CN104450750B - Resistance gene for degrading herbicide glyphosate and coded protein of resistance gene - Google Patents

Abstract

The invention belongs to the field of genetic engineering, and in particular relates to a resistance gene for degrading herbicide glyphosate and coded protein of the resistance gene. The gene and the protein are screened from a resistance gene which is capable of effectively degrading herbicide glyphosate; the gene is named glyphosate oxidase B4S7 gene, a nucleotide sequence is as shown in SEQ ID NO: 1, and the protein of the protein coded by virtue of the gene is as shown in SEQ ID NO: 2. Through DNA reshuffling on the coding gene of glycine oxidase mutant B3S1 of bacillus cereus as well as the enzyme-coupling activity screening based on T7 bacteriophage lytic-horseradish peroxidase/o-dianisidine, the glyphosate oxidase mutant B4S7 is finally obtained. The resistance gene disclosed by the invention preliminarily verifies the functions of the glyphosate oxidase B4S7 gene as well as application potential of the glyphosate oxidase B4S7 gene in glyphosate-resistance crops.

Description

A kind of resistant gene and its encoding proteins of degrading herbicide glyphosate

Technical field

The invention belongs to genetic engineering field, and in particular to a kind of resistant gene and its coding of degrading herbicide glyphosate Albumen, the gene is named as glyphosate oxidase B4S7 genes, and the gene and encoding proteins are that have to herbicide glyphosate There is acquisition in the resistant gene of efficient degradation activity.The invention further relates to a kind of resistant gene of efficient degradation herbicide glyphosate Purposes in plant herbicide glyphosate.

Background technology

Glyphosate (glyphosate) is a kind of non-selective broad spectrum weeding agent, and 1970 by Monsanto Chemicals (Monsanto Company, St.Louis, MO) is developed.Its trade name Roundup, Chinese name agriculture reaches, from commercialization Since, glyphosate is used widely in the whole world, and consumption increases year by year.

Glyphosate molecular formula is C₃H₈NO₅P, chemical entitled N- phosphoric acid methylene glycine (N- (phosphonomethyl) Glycine, GLP), it is a kind of derivative of glycine, relative molecular weight is 169.1.Sterling glyphosate is white crystal, nothing Taste, non-volatility, fusing point is (200 DEG C) high, and solubility is low (1.2%, 25 DEG C) in water, and is insoluble in absolute ethyl alcohol, benzene etc. and has Machine solvent.In agricultural application, in order to increase dissolubility of the glyphosate in water, absorptivity of the plant to glyphosate is improved, from And the herbicidal effect for having reached, glyphosate is usually processed into salt (such as glyphosate isopropyl amine salt, glyphosate potassium), and add Enter some surfactants and builder ingredient to improve its permeability on plant leaf blade.

In vivo, aromatic amino acid has important biological function, and it participates in micro-element, alkaloid and indoles The synthesis of many kinds of substance such as derivative, plays a significant role during protein synthesis, cell division etc., and synthetic quantity is not enough then Eubolism and the growth of plant can be had a strong impact on.Shikimic acid pathway is the important step of synthetic aromatic amino acid in plant Suddenly, 5- enol pyruvic acid shikimic acid -3- phosphate synthases (5-enolpyruvylshikimate-3-phosphate Synthase, EPSPS) be this approach key enzyme, its catalysis is by (the phosphoenol of PEP Pyruvate, PEP) enolpyruvyl acid moieties are transferred to 3- phosphoric acid shikimic acids (shikimate-3-phosphate, S3P) On 5- hydroxyls, synthesize 5- enol pyruvic acid shikimic acid -3- phosphoric acid.The latter is to form chorismic acid and and then synthesis aromatic series amino The precursor of acid.Used as the analogue of PEP (PEP), glyphosate competes the Binding Capacity of EPSPS with PEP Site, causes the activity inhibited of EPSPS, and so as to cause shikimic acid pathway to be obstructed, the synthesis of aromatic series propylhomoserin is blocked, finally Cause Plant death, herbicidal effect is reached with this.Therefore, crop is made to be endangered from herbicide while removing weeds using glyphosate Evil is significant for agricultural production.

Crop is obtained glyphosate resistance, and then be using gene work from an important means of the harm of herbicide Journey technology, is possible in the gene transfered plant body of degradation of glyphosate so as to obtain Herbicid resistant crop.

In natural environment, glyphosate can thoroughly be decomposed by some edaphons, and on microbiologic population influence compared with It is small.Since last century the eighties, constantly have microorganism and gene be reported can degrading herbicide glyphosate, and reported The gene in road derives from microorganism, up to the present not yet finds the glyphosate degradation gene (Duke 2011) of plant origin. Research shows that the degradation pathway of herbicide glyphosate mainly has two kinds：(1) the C-N keys generation amine methylphosphonic acid of fracture glyphosate And glyoxalic acid (glyoxylate) (AMPA)；(2) C-P keys generation methyl amimoacetic acid and the Phos (Duke 2011) of fracture glyphosate. Wherein, based on C-N lytic pathways.C-N keys can be broken so as to the enzyme of degradation of glyphosate has glyphosate oxidoreductase (glyphosate oxidoreductase, GOX) and two kinds of glycine oxidase (glycine oxidase, GO).GOX is in two Tenth century, the eighties were by Monsanto companies find and illustrate its mechanism of action (Barry et al.1995), and it is aerobic Under the conditions of herbicide glyphosate is aoxidized, at the same time co-factor FAD is reduced, formed reduction-state FAD and glyoxalic acid and amine Schiff bases (shiff base) between methyl acid phosphate, subsequent Schiff basic hydrolysis produces glyoxalic acid and amine methyl acid phosphate, reduction-state FAD reoxidized by oxygen, while oxo flavine ring is catalyzed another molecule glyphosate generation glyoxalic acid and amine first as intermediate Base phosphoric acid.Because wild type GOX is not high to the activity of glyphosate, Barry et al. has carried out external fixed to the encoding gene of GOX To evolution to improve oxidation activities of the GOX to glyphosate.Random mutation is carried out to the gene using fallibility PCR method, and is led to Glyphosate resistance plate screening mutant is crossed, final to obtain one plant of mutant of 10 times of activity raising v.247, it is to glyphosate Affinity is significantly improved (Km is changed into 2.6mM from the 20mM-30mM of wild type).Current GOX has been used to some resistance glyphosates Genetically modified crops, such as wheat (Triticum aestivum L.), beet (Beta vulgaris L.), radish (Brassica Rapa L.), but it is used together with CP4EPSP synthase genes.

Glycine oxidase (GO) is that another can be by being broken the enzyme of C-N key degradation of glyphosate.GO is a kind of four Aggressiveness lyochrom oxidizing enzyme, each monomer and molecule FAD non-covalents ground combine (Job et al.2002；Job et Al.2002), it is to pyrophosphoryl thiamine (vitamin B₁) biosynthesis play critically important effect (Settembre et al.2003).Similar with GOX, GO oxidative degradation glyphosates generate aminomethyl phosphonic acid and glyoxalic acid, except that, GO catalysis The reaction of oxidized glyphosate can simultaneously produce H₂O₂, this is primarily due to, and both oxidation mechanisms are different, and what GO took is a germplasm Sub- metastasis.For sequence similarity, the amino acid sequence similarity only 18.1% of GO and GOX, and with D- amino acid oxygen Change enzyme sequence similitude higher and increasingly similar in oxidizing property with D-AAO.Meanwhile, GO has extensive bottom Thing selectivity, its analogue to glycine and glycine has certain oxidation activity, makes these materials that de- amine to occur Oxidation, generates corresponding 2-ketoacid and hydrogen peroxide.Glyphosate as glycine analogue, equally can be by glycine oxygen Change enzyme institute oxidation Decomposition, this causes that glycine oxidase shows huge diving in the application of glyphosate resistance crop and development Power.2009, Pedotti et al. was based on the crystal structure of bacillus subtilis glycine oxidase (BsuGO), is set using rationality The method of meter has carried out in-vitro directed transformation to the enzyme.Fixed point saturation mutation is carried out respectively by 11 amino acid sites, and Beneficial mutation is superimposed, one plant is obtained and 175 times of (Km values reach 0.5mM), catalytic efficiency ((k is improved to glyphosate compatibility_cat/ K_m) improve 210 times of mutant G51S/A54R/H244A (Pedotti et al.2009).Wherein, Ser⁵¹Pendant hydroxyl group and Arg⁵⁴The phosphate group of guanidino group and glyphosate molecule there is electrostatic interaction so as to stabilize the knot of glyphosate and enzyme Close, optimize the conformation of enzyme active center, improve the compatibility of enzyme-to-substrate glyphosate.Ala²⁴⁴Though replacement do not change enzyme To the turn over number of substrate, the allosteric rearrangement of enzyme molecule avtive spot is but promoted, so as to cooperate with improve BsuGO mutant pair The catalysis activity of glyphosate.The G51S/A54R/H244A mutant genes have been transferred to alfalfa (Medicago sativa) and have obtained Obtained glyphosate resistance (Pollegioni et al.2011).Therefore, using glycine oxidase as the target of orthogenesis, Evolution transformation is carried out to it in laboratory conditions using protein engineering, its substrate to herbicide glyphosate is improved special The opposite sex and degrading activity have very great meaning for the application and popularization of glyphosate resistance crop from now on.

China is developing country maximum in the world, and agricultural production occupies critical role in national economy, and weeding The harm that the application of agent brings to crop yield and quality directly constrains the development of agricultural production with adverse effect, while to people Class health causes serious threat.In addition, research starting evening of the China on anti-fecundi-t, and foreign macro-organism Company has obtained some Glyphosate resistance genes and has constructed tight Intellectual Property Protection.Therefore, by screening and The method of improvement obtains the gene for being capable of preferable degradation of glyphosate, and releasing herbicide is to the inhibitory action of plant growth for China And world agriculture production is significant.

The content of the invention

It is an object of the invention to provide a kind of glyphosate enzyme mutant B4S7 obtained by directed evolution technologies And application of the gene in herbicide glyphosate.The gene carries out further orthogenesis by glyphosate oxidase B3S1 Transformation, obtains with reference to the high-throughput screening method based on T7 phage splitting cells.Glyphosate enzyme mutant B4S7's Full length gene is 1110bp, encodes 369 amino acid.The encoding gene is connected to pGEX-6p-1 (purchased from U.S. GE Healthcare companies) on plasmid vector, form recombinant plasmid pGEX-6p-B4S7.Then by recombinant plasmid transformed to E.coli BL21 (DE3) competent cell (is purchased from Invitrogen companies of the U.S.), is purified by GST affinity chromatographys system and obtains B4S7 eggs In vain, its oxidation activity to glyphosate is determined using dianisidine/horseradish peroxidase method.Result shows that grass is sweet Ks of the phosphine oxidase B 4S7 to substrate glyphosate_m=0.10mM, conversion number k_cat=3.62min^─1, catalytic efficiency k_cat/K_m=3.62 ×10^-4M^─1·min^─1.Therefore, B4S7 has glyphosate resistance very high, has in terms of glyphosate resistance crop very big Potential using value.

To achieve the above object, the present invention is using following technical scheme (technology path is shown in Fig. 1)：

With pGEX-6p-B3S1, pGEX-6P-B3S4, pGEX-6p-B3S6 and pGEX-6p-B4S7 plasmid as mutagenesis template (Zhan et al.2013), carries out DNA shuffling and builds mutant library.The mutated gene segment connection that will be obtained Onto pGEX-6p-1 plasmid vectors (Fig. 6), the recombinant plasmid transformed Escherichia coli Escherichia coli that will be formed are (referred to as： E.coli) DH5 α competent cells form mutant library.Cultivated to containing 600 μ L liquid LB with the toothpick picking monoclonal of sterilizing 96 deep-well plates of base (containing 100 μ g/ml ampicillins), shaken cultivation under the conditions of 37 DEG C.Added during thalli growth to logarithmic phase IPTG (isopropyl-β-D-thiogalactoside, final concentration 0.1mM) and T7 bacteriophages are (public purchased from U.S. Merck Millipore Department), shaken cultivation 6h under the conditions of 28 DEG C, make protein expression with discharge synchronously carry out.The μ L of supernatant 159 after cracking are drawn to be added to In 96 microwell plates, 20 μ L glyphosates, 20 μ L 0.32mg/mL dianisidines and 1 μ L 5unit/mL horseradish peroxides are subsequently added Thing enzyme, it is well mixed to react 8h under the conditions of 25 DEG C, the light absorption value of 450nm is determined using all-wave length ELIASA, select OD₄₅₀ Higher than the clone of control (B3S1, active soprano in template), the concentration for reducing substrate glyphosate carries out secondary screening and checking.Most The mutant that four activity are improved is obtained from 10000 clones eventually, enzyme activity determination result shows, mutant B4S7 is sweet to grass The active highest of phosphine, and the substrate affinity of glycine is further reduced, therefore, it is named as grass according to substrate preferences Sweet phosphine oxidizing ferment.

Compared with existing invention, Ks of the glyphosate oxidase B4S7 that the present invention is obtained to glyphosate_m=0.10mM is low In other glycine oxidases and glyphosate oxidoreductase for having reported, turn over number conversion number k_cat=3.62min^─1, urge Change efficiency k_cat/K_m=3.62 × 10⁴M^─1·min^─1.Illustrate that the enzyme has glyphosate degrading activity higher, especially its is right The compatibility of glyphosate is very high, i.e., the enzyme can just play its active degradation glyphosate when glyphosate concentration is very low, so as to reduce Residue of glyphosate in crop body, this is of great significance for glyphosate resistance crop tool.By determining pH to enzyme The influence of activity finds：The optimal pH of glyphosate oxidase B4S7 is 8.5, i.e., have most highly active in pH 8.5；When pH is small When 7, the activity decrease of B4S7；Under conditions of pH 7.0-11.0, B4S7 can maintain more than 80% activity.Therefore, B4S7 can be well adapted for neutral and alkalescence condition, and this is also suitable for it in plant interior expression.By temperature of the measurement to enzyme activity Property influence find：Between 0-30 DEG C, the enzymatic activity of B4S7 can keep more than 80%；When temperature is more than 30 DEG C, B4S7's Enzymatic activity begins to decline；When temperature is higher than 40 DEG C, enzymatic activity drastically declines.Therefore, B4S7 can be protected under low temperature and medium temperature condition Greater activity is held, this also meets the temperature range of plant normal growth.

Brief description of the drawings

Sequence table SEQ ID NO:Nucleotide sequence (the 1- of the 1 glyphosate oxidase B4S7 genes obtained for the present invention 1110bp), sequence length is 1110bp.Its corresponding coded sequence (i.e. CDS) is also 1-1110bp, and 369 amino are encoded altogether Acid.

Sequence table SEQ ID NO:2 is the protein sequence of glyphosate oxidase B4S7 gene codes.

Fig. 1:A kind of acquisition flow chart of the resistant gene of degradation of glyphosate.

Fig. 2：The plasmid map of recombinant plasmid pGEX-6p-B3S1.

Fig. 3：The plasmid map of recombinant plasmid pGEX-6p-B3S4.

Fig. 4：The plasmid map of recombinant plasmid pGEX-6p-B3S6.

Fig. 5：The plasmid map of recombinant plasmid pGEX-6p-B3S7.

Fig. 6:The plasmid map of expression vector pGEX-6p-1.

Fig. 7:Recombinant plasmid pGEX-6p-B4S7 collection of illustrative plates of the invention.

Fig. 8：Glyphosate oxidase B4S7 zymetology aerodynamic schematic diagrames.A figures in Fig. 8：The rice of glyphosate oxidase B4S7 Family name's equation curve figure, abscissa is glyphosate concentration of substrate, and ordinate is enzyme reaction rate；B figures in Fig. 8：Glyphosate The Lineweaver-Burk double reciprocal plots of enzyme B4S7, abscissa is concentration of substrate reciprocal value, and ordinate is reaction speed reciprocal Value.

Fig. 9:The zymologic property figure of glyphosate oxidase B4S7.Description of symbols in figure：A figures in Fig. 9：Glyphosate oxidase B4S7 optimal pH curve maps, abscissa is the buffer solution of different pH, and ordinate is relative activity；B figures in Fig. 9：Display grass is sweet Phosphine oxidase B 4S7 is different pH buffer solutions to pH tolerances, abscissa, and ordinate is relative activity；C figures in Fig. 9：Grass is sweet Phosphine oxidase B 4S7 optimum temperature curve maps, abscissa is different temperatures gradient, and ordinate is relative activity；D figures in Fig. 9： To temperature tolerance figure, abscissa is different treatment temperatures to glyphosate oxidase B4S7, and ordinate is relative activity.

Figure 10：It is transferred to growth curve chart of the Escherichia coli of B4S7 genes in 200mM glyphosates.Abscissa is given birth to for thalline For a long time, ordinate is the concentration of thalline.

Specific embodiment：

The following is the citing of embodiments of the present invention, further illustrated to of the invention, but be not to of the invention Limitation.

Embodiment 1 utilizes DNA shuffling technique construction mutant libraries

The present invention with mutant B3S1, B3S4, the B3S6 from Bacillus cercus glycine oxidase (BceGO) and B3S7 is template (Zhan et al.2013), and the DNA Shuffling Methods reported with reference to Stemmer simultaneously process DNA with supercritical ultrasonics technology Fragment, carries out DNA shuffling (Stemmer 1994；Miller et al.2002), it would be beneficial to mutational site recombinates, real Now to the further improvement of Bacillus cercus glycine oxidase, so as to obtain the grass with more preferable glyphosate degrading activity Sweet phosphine oxidizing ferment, is that the application of glyphosate resistance crop is taken a firm foundation, and reduces residual quantity of the glyphosate in plant, So that plant is reduced while glyphosate is released to its growth inhibition passes through threat of the food chain link to human health.

Comprise the following steps that described：

(1) amplification of templet gene

Extracting pGEX-6p-B3S1 (Fig. 2), pGEX-6p-B3S4 (Fig. 3), pGEX-6p-B3S6 (Fig. 4) and pGEX-6p- B3S7 (Fig. 5) plasmid (Zhan et al.2013), respectively as template, with plasmid vector pGEX-6p-1 (Fig. 6, purchased from the U.S. GE Healthcare companies) universal primer (6p-F/R) and Taq DNA polymerase carry out different mutants GO genes (thiO, GenBank acession number:KC203486 amplification).Primer sequence and PCR reaction systems are as follows：

6P-1F:5’-ATCCTCCAAAATCGGATCTGGAA-3’

6P-1R:5’-GGCAGATCGTCAGTCAGTCACG-3’

PCR reaction systems

PCR response procedures：

Predegeneration：94℃4min；94 DEG C of 30sec (denaturation), 52 DEG C of 30sec (annealing), 72 DEG C of 90sec (extension), totally 30 Individual circulation；72℃10min；15℃5min.After reaction terminates, the fragment expanded with 1% Ago-Gel recovery purifying. Using the micro ultraviolet specrophotometers of NanoDrop 1000 each fragment concentrations of measure, each fragment mixed in equal amounts, 37 DEG C steam moisture Hair, sample concentration to concentration is more than 30ng/ μ L.

(2) ultrasonication DNA fragmentation is utilized

Ultrasonic wave can produce stronger mechanical shearing to act on, and cause DNA structure to be broken.In addition, being produced during ul-trasonic irradiation Raw Cavitation effect and high temperature, can destroy the hydrogen bond of the DNA under aqueous environment, so as to cause DNA double helical structure Fracture.Therefore, the present embodiment carries out the break process of DNA fragmentation using supercritical ultrasonics technology (Miller et al.2002).Place's manage bar Part is：Setting power is 600W, frequency 25kHz, by the sample of 600 μ L as on ice, ultrasonication is carried out under condition of ice bath, Per ultrasonically treated 15s intervals 8s.A sample is taken per 10min, its size is detected using 2% agarose gel electrophoresis, until piece Section treatment to size is 100-200bp.Then, with the small fragment as template, reacted without primer PCR, response procedures such as table 1：

Table 1 is without primer PCR reaction system

Without primer PCR response procedures：94 DEG C of 3min (predegeneration)；94 DEG C of 30sec (denaturation), 40 DEG C of 30sec (annealing), 72 DEG C 20sec+1sec/ circulation (extensions), totally 70 circulate；72 DEG C of insulation 10min；15℃5min.After PCR reactions are completed, profit Primer size is detected with 1% agarose gel electrophoresis.

(3) there is primer PCR

It is template with the product without primer PCR, is carried out using glycine oxidase gene-specific primer BceGO-F/R Pcr amplification reaction, to obtain the genetic fragment of mutation.Reaction system such as table 2：

Table 2 has primer PCR reaction system

PCR reaction conditions are：94 DEG C of 3min (predegeneration)；94 DEG C of 30sec (denaturation), 59 DEG C of 30sec (annealing), 72 DEG C 70sec (extension), totally 30 circulations；72 DEG C of insulation 10min；15℃5min.After the completion of reaction, using 1% Ago-Gel The size of electrophoresis detection amplified production.

(4) structure of DNA shuffling mutated libraries

After the fragment purification that above-mentioned steps (three) are had obtained by primer PCR reaction, double digestion is carried out with BamHI and XhoI (double digestion system such as table 3), with the pGEX-6p-1 carriers formed through BamHI and XhoI double digestions 4 after digestion products recovery Even reaction is overnight (enzyme connects reaction system such as table 4) DEG C to carry out enzyme.

The double digestion system of table 3

The enzyme of table 4 connects reaction system

Take 1 μ L enzyme connect product things to mix with E.coli DH5 α competence, click on conversion, add 600 μ L SOB culture mediums (to match somebody with somebody Method processed：Tryptone 20g, dusty yeast 5g, measure 10mL 250mmol/L KCl solution, 5mL 2mol/L MgCl₂Solution, Add 900mL ddH₂After O stirring and dissolvings, pH to 7.0, plus ddH are adjusted₂O is settled to 1L, and high steam goes out at 121 DEG C after packing Bacterium 30min.) in 37 DEG C of recovery 1h.Then, the bacterium solution after recovery is coated into the LB containing ampicillin (100 μ g/ml) to consolidate Body flat board, 37 DEG C are inverted culture 12h to growing macroscopic monoclonal.10 clone detection insertion rates of random picking, and Sending sequencing company carries out sequence verification, obtains DNA shuffling mutated libraries.

The screening (high flux screening based on 96 orifice plates) of the mutant of the glyphosate oxidation activity high of embodiment 2

Screening technique of the method with reference to reports such as (Zhan et al.2013).Will control bacterium (pGEX- containing recombinant plasmid The E. coli DH5 α of 6p-B3S1) it is inoculated into containing (the addition of 600 μ L LB liquid mediums with the clone in mutation library Ampicillin to concentration be 100 μ g/ml) 96 deep-well plates in, while the bacterium in every hole is backed up into guarantor in ammonia benzyl resistant panel Deposit.The deep-well plates of good seal 96, after 37 DEG C of 200rpm shaken cultivations 16h, add per hole and contain ampicillin (100 μ g/ml), IPTG LB fluid nutrient mediums 200 the μ L, 28 DEG C of 200rpm of (final concentration 0.1mM) and T7 bacteriophages (Tarahovsky et al.1994) Shaken cultivation 6h makes protein induced expression and is discharged by phage splitting.(supernatant after cracking, contains to draw 159 μ L crude enzyme liquids Target protein) add 96 microwell plates in, and add 20 μ L substrates glyphosate (5mM), 20 μ L 3.2mg/mL dianisidine solution And 1 μ L 200U/mL horseradish peroxidase, 25 DEG C of reaction 8h.96 microwell plates are scanned followed by all-wave length ELIASA, is read The absorbance at 450nm is taken, chooses mutant of the light absorption value more than control bacterium, reducing glyphosate concentration carries out secondary screening and test Card, serves the final mutant for obtaining Hai Shenggong bioengineering limited company sequencing company and is sequenced and for zymetology Property analysis.

The protein expression of the mutant B4S7 of embodiment 3 and purifying

By the recombinant plasmid pGEX-6p-B4S7 (Fig. 7) of the genes of B4S7 containing mutant and E.coli BL21 (DE3) competence Mixing with cells, it is electroporated, amicillin resistance flat board (the μ g/mL of ampicillin concentration 100) is coated, 37 DEG C were cultivated Night is to growing macroscopic monoclonal.Picking monoclonal is seeded to 20mL LB liquid mediums (the μ g/ containing ampicillin 100 ML), 37 DEG C of 200rpm shaken cultivations are overnight.Amount switching bacterium solution to 2L fresh liquid LB culture mediums by 1% (contain ampicillin 100 μ g/mL), 37 DEG C of 200rpm shaken cultivations to OD600 are 0.6, add the derivant IPTG of final concentration of 0.1mM, 22 DEG C 160rpm Fiber differentiations 12h makes protein expression.Thalline is collected by centrifugation, and two are washed with 50mM sodium pyrophosphate buffer solutions (pH 7.0) It is secondary, then thalline is resuspended in the 50mM sodium pyrophosphate buffer solutions (pH 7.0) of 40mL precoolings.Existed using high pressure cell cracker (4-6 DEG C) carries out clasmatosis under cryogenic conditions, then, using 4 DEG C of 12000rpm centrifugation 30min of high speed freezing centrifuge, receives Collection supernatant is purified for target protein.

Sepharose 4Bs (purchased from U.S. GE Healthcare company) of the 1mL containing gst fusion protein affinity ligand is taken, It is added in the purification column for cleaning up, with sodium pyrophosphate buffer solution (pH 7.0) column scrubber Material Balance post of 50mM precoolings Bed.Column material after balance is added in the supernatant (crude enzyme liquid) after clasmatosis centrifugation, mixing is gently vibrated on ice 30min makes it fully be combined with the albumen containing GST labels.Then, column material is fallen with crude enzyme liquid mixture under the conditions of 4 DEG C Enter purification column, the crude enzyme liquid of outflow rejoins purification column, promotes gst fusion protein fully to be combined with column material twice repeatedly. Column material is washed with except foreigh protein removing and uncombined albumen, buffer solution with the 50mM sodium pyrophosphate buffer solutions (pH 7.0) of precooling Flow control is in about 1mL/min.After the completion of washing, 50mM Jiao's phosphorus that 600 μ L contain PreScission protease (150U) is added Sour sodium buffer solution (pH 7.0), stands overnight, and PreScission protease is fully cut GST labels, discharges the mesh without label Mark albumen.Add 50mM sodium pyrophosphate buffer solutions (pH 8.5) wash-out target proteins.It is pure using 12%SDS-PAGE electrophoresis detections The purity of albumen after change, and detect the dense of albumen after purification with Bradford reagents (Shanghai Sheng Gong bioengineering Co., Ltd) Degree.

The glycine oxidase B4S7 zymologic properties of embodiment 4 are determined

The enzyme activity determination of glycine oxidase and its mutant uses horseradish peroxidase method (Job et Al.2002), the H discharged by determining reaction₂O₂Amount reflection enzymatic activity.Using dianisidine as chromogenic substrate, glyphosate By glycine oxidase oxidation Decomposition, the H of generation₂O₂Dianisidine is aoxidized by horseradish peroxidase oxidation release oxygen Display is orange red, has absworption peak at 450nm, determines OD₄₅₀Standard curve is brought into so as to calculate enzymatic activity.

(1) glycine oxidase enzyme activity determination method

1 μm of oL of 1 μm of oL substrate (glycine or oxygen) or generation will be converted under (pH8.5) under the conditions of 25 DEG C, optimal pH to produce Thing H₂O₂The enzyme amount of required glycine oxidase is defined as an enzyme activity unit (unit).Enzyme activity reaction system (200 μ L) is wrapped Contain：20 μ L 50mM substrates (respectively glycine and glyphosate), 20 μ L 0.32mg/mL dianisidine solution, 1 μ L5U/mL Horseradish peroxidase, the glycine oxidase of a certain amount of purifying is supplemented to 50mM sodium pyrophosphate buffer solutions (pH8.5) Volume is 200 μ L.1h is reacted under 25 DEG C of constant temperatures, OD is determined_450nmLight absorption value, calculates enzyme activity.

(2) zymetology aerodynamic property is determined

Ibid, substrate is set to a series of concentration gradients to reaction system, and after reaction terminates, the 450nm that ELIASA is measured inhales Shading value Input Software GraphPad Prism 6.0 (being shareware), K is calculated using double counting backward techniques_m(Michaelis constant) and V_max(maximum reaction velocity) (see Fig. 8), then substitute into protein concentration and calculate catalytic constant k_cat。

(3) measure of optimum temperature and optimal pH

Optimal pH (see the A figures in Fig. 9)：Enzyme activity reaction system is used into 0.2mM Na respectively₂HPO₄- 0.1mM lemon acid bufferings Liquid (pH4.0-8.0) and 50mM sodium pyrophosphate buffer solutions (pH8.0-11.0) are supplemented to 200 μ L systems, after 25 DEG C of reaction 1h, survey Determine OD_450nmLight absorption value, 100% is defined as by maximum enzyme activity, calculates the relative activity under condition of different pH, determines the most suitable of enzyme pH。

Optimum temperature (see the C figures in Fig. 9)：Under conditions of pH8.5, enzyme activity reaction system is placed in condition of different temperatures 1h is reacted under (0-70 DEG C), OD is determined_450nmLight absorption value, 100% is defined as by maximum enzyme activity, is calculated under condition of different temperatures Relative activity, determines the optimum temperature of glycine oxidase.

(4) measure of pH tolerances and temperature tolerance

PH tolerances (see the B figures in Fig. 9)：Under the conditions of 0 DEG C, enzyme liquid is processed in the buffer solution (ibid) of different pH value 6h, 25 DEG C of reaction 1h, determines OD_450nmLight absorption value, 100% measure enzymatic activity is defined as by maximum enzyme activity, calculates relative enzyme activity, Tolerance of the research glycine oxidase to pH.

Temperature tolerance (see the D figures in Fig. 9)：Enzyme liquid is processed into 1h under different temperatures (ibid), then is placed in most suitable bar 1h is reacted under part and determines enzyme activity, maximum enzyme activity is defined as 100%, calculate relative enzyme activity, research glycine oxidase is to temperature The tolerance of degree.

Result shows Ks of the glyphosate oxidase B4S7 to substrate glyphosate_m=0.10mM, conversion number k_cat=3.62min^─1, Catalytic efficiency k_cat/K_m=3.62 × 10⁴M^─1·min^─1, illustrate that B4S7 has glyphosate resistance very high.Meanwhile, glyphosate The optimal pH of oxidase B 4S7 is 8.5, and under conditions of pH 7.0-11.0, B4S7 can maintain more than 80% activity.Cause This, B4S7 can be well adapted for neutral and alkalescence condition, and this is also suitable in plant interior expression.By temperature of the measurement to enzyme activity Property influence find：Between 0-30 DEG C, the enzymatic activity of B4S7 can keep more than 80%, therefore, B4S7 is in low temperature and middle temperature Under the conditions of can keep greater activity, this also meets the temperature range of plant normal growth.Thus, glyphosate oxidase B4S7 exists Glyphosate resistance crop aspect has very big potential using value.

The growth curve that embodiment 5 is transferred to the Escherichia coli of B4S7 genes is determined

Recombinant plasmid pGEX-6p-B4S7 containing B4S7 genes is transformed into competent escherichia coli cell.Positive gram of picking Longzi, accesses the LB fluid nutrient mediums containing ampicillin, 37 DEG C of 200rpm overnight incubations.Collects thalline, and use M9 liquid Culture medium (formula：NH₄Cl 0.5g, Na₂HPO₄3.0g, KH₂PO₄1.5g, is dissolved in 950ml distilled water.PH is adjusted to 7.4, And by volume constant volume to 1L, 121 DEG C of sterilizing 15min；After sterilizing, the following component that addition has been sterilized：200 μ L 1mol/L's MgSO₄·7H₂O, 5ml mass concentration are 20% glucose, the CaCl of 25 μ L 1mol/L₂Solution) wash twice.By resuspended bacterium Body is transferred to the liquid M9 culture mediums of 100mL, and (formula adds the μ g/mL of ampicillin 100 with foregoing, adds final concentration 200mM Glyphosate), 37 DEG C of 200rpm concussion and cultivates.With the big of pGEX-6p-1 containing empty plasmid and recombinant plasmid pGEX-6p-B3S1 Enterobacteria is used as control.A sample is taken per 6h, determine the light absorption value at 600nm, the life of the Escherichia coli for obtaining turning B4S7 genes Curve map (Figure 10) long.

Result shows, in 200mM glyphosates, the control Escherichia coli containing empty plasmid pGEX-6p-1 can not grow, and contain The control Escherichia coli Growth ability of recombinant plasmid pGEX-6p-B3S1 is faint, and contains the large intestine of recombinant plasmid pGEX-6p-B4S7 Apparently higher than control, this shows bacillus growth ability, and glyphosate oxidase B4S7 has certain glyphosate resistance, in glyphosate Resistance crop aspect has very big potential using value.

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Claims (1)

1. application of a kind of glyphosate oxidase B4S7 genes in resistance glyphosate genetically modified plants, it is characterised in that described The nucleotide sequence of glyphosate oxidase B4S7 genes such as SEQ ID NO：Shown in 1.