CN103525825A

CN103525825A - Clone of plant manganese poison-resistant important gene ShMDH1 and application thereof

Info

Publication number: CN103525825A
Application number: CN201310290864.7A
Authority: CN
Inventors: 田江; 廖红; 陈志坚
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: CN103525825B

Abstract

The invention discloses a clone of a key gene ShMDH1 promoting the manganese poison resistance of a plant and an application thereof. The nucleotide sequence of the ShMDH1 is shown by SEQ ID No:1, and the amino acid sequence of the coded protein is shown by SEQ ID No:2. The ShMDH1 gene regulates the synthesis and secretion of malic acid of stylosanthes guianensis, and plays an important role in enhancing the manganese resistance of the plant.

Description

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

Technical field

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

Background technology

More than 50% potential can anthropogenic soil be acid soil in the world, is mainly distributed in the torrid zone, subtropics and Temperate Region in China (Kochian et al., 2004).In acid soil, except lacking phosphorus and aluminium poison, manganese poisoning is also significant obstacle factor (de Carvalho et al., 1980 of restriction plant growth and output; Horst et al., 1988; Raghothama, 1999; Vance et al., 2003).Manganese is one of essential trace element of plant-growth (Mukhopadhyay and Sharma, 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, and the reduction of chlorophyll content, the photosynthetic symptom such as weaken finally suppress growth and output (Horst et al., 1999 of plant; Millaleo et al., 2010).

Research in the past shows that plant is in long-term evolution and artificially breeding process, by increasing synthetic and secretion, the aluminium in chelating rhizosphere of root system oxysuccinic acid; nickel; the metallic element such as copper and zinc, thus metal ion alleviated to the toxic action of plant-growth (Yang et al., 1997; Parker et al., 2001; Sasaki et al., 2004; Delhaize et al., 2012).By overexpression malate dehydrogenase (malic acid dehydrogenase) (MDH) gene in clover and tobacco, can improve the synthetic of transfer-gen plant root system oxysuccinic acid and secretion early stage, effectively strengthened transfer-gen plant resistance to aluminium ability (Tesfaye et al,, 2001; Wang et al., 2010), but about the mechanism of oxysuccinic acid secretion and resistance to manganese poisoning thereof, rarely have report.

Khuskhus originates from subtropical and tropical zones, has good nutritive value and feeding value, is the leguminous forage (Liu et al., 1997) of high-quality.Khuskhus has good adaptability to the acid soil that contains high manganese concentration, and it may pass through regulation and control plant gene expression in vivo, thereby strengthens the resistance to manganese ability (de Carvalho et al., 1980) of self.But, control the key gene of the resistance to manganese poisoning of khuskhus and not yet report.

Based on above-mentioned research background, applicant, by the method for two-dimensional electrophoresis, has identified the albumen of a manganese poisoning up-regulated expression, ShMDH1, and cloned the gene of this albumen of encoding, the expression of this gene is subject to the regulation and control of external source manganese poisoning, has 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 the synthetic function with secreting of regulating apple acid, 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 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 1038 based compositions, and its open reading frame (ORF) is 1-1038 bit base, and coding has the aminoacid sequence of sequence SEQ ID NO:2, and the protein that described aminoacid sequence forms 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 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 shMDH1can regulate and control to comprise the synthetic 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 provide contain 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 (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 shMDH1gene 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, for example khuskhus and soybean, also can be other plant, such as monocotyledons, 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 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 shMDH1gene or the recombinant vectors purposes in regulating plant oxysuccinic acid is 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 shMDH1the 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 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 for example to import acceptor Arabidopis thaliana and yeast cell by described recombinant expression vector.

Carry gene of the present invention shMDH1plant expression vector can by for example agriculture bacillus mediated conversion method, be transformed in Arabidopis thaliana, Yeast expression carrier can be transformed in yeast cell.

Advantage of the present invention and effect:

1. gene shMDH1though under malate dehydrogenase enzyme family in Arabidopis thaliana, clover, corn and paddy rice etc., be cloned and reported, it is the biological function aspect synthetic unclear at leguminous forage oxysuccinic acid.The present invention clone's gene shMDH1on khuskhus oxysuccinic acid is synthetic, have a significant impact, this adapts to the biological function important in inhibiting of acid soil manganese poisoning to illustrating the synthetic and secretion of oxysuccinic acid leguminous forage.

2. gene shMDH1not only affected the synthetic of oxysuccinic acid, this gene of overexpression has also increased Arabidopis thaliana and the restrain oneself ability of yeast cell 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: in water culture experiment, different manganese concentration is processed the impact on khuskhus growth; A: be that different manganese concentration is processed the impact on khuskhus chlorophyll content shown in figure; B: be that different manganese concentration is processed the impact on khuskhus biomass shown in figure; C: be that different manganese concentration is processed the impact on khuskhus leaf portion and root manganese concentration shown in figure; Wherein manganese concentration for the treatment of is respectively 5 μ M and 400 μ M; In test, 4 biology repetitions are established in each processing, and in figure, pillar is mean value and the standard error of 4 biology repeating datas; * represent same index significant difference between same gene type different treatment,p<0.05; * represents that same index difference between same gene type different treatment is extremely remarkable, 0. 001<p<0. 01.

Fig. 2: in water culture experiment, different manganese concentration is processed the impact on the accumulation of khuskhus root system oxysuccinic acid and secretion; A: different manganese concentration is processed the impact on the endogenous malic acid concentration of khuskhus root system; B: different manganese concentration is processed the impact on the secretion of khuskhus root system oxysuccinic acid; Wherein manganese concentration for the treatment of is respectively 5 μ M and 400 μ M; In test, 4 biology repetitions are established in each processing, and in figure, pillar is mean value and the standard error of 4 biology repeating datas; * represent same index significant difference between same gene type different treatment,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 that the empty carrier that 35S promoter drives contrasts the cell after onion epidermis plasmolysis: 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: it is right that manganese is processedshMDH1the regulation and control that gene is expressed at khuskhus root system; In figure, data are mean value and the standard error that 4 biology repeat; * number represent the significant difference of same index between different treatment,p<0.05.

Fig. 5: overexpressionshMDH1impact on synthetic and secretion and the resistance to manganese of transgenic arabidopsis oxysuccinic acid; A wherein:shMDH1the expression analysis of gene in transgenic arabidopsis; B:shMDH1different overexpression transgenic lines and wild-type Arabidopis thaliana oxysuccinic acid resultant quantity; C:shMDH1oxysuccinic acid secretory volume in different overexpression transgenic lines and wild-type Arabidopis thaliana; D:shMDH1different overexpression transgenic lines and the phenotype analytical of wild-type Arabidopis thaliana under manganese treatment condition, in figure, scale is 1 cm; E:shMDH1the comparison of different overexpression transgenic lines and wild-type Arabidopis thaliana biomass.F:shMDH1the comparison of different overexpression transgenic lines and wild-type Arabidopis thaliana manganese concentration; WT, the contrast strain of wild-type Arabidopis thaliana; OX,shMDH1overexpression strain; In test, 4 biology repetitions are established in each processing, 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 contrasting between strain,p<0.05; * represents that same index is extremely remarkable with the difference contrasting between strain in overexpression strain, 0. 001<p<0.01.

Fig. 6: overexpressionshMDH1impact on the synthetic and resistance to manganese of yeast cell oxysuccinic acid; A wherein: overexpressionshMDH1malic acid concentration in transgenosis bacterial strain and empty carrier contrast yeast cell; B: overexpressionshMDH1transgenic line and the phenotype analytical of empty carrier contrast yeast cell under manganese treatment condition; In test, 4 repetitions are established in each processing, 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 contrasting between strain,p<0.05.

Embodiment

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

embodiment 1

shMDH1the clone of gene

1, shMDH1gene clone

According to albumen two-dimensional electrophoresis, having identified one, to be subject to the albumen of manganese poisoning up-regulated expression be malate dehydrogenase (malic acid dehydrogenase).Then, according to the soybean of having 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 homology comparison, at design upstream, high conservative region homologous primer 5 '-ATTATTGCCGCTGAGGTTTTCAAGA-3 ' (SEQ ID NO:3) and downstream homologous primer 5 '-GATTCCCTTCAAGCAAGCATCG-3 ' (SEQ ID NO:4), use the total RNA of TRIzol method extraction column flowers and plants resistance to manganese genotype Fine-stem root system, then reverse transcription is carried out pcr amplification as template after becoming cDNA, PCR reaction system is 20 μ L, comprise each 0.5 μ L of 10 μ M upstream and downstream primers, 10 * PCR buffer, 2 μ L, 2.5 mM dNTP 1.6 μ L, Ex-Taq (TAKARA company) enzyme 0.12 μ L, cDNA template amount 1 μ L, then use sterilizing ddH ₂o supplies 20 μ L.PCR response procedures is: 94 ℃ 4 minutes; 94 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 30 seconds, 35 circulations of increasing; 72 ℃ are extended 10 minutes.The PCR product 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 product of 354 bp.(TAKARA company, specific descriptions are shown in: http://www.takara.com.cn/) carrier order-checking are identified to pMD18-T to reclaim product cloning.According to the result of order-checking, use Primer 5.0 primer-design softwares, the following amplification of design shMDH1the primer of gene: 5 ' end EST special primer 5 '-GAAAACCTCAGCAGCAATGGGAACAGT-3 ' (SEQ ID NO:5), 3 ' end EST special primer 5 '-CTGCTGAGGTTTTCAAGAAGGCAGG-3 ' (SEQ ID NO:6).

The RNA that root is processed in the khuskhus manganese poisoning of take is template, adopts the SMARTerTM RACE cDNA Amplification Kit test kit of Clontech company (U.S.) to build 5 ' and 3 ' RACE cDNA, carries out cDNA end rapid amplifying.Get respectively 5 '-CDS Primer A, the 5 ' TTTTTTTTTTTTTTTTTTTTTTTTTVN-3 ' (N=A in the 2 total RNA of μ g root and test kit, C, G, or T, V=A, G, or C) (SEQ ID NO:7) and 3 '-CDS Primer A, 5 '-AAGCAGTGGTATCAACGCAGAGTAC (T), 30 V N-3 ' (SEQ ID NO:8) combination, and in 5 ' RACE, add SMARTer II A Oligonucleotide, with SMARTScribe Reverse Transcriptase, carry out reverse transcription respectively subsequently, be built into 5 ' and 3 ' RACE cDNA.Above-mentioned shMDH1gene 5 ' EST special primer and 3 ' EST special primer respectively with test kit in UPM primer 5 '-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 ' (SEQ ID NO:21) pairing, 5 ' and 3 ' the RACE cDNA of take is template, respectively amplification shMDH15 ' end and 3 ' terminal sequence of gene, then reclaims with DNA gel the product that test kit reclaims respectively 5 '/3 ' end-PCR.Recovery obtains PCR product cloning, and to pMD18-T, (TAKARA company, specific descriptions are shown in: the evaluation of http://www.takara.com.cn/) checking order on carrier.To obtain above shMDH1the sequence of 5 ' and 3 ' end of gene and obtain above shMDH1partial cDNA Sequence with MEGA 4.1 softwares splicings, obtain khuskhus shMDH1full length cDNA sequence.

2, the structure of carrier

The structure of Arabidopis thaliana Overexpression vector: the khuskhus root cDNA of take is template, with upstream special primer 5 '-GGATCCAATGATTAAGCCTTCGATGC-3 ' (SEQ ID NO:9) and downstream special primer 5 '-ACGCGTTTACTGGTTGGCGAATT-3 ' (SEQ ID NO:10) amplification shMDH1oRF 1038 bp fragments, after PCR fragment recovery order-checking is errorless, by bamh I and mlui is carried out after double digestion fragment and object carrier, will shMDH1gene is connected to object carrier pYLRNAi (being so kind as to give by the Liu Yaoguang researcher of Agricultural University Of South China laboratory).

The structure of yeast Overexpression vector: the khuskhus root cDNA of take is template, uses upstream special primer

5 '-CGGGATCCAAAAAAATGTCTATTAAGCCTTCGATGCTC-3 ' (SEQ ID NO:11) and downstream special primer 5 '-GAATTCTTACTGGTTGGCGAATTTG-3 ' (SEQ ID NO:12) the 1052 bp object fragments that increase, after PCR fragment recovery order-checking is errorless, use bamh I and ecor I respectively enzyme cuts PCR product and object carrier pYES2 vector(Invitrogen company).

The structure of Subcellular Localization expression vector: the khuskhus root cDNA of take is template, with upstream special primer 5 '-TCTAGAGATGATTAAGCCTTCG-3 ' (SEQ ID NO:13) and downstream special primer 5 '-GGATCCCGCTGGTTGGCG-3 ' (SEQ ID NO:14) amplification shMDH1open reading frame fragment, after PCR fragment recovery order-checking is errorless, will shMDH1gene is connected to object carrier EGFP (being so kind as to give by the Wang Xiaojing of South China Normal University professor laboratory).

3, ShMDH1 Subcellular Localization and Gene Expression Profile Analysis thereof

1) ShMDH1 Subcellular Localization

To load ShMDH1 -eGFP carrier and EGFP empty carrier import onion epidermis cell by via Particle Bombardment Transformation method and carry out transient expression.Then use fluorescent microscope (LEICA DM5000B, Germany) to observe GFP and the PI fluorescent signal of onion epidermis cell.

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

2) shMDH1the expression pattern analysis of gene

Manganese pair shMDH1the impact of genetic expression:

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

Khuskhus eF-1athe primer of gene is:

EF-1aF: 5’- CACTTCAGGACGTGTACAAGATC -3’ (SEQ ID NO:15)

EF-1a R: 5’- CTTGGAGAGCTTCATGGTGCA -3’ (SEQ ID NO:16)

shMDH1the primer of gene is:

ShMDH1F: 5’- CCGCTGAGGTTTTCAAGAAGGCAGG -3’ (SEQ ID NO:17)

ShMDH1 R: 5’- CCTGCATGGCCACCTATGACCG -3’ (SEQ ID NO:18)

Result is as Fig. 4, from result, and relatively normal manganese (5 μ M), Excessive Manganese (400 μ M) is processed and has significantly been strengthened shMDH1expression in Fine-stem root, and Excessive Manganese processing is right 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 plant

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

2) acquisition of transgenic yeast bacterial strain

With reference to SC easy comp transformation kit(Invitrogen company) method, by the yeast expression building shMDH1vector plasmid and pYES2 empty carrier be transformed yeast INVSC1(Invitrogen company respectively).

2, the detection of transgenic line

1) detection of transgenic arabidopsis plant

By T1 for Arabidopis thaliana seed after hygromycin resistance screening, obtain T1 for transfer-gen plant, results seed; T2 is screened with hygromycin resistance for seed, and the T2 that acquisition seedling rate is 60%, for plant, gathers in the crops seed; T3 is screened with hygromycin resistance for seed, and obtaining seedling rate is 100% transgenic lines.Extract the RNA of this transgenic lines plant, reverse transcription becomes after cDNA, further uses quantitative PCR detection overexpression shMDH1the effect of gene.

Arabidopis thaliana house-keeping gene eF-1aas reference gene, relative expression quantity is goal gene shMDH1expression amount and the ratio of house-keeping gene expression amount.Khuskhus shMDH1detection primer be SEQ ID NO:17 and SEQ ID NO:18.

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

EF-1aF: 5’- GTCGATTCTGGAAAGTCGACC -3’ (SEQ ID NO: 19)

EF-1aR: 5’- AATGTCAATGGTGATACCACGC -3’ (SEQ ID NO: 20)

1.2) reaction system: comprise each 0.6 μ L of 10 μ M upstream and downstream primers, 2 * SYBR Green PCR master mix, 10 μ L, then supply 20 μ L with Mili-Q water.PCR response procedures is: 95 ℃ 1 minute; 95 ℃ 15 seconds, 58 ℃ 15 seconds, 72 ℃ 30 seconds, 35 circulations of increasing.

2) detection of transgenic yeast bacterial strain

By lacking 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 SEQ ID NO:11 and SEQ ID NO:12.PCR reaction system is 20 μ L, comprises each 0.5 μ L of 10 μ M upstream and downstream primers, 10 * PCR buffer, 2 μ L, and 2.5 mM dNTP 1.6 μ L, Taq enzyme 0.12 μ L, yeast liquid 1 μ L, then uses sterilizing ddH ₂o supplies 20 μ L.PCR response procedures is: 94 ℃ 4 minutes; 94 ℃ 30 seconds, 57 ℃ 30 seconds, 72 ℃ 60 seconds, 35 circulations of increasing; 72 ℃ are extended 10 minutes.The PCR product of amplification is by 1% agarose gel electrophoresis.

3, the resistance to manganese of transgenic arabidopsis is analyzed

1) root organic acid secretion

Overexpression shMDH1transgenic arabidopsis and wild-type Arabidopis thaliana are grown after 7 days on solid MS substratum, and seedling of the same size is transferred in liquid MS medium and continues to cultivate 7 days.Liquid MS medium is outwelled to the 0.5 mM CaCl that is 5.0 by pH value ₂solution cleans plant 3 times, then uses 0.5 mM CaCl ₂solution (pH 5.0) was collected after 24 hours, collected liquid and measured malic acid content.

2) manganese concentration is processed the impact on Arabidopis thaliana growth

T3 is for overexpression shMDH1transgenic arabidopsis and wild-type Arabidopis thaliana seed, after sterilization, are cultivated 7d in normal MS substratum, then seedling of the same size are moved to containing 0.1 mM, 2 mM and 4 mM MnSO ₄in solid MS substratum, process after 7d, results plant, measures plant fresh weight and manganese content.

Result as shown in Figure 5, from result, relative wild-type Arabidopis thaliana, overexpression shMDH1gene has significantly increased the secretion (Fig. 5 A-C) of malic acid concentration and the root system oxysuccinic acid of Arabidopis thaliana.And, overexpression shMDH1gene has significantly improved the resistance to manganese ability of Arabidopis thaliana.Relative wild-type plant, under Excessive Manganese is processed (2 mM and 4 mM), 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) yeast cell oxysuccinic acid is synthetic

To load shMDH1-the yeast of pYES2 and pYES2 empty carrier is cultivated after 1 d respectively in 100 mL liquid inducing cultures, and centrifugal 10 min of 6000 g collect thalline at 4 ℃, and 1 mL Millipore water cleans 2 times, freeze concentration machine (Labconco company) freeze-drying sample.Then the broken somatic cells wall of liquid nitrogen.Finally use 1.2 mL Millipore water dissolution, 4 ℃ of 12000 centrifugal 10 min of g, draws supernatant, excessively after 0.45 μ m millipore filtration, measures malic acid content.

2) manganese is processed the impact on yeast cell growth

Yeast overexpression shMDH1and containing the bacterial strain of empty carrier, to be cultured to OD600 in SC substratum be 0.6, after being diluted to 0.2, redilution is 0.02,0.002,0.0002 different OD concentration, containing 2% galactose respectively, 1% raffinose, 0.67% yeast nitrogen base w/o amino acids, 0.1% uracil dropout mix, 2% agar, 10 mM or 15 mM MnSO ₄substratum in cultivate 4 d, Taking Pictures recording result.

As shown in Figure 6, from result, pYES2 empty carrier contrast yeast strain, expresses result relatively shMDH1gene has significantly improved the malic acid concentration (Fig. 6 A) of yeast cell, and, significantly strengthened the resistance to manganese ability (Fig. 6 B) of yeast cell under Excessive Manganese (10 mM and 15 mM) is processed.

5, water culture experiment

Water culture experiment be take two khuskhus genotype ' TPRC2001-1 ' and ' Fine Stem ' is material, seed goes kind of skin to sterilize by 10% NaClO, be sown in quartz sand, 25 ℃ of darkroom vernalization, after germinateing, transplant and in greenhouse, carry out nutrient fluid cultivation to 14 L plastic tub.When the 2nd compound leaf of seedling launches completely (1 month), it is carried out to two manganese concentration and process, be respectively normal manganese concentration 5 μ M MnSO ₄, Excessive Manganese concentration 400 μ M MnSO ₄, gather in the crops sample after processing 10 d.Part Plant samples is for chlorophyll content, biomass and manganese content test.The 0.5 mM CaCl that part plant is 5.0 by pH value ₂after solution cleaning plant 3 times, with the above-mentioned nutritive medium of 40 mL of new preparation, collect the organic acid that plant root is secreted respectively, collect 6 h, collect rear collection liquid and measure for organic acid in-80 ℃ of preservations.

Result as shown in Figure 1-2.From result, relatively normal manganese concentration (5 μ M), Excessive Manganese (400 μ M) is processed 10 d and has significantly been reduced TPRC2001-1 chlorophyll concentration and Leaf biomass (Figure 1A), but Excessive Manganese processing has no significant effect (Figure 1B) to Fine-stem chlorophyll concentration and biomass.Excessive Manganese is processed the manganese concentration that has significantly increased TPRC2001-1 and Fine-stem leaf portion and root, but Excessive Manganese is processed the manganese concentration of lower Fine-stem leaf portion and root significantly lower than TPRC2001-1(Fig. 1 C).Relatively normal manganese concentration is processed, and Excessive Manganese is processed has significantly increased the malic acid concentration of Fine-stem root and the secretion of oxysuccinic acid, but Excessive Manganese is processed the secretion impact not obvious (Fig. 2 A and 2B) on the malic acid concentration of TPRC2001-1 root and oxysuccinic acid.

SEQ ID NO:1

ATGATTAAGCCTTCGATGCTCAGATCCCTCCACTCAGCCGTGTCCCGCGGCTCCTCTCACCTCGCTCGCCGTGGCTACTCCTCCGAGTCGACCCCCGATCGCAAGGTCGTCGTTCTCGGCGCCGCCGGCGGCATCGGCCAGCCCCTCTCCCTCCTCATGAAGCTCAACCCACTCGTTTCCAGCCTCTCCCTTTACGATATCGCCGGCACTCCCGGCGTCGCCGCCGATGTAAGCCACGTCAACACCAGATCTGAGGTGGTTGGATATCAAGGTGAAGGAGAGCTTGGAAAAGCTTTGGAGGGAGCTGATGTCGTTATTATCCCAGCTGGTGTGCCCAGGAAGCCTGGAATGACTCGTGATGATCTTTTCAACATTAATGCTGGCATTGTCAAGTCTCTCTGCACTGCTATTTCCAAGTACTGCCCCAAGGCCCTTGTTAACATGATCAGCAATCCTGTGAACTCCACTGTTCCCATTGCTGCTGAGGTTTTCAAGAAGGCAGGGACATATGATGAGAAGAGATTGTTTGGGGTCACCACCCTTGATGTTGTTAGGGCAAAAACTTTTTATGCTGGAAAGGCCAAAGTTCCAGTTGCTGAGGTCAATGTACCGGTCATAGGTGGCCATGCAGGCATTACTATTCTGCCATTATTTTCTCAAGCAACACCACAAGCCAATTTGGATCATGATGTCATAGTAGCTCTTACTAAGAGAACACAAGATGGAGGAACGGAAGTTGTTGAAGCTAAGGCTGGAAAGGGTTCTGCAACTTTGTCAATGGCTTATGCCGGAGCCCTTTTTGCCGATGCTTGTCTCAAGGGTCTTAATGGTGTCCCAGATGTTGTTGAGTGTTCATTTGTGCAATCTAATGTCACTGAACTTCCTTTCTTCGCTTCAAAGGTGAGGCTTGGGAAGAATGGCGTGGAAGAAGTTCTGGGGTTGGGGTCTCTTTCAGATTTTGAGAAAGAAGGCCTTGAAAAGCTTAAGCCTGAGCTCTTGTCATCTATTGAGAAGGGAATCAAATTCGCCAACCAGTAA

SEQ ID NO:2

MIKPSMLRSLHSAVSRGSSHLARRGYSSESTPDRKVVVLGAAGGIGQPLSLLMKLNPLVSSLSLYDIAGTPGVAADVSHVNTRSEVVGYQGEGELGKALEGADVVIIPAGVPRKPGMTRDDLFNINAGIVKSLCTAISKYCPKALVNMISNPVNSTVPIAAEVFKKAGTYDEKRLFGVTTLDVVRAKTFYAGKAKVPVAEVNVPVIGGHAGITILPLFSQATPQANLDHDVIVALTKRTQDGGTEVVEAKAGKGSATLSMAYAGALFADACLKGLNGVPDVVECSFVQSNVTELPFFASKVRLGKNGVEEVLGLGSLSDFEKEGLEKLKPELLSSIEKGIKFANQ。

SEQUENCE LISTING

<110> Agricultural University Of South China

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

<130>

<160> 21

<170> PatentIn version 3.2

<210> 1

<211> 1038

<212> DNA

<213>sequence

<400> 1

atgattaagc cttcgatgct cagatccctc cactcagccg tgtcccgcgg ctcctctcac 60

ctcgctcgcc gtggctactc ctccgagtcg acccccgatc gcaaggtcgt cgttctcggc 120

gccgccggcg gcatcggcca gcccctctcc ctcctcatga agctcaaccc actcgtttcc 180

agcctctccc tttacgatat cgccggcact cccggcgtcg ccgccgatgt aagccacgtc 240

aacaccagat ctgaggtggt tggatatcaa ggtgaaggag agcttggaaa agctttggag 300

ggagctgatg tcgttattat cccagctggt gtgcccagga agcctggaat gactcgtgat 360

gatcttttca acattaatgc tggcattgtc aagtctctct gcactgctat ttccaagtac 420

tgccccaagg cccttgttaa catgatcagc aatcctgtga actccactgt tcccattgct 480

gctgaggttt tcaagaaggc agggacatat gatgagaaga gattgtttgg ggtcaccacc 540

cttgatgttg ttagggcaaa aactttttat gctggaaagg ccaaagttcc agttgctgag 600

gtcaatgtac cggtcatagg tggccatgca ggcattacta ttctgccatt attttctcaa 660

gcaacaccac aagccaattt ggatcatgat gtcatagtag ctcttactaa gagaacacaa 720

gatggaggaa cggaagttgt tgaagctaag gctggaaagg gttctgcaac tttgtcaatg 780

gcttatgccg gagccctttt tgccgatgct tgtctcaagg gtcttaatgg tgtcccagat 840

gttgttgagt gttcatttgt gcaatctaat gtcactgaac ttcctttctt cgcttcaaag 900

gtgaggcttg ggaagaatgg cgtggaagaa gttctggggt tggggtctct ttcagatttt 960

gagaaagaag gccttgaaaa gcttaagcct gagctcttgt catctattga gaagggaatc 1020

aaattcgcca accagtaa 1038

<210> 2

<211> 345

<212> PRT

<213>sequence

<400> 2

Met Ile Lys Pro Ser Met Leu Arg Ser Leu His Ser Ala Val Ser Arg

1 5 10 15

Gly Ser Ser His Leu Ala Arg Arg Gly Tyr Ser Ser Glu Ser Thr Pro

20 25 30

Asp Arg Lys Val Val Val Leu Gly Ala Ala Gly Gly Ile Gly Gln Pro

35 40 45

Leu Ser Leu Leu Met Lys Leu Asn Pro Leu Val Ser Ser Leu Ser Leu

50 55 60

Tyr Asp Ile Ala Gly Thr Pro Gly Val Ala Ala Asp Val Ser His Val

65 70 75 80

Asn Thr Arg Ser Glu Val Val Gly Tyr Gln Gly Glu Gly Glu Leu Gly

85 90 95

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

100 105 110

Arg Lys Pro Gly Met Thr Arg Asp Asp Leu Phe Asn Ile Asn Ala Gly

115 120 125

Ile Val Lys Ser Leu Cys Thr Ala Ile Ser Lys Tyr Cys Pro Lys Ala

130 135 140

Leu Val Asn Met Ile Ser Asn Pro Val Asn Ser Thr Val Pro Ile Ala

145 150 155 160

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

165 170 175

Gly Val Thr Thr Leu Asp Val Val Arg Ala Lys Thr Phe Tyr Ala Gly

180 185 190

Lys Ala Lys Val Pro Val Ala Glu Val Asn Val Pro Val Ile Gly Gly

195 200 205

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

210 215 220

Ala Asn Leu Asp His Asp Val Ile Val Ala Leu Thr Lys Arg Thr Gln

225 230 235 240

Asp Gly Gly Thr Glu Val Val Glu Ala Lys Ala Gly Lys Gly Ser Ala

245 250 255

Thr Leu Ser Met Ala Tyr Ala Gly Ala Leu Phe Ala Asp Ala Cys Leu

260 265 270

Lys Gly Leu Asn Gly Val Pro Asp Val Val Glu Cys Ser Phe Val Gln

275 280 285

Ser Asn Val Thr Glu Leu Pro Phe Phe Ala Ser Lys Val Arg Leu Gly

290 295 300

Lys Asn Gly Val Glu Glu Val Leu Gly Leu Gly Ser Leu Ser Asp Phe

305 310 315 320

Glu Lys Glu Gly Leu Glu Lys Leu Lys Pro Glu Leu Leu Ser Ser Ile

325 330 335

Glu Lys Gly Ile Lys Phe Ala Asn Gln

340 345

<210> 3

<211> 25

<212> DNA

<213>sequence

<400> 3

attattgccg ctgaggtttt caaga 25

<210> 4

<211> 22

<212> DNA

<213>sequence

<400> 4

gattcccttc aagcaagcat cg 22

<210> 5

<211> 27

<212> DNA

<213>sequence

<400> 5

gaaaacctca gcagcaatgg gaacagt 27

<210> 6

<211> 25

<212> DNA

<213>sequence

<400> 6

ctgctgaggt tttcaagaag gcagg 25

<210> 7

<211> 27

<212> DNA

<213>sequence

<220>

<221> misc_feature

<222> (27)..(27)

<223> n is a, c, g, or t

<400> 7

tttttttttt tttttttttt tttttvn 27

<210> 8

<211> 28

<212> DNA

<213>sequence

<220>

<221> misc_feature

<222> (28)..(28)

<223> n is a, c, g, or t

<400> 8

aagcagtggt atcaacgcag agtactvn 28

<210> 9

<211> 26

<212> DNA

<213>sequence

<400> 9

ggatccaatg attaagcctt cgatgc 26

<210> 10

<211> 23

<212> DNA

<213>sequence

<400> 10

acgcgtttac tggttggcga att 23

<210> 11

<211> 38

<212> DNA

<213>sequence

<400> 11

cgggatccaa aaaaatgtct attaagcctt cgatgctc 38

<210> 12

<211> 25

<212> DNA

<213>sequence

<400> 12

gaattcttac tggttggcga atttg 25

<210> 13

<211> 22

<212> DNA

<213>sequence

<400> 13

tctagagatg attaagcctt cg 22

<210> 14

<211> 18

<212> DNA

<213>sequence

<400> 14

ggatcccgct ggttggcg 18

<210> 15

<211> 23

<212> DNA

<213>sequence

<400> 15

cacttcagga cgtgtacaag atc 23

<210> 16

<211> 21

<212> DNA

<213>sequence

<400> 16

cttggagagc ttcatggtgc a 21

<210> 17

<211> 25

<212> DNA

<213>sequence

<400> 17

ccgctgaggt tttcaagaag gcagg 25

<210> 18

<211> 22

<212> DNA

<213>sequence

<400> 18

cctgcatggc cacctatgac cg 22

<210> 19

<211> 21

<212> DNA

<213>sequence

<400> 19

gtcgattctg gaaagtcgac c 21

<210> 20

<211> 22

<212> DNA

<213>sequence

<400> 20

aatgtcaatg gtgataccac gc 22

<210> 21

<211> 45

<212> DNA

<213>sequence

<400> 21

ctaatacgac tcactatagg gcaagcagtg gtatcaacgc agagt 45

Claims

1. a plant key gene of resistance to manganese poisoning shMDH1, it is characterized in that its nucleotide sequence is as shown in SEQ ID NO:1.

One kind claimed in claim 1 shMDH1the protein of coding, 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 virus gene of resistance to manganese described in claim 1 shMDH1.

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

5. the virus gene of resistance to manganese described in claim 1 shMDH1application in preparation transgenic plant.

6. the virus gene of resistance to manganese described in claim 1 shMDH1in preparation, promote plant to adapt to acid soil preparation or improve the application on plant manganese tolerance.

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

8. expression vector claimed in claim 3 promotes plant to adapt to the preparation of acid soil or improves the application on plant manganese tolerance 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.