CN101230357A - Plant expression vector for improving aluminum-tolerance of plant - Google Patents
Plant expression vector for improving aluminum-tolerance of plant Download PDFInfo
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- CN101230357A CN101230357A CNA2007100664178A CN200710066417A CN101230357A CN 101230357 A CN101230357 A CN 101230357A CN A2007100664178 A CNA2007100664178 A CN A2007100664178A CN 200710066417 A CN200710066417 A CN 200710066417A CN 101230357 A CN101230357 A CN 101230357A
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Images
Abstract
The invention discloses a plane expression vector for increasing the ability of resisting the aluminum toxicity and the application thereof, which belongs to plant genetic engineering field. The vector contains the Escherichia coli malate dehydrogenase (EMDH); the upstream of the malate dehydrogenase (EMDH) is Rubisco small subunit photoinduced promoter. The test shows that, the activity of EMDH of transgene tobacco obtained by transforming the tobacco by the vector is 1 to 3.3 times of that of the wild tobacco. Under the stress of 100 to 300uM aluminum toxicity, the EMDH transferred tobacco can secrete more malic acid; the root grows well, which has an enhanced tolerance for aluminum toxicity. The specific vector of the invention can increases the tolerance for aluminum toxicity of the plant, and has a great potential of application in improving the crops in particular in the acid soil of South China.
Description
Technical field
The invention belongs to plant genetic engineering field, be specifically related to a kind of member and application that improves the plant expression vector of the anti-aluminium poison of plant ability.
Background technology
The southern soil of China mostly is red soil greatly, and nutrition is barren, is acid, makes the phosphorus in the soil and the phosphate fertilizer that is applied in the soil is soluble state, thereby plant is difficult to absorb, impaired development, and increment is low.In acid soil, phosphorus and aluminium and iron form the very low compound of solubleness.In this external acid soil, aluminium has become solubility, is easy to enter plant roots, the g and D that suppresses root, make root system can not absorb the moisture and the thing that absorbs nourishment, thereby caused the crop yield reduction, therefore growing plants often is subjected to the murder by poisoning of aluminium in acid soil.Sour earth is typical low productive soil, and wherein aluminium (Al) poison is considered to influence the main limiting factor of plant growth.The aluminum ion of micromole's level can produce plant and poison, and Al in the acid soil solution
3+Concentration be about 10～100 μ mol/L
-1(Ma JF.2000.Role of organicacid in detoxification of aluminum in higher plants.Plant Cell Physiol.41:383-390).The initial reaction of aluminium toxicity is to suppress the root growth of aluminium sensitive gene type plant, and then suppress plant to moisture, nutrient absorbing, the normal growth that influences plant is grown (Delhaize E, Ryan PR.1995.Aluminum toxicity and tolerance in plants.Plant Pysiol.107:315-332; Foy CD.1983.The pysiology of plant adaptation to minealstress.Iowa State J Res.57:355-392).Aluminum ion is diversified to the molecular mechanism of plant hazard in acid soil.On the one hand, too much free aluminum and exchangeability aluminum ion can grow by suppressing the tip of a root, cause the malabsorption of plant to other ion and moisture, thereby influence growth and development of plant.On the other hand, aluminium also can with tenuigenin in and on the film proteic phosphate, hydroxyl isopolarity group combine, influence the 26S Proteasome Structure and Function of film.
Measure commonly used is to lime in acid soil improving pH in soil in the agriculture production, yet often is the expense that can not afford liming the peasant of developing country, in addition, sows the acidity that lime can not be administered lower soil.Along with the fast development of biotechnology, people transform existing plant variety by genetic engineering means and make it to have anti-aluminium and become possibility.By excessive in plant or ectopic expression organic acid synthase gene, as malate dehydrogenase (malic acid dehydrogenase) and citrate synthase gene, can improve the intravital organic acid content of plant, and these organic acids are secreted in the soil by root system, boil together aluminum ion in the soil just can be removed the murder by poisoning of the aluminium of acid soil middle and high concentration to plant.So not only can improve the tolerance of transgenic plant, but also help absorption and the utilization of plant other essential nutrient elements such as phosphorus to the aluminium poison.
Result of study shows that well-grown plant has strong tolerance to the aluminium poison on acid soil, and plant is closely related with plant secretion organic acid ability in many cases to the resistance of aluminium poison, and in plant materials, organic acid also can reduce the toxic action of aluminium.Therefore organic acid can stop the absorption of plant for aluminium at the outside chela and the aluminum ion of plasma membrane.The known organic acid that can effectively remove the aluminium poison is a citric acid, oxalic acid, tartrate, oxysuccinic acid, propanedioic acid, succsinic acid and acetic acid.Oxysuccinic acid is a key product in the plant metabolism, participate in the many steps in the metabolic reaction, comprise process (Wang Zhong such as photosynthesis, pore and leaflet movement, dietetic alimentation, respiration, nitrogen assimilation and Fatty Acid Oxidation, Wang Sangen .2002. plant physiology (first version) Beijing such as Li Hesheng: Chinese agriculture press: 157-164).In the acidity or alkaline soil that lack phosphorus, many plants can secrete organic acids such as a large amount of citric acids, oxalic acid or oxysuccinic acid at root, with heavy metal ion such as chelating Aluminium in Soil, iron, alleviate the harm of aluminium poison and discharge phosphate anion for the plant absorbing utilization.
(malate dehydrogenase MDH) is one of the key enzyme of eukaryote and prokaryotic organism tricarboxylic acid cycle (TCA) to malate dehydrogenase (malic acid dehydrogenase).Its catalysis oxysuccinic acid in tricarboxylic acid cycle forms oxaloacetic acid, but also catalysis oxalic acid acetate forms oxysuccinic acid.Oxysuccinic acid synthetic is that at first the effect by phosphoric acid enol pyruvic acid carboxylase (PEPC) converts phosphoenolpyruvic acid to oxaloacetic acid in plant materials, by malate dehydrogenase (malic acid dehydrogenase) oxaloacetic acid is oxidized to oxysuccinic acid then.MDH and PEPC that good several types is arranged in the plant, and their expression is relevant with their function and tissue specificity.Contain specific MDH of root nodule and pepc gene in the root nodule of bean, their expression level organizes high 5-15 doubly than other.Isolated a species specific novel MDH in the root nodule of clover, this novel MDH has special dynamics makes it can produce a large amount of oxysuccinic acid in root nodule.Tesfaye etc. utilize tobacco cauliflower mosaic virus CaMV35S promotor to make up the plant expression vector of the specific MDH gene of root nodule, method by the Agrobacterium transfection obtains transgenic alfalfa (Tesfaye M, Temple SJ, Allan DL, et al.2001.Overexpression of Malate dehydrogenase in transgenic alfalfa enhancesorganic acid Synthesis and confers tolerance to aluminum.Plant Physiol.127:1836-1844).Their experimental result shows that the activity of transgenic alfalfa tip of a root MDH is 1.6 times of control plant, root organic acid content has improved 4.2 times, root excretory citric acid, oxalic acid, oxysuccinic acid, succsinic acid and acetic acid have promoted 7.1 times than not genetically modified contrast clover.When plant-growth is containing in the acid nutrient solution of aluminium, the biomass of plant accumulation that changes MDH is than not genetically modified clover height, when in water planting liquid that contains aluminium or soil, cultivating, and the growth difficulty of non-transgenic plant, the transgenic plant growing state is good, and the plain receptivity of phosphorus strengthens.Other organic acid concentration increases in plant root and the leaf, strengthens from root excretory organic acid concentration, and follows the raising of anti-aluminium poison ability.
Cui Xiaoying etc. import the clover embryo callus by agriculture bacillus mediated genetic transformation method with alfalfa Phylloxera type malate dehydrogenase M DH, and the transformant of screening is at 20 μ M Al
3+The elongation comparison of handling the 24h back root part in the solution improves 3.6%～22.5% according to plant, show the transgenic alfalfa of coercing overexpression malate dehydrogenase (malic acid dehydrogenase) under the processing (Cui Xiaoying that can better grow at the aluminium poison, Cui Yanbo .2004. overexpression malate dehydrogenase genes such as Deng Wei improve anti-the being subjected to property of clover to the aluminium poison. Molecular Plant Breeding .2 (5): 621-626).In sum, can to strengthen in the plant organic acid synthetic for overexpression MDH in plant, thereby be an available strategy that tackles acid soil and the harm of aluminium poison.
Because the blade of plant is the photosynthesis organ, photosynthate can provide a large amount of carbon skeletons for organic acid is synthetic, if overexpression malate dehydrogenase (malic acid dehydrogenase) (MDH) in the tenuigenin of plant leaf, just can more directly utilize the acid of photosynthate synthesizing apple to be secreted into the extracellular, enter into soil by root system then, the aluminum ion in the soil that boils together, the content of raising titanium pigment, promote the absorption of phosphorus, remove of the murder by poisoning of the aluminium of acid soil middle and high concentration plant.The plant expression vector of existing malate dehydrogenase gene all adopts constitutive promoter (CaMV35S), and the effect of CaMV35S does not have tissue specificity.Result of study with reporter gene shows that being expressed in the root of CaMV35S is the strongest, weak (Jefferson etc., 1987 in stem, blade, flower and fruit; EMBO, 6:3901-3907).1,5 diphosphoribulose carboxylase (Rubisco) is the protein of expression amount maximum in the plant, and this Protein content accounts for the 40-50% of soluble proteins in the vegetable cell.The small subunit of Rubisco (rbcS) is encoded by cell nucleus gene, the promotor of control rbcS genetic expression is a photoinduction type promotor (PrbcS), and the effect of PrbcS has very strong tissue specificity, needs inducing of optical signal, low-level expression is arranged in stem, and the expression in blade is the strongest.Show with the result of study of reporter gene PrbcS in blade specific activity CaMV35S high 3-4 doubly, so PrbcS is a kind of very strong photoinduction type promotor (Jefferson etc., 1987; EMBO, 6:3901-3907), PrbcS often is used to realize the high level expression of goal gene in blade in scientific effort.
Summary of the invention
One of purpose of the present invention is to provide a kind of dedicated carrier that improves the anti-aluminium poison of plant ability.
The carrier that is used to improve the anti-aluminium poison of plant ability provided by the present invention is the plant expression vector with photoinduction type promotor and malate dehydrogenase gene (being the EMDH gene).
In the above-mentioned carrier, described malate dehydrogenase gene EMDH derives from intestinal bacteria (Escherichiacoli).The described GenBank accession number that derives from colibacillary malate dehydrogenase gene EMDH is Y00129.
The upstream of described malate dehydrogenase gene EMDH is the photoinduction type promotor of Rubisco small subunit.
In the above-mentioned carrier, the carrier that sets out that is used to make up described plant expression vector is that pH2GW7 is (available from Flanders Interuniversity Institute for Biotechnology, VIB).
Plant expression vector of the present invention is pH2-35S-PrbcS-EMDH.
Another object of the present invention provides the application of above-mentioned carrier, and above-mentioned carrier by the agrobacterium mediation converted plant, is obtained the transfer-gen plant that anti-aluminium poison ability improves.
Above-mentioned plant comprises unifacial leaf and dicotyledons such as paddy rice, soybean, wheat and tobacco etc.
Experimental result of the present invention shows that the tobacco plant malate dehydrogenase enzymic activity of changeing the EMDH gene is 1～3.3 times of wild-type tobacco (not changeing the tobacco of EMDH gene).Under the coercing of aluminium poison, the tobacco that changes the EMDH gene can secrete more organic acid, and root growth is good, can improve plant to the resistance of aluminium poison with to the receptivity of phosphorus element.Dedicated carrier of the present invention can be in performance great role aspect the malicious tolerance of the aluminium that improves plant (as tobacco), for plant species improvement provides a new way.
Plant expression vector of the present invention is made up by following method and forms:
One, the amplification of malate dehydrogenase gene EMDH
1, from GenBank, search the full-length gene order of intestinal bacteria EMDH gene, and the following a pair of primer of implementation sequence:
EMDH5：caccATGGAAGTCGCAGTCCTCGGCGC
EMDH3：ggatccTCAATTACTTATTAACGAACTC
5 ' end primer EMDH5, end adds the CACC characteristic sequence, and forms the NcoI restriction enzyme site thus; 3 ' end primer EMDH3, end adds the BamHI restriction enzyme site; DNA is a template amplification with the bacillus coli gene group, obtains the total length of EMDH gene.
Two, the TOPO of EMDH gene clone
Reclaim and purifying EMDH gene fragment, by TOPO clone technology subclone to the pENTR-TOPO carrier
(available from invitrogen company)On, adopt alkaline lysis method of extracting plasmid DNA, detect and sequencing analysis acquisition recombinant plasmid pENTR-TOPO-EMDH (Fig. 4) by PCR.
Three, the structure of intermediate carrier pUC118-PrbcS-T
With restriction enzyme SphI with among the pUC118-PrbcS-T-rbcS-3C (by structures such as Sugita and provide, Sugita et al.1987, MGG, rbcS-3C 209:247-256) cuts out, reconnect the carrier DNA segment that does not contain rbcS-3C with ligase enzyme again, produce intermediate carrier pUC118-PrbcS-T;
Four, in intermediate carrier pUC118-PrbcS-T, introduce the NcoI restriction enzyme site
Utilize a pair of complementary primer NcoI5 and NcoI3 (Fig. 1), introduce the NcoI site by the point mutation technology at the chloroplast(id) positioning sequence initiation codon place of pUC118-PrbcS-T and produce intermediate carrier pUC118-PrbcS-*T;
Five, the structure of intermediate carrier pENTR*-Prbcs-*T
Utilize a pair of complementary primer HindIII5 and HindIII3 (Fig. 2), by the point mutation technology XmnI site in the entry vector pENTR-2B of Gateway (available from the invitrogen company) multiple clone site is changed into HindIII and produce intermediate carrier pENTR*-2B, cut pENTR*-2B and pUC118-PrbcS-*T with HindIII and EcoRI then, reclaim pENTR*-2B and be cut the promoter DNA segment PrbcS-*T that carrier segment pENTR* that the back produces and pUC118-PrbcS-*T are cut generation, pENTR* and PrbcS-*T are coupled together acquisition intermediate carrier pENTR*-Prbcs-*T (Fig. 3) with ligase enzyme.
Six, the structure of entry vector pENTR*-PrbcS-EMDH
With NcoI and BamHI cutting pENTR*-PrbcS-*T and pENTR-TOPO-EMDH, reclaim carrier pENTR*-PrbcS segment and EMDH gene segment, connect PENTR*-PrbcS and EMDH with ligase enzyme, obtain entry vector pENTR*-PrbcS-EMDH.
Seven, the structure of EMDH gene plant expression vector pH2-35S-PrbcS-EMDH
LR by the Gateway technology reacts the PrbcS-EMDH subclone in plant expression vector pH2GW7 (the purpose carrier of Gateway, Billy's stone VIB/Gent company), obtains the plant expression vector pH2-35S-PrbcS-EMDH of EMDH gene.
In above-mentioned carrier, the upstream of EMDH gene is added with photoinduction type promotor PrbcS.The photoinduction type promotor (PrbcS) that the present invention uses is the promotor of the rbcS-3C that separates from the genome of tomato, it is a dna segment by the 1.7kb of HindIII cutting generation, be subcloned on (Sugita et al.1987 among the pUC118, MGG, 209:247-256), the plasmid vector name of this subclone is called pUC118-PrbcS-T-rbcS-3C.For the product after the EMDH genetic expression can be positioned in the blade cell matter, the present invention cuts out the rbcS-3C among the pUC118-PrbcS-T-rbcS-3C with restriction enzyme SphI, reconnect the carrier DNA segment that does not contain rbcS-3C with ligase enzyme then, produce an intermediate carrier pUC118-PrbcS-T, utilize a pair of complementary primer (Fig. 1) then, introduce the NcoI site by the point mutation technology at the chloroplast(id) positioning sequence initiation codon place of pUC118-PrbcS-T and produce intermediate carrier pUC118-PrbcS-*T.
In order to use the plant expression vector of Gateway technique construction EMDH gene, it is basic framework that the present invention adopts the entry vector pENTR-2B of Gateway, utilize a pair of complementary primer (Fig. 2), by the point mutation technology XmnI site in the pENTR-2B multiple clone site is changed into HindIII and produce intermediate carrier pENTR*-2B, cut pENTR*-2B and pUC118-PrbcS-*T with HindIII and EcoRI then, reclaim pENTR*-2B and be cut the promoter DNA segment PrbcS-*T that carrier segment pENTR* that the back produces and pUC118-PrbcS-*T are cut generation, pENTR* and PrbcS-*T are coupled together Gateway entry vector pENTR*-Prbcs-*T (Fig. 3) who contains photoinduction type promotor (PrbcS) sequence of acquisition with ligase enzyme.
Description of drawings
The construction strategy of Fig. 1 intermediate carrier pUC118-PrbcS-*T
With SphI the rbcS-3C among the pUC118-PrbcS-T-rbcS-3C is excised, and make carrier from being connected to form pUC118-PrbcS-T, near the initiator codon of chloroplast(id) positioning sequence, introduce the NcoI site by the point mutation technology and produce intermediate carrier pUC118-PrbcS-*T.
The construction strategy of Fig. 2 intermediate carrier pENTR*-2B
By the point mutation technology XmnI in the pENTR-2B multiple clone site is changed into HindIII.
The construction strategy of Fig. 3 intermediate carrier pENTR*-Prbcs-*T
PrbcS-*T is subcloned on generation intermediate carrier pENTR*-PrbcS-*T among the pENTR*-2B.
The construction strategy of Fig. 4 pENTR-TOPO-EMDH
The amplification of Fig. 5 EMDH gene and TOPO clone
(A) be template amplification EMDH gene with the E.coli genome.The PCR product of 1-2:EMDH gene; 3:DNA Marker (500bp).(B) electrophoresis detection of recombinant plasmid pENTR-TOPO-EMDH.1: over against shining (molecular weight is 3.6 plasmid); 2-3: recombinant plasmid pENTR-TOPO-EMDH.(C) PCR of recombinant plasmid pENTR-TOPO-EMDH detects.1:DNA Marker (Φ X174/Hae III); 2: with pENTR-TOPO-EMDH is template, the PCR product that arrives with primer attL1 and EMDH3 primer amplification; 3: with pENTR-TOPO-EMDH is template, the PCR product that arrives with primer EMDH5 and EMDH3 primer amplification;
The construction strategy of Fig. 6 entry vector pENTR*-PrbcS-EMDH
The detection of Fig. 7 pENTR*-PrbcS-EMDH
(A) cut detection recombinant plasmid pENTR*-PrbcS-EMDH with BamH I and NcoI enzyme.1: λ DNA/HindIII Marker; 2: negative control plasmid (pUC118-PrbcS-T-rbcS-3C); 3-5: recombinant plasmid pENTR*-PrbcS-EMDH.(B) detect recombinant plasmid pENTR*-PrbcS-EMDH with PCR.1-3: with pENTR*-PrbcS-EMDH is template, the PCR product that increases with primer PrbcS5 and EMDH3; 4: with pENTR*-PrbcS-EMDH is template, the PCR product that increases with primer EMDH5 and EMDH3; 5: λ DNA/HindIII Marker.
Fig. 8 makes up the plant expression vector of EMDH gene with the LR reaction of Gateway
The detection of Fig. 9 EMDH plant expression vector pH2-35S-PrbcS-EMDH and the conversion of Agrobacterium
(A) electrophoresis detection of pH2-35S-PrbcS-EMDH plasmid.1.pH2GW7 plasmid DNA; 2-4.pH2-35S-PrbcS-EMDH plasmid DNA; 5.pENTR-TOPO-EMDH plasmid DNA.(B) PCR of pH2-35S-PrbcS-EMDH plasmid detects.1.DL2000DNA Marker; 2. be template with plasmid pH2-35S-PrbcS-EMDH, the PCR product that utilizes EMDH5 and EMDH3 primer amplification to obtain; 3-5. with plasmid pH2-35S-PrbcS-EMDH is template, the PCR product that utilizes PrbcS5 and EMDH3 primer amplification to obtain.(C) the Agrobacterium PCR that transforms the pH2-35S-PrbcS-EMDH plasmid detects.1.DL2000DNAMarker; 2. over against shining (with plasmid pH2-35S-PrbcS-EMDH is template, the PCR product that utilizes EMDH5 and EMDH3 primer amplification to obtain); 3-5. with the dried bacterium bacterium colony of farming is template, the PCR product that utilizes EMDH5 and EMDH3 primer amplification to obtain.
The insertion situation of Figure 10 EMDH in transgene tobacco detects
(A) electrophoresis detection of tobacco plant genomic dna.1. λ DNA/HindIII digest DNA Marker; 2-5. tobacco gene group DNA sample.(B) the genomic PCR of tobacco plant detects.1.500bp DNAMarker; 2. over against shining (with plasmid pH2-35S-PrbcS-EMDH is template, the PCR product that utilizes EMDH5 and EMDH3 primer amplification to obtain); 3-5. with the transgene tobacco genome is template, the PCR product that utilizes EMDH5 and EMDH3 primer amplification to obtain.6. negative contrast (not genetically modified wild-type tobacco RNA sample).
The transcriptional level of Figure 11 EMDH in transgene tobacco detects
(A) detection of tobacco RNA quality.1-3. change the RNA sample of EMDH tobacco plant; 4. the RNA sample of wild-type tobacco (negative contrast).(B) RT-PCR of transgene tobacco detects.1. negative contrast; 2-4. the RT-PCR product of transgene tobacco; 5. over against shining (with plasmid pH2-35S-PrbcS-EMDH is template, the PCR product that utilizes EMDH5 and EMDH3 primer amplification to obtain); 6 λ DNA/HindIII digest DNAMarker.
The enzyme assay of Figure 12 EMDH in transgene tobacco
The mensuration of Figure 13 EMDH transgene tobacco oxysuccinic acid secretory volume
Figure 14 EMDH transgene tobacco is coerced the dyeing situation of the tip of a root down at the aluminium poison
Figure 15 EMDH transgene tobacco is coerced the mensuration of root growth amount down at the aluminium poison
The growing state of Figure 16 EMDH transgene tobacco in the substratum that has the aluminium poison to coerce
A: do not containing the upward tobacco seedling of growth 17d of aluminum ions Blaydes substratum (pH4.0); B: after the 1mM aluminum ion is handled 1h, root is dyeed with 0.1% chrome azurol S at the tobacco seedling that does not contain the last growth of aluminum ions Blaydes substratum (pH4.0) 17d; C: containing 300 μ mol/LAl
3+Blaydes substratum (pH4.0) go up the tobacco seedling of growth after 17 days; D: containing 500 μ mol/L Al
3+Blaydes substratum (pH4.0) go up the tobacco seedling of growth after 17 days.
The growing state of Figure 17 EMDH transgene tobacco in the soil matrix that has the aluminium poison to coerce
Embodiment
Reagent and instrument
Reagent mainly is divided into molecular biology experiment reagent, plant genetic transforms required substratum and required all ingredients is identified and detected to transgenic plant.Various restriction enzymes, Taq archaeal dna polymerase, ThermoScript II, RNA enzyme inhibitors, dNTP etc. are precious biotechnology company limited (Dalian) product, plasmid extraction kit is available from vast Tyke Bioisystech Co., Ltd, and TRIzoL Reagent RNA extracts test kit, pENTR
TMDirectional TOPO
Cloning Kits and Gateway LR clonase Enzyme Mix kit are available from invitrogen company.All the other reagent are homemade analytical pure.
Instrument is molecular biology and genetically engineered laboratory common instrument.
All precious biotech firm synthesizes all primer sequences in Dalian.Method therefor is ordinary method if no special instructions in the embodiment of the invention.
The structure of embodiment 1, intermediate carrier pUC118-PrbcS-T
With plasmid extraction test kit (vast Imtech) purifying (by test kit specification sheets operation) pUC118-PrbcS-T-rbcS-3C (by structure such as Sugita and provide, Sugita et al.1987, MGG, 209:247-256), with restriction enzyme SphI (Fermentas) rbcS-3C among the pUC118-PrbcS-T-rbcS-3C is cut out, separate the carrier pUC118-PrbcS-T and the rbcS-3C fragment of having cut by agarose gel electrophoresis, reclaim the carrier pUC118-PrbcS-T of 4.6kb, (by the operation of test kit specification sheets) do not contain the carrier DNA segment of rbcS-3C to use the ligase enzyme test kit of precious biological (TaKaRa) to connect then, produce an intermediate carrier pUC118-PrbcS-T (Fig. 1), transform high-level efficiency (10 with the ligation mixture
8) competent escherichia coli cell (DH5 α, available from sky root biochemical technology company), be applied to and be added with penbritin (Amp transforming good intestinal bacteria, 100 μ g/ml) on the flat board, in 37 ℃ of incubated overnight, screening Amp resistance recon bacterium colony, from Amp resistance recon bacterium colony, extract plasmid, carry out enzyme with SphI and cut detection, the plasmid of successful connection only produces a 4.6kb band on agarose gel electrophoresis figure, select the plasmid vector pUC118-PrbcS-T of successful connection, again transformed into escherichia coli DH5 α, choose single bacterium colony and carry out liquid culture, use the test kit plasmid purification.
With plasmid purification pUC118-Prbcs-T is template, and (NcoI5 and NcoI3 Fig. 1), entrust TaKaRa synthetic according to a pair of complementary primer that is used for point mutation of chloroplast(id) positioning sequence design.In the point mutation reaction mixture, add the plasmid purification pUC118-PrbcS-T of 25ng as template, the point mutation primer NcoI5 and NcoI3, the 1 μ l dNTP (2.5mM) that add 125ng simultaneously, the KOD polysaccharase (Japanese Japan is spun) of 10 * KOD of 5 μ l reaction Buffer and 1 μ l, adding distilled water, to make the reaction final volume be 50 μ l.In 95 ℃ of heating 30 seconds, then according to 95 ℃, 30 seconds, 55 ℃, 1 minute, 68 ℃, 10 minutes program was carried out 15 round-robin reactions, prolongs reaction 10 minutes at 68 ℃ at last, the synthetic subchain that contains the mutational site on the PCR instrument.After reaction is finished reaction mixture was placed cooled on ice 2 minutes, add the restriction enzyme Dpn I (10U/ μ l) of 1 μ l and the Dpn I reaction buffer of 5 μ l to reaction mixture, in 37 ℃ of insulations 1 hour, degraded did not contain the fundamental chain in mutational site.Transform high-level efficiency (10 with reaction mixture
8) competent escherichia coli cell (DH5 α, available from sky root biochemical technology company), be applied to and be added with penbritin (Amp transforming good intestinal bacteria, 100 μ g/ml) on the flat board, screening Amp resistance recon bacterium colony, from Amp resistance recon bacterium colony, extract plasmid, carry out enzyme with NcoI (Fermentas) and cut detection, the successful plasmid that suddenlys change can be cut and produce the band of a 4.6kb by NcoI on agarose gel electrophoresis figure, select the successful plasmid vector pUC118-PrbcS-*T of sudden change, again transformed into escherichia coli DH5 α chooses single bacterium colony and carries out liquid culture, uses the test kit plasmid purification.After the chloroplast(id) positioning sequence initiation codon place of pUC118-PrbcS-T introduced the NcoI site, for making up the control by promotor PrbcS, the plant expression vector that the target protein of expression is positioned at the goal gene in the blade cell matter laid the foundation.
With plasmid purification pENTR-2B (available from invitrogen company) is template, is used for the complementary primer (Fig. 2) of point mutation according near the sequences Design the XmnI a pair of (HindIII5 and HindIII3), and it is synthetic to entrust the Shen, Shanghai can widely collect company.In the point mutation reaction mixture, add the plasmid purification pENTR-2B of 25ng as template, the point mutation primer HindIII5 and HindIII3, the 1 μ ldNTP (2.5mM) that add 125ng simultaneously, the KOD polysaccharase (Japanese Japan is spun) of 10 * KOD of 5 μ l reaction Buffer and 1ul, adding distilled water, to make the reaction final volume be 50 μ l.In 95 ℃ of heating 30 seconds, then according to 95 ℃, 30 seconds, 55 ℃, 1 minute, 68 ℃, 10 minutes program was carried out 15 round-robin reactions, prolongs reaction 10 minutes at 68 ℃ at last, the synthetic subchain that contains the mutational site on the PCR instrument.After reaction is finished reaction mixture was placed cooled on ice 2 minutes, add the restriction enzyme Dpn I (10U/ μ l) of 1 μ l and the DpnI reaction buffer of 5 μ l (brilliant beautiful) to reaction mixture, in 37 ℃ of insulations 1 hour, degraded did not contain the fundamental chain in mutational site.Transform high-level efficiency (10 with reaction mixture
8) competent escherichia coli cell (DH5 α, available from sky root biochemical technology company), be applied to and be added with penbritin (Amp transforming good intestinal bacteria, 100 μ g/ml) on the flat board, screening Amp resistance recon bacterium colony, from Amp resistance recon bacterium colony, extract plasmid, carry out enzyme with HindIII (brilliant beautiful) and cut detection, the successful plasmid that suddenlys change can be cut and produce the band of a 3.5kb by HindIII on agarose gel electrophoresis figure, select the successful plasmid vector pENTR*-2B of sudden change, again transformed into escherichia coli DH5 α chooses single bacterium colony and carries out liquid culture, uses the test kit plasmid purification.
The structure of embodiment 4, intermediate carrier pENTR*-Prbcs-*T
Cut the plasmid vector pENTR*-2B and the pUC118-PrbcS-*T of purifying with HindIII and EcoRI, separate the carrier that has cut and insert fragment by agarose gel electrophoresis, recovery pENTR*-2B is cut the carrier segment pENTR* (2.3kb) of back generation and the promoter DNA segment PrbcS-*T (1.7kb) that pUC118-PrbcS-*T is cut generation, uses the ligase enzyme test kit connection pENTR* and the PrbcS-*T of precious biological (TaKaRa) to produce intermediate carrier pENTR*-Prbcs-*T (Fig. 3) then.Transform high-level efficiency (10 with the ligation mixture
8) competent escherichia coli cell (DH5 α, available from sky root biochemical technology company), be applied on the flat board that is added with kantlex (Km, 50 μ g/ml) transforming good intestinal bacteria, in 37 ℃ of incubated overnight, screening Km resistance recon bacterium colony, extract plasmid from Km resistance recon bacterium colony, detect with HindIII and SphI double digestion, the plasmid of successful connection only produces two bands on agarose gel electrophoresis figure, article one, be the carrier band of 2.3kb, another is the insertion fragment PrbcS-*T of 1.7kb.Select the plasmid vector pENTR*-PrbcS-*T of successful connection, transformed into escherichia coli DH5 α chooses single bacterium colony and carries out liquid culture again, uses the test kit plasmid purification.
The amplification of embodiment 5, EMDH gene DNA and TOPO clone
The amplification of EMDH gene DNA and TOPO clone's strategy as shown in Figure 4, the first-selected full-length gene order of from GenBank, searching EMDH, and design a pair of primer, sequence is as follows:
EMDH5：5’-
CACCATGGAAGTCGCAGTCCTCGGCGC-3’
EMDH3：5’-GGATCCTCAATTACTTATTAACGAACTC-3’
5 ' end primer EMDH5 end adds the CACC characteristic sequence, and forms the NcoI restriction enzyme site thus; 3 ' end primer EMDH3 end adds the BamHI restriction enzyme site.
From E.coli, extract genomic dna.Preparation 100ml bacterium incubated overnight liquid, the centrifugal 10min of 5000rpm removes supernatant liquor; Add 9.5ml TE suspension precipitation, and add 0.5ml 10%SDS, 50 μ l (20mg/ml or 1mg dry powder) Proteinase Ks (TaKaRa company), mixing, 37 ℃ of insulation 1h; Add 1.5ml 5M NaCl, mixing; (4.1g NaCl is dissolved in 80ml H to add 1.5ml CTAB/NaCl solution
2O slowly adds 10g CTAB, adds water to 100ml), mixing, 65 ℃ of insulation 20min; Use equal-volume phenol: chloroform: primary isoamyl alcohol (25: 24: 1) extracting, the centrifugal 10min of 5000rpm moves to clean centrifuge tube with supernatant liquid; Use the equal-volume chloroform: primary isoamyl alcohol (24: 1) extracting, get supernatant liquor and move in the clean pipe; Add 1 times of volume Virahol, put upside down mixing, static 10min under the room temperature, deposit D NA; Pull the DNA precipitation out with glass rod, after the 70% ethanol rinsing, blot, be dissolved in 1ml and contain in the TE solution of RNase-20 ℃ of preservations; Can't pull out as the DNA precipitation, but 5000rpm is centrifugal, makes the DNA precipitation.Make PCR with EMDH gene upstream and downstream Auele Specific Primer EMDH5 and EMDH3, the KOD polymeric enzymatic amplification that spins (TOYOBO) with Japanese Japan obtains the total length 0.94kb (Fig. 4 A) of EMDH gene.Recovery and purifying EMDH full-length gene fragment, the product of EMDH is flat terminal dna fragmentation, therefore uses the pENTR of Invitrogen company
TMDirectional TOPO
The Cloning test kit, the flat terminal dna fragmentation that is recovered to is subcloned in the pENTR/D-TOPO carrier, experimental implementation is undertaken by the specification sheets of test kit, after spending the night, room temperature reaction uses reaction mixture transformed into escherichia coli competence DH5 α (available from sky root biochemical technology company), adopt alkaline lysis method of extracting plasmid DNA, through 1% agarose gel electrophoresis (Fig. 4 B), choose the big or small recombinant plasmid pENTR-TOPO-EMDH that conforms to theoretical value and do further PCR detection, use the upstream Auele Specific Primer attL1 and the EMDH3 that are positioned at the attL1 site to make PCR for first group, the recombinant plasmid of subclone success all can amplify the dna fragmentation (Fig. 4 C) about 1.1kb, make PCR with EMDH gene upstream and downstream Auele Specific Primer EMDH5 and EMDH3 for second group, the recombinant plasmid of subclone success all can amplify the dna fragmentation (Fig. 4 C) about 0.9kb, proves the full-length gene of EMDH gene through sequential analysis.
The structure of the ABC of cloning vector pENTR*-PrbcS-EMDH of the Gateway of embodiment 6, EMDH gene
Cut the plasmid vector pENTR*-PrbcS-*T and the pENTR-TOPO-EMDH (Fig. 6) of purifying with NcoI and BamHI, separate the carrier that has cut and insert fragment by agarose gel electrophoresis, recovery pENTR*-PrbcS-*T is cut the carrier segment pENTR*-PrbcS (4.0kb) of back generation and the dna segment (0.9kb) of the EMDH gene that pENTR-TOPO-EMDH is cut generation from gel, uses the ligase enzyme test kit connection pENTR*-PrbcS of precious biological (TaKaRa) and the dna segment of EMDH gene to produce entry vector pENTR*-PrbcS-EMDH (Fig. 6) then.Transform high-level efficiency (10 with the ligation mixture
8) competent escherichia coli cell (DH5 α, available from sky root biochemical technology company), be applied to and be added with kantlex (Km transforming good intestinal bacteria, 50 μ g/ml) on the flat board, in 37 ℃ of incubated overnight, screening Km resistance recon bacterium colony, from Km resistance recon bacterium colony, extract plasmid, detect with NcoI (Fermentas) and BamHI (Fermentas) double digestion, the plasmid of successful connection only produces two bands on agarose gel electrophoresis figure, article one, be the carrier band of 4.0kb, another is the EMDH gene fragment (Fig. 7 A) of 0.9kb.Select the plasmid vector pENTR*-PrbcS-EMDH of successful connection, make PCR with two groups of primers and detect.First group of Auele Specific Primer EMDH5 and EMDH 3 with the EMDH upstream and downstream carries out pcr amplification, selected plasmid can both amplify the EMDH band (Fig. 7 B) of a 0.9kb, second group with being arranged in the Auele Specific Primer PrbcS5 in PrbcS zone and the downstream Auele Specific Primer EMDH 3 of EMDH carries out pcr amplification, and selected plasmid can amplify the band (Fig. 7 B) of a 1.1kb.After being confirmed to be the plasmid of successful connection, transformed into escherichia coli DH5 α chooses single bacterium colony and carries out liquid culture again, with test kit plasmid purification pENTR*-PrbcS-EMDH.
The structure of embodiment 7, EMDH gene plant expression vector pH2-35S-PrbcS-EMDH
LR reaction by the Gateway technology is PrbcS-EMDH subclone in the plant expression vector pH2GW7 (the purpose carrier of Gateway, Belgian VIB/Gent company) (Fig. 8).Concrete way is: with the purpose carrier pH2GW7 of plasmid extraction test kit purifying Gateway, in the LR of Gateway reaction system, add pENTR*-PrbcS-EMDH and each 150ng of pH2GW7,1 μ l LR Clonase II Enzyme Mix (Invitrogen), be mixed and spend the night, PrbcS-EMDH is incorporated into the plant expression carrier plasmid pH2-35S-PrbcS-EMDH (Fig. 8) of acquisition EMDH among the pH2GW7 by the effect of intergrase in 25 ℃ of reactions.Transform high-level efficiency (10 with reaction mixture
8) competent escherichia coli cell (DH5 α, available from sky root biochemical technology company), be applied to and be added with spectinomycin (Spe transforming good intestinal bacteria, 50 μ g/ml) on the flat board, in 37 ℃ of incubated overnight, screening Spe resistance recon bacterium colony, from Spe resistance recon bacterium colony, extract plasmid (Fig. 9 A), select the big or small integrated plasmid pH2-35S-PrbcS-EMDH that conforms to the theoretical prediction value and carry out the PCR detection, first group of Auele Specific Primer EMDH5 and EMDH3 with the EMDH upstream and downstream carries out pcr amplification, selected plasmid can both amplify the EMDH band (Fig. 9 B) of a 0.9kb, second group with being arranged in the Auele Specific Primer PrbcS5 in PrbcS zone and the downstream Auele Specific Primer EMDH 3 of EMDH carries out pcr amplification, and selected plasmid can amplify the band (Fig. 9 B) of a 1.1kb.After being confirmed to be the plasmid of successful integration, transformed into escherichia coli DH5 α chooses single bacterium colony and carries out liquid culture again, uses the test kit plasmid purification.The selection markers gene that pH2GW7 carries is hygromycin gene (Hgr), the available like this plate screening transgenic plant that are added with Hgr.
The plant expression vector of embodiment 8, usefulness EMDH gene transforms Agrobacterium
The competent cell of preparation Agrobacterium, the electricity consumption impulse method changes the above-mentioned plant expression vector pH2-35S-PrbcS-EMDH that builds in the Agrobacterium (C58Cl (pPMP90)) over to, screens transformant being added with on the flat board of spectinomycin.The plasmid that takes a morsel adds in the Agrobacterium competent cell, gently mixing; The electricity that mixture is joined precooling transforms in the cup, at the bottom of knocking a glass body gently mixed liquid being dropped down onto glass; Electricity is transformed cup place electric conversion instrument (BIO-RAD) chute, with the electric shock cup of 1mm and 200 ohm, the parameter of 2.5kV/0.2cm shocks by electricity, take out electricity after the electric shock immediately and transform cup, add the 0.5mlSOC substratum rapidly, mixing is transferred in the centrifuge tube of 1.5ml; 28 ℃, the 200rpm shaking table is cultivated 3-5h; Under the room temperature, the centrifugal 1min of 7500rpm abandons most of supernatant, keeps 100 μ l with cell suspension; Agrobacterium has been coated on the LB solid medium of spectinomycin (Spe, 50 μ g/ml), cultivated for 28 ℃ and obtained single bacterium colony in 2 days; At first put into 20 μ l ddH with toothpick picking Agrobacterium bacterium colony
2Among the O, handle for 98 ℃ and take out the template of 10 μ l Agrobacterium lysates after 15 minutes as the PCR reaction.Make PCR with EMDH gene upstream and downstream Auele Specific Primer and detect, transform the EMDH gene band (Fig. 9 C) that successful bacterium colony all can amplify 0.9kb, the transformant bacterium colony of confirming through bacterium colony PCR is used to transform plant.
Embodiment 9, usefulness contain the Agrobacterium conversion tobacco of EMDH gene plant expression vector
The single colony inoculation of Agrobacterium that picking carries plasmid pH2-35S-PrbcS-EMDH (contains Spe, 100 μ g/ml) in the LB of 50ml substratum, 180rpm cultivates 24h, treats bacterium liquid OD for 28 ℃
600To about 1.0, centrifugal 10min (3000rpm), precipitation thalline.Suspend centrifugal 10min (3000rpm), precipitation thalline again with the MS liquid nutrient medium about 10ml.Repeat above operation 2～3 times.The MS liquid nutrient medium resuspension that adds certain volume at last makes the OD of thalline
600Value is 0.5.The aseptic seedling of preparation tobacco (Nicotiana tabacumcv.Xanth) by agriculture bacillus mediated, with leaf dish method transformation of tobacco, transforms Flos Pelargonii with the petiole method, obtains seedling by tissue culture then, and further screening obtains required transgenic plant.The blade of aseptic tobacco is cut into pieces the leaf dish, the petiole of Flos Pelargonii is cut into segment, in the Agrobacterium bacterium liquid for preparing, contaminate 15-20min, after blotting with aseptic thieving paper, being tiled in the last dark of callus inducing medium MS1 (MS+NAA02.1 μ g/ml+BAP0.02 μ g/ml) cultivated 2 days altogether, explant is transferred on the bud inducing culture MS4 (MS+NAA0.53 μ g/ml+BAP0.5 μ g/ml) that contains Totomycin (25 μ g/ml) carries out inducing of bud, about 15 days subcultures once.After having treated that blastogenesis becomes, change on the MS substratum that contains Totomycin (25ug/ml) and carry out inducing of root.
In order to confirm that transgene tobacco strain system by hygromycin selection contains the dna fragmentation of the goal gene that we import really, we do further to identify to the transgene tobacco that screens with PCR method.At first adopt the CTAB method to extract Plant Genome: to take by weighing and place the 1.5ml centrifuge tube about plant leaf 100mg, add liquid nitrogen and be ground to Powdered with special pestle; Add 900 μ l and be preheating to 2 * CTAB damping fluid (NaCl 1.4M, CTAB 2% for Tris-HCl pH 7.5 100mM, EDTA 20mM) of 65 ℃, 65 ℃ of degree heating in water bath take out cooling after 20 minutes; Add 500 μ l chloroforms-primary isoamyl alcohol mixed solution (24: 1) and shake up, 4 ℃ of centrifugal 10min (7500rpm) back is shifted supernatant and is managed to 1.5ml EP; Add 500 μ l chloroforms-primary isoamyl alcohol mixed solution (24: 1) once more and shake up 4 ℃ of centrifugal 10min (7500rpm); Take out supernatant and place new EP pipe, add 1/10 volume 3M pH5.2 sodium-acetate and equal-volume Virahol, shake up back 4 ℃ of centrifugal 20min (12000rpm); Abandon supernatant, after twice of 75% ethanol cleaning, drying is with the dissolving of TE damping fluid and the degradation of rna that contain RNase, the genome DNA sample that is obtained detected through gel electrophoresis concentration (Figure 10 A).With the plant genome DNA is template, upstream and downstream primer EMDH5 and EMDH3 with the EMDH gene do the PCR detection, the theoretical length of PCR product is 0.9kb, the plant that successfully changes goal gene over to all can amplify the band (Figure 10 B) of 0.9kb, and the transfer-gen plant of confirming through PCR is used for the RT-PCR analysis.
In order to investigate the transcribe situation of goal gene in transgene tobacco strain system, from transgenic plant, extract total RNA, be used for RT-PCR behind the green one-tenth cDNA that reverses and analyze, detect the transcriptional level of EMDH gene in transgenic plant.Adopt TRIzoL Reagent (Invitrogen) to extract RNA, get the about 0.1g of young leaf of plant, the TRIzoL extracting solution that adds 1ml grinds in mortar, room temperature moves into centrifuge tube after leaving standstill 5min, add the 0.2ml chloroform again, the vibration mixing, centrifugal 15min (12000rpm) shifts supernatant liquor to new pipe, add the 0.5ml Virahol, the mixing room temperature is placed 10min, and 4 ℃ of centrifugal 10min (12000rpm) abandon supernatant, precipitation is cleaned with 75% ethanol 1ml, 4 ℃ of centrifugal 5min (7500rpm) abandon ethanol vacuum-drying and precipitate or dry naturally, with 20 μ l diethylpyrocarbonate (DEPC) treating water dissolving RNA.The RNA sample that is obtained uses Reverse Transcriptase to carry out the synthetic of cDNA with detected through gel electrophoresis quality and concentration (Figure 11 A), get the about 0.1 μ g-5 μ g of the total RNA of plant, oligo (dT) 50ng, 10mM dNTP mix 1 μ l complements to 10 μ l with the DEPC treating water, behind the mixing, of short duration centrifugal it is collected in managed at the end, places 65 ℃ of heating 5min, ice bath 10min, add reaction mixture 9 μ l (5 * reaction buffer, 4 μ l, 25mMMgCl
24 μ l, 0.1M DTT 2 μ l, RNA enzyme inhibitors 1 μ l), with the said mixture mixing, of short duration centrifugal it is collected in managed at the end, 25 ℃ of insulation 2min add 1 μ l M-MuLV Reverse Transcriptase, with the said mixture mixing, of short duration centrifugal it is collected in managed at the end, 25 ℃ of insulation 20min, 42 ℃ of insulation 70min synthesize cDNA then.With cDNA is template, makes RT-PCR with the upstream and downstream primer EMDH5 of EMDH gene and EMDH3 and analyzes, and investigates the transcript whether goal gene is arranged in the transgene tobacco.The result proves that most of transgenic tobacco plant all has the transcript (Figure 11 B) of goal gene.
The activation analysis of embodiment 11, transgene tobacco malate dehydrogenase (malic acid dehydrogenase) (MDH)
Choosing through the RT-PCR analysis revealed has the transgenic tobacco plant of the transcript of goal gene to measure the activity of citrate synthase.Extracting soluble proteins from tobacco leaf is got the 1g tobacco leaf, adds 1ml albumen extract [100mM Tris-HCl (pH 7.5); 10% (V/V) glycerine; The 10mM mercaptoethanol; 1mM PMSF; 5% (W/V) PVP] grind, be transferred in the EP pipe the centrifugal 25min of 13000r/min (4 ℃).Supernatant is moved on in the new EP pipe, with the protein concn in the Bradford method mensuration plant supernatant.
The malate dehydrogenase enzyme assay press Tesfaye etc. (Plant physiology, 2001, method 127:1836-1844) is carried out, its principle is: oxaloacetic acid and NADH can generate L MALIC ACID and NAD under the effect of malate dehydrogenase (malic acid dehydrogenase).In this reaction, thereby by measuring the relative activity that wavelength is the reduction acquisition MDH of NADH under the 340NM.
The reaction system of measuring the MDH enzymic activity is: 0.05M Tris-HCl (pH 8.0), 4mM oxaloacetic acid, 0.1mM NADH.Add before the protein sample,, in reaction system, add 100 μ g tobacco protein samples then, detect the OD value at 340nm place once more with the absorption value (OD value) of uv-spectrophotometric instrument assaying reaction solution at wavelength 340nm place.The difference of calculating twice reading obtains MDH enzymic activity data.
We have selected three transgenic lines altogether and have measured its MDH enzymic activity, and the result shows that transgene tobacco MDH enzymic activity is 1～3.3 times (Figure 12) of wild-type tobacco, and the visible expression of EMDH in transgenic tobacco plant improved the MDH activity greatly.
Malic acid content adopts enzyme kinetics-determined by ultraviolet spectrophotometry (document).At first, the 0.35mL sample is mixed with 0.45mL damping fluid Tris-HCl (pH 9.0) and 30 μ L oxidized form of nicotinamide-adenine dinucleotide (NAD) solution (30mg/mL), and read the OD value (A1) that wavelength is the 340nm place.Then add 2 μ L malate dehydrogenase (malic acid dehydrogenase) suspension (16.5U/uL) in above-mentioned reaction soln and mix.After 15 minutes, read the OD value A2 at 340nm place.Measure OD value A1 ', the A2 ' of oxysuccinic acid standard model simultaneously.According to for the increased value (A2-A1) of test agent and oxysuccinic acid standardized solution absorbancy and (A2 '-A1 '), calculate content for oxysuccinic acid in the test agent.
Transgenosis seedling and wild-type tobacco seedling with big or small uniformity place AlCl respectively
3Concentration is the CaCl of 0,50,100,200,300 μ mol/L
2In the solution, collect CaCl after 24 hours
2Solution also concentrates root excretory oxysuccinic acid.Analytical results shows that the EMDH transgene tobacco all had higher oxysuccinic acid secretory volume (Figure 13) during each was handled.Wherein transgenic line E3 excretory oxysuccinic acid when 200 μ M/L aluminum ions are handled is 45 times of wild-type, and the excretory oxysuccinic acid is 15 times of wild-type tobacco when 300 μ mol/L aluminum ions are handled.
Embodiment 13, transgene tobacco detect the resistance of aluminium poison
Plant generally adopts tip of a root dyeing situation to weigh to the tolerance of aluminium poison.The painted dyestuff of the tip of a root has phenodin (hematoxylin) and chrome azurol (eriochrome cyanine S), and both all are suitable for susceptibility or the patience of plant identification to aluminium.Its principle is that dyestuff can enter cell interior by the cytolemma of death, and combine and produce blue (phenodin) or red (chrome azurol S) material with aluminium in residuing in dead cell nuclear, this reaction pair aluminium has specificity (the Cancado GMA of height, Loguercio LL, Martins PR.1999.Hematoxylin staining as a phenotypic indes for aluminum toleranceselection in tropical maize (Zea mays L.) .Theor Appl Genet.99:747-754; Ma JF, Zhen SJ, Li XF.1997.A rapid hydroponic screening for aluminumtolerance in barley.Plant soil.191:133-137), and tip of a root dye levels and aluminium are proportionate to the murder by poisoning degree of plant.
With big or small uniformity and take root good transgene tobacco and wild-type tobacco seedling, place AlCl respectively
3Concentration is respectively the CaCl of 0 (contrast), 50,100,200,300 μ mol/L
2Handle 24h in the solution (pH value 4.3), again through 0.1% chrome azurol S staining fluid dyeing 15min.Behind distilled water flushing, examine under a microscope and write down tip of a root dyeing situation.The result shows that the tip of a root of wild-type tobacco all by purple, the aluminum ion intoxicating phenomenon occurs, and the tip of a root of EMDH transgenic line E1, E3, E13 is not painted or have only very shallow painted (Figure 14).Even these results tentatively prove only overexpression EMDH in blade, also can strengthen the tolerance of transgenic plant to the aluminium poison.
The extension speed of root under the aluminium poison is coerced is to weigh plant to one of another kind of index of aluminium poison tolerance, so we also are determined at the aluminium poison and coerce down transgenic tobacco plant root growth speed.Transgene tobacco and wild-type tobacco seedling with big or small uniformity place AlCl respectively
3Concentration is the CaCl of 0,50,100,200,300 μ mol/L
2In the solution.After handling 24 hours, measure increment for examination plant root, triplicate, experimental result during as Figure 15 shown in.From experimental result as can be seen, when aluminium ion concentration was 200 μ mol/L and 300 μ mol/L, aluminum ion was 100% to the inhibiting rate of wild-type tobacco root growth, and the tip of a root of wild-type tobacco has become the fish hook shape, and a large amount of being completely cured appears in the root flavescence in solution.Yet the root of EMDH transgene tobacco but keeps healthy relatively state, and keeps higher increment.
Embodiment 15, the growing state analysis of transgenic tobacco plant under the aluminium poison is coerced
One, the growth experiment of transgene tobacco seedling on aluminiferous acidic culture
With big or small uniformity and transgene tobacco seedling that takes root good and wild-type tobacco seedling, transfer to AlCl respectively
3Concentration is in the Blaydes substratum (Parrot WA and BoutonJH.1990.Aluminum tolerance in alfalfa as expressed in tissue culture.Plant Sci.30:387-389) of 0,300,500 μ mol/L, observes its upgrowth situation behind the cultivation 17d.Go up the tobacco seedling of growth 17d shown in Figure 16 A not containing aluminum ions Blaydes substratum (pH4.0), carry out tip of a root dyeing with 0.1% chrome azurol S after the 1mM aluminum ion is handled 1h, the dyeing comparison of EMDH transgenosis strain E1 root system is according to plant wild-type light (Figure 16 B).The E1 seedling is containing 300 μ mol/L (Figure 16 C) and 500 μ mol/L (Figure 16 D) Al
3+Blaydes substratum (pH4.0) go up growth after 17 days, growing state is good, well developed root system; And wild-type tobacco growth under the aluminium poison is coerced obviously is obstructed, and root system development is bad.
Two, the potted plant experiment of transgene tobacco seedling on aluminiferous acidic matrix
With big or small uniformity and the transgene tobacco seedling that takes root good and the transfer of wild-type tobacco seedling in new pearlite interstitial substance, in the greenhouse, carry out pot experiment, per week waters the Blaydes substratum nutritive medium that contains AlPO4 and observes its upgrowth situation after twice, four month.EMDH transfer-gen plant E1, E3 well-grown, and bud has appearred.Yet the wild-type tobacco plant strain growth obviously is obstructed, and not only plant is short and small, and hypoevolutism, do not budding (Figure 17).To the plant height of measurement shows EMDH transgene tobacco in the soil matrix that the aluminium poison is coerced of tobacco plant height 1.2～1.5 times of wild-type tobacco.As seen the EMDH transgenic tobacco plant is coerced well-grown down at the aluminium poison, can also normally blossom and bear fruit.
Claims (6)
1. a carrier that is used to improve the anti-aluminium poison of plant ability is the plant expression vector with photoinduction type promotor and malate dehydrogenase gene.
2. carrier according to claim 1 is characterized in that: described malate dehydrogenase gene derives from intestinal bacteria.
3. carrier according to claim 2 is characterized in that: the described GenBank accession number that derives from colibacillary malate dehydrogenase gene (EMDH) is Y00129.
4. according to claim 1 or 2 or 3 described carriers, it is characterized in that: the upstream of described malate dehydrogenase gene is the photoinduction type promotor of Rubisco small subunit.
5. carrier according to claim 4 is characterized in that: the carrier that sets out that is used to make up described plant expression vector is pH2GW7.
6. carrier according to claim 5 is characterized in that: described plant expression vector is pH2-35S-PrbcS-EMDH.
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