CN1313614C - Glyphosate acetyl transferase gene and its application - Google Patents

Glyphosate acetyl transferase gene and its application Download PDF

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CN1313614C
CN1313614C CNB200510086626XA CN200510086626A CN1313614C CN 1313614 C CN1313614 C CN 1313614C CN B200510086626X A CNB200510086626X A CN B200510086626XA CN 200510086626 A CN200510086626 A CN 200510086626A CN 1313614 C CN1313614 C CN 1313614C
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glyphosate
acetyl transferase
plasmid
gene
recombinant plasmid
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CN1772908A (en
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林敏�
顿宝庆
陆伟
金丹
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Longping Biotechnology (Hainan) Co.,Ltd.
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Biotechnology Research Institute of CAAS
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Abstract

The present invention relates to a glyphosate N-acetyltransferase (GAT) gene and an application thereof. A novel glyphosate N-acetyltransferase gene is sieved by the present invention, and is respectively transferred to a recipient bacterium E. coli BL21 and mode plant tobaccos, and thus, the prepared genetic engineering strain and the transgenic tobaccos obtain the tolerance capability on the herbicide glyphosate.

Description

Glyphosate acetyl transferase gene and application thereof
Technical field:
The present invention relates to a kind of glyphosate acetyl transferase gene, the invention still further relates to the plasmid that contains glyphosate acetyl transferase gene, and the purposes of this gene aspect preparation transgenic engineered bacteria and cultivation transgenic plant.
Background technology:
Glyphosate (glyphosate) is go out natural disposition, the outstanding weedicide of inner sucting conduction type of a kind of wide spectrum, be one of weedicide kind of whole world usage quantity maximum (appoint out of the ordinary, Cao Song monk .1998. glyphosate and progress. agricultural chemicals .37 (7): 1-8).
But glyphosate is a kind of nonselective herbicide, and farm crop are had killing effect equally.Therefore, often only in orchard, plantation, bare place, use at present, or be applied in the no-tillage ground corn, soybean, cotton and broadcast preceding or broadcast aftertreatment, and the back directional process of emerging.This has just limited use range and the duration of service of glyphosate as weedicide greatly.
Have glyphosate resistance or degraded character farm crop in crop growth, using glyphosate, must cultivating, when saving farm production cost, can greatly improve agricultural production efficiency again.
Glyphosate suppresses phosphoenolpyruvic acid shikimic acid-3-phosphate synthase (EPSPS) activity in the plant shikimic acid metabolic process, and then the blocking-up die aromatischen Aminosaeuren biosynthesizing and make plant death (S.R.Padgette et al., inHerbicide-Resistent Crops:Agricultural, Environmental, Economic, Regulatory, andTechnical Aspects, S.O.Duke, Ed. (CRC Press, Boca Raton, FL, 1996), pp.53-84), all glyphosate resistant transgenic crops of current global commerce plantation are at EPSPS designed, are unique mechanism of action of present commercialization transgenosis glyphosate resistant crops.As U.S. Meng Shan Roudup Ready transgenosis resistance glyphosate kind of (Monsanto) company all.
Because glyphosate can be absorbed by plant materials, and may in influencing some meristematic tissue of growth and development of plants and output, accumulate (W.A.Pline, J.W.Wilcut, S.O.Duke, K.L.Edmisten, R.Wells, J.Agric.Food Chem.50, therefore 506 (2002)), use glyphosate to exist potential unfavorable factor to the people.
Glyphosate can effectively be detoxified by the N-acetylize, thereby loses herbicidal activity, and acetylizad glyphosate is not the useful effect substrate of EPSPS.Thereby utilize the acetylizad means of N-to have stronger glyphosate resistance by the render transgenic crop, and make glyphosate all can use in the plant whole growth cycle, be not subjected to the restriction of growth and development stage.
Summary of the invention:
The objective of the invention is to filter out a kind of new glyphosate acetyl transferase gene, and this gene is imported receptor biological, make the biological ability that obtains the tolerance glyphosate of resulting genetic engineering reorganization.
Technical scheme of the present invention is:
1. by exempting from the gene library that culture technique makes up glyphosate contaminated soil microorganism total DNA, the clone obtains glyphosate acetyl transferase gene with the function method for screening, and the sequence of this gene is shown in SEQ ID NO:1, and its size is 441bp;
2. the gene fragment shown in the above-mentioned SEQ ID NO:1 is connected to have and efficiently expresses in active pET28a (+) plasmid vector, make up recombinant plasmid pETGAT with complete glyphosate acetyl transferase gene;
3. the recombinant plasmid pETGAT that will contain glyphosate acetyl transferase gene imports does not have an acceptor e. coli bl21 of glyphosate resistance, and acquisition can tolerate the genetic engineering bacterial strain of glyphosate;
4. the gene fragment shown in the above-mentioned SEQ ID NO:1 is connected among the plant expression vector pBI121, makes up recombinant plasmid pBIGAT with complete glyphosate acetyl transferase gene;
5. the recombinant plasmid pBIGAT that will contain glyphosate acetyl transferase gene imports does not have among the tobacco bred NC89 of glyphosate resistance, and acquisition can tolerate the genetic engineering tobacco plant of glyphosate.
Experimental result shows that the recombination bacillus coli and the transgene tobacco that contain the acetyl transferase gene of gained of the present invention have obtained stronger glyphosate tolerance ability.
The present invention utilizes glyphosate contaminated soil microorganism total DNA to set up first gene library, directly obtained having the glyphosate acetyl transferase gene of careless glycosides phosphine tolerance by functional screening, the development and application of this enzyme will provide an optionally brand-new approach for supporting the application of glyphosate on plant.
Embodiment:
The plasmid of being lifted in following examples, bacterial strain etc. just are used for the present invention is described in further detail, and flesh and blood of the present invention are not limited.In fact, with gene and method that the present invention finds, those skilled in the art can obtain other multiple genetic engineering bacterial strain with glyphosate tolerance ability.
Plasmid, the bacterium source lifted among the embodiment are as follows:
PET28a (+) plasmid vector: be Novagen company commercially available prod;
Plant expression vector pBI121: be Clontech company commercially available prod;
E. coli jm109, recipient bacterium E.coli BL21: for sky, Beijing is the Time Inc. commercially available prod.
The structure of the total DNA gene library of embodiment 1 glyphosate contaminated soil
The 1. extraction of the total DNA of soil microorganisms (method is with reference to the microorganism journal, in April, 2003, and vol.43, No.2)
1) take by weighing 1 gram pedotheque, add 3ml DNA extraction liquid mixing, add 20ul Proteinase K (10mg/ml) again, 37 ℃ of temperature were bathed 30 minutes, and middle mixing for several times.
2) add 0.3ml 20%SDS, 65 ℃ of water-bath 2h put upside down mixing once every 15-20 minute.
3)-70 ℃ freezing, 65 ℃ dissolve then, 3 times repeatedly.
4) room temperature 6000xg is centrifugal 10 minutes, collects supernatant in a new 10ml centrifuge tube
5) precipitation adds 1ml DNA extraction liquid and 0.1ml 20%SDS again, vortex 10s, and 65 ℃ of water-baths 10 minutes, centrifugal 10 minutes of room temperature 6000xg merges supernatant liquor.
6) repeating step 4, merge supernatant liquor.
7) add equal-volume chloroform/primary isoamyl alcohol (24: 1v/v) mixing
8) 12000xg is centrifugal 10 minutes, and supernatant is transferred to a new 10ml centrifuge tube, adds 0.6v/v Virahol precipitation at room temperature 1h.
9) 16000xg is centrifugal 20 minutes, removes supernatant.
10) add the washing of 70% pre-cooled ethanol, precipitation is resuspended with the 100ul aseptic deionized water.
11) gel absorption method purify DNA
Utilize QXII DNA purifying to reclaim test kit (QIAEXII DNA Gel Extraction Kit) and carry out method for purifying and recycling, undertaken by the test kit explanation
(attached: DNA extraction liquid 100mmol/L Tris-HCl, 100mmol/L EDTA, 100mmol/L Na3PO4,1.5mol/L NaCl, 1%CTAB, pH8.0)
In 37 ℃ of water-baths, carry out partially digested with the rare 200X of an amount of Sau3AI to the total DNA of 50 ~ 100 μ g, endonuclease bamhi is electrophoresis on 0.4% agarose gel, behind ethidium bromide staining, under ultraviolet ray, downcut and contain the segmental agarose gel of 1 ~ 3kb, put into the centrifuge tube of 1.5ml, 55 ~ 65 ℃ of water-bath 7min of Binding Buffer that add 3 ~ 4 times of volumes thoroughly dissolve up to glue, place DNA/ agarose lysate to reclaiming under the column compartment temperature 10, the centrifugal 1min of 000rpm adds the Wash Buffer 750 μ l with the dehydrated alcohol dilution, under the room temperature 10, the centrifugal 1min of 000rpm adds 30 ~ 50 μ l ultrapure waters in centrifugal post, 10, the centrifugal 1min of 000rpm reclaims DNA.The PUC19 plasmid is cut precipitation with restriction enzyme BamHI and is reclaimed.With the 1-3kb endonuclease bamhi (0.3-0.9 μ g), T4 ligase enzyme (3U), the carrier pUC19 (1.0 μ g) that reclaim, 4 ℃ of connections are spent the night, and reaction volume is 10 μ l, will connect product electric shock transformed into escherichia coli Jm109, obtain the total DNA gene library of soil.
2. electric shock Transformed E .coli competent cell is made:
1) the single bacterium colony of picking E.coli JM109 is in the LB substratum, 225rpm, 37 ℃ of incubated overnight.
2) get 2%v/v bacterium liquid and be re-seeded in the new 100ml LB substratum, 300rpm, 37 ℃ are shaken bacterium to OD6000.5-0.7 (4-5 * 10 7Cells/ml).
3) cooled on ice 20min, below operation is carried out under near 0 ℃ as far as possible, and all containers all need cooled on ice.
4) bacterium liquid changes in two 50ml centrifuge tubes, and 4 ℃, 4000xg, centrifugal 15min abandons supernatant.
5) add the resuspended thalline of 50ml 10% glycerine.
6) 4 ℃, 4000xg, centrifugal 15min abandons supernatant.
7) add the resuspended thalline of 25ml 10% glycerine.
8) 4 ℃, 4000xg, centrifugal 15min abandons supernatant.
9) add the resuspended thalline of 10ml 10% glycerine.
10) 4 ℃, 4000xg, centrifugal 15min abandons supernatant.
11) add the resuspended thalline of 100-200 μ l 10% glycerine (precooling) (final concentration of cells 1-3 * 10 10Cells/ml), 40 μ l/ manage packing, are stored in behind the liquid nitrogen flash freezer in-70 ℃ of refrigerators.
3. the electric shock conversion method is set up gene library
1) gets two pipe E.coli JM109 competent cells (40 μ l) in dissolving on ice.
2) add 1-2 μ l respectively and connect product or empty carrier plasmid, mixing places about 1min on ice gently.
3) electric shock program (pre-set protocol-E.coli) is set.
4) mixed solution is changed in the 0.2cm electric shock cup of precooling, close the lid, put into the electric shock pond.
5) electric shock (Pulse).
6) take out the electric shock cup, add 1ml LB substratum immediately, gently mixing.
7) bacterium liquid is transferred in the 1.5ml centrifuge tube of precooling, 225rpm cultivates 1h for 37 ℃.
8) record shock parameters, time constant must be near 5milllseconds.
9) cultivate bacterium liquid coating penbritin flat board, 37 ℃ of cultivations.
The method of embodiment 2 functional screening glyphosate acetyl transferase genes
1. the bacterium that transforms is coated and added X-gal, on the LB solid medium of IPTG and Amp, obtain the recon bacterium colony about 10 of white after 16 hours, 000, they are changed on the M9 solid medium that adds 20mM glyphosate and Amp (60 μ g/ml), observing the bacterium colony that can grow after 72 hours is the clone who contains glyphosate acetyl transferase gene again.Positive colony upgrading grain, enzyme is cut checking, order-checking.
2. use alkaline lysis method of extracting plasmid DNA:
1) with the positive colony colony inoculation selected in 5ml liquid LB substratum, 37 ℃ of 200rpm joltings are spent the night
2) get the 1.5ml nutrient solution in the Eppendorf centrifuge tube, 12, the centrifugal 2min of 000rpm.
3) outwell supernatant fully, suspend with 100 μ l solution I (50mmol/L sucrose, 10mmol/LEDTA pH8.0,25mmol/LTris-HCl pH8.0) and precipitate thalline, place 5min on ice.
4) add the 200 μ l solution II (1%SDS, 0.2mol/L NaOH) of now joining, the mixing for several times that turns upside down is placed 5min on ice.
5) add 150 μ l solution III (5mol/L KAc+11.5ml Glacial acetic acid is mended and added water to 100ml), turn upside down for several times, place 5min on ice.
6) add 500 μ l phenol/chloroforms/primary isoamyl alcohol (25: 24: 1), mixing for several times turns upside down.
7) 12, the centrifugal 10min of 000rpm draws supernatant, adds the dehydrated alcohol of 2 times of volume precoolings, places 20min for-70 ℃.
8) 12, the centrifugal 15min of 000rpm abandons supernatant, the washing with alcohol precipitation with 70%.
9) after freezing the draining, add 50 μ l TE dissolving plasmid DNA.
10) get 2 μ l plasmid DNA, cut the testing goal fragment with the BamHI enzyme.
The sequence of embodiment 3 glyphosate acetyl transferase genes
From gene library, screen 1 strain positive recombinant, enzyme is cut the result and is shown the segment of having inserted an about 1Kb, sequencing analysis finds to contain the ORF sequence of a 441bp, with the nucleotide sequence homology of the active glyphosate acetyl transferase gene of known function (Genebank accession number AY597417) is: 74.60%.
The DNA nucleotide sequencing is given birth to worker biotech firm by Shanghai and is finished, Nucleotide and amino acid analysis software are mainly DNAman and (the National Center for Biotechnology Information of American National biotechnology information center, NCBI, http://www.ncbi.nlm.nih.gov/) Blast program.
The structure of embodiment 4 glyphosate acetyl transferase gene efficient expression vector plasmids
According to the ORF sequences Design primer of 441bp, pcr amplification obtains the GAT gene segment, is connected to then on the bacterial expression vector pET28a (+), makes up the recombinant plasmid pEGAT. that contains glyphosate acetyl transferase gene
The structure of embodiment 5 engineering strains and the mensuration of glyphosate tolerance ability
The recombinant plasmid pEGAT that will contain glyphosate acetyl transferase gene changes over to through chemical transformation does not have in the e. coli bl21 of glyphosate resistance, and specific practice is as follows:
1. the conversion of the preparation of competent cell and plasmid
1) preparation of competent cell (calcium chloride transformation)
A) picking list colony inoculation is to the test tube of the LB liquid nutrient medium that contains 5ml from activatory intestinal bacteria (Ecoli) BL21 flat board, and 37 ℃ of shaking culture made the OD600 value of bacterium liquid reach 0.4 to 0.6 in 2.5 to 3 hours, cooled on ice culture to 0 ℃
B) culture is poured in the centrifuge tube of aseptic 1.5ml
C) 4 ℃, the centrifugal 10min of 4000rpm
D) abandon supernatant, collect thalline
E) CaCl of the 0.1mol/l of the 0.5ml of usefulness precooling 2Resuspended thalline, centrifugal, abandon supernatant
F) CaCl of the 0.1mol/l of the 0.5ml of usefulness precooling 2Resuspended thalline, ice bath 15min, centrifugal, abandon supernatant
G) CaCl of the 0.1mol/l of the precooling of adding 200ul 2Resuspended thalline, ice bath is placed
2) plasmid transformed competence colibacillus cell
A) get the 0.5ul plasmid and be added in the pipe competent cell, rotate gently, place 30min on ice with the mixing content
B) centrifuge tube is placed 42 ℃ of water-bath heat shocks 90 seconds, do not rock centrifuge tube
C) news speed places centrifuge tube on ice, cooling 2min
D) the LB liquid nutrient medium of adding 800ul, 37 ℃, the 200rpm shaking table is cultivated 45min
E) nutrient solution of getting 50ul is coated on the LB solid plate that contains kantlex (50 μ g/ml), cultivates 12 to 16 hours for 37 ℃, checks to transform bacterium colony
F) select single bacterium colony, be inoculated in the test tube of the LB liquid nutrient medium that contains 5ml, 37 ℃ of shaking culture are extracted plasmid enzyme restriction and are verified as correct transformant, and order-checking proof then contains glyphosate acetyl transferase gene.
2. tolerate the mensuration of glyphosate ability:
With BL21 bacterial strain, the BL21 bacterial strain that contains pET28a (+) empty carrier is the contrast that contains the pEGAT recombinant bacterial strain, and picking list colony inoculation is in the LB of 20ml nutrient solution respectively, and 37 ℃ of 200rpm spend the night and shake training; Inoculum size with 2% is got 4 ℃ of 2ml bacterium liquid respectively, the centrifugal flush away substratum of 4000rpm, use the M9 liquid nutrient medium resuspended then, being inoculated into 100ml respectively contains in the 200mM glyphosate M9 liquid nutrient medium, make its initial OD 600 values be 0.05,37 ℃ of 200rpm shake 72 hours measurement results of training and show, the recombinant bacterial strain that the contains pEGAT obvious OD600 that grows reaches 0.42, and two control strains all do not have considerable change.
The structure of embodiment 6 glyphosate acetyl transferase gene plant expression carrier plasmids
According to the ORF sequences Design primer of 441bp, pcr amplification obtains the GAT gene segment, is connected to then on the plant expression vector pBI121, makes up the recombinant plasmid pBIGAT that contains glyphosate acetyl transferase gene
The structure of embodiment 7 transgene tobaccos and the mensuration of glyphosate tolerance ability
1. the competent preparation of agrobacterium tumefaciens lba4404
1) picking list bacterium colony from the flat board is inoculated into 5ml YEB liquid nutrient medium (containing Streptomycin sulphate Strep 125mg/L), and 28 ℃, 250rpm shaking culture spend the night.
2) get 2ml bacterium liquid, add in the 50ml YEB liquid nutrient medium (containing Strep125mg/L), 28 ℃, 250rpm shaking culture are to OD 600About about 0.6.
3) bacterium liquid is gone in the aseptic centrifuge tube of 50ml ice bath 30min, the centrifugal 5min of 5000rpm.
4) abandon supernatant, precipitation 2ml 20mM CaCl 2Heavily floating, every part 100 μ l branch installs in the 1.5ml centrifuge tube, preserves standby in the liquid nitrogen.
2. recombinant plasmid dna changes Agrobacterium over to
1) the pBIGAT plasmid DNA with about 1 μ g joins in the 100 μ l LBA4404 competent cells mixing, ice bath 5min respectively.
2) centrifuge tube is put freezing 8min in the liquid nitrogen, gone to temperature bath 5min in 37 ℃ of water-baths rapidly.
3) add 1ml YEB liquid nutrient medium, 250rpm recovery 4~5h on 28 ℃ of shaking tables.
4) get an amount of bacterium liquid and be applied on the YEB solid medium that contains Str (Streptomycin sulphate) 250~300mg/L, Rep (rifampin) 250~300mg/L and Kan (kantlex) 100mg/L, put 28 ℃ and cultivate 24~48h.
3. leaf dish method transformation of tobacco kind NC89
1) activation of Agrobacterium
The single bacterium colony of picking Agrobacterium is inoculated into (Kan 100mg/L, Str 100mg/L, Rif 300mg/L) in the 5ml YEB liquid nutrient medium from the flat board, and shaking culture is spent the night; Get 1ml bacterium liquid and be inoculated in the 50ml YEB liquid nutrient medium (Kan 100mg/L, Strep 100mg/L, Rif 300mg/L), thermal agitation is cultured to OD 600Be 0.4 ~ 0.5 (about 3 ~ 4h); The centrifugal 5min of 5000rpm, thalline MS 0Substratum (not containing hormone) is resuspended, makes OD 600Be 0.1 ~ 0.2.
2) genetic transformation of tobacco
A) cultivate altogether: the leaf piece that will infect is placed on the tobacco bud division culture medium (MS+IAA 0.5mg/L+6-BA 2mg/L) that is covered with 2 metafiltration paper, 25 ℃ of dark cultivations 4 days.
B) screening of resistant buds: will transfer on the resistant buds screening culture medium (MS+IAA0.5mg/L+6-BA 2mg/L+Kan 100mg/L+Carb 500mg/L) through the tobacco explant of cultivating altogether, and can sprout after 2~3 weeks.
C) take root: when treating resistant buds length, it is transferred on the root media (MS+Kan 100mg/L+Carb 500mg/L), promptly have adventive root to form after 1~2 week to the 1cm left and right sides.
4. tolerate the mensuration of glyphosate ability:
With identical seedling age non-transgenic tobacco NC89 is contrast, spray the different third ammonia salt of 5 ‰ careless glycosides phosphines (glyphosateisopropylamine salt) aqua, the glyphosate of non-transgenic contrast performance as a result is injured that reaction begins after one day to wilt and 3 weathers hop to it withered dying; The growth of always acting normally after positive transfer-gen plant sprays is not subjected to glyphosate and injures, and shows that transfer-gen plant has had the glyphosate tolerance ability.
Result of study shows that the recombination bacillus coli and the transgene tobacco that contain acetyl transferase gene have obtained stronger glyphosate tolerance ability.
Appendix
SEQ ID NO:1 glyphosate acetyl transferase gene nucleotide sequence
atgattgacg?tgaacccaat?taacgctgag?gatacttacg?agcttagaca?tagaattctt 60
agaccaaacc?aaccaatcga?ggcttgcatg?ttcgagtctg?atcttcttag?aggagctttc 120
catcttggag?gttactacgg?aggtaagctt?atttctattg?cttctttcca?tcaagctgag 180
cattctgagc?ttcaaggaca?aaagcaatac?caacttaggg?gaatggctac?tcttgaggga 240
tacagagagc?aaaaggctgg?ttcttctctt?atcaagcatg?ccgaggagat?cctcaggaag 300
aggggcgccg?accttctttg?gtgcaacgct?aggacttccg?cctctggata?ctacaagaag 360
cttggcttct?ctgagcaggg?agaggtgttc?gacactccac?ctgtgggacc?ccatatcctt 420
atgtacaaga?ggatcgcata?a 441

Claims (9)

1.SEQ the dna sequence dna shown in the ID NO:1.
2. the recombinant plasmid that contains dna sequence dna shown in the claim 1.
3. the described recombinant plasmid of claim 2 is to contain the plasmid of dna sequence dna shown in the claim 1 at escherichia coli expression.
4. the described recombinant plasmid of claim 2 is to contain the plasmid that dna sequence dna is expressed in tobacco shown in the claim 1.
5. the purposes of claim 2 or 3 described recombinant plasmids is preparation tolerance glyphosate gene engineering bacteria or preparation tolerance glyphosate transgenic plant.
6. genetic engineering bacterium that tolerates glyphosate, feature is to contain the described recombinant plasmid of claim 2.
7. a preparation method who tolerates the glyphosate gene engineering bacteria is to recipient bacterium with the described recombinant plasmid transformed of claim 2.
8. a method of cultivation that tolerates the glyphosate transgenic plant is to recipient plant with the described recombinant plasmid transformed of claim 2.
9. the method for cultivation of the described tolerance glyphosate of claim 8 transgenic plant, described recipient plant is a tobacco.
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WO2007024782A2 (en) 2005-08-24 2007-03-01 Pioneer Hi-Bred International, Inc. Compositions providing tolerance to multiple herbicides and methods of use thereof
JP2009000046A (en) * 2007-06-21 2009-01-08 Hitachi Zosen Corp Gene encoding enzyme involved in mevalonic acid pathway of eucommia ulmoides oliver
CN108414768B (en) * 2018-02-06 2020-10-30 中国农业科学院生物技术研究所 Gold-labeled detection test strip for glyphosate-resistant GAT transgenic crops
WO2022213520A1 (en) * 2021-04-08 2022-10-13 中国农业科学院生物技术研究所 Expression vector of glyphosate-resistant genes gr79 and gat, high glyphosate-resistant corn, and detection method therefor

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Publication number Priority date Publication date Assignee Title
CN1392261A (en) * 2001-06-19 2003-01-22 中山大学 Glyphosate degrading enzyme gene and carrier containing said gene
CN1431219A (en) * 2003-01-16 2003-07-23 中山大学 Resistance gene of glyphosate as well as carrier and transformant containing gene
WO2003092360A2 (en) * 2002-04-30 2003-11-13 Verdia, Inc. Novel glyphosate-n-acetyltransferase (gat) genes
CN1531594A (en) * 2000-10-30 2004-09-22 �����ɷ� Novel glyphosate N-acetyltransferase (GAT) genes

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Publication number Priority date Publication date Assignee Title
CN1531594A (en) * 2000-10-30 2004-09-22 �����ɷ� Novel glyphosate N-acetyltransferase (GAT) genes
CN1392261A (en) * 2001-06-19 2003-01-22 中山大学 Glyphosate degrading enzyme gene and carrier containing said gene
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