mineral and vitamin) (A.Rodriguez-NavarroJ.Ramos, " bacteriology magazine " (J.Bacteriol.), 159:940-945 (1984)) in cultivate.Add manganese chloride (Sigma company), tetramethyl ammonium chloride (tetramethylammonium chloride) (Sigma company), sodium chloride (Sigma company) or hygromycin-B (Sigma company) by explanation.

Wild type, L5709 (ena1. ∷ HIS3), RGY324 (gef1 ∷ HIS3ena1 ∷ HIS3) and RGY326 (nhx1 ∷ HIS3 ena1 ∷ HIS3) strain transform with pYES2 carrier (Invitrogen company) and plasmid pYES2-AVP1-E229D, it is described in R.G.Zhen etc., journal of biological chemistry (J.Biol.Chem.), 272:22340-22348 (1997).RGY343 (gef1 ∷ HIS3nhx1 ∷ HIS3) strain pRG151 (the GEF1-GFP) (R.A.Gaxiola etc. that are used for tissue chemical analysis, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 95:4046-4050 (1998)) with pRIN73[NHX1-(HA) ₃] (R.Nass and R.Rao, journal of biological chemistry (J.Biol.Chem.), 273:21054-21060 (1998)) transform.

Wild type and RGY296 (nhx1 ∷ HIS3) strain transforms (R.Ballester etc., cell (Cell), 59:681-686 (1989)) with the pAD4 carrier.RGY296 (nhx1 ∷ HIS3) transforms (seeing " clone of AtNHX1 ") with pRG308 (ADH1 ∷ AtNHX1).

The detection of sodium and potassium content in the cell

The cell incubated overnight is in SD-ura medium (Difco company; Synthetic medium contains 2% dextrose, no uracil).Overnight culture is injected YPGAL (1% yeast extract/2% peptone/2% galactose; Difco company) medium and be cultured to A ₆₀₀(absorbances during 600 nanometers) are 0.6.Under this OD (optical density), add NaCl to final concentration be 0.7M.Cultured cell 6 hours, centrifugal collection is with 1.1M sorbierite and 20mM magnesium chloride washed twice, then under 95 ℃, with water extracting 30 minutes.

Sodium ion and potassium ion quantity are to detect (referring to network address: rserv/uga.edu/rsnew/chemicalanalysis/) by induction coupling plasma-mass spectrum in Georgia university chemistry assay laboratory in the cell.By utilizing, estimated intracellular cation concentration (R.A.Gaxiola etc., EMBO magazine (EMBO J.), 11:3157-3164 (1992)) to being grown in water number in the cell that cell calculates in the 1M sodium chloride.

Immunofluorescence

RGY343 (gef1 ∷ HIS3 nhx1 ∷ HIS3) is to be incubated at SD-ura, SD-leu medium (Difco company; Synthetic medium contains 2% dextrose, no uracil and leucine) in to logarithmic growth mid-term, add 0.1 mg/ml hygromycin B, cultivated these cultures one hour at 30 ℃ then.In room temperature and not under the stirring condition, with 3.7% formaldehyde (Sigma company) fixed cell 45 minutes.Carried out spheroplast formation (spheroplast formation), can penetratingization (permeablization), washing and antibody cultivates (J.Pringle etc., in " yeast immunofluorescence method " (Immunofluorescence Methods for Yeast) (academic press that C.Guthrie and G.F.Fink edit, Santiago), Vol.194, pp.565-602 (1991)).The effect first antibody MAB HA11 be from Babco company (Richmond, CA).The crosslinked goat anti-mouse IgG (immunoglobulin G) of Cy3-is to derive from JacksonImmunoresearch company.4 ', 6-diamidino-2-phenylindone (4 ', 6-Diamidino-2-phenylindole) (Sigma company) adds film solid media so that mitochondria and nuclear DNA are dyeed.

Subcellular fractionation separates and Western analyzes

RGY343 (gef1 ∷ HIS3 nhx1 ∷ HIS3) is to be incubated at APG medium (pH7.0), lysate carries out fractionated (R.Nass and R.Rao through 10 step sucrose density gradients then, journal of biological chemistry (J.Biol.Chem.), 273:21054-21060 (1998)).Aliquot (100 microgram) to each fraction is carried out SDS/PAGE (SDS/ polyacrylamide gel electrophoresis), is transferred to nitrocellulose (R.Nass and R.Rao, journal of biological chemistry (J.Biol.Chem.), 273:21054-21060 (1998)) then.With monoclonal anti GFP (green fluorescent protein) antibody (1: 10,000 dilution, CLONTECH company), anti-hemagglutinin antibody is (1: 10,000 dilution, Boehringer Mannheim company) and the goat anti-mouse antibody (1: 5000) of peroxidase coupling Western blotting (Werstern blots) has been carried out the probe detection, and the chemiluminescence system (Amersham Pharmacia company) that strengthens with electrification luminous (ECL) develops the color.

The clone of AtNHX1

AtNHX1 is the phage cDNA library (J.J.Kieber etc. of clone A.thaliana, cell (Cell), 72:427-441 (1993)) (obtains) from arabidopsis living resources center, survey by carrying out probe with the sequence mark fragment (arabidopsis living resources center, DNA stores the center) of the expression that contains the part clone.Full-length clone (2.1kB) is connected to carrier pSK2 (Stratagene company) (at the NotI position), thereby produces plasmid pRG293.With pRG293 (as template) and GGCCCGGGATGGATTCTCTAGTGTCGAAACTGCCTTCG (No. 5 sequences) (the italic base is corresponding with the 1-30 nucleotide of open reading frame) and T7 oligonucleotides, and the AtNHX1 open reading frame is increased by PCR (polymerase chain reaction).The PCR product digests with XbaI and SalI and enzyme is cut and is connected in the pAD4 carrier then, obtains the pRG308 plasmid.Measure the reliability of the sequence of AtNHX1 open reading frame with checking PCR product.The open reading frame that full length sequence is initiated center (ATM021B04.4) report than the arabidopsis gene group is long, and has left gene library (GenBank) (number of asking for AF106324) in.

The clone of AVP1-D

Carrier pYES2 (Invitrogen company) is imported wild type, ena1, ena1nhx1 and ena1 gef1 mutant.Plasmid pYes2-AVP1-D (R.G.Zhen etc., journal of biological chemistry (J.Biol.Chem.), 272:22340-22348 (1997)) is imported ena1, ena1 nhx1 and ena1 gef1 mutant.5 times of serial dilutions (from 105 cells) thing of every strain is incubated at YPGAL (1% yeast extract/2% peptone/2% galactose) (containing or do not contain 0.5M sodium chloride) flat board, and cultivates 2 days in 30 ℃.The cell (wild type and the ena1 that transforms with the pYES2 carrier, and ena1, the ena1 nhx1 and the ena1 gef1 mutant that carry pYes2-AVD1-D) of exponential growth was exposed to 0.7M sodium chloride 6 hours.Prepare full cell extract, and recorded sodium ion and potassium concentration.

The result

The ena1 mutant of above-mentioned structure lacks cell membrane sodium outflow pump, therefore must rely on the toxicity that inner detoxification system is eliminated sodium.The growth of ena1 strain is to low na concn sensitivity (200mM), and this concentration does not suppress the growth of wild-type strain.Cross and express AVP1-D and can recover the tolerance of the responsive ena1 mutant of salt salt.AVP1-D needs the NHX1 and the GEF1 gene of function to the recovery of ena1 strain salt tolerance: ena1 nhx1AVP1-D and ena1 gef1 AVP1-D strain are salt-sensitive.

Fig. 5 A and Fig. 5 B are column type figure, expression wild-type yeast strain and carry the interior sodium ion of cell and the potassium content of the yeast strains of the multiple sudden change that influences the sodium tolerance, and wherein numerical value is the mean value of twice measurement, the stub line is represented standard deviation.Being exposed to the medium that replenishes with 0.7M sodium chloride after 6 hours, detected the wild type strain and carried multiple interior sodium ion of cell and the potassium content that influences the saltant of sodium tolerance.Can see at the intracellular sodium ions content of ena1 mutant strain than 8 times of wild type plant heights.(consistent) that can see the unanimity of the total cellular sodium ion in ena1 AVP1-D strain reduces, this minimizing agnogenio.Discover that this ena1AVP1-D strain is a salt tolerant, though sodium ions content is high 4 times than sodium ions content in the born of the same parents of wild type in its born of the same parents.In the ena1 AVP1-D strain that lacks gef1 or nhx1 (being ena1 gef1 or ena1 nhx1), sodium ions content is not reduced to the degree of sodium ions content in GEF1 NHX1 strain.Combine and top result, the model that heredity and physiological data and Nhx1, Gef1 and Avp-1 act synergistically with internal insulation sodium is consistent.As seen seeing in the drawings, arabidopsis vacuole hydrogen ion-pyrophosphatase (Avp1) is evident as yeast ena1 mutant strain and has given the salt tolerance.

Discover, potassium content and the positive correlation of sodium tolerance in the cell, and with the sodium ions content negative correlation (Fig. 4 B) of bacterial strain.The wild type potassium concentration is 100mM, but then is reduced to 20mm in the ena1 mutant strain.What is interesting is that expressing in the ena1 strain of AVP1-D gene excessively, potassium concentration almost returns to wild type level (Fig. 4 B) in the cell.Yet unless NHX1 and GEF1 have function, expressing excessively of AVP1-D can not recover the interior potassium ion of cell to wild type level (seeing double-mutant ena1 nhx1 or ena1 gef1 among Fig. 4 B).

As described herein, the detoxication of sodium ion needs the sodium ion/hydrogen ion permutoid (Nhx1) and the chloride channel (Gef1) of function in the yeast cells, and they are positioned at preceding vacuole chamber (R.A.Gaxiola etc. jointly, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 96:1480-1485 (1999)).When the arabidopsis (Araidopsis thaliana) of yeast NHX1 gene (AtNHX1) when homologue is cloned, detected its function in the nhx1 yeast mutant, discover that this AtNHX1 gene can partly suppress the responsive phenotype of cation of nhx1 mutant strain.Fig. 6 is the arrangement of amino acid sequence of the supposition of NhX1 homologue, and wherein the NhX1 homologue is from arabidopsis AtNHX1 (No. 1 sequence), human HsNHE-6 (No. 2 sequences) and yeast ScNHX1 (No. 3 sequences); Identical residue represents that with black surround short-term is represented the disappearance (gaps) in the sequence.The * that arranges above number the expression from human NHE1 ( ¹⁶³DVF-FLFLLPPI ¹⁷³) (No. 4 sequences) ammonia chlorine miaow piperazine (amiloride) binding site of reasoning out.

Function and the common location of embodiment 2:Gef1p Nhx1p in yeast

NHX1 and GEF1 gene, it plays an important role in the sodium detoxifcation, equally also is that other cation detoxifcation is needed.In the presence of poisonous cation, carried out containing gef1 and nhx1 (ena1) yeast mutation is the research of survival ability, research is the model that proposes according among Fig. 4 a and the 4b.

This isolation model is not only inferred and is existed function to connect between anion channel Gef1 and sodium permutoid Nhx1, and predicts that two kinds of albumen are positioned common chamber jointly.(school: " these two proteins " the bad understanding in the original text) because former studies show that, vacuole chamber (R.Nass and R.Rao before Nhx1 is positioned, journal of biological chemistry (J.Biol.Chem.), 273:21054-21060 (1998)), so also carried out experiment to determine whether Gef1 and Nhx1 albumen are positioned this chamber altogether.

Material and method

Use pRG151; GEF1-GFP and pRIN73; NHX1-(HA) ₃Plasmid transforms RGY419 (gef1 nhx1).Transformant is incubated at SD (Difco company; Synthetic medium with 2% dextrose).In order to detect these transformant to the cationic susceptibility of toxicity, 5 times of serial dilutions of above-mentioned system are (from 10 ⁵Cell begins) thing is incubated at YDP (1% yeast extract/2% peptone/2% dextrose) 2 days at 30 ℃, wherein adds 3mM manganese chloride, 0.45M tetramethyl-ammonium (TMA) as indicated, perhaps adds 0.05mg/ml hygromycin B (HYG).

For the common location of proof Gef1p and Nhx1p, two researchs have been carried out.

Measured the fluorescence distribution and the immunity of subcellular fraction in gef1 nhx1 cell and surveyed, wherein gef1 nhx1 cell is to transform with following two assemblies: GEF1-GFP merges and NHX1-(HA) ₃The fusion of mark.When cell grows to OD ₆₀₀=0.5 o'clock, add hygromycin B (Sigma company) and cultivated 40 minutes to final concentration 0.1mg/ml and in 30 ℃.Fixed cell and with anti-HA epitope and 4 ', the antibody staining of 6-diamidino-2-phenylindone (DAPI).Come observation of cell and utilize to remove to revolve algorithm (deconvolution algorithm) (Scanalytics company, Billerica MA) get optical cross section (B.K.Kennedy etc., cell (Cell), 89:381-391 (1997)) with the microscope of electric charge coupling; (stub=1m.).

Also measured Gef1p and the Nhx1p migration performance in saccharose gradient so that Nhx1 to be provided (HA) ₃Common location evidence with GEF1-GFP.With plasmid pRG151; GEF1-GFP and pRIN73; NHX1-(HA) ₃Transform RGY419 system (gef1 nhx1) and be incubated at APG medium (A.Rodriguez-Navarro and P.A.Rea, journal of biological chemistry (J.Biol.Chem.), 159:940-945 (1984)).Above-mentioned (such) is converted into spheroplast, through cytolysis (lysed), then with 10 step saccharose gradient (18-54%) fractionated (A.Sorin etc., journal of biological chemistry (J.Biol.Chem.), 272:9895-9901 (1997) and A.Antebi and G.R.Fink, cellular elements biology (Mol.Biol.Cell), 3:633-654 (1992)).Western blotting has shown the distribution of Gef1-GFP and Nhx1-HA.

The result

Discover that the Gef1 mutant is to 3mM manganese chloride, 0.45M tetramethyl ammonium chloride and 0.05 mcg/ml hygromycin B sensitivity.The Nhx1 mutant is also to tetramethyl ammonium chloride and hygromycin sensitivity.The nhx1 mutant can be the extreme sensitivity of hygromycin and analyzes the nhx1 function important instrument is provided.

Discover that the Nhx1 and the Gef1-GFP fusion of hemagglutinin (HA) mark are located altogether, as going to revolve shown in the microscope by outer fluorescence.Consistent signal after image revolves and turn 90 degrees is further supported this common location of two kinds of transport proteins in these cells.Nhx1 (HA) ₃Both location are altogether also supported in common migration in the sucrose density gradient of film preparation (membranepreparations) with GEF1-GFP albumen, and wherein film preparation is the cell available from presentation markup albumen.The settling property consistent with the settling property of preceding vacuole chamber (R.Nass and R.Rao, journal of biological chemistry (J.Biol.Chem.), 273:21054-21060 (1998)) that contains the membrane portions of two kinds of albumen.Gef1-GFP (but not being Nhx1) also is present in the Golgi body part, this and the consistent (R.A.Gaxiola etc. of previous research, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 95:4046-4050 (1998), B.Schwappach etc., journal of biological chemistry (J.Biol.Chem.), 273:15110-15118 (1998)).

The A.thaliana homologue of embodiment 3:NHX1 suppresses the ability of mutant yeast to hygromycin susceptibility

Yeast described herein is that the gene of identifying mediation salt tolerance in other biology provides important means.For checking the practicality of this system, identified sequence (having very high autoploidy) with saccharomyces cerevisiae NHX1 open reading frame from arabidopsis (seeing material and method), and use the sequence mark fragment of expressing (seeing material and method), to obtain the full-length clone of this arabidopsis gene.The comparison (alignment) of the amino acid sequence of the Nhx1 homologue of arabidopsis (AtNhx1), human (HsNhe6) and yeast (ScNhx1) has disclosed identical segment and the similitude (Fig. 6 A-C) in the membrane spaning domain of estimating of amino acid.Yet what need emphasize is that though these relations are arranged, the carboxyl terminal of AtNhx1 and ScNhx1 does not all demonstrate the autoploidy (Fig. 6 A-C) of height.

The feature of mammality sodium ion/hydrogen ion antiport carrier is that it can be suppressed by amiloride.The amiloride binding site of having determined to infer by point mutation body in human NHE1 antiport gene ( ¹⁶³Asparagus fern-figured silk fabrics-phenylpropyl alcohol-phenylpropyl alcohol-bright-phenylpropyl alcohol-bright-bright-dried meat-dried meat-different bright ¹⁷³( ¹⁶³DVFFLFLLPPI ¹⁷³) (No. 4 sequences) (L.Counillon etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 90:4508-4512 (1993)).AtNhx1, HsNhe-6 and ScNhx1 have sequence (Fig. 6) much at one.Yet in yeast culture, the trial that suppresses the activity of Nhx1 or AtNhx1 with amiloride all gets nowhere.

Yeast nhx1 mutant makes and can test the extreme susceptibility of hygromycin: in yeast, whether clone's arabidopsis AtNHX1ORF (open reading frame) can provide sodium ion/hydrogen ion function of exchange.Carrier pAD4 (R.Ballester etc., cell (Cell), 59:681-686 (1989)) is imported wild type and nhx1 system.With plasmid pRG308; ADH; AtNHX1 imports the nhx1 mutant.5 times of serial dilution things (105 cells begin) of described bacterial strain are incubated at YPD (-) or replenish with the YPD medium of 0.05mg/ml hygromycin (+) 2 days in 30 ℃.The serial dilution thing of identical fungus strain grows in APG medium (seeing that material is in method) (-), or grow in APG medium (A.Rodriguez-Navarro and the J.Ramos that replenishes with 0.4M sodium chloride, bacteriology magazine (J.Bacteriol.), 159:940-945 (1984))

This AtNHX1 gene can suppress the hygromycin susceptibility of nhx1 mutant.This AtNHX1 gene also suppresses the sodium chloride susceptibility of nhx1 mutant, but prerequisite is the availability decline of potassium ion.Yet the AtNHX1 gene can not be saved the Na of the double-mutant ena1 nhx1 that expresses the AVP1-D gene ^+-Responsive growth phenotype.

Embodiment 4: function obtains the generation of yeast AtNHX mutant strain

Material and method

Make up mutant library by the mutagenic treatment cloned genes, then can in the ena1 yeast, produce the gain-of-function mutation body of the AtNHX that strengthens the salt tolerance.This library can be used to transform salt sensitive yeast mutant ena1 and clones with the salt tolerant phenotype that strengthens.

Other demonstrates with the AtNHX1 gene has similar gene, as the gene of being reported by arabidopsis gene group promoter (AGI), also can express in mutant yeast to form the gain-of-function mutation strain.Think that at present the homologue of some AtNHX1 is a cell membrane transporter albumen, therefore, their functions in yeast usually are that pH is dependent, as desire successfully screening, then need medium to be made up of and pH accurate.The evaluation of cell membrane transporter albumen helps to design the plant (because the sodium that reduces is taken in) of the salt tolerance with enhancing.In addition, the plant cDNA expression library can be used for identifying that other participates in the transport protein family of sodium chloride detoxifcation in yeast.

In order to produce the gain-of-function mutation strain of AtNHX, can adopt Stratgene (Escherichia coli (Epicurian Coli) XL1-Red competent cell, the Cat#200129) method of the importing random mutation of Jian Liing.The gene that this method relates to being cloned on defective bacterial strain on 3 kinds of basic DNA reparation approach increases.Random mutation rate in this strain is approximately high 5000 times than wild type.The AtNHX gene library of sudden change can be transformed in the ena1 yeast mutants and at the salt tolerance screens.Yeast conversion adopts Schiestl and partner's reported method to carry out (20:1425 (1992), it is hereby expressly incorporated by reference for D.Gietz etc., nucleic acids research (Nucl.Acid Res.)).The method of another alternative XL1-Red random mutagenesis strategy is the PCR method (H.D.Madhani etc., cell (Cell), 91:673-684 (1997)) that Fink and partner describe.

Be test AtNHX1 homologue, can adopt at R.A.Gaxiola etc., identical bacterial strain and the condition described in institute of the NAS newspaper (Proc.Natl.Acad.Sci.USA), 96:1480-1485 (1999).Yet, also can adopt other method.When relating to cell membrane AtNHX1 homologue, the pH condition of measuring medium may be very crucial.

The result

The expression of crossing of A.Thaliana gain-of-function mutation body gene A VP1-D has increased detoxification ability in the yeast cells (R.A.Gaxiola etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 96:1480-1485 (1999)).According to hypothesis (though the present inventor is not limited to this hypothesis) latter is to flow into the vacuole chamber because hydrogen ion increases, and isolates thereby improve sodium ion by the Nhx1 permutoid.

Isolate research from the yeast cation and obtain conclusion

The importance that above-mentioned yeast research is isolated sodium ion in the yeast cells for preceding vacuolar pH value provides evidence.Plant H ⁺Cross expressing of-pyrophosphatase (Avp1) only is just to give yeast salt tolerance what those contained chloride channel (Gef1) that function is arranged and sodium ion/hydrogen ion permutoid (Nhx1).

These data are supported Nhx1 sodium ion/hydrogen ion permutoid and vacuole ATP enzyme and the GEF1 anion channel synergy model with vacuole chamber before cation is isolated from.Before some research promptings vacuole chambers can derive from cell membrane and late period Golgi body.These vesicles participate in the assembling of vacuole or carriage load probably to organelle.Can rational expectation, these preceding vacuole vesicles detoxify to cation by isolating, and reduce its concentration in cytoplasm and other organelle thus.

Yeast system described herein allows the salt tolerance of different heterologous proteins is carried out functional assessment ∷ chloride channel, proton pump, sodium ion/hydrogen ion permutoid and other cation/hydrogen ion permutoid or cation/bicarbonate symport albumen (symporters).The not only stable but also flexible operation of this system.Arabidopsis chloride channel (R.A.Gaxiola etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 95:4046-4050 (1998); M.Hechenberger etc., journal of biological chemistry (J.Biol.Chem.), 271:33632-33638 (1996)), the function of proton pump and sodium ion/hydrogen ion permutoid can analyze in corresponding yeast mutants.Though AtNHX1 can not suppress all yeast nhx1 mutation type surfaces, it can suppress some phenotype and and AtNHX1 and yeast NHX1 between this fact of dna homology coupling, show that this plant gene and yeast homologue have similar function.The AtNHX1 gene can suppress the nhx1 mutant to the susceptibility of hygromycin but faint sodium ion detoxifcation phenotype only is provided, and this may be that this transport protein has different regulation and control or has optionally result of different cation transfers in two kinds of biologies.

The ability that the adjusting of salt pair AtNHX1 and plant gene suppress yeast nhx1 mutant shows that the mechanism of cation detoxifcation may be similar in yeast and plant.In fact, former work shows that cell membrane sodium ion/hydrogen ion antiport carrier can mediate the accumulation of sodium in the vacuole in the salt-tolerant plant, and this antiport carrier utilizes by vacuole H ⁺-ATP enzyme (V-ATP enzyme) and/or hydrogen ion transhipment pyrophosphatase (V-pyrophosphatase; B.J.Barkla etc., Symp.Soc.Exp.Biol., 48:141-153 (1994); R.G.Zhen etc., " molecule and biochemical basis that the proton at tonoplast that pyrophosphoric acid can drive is transported " (The Molecular and Biochemical Basis ofPyrophosphate-Energized Proton Translocation at the VacuolarMembrane) (academic press, Santiago); M.Kirsh etc., molecular biology of plants (Plant Mol.Biol.), 32:543-547 (1996)) proton motive force that produces.

Discovery described herein, promptly gef1 and nhx1 mutant show the function that the resistance level of hygromycin is depended on vacuole and preceding vacuole organelle all to the hygromycin hypersensitization.The yeast mutants (rek1) that the potassium ion absorption suffers damage is to hygromycin hypersensitization (R.Madrid etc., journal of biological chemistry (J.Biol.Chem.), 273:14838-14844 (1998)); The potassium ion that reduces is taken in hyperpolarization cell membrane position and is driven alkaline kation such as the absorption of hygromycin.Reduce cell membrane H ⁺The sudden change of the proton pump activity of-ATP enzyme (Pmal) makes cell membrane potential depolarising and give the resistance to hygromycin (J.H.McCusker etc., molecular cytobiology (Mol.Cell.Biol.), 7:4082-4088 (1987)).Therefore, can the anticipated impact vacuole and the mutant of the pH of preceding vacuole chamber or film potential such as gef1 or nhx1 can influence the compartmentation (compartmentation) of hygromycin.

Have the genetically modified plants of raising vacuole hydrogen ion transhipment pump activity

Arabidopsis AtNHX1 expression of gene stress be induced this observed result further to support the effect (R.A.Gaxiola etc. of arabidopsis AtNHX1 gene in the salt homeostasis in the plant at salt, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 96:1480-1485 (1999)).Particularly, arabidopsis (Arabidopsisthaliana) has been used as the host plant model and has expressed the salt tolerance that causes in the plant so that crossing of these genes to be described.A recent research report shows, the expression of crossing of AtNHX1 gene has promoted the lasting growth of this plant in soil in transgenic arabidopsis (Arabidopsis thaliana) plant, this soil adds that with 200mM sodium chloride 1/8MS salt is irrigated and employing short-day periodic condition (M.Apse etc., science (Science), 285:1256-1258 (1999)).It should be noted that every adding 1/8MS salt then provides 2.5mM potassium, this reduced sodium chloride stress severe degree, and the short-day cycle reduced oxidative stress.

Embodiment 5: the expression of the enhancing of AtNHX1 gene in salt stress plant

Material and method

Under 19 ℃ and continuous illumination condition, A.thaliana plant (ecotypeColumbia) aseptic culture is (G.W.Haughn and C.Somerville on basic (unsupplemented) plant nutrition agar of no sucrose, molecular gene genetics (Mol.Gen.Genet.), 204:430-434 (1986)) 15 day.Add sodium chloride or potassium chloride to final concentration 250mM, cultivated plants then 6 hours.From handle through salt with the plant tissue of handling without salt separate total RNA (K.K.Niyogi and G.R.Fink, plant cell (Plant Cell), 4:721-733 (1992)), Hybond-N (Amersham company) film is with from plasmid pRG308's ³²The dna probe hybridization of P mark.65 ℃ of hybridization are spent the night.Wash (F.Ausebel etc., molecular biology fresh approach (Curr.Protocols in Mol.Biol.) (Wiley, New York) (1988)) with 0.2% standard citrate buffer solution (SSC)/0.1%SDS down at 65 ℃.Contrast (loading control) (I.Unfried etc., nucleic acids research (NucleicAcids Res.), 17:7513 (1989)) with the 18S probe as applied sample amount.Determine the relative amount of RNA with MACBAS 2.4 programs.

The result

Sodium chloride stress increase by 4.2 times of AtNHX1 mRNA. levels, and potassium chloride only impels 2.8 times of increases.The mRNA level that this kind caused by the sodium situation similar (R.Nass and R.Rao, journal of biological chemistry (J.Biol.Chem.), 273:21054-21060 (1998)) with yeast NHX1 gene that raises.RNA organizes trace (tissue blot) to hybridize with AtNHX1.The total RNA of 10 micrograms of the check plant of handling from plant in 15 day age (being exposed to 250mM sodium chloride or potassium chloride 6 hours) with without salt carries out electrophoresis on the sex change formaldehyde gel.This trace is hybridized with AtNHX1 ORF (open reading frame) internal probe.Contrast as applied sample amount with 18S ribosome probe.

Embodiment 6: the salt tolerance of crossing the genetically modified plants of expressing AtNHX1

Cross the genetically modified plants of expressing AtNHX1 and in the soil of irrigating, show lasting growth (M.Apsem etc., science (Science), 285:1256-1258 (1999)) with 200mM sodium chloride.

Embodiment 7: the salt tolerance of crossing the genetically modified plants of expressing 35SAVP1

Material and method

Two-in-series enhancer with 35S promoter designs transgenic arabidopsis (Arabidopsis thaliana) plant, to cross expression AVP1 wild type gene (R.Topfer etc., nucleic acids research (Nucl.Acid Res.), 15:5890 (1987)).The AVP1 coding is from the tonoplast proton pump (R.G.Zhen etc., journal of biological chemistry (J.Biol.Chem.), 272:22340-22348 (1997)) of the pyrophosphoric acid driving of arabidopsis.Previous studies show that, the AVP1 gene is to copy (Y.Kim etc. in the genome that is present in arabidopsis with list, plant physiology (Plant Physiol.), 106:375-382 (1994)), yet, one with chromosome on AVP1 homology but sequence inequality temporarily be called open reading frame F9K20.2 on BAC F9K20 by arabidopsis gene group promoter (AGI).

Utilize agriculture bacillus mediated Plant Transformation to produce and cross the genetically modified plants of expressing AVP1.Utilize the two-in-series enhancer of the 35S promoter of CaMV to come express transgenic AVP1 (R.Topfer etc., nucleic acids research (Nucl.Acid Res.), 15:5890 (1987)).15 strain wild types and 15 strain 35SAVP1 transgenosis strains were cultivated 16 days in 24 hours periods.During this period, per 4 days nutrient solutions (1/8M.S. salt) with dilution of plant water once.In the time of 17 days, add 200mM sodium chloride in the pouring liquid, and in the time of the 27th day, water with the nutrient solution that contains 250mM sodium chloride.After last sodium chloride is handled 10 days, plant is taken pictures.Adopt embodiment 6 described identical condition and processing.

The result

In T2 (second generation) stage, compare with the wild type plant, 5 not the 35SAVP1 plant of homology show the salt tolerance of enhancing.Yet the most noticeable phenotype is tangible in the T3 that isozygotys (third generation) plant.These genetically modified plants are bigger than wild-type plant.And the 35SAVP1 plant of isozygotying at 24 hours illumination states with exist 250mM sodium chloride to add under the condition of 1/8M.S. salt, shows and continues growth.When the 35SAVP1 plant grows under short condition diurnal periodicity (12 hours/day/periodicity of illumination), can be observed it and add lasting growth in the presence of the 1/8M.S. salt at 300mM sodium chloride.

Embodiment 8: wild type plant and 35SAVP1 transfer-gen plant are supported with the growth in the solution in hydroponics

The standard agricultural may force the agrotechnique that adopts other with the minimizing of fresh water sources.It is contemplated that,, plant and on crop production, to create the new epoch with seawater water for salt tolerant crop.It is reported, water planting increased the growth of plant and provide stress not root and bud material (D.M.Gibeaut etc., plant physiology (Plant Physiol.), 317-319 (1997)).Another significant advantage of water planting be it make people with than in soil more accurate way change ion component.These advantages to salt stress (salt stress) Physiologic Studies may be important.

Set up the water planting condition of arabidopsis thaliana, and tested its performance in the medium that sodium chloride concentration increases.

Material and method

In single test, wild type and 35SAVP1 genetically modified plants carry out water planting.Wild type and 35SAVP1 genetically modified plants are grown in the water culture thing, cultivate 65 days under 12 hour photoperiod.

In another experiment, wild type and 35SAVP1 genetically modified plants are grown in the water culture thing, cultivate 20 days under 12 hour photoperiod.From the 21st day, increase sodium chloride concentration gradually, adopt the mode that increased 50mM in per 4 days.Existing 200mM sodium chloride after 4 days, plant is taken pictures.

And in another water culture experiment, give (challenge) genetically modified plants commerce seawater prescription, comprising ion components whole in the seawater.35SAVP1,35SAtNHX1 single transgene and double transgenic plant and the wild type arabidopsis thaliana plant one growth 4 week (D.M.Gibeaut etc. under short-day cycle and the water planting condition that coexist, plant physiology (Plant Physiol.), 317-319 (1997)).Added the tropical ocean sea salt (network address: thatpetplace.com) that is equal to 50mM sodium chloride in per then 4 days.This artificial seawater mixture comprise be present in the real seawater all other main and micro-.The monitoring growth also can be monitored physiological parameter, for example, and sodium content and distribution.

The result

When wild type and 35SAVP1 genetically modified plants carried out water planting, there was significant difference in the size of both roots, leaf and stem, and wherein the 35SAVP1 genetically modified plants are partly much bigger.

(M.Apse etc. when increasing sodium chloride concentration, per 4 days increase 50mM gradually, science (Science), 285:1256-1258 (1999)), the 35SAVP1 genetically modified plants show very healthyly in the presence of 200mM sodium chloride, and wild type then shows the severe impairment effect to leaf and the stem that impinges upon them.

35SAVP1,35SAtNHX1 single transgene and double transgenic plant and the wild type arabidopsis thaliana plant one growth 4 week (D.M.Gibeaut etc. under short-day cycle and the water planting condition that coexist, plant physiology (Plant Physiol.), 317-319 (1997)), added the tropical ocean sea salt that is equal to 50mM sodium chloride in per then 4 days.Discover that 35SAVP1,35SAtNHX1 single transgene and the upgrowth situation of double transgenic plant in sea salt solution are better than wild type arabidopsis (Arabidopsis thaliana) plant.

Embodiment 9: in the tomato plant, cross the effect of expression arabidopsis (Arabidopsisthaliana) proton transport protein

Can detect these arabidopsiss (Arabidopsis thaliana) proton transport proteins (AVP1 and AtNHX1) effect in prior plant-tomato plant on agricultural of expressing.It is believed that the salt tolerance that increases the tomato plant has important economic impact possibly.

The tomato homologue of AVP1 and AtNHX1 can be separated, and corresponding chimera (S.Bidone etc., european journal of biological chemistry (Eur.J.Biochem.), the 253:20-26 (1998) that makes their high expresseds can be made up; A.Burbidge etc., experimental botany magazine (J.Exper.Botany), 48:2111-21112 (1997)).These genes can import callus by agriculture bacillus mediated infection.Can use tissue culture technique to regenerate and transform plant.Can detect salt tolerance and the physiologic parameters of plant, for example sodium content and distribution.

Can and carry out tomato with 35S AVP1 and transform (S.McCormick with 35S AtNHX1 assembly, " transform tomato " (Transformation oftomato with Agrobacterium tumefaciens) with Agrobacterium tumefaciens, K.Lindsey edit " in Plant Tissue Breeding handbook (the Plant Tissue Culture Manual) book, PP.1-9, the Kluwer academic press, Dordrecht, Holland (1991)).Can be by AVP1 and genetically modified existence of AtNHX1 and copy number in polymerase chain reaction and dna gel engram analysis T0 and the T1 genetically modified plants.In the seed of T1,, can identify heterozygote and homozygote plant through each offspring's compartment analysis.Can analyze salt tolerance and the physiologic parameters of homozygote plant, for example sodium content and distribution.Can design based on the degeneration oligonucleotide (oligous) that is present in the conserved sequence in AVP1 and the AtNHX1 homology source thing.These degeneration primers can be used for the RT-PCR reaction with cDNAs, and wherein cDNAs is the ribonucleic acid that the contains polyadenylic acid (poly (A)+RNA) make from tomato.The PCR fragment that produces can separate the cDNA clone (being Stratagene production number 936004) of total length as probe from commercial library.Caboche and partner have described similar strategy (A.Quesada etc., molecular biology of plants (Plant Mol.Biol.), 34:265-274 (1997)).

The result

The positive test the possibility of result shows that isolation model described herein also can be applicable to important crops.

Fig. 7 plants wild type plant (WT) in containing the high soil of salinity to sodium ion and potassium content figure in the representational transfer-gen plant of crossing expression AVP1 (1 ' and 2 ').Five strain wild-type plants (WT) and two strain AVP1 cross express transgenic system (1 ' and 2 ') plant and plant in soil under the cycle 10 hours light and shades.With nutrient solution (1/8MS salt) 6 weeks of irrigating plant of dilution, then to have added the dilution nutrient solution pouring of sodium chloride.The Nacl initial concentration is 100mM, and increases 50mM in per 4 days.There is 300mM sodium chloride in photo at that time corresponding to the 10th day plant.Exist 200mM sodium chloride after 24 hours, plant part and its fresh weight of weighing (fresh weigh) on the results ground.At 75 ℃ after following 48 hours, its dry weight of weighing.By atomic absorption detecting sodium ion and potassium content.Numerical value among Fig. 4 be mean value+/-SE (standard error) (n=4).As seeing as seen from the figure, sodium ion in the transgenic lines (1 ' and 2 ') and potassium content are significantly higher than sodium ion and the potassium content in the wild type counterparts.

Fig. 8 is that calcium is taken among Fig. 42 ' 35SAVP-1 transgenosis tonoplast vesicle (square) is taken in the vesicle of wild type (WT) among Fig. 4 to calcium curve map.Wild type plant (open circles (open circles)) and genetically modified plants 9 weeks of water planting under 10 hour photoperiod that are from 2 ' of Fig. 4.The tonoplast vesicle adds in the buffer solution, and this buffer solution comprises BTP-Hepes, 50mM potassium chloride, 1.5nM magnesium sulfate and the 10 μ M divalent calcium ions of 250mM sorbierite, 25mM pH8.0.Before adding 200 μ M pyrophosphoric acids, this mixture is incubated 10 minutes down at 20 ℃, to start reaction.Add calcium ion carrier A 23187 to final concentration 5 μ g/ml, with dissipation calcium ion gradient.At the appointed time filter aliquot (200 μ l), and as described in (11), wash with cold buffer liquid.As can be seen, from 2 ' genetically modified plants that are have bigger calcium pickup than wild-type plant.

Above-mentioned data are consistent with following hypothesis: cross the genetically modified plants express AVP-1 and have the proton pump sexuality of enhancing at its cell membrane, and the hydrogen ion that strengthens of the effect of the ion transporter that drives by hydrogen ion is supplied with and caused ion accumulation higher in the vacuole.For further supporting this theory, measured the calcium ion picked-up ability of wild type and transgenosis tonoplast vesicle.

Many documentary evidences are arranged, and calcium ion enters plant vacuole (K.S.Schumaker, H.Sze, plant physiology (PlantPhysiol.), 79,1111-1117 (1985)) by calcium ion/hydrogen ion antiport carrier.In addition, the gene of coding arabidopsis (A.thaliana) calcium ion/hydrogen ion antiport support C AX1 and CAX2 separated and sign (K.D.Hirschi, R.-G.Zhen, K.W.Cunningham, P.A.Rea, G.R.Fink, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 93,8782-8786 (1996)).Fig. 8 shows that calcium pickup is high by 36% from the vesicle of wild type in 35SAVP-1 transgenosis tonoplast vesicle.Utilization Calcium ionophore A23 can reduce to background level with the counting of 45 calcium ions, and this shows the seal (Fig. 8) (K.S.Schumaker, H.Sze, plant physiology (PlantPhysiol.), 79,1111-1117 (1985)) of this vesicle.

Though be not limited to this theory, in Fig. 9 A and 9B, described arid and the corresponding to model of freeze injury tolerance with the enhancing of crossing the genetically modified plants of expressing the AVP-1 gene.This model description: how the increase of AVP-1 pump quantity can provide more hydrogen ion in the vacuole of genetically modified plants, makes secondary transport protein can import more cation and enters the vacuole chamber.The cation of higher quantity is given bigger osmotic pressure (seeing Figure 14), and it causes the ability of the higher reservation water of plant, and this helps the low flow of water of plant opposing soil.

Embodiment 10: the dual genetically modified plants with 35S AVP1 and 35S AtNHX1

The hydrogen ion availability that increases possibly in the vacuole chamber is expressed in crossing of the tonoplast proton pump AVP1 that pyrophosphoric acid drives, and AtNHX1 sodium ion/hydrogen ion antiport carrier utilizes these hydrogen ions, so that sodium ion is isolated in the vacuole.Therefore, these transport proteins maximizes the isolating power of vacuole probably than high expressed.

For producing the transgenic arabidopsis plant of expressing AVP1 and two kinds of genes of AtNHX1, can with T3 35S AVP1 plant as maternal and with T3 35S AtNHX1 plant as male parent.Maternal plant can manually be castrated, and results are from the fresh flower pesticide of donor plant.Use these flower pesticide, contact with column cap, then can give the plant pollination of castrating by making flower pesticide.The flower of pollination can make marks, and with any on the same female plant stay open or do not open spent and removed, any when avoiding gathering in the crops obscures.The seed of results should use for 50% sodium hypochlorite solution sterilization and violent the mixing 5 minutes, and water fully washs then.Seed through sterilizing can be kept in the soft agar under 4 ℃ and spend the night.They can be loose to plant in the kanamycin that solidifies-hygromycin then and select medium.This 35S AVP1 assembly contains neomycin phosphotransferase II gene, and it gives plant kanamycin tolerance, and 35S AtNHX1 assembly contains modified hygromycin B phosphotransferase, and it gives plant hygromycin tolerance.The seedling of resistance can be transplanted in soil and the water ballast medium to check their salt tolerant phenotype.

Utilize the identical two-in-series enhancer (R.Topfer etc. of above-mentioned 35A promotor, nucleic acids research (Nucl.Acid Res.), 15:5890 (1997)) can design transgenic arabidopsis (Arabdopsis thaliana) plant (R.G.Zhen etc. that express A.thaliane gain-of-function mutation body gene A VP1-D, journal of biological chemistry (J.Biol.Chem.), 272:22340-22348 (1997)).The plant that crosses expressive function acquisition mutant gene may show the phenotype of enhancing.These plants can characterize simultaneously with 35SAVP1,35S AtNHX list and double transgenic plant.The gain-of-function mutation body gene A VP1-D of A.thaliana can subclone advances to carry the pRT103 plasmid (R.Topfer etc., nucleic acids research (Nucl.Acid Res.), 15:5890 (1987)) of the polyadenylation signal of 35S promoter and CaMV.The HindIII fragment that contains chimeric 35SAVP-D gene can be advanced pBIBhyg (D.Becker, nucleic acids research (Nucl.Acid Res.), 18:203 (1990)) by subclone.T-DNA (transforming DNA) carrier that generates can transform by electroporation and enter Agrobacterium tumefaciems GV3101 strain, and the vacuum that can be used for the A.thaliana ecotype Columbia of back is infiltrated (infiltration) (N.Bechtold etc., C.R.Jeances Acad.Sci.Ser.III Sci.Vie, 316:1194-1199 (1993)).Can confirm to integrate by southern blotting technique of T3 plant and the expression that on the RNA of positive T3 plant trace, detects.

Embodiment 11: the transhipment comparative studies of the root vacuole of wild type and 35SAVP1 genetically modified plants

Whether the vacuole that can study to determine 35S AVP1 genetically modified plants shows the height proton transport activity more that depends on pyrophosphoric acid.These are measured and availablely to carry out from the root and the bud tissue of hydroponic plant respectively.This transgenosis can show the tissue specificity regulation and control, and is irrelevant with 35S promoter.

Be the hydrogen ion transport activity that the pyrophosphoric acid of wild type and 35S AVP1 genetically modified plants relatively relies on, can prepare the vesicle that is rich in tonoplast from the sealing of the root of above-mentioned plant and leaf.Can adopt described homogenizing of Rea and Turner and differential centrifugation program (P.A.Rea etc., " tonoplast ATP and inorganic pyrophosphatase " (TonoplastAdenosine Triphosphate and Inorganic Pyrophosphatase), in " plant biochemistry method " (Meth.Plant Biochem) book, pp.385-405, academic publishing Co., Ltd, London (1990)).In the analysis medium that contains tonoplast enrichment vesicle, available acridine orange (2.5 μ M) comes fluorometric analysis hydrogen ion transhipment (R.G.Zhen etc., journal of biological chemistry (J.Biol.Chem.), 272:22340-22348 (1997)) as striding film pH difference indicator.Does this analysis medium contain trishydroxymethylaminomethane-pyrophosphoric acid (Tris-PPi), the potassium chloride of 50mM, the acridine orange of 2.5 μ M, 5mM Tris (trishydroxymethylaminomethane)-Mes (2-(N-morpholine)-ethylsulfonic acid of 300 μ M?) (pH8.0).Can add 1.3mM magnesium sulfate and start acidifying in the vesica, and come cessation reaction to 2.5 μ M with interpolation proton carrier FCCP (carbonyl cyanogen is to the trifluoromethoxy phenylhydrazone).Can be respectively 495 and the excitation-emission wavelength measurement fluorescence of 540nM, wherein adopt the slit width (R.G.Zhen etc., journal of biological chemistry (J.Biol.Chem.), 269:23342-23350 (1994)) of 5nM.The further test of available interpolation specific inhibitor aminomethylene di 2 ethylhexyl phosphonic acid (aminomethylenediphosphonate) supports that the hydrogen ion transhipment is the viewpoint (R.G.Zhen etc. that AVP1 drives, plant physiology (Plant Physiol.), 104:153-159 (1994)).

Embodiment 12: the leaf and the sodium ion in the stem/potassium ion ratio that detect genetically modified plants

The toxic concentration of sodium chloride at first accumulates in fully extended leaf, wherein sodium chloride by compartmentation in vacuole.Be exposed to sodium chloride and can disturb or reduce the potassium ion picked-up, this causes potassium ion to lack and growth inhibition (S.J.Wu etc., plant cell (Plant Cell), 8:617-627 (1996).The cytosol consequence of potassium content that reduces and higher sodium ions content is the enzyme that suppresses important.. the example of these enzymes is 3 ' (2 ') of yeast, 5 '-nucleoside diphosphate acid enzyme, and when potassium content was low, its activity was to sodium ion responsive (J.R.Murguia etc., science (Science), 267:232-234 (1995)) more.

Can measure verify that genetically modified plants as herein described have an enhancing sodium ion is isolated in its leaf cell or other local vacuole ability.Be the ratio of sodium ion/potassium ion in detection leaf and the stem, but water planting wild type and 35S AVP1/35S AtNHX1 are two and single transgene plant (D.M.Gibeaut etc., plant physiology (Plant Physiol.), 317-319 (1997)).Growth medium (growthmedia) can gradually sodium chloride be added, from 50mM, until 250mM (M.Apse etc., science (Science), 285:1256-1258 (1999)).At each time point. can collect rosette and the stem of treated plant, and weigh.Sample should be in 80 ℃ of baking ovens dry and its dry weight of weighing.This drying sample should boil in the water of determining volume, and by its sodium ion of atomic absorption spectroscopy determination and potassium content (M.Apse, etc., science (science), 285:1256-1258 (1999); R.Gaxiola etc., EMBO magazine (EMBO J.), 11:3157-3164 (1992)).

Embodiment 13: determine that whether the bigger reason of 35S AVP1 genetically modified plants is because its cell is bigger or because it has more cell, also or have both at the same time

Bud meristematic tissue label index can compare with the bud meristematic tissue label index of wild-type plant.Can carry out form and anatomic observation, promptly measure and count the cell of leaf, root and stem.For determine 35S AVP1 genetically modified plants whether because its to have a more cell bigger, their bud meristematic tissue label index can be compared with the bud meristematic tissue label index of wild type.

For measuring the synthetic or cell increment of DNA, can adopt 5-bromo-2 '-BrdU (BrdU), it can replace thymidine to mix DNA.The cell that has mixed 5-bromo-2 '-BrdU in DNA can detect as second antibody with the monoclone antibody and anti-mouse Ig (the immunoglobulin)-alkaline phosphatase of anti-5-bromo-2 '-BrdU monoclone antibody.In conjunction with the monoclone antibody of anti-5-bromo-2 '-BrdU can use the optical microscopy visualization, and set up DAPI (DPAI) painted and 5-bromo-2 '-BrdU positive (positives) between ratio.The method is Chiatante and the presentation method (M.Levi etc. of partner institute, plant physiology (Physiol.Plant), 71:68-72 (1987)) and the improvement of 5-bromo-2 '-BrdU mark and detection kit II (from Boehringer Mannheim company).This plant can be exposed to the different time of medium of 5-bromo-2 '-BrdU mark, can fix then, paraffin embedding and the section, as Meyerowitz and the described (G.Drews etc. of partner, molecular biology of plants report (Plant Mol.Biol.Rep.), 5:242-250 (1998)).

Be to observe leaf texture, flesh tissue can be embedded in 5% the agarose, and with the microsection cutter it is cut into slices.Preliminary root is observed, and seedling can be fixed 4 hours in room temperature in 50% ethanol, 5% acetate and 3.7% formaldehyde, dewater in the classification ethanol series, with dimethylbenzene it is permeated, and soaks with paraffin again..8 available 0.05% Toluidine blue staining of Wei Mi section, and can count at the microscopically pair cell.As the replacement method of visualization and definite cell size, can adopt the described method of Greenberg and partner (Rate etc., plant cell (The Plant Cell), 11:1695-1708 (1999)).

The result

Show that by microscopic examination the cell of transgenic arabidopsis plant is not bigger, but its cell number is more than wild type.Perusal finds, in AVP-12 ' is, Duos 8 leaves than wild type is average in rosette.The F1 plant is to be that 1 ' and 2 ' hybridization produces by the genetically modified plants strain, and its rosette has bigger and the more leaf of number than wild-type plant.Figure 10 is wild type and the leaf of crossing transgenosis (1 ' and the 2 ') arabidopsis thaliana express AVP1, and these plants are at 20 ℃ and at 16 hours full fluorescence in vain/grow under 8 hours dark cycles.When plant begins bolting, carefully the leaf of describing is downcut (sectored) with scalpel, arrange by size then for comparing.Though genetically modified plants have bigger and the leaf of more number more than wild-type plant, do not see that genetically modified plants have bigger cell.These data are consistent with following hypothesis: cross at AVP-1 and divide liveliness proof higher in the genetically modified plants of expression.

The dry weight of water planting transgenic arabidopsis plant, when to similar growing state under wild-type plant relatively the time, the increase that further shows the cell quality has nothing to do with any increase of vegetation water picked-up.Can see the increase of quality dry weight (drymass weight) in root, rosette and stem structure, shown in following table 1, wherein numerical value is the mean value of 6 strain plants.

Table 1

The dry weight of the arabidopsis thaliana part that water planting is supported

Plant parts	Wild type (WT)	1 ' transgenosis strain	2 ' transgenosis strain
				Phase	0.03 gram	0.05 gram	0.14 gram
Rosette	0.10 gram	0.20 gram	0.60 gram
				Stem	0.40 gram	0.50 gram	0.80 gram

The expression excessively of embodiment 14:AVP-1 is to the influence of hormonal activity

Can cross branch liveliness proof that expression is used for producing increase simultaneously with AVP-1 and/or the organ of bud takes place by increasing of hormone.

Expressed the influence of AVP-1 for judging, cut true leaf with scalpel from wild type and transgenosis (1 ', the 2 ') arabidopsis thaliana of expressing AVP-1 excessively carefully hormonal activity.In the plate leaf is positioned on the water saturated 5 metafiltration paper of distillation.This plate is being cultivated under hour dark cycle under the cold white fluorescence and 16 hours illumination/8 in 20 ℃.Can see that from Figure 11 A (leaf plate) and 11B (, thereby placing the root architecture of more clearly describing to grow) leaf of genetically modified plants is still greener and have a rootability of enhancing from leaf from the leaf in Figure 11 A plate.The rootability that strengthens shows that the plant growing cellulose content of increase and the aging that delays are consistent with the basic element of cell division level of increase.

The expression excessively of embodiment 15:AVP-1 is to the influence of callus induction in the petunia explant

Method

The petunia explant is incubated at the MS medium, it comprises MS salt (Gibco BRL company), add the inositol of the thiamine of the hydrochloric acid pyridoxic acid (pyrodoxinHCl) of the nicotinic acid of 1mg/L, 1mg/L, 1mg/L, 100mg/L, 3% sucrose, 1mg/L 2, the 6-BA (6-benzyl aminopurine) of 4-D (2,4 dichlorophenoxyacetic acid) and 0.5mg/L.This medium solidifies with 0.7% agar before autoclaving, and regulates pH value to 5.8.This culture is also cultivated in the dark in 25 ℃ in incubator.

The result

As shown in figure 15, transgenic petunia explant (35-S AVP-1) shows the callus induction of obvious enhancing after cultivating for 6 weeks.

The expression excessively of embodiment 16:AVP-1 is to the influence of shoot regeneration in the blade sections

The growth that the known blade sections that grows in the appropriate culture medium can produce bud.Carried out research to determine the influence of expressing shoot regeneration in the petunia blade of crossing of 35-S AVP-1.

The explant that is used as shoot regeneration from the blade of regeneration conversion (35-S AVP-1) and contrast petunia.With sharp knife blade leaf is cut into about 1 centimetre of wide sheet.This explant is incubated in the MS medium, this MS medium comprises MS salt (Gibco company), B5 vitamin (1mg/L nicotinic acid, 1mg/L hydrochloric acid pyridoxic acid, 1mg/L thiamine and 100mg/L inositol), 3% sucrose, 2mg/L 6-benzyl aminopurine and 0.01mg/L naphthazole acid (naptnaleneacitic acid), 0.7% agar, pH5.8.The fragment of this cultivation and was cultivated under hour dark cycle of illumination/8 at 16 hours under cold white fluorescence, 25 ℃.

As shown in figure 12, the petunia leaf that transforms with AVP-1 gene (35S AVP-1 plate) under constitutive promoter shows than wild type petunia blade sections (WT) and has stronger shoot regeneration ability, and the result of this and transgenic arabidopsis plant is similar.These results and following identical of views: expressing excessively of this vacuolar proton pump will improve the shoot regeneration ability of any plant.

Show the very conservative (paddy=86% homogeneity of this gene as the autoploidy comparative analysis (blastearch) that probe carries out with the AVP-1 open reading frame; Tobacco=89% homogeneity; Barley=86% homogeneity; Grape=82% homogeneity; Barley=86% homogeneity (school: repeat); And corn=90%).The latter shows that the mistake of AVP-1 gene is expressed in other vegetation type can produce such result similarly.

The expression excessively of embodiment 17:AVP-1 is to the influence of the bud and the root regeneration of arabidopsis cotyledon

The known proper culture medium of using, bud and Gen Ke are by regeneration of cotyledons.At wild type (WT) and AVP-1 transgenic arabidopsis (1 ' and 2 '), bud and root regeneration have been identified from cotyledon.

The cotyledon of 5 ages in days is as the explant of regeneration.Explant is incubated at bud inducing culture (SIM), and condition of culture is 20 ℃, 16 hours illumination/8 hour dark cycles.This SIM comprises MS salt (Gibco company), B5 vitamin (comprising 1mg/L nicotinic acid, 1mg/L hydrochloric acid pyridoxic acid, 1mg/L thiamine and 100mg/L inositol), 3% sucrose, 1mg/L 6-(γ, γ-dimethylallyl amino) purine nucleosides (6-(gama-gama-dimethylallylamino) purine riboside) (2-phosphoric acid) and the acid of 0.1mg/L naphthazole, and solidify with 0.7% agar.The pH of medium is transferred to 5.8.Behind autoclaving, to 2-phosphoric acid (2-iP) filtration sterilization and add in this medium.

As in Figure 13 A and 13B finding, from the frequency of the explant regeneration buds of genetically modified plants (1 ' and 2 ') and root be higher than and the time early than wild type (WT), this is consistent with higher meristematic tissue competence.The evidence that transforms difference between cotyledon and the wild type cotyledon is very obvious at 23 days (Figure 13 A), and at back 37 days of cultivation more obviously (Figure 13 B).

Embodiment 18:AVP-1 cross to express the influence from the shoot regeneration of the root of arabidopsis thaliana and cotyledon explant

Crossing expression from wild type (WT) and transgenosis (1 ' and 2 ') AVP-1 Intend The south mustardRoot of plant and cotyledon (5 age in days) explant places the bud inducing culture, as described in the embodiment 16.As shown in Figure 6, produce new construction from the explants of genetically modified plants (1 ' and 2 ') early than wild type, this is consistent with higher meristematic tissue competence.

Embodiment 19: the separation of mutant in transport protein

Aspect the Analysis of Complex biological character, the genetics method is (R.Serrano etc., plant science comment (Crit.Rev.Plant Sci.), 13:121-138 (1994)) very effectively.For the plant biological scholar, reverse genetics is very important new tool.Being knocked out by a series of marks of Sussman and partner preparation is (P.Krysan etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 93:8145-8150 (1996)) be that the genetic disorder (gene disruption) of analyzing arabidopsis has been opened very important approach.

The arabidopsis of Univ. of Wisconsin-Madison knocks out facility, and (network address: biotech.wisc.edu/NewServicesAndResearch/Arabidopsis) can be used at 60,480 arabidopsiss (ecotype WS) is that the T-DNA that searches in (it transforms with T-DNA carrier pD991) in AtCLC-c, AtCLC-d, AVP1, AtNHX1 and homologue thereof inserts segment.The phenotype of the plant that said gene knocks out will for understand these transport proteins normal and stress situation under physiological action give a clue.The elementary sign of gene knockout plant comprises its salt tolerance and its sodium ion/potassium ion ratio of detecting.By hybridization produce dual knock out plant help further to understand between the transport protein interaction and with the hybridization of 35S AVP1 and 35S AtNHX1 genetically modified plants.

Knock out for searching arabidopsis gene, can design PCR (polymerase chain reaction) primer with reference to the guidance that the University of Wisconsin website describes in detail.The test primer can be delivered to University of Wisconsin Madison, wherein can carry out 62 PCR reactions, it is to give us to be used for the southern blotting technique analysis.With positive PCR product order-checking.Have T-DNA to insert this gene if this sequence shows, then this gene-specific primer with another group PCR reaction with 9 in determining 225 may storehouses which contain this gene knockout (knockout).After having identified interested storehouse, screening scanning 25 pipe seeds are carried the plant that T-DNA knocks out with searching.

Embodiment 20: the cation detoxifcation in the plant cell

Research described herein advantageously illustrates in conjunction with other evidence, yeast has identical approach from Golgi body net transitional vesicle to vacuole and signal (R.Gaxiola etc. with plant, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 96:1480-1485 (1999); F.Marty, " biology of vacuole takes place: from microscopical observation " (" The Biogenesis of Vacuoles:Insights from Microscopy "), be published in " plant vacuole " (The Plant Vacuole), 1-42, R.A.Leigh and D.Sanders, the academic press, Santiago (1997); D.C.Bassham etc., plant physiology (Plant Physiol.), 117:407-415 (1998)).

Do not wish to be limited to by theoretical, the present inventor believes that mostly in two systems, preceding vacuole chamber is dynamic entity, its cation in the cytoplasm that can detoxify, and its load (cargo) is transported to vacuole or directly to the extracellular.Gef1 chloride channel and Nhx1 sodium ion/hydrogen ion permutoid all are located in the preceding vacuole chamber of yeast (R.A.Gaxiola etc., institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 96:1480-1485 (1999)).Monitored the behavior of gef1-GFP chimera in the live body yeast cells, shown that its location changes with environmental condition.In addition, shown that two kinds of CLC-c and CLC-d in 4 kinds of A.thaliana CLC chloride ion channels can suppress the gef1 mutant phenotype, location (the R.A.Gaxiola etc. that this hint is similar, institute of NAS newspaper (Proc.Natl.Acad.Sci.USA), 95:4046-4050 (1998)).

How this cation detoxication reaches generation wherein in plant cell in order to verify, can monitor in vivo AVP1, AtNHX1 and AtCLC-c and-the chimeric cell of GFP (green fluorescent protein) of d in location (B.Hong etc., plant physiology (Plant Physiol.), 119:1165-1175 (1999)).Also available confocal microscope is determined the common location of different transport proteins.For reaching this purpose, need variant (versions) or antibody (Guiltinan M.J. etc., cell biology method (Meth.Cell Biol.), the 49:143-151 (1995) of the HA mark of the transport protein of studying; JauhG.-Y. etc., plant cell (Plant Cell), 11:1867-1882 (1999); MullenR.T. etc., plant magazine (Plant J.), 12:313-322 (1997)).

For making up the GFP chimera, that can adopt Davis and Viestra report has soluble g FP variant (versionsGFP) (S.J.Davies and a R.D.Viestra that improves fluorescence in arabidopsis (A.thaliana), the soluble derivative that is used for the green fluorescent protein (GFP) of arabidopsis (Arabidopsisthaliana), network address http://brindabella.mrc-lmb.cam.ac.uk/IndexGFP.html (1998)).Can obtain two types GFP chimera, promptly a kind of by the natural promoter regulation and control, another kind is regulated and control by 35S promoter.Pass through electroporation, the chimeric T-NDA carrier of GFP that contains that generates is transformed into Agrobacterium tumefaciems GV3101 bacterial strain, and the vacuum that is used for arabidopsis (Arabidopsis thaliana) ecotype Columbia is subsequently infiltrated (Bechtold N. etc., C.R.Jeances Acad.Sci.Ser.III Sci.Vie, 316:1194-1199 (1993)).For mark hemagglutinin (HA) epitope, can adopt the PCR strategy, this strategy is for yeast designs, but through transformation can be used for being marked at express in the yeast vector plant gene.Futcher and partner have designed the carrier that contains the URA3 yeast genes, and this gene is outflanked (B.L.Schneider etc., yeast (Yeast), 11:1265-1274 (1995)) by the recurring unit in the same way of epitope mark (HA).By PCR, this mark-URA3-marker cassette can increase, thereby the PCR fragment that generates has autoploidy at each end with genes of interest.Can utilize reorganization in the body in the yeast then, guide the PCR-chimera to be integrated into to contain the plasmid of plant purpose ORF (open reading frame), use URA ⁺(bird pyrimidine wild type) Phenotypic Selection transformant.When positive transformant was cultivated in the presence of the 5-fluororotic acid, the URA3 gene can be by " rejecting ".The carrier that carries this plant gene has the selected marker different with the URA3 gene.

In a word, in the important economically crop, the operation of vacuole pyrophosphatase be can be crop improve important approach is provided.Plant drought-enduring and anti-freeze injury can be the area of falling into disuse owing to arid or few rainfall the new methods of cultivation is provided, and for the peasant provides protection, makes the influence of its freeze injury of avoiding not expected (sleet etc.).This class crop also may grow in the too high soil of for wild type crop salinity.

Though the present invention is described with reference to its preferred specific embodiment, to those skilled in the art, can make multiple change and/or modification, and not depart from the spirit or scope of the present invention.All these are changed all in appended patent application right claimed range of the present invention.All lists of references of quoting in this manual are hereby expressly incorporated by reference document.

Claims (74)

1. make the method for the genetically modified plants of branch liveliness proof with raising, comprise one or more cells that can change the exogenous nucleic acid introduced plant that the vacuole pyrophosphatase is expressed in the described plant, increase vacuole pyrophosphatase activity, and in described plant, produce transformant, produce the genetically modified plants of branch liveliness proof thus with raising.

2. method according to claim 1 further comprises from described transformant aftergrowth, to produce the genetically modified plants that genetically modified plants and selection have the branch liveliness proof of raising, produces the genetically modified plants of the branch liveliness proof with improvement thus.

3. method according to claim 1, wherein said exogenous nucleic acid coding AVP1 or its homologue.

4. method according to claim 3, wherein said AVP1 or AVP1 homologue are to be present in for crossing to express in the assembly that AVP1 designs.

5. method according to claim 4, wherein said assembly comprise described AVP1 gene or AVP1 homologue, are can be operatively connected in chimeric promoters, and described promotor is to design for crossing expression AVP1 or its homologue.

6. method according to claim 5, wherein said AVP1 gene or its homologue are can be operatively connected in chimeric promoters, and described chimeric promoters is selected from by tissue-specific promoter, constitutive promoter, inducible promoter and its group of forming.

7. method according to claim 5, wherein said AVP1 gene or AVP1 homologue are the two-in-series enhancers that can be operatively connected in 35S promoter.

8. method according to claim 3, wherein said AVP1 or its homologue are to derive from wild-type plant.

9. method according to claim 3, wherein said AVP1 or its homologue are to derive from genetically modified plants.

10. method according to claim 3, wherein said AVP1 or its homologue are to derive from the mutant plant.

11. being synthetic, method according to claim 3, wherein said AVP1 or its homologue obtain.

12. method according to claim 3, wherein said AVP1 or its homologue are natural and synthetic obtains.

13. genetically modified plants by method generation according to claim 1.

14. make the method for the genetically modified plants of meristematic tissue vigor with raising, the one or more cells that comprise the exogenous nucleic acid introduced plant that can change the expression of vacuole pyrophosphatase, be increased in the vacuole pyrophosphatase activity in the described plant, and in described plant, produce transformant, produce the genetically modified plants of meristematic tissue vigor thus with raising.

15. method according to claim 14 further comprises from described transformant aftergrowth, to produce the genetically modified plants that genetically modified plants and selection have the branch liveliness proof of raising, produces the genetically modified plants of the branch liveliness proof with improvement thus.

16. method according to claim 14, wherein said exogenous nucleic acid coding AVP1 or its homologue.

17. method according to claim 16, wherein said AVP1 or AVP1 homologue are to be present in for crossing to express in the assembly that AVP1 designs.

18. method according to claim 17, wherein said assembly comprise described AVP1 gene or AVP1 homologue, are can be operatively connected in chimeric promoters, described promotor is to design for crossing expression AVP1 or its homologue.

19. method according to claim 18, wherein said AVP1 gene or its homologue are can be operatively connected in chimeric promoters, and described chimeric promoters is selected from by tissue-specific promoter, constitutive promoter, inducible promoter and its group of forming.

20. method according to claim 18, wherein said AVP1 gene or AVP1 homologue are the two-in-series enhancers that can be operatively connected in 35S promoter.

21. method according to claim 16, wherein said AVP1 or its homologue are to derive from wild-type plant.

22. method according to claim 16, wherein said AVP1 or its homologue are to derive from genetically modified plants.

23. method according to claim 16, wherein said AVP1 or its homologue are to derive from the mutant plant.

24. being synthetic, method according to claim 16, wherein said AVP1 or its homologue obtain.

25. method according to claim 16, wherein said AVP1 or its homologue are natural and synthetic obtains.

26. make the method for the genetically modified plants of biomass with raising, the one or more cells that comprise the nucleic acid assembly introduced plant that can change the expression of vacuole pyrophosphatase, be increased in the vacuole pyrophosphatase activity in the described cell, thereby the generation transformant, generation has the genetically modified plants of the biomass of raising thus.

27. method according to claim 26 further comprises from described transformant aftergrowth, to produce the genetically modified plants that genetically modified plants and selection have the biomass of raising.

28. genetically modified plants by method generation according to claim 26.

29. preparation has the method for genetically modified plants of the biomass of raising than its corresponding wild type plant, the biomass of wherein said raising relates to the increase of the described biomass of plant part, described plant part is selected from the group of being made up of leaf, stem, root, seed, flower and fruit, described method comprises one or more cells of the exogenous nucleic acid introduced plant that can change the expression of vacuole pyrophosphatase, strengthen the activity of the described vacuole pyrophosphatase in described plant, thereby the generation transformant, generation has the genetically modified plants of the biomass of raising thus.

30. method according to claim 29 further comprises from described transformant aftergrowth, to produce genetically modified plants and to select the genetically modified plants bigger than its corresponding wild-type plant, produces the genetically modified plants of the biomass with raising thus.

31. method according to claim 29, wherein said exogenous nucleic acid coding AVP1 or its homologue.

32. method according to claim 31, wherein said AVP1 or its homologue are to be present in for crossing to express in the assembly that AVP1 or its homologue design.

33. method according to claim 32, wherein said assembly comprise described AVP1 gene or its homologue, it can be operatively connected in chimeric promoters, and described promotor is to design for crossing expression AVP1.

34. method according to claim 33, wherein said AVP1 gene or its homologue are can be operatively connected in chimeric promoters, and described chimeric promoters is selected from by tissue-specific promoter, constitutive promoter, inducible promoter and its group of forming.

35. method according to claim 32, wherein said AVP1 gene or its homologue are the two-in-series enhancers that can be operatively connected in 35S promoter.

36. method according to claim 31, wherein said AVP1 or its homologue are to derive from wild-type plant.

37. method according to claim 31, wherein said AVP1 or its homologue are to derive from genetically modified plants.

38. method according to claim 31, wherein said AVP1 or its homologue are to derive from the mutant plant.

39. method according to claim 29, wherein said genetically modified plants are to grow in the soil.

40. method according to claim 29, wherein said genetically modified plants are aquatic cultivations.

41. method according to claim 29, wherein the cell from described genetically modified plants is to grow in the culture.

42. genetically modified plants by method generation according to claim 29.

43. produce the method for the genetically modified plants with stem structure thicker than wild type, the one or more cells that comprise the exogenous nucleic acid introduced plant that can change the expression of vacuole pyrophosphatase, strengthen the vacuole pyrophosphatase activity in described plant, thereby the generation transformant produces the genetically modified plants with chap stem structure thus.

44. according to the described method of claim 43, wherein said stem structure is to be selected from the group of being made up of layer of wood, bark and cambium.

45. according to the described method of claim 43, further comprise,, produce genetically modified plants thus with chap stem structure to produce the genetically modified plants that genetically modified plants and selection have the chap stem structure from described transformant aftergrowth.

46. preparation has the method for genetically modified plants of the root architecture of increase than its corresponding wild type plant, the one or more cells that comprise the exogenous nucleic acid introduced plant that can change the expression of vacuole pyrophosphatase, strengthen the vacuole pyrophosphatase activity in described plant, thereby the generation transformant, generation has the genetically modified plants of the root architecture of increase thus.

47. according to the described method of claim 46, wherein said exogenous nucleic acid coding AVP1 or its homologue.

48. by the genetically modified plants that produce according to the described method of claim 46.

49. preparation has the method for genetically modified plants of the shoot regeneration ability of increase than its corresponding wild type plant, the one or more cells that comprise the nucleic acid introduced plant that can change the expression of vacuole pyrophosphatase, strengthen the vacuole pyrophosphatase activity in described plant, thereby the generation transformant, generation has the genetically modified plants of the shoot regeneration ability of increase thus.

50. according to the described method of claim 49, further comprise,, produce the genetically modified plants of shoot regeneration ability thus with improvement with the genetically modified plants that produce genetically modified plants and select to have the shoot regeneration ability of increase from described transformant aftergrowth.

51. according to the described method of claim 49, wherein said exogenous nucleic acid coding AVP1 or its homologue.

52. according to the described method of claim 51, wherein said AVP1 or AVP1 homologue are to be present in for crossing to express in the assembly that AVP1 designs.

53. according to the described method of claim 52, be can be operatively connected in chimeric promoters comprising the described assembly of described AVP1 gene or AVP1 homologue, described promotor is to design for crossing expression AVP1 or its homologue.

54. according to the described method of claim 53, wherein said AVP1 gene or its homologue are can be operatively connected in chimeric promoters, and described chimeric promoters is selected from by tissue-specific promoter, constitutive promoter, inducible promoter and its group of forming.

55. according to the described method of claim 53, wherein said AVP1 gene or AVP1 homologue are the two-in-series enhancers that can be operatively connected in 35S promoter.

56. according to the described method of claim 52, wherein said AVP1 or its homologue are to derive from wild-type plant.

57. according to the described method of claim 52, wherein said AVP1 or its homologue are to derive from genetically modified plants.

58. according to the described method of claim 52, wherein said AVP1 or its homologue are to derive from the mutant plant.

59. according to the described method of claim 52, wherein said AVP1 or its homologue are that synthetic obtains.

60. according to the described method of claim 52, wherein said AVP1 or its homologue are natural and synthetic obtains.

61. by the genetically modified plants that produce according to the described method of claim 49.

62. preparation has the method for genetically modified plants of the root regeneration ability of increase than its corresponding wild type plant, the one or more cells that comprise the nucleic acid introduced plant that can change the expression of vacuole pyrophosphatase, strengthen the vacuole pyrophosphatase activity in described plant, thereby the generation transformant, generation has the genetically modified plants of the root architecture of increase thus.

63. according to the described method of claim 62, further comprise,, produce the genetically modified plants of root regeneration ability thus with improvement with the genetically modified plants that produce genetically modified plants and select to have the root regeneration ability of increase from described transformant aftergrowth.

64. according to the described method of claim 62, wherein said exogenous nucleic acid coding AVP1 or its homologue.

65. according to the described method of claim 64, wherein said AVP1 or AVP1 homologue are to be present in for crossing to express in the assembly that AVP1 designs.

66. according to the described method of claim 65, be can be operatively connected in chimeric promoters comprising the described assembly of described AVP1 gene or AVP1 homologue, described promotor is to design for crossing expression AVP1 or its homologue.

67. according to the described method of claim 66, wherein said AVP1 gene or its homologue are can be operatively connected in chimeric promoters, and described chimeric promoters is selected from by tissue-specific promoter, constitutive promoter, inducible promoter and its group of forming.

68. according to the described method of claim 64, wherein said AVP1 gene or AVP1 homologue are the two-in-series enhancers that can be operatively connected in 35S promoter.

69. according to the described method of claim 64, wherein said AVP1 or its homologue are to derive from wild-type plant.

70. according to the described method of claim 64, wherein said AVP1 or its homologue are to derive from genetically modified plants.

71. according to the described method of claim 64, wherein said AVP1 or its homologue are to derive from the mutant plant.

72. according to the described method of claim 64, wherein said AVP1 or its homologue are that synthetic obtains.

73. according to the described method of claim 64, wherein said AVP1 or its homologue are natural and synthetic obtains.

74. by the genetically modified plants that produce according to the described method of claim 62.

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