CN102161996B - Jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants - Google Patents

Abstract

The invention relates to the technical field of genetic engineering, in particular relating to a jujube tree ascorbate peroxidase gene and application thereof in improving stress resistance of plants. The nucleotide sequence of the jujube tree ascorbate peroxidase gene and the amino acid sequence of the coding protein of the jujube tree ascorbate peroxidase gene are shown in sequence tables of SEQ ID NO:1 and SEQ ID NO:2. In the invention, the jujube tree ascorbate peroxidase gene is obtained to construct a plant expression vector successfully transferred into an arabidopsis strain, and the salt tolerance of the jujube tree ascorbate peroxidase gene is evaluated, thereby proving that the jujube tree ascorbate peroxidase gene can be used for improving the salt tolerance of the plants. In addition, a prokaryotic expression vector constructed by the invention can be used for realizing induction and expression in escherichia coli. According to the invention, the first-time separation of the jujube tree ZjAPX (Ascorbate Peroxidase) gene is realized, and the gene can be used as a target gene transferred to the plants so as to improve the stress resistance, especially the salt tolerance of the plants, and has guiding significance in improving varieties of the plants, thereby providing test data and laying a theoretical basis for sufficiently utilizing the excellent-resistance gene of jujube trees to improve the stress resistance of the plants.

Description

Jujube tree Ascorbate peroxidase gene and the application in improving stress resistance of plant thereof

Technical field

The present invention relates to gene engineering technology field, be specially a kind of jujube tree Ascorbate peroxidase gene and the application in improving stress resistance of plant thereof.

Background technology

Ascorbate peroxidase enzyme (ascorbate peroxidase, APX) claims again vitamin C peroxidase, is present in higher plant, Eukaryotic Algae and some cyanobacteria, also detects the APX activity in some insect.In higher plant, xitix (ascorbic acid, AsA)-gsh (glutathione, GSH) circulation has extremely important effect for Scavenger of ROS.Wherein ascorbate peroxidase enzyme (APX) is the key enzyme in AsA-GSH circulation.The maximum characteristics that APX participates in oxygen scavenging activity are that substrate A sA is had high specificity, and can play a role under the AsA of lower level.The functional study of Ascorbic Acid peroxidase shows that APX has played significant effect to the resistance that improves plant.Therefore, clone plant APX gene also strengthens the expression of this gene by transgenic method, for improving stress resistance of plant and the improvement plant variety has important practical significance.At present, the APX gene of existing various plants is cloned and is applied to improve the resistance of plant.

1976, Foyer and Hailiwell at first found a kind of peroxidase take AsA as electron donor.1980, Nakano and Asada determined that formally this enzyme is APX, is present in the matrix of chloroplast(id).At present, many investigators have carried out the molecular cloning of the various isozyme genes of APX in various plants, but focus mostly in herbaceous plant, as: spinach, pea, duckweed, cotton, cucumber, Semen Ricini, Sunflower Receptacle, tealeaves, wheat, corn, tobacco, summer squash etc.Because xylophyta genetic background is complicated, only the APX gene of the APX gene of apple and grape mildew-resistance is cloned so far.Yet, also there is not relevant report for the research of the APX gene of ancient, the important economic tree-jujube tree of China, up to the present, yet there are no the report of from jujube tree, isolating the APX gene and being applied to improve stress resistance of plant.

Jujube tree originates in China Huanghe valley, it is one of distinctive nonwood forest trees of China, jujube tree is cold-resistant, drought resisting, the ability of resisting the bad factor in the various external worlds is strong, and especially barren for soil, drought has very strong endurance, in Loess Plateau such as northwest, Shanxi, Taihang Mountain, northerns Shensi, jujube tree generally is grown under the poor environments such as soil is quite barren, the hillside, hills of arid short of rain, Gao Ya limit, and can both the normal growth result.This extremely drought-enduring, barren-resistant characteristics significantly are better than other farm crop, therefore the resistance gene of jujube tree is very valuable genetic resources, understand the degeneration-resistant mechanism of the jujube tree with good resistance and molecular structure, expression, function and the regulation and control of adverse circumstance genes involved, can be the resistance that takes full advantage of these good resistant genes raising plants certain experimental data and based theoretical is provided.

Summary of the invention

The object of the present invention is to provide a kind of jujube tree Ascorbate peroxidase gene and the application in improving stress resistance of plant thereof.

The present invention adopts following technical scheme to realize: a kind of jujube tree Ascorbate peroxidase gene, its nucleotide sequence is shown in sequence table SEQ ID NO:1.

By the albumen of described a kind of jujube tree Ascorbate peroxidase gene coding, its aminoacid sequence is shown in sequence table SEQ ID NO:2.

The application of a kind of jujube tree Ascorbate peroxidase gene in improving stress resistance of plant.

The present invention is from the fruitful branch (being that jujube hangs) of tall bottle with spout jujube tree (Zizyphus jujuba Mill. HUPING), utilize the CTAB method to extract total RNA, method separation and purification mRNA from the total RNA that extracts according to the mRNA purification kit, the construction cDNA library, analyze and compare by NCBI Blast after the order-checking, the complete sequence that therefrom to screen a cDNA sequence be the jujube tree Ascorbate peroxidase gene is with its called after ZjAPX(Zizyphus jujube Mill. ascorbate peroxidase).Bioinformatic analysis shows ZjAPXThe cDNA sequence total length of gene is 753bp, and 251 amino acid of encoding wherein comprise 32 acidic amino acids, and 20 basic aminoacidss, the relative molecular weight of protein are 62.55KD, and theoretical iso-electric point is 5.10.

According to the nucleotide sequence of jujube tree Ascorbate peroxidase gene, the design synthetic primer, the sequence of its upstream primer APX1 is: 5'-T GAATTCAGATGGGGAAGTGCTAC-3' comprises EcoRI restriction enzyme site (being the underscore part) is protected base T and AG for three, and jujube tree ascorbate peroxidase enzyme ZjAPXThe initiator codon ATG(of gene is part shown in the square frame); The sequence of downstream primer APX2 is: 5'-AT CCCGGGTAGCATCAGCAAATC-3' comprises SmaI restriction enzyme site (being the underscore part) is protected base AT and T for three.Go out by round pcr success subclone ZjAPXThe purpose fragment of gene is with what obtain ZjAPXThe goal gene fragment is through restriction endonuclease EcoRI and SmaI is connected on plant expression vector pEZR (K)-LNY after modifying, and the plant expression vector pEZR (K) that the dull and stereotyped resistance screening of the LB through containing kantlex, PCR identify, enzyme is cut evaluation, the order-checking proof is carried goal gene-LNY-ZjAPX successfully constructs.To carry the plant expression vector pEZR (K) of goal gene-LNY-ZjAPX by freeze-thaw method and change in the competent cell of agrobacterium tumefaciens lba4404, the dull and stereotyped resistance screening of the YEB through containing kantlex, PCR assay certificate engineering bacteria pEZR (K)-LNY-ZjAPX-L successfully construct.Agriculture bacillus mediated ZjAPXThe gene transformation Arabidopis thaliana, 1/2 MS Screening of Media through containing kantlex obtains to contain goal gene ZjAPXTransgenic arabidopsis plant T ₀In generation, identify that through PCR goal gene confirms ZjAPXSuccessfully change Arabidopis thaliana over to.With the 1/2MS medium treatment transgenic arabidopsis that contains 0.1 M NaCl, to transfer-gen plant and T thereof ₁In generation, carried out Evaluation of Salt Tolerance, observes its upgrowth situation, find transgenic arabidopsis under salt stress is processed growing way and root system significantly better than wild Arabidopis thaliana.

In addition, in order to further investigate the jujube tree Ascorbate peroxidase gene ZjAPXAnd proteins encoded ZjAPX, the present invention has made up recombinant prokaryotic expression vector pGEX-4T-2-ZjAPX, realize its prokaryotic expression in e. coli bl21 (DE3), for the aspect researchs such as the structure of this gene coded protein, function, regulation and control provide certain experimental data.

Beneficial effect of the present invention is: isolate first the jujube tree Ascorbate peroxidase gene ZjAPX, this gene can be used as goal gene and changes plant over to, to improve the resistance, particularly salt tolerance of plant, has important practical significance for the improvement plant variety.Further investigation for the degeneration-resistant mechanism of the jujube tree with good resistance, help molecular structure, expression, function and the regulation and control of clear and definite adverse circumstance genes involved, provide certain experimental data and based theoretical for further taking full advantage of the resistance that these good resistant genes improve plant.

Description of drawings

Fig. 1 is ZjAPXWith known APXThe amino acid systematic evolution tree cluster analysis of genoid;

Fig. 2 is goal gene ZjAPXPcr amplification figure as a result;

Fig. 3 is that the PCR of recombinant plasmid pEZR (K)-LNY-ZjAPX identifies collection of illustrative plates;

Fig. 4 is that the PCR of engineering bacteria pEZR (K)-LNY-ZjAPX-L identifies collection of illustrative plates;

Fig. 5 is for turning ZjAPXThat resistance screening of the card of gene Arabidopis thaliana is dull and stereotyped;

Fig. 6 is for turning ZjAPXGene Arabidopis thaliana plant;

Fig. 7 is for turning ZjAPXThe PCR detected result figure of gene Arabidopis thaliana plant;

Fig. 8 is transgenic arabidopsis and the wild-type Arabidopis thaliana of processing through 0.1 M NaCl 30 days;

Fig. 9 is transgenic arabidopsis and the wild-type Arabidopis thaliana of processing through 0.1 M NaCl 40 days;

Figure 10 is that the PCR of prokaryotic expression carrier pGEX-4T-2-ZjAPX identifies collection of illustrative plates;

Figure 11 is prokaryotic expression carrier pGEX-4T-2-ZjAPX's BamHI and SmaThe I double digestion is identified collection of illustrative plates;

Figure 12 is pGEX-4T-2-ZjAPX-B SDS-PAGE electrophoresis result figure after IPTG induces.

Wherein, the M among Fig. 2,3,4,7,10,11 is DL 2000 Marker, and band is followed successively by 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp.

Embodiment

Below in conjunction with embodiment, the invention will be further elaborated.Experimental technique among the following embodiment if no special instructions, is ordinary method.

Embodiment 1: the jujube tree Ascorbate peroxidase gene ZjAPXAcquisition

Gather the fruitful branch (being that jujube hangs) of tall bottle with spout jujube tree (Ziziphus jujuba Mill. HUPING) the about 2mm of length spring, utilize the CTAB method to extract total RNA, according to the mRNA purification kit (available from U.S. Promega, article No.: Z5200) method separation and purification mRNA from the total RNA that extracts, utilize cDNA library make up test kit (available from American I nvitrogen, article No.: 18248-039) construction cDNA library.Library cDNA is connected with cloning vector pSPORT1, connects product and be converted into E.coliDH5 α competent cell, coat contain amicillin resistance the LB flat board in 37 ℃ of overnight incubation, blue hickie screening recombinant plasmid, random choose some positive plasmid will be cut the plasmid that assay certificate has Insert Fragment through enzyme, entrust Shanghai to give birth to the worker and check order.

Utilize NCBI (www.ncbi.nlm.nih.gov/) on-line analysis software to carry out the Blast compare of analysis sequencing result, the result shows that one of them cDNA sequence is the ascorbate peroxidase enzyme homologous gene complete sequence of jujube tree, with its called after ZjAPX( ZIzyphus jUjube Mill aScorbate pEro xIdase ).Bioinformatic analysis is the result show: ZjAPXThe cDNA sequence total length of gene is 753bp, and 251 amino acid of encoding wherein include 32 acidic amino acids, 20 basic aminoacidss; The relative molecular weight that calculates protein with ProtParam is 62.55 KD, and theoretical iso-electric point is 5.10, and instability index is 57.06, belongs to labile protein; The cross-film of carrying out protein sequence with TMHM-M-2.0 is distinguished to analyse and is shown that this albumen is not transmembrane protein, belong to the outer albumen of film, be connected to the conservative analysis that http://www.expasy.org/prosite carries out encoding sequence, the protein that shows this sequence encoding belongs to typical plant heme peroxiredoxins albumen, comprises a Peroxidase activity position " APimLRLaWHSA " and a peroxidase heme part signal " DIVALSGGHTL " in the sequence; Show by carrying out sequence homology analysis in ncbi database Blast X search, this proteins encoded has high homology with the ascorbate peroxidase enzyme APX of at present known other plant, with the homology of cocoa tree APX, upland cotton APX be 93%, with the homology of corn APX be 92%, with the homology of Arabidopis thaliana APX be 86%; With DNAStar its aminoacid sequence and other are belonged to the ascorbate peroxidase enzyme APX structure evolutionary tree of planting, as shown in Figure 1, the APX quilt of 11 source same species is poly-to be 7 monoids, jujube (Ziziphus jUjube), Arabidopis thaliana (Arabidopsis thaliana) is poly-separately to be a class, oranges and tangerines (Citrus maxima), sweet potato (Ipomoea batatas) is poly-to be a class, tobacco (Nicotiana tabacum), Solanum (Solanum lycopersicum) is poly-to be a class, hevea (Hevea brasiliensis), dragon spruce (Picea sitchensis) is poly-to be a class, spinach (Spinacia oleracea), corn (Zea mays) is poly-to be a class, poly-with upland cotton (Gossypium hirsutum) again simultaneously is a large class, this cluster analysis result shows that also the sibship of jujube ascorbate peroxidase enzyme APX and other species is all far away, but its protein similar is high.Above-mentioned bioinformatic analysis comprises that nucleotide sequence analysis, amino acid sequence analysis, sequence similarity analysis, evolutionary tree cluster analysis are routine techniques means well known to those skilled in the art, clone the sequence that the nucleotide sequence that obtains is the jujube tree Ascorbate peroxidase gene by multiple compare of analysis proved invention, the aminoacid sequence of its coding is the sequence of its proteins encoded jujube tree ascorbate peroxidase enzyme, and this protein function is determined by unique.

According to the accurate nucleotide sequence design primer of the jujube tree Ascorbate peroxidase gene of above-mentioned acquisition and to entrust Shanghai to give birth to the worker synthetic, be used for the subclone of APX gene.The sequence of upstream primer APX1 is: 5'-T GAATTCAGATGGGGAAGTGCTAC-3' comprises EcoRI restriction enzyme site (being the underscore part) is protected base T and AG for three, and jujube tree ascorbate peroxidase enzyme ZjAPXThe initiator codon ATG(of gene is part shown in the square frame); The sequence of downstream primer APX2 is: 5'-AT CCCGGGTAGCATCAGCAAATC-3' comprises SmaI restriction enzyme site (being the underscore part) is protected base AT and T for three.

Carry with cloning vector pSPORT1-ZjAPX ZjAPXBe template, take APX1 and APX2 as primer, amplification obtains to contain the jujube tree Ascorbate peroxidase gene of complete open reading frame.The PCR reaction system is: 10 * PCR buffer(contains Mg ²⁺) 5 μ l, dNTP 0.5 μ l, each 2.5 μ l of upstream and downstream primer (20pmol/L), rTaq polysaccharase 0.8 μ l, template DNA (5.2 μ g/ μ l) 2 μ l, sterilization ultrapure water 36.7 μ l, cumulative volume 50 μ l.The pcr amplification condition is: 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ extend 2min totally 40 circulations, last 72 ℃ are extended 5min.Get 5ml PCR product, 1% agarose gel electrophoresis analysis confirmation purpose clip size.According to the operation of DNA purification kit, purified pcr product.

Embodiment 2: ZjAPXThe structure of gene plant expression vector

1. experiment material

1.1 carrier and bacterial strain

Plant expression vector pEZR (K)-LNY, intestinal bacteria ( E.coli) DH5 α, carry ZjAPXThe recombinant plasmid pSPORT1-ZjAPX of gene preserves by Shanxi Province Academy of Agricultural Sciences biotech research center vegetable cell and fetology research department.

1.2 reagent and substratum

Restriction enzyme EcoRI, SmaI, HindIII, BamHI, DNA Ligation Kit, DL2000 DNA Marker, sepharose DNA Fragment Purification test kit are all available from precious biotechnology (Dalian) company limited.

Kantlex (100mg/ml)

Phenol: chloroform: primary isoamyl alcohol (V: V: V=25: 24: 1)

Glucose solution (1M)

EDTA(0.5M, pH8.0)

10%SDS(pH7.2)

NaOH(2M)

Tris-HCl(1M, pH8.0)

Sodium-acetate (3M, pH5.2)

TE solution (10mM Tris-HCl, 1mM EDTA, pH8.0)

10 * TBE electrophoretic buffer:

Boric acid 55.2g/L

Tris 108g/L

EDTA(0.5 M, pH8.0) 40ml/L

LB substratum: (solid medium adds agar 15g/L)

Peptone 10g/L

Yeast extract 5g/L

NaCl 10g/L

2. experimental technique

2.1 intestinal bacteria ( E.coli) making of DH5 α competent cell

Use CaCl ₂The standby intestinal bacteria of legal system ( E.coli) DH5 α competent cell, concrete grammar is seen fine works molecular biology experiment guide.

2.2 a small amount of of plant expression vector pEZR (K)-LNY and recombinant plasmid pSPORT1-ZjAPX preparation

The pSPORT1-ZjAPX plasmid E.coliDH5 α bacterium liquid adds 1 μ l penbritin (100mg/ml) with the LB liquid nutrient medium of 2ml and connects 37 ℃ of incubated overnight gained behind the bacterium.PEZR (K)-LNY plasmid E.coliDH5 α bacterium liquid adds 1 μ l kantlex (100mg/ml) with the LB liquid nutrient medium of 2ml and connects 37 ℃ of incubated overnight gained behind the bacterium.

Alkaline lysis rapid extraction plasmid pEZR (K)-LNY and pSPORT1-ZjAPX, concrete steps are seen fine works molecular biology experiment guide.

The plasmid of drying is dissolved among the 20 μ l TE, and-20 ℃ save backup.

2.3 goal gene ZjAPXThe amplification of sequence

Take cloning vector pSPORT1-ZjAPX as template, carry out pcr amplification to obtain to contain the jujube tree Ascorbate peroxidase gene of complete open reading frame with the Auele Specific Primer APX1 that designs and APX2.At first utilize the grads PCR technology that different annealing temperatures is set, finding out optimum annealing temperature is 54 ℃, then makes pcr amplification according to following condition.

Pcr amplification ZjAPXGene, reaction system is: 10 * PCR buffer, 5 μ l(contain Mg ²⁺), dNTP 0.5 μ l, each 2.5 μ l of upstream and downstream primer (20pmol/L), rTaq polysaccharase 0.8 μ l, template DNA (5.2 μ g/ μ l) 2 μ l, sterilization ultrapure water 36.7 μ l, cumulative volume 50 μ l; Amplification condition is: 95 ℃ of denaturation 5min, 94 ℃ of sex change 1min, 54 ℃ of annealing 1min, 72 ℃ extend 2min totally 40 circulations, last 72 ℃ are extended 5min.

2.4 goal gene ZjAPXThe preparation of fragment and carrier pEZR (K)-LNY fragment

The goal gene that pcr amplification obtains ZjAPXThe multiple clone site of fragment two ends and carrier pEZR (K)-LNY all has EcoRI and SmaThe I restriction enzyme site utilizes EcoRI and SmaI is the double digestion goal gene respectively ZjAPXPCR product and carrier pEZR (K)-LNY, make it have identical sticky end, ready for connecting.

Goal gene ZjAPXThe endonuclease reaction system of PCR product:

10×T buffer 5μl

0.1% BSA 5μl

EcoRⅠ 1μl

SmaⅠ 1μl

PCR product 10 μ l

S.D.W 28μl

Cumulative volume 50 μ l

The volume of PCR product is decided according to its concentration, and minimum enzyme is cut the goal gene fragment of 1～2 μ g, and adding ultrapure water to cumulative volume is 50 μ l.

The endonuclease reaction system of carrier pEZR (K)-LNY:

10×T buffer 5μl

0.1% BSA 5μl

EcoRⅠ 1μl

SmaⅠ 1μl

Carrier 5 μ l

S.D.W 33μl

Cumulative volume 50 μ l

Enzyme is cut the rear 5ml double digestion product of getting respectively, and 1.0% agarose gel electrophoresis is analyzed, and confirms that clip size and expection match.

Operate goal gene fragment and the carrier segments of purifying behind double digestion according to the explanation of sepharose DNA Fragment Purification test kit.

2.5 enzyme is cut the connection of product

With the pEZR (K) of T4 dna ligase after with purifying-LNY carrier with ZjAPXGene fragment connects.Dna fragmentation and goal gene with carrier pEZR (K)-LNY ZjAPXDna fragmentation to be mixed with into volume be dna solution (mole ratio of carrier DNA and goal gene DNA is generally 1:5) about 5 μ l, add the Solution I among isopyknic DNA Ligation Kit in the above-mentioned dna solution, fully mixing.Ligation system cumulative volume is about 10 μ l, and 16 ℃ of reactions are spent the night.

2.6 connect the conversion of product

Join in the bacillus coli DH 5 alpha competent cell of 100 μ l connecting product, mixing is placed 30min on ice gently.Put into the circulator bath heat-shocked 90s of pre-heating to 42 ℃, fast the EP pipe is transferred in the ice bath, make cell cooling 3～5min.The LB substratum that adds 400 μ l is transferred to the EP pipe on 37 ℃ the shaking table, and temperature is bathed 40min and made bacteria resuscitation, and Resuscitation Period should leniently shake cell (rotating speed is lower than 225r/min).Get the transformed bacteria liquid of proper volume and transfer to the LB flat board that contains kantlex, smoothen.Flat board is placed room temperature until liquid is absorbed, be inverted flat board, 37 ℃ leave standstill overnight incubation.

2.7 the evaluation of converted product

(1) PCR identifies

Bacterium to be transformed transforms in single bacterium colony to 50 μ l ultrapure water with aseptic toothpick picking after the LB flat board that contains kalamycin resistance grows bacterium colony, and boiling water bath 5min makes the bacterium cracking.Get 1 μ l split product and carry out the PCR positive identification as template, its reaction system is: 10 * PCR buffer, 1.5 μ l(contain Mg ²⁺), dNTP 0.2 μ l, each 0.5 μ l of upstream and downstream primer (20pmol/L), rTaq polysaccharase 0.1 μ l, template DNA (2.6 μ g/ μ l) 1 μ l, add sterilization ultrapure water 11.2 μ l to cumulative volume be 1 μ l; Amplification condition is the same.The transformed bacteria that PCR is accredited as the positive is streak culture.

(2) enzyme is cut evaluation

After PCR being accredited as the transformed bacteria usefulness alkali cracking subtraction extraction plasmid DNA of the positive, use HindIII and BamHI is carried out double digestion to recombinant plasmid pEZR (K)-LNY-ZjAPX and is identified.

Recombinant plasmid pEZR (K)-LNY-ZjAPX's HindIII and BamHI double digestion reaction system:

10×K buffer 1μl

Hind III 0.5μl

BamHⅠ 0.5μl

Recombinant plasmid 1 μ l

S.D.W 7μl

Cumulative volume 10 μ l

Agarose gel electrophoresis endonuclease reaction liquid, the size of checking endonuclease bamhi is preserved and to be carried the positive bacterium colony of purpose fragment, and positive strain is sent to Shanghai gives birth to worker's evaluation of checking order.

3.1 ZjAPXThe amplification of cDNA

Utilize specificity upstream and downstream primer APX1 and the APX2 of design to carry out pcr amplification, product is electrophoresis in 1.0% sepharose, after EB dyeing, under ultraviolet lamp, observe the band of a treaty 750bp, as shown in Figure 2, coincide with designed amplification purpose clip size.M:DL2000 DNA Marker among the figure; The 1:PCR product.

The preparation of goal gene fragment and carrier segments

Goal gene jujube tree PCR product and pEZR (K)-LNY plasmid are used respectively EcoRI and SmaI is carried out double digestion, and enzyme is cut product through agarose gel electrophoresis, and purifying goal gene fragment and carrier segments are used for connecting.

The evaluation of recombinant plasmid

The positive single bacterium colony of picking carries out PCR and identifies from the LB flat board that contains kantlex, as shown in Figure 3, and M:DL2000 DNA Marker among the figure; 1,2,3,4: positive colony PCR product, size is about 750bp, conforms to expected results, tentatively infers the construction of recombinant plasmid success, the size that agarose gel electrophoresis detects recombinant plasmid is about 12.4kb.Sequencing result shows that the plasmid of positive transformed bacteria contains the purpose fragment really, and reading frame is correct, and recombinant plasmid pEZR (K)-LNY-ZjAPX successfully constructs.

Embodiment 3: agriculture bacillus mediated ZjAPXThe gene transformation Arabidopis thaliana

1. experiment material

1.1 vegetable material

Wild-type Arabidopis thaliana seed is provided by Shanxi Province Academy of Agricultural Sciences biotech research center vegetable cell and fetology research department, and culture medium is available from garden crop institute of Shanxi Province Academy of Agricultural Sciences.

1.2 carrier and bacterial strain

Plant expression vector pEZR (K)-LNY-ZjAPX, agrobacterium tumefaciens lba4404 are preserved by Shanxi Province Academy of Agricultural Sciences biotech research center vegetable cell and fetology research department.

1.3 reagent and substratum

Kantlex is given birth to the worker available from Shanghai, and agents useful for same is all available from the huge chemical industry in Tianjin in YEB, the 1/2MS substratum.

The YEB substratum:

Peptone 5g/L, extractum carnis 5g/L, yeast extract paste 1g/L, sucrose 5g/L, sal epsom 0.5g/L.Annotate: it is 1.0% agar powder that solid medium need add final concentration.

1/2 MS substratum:

Method according to preparation MS substratum is prepared 1/2 MS substratum, and the amount of macroelement, trace element, organic element, molysite respectively reduces by half, sucrose 30g/L, agar powder 6g/L.

2. experimental technique

2.1 the cultivation of Arabidopis thaliana

The Arabidopis thaliana seed is first at 95% alcohol immersion, 30～60s; Change again 2.65% clorox sterilization 5min over to, turn upside down therebetween and wash, the centrifugal 2min of 5000rpm; Sterilization washing three times; Then seed is suspended in 0.1% the agar powder colloidal sol; Draw suspension with the rifle head Seed Points is sowed on the 1/2 MS solid medium, sealing; Behind 4 ℃ of dark vernalization 2d, the Arabidopis thaliana seed at 22 ℃, is cultivated under the 16h/8h photoperiod condition; When Arabidopis thaliana grows 2 true leaves, be transplanted to and continue in the nutraceutical matrix to cultivate.

2.2 the making of agrobacterium tumefaciens lba4404 competent cell

The concrete steps of preparation agrobacterium tumefaciens lba4404 competent cell are seen fine works molecular biology experiment guide.

2.3 structure and the screening of engineering bacteria pEZR (K)-LNY-ZjAPX-L

Change the recombinant plasmid vector pEZR (K) that successfully constructs-LNY-ZjAPX over to Agrobacterium LBA4404 by freeze-thaw method, then extract the plasmid DNA of positive colony, and identify by PCR whether recombinant plasmid vector changes Agrobacterium over to.

The well-grown some single bacterium colonies of random choose on the screening flat board of recombinant plasmid pEZR (K)-LNY-ZjAPX are arranged from the conversion of 28 ℃ of cultivations, respectively get in 1/2nd bacterium colony to the 50 μ l ultrapure waters, boiling water bath 5min makes the bacterium cracking, gets 1 μ l and carries out the PCR evaluation as template.With identify correct positive engineering bacteria again streak culture preservation with for subsequent use.

The cultivation of engineering bacteria pEZR (K)-LNY-ZjAPX-L

Select and identify correct positive colony in 10ml YEB liquid nutrient medium (containing the kantlex that final concentration is 50 μ g/ml) incubated overnight, the centrifugal 5min of 6000rpm collects thalline, with substratum suspension thalline to OD ₆₀₀Value is about 0.8, is used for the arabidopsis thaliana transformation plant.

2.5 Agrobacterium is infected Arabidopis thaliana

When Arabidopis thaliana Plantlet formation bud, namely can be used for Agrobacterium-mediated Transformation.The conversion medium that will contain Agrobacterium is poured centrifuge tube into, the bud of Arabidopis thaliana plant is partly immersed conversion medium 3～5 s after, the plant of contaminating is put in the vinyl disc, the dark place is placed and is spent the night.Under normal operation, the Arabidopis thaliana plant after transforming is cultivated in continuation.When the angle of Arabidopis thaliana during withered and yellow, the wish cracking of fruit, the results seed, this is T ₀For seed.

2.6 turn ZjAPXThe screening of gene Arabidopis thaliana

The T of transgenic arabidopsis plant with sterilization ₀In 1/2 MS screening culture medium (containing the kantlex that final concentration is 50 μ g/ml), 4 ℃ of dark vernalization 2d are placed on 22 ℃, cultivate under the 16h/8h photoperiod condition for planting seed.It is normal to transform the longer growth of seedling of successful Arabidopis thaliana seed, unconverted successful seedling leaf jaundice, poor growth.When the normal Arabidopis thaliana of growth when growing 2 true leaves, be transplanted to and continue in the culture medium to be cultured to that the angle fruit is withered and yellow, the wish cracking, collect T ₁For seed.

2.7 turn ZjAPXThe Stress treatment of gene Arabidopis thaliana

With T ₁For the sowing of the Arabidopis thaliana of seed, be transplanted in the matrix, cultivate under the photoperiod at 16h/8h, and water in good time, be cultured to that the angle fruit is withered and yellow, the wish cracking, collect T ₂For seed; And take T ₁Blade extracts DNA, detects with PCR method.With wild-type and T ₂Sow simultaneously containing on the 1/2 MS substratum of 0.1 M NaCl for seed, observe its growing state.

3. experimental result

3.1 the conversion results of Agrobacterium

Change the plant expression vector pEZR (K) that builds-LNY-ZjAPX over to Agrobacterium LBA4404, at YEB solid medium (containing the kantlex that final concentration is 50 μ g/ml) screening transformant, from screening the dull and stereotyped upper well-grown some single bacterium colonies of random choose, carrying out PCR detects, as shown in Figure 4, identical with expected results, show that the recombinant plant expression vector successfully changes Agrobacterium LBA4404 over to.

Turn ZjAPXAcquisition and the Resistance Identification of gene Arabidopis thaliana plant

After infecting Arabidopis thaliana as stated above, with the T of transgenic arabidopsis of sterilization ₀, at 22 ℃, cultivate under the 16h/8h photoperiod behind 4 ℃ of dark vernalization 2d in 1/2 MS screening culture medium (containing the kantlex that final concentration is 50 μ g/ml) for planting seed.Observe and find, transform successful Arabidopsis thaliana Seedlings and have the characteristic of anti-kantlex, growth is normal, and has true leaf to form; And unconverted successful Arabidopsis thaliana Seedlings does not have anti-kantlex, and plant is short and small, and the leaf jaundice does not have true leaf to form, as shown in Figure 5 yet.

The transgenic arabidopsis plant that will have resistance moves into culture medium, cultivates under the photoperiod at 16h/8h, and its upgrowth situation is good, as shown in Figure 6, is cultured to that plant angle fruit is withered and yellow, during the wish cracking, gathers in the crops its seed.

The PCR detected result proves to be for 2,3,4,6, No. 8 to turn as shown in Figure 7 ZjAPXStrain.

3.3 turn ZjAPXThe salt tolerance experiment of gene Arabidopis thaliana plant

Choose the transgenic arabidopsis seed and wild-type Arabidopis thaliana seed carries out the NaCl Stress treatment in contrast, treatment condition are: NaCl is treated to and adds 0.1M NaCl in the 1/2MS substratum, and control treatment is the 1/2MS substratum, observes its upgrowth situation.Experimental result shows that 30 days transgenic arabidopsis of salt stress processing is all good than wild-type Arabidopis thaliana growing way, shows obvious salt tolerance, as shown in Figure 8; Process after 40 days, the plant of transgenic arabidopsis is obviously healthy and strong than wild-type plant strain growth, well developed root system, as shown in Figure 9.

Embodiment 4: ZjAPXProkaryotic expression

1. experiment material

1.1 carrier and bacterial strain

The tall bottle with spout jujube that jujube tree Ascorbate peroxidase gene cDNA sequence makes up from this seminar ( Ziziphus jujubaMill. HUPING) screening obtains in the fruitful branch cDNA library, is cloned on the pSPORT1 carrier; Cloning vector pSPORT1, prokaryotic expression carrier pGEX-4T-2, e. coli bl21 (DE3) are preserved by Shanxi Province Academy of Agricultural Sciences biotech research center vegetable cell and fetology research department.

1.2 reagent and instrument

Sepharose DNA reclaims test kit QIAquick available from QIAGEN company; Restriction enzyme BamHI, SmaI, T4 dna ligase, DNA Marker are all available from precious biotechnology (Dalian) company limited; Kantlex is available from Shanghai Sangon Biological Engineering Technology And Service Co., Ltd; The chemical reagent such as IPTG, acrylamide is available from Shanghai biotechnology Engineering Co., Ltd; The testing installations such as electrophoresis apparatus, constant-temperature metal bath, constant incubator, photographic camera provide by Shanxi Province Academy of Agricultural Sciences biotech research center vegetable cell and fetology research department.

2. experimental technique

2.1 the design of PCR primer is with synthetic

According to jujube tree ascorbate peroxidase enzyme cDNA sequence, select wherein to encode 216 amino acid whose 681bp nucleotide sequences, the design Auele Specific Primer, upstream primer K1 adds BamHThe I restriction enzyme site, downstream primer K2 adds SmaThe I restriction enzyme site, primer sequence is as follows:

K1：5'-AT GGATCCATGCTACGCCTTGCATG-3'；

K2：5'-AT CCCGGGTTAAGCATCAGCAAATC-3'；

Above primer is synthetic by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd.

2.2 the amplification of goal gene sequence

Carry the purpose fragment take cloning vector pSPORT1() be template, carry out pcr amplification with the Auele Specific Primer K1 and the K2 that design.At first utilize the grads PCR technology that different temperature is set, finding out optimum annealing temperature is 58 ℃.The PCR reaction system is: 10 * PCR buffer, 5 μ l(contain Mg ²⁺), dNTP 1 μ l, upstream and downstream primer K1 and K2(20 pmol/L) each 1 μ l, rTaq polysaccharase 0.3 μ l, template DNA (5.2 μ g/ μ l) 1 μ l, sterilization ultrapure water 40.7 μ l, cumulative volume 50 μ l; Amplification condition is: 95 ℃ of denaturation 5min, 94 ℃ of sex change 30s, 58 ℃ of annealing 30s, 72 ℃ extend 1min totally 30 circulations, last 72 ℃ are extended 10min.

2.3 the structure of recombinant prokaryotic expression vector

After obtaining the PCR product, through agarose electrophoresis checking clip size, then use BamHI and SmaI double digestion goal gene fragment and pGEX-4T-2 carrier carry out purifying with QIAquick gel recycling box to the double digestion product again.Under the effect of T4 dna ligase, with the goal gene directed cloning to the pGEX-4T-2 carrier BamHI and SmaThe I restriction enzyme site, then with recombinant plasmid transformed to e. coli bl21 (DE3) competent cell.

2.4 the evaluation of recombinant prokaryotic expression vector

Carry the feature of ampicillin resistance gene according to the pGEX-4T-2 carrier, there is e. coli bl21 (DE3) competent cell of recombinant plasmid to be coated on the flat board that contains penbritin conversion, the single bacterium colony of picking resistance carries out pcr amplification and identifies that reaction conditions and response procedures are the same.Agarose gel electrophoresis is selected positive bacterium colony, extracts plasmid.Use restriction enzyme BamHI and SmaI is carried out enzyme to recombinant plasmid and is cut evaluation, the agarose gel electrophoresis observations.Positive strain is sent to Shanghai Sangon Biological Engineering Technology And Service Co., Ltd's order-checking, the direction of insertion of goal gene and the exactness of reading frame in the checking recombinant plasmid.

2.5 prokaryotic expression

2.5.1 the competent preparation of e. coli bl21 (DE3)

Use CaCl ₂Standby e. coli bl21 (DE3) competent cell of legal system, concrete grammar is seen fine works molecular biology experiment guide.Be distributed into 100 μ l or 200 μ l aliquots, freezing in the liquid nitrogen ,-70 ℃ of storages are for subsequent use.

2.5.2 plasmid extraction

The alkali cracking subtraction extracts the prokaryotic expression plasmid of successful connection.

2.5.3 transform

Above-mentioned prokaryotic expression plasmid is changed in e. coli bl21 (DE3) competence of its expression vector, concrete steps are:

(1) get the competent cell of 100 μ l of-70 ℃ of frozen or fresh preparations and transfer in the aseptic Eppendorf tube, every pipe adds prokaryotic expression plasmid DNA(volume≤10 μ l, and DNA≤50ng), rotate gently with the mixing content places 30 min in ice.

(2) centrifuge tube is put on the test-tube stand in the circulator bath of pre-heating to 42 ℃, places 90 s, do not shake test tube.

(3) fast pipe is transferred in the ice bath, made cell cooling 1～2min.

(4) every centrifuge tube adds 400 μ l LB liquid nutrient mediums, with water-bath substratum is warmed to 37 ℃, then centrifuge tube is transferred on 37 ℃ of shaking tables, and incubation 45min makes bacteria resuscitation.

(5) getting the competent cell that transformed of part is coated on and contains on the LB plate culture medium of penbritin that final concentration is 50 μ g/ml.

(6) be inverted plate, in 37 ℃ of cultivations, bacterium colony can occur behind 12～16h.

2.5.4 abduction delivering

(1) gets the Erlenmeyer flask of certain capacity, (final concentration is 50 μ g/ml) to add respectively 5ml LB liquid nutrient medium and 2.5 μ l penbritins, get the bacterium liquid that shakes from the day before yesterday respectively in the 50 μ l access Erlenmeyer flask, and with empty plasmid in contrast, 37 ℃ of shaking table joltings are to OD ₆₀₀Value is 0.5.

(2) get test tube with above-mentioned Erlenmeyer flask equivalent, add respectively bacterium liquid 2ml, adding 20 μ l IPTG(IPTG final concentrations in each test tube is 1mM) induce 37 ℃ of shaking table jolting 4h.

(3) bacterium liquid is poured in 2.0 the pipe into the centrifugal 3min of 12000 rpm.

(4) remove supernatant liquor, add loading buffer 200 μ l, add beta-mercaptoethanol 20 μ l, boil 10min after the resuspended precipitation.

(5) put into the ice chest cooling, 4 ℃ of preservations, the SDS-PAGE gel electrophoresis is for subsequent use.

2.6 SDS-PAGE gel electrophoresis

(1) configuration 30% acrylamide separation gel is recorded separation gel between two sheet glass that assemble, and to apart from locating about the 3cm of sheet glass upper end, uses ddH ₂The O sealing is so that glue face level.

(2) remove upper strata ddH after the gelling admittedly to be separated of the 20min left and right sides ₂O blots residual moisture with filter paper.

(3) the concentrated glue of preparation 5%, perfusion are extremely apart from locating about the 2mm of sheet glass upper end.

(4) insert comb, extract comb after gelling to be concentrated is solid.

(5) respectively get 20 μ l sample point samples, albumen Marker loading 5-8 μ l.

(6) low pressure 70V electrophoresis to sample arrives in the separation gel.

(7) high pressure 150V electrophoresis is extremely apart from bottom 1cm place.

(8) the fixing 1.5h of stationary liquid.

(9) staining fluid dyeing 2h.

(10) place the shaking table decolouring to spend the night.

3. experimental result

3.1 ZjAPXThe amplification of cDNA

After the specificity upstream and downstream primer K1 of utilization design and K2 carry out pcr amplification, product is electrophoresis in 1.0% sepharose, after EB dyeing, observing one under the ultraviolet lamp between 750bp and 500bp, near the band of 750bp, coincideing with designed amplification purpose clip size.

3.2 the structure of recombinant prokaryotic expression vector

Utilize Auele Specific Primer K1 and K2 to carry out pcr amplification, product and pGEX-4T-2 plasmid are used BamHI and SmaI double digestion, product are through agarose gel electrophoresis, and the PCR product is about 750bp, and the pGEX-4T-2 fragment is about 4.9kb, conform to expected results.Purifying goal gene fragment and carrier segments connect respectively.

3.3 recombined pronucleus expression plasmid identification

Be applied on the plate culture medium that contains penbritin connecting product, the positive single bacterium colony of picking carries out the PCR evaluation after 37 ℃ of incubated overnight, and the result as shown in figure 10; There is the positive bacterium colony of purpose fragment to extract plasmid to proof, carries out restriction enzyme BamHI and SmaThe I double digestion identifies, the result as shown in figure 11, the about 750bp of double digestion fragment conforms to expected results, tentatively infer construction of recombinant plasmid successfully, and the size of recombinant plasmid is about about 5.6kb.Sequencing result shows in the plasmid of this transformed bacteria and really contains the purpose fragment, and reading frame is correct, the success of recombined pronucleus expression plasmid construction.

3.4 induce prokaryotic expression

Engineering bacteria pGEX-4T-2-ZjAPX-B, under 37 ℃ of conditions, after inducing 4h, IPTG takes a sample, the SDS-PAGE electrophoresis result as shown in figure 12, observe contain plasmid pGEX-4T-2-ZjAPX e. coli bl21 (DE3) under the condition that IPTG induces, compare at specific proteins band of about 50KD place generation with the empty thalline pGEX-4T-2-B of contrast, the GST size is about 26KD, target protein ZjAPX size is 23.76KD, so the fusion rotein size is about 49.76KD, show the success of inducing jujube tree ascorbate peroxidase expression of enzymes.

Claims (3)

1. a jujube tree Ascorbate peroxidase gene is characterized in that: have the nucleotide sequence shown in sequence table SEQ ID NO:1.

2. by the albumen of a kind of jujube tree Ascorbate peroxidase gene coding claimed in claim 1, it is characterized in that: have the aminoacid sequence shown in sequence table SEQ ID NO:2.

3. the application of a kind of jujube tree Ascorbate peroxidase gene as claimed in claim 1 in improving plant salt endurance.

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