CN102234652B - Wheat salt-resistant and oxidation-resistant gene TaFLS and application thereof - Google Patents

Abstract

The invention discloses a wheat salt-resistant gene, i.e., a wheat secondary metabolism flavone synthetic route gene TaFLS and a plant expression vector pSTART-TaFLS containing the gene TaFLS. The invention further discloses application of an expression vector of the gene to the cultivation of salt-resistant and oxidation-resistant plants. As proved by an experiment, the salt resistance and the oxidation resistance of a transgenic plant are remarkably enhanced.

Description

Anti-oxidant gene TaFLS of wheat salt tolerance and application thereof

Technical field

The invention belongs to technical field of biological genetic engineering, relate in particular to the anti-oxidant gene of wheat salt tolerance, be i.e. wheat secondary metabolism flavonoid route of synthesis gene TaFLS and application thereof.

Background technology

The soil salinization has a strong impact on crop yield.And industrial expansion makes the soil salinization more serious, has become a global problem.China's soil salinization is even more serious, and is populous, and crisis in food is particularly outstanding.Therefore, cultivate the salt tolerant new crop varieties and become China when the urgent task of previous ten minutes.

Utilizing the transgene improvement plant technology that new proterties is changed in the crop, develop efficiently the transgenic plant new variety and be used for the plantation in the saltings with this, is a technology with broad prospect of application.

At present, utilize genetic engineering technique to carry out the research of plant salt tolerance aspect and obtained bigger progress, cloned a large amount of genes involveds, and, be used for the salt tolerant Mechanism Study in these gene transferred plants.Some experiments show, in gene transferred plant relevant in plant itself and the other biological with salt tolerant, its allos transcribe can the render transgenic plant with translation product saline-alkaline tolerance improve.

Flavonoid has multiple function in plant; Except serving as the signaling molecule that pigment and plant and mikrobe do mutually; Inducing plant root and fungal component interact and resist outside the invasion and attack of germ, insect and some phytophagous animal, can also stop the injury of abiotic stress such as high salt to plant.For example; To two kinds of different water rice varieties (salt non-sensitive type FL478; Salt responsive type IR29) carry out the salt stress expression pattern analysis, discovery salt responsive type IR29 flavonoid route of synthesis under salt stress is induced PAL, CHS, CHI, F3 ' H and the equal up-regulated expression of DFR; Salt tolerance type and salt responsive type sugarcane two article tie up to that solubility polyphenols, cyanidin(e) and flavones content all raise under the salt stress, and the ascensional range of tolerance type strain is respectively 2.5,2.8 and 3.0 times of responsive type.

High salt is one of modal abiotic stress, thereby very necessity of plant salt tolerance mechanism is understood by system.For many years; Big quantity research has more in depth been illustrated the salt stress acknowledgement mechanism of plant on DNA, RNA and protein level; But on the secondary metabolism level, still know little about it; Particularly the anti-contrary relation of flavonoid metabolism and plant does not still have report, thereby is necessary the mechanism of action of flavonoid in the plant salt stress response inquired into.And through also significant to the quality of wheat improvement to the content of flavonoid biosynthetic pathway regulation and control wheat flavonoid.

Summary of the invention

To the deficiency of prior art, the purpose of this invention is to provide the anti-oxidant gene of a kind of wheat salt tolerance, i.e. wheat secondary metabolism flavonoid route of synthesis gene TaFLS and application thereof.

Technical program of the present invention lies in from wheat separating obtaining wheat secondary metabolism flavonoid route of synthesis gene TaFLS, then this gene is forwarded in the farm crop such as wheat to realize that farm crop plant recycling time matter soil in salinization soil.

The anti-oxidant gene name of wheat salt tolerance provided by the invention is called wheat secondary metabolism flavonoid route of synthesis gene TaFLS, and the nucleotide sequence of said gene cDNA is shown in SEQ ID No.1.

The present invention also provides the plant expression vector pSTART-TaFLS that contains the anti-oxidant gene TaFLS of above-mentioned wheat salt tolerance.

The application of gene TaFLS according to the invention in cultivating anti-salt oxidation resistant plant.Wherein said plant optimization is common wheat or Arabidopis thaliana.

For the ease of transgenic plant or clone are screened; Can process the plant expression vector (pSTART) that contains said gene TaFLS; As the mark (GUS etc.) that can bring Selection In perhaps has the antibiotic marker thing (Totomycin, kantlex, qingfengmeisu qiong etc.) of resistance etc.

In fact, anyly can foreign gene be imported the carrier of expressing in the plant and can be applied to the present invention, the preferred carrier of the present invention is pSTART.

Gene of the present invention can be widely used in cultivates the anti-oxidant variety of crops of anti-salt.

Beneficial effect of the present invention: utilize existing plant gene engineering technology; The present invention clones first and has obtained wheat secondary metabolism flavonoid route of synthesis gene TaFLS; And change this gene over to Arabidopis thaliana, prove that through comparative analysis salt tolerant, the resistance of oxidation of transfer-gen plant obviously improve.

Description of drawings

The amplification of Fig. 1 TaFLS full length gene cDNA sequence

-: negative control; M: molecular weight marker; The arrow indication is the purpose fragment.

The RT-PCR that Fig. 2 salt stress goes down the hill to melt TaFLS and these other genes involveds of path in No. 3 (SR3) and Jinan 177 (JN177) root system analyzes.

Abduction delivering and the purifying target protein of Fig. 3 pET32a-TaFLS in intestinal bacteria DE3

1: intestinal bacteria are not induced through IPTG; 2: 16 ℃ of processes of intestinal bacteria IPTG induces 24h, and the ultrasonication cell is through the centrifuged deposit total protein; 3: supernatant behind the ultrasonication cell centrifugation; 4: the target protein that obtains behind the supernatant process HIS magnetic beads for purifying; M: protein molecular weight Marker; Arrow is represented albumen behind the purifying.

Fig. 4 plant expression vector pSTART-TaFLS makes up

The segmental high-fidelity enzymatic amplification of A:TaFLS, the arrow indication is the purpose fragment;

The linear pSTART plasmid band that B:pSTART double digestion XbaI and BamHI, arrow expressed enzyme cut;

C:pSTART-TaFLS expression vector double digestion XbaI+BamHI checking, the TaFLS band that the arrow expressed enzyme is cut;

The PCR of Fig. 5 pSTART-TaFLS transgenic line identifies.

The A:DNA level detection is extracted DNA from different transgenic arabidopsis strain systems (as 3,4,5,6,13,14,15,16,23), and with the TaFLS band of TaFLS primer amplified, arrow is represented amplified fragments;

The B:mRNA expression level is analyzed, and extracts RNA from different transgenic arabidopsis strain systems (as 3,4,5,6,13,14,15,16,23), and with the TaFLS band of TaFLS primer amplified, AtActin is Arabidopis thaliana Actin genetic expression;

The digitized representation transgenic line, pSTART empty carrier transfer-gen plant is changeed in the VC representative, and Col0 represents the wild Arabidopis thaliana of Col type ,-represent negative control, M to represent molecular weight marker.

Fig. 6 TaFLS vitro enzyme is lived and is analyzed

The HPLC of Fig. 6 A:TaFLS substrate DHQ and DHK and product Q and K standard substance analyzes;

Fig. 6 B: e. coli protein extract and the HPLC after DHQ and the DHK vitro reactions to transform the pET32a empty carrier analyze, and can only detect the substrate peak, product Q and K peak do not occur;

Fig. 6 C: analyze with the HPLC after conversion PET32a-TaFLS e. coli protein extract and DHQ and the DHK vitro reactions, can detect substrate DHQ, DHK and product Q, K peak, show that TaFLS can catalysis DHQ and DHK.

Fig. 7 H ₂O ₂Handle the phenotype analytical that system of transgenic arabidopsis strain down and contrast strain are

VC: the transgenic contrast strain system that changes the pSTART empty carrier over to;

OE1/OE2: two strain systems that change the pSTART-TaFLS gene over to;

A:1/2MS+0mM H ₂O ₂B:1/2MS+0.3mM H ₂O ₂C:1/2MS+0.5mM H ₂O ₂D:1/2MS+4mM H ₂O ₂E and F: transgenic line OE1/OE2 H ₂O ₂Handle the analyses of back main root length and lateral root number statistical; G:8mM H ₂O ₂Handle seed germination rate down.Data show that the TaFLS transgenic arabidopsis is at H ₂O ₂Energy for growth under coercing, germination rate all are superior to contrasting strain system.

Fig. 8 NaCl coerces down the phenotype analytical of transgenic arabidopsis strain system and contrast strain system

VC: the transgenic contrast strain system that changes the pSTART empty carrier over to;

OE1/OE2: the strain system that changes the pSTART-TaFLS gene over to;

A:1/2MS+0mM NaCl; B:1/2MS+100mM NaCl; C:1/2MS+150mM NaCl; D:1/2MS+200mM NaCl; E: transgenic line OE1/OE2 NaCl handles back main root length statistical study; Data show that the energy for growth of TaFLS transgenic arabidopsis under NaCl coerces is superior to contrasting strain system.

Embodiment

The clone of embodiment 1, TaFLS

1.1 extract wheat Total RNA

1. organization material is put into the mortar of liquid nitrogen precooling, abundant grind into powder in liquid nitrogen;

2. treat that the liquid nitrogen volatilization is dried, transfer to immediately in the centrifuge tube of 2ml that every 100mg material adds the TRIzol extracting solution of the Invitrogen company of 1ml approximately; After the thawing, inhale repeatedly with the application of sample rifle and to blow thermal agitation mixing sample; Make the abundant cracking of sample, room temperature was placed 5 minutes;

3. add 0.2ml chloroform (chloroform), thermal agitation mixing 15 seconds, room temperature was placed 10 minutes;

4.4 ℃, centrifugal 15 minutes of 12000rpm;

5. with the careful sucking-off of pipettor upper strata water, add in the centrifuge tube of new 1.5ml, add the Virahol (1: 1 volume) of 500 μ l, abundant mixing ,-20 ℃, deposition 30min or spend the night;

6.4 ℃, the centrifugal 10min of 12000rpm, careful abandoning supernatant;

7.RNA deposition is with 75% washing with alcohol of 1ml.4 ℃, the centrifugal 10min collecting precipitation of 8000rpm;

8. repeat with RNA deposition of 75% washing with alcohol;

9. remove supernatant, RNA is deposited in and dries about 10-15 minute on the aseptic technique platform, and it is transparent that RNA shows slightly, and the RNase-free water that adds proper volume (30-50 μ l) fully dissolves (can be placed on-80 ℃ of prolonged preservation);

10. ultraviolet spectrophotometer and 1%Agrose detected through gel electrophoresis RNA concentration and quality.

Annotate: a) with the output of UV spectrophotometer measuring RNA, the absorbancy at the 260nm place, 1OD=40ug/ml.According to light absorption value, detect the purity of RNA, the OD of pure rna at 260nm and 280nm place ₂₆₀/ OD ₂₈₀Ratio should be near 2.0 (ratio be preferably between 1.9～2.1).

B) with quality and the size of 1%Agrose gel electrophoresis inspection side RNA.Draw the RNase-free water that 1ul RNA adds 3 μ l, add 65 ℃ of sex change of 1 μ l sample-loading buffer 5 minutes.With EB dyeing, other gets the 1kb DNAMarker of 3 μ l as contrast behind the electrophoresis.

1.2 synthetic (the Affymetrix one-cycle cDNA Synthesis Kit) of the first chain cDNA

1.Poly-A the preparation of RNA controls (with Poly-A control dilution buffer dilution Poly-Acontrol stock), according to following dilution proportion:

2. reaction system

The reaction reagent volume

RNA sample (1ug/ul) 8 μ l

Diluted?poly-A?RNA?controls 2μl

T7-ol?igo(dT)Primer，50uM 2μl

RNase-free?water 0μl

TV 12 μ l

With mentioned reagent sample mixing gently, 70 ℃ of temperature were bathed 10 minutes, placed centrifugal a moment on ice at least 2 minutes;

3. prepare remaining ingredient according to following table during:

The reaction reagent volume

5×1st?Strand?Reaction?Mix 4μl

DTT(0.1M) 2μl

dNTP(10mM) 1μl

TV 7 μ l

Mixing, 42 ℃, temperature was bathed 2 minutes; 2uL (8～16ug is initial).

4. mixed solution is added and accomplished in 12 μ l RNA/T7-oligo (dT) solution of reaction, flick the tube wall mixing, slight centrifugal 50 seconds, add 1 μ l SuperScript II RT (200U/ μ l), making total reaction volume is 20 μ l.Put into 42 ℃ of incubations behind the mixing immediately 1 hour, 4 ℃ were cooled off 5 minutes.

1.3 synthetic (the Affymetrix one-cycle cDNA Synthesis Kit) of the second chain cDNA

1. 1st cDNA synthetic product is placed on ice, adds following reagent:

The reagent component volume

RNase-Free?water 91μl

5×2nd?Strand?Buffer 30μl

dNTP(10mM) 3μl

E.coli?DNA?Ligase(10U/ul) 1μl

E.coli?DNA?Polymerase(10U/ul)?4μl

E.coli?RNase?H(10U/ul) 1μl

TV 130 μ l

Mixing, reacted 2 hours by 16 ℃;

2. add 2 μ l T4DNA Polymerase, mixing, 16 ℃, 5 minutes;

3. add 10 μ l EDTA (0.5M), mixing, termination reaction.-20 ℃ of preservations.

1.4 the clone of ORFs and sequencing

1. primer sequence: according to sequencing result, design gene upstream and downstream primer, the ORFs of amplification gene.

2.PCR reaction system (50 μ L):

2×GC?buffer?I 10μl

Template cDNA 1ul

dNTPs(2.5mM?each) 0.5μl

Primer1(10μM) 1μl

Primer2(10μM) 1μl

LA?Taq(TaKaRa) 0.5ul

DdH ₂O adds to final volume 50 μ l

3.PCR response procedures is: 94 ℃ of preparatory sex change 5min; 94 ℃ of sex change 45sec, 55 ℃ of renaturation 45sec, 72 ℃ are extended 1min, circulate 35 times; 72 ℃ are extended 7min.

4. the recovery of amplified fragments, with the T carrier to be connected method for transformation the same, order-checking is accomplished by Shanghai Invirtron company.The result sees Fig. 1.

1.5 gene expression analysis (RT-PCR and real-time PCR)

A. coerce down the extraction of RNA

No. 3 (SR3) and Jinan 177 (JN177) normal seed germination are melted in the mountain, and (about 3 time-of-weeks) began to apply salt stress (200mM NaCl) when the Hangload nutrient solution was cultured to the about 10cm of plant height.Young tender root system is got in after processing 0,1,6,12,24 hour respectively, and the Trizol method is extracted RNA, and method is the same.

B. rt (RT) produces cDNA

Rt produces cDNA, and method is the same.

C.PCR reaction and electrophoresis

1. be template with cDNA, carry out the PCR reaction.Primer is following

TaAct-S：5’-GTTCCAATCTATGAGGGATACACGC-3’

TaAct-A：5’-GAACCTCCACTGAGAACAACATTACC-3’

2.PCR system:

ddH ₂O 13.5μl

10×Taq?buffer(Mg2+free) 2μl

MgCl ₂(25mM) 1.2μl

Primer1(10μM) 1μl

Primer2(10μM) 1μl

dNTP(10mM?each) 0.2μl

rTaq?polymerase(5U/μl) 0.1μl

Rt cDNA template 1 μ l

Total?Volume 20μl

3.PCR program:

95 ℃ of 5min, 95 ℃ of 30s of 25～30 cycles, 55 ℃ of 30s, 72 ℃ of 35s; 72 ℃ of 7min. confirm the cycle number of PCR, the add-on of adjustment cDNA template according to the amplification situation of confidential reference items Actin.4.1% agarose gel electrophoresis.

The result sees Fig. 2.

Embodiment 2, prokaryotic expression analysis

2.1 construction of prokaryotic expression vector

Used expression vector is Pet32a, and recipient bacterium is DE3.Select for use Hind III and EcoR I respectively pET32a to be carried out double digestion with the pMD18-T carrier that contains goal gene; Reclaim big fragment of carrier and goal gene small segment respectively; Connect back transformed into escherichia coli DH10B competent cell with the T4 dna ligase; Promptly obtain having the prokaryotic expression carrier of goal gene behind the evaluation recon, transformation receptor bacterium DE3 competence is used for protein expression then.

1.PET32a cut with the enzyme of pMD18-T

The plasmid of alkaline lysis method of extracting pET32a and pMD18-T is respectively got 10ul and is carried out HindIII and EcoR I double digestion.The plasmid process for extracting as above, plasmid double digestion system is following:

EcoRI 1μl

HindIII 1μl

10×K?buffer 1μl

Plasmid 300ng

DdH ₂O to 20 μ l

Cut more than 4 hours in 37 ℃ of thermostat water bath enzymes.

2. detection of the agarose gel electrophoresis of digested plasmid DNA and recovery method are the same.

3. gene is cut big segmental connection of the pET24a that obtains with enzyme

Ligation is formed: 10 * ligase enzyme damping fluid, 1 μ l

T4 dna ligase 1 μ l

Reclaim gene purpose segment 6 μ l

Reclaim product pET24a 2 μ l

Add dd H ₂O is to end reaction volume 10 μ l, 16 ℃ of connections spend the night (mol ratio that reclaims goal gene and pET24a is 4: 1).

4. transformed into escherichia coli, method is the same.

5. the evaluation of recon

1. the enzyme of plasmid is cut evaluation

Picking list bacterium colony is inoculated in 5ml respectively and contains 37 ℃ of concussion overnight cultures in the LB liquid nutrient medium of kan, and alkaline denaturation extracts plasmid, chooses suitable enzyme and carries out enzyme and cut, and 1% agarose gel electrophoresis detects the fragment that whether contains expection molecular weight size.

2. the PCR of plasmid checking

Carry out pcr amplification with gene specific primer, 1% agarose gel electrophoresis detects the fragment that whether contains expection molecular weight size.

6. transformation receptor bacterium DE3

The prokaryotic expression carrier thermal shock method transformed into escherichia coli DE3 competent cell that builds, screening is the same with authentication method.

2.2 the preparation of sample

Extracting solution of protein: 400mg SDS

100mg?DTT

2.5ml?0.5M?Tris-HCl(pH6.8)

2ml?glycerol

The 3mg bromjophenol blue

After the mixing, adding distil water is to 10ml.

1. will contain and express segmental positive colony and be inoculated in 5ml and contain in the LB liquid nutrient medium of kantlex 30 μ g/ml, 37 ℃ shaking culture 3-5 hour.Be about at 1.0 o'clock to OD600, sucking-off 1ml bacterium liquid gives over to control group.

2. the IPTG that adds 1M in remaining nutrient solution makes final concentration be respectively 0.6mM.Cultivate the expression that 30h induces inducible protein for 20 ℃.

3. do not induce with inductive bacterium liquid all to add in the 1.5ml centrifuge tube, the centrifugal 1min of 10000rpm collects thalline, abandons supernatant, adds 100 μ l protein extracts, vortex vibration mixing, and boiling water bath 10min, the centrifugal 15min of 13000rpm, supernatant is subsequent use.

2.3 SDS-PAGE electrophoresis

1. medicine preparation:

(1) 10 * electrode buffer: get Tris 30.3g, Glycine 144g, SDS 10g is settled to 1000ml after the adding distil water dissolving;

(2) 40% acrylic amide mother liquors (Acr): get 40g Acr, adding distil water dissolving back constant volume is to 100ml;

(3) 2% methylene diacrylamides (Bis); Get 2g Bis-Acr, adding distil water dissolving back constant volume is to 100ml;

(4) 3mol/L Tris-HCl (pH 8.8): get 36.3g Tris and dissolve in zero(ppm) water, be settled to 100ml, with the HCl accent pH to 8.8 of 1mol/L;

(5) 1mol/L Tris-HCl (pH 6.8): get 12.1g Tris and dissolve in zero(ppm) water, be settled to 100ml, with the HCl accent pH to 6.8 of 1mol/L;

(6) 10%SDS: take by weighing the dissolving of SDS powder 10g adding distil water and be settled to 100ml;

(7) 10%AP: take by weighing the dissolving of ammonium persulphate 1g adding distil water and be settled to 10ml;

(8) TEMED: available from Amersham company;

(9) fixed and stained liquid: get the 20ml Glacial acetic acid min. 99.5,80ml methyl alcohol adding distil water is settled to 200ml, takes by weighing 200mg Xylene Brilliant Cyanine G R-250 and is dissolved in the above-mentioned solution.

2.SDS-PAGE the preparation of gel

(1) after the sheet glass clean dry, with 1% agar shrouding.Sheet glass is put into (band ear offset plate outside) on the gum-making rack, fixes with clip, adds the agar of an amount of fusing downwards in the plastic channel of end closure, leaves standstill, and solidifies until agar.

(2) join glue and prepare 12% separation gel and 4% concentrated glue according to the ratio in the following table.

Polyacrylamide gel is formed 12% separation gel 26ml 4% and is concentrated glue 10ml

40％Acr 7.62ml 0.986ml

2％Bis 4.16ml 0.54ml

3M?Tris-HCl(pH?8.8) 3.28ml ----

1M?Tris-HCl(pH6.8) ---- 1.25ml

10％SDS 0.26ml 0.1ml

Zero(ppm) water 10.56ml 7.14ml

TEMED 10.4μl 10μl

10％APS 98μl 75μl

(3) pour separation gel into after, add one deck propyl carbinol at Jiao Mianshang, leave standstill, solidify until separation gel.

(4) be inverted offset plate, flow out propyl carbinol, with distilled water flushing glue face, exhaust unnecessary water with filter paper, preparation concentrates glue, pours into behind the mixing between the sheet glass, and comb is placed between sheet glass, notes not producing bubble, leaves standstill to gelling.

(5) offset plate is taken off from gum-making rack, remove the residual agar in clean offset plate bottom; Offset plate is fixed to (band ear offset plate is interior) on the electrophoresis chamber, upright electrophoresis chamber, clip is offset plate fixedly.

(6) extract comb, groove adds electrophoretic buffer under electrophoresis chamber, blows out the bubble that gets between the sheet glass.8 μ l sample liquid are clicked and entered in the point sample hole.Then electrophoresis chamber is put into 4 ℃ of chromatography cabinets, note wanting level, 10mA current stabilization electrophoresis to indicator to migrate to gel bottom continued electrophoresis 30 minutes, stop electrophoresis again.

(7) electrophoresis finishes, and takes off offset plate, opens sheet glass, exposes glue, cuts concentrated glue with blade, cuts away separation gel and marks for one jiao, dyes.

(8) dyeing and decolouring.Take off glue, put and swaying dyeing in the 200ml staining fluid and spend the night, transfer to then and sway decolouring 2-4 hour in the vinyl disc that zero(ppm) water is housed, change zero(ppm) water therebetween for several times, till clean to the background decolouring.

(9) saving result.Decolour the electrophoretogram of clean glue with gel imaging appearance system for photographing and analyze.

3. fusion rotein purifying and the detection of egg enzyme property

(1) contains Bacillus coli cells 37 ℃ of cultivations in containing the LB substratum of 50mg/L kantlex of recombinant plasmid.Until OD ₆₀₀Be about 0.6, add IPTG, making final concentration is 0.5mM, and 37 degree were cultivated 5-6 hour.Inducible protein is expressed.

(2) collect bacterial cell, and 40mL initial buffer liquid (the 20mM potassium phosphate buffer, pH7.4,0.5mMNaCl) resuspended.

(3) add 5mg N,O-Diacetylmuramidase room temperature 30min lysing cell.

(4) ice bath 20200W ultrasonic disruption cell 10s, each 10s at interval.Centrifugal 15min 4 degree of 12000rpm.

(5) with NiSO4 balance Hi-Trap (Amersham) pillar of 0.1mM.Method for purifying proteins is with reference to (chu, 2003) such as chu.The BSA method is measured protein content with reference to Bradford ' s (1976).

(6) protein product that obtains is used the 15%SDS-PAGE electrophoresis.The Xylene Brilliant Cyanine G R-250 observation of dyeing.The result sees Fig. 3.

The structure of embodiment 3, carrier and conversion Agrobacterium

3.1 the structure of 35S promoter plant expression vector

Plant expression vector pSTART is the binary vector that contains 35S promoter and NPT II gene, on its MCS, contains restriction enzyme XbaI and BamHI site.Use restriction enzyme XbaI and BamHI double digestion carrier pSTART and goal gene segment respectively.The carrier of complete degestion reclaims, links to each other with the cDNA fragment of double digestion then through glue after electrophoretic separation on 1% sepharose, makes up to obtain plant expression vector.Enzyme is cut system, and transformed into escherichia coli DH10B competence is identified recon.Connection, recovery, conversion and authentication method are the same.The result sees Fig. 4.

3.2 the competent preparation of Agrobacterium AGL1/EHA105

(1) goes up the single bacterium colony of picking agrobacterium tumefaciens from YEP dull and stereotyped (containing 50 μ g/ml Rifampins), be inoculated in the YEP liquid nutrient medium that contains 50 μ g/ml Rifampins 200rpm/min, 28 ℃ of overnight cultures.

(2) getting 2ml incubated overnight liquid is inoculated in 50ml and contains in the identical antibiotic YEP liquid nutrient medium and be cultured to OD under the same conditions ₆₀₀Reach 0.5.

(3) bacterium liquid ice bath 30min, 4 ℃, the centrifugal 10min of 5000rpm collects thalline.

(4) thalline is resuspended among the NaCl of 10ml 0.15mol/L of ice bath centrifugal collection thalline.

(5) resuspending is divided in bacterium liquid in the 1.5mlEppendorf pipe with 200 μ l/ pipe in the CaCl2 solution of 1ml 20mmol/L ice precooling, puts quick-frozen 1min in the liquid nitrogen, and-70 ℃ of preservations are subsequent use.

3.3 freeze-thaw method transforms agrobacterium tumefaciens AGL1/EHA105

(1) at room temperature melts the Agrobacterium competent cell, add 1 μ g expression vector DNA, ice bath 30min behind the mixing.

(2) put liquid nitrogen flash freezer 1min, move to 37 ℃ of insulation 3min rapidly.

(3) the YEP 800 μ l of adding antibiotic-free, 3hr are cultivated in 28 ℃ of concussions.

(4) the centrifugal 30s of 7000rpm collects thalline, is applied on the YEP flat board that contains 50 μ g/ml Rifampins, 50 μ g/ml Kan, is inverted dark the cultivation 2-3 days for 28 ℃.

3.4 thalline PCR identifies

Thalline PCR method and program are the same.

Embodiment 4, TaFLS vitro enzyme are lived and are analyzed

The reaction system (is example with substrate DHQ) 4.1 enzyme is lived

400 μ l reaction systems:

10* α-Tong Wuersuan (100mM) 40 μ l

10* ascorbic acid (100mM) 40 μ l

20* ferrous sulfate (5mM wherein contains the 200mM ascorbic acid) 20 μ l

10* glycocoll (500mM) 40 μ l

100*DHQ(10mM) 4μl

TaFLS protein 20 0 μ g

HEPES (50mM) complement is that volume is in 400 μ l

4.2 enzyme reaction condition: 25 ℃ of 90min

Reaction finishes the back and adds ETHYLE ACETATE termination reaction and extracting, and then 45 ℃ of vacuum are drained, and obtain the purpose product, with 80% chromatogram dissolve with methanol, advance the HPLC liquid phase analysis

4.3HPLC condition: high performance liquid chromatograph (Japanese shimadzu LC-ATvp)

Chromatographic column (Agela Venusil ASB C18)

A: B=45: 55 (V/V) (A:4.5% formic acid B:100% methyl alcohol)

Detect wavelength: 280nm

The result sees Fig. 5.

Embodiment 5, gene function checking-Arabidopis thaliana transform and screening

5.1 Arabidopis thaliana plantation

Seed is put into the EP pipe, soak 5min in 70% ethanol, then sanitising agent (20% drift ice (white cat, Shanghai), 0.1%Triton) washing 10-15min, aseptic water washing 4 times, 4 ℃ of vernalization 72h.In the seed that disinfects, add 0.5%agarose and (be cooled to 40 degree in order to avoid scald seed dead; Adding agarose is to scatter in order to help seed), be laid on the 1/2MS solid medium, dry up on the super clean bench; (can blow for a moment) in order to avoid on the petridish lid steam is arranged in the seed germination process more.Change in the phytotron and cultivate about a week, can transplant.With pack into the pot of suitable size of artificial soil; 70 degree oven dry (were killed worm's ovum etc., otherwise are understood snake) more than 2 hours, then pot were placed in the nutritive medium; It is fully absorbed water; To in the artificial soil that is full of nutritive medium, cover preservative film at 7-10 days seedling replanting of growth on the 1/2MS solid medium, and change in the phytotron and cultivate.Throw off preservative film after 1-2 days.Watered primary water (watering in iron pan under the POT) at a distance from several days.

5.2 Arabidopis thaliana transforms

(1) when Arabidopis thaliana (Colombia's wild-type) inflorescence forms, the inflorescence top is cut the generation of surveying inflorescence to induce.Before transforming material is irrigated nutritive medium.

(2) transform previous day, get 2ml activatory Agrobacterium AGL 1 and be added to and contain in the corresponding antibiotic 200ml YEP substratum, incubated overnight is to OD ₆₀₀=1.0-1.2.

(3) centrifugal collection thalline, and be resuspended in the dip-dyeing solution (5% sucrose, 0.04% Silwet L-77), make OD ₆₀₀=0.8.

(4) inflorescence was immersed dip-dyeing solution 30 seconds, the swing inflorescence makes and forms a skim on the inflorescence therebetween.

(5) cover inflorescence with preservative film, secretly cultivate after one day and throw off preservative film, cultivate as for 19-22 ℃ of culturing room.

(6) contaminated once with method again at a distance from 5-7 days.

(7) gather in the crops seed after about one month.

5.3 it is screening that Arabidopis thaliana transforms positive strain

(1) results T0 for seed disinfection after (75% ethanol 5min, detergent wash 10-15min, aseptic water washing 3-5 time), be laid on the MS screening culture medium that contains 50 μ g/mL Kan or 50 μ g/mL Hygo (method is the same).

(2) 4 ℃ of vernalization 48h move on to phytotron growth 7-10 days.The resistance seedling is moved on to continued growth in the soil.

(3) etc. the most petals of plant pod has as a result been tied up the plant individual plant with marline, so that individual plant receipts seed T1 is for seed.

(4) pcr amplification is a template with the genomic dna of transformation plant, uses gene specific primer.PCR reaction system and reaction conditions see before.

(5) the T1 seed treatment is the same, and selecting the resistance ratio in for plant at T2 is single independent strain system that inserts of 3: 1.5.4 the transgenic positive plant is identified

(1) the CTAB method is extracted genomic dna.

(2) PCR identifies positive plant.

Pcr amplification is a template with the genomic dna of transformation plant, and primer is used gene specific primer respectively.PCR reaction system and reaction conditions see before.

The result sees Fig. 6.

Embodiment 6, the degeneration-resistant phenotype analytical of Arabidopis thaliana transfer-gen plant

6.1 sprout experiment

The Arabidopis thaliana seed with 70% alcohol vibration sterilization 5min, is used the resuspended 1min of absolute ethyl alcohol then, is layered on the aseptic exsiccant filter paper, treat the seed-coat drying after, put the 1/2MS solid medium and (add H ₂O ₂) on the flat board, place 50 seeds for every kind, seal with sealing film, put into that 4 ℃ of refrigerators are dark to be cultivated 2 days, put into illumination box normal cultured 5d then, add up the open ratio of cotyledon.Experiment repetition 3 times.

6.2 root growth experiment

Seed behind the surface sterilization is taped against on the MS substratum, and 4 ℃ of dark 2d that cultivate change illumination box over to and cultivate 2-3d, again the plant that sprouts are put into the 1/2MS (NaCl/H that adds different concns ₂O ₂) vertically cultivate, to take pictures after 7 days, Image J software statistics root is long.Experiment repetition 3 times.

The result sees Fig. 7, Fig. 8.

Claims (2)

1. anti-oxidant gene TaFLS of wheat salt tolerance, it is characterized in that: the nucleotide sequence of said gene cDNA is shown in SEQ ID No.1.

2. the said gene TaFLS of claim 1 is cultivating anti-oxidant Arabidopis thaliana of anti-salt or Application in Wheat.

Images