CN103468717A - Populus deltoids*populus nigra basic-domain leucine-zipper (PdbZIP) gene and application thereof - Google Patents

Abstract

The invention discloses a stress related gene PdbZIP of populus deltoids*populus nigra (Populus deltoids*Populus nigra). The gene is very important in a salt-resistant process of a plant. The provided bZIP gene is named PdbZIP and has a base sequence shown in SEQ ID NO:3 in a sequence table. A promoter is added before initial nucleotide is transcribed when the stress related gene PdbZIP disclosed by the invention is built in an expression vector pCAMBIA1304; meanwhile, an optional marker green fluorescent protein (GFP) is added, so as to identify and screen transgenic plant cells or plants; the expression vector with the stress related gene PdbZIP disclosed by the invention can transform a plant host by many methods, and is used for cultivating salt-resistant plant variety. The gene disclosed by the invention has a wide application prospect in cultivation of the salt-resistant plant.

Description

European-American Poplar PdbZIP gene and application thereof

Technical field

The invention belongs to gene engineering technology field, be specifically related to a kind of PdbZIP gene and application thereof from European-American Poplar (Populus deltoides * Populus nigra).

Background technology

European-American Poplar (Populus deltoides * Populus nigra) is one of the most arable short industrial cut stock intensive farming in felling cycle seeds of mid latitudes.China has introduced many good Carolina poplar colones for building large-area fast-growing, high-yield woods and obtaining good economic and social benefit in recent years.But high salt etc. have limited its further popularization.Therefore, in the time of being introduced into high salt water-deficient area, the strain of screening and cultivating anti-salt is prerequisite.Along with molecular biological development, utilize the anti-molecules of salt mechanism of Protocols in Molecular Biology research European-American Poplar to become the important channel addressed this problem, existing a plurality of genes are found relevant with the resistance that improves willow at present.Positive alkaline leucine zipper (basic-domain leucine-zipper, bZIP) transcription factor ubiquity in eukaryote participates in the growth of seed maturity, flower, the defense response of degeneration-resistant and Resistant in plant.The bZIP gene of various plants is widely studied in recent years, but have not been reported in the research of European-American Poplar.

Summary of the invention

The present invention, for solving the above-mentioned problems in the prior art, provides a kind of bZIP gene relevant to adverse circumstance in European-American Poplar, and this gene is cloned in European-American Poplar, is a member of bZIP gene family, called after PdbZIP.It has vital role in anti-reactant salt.

European-American Poplar disclosed by the invention (Populus deltoides * Populus nigra) adverse circumstance genes involved PdbZIP, it has the base sequence as shown in SEQ ID NO:3.Its sequence is by 408 based compositions, holds the open reading frame sequence that the 1st to the 408th residue is this gene from 5 ', and its expression is subject to inducing of salt stress.

Another aspect of the present invention is to disclose gene mentioned above, has replacement, disappearance or the interpolation of one or several base of base sequence process as shown in SEQ ID NO:3 and has the base sequence that has identical activity with the albumen of SEQ ID NO:3 coding; Or there is the homology more than 90% with SEQ ID NO:3, and the base sequence of coding identical function protein.

Another aspect of the present invention is to disclose the amplimer of gene mentioned above, has the base sequence as shown in SEQ ID NO:1～2.

Another aspect of the present invention is to disclose the protein of the PdbZIP genes encoding of European-American Poplar mentioned above (Populus deltoides * Populus nigra), there is the amino acid residue sequence as shown in SEQ ID NO:4, the protein that it is comprised of 135 amino-acid residues.

Another aspect of the present invention is to disclose the derived protein of protein mentioned above, it is characterized in that: have amino acid residue sequence process one or several amino acid whose replacement, disappearance or the interpolation as shown in SEQ ID NO:4 and have the amino acid residue sequence that has identical activity with the albumen of SEQ ID NO:4 coding.

Another aspect of the present invention is openly to contain the expression vector of gene mentioned above.Anti contravariance related gene PdbZIP provided by the present invention, used a kind ofly can guide the expression vector transformed plant of foreign gene in expression of plants, can obtain the transfer-gen plant that drought stress resistance is strengthened.In preferred situation, described expression vector is plasmid pCAMBIA1304.Anti contravariance related gene PdbZIP of the present invention is in being building up to expression vector the time, can add that before its transcription initiation Nucleotide any strong promoter or inducible promoter all can, simultaneously must be identical with the reading frame of encoding sequence, guarantee the translation of whole sequence.For the ease of transgenic plant cells or plant are identified and are screened, can when carrier construction, to carrier, be processed, but for example add selective marker, common spendable mark is to the gene of antibiotics resistance enzyme and Biosafety mark, can be also that GUS, GFP etc. can produce the enzyme of colour-change or the gene of luminophor etc.The expression vector that carries adverse circumstance genes involved PdbZIP of the present invention can pass through several different methods conversion of plant host, for cultivating the plant variety of anti-salt.

Another aspect of the present invention is to disclose the clone of a kind of the present invention of containing gene mentioned above.In preferred technical scheme, cell mentioned above is intestinal bacteria Top10 cell or Agrobacterium EH105 cell.

Another aspect of the present invention is to disclose gene mentioned above application in cultivating salt-resistant plant.The plant host transformed can be that monocotyledons can be also dicotyledons.Enumerated agriculture bacillus mediated transformation of Arabidopsis thaliana method in the embodiment of the present invention, and obtained transfer-gen plant, the proof that test-results is strong turn PdbZIP gene Arabidopis thaliana and possess stronger salt resistance ability.

Character of innovation of the present invention is: the amplification of success of the present invention positive alkaline leucine zipper (basic-domain leucine-zipper, bZIP) transcription factor PdbZIP gene, the fields such as cultivation that it can significantly improve the expression of downstream adversity gene and then can be widely used in the anti-salt kind of willow.

The accompanying drawing explanation

The expression schematic diagram that Fig. 1 is the PdbZIP gene, wherein: X-coordinate is time (0,2,4,6h), ordinate zou is relative expression quantity, from the diagram result, can see that PdbZIP gene expression amount under salt stress significantly improves, and inconsistent at the different time points expression amount.This result proof PdbZIP gene is induced to express at salt stress.

Fig. 2 is PdbZIP gene electrophorogram, wherein: the electrophoretic band of the 1st, PdbZIP gene; M is DNA marker DL2000, from the diagram result, shows by pcr amplification, can obtain the total length (contrast DNA marker, the electrophoretic band size is 408bp) of European-American Poplar PdbZIP gene, and this result proof PdbZIP gene fragment length is correct.

Fig. 3 is bacterium liquid PCR method screening recombinant plasmid, and swimming lane 1-9 is the result with PdbZIP gene masculine clone; M is DNA marker DL2000, from the diagram result, shows that the PdbZIP gene successfully is connected to (contrast DNA marker, the electrophoretic band size is 408bp) the pCAMBIA1304 expression vector.

The maximal photochemistry efficiency that Fig. 4 is the mensuration blade is figure as a result.At 200mmolL ^-1after NaCl processes 24h, the maximal photochemistry efficiency of wild-type and transgenic line has all reduced, but the relative wild-type of the maximal photochemistry efficiency of transgenic line reduces less.Wherein the maximal photochemistry efficiency of wild-type has reduced by 25%, T ₂-3 have reduced by 18%, T ₂-6 have reduced by 10%, T ₂-14 have reduced by 15%.

Embodiment

Following non-limiting example can make the present invention of those of ordinary skill in the art's comprehend, but does not limit the present invention in any way.

In test, use reagent to reclaim test kit all purchased from sky root biotech firm as T4-DNA ligase enzyme, intestinal bacteria competence Top10, pGEM-T cloning vector and DNA gel, ExTaq enzyme and RNA reverse transcription test kit are purchased from precious biological (TaKaRa) engineering corporation in Dalian, and common agents is purchased from Baeyer enlightening company.The PCR instrument is purchased from Biometra, and whizzer is purchased from SIGMA company, and ultraviolet gel imaging instrument is purchased from Bio-rad company, and Ultralow Temperature Freezer is purchased from Sanyo, and primer synthesizes and order-checking is completed by Beijing six directions Hua Da genome company.

In the test method that the present invention uses, if no special instructions, be those skilled in the art's common practise, various buffered soln, detection reagent etc., if no special instructions, all can be obtained or be prepared by the normal experiment method by commercial sources.

The extraction of embodiment 1 total RNA

One. the present invention's European-American Poplar 107 materials used are taken from Baoding, that annual cutting cuttage is cultivated in the Beijing Forestry University nursery, after cuttage, for the maintenance ground moistening, within every three days, water once permeable, after growing young leaves, by similarly 250mM NaCl sprinkling processing for (growing 3 months) European-American Poplar seedling of growing way, and respectively 0,2,4,6h wins the top leaf and is placed in liquid nitrogen, then in-80 ℃ of Ultralow Temperature Freezers, save backup.Be used for carrying out the extraction of total RNA and synthesizing of cDNA.

Two. extract the flow process of total RNA the European-American Poplar blade that utilizes the CTAB method to coerce from 250mM NaCl:

(1) extract the front DTT of 0.1mM final concentration, 65 ℃ of preheatings of adding of RNA in 2 * CTAB damping fluid.

(2) by the 0.5g liquid nitrogen grinding, good material is placed in the Eppendorf pipe of 2mL, adds the Extraction buffer of 900 μ L preheatings, fully shake up, 65 ℃ of water-bath 15min, during concussion 2-3 time.

(3) the Eppendorf pipe is taken out and is cooled to room temperature, add trichloromethane 900 μ L, vibration 10min.4 ℃, 12000r.min ^-1centrifugal 15min.

(4) get approximately 600 μ L supernatants, add isopyknic trichloromethane vibration 6min.4 ℃, 12000r.min ^-1centrifugal 15min.

(5) get approximately 400 μ L supernatants, add the i.e. LiCl solution of 100 μ L of 1/4 volume, mix gently, place 3h for-20 ℃.Take out, 4 ℃, 12000rmin ^-1centrifugal 15min.

(6) abandon supernatant, then add 70% ethanol 700 μ L, 500 μ L and wash respectively 2 times.Sop up ethanol fully, be dissolved in 20～30 μ LddH after room temperature dries up ₂standby in O.

Three. the total RNA of blade that the aforesaid method of take obtains is template, use M-MLV Reverse Transcriptase test kit, reverse transcription is carried out in operation according to the test kit working instructions, synthetic cDNA the 1st chain, the analysis as template for quantitative fluorescent PCR or pcr amplification.

Embodiment 2 utilizes fluorescence quantitative PCR method to detect the expression of European-American Poplar PdbZIP gene in adverse circumstance

According to the conserved sequence of European-American Poplar PdbZIP gene, PdbZIP1(SEQ ID NO.1) and downstream primer design special primer upstream primer:: PdbZIP2(SEQ ID NO.2).

The primer title	The sequence title	Base sequence (5 '--3 ')
			PdbZIP1	SEQ ID NO.1	ATGAGCCGCA TCTTCACAAC TCCTGAA
PdbZIP2	SEQ ID NO.2	TTAGCCTAGC AAATCTGAAG AACTTGTAAT

Will be by embodiment 1 method, respectively 0,2,4,6h wins the prepared cDNA sample of top leaf, usings it as template, take PdbZIP1(SEQ ID NO.1) and PdbZIP2(SEQ ID NO.2) be primer, carry out respectively the quantitative fluorescent PCR analysis.

Described quantitative fluorescent PCR reaction system (20 μ L):

It is three steps that the quantitative fluorescent PCR reaction is divided into, and respectively: in the sex change stage, the cycle stage, in the melting curve stage, concrete reactions steps is: 95 ℃ of 10min; 95 ℃ of 15s; 60 ℃ of 1min(totally 40 circulations); 95 ℃ of 15s; 60 ℃ of 1h; 95 ℃ of 15s.

Result is as shown in the expression schematic diagram of Fig. 1 PdbZIP gene, wherein: X-coordinate is time (0,2,4,6h), ordinate zou is relative expression quantity, proof is when 250mM NaCl Stress treatment, the PdbZIP gene is induced to express obviously, when processing 4h, expression amount reaches maximum value, is about 6 times of control value.

Embodiment 3 utilizes PCR method clone European-American Poplar PdbZIP gene

According to the conserved sequence of European-American Poplar PdbZIP gene, PdbZIP1(SEQ ID NO.1) and downstream primer design special primer upstream primer:: PdbZIP2(SEQ ID NO.2).

The primer title	The sequence title	Base sequence (5 '--3 ')
			PdbZIP1	SEQ ID NO.1	ATGAGCCGCA TCTTCACAAC TCCTGAA
PdbZIP2	SEQ ID NO.2	TTAGCCTAGC AAATCTGAAG AACTTGTAAT

The described method of embodiment 1 of usining prepares synthetic cDNA as template, take PdbZIP1(SEQ ID NO.1) and PdbZIP2(SEQ ID NO.2) be primer, carry out the PCR reaction;

Described PCR reaction system is (25 μ l):

Described PCR response procedures is as follows: carry out 35 circulations after 95 ℃ of denaturation 5min, each circulation is 95 ℃ of sex change 50s, 63 ℃ of annealing 90s, and 72 ℃ are extended 2min, last, and sample extends 10min at 72 ℃.The PCR product obtained is detected with 1% agarose gel electrophoresis, the target stripe of 400bp left and right size is tapped rubber, utilize test kit to complete purifying and reclaim, send the order-checking of order-checking company.Sequencing result is as SEQ ID NO.3, and its corresponding amino acid residue sequence is SEQ ID NO.4.

The structure of embodiment 4 European-American Poplar PdbZIP expression vectors

The PdbZIP gene fragment that embodiment 2 is obtained is connected with pMD18-T Vector, this is connected to product and transform intestinal bacteria TOP10 competent cell, extract plasmid, utilize primer PdbZIP1(SEQ ID NO.1) and PdbZIP2(SEQ ID NO.2) carry out PCR and enzyme is cut detection, filter out the positive colony that forward inserts, called after pMD18-T-PdbZIP positive colony, pMD18-T-PdbZIP and pCAMBIA1304 are carried out to enzyme with BglII with the SpeI restriction enzyme cuts and is connected simultaneously, to connect in the positive colony Transformed E H105 Agrobacterium (purchased from the precious biotech firm in Dalian) obtained.Concrete operation step is as follows:

One. transform intestinal bacteria (be about to the PdbZIP gene fragment and transform intestinal bacteria TOP10 competent cell with the product that is connected of pMD18-T Vector)

(1) take out intestinal bacteria TOP10 competent cell from Ultralow Temperature Freezer and be placed in thawing on ice;

(2) with shifting to an earlier date cooling aseptic suction nozzle, cell suspension is divided in the centrifuge tube that installs to new precooling, dispensed loading amount is every pipe 50 μ l;

(3) add the pMD18-T-PdbZIP that connects product 10 μ l in centrifuge tube, gently revolve and mix, place 30min on ice;

(4) centrifuge tube is put into to heat shock 90s in 42 ℃ of water-baths, does not shake;

(5) fast centrifuge tube is put on ice, makes the cooling 2-3min of cell;

(6) on Bechtop to the LB substratum that adds 500 μ l sterilizings in every pipe, be placed on 37 ℃ of shaking tables 180rpm, shaking culture 45min;

(7) getting 30 μ l X-gal evenly is applied on the LB nutrient agar that contains kantlex;

(8) after X-gal absorbs fully, get the competent cell that 120 μ l have transformed and transfer on the LB nutrient agar that contains kantlex, gently cell evenly is coated with and opens with spreader;

(9) flat board being placed at room temperature several minutes makes liquid be absorbed fully by substratum;

(10) be inverted flat board, under 37 ℃, cultivate 12-16h; Picking intestinal bacteria positive colony, utilize primer PdbZIP1(SEQ ID NO.1) and PdbZIP2(SEQ ID NO.2) carry out PCR evaluation (the PCR system is as embodiment 3), the PCR product obtained is detected with 1% agarose gel electrophoresis, target stripe is tapped rubber, utilize test kit to complete purifying and reclaim, and serve sea and give birth to the order-checking of work biotech firm.Sequencing result is consistent with SEQ ID NO.3, thereby proof goal gene PdbZIP transforms in intestinal bacteria TOP10.

Two. extract the plasmid operation step as follows:

(1) get each 5mL of bacterium liquid containing the complete colibacillary goal gene PdbZIP of conversion of expression vector pCAMBIA1304 bacterium liquid (purchased from Shanghai north promise bio tech ltd) and aforesaid method one preparation, the centrifugal 30s of 12000rpm room temperature collects thalline, absorbs supernatant as far as possible; Utilize that it root plasmid is little to be carried middle amount test kit and carry out following operation.

(2) add 500 μ L solution P1 in centrifuge tube, concuss makes thalline fully broken;

(3) add 500 μ L solution P2 in centrifuge tube, gentle spins upside down 6-8 time, and thalline is fully dissolved, and now solution becomes limpid;

(4) add 700 μ L solution P3 in centrifuge tube, spinning upside down 6-8 time of gentleness, fully mix immediately, now in centrifuge tube, there will be white flocks;

(5) by the above-mentioned solution that mixes at the centrifugal 10min of 12000rpm room temperature;

(6) add 500 μ L balance liquid BL in adsorption column CP4, the centrifugal 1min of 12000rpm room temperature, outwell collection liquid, and adsorption column is put back in collection tube;

(7) supernatant is proceeded in adsorption column CP4 to the centrifugal 1min of 12000rpm with the pipettor gradation;

(8) add 500 μ L protein liquid removal PD in adsorption column, the centrifugal 1min of 12000rpm, then outwell collection liquid, and adsorption column is relay and reclaims in collector;

(9) add 700 μ L rinsing liquid PW in adsorption column, the centrifugal 1min of 12000rpm, outwell collection liquid, and adsorption column is relay and reclaims in collector;

(10) add 500 μ L rinsing liquid PW in adsorption column, the centrifugal 1min of 12000rpm, outwell collection liquid, and adsorption column is relay and reclaims in collector;

(11) centrifuge tube is placed on to the empty centrifugal 2min of 12000rpm in whizzer, removes rinsing liquid remaining in adsorption column;

(12) collection tube is put into to the centrifuge tube of a clean 1.5mL, uncapped in 37 ℃ of incubators and place 10min, purpose is that alcohol is volatilized totally fully;

(13) to the deionized water that adds 50 μ L in collection tube, standing 2min, the centrifugal 2min of 12000rpm, collect plasmid pMD18-T-PdbZIP and pCAMBIA1304 standby.

Three. the plasmid pMD18-T-PdbZIP extracted by the step 2 method is carried out to enzyme with pCAMBIA1304 with BglII and SpeI restriction enzyme simultaneously and cuts and be connected, enzyme cut and the attended operation step as follows:

(1) enzyme is cut system (20 μ l)

The endonuclease reaction temperature is 37 ℃, and the enzyme time of cutting is 4-6h.Enzyme is cut to product and carry out agarose gel electrophoresis.The PdbZIP gene enzyme is cut electrophorogram as a result, and as Fig. 2: swimming lane 1 wherein is simultaneously to the result of plasmid enzyme restriction with BglII and SpeI restriction enzyme; Swimming lane M is DNA marker DL2000, show and pass through double digestion from the diagram result, can obtain total length (the contrast DNA marker DL2000 of European-American Poplar PdbZIP gene, the electrophoretic band size is 408bp), this result proof PdbZIP gene fragment successfully is connected on cloning vector pCAMBIA1304.

(2) linked system (10 μ l), 16 ℃ of reaction 10h.

Plasmid pMD18-T-PdbZIP enzyme is cut the recovery product 4 μ l of acquisition

Ligation Solution Ⅰ 5μl

Recovery product 1 μ l after the pCAMBIA1304 enzyme is cut

Four. the PCR of the connection product that method three obtains detects

The connection product that will be obtained by method three transforms competent escherichia coli cell Top10 according to the heat shock method, converted product is coated on the LB flat board that contains the 100mg/L kantlex, cultivate approximately after 12 o'clock for 37 ℃, the single bacterium colony of picking white, with bacterium liquid PCR method screening recombinant plasmid, result is as Fig. 3, and swimming lane 1-9 is the result with PdbZIP gene masculine clone; M is DNA marker DL2000, show that from the diagram result PdbZIP gene successfully is connected to (contrast DNA marker the pCAMBIA1304 expression vector, the electrophoretic band size is 408bp), send Beijing six directions Hua Da genome company to carry out the order-checking of DNA sequence dna the positive colony identified, the correct positive colony of screening sequencing result.The expression vector called after

pCAMBIA1304-35S:PdbZIP:GFP。

Expression vector pCAMBIA1304-35S:PdbZIP:GFP prepared by the present invention, can pass through several different methods conversion of plant host, the plant variety that can cultivate anti-salt.The plant host transformed can be that monocotyledons can be also dicotyledons.

The present invention has prepared expression vector pCAMBIA1304-35S:PdbZIP:GFP and has transformed the positive colony of intestinal bacteria Top10 cell, thereby add 35s strong promoter and GFP fluorescent probe before anti contravariance related gene PdbZIP transcription initiation Nucleotide, thereby make in its transfer-gen plant drought stress resistance strengthened in preparation (such as: the cultivation of the anti-salt kind of willow etc.) have wide practical use, and can be convenient to transgenic plant cells or plant are identified and screened.

The functional verification of embodiment 5 European-American Poplar PdbZIP genes

One. the preparation of the EH105 Agrobacterium that contains plant expression vector, operation steps is as follows:

(1) picking Agrobacterium EH105 bacterium colony being inoculated in 5mlYEB liquid nutrient medium (containing Rifampin Rif80mg/L), 200r/min, 28 ℃ of shaking culture are spent the night;

(2) get that 2ml bacterium liquid is inoculated into that 50mlYEB liquid nutrient medium (containing Rifampin Rif80mg/L) continues to cultivate until OD600=0.5, ice bath 30min;

(3) get 2.5ml centrifugal, centrifugal twice altogether, condition is 4 ℃ at every turn, 5000r/min, and 10min, supernatant discarded, collect thalline;

(4) by the concentration of 10ml precooling, be that 0.15mol/L NaCl solution suspends, collects thalline;

(5) by the concentration of 1ml precooling, be the resuspended thalline of 20mmol/L CaCl2 solution again;

(6) divide and install to (operation on ice) in the 1.5ml centrifuge tube with the bacterium liquid of every pipe 200 μ l, after liquid nitrogen flash freezer 1min, place-80 ℃ standby;

(7) getting competent cell is slowly melting on ice;

(8) the pCAMBIA1304-35S:PdbZIP:GFP plasmid DNA of getting 20 μ l joins in 200 μ l Agrobacterium competent cells, fully mixes rear ice bath 30min;

(9) then in liquid nitrogen, after quick-frozen 1min, be placed in rapidly 37 ℃ of water bath heat preservation 5min, and then ice bath 2min;

(10) add the empty YEB liquid nutrient medium of 800 μ l, 200r/min,, cultivate 4-5h by 28 ℃;

(11) 12000r/min, the centrifugal 30s of room temperature, remove the part supernatant;

(12) thalline of collection being coated on uniformly to the YEB solid selects on substratum (kantlex and Rifampin);

(13) cultivate 2-3d under 28 ℃ of dark conditions, obtain the EH105 Agrobacterium that contains plant expression vector.

Two. agriculture bacillus mediated transformation of Arabidopsis thaliana, concrete grammar is as follows:

(1) preparation transforms suspension, adds 25g sucrose in 500ml distilled water, and the concentration that makes sucrose is 50g/l, and autoclaving transforms in forward direction conversion suspension and adds MES0.25g, Silwet L-77100 μ l.

(2) transform and water sufficient water to Arabidopis thaliana (plant origin is in the Yin Wei of Beijing Forestry University human relations academician laboratory) the day before yesterday, the fruit pod is cut.

(3) take out EH105 Agrobacterium that prepare according to the step 1 method, that contain plant expression vector from-80 ℃ of refrigerators, be inoculated in YEB solid medium (adding 100mg/L Km80mg/L Rif) upper, 28 ℃, secretly cultivate 36-48h.

(4) picking list bacterium colony, the 5ml YEB(be inoculated in contains 100mg/L Km80mg/L rif) in, being placed on shaking table, rotating speed 200r/min,, secretly cultivate 36-48h by 28 ℃.

(5) get 5ml bacterium liquid and join in the 500mlYEB substratum, be placed on shaking table, turn/min of rotating speed 200,28 ℃, the dark cultivation, be cultured to logarithmic phase, OD ₆₀₀for 0.8-1.5.

(6) bacterium liquid is placed in to the aseptic centrifuge tube of 50mL, 5000 turn/min, centrifugal 15min.

(7) remove liquid, the thalline of centrifugation is suspended with suspension, jolting mixes, bacterium liquid OD to be suspended ₆₀₀during for 1.0-1.5, be used for transforming.

(8) the Arabidopis thaliana floral organ is immersed to about 2min in agrobacterium suspension, brush lightly too much bacterium liquid, and use Adsorption of Filter Paper, then with preservative film, the Arabidopis thaliana plant is wrapped up to moisturizing, plant lies against the dark 24h of place.

(9) 24h is placed in phytotron and cultivates, and after 2-3d, preservative film is removed, and with waddy, supports drooping branch, notes keeping ground moistening, by normal rule, waters.

(10) after seed maturity, be the T1 seed, the Arabidopis thaliana over-ground part is cut, smooth out with the fingers lower fruit pod, cross micro mesh sieve and remove the impurity such as pericarp, put into 37 ℃ of baking oven 3-4d dryings, then be placed in 4 ℃ of Refrigerator stores.

Three. the kalamycin resistance screening

Utilize the method for above-mentioned steps two, by after the agrobacterium mediation converted Arabidopis thaliana, the transfer-gen plant of acquisition is put in growth cabinet and cultivated.After it bears seeds, results T1 seed, with 70% alcohol disinfecting 5min, then with 2.6% the clorox 10min that sterilizes, finally with aqua sterilisa, rinse 3-4 times, inoculate to the 1/2MS screening culture medium (kantlex concentration is 60mg/ml), carry out antibiotic-screening, be placed in illumination box, control 25 ℃/15 ℃ of temperature (daytime/night), light application time is 16h/8h(daytime/night), cultivate 2 weeks, filter out the transgenic arabidopsis with that resistance of card.The material that the T2 generation (T2-3, T2-6, T2-14) obtained is detected as follow-up physical signs maximal photochemistry efficiency (Fv/Fm).

Four. maximal photochemistry efficiency (Fv/Fm) detects

T2 generation 3 strains and wild-type (wild type with growth 60d, WT) Arabidopis thaliana is material and processes with 200mmolL-1NaCl, the CIRAS-2 Portable photosynthesis system that adopts Britain PP Systems company to produce, measure the maximal photochemistry efficiency (Fv/Fm) of blade under room temperature (25 ℃) and atmosphere CO 2 concentration, light intensity is 800 μ molm-2s-1, each is processed and measures 3 repetitions, and result is as shown in following table and Fig. 4:

	Contrast (contral) Fv/Fm	Salt is processed (200mmolL -1NaCL）Fv/Fm
			Wild-type (wild type)	0.8	0.6
T2-3	0.8	0.65
			T2-6	0.8	0.72
T2-14	0.8	0.7

At 200mmolL ^-1after NaCl processes 24h, the maximal photochemistry efficiency of wild-type and transgenic line has all reduced, but the relative wild-type of the maximal photochemistry efficiency of transgenic line reduces less.Wherein the maximal photochemistry efficiency of wild-type has reduced by 25%, T ₂-3 have reduced by 18%, T ₂-6 have reduced by 10%, T ₂-14 have reduced by 15%.The proof that test-results is strong turn PdbZIP gene Arabidopis thaliana and possess stronger salt resistance ability.

The above; it is only preferably embodiment of the present invention; but protection scope of the present invention is not limited only to this; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses; be equal to replacement or changed according to the technical program and inventive concept thereof, within all should being encompassed in protection scope of the present invention.

Claims (10)

1. the PdbZIP gene of European-American Poplar (Populus deltoides * Populus nigra), is characterized in that: have the base sequence as shown in SEQ ID NO:3.

2. gene according to claim 1 is characterized in that: have base sequence as shown in SEQ ID NO:3 through replacement, disappearance or the interpolation of one or several base and have the base sequence that has identical activity with the albumen of SEQ ID NO:3 coding; Or there is the homology more than 90% with SEQ ID NO:3, and the base sequence of coding identical function protein.

3. the primer of the described gene of claim 1 that increases, have the base sequence as shown in SEQ ID NO:1～2.

4. the protein of genes encoding as claimed in claim 1, it has the amino acid residue sequence as shown in SEQ ID NO:4.

5. the derived protein of protein according to claim 4 is characterized in that: have amino acid residue sequence as shown in SEQ ID NO:4 through one or several amino acid whose replacement, disappearance or interpolation and have the amino acid residue sequence that has identical activity with the albumen of SEQ ID NO:4 coding.

6. the expression vector that contains the described gene of claim 1.

7. expression vector according to claim 6, it is characterized in that: described expression vector is plasmid pCAMBIA1304.

8. the clone that contains the described gene of claim 1.

9. clone according to claim 8, it is characterized in that: described cell is intestinal bacteria Top10 cell or Agrobacterium EH105 cell.

10. the application of gene claimed in claim 1 in cultivating salt-resistant plant.

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