CN109837265A - The method for improving drought tolerance in plants ability - Google Patents

Abstract

The present application relates to the field of transgenes and gene editing, and in particular to transgenes and gene editing in plants such as crops. The present invention provides a method for improving plant drought tolerance by expressing ACC deaminase specifically. The present invention improves drought tolerance in plants, such as crops, especially maize.

Description

The method for improving drought tolerance in plants ability

Technical field

The present invention provides a kind of methods that drought resistance in plants is improved by particular expression acc deaminase.

Background technique

The sustainable growth of population brings two challenges for agricultural sustainable development.One is more populations need More crop products are consumed, and production estimation needs a large amount of water resource.And another challenge is, population increases can be competing More available water is occupied to striving property, this can be further exacerbated by the notch of agricultural water.Guarantee the duration of agricultural production, just Need to increase crops water resource utilization efficiency.Drought-enduring plant is cultivated, is an important channel for maintaining agricultural sustainability.

Plant adapts to drought stress and has evolved three kinds of different mechanism [1].Regulation can generally be passed through by escaping drought plant Growth period will carry out escape drought stress by lagging or in advance with water more period (for example, florescence).Keep away drought Plant can generally be closed in advance when drought stress by regulating and controlling stomata, be reduced rising.Stomata is closed in advance to be subtracted simultaneously The intake of few carbon dioxide, influences plant growth.Drought-resistant plant is under certain drought condition, by adjusting the water of itself Gesture reduces the concentration of active oxygen, to be resistant to arid and maintain certain growth.Three kinds of different mechanisms have phase on different plants The evolution value answered；Escaping drought plant can generally change at florescence under drought condition or growth period.Keeping away drought mechanism can To be identified the case where closing by stomata；Often in the case where mild drought, keeps away drought mechanism and play great role.No With plant under different drought stress conditions, one or more mechanism can be used；From crop, drought resistance mechanism more has Application value.

The molecular mechanism of plant drouhgt stress response is extremely complex, according to the differentiation of signal path, can divide into and fall off Two kinds of approach that sour (ABA) is relied on and do not depended on.Abscisic acid is a kind of plant growth regulator, and is produced from root under drought condition It gives birth to and is enriched with, blade is entered by conduit and regulates and controls the flow of water or oxidation resistance.In addition some researches show that can also produce in blade Raw abscisic acid.Abscisic acid generally passes through in conjunction with its receptor and the interaction that changes receptor with phosphoprotein phosphatase, and then under regulating and controlling The phosphorylation state of phosphokinase is swum, activation relies on the signal path of abscisic acid.The signal path of abscisic acid is not depended on often Rely on the transcripton of a kind of special AP2 class, responses of drought stress element conjugated protein (DREB, Dehydration depended element binding protein).Two kinds of mechanism can cooperate with the enhancing for participating in drought resistance between each other；Wherein other growths Regulatory factor, such as ethylene participate in the dialogue for adjusting two kinds of mechanism.

Ethylene has also assisted in response of the plant to drought stress.It often will affect the synthesis of ethylene under drought stress.Plant Internal ethylene synthase originates in methionine (Methionine, Met)；In the presence of atriphos (ATP), Ademetionine Methionine is transformed into s-adenosylmethionine (SAM) by synzyme first.Then in (the ACC synthesis of 1-aminocyclopropane-1-carboxylic acid synzyme Enzyme) SAM is transformed into 1- 1-aminocyclopropane-1-carboxylic acid (ACC) and methylthioadenosine (MTA) under effect.MTA can pass through hydrolysis and egg Propylhomoserin approach recombines methionine.ACC is the direct precursor of Synthesis pathway, and ACC synzyme is the speed limit of ethylene synthase Step.Acc oxidase is catalyzed ACC and generates ethylene, this process is also critically important.ACC synzyme 6 is knocked out in corn, it is suppressed that Drought-induced leaf senile；The expression that ACC synzyme 6 is reduced using RNA silent technology, is verified, gene by field experiment The strain of silencing compares and compares the yield [2] significantly improved under drought stress.Ethylene Signal transmitting is overexpressed in corn Negative regulatory factor, ZmARGOS8 also improves the drought resistance [3] of plant.Acc deaminase (ACC deaminase) can drop It solves ACC and generates ammonia and batanone acid, reduce the synthesis of ethylene.Acc deaminase is widely present in fungi, bacterium and yeast；Have Plant can be improved to the tolerance of arid when same plant symbiosis in rhizospheric microflora of soil.In tomato and capsicum Rhizosphere applies Pi Shi achromobacter ARV8, improves drought resistance [4]；Similar result is also sent out in corn, wheat and pea It is existing.After knocking out the acc deaminase gene in these plants probiotics, it is found that it substantially drops the effect of plant drought resistance Low, this just illustrates that acc deaminase plays an important role in plant drought.It is now recognized that possible mechanism is to work as probiotics When with root system interaction, the ACC of plant can be secreted into rhizosphere, by the acc deaminase degradation ACC in microorganism, indirectly The synthesis of ground reduction Plant Ethylene.In microorganism acc deaminase plant drought resistance positive evidence there is no too many report and It proves.Another aspect acc deaminase homologous gene in plant does not have been reported that.Method of the present invention by being overexpressed, verifying And it screens a kind of microbe-derived acc deaminase and the drought-resistance ability of plant can be improved, and also predicted and pass through plant Drought-resistance ability can be improved by being suitably transformed in the aminopherase of itself.

Summary of the invention

Some embodiments of the invention are as follows:

1.ACC deaminase or the polynucleotide sequence for encoding acc deaminase are used to improve the purposes of drought tolerance in plants ability.

2. according to purposes described in embodiment 1, wherein the acc deaminase is Ka Liduoniya bulkholderia cepasea The acc deaminase of (Burkholderia caledonica) LMG19076.

3. according to purposes described in embodiment 2, wherein the amino acid sequence of the acc deaminase such as SEQ ID NO:7 It is shown.

4. according to purposes described in embodiment 1, wherein the polynucleotide sequence of the coding acc deaminase is originated from noise made in coughing or vomiting The acc deaminase gene of more Buddhist nun Asia bulkholderia cepasea LMG19076.

5. according to purposes described in embodiment 4, wherein the polynucleotide sequence such as SEQ ID of the coding acc deaminase Shown in NO:5 or SEQ ID NO:6.

6. have ACC identification and deamination function modified plant aminopherase or coding have ACC identification and The polynucleotide sequence of the modified plant aminopherase of deamination function is used to improve the purposes of drought tolerance in plants ability.

7. according to purposes described in embodiment 6, wherein the modified plant for having ACC identification and deamination function Aminopherase is originated from the aminopherase dependent on 5' phosphopyridoxal pyridoxal phosphate of plant endo.

8. according to purposes described in embodiment 7, wherein the ammonia dependent on 5' phosphopyridoxal pyridoxal phosphate of the plant endo Based transferase is corn aminopherase, rice aminopherase or arabidopsis aminopherase.

9. according to purposes described in embodiment 8, wherein the amino acid sequence of the corn aminopherase such as SEQ ID Shown in NO:15.

10. according to purposes described in embodiment 8, wherein the amino acid sequence of the rice aminopherase such as SEQ ID Shown in NO:19.

11. according to purposes described in embodiment 8, wherein the amino acid sequence such as SEQ of the arabidopsis aminopherase Shown in ID NO:17.

12. according to purposes described in embodiment 6, wherein the coding has the engineered of ACC identification and deamination function Plant aminopherase polynucleotide sequence be originated from plant endo the aminopherase dependent on 5' phosphopyridoxal pyridoxal phosphate Gene.

13. according to purposes described in embodiment 12, wherein the plant endo dependent on 5' phosphopyridoxal pyridoxal phosphate Aminotransferase gene is corn aminotransferase gene, rice aminotransferase gene or arabidopsis aminotransferase gene.

14. according to purposes described in embodiment 13, wherein the encoder block sequence of the corn aminotransferase gene is such as Shown in SEQ ID NO:14.

15. according to purposes described in embodiment 13, wherein the encoder block sequence of the rice aminotransferase gene is such as Shown in SEQ ID NO:18.

16. according to purposes described in embodiment 13, wherein the encoder block sequence of the arabidopsis aminotransferase gene As shown in SEQ ID NO:16.

17. the purposes according to any one of embodiment 1-16, wherein the plant is dicotyledon or list Leaf plant, such as grass family (Gramineae) plant, such as Zea (Zea) plant；Preferably, the plant be selected from by The group of the following composition: corn, wheat, rice, sorghum, arabidopsis, soybean, tomato, sunflower, spinach and rape.

18. a kind of method for improving drought tolerance in plants ability comprising be overexpressed coding acc deaminase in the plant Polynucleotide sequence or coding have the polynucleotides sequence of the modified plant aminopherase of ACC identification and deamination function Column.

19. the polynucleotide sequence of the coding acc deaminase is originated from more in noise made in coughing or vomiting according to method described in embodiment 18 The acc deaminase gene of Buddhist nun Asia bulkholderia cepasea LMG19076.

20. according to method described in embodiment 19, wherein the polynucleotide sequence such as SEQ of the coding acc deaminase Shown in ID NO:5 or SEQ ID NO:6.

21. according to method described in embodiment 18, wherein the coding has the engineered of ACC identification and deamination function Plant aminopherase polynucleotide sequence be originated from plant endo the aminopherase dependent on 5' phosphopyridoxal pyridoxal phosphate Gene.

22. according to method described in embodiment 21, wherein the plant endo dependent on 5' phosphopyridoxal pyridoxal phosphate Aminotransferase gene is corn aminotransferase gene, rice aminotransferase gene or arabidopsis aminotransferase gene.

23. according to method described in embodiment 22, wherein the encoder block sequence of the corn aminotransferase gene is such as Shown in SEQ ID NO:14.

24. according to method described in embodiment 22, wherein the encoder block sequence of the rice aminotransferase gene is such as Shown in SEQ ID NO:18.

25. according to method described in embodiment 22, wherein the encoder block sequence of the arabidopsis aminotransferase gene As shown in SEQ ID NO:16.

26. the method according to any one of embodiment 18-25, wherein the overexpression is by plant ubiquitin promoter Or Plant Actin promoter drives.

27. according to method described in embodiment 26, wherein the plant ubiquitin promoter is maize ubiquitin promoter prZmUbi1。

28. according to method described in embodiment 27, wherein the sequence such as SEQ of the maize ubiquitin promoter prZmUbi1 Shown in ID NO:4.

29. according to method described in embodiment 26, wherein the Plant Actin promoter is rice actin Promoter prAct1.

30. according to method described in embodiment 29, wherein the sequence of the rice actin promoters prAct1 is such as Shown in SEQ ID NO:9.

31. the method according to any one of embodiment 18-25, wherein the overexpression is started by plant induction type Son or plant organ specificity promoter drive.

32. according to method described in embodiment 31, wherein the plant inducer conductivity type promoter is that arabidopsis is drought-induced Promoter prAtRD29A or other drought-induced promoters.

33. according to method described in embodiment 31, wherein the plant organ specificity promoter is that green organs are special Specific Promoters prOsPSI33kd, stomata specificity promoter prAt1G22690 or similar promoter.

34. the method according to any one of embodiment 18-33, wherein the plant is dicotyledon or list Leaf plant, such as gramineae plant, such as Zea plant；Preferably, the plant is selected from the group being made of the following: Corn, wheat, rice, sorghum, arabidopsis, soybean, tomato, sunflower, spinach and rape.

35. a kind of improve drought tolerance in plants by carrying out mutation or gene editing to the aminotransferase gene in plant The method of ability comprising be based on protein sequence and structural analysis, and/or nature/induced mutation information, be transformed in plant Aminotransferase gene make its encoded enzyme have degradation ACC function.

36. according to method described in embodiment 35, wherein the aminotransferase gene in plant is in plant The aminotransferase gene dependent on 5' phosphopyridoxal pyridoxal phosphate of source property.

37. according to method described in embodiment 36, wherein the plant endo dependent on 5' phosphopyridoxal pyridoxal phosphate Aminotransferase gene is corn aminotransferase gene, rice aminotransferase gene or arabidopsis aminotransferase gene.

38. according to method described in embodiment 37, wherein the encoder block sequence of the corn aminotransferase gene is such as Shown in SEQ ID NO:14.

39. according to method described in embodiment 37, wherein the encoder block sequence of the rice aminotransferase gene is such as Shown in SEQ ID NO:18.

40. according to method described in embodiment 37, wherein the encoder block sequence of the arabidopsis aminotransferase gene As shown in SEQ ID NO:16.

41. the method according to any one of embodiment 35-40, wherein the plant is dicotyledon or list Leaf plant, such as gramineae plant, such as Zea plant；Preferably, the plant is selected from the group being made of the following: Corn, wheat, rice, sorghum, arabidopsis, soybean, tomato, sunflower, spinach and rape.

Detailed description of the invention

Fig. 1 shows the map of binary vector 20049.

Fig. 2 shows the comparison of the dry weight of two groups of JHAX aerial parts.The column on the left side is the wild type for carrying out Osmotic treatment JHAX；The column on the right is the wild type JHAX normally to water.

Fig. 3 shows Ka Liduoniya bulkholderia cepasea acc deaminase, arabidopsis aminopherase, rice amino The albumen homology of transferase and corn aminopherase compares.

Fig. 4 shows the map of binary vector 19982.

Fig. 5 shows the map of binary vector 19953.

Sequence table information

SEQ ID NO:1 is prHvLPT2 (the special lipid transfer proteins promoter of barley aleurone).

SEQ ID NO:2 is cRFP (red fluorescent protein gene).

SEQ ID NO:3 is tPI-15 (tomato protease inhibitor II terminator).

SEQ ID NO:4 is prZmUbi1 (maize ubiquitin promoter).

SEQ ID NO:5 is that cBcaLMG19076 (comes from Ka Liduoniya Burkholder after Maize codon optimization The acc deaminase gene of Salmonella LMG19076, encoder block sequence).

SEQ ID NO:6 is that (ACC from Ka Liduoniya bulkholderia cepasea LMG19076 is de- by cBcaLMG19076 Adnosine deaminase gene, encoder block sequence).

SEQ ID NO:7 is that (ACC from Ka Liduoniya bulkholderia cepasea LMG19076 is de- by cBcaLMG19076 Adnosine deaminase gene, protein sequence).

SEQ ID NO:8 is tUbi1 (maize ubiquitin terminator).

SEQ ID NO:9 is prAct1 (rice actin promoters).

SEQ ID NO:10 is cPAT (the phosphine oxamate acetyl transferase gene of soybean codon optimization).

SEQ ID NO:11 is t35S (cauliflower mosaic virus 35S terminator).

SEQ ID NO:12 is cPMI (6- Phophomannose isomerase gene).

SEQ ID NO:13 is tNOS (Agrobacterium rouge alkali synthetase gene terminator).

SEQ ID NO:14 is cGRMZM2G151440 (corn aminotransferase gene, encoder block sequence).

SEQ ID NO:15 is cGRMZM2G151440 (corn aminotransferase gene, protein sequence).

SEQ ID NO:16 is cAtACD1 (arabidopsis aminotransferase gene, encoder block sequence).

SEQ ID NO:17 is cAtACD1 (arabidopsis aminotransferase gene, protein sequence).

SEQ ID NO:18 is cLOC_Os02g53330 (rice aminotransferase gene, encoder block sequence).

SEQ ID NO:19 is cLOC_Os02g53330 (rice aminotransferase gene, protein sequence).

SEQ ID NO:20 is ZmADH1 forward primer.

SEQ ID NO:21 is ZmADH1 reverse primer.

SEQ ID NO:22 is ZmADH1 probe.

SEQ ID NO:23 is PMI forward primer.

SEQ ID NO:24 is PMI reverse primer.

SEQ ID NO:25 is PMI probe.

SEQ ID NO:26 is Red forward primer.

SEQ ID NO:27 is Red reverse primer.

SEQ ID NO:28 is Red probe.

SEQ ID NO:29 is ZmEF forward primer.

SEQ ID NO:30 is ZmEF reverse primer.

SEQ ID NO:31 is ZmEF probe.

SEQ ID NO:32 is BcaLMG19076-ACCD forward primer.

SEQ ID NO:33 is BcaLMG19076-ACCD reverse primer.

SEQ ID NO:34 is BcaLMG19076-ACCD probe.

SEQ ID NO:35 is that cBunMTI641-ACCD (encloses bulkholderia cepasea from grass roots The acc deaminase gene of (Paraburkholderia unamae) MTI-641, encoder block sequence).

SEQ ID NO:36 is that (ACC for enclosing bulkholderia cepasea MTI-641 from grass roots is de- by cBunMTI641-ACCD Adnosine deaminase gene, protein sequence).

SEQ ID NO:37 is that cBunMTI641-ACCD (encloses Burkholder from grass roots after Maize codon optimization The acc deaminase gene of Salmonella MTI-641, encoder block sequence).

SEQ ID NO:38 is that cRlePB172-ACCD (comes from rhizobium leguminosarum (Rhizobium leguminosarum) The acc deaminase gene of PB172, encoder block sequence).

SEQ ID NO:39 is cRlePB172-ACCD (acc deaminase gene from rhizobium leguminosarum PB172, albumen Sequence).

SEQ ID NO:40 be cRlePB172-ACCD (Maize codon optimization after from rhizobium leguminosarum PB172's Acc deaminase gene, encoder block sequence).

Specific embodiment

Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will Understand, the following example is merely to illustrate the present invention, and should not be taken as limiting the scope of the invention.It is not specified in embodiment specific Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is It can be with conventional products that are commercially available.

Embodiment 1. is overexpressed the acc deaminase from Ka Liduoniya bulkholderia cepasea in corn (BcaLMG19076-ACCD, SEQ ID NO:5-7)

1.1. the building of binary vector 20049

From the acc deaminase recalled in ncbi database in Ka Liduoniya bulkholderia cepasea LMG19076 (BcaLMG19076-ACCD, SEQ ID NO:6-7)；Its sequence is subjected to Maize codon optimization (SEQ ID NO:5), design Both-end has BsmBI restriction enzyme site, and synthesizes at Jin Sirui (GenScript).BamHI/SpeI segment is connected into after BsmBI digestion In the intermediate vector of change.Spectinomycin positive colony (Spec+) passes through PvuI/NotI digestion verification.Correctly clone is further It is errorless by sequence verification junction.Maize ubiquitin promoter (SEQ ID NO:4), the expression for BcaLMG19076-ACCD (Fig. 1).

1.2. the Agrobacterium-mediated Transformation of corn

Corn transformation kind is JHAX in experiment.Using conversion method for agrobacterium, concrete operations see reference document [5].It carries Containing 6- Phophomannose isomerase gene (PMI, SEQ ID NO:12) expression cassette as the selection markers in plant in body；Choosing The plant for selecting the PMI positive carries out copy number analysis (using SEQ ID NO:23-25).

1.3.T0 it is identified for transgene copy number

10 milligrams of blades are collected from each PMI positive seedling, genomic DNA [6] are extracted using improved Tris-SDS method.It is interior The TaqMan primer and probe of ginseng gene and target gene utilizes the Primer of Applied Biosystem company, the U.S. 3.0 software design of Express, and synthesized by Invitrogen company, sequence information is shown in Tables 1 and 2.

The primer and probe sequence (copy number) of 1. reference gene real-time fluorescence quantitative PCR of table

The primer and probe sequence (copy number) of 2. target gene real-time fluorescence quantitative PCR of table

Real-time fluorescence quantitative PCR (qPCR) reaction system and reaction condition

Reaction system: it is 25 μ l that qPCR, which reacts total system, wherein containing 12.5 μ l JumpStart^TM Taq ReadyMix^TM, 5 μ l plant genome DNAs, (primer is final concentration of for each 0.4 μ l reference gene and target gene primed probe group 300nM, the final concentration of 100nM of probe), the pure water of the DMSO of 0% to 10% concentration (v/v) and nuclease free.

Response procedures: TaqMan real-time fluorescence quantitative PCR uses the Applied of standard system 7900 Fast real-time PCR systems.Table 3 is PCR response procedures.After PCR, result is analyzed using 2.4 software of SDS.

Table 3.PCR response procedures

The T0 plantlet of transplant of transgenosis is copied to greenhouse (table 4) with 1.

4. transgene copy number of table and expression analysis

1.4.T2 for seed growing

The T0 for copying transgenosis with 1 is selfed in the greenhouse generates T1 for seed.Transgenosis is detected by Taqman to copy Shellfish number (with 1.3) retains the T1 with 1 copy transgenosis for plant.It, will be newest a piece of complete when corn growth is to V9 period The blade sampling being unfolded entirely, uses TRIzol^TMNormal process extract RNA.The method that gene expression detection equally uses Taqman, The TaqMan primer and probe sequence information of reference gene and target gene is shown in Table 5 and table 6.Select ZmEF gene as internal reference, In triplicate, while three biology repeat each sample；With 2^-ΔΔCtMethod carries out relative quantitative assay to gene expression.Such as Table 4, BcaLMG19076-ACCD selectively for there is higher expression in plant, (non-transgenic control expression quantity is T1 Zero).

The primer and probe sequence (gene expression) of 5. reference gene real-time fluorescence quantitative PCR of table

The primer and probe sequence (gene expression) of 6. target gene real-time fluorescence quantitative PCR of table

When T1 transgenic plant is bloomed, artificial emasculation；Female fringe, which is awarded, with wild type JHAX pollen generates T2 for seed (table 4). T2 seed can isolate transgenic positive plant and nontransgenic plants.T2 is used for Drought Stress Tolerance Analysis of A for plant.

Embodiment 2. is overexpressed BcaLMG19076-ACCD and improves corn water application efficiency

Water application efficiency (Water Use Efficiency) is water required for accumulation units's weight dry matter；It needs Water it is fewer, reacted the raising that plant keeps away non-irrigated ability.Under arid initial stage and/or mild drought, it is stronger to keep away non-irrigated ability, can So that plant preferably copes with drought stress.

The T2 of 3 transgenic events is selected to carry out water application efficiency analysis (table 7) for seed.Pass through the red of seed aleurone Transgenosis and non-transgenic T2 are respectively chosen 50 for seed by color fluorescent marker (RFP).Prepare the square basin of 9cm × 9cm, is packed into The compost of similar weight.T2 seed sowing is in square basin middle position, 1 centimetre of depth.Normal watering and fertilising are until length to V3 phase. Choose 30 transgenic plants and 30 nontransgenic plants similar in size；After full water is placed 2 hours, covered with plastic lid, Then control water arid.Remaining seedling harvests aerial part, weighs starting fresh weight (initial FW) and starting dry weight (initial DW).Before Osmotic treatment, basin and total plant mass are weighed；After 7 days control water, then weigh basin and plant gross weight；The difference of the two is exactly tired Count water usage.It harvests above-ground plant parts simultaneously and weighs final fresh weight (final FW) and final dry weight (final DW). Total water consumption=accumulative water usage-(final fresh weight-starting fresh weight), water application efficiency=total water conservancy use/(final dry weight-rises Beginning dry weight).It is for statistical analysis using T-test.

T2 plant from A009A and A014A, transgenic plant improve significantly corn water application efficiency (table 7, P < 0.05).In T2 plant from A050A, it is overexpressed BcaLMG19076-ACCD and also significantly enhances water application efficiency (P= 0.07).Because being adjusted on hormone levels, the difference of different transformation event Waste water utilization efficiency is in plant Ethylene contents difference is directly related, and this difference can be the regulation (table 4) by BcaLMG19076-ACCD expression, It can be by post-transcriptional control.

7. water application efficiency of table compares

Embodiment 3. is overexpressed BcaLMG19076-ACCD and increases drought resistance of maize

Drought resistance is plant by adjusting itself flow of water, reduces activity keto concentration, reduces membranolysis, coerces in arid Compel lower maintenance certain growth and yield.Severe drought will lead to crops total crop failure, and severe is often avoided in actual production Arid.Therefore under medium drought, increase production rate-maintenance capability by improving crop drought resistance, it is most important.Mild drought exists The stress conditions of field underproduction 40-60% are defined as on corn.

In the greenhouse, the experiment of pairing arid is used for the difference of test plants drought-resistant ability.Since biomass is the same as the aobvious of yield Correlation, match arid experiment in 40-60% biomass reduction drought stress processing is accordingly defined as mild drought.? Under the conditions of this, (table 8) is tested to the T2 plant from two transformation events.In parallel, 10 couples of wild type JHAX are being just Often watering, while 10 couples of wild type JHAX carry out Osmotic treatment.After stress, the dry weight of two groups of JHAX aerial parts is measured. Osmotic treatment results in 47% reduction (Fig. 2), as medium drought.

T2 seed is sorted by RFP, and 30 transgenosis and 30 non-transgenic seeds are chosen in each transformation event, are broadcast Kind is into germination disk.Normal watering, fertilizing is until the V3 phase, selects the similar transgenosis of plant size and nontransgenic plants, two-by-two Pairing, is transplanted in 34 × 28 centimetres of big basin.After transplanting, full water is poured.Then control water carries out Osmotic treatment always, until same In one transformation event 90% leaf rolling degree of non-transgenic to ' σ ' type when stop Osmotic treatment.Harvest aerial part, every seedling An independent sack；In an oven 80 DEG C drying 2 days after record dry weight.It is examined using T and carries out significance analysis.

It is overexpressed BcaLMG19076-ACCD and improves drought-resistant maize ability (table 8) also significantly.

Biomass compares under 8. mild drought of table

Embodiment 4. plant modification aminopherase increases drought resistance of maize

Acc deaminase is a kind of deamination for depending on 5' phosphopyridoxal pyridoxal phosphate (5 '-phosphate of pyridoxal (PLP)) Enzyme, the homologous gene on plant are never explicitly reported.There are many aminopherases in plant (aminotransferase), the amino group on amino acid can be shifted.GRMZM2G151440 is exactly one of represents The aminopherase dependent on 5' phosphopyridoxal pyridoxal phosphate of property.By protein sequence and structure alignment, gene editing, egg can be passed through Endogenous aminopherase is transformed in the methods of white matter engineering, it is made to have ACC identification and deamination function, anti-to further increase crop Non-irrigated ability.Similar also rice aminotransferase gene (LOC_Os02g53330) and arabidopsis AtACD1 gene (AT1G48420), can be used for being transformed and improving plant drought ability.

Embodiment 5., which is overexpressed BcaLMG19076-ACCD and RlePB172-ACCD, not can increase drought resistance of maize

Select the acc deaminase gene BunMTI641-ACCD (SEQ that bulkholderia cepasea MTI-641 is enclosed from grass roots ID NO:35-36) and acc deaminase gene RlePB172-ACCD (SEQ ID NO:38- from rhizobium leguminosarum PB172 39).Optimize (respectively SEQ ID NO:37 and SEQ ID NO:40) by Maize codon, is connected into plant expressing vector respectively 19982 (Fig. 4) and 19953 (Fig. 5).The same with BcaLMG19076-ACCD, T2 has carried out pairing arid experiment test for plant. The result shows that the effect (table 9) for improving plant drought resistance is not implemented in both acc deaminase genes.

Biomass compares under 9. mild drought of table

Conclusion: not every acc deaminase can improve plant drought resistance.

Bibliography

[1]Fang YJ,Xiong LZ.General mechanisms of drought response and their application in drought resistance improvement in plants.Cell Mol Life Sci.2015Feb；72(4):673-89.

[2]Habben JE,Bao X,Bate NJ,DeBruin JL,Dolan D,Hasegawa D,Helentjaris TG,Lafitte RH,Lovan N,Mo H,Reimann K,Schussler JR.Transgenic alteration of ethylene biosynthesis increases grain yield in maize under field drought- stress conditions.Plant Biotechnol J.2014Aug；12(6):685-93.

[3]Shi J,Habben JE,Archibald RL,Drummond BJ,Chamberlin MA,Williams RW,Lafitte HR,Weers BP.Overexpression of ARGOS Genes Modifies Plant Sensitivity to Ethylene,Leading to Improved Drought Tolerance in Both Arabidopsis and Maize.Plant Physiol.2015Sep；169(1):266-82.

[4]Shimon M,Tsipora T,Bernard RG.Plant growth-promoting bacteria that confer resistance to water stress in tomatoes and peppers.Plant Science 166 (2004)525–530.

[5]Ishida Y,Hiei Y,Komari T.Agrobacterium-mediated transformation of maize.Nat Protoc.2007；2(7):1614-21.

[6]Li H,Li J,Cong XH,Duan YB,Li L,Wei PC,Lu XZ,Yang JB.Improvement of Agrobacterium-mediated transformation in Hi-II maize(Zea mays)using standard binary vectors.Plant Cell Rep.2008Feb；27(2):297-305.

Sequence table

<110>Syngenta Share-holding Co., Ltd (Syngenta Participations AG)

Syngenta biotechnology (China) Co., Ltd (Syngenta Biotechnology China Co., Ltd.)

<120>method of drought tolerance in plants ability is improved

<130> 81748-CN-REG-ORG-NAT-1

<141> 2018-12-05

<160> 40

<170> PatentIn version 3.5

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<213>artificial sequence

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<213>artificial sequence

<220>

<223>cRed(red fluorescent protein gene)

<400> 2

atggcctcct ccgagaacgt catcaccgag ttcatgcgct tcaaggtgcg catggagggc 60

accgtgaacg gccacgagtt cgagatcgag ggcgagggcg agggccgccc ctacgagggc 120

cacaacaccg tgaagctgaa ggtgaccaag ggcggccccc tgcccttcgc ctgggacatc 180

ctgtcccccc agttccagta cggctccaag gtgtacgtga agcaccccgc cgacatcccc 240

gactacaaga agctgtcctt ccccgagggc ttcaagtggg agcgcgtgat gaacttcgag 300

gacggcggcg tggcgaccgt gacccaggac tcctccctgc aggacggctg cttcatctac 360

aaggtgaagt tcatcggcgt gaacttcccc tccgacggcc ccgtgatgca gaagaagacc 420

atgggctggg aggcctccac cgagcgcctg tacccccgcg acggcgtgct gaagggcgag 480

acccacaagg ccctgaagct gaaggacggc ggccactacc tggtggagtt caagtccatc 540

tacatggcca agaagcccgt gcagctgccc ggctactact acgtggacgc caagctggac 600

atcacctccc acaacgagga ctacaccatc gtggagcagt acgagcgcac cgagggccgc 660

caccacctgt tcctgtag 678

<210> 3

<211> 318

<212> DNA

<213>artificial sequence

<220>

<223>tPI-15(tomato protease inhibitor II terminator)

<400> 3

agacttgtcc atcttctgga ttggccaact taattaatgt atgaaataaa aggatgcaca 60

catagtgaca tgctaatcac tataatgtgg gcatcaaagt tgtgtgttat gtgtaattgc 120

tagttatctg aataaaagag aaagagatca tccatatttc ttatcctaaa tgaatgtcac 180

gtgtctttat aattctttga tgaaccagat gcatttcatt aaccaaatcc atatacatat 240

aaatattaat catatataat taatatcaat tgggttagca aaacaaatct agtctaggtg 300

tgttttgcga atgcggcc 318

<210> 4

<211> 1993

<212> DNA

<213>artificial sequence

<220>

<223>prZmUbi1(maize ubiquitin promoter)

<400> 4

ctgcagtgca gcgtgacccg gtcgtgcccc tctctagaga taatgagcat tgcatgtcta 60

agttataaaa aattaccaca tatttttttt gtcacacttg tttgaagtgc agtttatcta 120

tctttataca tatatttaaa ctttactcta cgaataatat aatctatagt actacaataa 180

tatcagtgtt ttagagaatc atataaatga acagttagac atggtctaaa ggacaattga 240

gtattttgac aacaggactc tacagtttta tctttttagt gtgcatgtgt tctccttttt 300

ttttgcaaat agcttcacct atataatact tcatccattt tattagtaca tccatttagg 360

gtttagggtt aatggttttt atagactaat ttttttagta catctatttt attctatttt 420

agcctctaaa ttaagaaaac taaaactcta ttttagtttt tttatttaat aatttagata 480

taaaatagaa taaaataaag tgactaaaaa ttaaacaaat accctttaag aaattaaaaa 540

aactaaggaa acatttttct tgtttcgagt agataatgcc agcctgttaa acgccgtcga 600

cgagtctaac ggacaccaac cagcgaacca gcagcgtcgc gtcgggccaa gcgaagcaga 660

cggcacggca tctctgtcgc tgcctctgga cccctctcga gagttccgct ccaccgttgg 720

acttgctccg ctgtcggcat ccagaaattg cgtggcggag cggcagacgt gagccggcac 780

ggcaggcggc ctcctcctcc tctcacggca ccggcagcta cgggggattc ctttcccacc 840

gctccttcgc tttcccttcc tcgcccgccg taataaatag acaccccctc cacaccctct 900

ttccccaacc tcgtgttgtt cggagcgcac acacacacaa ccagatctcc cccaaatcca 960

cccgtcggca cctccgcttc aaggtacgcc gctcgtcctc cccccccccc cctctctacc 1020

ttctctagat cggcgttccg gtccatggtt agggcccggt agttctactt ctgttcatgt 1080

ttgtgttaga tccgtgtttg tgttagatcc gtgctgctag cgttcgtaca cggatgcgac 1140

ctgtacgtca gacacgttct gattgctaac ttgccagtgt ttctctttgg ggaatcctgg 1200

gatggctcta gccgttccgc agacgggatc gatttcatga ttttttttgt ttcgttgcat 1260

agggtttggt ttgccctttt cctttatttc aatatatgcc gtgcacttgt ttgtcgggtc 1320

atcttttcat gctttttttt gtcttggttg tgatgatgtg gtctggttgg gcggtcgttc 1380

tagatcggag tagaattctg tttcaaacta cctggtggat ttattaattt tggatctgta 1440

tgtgtgtgcc atacatattc atagttacga attgaagatg atggatggaa atatcgatct 1500

aggataggta tacatgttga tgcgggtttt actgatgcat atacagagat gctttttgtt 1560

cgcttggttg tgatgatgtg gtgtggttgg gcggtcgttc attcgttcta gatcggagta 1620

gaatactgtt tcaaactacc tggtgtattt attaattttg gaactgtatg tgtgtgtcat 1680

acatcttcat agttacgagt ttaagatgga tggaaatatc gatctaggat aggtatacat 1740

gttgatgtgg gttttactga tgcatataca tgatggcata tgcagcatct attcatatgc 1800

tctaaccttg agtacctatc tattataata aacaagtatg ttttataatt attttgatct 1860

tgatatactt ggatgatggc atatgcagca gctatatgtg gattttttta gccctgcctt 1920

catacgctat ttatttgctt ggtactgttt cttttgtcga tgctcaccct gttgtttggt 1980

gttacttctg cag 1993

<210> 5

<211> 1017

<212> DNA

<213>artificial sequence

<220>

<223>Ka Liduoniya bulkholderia cepasea LMG is come from after the optimization of cBcaLMG19076(Maize codon 19076 acc deaminase gene, encoder block sequence)

<400> 5

atgaatctcc agaggttccc acgctacccc ctgactttcg gccccactcc catccagcca 60

ctcaagcgcc tgtcggacca tctcggcggc aaggtgcacc tgtacgctaa gcgggaggac 120

tgcaactcgg gcttcgcgtt cggtggcaat aagacacgca agctcgagta cctgatccca 180

gaggctctcg ctcagggctg cgacaccctg gtgtcgatcg gtggcattca gtctaaccag 240

acgaggcagg ttgctgctgt ggctgctcat ctcggcatga agtgcgtgct ggtccaggag 300

aactgggtca attacagcga cgccgtgtac gatcgggtcg gcaatatcca gatgtcgagg 360

attctcggcg ctgacgtcag gctggttcca gacggcttcg atatcgggtt caggaagtca 420

tgggaggatg ctctggagtc cgtgcgggct gctggcggga agccatacgc tattccagct 480

ggctgcagcg accaccctct cggcgggctg ggcttcgtcg ggttcgctga ggaggttcgc 540

cagcaggagg ctgagctcgg cttcaagttc gattacatcg tggtctgctc cgttaccggc 600

agcacgcagg ctggcatggt tgtggggttc gctgctgacg gcagggcgga tagggtcatc 660

gggattgacg cgtctgctaa gcccgcccag acaagggagc agatcactcg gattgcttca 720

aggacggctg agaaggtcgg cctcgggagg gacatcatgg ccaaggacgt cgttctggat 780

gagaggttcg gtggcccaga gtacggcctc cctaacgatg ggacactcca ggctatcagg 840

ctgtgcgcta ggcaggaggg cgttctgact gacccggtgt acgagggcaa gtccatgcat 900

gggatgattg atatggtgcg gaacggcgag ttccccgagg ggagccgcgt cctctacgct 960

catctcggcg gggtgccagc cctcaacggg tactctttca tcttcaggaa tgggtag 1017

<210> 6

<211> 1017

<212> DNA

<213>Ka Liduoniya bulkholderia cepasea

<220>

<223>acc deaminase gene of the cBcaLMG19076(from Ka Liduoniya bulkholderia cepasea LMG 19076, Encoder block sequence)

<400> 6

atgaatctgc agcgatttcc ccgctatccg ctcaccttcg ggccaacacc catccagccg 60

ctcaagcggc tgagcgatca tctgggcggc aaggtgcatc tctatgccaa gcgcgaggac 120

tgcaacagcg gcttcgcgtt cggcggcaac aagacgcgca agctcgaata cctgattccc 180

gaagcactcg cgcaaggctg cgatacgctc gtgtcgatcg gcggcatcca gtcgaaccag 240

acgcgccagg tcgcggcggt tgccgcgcat ctcggcatga agtgcgtact cgtgcaggaa 300

aactgggtca actattcgga cgccgtgtac gaccgcgtcg gcaacattca gatgtcgcgc 360

attctgggcg cggacgtccg cctcgtgccg gacggcttcg acattggctt tcgtaagagc 420

tgggaagacg cgctcgaaag cgtgcgcgcc gcgggcggca agccatacgc gattccggca 480

ggctgctccg accatccgct gggcggtctc ggctttgtcg gtttcgccga agaagtgcgc 540

cagcaggaag cggaactcgg cttcaagttc gactacatcg tggtgtgctc ggtgacgggc 600

agcacgcagg caggcatggt ggtgggtttc gccgccgacg gccgcgcaga tcgcgtgatc 660

ggcatcgacg cgtccgcgaa accggcgcag acccgcgagc agatcacgcg aatcgcgagt 720

cgcaccgcag agaaagtggg cctcggacga gatatcatgg ctaaggacgt cgtgctcgac 780

gaacgcttcg gcggccccga atacggcctg ccgaacgacg gcacgctaca ggcgatccgc 840

ctgtgcgccc gccaggaagg cgtgctcacc gatccggtgt acgaaggcaa gtcgatgcac 900

ggaatgatcg acatggtgcg caacggcgaa tttcccgagg gctcgcgcgt gctctatgcg 960

cacctcggcg gcgtgcctgc cctgaacggc tatagcttta tctttcgcaa cggttaa 1017

<210> 7

<211> 338

<212> PRT

<213>Ka Liduoniya bulkholderia cepasea

<220>

<223>acc deaminase gene of the cBcaLMG19076(from Ka Liduoniya bulkholderia cepasea LMG 19076, Protein sequence)

<400> 7

Met Asn Leu Gln Arg Phe Pro Arg Tyr Pro Leu Thr Phe Gly Pro Thr

1 5 10 15

Pro Ile Gln Pro Leu Lys Arg Leu Ser Asp His Leu Gly Gly Lys Val

20 25 30

His Leu Tyr Ala Lys Arg Glu Asp Cys Asn Ser Gly Phe Ala Phe Gly

35 40 45

Gly Asn Lys Thr Arg Lys Leu Glu Tyr Leu Ile Pro Glu Ala Leu Ala

50 55 60

Gln Gly Cys Asp Thr Leu Val Ser Ile Gly Gly Ile Gln Ser Asn Gln

65 70 75 80

Thr Arg Gln Val Ala Ala Val Ala Ala His Leu Gly Met Lys Cys Val

85 90 95

Leu Val Gln Glu Asn Trp Val Asn Tyr Ser Asp Ala Val Tyr Asp Arg

100 105 110

Val Gly Asn Ile Gln Met Ser Arg Ile Leu Gly Ala Asp Val Arg Leu

115 120 125

Val Pro Asp Gly Phe Asp Ile Gly Phe Arg Lys Ser Trp Glu Asp Ala

130 135 140

Leu Glu Ser Val Arg Ala Ala Gly Gly Lys Pro Tyr Ala Ile Pro Ala

145 150 155 160

Gly Cys Ser Asp His Pro Leu Gly Gly Leu Gly Phe Val Gly Phe Ala

165 170 175

Glu Glu Val Arg Gln Gln Glu Ala Glu Leu Gly Phe Lys Phe Asp Tyr

180 185 190

Ile Val Val Cys Ser Val Thr Gly Ser Thr Gln Ala Gly Met Val Val

195 200 205

Gly Phe Ala Ala Asp Gly Arg Ala Asp Arg Val Ile Gly Ile Asp Ala

210 215 220

Ser Ala Lys Pro Ala Gln Thr Arg Glu Gln Ile Thr Arg Ile Ala Ser

225 230 235 240

Arg Thr Ala Glu Lys Val Gly Leu Gly Arg Asp Ile Met Ala Lys Asp

245 250 255

Val Val Leu Asp Glu Arg Phe Gly Gly Pro Glu Tyr Gly Leu Pro Asn

260 265 270

Asp Gly Thr Leu Gln Ala Ile Arg Leu Cys Ala Arg Gln Glu Gly Val

275 280 285

Leu Thr Asp Pro Val Tyr Glu Gly Lys Ser Met His Gly Met Ile Asp

290 295 300

Met Val Arg Asn Gly Glu Phe Pro Glu Gly Ser Arg Val Leu Tyr Ala

305 310 315 320

His Leu Gly Gly Val Pro Ala Leu Asn Gly Tyr Ser Phe Ile Phe Arg

325 330 335

Asn Gly

<210> 8

<211> 1035

<212> DNA

<213>artificial sequence

<220>

<223>tUbi1(maize ubiquitin terminator)

<400> 8

gtcatgggtc gtttaagctg ccgatgtgcc tgcgtcgtct ggtgccctct ctccatatgg 60

aggttgtcaa agtatctgct gttcgtgtca tgagtcgtgt cagtgttggt ttaataatgg 120

accggttgtg ttgtgtgtgc gtactaccca gaactatgac aaatcatgaa taagtttgat 180

gtttgaaatt aaagcctgtg ctcattatgt tctgtctttc agttgtctcc taatatttgc 240

ctgcaggtac tggctatcta ccgtttctta cttaggaggt gtttgaatgc actaaaacta 300

atagttagtg gctaaaatta gttaaaacat ccaaacacca tagctaatag ttgaactatt 360

agctattttt ggaaaattag ttaatagtga ggtagttatt tgttagctag ctaattcaac 420

taacaatttt tagccaacta acaattagtt tcagtgcatt caaacacccc cttaatgtta 480

acgtggttct atctaccgtc tcctaatata tggttgattg ttcggtttgt tgctatgcta 540

ttgggttctg attgctgcta gttcttgctg aatccagaag ttctcgtagt atagctcaga 600

ttcatattat ttatttgagt gataagtgat ccaggttatt actatgttag ctaggttttt 660

tttacaagga taaattatct gtgatcataa ttcttatgaa agctttatgt ttcctggagg 720

cagtggcatg caatgcatga cagcaacttg atcacaccag ctgaggtaga tacggtaaca 780

aggttcttaa atctgttcac caaatcattg gagaacacac atacacattc ttgccagtct 840

tggttagaga aatttcatga caaaatgcca aagctgtctt gactcttcac ttttggccat 900

gagtcgtgac ttagtttggt ttaatggacc ggttctccta gcttgttcta ctcaaaactg 960

ttgttgatgc gaataagttg tgatggttga tctctggatt ttgttttgct ctcaatagtg 1020

gacgagatta gatag 1035

<210> 9

<211> 2181

<212> DNA

<213>artificial sequence

<220>

<223>prAct1(rice actin promoters)

<400> 9

tgcagcccat ccctcagccg cctttcacta tcttttttgc ccgagtcatt gtcatgtgaa 60

ccttggcatg tataatcggt gaattgcgtc gattttcctc ttataggtgg gccaatgaat 120

ccgtgtgatc gcgtctgatt ggctagagat atgtttcttc cttgttggat gtattttcat 180

acataatcat atgcatacaa atatttcatt acactttata gaaatggtca gtaataaacc 240

ctatcactat gtctggtgtt tcattttatt tgcttttaaa cgaaaattga cttcctgatt 300

caatatttaa ggatcgtcaa cggtgtgcag ttactaaatt ctggtttgta ggaactatag 360

taaactattc aagtcttcac ttattgtgca ctcacctctc gccacatcac cacagatgtt 420

attcacgtct taaatttgaa ctacacatca tattgacaca atattttttt taaataagcg 480

attaaaacct agcctctatg tcaacaatgg tgtacataac cagcgaagtt tagggagtaa 540

aaaacatcgc cttacacaaa gttcgcttta aaaaataaag agtaaatttt actttggacc 600

acccttcaac caatgtttca ctttagaacg agtaatttta ttattgtcac tttggaccac 660

cctcaaatct tttttccatc tacatccaat ttatcatgtc aaagaaatgg tctacataca 720

gctaaggaga tttatcgacg aatagtagct agcatactcg aggtcattca tatgcttgag 780

aagagagtcg ggatagtcca aaataaaaca aaggtaagat tacctggtca aaagtgaaaa 840

catcagttaa aaggtggtat aaagtaaaat atcggtaata aaaggtggcc caaagtgaaa 900

tttactcttt tctactatta taaaaattga ggatgttttt gtcggtactt tgatacgtca 960

tttttgtatg aattggtttt taagtttatt cgcttttgga aatgcatatc tgtatttgag 1020

tcgggtttta agttcgtttg cttttgtaaa tacagaggga tttgtataag aaatatcttt 1080

aaaaaaaccc atatgctaat ttgacataat ttttgagaaa aatatatatt caggcgaatt 1140

aattctcaca atgaacaata ataagattaa aatagctttc ccccgttgca gcgcatgggt 1200

attttttcta gtaaaaataa aagataaact tagactcaaa acatttacaa aaacaacccc 1260

taaagttcct aaagcccaaa gtgctatcca cgatccatag caagcccagc ccaacccaac 1320

ccaacccaac ccaccccagt ccagccaact ggacaatagt ctccacaccc ccccactatc 1380

accgtgagtt gtccgcacgc accgcacgtc tcgcagccaa aaaaaaaaaa agaaagaaaa 1440

aaaagaaaaa gaaaaaacag caggtgggtc cgggtcgtgg gggccggaaa cgcgaggagg 1500

atcgcgagcc agcgacgagg ccggccctcc ctccgcttcc aaagaaacgc cccccatcgc 1560

cactatatac ataccccccc ctctcctccc atccccccaa ccctaccacc accaccacca 1620

ccacctccac ctcctccccc ctcgctgccg gacgacgagc tcctcccccc tccccctccg 1680

ccgccgccgc gccggtaacc accccgcccc tctcctcttt ctttctccgt tttttttttc 1740

cgtctcggtc tcgatctttg gccttggtag tttgggtggg cgagaggcgg cttcgtgcgc 1800

gcccagatcg gtgcgcggga ggggcgggat ctcgcggctg gggctctcgc cggcgtggat 1860

cgatccggcc cggatctcgc ggggaatggg gctctcggat gtagatctgc gatccgccgt 1920

tgttggggga gatgatgggg ggtttaaaat ttccgccatg ctaaacaaga tcaggaagag 1980

gggaaaaggg cactatggtt tatattttta tatatttctg ctgcttcgtc aggcttagat 2040

gtgctagatc tttctttctt ctttttgtgg gtagaatttg aatccctcag cattgttcat 2100

cggtagtttt tcttttcatg atttgtgaca aatgcagcct cgtgcggagc ttttttgtag 2160

gtagaagctg gctgacgccg g 2181

<210> 10

<211> 552

<212> DNA

<213>artificial sequence

<220>

<223>the phosphine oxamate acetyl transferase gene of cPAT(soybean codon optimization)

<400> 10

atgtctccag agagaaggcc agttgagatt agacctgcta ctgcggccga tatggcagct 60

gtttgtgata ttgttaacca ttatattgag acttctactg ttaacttcag aactgagcca 120

caaactcctc aagagtggat tgatgatctt gagagacttc aagatagata cccttggctt 180

gttgctgagg ttgagggagt tgttgctgga attgcttatg ctggaccttg gaaggctaga 240

aacgcttatg attggactgt tgagtctact gtttatgttt ctcatagaca tcaaagactt 300

ggacttggat ctactcttta tactcatctt cttaagtcta tggaggctca aggattcaag 360

tctgttgttg ctgttattgg acttccaaac gatccatctg ttagacttca tgaggctctt 420

ggatatactg ctagaggaac tcttagagct gctggatata agcatggagg atggcatgat 480

gttggattct ggcaaagaga tttcgagctt ccagctccac caagaccagt tagaccagtt 540

actcaaattt ga 552

<210> 11

<211> 70

<212> DNA

<213>artificial sequence

<220>

<223>t35S(cauliflower mosaic virus 35S terminator)

<400> 11

cttagtatgt atttgtattt gtaaaatact tctatcaata aaatttctaa ttcctaaaac 60

caaaatccag 70

<210> 12

<211> 1176

<212> DNA

<213>artificial sequence

<220>

<223>cPMI(6- Phophomannose isomerase gene)

<400> 12

atgcaaaaac tcattaactc agtgcaaaac tatgcctggg gcagcaaaac ggcgttgact 60

gaactttatg gtatggaaaa tccgtccagc cagccgatgg ccgagctgtg gatgggcgca 120

catccgaaaa gcagttcacg agtgcagaat gccgccggag atatcgtttc actgcgtgat 180

gtgattgaga gtgataaatc gactctgctc ggagaggccg ttgccaaacg ctttggcgaa 240

ctgcctttcc tgttcaaagt attatgcgca gcacagccac tctccattca ggttcatcca 300

aacaaacaca attctgaaat cggttttgcc aaagaaaatg ccgcaggtat cccgatggat 360

gccgccgagc gtaactataa agatcctaac cacaagccgg agctggtttt tgcgctgacg 420

cctttccttg cgatgaacgc gtttcgtgaa ttttccgaga ttgtctccct actccagccg 480

gtcgcaggtg cacatccggc gattgctcac tttttacaac agcctgatgc cgaacgttta 540

agcgaactgt tcgccagcct gttgaatatg cagggtgaag aaaaatcccg cgcgctggcg 600

attttaaaat cggccctcga tagccagcag ggtgaaccgt ggcaaacgat tcgtttaatt 660

tctgaatttt acccggaaga cagcggtctg ttctccccgc tattgctgaa tgtggtgaaa 720

ttgaaccctg gcgaagcgat gttcctgttc gctgaaacac cgcacgctta cctgcaaggc 780

gtggcgctgg aagtgatggc aaactccgat aacgtgctgc gtgcgggtct gacgcctaaa 840

tacattgata ttccggaact ggttgccaat gtgaaattcg aagccaaacc ggctaaccag 900

ttgttgaccc agccggtgaa acaaggtgca gaactggact tcccgattcc agtggatgat 960

tttgccttct cgctgcatga ccttagtgat aaagaaacca ccattagcca gcagagtgcc 1020

gccattttgt tctgcgtcga aggcgatgca acgttgtgga aaggttctca gcagttacag 1080

cttaaaccgg gtgaatcagc gtttattgcc gccaacgaat caccggtgac tgtcaaaggc 1140

cacggccgtt tagcgcgtgt ttacaacaag ctgtaa 1176

<210> 13

<211> 253

<212> DNA

<213>artificial sequence

<220>

<223>tNOS(Agrobacterium rouge alkali synthetase gene terminator)

<400> 13

gatcgttcaa acatttggca ataaagtttc ttaagattga atcctgttgc cggtcttgcg 60

atgattatca tataatttct gttgaattac gttaagcatg taataattaa catgtaatgc 120

atgacgttat ttatgagatg ggtttttatg attagagtcc cgcaattata catttaatac 180

gcgatagaaa acaaaatata gcgcgcaaac taggataaat tatcgcgcgc ggtgtcatct 240

atgttactag atc 253

<210> 14

<211> 1260

<212> DNA

<213>corn

<220>

<223>cGRMZM2G151440(corn aminotransferase gene, encoder block sequence)

<400> 14

atgaccgtca cttcgactct cccactcctc tgtcgcttcc gccgacacct gagtcttgtc 60

gctgccgccg ggatggccgg agtcaccgcc accgggatgg ccggagttcc cgccaccttc 120

tcctcagcca ccgctcagat cgggggcttc ctctcgaaga agccttacgc gccgccgttg 180

tgggccacgc acctctcccc catgccttgc cataccttct cgctcggcca tttcccaaca 240

ccgattcaca agtggaatct gcccaatttg ccggaaggaa cggaagtgtg gatcaagcga 300

gacgatttat caggcatgca gttgagtgga aacaaggtcc ggaagctgga gttcttgatg 360

gcagatgccg tagcacaagg cgcagactgc gttattactg ttgggggtat acagagtaac 420

cactgccgtg ccacagccgt ggctgctaag tatctcaatc ttgattgcta cctgatacta 480

cgtacctcca agcttcttgt ggataaggac cctggtttgg ttggcaatct tcttgtcgag 540

agactagttg gggcacacgt tgatcttgtg tcaaaagaag aatatggaaa aattggtagt 600

gtggctttag ctgacctgct gaaaaaaagg cttctggaag aagggaggaa gccgtatgtg 660

attcctgttg gtggatcaaa ctcattggga acttggggat atattgaggc aataagggag 720

attgagcagc aaattcagca atcttctgat gttcagtttg atgatattgt tgttgcatgt 780

ggcagtggtg gaaccattgc tggccttgct ttaggatcca gattgagcag cttaaataca 840

aaagttcatg cattctctgt ttgtgatgac cctgaatact tctatgacta tgtccaaggc 900

ctgattgatg gacttaattc tggtttcgat tcacatgata tagttagcat ggaaaatgct 960

aaggggttag gctatgccat gaacacagct gaggagctta agtttgtcaa agacatagct 1020

gcatcaacag gcattgtcct tgatccagtc tacagtggga aggcggttta tggtttgcta 1080

aaagacatgg ctggcaatcc agccaaatgg aaaggtcgaa aagttctgtt tatccacaca 1140

ggtggtcttc ttgggttgta tgataaggct gaccagttgt catctttggc tgggagctgg 1200

cgcagaatgg atcttgaaga ttctgttcca cgcaaagatg gcactggtaa gatgttctga 1260

<210> 15

<211> 419

<212> PRT

<213>corn

<220>

<223>cGRMZM2G151440(corn aminotransferase gene, protein sequence)

<400> 15

Met Thr Val Thr Ser Thr Leu Pro Leu Leu Cys Arg Phe Arg Arg His

1 5 10 15

Leu Ser Leu Val Ala Ala Ala Gly Met Ala Gly Val Thr Ala Thr Gly

20 25 30

Met Ala Gly Val Pro Ala Thr Phe Ser Ser Ala Thr Ala Gln Ile Gly

35 40 45

Gly Phe Leu Ser Lys Lys Pro Tyr Ala Pro Pro Leu Trp Ala Thr His

50 55 60

Leu Ser Pro Met Pro Cys His Thr Phe Ser Leu Gly His Phe Pro Thr

65 70 75 80

Pro Ile His Lys Trp Asn Leu Pro Asn Leu Pro Glu Gly Thr Glu Val

85 90 95

Trp Ile Lys Arg Asp Asp Leu Ser Gly Met Gln Leu Ser Gly Asn Lys

100 105 110

Val Arg Lys Leu Glu Phe Leu Met Ala Asp Ala Val Ala Gln Gly Ala

115 120 125

Asp Cys Val Ile Thr Val Gly Gly Ile Gln Ser Asn His Cys Arg Ala

130 135 140

Thr Ala Val Ala Ala Lys Tyr Leu Asn Leu Asp Cys Tyr Leu Ile Leu

145 150 155 160

Arg Thr Ser Lys Leu Leu Val Asp Lys Asp Pro Gly Leu Val Gly Asn

165 170 175

Leu Leu Val Glu Arg Leu Val Gly Ala His Val Asp Leu Val Ser Lys

180 185 190

Glu Glu Tyr Gly Lys Ile Gly Ser Val Ala Leu Ala Asp Leu Leu Lys

195 200 205

Lys Arg Leu Leu Glu Glu Gly Arg Lys Pro Tyr Val Ile Pro Val Gly

210 215 220

Gly Ser Asn Ser Leu Gly Thr Trp Gly Tyr Ile Glu Ala Ile Arg Glu

225 230 235 240

Ile Glu Gln Gln Ile Gln Gln Ser Ser Asp Val Gln Phe Asp Asp Ile

245 250 255

Val Val Ala Cys Gly Ser Gly Gly Thr Ile Ala Gly Leu Ala Leu Gly

260 265 270

Ser Arg Leu Ser Ser Leu Asn Thr Lys Val His Ala Phe Ser Val Cys

275 280 285

Asp Asp Pro Glu Tyr Phe Tyr Asp Tyr Val Gln Gly Leu Ile Asp Gly

290 295 300

Leu Asn Ser Gly Phe Asp Ser His Asp Ile Val Ser Met Glu Asn Ala

305 310 315 320

Lys Gly Leu Gly Tyr Ala Met Asn Thr Ala Glu Glu Leu Lys Phe Val

325 330 335

Lys Asp Ile Ala Ala Ser Thr Gly Ile Val Leu Asp Pro Val Tyr Ser

340 345 350

Gly Lys Ala Val Tyr Gly Leu Leu Lys Asp Met Ala Gly Asn Pro Ala

355 360 365

Lys Trp Lys Gly Arg Lys Val Leu Phe Ile His Thr Gly Gly Leu Leu

370 375 380

Gly Leu Tyr Asp Lys Ala Asp Gln Leu Ser Ser Leu Ala Gly Ser Trp

385 390 395 400

Arg Arg Met Asp Leu Glu Asp Ser Val Pro Arg Lys Asp Gly Thr Gly

405 410 415

Lys Met Phe

<210> 16

<211> 1206

<212> DNA

<213>arabidopsis

<220>

<223>cAtACD1(arabidopsis aminotransferase gene, encoder block sequence)

<400> 16

atgagaggac gaagcttgac actctcaaga gtaaagctcg agcttgcgag aagaagcatg 60

tctgcaacat ccgtaccttc aatggcggat tttctcacca aaaaacctta ctctcctcct 120

tcttgggctt ctcatcttcg tccgcttcct tctcacactt tctccctcgc tcaccttcct 180

actccgatcc atcgatggaa tcttcctggt cttcctaatg gcacagaact ctggatcaag 240

cgagatgatt tcaccggaat ggaattgagt ggaaacaaag tacgaaaact cgaattctta 300

atggcggaag ctgttgatca acacgctgat actgtaatca ctatcggcgg tattcagagc 360

aatcattgtc gtgctacagc cactgcatct aactatctta atctcaattc tcatcttatt 420

ctccgtactt ccaagcttct tgctgatgaa gatcctggat tggttgggaa tctccttgtc 480

gagcgtctcg ttggagctaa tgttcatcta atctctaaag aagagtattc ttccattggg 540

agtgaggctc ttactaatgc tctgaaagag aaactggaaa aagaaggaaa gaaaccctat 600

gttattccag tcggtggatc gaactctttg ggaacttggg gttatataga agcagcaagg 660

gaaattgagg agcagctgaa ttatagaccc gatgacctga aatttgatga tattgtggta 720

gcatgtggca gtggtggtac aattgctggt atttcattgg ggtcttggtt gggagctcta 780

aaagccaagg ttcatgcttt ctcggtttgc gatgatcctg attacttcta tgactttgtc 840

caagggcttc tggatggact tcacgctggt gttaactctc gtgatatcgt caacatccac 900

aatgccaaag gaaaaggata tgccatgaac acgtcagagg agcttgagtt tgtaaagaaa 960

gtagcaagtt caactggtgt tattcttgat ccggtttaca gtgggaaagc tgcgtatggt 1020

ttgataaatg agatcaccaa agatcccaaa tgttgggagg gaaggaagat attgttcata 1080

cacactggtg ggcttcttgg gttgtatgat aaggttgatc aaatggcatc tctgatgggt 1140

aattggtccc ggatggatgt ttcagaatcc gttccaagaa aagatggtgt tgggaaaatg 1200

ttctag 1206

<210> 17

<211> 401

<212> PRT

<213>arabidopsis

<220>

<223>cAtACD1(arabidopsis aminotransferase gene, protein sequence)

<400> 17

Met Arg Gly Arg Ser Leu Thr Leu Ser Arg Val Lys Leu Glu Leu Ala

1 5 10 15

Arg Arg Ser Met Ser Ala Thr Ser Val Pro Ser Met Ala Asp Phe Leu

20 25 30

Thr Lys Lys Pro Tyr Ser Pro Pro Ser Trp Ala Ser His Leu Arg Pro

35 40 45

Leu Pro Ser His Thr Phe Ser Leu Ala His Leu Pro Thr Pro Ile His

50 55 60

Arg Trp Asn Leu Pro Gly Leu Pro Asn Gly Thr Glu Leu Trp Ile Lys

65 70 75 80

Arg Asp Asp Phe Thr Gly Met Glu Leu Ser Gly Asn Lys Val Arg Lys

85 90 95

Leu Glu Phe Leu Met Ala Glu Ala Val Asp Gln His Ala Asp Thr Val

100 105 110

Ile Thr Ile Gly Gly Ile Gln Ser Asn His Cys Arg Ala Thr Ala Thr

115 120 125

Ala Ser Asn Tyr Leu Asn Leu Asn Ser His Leu Ile Leu Arg Thr Ser

130 135 140

Lys Leu Leu Ala Asp Glu Asp Pro Gly Leu Val Gly Asn Leu Leu Val

145 150 155 160

Glu Arg Leu Val Gly Ala Asn Val His Leu Ile Ser Lys Glu Glu Tyr

165 170 175

Ser Ser Ile Gly Ser Glu Ala Leu Thr Asn Ala Leu Lys Glu Lys Leu

180 185 190

Glu Lys Glu Gly Lys Lys Pro Tyr Val Ile Pro Val Gly Gly Ser Asn

195 200 205

Ser Leu Gly Thr Trp Gly Tyr Ile Glu Ala Ala Arg Glu Ile Glu Glu

210 215 220

Gln Leu Asn Tyr Arg Pro Asp Asp Leu Lys Phe Asp Asp Ile Val Val

225 230 235 240

Ala Cys Gly Ser Gly Gly Thr Ile Ala Gly Ile Ser Leu Gly Ser Trp

245 250 255

Leu Gly Ala Leu Lys Ala Lys Val His Ala Phe Ser Val Cys Asp Asp

260 265 270

Pro Asp Tyr Phe Tyr Asp Phe Val Gln Gly Leu Leu Asp Gly Leu His

275 280 285

Ala Gly Val Asn Ser Arg Asp Ile Val Asn Ile His Asn Ala Lys Gly

290 295 300

Lys Gly Tyr Ala Met Asn Thr Ser Glu Glu Leu Glu Phe Val Lys Lys

305 310 315 320

Val Ala Ser Ser Thr Gly Val Ile Leu Asp Pro Val Tyr Ser Gly Lys

325 330 335

Ala Ala Tyr Gly Leu Ile Asn Glu Ile Thr Lys Asp Pro Lys Cys Trp

340 345 350

Glu Gly Arg Lys Ile Leu Phe Ile His Thr Gly Gly Leu Leu Gly Leu

355 360 365

Tyr Asp Lys Val Asp Gln Met Ala Ser Leu Met Gly Asn Trp Ser Arg

370 375 380

Met Asp Val Ser Glu Ser Val Pro Arg Lys Asp Gly Val Gly Lys Met

385 390 395 400

Phe

<210> 18

<211> 1698

<212> DNA

<213>rice

<220>

<223>cLOC_Os02g53330(rice aminotransferase gene, encoder block sequence)

<400> 18

atggcgcgtg gggcccacca ggcaccaggt gggttttgga ctgtagctgc tgccccgacg 60

cgctgctcgc tgccccactc tctccccatt cccctccacg ccgccgccgc cgccgccgcg 120

tggatggccg gagtttccgc cgcctccgcc gccgggaaga tcgggagctt cctctccaag 180

aggccgtacg cgccgccgtc ctgggcctcg cacctgtccc ccgccccctc gcagaccttc 240

tcgctcggcc atttcccgac gccgatccac aagtggaatc tgcccaattt gccgaatggc 300

acggaggtgt ggatcaagcg agacgacatc tcaggcatgc agttgagcgg gaacaaggtc 360

cggaagctcg agttcctgat ggcagatgcc gtcgcgcagg gcgctgactg cgttataact 420

gtaggtggca tccagagcaa tcactgccgt gccaccgcag tggctgcaaa gtatataaat 480

cttgattgtt atctgatact gcgcacatcc aagcttcttg tggataagga ccctggtttg 540

gttgggaatc tccttgttga gagattggtg ggagcgcata ttgatcttgt ttcaaaagaa 600

gaatatggaa aaattggcag tgtggcttta gcggacttgc tgaaaaagaa gcttttggaa 660

gaaggccgaa aaccatatgt tattcctgtt ggtggatcaa actctctagg aacttgggga 720

tatatagaag caataaggga gattgaacat caaattcaga tatcaggtga tgttcagttt 780

gacgacattg ttgttgcatg tggcagcggt ggaaccattg ctggtcttgc tttaggatca 840

aaattgagta gcttaaaggc aaaagtccat gctttctctg tttgtgatga tcctggatat 900

ttccattcct atgttcaaga cctgattgat ggacttcatt ctgatttgcg ttcacatgat 960

ttagtcaaca ttgaaaatgc taagggctta ggttatgcca tgaacacagc tgaggagctt 1020

aagtttgtta aagatatagc tacagcaaca ggcattgtcc ttgatccagt ctacagtgga 1080

aaggcagcat atggaatgct gaaagacatg ggtgctaatc cagctaagtg ggaagggcga 1140

aaaattcttt ttgtacatac aggtggtctt cttggtttgt atgataaggt tgatgagtta 1200

tcatctttga gtgggagttg gcgcagaatg gatcttgaag aatctgttcc acgcaaagat 1260

ggaactgtcc catcacaggt aattcatgaa gaattcagag aaggctgcag aatcattacc 1320

tgcttggact tggattcagc ttgcaggatc tccagtacct ccttatcggg gttgatccca 1380

tacctcgcct gcacgttcat ctccataaac tccagcagag gaatcaaagc tacacaaagt 1440

caaaaaccaa cggtaaatca gatgcaaaac ctgtcaatct tttctgaacc agaatttgct 1500

gaacaagaga agaaactgaa aaggcaaaag aaaaaaaaaa tgcaatgccc ctacatacgt 1560

gagccggtgc tcttccagag ctccttgtgg ctggcatctg ctcctttggg aaggacatgg 1620

acgaggatga tccggtcgcc ggccttcacc aggttatcca ccgcccactt cgccgcggcc 1680

ttgctcgacg gcgagtag 1698

<210> 19

<211> 565

<212> PRT

<213>rice

<220>

<223>cLOC_Os02g53330(rice aminotransferase gene, protein sequence)

<400> 19

Met Ala Arg Gly Ala His Gln Ala Pro Gly Gly Phe Trp Thr Val Ala

1 5 10 15

Ala Ala Pro Thr Arg Cys Ser Leu Pro His Ser Leu Pro Ile Pro Leu

20 25 30

His Ala Ala Ala Ala Ala Ala Ala Trp Met Ala Gly Val Ser Ala Ala

35 40 45

Ser Ala Ala Gly Lys Ile Gly Ser Phe Leu Ser Lys Arg Pro Tyr Ala

50 55 60

Pro Pro Ser Trp Ala Ser His Leu Ser Pro Ala Pro Ser Gln Thr Phe

65 70 75 80

Ser Leu Gly His Phe Pro Thr Pro Ile His Lys Trp Asn Leu Pro Asn

85 90 95

Leu Pro Asn Gly Thr Glu Val Trp Ile Lys Arg Asp Asp Ile Ser Gly

100 105 110

Met Gln Leu Ser Gly Asn Lys Val Arg Lys Leu Glu Phe Leu Met Ala

115 120 125

Asp Ala Val Ala Gln Gly Ala Asp Cys Val Ile Thr Val Gly Gly Ile

130 135 140

Gln Ser Asn His Cys Arg Ala Thr Ala Val Ala Ala Lys Tyr Ile Asn

145 150 155 160

Leu Asp Cys Tyr Leu Ile Leu Arg Thr Ser Lys Leu Leu Val Asp Lys

165 170 175

Asp Pro Gly Leu Val Gly Asn Leu Leu Val Glu Arg Leu Val Gly Ala

180 185 190

His Ile Asp Leu Val Ser Lys Glu Glu Tyr Gly Lys Ile Gly Ser Val

195 200 205

Ala Leu Ala Asp Leu Leu Lys Lys Lys Leu Leu Glu Glu Gly Arg Lys

210 215 220

Pro Tyr Val Ile Pro Val Gly Gly Ser Asn Ser Leu Gly Thr Trp Gly

225 230 235 240

Tyr Ile Glu Ala Ile Arg Glu Ile Glu His Gln Ile Gln Ile Ser Gly

245 250 255

Asp Val Gln Phe Asp Asp Ile Val Val Ala Cys Gly Ser Gly Gly Thr

260 265 270

Ile Ala Gly Leu Ala Leu Gly Ser Lys Leu Ser Ser Leu Lys Ala Lys

275 280 285

Val His Ala Phe Ser Val Cys Asp Asp Pro Gly Tyr Phe His Ser Tyr

290 295 300

Val Gln Asp Leu Ile Asp Gly Leu His Ser Asp Leu Arg Ser His Asp

305 310 315 320

Leu Val Asn Ile Glu Asn Ala Lys Gly Leu Gly Tyr Ala Met Asn Thr

325 330 335

Ala Glu Glu Leu Lys Phe Val Lys Asp Ile Ala Thr Ala Thr Gly Ile

340 345 350

Val Leu Asp Pro Val Tyr Ser Gly Lys Ala Ala Tyr Gly Met Leu Lys

355 360 365

Asp Met Gly Ala Asn Pro Ala Lys Trp Glu Gly Arg Lys Ile Leu Phe

370 375 380

Val His Thr Gly Gly Leu Leu Gly Leu Tyr Asp Lys Val Asp Glu Leu

385 390 395 400

Ser Ser Leu Ser Gly Ser Trp Arg Arg Met Asp Leu Glu Glu Ser Val

405 410 415

Pro Arg Lys Asp Gly Thr Val Pro Ser Gln Val Ile His Glu Glu Phe

420 425 430

Arg Glu Gly Cys Arg Ile Ile Thr Cys Leu Asp Leu Asp Ser Ala Cys

435 440 445

Arg Ile Ser Ser Thr Ser Leu Ser Gly Leu Ile Pro Tyr Leu Ala Cys

450 455 460

Thr Phe Ile Ser Ile Asn Ser Ser Arg Gly Ile Lys Ala Thr Gln Ser

465 470 475 480

Gln Lys Pro Thr Val Asn Gln Met Gln Asn Leu Ser Ile Phe Ser Glu

485 490 495

Pro Glu Phe Ala Glu Gln Glu Lys Lys Leu Lys Arg Gln Lys Lys Lys

500 505 510

Lys Met Gln Cys Pro Tyr Ile Arg Glu Pro Val Leu Phe Gln Ser Ser

515 520 525

Leu Trp Leu Ala Ser Ala Pro Leu Gly Arg Thr Trp Thr Arg Met Ile

530 535 540

Arg Ser Pro Ala Phe Thr Arg Leu Ser Thr Ala His Phe Ala Ala Ala

545 550 555 560

Leu Leu Asp Gly Glu

565

<210> 20

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>ZmADH1 forward primer

<400> 20

gaacgtgtgt tgggtttgca t 21

<210> 21

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>ZmADH1 reverse primer

<400> 21

tccagcaatc cttgcacctt 20

<210> 22

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>ZmADH1 probe

<400> 22

tgcagcctaa ccatgcgcag ggta 24

<210> 23

<211> 29

<212> DNA

<213>artificial sequence

<220>

<223>PMI forward primer

<400> 23

gctgtaagag cttactgaaa aaattaaca 29

<210> 24

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>PMI reverse primer

<400> 24

cgatctgcag gtcgacgg 18

<210> 25

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>PMI probe

<400> 25

tgccgccaac gaatcaccgg 20

<210> 26

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>Red forward primer

<400> 26

aagtccatct acatggccaa gaa 23

<210> 27

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>Red reverse primer

<400> 27

gtgggaggtg atgtccagct t 21

<210> 28

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>Red probe

<400> 28

cagctgcccg gctactacta cgtggac 27

<210> 29

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>ZmEF forward primer

<400> 29

gcgccgtcac cgtatcc 17

<210> 30

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>ZmEF reverse primer

<400> 30

gctcgtcggg cgtcagta 18

<210> 31

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>ZmEF probe

<400> 31

atcagaggcg agcagaaacc acaccac 27

<210> 32

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>BcaLMG19076-ACCD forward primer

<400> 32

ctagtgcgcc agctcgtca 19

<210> 33

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>BcaLMG19076-ACCD reverse primer

<400> 33

ggagagaggg caccagacg 19

<210> 34

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>BcaLMG19076-ACCD probe

<400> 34

caggcacatc ggcagcttaa acgac 25

<210> 35

<211> 1017

<212> DNA

<213>grass roots encloses bulkholderia cepasea

<220>

<223>cBunMTI641-ACCD(encloses the acc deaminase gene of bulkholderia cepasea MTI-641 from grass roots, coding Frame sequence)

<400> 35

atgaacctcc agcgcttccc ccggtacccc ctcacgttcg ggccgacgcc aatccagccg 60

ctcaagcgcc tgagccagca tctcggcggc aaggtcgagc tctatgcgaa acgcgaggac 120

tgcaacagcg ggctcgcgtt cggcggcaac aagacgcgca agctcgaata cctgattccc 180

gacgcacttg cgcaaggttg cgacacgctg gtgtcgatcg gcggcattca gtcgaaccag 240

actcgccagg tcgcggccgt tgccgcgcat ctcggcatga agtgcgtact tgttcaggag 300

aactgggtaa attactccga cgccgtctac gaccgcgtcg gcaacatcca gatgtcacgc 360

atgatgggcg cggacgtgcg gctcgtggcg gacggcttcg atatcggcat tcgcccgagc 420

tgggaggagg cgctcgaaag cgtgcgccag gcgggcggca aaccgtacgc gataccggca 480

gggtgctccg agcatccgct cgggggcctc ggcttcgtgg gctttgccga ggaagtgcgg 540

cagcaggaag ccgagctcgg cttcaagttt gactacatcg tcgtgtgctc ggtcacgggc 600

agcacgcagg cggggatggt ggtgggcttc gccgccgacg gccgcgcgaa tcgcgtgatc 660

ggtatcgacg cctccgctac gcccgaaaaa acgcacgcac agatcacgcg catcgcgcgt 720

cacacggcgg gtctggtcga cctcagccgc gatatcggcg agcaggacgt gattctcgac 780

acgcgctacg gcgggcccga atacgggctg ccgaacgagg gcacgctgga ggcgattcgc 840

ctgtgcgcgc ggatggaagg catgctgacc gacccggtgt acgaaggcaa gtcgatgcac 900

ggcatgatcg acaaagtgcg cctcggtgag ttcgagccgg gttcgaaggt gctgtatgcc 960

catctgggcg gcgtgcccgc gttgagcgca tacagcttca ttttccgcga cggttga 1017

<210> 36

<211> 338

<212> PRT

<213>grass roots encloses bulkholderia cepasea

<220>

<223>cBunMTI641-ACCD(encloses the acc deaminase gene of bulkholderia cepasea MTI-641, albumen from grass roots Sequence)

<400> 36

Met Asn Leu Gln Arg Phe Pro Arg Tyr Pro Leu Thr Phe Gly Pro Thr

1 5 10 15

Pro Ile Gln Pro Leu Lys Arg Leu Ser Gln His Leu Gly Gly Lys Val

20 25 30

Glu Leu Tyr Ala Lys Arg Glu Asp Cys Asn Ser Gly Leu Ala Phe Gly

35 40 45

Gly Asn Lys Thr Arg Lys Leu Glu Tyr Leu Ile Pro Asp Ala Leu Ala

50 55 60

Gln Gly Cys Asp Thr Leu Val Ser Ile Gly Gly Ile Gln Ser Asn Gln

65 70 75 80

Thr Arg Gln Val Ala Ala Val Ala Ala His Leu Gly Met Lys Cys Val

85 90 95

Leu Val Gln Glu Asn Trp Val Asn Tyr Ser Asp Ala Val Tyr Asp Arg

100 105 110

Val Gly Asn Ile Gln Met Ser Arg Met Met Gly Ala Asp Val Arg Leu

115 120 125

Val Ala Asp Gly Phe Asp Ile Gly Ile Arg Pro Ser Trp Glu Glu Ala

130 135 140

Leu Glu Ser Val Arg Gln Ala Gly Gly Lys Pro Tyr Ala Ile Pro Ala

145 150 155 160

Gly Cys Ser Glu His Pro Leu Gly Gly Leu Gly Phe Val Gly Phe Ala

165 170 175

Glu Glu Val Arg Gln Gln Glu Ala Glu Leu Gly Phe Lys Phe Asp Tyr

180 185 190

Ile Val Val Cys Ser Val Thr Gly Ser Thr Gln Ala Gly Met Val Val

195 200 205

Gly Phe Ala Ala Asp Gly Arg Ala Asn Arg Val Ile Gly Ile Asp Ala

210 215 220

Ser Ala Thr Pro Glu Lys Thr His Ala Gln Ile Thr Arg Ile Ala Arg

225 230 235 240

His Thr Ala Gly Leu Val Asp Leu Ser Arg Asp Ile Gly Glu Gln Asp

245 250 255

Val Ile Leu Asp Thr Arg Tyr Gly Gly Pro Glu Tyr Gly Leu Pro Asn

260 265 270

Glu Gly Thr Leu Glu Ala Ile Arg Leu Cys Ala Arg Met Glu Gly Met

275 280 285

Leu Thr Asp Pro Val Tyr Glu Gly Lys Ser Met His Gly Met Ile Asp

290 295 300

Lys Val Arg Leu Gly Glu Phe Glu Pro Gly Ser Lys Val Leu Tyr Ala

305 310 315 320

His Leu Gly Gly Val Pro Ala Leu Ser Ala Tyr Ser Phe Ile Phe Arg

325 330 335

Asp Gly

<210> 37

<211> 1017

<212> DNA

<213>artificial sequence

<220>

<223>enclose bulkholderia cepasea MTI-641's from grass roots after the optimization of cBunMTI641-ACCD(Maize codon Acc deaminase gene, encoder block sequence)

<400> 37

atgaatctcc agcggttccc tcggtacccc ctcactttcg gccccacacc catccagcca 60

ctgaagcgcc tgtcccagca cctcggcggc aaggtggagc tctacgccaa gcgcgaggac 120

tgcaactcgg gcctggcgtt cggtggcaat aagacaagga agctcgagta cctgatccca 180

gacgctctcg ctcagggctg cgatacactg gtgtcgatcg gcgggattca gtctaaccag 240

actaggcagg tggctgctgt cgctgctcac ctcggcatga agtgcgtcct ggttcaggag 300

aactgggtca attacagcga cgccgtctac gatagggttg gcaatatcca gatgtcgcgc 360

atgatggggg ctgacgttag gctcgtggct gacggcttcg atatcgggat tcggccatca 420

tgggaggagg ctctggagtc cgtccgccag gcgggcggga agccatacgc tatcccagct 480

ggctgcagcg agcatcctct cggcgggctg ggcttcgtgg ggttcgctga ggaggtccgc 540

cagcaggagg ctgagctcgg cttcaagttc gactacatcg tggtctgctc cgtgaccggc 600

agcacgcagg ctggcatggt tgtggggttc gctgctgacg gcagggcgaa cagggtcatc 660

gggattgatg cttccgccac acccgagaag actcacgccc agatcaccag gattgctagg 720

catacggctg gcctcgttga cctgagcagg gatatcggcg agcaggacgt gattctcgat 780

acccgctacg gtggcccaga gtacggcctg cctaacgagg ggacgctcga ggctatcagg 840

ctgtgcgctc ggatggaggg catgctgacc gacccggtgt acgagggcaa gtctatgcac 900

gggatgattg ataaggtcag gctcggcgag ttcgagccag ggtcaaaggt tctgtacgct 960

cacctcggcg gggttccagc tctctcggct tactcgttca tcttcaggga cggctga 1017

<210> 38

<211> 1017

<212> DNA

<213>rhizobium leguminosarum

<220>

<223>acc deaminase gene of the cRlePB172-ACCD(from rhizobium leguminosarum PB172, encoder block sequence)

<400> 38

atgaatctgc agcgatttcc ccgctatccg ctcaccttcg ggccaacacc catccagccg 60

ctcaagcggc tgagcgatca tctgggcggc aaggtgcatc tctatgccaa gcgcgaggac 120

tgcaacagcg gcttcgcgtt cggcggcaac aagacgcgca agctcgaata cctgattccc 180

gaagcactcg cgcaaggctg cgatacgctc gtgtcgatcg gcggcatcca gtcgaaccag 240

acgcgccagg tcgcggcggt tgccgcgcat ctcggcatga agtgcgtact cgtgcaggaa 300

aactgggtca actattcgga cgccgtgtac gaccgcgtcg gcaacattca gatgtcgcgc 360

attctgggcg cggacgtccg cctcgtgccg gacggcttcg acattggctt tcgtaagagc 420

tgggaagacg cgctcgaaag cgtgcgcgcc gcgggcggca agccatacgc gattccggca 480

ggctgctccg accatccgct gggcggtctc ggctttgtcg gtttcgccga agaagtgcgc 540

cagcaggaag cggaactcgg cttcaagttc gactacatcg tggtgtgctc ggtgacgggc 600

agcacgcagg caggcatggt ggtgggtttc gccgccgacg gccgcgcaga tcgcgtgatc 660

ggcatcgacg cgtccgcgaa accggcgcag acccgcgagc agatcacgcg aatcgcgagt 720

cgcaccgcag agaaagtggg cctcggacga gatatcatgg ctaaggacgt cgtgctcgac 780

gaacgcttcg gcggccccga atacggcctg ccgaacgacg gcacgctaca ggcgatccgc 840

ctgtgcgccc gccaggaagg cgtgctcacc gatccggtgt acgaaggcaa gtcgatgcac 900

ggaatgatcg acatggtgcg caacggcgaa tttcccgagg gctcgcgcgt gctctatgcg 960

cacctcggcg gcgtgcctgc cctgaacggc tatagcttta tctttcgcaa cggttaa 1017

<210> 39

<211> 338

<212> PRT

<213>rhizobium leguminosarum

<220>

<223>cRlePB172-ACCD(is from the acc deaminase gene from rhizobium leguminosarum PB172, protein sequence)

<400> 39

Met Asn Leu Gln Arg Phe Pro Arg Tyr Pro Leu Thr Phe Gly Pro Thr

1 5 10 15

Pro Ile Gln Pro Leu Lys Arg Leu Ser Asp His Leu Gly Gly Lys Val

20 25 30

His Leu Tyr Ala Lys Arg Glu Asp Cys Asn Ser Gly Phe Ala Phe Gly

35 40 45

Gly Asn Lys Thr Arg Lys Leu Glu Tyr Leu Ile Pro Glu Ala Leu Ala

50 55 60

Gln Gly Cys Asp Thr Leu Val Ser Ile Gly Gly Ile Gln Ser Asn Gln

65 70 75 80

Thr Arg Gln Val Ala Ala Val Ala Ala His Leu Gly Met Lys Cys Val

85 90 95

Leu Val Gln Glu Asn Trp Val Asn Tyr Ser Asp Ala Val Tyr Asp Arg

100 105 110

Val Gly Asn Ile Gln Met Ser Arg Ile Leu Gly Ala Asp Val Arg Leu

115 120 125

Val Pro Asp Gly Phe Asp Ile Gly Phe Arg Lys Ser Trp Glu Asp Ala

130 135 140

Leu Glu Ser Val Arg Ala Ala Gly Gly Lys Pro Tyr Ala Ile Pro Ala

145 150 155 160

Gly Cys Ser Asp His Pro Leu Gly Gly Leu Gly Phe Val Gly Phe Ala

165 170 175

Glu Glu Val Arg Gln Gln Glu Ala Glu Leu Gly Phe Lys Phe Asp Tyr

180 185 190

Ile Val Val Cys Ser Val Thr Gly Ser Thr Gln Ala Gly Met Val Val

195 200 205

Gly Phe Ala Ala Asp Gly Arg Ala Asp Arg Val Ile Gly Ile Asp Ala

210 215 220

Ser Ala Lys Pro Ala Gln Thr Arg Glu Gln Ile Thr Arg Ile Ala Ser

225 230 235 240

Arg Thr Ala Glu Lys Val Gly Leu Gly Arg Asp Ile Met Ala Lys Asp

245 250 255

Val Val Leu Asp Glu Arg Phe Gly Gly Pro Glu Tyr Gly Leu Pro Asn

260 265 270

Asp Gly Thr Leu Gln Ala Ile Arg Leu Cys Ala Arg Gln Glu Gly Val

275 280 285

Leu Thr Asp Pro Val Tyr Glu Gly Lys Ser Met His Gly Met Ile Asp

290 295 300

Met Val Arg Asn Gly Glu Phe Pro Glu Gly Ser Arg Val Leu Tyr Ala

305 310 315 320

His Leu Gly Gly Val Pro Ala Leu Asn Gly Tyr Ser Phe Ile Phe Arg

325 330 335

Asn Gly

<210> 40

<211> 1017

<212> DNA

<213>artificial sequence

<220>

<223>cRlePB172-ACCD(Maize codon optimization after from the acc deaminase from rhizobium leguminosarum PB172 Gene, encoder block sequence)

<400> 40

atgaatctcc agaggttccc acgctacccc ctgactttcg gccccactcc catccagcca 60

ctcaagcgcc tgtcggacca tctcggcggc aaggtgcacc tgtacgctaa gcgggaggac 120

tgcaactcgg gcttcgcgtt cggtggcaat aagacacgca agctcgagta cctgatccca 180

gaggctctcg ctcagggctg cgacaccctg gtgtcgatcg gtggcattca gtctaaccag 240

acgaggcagg ttgctgctgt ggctgctcat ctcggcatga agtgcgtgct ggtccaggag 300

aactgggtca attacagcga cgccgtgtac gatcgggtcg gcaatatcca gatgtcgagg 360

attctcggcg ctgacgtcag gctggttcca gacggcttcg atatcgggtt caggaagtca 420

tgggaggatg ctctggagtc cgtgcgggct gctggcggga agccatacgc tattccagct 480

ggctgcagcg accaccctct cggcgggctg ggcttcgtcg ggttcgctga ggaggttcgc 540

cagcaggagg ctgagctcgg cttcaagttc gattacatcg tggtctgctc cgttaccggc 600

agcacgcagg ctggcatggt tgtggggttc gctgctgacg gcagggcgga tagggtcatc 660

gggattgacg cgtctgctaa gcccgcccag acaagggagc agatcactcg gattgcttca 720

aggacggctg agaaggtcgg cctcgggagg gacatcatgg ccaaggacgt cgttctggat 780

gagaggttcg gtggcccaga gtacggcctc cctaacgatg ggacactcca ggctatcagg 840

ctgtgcgcta ggcaggaggg cgttctgact gacccggtgt acgagggcaa gtccatgcat 900

gggatgattg atatggtgcg gaacggcgag ttccccgagg ggagccgcgt cctctacgct 960

catctcggcg gggtgccagc cctcaacggg tactctttca tcttcaggaa tgggtag 1017

Claims (18)

1.ACC deaminase or the polynucleotide sequence for encoding acc deaminase are used to improve the purposes of drought tolerance in plants ability.

2. purposes according to claim 1, wherein the acc deaminase is Ka Liduoniya bulkholderia cepasea LMG 19076 acc deaminase.

3. purposes according to claim 2, wherein the amino acid sequence of the acc deaminase is as shown in SEQ ID NO:7.

4. purposes according to claim 1, wherein the polynucleotide sequence of the coding acc deaminase is originated from Ka Liduoni The acc deaminase gene of sub- bulkholderia cepasea LMG 19076.

5. purposes according to claim 4, wherein the polynucleotide sequence such as SEQ ID NO of the coding acc deaminase: Shown in 5 or SEQ ID NO:6.

6. purposes according to any one of claims 1-5, wherein the plant is selected from the group being made of the following: beautiful Rice, wheat, rice, sorghum, arabidopsis, soybean, tomato, sunflower, spinach and rape.

7. a kind of method for improving drought tolerance in plants ability comprising be overexpressed the multicore of coding acc deaminase in the plant Nucleotide sequence.

8. according to the method described in claim 7, the polynucleotide sequence of the coding acc deaminase is originated from Ka Liduoni Abel The acc deaminase gene of Ke Huoerde Salmonella LMG 19076.

9. according to the method described in claim 8, wherein it is described coding acc deaminase polynucleotide sequence such as SEQ ID NO: Shown in 5 or SEQ ID NO:6.

10. the method according to any one of claim 7-9, wherein the overexpression is by plant ubiquitin promoter or plant Actin promoter drives.

11. according to the method described in claim 10, wherein the plant ubiquitin promoter is maize ubiquitin promoter prZmUbi1。

12. according to the method for claim 11, wherein the sequence of the maize ubiquitin promoter prZmUbi1 such as SEQ ID Shown in NO:4.

13. according to the method described in claim 10, wherein the Plant Actin promoter is rice actin starting Sub- prAct1.

14. according to the method for claim 13, wherein the sequence such as SEQ of the rice actin promoters prAct1 Shown in ID NO:9.

15. the method according to any one of claim 7-9, wherein the overexpression is by plant inducible promoter or plant Object organ specific promoters drive.

16. according to the method for claim 15, wherein the plant inducer conductivity type promoter is the drought-induced starting of arabidopsis Sub- prAtRD29A or other drought-induced promoters.

17. according to the method for claim 15, wherein the plant organ specificity promoter is green organs specificity Promoter prOsPSI33kd, stomata specificity promoter prAt1G22690 or similar promoter.

18. the method according to any one of claim 7-17, wherein the plant is selected from the group being made of the following: Corn, wheat, rice, sorghum, arabidopsis, soybean, tomato, sunflower, spinach and rape.