CN103525825B - The clone of the resistance to manganese poisoning important gene ShMDH1 of one kind of plant and application thereof - Google Patents

Abstract

The invention discloses clone and application thereof that one promotes the key gene <i>ShMDH1</iGrea tT.GreaT.GT of the resistance to manganese poisoning of plant.Is the nucleotide sequence of this <i>ShMDH1</iGrea tT.GreaT.GT as SEQ? ID? is the aminoacid sequence of coded protein as SEQ shown in NO:1? ID? shown in NO:2.The synthesis of <i>ShMDH1</iGrea tT.GreaT.GT gene regulating khuskhus oxysuccinic acid and secretion, have vital role to the resistance to manganese ability strengthening plant.

Description

The clone of the resistance to manganese poisoning important gene ShMDH1 of one kind of plant and application thereof

Technical field

The present invention relates to plant biotechnology field, be specifically related to the gene of resistance to manganese poisoning shMDH1clone and application.

Background technology

In the world more than 50% potential can anthropogenic soil be acid soil, be mainly distributed in the torrid zone, subtropics and Temperate Region in China (Kochianetal., 2004).In acid soil, except scarce phosphorus and aluminium poison, manganese poisoning is also significant obstacle factor (deCarvalhoetal., 1980 of restriction plant growth and output; Horstetal., 1988; Raghothama, 1999; Vanceetal., 2003).Manganese is one of the required trace element of plant-growth (MukhopadhyayandSharma, 1991).But Excessive Manganese also can cause toxic action to plant.The symptom of manganese poisoning generally shows as blade and occurs brown manganese oxidation spot, the reduction of chlorophyll content, the photosynthetic symptom such as to weaken, final growth and output (Horstetal., 1999 suppressing plant; Millaleoetal., 2010).

Research in the past shows that plant is in long-term evolution and artificially breeding process, by increasing synthesis and the secretion of root system oxysuccinic acid, the aluminium in chelating rhizosphere; nickel; the metallic element such as copper and zinc, thus alleviate metal ion to toxic action (Yangetal., 1997 of plant-growth; Parkeretal., 2001; Sasakietal., 2004; Delhaizeetal., 2012).Early stage by clover and tobacco overexpression malate dehydrogenase (malic acid dehydrogenase) (MDH) gene, synthesis and the secretion of transfer-gen plant root system oxysuccinic acid can be improved, effectively enhance transfer-gen plant resistance to aluminium ability (Tesfayeetal, 2001; Wangetal., 2010), but rarely have report about the mechanism of oxysuccinic acid secretion and resistance to manganese poisoning thereof.

Khuskhus originates from subtropical and tropical zones, has good nutritive value and feeding value, is the leguminous forage (Liuetal., 1997) of high-quality.Khuskhus has good adaptability to the acid soil containing high manganese concentration, and it by regulation and control plant gene expression in vivo, thus can strengthen the resistance to manganese ability (deCarvalhoetal., 1980) of self.But the key gene controlling the resistance to manganese poisoning of khuskhus is not yet reported.

Based on above-mentioned research background, applicant, by the method for two-dimensional electrophoresis, identifies the albumen of a manganese poisoning up-regulated expression, ShMDH1, and cloned the gene of this albumen of coding, the expression of this gene, by the regulation and control of external source manganese poisoning, has a higher similarity with other malate dehydrogenase (malic acid dehydrogenase) of plant.This gene of overexpression in Arabidopis thaliana and yeast, result shows shMDH1the albumen of genes encoding has regulating apple acid synthesis and the function of secretion, finally improves the resistance to manganese poisoning ability of transgenic line.

Summary of the invention

The object of the invention is for the deficiencies in the prior art, a kind of malate dehydrogenase gene is provided shMDH1, 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:

Malate dehydrogenase gene provided by the present invention shMDH1, can derive from khuskhus, it comprises or has and is selected from following nucleotide sequence:

(1) nucleotide sequence shown in SEQIDNO: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 SEQIDNO: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 SEQIDNO:2, or, at least 50% is had with the aminoacid sequence shown in SEQIDNO: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).

SEQIDNO:1 is by 1038 based compositions, and its open reading frame (ORF) is 1-1038 bit base, and coding has the aminoacid sequence of sequence SEQIDNO:2, and the protein of described aminoacid sequence composition is called ShMDH1 albumen in the present invention.

Malate dehydrogenase gene provided by the invention shMDH1the protein of coding, it comprises or has and is selected from following aminoacid sequence:

(1) aminoacid sequence shown in SEQIDNO:2;

(2) substituting, lack and/or inserting and the aminoacid sequence different from the aminoacid sequence shown in SEQIDNO:2 due to one or more (such as 1-25 is individual, 1-20,1-15 are individual, 1-10,1-5, and 1-3 is individual) amino-acid residue;

(3) have at least 50% with the aminoacid sequence shown in SEQIDNO: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 shMDH1can regulate and control to comprise the synthesis of oxysuccinic acid in its transgenic organism with protein.

Increase above-mentioned shMDH1the 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 shMDH1the expression vector of gene, available existing plant expression vector construction contains shMDH1the 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 shMDH1gene 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 khuskhus and soybean, also can be other plant, such as monocotyledons is 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 shMDH1gene or the purposes of recombinant vectors in the synthesis of regulating plant oxysuccinic acid, comprise 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 shMDH1the 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 shMDH1import in Arabidopis thaliana and yeast cell, obtain transgenic line; The resistance to manganese abilities of described transgenic line etc. are higher than described object adjoining tree and bacterial strain.

Described gene shMDH1can be such as import acceptor Arabidopis thaliana and yeast cell by described recombinant expression vector.

Carry gene of the present invention shMDH1plant expression vector be transformed in Arabidopis thaliana by such as Agrobacterium-medialed transformation method, Yeast expression carrier can be transformed in yeast cell.

Advantage of the present invention and effect:

1. gene shMDH1though belonging to malate dehydrogenase enzyme family be cloned in Arabidopis thaliana, clover, corn and paddy rice etc. and reported, its biological function in the synthesis of leguminous forage oxysuccinic acid is also unclear.The gene of the present invention clone shMDH1have significant impact to the synthesis of khuskhus oxysuccinic acid, this is to illustrating oxysuccinic acid synthesis and secreting the biological function important in inhibiting adapting to acid soil manganese poisoning leguminous forage.

2. gene shMDH1not only have impact on the synthesis of oxysuccinic acid, this gene of overexpression also add Arabidopis thaliana and yeast cell and restrains oneself ability to manganese 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 impact that in water culture experiment, the process of different manganese concentration grows khuskhus; Be that the process of different manganese concentration is on the impact of khuskhus chlorophyll content shown in A: figure; Be that the process of different manganese concentration is on the impact of khuskhus biomass shown in B: figure; The impact being the process of different manganese concentration shown in C: figure on khuskhus leaf portion and root manganese concentration; Wherein manganese concentration for the treatment of is respectively 5 μMs and 400 μMs; In test, each process establishes 4 biology to repeat, and in figure, pillar is mean value and the standard error of 4 biology repeating datas; * same index significant difference between same gene type different treatment is represented, p<0.05; * represents that same index difference between same gene type different treatment is extremely remarkable, 0.001< p<0.01.

Fig. 2: the impact that in water culture experiment, the process of different manganese concentration accumulates khuskhus root system oxysuccinic acid and secretes; A: the process of different manganese concentration is on the impact of the endogenous malic acid concentration of khuskhus root system; B: the impact that the process of different manganese concentration is secreted khuskhus root system oxysuccinic acid; Wherein manganese concentration for the treatment of is respectively 5 μMs and 400 μMs; In test, each process establishes 4 biology to repeat, and in figure, pillar is mean value and the standard error of 4 biology repeating datas; * same index significant difference between same gene type different treatment is represented, p<0.05; * represents that same index difference between same gene type different treatment is extremely remarkable, 0.001< p<0.01.

Fig. 3: shMDH1subcellular Localization; Fig. 3 A-C is the cell after the empty vector control onion epidermis plasmolysis of 35S promoter driving: A is GFP fluorescent signal; B is PI signal; C is the fusion results of A and B; D-F is ShMDH1 -the cellular localization of GFP after onion epidermis plasmolysis, D is ShMDH1 -gFP fluorescent signal; E is PI signal; F is the fusion results of D and E.

Fig. 4: manganese process pair shMDH1the regulation and control that gene is expressed at khuskhus root system; In figure, data are mean value and the standard error of 4 biology repetitions; * number significant difference of the same index of expression between different treatment, p<0.05.

Fig. 5: overexpression shMDH1on the synthesis of transgenic arabidopsis oxysuccinic acid and the impact of secretion and resistance to manganese; Wherein A: shMDH1the expression analysis of gene in transgenic arabidopsis; B: shMDH1different overexpression transgenic line and wildtype Arabidopsis thaliana oxysuccinic acid resultant quantity; C: shMDH1oxysuccinic acid secretory volume in different overexpression transgenic line and wildtype Arabidopsis thaliana; D: shMDH1different overexpression transgenic line and the phenotype analytical of wildtype Arabidopsis thaliana under manganese treatment condition, in figure, scale is 1cm; E: shMDH1the comparison of different overexpression transgenic line and wildtype Arabidopsis thaliana biomass.F: shMDH1the comparison of different overexpression transgenic line and wildtype Arabidopsis thaliana manganese concentration; WT, the contrast strain of wildtype Arabidopsis thaliana; OX, shMDH1overexpression strain; In test, each process establishes 4 biology to repeat, and in figure, pillar is mean value and the standard error of 4 biology repeating datas; * represent that same index is at overexpression strain and the significant difference contrasted between strain, p<0.05; * represents that same index is extremely remarkable with the difference contrasted between strain in overexpression strain, 0.001< p<0.01.

Fig. 6: overexpression shMDH1on the synthesis of yeast cell oxysuccinic acid and the impact of resistance to manganese; Wherein A: overexpression shMDH1malic acid concentration in transgenic strain and empty vector control yeast cell; B: overexpression shMDH1transgenic line and the phenotype analytical of empty vector control yeast cell under manganese treatment condition; In test, 4 repetitions are established in each process, and in figure, pillar is mean value and the standard error of 4 repeating datas; * represent that same index is at overexpression strain and the significant difference contrasted between strain, p<0.05.

Embodiment

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

embodiment 1

shMDH1the clone of gene

1, shMDH1gene clone

Identifying the albumen by manganese poisoning up-regulated expression according to albumen two-dimensional electrophoresis is malate dehydrogenase (malic acid dehydrogenase).Then, according to the soybean reported (XM_003527163), clover (AF020271), the nucleotide sequence of Root or stem of Littleleaf Indianmulberry (BT146072) and castor-oil plant (XM_002524216) gene carries out tetraploid rice, in design upstream, high conservative region homologous primer 5 '-ATTATTGCCGCTGAGGTTTTCAAGA-3 ' (SEQIDNO:3) and downstream homologous primer 5 '-GATTCCCTTCAAGCAAGCATCG-3 ' (SEQIDNO:4), use TRIzol method extraction column flowers and plants resistance to manganese genotype Fine-stem root system total serum IgE, then reverse transcription carries out pcr amplification as template after becoming cDNA, PCR reaction system is 20 μ L, comprise 10 μMs of each 0.5 μ L of upstream and downstream primer, 10 × PCRbuffer2 μ L, 2.5mMdNTP1.6 μ L, Ex-Taq (TAKARA company) enzyme 0.12 μ L, cDNA template amount 1 μ L, then sterilizing ddH is used ₂o supplies 20 μ L.PCR response procedures is: 94 DEG C 4 minutes; 94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 30 seconds, 35 circulations of increasing; 72 DEG C extend 10 minutes.The PCR primer of amplification is separated by 1% agarose gel electrophoresis, and imaging in gel imaging system.Using DNA gel to reclaim test kit recovery length is the PCR primer of 354bp.Reclaim product cloning to pMD18-T (TAKARA company, specific descriptions are shown in: http://www.takara.com.cn/) carrier and qualification of checking order.According to the result of order-checking, use Primer5.0 primer-design software, the following amplification of design shMDH1the primer of gene: 5 ' end EST special primer 5 '-GAAAACCTCAGCAGCAATGGGAACAGT-3 ' (SEQIDNO:5), 3 ' end EST special primer 5 '-CTGCTGAGGTTTTCAAGAAGGCAGG-3 ' (SEQIDNO:6).

With the RNA of khuskhus manganese poisoning process root for template, adopt SMARTerTMRACEcDNAAmplificationKit test kit structure 5 ' and the 3 ' RACEcDNA of Clontech company (U.S.), carry out cDNA end rapid amplifying.Get 5 '-CDSPrimerA5 ' the TTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (N=A in 2 μ g root total serum IgE and test kit respectively, C, G, orT, V=A, G, orC) (SEQIDNO:7) and 3 '-CDSPrimerA5 '-AAGCAGTGGTATCAACGCAGAGTAC (T) 30VN-3 ' (SEQIDNO:8) combine, and SMARTerIIAOligonucleotide is added in 5 ' RACE, carry out reverse transcription with SMARTScribeReverseTranscriptase respectively subsequently, be built into 5 ' and 3 ' RACEcDNA.Above-mentioned shMDH1gene 5 ' EST special primer and 3 ' EST special primer match with UPM the primer 5 '-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 ' (SEQIDNO:21) in test kit respectively, with 5 ' and 3 ' RACEcDNA for template, increase respectively shMDH15 ' end and 3 ' terminal sequence of gene, then reclaims with DNA gel the product that test kit reclaims 5 '/3 ' end-PCR respectively.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 shMDH15 ' of gene and 3 ' sequence of holding and obtaining above shMDH1partial cDNA Sequence MEGA4.1 software splicing obtain khuskhus shMDH1full length cDNA sequence.

2, the structure of carrier

The structure of Arabidopis thaliana Overexpression vector: with khuskhus root cDNA for template, with upstream specific primer 5 '-GGATCCAATGATTAAGCCTTCGATGC-3 ' (SEQIDNO:9) and downstream special primer 5 '-ACGCGTTTACTGGTTGGCGAATT-3 ' (SEQIDNO:10) amplification shMDH1oRF1038bp fragment, after PCR fragment recovery order-checking is errorless, passes through bamhI and mluafter I pair of fragment and object carrier carry out double digestion, will shMDH1gene is connected to object carrier pYLRNAi (being so kind as to give by Agricultural University Of South China Liu Yaoguang researcher laboratory).

The structure of yeast Overexpression vector: with khuskhus root cDNA for template, use upstream specific primer

Increase 1052bp object fragment for 5 '-CGGGATCCAAAAAAATGTCTATTAAGCCTTCGATGCTC-3 ' (SEQIDNO:11) and downstream special primer 5 '-GAATTCTTACTGGTTGGCGAATTTG-3 ' (SEQIDNO:12), after PCR fragment recovery order-checking is errorless, use bamh I He ecorI respectively enzyme cuts PCR primer and object carrier pYES2vector(Invitrogen company).

The structure of Subcellular Localization expression vector: with khuskhus root cDNA for template, with upstream specific primer 5 '-TCTAGAGATGATTAAGCCTTCG-3 ' (SEQIDNO:13) and downstream special primer 5 '-GGATCCCGCTGGTTGGCG-3 ' (SEQIDNO:14) amplification shMDH1open reading frame fragment, after PCR fragment recovery order-checking is errorless, will shMDH1gene is connected to object carrier EGFP (teaching laboratory by South China Normal University Wang Xiaojing to be so kind as to give).

3, ShMDH1 Subcellular Localization and Gene Expression Profile Analysis thereof

1) ShMDH1 Subcellular Localization

ShMDH1 will be loaded -eGFP carrier and EGFP empty carrier import onion epidermis cell by Gene Knock-out Mice and carry out transient expression.Then fluorescent microscope (LEICADM5000B, Germany) is used to observe GFP and the PI fluorescent signal of onion epidermis cell.

As shown in Figure 3, from result, ShMDH1 is positioned other positions in onion epidermis cell except cell walls to result.

2) shMDH1the expression pattern analysis of gene

Manganese pair shMDH1the impact of genetic expression:

Two khuskhus genotype ' TPRC2001-1 ' and ' FineStem ', seed is sterilized through 10%NaClO after removing seed coat, is sown in quartz sand, 25 DEG C of darkroom vernalization, transplants and carry out nutrient fluid cultivation in greenhouse after germinateing to 14L plastic tub.When seedling the 2nd compound leaf launches completely (1 month), the process of two manganese concentration is carried out to it, be respectively normal manganese concentration 5 μMs of MnSO ₄, Excessive Manganese concentration 400 μMs of MnSO ₄, gather in the crops sample after process 10d, extract root RNA, reverse transcription becomes cDNA, uses quantitative PCR detection further shMDH1expression pattern.The house-keeping gene of khuskhus eF-1aas internal reference.Primer for quantitative PCR detection gene expression amount is respectively:

Khuskhus eF-1athe primer of gene is:

EF-1aF:5’-CACTTCAGGACGTGTACAAGATC-3’(SEQIDNO:15)

EF-1aR:5’-CTTGGAGAGCTTCATGGTGCA-3’(SEQIDNO:16)

shMDH1the primer of gene is:

ShMDH1F:5’-CCGCTGAGGTTTTCAAGAAGGCAGG-3’(SEQIDNO:17)

ShMDH1R:5’-CCTGCATGGCCACCTATGACCG-3’(SEQIDNO:18)

Result is as Fig. 4, and from result, relatively normal manganese (5 μMs), Excessive Manganese (400 μMs) process significantly enhances shMDH1expression in Fine-stem root, and Excessive Manganese process pair shMDH1expression impact in TPRC2001-1 root is not obvious.

embodiment 2

The research of transgenic line

1, the acquisition of transgenic line

1) acquisition of transgenic Arabidopsis plants

By the overexpression built shMDH1vector plasmid is converted in agrobacterium tumefaciens Gv3101, adopts agriculture bacillus mediated Arabidopis thaliana inflorescence infection protocol to obtain transgenic Arabidopsis plants.

2) acquisition of transgenic yeast bacterial strain

With reference to SCeasycomptransformationkit(Invitrogen company) method, by the yeast expression built shMDH1vector plasmid and pYES2 empty carrier be transformed yeast INVSC1(Invitrogen company respectively).

2, the detection of transgenic line

1) detection of transgenic Arabidopsis plants

By T1 for Arabidopis thaliana seed through hygromycin resistance screening after, obtain T1 for transfer-gen plant, results seed; T2 is screened for seed hygromycin resistance, obtain seedling rate be the T2 of 60% for plant, results seed; Screened for seed hygromycin resistance by T3, obtaining seedling rate is 100% transgenic lines.Extract the RNA of this transgenic lines plant, after reverse transcription becomes cDNA, use quantitative PCR detection overexpression further shMDH1the effect of gene.

Arabidopis thaliana house-keeping gene eF-1aas reference gene, gene for the purpose of relative expression quantity shMDH1expression amount and the ratio of house-keeping gene expression amount.Khuskhus shMDH1detection primer be SEQIDNO:17 and SEQIDNO:18.

1.1) Arabidopis thaliana eF-1athe primer of gene is:

EF-1aF:5’-GTCGATTCTGGAAAGTCGACC-3’(SEQIDNO:19)

EF-1aR:5’-AATGTCAATGGTGATACCACGC-3’(SEQIDNO:20)

1.2) reaction system: comprise 10 μMs of each 0.6 μ L, 2 × SYBRGreenPCRmastermix10 μ L of upstream and downstream primer, then supply 20 μ L with Mili-Q water.PCR response procedures is: 95 DEG C 1 minute; 95 DEG C 15 seconds, 58 DEG C 15 seconds, 72 DEG C 30 seconds, 35 circulations of increasing.

2) detection of transgenic yeast bacterial strain

By scarce urinary ammonia acid screening culture medium screening positive transformant, and detect the goal gene in yeast cell DNA by PCR shMDH1, khuskhus shMDH1detection primer be SEQIDNO:11 and SEQIDNO:12.PCR reaction system is 20 μ L, and comprise 10 μMs of each 0.5 μ L, 10 × PCRbuffer2 μ L, 2.5mMdNTP1.6 μ L, Taq enzyme 0.12 μ L of upstream and downstream primer, yeast liquid 1 μ L, then uses sterilizing ddH ₂o supplies 20 μ L.PCR response procedures is: 94 DEG C 4 minutes; 94 DEG C 30 seconds, 57 DEG C 30 seconds, 72 DEG C 60 seconds, 35 circulations of increasing; 72 DEG C extend 10 minutes.The PCR primer of amplification is by 1% agarose gel electrophoresis.

3, the resistance to manganese of transgenic arabidopsis is analyzed

1) root organic acid secretion

Overexpression shMDH1after transgenic arabidopsis and wildtype Arabidopsis thaliana grow 7 days on solidified MS media, seedling of the same size is transferred in liquid MS medium and continues cultivation 7 days.Being outwelled by liquid MS medium, is the 0.5mMCaCl of 5.0 by pH value ₂solution cleaning plant 3 times, then uses 0.5mMCaCl ₂after solution (pH5.0) collects 24 hours, collect liquid and measure malic acid content.

2) manganese concentration process impact that Arabidopis thaliana is grown

T3 is for overexpression shMDH1transgenic arabidopsis and wildtype Arabidopsis thaliana seed, after sterilization, are cultivated 7d, are then moved to seedling of the same size containing 0.1mM, 2mM and 4mMMnSO in normal MS substratum ₄after processing 7d in solidified MS media, results plant, measures plant fresh weight and Fe content.

Result as shown in Figure 5, from result, versus wild type Arabidopis thaliana, overexpression shMDH1gene significantly increases the malic acid concentration of Arabidopis thaliana and the secretion (Fig. 5 A-C) of root system oxysuccinic acid.Further, overexpression shMDH1gene significantly improves the resistance to manganese ability of Arabidopis thaliana.Versus wild type plant, under Excessive Manganese process (2mM and 4mM), transgenic arabidopsis has higher biomass and lower manganese concentration (Fig. 5 D-F).

4, the resistance to manganese of transgenic yeast cell is analyzed

1) synthesis of yeast cell oxysuccinic acid

To load shMDH1-after the yeast of pYES2 and pYES2 empty carrier cultivates 1d respectively in 100mL liquid inducing culture, at 4 DEG C, the centrifugal 10min of 6000g collects thalline, and 1mLMillipore water cleans 2 times, freeze concentration machine (Labconco company) freeze-drying sample.Then the broken somatic cells wall of liquid nitrogen.Finally use 1.2mLMillipore water dissolution, 4 DEG C of centrifugal 10min of 12000g, draw supernatant, after crossing 0.45 μm of millipore filtration, measure malic acid content.

2) manganese process is on the impact of yeast cell growth

Yeast overexpression shMDH1and to be cultured to OD600 containing the bacterial strain of empty carrier in SC substratum be 0.6, after being diluted to 0.2, redilution is 0.02,0.002,0.0002 different OD concentration, respectively containing 2%galactose, 1%raffinose, 0.67%yeastnitrogenbasew/oaminoacids, 0.1%uracildropoutmix, 2%agar, 10mM or 15mMMnSO ₄substratum in cultivate 4d, Taking Pictures recording result.

As shown in Figure 6, from result, relative pYES2 empty vector control yeast strain, expresses result shMDH1gene significantly improves the malic acid concentration (Fig. 6 A) of yeast cell, and, significantly enhance the resistance to manganese ability (Fig. 6 B) of yeast cell under Excessive Manganese (10mM and 15mM) process.

5, water culture experiment

Water culture experiment with two khuskhus genotype ' TPRC2001-1 ' and ' FineStem ' for material, seed is sterilized through 10%NaClO after removing seed coat, be sown in quartz sand, 25 DEG C of darkroom vernalization, transplant after germinateing and carry out nutrient fluid cultivation in greenhouse to 14L plastic tub.When seedling the 2nd compound leaf launches completely (1 month), the process of two manganese concentration is carried out to it, be respectively normal manganese concentration 5 μMs of MnSO ₄, Excessive Manganese concentration 400 μMs of MnSO ₄, after process 10d, gather in the crops sample.Part Plant samples is used for chlorophyll content, biomass and manganese content test.Part plant pH value is the 0.5mMCaCl of 5.0 ₂after solution cleaning plant 3 times, collect the organic acid of plant root secretion respectively with the above-mentioned nutritive medium of 40mL of new preparation, collect 6h, collect rear collection liquid and measure for organic acid in-80 DEG C of preservations.

Result as shown in Figure 1-2.From result, relatively normal manganese concentration (5 μMs), Excessive Manganese (400 μMs) process 10d significantly reduces TPRC2001-1 chlorophyll concentration and Leaf biomass (Figure 1A), but Excessive Manganese process has no significant effect (Figure 1B) Fine-stem chlorophyll concentration and biomass.Excessive Manganese process significantly increases the manganese concentration of TPRC2001-1 and Fine-stem leaf portion and root, but under Excessive Manganese process the manganese concentration of Fine-stem leaf portion and root significantly lower than TPRC2001-1(Fig. 1 C).Relatively normal manganese concentration process, Excessive Manganese process significantly increases the malic acid concentration of Fine-stem root and the secretion of oxysuccinic acid, but the secretion of Excessive Manganese process on the malic acid concentration of TPRC2001-1 root and oxysuccinic acid affects not obvious (Fig. 2 A and 2B).

SEQIDNO:1

ATGATTAAGCCTTCGATGCTCAGATCCCTCCACTCAGCCGTGTCCCGCGGCTCCTCTCACCTCGCTCGCCGTGGCTACTCCTCCGAGTCGACCCCCGATCGCAAGGTCGTCGTTCTCGGCGCCGCCGGCGGCATCGGCCAGCCCCTCTCCCTCCTCATGAAGCTCAACCCACTCGTTTCCAGCCTCTCCCTTTACGATATCGCCGGCACTCCCGGCGTCGCCGCCGATGTAAGCCACGTCAACACCAGATCTGAGGTGGTTGGATATCAAGGTGAAGGAGAGCTTGGAAAAGCTTTGGAGGGAGCTGATGTCGTTATTATCCCAGCTGGTGTGCCCAGGAAGCCTGGAATGACTCGTGATGATCTTTTCAACATTAATGCTGGCATTGTCAAGTCTCTCTGCACTGCTATTTCCAAGTACTGCCCCAAGGCCCTTGTTAACATGATCAGCAATCCTGTGAACTCCACTGTTCCCATTGCTGCTGAGGTTTTCAAGAAGGCAGGGACATATGATGAGAAGAGATTGTTTGGGGTCACCACCCTTGATGTTGTTAGGGCAAAAACTTTTTATGCTGGAAAGGCCAAAGTTCCAGTTGCTGAGGTCAATGTACCGGTCATAGGTGGCCATGCAGGCATTACTATTCTGCCATTATTTTCTCAAGCAACACCACAAGCCAATTTGGATCATGATGTCATAGTAGCTCTTACTAAGAGAACACAAGATGGAGGAACGGAAGTTGTTGAAGCTAAGGCTGGAAAGGGTTCTGCAACTTTGTCAATGGCTTATGCCGGAGCCCTTTTTGCCGATGCTTGTCTCAAGGGTCTTAATGGTGTCCCAGATGTTGTTGAGTGTTCATTTGTGCAATCTAATGTCACTGAACTTCCTTTCTTCGCTTCAAAGGTGAGGCTTGGGAAGAATGGCGTGGAAGAAGTTCTGGGGTTGGGGTCTCTTTCAGATTTTGAGAAAGAAGGCCTTGAAAAGCTTAAGCCTGAGCTCTTGTCATCTATTGAGAAGGGAATCAAATTCGCCAACCAGTAA

SEQIDNO:2

MIKPSMLRSLHSAVSRGSSHLARRGYSSESTPDRKVVVLGAAGGIGQPLSLLMKLNPLVSSLSLYDIAGTPGVAADVSHVNTRSEVVGYQGEGELGKALEGADVVIIPAGVPRKPGMTRDDLFNINAGIVKSLCTAISKYCPKALVNMISNPVNSTVPIAAEVFKKAGTYDEKRLFGVTTLDVVRAKTFYAGKAKVPVAEVNVPVIGGHAGITILPLFSQATPQANLDHDVIVALTKRTQDGGTEVVEAKAGKGSATLSMAYAGALFADACLKGLNGVPDVVECSFVQSNVTELPFFASKVRLGKNGVEEVLGLGSLSDFEKEGLEKLKPELLSSIEKGIKFANQ。

SEQUENCELISTING

<110> Agricultural University Of South China

The clone of the resistance to manganese poisoning important gene ShMDH1 of <120> mono-kind of plant and application thereof

<130>

<160>21

<170>PatentInversion3.2

<210>1

<211>1038

<212>DNA

<213> sequence

<400>1

atgattaagccttcgatgctcagatccctccactcagccgtgtcccgcggctcctctcac60

ctcgctcgccgtggctactcctccgagtcgacccccgatcgcaaggtcgtcgttctcggc120

gccgccggcggcatcggccagcccctctccctcctcatgaagctcaacccactcgtttcc180

agcctctccctttacgatatcgccggcactcccggcgtcgccgccgatgtaagccacgtc240

aacaccagatctgaggtggttggatatcaaggtgaaggagagcttggaaaagctttggag300

ggagctgatgtcgttattatcccagctggtgtgcccaggaagcctggaatgactcgtgat360

gatcttttcaacattaatgctggcattgtcaagtctctctgcactgctatttccaagtac420

tgccccaaggcccttgttaacatgatcagcaatcctgtgaactccactgttcccattgct480

gctgaggttttcaagaaggcagggacatatgatgagaagagattgtttggggtcaccacc540

cttgatgttgttagggcaaaaactttttatgctggaaaggccaaagttccagttgctgag600

gtcaatgtaccggtcataggtggccatgcaggcattactattctgccattattttctcaa660

gcaacaccacaagccaatttggatcatgatgtcatagtagctcttactaagagaacacaa720

gatggaggaacggaagttgttgaagctaaggctggaaagggttctgcaactttgtcaatg780

gcttatgccggagccctttttgccgatgcttgtctcaagggtcttaatggtgtcccagat840

gttgttgagtgttcatttgtgcaatctaatgtcactgaacttcctttcttcgcttcaaag900

gtgaggcttgggaagaatggcgtggaagaagttctggggttggggtctctttcagatttt960

gagaaagaaggccttgaaaagcttaagcctgagctcttgtcatctattgagaagggaatc1020

aaattcgccaaccagtaa1038

<210>2

<211>345

<212>PRT

<213> sequence

<400>2

MetIleLysProSerMetLeuArgSerLeuHisSerAlaValSerArg

151015

GlySerSerHisLeuAlaArgArgGlyTyrSerSerGluSerThrPro

202530

AspArgLysValValValLeuGlyAlaAlaGlyGlyIleGlyGlnPro

354045

LeuSerLeuLeuMetLysLeuAsnProLeuValSerSerLeuSerLeu

505560

TyrAspIleAlaGlyThrProGlyValAlaAlaAspValSerHisVal

65707580

AsnThrArgSerGluValValGlyTyrGlnGlyGluGlyGluLeuGly

859095

LysAlaLeuGluGlyAlaAspValValIleIleProAlaGlyValPro

100105110

ArgLysProGlyMetThrArgAspAspLeuPheAsnIleAsnAlaGly

115120125

IleValLysSerLeuCysThrAlaIleSerLysTyrCysProLysAla

130135140

LeuValAsnMetIleSerAsnProValAsnSerThrValProIleAla

145150155160

AlaGluValPheLysLysAlaGlyThrTyrAspGluLysArgLeuPhe

165170175

GlyValThrThrLeuAspValValArgAlaLysThrPheTyrAlaGly

180185190

LysAlaLysValProValAlaGluValAsnValProValIleGlyGly

195200205

HisAlaGlyIleThrIleLeuProLeuPheSerGlnAlaThrProGln

210215220

AlaAsnLeuAspHisAspValIleValAlaLeuThrLysArgThrGln

225230235240

AspGlyGlyThrGluValValGluAlaLysAlaGlyLysGlySerAla

245250255

ThrLeuSerMetAlaTyrAlaGlyAlaLeuPheAlaAspAlaCysLeu

260265270

LysGlyLeuAsnGlyValProAspValValGluCysSerPheValGln

275280285

SerAsnValThrGluLeuProPhePheAlaSerLysValArgLeuGly

290295300

LysAsnGlyValGluGluValLeuGlyLeuGlySerLeuSerAspPhe

305310315320

GluLysGluGlyLeuGluLysLeuLysProGluLeuLeuSerSerIle

325330335

GluLysGlyIleLysPheAlaAsnGln

340345

<210>3

<211>25

<212>DNA

<213> sequence

<400>3

attattgccgctgaggttttcaaga25

<210>4

<211>22

<212>DNA

<213> sequence

<400>4

gattcccttcaagcaagcatcg22

<210>5

<211>27

<212>DNA

<213> sequence

<400>5

gaaaacctcagcagcaatgggaacagt27

<210>6

<211>25

<212>DNA

<213> sequence

<400>6

ctgctgaggttttcaagaaggcagg25

<210>7

<211>27

<212>DNA

<213> sequence

<220>

<221>misc_feature

<222>(27)..(27)

<223>nisa,c,g,ort

<400>7

tttttttttttttttttttttttttvn27

<210>8

<211>28

<212>DNA

<213> sequence

<220>

<221>misc_feature

<222>(28)..(28)

<223>nisa,c,g,ort

<400>8

aagcagtggtatcaacgcagagtactvn28

<210>9

<211>26

<212>DNA

<213> sequence

<400>9

ggatccaatgattaagccttcgatgc26

<210>10

<211>23

<212>DNA

<213> sequence

<400>10

acgcgtttactggttggcgaatt23

<210>11

<211>38

<212>DNA

<213> sequence

<400>11

cgggatccaaaaaaatgtctattaagccttcgatgctc38

<210>12

<211>25

<212>DNA

<213> sequence

<400>12

gaattcttactggttggcgaatttg25

<210>13

<211>22

<212>DNA

<213> sequence

<400>13

tctagagatgattaagccttcg22

<210>14

<211>18

<212>DNA

<213> sequence

<400>14

ggatcccgctggttggcg18

<210>15

<211>23

<212>DNA

<213> sequence

<400>15

cacttcaggacgtgtacaagatc23

<210>16

<211>21

<212>DNA

<213> sequence

<400>16

cttggagagcttcatggtgca21

<210>17

<211>25

<212>DNA

<213> sequence

<400>17

ccgctgaggttttcaagaaggcagg25

<210>18

<211>22

<212>DNA

<213> sequence

<400>18

cctgcatggccacctatgaccg22

<210>19

<211>21

<212>DNA

<213> sequence

<400>19

gtcgattctggaaagtcgacc21

<210>20

<211>22

<212>DNA

<213> sequence

<400>20

aatgtcaatggtgataccacgc22

<210>21

<211>45

<212>DNA

<213> sequence

<400>21

ctaatacgactcactatagggcaagcagtggtatcaacgcagagt45

Claims (8)

1. the plant key gene of a resistance to manganese poisoning ShMDH1, is characterized in that its nucleotide sequence is as shown in SEQIDNO:1.

2. a protein for ShMDH1 coding according to claim 1, is characterized in that its aminoacid sequence is as shown in SEQIDNO:2.

3. an expression vector, is characterized in that containing the key gene of resistance to manganese poisoning ShMDH1 described in claim 1.

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

5. the application of the key gene of resistance to manganese poisoning ShMDH1 described in claim 1 in preparation transgenic plant, described plant is Arabidopis thaliana or khuskhus.

6. the key gene of resistance to manganese poisoning ShMDH1 described in claim 1 promotes plant to adapt to acid soil preparation in preparation or improves the application on plant manganese tolerance, and described plant is Arabidopis thaliana or khuskhus.

7. the application of expression vector according to claim 3 in preparation transgenic plant, described plant is Arabidopis thaliana or khuskhus.

8. the application of expression vector according to claim 3 on the preparation or raising plant manganese tolerance of preparation promotion plant adaptation acid soil, described plant is Arabidopis thaliana or khuskhus.