CN103468717B - 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 it and have further promoted.Therefore,, be introduced into high salt water-deficient area time, 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 addressing this problem, existing multiple 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, and from the 1st to the 408th open reading frame sequence that residue is this gene of 5 ' end, its expression is subject to the induction of salt stress.

Another aspect of the present invention is openly 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 with the albumen of SEQ ID NO:3 coding with identical activity; Or there is more than 90% homology with SEQ ID NO:3, and the base sequence of coding identical function protein.

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

Another aspect of the present invention is the openly 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 made up of 135 amino-acid residues.

Another aspect of the present invention is the openly 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 with the albumen of SEQ ID NO:4 coding with identical activity.

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, uses 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 the time being building up in expression vector, before its transcription initiation Nucleotide, can add that any strong promoter or inducible promoter all can, simultaneously must be identical with the reading frame of encoding sequence, ensure the translation of whole sequence.For the ease of transgenic plant cells or plant are identified and are screened, can in the time of carrier construction, process carrier, for example adding can selective marker, common spendable mark is gene and the Biosafety mark to antibiotics resistance enzyme, 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 the openly 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 that gene openly mentioned above is in the application of cultivating in salt-resistant plant.The plant host transforming can be that monocotyledons can be also dicotyledons.In the embodiment of the present invention, enumerated agriculture bacillus mediated transformation of Arabidopsis thaliana method, 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 present invention positive alkaline leucine zipper (basic-domain leucine-zipper that successfully increased, bZIP) transcription factor PdbZIP gene, it can significantly improve the expression of downstream adversity gene and then can be widely used in the fields such as the cultivation of the anti-salt kind of willow.

Brief description of the drawings

Fig. 1 is the expression schematic diagram of PdbZIP gene, wherein: X-coordinate is time (0,2,4,6h), ordinate zou is relative expression quantity, can see that from diagram result PdbZIP gene expression amount under salt stress significantly improves, and inconsistent at different time points expression amount.This result proves that 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, shows by pcr amplification from diagram result, can obtain the total length (contrast DNA marker, electrophoretic band size is 408bp) of European-American Poplar PdbZIP gene, and this result proves that 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, shows that from diagram result PdbZIP gene is successfully connected to (contrast DNA marker, electrophoretic band size is 408bp) pCAMBIA1304 expression vector.

Fig. 4 is the maximal photochemistry efficiency result figure that measures blade.At 200mmolL ^-1naCl processes after 24h, and 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 if T4-DNA ligase enzyme, intestinal bacteria competence Top10, pGEM-T cloning vector and DNA gel recovery test kit are all purchased from Tian Gen biotech firm, 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.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 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 Beijing Forestry University nursery, after cuttage, be to keep ground moistening to water once permeable in every three days, 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 top leaf and is placed in liquid nitrogen, then in-80 DEG C of Ultralow Temperature Freezers, save backup.For carrying out the extraction of total RNA and synthesizing of cDNA.

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

(1) extract the front DTT that adds 0.1mM final concentration in 2 × CTAB damping fluid of RNA, 65 DEG C of preheatings.

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

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

(4) get approximately 600 μ L supernatants, add isopyknic trichloromethane vibration 6min.4 DEG C, 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 DEG C.Take out, 4 DEG C, 12000rmin ^-1centrifugal 15min.

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

Three. the total RNA of blade obtaining taking aforesaid method is as template, use M-MLV Reverse Transcriptase test kit, carry out reverse transcription according to the operation of 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, design special primer upstream primer: PdbZIP1(SEQ ID NO.1) and downstream primer: PdbZIP2(SEQ ID NO.2).

Primer title	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, using it as template, taking PdbZIP1(SEQ ID NO.1) and PdbZIP2(SEQ ID NO.2) as primer, carry out respectively quantitative fluorescent PCR analysis.

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

Quantitative fluorescent PCR reaction is divided into three steps, respectively: in the sex change stage, the cycle stage, in the melting curve stage, concrete reactions steps is: 95 DEG C of 10min; 95 DEG C of 15s; 60 DEG C of 1min(totally 40 circulations); 95 DEG C of 15s; 60 DEG C of 1h; 95 DEG C 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, prove in the time of 250mM NaCl Stress treatment, PdbZIP gene is induced to express obviously, in the time 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, design special primer upstream primer: PdbZIP1(SEQ ID NO.1) and downstream primer: PdbZIP2(SEQ ID NO.2).

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

Prepare synthetic cDNA as template using method described in embodiment 1, taking PdbZIP1(SEQ ID NO.1) and PdbZIP2(SEQ ID NO.2) as primer, carry out PCR reaction;

Described PCR reaction system be (25 μ l):

Described PCR response procedures is as follows: after 95 DEG C of denaturation 5min, carry out 35 circulations, each circulation is 95 DEG C of sex change 50s, 63 DEG C of annealing 90s, and 72 DEG C are extended 2min, last, and sample extends 10min at 72 DEG C.The PCR product obtaining 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 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) obtaining.Concrete operation step is as follows:

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

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

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

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

(4) centrifuge tube is put into heat shock 90s in 42 DEG C of water-baths, 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 DEG C of shaking tables 180rpm, shaking culture 45min;

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

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

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

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

Two. extract plasmid operation step as follows:

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

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

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

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

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

(6) in adsorption column CP4, add 500 μ L balance liquid BL, the centrifugal 1min of 12000rpm room temperature, outwells 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 pipettor gradation;

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

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

(10) in adsorption column, add 500 μ L rinsing liquid PW, the centrifugal 1min of 12000rpm, outwells collection liquid, and adsorption column is relay and reclaimed 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 the centrifuge tube of a clean 1.5mL, uncapped and place 10min in 37 DEG C of incubators, object is that alcohol is volatilized totally completely;

(13) to the deionized water that adds 50 μ L in collection tube, leave standstill 2min, the centrifugal 2min of 12000rpm, collects plasmid pMD18-T-PdbZIP and pCAMBIA1304 for subsequent use.

Three. the plasmid pMD18-T-PdbZIP extracting by 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 attended operation step as follows:

(1) enzyme is cut system (20 μ l)

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

(2) (l), 16 DEG C are reacted 10h to 10 μ to linked system.

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 pCAMBIA1304 enzyme is cut

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

The connection product being obtained by method three is transformed to competent escherichia coli cell Top10 according to heat shock method, converted product is coated on the LB flat board that contains 100mg/L kantlex, cultivate after approximately 12 o'clock for 37 DEG C, 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 diagram result PdbZIP gene is successfully connected to (contrast DNA marker pCAMBIA1304 expression vector, 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 identifying, the correct positive colony of screening sequencing result.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 transforming 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 (for example: the cultivation of the anti-salt kind of willow etc.) in its transfer-gen plant drought stress resistance being strengthened in preparation had wide practical use, and can be convenient to transgenic plant cells or plant identify and screen.

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 DEG C 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 DEG C at every turn, 5000r/min, and 10min, supernatant discarded, collects thalline;

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

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

(6) divide and install to (operation on ice) in 1.5ml centrifuge tube with the bacterium liquid of every pipe 200 μ l, after liquid nitrogen flash freezer 1min ,-80 DEG C of placements are for subsequent use;

(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 DEG C 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,, cultivates 4-5h by 28 DEG C;

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

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

(13) under 28 DEG C of dark conditions, cultivate 2-3d, 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, fruit pod is cut.

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

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

(5) get 5ml bacterium liquid and join in 500mlYEB substratum, be placed on shaking table, turn/min of rotating speed 200,28 DEG C, secretly cultivate, 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) Arabidopis thaliana floral organ is immersed to about 2min in agrobacterium suspension, brush lightly too much bacterium liquid, and with Adsorption of Filter Paper, then with preservative film, 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 supports drooping branch with waddy, notes keeping ground moistening, waters by normal rule.

(10) after seed maturity, be T1 seed, 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 DEG C of baking oven 3-4d dry, be then placed in 4 DEG C of Refrigerator stores.

Three. kalamycin resistance screening

Utilize the method for above-mentioned steps two, by after 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 rinse 3-4 times with aqua sterilisa, inoculate to 1/2MS screening culture medium (kantlex concentration is 60mg/ml), carry out antibiotic-screening, be placed in illumination box, control 25 DEG C/15 DEG C 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) obtaining is detected as follow-up physical signs maximal photochemistry efficiency (Fv/Fm).

Four. maximal photochemistry efficiency (Fv/Fm) detects

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

	Contrast (contral) Fv/Fm	Salt processing (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 ^-1naCl processes after 24h, and 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; any be familiar with those skilled in the art the present invention disclose technical scope in; 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 (2)

1. European-American Poplar ( populus deltoides × Populus nigra) PdbZIP gene, it is characterized in that: be the base sequence shown in SEQ ID NO:3.
2. 2. a primer for gene described in the claim 1 that increases is the base sequence shown in SEQ ID NO:1 ~ 2.

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

   4. contain the expression vector of gene described in claim 1.

   5. expression vector according to claim 4, is characterized in that: the carrier that sets out of described expression vector is pCAMBIA1304.

   6. gene claimed in claim 1 is in the application of cultivating in salt-resistant plant, and described plant is dicotyledons.