CN102925453B - 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 application.

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 have the potential arable land of more than 50% to be acid soil, be mainly 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 collaborative suppression to plant-growth and output of the shortage of available phosphorus and aluminium toxicity.

In the evolutionary process of nature and artificial selection for a long time, plant defines the coadaptation mechanism of a series of form, physiology and molecule, overcomes the obstruction factors such as low ph value, available phosphorus shortage and the aluminium toxicity existed in acid soil.It is reported, plant adapts to the mechanism of aluminium toxicity, mainly comprises inside and restrains oneself and outer row's mechanism (such as, the secretion of oxysuccinic acid); Adapt to the mechanism of low-phosphorus stress, mainly comprise (Raghothama, 1999 such as induction of the change of root morphology and architecture, organic acid secretion, the induction of acid phosphatase and secretion, high affinity P i transportors; Vance et al., 2003).Due to the shortage of available phosphorus and aluminium toxicity simultaneously and be present in acid soil, implied that plant may exist common adaptation mechanism, overcome shortage and the aluminium toxicity of available phosphorus.It is worth mentioning that, find in the research of the plants such as rape, Kidney bean, soybean, Lupinus albus, low-phosphorus stress significantly strengthens the secretion of root system oxysuccinic acid, and promotes that it is to the activation of external source insoluble phosphorus and utilization (Hoffland et al., 1989; Hinsinger, 2001; Shen et al., 2002; Liao et al., 2006; Sas et al., 20010); Under Acid-Al stress, the secretory volume of the oxysuccinic acid of Arabidopis thaliana, wheat, triticale and soybean significantly increases, thus alleviates aluminium to suppression (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 illustrate that plant adapts to the shortage of available phosphorus and the node of aluminium toxicity in acid soil.The transhipment subbase being cloned into first coding secretion root system oxysuccinic acid for 2004 since Sasaki etc. from wheat because of ALMT1 since, Arabidopis thaliana, grape, rape and barley have been cloned into similar oxysuccinic acid transhipment.Further research shows, the expression of these organic acids transhipment controls process (Raman et al., 2005 of plant secretion oxysuccinic acid; Hoekenga et al., 2006; Ligaba et al., 2006; Furukawa et al., 2007).

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

For above-mentioned research background, applicant by the method for homologous clone, cloned one by external source pH, phosphorus and aluminium coordinated regulation, the oxysuccinic acid of tip of a root specifically expressing transhipment, gmAlMT1.By expression systems such as whole strain, hair root and frog's eggs, prove that the albumen of GmAlMT1 genes encoding has the function controlling oxysuccinic acid secretion, final raising transfer-gen plant improves the ability of the poison of resistance to aluminium.

Summary of the invention

The object of the invention is for the deficiencies in the prior art, provide a kind of oxysuccinic acid transport subbase because of gmALMT1.

Another object of the present invention is to provide the protein of said gene coding.

A further object of the present invention is to provide 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 SEQ ID NO:1;

(2) nucleotide sequence of hybridizing under low stringent condition such as grade, medium stringency condition, preferably high stringency condition with the complementary sequence of the nucleotide sequence of (1);

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

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

(5) to encode the nucleotide sequence of one of following aminoacid sequence: the aminoacid sequence shown in SEQ ID NO:2, or, due to one or more (such as 1-25, 1-20, 1-15, 1-10, 1-5, 1-3) the substituting of amino-acid residue, lack and/or insert and the aminoacid sequence different from the aminoacid sequence shown in SEQ ID NO:2, or, at least 50% is had with the aminoacid sequence shown in SEQ ID NO:2, at least 60%, at least 70%, at least 75%, preferred 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,

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

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

SEQ ID NO:1 is by 1461 based compositions, its open reading frame (ORF) is 1-1461 bit base, coding has the aminoacid sequence of sequence SEQ ID NO:2, and the protein of described aminoacid sequence composition 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 SEQ ID NO:2;

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

(3) have at least 50% with the aminoacid sequence shown in SEQ ID NO:2, at least 60%, at least 70%, the aminoacid sequence of at least 75%, preferably 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.

Increase above-mentioned gmALMTT1the primer pair of full length gene or its arbitrary fragment belongs to protection scope of the present invention.

The present invention also provides containing 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., as pYLRNAi (be so kind as to give by Liu Yaoguang researcher laboratory, specifically describe and see document: Hu Xuxia and Liu Yaoguang, 2006, Molecular Plant Breeding) or other derivative plant expression vector.

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, such as leguminous plants cell, or microorganism cells, such as bacterium or fungal cell, such as yeast cell.Described cell can be separated, in vitro, cultivate or the part of 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, such as soybean, also can be other plant, such as monocotyledons be as paddy rice, wheat, barley, corn, Chinese sorghum, sugarcane, oat or rye etc., or other dicotyledonss are 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 Plant cell regeneration transgenic plant of the present invention, or by 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 preparation transgenic plant and preparation promotes that plant adapts to the preparation of acid soil.

The invention still further relates to the method that regulating plant adapts to acid soil, the method comprises preparation containing of the present invention gmALMT1the plant of gene or recombinant vectors, such as, described method can comprise from Plant cell regeneration transgenic plant of the present invention or by plant of the present invention and another plant hybridization.

A preferred embodiment provided by the present invention is by said gene gmALMT1import in soybean, obtain transgenosis composite plant; The resistance to aluminium abilities of root of described transgenosis composite plant etc. are higher than described object control plant.

Described gene gmALMT1such as can import recipient plant by described recombinant expression vector.

Carry gene of the present invention gmALMT1plant expression vector by such as agriculture bacillus mediated Regenerated from Hypocotyl Explants method be transformed into soya cells or tissue in.

Advantage of the present invention and effect:

1. gene gmALMT1belonging to oxysuccinic acid translocator family at Arabidopis thaliana, though be cloned and reported in wheat and rape, its biological function in the secretion of leguminous crop oxysuccinic acid is also unclear.The gene of the present invention clone gmALMT1have significant impact to the secretion of soybean oxysuccinic acid, this is to illustrating the biological function important in inhibiting of oxysuccinic acid transporter gene at leguminous crop adaptation acid soil.

2. gene gmALMT1not only have impact on the transhipment of tip of a root oxysuccinic acid, this gene of overexpression also add soybean and Arabidopis thaliana and restrains oneself ability to aluminium poison; The functional study of this gene has far-reaching Research Significance for the molecule mechanism of resolving leguminous crop adaptation acid soil.

Accompanying drawing explanation

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

Fig. 2: in water culture experiment, low ph value, aluminium and phosphorus nutrient are on the impact of the relative root growth of Soybean Root.

Fig. 3: in water culture experiment, low ph value, aluminium and phosphorus nutrient are on the impact of soybean root system malic acid content and oxysuccinic acid secretory volume.

Fig. 4: gmALMT1subcellular Localization.

Fig. 5: aluminium and pH value pair gmALMT1in the regulation and control of root system expression specificity.

Fig. 6: aluminium, pH value and phosphorus nutrient pair gmALMT1the impact of genetic expression.

The electrophysiologic activity of Fig. 7: GmALMT1 translocator.

Fig. 8: interfere and process LAN gmALMT1on the secretion of genetically engineered soybean hairly root oxysuccinic acid and the impact of Aluminum toxicity.

Fig. 9: process LAN gmALMT1the secretion of gene pairs transgenic arabidopsis oxysuccinic acid and the impact of Aluminum toxicity.

Embodiment

In following embodiment, if no special instructions, ordinary method is.

embodiment 1

gmALMT1the clone of gene

1, gene ( gmALMT1) clone

Wheat TaALMT1-1(AB081803 according to having reported) and TaALMT1-2(AB081804), Arabidopis thaliana AtALMT1(At1g08430) and rape BnAlMT1(AB194300) BnAlMT2(AB194301) aminoacid sequence of gene carries out tetraploid rice, at design upstream, high conservative region degenerated primer 5 '-TGGGCIRTIHTIACIGTIGT-3 ' (SEQ ID NO:3) and downstream degenerated primer 5 '-CCIGCCCAIAYIGGRMA-3 ' (SEQ ID NO:4), and to extract phosphorus efficiency genotype HN98 soybean tip of a root total serum IgE according to TRIzol single stage method, then the cDNA that reverse transcription obtains carries out pcr amplification as template, PCR reaction system is 50 μ L, comprise 10 μMs of each 1 μ L of forward and reverse primer, 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, then sterilizing ddH is used ₂o supplies 50 μ L.PCR response procedures is: 94 DEG C 1 minute; 94 DEG C 30 seconds, 52 DEG C 30 seconds, 72 DEG C 2 minutes, 30 circulations of increasing; 72 DEG C extend 10 minutes.The PCR primer of amplification passes through 1% agarose gel electrophoresis, after Golden view nucleic acid staining dye, in gel imaging system imaging.It is the PCR primer of 405bp that DNA gel reclaims test kit recovery length.Recovery obtains PCR primer and is cloned into and pMD18-T (TAKARA company, specific descriptions are shown in: http://www.takara.com.cn/) carrier carries out order-checking qualification.According to the result of order-checking, use Primer 5.0 primer-design software, 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 instructions.Get 5 μ L PCR primer and carry out electrophoresis detection, the two PCR reactions taken turns obtain special product, and concrete operations are carried out with reference to CLONTECH ' s AdvantageT M 2 PCR Kit operation instructions.Then reclaim test kit with DNA gel to reclaim with reference to reclaiming the product of 3 '/5 '-RACE PCR.Recovery obtains PCR primer and is cloned into and pMD18-T (TAKARA company, specific descriptions are shown in: http://www.takara.com.cn/) carrier carries out order-checking qualification.To obtain above gmALMT13 ' of gene and 5 ' sequence of holding and obtaining above gmALMT1partial cDNA Sequence splicing obtain soybean gmALMT1full length cDNA sequence.

2, the structure of carrier

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

With downstream special primer

5 '-CAGCACGCGTCTATTTACAAATTGAATGTTCCGAGGG-3 ' (SEQ ID NO:10) increases gmALMT1oRF 1461 bp fragment, after PCR fragment recovery order-checking is errorless, passes through bgliI He mluafter I pair of fragment and object carrier carry out double digestion, will gmALMT1gene is connected to object carrier pYLRNAi.

The structure of interference vector: with soybean tip of a root cDNA for template, use upstream specific primer

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

The structure of Subcellular Localization expression vector: conventionally, extracts soybean root RNA, and its reverse transcription is become cDNA, with this cDNA for template, use upstream specific primer

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

With downstream special primer

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

Amplification gmALMT1open reading frame fragment, after PCR fragment recovery order-checking is errorless, will gmALMT1gene is connected to object carrier EGFP.

The structure of electro physiology expression vector: conventionally, extracts soybean root RNA, and its reverse transcription is become cDNA, with this cDNA for template, use upstream specific primer

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

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

Amplification gmALMT1open reading frame fragment, after PCR fragment recovery order-checking is errorless, passes through bgliI and speafter I carries out double digestion to recovery fragment and object carrier, will gmALMT1gene is connected to object carrier T7TS.

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

1) Subcellular Localization of GmALMT1

By Gene Knock-out Mice, will be loaded with gmALMT1eGFP carrier and EGFP empty carrier import onion epidermis cell and soybean protoplast and carry out transient expression.Then Laser Scanning Confocal Microscope (TCS SP2 is used; Leica) and fluorescent microscope (LEICA DM5000B, Germany) observe GFP and the PI fluorescent signal of onion epidermis cell and soybean protoplast respectively.The results are shown in Figure the Subcellular Localization that 4 are GmALMT1.Wherein A-C is onion epidermis contrast: the zero load that 35S promoter drives, and A is GFP fluorescent signal; B is 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 -gFP fluorescent signal; E is PI signal; F is the fusion results of D and E.G is 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 -gFP fluorescent signal; I is the fusion results of H and light field.Except A-F scale is 100 μm in figure; G-I scale is 20 μm.

2) gmALMT1the expression pattern analysis of gene

gmALMT1the tissue specificity analysis of genetic expression:

The consistent seedling of growth, after normal nutrition liquid germinates 3 days, is transferred to containing 50 μMs of AlCl by soybean seeds ₃0.5 mM CaCl ₂in solution (pH value is 4.3), process 6 hours, extracts 0-2 cm, the RNA of 2-4 cm, 4-7 cm root segment respectively.

Aluminum concentration pair gmALMT1the impact of genetic expression:

The consistent seedling of growth, after normal nutrition liquid germinates 3 days, is transferred to containing 0,50,100 μM of AlCl by soybean seeds ₃0.5 mM CaCl ₂in solution (pH value is 4.3), process 6 hours, extracts the RNA of 0-2 cm root segment.

Low ph value and aluminium pair gmALMT1the impact of genetic expression:

Homogeneous seedling, after normal nutrition liquid germinates 3 days, is transferred to 0.5 mM CaCl by soybean seeds ₂in solution (pH value is 4.3), process 0,6,12 hours, then transfers to containing 50 μMs of AlCl ₃0.5 mM CaCl ₂process 2,4,6,8,10,12 and 24 hours in solution (pH value is 4.3).Each time point gathers the root segment of 0-2 cm respectively, extracting RNA.

Aluminium, pH value and phosphorus pair gmALMT1the impact of genetic expression:

Homogeneous seedling is transferred to different process, i.e.+Al/+P/pH4.3 after germinateing 3 days in normal nutrition liquid and scarce phosphorus nutrition liquid respectively by soybean seeds, + 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 is respectively 320 μMs (+P) and 0 μM (-P); Aluminium process is respectively 38 μMs of Al ³⁺(+Al) and 0 μM of Al ³⁺(-Al).The activity of aluminium is calculated with GEOCHEM-EZ.Process the root segment gathering 0-2 cm after 6 hours, extracting RNA.

Above-mentioned RNA reverse transcription is become cDNA, uses quantitative PCR detection further gmALMT1expression pattern.The house-keeping gene of soybean tefS1as internal reference.Primer 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.Wherein A: 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.In figure, data are 4 mean values and standard error repeated.

Fig. 6 is pH value, aluminium and P availability pair gmALMT1the impact of expression pattern.Wherein A is under the condition of high phosphorus, pH value pair gmALMT1the impact that gene is expressed at the tip of a root; B is under low-phosphorous condition, pH value pair 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.In figure, data are 4 mean values and standard error repeated.Asterisk represents that the significance of same index between two genotype compares: * represents conspicuous level pduring <0.05, significant difference; * represents conspicuous level 0. 001 < pduring <0.01, the significance of difference is between significantly and extremely significantly; * * represents conspicuous level pduring <0.001, difference is extremely remarkable; Ns represents that difference is not remarkable.

4, the electrophysiologic study of GmALMT1 function

The GmALMT1-TST7 carrier reverse transcription built is become cRNA.Select good frog's egg cell, injection about 30 ng GmALMT1-TST7, controlled trial injects 30 nL water.Inject to frog's egg the sodium malate that 50 nL concentration are 0 to 100 mM simultaneously, make endogenous oxysuccinic acid activity be respectively 1.3,2.4 and 4.5 ({ mal ^2-i).PH of cushioning fluid is adjusted to 7.5 or 4.5, detects with Two-electrode voltage-clamp.PCLAMP 10.0 (Axon company of the U.S.) and Digidatas 1320A software is utilized to carry out image procossing and data analysis.Fig. 7 is the electrophysiologic study of GmALMT1 function.A, the active impact on GmALMT1 channel current of oxysuccinic acid; B, pH value is on the impact of GmALMT1 channel current; C, when pH value is 7.5, the active impact on the I-V curve of GmALMT1 of oxysuccinic acid; D, when pH value is 4.5, the active impact on the I-V curve of GmALMT1 of oxysuccinic acid.

the research of embodiment 2 transgenosis compound plant

1, the acquisition of transgenic line

1) acquisition of genetically engineered soybean compound plant

The expression vector built and interference vector are converted in Agrobacterium rhyzogenesK599, adopt agriculture bacillus mediated soybean hypocotyl injection to obtain transgenosis compound plant (root be transgenic hairy root, overground part be non-transgenic), follow-up phenotypic evaluation all uses this strain.Empty vector control: according to the method described above, by expression vector pYLRNAi same method soybean transformation, obtains and turns pYLRNAi zero load contrast strain (CK).

2) acquisition of transgenic Arabidopsis plants

By the expression vector Plastid transformation that builds in agrobacterium tumefaciens Gv3101, agriculture bacillus mediated Arabidopis thaliana inflorescence infection protocol is adopted to obtain transgenic Arabidopsis plants.Empty vector control: according to the method described above, by expression vector pYLRNAi same method arabidopsis thaliana transformation, obtains and turns pYLRNAi zero load contrast strain (CK).

2, the detection of transgenic line

1) detection of genetically engineered soybean compound plant

Hairly root forms latter 21 days, adopts root samples and extracts RNA, after reverse transcription becomes cDNA, use the effect of quantitative PCR detection process LAN and interference further.

2) detection of transgenic Arabidopsis plants

Gather T1 for Arabidopis thaliana seed after hygromycin resistance screening, T1 is for transfer-gen plant in acquisition; Gather T2 to screen for seed hygromycin resistance, obtaining seedling rate is that the T2 of 60% is for plant; Gather T3 for seed, with hygromycin resistance screening, obtaining seedling rate is 100% transgenic lines.Extract the RNA of this transgenic lines plant, after reverse transcription becomes cDNA, use the effect of quantitative PCR detection process LAN further.

Soybean house-keeping gene tefS1with the tubulin of Arabidopis thaliana as reference gene, gene for the purpose of relative expression quantity gmALMT1expression amount and the ratio of house-keeping gene expression amount.Soybean house-keeping gene tefS1detection primer be SEQ ID NO:19 and SEQ ID NO:20; gmALMT1detection 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 DEG C of denaturation 1 min, then by 95 DEG C of sex change 15 s, 55-60 DEG C of annealing 15s, carries out 35-40 circulation, and last 72 DEG C extend 30 s.Quantitative PCR confirms to obtain effective different transgenic line.

3, transgenosis compound plant phenotype is analyzed

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

Positive transgenic soybean compound strain and contrast strain are transferred to 4.3 mM CaCl ₂cultivate in solution (pH value is 4.3) after 6 hours, collect root exudates.By 20 mL secretory product with after Zeo-karb process, with freeze-drying simmer down to 1mL.With the malic acid content in HPLC Instrument measuring sample after 0.45mm membrane filtration.

2) brazilwood extract dyeing detects the Aluminum toxicity of transgenic hairy root

By positive transgenic soybean compound strain and contrast strain after aluminium process in 6 hours, cut its tip of a root position, dye about half an hour with phenodin dye liquor.Take out the tip of a root, rinse the phenodin dyestuff of surface attachment with clear water, under Stereo microscope, observe staining conditions.

Fig. 8 is gmALMT1the impact of gene pairs soybean compound plant oxysuccinic acid secretory volume and Aluminum toxicity thereof.Wherein A: gmALMT1gene is at the expression analysis of genetically engineered soybean hairly root; B: gmALMT1the excessive oxysuccinic acid secretory volume with interfering strain and contrast hairly root; C: gmALMT1the excessive accumulation with interfering strain and contrast hairly root surfaces of aluminum.CK, transforms unloaded contrast strain; OX, gmALMT1overexpression strain; RNAi, gmALMT1interference strain.Asterisk represents that same index compares with the significance contrasted between strain in excessive and interference strain: * represents conspicuous level pduring <0.05, significant difference; * represents conspicuous level 0. 001 < pduring <0.01, the significance of difference is between significantly and extremely significantly; * * represents conspicuous level pduring <0.001, difference is extremely remarkable; Ns represents that difference is not remarkable.

4, transgenic arabidopsis Aluminum toxicity is analyzed

1) root organic acid secretion

Seedling of the same size, after MS substratum sprouts a week, is transferred in liquid MS medium and continues cultivation 10 days by transgenosis and wildtype Arabidopsis thaliana.Being cultivated by liquid MS and outwell, is the 0.5 mM CaCl of 4.3 by pH value ₂solution cleaning plant 3 times, changes 0.5 mM CaCl into by nutrient solution ₂solution (pH value is 4.3), cultivated after 24 hours, collected nutrient solution and measured malic acid content.

2) aluminium is on the impact of root growth

Seedling of the same size, after MS substratum sprouts 4 days, is transferred to containing 0 or 400 μM of AlCl by transgenosis and wildtype Arabidopsis thaliana ₃caCl ₂solid medium (CaCl ₂concentration is 0.5 mM, and pH value is 4.3) and to measure main root root long.Aluminium process, after 2 days, measures main root root long again.Calculate the allometry of root.

Fig. 9 is gmALMT1the expression of gene is on the impact of transgenic arabidopsis Aluminum toxicity.Wherein A: gmALMT1the expression analysis of gene in transgenic arabidopsis; B: gmALMT1the oxysuccinic acid secretory volume of different transgenic line and wildtype Arabidopsis thaliana root; C: gmALMT1different transgenic line and the phenotype analytical of wildtype Arabidopsis thaliana under aluminium treatment condition.D: gmALMT1the comparison of different transgenic lines root growth amount relative to wildtype Arabidopsis thaliana.CK, transforms unloaded contrast strain; OX, gmALMT1overexpression strain; Asterisk represents that same index compares with the significance contrasted between strain in overexpression strain: * represents conspicuous level pduring <0.05, significant difference; * represents conspicuous level 0. 001 < pduring <0.01, the significance of difference is between significantly and extremely significantly; * * represents conspicuous level pduring <0.001, difference is extremely remarkable; Ns represents that difference is not remarkable.

5, field test and water culture experiment

Fig. 1 is total root length and the biomass of soybean in field test.A: the total root of field test soybean is long; B: soybean plant strain dry weight; In test, 6 repetitions are established in each process, and in figure, pillar is mean value and the standard error of each process of test 6 repeating datas.Asterisk represents the result that same index is carried out the significance of difference and compared between same process different genotype, and namely * represents conspicuous level pduring <0.05, significant difference; * represents conspicuous level 0.001< pduring <0.01, the significance of difference is between significantly and extremely significantly; * * represents conspicuous level pduring <0.001, difference is extremely remarkable.

Fig. 2 is the impact that in water culture experiment, aluminium, pH value and phosphorus nutrient grow Soybean Root.Shown in A: figure for high phosphorus with add the impact of aluminium on the relative root growth of soybean under low-phosphorous condition; Shown in B: figure for there is no an aluminium process condition under, phosphorus and the impact of pH value on the relative root growth of soybean.Wherein pH is respectively 4.3 and pH 5.8; Phosphorus process is respectively 320 μMs (+P) and 0 μM (-P); Aluminium process is respectively 38 μMs of Al ³⁺(+Al) and 0 μM of Al ³⁺(-Al), the activity of aluminium is calculated by GEOCHEM-EZ.In test, 4 repetitions are established in each process, and in figure, pillar is mean value and the standard error of each process of test 4 repeating datas.Asterisk represents the result that same index is carried out the significance of difference and compared between same process different genotype, and * represents conspicuous level pduring <0.05, significant difference; * represents conspicuous level 0. 001 < p<0. 01 time, the significance of difference is between significantly and extremely significantly; * * represents conspicuous level pduring <0.001, difference is extremely remarkable.

Fig. 3 is the impact that in water culture experiment, aluminium, pH value and phosphorus nutrient accumulate soybean root system oxysuccinic acid and secrete.A: under high phosphorus condition, the impact that pH value and aluminium are secreted soybean oxysuccinic acid; B: under low-phosphorous condition, what pH value and aluminium were secreted soybean oxysuccinic acid affects C: under high phosphorus condition, and what pH value and aluminium accumulated soybean oxysuccinic acid affects D: under low-phosphorous condition, the impact that pH value and aluminium accumulate soybean oxysuccinic acid.In test, 4 repetitions are established in each process, and in figure, pillar is mean value and the standard error of each process of test 4 repeating datas.Asterisk represents the result that same index is carried out the significance of difference and compared between same process different genotype, and * represents conspicuous level pduring <0.05, significant difference; * represents conspicuous level 0. 001 < p<0. 01 time, the significance of difference is between significantly and extremely significantly; * * represents conspicuous level pduring <0.001, difference is extremely remarkable.

SEQ ID NO:1

ATGGATATAGAGTCAACAACCCAAGCAAACAAGGGTGGATTTTTATCTCATTTGGGGAACTGCCTCCAGGATTTGCCTTGGAACTTCAAGTCCAAGGTTATCAACATCACAAGGAGCATAACAAAGATTGGAAAAGATGACCCCAGACGAGTAATTCACTCATTAAAAGTGGCAGTTGCTCTCACATCGGTGTCATTGGTTTACTACTCGAGGCCTCTTTATGATGGCTTCGGAGTTGCCGGAATGTGGGCTGTTCTGACAGTGGTGGTAGTGTTTGAATTCAGTGTAGGTGCAACCCTCAGCAAAGGTTTAAATAGAGGATTTGCTACATTATTAGCTGGTGCTTTAGGGGTTGGAGGGCAACACTTAGCTACAGCTTTTGGAGGAAGAGCAGAACCTATTGTCCTTGGGATCCTTGTCTTCATTTTAGCAGCAGGAGCTACTTTTTTCAGATTTTTTCCAAAGATCAAGCAAAGATATGATTATGGGATTGTGGTATTTATATTGACATTTTGTTTGGTCGCTGTCTCGGGTTATAGAGTGGAAGAACTCTTCGAGCTTGCCCATCAGAGACTTTCAACAATTTTATTAGGAGCAGCAGCTTGCATGGTCATCTCCATTTTCATTTGTCCAGTATGGGCAGGTGAAGACTTTCACAAGTTGGTGGCTTCCAATATAGAAAAGTTAGCAAATTACCTACAAGGATTCGAAACCGAATATTTTCATTGCTCAGAGGATACAAAAAAGTGTGAGAAGTCAGTTCTTGAAGGATATAAAAGTGTTCTTAATTCTAAAGCAAGTGAGGAGTCCTTGGCAAATTTGGCAAGGTGGGAACCAGGACATGGCCGTTTTCGTCTTCGCCATCCTTGGGAGCAGTACTTGAAGATTGGAGCACTTACTCGGGAATGTGCTTACAAGATTGAAACCATTAACAACTACCTCAACCCGGAAATCCAAGTATCTTTGGAATTCAAGTGTAAAGTTCAGGAACCATGCACAAAGATGACCTCAGAGTCCAACAAGGCATTAAAGGCAATATCTTCATCAATCAAAAAAATGACACACCCATCAACTGCCAAAGTCCACATAGAAAATTCAAAAACTGCAGTTGAAGACCTCAAAGTTGCCCTTGAAATCGTTTCTTTGGAAGATACTGATCTCCTATCCATAATCCCAGTTGCCACAGTTGCATCAATACTCGAAGAAATCACCAAATCAGTGGAGAAAATATATGAGTCTGTTTCTGAGCTTTCTCACTTAGCCCATTTCAAGAGTGTTGTTGAACCCAATGTCTCACCAGAGAAGCCACCCCTTCTTCATCGAGGTATCATAAAACCTGTTGTGGATATTGATAACACCGTCGACCATGTTGAAATTACAATCCCAGAGATAACTACAGACTCTCCAGAGAAAGAAAAGGCACCAACAACAAAACCCTCGGAACATTCAATTTGTAAATAG

SEQ ID NO:2

MDIESTTQANKGGFLSHLGNCLQDLPWNFKSKVINITRSITKIGKDDPRRVIHSLKVAVALTSVSLVYYSRPLYDGFGVAGMWAVLTVVVVFEFSVGATLSKGLNRGFATLLAGALGVGGQHLATAFGGRAEPIVLGILVFILAAGATFFRFFPKIKQRYDYGIVVFILTFCLVAVSGYRVEELFELAHQRLSTILLGAAACMVISIFICPVWAGEDFHKLVASNIEKLANYLQGFETEYFHCSEDTKKCEKSVLEGYKSVLNSKASEESLANLARWEPGHGRFRLRHPWEQYLKIGALTRECAYKIETINNYLNPEIQVSLEFKCKVQEPCTKMTSESNKALKAISSSIKKMTHPSTAKVHIENSKTAVEDLKVALEIVSLEDTDLLSIIPVATVASILEEITKSVEKIYESVSELSHLAHFKSVVEPNVSPEKPPLLHRGIIKPVVDIDNTVDHVEITIPEITTDSPEKEKAPTTKPSEHSICK

Claims (2)

1. oxysuccinic acid transhipment subbase because of GmALMT1 prepare regulating and controlling soybean or Arabidopis thaliana adapt to low-phosphorous in acid soil, aluminium is malicious and application in the preparation of low ph value, described oxysuccinic acid transhipment subbase is because the nucleotide sequence of GmALMT1 is as shown in SEQ ID NO:1.

2. the oxysuccinic acid transhipment subbase protein of encode because of GmALMT1 prepare regulating and controlling soybean or Arabidopis thaliana adapt to low-phosphorous in acid soil, aluminium is malicious and application in the preparation of low ph value, described oxysuccinic acid transports subbase because the nucleotide sequence of GmALMT1 is as shown in SEQ ID NO:1.