CN102766609A - Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof - Google Patents
Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof Download PDFInfo
- Publication number
- CN102766609A CN102766609A CN2012101965027A CN201210196502A CN102766609A CN 102766609 A CN102766609 A CN 102766609A CN 2012101965027 A CN2012101965027 A CN 2012101965027A CN 201210196502 A CN201210196502 A CN 201210196502A CN 102766609 A CN102766609 A CN 102766609A
- Authority
- CN
- China
- Prior art keywords
- sequence
- gmepsps
- plant
- gene
- glyphosate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Abstract
The invention discloses a glyphosate resistant protein, and a coding gene and application thereof. The protein provided by the invention is (a) or (b) or (c) as follows: (a) a protein composed of an amino acid sequence shown as 73rd-504th site of a sequence 1 in the sequence table; (b) a protein composed of the amino acid sequence shown in the sequence 1 in the sequence table; and (c) a protein, derived from the sequence 1 and with glyphosate resistance, obtained by substitution and / or deletion and / or addition of one or several amino acid residues on the amino acid sequence of (a) or (b). The GmEPSPS protein and the coding gene thereof provided by the present invention have significant resistance effect on a glyphosate herbicide. Experiments prove that GmEPSPS transgenic tobacco and GmEPSPS transgenic maize can still grow normally under 0.15% of glyphosate, but non-transgenic tobacco and maize are obviously suppressed in growth, and have leaves yellowing and withered. It shows that GmEPSPS can stably and efficiently express in plants, and be further used in production of glyphosate resistant transgenic plants.
Description
Technical field
The invention belongs to plant genetic engineering field; Relate to a kind of resistance glyphosate epsp synthase and encoding sox and application; Be particularly related to a kind of artificial reconstructed synthetic resistance glyphosate epsp synthase and encoding sox GmEPSPS thereof, and contain this expression carrier and the application aspect the development of antiweed transgenic plant thereof.
Background technology
Glyphosate 62 IPA Salt is a kind of interior non-selective, wide spectrum steriland herbicide of conduction of inhaling; Have that physico-chemical property is stable, efficient, low toxicity, low residue, be prone to by microbiological degradation; Do not destroy advantages such as edatope; Be widely used in the agriculture prodn, become at present the maximum pesticide species of turnout in the world.Its dependent interaction mechanism is because Glyphosate 62 IPA Salt is the analogue of PEP (PEP); Therefore Glyphosate 62 IPA Salt, shikimic acid-3-phosphoric acid, epsp synthase combine to form EPSP-S3P-Glyphosate 62 IPA Salt complex body (this complex body is highly stable) easily; Prevention PEP combines with epsp synthase; Glyphosate 62 IPA Salt is with regard to the activity of 5-enol form acetone shikimic acid in the competitive inhibition shikimic acid pathway-3-phosphate synthase (EPSPS) like this; Disturb the biosynthesizing of the interior die aromatischen Aminosaeuren of organism and some aromatic compounds, cause biological death.
Plant can obtain the ability of resistance glyphosate through transform importing the EPSPS gene that Glyphosate 62 IPA Salt is had a tolerance.The EPSPS of agrobacterium tumefaciens CP4, Pseudomonas fluorescens G2 and Salmonellas CT7 is verified widely in plant and is used.Development along with the anti-herbicide gene clone; The resistance glyphosate genetically modified crops are also come out one after another and large scale application; These genetically modified crops have following advantage: (1) used weedicide kind wide spectrum, selectivity are strong, environmentally friendly, easy to use, are specially adapted to minimal till and no-tillage system, have strengthened the moisture maintenance; Significantly reduced depletion, and formed organic substance and pin soil carbon and reduce Carbon emission; Reduce mechanical cultivation simultaneously, the fuel economy energy; (2) reduce the total usage quantity of weedicide, the controlling weeds expense is descended, reduce agricultural cost effectively, increase the economic benefit of agricultural-food; (3) solved the special weeds problem that conventional weedicide is difficult to prevent and treat, like the wild-rice in rice field, red rice, wide leaf arm shape grass, Pharbitis purpurea; The bromegrass of wheat paddock, nutgrass flatsedge; The Tradescantia albiflora in soybean field, pure leaf Cassia tora and parasitic weeds; (4) solve of the injury of soil long residual herbicide to succession crop.The employed Glyphosate 62 IPA Salt of genetically modified crops does not have pedo relict, so fool proof to succession crop.
From genetically modified crops commercialization first in 1996 so far, the herbicide-resistant proterties is the main proterties of genetically modified crops all the time.In 2011; Genetically modified crops herbicide-resistant proterties has been used in soybean, corn, rape, cotton, beet and the clover; Cultivated area is 9,390 ten thousand hectares; Account for 59% of global genetically modified crops cultivated area, exceed much than the pest-resistant cultivar that accounts for global genetically modified crops area 15% (2,390 ten thousand hectares), this wherein major part be resistance glyphosate genetically modified crops kinds.Therefore the national Glyphosate 62 IPA Salt output that comprises the U.S. and China in recent years sharply increases also seed selection with serial transgenic resistance glyphosate kind, and popularization has direct relation with big area.
China has obtained certain progress in the research aspect the transgenic Glyphosate 62 IPA Salt crop, does not change the report that the Antiglyphosate gene crop gets into the commercialization stage but still have at present.Chinese invention patent 03826892.2 disclosed a kind of Antiglyphosate gene (EPSPS gene) that derives from Pseudomonas fluorescens (Pseudomonas fluorescens); But because it derives from bacterium; Bacterium codon preference and plant have than big-difference; To directly transform plant from the EPSPS gene of bacterium, its expression efficiency is lower, therefore; This area is necessary the Antiglyphosate gene from mikrobe is carried out artificial reconstructed, with guarantee Antiglyphosate gene can be in plant materials high efficiency stable expression.
Summary of the invention
The purpose of this invention is to provide a kind of resistance glyphosate albumen and encoding sox and application.
Protein provided by the present invention, title are GmEPSPS, (a) or (b) or (c):
(a) protein of forming by the aminoacid sequence shown in the 73-504 position of sequence in the sequence table 1;
(b) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(c) with (a) or aminoacid sequence (b) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have glyphosate resistance by sequence 1 deutero-protein.
Wherein, the albumen that (a) is limited is the one-tenth body protein of GmEPSPS, does not contain transit peptide sequence; (b) albumen that is limited is the midbody albumen that 5 ' end has the GmEPSPS of petunia CTP4 chloroplast transit peptides.
Above-mentioned (c) but in the protein synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Proteinic encoding sox in above-mentioned (c) can pass through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the sequence table 2, and/or carries out the missense mutation of one or several base pair.
For the ease of the proteic purifying of GmEPSPS, label as shown in the table on proteinic N-terminal that can the amino acid residue sequence of sequence 1 is formed in by sequence table or C-terminal connect.
Table: the sequence of label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned (a) but and the GmEPSPS albumen synthetic (b), also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.The proteic encoding sox of GmEPSPS in above-mentioned (c) can be through the codon that in the dna sequence dna shown in the Nucleotide of 5 ' terminal 461-1975 position, lacks one or several amino-acid residue with sequence in the sequence table 2; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table.
The proteic nucleic acid molecule of said GmEPSPS of encoding also belongs to protection scope of the present invention.
Said nucleic acid molecule can be DNA, like cDNA, genomic dna or recombinant DNA; Said nucleic acid molecule also can be RNA, like mRNA, hnRNA or tRNA etc.
In one embodiment of the invention, said nucleic acid molecule is specially the coding proteic gene of said GmEPSPS (called after GmEPSPS); Said GmEPSPS gene is following 1) to 5) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the 677th to 1975 Nucleotide of sequence 2 in the sequence table;
2) encoding sequence is the dna molecular shown in the 461st to 1975 Nucleotide of sequence 2 in the sequence table;
3) dna molecular shown in the 7th of sequence 2 the to 1975 Nucleotide in the sequence table;
4) dna molecular shown in the sequence 2 in the sequence table;
5) under stringent condition with 1)-4) and in arbitrary qualification the dna molecule hybridize and the proteic dna molecular of said GmEPSPS of encoding.
Above-mentioned stringent condition can be with 6 * SSC, and the solution of 0.5%SDS 65 ℃ of hybridization down, is used 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Wherein, sequence 2 is made up of 1981 Nucleotide, and the 461-1975 position is an encoding sequence, the protein shown in the sequence 1 in the code sequence tabulation.
The recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain above-mentioned nucleic acid molecule also belong to protection scope of the present invention.
Said recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.
Said recombinant expression vector can be used existing plant expression vector construction.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment, like pBI121, pBin19, pCAMBIA2301, pCAMBIA3301, pCAMBIA1301-UbiN, pCAMBIA1300 or other plant expression vector of deriving.Said plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor.When using said gene constructed recombinant expression vector; Before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter; For example cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter Rd29A etc., they can use separately or be used in combination with other plant promoter; In addition; When using gene constructed recombinant expression vector of the present invention; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of transgenic plant cells or plant being identified and screening; Can process used recombinant expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change with resistance as adding the coding that in plant, to express.Also can not add any selected marker, directly with adverse circumstance screening transformed plant.
In one embodiment of the invention, the promotor of the said GmEPSPS genetic transcription of startup is specially 35S promoter (like the CaMV35S promotor) or corn Ubi-1 promotor in the said recombinant expression vector.More concrete, said recombinant expression vector is (1) or (2) as follows:
(1) inserts the recombinant plasmid that said GmEPSPS gene obtains at the MCS place of pCAMBIA3300-35S-pROKII MCS-nos-bar (hereinafter to be referred as CPB) carrier; The promotor that starts said GmEPSPS genetic transcription in the said recombinant plasmid is the CaMV35S promotor.Said MCS is specially XbaI and SacI.
(2) insert the recombinant plasmid that said GmEPSPS gene obtains at the MCS place of pCAMBIA3300-Ubi-pROKII MCS-nos-bar (application of Li Bei .FLP_frt locus specificity recombination system in corn and the acquisition .2008 Shandong University Ph D dissertation of commentaries on classics TsVP drought-resistant corn) (hereinafter to be referred as CUB) carrier; The promotor that starts said GmEPSPS genetic transcription in the said recombinant plasmid is a corn Ubi-1 promotor.Said MCS is specially XbaI and Sac I.
Said CUB carrier (pCAMBIA3300-Ubi-pROKII MCS-nos-bar): the application of Li Bei .FLP_frt locus specificity recombination system in corn and the acquisition .2008 Shandong University Ph D dissertation of commentaries on classics TsVP drought-resistant corn.
Said CPB carrier (pCAMBIA3300-35S-pROKII MCS-nos-bar): be the plasmid that on CUB vector plasmid basis, makes up, through Hind III and Xba I difference said CUB carrier of double digestion and pBI121 (Beijing DingGuo ChangSheng Biology Technology Co., Ltd, production number is MCV032); 35S promoter fragment after reclaiming purification kit and reclaim enzyme respectively and cut with gel; And the skeleton fragment of CUB carrier, and carry out ligation, the purpose plasmid that obtains is changed in the intestinal bacteria; Resistance screening; The picking positive monoclonal carries out liquid culture with positive monoclonal, extracts the positive colony plasmid and carries out Hind III and the evaluation of Xba I double digestion; To cut the recombinant plasmid sample presentation order-checking of identifying that preliminary judgement is correct through enzyme, obtain CPB carrier (pCAMBIA3300-35S-pROKII MCS-nos-bar).
Said expression cassette is by the promotor that can start said GmEPSPS genetic expression, said GmEPSPS gene, and transcription termination sequence is formed.
In one embodiment of the invention, said reorganization bacterium is specially the recombination bacillus coli that carries said GmEPSPS gene.
Said GmEPSPS albumen, or said nucleic acid molecule, or said recombinant expression vector, expression cassette or reorganization bacterium are at following a1) or a2) in application also belong to protection scope of the present invention:
A1) regulation and control plant or mikrobe resistance glyphosate function;
A2) seed selection anti-glyphosate plants or mikrobe kind.
In one embodiment of the invention, said regulation and control plant or mikrobe resistance glyphosate function are specially and improve plant or mikrobe resistance glyphosate function.
Another object of the present invention provides a kind of method of cultivating the transgenic plant of resistance glyphosate function raising.
This method comprises the step in the proteic gene importing of the said GmEPSPS of the coding purpose plant; Said transgenic plant are compared with said purpose plant, and the resistance glyphosate function improves.
A further object of the present invention provides a kind of method of cultivating the transgenic microorganism of resistance glyphosate function raising.
This method comprises the step in the proteic gene importing of the said GmEPSPS of the coding purpose mikrobe; Said transgenic microorganism is compared with said purpose mikrobe, and the resistance glyphosate function improves.
In above-mentioned two methods, the proteic gene of said GmEPSPS of encoding is said GmEPSPS gene, is following 1) to 5) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the 677th to 1975 Nucleotide of sequence 2 in the sequence table;
2) encoding sequence is the dna molecular shown in the 461st to 1975 Nucleotide of sequence 2 in the sequence table;
3) dna molecular shown in the 7th of sequence 2 the to 1975 Nucleotide in the sequence table;
4) dna molecular shown in the sequence 2 in the sequence table;
5) under stringent condition with 1)-4) and in the dna molecule hybridize and the proteic dna molecular of said GmEPSPS of encoding of arbitrary qualification.
Above-mentioned stringent condition can be with 6 * SSC, and the solution of 0.5%SDS 65 ℃ of hybridization down, is used 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
Said GmEPSPS gene specifically can import in said purpose plant or the said purpose mikrobe through above-mentioned arbitrary said recombinant expression vector, obtains said transgenic plant or transgenic microorganism.Import said purpose plant specifically can be through using conventional biological methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity led, agriculture bacillus mediated, particle gun with said recombinant expression vector transformed plant cells or tissue, and the plant transformed tissue cultivating become plant.
Said plant can be monocotyledons, also can be dicotyledons.In the present invention, said monocotyledons is specially corn, like corn variety Zheng 58 etc.; Said dicotyledonous plant is specially tobacco, like tobacco bred W38 etc.
In one embodiment of the invention, said mikrobe is specially intestinal bacteria, like bacillus coli DH 5 alpha.
The present invention is according to 5-enol pyruvic acid shikimic acid-3-phosphate synthase (the 5-enolpyrul-shikimate-3-phosphate synthase that derives from Pseudomonas fluorescens; EPSPS) mutator gene; Remove two sections base sequences of original gene ORF sequence 5 ' end 147bp and 3 ' end 531bp; Only stay one section base sequence of the 654bp of intercooler core structural domain sequence, splice with one section base sequence from Longstamen Onion Bulb EPSPS gene 591bp; Guaranteeing that the epsp synthase Argine Monohydrochloride sequence that splicing is transformed forms under the overall constant situation, carrying out base substitution, tentatively obtaining the dna sequence dna of a transformation with the codon of plant-preference; Get rid of exist in the dna sequence dna cause the unsettled AT of being rich in sequence of plant transcription and restriction enzyme site commonly used (BamH I, EcoR I, Hind III, Nco I, Xho I, Xba I etc.), the method through permutation cipher corrects elimination then; And add the preceding terminal sequence of paddy rice ACT1-intron, petunia CTP4 chloroplast transit peptides and corn EPSP gene at 5 ' end, confirm and EPSPS gene that chemosynthesis is transformed, its nucleotide sequence shown in sequence in the sequence table 2, called after GmEPSPS.
Gene GmEPSPS of the present invention is operably connected with expression vector, and transformed into escherichia coli, Preliminary detection synthetic GmEPSPS of the present invention gene expression product is to the tolerance of Glyphosate 62 IPA Salt fast.Gene GmEPSPS of the present invention is operably connected with plant expression vector, and expression vector is imported in the corresponding Agrobacterium, and then carry out genetic transformation, cultivate resistance glyphosate transgene tobacco, resistance glyphosate transgenic corns through agrobacterium-mediated transformation.Also can carry out genetic transformation, make it possess resistance glyphosate property other farm crop or fruit tree etc.
The present invention has advantage and beneficial effect at least:
The artificial reconstructed synthetic Antiglyphosate gene of the present invention GmEPSPS sequence is compared with original EPSPS gene order, has strengthened its expression in plant greatly.Use plant-preference property codon; The inverted repeats that is rich in AT sequence and existence and indefinite eukaryotic DNA intron sequences in the original DNA sequence have been reduced; Improved GmEPSPS gene G+C content is 59.93%, is respectively 32.36% and 27.46% with the homology of the dna sequence dna of original two EPSPS genes (Pseudomonas fluorescens EPSPS mutator gene and Longstamen Onion Bulb EPSPS gene).Antiglyphosate gene GmEPSPS of the present invention can be in vegetable cell the expression of efficient stable.
Behind Antiglyphosate gene GmEPSPS importing tobacco and corn, can obtain the GmEPSPS transformant of genetic stability, improve the glyphosate resistance of plant.In addition, farm crop such as this gene also can soybean transformation, cotton, paddy rice, vegetables, it is active to make it possess corresponding resistance glyphosate; Thereby be suitable for and minimal till and no-tillage system; Strengthen the moisture maintenance, significantly reduced depletion, had important economic value and wide application prospect.
Description of drawings
Fig. 1 is the GmEPSPS gene composition sequence pie graph that artificial splicing is transformed.
Fig. 2 makes up synoptic diagram for plant expression vector CPB-GmEPSPS-Bar.
Fig. 3 makes up synoptic diagram for plant expression vector CUB-GmEPSPS-Bar.
Fig. 4 is T
0PCR detected result for goal gene GmEPSPS in the transformant.Wherein, M:100bp DNA Mark; 1-4: change CPB-GmEPSPS-Bar tobacco plant CPB-GB-1 to CPB-GB-4; The CK1:CPB-GmEPSPS-Bar plasmid; CK2: the transgene tobacco that changes the CPB empty carrier; CK3: transgene tobacco not.
Fig. 5 is T
0PCR detected result for corn transformant goal gene GmEPSPS.Wherein, M:100bp DNA Mark; 1-4: change CUB-GmEPSPS-Bar milpa CUB-GB-1 to CUB-GB-4; The CK1:CUB-GmEPSPS-Bar plasmid; CK2: the transgenic corns that changes the CUB empty carrier; CK3: transgenic corns not.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
CUB carrier (pCAMBIA3300-Ubi-pROKII MCS-nos-bar): the application of Li Bei .FLP_frt locus specificity recombination system in corn and the acquisition .2008 Shandong University Ph D dissertation of commentaries on classics TsVP drought-resistant corn.
Said CPB carrier (pCAMBIA3300-35S-pROKII MCS-nos-bar): be the plasmid that on CUB vector plasmid basis, makes up, through Hind III and Xba I difference said CUB carrier of double digestion and pBI121 (Beijing DingGuo ChangSheng Biology Technology Co., Ltd, production number is MCV032); 35S promoter fragment after reclaiming purification kit and reclaim enzyme respectively and cut with gel; And the skeleton fragment of CUB carrier, and carry out ligation, the purpose plasmid that obtains is changed in the intestinal bacteria; Resistance screening; The picking positive monoclonal carries out liquid culture with positive monoclonal, extracts the positive colony plasmid and carries out Hind III and the evaluation of Xba I double digestion; To cut the recombinant plasmid sample presentation order-checking of identifying that preliminary judgement is correct through enzyme, obtain CPB carrier (pCAMBIA3300-35S-pROKII MCS-nos-bar).
Agrobacterium LBA4404: the Beijing DingGuo ChangSheng Biology Technology Co., Ltd, production number is MCC026.
Tobacco W38 (Nicotiana tobacum cv.Wisconsin 38): Sui is along photograph, and Li Linli wishes Qin Long etc. and wax plum agglutinin gene is cloned and aphid, slug resistance is analyzed. Scientia Agricultura Sinica, 2011 (2): 358-368)
Corn inbred line Zheng 58: Cao Guohui, Shi Hongliang, Wang Bangtai etc. the molecule marker of the anti-graywall gene of corn. the breeding of molecule plant, 2009 (4): 709-715
The transformation of embodiment 1, GmEPSPS gene is synthetic
According to 5-enol pyruvic acid shikimic acid-3-phosphate synthase (the 5-enolpyrul-shikimate-3-phosphate synthase that derives from Pseudomonas fluorescens (Pseudomonas fluorescens); EPSPS) mutator gene; Remove two sections base sequences of original gene ORF sequence 5 ' end 147bp and 3 ' end 531bp; Only stay one section base sequence of the 654bp of intercooler core structural domain sequence, splice with one section base sequence from Longstamen Onion Bulb EPSPS gene 591bp; Guaranteeing that the epsp synthase Argine Monohydrochloride sequence that splicing is transformed forms under the overall constant situation, carrying out base substitution, tentatively obtaining the dna sequence dna of a transformation with the codon of plant-preference; Get rid of exist in the dna sequence dna cause the unsettled AT of being rich in sequence of plant transcription and restriction enzyme site commonly used (BamH I, EcoR I, Hind III, Nco I, Xho I, Xba I etc.), the method through permutation cipher corrects elimination then; And add the preceding terminal sequence (the 677-736 position of sequence 2) of paddy rice ACT1-intron sequence (the 7-460 position of sequence 2), petunia CTP4 chloroplast transit peptide sequence (the 461-676 position of sequence 2) and corn EPSP gene at 5 ' end; The structure of subsequent recombination expression vector for ease; Introduce Xba I and Sac I restriction enzyme site simultaneously respectively at the sequence two ends; Confirm the EPSPS gene (GmEPSPS gene composition sequence pie graph is seen Fig. 1) that also chemosynthesis is transformed; Its nucleotide sequence shown in sequence in the sequence table 2, called after GmEPSPS, the 677-1975 position of sequence 2 is the encoding sequence of said GmEPSPS gene; Protein (GmEPSPS becomes body protein, does not contain the transit peptides aminoacid sequence) shown in the 73-504 amino acids of sequence 1 in the code sequence tabulation; Protein shown in the sequence 1 (GmEPSPS midbody albumen, 5 ' end contains the aminoacid sequence of petunia CTP4 chloroplast transit peptides) in the 461-1975 position code sequence tabulation of sequence 2.
Improved GmEPSPS gene G+C content is 59.93%, is respectively 32.36% and 27.46% with the homology of the dna sequence dna of original two EPSPS genes (Pseudomonas fluorescens EPSPS mutator gene and Longstamen Onion Bulb EPSPS gene).The aminoacid sequence of improved GmEPSPS genes encoding (its aminoacid sequence is shown in sequence in the sequence table 1) shows that with original two EPSPS genes (Pseudomonas fluorescens EPSPS mutator gene and Longstamen Onion Bulb EPSPS gene) amino acid sequence coded compare of analysis homology is respectively 55.64% and 62.57%.
The synthetic work of new EPSPS gene GmEPSPS (sequence 2) is accomplished by sky, Beijing bounties Gene Tech. Company Limited.
The structure of embodiment 2, resistance glyphosate fusion gene plant expression vector
GmEPSPS gene in the artificial synthesized sequence table shown in the sequence 2; With Xba I and the said GmEPSPS gene of Sac I difference double digestion; Simultaneously with Xba I and Sac I difference double digestion plant expression vector CPB and CUB; GmEPSPS fragment after reclaiming purification kit and reclaim enzyme respectively and cut with gel, and the skeleton fragment of CPB carrier and CUB carrier.The GmEPSPS fragment is carried out ligation with the skeleton fragment of CPB carrier and CUB carrier respectively, obtains the purpose plasmid.The purpose plasmid is changed in the intestinal bacteria; Resistance screening, the picking positive monoclonal carries out liquid culture with positive monoclonal; Extract the positive colony plasmid and carry out Xba I and the evaluation of Sac I double digestion, will cut the recombinant plasmid sample presentation order-checking of identifying that preliminary judgement is correct through enzyme.
Inserted the GmEPSPS gene fragment shown in the sequence 2 in the sequence table between Xba I that is illustrated in support C PB carrier through checking order and Sac I restriction enzyme site; The proof plasmid construction is correct; With its called after CPB-GmEPSPS-Bar; Building process is as shown in Figure 2, and the promotor that starts said GmEPSPS genetic transcription among the said recombinant eukaryotic expression plasmid CPB-GmEPSPS-Bar is the CaMV35S promotor, and marker gene is the anti-herbicide gene Bar of streptomyces hygroscopicus.
Inserted the GmEPSPS gene fragment shown in the sequence 2 in the sequence table between Xba I that is illustrated in support C UB carrier through checking order and Sac I restriction enzyme site; The proof plasmid construction is correct; With its called after CUB-GmEPSPS-Bar; Building process is as shown in Figure 3, and the promotor that starts said GmEPSPS genetic transcription among the said plant recombination expression vector CUB-GmEPSPS-Bar is a corn Ubi-1 promotor, and marker gene is the anti-herbicide gene Bar of streptomyces hygroscopicus.
The M9 culture medium preparation:
(1) MgSO of elder generation's preparation 1M
4: take by weighing MgSO
47H
2O 2.46g adds distilled water 10mL dissolving, and autoclaving is subsequent use;
(2) CaCl of preparation 1M
2: CaCl
26H
2O 2.191g adds distilled water 10mL dissolving, and autoclaving is subsequent use;
(3) prepare 5 * M9 salts solution again:
Na
2PO
4·7H
2O?12.8g
KH
2PO
4?3.0g
NaCl?0.5g
NH
4Cl?1.0g
Add distilled water 200ml dissolving, sterilized 15 minutes for 121 ℃,
Remarks: above three kinds of preparations respectively, bottling can be sent to high pressure together respectively.
(4) glucose solution of preparation 20%: 4g glucose adds distilled water 20ml dissolving, 0.22 micron filter filtration sterilization;
(5) aseptic technique preparation M9 substratum
5 * M9 salts solution 200ml
The MgSO of 1M
42ml
20% glucose solution 20ml
The CaCl of 1M
20.1ml
Add the sterilization distilled water to 1000ml.
The plant recombination expression vector CPB-GmEPSPS-Bar that embodiment 2 is made up changes bacillus coli DH 5 alpha over to, picking list colony inoculation in the LB substratum of 30ml, 37 ℃, 250r/min incubated overnight; Inoculum size with 1% is inoculated into respectively in the M9 liquid nutrient medium that contains 0mM, 60mM Glyphosate 62 IPA Salt, through behind 37 ℃, the shaking culture of 48h, measures the OD of culture
600Value.The bacillus coli DH 5 alpha that setting simultaneously changes the CPB empty carrier over to is as negative control.The experiment triplicate, results averaged.
The result is as shown in table 1, through behind 37 ℃, the shaking culture of 250r/min, 48h, is not containing on the M9 substratum of Glyphosate 62 IPA Salt, and three kinds of intestinal bacteria are normal growth all; In containing the M9 substratum of 60mM Glyphosate 62 IPA Salt, negative control almost can not be grown, OD
600Value is 0.05; The OD of positive control
600Value is 1.14, changes the OD of the bacillus coli DH 5 alpha of CPB-GmEPSPS-Bar over to
600Value is 1.36.The result shows that reforming composite new gene GmEPSPS has the ability that transforms the host e. coli resistance glyphosate that improves.
Two kinds of colibacillary OD of table 1
600Value
| |
|
|
MV | |
| Negative control | 0.03 | 0.07 | 0.05 | 0.05 |
| DH5α-GmEPSPS | 1.21 | 1.69 | 1.18 | 1.36 |
Annotate: the DH5 α-GmEPSPS in the table promptly representes to change over to the bacillus coli DH 5 alpha of CPB-GmEPSPS-Bar.
Expression and the functional verification in transgene tobacco of embodiment 4, GmEPSPS gene
One, changes the acquisition of GmEPSPS genetic tobacco
1, the preparation of reorganization Agrobacterium
The plant recombination expression vector CPB-GmEPSPS-Bar (or CPB empty carrier) that embodiment 2 is built passes through freeze-thaw method (with reference to Holsters M; De Waele D; DepickerA.Transfection and transformation of Agrobacterium tumefaciens.Mol Gen Genet; 1978,183:181-187) change Agrobacterium LBA4404 over to.Use the primer of forming by 5 '-ACAAGATCAGGAAGAGGGGAAAAGG-3 ' (the 266-290 position of sequence 2) and 5 '-GGAGCCGAACACGAGGAAGC-3 ' (reverse complementary sequence of the 549-568 position of sequence 2) to identify (the purpose stripe size is 303bp) to the reorganization Agrobacterium after transforming to carrying out PCR.To show the Agrobacterium LBA4404 called after LBA4404/CPB-GmEPSPS-Bar that contains the GmEPSPS gene through evaluation; With the Agrobacterium LBA4404 called after LBA4404/CPB that changes the CPB empty carrier over to.
2, Agrobacterium conversion tobacco
Substratum altogether: on the basis of MS substratum, adding final concentration is the 6-BA of 1mg/L, and final concentration is the NAA of 0.1mg/L, and pH 5.8.
Division culture medium: on the basis of MS substratum, adding final concentration is the 6-BA of 1mg/L, and final concentration is the NAA of 0.1mg/L, and final concentration is the careless fourth phosphine of 5mg/L, and final concentration is the cephamycin of 400mg/L, and pH 5.8.
Root media: on the basis of 1/2MS substratum, adding final concentration is the NAA of 0.5mg/L, and final concentration is the careless fourth phosphine of 5mg/L, and pH 5.8.
1) chooses aseptic seedling (the switching back 10-15 days) blade of eugonic tobacco W38 (Nicotiana tobacum cv.Wisconsin 38), be cut into 1cm with sharp knife blade
2Small pieces discard middle arteries and veins.
2) tobacco leaf that cuts is soaked in the OD that gets ready
600In the Agrobacterium bacterium liquid (LBA4404/CPB-GmEPSPS-Bar and LBA4404/CPB) for the preparation of the step 1 of 0.6-0.8, guarantee that blade cut wound edge contacts with bacterium liquid fully, immersion 5min.
3) the bacterium liquid that inclines will infect blade and transfer to inhale on the sterilization thieving paper and remove unnecessary bacterium liquid.
4) blade is transferred in the common substratum, vacuum side of blade was cultivated two days in the dark up altogether.
5) blade after will cultivating altogether is transferred on the division culture medium illumination cultivation.
6) budlet to be differentiated grows to 2-3cm, and its cutting-out is transferred on the root media.For the plant of guaranteeing to obtain belongs to different transformation plants, each explant is only got an indefinite bud.Can take root into seedling in the 7-10d.
7) transgene tobacco is transplanted: the seedling of will taking root changes in the greenhouse, throws off evening and seals film, and seal up daytime, behind the refining seedling 2-3d, seedling is taken out, and flush away is attached to the substratum on the root in water, carefully plants in the ready flowerpot, and it is alive that In Shade 3-5d can become.Acquisition has the T of careless fourth phosphine resistance
0For transgenic tobacco plant.
3, the Molecular Identification of transgenic tobacco plant
T to step 2 acquisition
0The transgene tobacco that generation changes the CPB-GmEPSPS-Bar recombinant expression vector over to carries out the PCR detection; Genomic dna with the transgene tobacco seedling is a template, and it is 5 '-ACAAGATCAGGAAGAGGGGAAAAGG-3 ' (the 266-290 position of sequence 2) and 5 '-GGAGCCGAACACGAGGAAGC-3 ' (reverse complementary sequence of the 549-568 position of sequence 2) that PCR detects the primer that uses.Simultaneously with not genetically modified tobacco bred W38 as negative control, the T that obtains with step 2
0In generation, changes CPB empty carrier tobacco over to and contrasts as another.
The PCR detected result is as shown in Figure 4, change over to the CPB-GmEPSPS-Bar recombinant expression vector transgene tobacco not homophyletic system all amplification obtained the purpose fragment that length is about 303bp; And it is identical with not transgene tobacco as negative control to change the transgene tobacco of CPB empty carrier over to, does not all amplify the purpose fragment.
Two, the functional verification of transgene tobacco
The Glyphosate 62 IPA Salt aqueous solution with 0.15% (0.15g/100ml) sprays the T that step 1 obtains
0In generation, changeed the blade of GmEPSPS genetic tobacco positive plant, observes plant strain growth state and leaf morphology after 2 weeks.The transgenic tobacco plant that changes the CPB empty carrier over to is set simultaneously, and the non-transgenic tobacco is as contrast.Whenever the plant that grows tobacco is chosen 10 strains and experimentizes.The experiment triplicate.
The result shows, changeing the GmEPSPS genetic tobacco all can normal growth, and changes the transgenic tobacco plant of CPB empty carrier over to the growth of non-transgenic tobacco is obviously suppressed, and turning to be yellow in various degree, withering appears in blade.
Expression and the functional verification in transgenic corns of embodiment 5, GmEPSPS gene
One, changes the acquisition of GmEPSPS gene corn
1, the preparation of reorganization Agrobacterium
Used plant recombination expression vector is the CUB-GmEPSPS-Bar that embodiment 2 makes up, and other concrete operation methods and step are with in embodiment 4 step 11.
2, Agrobacterium-mediated Transformation corn
Adopt agriculture bacillus mediated corn stem apex method for transformation (with reference to Li Bei; The application of FLP_frt locus specificity recombination system in corn and the acquisition of commentaries on classics TsVP drought-resistant corn; Ph D dissertation; 2008) transformation receptor corn inbred line Zheng 58 seedling shoot apical meristem is transplanted in the flowerpot after cultivating 3d altogether., 0.3% weedicide grass fourth phosphine obtains anti-careless fourth phosphine transfer-gen plant after spraying screening.
3, the Molecular Identification of transgenic corn plant
T to step 2 acquisition
0The transgenic corns that generation changes the CUB-GmEPSPS-Bar recombinant expression vector over to carries out the PCR detection; Genomic dna with the transgenic corns of 7-8 leaf phase is a template, and it is 5 '-ACAAGATCAGGAAGAGGGGAAAAGG-3 ' (the 266-290 position of sequence 2) and 5 '-GGAGCCGAACACGAGGAAGC-3 ' (reverse complementary sequence of the 549-568 position of sequence 2) that PCR detects the primer that uses.Simultaneously with not genetically modified corn inbred line Zheng 58 as negative control, the T that obtains with step 2
0The corn that generation changes the CUB empty carrier over to contrasts as another.
The PCR detected result is as shown in Figure 5, change over to the CUB-GmEPSPS-Bar recombinant expression vector transgenic corns not homophyletic system all amplification obtained the purpose fragment that length is about 303bp; And it is identical with not transgenic corns as negative control to change the transgenic corns of CUB empty carrier over to, does not all amplify the purpose fragment.
Two, the functional verification of transgenic corns
The Glyphosate 62 IPA Salt aqueous solution with 0.15% (0.15g/100ml) is avoided the T that master pulse smearing step one obtains
0In generation, changeed the blade of GmEPSPS gene corn positive plant, observes plant strain growth state and leaf morphology after 2 weeks.The transgenic corn plant change the CUB empty carrier over to and non-transgenic corn are set simultaneously as contrast.Every kind of milpa chosen 10 strains and experimentizes.The experiment triplicate.
The result shows, changeing the GmEPSPS gene corn all can normal growth, and changes the transgenic corn plant of CUB empty carrier over to the growth of non-transgenic corn is obviously suppressed, and yellow leaf, withers.
Though, the present invention has been done detailed description in the preceding text with general explanation and specific embodiments, on basis of the present invention, can to some modifications of do or improvement, this will be apparent to those skilled in the art.Therefore, these modifications or the improvement on the basis of not departing from spirit of the present invention, made all belong to the scope that requirement of the present invention is protected.
Claims (10)
1. protein is (a) or (b) or (c) as follows:
(a) protein of forming by the aminoacid sequence shown in the 73-504 position of sequence in the sequence table 1;
(b) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;
(c) with (a) or aminoacid sequence (b) through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have glyphosate resistance by sequence 1 deutero-protein.
2. coding claim 1 said proteinic nucleic acid molecule.
3. nucleic acid molecule according to claim 2 is characterized in that: said nucleic acid molecule is the said proteinic gene of coding claim 1; Said gene is following 1) to 5) in arbitrary described dna molecular:
1) encoding sequence is the dna molecular shown in the 677th to 1975 Nucleotide of sequence 2 in the sequence table;
2) encoding sequence is the dna molecular shown in the 461st to 1975 Nucleotide of sequence 2 in the sequence table;
3) dna molecular shown in the 7th of sequence 2 the to 1975 Nucleotide in the sequence table;
4) dna molecular shown in the sequence 2 in the sequence table;
5) under stringent condition with 1)-4) and in the dna molecule hybridize and the said protein DNA molecule of coding claim 1 of arbitrary qualification.
4. the recombinant vectors, expression cassette, transgenic cell line or the reorganization bacterium that contain claim 2 or 3 said nucleic acid molecule.
5. recombinant vectors according to claim 4 is characterized in that: said recombinant vectors is recombinant expression vector or recombinant cloning vector.
6. recombinant vectors according to claim 5 is characterized in that: the promotor that starts said genetic transcription in the said recombinant expression vector is 35S promoter or corn Ubi-1 promotor.
7. described protein of claim 1, or claim 2 or 3 described nucleic acid molecule, or claim 4 or 5 or 6 described recombinant expression vectors, expression cassette or reorganization bacterium are at following a1) or a2) in application:
A1) regulation and control plant or mikrobe resistance glyphosate function;
A2) seed selection anti-glyphosate plants or mikrobe kind.
8. a method of cultivating the transgenic plant of resistance glyphosate function raising comprises the said proteinic gene of coding claim 1 is imported the step in the purpose plant; Said transgenic plant are compared with said purpose plant, and the resistance glyphosate function improves.
9. a method of cultivating the transgenic microorganism of resistance glyphosate function raising comprises the said proteinic gene of coding claim 1 is imported the step in the purpose mikrobe; Said transgenic microorganism is compared with said purpose mikrobe, and the resistance glyphosate function improves.
10. application according to claim 7, or claim 8 or 9 described methods, it is characterized in that: said plant is monocotyledons or dicotyledons; Said mikrobe is intestinal bacteria.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210196502 CN102766609B (en) | 2012-06-14 | 2012-06-14 | Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201210196502 CN102766609B (en) | 2012-06-14 | 2012-06-14 | Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102766609A true CN102766609A (en) | 2012-11-07 |
| CN102766609B CN102766609B (en) | 2013-10-23 |
Family
ID=47094186
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201210196502 Expired - Fee Related CN102766609B (en) | 2012-06-14 | 2012-06-14 | Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102766609B (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886521A (en) * | 2016-04-19 | 2016-08-24 | 北京市农林科学院 | Glyphosate-resistant selective marker gene and application thereof in maize transgenic technology |
| CN106854237A (en) * | 2016-12-29 | 2017-06-16 | 广西大学 | Functional protein POX08415 and its encoding gene and application |
| CN108330115A (en) * | 2018-04-13 | 2018-07-27 | 重庆市农业科学院 | A kind of resistance glyphosate epsp synthase MC1-EPSPS and its encoding gene and application |
| CN110272880A (en) * | 2019-05-22 | 2019-09-24 | 华中农业大学 | A kind of saltant type glyphosate degrading enzyme and its clone, expression and application |
| CN111154787A (en) * | 2019-03-21 | 2020-05-15 | 浙江大学 | Rice EPSP synthetase mutant gene, mutant and application thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101421402A (en) * | 2006-01-11 | 2009-04-29 | 分子作物育种代理有限公司 | Method of producing transgenic graminaceous cells and plants |
| CN101500421A (en) * | 2006-06-06 | 2009-08-05 | 孟山都技术有限公司 | Methods for weed control |
| CN101508996A (en) * | 2002-07-18 | 2009-08-19 | 孟山都技术有限公司 | Methods for using artificial polynucleotides and compositions thereof to reduce transgene silencing |
-
2012
- 2012-06-14 CN CN 201210196502 patent/CN102766609B/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101508996A (en) * | 2002-07-18 | 2009-08-19 | 孟山都技术有限公司 | Methods for using artificial polynucleotides and compositions thereof to reduce transgene silencing |
| CN101421402A (en) * | 2006-01-11 | 2009-04-29 | 分子作物育种代理有限公司 | Method of producing transgenic graminaceous cells and plants |
| CN101500421A (en) * | 2006-06-06 | 2009-08-05 | 孟山都技术有限公司 | Methods for weed control |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105886521A (en) * | 2016-04-19 | 2016-08-24 | 北京市农林科学院 | Glyphosate-resistant selective marker gene and application thereof in maize transgenic technology |
| CN105886521B (en) * | 2016-04-19 | 2019-04-16 | 北京市农林科学院 | Resistance glyphosate riddled basins and its application in corn gene technology |
| CN106854237A (en) * | 2016-12-29 | 2017-06-16 | 广西大学 | Functional protein POX08415 and its encoding gene and application |
| CN106854237B (en) * | 2016-12-29 | 2019-08-02 | 广西大学 | Functional protein POX08415 and its encoding gene and application |
| CN108330115A (en) * | 2018-04-13 | 2018-07-27 | 重庆市农业科学院 | A kind of resistance glyphosate epsp synthase MC1-EPSPS and its encoding gene and application |
| CN108330115B (en) * | 2018-04-13 | 2021-08-13 | 重庆市农业科学院 | Glyphosate-resistant EPSP synthetase MC1-EPSPS, and coding gene and application thereof |
| CN111154787A (en) * | 2019-03-21 | 2020-05-15 | 浙江大学 | Rice EPSP synthetase mutant gene, mutant and application thereof |
| CN111154787B (en) * | 2019-03-21 | 2020-10-13 | 浙江大学 | Rice EPSP synthetase mutant gene, mutant and application thereof |
| CN110272880A (en) * | 2019-05-22 | 2019-09-24 | 华中农业大学 | A kind of saltant type glyphosate degrading enzyme and its clone, expression and application |
| CN110272880B (en) * | 2019-05-22 | 2021-01-01 | 华中农业大学 | Mutant glyphosate degrading enzyme and cloning, expression and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN102766609B (en) | 2013-10-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101173002B (en) | Plants stress tolerance correlation transcription factor GmWRKY54, encoding gene and application thereof | |
| CN101747419B (en) | Protein related to salt tolerance, coding gene thereof and application thereof | |
| US10633669B2 (en) | Herbicide-resistant protein, encoding gene and use thereof | |
| CN102766609B (en) | Glyphosate resistant EPSP synthetase GmEPSPS, and coding gene and application thereof | |
| US20210163973A1 (en) | Plant constitutive expression promoter and applications thereof | |
| US10655140B2 (en) | Herbicide-resistant protein, encoding gene and use thereof | |
| CN100486994C (en) | Nitrate transport protein of diatom and its coding gene and application | |
| CN102286524B (en) | Plant transformation vector, and construction method and application thereof | |
| CN102399268B (en) | Plant stress tolerance-related transcription factor GmNAC11, coding gene and application thereof | |
| US10562944B2 (en) | Herbicide-resistant protein, encoding gene and use thereof | |
| CN104745600B (en) | Applications of the paddy gene OsVHA1 in delaying plant leaf blade aging and improving plant salt endurance | |
| WO2018175900A2 (en) | Synthetic desalination genetic circuit in plants | |
| CN102776158B (en) | Anti-glyphosate EPSP (enolpyruvyl shikimate phosphate) synthase GmEPSPS-2 as well as encoding gene and application thereof | |
| CN103409445A (en) | Glyphosate-resistance gene MTP-SMG2-EPSPS and application thereof in cultivation of glyphosate-resistance corn | |
| CN108330115A (en) | A kind of resistance glyphosate epsp synthase MC1-EPSPS and its encoding gene and application | |
| CN112725365B (en) | BNAM79EPSPS glyphosate-resistant gene and application thereof | |
| CN101704882B (en) | Plant yellow dwarf resistance-associated protein, coding gene and application thereof | |
| CN102618572A (en) | Method for cultivating drought-enduring plants | |
| CN102559703B (en) | Glyphosate-resistant herbicide gene AroA-Ra from grape crown gall antagonistic bacteria rahnella aquatilis and application thereof | |
| CN102146126A (en) | Protein related to insect resistance and encoding gene and application thereof | |
| CN101704884A (en) | Plant drought resistance and salt tolerance associated protein EeABF6, coding gene and application thereof | |
| CN105566468A (en) | Plant fertility related protein and applications thereof | |
| CN101979407B (en) | Plant drought and salt tolerance-related protein TaCRF2 and coding gene thereof and application | |
| CN102718851B (en) | Plant stress tolerance related protein GmG2 and encoding gene and application thereof | |
| US20230313212A1 (en) | Plastid transformation by complementation of nuclear mutations |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20131023 Termination date: 20180614 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |