CN101570761B - Plant expression vector of arabidopsis glutathione dependent formaldehyde dehydrogenase gene, construction method and application thereof - Google Patents
Plant expression vector of arabidopsis glutathione dependent formaldehyde dehydrogenase gene, construction method and application thereof Download PDFInfo
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- CN101570761B CN101570761B CN2009100945908A CN200910094590A CN101570761B CN 101570761 B CN101570761 B CN 101570761B CN 2009100945908 A CN2009100945908 A CN 2009100945908A CN 200910094590 A CN200910094590 A CN 200910094590A CN 101570761 B CN101570761 B CN 101570761B
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Abstract
The invention relates to a plant expression vector pH2-35S-PrbcS-adh of arabidopsis glutathione-dependent formaldehyde dehydrogenase gene, which is the plant expression vector of light-induced promoter (PrbcS) containing tomato Rubisco 3C small subunit gene and the arabidopsis glutathione-dependent formaldehyde dehydrogenase (FALDH) gene adh. The gene adh is amplified from arabidopsis, and the over expression of the gene adh in tobacco lamina cytoplasm is controlled by the light-induced promoter, so that the efficiency of decomposing formaldehyde by tobacco cell can be improved, thus enhancing the capability of absorbing and enduring exogenous formaldehyde by plant. The experiment proves that enzyme activity of adh transgenic tobacco FALDH is increased by about 3-5 times compared with that of wild type; after being cultured for 30 days on an MS solid culture medium containing 10mmol/L of formaldehyde, the growing situation of the adh transgenic tobacco is obviously superior to that of wild type. In addition, when 2mM formaldehyde liquid is used for treating the plant, the formaldehyde absorption rate of the adh transgenic tobacco is obviously higher than that of wild type.
Description
Technical field:
The invention belongs to plant genetic engineering field, be specifically related to a kind of plant expression vector pH2-35S-PrbcS-adh that improves plant absorbing and tolerance methanal ability, and construction process and application.
Background technology:
Formaldehyde is a kind of colourless, and the gas of intense stimulus smell is arranged, soluble in water, pure and mild ether.Its response capacity is extremely strong, energy and protein, and nucleic acid and lipid produce nonspecific reaction (Feldman etc.; Prog Nucleic AcidRes Mol Biol, 1973,13:1-49); be a kind of very active compound, therefore very high toxicity all arranged concerning all biologies.Formaldehyde is widely used in the industrial production, is the raw material of making resin, tackiness agent, paint, plastics, regenerated fiber, is the most widely used chemical raw material of adhesives industries.Along with the raising of expanding economy and living standards of the people, the building and ornament materials that various raw materials are made have entered into various indoor public places and family, make formaldehyde become room air pollution and generally acknowledge the most representative chemical substance.People move in the new home and will carry out upholstery and upgrade furniture, and it is the main source of room air formaldehyde pollution just.The serious place of formaldehyde pollution hazard is: room, office, meeting room, hotel, karaoke chamber, furniture market, building materials market etc.Free concentration of formaldehyde is 0.08 (mg/ cubic meter) at the permissible value of China's regulation in the room air, has investigation to show that new hotel guest-room formaldehyde in indoor air concentration is up to the 0.36mg/ cubic meter, and average out to 0.173mg/ cubic meter is 13 times of outdoor contrast.And the furniture market is up to the 0.873mg/ cubic meter, and average out to 0.432mg/ cubic meter is 33 times of outdoor contrast.Build up the laboratory of decorating of passing through, the formaldehyde mean concns is outdoor 62.5 times up to the 0.5mg/ cubic meter in the air.Sampling Detection discovery unit and resident family's indoor environmental pollution are serious, have looked into 11 families and have had only 1 family qualified, wherein have 1 family formaldehyde to exceed standard 25 times unexpectedly.
Formaldehyde is than high toxic material, when formaldehyde in indoor air content is the 0.1mg/ cubic meter, peculiar smell and sense of discomfort is just arranged; When reaching 0.5 mg/ cubic meter, can stimulate eyes, cause and shed tears; When reaching 0.6 mg/ cubic meter, can cause throat discomfort or pain.When concentration is higher, the vomiting that can cause nausea, it is uncomfortable in chest to cough, asthma even pulmonary edema; When reaching the 30mg/ cubic meter, meeting is the causing death immediately.Long-term contact low dosage formaldehyde can cause chronic respiratory tract disease, cause snuff cancer, colorectal carcinoma, brain tumor, menoxenia, nuclear transgenation, in the dna single chain commissure and DNA and protein commissure and suppress dna damage reparation, pregnancy syndrome, cause neonatal chromosome disorder, leukemia, the comprehensive disease of so-called finishing (sick-house) that Here it is.With respect to benzene,toluene,xylene, the objectionable impuritiess such as TDI, VOC in the paint, formaldehyde has latency period long (3-15), hides characteristics such as dark, that distribution is wide, volatile, it is difficult to administer, harm is big.
Have and studies show that external source formaldehyde can be integrated in the metabolism of photosynthetic cells as carbon source.Such as, common extension orchid exists
14Produce by one carbon metabolism when growing on the formaldehyde of C mark
14The product of C mark is as Serine and Yelkin TTS.Formaldehyde can and Triptide, arginine, N and tetrahydrofolic acid (THFA) carry out metabolism by different approach after forming adducts.Several Eukaryotic biochemistry and genetics research are shown that glutathione dependent formaldehyde dehydrogenase (FALDH) is one of metabolic key enzyme of formaldehyde.The institute that this enzyme extensively is present in plant materials in a organized way in, its catalytic reaction be a substrate generation thioformyl Triptide (S-formylglutathione) with the adducts thiol methyl Triptide (S-hydroxymethylglutathione) of formaldehyde and Triptide.Also have sulfo-formyl Triptide lytic enzyme in plant, it can be decomposed into formic acid and Triptide to S-formylglutathione.These two enzymes are formed the oxidative pathway of a formaldehyde to formic acid.
Plant FALDH is at present from from pea seed, separates obtaining in Arabidopis thaliana and the corn, and their molecule and dynamic characteristic are very close.The FALDH iso-electric point of different sources constitutes by two identical subunits between 5.3-5.6, and the molecular weight that SDS-PAGE detects subunit is about about 40-45kDa.PH is 8.0 o'clock, derives from pea seed, and the FALDH of Arabidopis thaliana and corn is respectively 2uM to the Km value of S-hydroxymethylglutathione, 1.2uM, and 12uM, Kcat is respectively 380min
-1, 1351min
-1, 473min
-1
People such as Achkor carry out genetic manipulation in order to detect the function of formaldehyde dehydrogenase in plant to the gene that derives from Arabidopis thaliana FALDH, have obtained the Arabidopis thaliana transgenic line of different FALDH levels.The Arabidopis thaliana of this enzyme of overexpression improves 25% to the ingestion efficiency of external source formaldehyde, and the plant that the FALDH level reduces (owing to suppressing phenotype or antisense expression altogether) is compared with the wild-type Arabidopis thaliana, and formaldehyde detoxification speed is significantly slow, ability drop.These results show the ability of plant absorbing formaldehyde relevant (Achkor etc., Plant Physiol, 2003,132 (4): 2248-2255) with the FALDH activity level.
Higher plant all has formaldehyde dissimilation approach, but the key enzyme FALDH expression level in higher plant in this approach is lower, can't tackle the harm of external environment middle and high concentration formaldehyde.Constitutive promoter CaMV 35S overexpression FALDH is used in studies have shown that of Achkor etc. in Arabidopis thaliana, can improve the ability that transgenic arabidopsis absorbs liquid formaldehyde in the external environment.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 glutathione dependent formaldehyde dehydrogenase gene of plant absorbing and tolerance methanal ability, this carrier is the plant expression vector that contains arabidopsis glutathione dependent formaldehyde dehydrogenase gene (being the adh gene); This construction of carrier is provided simultaneously, and the application of this carrier in the transgenic plant of preparation rapid absorption and tolerance methanal.
Above-mentioned purpose of the present invention is to be achieved by following technical scheme:
The carrier that is used to improve the anti-absorption of plant and is subjected to the formaldehyde ability provided by the present invention is the plant expression vector with photoinduction promoter and glutathione dependent formaldehyde dehydrogenase gene cDNA.
In the above-mentioned carrier, the cDNA of described glutathione dependent formaldehyde dehydrogenase gene adh derives from Arabidopis thaliana (Arabidopsis thaliana).The described GenBank accession number that derives from the glutathione dependent formaldehyde dehydrogenase gene adh of Arabidopis thaliana is 834417.
The upstream of above-mentioned glutathione dependent formaldehyde dehydrogenase cDNA is the photoinduction type promotor of Rubisco 3C small subunit.
In the above-mentioned carrier, the initial vector of plant expression vector is that pH2GW7 is (available from FlandersInteruniversity Institute for Biotechnology, VIB) under being used to make up.
Above-mentioned plant expression vector pH2-35S-PrbcS-adh of the present invention is made up by following method and forms:
(1) from GenBank, search the cDNA sequence of Arabidopis thaliana adh gene, and the following a pair of primer of implementation sequence:
5’adh：CA
CCATGGCGACTCAAGGTCAGGTTATC
3’adh：
GAATTCTCATTTGCTGGTATCGAGG
Upstream primer 5 ' adh, 5 ' end adds the CACC characteristic sequence, and forms Nco I restriction enzyme site thus; Downstream primer 3 ' adh, end adds the EcoRI restriction enzyme site.With the Arabidopis thaliana first chain cDNA is the masterplate amplification, obtains the full-length cDNA of adh gene.
(2) reclaim also purifying adh full-length gene cDNA fragment, and be connected on the pMD-18T carrier, adopt alkaline lysis method of extracting plasmid DNA, cut detection by PCR detection and enzyme and obtain recombinant plasmid pMD-adh.
(3) make up intermediate carrier pENTR
TM-adh cuts pMD18-adh and pENTR with Sal I and EcoR I enzyme
TM2B (Invitrogen) obtains target gene fragment adh and carrier segments pENTR
TM, connect after the recovery, the transformed competence colibacillus cell, obtain recombinant vectors pENTR
TM-adh.
(4) make up entry vector pENTR*-PrbcS-adh, with Nco I and EcoR I digestion pENTR*-PrbcS-*T-GFP (construction process of pENTR*-PrbcS-*T-GFP is seen another patent application of the applicant, and application number is 200710066422.9) and pENTR
TM-adh reclaims carrier pENTR*-PrbcS fragment and adh gene cDNA fragment, connects then, conversion, extracting plasmid carry out that PCR detects and enzyme is cut detection, obtains recombinant plasmid pENTR*-PrbcS-adh.
(5) make up plant expression vector pH2-35S-PrbcS-adh, LR by the Gateway technology reacts the PrbcS-adh subclone to plant expression vector pH2GW7 (the purpose carrier of Gateway, Belgium VIB/Gent company) in, obtains the plant expression vector pH2-35S-PrbcS-adh of adh gene.
The application of recombinant vectors pH2-35S-PrbcS-adh of the present invention in the transfer-gen plant of preparation tolerance methanal.
The present invention utilizes photoinduction type promotor PrbcS to make up the plant expression vector of adh gene, so that overexpression glutathione dependent formaldehyde dehydrogenase in the tenuigenin of transgenic plant blade, because this enzyme is the key enzyme of formaldehyde pathways metabolism in the vegetable cell, therefore strengthen the metabolic efficient of formaldehyde in the vegetable cell, and then improved the ability of plant absorbing and tolerance external source formaldehyde.
Description of drawings:
The construction strategy of Fig. 1 intermediate carrier pMD-adh;
The detection of Fig. 2 intermediate carrier pMD-adh; A:PCR amplification adh gene fragment, M: λ DNA/HindIII, 1～4: amplified production; B:pMD-adh plasmid electrophoresis detection, M: λ DNA/HindIII, 1:pMD-hps/phi (control plasmid), 2～5:pMD-adh; The C:EcoRI enzyme is cut and is detected the pMD-adh plasmid, M: λ DNA/HindIII, and 1～3:EcoRI enzyme is cut the pMD-adh product, and the 4:sphI enzyme is cut pMD-hps/phi product (control plasmid); D:PCR checks pMD18-adh, M: λ DNA/HindIII, 1～5: amplified production.
Fig. 3 intermediate carrier pENTR
TMThe construction strategy of-adh;
Fig. 4 intermediate carrier pENTR
TMThe detection of-adh; A:pENTR
TM-adh plasmid electrophoresis detection, 1: plasmid pMD-dof1 (control plasmid), 2～4:pENTR
TM-adh sample; B:BamHI+EcoR I double digestion detects pENTR
TM-adh, M:D2000DNA marker, 1～3: enzyme is cut product; C:PCR checks pENTR
TM-adh, M:D2000 DNA marker, 1～3: amplified production.
The construction strategy of the ABC of cloning vector pENTR*-PrbcS-adh of the Gateway of Fig. 5 adh gene;
The detection of the ABC of cloning vector pENTR*-PrbcS-adh of Fig. 6; A:pENTR*-PrbcS-adh plasmid electrophoresis detection, 1:pENTR*-PrbcS-*T-hps/phi (control plasmid), 2～3:pENTR*-PrbcS-adh plasmid sample; The B:EcoRV enzyme is cut and is detected pENTR*-PrbcS-adh sample, M: λ DNA/HindIII, 1～2:pENTR*-PrbcS-adh, 3:pENTR
TM-adh; C:PCR checks pENTR*-PrbcS-adh, M: λ DNA/HindIII, 1～2:pENTR*-PrbcS-adh template, 3:pENTR
TM-adh template.
Fig. 7 makes up the strategy of the plant expression vector of adh gene with the LR reaction of Gateway;
The detection of Fig. 8 adh plant expression vector pH2-35S-PrbcS-adh and the detection of Agrobacterium-mediated Transformation daughter colony; A: electrophoresis detection recombinant plasmid, M: λ DNA/HindIII, 1～2:pH2-35S-PrbcS-adh, 3:pENTR*-PrbcS-adh, 4:pH2GW7; B:EcoR V enzyme is cut and is detected pH2-35S-PrbcS-adh, M:D2000DNA marker, 1:pENTR*-PrbcS-adh, 2～3:pH2-35S-PrbcS-adh; C:PCR detects pH2-35S-PrbcS-adh, 1～2:pH2-35S-PrbcS-adh amplified production, and 3:pENTR*-PrbcS-adh amplified production, 4:pH2GW7 are amplification template; D: bacterium colony PCR detects the changing effect of pH2-35S-PrbcS-adh, M:500bp DNA marker, 1～2: bacterium colony PCR sample, 3: over against shining (template is pENTR*-PrbcS-adh), 4: negative contrast (template is pH2GW7).
Insertion situation and the transcriptional level of Fig. 9 adh in transgene tobacco detects; A: change adh tobacco gene group pcr amplification, M: λ DNA/HindIII, 1～6: transgene tobacco strain system, 7: wild-type tobacco, 8: over against shining (template is the pH2-35S-PrbcS-adh plasmid); B: change adh tobacco RT-PCR amplification, M: λ DNA/HindIII, F3, F4, F5, F6, F16, F25: transgene tobacco strain system, WT: wild-type tobacco.
The enzyme assay of Figure 10 FALDH in transgene tobacco; F3, F4, F6, F16, F25: transgene tobacco strain system; WT: wild-type tobacco.
Figure 11 transgene tobacco absorbs the liquid formaldehyde rate determination; F6, F16: transgene tobacco strain system; WT: wild-type tobacco.
Figure 12 transgene tobacco is for the resistance of formaldehyde in the substratum.
A: wild-type tobacco; B: transgene tobacco.
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, Gateway LR clonaseEnzyme Mix kit 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.
Amplification of embodiment 1:FALDH gene cDNA and TA clone
At first search the full-length gene order of adh from GenBank, and design a pair of primer, sequence is as follows:
5’adh：CA
CCATGGCGACTCAAGGTCAGGTTATC
3’adh：
GAATTCTCATTTGCTGGTATCGAGG
Upstream primer 5 ' adh end adds the CACC characteristic sequence, and forms the NcoI restriction enzyme site thus; Downstream primer 3 ' adh end adds the EcoRI restriction enzyme site.
From Arabidopis thaliana (Arabidopsis thaliana) 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, carries out PCR with adh gene upstream and downstream Auele Specific Primer 5 ' adh and 3 ' adh, and amplification obtains the about 1.1kb of full-length cDNA (Fig. 2 A) of adh.Reclaim and purifying adh full-length gene fragment, and be connected on pMD-18T (the precious biotech firm in the Dalian) carrier (Fig. 1), 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 PCR detection and double digestion detection (Fig. 2 B).With the recombinant plasmid is template, the PCR product (Fig. 2 D) of the 1.1kb that arrives with primer 5 ' adh and 3 ' adh primer amplification.Multiple clone site according to positive recombinant plasmid pMD-adh carrier two ends, with EcoRI single endonuclease digestion recombinant plasmid, detect enzyme through 1% agarose gel electrophoresis and cut product, the recombinant plasmid pMD-adh enzyme of successful connection is cut product and is the band (Fig. 2 C) about 3.9kb in theory.
Embodiment 2: make up intermediate carrier pENTR
TM-adh
With SalI and EcoR I double digestion pMD-adh and pENTR
TM2B-ccdB (Fig. 3) separates the carrier that has cut and inserts fragment by agarose gel electrophoresis, reclaims pMD-adh respectively and is cut adh gene fragment (1.1kb) and the pENTR that the back produces
TM2B-ccdB is cut the carrier segments pENTR that the back produces
TM2B uses the ligase enzyme test kit of precious biological (TaKaRa) to connect pENTR then
TMThe dna segment of 2B and adh gene produces intermediate carrier pENTR
TM-adh (Fig. 3).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 extracts plasmid from Km resistance recon bacterium colony, select the plasmid vector pENTR of successful connection
TM-adh carries out electrophoresis detection, and its size is 3.4kb, has plasmid pMD-dof1 in contrast, and size is 3.2kb (Fig. 4 A).With pENTR
TM-adh is a template, utilizes adh gene upstream and downstream primer to carry out pcr amplification, and amplification obtains the band (Fig. 4 C) about 1.1kb.Detect with BamHI (Takara) and EcoR I (Takara) double digestion, the plasmid of successful connection only produces two bands on agarose gel electrophoresis figure, be respectively 2.4kb and 1.0kb (Fig. 4 B).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
TM-adh.
Embodiment 3: the structure of the clone pENTR*-PrbcS-adh that crosses the threshold
With Nco I and EcoR I double digestion pENTR
TM-adh and pENTR*-PrbcS-*T-GFP plasmid (Fig. 5), separate the carrier that has cut and insert fragment by agarose gel electrophoresis, from gel, reclaim pENTR*-PrbcS-*T-GFP and be cut carrier segment pENTR*-PrbcS (4.0kb) and the pENTR that the back produces
TM-adh is cut the dna segment (1.1kb) of the adh gene of generation, uses the ligase enzyme test kit connection pENTR*-PrbcS of precious biological (TaKaRa) and the dna segment of adh gene to produce entry vector pENTR*-PrbcS-adh (Fig. 5) 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 extracts plasmid from Km resistance recon bacterium colony, select the plasmid vector pENTR*-PrbcS-adh of successful connection, carry out electrophoresis detection, its size is 4.9kb, has plasmid pENTR*-PrbcS-*T-hps/phi in contrast, and size is 5.3kb (Fig. 6 A).With pENTR*-PrbcS-adh is template, utilizes adh gene upstream and downstream primer to carry out pcr amplification, and amplification obtains the band about 1.1kb, control plasmid pENTR
TM-adh amplified production also is the band (Fig. 6 C) of a 1.1kb.Cut detection pENTR*-PrbcS-adh, the plasmid of successful connection and control plasmid pENTR with EcoRV (Takara) enzyme
TM-adh is all at the band (Fig. 6 B) that occurs on the agarose gel electrophoresis figure about a 200bp.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-adh.
Embodiment 4: the structure of plant expression vector pH2-35S-PrbcS-adh
LR reaction by the Gateway technology is PrbcS-adh subclone in the plant expression vector pH2GW7 (the purpose carrier of Gateway, Belgian VIB/Gent company) (Fig. 7).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-adh and each 150ng of pH2GW7,1 μ l LR Clonase II Enzyme Mix (Invitrogen), be mixed and spend the night, PrbcS-adh is incorporated into the plant expression carrier plasmid pH2-35S-PrbcS-adh (Fig. 7) of acquisition adh 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 is available from sky root biochemical technology company), be applied on the flat board that is added with spectinomycin (Spe, 50 μ g/ml) transforming good intestinal bacteria, in 37 ℃ of incubated overnight, screening Spe resistance recon bacterium colony.From Spe resistance recon bacterium colony, extract plasmid, select the plasmid pH2-35S-PrbcS-adh (Fig. 8 A) of the size successful integration similar with control plasmid pH2GW7.PH2-35S-PrbcS-adh carries out PCR and detects, the Auele Specific Primer of photoinduction promoter 5 ' PrbcS-2 is used in the upstream, the downstream uses 3 ' adh primer to carry out pcr amplification, uses pENTR*-PrbcS-adh as template over against shining test, and negative contrast uses pH2GW7 as template.1.3kb purpose band all appears in pH2-35S-PrbcS-adh and pENTR*-PrbcS-adh amplified production, and negative contrast does not have amplified production (Fig. 8 C).Further cut the exactness of checking recombinant plasmid then with enzyme, singly cut with EcoRV and detect pH2-35S-PrbcS-adh, enzyme is cut result and EcoRV, and singly to cut pENTR*-PrbcS-adh identical, obtains a band (Fig. 8 B) about 200bp.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 Totomycin.
Embodiment 5: the plant expression vector with the adh gene transforms Agrobacterium
The competent cell of preparation Agrobacterium, the electricity consumption impulse method changes the above-mentioned plant expression vector pH2-35S-PrbcS-adh 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 5 minutes as the PCR reaction.PCR detects the pH2-35S-PrbcS-adh conversion results, the upstream and downstream primer is respectively 5 ' adh and 3 ' adh, over against using the pENTR*-PrbcS-adh plasmid according to the amplification system template, the pH2GW7 plasmid is used in negative contrast, amplification segment theoretical length is 1.1kb, the PCR product shows that through electrophoretic analysis its clip size conforms to the theoretical prediction value, shows that plasmid has changed Agrobacterium (Fig. 8 D) over to
Embodiment 6: with the Agrobacterium conversion tobacco that contains adh gene plant expression vector
The single colony inoculation of Agrobacterium that picking carries plasmid pH2-35S-PrbcS-adh (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+BAP 0.02 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.
Insertion situation and the transcriptional level of embodiment 7:adh gene in transgene tobacco detects
In order to confirm to contain really the dna fragmentation of the goal gene of importing, the transgene tobacco that screens is done further to identify with PCR method by the transgene tobacco strain system of hygromycin selection.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 pH7.5100mM, EDTA20mM) 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 1.5mlEP; 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 of acquisition.To change adh tobacco resistance seedling genome as template, the upstream and downstream primer is respectively 5 ' PrbcS-2 (PrbcS Auele Specific Primer), and 3 ' adh carries out pcr amplification detection adh and whether inserts the tobacco gene group.The amplified production size is about 1.3kb, and consistent with the expection supposition, the illustration purpose gene has all inserted the genome of these transgenic lines, and target stripe (Fig. 9 A) does not appear in wild-type tobacco genome PCR product.
In order to investigate the transcribe situation of goal gene in transgene tobacco strain system, from transgenic plant, extract total RNA, reverse transcription is used for the RT-PCR analysis after becoming cDNA, detects the transcriptional level of adh 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 detected through gel electrophoresis quality and concentration.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 * reactionbuffer, 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 ReverseTranscriptase, 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, carries out RT-PCR with the upstream and downstream primer of adh gene and analyzes, and investigates the transcript whether goal gene is arranged in the transgene tobacco.The result proves that transgenic tobacco plant all has the transcript of goal gene, and the tobacco of wild-type does not then have (Fig. 9 B).
Embodiment 8: the enzyme activity assay of transgene tobacco formaldehyde dehydrogenase (FALDH)
Extracting soluble proteins from tobacco leaf is got the 0.2g tobacco leaf, adds 1ml albumen extract [100mMTris-HCl (pH7.5); 10% (V/V) glycerine; The 10mM mercaptoethanol; 1mM PMSF; 5% (W/V) PVP] grind, be transferred in the EP pipe the centrifugal 25min of 13000rpm (4 ℃).Supernatant is moved on in the new EP pipe, with the protein concn in the Bradford method mensuration plant supernatant.
The FALDH enzymic activity 25 ℃ by the generations that detect NADH calculate (Koivusalo etc., FEBSLett, 1989,257:105-109).Reaction system (800 μ l) is: 0.1M sodiumphosphate (pH8.0), 1mM HCHO, 1mM glutathione, 2.4mM NAD, protein sample.
Change the strain of adh tobacco and tie up to 25 ℃ of continuous lights cultivations after 15 days, detect FALDH enzymic activity (Figure 10).Transgene tobacco activity ratio wild-type tobacco improves about about 3～5 times, this show adh in tobacco overexpression have active FALDH.
Embodiment 9: transgene tobacco absorbs the liquid formaldehyde rate determination
Formaldehyde can with Nash reagent (Ammonium Acetate: 15%, Glacial acetic acid: 0.3%, methyl ethyl diketone: 0.2%) chemical reaction takes place and produce coloured material, the maximum absorption wavelength of this material is 410nm, therefore can the preparation standard curve, can calculate the content of formaldehyde in the reaction solution according to the HCHO-Nash typical curve, utilize this method can measure the uptake rate of plant PARA FORMALDEHYDE PRILLS(91,95).Get 0.3g left and right sides vegetable material and put into little triangular flask, add 10～20ml treatment solution (HCHO 2-4mM, KHCO
35mM, MES 0.1%), handle certain hour.Take out 20～100 μ l treatment solutions, add water to 1mL, add 1mL Nash reagent again and after 30 minutes, measure OD 30 ℃ of insulations
410, calculate the remaining concentration of formaldehyde of treatment solution (Figure 11) according to typical curve again.The result shows, it is about 5% and 8% that 2 transgene tobacco strains tie up to the ratio of handling residual formaldehyde after 48 hours in the 2mmol/L formaldehyde solution, and the ratio of wild-type residual formaldehyde is 25%, and the speed that this explanation transgene tobacco absorbs formaldehyde in the liquid is better than wild-type.
Embodiment 10: the resistance of transgene tobacco PARA FORMALDEHYDE PRILLS(91,95) detects
Transgenosis and wild-type seedling are moved into MS solid medium (concentration of formaldehyde 10mmol/L), after 25 ℃ of continuous lights are cultivated about 30 days, observe the tobacco phenotype and change (Figure 12), it is obviously more excellent than wild-type growing way to change the adh tobacco as can be seen, this shows that FALDH helps to improve the resistance of tobacco for liquid formaldehyde in the solid medium very much, compare with wild-type, the growth of transgenosis strain is subjected to the influence of formaldehyde very little.
Claims (1)
1. make up the method for the plant expression vector pH2-35S-PrbcS-adh of arabidopsis glutathione dependent formaldehyde dehydrogenase gene, described carrier contains glutathione dependent formaldehyde dehydrogenase gene adh cDNA, the cDNA of glutathione dependent formaldehyde dehydrogenase gene adh derives from Arabidopis thaliana, the GenBank accession number is 834417, the upstream is connected with the photoinduction type promotor PrbcS of Rubisco 3C small subunit, and the initial vector of plant expression vector is pH2GW7 under being used to make up; This method comprises the steps:
(1) from GenBank, search the cDNA sequence of Arabidopis thaliana adh gene, and the following a pair of primer of implementation sequence:
5’adh：CA
CCATGGCGACTCAAGGTCAGGTTATC
3’adh：
GAATTCTCATTTGCTGGTATCGAGG
Upstream primer 5 ' adh, 5 ' end adds the CACC characteristic sequence, and forms Nco I restriction enzyme site thus; Downstream primer 3 ' adh, end add the EcoRI enzyme and cut the position, are the masterplate amplification with the Arabidopis thaliana first chain cDNA, obtain the full-length cDNA of adh gene;
(2) reclaim also purifying adh full-length gene cDNA fragment, and be connected on the pMD-18T carrier, adopt alkaline lysis method of extracting plasmid DNA, cut detection by PCR detection and enzyme and obtain recombinant plasmid pMD-adh;
(3) make up intermediate carrier pENTR
TM2B-adh cuts pMD18-adh and pENTR with SalI and EcoR I enzyme
TM2B (Invitrogen) obtains target gene fragment adh and carrier segments pENTR
TM, connect after the recovery, the transformed competence colibacillus cell, obtain recombinant vectors pENTR
TM-adh;
(4) make up entry vector pENTR*-PrbcS-adh, with Nco I and EcoR I digestion pENTR*-PrbcS-*T-GFP and pENTR
TM-adh reclaims carrier pENTR*-PrbcS fragment and adh gene cDNA fragment, connects then, conversion, extracting plasmid carry out that PCR detects and enzyme is cut detection, obtains recombinant plasmid pENTR*-PrbcS-adh;
(5) make up plant expression vector pH2-35S-PrbcS-adh, the LR reaction by the Gateway technology the PrbcS-adh subclone in plant expression vector pH2GW7, the plant expression vector pH2-35S-PrbcS-adh of acquisition adh gene.
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| CN101302530A (en) * | 2007-12-05 | 2008-11-12 | 昆明理工大学 | Gateway cloning entry vector, construction method and use thereof |
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Non-Patent Citations (2)
| Title |
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| Hakima Achkor et al..Enhanced Formaldehyde Detoxification by Overexpression of Glutathione-Dependent Formaldehyde Dehydrogenase from Arabidopsis.《Plant Physiology》.American Society of Plant Biologists,2003,第 132 卷2248-2255. * |
| 陈丽梅等.耐受甲醛并能利用甲醇细菌的分离及其基因组文库的构建.《安徽农业科学》.2008,第 36 卷(第 3 期),978-981. * |
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