CN102925453A - Malic acid transporter gene GmALMT1 and application thereof - Google Patents

Abstract

The invention discloses a malic acid transporter gene GmALMT1 and an application thereof. The nucleotide sequence of the malic acid transporter gene GmALMT1 is shown as SEQ ID NO.1. An amino acid sequence of an encoded protein is shown as SEQ ID NO.2. The GmALMT1 gene provided by the invention can be used for regulating soybean root tip malic acid secretion.

Description

A kind of oxysuccinic acid transhipment subbase is because of GmALMT1 and application thereof

Technical field

The present invention relates to plant biotechnology field, be specifically related to oxysuccinic acid transhipment subbase because of GmALMT1And use.

Background technology

Acid soil extensively distributes in the world, the whole world acid soil total area accounts for the 30-40% of world's cultivated area, and the potential arable land more than 50% is arranged is acid soil, mainly is distributed in the torrid zone, subtropics and Temperate Region in China (Kochian et al., 2004).Acid soil not only shows H to the effect that crop growth suppresses ⁺Excessive concentration damage to crops itself, and usually show as the shortage of available phosphorus and aluminium toxicity to the collaborative inhibition of plant-growth and output.

In the evolutionary process of long-term nature and artificial selection, plant has formed the coadaptation mechanism of a series of forms, physiology and molecule, overcomes the obstruction factors such as low pH value, available phosphorus shortage and aluminium toxicity that exist in the acid soil.It is reported that plant adapts to the mechanism of aluminium toxicity, comprise that mainly inside restrains oneself and efflux mechanism (for example, the secretion of oxysuccinic acid); The mechanism that adapts to low-phosphorus stress, mainly comprise root morphology and architecture change, organic acid secretion, acid phosphatase Enzyme induced formation and secretion, high affinity phosphorus transporter (Raghothama, 1999 such as induce; Vance et al., 2003).Because the shortage of available phosphorus and aluminium toxicity while also are present in the acid soil, have hinted that may there be common adaptation mechanism in plant, overcome shortage and the aluminium toxicity of available phosphorus.It is worth mentioning that, in the research of the plants such as rape, Kidney bean, soybean, Lupinus albus, find that low-phosphorus stress is significantly strengthened the secretion of root system oxysuccinic acid, and promote it to activation and utilization (Hoffland et al., 1989 of external source insoluble phosphorus; Hinsinger, 2001; Shen et al., 2002; Liao et al., 2006; Sas et al., 20010); Under aluminium was coerced, the secretory volume of the oxysuccinic acid of Arabidopis thaliana, wheat, triticale and soybean significantly increased, thereby alleviated aluminium to inhibition (Delhaize et al., 1993 of root growth; Pellet et al., 1996; Ritchey et al., 1998; Ma, 2000).The secretion that these results disclose regulating apple acid may be that the explanation plant adapts to the shortage of available phosphorus in the acid soil and the node of aluminium toxicity.Since Sasaki etc. was cloned into first coding secretion root system oxysuccinic acid in 2004 from wheat transhipment subbase is because of ALMT1, on Arabidopis thaliana, grape, rape and barley, be cloned into similar oxysuccinic acid transhipment.Further studies show that process (Raman et al., 2005 of the expression control plant secretion oxysuccinic acid of these organic acid transhipment; Hoekenga et al., 2006; Ligaba et al., 2006; Furukawa et al., 2007).

Soybean is important cash crop, has very important status in China's agriculture production, and it is a kind of traditional grain, oil, raise the leguminous crop of dual-purpose.Early-stage Study finds that the soybean genotype that adapts to southern acid soil has higher phosphorus efficient and anti-aluminium toxicity ability, but the key gene of controlling its process is not yet reported.

For above-mentioned research background, the applicant has cloned one and has been subjected to oxysuccinic acid transhipment external source pH, phosphorus and aluminium coordinated regulation, tip of a root specifically expressing by the method for homologous clone, GmAlMT1By expression systems such as whole strain, hair root and frog's eggs, prove that the albumen of GmAlMT1 genes encoding has the function of control oxysuccinic acid secretion, finally improves the ability that transfer-gen plant improves the poison of anti-aluminium.

Summary of the invention

The objective of the invention is for the deficiencies in the prior art, provide a kind of oxysuccinic acid transhipment subbase because of GmALMT1

Another object of the present invention provides the protein of said gene coding.

Further purpose of the present invention provides the application of the protein of said gene and coding thereof.

Above-mentioned purpose of the present invention is achieved by the following technical programs:

Oxysuccinic acid provided by the present invention transhipment subbase because of GmALMT1, can derive from soybean, it comprises or has and is selected from following nucleotide sequence:

(1) nucleotide sequence shown in the SEQ ID NO:1;

(2) with the complementary sequence of the nucleotide sequence of (1) at the low nucleotide sequence of hybridizing under stringent condition, medium stringent condition, the preferably high stringency condition that waits;

(3) have at least 50%, at least 60%, at least 70%, at least 75% with the nucleotide sequence of (1), the nucleotide sequence of preferred at least 80%, more preferably at least 85%, particularly preferably at least 90%, especially at least 95% or 98% or 99% identity;

(4) different nucleotide sequence from the protein of the nucleotide sequence coded same acid sequence of (1) but on sequence;

(5) nucleotide sequence of one of following aminoacid sequence of coding: the aminoacid sequence shown in the SEQ ID NO:2, perhaps, because one or more (for example 1-25,1-20,1-15,1-10,1-5,1-3) amino-acid residue substitutes, lack and/or insert and the aminoacid sequence different from the aminoacid sequence shown in the SEQ ID NO:2, perhaps, have at least 50% with the aminoacid sequence shown in the SEQ ID NO:2, at least 60%, at least 70%, at least 75%, preferably at least 80%, more preferably at least 85%, more preferably at least 90%, especially the aminoacid sequence of at least 95% or 98% or 99% identity;

The active fragments of any one nucleotide sequence in (6) (1)-(5);

(7) with the nucleotide sequence of any one nucleotide sequence complementation in (1)-(5).

SEQ ID NO:1 is by 1461 based compositions, and its open reading frame (ORF) is the 1-1461 bit base, and coding has the aminoacid sequence of sequence SEQ ID NO:2, and the protein that described aminoacid sequence forms is called GmALMT1 albumen in the present invention.

Oxysuccinic acid provided by the invention transhipment subbase because of GmALMT1The protein of coding, it comprises or has and is selected from following aminoacid sequence:

(1) aminoacid sequence shown in the SEQ ID NO:2;

(2) because the substituting of one or more (for example 1-25,1-20,1-15,1-10,1-5,1-3) amino-acid residue, disappearance and/or insert and the aminoacid sequence different from the aminoacid sequence shown in the SEQ ID NO:2;

(3) have at least 50%, at least 60%, at least 70%, at least 75% with the aminoacid sequence shown in the SEQ ID NO:2, the aminoacid sequence of preferred at least 80%, more preferably at least 85%, particularly preferably at least 90%, especially at least 95% or 98% or 99% identity;

(4) active fragments of (1) or (2) or (3) described aminoacid sequence;

(5) aminoacid sequence of polynucleotide molecule coding of the present invention.

Gene provided by the invention GmALMT1Can regulate and control to comprise the transhipment of oxysuccinic acid in its transgenic organism with protein.

It is above-mentioned to increase GmALMTT1The primer of full length gene or its arbitrary fragment is to belonging to protection scope of the present invention.

The present invention also provide contain above-mentioned GmALMT1The expression vector of gene, available existing plant expression vector construction contains GmALMT1The recombinant expression vector of gene.Described plant expression vector comprises double base agrobacterium vector etc., such as pYLRNAi (by being so kind as to give in Liu Yaoguang researcher laboratory, specifically describing and see document: Hu Xuxia and Liu Yaoguang, 2006, Molecular Plant Breeding) or other plant expression vector of deriving.

The present invention also provides a kind of genetic engineering bacterium, and it contains above-mentioned expression vector.

The invention still further relates to cell, it comprises of the present invention GmALMT1Gene or recombinant vectors.Described cell can be vegetable cell, for example leguminous plants cell, perhaps microorganism cells, for example bacterium or fungal cell, for example yeast cell.Described cell can be a part that separate, that exsomatize, that cultivate or plant.

The invention still further relates to plant or plant part, vegetable material, plant seed, it comprises cell of the present invention.Described plant can be leguminous plants, soybean for example, also can be other plant, such as monocotyledons such as paddy rice, wheat, barley, corn, Chinese sorghum, sugarcane, oat or rye etc., perhaps other dicotyledonss such as tobacco, Sunflower Receptacle, beet, capsicum, potato, tomato etc.Also relate to the transgenic seed from described plant.

The invention still further relates to the method for producing plant, the method comprises: from vegetable cell regeneration of transgenic plant of the present invention, perhaps with plant of the present invention and another plant hybridization.

The invention still further relates to the plant that method of the present invention is produced.

The invention still further relates to of the present invention GmALMT1Gene or the recombinant vectors purposes in the secretion of regulating plant oxysuccinic acid comprises that preparation transgenic plant and preparation promote plant to adapt to the preparation of acid soil.

The invention still further relates to the method that regulating plant adapts to acid soil, it is of the present invention that the method comprises that preparation contains GmALMT1The plant of gene or recombinant vectors, for example, described method can comprise from vegetable cell regeneration of transgenic plant of the present invention or with plant of the present invention and another plant hybridization.

A preferred embodiment provided by the present invention is with said gene GmALMT1Import in the soybean, obtain the transgenosis composite plant; The anti-aluminium ability of the root of described transgenosis composite plant etc. is higher than described purpose control plant.

Described gene GmALMT1Can for example import recipient plant by described recombinant expression vector.

Carry gene of the present invention GmALMT1Plant expression vector can be transformed into by for example agriculture bacillus mediated Regenerated from Hypocotyl Explants method soya cells or the tissue in.

Advantage of the present invention and effect:

1. gene GmALMT1Affiliated oxysuccinic acid translocator family is at Arabidopis thaliana, though be cloned and report in wheat and the rape, its biological function aspect the secretion of leguminous crop oxysuccinic acid is also unclear.The present invention clone's gene GmALMT1Secretion has significant impact on the soybean oxysuccinic acid, and this adapts to the biological function important in inhibiting of acid soil at leguminous crop to illustrating the oxysuccinic acid transporter gene.

2. gene GmALMT1Not only affected the transhipment of tip of a root oxysuccinic acid, this gene of overexpression has also increased soybean and Arabidopis thaliana to the ability of restraining oneself of aluminium poison; The functional study of this gene has far-reaching Research Significance for the molecule mechanism of resolving leguminous crop adaptation acid soil.

Description of drawings

Fig. 1: the long and dry weight of the total root of soybean in the field test.

Fig. 2: the impact of low pH value, aluminium and the relative root growth of phosphorus Usefulness Pair Soybean Root in the water culture experiment.

Fig. 3: the impact of low pH value, aluminium and phosphorus Usefulness Pair soybean root system malic acid content and oxysuccinic acid secretory volume in the water culture experiment.

Fig. 4: GmALMT1Subcellular Localization.

Fig. 5: aluminium and pH value are right GmALMT1Regulation and control at the root system expression specificity.

Fig. 6: aluminium, pH value and phosphorus Usefulness Pair GmALMT1The impact of genetic expression.

Fig. 7: the electrophysiologic activity of GmALMT1 translocator.

Fig. 8: interfere and excessively expression GmALMT1On the secretion of genetically engineered soybean hairly root oxysuccinic acid and the impact of anti-aluminium.

Fig. 9: cross and express GmALMT1The secretion of gene pairs transgenic arabidopsis oxysuccinic acid and the impact of anti-aluminium.

Embodiment

Among the following embodiment, if no special instructions, be ordinary method.

Embodiment 1

GmALMT1Gene cloning

1, gene ( GmALMT1) the clone

According to the wheat TaALMT1-1(AB081803 that has reported) and TaALMT1-2(AB081804), Arabidopis thaliana AtALMT1(At1g08430) and rape BnAlMT1(AB194300) BnAlMT2(AB194301) aminoacid sequence of gene carries out homology relatively, at high conservative zone design upstream degenerated primer 5 '-TGGGCIRTIHTIACIGTIGT-3 ' (SEQ ID NO:3) and downstream degenerated primer 5 '-CCIGCCCAIAYIGGRMA-3 ' (SEQ ID NO:4), and to extract the total RNA of the phosphorus efficiency genotype HN98 soybean tip of a root according to the TRIzol single stage method, then the cDNA that obtains of reverse transcription carries out pcr amplification as template, the PCR reaction system is 50 μ L, comprise each 1 μ L of the forward and reverse primer of 10 μ M, 10 * PCR buffer, 5 μ L, 2.5 mM dNTP 4 μ L, Taq enzyme 0.5 μ L, cDNA template amount 2.5 μ L are then with sterilization ddH ₂O supplies 50 μ L.The PCR response procedures is: 94 ℃ 1 minute; 94 ℃ 30 seconds, 52 ℃ 30 seconds, 72 ℃ 2 minutes, 30 circulations of increasing; 72 ℃ were extended 10 minutes.The PCR product of amplification is by 1% agarose gel electrophoresis, behind Golden view nucleic acid staining dye, in the gel imaging system imaging.It is the PCR product of 405bp that dna gel reclaims test kit recovery length.Recovery obtains the PCR product cloning, and (TAKARA company, specific descriptions are seen: the evaluation of http://www.takara.com.cn/) checking order on the carrier to pMD18-T.According to the result of order-checking, use Primer 5.0 primer-design softwares, design following RACE PCR primer:

3 ' special primer 5 '-GCCTCTTTATGATGGCTTCGGAGTTG-3 ' (SEQ ID NO:5)

3 ' nested primer 5 '-AATGTGGGCTGTTCTGACAGTGGTG-3 ' (SEQ ID NO:6)

5 ' special primer 5 '-CAAGTACTGCTCCCAAGGATGGCGA-3 ' (SEQ ID NO:7)

5 ' nested primer 5 '-GACGGGACAAATGAAAGTGGAGATGACC-3 ' (SEQ ID NO:8).

Carry out according to CLONTECH ' s Advantage TM 2 PCR Kit operation instructionss.Get 5 μ L PCR products and carry out electrophoresis detection, the two PCR reactions of taking turns obtain special product, and concrete operations are carried out with reference to CLONTECH ' s AdvantageT M 2 PCR Kit operation instructionss.Then reclaiming test kit with dna gel reclaims with reference to the product recovery to 3 '/5 '-RACE PCR.Recovery obtains the PCR product cloning, and (TAKARA company, specific descriptions are seen: the evaluation of http://www.takara.com.cn/) checking order on the carrier to pMD18-T.With above acquisition GmALMT1The sequence of 3 ' and 5 ' end of gene and obtain previously GmALMT1Partial cDNA Sequence splicing obtain soybean GmALMT1Full length cDNA sequence.

2, Vector construction

The structure of Overexpression vector: take soybean tip of a root cDNA as template, with upstream special primer 5 '-GGAAGATCTCATGGATATAGAGTCAACAACCCAAGC-3 ' (SEQ ID NO:9)

With the downstream special primer

5 '-CAGCACGCGTCTATTTACAAATTGAATGTTCCGAGGG-3 ' (SEQ ID NO:10) amplification GmALMT1ORF 1461 bp fragments, after PCR fragment recovery order-checking is errorless, by BglII and MluAfter I is carried out double digestion to fragment and purpose carrier, will GmALMT1Gene is connected to purpose carrier pYLRNAi.

The structure of interference vector: take soybean tip of a root cDNA as template, use the upstream special primer

5 '-GGATCCTGGTCGCTGTCTCG-3 ' (SEQ ID NO:11) and downstream special primer 5 '-AAGCTTACATTCCCGAGTAAGTGCT-3 ' (SEQ ID NO:12) amplification 392 bp purpose fragments are used BamHI and HindIII respectively enzyme is cut PCR product and purpose carrier pYLRNAi, connection carrier behind the recovery product purification, transforming intestinal bacteria Top10F ' (is so kind as to give by Liu Yaoguang researcher laboratory, specific descriptions are seen document: Wang et al., 2006, The Plant Cell), check order errorless after, with upstream special primer 5 '-CTGCAGACATTCCCGAGTAAGTGCT-3 ' (SEQ ID NO:13) and downstream special primer 5 '-ACGCGTTGGTCGCTGTCTCG-3 ' (SEQ ID NO:14) reverse fragment that increases, use PstI and MluBehind the reverse fragment of I double digestion and the carrier with the forward fragment, reverse fragment is connected among the purpose carrier pYLRNAi that includes the forward fragment, transform intestinal bacteria Top10F ', the errorless rear transforming agrobacterium rhizogenes K599 that checks order (is so kind as to give by University of Queensland pulse family comprehensive rearch centre, be stored in root system biological study centralab of Agricultural University Of South China, specific descriptions are seen document Kereszt A, et al., 2007), be used for agriculture bacillus mediated soybean hypocotyl injection and carry out the hair root conversion.

The structure of Subcellular Localization expression vector: according to ordinary method, extract soybean root RNA, and its reverse transcription is become cDNA, take this cDNA as template, use the upstream special primer

5’- TCTAGAGATGGATATAGAGTCAACAACC -3’(SEQ ID NO:15)

With the downstream special primer

5’- GGTACCAGACAAATTGAATGTTCCGAG -3’(SEQ ID NO:16)

Amplification GmALMT1The open reading frame fragment, after PCR fragment recovery order-checking is errorless, will GmALMT1Gene is connected to purpose carrier EGFP.

The structure of electricity physiology expression vector: according to ordinary method, extract soybean root RNA, and its reverse transcription is become cDNA, take this cDNA as template, use the upstream special primer

5’-CTTGCAGATCTGCCGCCACCATGGATATAGAGTCA-3’(SEQ ID NO:17)

With downstream special primer 5 '-GCAAGACTAGTCTATTTACAAATTGA-3 ' (SEQ ID NO:18)

Amplification GmALMT1The open reading frame fragment, after PCR fragment recovery order-checking is errorless, by BglII and SpeI will to after reclaiming fragment and purpose carrier and carrying out double digestion GmALMT1Gene is connected to purpose carrier T7TS.

3, the expression pattern analysis of the Subcellular Localization of GmALMT1 and gene thereof

1) Subcellular Localization of GmALMT1

By the via Particle Bombardment Transformation method, will load GmALMT1The EGFP carrier and the EGFP empty carrier imports onion epidermis cell and soybean protoplast is carried out transient expression.Then use Laser Scanning Confocal Microscope (TCS SP2; Leica) and fluorescent microscope (LEICA DM5000B, Germany) observe respectively GFP and the PI fluorescent signal of onion epidermis cell and soybean protoplast.The results are shown in Figure 4 for the Subcellular Localization of GmALMT1.Wherein A-C is the onion epidermis contrast: the zero load that 35S promoter drives, and A is the GFP fluorescent signal; B is the PI signal; C is the fusion results of A and B; D-F is GmALMT1 -GFP is in the Subcellular Localization of onion epidermis, and D is GmALMT1 -The GFP fluorescent signal; E is the PI signal; F is the fusion results of D and E.G is the soybean protoplast contrast: the zero load that 35S promoter drives; H-I is GmALMT1 -GFP is in the Subcellular Localization of soybean protoplast, and H is GmALMT1 -The GFP fluorescent signal; I is the fusion results of H and light field.Removing the A-F scale among the figure is 100 μ m; The G-I scale is 20 μ m.

2) GmALMT1The expression pattern analysis of gene

GmALMT1The tissue specificity analysis of genetic expression:

Soybean seeds is transferred to the consistent seedling of growth and is contained 50 μ M AlCl after normal nutritive medium germinateed 3 days ₃0.5 mM CaCl ₂Processed 6 hours in the solution (the pH value is 4.3), extract respectively 0-2 cm, 2-4 cm, the RNA of 4-7 cm root segment.

Aluminum concentration pair GmALMT1The impact of genetic expression:

Soybean seeds is transferred to the consistent seedling of growth and is contained 0,50,100 μ M AlCl after normal nutritive medium germinateed 3 days ₃0.5 mM CaCl ₂Processed 6 hours in the solution (the pH value is 4.3), extract the RNA of 0-2 cm root segment.

Low pH value and aluminium pair GmALMT1The impact of genetic expression:

Soybean seeds is transferred to 0.5 mM CaCl with the seedling of homogeneous after normal nutritive medium germinateed 3 days ₂Processing is 0,6,12 hours in the solution (the pH value is 4.3), then transfers to contain 50 μ M AlCl ₃0.5 mM CaCl ₂Process 2,4,6,8 in the solution (the pH value is 4.3), 10,12 and 24 hours.Each time point gathers respectively the root segment of 0-2 cm, extracting RNA.

Aluminium, pH value and phosphorus pair GmALMT1The impact of genetic expression:

Soybean seeds is transferred to different processing with the seedling of homogeneous after germinateing 3 days respectively in normal nutritive medium and scarce phosphorus nutrition liquid, namely+and Al/+P/pH4.3, + Al/-P/pH4.3,-Al/+P/pH4.3 ,-Al/+P/pH5.8 ,-Al/-P/pH4.3 and-Al/-P/pH5.8.Wherein pH is respectively 4.3 and pH 5.8; Phosphorus process be respectively 320 μ M (+P) and 0 μ M (P); Aluminium is processed and is respectively 38 μ M Al ³⁺(+Al) and 0 μ M Al ³⁺(-Al).Calculate the activity of aluminium with GEOCHEM-EZ.Process the root segment that gathers 0-2 cm after 6 hours, extracting RNA.

Above-mentioned RNA reverse transcription is become cDNA, further use quantitative PCR detection GmALMT1Expression pattern.The house-keeping gene of soybean TefS1As confidential reference items.The primer that is used for quantitative PCR gene expression detection amount is respectively:

Soybean TefS1The primer of gene is:

TefS1 F: 5’- TGCAAAGGAGGCTGCTAACT -3’ (SEQ ID NO:19)

TefS1 R: 5’- CAGCATCACCGTTCTTCAAA -3’ (SEQ ID NO:20)

GmALMT1The primer of gene is:

GmALMT1F: 5’-GAGCACTTACTCGGGAATGTG-3’ (SEQ ID NO:21)

GmALMT1 R: 5’-GGACTTTGGCAGTTGATGGG-3’ (SEQ ID NO:22)

Fig. 5 is low pH value and aluminium pair GmALMT1The impact of expression pattern.A wherein: GmALMT1Gene is at the expression amount of the different sections of root; B: aluminum concentration pair GmALMT1The impact of genetic expression; C: low pH value and aluminium pair GmALMT1The impact of genetic expression.Data are mean value and the standard error of 4 repetitions among the figure.

Fig. 6 is the Usefulness Pair of pH value, aluminium and phosphorus GmALMT1The impact of expression pattern.Wherein A is under the condition of high phosphorus, and the pH value is right GmALMT1The impact that gene is expressed at the tip of a root; B is that the pH value is right under the low-phosphorous condition GmALMT1The impact that gene is expressed at the tip of a root; C is P Al interactions pair GmALMT1The impact that gene is expressed at the tip of a root.Data are mean value and the standard error of 4 repetitions among the figure.Asterisk represents the significance of same index between two genotype relatively: * represents conspicuous level P＜0.05 o'clock, significant difference; * represent conspicuous level 0. 001＜ P＜0.01 o'clock, the significance of difference between significantly with extremely remarkable between; * * represents conspicuous level P＜0.001 o'clock, difference was extremely remarkable; Ns represents that difference is not remarkable.

4, the electrophysiologic study of GmALMT1 function

The GmALMT1-TST7 carrier reverse transcription that builds is become cRNA.Select good frog's egg cell, inject about 30 ng GmALMT1-TST7, controlled trial is injected 30 nL water.Simultaneously injecting 50 nL concentration to frog's egg is the sodium malate of 0 to 100 mM, makes endogenous oxysuccinic acid activity be respectively 1.3,2.4 and 4.5 ({ mal ^2-I).PH of cushioning fluid is adjusted into 7.5 or 4.5, detects with the two electrodes voltage clamp.Utilizing pCLAMP 10.0 (U.S. Axon company) and Digidatas 1320A software to carry out image processes and data analysis.Fig. 7 is the electrophysiologic study of GmALMT1 function.A, the oxysuccinic acid activity is on the impact of GmALMT1 channel current; B, the pH value is on the impact of GmALMT1 channel current; C, the pH value is 7.5 o'clock, the oxysuccinic acid activity is on the impact of the I-V curve of GmALMT1; D, the pH value is 4.5 o'clock, the oxysuccinic acid activity is on the impact of the I-V curve of GmALMT1.

The research of the compound plant of embodiment 2 transgenosiss

1, the acquisition of transgenic line

1) acquisition of the compound plant of genetically engineered soybean

The expression vector and the interference vector that build are converted in the Agrobacterium rhyzogenesK599, adopt agriculture bacillus mediated soybean hypocotyl injection to obtain the compound plant of transgenosis (root is that transgenosis hairly root, overground part are non-transgenic), follow-up phenotypic evaluation is all used this strain.Empty carrier contrast: according to the method described above, with expression vector pYLRNAi same method soybean transformation, obtain to turn the unloaded contrast of pYLRNAi strain (CK).

2) acquisition of transgenic arabidopsis plant

To agrobacterium tumefaciens Gv3101, adopt agriculture bacillus mediated Arabidopis thaliana inflorescence infection protocol to obtain the transgenic arabidopsis plant expression vector Plasmid Transformation that builds.Empty carrier contrast: according to the method described above, with expression vector pYLRNAi same method arabidopsis thaliana transformation, obtain to turn the unloaded contrast of pYLRNAi strain (CK).

2, the detection of transgenic line

1) detection of the compound plant of genetically engineered soybean

Hairly root forms rear 21 days, adopts root sample extraction RNA, after reverse transcription becomes cDNA, further crosses the effect of expressing and interfering with quantitative PCR detection.

2) detection of transgenic arabidopsis plant

Collection T1 after the hygromycin resistance screening, obtains T1 for transfer-gen plant for the Arabidopis thaliana seed; Gather T2 and screen with hygromycin resistance for seed, obtain seedling rate and be 60% T2 for plant; Gather T3 for seed, with the hygromycin resistance screening, obtaining seedling rate is 100% transgenic lines.Extract the RNA of this transgenic lines plant, after reverse transcription becomes cDNA, further cross the effect of expression with quantitative PCR detection.

The soybean house-keeping gene TefS1With the tubulin of Arabidopis thaliana as reference gene, relative expression quantity is goal gene GmALMT1Expression amount and the ratio of house-keeping gene expression amount.The soybean house-keeping gene TefS1The detection primer be SEQ ID NO:19 and SEQ ID NO:20; GmALMT1The detection primer be SEQ ID NO:21 and SEQ ID NO:22.

1) primer of Arabidopis thaliana tubulin gene is:

tubulin F: 5’- TGTCGTCCAACCTTACAACTCACT -3’ (SEQ ID NO:23)

tubulin R: 5’- TCTCCAGGGTCCTCCATTCC -3’ (SEQ ID NO:24)

2) reaction system:

2×SYBR Green PCR master mix： 10 μL

Upstream primer. (10 mol/L): 0.6 μ L

Downstream primer. (10 mol/L): 0.6 μ L

Mili-Q water: mend to 20 μ L

The cDNA:2 μ L of dilution

3) reaction conditions:

95 ℃ of denaturation 1 min, then by 95 ℃ of sex change 15 s, 55-60 ℃ of annealing 15s carries out 35-40 circulation, and last 72 ℃ are extended 30 s.Quantitative PCR confirms to obtain effective different transgenic line.

3, the compound plant phenotype analytical of transgenosis

1) mensuration of hairly root root system oxysuccinic acid secretory volume

The compound strain of positive genetically engineered soybean and contrast strain are transferred to 4.3 mM CaCl ₂Cultivate in the solution (the pH value is 4.3) after 6 hours, collect root exudates.After 20 mL secretory product are processed with Zeo-karb, with freeze-drying simmer down to 1mL.Behind the 0.45mm membrane filtration, use the malic acid content in the HPLC Instrument measuring sample.

2) brazilwood extract dyeing detects the anti-aluminium of transgenosis hairly root

The compound strain of positive genetically engineered soybean and contrast strain after 6 hours aluminium is processed, are cut its tip of a root position, with phenodin dye liquor dyeing about half an hour.Take out the tip of a root, with the phenodin dyestuff of flushing with clean water surface attachment, under Stereo microscope, observe the dyeing situation.

Fig. 8 is GmALMT1The impact of gene pairs soybean compound plant oxysuccinic acid secretory volume and anti-aluminium thereof.A wherein: GmALMT1Gene is at the expression analysis of genetically engineered soybean hairly root; B: GmALMT1Excessive and interfere strain and contrast hairly root oxysuccinic acid secretory volume; C: GmALMT1Excessive and interfere strain and contrast hairly root surfaces of aluminum accumulation.CK transforms unloaded contrast strain; OX, GmALMT1The overexpression strain; RNAi, GmALMT1The interference strain.Asterisk represents the significance of same index between excessive and interference strain and contrast strain relatively: * represents conspicuous level P＜0.05 o'clock, significant difference; * represent conspicuous level 0. 001＜ P＜0.01 o'clock, the significance of difference between significantly with extremely remarkable between; * * represents conspicuous level P＜0.001 o'clock, difference was extremely remarkable; Ns represents that difference is not remarkable.

4, the anti-aluminium analysis of transgenic arabidopsis

1) root organic acid secretion

Transgenosis and wild-type Arabidopis thaliana are transferred to seedling of the same size in the liquid MS medium and to be continued to cultivate 10 days after the MS substratum is sprouted a week.Liquid MS cultivation is outwelled, and is 4.3 0.5 mM CaCl with the pH value ₂Solution cleans plant 3 times, changes nutrient solution into 0.5 mM CaCl ₂Solution (the pH value is 4.3) was cultivated after 24 hours, collected nutrient solution and measured malic acid content.

2) aluminium is on the impact of root growth

Transgenosis and wild-type Arabidopis thaliana are transferred to seedling of the same size and are contained 0 or 400 μ M AlCl after the MS substratum was sprouted 4 days ₃CaCl ₂Solid medium (CaCl ₂Concentration is 0.5 mM, and the pH value is 4.3) and to measure the main root root long.Aluminium was processed after 2 days, and it is long again to measure the main root root.Calculate the allometry of root.

Fig. 9 is GmALMT1The expression of gene is on the impact of the anti-aluminium of transgenic arabidopsis.A wherein: GmALMT1The expression analysis of gene in transgenic arabidopsis; B: GmALMT1The oxysuccinic acid secretory volume of different transgenic lines and wild-type Arabidopis thaliana root; C: GmALMT1Different transgenic lines and the phenotype analytical of wild-type Arabidopis thaliana under the aluminium treatment condition.D: GmALMT1The comparison of the relative root growth amount with the wild-type Arabidopis thaliana of different transgenic lines.CK transforms unloaded contrast strain; OX, GmALMT1The overexpression strain; Asterisk represents the significance of same index between overexpression strain and contrast strain relatively: * represents conspicuous level P＜0.05 o'clock, significant difference; * represent conspicuous level 0. 001＜ P＜0.01 o'clock, the significance of difference between significantly with extremely remarkable between; * * represents conspicuous level P＜0.001 o'clock, difference was extremely remarkable; Ns represents that difference is not remarkable.

5, field test and water culture experiment

Fig. 1 is the long and biomass of total root of soybean in the field test.A: the total root of field test soybean is long; B: soybean plant strain dry weight; 6 repetitions are established in each processing in the test, and pillar is respectively processed mean value and the standard error of 6 repeating datas among the figure for test.Asterisk represents that same index carries out significance of difference result relatively between same processing different genotype, and namely * represents conspicuous level P＜0.05 o'clock, significant difference; * represent conspicuous level 0.001＜ P＜0.01 o'clock, the significance of difference between significantly with extremely remarkable between; * * represents conspicuous level P＜0.001 o'clock, difference was extremely remarkable.

Fig. 2 is aluminium in the water culture experiment, pH value and phosphorus Usefulness Pair Soybean Root affects on the growth.A: add aluminium to the impact of the relative root growth of soybean under high phosphorus and the low-phosphorous condition shown in the figure; B: be not have under the condition of aluminium processing phosphorus and the impact of pH value on the relative root growth of soybean shown in the figure.Wherein pH is respectively 4.3 and pH 5.8; Phosphorus process be respectively 320 μ M (+P) and 0 μ M (P); Aluminium is processed and is respectively 38 μ M Al ³⁺(+Al) and 0 μ M Al ³⁺(-Al), the activity of aluminium is calculated by GEOCHEM-EZ.4 repetitions are established in each processing in the test, and pillar is respectively processed mean value and the standard error of 4 repeating datas among the figure for test.Asterisk represents that same index carries out significance of difference result relatively between same processing different genotype, and * represents conspicuous level P＜0.05 o'clock, significant difference; * represent conspicuous level 0. 001＜ P＜0. 01 o'clock, the significance of difference between significantly with extremely remarkable between; * * represents conspicuous level P＜0.001 o'clock, difference was extremely remarkable.

Fig. 3 is the impact of aluminium in the water culture experiment, pH value and the accumulation of phosphorus Usefulness Pair soybean root system oxysuccinic acid and secretion.A: under the high phosphorus condition, pH value and aluminium are on the impact of soybean oxysuccinic acid secretion; B: under the low-phosphorous condition, pH value and aluminium are on the C that affects of soybean oxysuccinic acid secretion: under the high phosphorus condition, pH value and aluminium affect D to what the soybean oxysuccinic acid accumulated: under the low-phosphorous condition, and the impact that pH value and aluminium accumulate the soybean oxysuccinic acid.4 repetitions are established in each processing in the test, and pillar is respectively processed mean value and the standard error of 4 repeating datas among the figure for test.Asterisk represents that same index carries out significance of difference result relatively between same processing different genotype, and * represents conspicuous level P＜0.05 o'clock, significant difference; * represent conspicuous level 0. 001＜ P＜0. 01 o'clock, the significance of difference between significantly with extremely remarkable between; * * represents conspicuous level P＜0.001 o'clock, difference was extremely remarkable.

SEQ ID NO:1

ATGGATATAGAGTCAACAACCCAAGCAAACAAGGGTGGATTTTTATCTCATTTGGGGAACTGCCTCCAGGATTTGCCTTGGAACTTCAAGTCCAAGGTTATCAACATCACAAGGAGCATAACAAAGATTGGAAAAGATGACCCCAGACGAGTAATTCACTCATTAAAAGTGGCAGTTGCTCTCACATCGGTGTCATTGGTTTACTACTCGAGGCCTCTTTATGATGGCTTCGGAGTTGCCGGAATGTGGGCTGTTCTGACAGTGGTGGTAGTGTTTGAATTCAGTGTAGGTGCAACCCTCAGCAAAGGTTTAAATAGAGGATTTGCTACATTATTAGCTGGTGCTTTAGGGGTTGGAGGGCAACACTTAGCTACAGCTTTTGGAGGAAGAGCAGAACCTATTGTCCTTGGGATCCTTGTCTTCATTTTAGCAGCAGGAGCTACTTTTTTCAGATTTTTTCCAAAGATCAAGCAAAGATATGATTATGGGATTGTGGTATTTATATTGACATTTTGTTTGGTCGCTGTCTCGGGTTATAGAGTGGAAGAACTCTTCGAGCTTGCCCATCAGAGACTTTCAACAATTTTATTAGGAGCAGCAGCTTGCATGGTCATCTCCATTTTCATTTGTCCAGTATGGGCAGGTGAAGACTTTCACAAGTTGGTGGCTTCCAATATAGAAAAGTTAGCAAATTACCTACAAGGATTCGAAACCGAATATTTTCATTGCTCAGAGGATACAAAAAAGTGTGAGAAGTCAGTTCTTGAAGGATATAAAAGTGTTCTTAATTCTAAAGCAAGTGAGGAGTCCTTGGCAAATTTGGCAAGGTGGGAACCAGGACATGGCCGTTTTCGTCTTCGCCATCCTTGGGAGCAGTACTTGAAGATTGGAGCACTTACTCGGGAATGTGCTTACAAGATTGAAACCATTAACAACTACCTCAACCCGGAAATCCAAGTATCTTTGGAATTCAAGTGTAAAGTTCAGGAACCATGCACAAAGATGACCTCAGAGTCCAACAAGGCATTAAAGGCAATATCTTCATCAATCAAAAAAATGACACACCCATCAACTGCCAAAGTCCACATAGAAAATTCAAAAACTGCAGTTGAAGACCTCAAAGTTGCCCTTGAAATCGTTTCTTTGGAAGATACTGATCTCCTATCCATAATCCCAGTTGCCACAGTTGCATCAATACTCGAAGAAATCACCAAATCAGTGGAGAAAATATATGAGTCTGTTTCTGAGCTTTCTCACTTAGCCCATTTCAAGAGTGTTGTTGAACCCAATGTCTCACCAGAGAAGCCACCCCTTCTTCATCGAGGTATCATAAAACCTGTTGTGGATATTGATAACACCGTCGACCATGTTGAAATTACAATCCCAGAGATAACTACAGACTCTCCAGAGAAAGAAAAGGCACCAACAACAAAACCCTCGGAACATTCAATTTGTAAATAG

SEQ ID NO:2

MDIESTTQANKGGFLSHLGNCLQDLPWNFKSKVINITRSITKIGKDDPRRVIHSLKVAVALTSVSLVYYSRPLYDGFGVAGMWAVLTVVVVFEFSVGATLSKGLNRGFATLLAGALGVGGQHLATAFGGRAEPIVLGILVFILAAGATFFRFFPKIKQRYDYGIVVFILTFCLVAVSGYRVEELFELAHQRLSTILLGAAACMVISIFICPVWAGEDFHKLVASNIEKLANYLQGFETEYFHCSEDTKKCEKSVLEGYKSVLNSKASEESLANLARWEPGHGRFRLRHPWEQYLKIGALTRECAYKIETINNYLNPEIQVSLEFKCKVQEPCTKMTSESNKALKAISSSIKKMTHPSTAKVHIENSKTAVEDLKVALEIVSLEDTDLLSIIPVATVASILEEITKSVEKIYESVSELSHLAHFKSVVEPNVSPEKPPLLHRGIIKPVVDIDNTVDHVEITIPEITTDSPEKEKAPTTKPSEHSICK

Claims (10)

1. oxysuccinic acid transporter gene GmALMT1, it is characterized in that its nucleotide sequence is shown in SEQ ID NO:1.

2. described oxysuccinic acid transporter gene of claim 1 GmALMT1The protein of coding is characterized in that its aminoacid sequence is shown in SEQ ID NO:2.

3. an expression vector is characterized in that containing oxysuccinic acid transporter gene claimed in claim 1 GmALMT1

4. a genetic engineering bacterium is characterized in that containing expression vector claimed in claim 3.

5. the described oxysuccinic acid transporter gene of claim 1 GmALMT1Application in the preparation transgenic plant.

The described oxysuccinic acid of claim 1 transhipment subbase because of GmALMT1Application in the preparation of preparation promotion plant adaptation acid soil.

7. the application of expression vector claimed in claim 3 in the preparation transgenic plant.

8. the application of expression vector claimed in claim 3 in the preparation of preparation promotion plant adaptation acid soil.

9. the application described in according to claim 5 is characterized in that described plant is dicotyledons.

10. application according to claim 9 is characterized in that described dicotyledons is soybean.

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