CN101265479A - Plant expression vector of citric acid synthesis gene and application thereof - Google Patents
Plant expression vector of citric acid synthesis gene and application thereof Download PDFInfo
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- CN101265479A CN101265479A CNA2007100664197A CN200710066419A CN101265479A CN 101265479 A CN101265479 A CN 101265479A CN A2007100664197 A CNA2007100664197 A CN A2007100664197A CN 200710066419 A CN200710066419 A CN 200710066419A CN 101265479 A CN101265479 A CN 101265479A
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Abstract
The invention discloses special vector pPZP211-PrbcS-cs which can enhance the aluminum toxicity resistance ability of plants. The vetor is plant expression vector containing tobacco citrate synthetase gene cs. The invention clones cs gene from the tobacco, a photoinduction promoter (the promoter of Rubisco small subunit) is used for controlling excessive expression of the gene cs in tobacco. Citric acid is synthesized and excreted out of cells. The citric acid enters the soil by roots and wraps the aluminium ion in soil with chemical bonds. Therefore, the damage to the plants, caused by high concentration aluminum in soil, is alleviated. The laboratory result shows that citrate synthase of the tobacco lamina with transferring cs gene is 1 to 5.5 times the activity of wild tobacco. Under the intimidation of the aluminum toxicity which is 100 to 300 micrometers, the tobacco with the transferring cs gene can excrete more organic acid, so that the roots grow better and the resistance ability against aluminum toxicity is strengthened remarkably. The special vector provided by the invention can enhance the aluminum toxicity resistance ability of the plants; the special vector has wide application potential in genetic improvement of plant varieties, more particularly for the crop varieties planted in the acid soil of southern China.
Description
Technical field:
The invention belongs to plant genetic engineering field, relate to a kind of plant expression vector of citric acid synthesis gene, this carrier can improve the anti-aluminium poison of plant ability.
Background technology:
Aluminium is one of modal element in the earth's crust, contain abundant, account for 7% of earth's crust weight, be only second to oxygen and silicon, occupy the 3rd, aluminium simultaneously also be the abundantest metallic element of content in the earth's crust (Zhao and great waves .1996. aluminium element are given birth to tea tree and the relation of kind. tropical crops science and technology. (3): 33-35).In neutrality or weak acid environment, aluminium mainly is that the form with the silicate of stationary state and oxide compound is present in the soil.In sour environment, insoluble aluminide just changes the aluminium of dissolved state into, enters in the soil then to plant toxigenicity (Hideaki is biology of aluminum toxicity and tolerance in higherplants.Int Rev Cytol.200:1-46 M.2000.Cell).Simultaneously the aluminum ion in the acid soil also forms the aluminum phosphate compound of low-solubility with phosphate anion, thereby causes that available phosphorus content reduces greatly in the soil.The aluminium of solubility can be divided into following a few class: free aluminium, Al (H
2O)
6 3+, polymeric aluminum Al
13And and lower molecular weight aluminum compound (Kochian LV.1995.Cellular mechanisms of aluminum toxicity and resistancein plants.Annu Rev Plant Physiol Mol Biol.46:479-487).Along with the rising of soil pH value, Al (H
2O)
6 3+Change Al (OH) into
2+, Al (OH)
2 +In neutral soil, main Al (OH) with indissoluble
3Form exists; Next at alkaline condition mainly is with aluminate negatively charged ion Al (OH)
4 -Form have (Hideaki is biology of aluminum toxicity and tolerance in higher plants.Int Rev Cytol.200:1-46 M.2000.Cell).Its toxicity of the aluminium of different shape has evident difference again.It is generally acknowledged that at present aluminium mainly is with Al (H in the pH value is lower than 4.5 acid soil
2O)
6 3+Form exists, promptly usually said Al
3+, and the aluminium of this form is considered to the form the strongest to phytotoxicity.
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 molL
-1(Ma JF.2000.Role of organic acid in detoxificationof 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 therefore suppress plant to moisture, nutrient absorbing, the normal growth that influences plant is grown (Delhaize E, Ryan PR.1995.Aluminum toxicityand tolerance in plants.Plant Pysiol.107:315-332; Foy CD.1983.Thepysiology of plant adaptation to mineal stress.Iowa State J Res.57:355-392).The aluminium toxicity symptom of easy identification is exactly the inhibition of root growth, and the tip of a root is the initial site that aluminium toxicity takes place.Ryan finds to have only the direct tip of a root to corn to carry out the aluminium poison and coerce processing, and the growth of root just can be suppressed; If to the elongation zone or remove the whole of tip of a root
Handle, growth to root does not influence (Ryan PR, Ditomaso JM, Kochian LV.1993.Aluminumtoxicity in roots:an investigation of special sensitivity and to role of the cap.J ExpBot.44:437-446).
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, and then influence the 26S Proteasome Structure and Function of film.Plant cell membrane plays an important role in the process of plant absorbing moisture and nutrient, and aluminium can act on lipid and albumen passage, makes these processes be suppressed.In addition, aluminium can also start the metabolism that second messenger on the film upsets plant, can influence the normal physiological function of plant as the interaction of aluminium and calcium.Complexing action can also take place with a lot of biomacromolecules in aluminium, influences their activity.Aluminium can combine with calmodulin, changes its structure and its function is exerted an influence; Aluminium can with the DNA combination, increase double-spiral structure stability, stop dna replication dna, influence cell fission; Aluminium can form the Al-ATP mixture with ATP, and the vegetable cell energy metabolism is restricted.
If exist in the plant rhizosphere solution and the very strong ligand of Al complex ability, promptly can reduce free Al ion in the solution, thereby reduce itself and root cells bonded chance, the murder by poisoning of alleviation Al.Some organic acids such as citric acid, oxalic acid, oxysuccinic acid, tartrate, Whitfield's ointment, propanedioic acid etc. can form stabilized complex with the Al complexing, and these mixtures are lower to the toxicity of plant, or even nontoxic.Organic acid combines with Al and forms the Al-organic acid complex compound low to phytotoxicity, thereby has reduced Al ionic concentration in the cell, has reduced Al and cellularstructure material bonded chance, thereby the protection cell is avoided destroying; And enter the organic acid of soil and the Al complexing in the rhizosphere through the root system secretion, reduce Al ionic concentration in the rhizosphere, thereby can alleviate of the harm of aluminium poison plant.The ability of organic acid alleviation aluminium poison depends on stability factor (the Hue NV of the Al-organic acid mixture of organic acid and Al formation, F.1986.Effect of organic acids onaluminum toxicty in subsoils.Soil Sci Am is J.50:28-34 for Craddock GR, Adams).Studies show that, add with the citric acid of Al equimolar amount and can remove Al poison (Ma JF Hiradae S, Nomotto K, Iwashita T, MataumotoH.1997a.Internal detoxification mechanism of Al in hydrangea.Identification of Alfrom in the leace.Plant physiol.113:1033-1039; Li XF, Ma JF, Matsumoto be secretion of both citrate and malate in rye.Plant and Soil.242:235-243 H.2002.Aluminum-induced).It is respectively that (Hiradate S, Matsumoto be aluminum withbuckwheat.Nature.390:569-570 H.1997b.Detoxifying for Ma JF, Zheng SJ for 3 times and 5～8 times of Al that but oxalic acid and oxysuccinic acid are removed the needed amount of aluminium poison; Li XF, Ma JF, Matsumoto be secretion of both citrate and malate in rye.Plant Soil.242:235-243 H.2002.Aluminum-induced).The ability of organic acid releasing aluminium poison is main relevant with their chemical structure, promptly-OH ,-position (the Hue NV of COOH functional group on main C chain, F.1986.Effect oforganic acids on aluminum toxicty in subsoils.Soil Sci Am is J.50:28-34 for Craddock GR, Adams).Separating the strongest its chemical structure characteristic of organic acid of aluminium poison ability is that two couples of OH/COOH are positioned on two adjacent C atoms (as citric acid, tartrate), perhaps two COOH directly connect (as oxalic acid), and these organic acids can form stable five-ring or six-membered ring structure with aluminium.Buckwheat root system under Al coerces can be secreted a large amount of oxalic acid, oxalic acid and Al complexing have effectively alleviated the toxic action of Al to root system, simultaneously also can effectively eliminate intravital aluminium poison (Ma JF at a large amount of oxalic acid of buckwheat cylinder accumulation, Hiraaadate S, Matsumoto be aluminumresistance in buckwheat:II.Oxalic acid detoxifies aluminum internally.Plant Physiol.117:753-759 H.1998.High; Zheng SJ, Ma JF, Matsumoto be aluminum resistance inbuckwheat:I.Al-induced specific secretion of oxalic acid from root tips.PlantPhysiol.117:745-751 H.1998.High).
There are 39.5 hundred million hm in the whole world
2Acid soil, wherein soil area in arable land is 1.79 hundred million hm
2Mainly be distributed in the torrid zone, subtropics and area, temperate zone, especially developing country (Kochian LV.1995.CelluarMechanisms of aluminum toxicity and resistance in plant.Annu Rew Plant physiolplant mol Biol.46:137).China's acid soil spreads all over 15 provinces and regions, south, and the total area reaches 2,030 ten thousand hm
2, account for 21% of the national soil total area.Aluminium is to murder by poisoning and the low important factor that has become the restriction plant growth of the absorbable available phosphorus content of plant of plant on the acid soil.All the time, people by a large amount of limings, improve pH in soil usually, make the free aluminum precipitation, remove aluminium toxicity.Yet this method is cured the symptoms, not the disease, and can only improve topsoil, and the acidifying of deep soil is not but had substantial change, and the cost height, and environmental pollution is serious.So screening and cultivate the drugs of anti-aluminium kind to be used for agriculture production be the key of dealing with problems.
Along with the fast development of biotechnology, people transform existing kind by genetic engineering means and make it to have anti-aluminium and become possibility.Studies have shown that in the past as citrate synthase and malate synthetase gene, can improve the intravital organic acid content of plant by excessive in plant or ectopic expression organic acid synthase gene.These organic acids are secreted in the soil by root system, and the aluminum ion in the soil that boils together 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.
Citrate synthase (CS) is one and participates in the key enzyme that oxaloacetic acid (OAA) and acetyl-CoA condensation produce citric acid, and this biochemical reaction circulates at TCA, play an important role in the glyoxylate cycle approach of the betaoxidation of lipid acid and photorespiration.Because citric acid is an important intermediate product in multiple biochemical reaction such as amino acid and the lipid acid route of synthesis, its synthetic and decomposition is subjected to strict control.The enzyme and the meta-bolites of overall thinking citric acid upstream and downstream are very important when trial is carried out genetic manipulation to the citric acid metabolism.The Decomposition of the generation of increase citric acid or minimizing citric acid can strengthen the accumulation of citric acid or flow, increase enzyme synthetic relevant such as the activity of CS, MDH (malate dehydrogenase (malic acid dehydrogenase)) and PEPC (phosphoric acid enol pyruvic acid carboxylase) with citric acid, or minimizing and the citric acid activity of decomposing relevant enzyme such as aconitase (ACO) and isocitric enzyme (IDH) can strengthen the accumulation of citric acid, and the increase of citric acid growing amount may promote the secretion of citric acid.
Because the synthetic carbon skeleton that needs of citric acid, the blade of plant is the photosynthesis organ, and the photosynthate that produces in blade can provide a large amount of carbon skeletons for citric acid synthetic.The plant expression vector of existing cs 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), often is used to realize the high level expression of goal gene in blade.
Summary of the invention:
The object of the present invention is to provide a kind of plant expression vector that improves the citric acid synthesis gene of the anti-aluminium poison of plant ability, this carrier is the plant expression vector that contains citrate synthase gene cs; This construction of carrier is provided simultaneously, and the application of this carrier in the transfer-gen plant of preparation tolerance aluminium poison.
In order to realize above-mentioned purpose of the present invention, the invention provides following technical scheme:
A kind of recombinant plant expression vector contains tobacco cs gene in this plant expression vector.
The initial vector of described plant expression vector is pPZP211; Described cs upstream region of gene is added with the promotor PrbcS of Rubisco small subunit.
Recombinant vectors of the present invention is pPZP211-PrbcS-cs, and it contains promotor PrbcS, is right after the cs gene thereafter.
So-called cs gene, its GenBank accession number is: X84226.
Above-mentioned recombinant vectors pPZP211-PrbcS-cs of the present invention is made up by following method and forms:
(1) from GenBank, search the full-length gene order of tobacco cs, and the following a pair of primer of implementation sequence:
cA1：5’-CA
CCATGGTGTTCTATCGCGGCGTTTC-3’
cA2：5’-TCTAGATCATGCTTTCTTGCAAATGGTTC-3’
5 ' end primer cA1, end adds the CACC characteristic sequence, and forms the NcoI restriction enzyme site thus; 3 ' end primer cA2, end adds the XbaI enzyme cutting site; The first chain cDNA is a template amplification with tobacco, obtains the full-length cDNA of cs;
(2) reclaim also purifying cs full-length gene fragment, and be connected on the pMD18-T carrier, adopt alkaline lysis method of extracting plasmid DNA, cut detection by PCR detection and enzyme and obtain recombinant plasmid pMD-cs;
(3) make up intermediate carrier pUC118-PrbcS-*T, with SphI pUC118-PrbcS-T-rbcS-3C (by structures such as Sugita and provide, Sugita et al., 1987, MGG, rbcS-3C excision 209:247-256), 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;
(4) the PrbcS promotor being connected to 5 ' of cs gene holds, with NcoI and XbaI double digestion pMD-cs and pUC118-PrbcS-*T, reclaim cs gene fragment and the big fragment pUC118-PrbcS of carrier, connect then, conversion, extracting plasmid carry out that PCR detects and enzyme is cut detection, obtains recombinant plasmid pUC118-PrbcS-cs;
(5) with HindIII and XbaI double digestion pUC118-PrbcS-cs and pPZP211 (by structures such as Hajdukiewicz and provide, Hajdukiewicz et al., Plant Mol.Biol.25,989 (1994)), reclaim purifying small segment PrbcS-cs and big fragment pPZP211, connect then and transform, select mono-clonal and extracting plasmid, carry out with two groups of primers then that PCR detects and enzyme is cut detection, acquisition recombinant vectors pPZP211-PrbcS-cs, it contains promotor PrbcS, is right after the cs gene thereafter.
The present invention provides the construction process of recombinant plant expression vector of the present invention simultaneously:
(1) from GenBank, search the full-length gene order of tobacco cs, and the following a pair of primer of implementation sequence:
cA1：5’-CA
CCATGGTGTTCTATCGCGGCGTTTC-3’
cA2：5’-
TCTAGATCATGCTTTCTTGCAAATGGTTC-3’
5 ' end primer cA1, end adds the CACC characteristic sequence, and forms the NcoI restriction enzyme site thus; 3 ' end primer cA2, end adds the XbaI enzyme cutting site; The first chain cDNA is a template amplification with tobacco, obtains the full-length cDNA of cs;
(2) reclaim also purifying cs full-length gene fragment, and be connected on the pMD18-T carrier, adopt alkaline lysis method of extracting plasmid DNA, cut detection by PCR detection and enzyme and obtain recombinant plasmid pMD-cs;
(3) make up intermediate carrier pUC118-PrbcS-*T, with SphI pUC118-PrbcS-T-rbcS-3C (by structures such as Sugita and provide, Sugita et al., 1987, MGG, rbcS-3C excision 209:247-256), 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;
(4) the PrbcS promotor being connected to 5 ' of cs gene holds, with NcoI and XbaI double digestion pMD-cs and pUC118-PrbcS-*T, and recovery purifying cs gene fragment and the big fragment pUC118-PrbcS of carrier, connect then, conversion, extracting plasmid carry out PCR and detect, and obtains recombinant plasmid pUC118-PrbcS-cs;
(5) with HindIII and XbaI double digestion pUC118-PrbcS-cs and pPZP211 (by structures such as Hajdukiewicz and provide, Hajdukiewicz et al., Plant Mol.Biol.25,989 (1994)), reclaim purifying small segment PrbcS-cs and big fragment pPZP211, connect conversion then, select mono-clonal and shake bacterium and extracting plasmid, carry out PCR with two groups of primers then and detect acquisition recombinant vectors pPZP211-PrbcS-cs, it contains promotor PrbcS, is right after the cs gene thereafter.
In addition, also provide the application of recombinant vectors of the present invention in the transfer-gen plant of preparation tolerance aluminium poison, especially the application in the transgene tobacco of preparation tolerance aluminium poison.
The present invention utilizes photoinduction type promotor PrbcS to make up the plant expression vector of cs gene, so that overexpression CS gene in the tenuigenin of transgenic plant blade, the carbon skeleton synthesizing citric acid that directly utilizes photosynthate to produce, make it to be secreted into the extracellular, enter into soil by root system at last, boil together aluminum ion in the soil is removed the murder by poisoning of the aluminium of acid soil middle and high concentration to plant, improves the ability of the transgenic plant poison of anti-aluminium.
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. among the pUC118,1987, MGG, 209:247-256), the plasmid vector name of this subclone is called pUC118-PrbcS-T-rbcS-3C.For the product after the cs 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) again, 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.
Plant expression vector is that pPZP211 is (by structures such as Hajdukiewicz and Hajdukiewicz et al, 1994 are provided among the present invention, PMB, 25:984-), also can select other Agrobacterium binary vector for use, as the pCAMBIA carrier series and the derivative vector thereof of CAMBIA company.
Above-mentioned carrier is imported plant tissue by agriculture bacillus mediated or other physics, chemical process and be incorporated in the Plant Genome, obtain the transgenic plant that aluminium poison tolerance is improved.Dedicated carrier of the present invention is intended to be used to improve the tolerance of plant to the aluminium poison, unifacial leaf and dicotyledons all is suitable for, as tobacco, paddy rice, soybean, wheat etc.
Experimental result of the present invention shows that the tobacco plant citrate synthase activity of changeing the cs gene is 1～5.5 times of wild-type tobacco.Under the coercing of aluminium poison, the tobacco that changes the cs gene can secrete more organic acid, and root growth is good, can improve the resistance of plant to the aluminium poison.Dedicated carrier of the present invention can be brought into play great role raising plant (as tobacco) aspect the tolerance of aluminium poison, particularly in the southern china characteristic of acid red soil, can significantly promote plant to the tolerance of aluminium poison and improve the absorb efficient of plant, thereby also provide a new way for plant species improvement to the phosphorus element.
Description of drawings:
Fig. 1: the structure of intermediate carrier pUC118-PrbcS-*T.With SphI the rbcS-3C among the pUC118-PrbcS-T-rbcS-3C is excised, and make carrier, near the initiator codon of chloroplast(id) positioning sequence, introduce the NcoI site by the point mutation technology from being connected to form pUC118-PrbcS-T;
Fig. 2: pPZP211-PrbcS-cs plant expression vector construction strategy synoptic diagram;
Fig. 3: obtain the cs gene cDNA by the RT-PCR amplification.(A) the total RNA of tobacco; (B) RT-PCR of tobacco RNA.M，λDNA/HindIII?digest?Marker。1～4, cs Gene RT-PCR product;
Fig. 4: the enzyme of recombinant plasmid pMD-cs is cut detection.1-2 is with XbaI and the two enzyme recombinant plasmid pMD-cs of NcoI; M, DNA/HindIII digest Marker;
Fig. 5: the detection of recombinant plasmid pUC118-PrbcS-cs.(A) PCR detects.M, λ DNA/HindIIIdigest Marker; 1～4, be the PCR product of template amplification with pUC118-PrbcS-cs; (B) enzyme is cut detection.M, λ DNA/HindIII digest Marker; 1-2 cuts the pUC118-PrbcS-cs recombinant plasmid with the EcoRI enzyme;
Fig. 6: the detection of recombinant plasmid pPZP211-PrbcS-cs; (A) PCR detects.M,
X174DNAMarker; 1～3, be the PCR product of template amplification cs full length gene with the pUC118-PrbcS-cs recombinant plasmid; 4～6, be template with the pPZP211-PrbcS-cs recombinant plasmid, with a downstream primer cA2 amplification PCR products that is positioned at a primer (rbcS5) and the cs gene of PrbcS promotor inside; (B) M,
X174DNAMarker; 1～3, cut the pPZP211-PrbcS-cs recombinant plasmid with the EcoRI enzyme;
Fig. 7: the cs gene in transgene tobacco the insertion situation and the detection of transcriptional level.(A) the cs gene inserts the detection of situation.M, DNAmarker; P, positive control (is the PCR product of template amplification with the pPZP211-PrbcS-cs recombinant plasmid); CS1, CS8, CS11 are the PCR product of template amplification with the genomic dna of transgene tobacco; CK, negative contrast (not genetically modified wild-type tobacco); (B) detection of cs gene transcription level; M, DNA marker; P, positive control, CS1, CS8, CS11, the RT-PCR product of transgene tobacco strain system;
Fig. 8: the determination of activity of citrate synthase in the transgene tobacco blade.CS1, CS8, CS11, transgene tobacco strain system; CK, negative contrast (not genetically modified wild-type tobacco);
Fig. 9: transgene tobacco detects the tolerance of aluminium poison.(A) transgene tobacco is through 100 μ M AlCl
3Handle that the tip of a root dyes with chrome azurol S after 3 hours; (B) transgene tobacco is through 300 μ M AlCl
3Handle that the tip of a root dyes with chrome azurol S after 3 hours.CS1, CS8, CS11, transgene tobacco strain system; CK, negative contrast;
Figure 10: transgene tobacco T
2Coerce the mensuration of root extension speed down for plant at the aluminium poison.CS8, CS10, CS19, CS19, transgene tobacco strain system; CK; Negative contrast;
Figure 11: the upgrowth situation of transgene tobacco under aluminium is coerced.(A) transgene tobacco is at 100 μ M AlCl
3Upgrowth situation in the matrix; (B) transgene tobacco is at 300 μ M AlCl
3Upgrowth situation in the matrix.CS8, CS11 difference, transgene tobacco strain system; CK, negative contrast.
Below in conjunction with accompanying drawing, further illustrate essentiality content of the present invention with embodiments of the invention, but do not limit the present invention with this.
Embodiment:
Reagent and instrument related among the following embodiment are:
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 Japanese precious biotechnology company limited (Dalian) product, plasmid extraction kit is available from vast Tyke Bioisystech Co., Ltd, and TRIzoL Reagent RNA extracts reagent 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 Tyke) 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, it root biochemical technology), 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 ldNTP (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, it root biochemical technology), 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.
The structure of embodiment 3, plant expression vector pPZP211-PrbcS-cs
The construction strategy of plant expression vector pPZP211-PrbcS-cs as shown in Figure 2, the first-selected full-length gene order of from GenBank, searching tobacco cs, and design a pair of special primer, the first chain cDNA is a template amplification with tobacco, obtain the full-length cDNA of cs, reclaim and purifying cs full-length gene fragment, be connected on the pMD18-T carrier and obtain pMD-cs.With NcoI and XbaI double digestion pMD-cs and pUC118-PrbcS-*T, reclaim purifying cs gene fragment and the big fragment pUC118-PrbcS of carrier, connect, obtain recombinant plasmid pUC118-PrbcS-cs then.With HindIII and XbaI double digestion pUC118-PrbcS-cs and pPZP211, reclaim purifying small segment PrbcS-cs and big fragment pPZP211, connect then, obtain recombinant vectors pPZP211-PrbcS-cs, it contains promotor PrbcS, is right after the cs gene thereafter.Concrete building process is as follows:
One, the cDNA of tobacco citrate synthase gene cs amplification and TA clone
Search the full-length gene order of tobacco cs from GenBank, and design a pair of primer, sequence is as follows:
cA1：5’-CA
CCATGGTGTTCTATCGCGGCGTTTC-3’
cA2：5’-
TCTAGATCATGCTTTCTTGCAAATGGTTC-3’
5 ' end primer cA1, end adds the CACC characteristic sequence, and forms the NcoI restriction enzyme site thus; 3 ' end primer cA2, end adds the XbaI enzyme cutting site.
From tobacco (Nicotiana tabacum cv.Xanth) seedling, extract total RNA with TRIzoL Reagent (Invitrogen), 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.Use M-MuLV Reverse Transcriptase Kit (TaKaRa) to carry out the synthetic of cDNA, 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, 25mM MgCl
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 PCR with cs gene upstream and downstream Auele Specific Primer cA1 and cA2, and amplification obtains the full-length cDNA 1.4kb (Fig. 3) of cs.Reclaim and purifying cs full-length gene fragment, and be connected on pMD18-T (the precious biotech firm in the Dalian) carrier, transformed into escherichia coli competence DH5 α (day root biochemical technology), adopt alkaline lysis method of extracting plasmid DNA, through 1% agarose gel electrophoresis, choose the big or small recombinant plasmid that conforms to theoretical value and do further double digestion detection.Multiple clone site according to positive recombinant plasmid pMD-cs carrier two ends, with NcoI and XbaI double digestion recombinant plasmid, detect enzyme through 1% agarose gel electrophoresis and cut product, the DNA that the recombinant plasmid pMD-cs of successful connection contains about 1.4kb inserts fragment (Fig. 4), proves the cDNA of tobacco cs full-length gene through sequential analysis.
Two, the structure of recombinant plasmid pUC118-PrbcS-cs
For the PrbcS promotor being connected to 5 ' end of cs gene, with NcoI and XbaI double digestion pMD-cs and pUC118-PrbcS*T (this carrier is the donor of PrbcS promotor), reclaim cs gene cDNA fragment and the big fragment pUC118-PrbcS of carrier (about 4.2kb) of purifying 1.4kb, use the ligase enzyme test kit of precious biological (TaKaRa) to connect cs gene cDNA fragment and the big fragment pUC118-PrbcS of carrier, transform high-level efficiency (10 then with the ligation mixture
8) competent escherichia coli cell (DH5 α, it root biochemical technology), be applied on the flat board that is added with penbritin (Amp, 100 μ g/ml) transforming good intestinal bacteria, screening Amp resistance recon bacterium colony extracts from Amp resistance recon bacterium colony that plasmid carries out PCR and enzyme is cut evaluation.With the recombinant plasmid is template, is positioned at one that PrbcS starts inner primer and the downstream primer cA2 of cs gene carries out pcr amplification, and the theoretical length of PCR product is 1.6kb.The PCR product is through 1% agarose gel electrophoresis, and the result shows that the molecular weight of amplified production is 1.6kb, and actual size is consistent with theoretical value, shows the cs gene correctly to be inserted into (Fig. 5 A) on the carrier that contains photoinduction promoter PrbcS.Then the positive recombinant plasmid of pcr analysis is carried out enzyme cuts detection, connect correct recombinant plasmid through the EcoRI enzyme cut obtain one about 1.0kb small segment and the big fragment of a 4.6kb, enzyme is cut product and is detected through 1% agarose gel electrophoresis, the result conforms to theory (Fig. 5 B), obtains the correct recombinant plasmid pUC118-PrbcS-cs that connects thus.
Three, the structure of pPZP211-PrbcS-cs
With HindIII and XbaI double digestion pUC118-PrbcS-cs and pPZP211 (by structures such as Hajdukiewicz and provide, Hajdukiewicz et al, 1994, PMB, 25:984-), reclaim purifying purpose fragment PrbcS-cs (about 2.9kb) and the big fragment of pPZP211 carrier (length is about 9.3kb), use the ligase enzyme test kit of precious biological (TaKaRa) to connect purpose fragment PrbcS-cs and the big fragment of pPZP211 carrier then, transform high-level efficiency (10 with the ligation mixture
8) competent escherichia coli cell (DH5 α, it root biochemical technology), 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 extracts plasmid from Spe resistance recon bacterium colony, carry out PCR and enzyme then and cut detection.PCR detect to adopt two groups of primers to carry out, and first group is positioned at a primer (rbcS5) of PrbcS promotor inside with one and the downstream primer cA2 of cs gene makes PCR, and the theoretical length of PCR product is 1.6kb; Second group upstream and downstream primer is respectively primer cA1, the cA2 of amplification cs full-length gene, and the theoretical length of PCR product then is 1.4kb.Two groups of PCR products show its clip size conform to the theoretical prediction value respectively (Fig. 6 A) through 1% agarose gel electrophoresis analysis.This shows that the PrbcS-cs segment correctly is inserted among the plant expression vector pPZP211.All comprise an EcoRI site after cs gene inside and the NOS terminator, cut detection so adopt EcoRI to carry out enzyme, if direction of insertion is correct, enzyme is cut product the small segment that length is about 1.4kb can occur.The size that agarose gel electrophoresis detected result demonstration enzyme is cut the product small segment matches (Fig. 6 B) with theoretical supposition, obtains the plant expression vector pPZP211-PrbcS-cs of cs gene thus, and this carrier has promotor PrbcS, is right after the cs gene thereafter.
The competent cell of preparation Agrobacterium, the electricity consumption impulse method changes the plant expression vector Agrobacterium pPZP211-PrbcS-cs 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 lddH 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 cs gene upstream and downstream Auele Specific Primer and detect, transform the cs gene band that successful bacterium colony all can amplify 1.4kb, the transformant bacterium colony of confirming through bacterium colony PCR is used to transform plant.
Embodiment 5, usefulness plant expression vector pPZP211-PrbcS-cs transformation of tobacco
The single colony inoculation of Agrobacterium that picking carries the pPZP211-PrbcS-cs plasmid (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 tabacum cv.Xanth) by agriculture bacillus mediated, with leaf dish method transformation of tobacco, 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, 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 kantlex (50 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 kantlex (50 μ g/ml) and carry out inducing of root.Obtain 18 independently transgenic lines altogether by the kantlex screening, then the transcriptional level of cs in these transfer-gen plants analyzed.
In order to confirm to contain really the cDNA fragment of the goal gene of the present invention's importing, the transgene tobacco that screens is done further to identify with PCR method by the transgene tobacco strain system of kantlex screening.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 7.5100mM, 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.With the plant genome DNA is template, making PCR with the downstream primer cA2 of primer that is positioned at PrbcS promotor transcription initiation site place and cs gene detects, the theoretical length of PCR product is 1.5kb, the plant that successfully changes goal gene over to all can amplify the band (Fig. 7 A) of 1.5kb, 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 cs 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.Use Reverse Transcriptase to carry out the synthetic of cDNA, 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 a downstream primer cA2 who is positioned at the primer of PrbcS promotor inside and draws the cs gene 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 (Fig. 7 B) of goal gene.
The activation analysis of citrate synthase in embodiment 7, the transgene tobacco (CS)
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; 1mMPMSF; 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.
(Plant Physiology such as Anoop are pressed in the determination of activity of citrate synthase, 2003, method 132:2205-2217) is carried out, principle is that L MALIC ACID generates oxaloacetic acid under the effect of malate dehydrogenase (malic acid dehydrogenase), and oxaloacetic acid is extremely unstable, under the effect of citrate synthase and acetyl-CoA, can generate citric acid.In this reaction, thereby by measuring the relative activity that wavelength is the increasing amount acquisition CS of the NADH of 340nm place.
The reaction system of measuring the CS enzymic activity is: 0.05M Tris-HCl (PH 7.5), and 10mM L-Malate, 10U/mLMDH, 0.5mMNAD adds 100 μ g tobacco protein samples in the reaction system of 1ml.On the uv-spectrophotometric instrument, survey the absorption value (OD value) at its 340nm place, treat that reading is stablized after, add 10 μ L4mM CoA (acetyl-CoA), the absorption value at detection 340nm place within 1min.Calculate the difference of twice reading, obtain CS enzymic activity data, the result shows that transgene tobacco CS enzymic activity is 1～5.5 times (Fig. 8) of wild-type tobacco, and the visible expression of cs in transgenic tobacco plant improved the activity of CS greatly.
The anti-aluminium of plant generally adopts tip of a root dyeing situation isophenous index to weigh.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 tolerance selection intropical maize (Zea mays L.) .Theor Appl Genet.99:747-754; Ma JF, Zhen SJ, Li XF.1997.A rapid hydroponic screening for aluminum tolerance 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.
The transgene tobacco seedling of getting big or small uniformity and taking root good places AlCl respectively
3Concentration is respectively in the calcium chloride solution (pH value 4.3) of 0 (contrast), 50,100,300 μ mol/L and handled three hours, again with 0.1% the chrome azurol S staining fluid 15min that dyes.Behind distilled water flushing, examine under a microscope tip of a root dyeing situation.The root system of experimental result demonstration wild-type tobacco and the tip of a root are all by purple, and the tobacco seedling of commentaries on classics cs gene is through the AlCl of 100 μ M
3It is painted substantially or have only very shallow painted (Fig. 9 A) to handle the back root system and the tip of a root.AlCl in high density
3After (300 μ M) handled, its root system of plant CS-8 that the CS expression level is low was painted a bit, but coloring degree is also light than wild-type (CK), and visible transgene tobacco has tangible dependency (Fig. 9 B) to the tolerance degree of aluminium and the activity level of CS.These results tentatively prove overexpression cs in blade, also can strengthen the tolerance of transgenic plant to the aluminium poison.
Embodiment 9, transgenic tobacco plant are coerced the mensuration of root extension speed down at 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, and therefore also being determined at the aluminium poison coerces down transgenic tobacco plant root growth speed.Therefore choose the seedling of transgene tobacco size uniformity, place AlCl respectively
3Concentration is in the calcium chloride solution of 0,50,100,300 μ mol/L.After handling 24 hours, measure the elongation of transfer-gen plant root, triplicate, experimental result is as shown in figure 10.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 (CK) root elongation, yet the cs transfer-gen plant still keeps certain growth vigor, and root system is kept certain elongation.
Embodiment 10, transgenic tobacco plant excretory citric acid content are measured
Get the transgenic tobacco plant and the wild-type tobacco seedling of big or small uniformity, place AlCl respectively
3Concentration is to handle 3h in the calcium chloride solution of 100,300 μ mol/L, then collects calcium chloride solution, concentrates back efficient liquid phase chromatographic analysis citric acid content.Analytical results shows that the synthetic citric acid is transported to root in a large number in blade, and cs transgenic tobacco plant root system excretory citric acid content is about 1.5～2 times of wild-type adjoining tree.
Embodiment 11: transgene tobacco is in the growing state analysis under aluminium is coerced
Will be through AlCl
3The tobacco plant seedling of handling is transplanted to and carries out pot experiment in the new pearlite interstitial substance in the greenhouse, and the per week pouring contains AlPO
4Nutritive medium (pH4.3, prescription is used AlPO with the prescription of MS substratum
4Replace KH wherein
2PO
4) twice, 50ml.Only water after two months, observe the upgrowth situation of tobacco plant.After four months, the cs transfer-gen plant blossoms and bears fruit, however wild-type tobacco (CK) plant strain growth obviously be obstructed, not only plant is short and small, but also does not begin reproductive growth.Find through 100 μ mol/L LAlCl by measurement the tobacco plant height
3The plant height of the cs transgene tobacco of handling is 2.09～2.1 times (Figure 11 A) of wild-type tobacco; Through 300 μ mol/L AlCl
3The plant height of the cs transgene tobacco of handling is 1.8～2 times (Figure 11 B) of wild-type tobacco plant height.As seen the citric acid of transgene tobacco root excessive secretion can be in containing the acidic medium of aluminum phosphate the aluminium chelate ion, can remove the harm of aluminium poison, help the absorption of tobacco for the phosphorus element.So the cs transgenic tobacco plant is coerced well-grown down at the aluminium poison, can normally blossom and bear fruit.
Claims (9)
1, a kind of recombinant plant expression vector wherein contains tobacco cs gene in plant expression vector.
2, the recombinant vectors of claim 1, the initial vector of wherein said plant expression vector are pPZP211.
3, the recombinant vectors of claim 1, wherein said cs upstream region of gene is added with the promotor PrbcS of Rubisco small subunit.
4, the recombinant vectors of claim 1 is pPZP211-PrbcS-cs, has promotor PrbcS on initial vector pPZP211, is right after the cs gene thereafter.
5, the recombinant vectors of claim 1 is formed by following method structure:
(1) from GenBank, search the full-length gene order of tobacco cs, and the following a pair of primer of implementation sequence:
cA1：5’-CA
CCATGGTGTTCTATCGCGGCGTTTC-3’
cA2：5’-
TCTAGATCATGCTTTCTTGCAAATGGTTC-3’
5 ' end primer cA1, end adds the CACC characteristic sequence, and forms the NcoI restriction enzyme site thus; 3 ' end primer cA2, end adds the XbaI enzyme cutting site; The first chain cDNA is a template amplification with tobacco, obtains the full-length cDNA of cs;
(2) reclaim also purifying cs full-length gene fragment, and be connected on the pMD18-T carrier, adopt alkaline lysis method of extracting plasmid DNA, cut detection by PCR detection and enzyme and obtain recombinant vectors pMD-cs;
(3) make up intermediate carrier pUC118-PrbcS-*T, with SphI pUC118-PrbcS-T-rbcS-3C (by structures such as Sugita and provide, Sugita et al., 1987, MGG, rbcS-3C excision 209:247-256), 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;
(4) the PrbcS promotor being connected to 5 ' of cs gene holds, with NcoI and XbaI double digestion pMD-cs and pUC118-PrbcS-*T, reclaim cs gene fragment and the big fragment pUC118-PrbcS of carrier, connect then, conversion, extracting plasmid carry out that PCR detects and enzyme is cut detection, obtains recombinant plasmid pUC118-PrbcS-cs;
(5) with HindIII and XbaI double digestion pUC118-PrbcS-cs and pPZP211 (by structures such as Hajdukiewicz and provide, Hajdukiewicz et al., Plant Mol.Biol.25,989 (1994)), reclaim purifying small segment PrbcS-cs and big fragment pPZP211, connect then and transform, select mono-clonal and extracting plasmid, carry out with two groups of primers that PCR detects and enzyme is cut detection, acquisition recombinant vectors pPZP211-PrbcS-cs, it contains promotor PrbcS, is right after the cs gene thereafter.
6, a kind of cs gene, its GenBank accession number is: X84226.
7, the construction process of the recombinant plant expression vector of claim 1 is characterized in that:
(1) from GenBank, search the full-length gene order of tobacco cs, and the following a pair of primer of implementation sequence:
cA1：5’-CA
CCATGGTGTTCTATCGCGGCGTTTC-3’
cA2：5’-
TCTAGATCATGCTTTCTTGCAAATGGTTC-3’
5 ' end primer cA1, end adds the CACC characteristic sequence, and forms the NcoI restriction enzyme site thus; 3 ' end primer cA2, end adds the XbaI enzyme cutting site; The first chain cDNA is a template amplification with tobacco, obtains the full-length cDNA of cs;
(2) reclaim also purifying cs full-length gene fragment, and be connected on the pMD18-T carrier, adopt alkaline lysis method of extracting plasmid DNA, cut detection by PCR detection and enzyme and obtain recombinant vectors pMD-cs;
(3) make up intermediate carrier pUC118-PrbcS-*T, with SphI pUC118-PrbcS-T-rbcS-3C (by structures such as Sugita and provide, Sugita et al., 1987, MGG, rbcS-3C excision 209:247-256), 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;
(4) the PrbcS promotor being connected to 5 ' of cs gene holds, with NcoI and XbaI double digestion pMD-cs and pUC118-PrbcS*T, and recovery purifying cs gene fragment and the big fragment pUC118-PrbcS of carrier, connect then, conversion, extracting plasmid carry out pcr amplification, obtains recombinant plasmid pUC118-PrbcS-cs;
(5) with HindIII and XbaI double digestion pUC118-PrbcS-cs and pPZP211 (by structures such as Hajdukiewicz and provide, Hajdukiewicz et al., Plant Mol.Biol.25,989 (1994)), reclaim purifying small segment PrbcS-cs and big fragment pPZP211, connect then and transform, select mono-clonal and shake bacterium and extracting plasmid, carry out PCR then and detect, obtain recombinant vectors pPZP211-PrbcS-cs, it contains promotor PrbcS, is right after the cs gene thereafter.
8, the application of the recombinant vectors of one of claim 1-5 in the transfer-gen plant of preparation tolerance aluminium poison.
9, the application of the recombinant vectors of one of claim 1-5 in the transgene tobacco of preparation tolerance aluminium poison.
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|---|---|---|---|---|
| CN102229948A (en) * | 2011-05-09 | 2011-11-02 | 中国农业科学院油料作物研究所 | Method for increasing oil content in tobacco |
| CN102883596A (en) * | 2010-04-09 | 2013-01-16 | 明知大学产学协力团 | Aboveground organ specific promoters for transforming plants and uses thereof |
| CN104561026A (en) * | 2013-10-29 | 2015-04-29 | 中国农业大学 | Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants |
| CN113502276A (en) * | 2021-08-03 | 2021-10-15 | 昆明理工大学 | Application of isocitrate dehydrogenase in improving formaldehyde absorption and metabolism capacity of plants |
-
2007
- 2007-12-05 CN CNA2007100664197A patent/CN101265479A/en active Pending
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102883596A (en) * | 2010-04-09 | 2013-01-16 | 明知大学产学协力团 | Aboveground organ specific promoters for transforming plants and uses thereof |
| CN102229948A (en) * | 2011-05-09 | 2011-11-02 | 中国农业科学院油料作物研究所 | Method for increasing oil content in tobacco |
| CN104561026A (en) * | 2013-10-29 | 2015-04-29 | 中国农业大学 | Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants |
| CN104561026B (en) * | 2013-10-29 | 2017-10-27 | 中国农业大学 | Application of peanut AhFRDL1 gene in improving aluminum toxicity stress resistance of plants |
| CN113502276A (en) * | 2021-08-03 | 2021-10-15 | 昆明理工大学 | Application of isocitrate dehydrogenase in improving formaldehyde absorption and metabolism capacity of plants |
| CN113502276B (en) * | 2021-08-03 | 2022-07-15 | 昆明理工大学 | Application of isocitrate dehydrogenase in improving formaldehyde absorption and metabolism capacity of plants |
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