CN102108360A - EPSP synthase gene descended from Pseudomonas fluorescens and application thereof - Google Patents

Abstract

The invention discloses an 5-enolpyruvyl-shikimate-3-phosphate (EPSP) synthase gene descended from Pseudomonas fluorescens and an application thereof. The nucleotide sequence of the gene is shown in SEQ ID NO 1, the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO 2; and the gene contains 1335 basic groups and the number of the encoded amino acids is 445. The EPSP synthase gene descended from Pseudomonas fluorescens has relatively high homology with the reported EPSP synthase gene descended from Agrobacterium rhizogenes CP4, has higher glyphosate tolerance and can be used in the cultivation of the transgenic crop.

Description

A kind of epsp synthase gene and application thereof that derives from Pseudomonas fluorescens

Technical field

The invention belongs to the microbiological genetic engineering field, be specifically related to a kind of epsp synthase gene and application thereof that derives from Pseudomonas fluorescens.

Background technology

(N-phosphonomethyl-glycine glyphosate) is Monsanto company product to glyphosate

In main active ingredient, this weedicide is go out natural disposition, the outstanding weedicide of inner sucting conduction type of a kind of wide spectrum, is one of weedicide of whole world usage quantity maximum.But this weedicide but is a kind of nonselective herbicide, and farm crop are had killing effect equally.For in agriculture production, using glyphosate, must cultivate farm crop with glyphosate resistance or degradation property.

5-enol form acetone shikimic acid in the glyphosate competitive inhibition plant shikimic acid metabolic process-3-phosphate synthase (EPSP) activity, and then the blocking-up die aromatischen Aminosaeuren (comprising tryptophane, tyrosine, phenylalanine etc.) biosynthesizing and make plant death, all glyphosate resistant transgenic crops of current global commerce plantation are at EPSP designed, are unique mechanism of action of present commercialization transgenosis glyphosate resistant crops.It is the encoding gene of glyphosate action target EPSP synthase that the bacteriogenic aroA mutant of applied chemistry mutagenesis, the research of resistance mechanism have been proved conclusively the aroA gene.Part patent surplus companies such as U.S. Monsanto and Calegene have applied for 100 at aspects such as the encoding gene aroA of EPSP synthase and resistance glyphosate transgenic plant thereof, obtain crop series of productss such as transgenosis resistance glyphosate soybean, corn, rape, beet and cotton, wherein multiple genetically modified crops such as soybean have entered commercialization production.

The country of plantation antiweed genetically modified crops is more and more at present, and area also increases sharply, and cultivated area constantly enlarges, and accounts for more than 78% of genetically modified crops of whole world plantation.Show according to data in 2002: the glyphosate tolerant soybean remains the staple transgenic crop of seven states such as the U.S., Argentina, Canada, Mexico, Romania, Uruguay and South Africa.Glyphosate tolerant weedicide soybean is planted 3,650 ten thousand hectares in the world, accounts for 62% of the total transgenic crop cultivated area in the whole world.Yet now the Antiglyphosate gene of main research mainly is the I type, thereby the epsp synthase gene of excavating novel II class will be necessary.

Summary of the invention

Technical problem to be solved by this invention is to provide a kind of epsp synthase gene and application thereof that derives from Pseudomonas fluorescens, and and this gene carried out functional verification, have higher relatively glyphosate tolerant to prove it.

The present invention realizes above-mentioned purpose by the following technical solutions:

A kind of epsp synthase gene that derives from Pseudomonas fluorescens, its nucleotide sequence are shown in SEQ ID NO 1, and the aminoacid sequence of its encoded protein matter is shown in SEQ ID NO 2.

Through sequence comparing analysis (referring to Fig. 3), show that this EPSP synthase belongs to II type EPSP synthase.

Described epsp synthase gene adopts synthetic.Gather the pedotheque that glyphosate frequently uses orcharding, separate the total DNA of Pseudomonas fluorescens (Pseudomonas fluorescens), make up genomic library with exempting from cultural method, and screening glyphosate resistance transformant; Transformant is put screening resistance transformant on the M9 solid medium that contains the 100mM glyphosate, promptly get EPSP synthase of the present invention.

The EPSP synthase of above-mentioned gained is carried out the full nucleotide sequencing of the dna segment of glyphosate highly-tolerant.Analytical results shows: the segment size of insertion is 2789, has wherein comprised the reading frame of 1335bp, totally 445 of amino acids coding, and nucleotide sequence and aminoacid sequence are respectively shown in SEQ ID NO 1 and SEQ IDNO 2.

After epsp synthase gene of the present invention cut with Nco I and Xho I enzyme, be connected into that carrier pET-28a (NEB company) that same enzyme cuts obtains recombinant plasmid pET-AroA-P.fluorescens and with its transformed into escherichia coli (Escherichia coli) BL21 (DE3) (Novagen company).

This EPSP synthase is carried out the glyphosate tolerance experiment, and the result shows: this EPSP synthase has very strong glyphosate tolerance activity.

This EPSP synthase is carried out the mensuration of enzyme activity determination and kinetic parameter, and enzymic activity is 59.63 ± 0.09nkat/mg as a result, K _i/ K _mBe 0.167 ± 0.025.According to this kinetic parameter as can be known: EPSP synthase of the present invention not only has higher glyphosate resistance, but also is keeping the affinity stronger with PEP, and these features will provide possibility for the cultivation that is used for genetically modified crops.

Epsp synthase gene of the present invention has been filled up the blank of Pseudomonas fluorescens in the II type epsp synthase gene, also provides theoretical support for being applied to genetically engineered simultaneously.

Description of drawings

Fig. 1 is the glyphosate tolerant experimental result of EPSP synthase of the present invention, wherein:

A representative intestinal bacteria ER2799 (carrying the p251-AroA-P.fluorescens plasmid), ER2799 (carrying the p251-AroA-E.coli plasmid) and intestinal bacteria ER2799 growing state when glyphosate concentration is 0mM;

B representative intestinal bacteria ER2799 (carrying the p251-AroA-P.fluorescens plasmid), ER2799 (carrying the p251-AroA-E.coli plasmid) and intestinal bacteria ER2799 growing state when glyphosate concentration is 50mM;

C representative intestinal bacteria ER2799 (carrying the p251-AroA-P.fluorescens plasmid), ER2799 (carrying the p251-AroA-E.coli plasmid) and intestinal bacteria ER2799 growing state when glyphosate concentration is 100mM.

Fig. 2 is the comparison of the aminoacid sequence of the EPSP synthase that comes from Agrobacterium CP4 that comes from colibacillary EPSP synthase and II type of EPSP synthase of the present invention and I type.

Fig. 3 is the phylogeny comparative result of EPSP synthase of the present invention and typical type I and II type EPSP synthase.

Fig. 4 for EPSP synthase 3D structure of the present invention and with the comparison of the 3D structure of the EPSP synthase that comes from Agrobacterium CP4.

Fig. 5 is the SDS-PAGE electrophorogram of EPSP synthase of the present invention, the 1 EPSP synthase protein of representing with HisTrapHP test kit purifying that derives from Pseudomonas fluorescens wherein; 2 representatives are represented molecular weight of albumen MARKER at the EPSP synthase protein, 3 that derives from Pseudomonas fluorescens of e. coli bl21 (DE3) overexpression.

Embodiment

Describe technical scheme of the present invention in detail below in conjunction with accompanying drawing.Embodiment is only unrestricted in order to technical scheme of the present invention to be described, although the present invention is had been described in detail with reference to preferred embodiment, those of ordinary skill in the art is to be understood that, can make amendment or be equal to replacement the technical scheme of invention, and not breaking away from the spirit and scope of technical solution of the present invention, it all should be encompassed in the claim scope of the present invention.

If the used reagent of the present invention is unexplained reference, all available from Sigma-aldrich (Sigma-Aldrich) company.

The present invention relates to molecular biology experiment, as not dated especially, all with reference to from " molecular cloning " book (J. Sa nurse Brooker, E.F. be Ritchie, T. Manny A Disi work not, 1994, Science Press.)

Embodiment 1

The dna segment clone of glyphosate highly-tolerant

1, glyphosate frequently uses the collection of pedotheque in the paddy rice ground

From the soil that used four times and used continuously the orchard more than 10 years in 1 year at least, gather pedotheque.

2, the screening of resistance glyphosate bacterial strain

Take by weighing glyphosate and frequently use orchard soil sample 1g, add 0.9% (w/v) sodium chloride solution 1ml, 5000 rev/mins of concussion mixings, 3000 rev/mins light centrifugal, outwell supernatant, add 0.9% (wt/vol) sodium chloride solution 1ml again, 5000 rev/mins of concussion mixings, 10min leaves standstill on ice, draws 150 μ l solution coat and cultivates 24h with containing in the LB solid medium of 60mM glyphosate.In the test tube with longer single colony inoculation and adding 1.6ml LB liquid nutrient medium, cultivate 48h for 28 ℃, the nutrient solution of drawing 150 μ l then be coated with once more with the LB solid medium that contains the 100mM glyphosate in cultivate 24h, at last an only well-grown bacterium colony Pseudomonas fluorescens (Pseudomonas fluorescens) is elected further and studies.

3, the extraction of the total DNA of bacterial strain and evaluation

To separate the bacterium individual plant of acquisition among the embodiment 2 at 10ml liquid LB substratum (5g/L yeast extract, 5g/L NaCl, 10g/L Tryptones, phosphoric acid buffer pH=7.5) cultivates 16h in, bacterial culture fluid obtains bacterial sediment with 6,000 rev/mins of centrifugal 5min of speed.These are deposited in-20 ℃ of freezing 1h down.(pH=8.0) solution cleans once for 10mM Tris-HCl, 1mM EDTA to use TE afterwards.Add and contain the sterilized water suspension that 20 μ l concentration are 10mg/mL N,O-Diacetylmuramidase (Sigma-Aldrich), cultivate 1h at 37 ℃ of following shaking tables.Add 50 μ L 0.5M EDTA, 50 μ l 10% (w/v) SDS and 50 μ l concentration are the NaCl of the 5M mixing that vibrates gently.Add 10 μ l concentration again and swash K (Takara Japan) for 20mg/mL albumen, reactant is cultivated 1h down at 37 ℃.With with cultivate the quite phenol of (1 times of volume) of bacterium liquid liquid volume: chloroform: primary isoamyl alcohol (25: 24: 1) extracts DNA.The chloroform of water with suitable water volume 1/2 (0.5 times of volume): primary isoamyl alcohol (24: 1) extraction.Centrifugal 5min behind the vibration mixing.Aqueous phase adds the primary isoamyl alcohol with water volume suitable (1 times of volume).Centrifugal 15min behind the vibration mixing.Get precipitation, with 70% (v/v) alcohol flushing DNA, drying, resuspension in the TE damping fluid afterwards.The total DNA of gained is standby under being stored in 4 ℃.

With extractive total DNA is template, utilizes the 16sRNA Auele Specific Primer of Pseudomonas fluorescens to increase.The primer of amplification is 16SR (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 16SF (5 '-ACGGCTACCT TGTTACGACTTC-3 ').With KOD Plus (Toyobo Japan) is the TaqDNA polysaccharase, and amplification condition is followed successively by: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 120s, 30 circulations of increasing.After the loop ends, add the rtaq enzyme (Dalian Bao Bio-Engineering Company) of 2U, 72 ℃ are extended 90s, the long 1500bp of amplified fragments.After PCR finished, 1% (w/v) sepharose reclaimed, and gets 10 μ l and directly links to each other with the T/A cloning vector (Dalian Bao Bio-Engineering Company), and 4 ℃ of connections are spent the night.Again this carrier is transformed in the DH5 α competence.Utilize the ABI3700 kapillary automatization sequenator order-checking of ABI Prism Big Dye, the sequence that records is analyzed by Blast program and GenBank amplifying nucleic acid data, the 16s rRNA that obtains in bacterial strain and the Pseudomonas fluorescens has 99% homology, thereby the confirmation isolated strains is a Pseudomonas fluorescens.

4, the structure of Pseudomonas fluorescens genomic library

Pseudomonas fluorescens DNA cuts in 10 μ l reaction systems with Sau3A I enzyme and carries out partially digested test, the Sau3AI enzyme was by dilution in 1: 100,37 ℃ respectively enzyme cut 10min, 20min, 30min, 40min, 50min, add 10 * loading buffer, 1 μ l termination reaction behind the 60min, the suitableeest endonuclease reaction time of electrophoresis detection.Then selecting identical system enzyme to cut 30min carries out a large amount of enzymes and cuts.Behind 0.7% (w/v) agarose gel electrophoresis, downcutting length is the dna fragmentation of 2-4kb, reclaims with gel reagents box (Shanghai bio-engineering corporation).Plasmid vector pACYC184 (NEB company) carries out terminal dephosphorylation with SAP alkaline phosphatase behind the BamHI complete degestion, to reduce carrier from connecting.Bacterial strain DNA (200ng) after the above-mentioned recovery and terminal dephosphorylized plasmid vector pACYC184 (150ng) are connected 16h with the T4ligase of 2U under 4 ℃.

Connect product propyl carbinol post precipitation, ethanol centrifuge washing with 70% (v/v), ultrapure water with 10 μ l dissolves at last, with the connector transformed into escherichia coli DH5 α competent cell that shocks by electricity, shock parameters is: electricimpulse is 2.5 μ L, voltage 2.5kV, resistance 200 Ω, the electric shock time is 4.5s.After the recovery, bacterium liquid is coated on LB (the containing 50 μ g/mL penbritins) flat board, cultivated 12-16h for 37 ℃.Then extract test kit (U.S. Omega company) in a large number and carry out the plasmid extraction with plasmid.So just be built into the genomic library that includes goal gene.

5, screening glyphosate resistance transformant

With the plasmid that extracts in a large number change over to intestinal bacteria ER2799 (NEB company) be coated with the M9 flat board that contains the 50mM glyphosate on cultivate 48h, finding has three colony growths good.These three bacterium colonies are inoculated into respectively on the M9 flat board that contains 100mM, 150mM glyphosate cultivate, discovery has only 1 clone to grow containing on the M9 flat board of 100mM glyphosate, the plasmid called after pAroA-P.fluorescens that it is contained, epsp synthase gene promptly of the present invention.Cloning extractive plasmid pAroA-P.fluorescens from this changes over to the intestinal bacteria ER2799 (NEB company) once more, transformant is checked resistance with aseptic toothpick point with containing on the M9 solid medium of 100mM glyphosate, the result proves that the transformant that this clone is produced all has the resistance glyphosate characteristic, shows that the resistance glyphosate characteristic causes owing to changing pAroA-P.fluorescens over to really.

6, glyphosate tolerance experiment

With P 1 (5 '-gagagaggatccatggaaaccgctgtgaacgctgatga-3 ') and

P2 (5 '-gagctctcacaattgagcctcttgggcaac-3 ') be primer, with pAroA-P.fluorescens the pcr amplification that template is carried out epsp synthase gene AroA-P.fluorescens.With KOD Plus (Toyobo Japan) is the Taq archaeal dna polymerase, and amplification condition is followed successively by: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 90s, 30 circulations of increasing.After the loop ends, add the rtaq enzyme (Dalian Bao Bio-Engineering Company) of 2U, 72 ℃ are extended 90s, the long 1335bp of amplified fragments (as follows).Select pG251 (CN1338515NCBI) as expression vector, cut and carry out the glue recovery with restriction endonuclease BamH I (Dalian Bao Bio-Engineering Company) enzyme.The vector plasmid that enzyme is cut carries out the dephosphorylation operation, to reduce plasmid from connecting, and alkaline phosphatase is carried out inactivation operate back (Sambrook molecular cloning handbook 1989), be connected with the dna fragmentation (being that length is the epsp synthase gene AroA-P.fluorescens of 1335bp) of external source again.Before the connection, with bacterial genomes dna fragmentation and the pG251 carrier DNA of the part enzymolysis that reclaims, with the method estimated concentration of agarose gel electrophoresis, the concentration of guaranteeing foreign DNA in the ligation is carrier concn 3-5 times at least.16 ℃ of temperature of reaction, the tie-time is 10-12h.Again this carrier is transformed in the DH5 α competence.Utilize the ABI3700 kapillary automatization sequenator order-checking of ABI Prism Big Dye, determine the i.e. exactness of (p251-AroA-P.fluorescens) of recombinant plasmid.Adopt the primer that uses the same method

P3:(5 '-CGGGATCCGTTAATGCCGAAATTTTGCTTAATC-3 ') and

P4:(5 '-CGGAGCTCAGGTCC GAAAAAAAACGCCGAC-3 ') the colibacillary epsp synthase gene AroA-E.coli of amplification is connected to pG251 equally and obtains the p251-AroA-E.coli recombinant plasmid

Intestinal bacteria ER2799 (carrying the p251-AroA-P.fluorescens plasmid) and ER2799 (carrying the p251-AroA-E.coli plasmid) are inoculated in the M9 liquid nutrient medium that contains the 0-100mM glyphosate, after cultivating through 37 ℃, the shaking table of 36h, the OD660 that measures culture carries out tolerance test, simultaneously there not to be the negative contrast of intestinal bacteria ER2799 of inserting pulsating plasmid.

Result: intestinal bacteria ER2799 (carrying the p251-AroA-P.fluorescens plasmid) and ER2799 (carrying the p251-AroA-E.coli plasmid) are inoculated in the M9 liquid nutrient medium that contains the 0-100mM glyphosate, after cultivating through 37 ℃, the shaking table of 36h, find that negative control almost can not grow in M9; And ER2799 (carrying the p251-AroA-E.coli plasmid) seriously is suppressed in the M9 of 50mM glyphosate liquid nutrient medium; And ER2799 (carrying the p251-AroA-P.fluorescens plasmid) can also grow in containing the M9 liquid nutrient medium of 100mM glyphosate (referring to Fig. 1).

Embodiment 2

The sequential analysis of the dna segment of glyphosate highly-tolerant and the functional verification of EPSP synthase thereof

1, the sequential analysis of the dna segment of glyphosate highly-tolerant

Utilize sequencing method progressively to screening the glyphosate highly-tolerant plasmid pAroA-P.fluorescens that obtains among the embodiment 1, epsp synthase gene complete sequence promptly of the present invention carries out dna sequencing.Analytical results shows: the segment size of insertion has wherein comprised one 1335 reading frame for 2789bp, and its nucleotide sequence is shown in SEQ ID NO 1, and amino acid sequence coded is shown in SEQ ID NO 1.1335 bases of this plasmid total length, totally 445 of amino acids coding.

The amino acid sequence homology analytical results shows: the EPSP synthase that comes from Agrobacterium CP4 of the aminoacid sequence of AroA-P.fluorescens epsp synthase gene of the present invention and II type has 47.62% homology (referring to Fig. 2), and with the homology less than 30% of colibacillary EPSP synthase of coming from of I type, illustrate that AroA-P.fluorescens EPSP synthase of the present invention belongs to the EPSP of II.The phylogeny comparative result of AroA-P.fluorescens EPSP synthase of the present invention and typical type I and II type EPSP synthase is referring to Fig. 3.

With online software Swiss-model AroA-P.fluorescens EPSP synthase of the present invention is carried out 3D structure prediction (referring to Fig. 4), the result shows that its 3D structure is closely similar with the 3D structure of the EPSP synthase that comes from Agrobacterium CP4.But have a zone to exist very significantly amino acid difference between them, this zone is positioned near the Glu-354 and Arg-357 of high conservative.Glu-354 and Arg-357 are the parts of a ring of being made up of 12 amino acid (347-358), and this ring not only affects PEP or glyphosate combination, and affect this ball-like structure from open to the outside world to " closing " state.

Embodiment 3

The synthetic of the epsp synthase gene of glyphosate highly-tolerant

With method for synthesizing gene (Nucleic Acids Research, 2004,32, e98) can be with the epsp synthase gene of above-mentioned glyphosate highly-tolerant clone.The primer of design is:

1.AroA-1：Tm＝54，60mer

ATG，GAA，ACC，GCT，GTG，AAC，GCT，GAT，GAG，TTG，ACC，TTC，CTT，GCC，GAA，CCG，GGT，GGC，CGC，TTG

2.AroA-2：Tm＝54，60mer

GGA，GAT，CGA，CTT，ATC，GCC，CGG，CAC，CCG，AAT，GCT，TCC，GCT，CAA，GCG，GCC，ACC，CGG，TTC，GGC

3.AroA-3：Tm＝54，60mer

CCG，GGC，GAT，AAG，TCG，ATC，TCC，CAT，CGT，TCG，ATC，ATG，CTG，GGT，TCG，TTG，GCT，GGG，GGT，GTG

4.AroA-4：Tm＝54，60mer

CAG，GGC，ATC，TTC，ACC，TTC，AAG，GAA，ACC，TTC，GAC，CTC，AGT，CAC，ACC，CCC，AGC，CAA，CGA，ACC

5.AroA-5：Tm＝54，60mer

CTT，GAA，GGT，GAA，GAT，GCC，CTG，GCG，ACC，TTG，CAG，GCA，TTT，CGC，GAC，ATG，GGC，GTG，GTG，ATT

6.AroA-6：Tm＝54，60mer

CAC，GCC，ATG，AAT，GGT，CAC，GCG，CCC，ATG，ATG，CGG，GCC，CTC，AAT，CAC，CAC，GCC，CAT，GTC，GCG

7.AroA-7：Tm＝54，60mer

CGC，GTG，ACC，ATT，CAT，GGC，GTG，GGG，CTG，CAT，GGG，TTG，AAG，CCG，GCG，CCG，GGG，CCG，ATT，TAC

8.AroA-8：Tm＝54，60mer

ACC，GGA，CAG，CAA，ACG，CAT，GGA，AGT，ACC，TGA，GTT，ACC，CAG，GTA，AAT，CGG，CCC，CGG，CGC，CGG

9.AroA-9：Tm＝54，60mer

TCC，ATG，CGT，TTG，CTG，TCC，GGT，TTG，CTG，GCG，GCG，CAG，AGC，TTC，GAC，AGC，GTG，CTG，ACC，GGT

10.AroA-10：Tm＝54，60mer

GGC，CAC，ACG，ACT，CAT，CGG，GCG，CTT，GGA，CAG，GAA，TGC，GTC，ACC，GGT，CAG，CAC，GCT，GTC，GAA

11.AroA-11：Tm＝54，60mer

CGC，CCG，ATG，AGT，CGT，GTG，GCC，AGG，CCG，CTG，CGG，GAA，ATG，GGA，GCG，GTG，ATC，GAG，ACG，GGG

12.AroA-12：Tm＝54，60mer

CTG，GCC，ACC，CCG，AAT，GGT，CAG，CGG，CGG，ACG，CCC，TTC，CGG，CCC，CGT，CTC，GAT，CAC，CGC，TCC

13.AroA-13：Tm＝54，60mer

CTG，ACC，ATT，CGG，GGT，GGC，CAG，TCG，TTG，AAA，GGC，CTG，GCT，TAT，GCA，ATG，CCG，ATG，GCC，AGT

14.AroA-14：Tm＝54，60mer

GTA，CAA，CCC，AGC，CAA，CAA，CAG，GCA，GGA，TTT，GAC，CTG，GGC，ACT，GGC，CAT，CGG，CAT，TGC，ATA

15.AroA-15：Tm＝54，60mer

CTG，TTG，TTG，GCT，GGG，TTG，TAC，GCT，GAA，GGT，AAA，ACC，GCG，GTG，ACC，GAG，CCT，GCG，CCG，ACC

16.AroA-16：Tm＝54，60mer

ATA，ACC，AAA，GCC，ACG，CAA，CAT，GCG，CTC，GGT，GTG，GTC，ACG，GGT，CGG，CGC，AGG，CTC，GGT，CAC

17.AroA-17：Tm＝54，60mer

ATG，TTG，CGT，GGC，TTT，GGT，TAT，CCG，GTG，GCG，GTG，GAG，GGC，GCG，ACG，GCG，TCG，GTG，GAG，TCC

18.AroA-18：Tm＝54，60mer

CCC，CGG，CAC，TTC，TAT，ATG，AGC，AGC，CGT，CAG，CGC，ATG，ACC，GGA，CTC，CAC，CGA，CGC，CGT，CGC

19.AroA-19：Tm＝54，60mer

GCT，CAT，ATA，GAA，GTG，CCG，GGG，GAT，ATT，TCT，TCT，TCA，GCG，TTC，TTT，CTG，GTG，GCA，GCT，TCG

20.AroA-20：Tm＝54，60mer

ACC，CAC，ATG，CTC，CAG，CAG，CAA，CTC，AGA，ACC，CTC，GGC，AAT，CGA，AGC，TGC，CAC，CAG，AAA，GAA

21.AroA-21：Tm＝54，60mer

TTG，CTG，CTG，GAG，CAT，GTG，GGT，GTC，AAT，CCG，ACG，CGT，ACC，GGC，GTG，ATC，GAT，ATC，CTG，CGG

22.AroA-22：Tm＝54，60mer

TTC，ACG，CTG，GTT，CTC，CAG，GGT，AAT，ATC，CGC，GCC，CAT，CAG，CCG，CAG，GAT，ATC，GAT，CAC，GCC

23.AroA-23：Tm＝54，60mer

ACC，CTG，GAG，AAC，CAG，CGT，GAA，GTA，GGT，GGT，GAG，CCT，GTC，GCG，GAT，CTG，CGC，GTA，AGA，GCG

24.AroA-24：Tm＝54，60mer

CAC，CAA，CGC，TTC，AGG，AAT，CTC，GAT，CCC，TTT，CAA，CGC，TAC，CGC，TCT，TAC，GCG，CAG，ATC，CGC

25.AroA-25：Tm＝54，60mer

GAG，ATT，CCT，GAA，GCG，TTG，GTG，CCG，TTG，GCG，ATC，GAT，GAG，TTT，CCG，GTG，TTG，TTC，GTC，GCG

26.AroA-26：Tm＝54，60mer

AGC，ACC，ACG，TAA，CAC，CGT，TCG，TCC，CTC，GGC，ACA，GGC，CGC，CGC，GAC，GAA，CAA，CAC，CGG，AAA

27.AroA-27：Tm＝54，60mer

CGA，ACG，GTG，TTA，CGT，GGT，GCT，TCA，GAG，CTG，CGC，GTG，AAG，GAG，TCC，GAC，CGT，ATC，CAG，GTC

28.AroA-28：Tm＝54，60mer

TTC，ACA，CTT，CAC，GCC，CAA，CGC，CAG，CAG，ACC，ATC，GGC，CAT，GAC，CTG，GAT，ACG，GTC，GGA，CTC

29.AroA-29：Tm＝54，60mer

GCG，TTG，GGC，GTG，AAG，TGT，GAA，CCG，ACC，CCT，GAT，GGG，ATT，ATC，ATT，GAG，GGT，GGC，CCG，ATG

30.AroA-30：Tm＝54，60mer

AAT，GCG，GTG，ATC，GCC，ATG，GGC，ATG，CAC，CTC，ACC，ACC，GCC，CAT，CGG，GCC，ACC，CTC，AAT，GAT

31.AroA-31：Tm＝54，60mer

GCC，CAT，GGC，GAT，CAC，CGC，ATT，GCC，ATG，GCG，TTC，AGC，GTG，GCT，TCC，CTA，CGG，GCG，GCG，GCG

32.AroA-32：Tm＝54，60mer

AGA，CGT，AGC，AAC，ATT，GGC，GCA，GTC，ACG，GAT，ACG，AAT，CGG，CGC，CGC，CGC，CCG，TAG，GGA，AGC

33.AroA-33：Tm＝54，60mer

TGC，GCC，AAT，GTT，GCT，ACG，TCT，TTT，CCG，AAT，TTT，CTT，ACA，CTG，TGT，GCC，CAC，GTC，GGC，ATC

34.AroA-34：Tm＝54，48mer

TCA，CAA，TTG，AGC，CTC，TTG，GGC，AAC，ACG，GAT，GCC，GAC，GTG，GGC，ACA，CAG

Utilize PCR to carry out the epsp synthase gene amplification, in 100 μ l reaction systems, the AroA-2-AroA-33 addition of totally 32 primers is 2ng, and outside primer AroA-1 and AroA-34 addition are 30ng, and amplification condition is: 94 ℃ of preheating 1min; 94 ℃, 30s, 50 ℃, 30s, 72 ℃, 2min, the Taq archaeal dna polymerase that uses is KOD FX taq enzyme (Toyobo company, Japan), totally 25 circulations.

After PCR finished, the 1wt% agarose gel reclaimed, and got 10 μ l directly link to each other with the T/A cloning vector (Dalian treasured biotech firm).4 ℃ of connections are spent the night, and efficiently transform in the DH5 α competence.Obtain positive colony.

Embodiment 4

The epsp synthase gene of glyphosate highly-tolerant is expressed

The positive colony of the EPSP gene of synthetic of the present invention adopts following primer:

P5 (5 '-gagagaccatgatggaaaccgctgtgaacgctgatga-3 ') and

P6(5’-gtctcgagtcacaattgagcctcttgggcaac-3’)

Carrying out pcr amplification, is the Taq archaeal dna polymerase with KOD Plus (Toyobo Japan), and amplification condition is followed successively by: 94 ℃ of 30s, 55 ℃ of 30s, 72 ℃ of 90s, 30 circulations of increasing.After the loop ends, add the rtaq enzyme (Dalian Bao Bio-Engineering Company) of 2U, 72 ℃ are extended 90s, the long 1335bp of amplified fragments.After the PCR product is cut with Nco I and Xho I enzyme, be connected into that carrier pET-28a (NEB company) that same enzyme cuts obtains recombinant plasmid pET-AroA-P.fluorescens and with its transformed into escherichia coli BL21 (DE3) (Novagen company), transformant be coated in the LB solid medium cultivate 24h.It is carried out the protein expression purifying, the SDS-PAGE electrophoresis detection with gel-protein purification test kit HisTrap HP (Amersham Biosciences company).

Through the SDS-PAGE electrophoresis detection, the about 48kDa of albumen size conforms to predictor (referring to Fig. 5).

Embodiment 5

The mensuration of EPSP enzyme activity determination and kinetic parameter

1. measuring method

The inorganic phosphorus typical curve: the 10mM inorganic phosphorus was by dilution in 1: 10, get 0,1,2,3 respectively ... in 20 μ l and the 1.5ml Eppendorf centrifuge tube, add pure water to 100 μ l mixing, add MAT solution 0.8ml mixing, timing adds the rapid mixing of SC solution 100 μ l after three minutes, room temperature is measured the OD660 value after leaving standstill 20min.Triplicate.With the inorganic phosphorus concentration is X-coordinate, and the OD660 value obtains the inorganic phosphorus typical curve for total ordinate zou mapping.

1) enzyme activity determination: protein quantification adopts Xylene Brilliant Cyanine G G-250 staining (Bradford, 1976).Adding following solution on ice with in the 1.5ml Eppendorf centrifuge tube: 10mM PEP solution 2 μ l, 10mMS3P solution 2 μ l, 0.5M HEPES solution 2 μ l, 1mM (NH ₄) ₆MO ₇O ₂₄4H ₂O solution 2 μ l and distilled water 12 μ l mixings, bathe with 28 ℃ of temperature and respectively to manage sample room behind the 5min and add 5 μ l purifying protein and timing every 2s, behind the 2min more at interval 2s add 200 μ l MAT solution successively, behind the colour developing 3min more at interval 2s add the rapid mixing of 20 μ l 34%SC solution successively, measure the OD660 value behind the color development at room temperature 20min.Contrast is except that not adding the purifying protein all the other same sample hoses.After the OD660 value of sample hose and control tube was subtracted each other, contrast inorganic phosphorus typical curve can be tried to achieve the inorganic phosphorus molar weight that reaction discharges, and just obtained the enzyme activity (nkat/mg) of enzyme again divided by reaction times and zymoprotein amount.

2) half amount of suppression (IC ₅₀) measure: add 0,10 in the above-mentioned reaction solution ^-3, 10 ^-2, 10 ^-1, 1,10,100, the 500mM glyphosate, gained specific activity of enzyme data are X-axis with glyphosate concentration, adopt logarithmic coordinates, be that Y-axis is mapped with speed of response (nkat/mg).

3) K _m(PEP) measure: the S3P strength of solution is constant at 1mM, measures enzyme reaction rate by above-mentioned reaction system down in different PEP concentration (0.05,0.067,0.1,0.2,0.5,1.0mM), the numerical value of surveying is pressed V-v/[S] (Eadic-Hofstee) method map.

4) K _i(glyphosate) measure: measure down in different glyphosate concentration (0,10,50,100 μ M) that PEP concentration is 0.05,0.067,0.1,0.2,0.5, the enzyme reaction rate of EPSP during 1.0mM.Take double-reciprocal plot, obtain 1/V-1/[S] straight line, again with each collinear slope as ordinate zou, the concentration of glyphosate obtains a new straight line as X-coordinate, the intersection point of this straight line and X-axis is K _i(glyphosate) value.

2. measurement result

EPSP synthase activity of the present invention is 59.63 ± 0.09nkat/mg, and its kinetic parameter is as shown in table 1.

The kinetic parameter of table 1 EPSP synthase of the present invention

According to the kinetic parameter of above-mentioned EPSP synthase as can be known: EPSP synthase of the present invention not only has higher glyphosate resistance, but also keeping the affinity stronger with PEP, these characteristics provide possibility for the cultivation that EPSP synthase of the present invention is used for genetically modified crops.

Attached: related Nucleotide/aminoacid sequence table among the application:

＜110〉Academy of Agricultural Sciences, Shanghai City

＜120〉a kind of epsp synthase gene and application thereof that derives from Pseudomonas fluorescens

<160>2

<170>PatentIn?version?3.3

<210>SEQ?ID?No?1

<211>1335

<212>DNA

＜213〉Pseudomonas fluorescens (Pseudomonas fluorescens.)

<400>

ATGGA?AACCG?CTGTG?AACGC?TGATG?AGTTG?ACCTT?CCTTG?CCGAA?CCGGG?TGGCC?GCTTG 60

AGCGG?AAGCA?TTCGG?GTGCC?GGGCG?ATAAG?TCGAT?CTCCC?ATCGT?TCGAT?CATGC?TGGGT?120

TCGTT?GGCTG?GGGGT?GTGAC?TGAGG?TCGAA?GGTTT?CCTTG?AAGGT?GAAGA?TGCCC?TGGCG?180

ACCTT?GCAGG?CATTT?CGCGA?CATGG?GCGTG?GTGAT?TGAGG?GCCCG?CATCA?TGGGC?GCGTG?240

ACCAT?TCATG?GCGTG?GGGCT?GCATG?GGTTG?AAGCC?GGCGC?CGGGG?CCGAT?TTACC?TGGGT?300

AACTC?AGGTA?CTTCC?ATGCG?TTTGC?TGTCC?GGTTT?GCTGG?CGGCG?CAGAG?CTTCG?ACAGC?360

GTGCT?GACCG?GTGAC?GCATT?CCTGT?CCAAG?CGCCC?GATGA?GTCGT?GTGGC?CAGGC?CGCTG?420

CGGGA?AATGG?GAGCG?GTGAT?CGAGA?CGGGG?CCGGA?AGGGC?GTCCG?CCGCT?GACCA?TTCGG?480

GGTGG?CCAGT?CGTTG?AAAGG?CCTGG?CTTAT?GCAAT?GCCGA?TGGCC?AGTGC?CCAGG?TCAAA?540

TCCTG?CCTGT?TGTTG?GCTGG?GTTGT?ACGCT?GAAGG?TAAAA?CCGCG?GTGAC?CGAGC?CTGCG?600

CCGAC?CCGTG?ACCAC?ACCGA?GCGCA?TGTTG?CGTGG?CTTTG?GTTAT?CCGGT?GGCGG?TGGAG?660

GGCGC?GACGG?CGTCG?GTGGA?GTCCG?GTCAT?GCGCT?GACGG?CTGCT?CATAT?AGAAG?TGCCG?720

GGGGA?TATTT?CTTCT?TCAGC?GTTCT?TTCTG?GTGGC?AGCTT?CGATT?GCCGA?GGGTT?CTGAG?780

TTGCT?GCTGG?AGCAT?GTGGG?TGTCA?ATCCG?ACGCG?TACCG?GCGTG?ATCGA?TATCC?TGCGG?840

CTGAT?GGGCG?CGGAT?ATTAC?CCTGG?AGAAC?CAGCG?TGAAG?TAGGT?GGTGA?GCCTG?TCGCG?900

GATCT?GCGCG?TAAGA?GCGGT?AGCGT?TGAAA?GGGAT?CGAGA?TTCCT?GAAGC?GTTGG?TGCCG 960

TTGGC?GATCG?ATGAG?TTTCC?GGTGT?TGTTC?GTCGC?GGCGG?CCTGT?GCCGA?GGGAC?GAACG 1020

GTGTT?ACGTG?GTGCT?TCAGA?GCTGC?GCGTG?AAGGA?GTCCG?ACCGT?ATCCA?GGTCA?TGGCC 1080

GATGG?TCTGC?TGGCG?TTGGG?CGTGA?AGTGT?GAACC?GACCC?CTGAT?GGGAT?TATCA?TTGAG 1140

GGTGG?CCCGA?TGGGC?GGTGG?TGAGG?TGCAT?GCCCA?TGGCG?ATCAC?CGCAT?TGCCA?TGGCG 1200

TTCAG?CGTGG?CTTCC?CTACG?GGCGG?CGGCG?CCGAT?TCGTA?TCCGT?GACTG?CGCCA?ATGTT 1260

GCTAC?GTCTT?TTCCG?AATTT?TCTTA?CACTG?TGTGC?CCACG?TCGGC?ATCCG?TGTTG?CCCAA 1320

GAGGC?TCAAT?TGTGA?1335

<210>SEQ?ID?No?2

<211>445

<212>PRT

＜213〉Pseudomonas fluorescens (Pseudomonas fluorescens.)

1 Met?Glu?Thr?Ala?Val?Asn?Ala?Asp?Glu?Leu?Thr?Phe?Leu?Ala?Glu?Pro?Gly?Gly?Arg?Leu

21 Ser?Gly?Ser?Ile?Arg?Val?Pro?Gly?Asp?Lys?Ser?Ile?Ser?His?Arg?Ser?Ile?Met?Leu?Gly

41 Ser?Leu?Ala?Gly?Gly?Val?Thr?Glu?Val?Glu?Gly?Phe?Leu?Glu?Gly?Glu?Asp?Ala?Leu?Ala

61 Thr?Leu?Gln?Ala?Phe?Arg?Asp?Met?Gly?Val?Val?Ile?Glu?Gly?Pro?His?His?Gly?Arg?Val

81 Thr?Ile?His?Gly?Val?Gly?Leu?His?Gly?Leu?Lys?Pro?Ala?Pro?Gly?Pro?Ile?Tyr?Leu?Gly

101?Asn?Ser?Gly?Thr?Ser?Met?Arg?Leu?Leu?Ser?Gly?Leu?Leu?Ala?Ala?Gln?Se?rPhe?Asp?Ser

121?Val?Leu?Thr?Gly?Asp?Ala?Phe?Leu?Ser?Lys?Arg?Pro?Met?Ser?Arg?Val?Ala?Arg?Pro?Leu

141?Arg?Glu?Met?Gly?Ala?Val?Ile?Glu?Thr?Gly?Pro?Glu?Gly?Arg?Pro?Pro?Leu?Thr?Ile?Arg

161?Gly?Gly?Gln?Ser?Leu?Lys?Gly?Leu?Ala?Tyr?Ala?Met?Pro?Met?Ala?Ser?Ala?Gln?Val?Lys

181?Ser?Cys?Leu?Leu?Leu?Ala?Gly?Leu?Tyr?Ala?Glu?Gly?Lys?Thr?Ala?Val?Thr?Glu?Pro?Ala

201?Pro?Thr?Arg?Asp?His?Thr?Glu?Arg?Met?Leu?Arg?Gly?Phe?Gly?Tyr?Pro?Val?Ala?Val?Glu

221?Gly?Ala?Thr?Ala?Ser?Val?Glu?Ser?Gly?His?Ala?Leu?Thr?Ala?Ala?His?Ile?Glu?Val?Pro

241?Gly?Asp?Ile?Ser?Ser?Ser?Ala?Phe?Phe?Leu?Val?Ala?Ala?Ser?Ile?Ala?Glu?Gly?Ser?Glu

261?Leu?Leu?Leu?Glu?His?Val?Gly?Val?Asn?Pro?Thr?Arg?Thr?Gly?Val?Ile?Asp?Ile?Leu?Arg

281?Leu?Met?Gly?Ala?Asp?Ile?Thr?Leu?Glu?Asn?Gln?Arg?Glu?Val?Gly?Gly?Glu?Pro?Val?Ala

301?Asp?Leu?Arg?Val?Arg?Ala?Val?Ala?Leu?Lys?Gly?Ile?Glu?Ile?Pro?Glu?Ala?Leu?Val?Pro

321?Leu?Ala?Ile?Asp?Glu?Phe?Pro?Val?Leu?Phe?Val?Ala?Ala?Ala?Cys?Ala?Glu?Gly?Arg?Thr

341?Val?Leu?Arg?Gly?Ala?Ser?Glu?Leu?Arg?Val?Lys?Glu?Ser?Asp?Arg?Ile?Gln?Val?Met?Ala

361?Asp?Gly?Leu?Leu?Ala?Leu?Gly?Val?Lys?Cys?Glu?Pro?Thr?Pro?Asp?Gly?Ile?Ile?Ile?Glu

381?Gly?Gly?Pro?Met?Gly?Gly?Gly?Glu?Val?His?Ala?His?Gly?Asp?His?Arg?Ile?Ala?Met?Ala

401?Phe?Ser?Val?Ala?Ser?Leu?Arg?Ala?Ala?Ala?Pro?Ile?Arg?Ile?Arg?Asp?Cys?Ala?Asn?Val

421?Ala?Thr?Ser?Phe?Pro?Asn?Phe?Leu?Thr?Leu?Cys?Ala?His?Val?Gly?Ile?Arg?Val?Ala?Gln

441?Glu?Ala?Gln?Leu?-

Claims (3)

1. an epsp synthase gene that derives from Pseudomonas fluorescens is characterized in that, its nucleotide sequence is shown in SEQ ID NO 1.

2. the epsp synthase gene that derives from Pseudomonas fluorescens according to claim 1 is characterized in that, the aminoacid sequence of its encoded protein matter is shown in SEQ ID NO 2.

3. claim 1 or the 2 described application of epsp synthase gene in glyphosate tolerant that derive from Pseudomonas fluorescens.

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