CN103525838A

CN103525838A - Malic enzyme gene SgME1 as well as application thereof

Info

Publication number: CN103525838A
Application number: CN201310290850.5A
Authority: CN
Inventors: 田江; 廖红; 孙丽莉
Original assignee: South China Agricultural University
Current assignee: South China Agricultural University
Priority date: 2013-07-11
Filing date: 2013-07-11
Publication date: 2014-01-22
Anticipated expiration: 2033-07-11
Also published as: CN103525838B

Abstract

The invention discloses a gene SgME1 which codes malic enzyme as well as an application of the gene SgME1. The nucleotide sequence of the malic enzyme gene SgME1 is shown as SEQIDNO:1, and the amino acid sequence of coded proteins is shown as SEQIDNO:2. The SgME1 gene has the functions of regulating and controlling synthesis of malic acid of stylosanthes guianensis, and over-expressing the SgME1 to accelerate synthesis of malic acid of yeasts, hairy roots of beans and Arabidopsis and increasing the aluminum tolerance of the SgME1.

Description

A kind of malic enzyme gene SgME1 and application thereof

Technical field

The present invention relates to plant biotechnology field, be specifically related to malic enzyme gene sgME1and application.

Background technology

Aluminium toxicity is one of the major obstacle factor of acid soil upper limit fabrication growth (Kochian et al., 2004).PH is less than at 5 o'clock, and the aluminium in soil just exists with activity form, can by root system of plant, be absorbed (Kinraide, 1991) rapidly, and the growth of the tip of a root is produced to toxic action, finally causes the reduction of crop yield.

Aluminum ion can interact with cell walls and synplasm, and then upsets the normal biochemical reactions of vegetable cell (Kochian et al., 2005).Research shows, aluminium is easily combined on electronegative cell walls pectin substance, thereby reduces the ductility of cell walls, the elongation of restrictive cell (Yang et al., 2011).Aluminium except with cell wall-bound, can also be accumulated in synplasm, upset formation (the Sivaguru et al. of cytoskeleton, 1999) and suppress division (the Roy et al. of cell, 1989) etc., and can make lipid peroxidation (Yamamoto et al., 2001), thereby affect root growth and development.

The mechanism that plant adapts to aluminium poison mainly comprises that inside restrains oneself and outside Rejection mechanism (Kochian et al., 2005; Ma et al., 2001).Inside is restrained oneself mechanism and is mainly referred to aluminium and organic acid or other compound chelating that enters into tenuigenin, forms stable complex compound, and is isolated in vacuole, plays the effect (Ma et al., 1998) of removing aluminium toxicity.Outside Rejection mechanism is mainly to secrete organic acid aluminium chelate ion by root system, forms stable organic acid-aluminium mixture, reduces the absorption to aluminium, thereby plays the effect of alleviating aluminium toxicity.A large amount of evidences show, the secretion of oxysuccinic acid, citric acid and oxalic acid etc. can effectively reduce the murder by poisoning ability of aluminium.At present, the translocator oxysuccinic acid of mediation oxysuccinic acid and citrate exudation is transported sub-ALMT1 and the successful separating clone of the sub-MATE of citrate transporter, and the expression of these genes is subject to the regulation and control of aluminium, overexpression aLMT1with mATEpromote respectively the secretion of oxysuccinic acid and citric acid, and can improve resistance to aluminium (Sasaki et al., 2004 of plant; Magalhaes et al., 2007).Meanwhile, under aluminium is coerced, by regulation and control encode tricarboxylic acid cycle key gene ( pEPC, cS, mDHwith iCDH) expression, be conducive to the synthetic of citric acid and oxysuccinic acid and secretion, by organic acid, secrete alleviation aluminium toxicity (Rangel et al., 2010; Wang et al., 2010).

Khuskhus is pioneer's leguminous crop of the adaptation Perenniporia martius growth of generally acknowledging, is the important high-quality legume forage of south China (Liu et al., 1997), belong to pulse family khuskhus belong to ( stylosanthesspp.), because of rich in proteins, carbohydrate and fiber, at subtropical and tropical zones establishing in large scale such as Australia, South America, South Africa, South East Asia, and there is higher yield and quality (Noble et al., 2000).It is reported, 60% acid soil is distributed in subtropical and tropical zones in the world, therefore, be adapted to Perenniporia martius growing plants and likely there is unique resistance to aluminium mechanism (Metali et al., 2012), the khuskhus of long-term planting on Perenniporia martius acid soil may have the ability of potential adaptation acid soil growth.

By early stage resistance to aluminium, screen, we find that phosphorus poor efficiency khuskhus genotype Fine-stem coerces sensitivity to aluminium, phosphorus efficiency khuskhus genotype TPRC2001-1 has the good ability of restraining oneself (Du et al. to aluminium poison, 2009), and the resistance to aluminium ability of TPRC2001-1 is suitable with resistance to aluminium crop paddy rice, yet rarely has the research to the resistance to aluminium mechanism of khuskhus.

For above-mentioned research background, applicant is by the method for Two-Dimensional Gel Electrophoresis, from the resistance to aluminium genotype of khuskhus TPRC2001-1, be separated to a Protein S gME1 who is subject to the coding malic enzyme of aluminium regulation and control, and utilize RACE technology, obtain the full length gene sequence of this albumen of coding.Utilize the expression systems such as yeast, Kidney bean hairly root and Arabidopis thaliana, prove sgME1the albumen of genes encoding has the function of synthesizing apple acid, finally improves the ability of the transgenic line poison of resistance to aluminium.

Summary of the invention

The object of the invention is for the deficiencies in the prior art, a kind of malic enzyme gene is provided sgME1, another object of the present invention is to provide the protein of said gene coding, and 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:

Malic enzyme gene provided by the present invention sgME1, can derive from khuskhus, it comprises or has and is selected from following nucleotide sequence:

(1) nucleotide sequence shown in 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, 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 preferred nucleotide sequence of 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 in sequence;

(5) the encode nucleotide sequence of one of following aminoacid sequence: the aminoacid sequence shown in SEQ ID NO:2, or, for example, due to one or more (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 SEQ ID NO:2, or, have at least 50% with the aminoacid sequence shown in 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,

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

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

SEQ ID NO:1 is by 1758 based compositions, and its open reading frame (ORF) is 1-1758 bit base, and coding has the aminoacid sequence of sequence SEQ ID NO:2, and the protein that described aminoacid sequence forms is called SgME1 albumen in the present invention.

Malic enzyme gene provided by the invention sgME1the protein of coding, it comprises or has and is selected from following aminoacid sequence:

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

(2) for example, substituting, lacking and/or inserting and the aminoacid sequence different from the aminoacid sequence shown in SEQ ID NO:2 due to one or more (1-25 is individual, 1-20, and 1-15 is individual, and 1-10,1-5,1-3 is individual) amino-acid residue;

(3) have at least 50%, at least 60%, at least 70%, at least 75% with the aminoacid sequence shown in SEQ ID NO:2, the preferred aminoacid sequence of 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 sgME1can regulate and control to comprise the synthetic of oxysuccinic acid in its transgenic organism with protein.

Increase above-mentioned sgME1the primer pair of full length gene or its arbitrary fragment belongs to protection scope of the present invention.

The present invention also provide contain above-mentioned sgME1the expression vector of gene, available existing plant expression vector construction contains sgME1the recombinant expression vector of gene.Described plant expression vector comprises double base agrobacterium vector etc., as pYLRNAi (by being so kind as to give in Liu Yaoguang researcher laboratory, specifically describe and to 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 sgME1gene or recombinant vectors.Described cell can be vegetable cell, for example leguminous plants cell, or microorganism cells, for example bacterium or fungal cell, for example yeast cell.Described cell can be a part separated, in vitro, 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, as paddy rice, wheat, barley, corn, Chinese sorghum, sugarcane, oat or rye etc., or other dicotyledonss be 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, 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 sgME1gene or the recombinant vectors purposes in regulating plant oxysuccinic acid synthetic, 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 sgME1the plant of gene or recombinant vectors, for example, described method can comprise from vegetable cell regeneration of 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 sgME1import in Kidney bean hair root and Arabidopis thaliana, obtain transgenic line; The resistance to aluminium abilities of described transgenic line etc. are higher than described object control plant.

Described gene sgME1can for example by described recombinant expression vector, import recipient plant.

Carry gene of the present invention sgME1plant expression vector can be transformed in Kidney bean hairly root and Arabidopis thaliana by for example agriculture bacillus mediated Regenerated from Hypocotyl Explants method.

Advantage of the present invention and effect:

1. gene sgME1under malic enzyme family at Arabidopis thaliana, though be cloned in wheat and paddy rice and reported, it is participating in the biological function of oxysuccinic acid aspect synthetic unclear.The present invention clone's gene sgME1khuskhus oxysuccinic acid is synthesized and has significant impact, and this adapts to the biological function important in inhibiting of acid soil to illustrating malic enzyme gene at leguminous crop.

2. gene sgME1not only affected the synthetic of oxysuccinic acid, this gene of overexpression has also increased yeast, Kidney bean hair root and the Arabidopis thaliana ability of restraining oneself 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: 50 μ M aluminium are processed the impact on khuskhus and rice root elongation; In test, 4 repetitions are established in each processing, and in figure, pillar and error line are respectively mean value and the standard error (lower same) of 4 repeating datas.Upper and lower case letter represents respectively the significance analysis of 24 and 72 hours root relative elongations, and different letter representations exist psignificant difference between=0.05 horizontal genotype.

Fig. 2: aluminium is processed the impact on khuskhus root aluminium content; * number represent that same processing carries out the result of significance of difference comparison between different genotype, * * represents conspicuous level 0.001 < P < 0.01 o'clock, and difference is extremely remarkable.

Fig. 3: aluminium is processed the impact on root malic acid concentration and oxysuccinic acid secretion; A, 100 μ M AlCl3process 24 hours back root part malic acid concentrations; B, aluminium is processed 72 hours back root part malic acid concentrations; C, aluminium is processed oxysuccinic acid secreting rate after 24 hours; D, aluminium is processed oxysuccinic acid secreting rate after 72 hours; In figure, pillar and error line are respectively mean value and the standard error that 4 repeating datas are respectively processed in test; Asterisk represents the result of same gene type significance of difference comparison between different treatment, * * represent conspicuous level 0.001<p<0.01 o'clock, difference was extremely remarkable.

Fig. 4: obtain malic enzyme SgME1 by bidirectional electrophoresis technique isolation identification; A, does not add aluminium and processes after 24 hours, and SgME1 protein site is at the albumen figure of TPRC2001-1 root; B, adds the albumen figure that aluminium is processed SgME1 after 24 hours; C, does not add the albumen figure that aluminium is processed SgME1 after 72 hours; D, adds the albumen figure that aluminium is processed SgME1 after 72 hours; Box indicating SgME1 protein site.

Fig. 5: SgME1 merges GFP onion epidermis Subcellular Localization; What front two rows showed respectively is the picture that pBEGFP empty carrier transforms onion epidermis cell plasmolysis front and back; Rear two rows show that respectively SgME1 merges the picture that GFP carrier transforms onion epidermis cell plasmolysis front and back; Fractographic content have respectively green fluorescence passage (GFP fluorescence), light microscopic passage (light field), red fluorescence passage (PI dyeing) and overlapping after picture (fusion).

Fig. 6:sgME1transformed yeast, Kidney bean hair root and the impact of Arabidopis thaliana on endogenous malic acid concentration; A, pYES2 empty carrier and pYES2-sgME1endogenous malic acid concentration after transformed yeast; B, pYL empty carrier and pYL-sgME1transform the endogenous malic acid concentration after Kidney bean hair root; C, pYL empty carrier and pYL-sgME1the endogenous malic acid concentration of arabidopsis thaliana transformation.CK, transforms unloaded contrast strain; OX,sgME1the overexpression strain of gene.Asterisk represents the result of same index significance of difference comparison between different transformation plants, and * represents conspicuous levelp<0.05 o'clock, significant difference, * * represent conspicuous level 0.001<p<0.01 o'clock, difference was extremely remarkable.

Fig. 7: it is right that aluminium is processedsgME1the impact of expression amount; A, aluminium process 24 hours rightsgME1impact at khuskhus root expression amount; B, aluminium process 72 hours rightsgME1impact at khuskhus root expression amount; * represent conspicuous levelp<0.05 o'clock, significant difference.

Fig. 8: overexpressionsgME1impact on yeast, Kidney bean hair root and the resistance to aluminium of transformation of Arabidopsis thaliana strain; A, transforms pYES2 empty carrier and pYES2-sgME1yeast at the phenotype analytical of aluminium treatment condition; B,sgME1different Kidney bean hair root transgenic lines and empty carrier are to impinging upon the phenotype analytical under aluminium treatment condition; C,sgME1different Kidney bean hair root transgenic lines and empty carrier contrast the comparison of relative root elongation.D,sgME1different Arabidopis thaliana transgenic lines and empty carrier are to impinging upon the phenotype analytical under aluminium treatment condition; E,sgME1different Arabidopis thaliana transgenic lines and empty carrier contrast the comparison of relative root elongation.CK, transforms unloaded contrast strain; OX,sgME1the overexpression strain of gene.Asterisk represents the result of same index significance of difference comparison between different transformation plants, and * represents conspicuous levelp<0.05 o'clock, significant difference.

Embodiment

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

embodiment 1

sgME1the clone of gene

1. the separation of SgME1 albumen and evaluation

With reference to (2011) method extraction column flowers and plants root protein matter such as Chen Zhijian, carry out two-dimensional electrophoresis.Each test is processed 3 repetitions is set, and screening does not add aluminium contrast and adds the protein site that between aluminium processing, significant difference is expressed, and carries out MALDI-TOF/TOF MS mass spectroscopy with ABI 4700 TOF-TOF (Applied Biosystems company, the U.S.).

Result as shown in Figure 4, from result: compared with the control, coerce semi-invariant significant variation not of the SgME1 albumen of 24 hours khuskhus roots at aluminium.But coerce after 72 hours at aluminium, the semi-invariant of SgME1 albumen significantly increases at root, the reinforcement that the expression amount of SgME1 albumen is coerced by long-term aluminium is described.

2. sgME1the clone of gene

According to Kidney bean (J03825.1), soybean (XM_003526456.1), the conserved domain design primer of grape (XM_003631725.1) and clover (XM_003630679.1) ME gene sgME1-EST-F (SEQ ID NO:3) and sgME1-EST-R (SEQ ID NO:4).20 μ L PCR reaction systems are, sterilizing ddH ₂o 13.68 μ L, 10 * PCR buffer, 2 μ L, 2.5 mM dNTP 1.6 μ L, each 0.8 μ L of the forward and reverse primer of 10 μ M, ExTaq enzyme (TAKARA company, Japan) 0.12 μ L, cDNA template 1 μ L.Response procedures is, 95 ℃ of sex change 1 min, and 94 ℃ of 30 s then, 58 ℃ of 30 s, 72 ℃ of 30 s, carries out 30 circulations, and last 72 ℃ are extended 10 min.After pcr amplification, obtain the segment of 443 bp, by being added with Golder view nucleic acid dye (Ding Guo company, China) 1% sepharose (Genetech company, the U.S.) after electrophoresis and gel imaging system imaging confirm that segment size is correct, with reference to Tiangen company (Germany) gel, reclaim test kit explanation object segment is cut to glue recovery, be connected into pGEM-T carrier (Promega company, the U.S.) order-checking.

According to SgME1 est sequence design 5 ' end RACE primer sgME1-RACE5 '-R (SEQ ID NO:5) and 3 ' terminal specific primer sgME1-RACE3 '-F (SEQ ID NO:6), the cDNA of the khuskhus TPRC2001-1 root system after the aluminium of take is processed is template, adopt the SMARTerTM RACE cDNA Amplification Kit test kit of Clotech company (U.S.) to carry out cDNA end rapid amplifying, obtain 5 ' end and 3 ' end cDNA sequence.Adopt MEGA 4.1 softwares, by two terminal sequences and est sequence splicing, obtain full length sequence.

SEQ ID NO:3：5’- GTTTGAAGACTTCGCGAATCATAATG -3’

SEQ ID NO:4：5’- CTTTGGTAAATGTCTTCCCTACTCC -3’

SEQ ID NO:5：5’ - CCACTTCTTTGGTAAATGTCTTCCCTACTCCAG -3’

SEQ ID NO:6：5’- CTTATACAGTTTGAAGACTTCGCGAATCATAATG -3’

3. the structure of carrier

(1) structure of Subcellular Localization expression vector

The pBEGFP that transient expression carrier adopts 35S to drive, uses sgME1-GFP-F (SEQ ID NO:7) and sgME1-GFP-R (SEQ ID NO:8) amplification SgME1 total length ORF, the 5 ' end at primer adds respectively kpni and bamHbefore two restriction enzyme sites of I and ATG, add 4 protection bases, guarantee that target protein and the GFP albumen of coding can correctly merge.PCR product is cut glue and is reclaimed and be successfully connected into after pGEM-T carrier, adopts plasmid to extract in a small amount test kit (Tiangen company, Germany) and extracts plasmid.With kpni and bamHi double digestion pBEGFP and being mounted with sgME1the T vector plasmid of gene, connects and transforms DH10B after recovery object fragment, and order-checking is confirmed sgME1the sequence of gene is correct.

(2) structure of Yeast expression carrier

With containing respectively bamHi and ecoRi restriction enzyme site sgME1-pYES2-F (SEQ ID NO:9) and sgME1-pYES2-R (SEQ ID NO:10), passes through pcr amplification sgME1oRF sequence, and be connected into pGEM-T carrier.With bamHi and ecoRi is to pYES2 (Invitrogen company, the U.S.) and be mounted with sgME1the T vector plasmid of gene carries out double digestion, after recovery object fragment, connects and transforms DH10B.After order-checking accurately, select positive colony and shake in a large number bacterium extracting plasmid, acquisition is mounted with sgME1the pYES2 carrier of gene.

(3) structure of SgME1 Overexpression vector

With containing respectively saci and mlui restriction enzyme site sgME1-pYL-F (SEQ ID NO:11) and sgME1-pYL-R (SEQ ID NO:12), passes through pcr amplification sgME1oRF sequence, and be connected into pGEM-T carrier.With saci and mlui is to pYL and being mounted with sgME1the T vector plasmid of gene carries out double digestion, after recovery object fragment, connects and transforms DH10B.Select positive colony and shake in a large number bacterium extracting plasmid, obtain the pYL carrier that is mounted with SgME1 gene.After order-checking confirmation is errorless, adopt freeze-thaw method to transform respectively Agrobacterium K599 and Gv3101, for the conversion of Kidney bean hair root and the whole strain of Arabidopis thaliana, empty carrier is converted into contrast.

SEQ ID NO:7：5’- GGTACCAGTCATGTCAAGCACATTGG -3’

SEQ ID NO:8：5’- GGATCCCAACGGTAGTTTCTGTAGCCT -3’

SEQ ID NO:9：5’- GGATCCAACACAATGTCCTCAAGCACATTGGTGA ATGGTGG -3’

SEQ ID NO:10：5’- CACTGCTACTATGAATTCTTTGGTTTAGTTCA -3’

SEQ ID NO:11：5’- TTGTTAGAGCTCATGTCAAGCACATTG -3’

SEQ ID NO:12：5’- ACGCGTTCAACGGTAGTTTCTGTAGC -3’

4. the expression pattern analysis of the Subcellular Localization of SgME1 and gene thereof

(1) Subcellular Localization of SgME1

By via Particle Bombardment Transformation method, will load sgME1eGFP carrier and EGFP empty carrier imports onion epidermis cell and soybean protoplast is carried out transient expression.Then use fluorescent microscope (LEICA DM5000B, Germany) to observe GFP and the PI fluorescent signal of onion epidermis cell.

Result as shown in Figure 5.From result, the fluorescence of SgME1-GFP is mainly distributed in tenuigenin, illustrates that SgME1 protein localization is in tenuigenin.

(2) sgME1the expression pattern analysis of gene

Method with reference to the SYBR Green of TAKARA company (Japan) quantification kit specification sheets is carried out quantitative PCR, with Rotor-Gene 3000 qRT-PCR systems (Corbett Research, Australia) operation.Using 100 times of cDNA diluted samples as quantitative PCR reaction template, choose 4 times of suitable cDNA stoste dilutions as 1 times of standard specimen production standard curve.20 μ L reaction systems are, 2 * SYBR Green PCR master mix, 10 μ L, Mili-Q water 6.4 μ L, each 0.8 μ L of the forward and reverse primer of 10 μ M, the cDNA template 2 μ L of dilution.Response procedures is, 95 ℃ of sex change 1 min, and 94 ℃ of 15 s then, 60 ℃ of 15 s, 72 ℃ of 30 s, carries out 40 circulations.With the expression amount of Real-Time Analysis Software 6.0 (Corbett Research, Australia) each sample of computational analysis, the ratio of the expression amount that relative expression quantity is goal gene and house-keeping gene expression amount, the primer of house-keeping gene is sgEF-1a-F (SEQ ID NO:13) with sgEF- 1a-R (SEQ ID NO:14) sgME1the quantitative primer of gene is sgME1-F (SEQ ID NO:15) with sgME1-R (SEQ ID NO:16).

SEQ ID NO:13：5’- CACTTCAGGACGTGTACAAGATC -3’

SEQ ID NO:14：5’- CTTGGAGAGCTTCATGGTGCA -3’

SEQ ID NO:15：5’- GTTTGAAGACTTCGCGAATCATAATG -3’

SEQ ID NO:16：5’- CTATCAACTCAGCAATACCAGTTCC -3’

As shown in Figure 7, with protein expression pattern similarity, aluminium is processed 24 h couple to result sgME1gene is little in the expression impact of TPRC2001-1 root, and aluminium is processed 72 h and significantly strengthened sgME1gene is in the expression of TPRC2001-1 root, but little to Fine-stem root SgME1 Gene Expression.

embodiment 2

The research of transgenic line

1. the acquisition of transgenic line

(1) acquisition of yeast transgenic line

With reference to SC easy comp transformation kit (Invitrogen company, the U.S.), by pYES2- sgME1with pYES2 empty carrier difference transformed yeast INVSC1.

(2) acquisition of transgenosis Kidney bean hair root

The expression vector building is converted in Agrobacterium rhyzogenesK599, adopts the in vitro hair root revulsion of agriculture bacillus mediated Kidney bean to obtain transgenosis hair root.

(3) acquisition of transgenic arabidopsis plant

The expression vector plasmid building is converted in agrobacterium tumefaciens Gv3101, adopts agriculture bacillus mediated Arabidopis thaliana inflorescence infection protocol to obtain transgenic arabidopsis plant.

2. the detection of transgenic line

(1) detection of yeast transgenic line

By lacking urinary ammonia acid screening culture medium screening positive transformant, and detect by PCR.

(2) detection of transgenosis Kidney bean hair root

The YITIAOGEN growing from explant is a system, and after subculture 3 times, hairly root extracts RNA, and reverse transcription becomes after cDNA, further by quantitative PCR detection, crosses the effect of expression.

(3) detection of transgenic arabidopsis plant

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

Kidney bean house-keeping gene pvEF-1atubulin(SEQ ID NO:19 and the SEQ ID NO:20 of (SEQ ID NO:17 and SEQ ID NO:18) and Arabidopis thaliana) conduct is respectively as the reference gene of Kidney bean hair root and Arabidopis thaliana detection by quantitative, and relative expression quantity is goal gene sgME1expression amount and the ratio of reference gene expression amount. sgME1detection primer be SEQ ID NO:15 and SEQ ID NO:16.

SEQ ID NO:17：5’- TGAACCACCCTGGTCAGATT -3’

SEQ ID NO:18：5’- TCCAGCATCACCATTCTTCA -3’

SEQ ID NO:19：5’- TGTCGTCCAACCTTACAACTCACT -3’

SEQ ID NO:20：5’- TCTCCAGGGTCCTCCATTCC -3’

3. the variation of transgenic line malic acid concentration

(1) mensuration of yeast cell malic acid content

The yeast that loads pYES2-SgME1 and pYES2 empty carrier is cultivated after 1 d respectively in 100 mL liquid inducing cultures, transferred in 50 mL centrifuge tubes, at 4 ℃, centrifugal 10 min of 6000 g collect bacterial sediment.With 1 mL Millipore water, clean 2 times, siphon away after water, with freeze concentration machine (Labconco company, the U.S.) freeze-drying sample.Then use liquid nitrogen grinding sample, broken somatic cells wall.Finally by 1.2 mL Millipore water dissolution and shift in sample to 1.5 mL centrifuge tube, at 4 ℃, centrifugal 10 min of 12000 g, carefully draw supernatant, cross 0.45 μ m millipore filtration, adopt high performance liquid chromatograph to measure malic acid content.

(2) mensuration of transgenosis Kidney bean hairly root malic acid content

Take 0.3 g hair root and be placed in 30 mL organic acids and collect liquid, after 60 rpm dark culturing 1 d, extract the endogenous organic acid of hair root sample, and adopt high performance liquid chromatograph to measure malic acid content at 28 ℃.

(3) mensuration of transgenic arabidopsis malic acid content

Transgenosis and wild-type Arabidopis thaliana are sprouted after 5 days at MS substratum, and seedling of the same size is transferred in liquid MS medium and continues to cultivate 10 days.Liquid MS is cultivated and outwelled, the 0.5 mM CaCl that is 4.2 by pH value ₂solution cleans plant 3 times, changes nutrient solution into 0.5 mM CaCl ₂solution (pH value is 4.2), cultivated after 24 hours, measured the content of the endogenous oxysuccinic acid of Arabidopis thaliana.

Result as shown in Figure 6.From result, overexpression in yeast, Kidney bean hair root and Arabidopis thaliana sgME1significantly improved the content of oxysuccinic acid in transgenic line, synthetic that SgME1 can regulating apple acid has been described.

4. the impact of aluminium on transgenic line growth

(1) aluminium is processed the impact on yeast growth

By the yeast that loads pYES2-SgME1 and pYES2 empty carrier overnight incubation in liquid screening substratum respectively, after reaching 0.6, OD600 is adjusted to 0.2, then be diluted to 0.02,0.002,0.0002, each OD value bacterium liquid is drawn respectively 10 μ L, and point is on inducing culture.Cultivate 4 d for 30 ℃, regularly take pictures every day, observes the situation of yeast growth.

(2) aluminium is processed the impact on transgenosis growth of hair root

Cut 1-2 cm hair root and be laid on aluminium processing substratum, aluminium is processed and is started to sweep root and calculate relative root long every day.

(3) aluminium is processed the impact on transgenosis growth of hair root

Transgenosis and wild-type Arabidopis thaliana are sprouted after 4 days at MS substratum, and seedling of the same size is transferred to containing 0 or 400 μ M AlCl ₃caCl ₂solid medium (CaCl ₂concentration is 4.3 mM, and pH value is 4.2), and it is long to measure root.Aluminium was processed after 2 days, again measured root long, calculated the relative elongation of root.

Result as shown in Figure 8, from result, in yeast, pYES2 empty carrier contrasts yeast strain relatively, being mounted with pYES2-SgME1 yeast strain processes under 2 d and 4 d and grows all than empty carrier control strain good (Fig. 8 A) at aluminium, showing to express SgME1 in yeast can increase the synthetic of yeast cell oxysuccinic acid, and has improved the restrain oneself ability of yeast to aluminium toxicity.In Kidney bean hair root, 3 transgenosis Kidney bean hair roots and empty carrier contrast strain are carried out to resistance to aluminium analysis (Fig. 8 B and C), result shows, aluminium is processed the growth that has significantly suppressed Kidney bean hair root, but to the growth-inhibiting degree of transgenic line root lower than contrast strain, the root relative elongation of transgenic line is to contrast strain more than 1.5 times, and overexpression is described sgME1gene can improve the resistance to aluminium of transgenosis Kidney bean hair root.In Arabidopis thaliana, aluminium is processed the growth that has significantly suppressed Arabidopis thaliana root, but to the growth-inhibiting degree of transgenic line root lower than contrast strain, the root relative elongation of transgenic line is 2 times (Fig. 8 D and E) of contrast strain, illustrates that overexpression SgME1 gene can improve the resistance to aluminium of transgenic arabidopsis

5. water culture experiment

1) the resistance to aluminium of khuskhus and paddy rice is analyzed

Select full rice paddy seed, the 5% clorox water after 10 min of sterilizing is rinsed well, after distilled water immersion 24 h, moves into CaSO ₄in, vernalization under dark condition in 28 ℃ of incubators, during keep seed moistening.Stylo seed is peelled off kind of a shell, after 80 ℃ of water-bath 3 min, is seeded in CaSO ₄in, vernalization in 28 ℃ of incubators.When khuskhus and rice root are grown to 1 cm, carry out 50 μ M l aluminium and process, it is long that scan root every day, and it is long to calculate relative root, and each processing arranges 4 biology and repeats.

2) cultivation of khuskhus seedling.Khuskhus is peelled off to kind of a shell, after 80 ℃ of water-bath 3 min, 75% alcohol disinfecting 30 s, then, with 10% clorox, 10 min that sterilize, aqua sterilisa cleans 3-5 time, is then seeded in 28 ℃ of dark culturing 1.5 d on MS substratum.The khuskhus of sprouting is moved in the centrifuge tube (cutting bottom) that is added with agaropectin, be placed in hydroponic box and in greenhouse, carry out 1/2 Hoagland nutrient fluid cultivation, culture temperature is 28 ℃/25 ℃ (daytime/night), humidity 75%.Rough leaf is transplanted to 14 L plastic tub after launching, and within every 7 days, changes 1 nutritive medium, and every 2 days with KOH or H ₂sO ₄regulate pH value to 5.8.

3) aluminium is processed and results.Khuskhus carries out aluminium processing after growing 1 month in normal nutritive medium, and control treatment is 0.5 mM CaCl ₂, aluminium is treated to 0.5 mM CaCl ₂add 100 μ M AlCl ₃.Each processing arranges 4 biology and repeats, after processing 24 h and 72 h, gather in the crops respectively sample, solution is collected and is used for measuring organic acid secretion, and plant sample is dried the mensuration for aluminium content, and sample segment is used for measuring the extraction of endogenous organic acid, root system total protein and RNA in-80 ℃ of preservations.

Result as shown in Figure 1.TPRC2001-1 is under short-term (24 h) aluminium (50 μ M Al) is coerced, and XN1 is the same with paddy rice, has the resistance to aluminium ability stronger than Fine-stem; And under short-term high alumina is processed, its resistance to aluminium ability and Fine-stem difference are not remarkable, but far below XN1.And under long-term aluminium is coerced, it is suitable that TPRC2001-1 has with XN1, the resistance to aluminium ability stronger than Fine-stem.

The khuskhus of growing 1 month is carried out to 100 mM aluminium and process, relatively the impact of different aluminum treatment time on different khuskhus genotype root aluminium content.Result as shown in Figure 2, aluminium is processed the aluminium content that 24 h and 72 h have all significantly increased khuskhus TPRC2001-1 and Fine-stem root, and the root aluminium content of TPRC2001-1 is significantly higher than Fine-stem, be respectively 2.5 times of Fine-stem and 2.3 times, illustrate that TPRC2001-1 root can accumulate aluminium, its resistance to aluminium mechanism may be mainly that mechanism is restrained oneself in inside.

This test has further been studied aluminium toxicity to the synthetic impact with secreting of khuskhus root oxysuccinic acid.As shown in Figure 3, it is not obvious to the concentration affects of khuskhus TPRC2001-1 and Fine-stem root system oxysuccinic acid that aluminium is processed 24 h to result.Aluminium is processed the concentration that 72 h have significantly promoted TPRC2001-1 root system oxysuccinic acid, and showing as TPRC2001-1 root system malic acid concentration is its 2.6 times of not adding aluminium control treatment.From Fig. 3, it can also be seen that, it is not remarkable on the secretion impact of two khuskhus genotype root system oxysuccinic acid that aluminium is processed 24 h.But aluminium is processed the secretion that 72 h significantly increase TPRC2001-1 root system oxysuccinic acid, for it does not add 5 times of aluminium control treatment, it is 3.5 times that aluminium is processed lower Fine-stem.

SEQ ID NO:1

ATGTCAAGCACATTGGTGAATGGTGGTGTTGAAGAAGCTTCTGCTACTGTTGATGGTGGTGTTAGAGACTTTTATGGTGAAGAAAGAGCCACGGAGGACCAGCTTATCACTCCTTGGAACTTCTCTGTCTCAAGTGGCAGCACTCTGCTGCGCGATCCGCGCTACAACAAAGGCCTCGCCTTCACCGAGAAAGAAAGAGATGCTCATTACCTGCGCGGCCTTTTGCCCCCAGCAGTATTCAATCAAGAACTTCAGGAGAAGAGGTTGATGTATAACCTTCGCCAGTATGAAGTTCCTCTGCATAGGTACATGGCCTTGATGGATCTTCAGGAGAGGAATGAGAGGCTGTTTTACAAGGTTCTGATTGATAACGTGGAGGAGCTGCTTCCTGTTGTTTACACTCCAACTGTTGGAGAAGCCTGCCAGAAATATGGAAGCATTTTCCGCCGCCCGCAGGGTCTATACATCAGTTTGAAAGAGAAAGGCAAGATCCTTGAGGTACTGAAAAACTGGCCAGAGAAGAACATTCAAGTTATTGTTGTCACTGATGGCGAGCGTATATTAGGACTTGGAGATCTTGGCTGCCAGGGAATGGGGATTCCTGTTGGGAAGCTTTCGCTCTATACTGCATTGGGAGGCGTTCGTCCTTCATCGTGCCTGCCTATAACCATTGATGTTGGCACAAACAATGAAAAGTTGCTGAATGATGAGTTTTACATCGGTCTTAGACAAAAGCGTGCAACTGGGAAGGAATATGCTGAGCTTCTAGATGAGTTCATGTGCGCCGTTAAGAAGAACTATGGGGAGAAAGTTCTTATACAGTTTGAAGACTTCGCGAATCATAATGCATTCGATCTGCTCGCAAAATACAGTTCATCTCATCTTGTTTTCAATGATGATATTCAGGGTACAGCATCTGTGGTATTAGCAGGATTGCTTGCTTCCTTAAAATTAGTTGGGGGAAACTTAGCTGACCATACCTTCTTATTTTTGGGTGCTGGAGAGGCTGGAACTGGTATTGCTGAGTTGATAGCACTTGAGATATCAAAACAGACAAAAGCTCCAGTGGAAGAAACCCGCAAAAAGATTTGGCTTGTTGACTCTAAGGGTCTTATTGTTAGCTCTCGCCTCGGATCACTTCAGCACTTCAAAAAGCCTTGGGCACATGAGCATGAACCTGTGAAGGAGCTTGTTGACGCTGTCAAGGCAATCAAGCCAACAGTGTTGATTGGATCATCTGGAGTAGGGAAGACATTTACCAAAGAAGTGGTTGAAACCATGGCATCATTGAATAAGAAACCACTCATTCTTGCTCTCTCGAATCCAACATCGCAATCCGAGTGCACTGCAGAAGAAGCTTATACATGGAGCAAGGGCCAAGCAATCTTTGCTAGCGGTAGCCCATTTGATCCTGTTGAATATGAAGGAAAAGTCTTTGTTCCTGGACAGGCTAACAACGCTTACATATTCCCTGGTTTCGGCTTGGGTTTGATCATGTCTGGAGCAATCCGCGTCCGCGATGAGATGCTCTTAGCAGCCTCTGAAGCTTTGGCTGCACAAGTGTCACAGGAGAACTATGATAAAGGACTGATTTACCCTCCATTCACAAATATCAGAAAGATATCAGCGCACATTGCTGCTAATGTGGCTGCCAAGGCATATGAACTTGGTCTTGCCTCTAATCTACCTCAACCTAAGGATCTTGTCAAATATGCAGAAAGTTGCATGTACAGCCCAGGCTACAGAAACTACCGTTGA

SEQ ID NO:2

MSSTLVNGGVEEASATVDGGVRDFYGEERATEDQLITPWNFSVSSGSTLLRDPRYNKGLAFTEKERDAHYLRGLLPPAVFNQELQEKRLMYNLRQYEVPLHRYMALMDLQERNERLFYKVLIDNVEELLPVVYTPTVGEACQKYGSIFRRPQGLYISLKEKGKILEVLKNWPEKNIQVIVVTDGERILGLGDLGCQGMGIPVGKLSLYTALGGVRPSSCLPITIDVGTNNEKLLNDEFYIGLRQKRATGKEYAELLDEFMCAVKKNYGEKVLIQFEDFANHNAFDLLAKYSSSHLVFNDDIQGTASVVLAGLLASLKLVGGNLADHTFLFLGAGEAGTGIAELIALEISKQTKAPVEETRKKIWLVDSKGLIVSSRLGSLQHFKKPWAHEHEPVKELVDAVKAIKPTVLIGSSGVGKTFTKEVVETMASLNKKPLILALSNPTSQSECTAEEAYTWSKGQAIFASGSPFDPVEYEGKVFVPGQANNAYIFPGFGLGLIMSGAIRVRDEMLLAASEALAAQVSQENYDKGLIYPPFTNIRKISAHIAANVAAKAYELGLASNLPQPKDLVKYAESCMYSPGYRNYR。

SEQUENCE LISTING

<110> Agricultural University Of South China

<120>a kind of malic enzyme gene SgME1 and application thereof

<130>

<160> 20

<170> PatentIn version 3.2

<210> 1

<211> 1758

<212> DNA

<213>sequence

<400> 1

atgtcaagca cattggtgaa tggtggtgtt gaagaagctt ctgctactgt tgatggtggt 60

gttagagact tttatggtga agaaagagcc acggaggacc agcttatcac tccttggaac 120

ttctctgtct caagtggcag cactctgctg cgcgatccgc gctacaacaa aggcctcgcc 180

ttcaccgaga aagaaagaga tgctcattac ctgcgcggcc ttttgccccc agcagtattc 240

aatcaagaac ttcaggagaa gaggttgatg tataaccttc gccagtatga agttcctctg 300

cataggtaca tggccttgat ggatcttcag gagaggaatg agaggctgtt ttacaaggtt 360

ctgattgata acgtggagga gctgcttcct gttgtttaca ctccaactgt tggagaagcc 420

tgccagaaat atggaagcat tttccgccgc ccgcagggtc tatacatcag tttgaaagag 480

aaaggcaaga tccttgaggt actgaaaaac tggccagaga agaacattca agttattgtt 540

gtcactgatg gcgagcgtat attaggactt ggagatcttg gctgccaggg aatggggatt 600

cctgttggga agctttcgct ctatactgca ttgggaggcg ttcgtccttc atcgtgcctg 660

cctataacca ttgatgttgg cacaaacaat gaaaagttgc tgaatgatga gttttacatc 720

ggtcttagac aaaagcgtgc aactgggaag gaatatgctg agcttctaga tgagttcatg 780

tgcgccgtta agaagaacta tggggagaaa gttcttatac agtttgaaga cttcgcgaat 840

cataatgcat tcgatctgct cgcaaaatac agttcatctc atcttgtttt caatgatgat 900

attcagggta cagcatctgt ggtattagca ggattgcttg cttccttaaa attagttggg 960

ggaaacttag ctgaccatac cttcttattt ttgggtgctg gagaggctgg aactggtatt 1020

gctgagttga tagcacttga gatatcaaaa cagacaaaag ctccagtgga agaaacccgc 1080

aaaaagattt ggcttgttga ctctaagggt cttattgtta gctctcgcct cggatcactt 1140

cagcacttca aaaagccttg ggcacatgag catgaacctg tgaaggagct tgttgacgct 1200

gtcaaggcaa tcaagccaac agtgttgatt ggatcatctg gagtagggaa gacatttacc 1260

aaagaagtgg ttgaaaccat ggcatcattg aataagaaac cactcattct tgctctctcg 1320

aatccaacat cgcaatccga gtgcactgca gaagaagctt atacatggag caagggccaa 1380

gcaatctttg ctagcggtag cccatttgat cctgttgaat atgaaggaaa agtctttgtt 1440

cctggacagg ctaacaacgc ttacatattc cctggtttcg gcttgggttt gatcatgtct 1500

ggagcaatcc gcgtccgcga tgagatgctc ttagcagcct ctgaagcttt ggctgcacaa 1560

gtgtcacagg agaactatga taaaggactg atttaccctc cattcacaaa tatcagaaag 1620

atatcagcgc acattgctgc taatgtggct gccaaggcat atgaacttgg tcttgcctct 1680

aatctacctc aacctaagga tcttgtcaaa tatgcagaaa gttgcatgta cagcccaggc 1740

tacagaaact accgttga 1758

<210> 2

<211> 585

<212> PRT

<213>sequence

<400> 2

Met Ser Ser Thr Leu Val Asn Gly Gly Val Glu Glu Ala Ser Ala Thr

1 5 10 15

Val Asp Gly Gly Val Arg Asp Phe Tyr Gly Glu Glu Arg Ala Thr Glu

20 25 30

Asp Gln Leu Ile Thr Pro Trp Asn Phe Ser Val Ser Ser Gly Ser Thr

35 40 45

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

50 55 60

Glu Arg Asp Ala His Tyr Leu Arg Gly Leu Leu Pro Pro Ala Val Phe

65 70 75 80

Asn Gln Glu Leu Gln Glu Lys Arg Leu Met Tyr Asn Leu Arg Gln Tyr

85 90 95

Glu Val Pro Leu His Arg Tyr Met Ala Leu Met Asp Leu Gln Glu Arg

100 105 110

Asn Glu Arg Leu Phe Tyr Lys Val Leu Ile Asp Asn Val Glu Glu Leu

115 120 125

Leu Pro Val Val Tyr Thr Pro Thr Val Gly Glu Ala Cys Gln Lys Tyr

130 135 140

Gly Ser Ile Phe Arg Arg Pro Gln Gly Leu Tyr Ile Ser Leu Lys Glu

145 150 155 160

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

165 170 175

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

180 185 190

Leu Gly Cys Gln Gly Met Gly Ile Pro Val Gly Lys Leu Ser Leu Tyr

195 200 205

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

210 215 220

Asp Val Gly Thr Asn Asn Glu Lys Leu Leu Asn Asp Glu Phe Tyr Ile

225 230 235 240

Gly Leu Arg Gln Lys Arg Ala Thr Gly Lys Glu Tyr Ala Glu Leu Leu

245 250 255

Asp Glu Phe Met Cys Ala Val Lys Lys Asn Tyr Gly Glu Lys Val Leu

260 265 270

Ile Gln Phe Glu Asp Phe Ala Asn His Asn Ala Phe Asp Leu Leu Ala

275 280 285

Lys Tyr Ser Ser Ser His Leu Val Phe Asn Asp Asp Ile Gln Gly Thr

290 295 300

Ala Ser Val Val Leu Ala Gly Leu Leu Ala Ser Leu Lys Leu Val Gly

305 310 315 320

Gly Asn Leu Ala Asp His Thr Phe Leu Phe Leu Gly Ala Gly Glu Ala

325 330 335

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

340 345 350

Lys Ala Pro Val Glu Glu Thr Arg Lys Lys Ile Trp Leu Val Asp Ser

355 360 365

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

370 375 380

Lys Pro Trp Ala His Glu His Glu Pro Val Lys Glu Leu Val Asp Ala

385 390 395 400

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

405 410 415

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

420 425 430

Lys Pro Leu Ile Leu Ala Leu Ser Asn Pro Thr Ser Gln Ser Glu Cys

435 440 445

Thr Ala Glu Glu Ala Tyr Thr Trp Ser Lys Gly Gln Ala Ile Phe Ala

450 455 460

Ser Gly Ser Pro Phe Asp Pro Val Glu Tyr Glu Gly Lys Val Phe Val

465 470 475 480

Pro Gly Gln Ala Asn Asn Ala Tyr Ile Phe Pro Gly Phe Gly Leu Gly

485 490 495

Leu Ile Met Ser Gly Ala Ile Arg Val Arg Asp Glu Met Leu Leu Ala

500 505 510

Ala Ser Glu Ala Leu Ala Ala Gln Val Ser Gln Glu Asn Tyr Asp Lys

515 520 525

Gly Leu Ile Tyr Pro Pro Phe Thr Asn Ile Arg Lys Ile Ser Ala His

530 535 540

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

545 550 555 560

Asn Leu Pro Gln Pro Lys Asp Leu Val Lys Tyr Ala Glu Ser Cys Met

565 570 575

Tyr Ser Pro Gly Tyr Arg Asn Tyr Arg

580 585

<210> 3

<211> 26

<212> DNA

<213>sequence

<400> 3

gtttgaagac ttcgcgaatc ataatg 26

<210> 4

<211> 25

<212> DNA

<213>sequence

<400> 4

ctttggtaaa tgtcttccct actcc 25

<210> 5

<211> 33

<212> DNA

<213>sequence

<400> 5

ccacttcttt ggtaaatgtc ttccctactc cag 33

<210> 6

<211> 34

<212> DNA

<213>sequence

<400> 6

cttatacagt ttgaagactt cgcgaatcat aatg 34

<210> 7

<211> 26

<212> DNA

<213>sequence

<400> 7

ggtaccagtc atgtcaagca cattgg 26

<210> 8

<211> 27

<212> DNA

<213>sequence

<400> 8

ggatcccaac ggtagtttct gtagcct 27

<210> 9

<211> 41

<212> DNA

<213>sequence

<400> 9

ggatccaaca caatgtcctc aagcacattg gtgaatggtg g 41

<210> 10

<211> 32

<212> DNA

<213>sequence

<400> 10

cactgctact atgaattctt tggtttagtt ca 32

<210> 11

<211> 27

<212> DNA

<213>sequence

<400> 11

ttgttagagc tcatgtcaag cacattg 27

<210> 12

<211> 26

<212> DNA

<213>sequence

<400> 12

acgcgttcaa cggtagtttc tgtagc 26

<210> 13

<211> 23

<212> DNA

<213>sequence

<400> 13

cacttcagga cgtgtacaag atc 23

<210> 14

<211> 21

<212> DNA

<213>sequence

<400> 14

cttggagagc ttcatggtgc a 21

<210> 15

<211> 26

<212> DNA

<213>sequence

<400> 15

gtttgaagac ttcgcgaatc ataatg 26

<210> 16

<211> 25

<212> DNA

<213>sequence

<400> 16

ctatcaactc agcaatacca gttcc 25

<210> 17

<211> 20

<212> DNA

<213>sequence

<400> 17

tgaaccaccc tggtcagatt 20

<210> 18

<211> 20

<212> DNA

<213>sequence

<400> 18

tccagcatca ccattcttca 20

<210> 19

<211> 24

<212> DNA

<213>sequence

<400> 19

tgtcgtccaa ccttacaact cact 24

<210> 20

<211> 20

<212> DNA

<213>sequence

<400> 20

tctccagggt cctccattcc 20

Claims

1. the gene of the malic enzyme of encoding sgME1, it is characterized in that its nucleotide sequence is as shown in SEQ ID NO:1.

2. a malic enzyme claimed in claim 1, is characterized in that its aminoacid sequence is as shown in SEQ ID NO:2.

3. an expression vector, is characterized in that containing the gene of malic enzyme of encoding described in claim 1 sgME1.

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

5. malic enzyme gene described in claim 1 sgME1application in preparation transgenic plant.

6. malic enzyme gene described in claim 1 sgME1in preparation, promote plant adapt to the preparation of acid soil or improve the application in Aluminum Tolerance in Plants ability.

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

8. expression vector claimed in claim 3 promotes plant adapt to the preparation of acid soil or improve the application in Aluminum Tolerance in Plants ability in preparation.

9. according to the application described in any one in claim 5-8, it is characterized in that described plant is dicotyledons or monocotyledons.

10. application according to claim 9, is characterized in that described dicotyledons is leguminous plants, tobacco, Sunflower Receptacle, beet, capsicum, potato or tomato; Described monocotyledons is paddy rice, wheat, barley, corn, Chinese sorghum, sugarcane, oat or rye.