CN103305526B - Application of protein TaDREB (triticum aestivum L. dehydration responsive element binding) 6 to preparation of high temperature resistant transgenic plant - Google Patents

Abstract

The invention discloses application of a protein TaDREB (triticum aestivum L. dehydration responsive element binding) 6 to preparation of a high temperature resistant transgenic plant. The invention provides a method for cultivating a transgenic plant. The method is characterized by transferring a coding gene of the TaDREB6 protein to a target plant to obtain a transgenic plant with high temperature resistance higher than that of the target plant, wherein the protein TaDREB6 is (a) or (b); and (a) is a protein formed by amino acid sequences shown in a sequence 1 in a sequence table, and (b) is a protein which is obtained by carrying out substitution and/or deletion and/or addition of one or more amino acid residues on the amino acid sequences in the sequence 1, is related to the high temperature resistance of the plant and is derived from the sequence 1. The protein TaDREB6 provides a foundation for expression of genes related to artificial control of stress resistance and stress tolerance, and can play an important role in breeding for culturing the plant with enhanced stress resistance and stress tolerance.

Description

The application of TaDREB6 albumen in the high temperature resistant transgenic plant of preparation

Technical field

The present invention relates to the application of TaDREB6 albumen in the high temperature resistant transgenic plant of preparation.

Background technology

The environment stresses such as arid, high salt and low temperature are the obstruction factors affecting wheat growth, growth.Therefore, understand wheat to the response of adverse environmental factor and signal transduction mechanism, improve the resistance of wheat breed, become one of vital task of wheat genetic research and wheat breed improvement.

A series of responsing reaction can be produced in plant materials, along with many Physiology and biochemistries and change developmentally under environment stress.Specify the reaction mechanism of plant to adverse circumstance, science argument will be provided for adversity gene engineering research and application.At present, plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, and exploration biotechnology improves plant growth characteristics, its objective is and improves plant to the adaptive faculty of adverse circumstance.

Under the adverse environmental factor of the environment-stress such as arid, high salt and low temperature, plant can make corresponding adjustment in molecule, cell and integral level, to reduce the injury existence that environment causes to the full extent.Many genes are expressed by stress-inducing, the product of these genes can not only participate in the stress response of plant directly, and the expression of other genes involved can be regulated or participate in signal transduction path, thus plant is avoided or reduces injury, strengthen the resistance to stressful environmental.To coerce relevant gene product and can be divided into two large classes: the product of first kind genes encoding comprises ionophorous protein, aquaporin, osmotic factor (sucrose, proline(Pro) and trimethyl-glycine etc.) synthetic enzyme etc. participate in the gene product that plant stress is replied directly; The product of Equations of The Second Kind genes encoding comprises participation and coerces relevant signal transmission and the protein factor of Gene expression and regulation, as protein kinase, transcription factor etc.Wherein, play an important role in the gene expression regulation that transcription factor is replied at plant stress.

Transcription factor also referred to as trans-acting factor, be can with the DBP of cis-acting elements generation specific effect in eukaryotic gene promoter region, by between them and and other associated protein between interaction, activate or suppress transcribe.The DNA land of transcription factor determines the specificity that it is combined with cis-acting elements, and transcription regulatory region determines it and plays activation or restraining effect to genetic expression.In addition, himself activity is also subject to the impact of the effect such as nuclear location and oligomerization.

At present known in plant to coerce relevant transcription factor and mainly contain: AP2 (APETALA2)/EREBP (the element responsive to ethylene associated proteins with AP2 structural domain, ethylene responsive element bindingprotein) transcription factor family, bZIP (basic region/leucinezipper motif transcription factors) class transcription factor containing basic region and leucine zipper, WRKY transcription factor family containing conservative WRKY aminoacid sequence, MYC family containing basic helix-loop-helix (bHLH) and leucine zipper and there is the MYB family of tryptophane bunch (Trp cluster).

DREB (dehydration response element conjugated protein, DRE-binding protein) class transcription factor is a member in AP2 family in EREBP-like subfamily.DREB and EREBP class transcription factor they on aminoacid sequence, there is no significant homogeny, but all contain one section of very conservative DNA calmodulin binding domain CaM (EREBP/AP2 structural domain) be made up of about 58 amino acid.Protein tridimensional analysis shows, 3 beta sheets are contained in this region, plays a crucial role to all kinds of cis-acting elements of identification.Wherein be arranged in the difference of two amino-acid residues of the 14th, 19 of second beta sheet, determine the specific combination of this kind of transcription factor and different cis-acting elements.

Stress tolerance due to plant is the complex character regulated and controled by polygene, relies on importing individual feature protein gene to be difficult to the comprehensive raising realizing stress resistance of plant.Therefore, utilize a transcription factors critical to promote the expression of multiple functional gene, thus strengthen the resistance of plant, become the engineered study hotspot of plant stress-resistance.

Summary of the invention

The object of this invention is to provide the application of TaDREB6 albumen in the high temperature resistant transgenic plant of preparation.

The invention provides a kind of method of cultivating transgenic plant, be that the encoding gene of TaDREB6 albumen is imported in object plant, obtain the transgenic plant of heat-resisting ability higher than described object plant; Described TaDREB6 albumen is following (a) or (b): the protein that (a) is made up of the aminoacid sequence shown in sequence in sequence table 1; (b) by the aminoacid sequence of sequence 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and the protein that by sequence 1 derived relevant to plant heat-resisting ability.

The encoding gene of described TaDREB6 albumen can be following 1) to 5) in arbitrary described DNA molecular:

1) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end the 19th to 783 Nucleotide;

2) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end the 19th to 814 Nucleotide;

3) DNA molecular shown in sequence 2 of sequence table;

4) under strict conditions with 1) or 2) or 3) DNA sequence dna that limits hybridizes and the DNA molecular of coded plant stress tolerance correlative protein;

5) with 1) or 2) or 3) DNA sequence dna that limits has more than 90% homology, and the DNA molecular of coded plant stress tolerance correlative protein.

Described stringent condition is in the solution of 0.1 × SSPE (or 0.1 × SSC), 0.1%SDS, hybridizes and wash film under 65 DEG C of conditions.

The encoding gene of described TaDREB6 albumen imports in described object plant by recombinant vectors; Described recombinant vectors is the recombinant plasmid multiple clone site that the encoding gene of described TaDREB6 albumen inserts plant expression vector obtained.The cell or tissue of the recombinant vectors carrying described gene by using the conventional biology methods such as Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated, particle gun to transform described biology.

Available existing plant expression vector construction contains the recombinant expression vector of described gene.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any one enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can be used alone or are combined with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of qualification and screening, can processing plant expression vector used, enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the chemical resistance reagent marker gene etc. of colour-change can be produced as added coding.Also any selected marker can not be added, directly according to phenotypic screen.

Described plant expression vector specifically can be pBI121 plasmid.

Described object plant is monocotyledons or dicotyledons.Described dicotyledons is Arabidopis thaliana, as Columbia ecotype Arabidopis thaliana.

The present invention also protects the application of described TaDREB6 albumen in regulating plant heat-resisting ability.Described plant is monocotyledons or dicotyledons.Described dicotyledons is Arabidopis thaliana, as Columbia ecotype Arabidopis thaliana.

The present invention also protects the encoding gene of described TaDREB6 albumen cultivating the application in high temperature resistant transgenic plant.Described plant is monocotyledons or dicotyledons.Described dicotyledons is Arabidopis thaliana, as Columbia ecotype Arabidopis thaliana.

The present invention also protects described TaDREB6 albumen as the application of transcription factor.

Arbitrary described high temperature can be more than 40 DEG C above, specifically can be 43 DEG C.

TaDREB6 gene is expressed under the induction of arid, high salt, high temperature, low temperature, dormin, ethene, Whitfield's ointment, methyl jasmonate, special regulation and control can contain the transcriptional expression of the gene of DRE/CRT cis element (core sequence: CCGAC).By TaDREB6 gene transfered plant, drought resistance and the high thermal resistance of plant can be improved.The present invention is that manual control expression that the is degeneration-resistant and gene of resistance to retrocorrelation provides the foundation, and plays an important role in the plant breeding of cultivating resistance and resistance of reverse enhancing.

Accompanying drawing explanation

Fig. 1 is the express spectra that TaDREB6 gene is expressed by stress-inducing.

Fig. 2 is the structural representation of recombinant plasmid pBI121-TaDREB6.

Fig. 3 is the plant phenotype after pyroprocessing (43 DEG C, 2 hours).

Fig. 4 is the plant phenotype after pyroprocessing (43 DEG C, 15 hours).

Fig. 5 proves the Binding in vivo specificity of transcription factor with yeast-one-hybrid system and activates the principle schematic of characteristic.

Fig. 6 is TaDREB6 gene Subcellular Localization.

Embodiment

Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.

The kind that little Bai wheat (Triticum aestivum cv.Xiaobaimai) is common wheat (Triticum aestivum L.), the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science, and the public also can obtain (being numbered ZM242) from national germplasm resource bank, mention that the document of little Bai wheat is: the screening of Sun Haitao etc., wheat TaDREB6 transcription factor interact protein, Scientia Agricultura Sinica, 2011,44 (22): 4740-4747., mention that the document of little Bai wheat is: Isolation and molecular characterization of the Triticum aestivum L.ethylene-responsive factor 1 (TaERF1) that increases multiple stresstolerance, Plant Mol Biol (2007) 65:719-732, Zhao Shi Xu, Lan Qin Xia, MingChen, Xian Guo Cheng, Rui Yue Zhang, Lian Cheng Li, Yun Xing Zhao, Yan Lu, Zhi YongNi, Li Liu, Zhi Gang Qiu, You Zhi Ma).

The discovery of embodiment 1, TaDREB6 albumen and encoding gene thereof

One, the separation of mRNA

The hydroponics growing wheat of about 10 days (kind is little Bai wheat) seedling in tri-leaf period is carried out Osmotic treatment 2 hours (treatment process: by taking-up careful for seedling, note not hindering root, blot with the moisture of clean thieving paper by blade and root, again seedling is placed on clean thieving paper, In Shade 2 hours), with liquid nitrogen flash freezer ,-80 DEG C save backup.Quikprep Micro mRNA Purification Kit (Pharmacia) is adopted to carry out the separation of mRNA.

Two, the structure of cDNA library and titer determination

1, the structure of cDNA library

Adopt Timesaver ^tMcDNA Synthesis Kit (Pharmacia) synthesizes cDNA double-strand by being separated the mRNA obtained in step one, and adds EcoRI/NotI adaptor; Adopt iII Gold Cloning Kit (Stratagene) carries out the structure of cDNA library, obtains 500ul storehouse liquid altogether.

2, the mensuration of titre

(1) get 1ul storehouse liquid SM Buffer and dilute 1000 times;

(2) get 1ul, 10ul, 100ul diluent respectively to enter in three 10ml centrifuge tubes, add 100ul competence Host Strains XL1-Blue MRF ' (OD respectively ₆₀₀be 1.0), in 37 DEG C of temperature bath 20min;

(3) add 3ml respectively and push up glue (50 DEG C) mixing, be laid on immediately on solid NZY flat board, solidify rear inversion, 37 DEG C of overnight incubation;

(4) according to dull and stereotyped plaque number, average, be storage capacity.

Calculation formula:

As calculated, the titre of this cDNA library is 3.0 × 10 ⁶individual plaque.

Three, the screening of cDNA library

1, the preparation of probe

According to AP2 conserved regions primers WAPF and WAPR of the dreb gene of having cloned, with the cDNA of common wheat for template carries out pcr amplification, program and system as follows:

Primer sequence: WAPF:5 '-ACC GCG GTG TGA GGC AGA GGA-3 ';

WAPR：5’-TGA GAA GTT GAC ACG TGC TTT GGC-3’。

Reaction system (50 μ l): template (60ng/ul) 0.5 μ l, dNTP (10mM) 1 μ l, primer (25 μMs) 1 μ l, 10 × buffer 5 μ l, ddH ₂042.1 μ l, Taq (5U/ μ l) 0.4 μ l.

Amplification condition (PTC-200):

94 DEG C of 2min; 94 DEG C of 30sec, 50 DEG C of 45sec, 72 DEG C of 45sec, 72 DEG C of 2min, 30 circulations.

Get amplified production 2ul electrophoresis detection in 1.2% sepharose, Ethidum Eremide dyes, and whether ultraviolet gel imaging instrument scanning, have a bright band in the position of 180bp.

2, the recovery of probe

Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV805A) is adopted to reclaim purified probes.

(1) under ultraviolet lamp, cut rapidly the band containing target DNA fragment, probably in the position of 180bp, exhaust gel surface liquid with paper handkerchief and shred.Calculated for gel weight (recording 1.5ml centrifuge tube weight in advance), this weight is as a gel volume (as 100mg=100ul); Add the DR-I solution of 3 gel volumes, in 75 DEG C of heating after mixing, be interrupted mixing, until gel piece melts (about 6-8min) completely.

(2) add the DR-II solution of 0.5 DR-I volume, then to add Virahol to final concentration be 20%, mix.

(3) mixed solution in aspiration step (2), transfer to DNA preparation pipe (being placed in 2ml centrifuge tube), the centrifugal 1min of 3600rpm, abandons filtrate.

(4) putting back centrifuge tube by preparing pipe, adding 0.5ml RinseA solution, the centrifugal 30s of 3600rpm, abandons filtrate.

(5) putting back centrifuge tube by preparing pipe, adding 0.7ml RinseB solution, the centrifugal 30s of 3600rpm, abandons filtrate.Use 0.7ml RinseB solution washing in the same way more once.

(6) centrifuge tube is placed in by preparing pipe, the centrifugal 1min of top speed.

(7) be placed in clean 1.5ml centrifuge tube by preparing pipe, prepare film centre at DNA and add 25ul water, room temperature leaves standstill 1min, the centrifugal 1min eluted dna of top speed.

(8) what elute is target DNA, saves backup.

Wherein, DR-I solution: gel melt agent (containing DNA protective material, preventing DNA from high temperature degrading).The airtight storage of room temperature (TaKaRa company, Code No.:DV805A).

DR-II solution: high from liquid sequence solutions (impelling the DNA fragmentation being greater than 100bp to be selectively bound to DNA to prepare on film).The airtight storage of room temperature.If there is precipitation, should re-use after 70 DEG C of incubations dissolve and are cooled to room temperature (TaKaRa company, Code No.:DV805A).

RinseA solution: washings.The airtight storage of room temperature (TaKaRa company, Code No.:DV805A).

RinseB solution: washings.The airtight storage of room temperature (TaKaRa company, Code No.:DV805A).

3, transferring film

(1) get 1ul storehouse liquid, in the culture dish of diameter 150mm, cultivate phage, general 6.0 × 10 ³pfu.

(2) plaque needs after cultivating to be placed on 4 DEG C of coolings, faces the used time and takes out and be placed in super clean bench and dry up, and when preventing transferring film, top gelatinous envelope sticks up.

(3) by Hybrond-N ⁺film is cut into circle, and diameter group is that the culture dish of 150mm is slightly little, shows numbering and date (corresponding with culture dish) with pencil on film.

(4) clamp film both sides with tweezers, have upward literal, centre first contacts flat board, comes up, and does not move, and does not have bubble, and film is shakeout naturally, after shakeouing completely, and timing.

(5) on film, prick three asymmetric holes with syringe needle, the position marking pen corresponding at the culture dish back side carries out mark.

(6) after 3min, with tweezers from uncover film gently, be not with top lifting glue.

(7) film is put into rapidly culture dish (putting one deck filter paper and 15ml sex change liquid) the sex change 7min filling sex change liquid, have down literal, note avoiding solution to arrive the upper surface of film.

(8) film is transferred to fill and (puts one deck filter paper and 15ml neutralizer) in the culture dish of neutralizer and neutralize twice, each 3min.

(9) then film is proceeded in rinsing liquid and wash 30min, can shake gently.

(10) take out film, clean filter paper blots, has down literal.

(11) wrapped by film with preservative film, on UV-crosslinked instrument, crosslinked 1min, deposits for subsequent use for 4 DEG C.

Sex change liquid: NaCl 1.5M (87.75g/1000ml)

NaOH 0.5M (20g/1000ml)

Neutralizer: NaCl 1.5M (87.75g/1000ml)

Tris 0.5M (60.57g/1000ml)

EDTA 0.001M (2ml 0.5M stock solution/1000ml)

Add 700-800ml water dissolution, with HCl adjust pH to 8.0, be finally settled to 1000ml.

Rinsing liquid: Tris-HCl (pH7.5) 0.2M (200ml 1.0M stock solution/1000ml)

2×SSC(100ml 20×SSC/1000ml)

4, prehybridization and hybridization

According to the quantity preparation prehybridization solution of the film that will hybridize, the composition of every 10ml prehybridization solution is composed as follows.

ddH ₂O 6.5ml

5×HSB 2ml

DenHardt’sIII 1ml

* ssDNA (salmon sperm dna, Sigma) (10mg/ml) 0.5ml

* ssDNA needs sex change before adding: put 10min on ice immediately after boiling 10min.

Prehybridization solution is mixed, puts into nylon membrane after being preheated to clarification in 65 DEG C, 65 DEG C of prehybridization 5-6h (new film); After probe mark is complete, add isopyknic 0.4N NaOH solution, leave standstill 10min in room temperature after mixing and make probe sex change, then join in prehybridization solution, 65 DEG C of hybridized overnight.

5, wash-out

Film is washed under 55 DEG C of-65 DEG C of conditions.

I washes: 2 × SSC/0.5%SDS, washes twice, each 15min;

II washes: 0.1 × SSC/0.1%SDS, washes detection signal strength in membrane process, determines elution time.

Blot washed film with filter paper, preservative film is wrapped, pressure X-mating plate.

6, two of positive colony screening is taken turns

(1) X-ray is alignd with film, determine position, describe the position of three asymmetric points on film.

(2) on projection reading set, put X-ray and corresponding culture dish, according to asymmetric point, culture dish is located.

(3) take out the positive hybridization spot determined with the 1ml rifle head of decaptitating, be placed in 1ml SM buffered soln, add 50ul chloroform.

(4) vibrate 30 seconds, room temperature places 1 hour, centrifugal, gets supernatant.

(5) get 10-50ul supernatant bed board again, cultivate phage and carry out postsearch screening.

(6) step of postsearch screening is the same: transferring film, prehybridization and hybridization, wash-out, pressure X-mating plate, obtain single positive plaque.

Four, TaDREB6 is obtained

1, (Mass excision) is deleted in cluster

(1) XL1-Blue MRF ' and XLOLR bacterium is prepared.Cultivate XL1-Blue MRF ' and XLOLR bacterium with LB liquid medium, 30 DEG C are spent the night, and add 0.2% (w/v) maltose, 10mM MgSO in substratum ₄and microbiotic, be respectively 12.5 μ g/ml tsiklomitsins and 50 μ g/ml kantlex.

(2) second days, the centrifugal 10min of 1000 × g collected thalline, uses 10mM MgSO ₄resuspended, make OD ₆₀₀reach 1.0.

(2) add in the sterile centrifugation tube of a 10ml:

1 μ l storehouse liquid (the structure part see cDNA library) is (about containing 6.0 × 10 ³phage particle)

XL1-Blue MRF ' 200 μ l (OD ₆₀₀be 1.0)

ExAssist assistant phage 2 μ l (> 1 × 10 ¹⁰pfu/ml)

(3) 37 DEG C of incubation 15min.

(4) 20ml liquid NZY substratum is added, 37 DEG C of shaking culture 2.5-3h.

(5) 65-70 DEG C of heating 20min.

The centrifugal 10min of (6) 1000 × g, supernatant moves in a new pipe.

(7) in the centrifuge tube of a 1.5ml, mix 200 μ l XLOLR bacterium and 1 μ l supernatant.

(8) 37 DEG C of incubation 15min.

(9) get 10 μ l, 100 μ l bacterium liquid are applied on LB solid medium (containing ammonia benzyl 50 μ g/ml) respectively, 37 DEG C of overnight incubation.

2, the inspection of cDNA library Insert Fragment

(1) the single bacterium colony deleted in cluster in random picking step 1, extracts their plasmid DNA.

(2) with restriction restriction endonuclease EcoR I (Takara) digestion, reaction system 10 μ l:

(3) 37 DEG C of digestion 2h, 0.8% agarose gel electrophoresis, find that the carrier of more than 95% has Insert Fragment, illustrate that the phage of more than 95% contains recon, therefore the actual recon contained in library is 2.85 × 10 ⁶(titre of cDNA library is 3.0 × 10 ⁶).The Insert Fragment of the recon of more than 50%, between 800bp-4Kb, illustrates that the library built is more complete.

3, (Single-clone excision) is deleted in mono-clonal

(1) the single positive plaque step 6 postsearch screening cDNA library of the screening part of cDNA library (see three) will be obtained under flat board is scratched, put in a centrifuge tube that is aseptic, that be added with 500 μ l SM damping fluids and 20 μ l chloroforms, vortex oscillation 10sec, 4 DEG C of storages.

(2) cultivate XL1-Blue MRF ' and XLOLR bacterium with LB liquid medium, 30 DEG C are spent the night, and add 0.2% (w/v) maltose, 10mM MgSO in substratum ₄and microbiotic, be respectively 12.5 μ g/ml tsiklomitsins and 50 μ g/ml kantlex.

(3) second days, the centrifugal 10min of 1000 × g collected thalline, uses 10mM MgSO ₄resuspended, make OD ₆₀₀reach 1.0.

(4) add in the sterile centrifugation tube of a 10ml:

XL1-Blue MRF ' 200 μ l (OD ₆₀₀be 1.0)

Phage stock solution 250 μ l is (at least containing 1 × 10 ⁵phage particle)

ExAssist assistant phage 1 μ l (> 1 × 10 ¹⁰pfu/ml)

(5) 37 DEG C of incubation 15min.

(6) 3ml liquid NZY substratum is added, 37 DEG C of shaking culture 2.5-3h.

(7) in 65-70 DEG C of water-bath centrifuge tube 20min, the then centrifugal 15min of 1000 × g.

(8) supernatant is moved in a new centrifuge tube, be phagemid suspension.

(9) in the centrifuge tube of a 1.5ml, add XLOLR bacterium that 200 μ l steps (3) prepare and the phagemid suspension 100 μ l that step (8) prepares, add 300 μ l liquid NZY substratum again, 37 DEG C of incubation 45-60min.

(10) getting 50 μ l bacterium liquid is applied on LB solid medium (containing ammonia benzyl 50 μ g/ml), 37 DEG C of overnight incubation.

(11) second days picking positive colonies, use LB liquid medium overnight incubation, extract plasmid, cut, electrophoresis detection Insert Fragment length with EcoRI enzyme.

(12) choose the clone that Insert Fragment is greater than 800bp to check order, on ABI733 sequenator (GenecoreBiological Company), dideoxy nucleotide chain cessation method is adopted to measure sequence, the complete sequence obtained is compared with RiboaptDBs such as EMBL Bank and GENEBANK, analyzes with DNASIS software.Find that No. 42 clones have a conservative AP2/EREBP structural domain.And gene structure is complete.

(13) nucleotide sequence of No. 42 clones and the aminoacid sequence of correspondence is analyzed.

By above-mentioned steps, find new gene order, and obtain its corresponding protein sequence.By the albumen of protein called after TaDREB6 shown in the sequence 1 of sequence table, being made up of 254 amino-acid residues, is conservative AP2/EREBP structural domain from N-terminal the 76th to 133 amino acids residues.By the encoding gene called after TaDREB6 gene of TaDREB6 albumen, its open reading frame is that the sequence 2 of sequence table is from 5 ' end the 19th to 783 Nucleotide.

Embodiment 2, real-time fluorescence quantitative PCR analyze the expression characterization of TaDREB6 gene

One, seedling age is the little Bai wheat seedling of 10 days, carries out following process respectively:

(1) Osmotic treatment: the wheat seedling of water planting is taken out the moisture blotted on root, be placed on dry filter paper, arid is cultivated after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours or 24 hours and is taken out material, and with liquid nitrogen flash freezer ,-80 DEG C save backup.

(2) salt marsh process: the wheat seedling of water planting is placed in 2% by NaCl and Na ₂sO ₄(NaCl and Na in the sodium salt solution of composition ₂sO ₄mass percent be 3: 2) in, illumination cultivation takes out material after 30 minutes, 1 hour, 4 hours, 6 hours, 12 hours, 24 hours or 48 hours respectively, and with liquid nitrogen flash freezer ,-80 DEG C save backup.

(3) subzero treatment: wheat seedling is placed in 4 DEG C of incubators, illumination cultivation is taken out after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours or 24 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(4) pyroprocessing: at wheat seedling being placed in 42 DEG C, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours or 24 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(5) dormin process: dormin solution wheat seedling being placed in 200 μMs, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours or 48 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(6) methyl jasmonate treatment: methyl jasmonate solution wheat seedling being placed in 50 μMs, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours or 24 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(7) ethene process: wheat seedling is placed in the plastics bag containing ethene, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 6 hours, 12 hours, 24 hours or 48 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(8) Whitfield's ointment process: salicylic acid solution wheat seedling being placed in 50 μMs, illumination cultivation is taken out respectively after 30 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours or 24 hours and is used liquid nitrogen flash freezer, and-80 DEG C save backup.

(9) process contrasted: the wheat seedling-80 DEG C directly got without any process is in contrast frozen.

Two, real-time fluorescence quantitative PCR analyzes the expression characterization of TaDREB6 gene

1, Quikprep Micro mRNA Purification Kit (Pharmacia) is adopted to carry out the separation of mRNA in each sample of step one.

2, R103-Quant_Reverse_Transcriptase (TIANGEN) is adopted to be cDNA by mRNA reverse transcription.

3, real-time fluorescence quantitative PCR

Primer pair for detecting TaDREB6 gene is as follows:

TaDREB6RTF：5’-GCACTTGTTGATGTCCTGAAT-3’；

TaDREB6RTR：5’-ATGACATGCCCTTGTCTGC-3’。

Primer pair for detecting reference gene (actin gene) is as follows:

actin-2F：5’-CTCCCTCACAACAACCGC-3’；

actin-2R：5’-TACCAGGAACTTCCATACCAAC-3’。

Using the expression amount of TaDREB6 gene in check sample as 1, in each sample, the relative expression quantity of TaDREB6 gene is shown in Fig. 1 compared with check sample.

The acquisition of embodiment 3, transgenic plant and resistance of reverse qualification thereof

One, the structure of recombinant expression vector

1, the total serum IgE of little Bai wheat is extracted and reverse transcription is cDNA.

2, with the cDNA of step 1 for template, with TaDREB6-121F and TaDREB6-121R composition primer pair carry out pcr amplification, obtain pcr amplification product.

TaDREB6-121F：5’-GGG GGATCCATGGAGACCGGGGGTAGCAA-3’；

TaDREB6-121R：5’-GGG CTCGAGCCGCATGACTACAACCCTCAT-3’。

PCR reaction system (50 μ l): template (60ng/ul) 0.5 μ l, dNTP (10mM) 1 μ l, upstream primer (25 μMs) 0.5 μ l, downstream primer (25 μMs) 0.5 μ l, 2 × GC buffer5 μ l, Taq (5U/ μ l) 0.4 μ l, ddH ₂042.1 μ l.

Pcr amplification condition (PTC-200): 94 DEG C of 2min; 94 DEG C of 40sec, 62 DEG C of 40sec, 72 DEG C of 90sec, 35 circulations.

3, use the pcr amplification product of restriction enzyme BamHI and XhoI double digestion step 2, obtain digestion products.

4, with restriction enzyme BamHI and XhoI double digestion pBI121 plasmid (Clontech company), carrier framework (about 12654bp) is reclaimed.

5, the digestion products of step 3 is connected with the carrier framework of step 4, obtains recombinant plasmid pBI121-TaDREB6.According to sequencing result, structrual description carries out to recombinant plasmid pBI121-TaDREB6 as follows: between BamHI and the XhoI restriction enzyme site of pBI121 plasmid, insert the sequence 2 of sequence table from the TaDERB6 gene (open reading frame is that the sequence 2 of sequence table is from 5 ' end the 19th to 783 Nucleotide) shown in 5 ' end the 19th to 814 Nucleotide.The structural representation of recombinant plasmid pBI121-TaDREB6 is shown in Fig. 2.

Two, the acquisition of transgenic plant

1, with recombinant plasmid pBI121-TaDREB6 transformation Agrobacterium C58 (Beijing Baeyer enlightening biotech company), recombinational agrobacterium is obtained.

2, recombinational agrobacterium is inoculated in YEP liquid nutrient medium, 28 DEG C, 3000rpm cultivate about 30 hours.

3, the bacterium liquid of step 2 is gone in YEP liquid nutrient medium (containing 50 μ g/ml kantlex and 50 μ g/ml Rifampins), 28 DEG C, 300rpm cultivates about 14 hours (bacterium liquid OD600 reaches 1.5-3.0).

4, by the bacterium liquid 4 DEG C of step 3, the centrifugal 10min of 4000g, being also diluted to OD600 with 10% aqueous sucrose solution (silwet-L77 containing 0.02% volume ratio) is about 0.8-1.0 to collect thalline.

5, whole strain Columbia ecotype Arabidopis thaliana (Col-0, SALK company) is tipped upside down in the container of the bacterium liquid filling step 4 together with flowerpot, make flower soak about 50s, after immersion, take out flowerpot, be sidelong in pallet, cover black plastic cloth; Open plastic cloth after 24 hours, upright placing flowerpot, carry out normal illumination cultivation, results T ₁for seed.

T ₁in MS substratum (kantlex containing 50 μ g/L), screen resistant plant for after seed harvest, resistant plant is transplanted in soil, results T ₂to be cultivated as plant (T for seed ₂for plant).

Extract T respectively ₁for plant and T ₂for the blade of plant total serum IgE and reverse transcription is cDNA, carry out PCR qualification with the primer pair of TaDREB6-121F and TaDREB6-121R composition to the cDNA from each sample, PCR is accredited as positive plant and transfer-gen plant.For a certain T ₁for plant, if its T ₂be accredited as the positive for the equal PCR of plant, then this plant is the transfer-gen plant isozygotied, and this plant and offspring thereof are 1 transgenic line.

T ₂t is produced for transfer-gen plant selfing ₃for seed.

Three, the acquisition of empty carrier plant is turned

Replace recombinant plasmid pBI121-TaDREB6 with pBI121 plasmid, other same step 2, obtains the T turning empty carrier plant ₃for seed, as transfer-gen plant T ₃for the contrast of seed.

Four, the high temperature resistant qualification of transgenic plant

1, by the T of a transgenic line ₃for plant, a T turning empty carrier strain ₃carry out high temperature resistant qualification (each strain adopts parallel processing, arranges and repeats experiment for three times, results averaged) respectively for plant and Columbia ecotype Arabidopis thaliana (strain of each strain 60), concrete steps are as follows:

Seed germination was placed 2 hours after one week in 43 DEG C of high temperature, then recovered 25 DEG C and normally cultivated.

Recover normal cultivation take pictures after 1 week and add up survival rate.Fig. 3 is shown in by photo.Columbia ecotype Arabidopis thaliana major part is dead, and survival rate is only 47%.The survival rate of transgenic line plant is up to 71%.The phenotype and the survival rate that turn empty carrier plant are all consistent with Columbia ecotype Arabidopis thaliana.

2, by the T of a transgenic line ₃for plant, a T turning empty carrier strain ₃carry out high temperature resistant qualification (each strain adopts parallel processing, arranges and repeats experiment for three times, results averaged) respectively for plant and Columbia ecotype Arabidopis thaliana (strain of each strain 60), concrete steps are as follows:

Plant 25 DEG C is normally cultivated, and is then placed 15 hours in 43 DEG C of high temperature by the seedling of sprouting after 4 weeks, then recovers 25 DEG C and normally cultivates.

Recover normal cultivation take pictures after 1 week and add up survival rate.Fig. 4 is shown in by photo.Columbia ecotype Arabidopis thaliana major part is dead, and survival rate is only 15%.The survival rate of transgenic line plant is up to 25%.The phenotype and the survival rate that turn empty carrier plant are all consistent with Columbia ecotype Arabidopis thaliana.

The activation characteristic of embodiment 4, TaDREB6

The cardinal principle of the activation characteristic of transcription factor is proved as shown in Figure 5 with yeast-one-hybrid system, DRE cis-acting elements and mutant DRE cis-acting elements are building up to respectively basic promotor Pmin (minimal promoter) upstream of pHISi-1 carrier and pLacZi carrier, Pmin promotor downstream connects reporter gene (His3, LacZ and URA3).After the expression vector YEP-GAP (not containing mobilizing function) of the goal gene being connected with encoding transcription factors is transformed into the yeast cell being connected with DRE cis-acting elements and mutant DRE cis-acting elements respectively, if the reporter gene be connected with in the yeast cell of mutant DRE cis-acting elements can not be expressed, and the reporter gene be connected with in the yeast cell of specific DRE cis-acting elements can be expressed, illustrate that this transcription factor can be combined with DRE cis-acting elements, and there is mobilizing function, have activated Pmin promotor, impel reporter gene to express.Thus demonstrate Binding in vivo specificity and the mobilizing function of object transcription factor.

One, by gene constructed for TaDREB6 on expression vector YEP-GAP

1, the total serum IgE of little Bai wheat is extracted and reverse transcription is cDNA.

2, with the cDNA of step 1 for template, with TaDREB6-EI and TaDREB6-XI composition primer pair carry out pcr amplification, obtain pcr amplification product.

TaDREB6-EI：5’-GGGGAATTCATGGAGACCGGGGGTAGCAA-3’；

TaDREB6-XI：5’-GGGCTCGAGAAGACTAAACCCATCATCACCATCTTGG-3’。

PCR reaction system (50 μ l): template (60ng/ul) 0.5 μ l, dNTP (10mM) 1 μ l, upstream primer (25 μMs) 0.5 μ l, downstream primer (25 μMs) 0.5 μ l, 10 × buffer5 μ l, Taq (5U/ μ l) 0.4 μ l, ddH ₂o 42.1 μ l.

Pcr amplification condition (PTC-200): 94 DEG C of 2min; 94 DEG C of 30sec, 58 DEG C of 45sec, 72 DEG C of 70sec, 30 circulations.

3, use the pcr amplification product of restriction enzyme EcoRI and XhoI double digestion step 2, obtain digestion products.

4, with restriction enzyme EcoRI and XhoI double digestion YEP-GAP plasmid (Liu Q, Kasuga M, SakumaY, Abe H, Miura S, Yamaguchi-Shinozaki K, Shinozaki K., 1998), carrier framework is reclaimed.

5, the digestion products of step 3 is connected with the carrier framework of step 4, obtains recombinant plasmid YEP-GAP-TaDREB6.

Two, the Binding in vivo specificity of TaDREB6 and the checking of activation characteristic

1, the structure of yeast reporter

Fragment 5 '-GAATTC-DRE-DRE-DRE-DRE-GTCGAC-3 ' (core sequence of DRE: TACCGACAT) containing 4 DRE elements is building up to respectively the Pmin of phis-1 carrier (MATCHMAKER One-Hybrid System, Clontech company) _hIS3promotor and pLacZi carrier (MATCHMAKER One-Hybrid System, Clontech company) P _cYCIpromotor upstream, obtains recombinant vectors pHis-1-DRE and pLacZi-DRE respectively, respectively pHis-1-DRE and pLacZi-DRE carrier is cut into wire with Xho I and Nco I restriction endonuclease.First by wire pHis-1-DRE vector to yeast cell (YM4271 strain, MATCHMAKER One-Hybrid System, Clontech company) in, obtain can on SD/His-substratum the yeast transformant (Yeast transformant) of normal growth.Then with this yeast transformant for host cell, continue to transform the pLacZi-DRE carrier repeating DRE elements containing 4.Lack so at the same time on the SD/His-/Ura-substratum of Histidine and uridylic, select to obtain normal dual yeast reporter containing pHis-1-DRE and pLacZi-DRE; The core sequence CCGAC of 4 DRE elements is mutated into TTTTT (MDRE), i.e. 5 '-GAATTC-MDRE-MDRE-MDRE-MDRE-GTCGAC-3 ', by the sub-construction process of normal dual yeast reporter, then build dual yeast reporter of the mutant containing 4 MDRE boxes.

2, PEG/LiAc method transformed yeast and interpretation of result

(1) inoculation yeast bacterial strain (YM4271 strain, MATCHMAKER One-Hybrid System, Clontech company) in 1ml YPD liquid nutrient medium, concuss 2 minutes, after dispersion agglomerate, suspension is gone in the triangular flask containing 50ml YPD liquid nutrient medium, 30 DEG C/250rpm shakes and spends the night, and surveys OD600=1.7-1.8 (counting about 4 × 10 ⁷individual/mL).

(2) get 30ml step (1) overnight culture to receive in the fresh YPD substratum of 300ml, 30 DEG C/250rpm cultivates, and about 3 is little of OD600=0.5 ± 0.1, the centrifugal 5min of room temperature 1000g, collects thalline, abandons supernatant, suspend with 1/2 volume 1 × TE, 1000g/5min is centrifugal.

(3) supernatant is abandoned in suction, and with the freshly prepared 1 × TE/LiAc solution suspension of 1.5ml, vibration mixing is for subsequent use.

(4) take out 0.1ml competent yeast to transform, add following solutions successively: 0.1 μ g recombinant plasmid YEP-GAP-TaDREB6,0.1mg ssDNA (salmon sperm dna, Sigma), 0.6mlPEG/LiAc at a high speed vibration 1 minute, 30 DEG C/200rpm shaking culture 30 minutes.

(5) add 70ul DMSO (sigma#D8779), be inverted mixing gently, 42 DEG C of heat shocks 30 minutes, vibrate therebetween gently, ice bath 2 minutes, the centrifugal 5min of room temperature 1000g.

(6) supernatant is abandoned in suction, adds 0.5ml 1 × TE buffer suspension cell.

(7) suspension is dipped with transfering loop, respectively at the SD/His containing 0,15mmol/L 3-AT ^-/ Ura ^-/ Trp ^-on selective medium, setting-out is cultivated.

(8) normal dual yeast reporter that dull and stereotyped half culturing step 1 builds, dual yeast reporter of the mutant that second half culturing step 1 builds, to do check analysis.

(9) be placed upside down in incubator, cultivate 3-4 days for 30 DEG C.

(10) found that the SD/His at 0mmol/L 3-AT ^-/ Ura ^-/ Trp ^-culture medium flat plate on yeast reporter of normal yeast reporter and sudden change have growth, but the diameter of yeast reporter of sudden change is obviously little; And at the SD/His of 15mmol/L3-AT ^-/ Ura ^-/ Trp ^-culture medium flat plate on normal yeast reporter can normal growth, but yeast reporter of sudden change is by supression not growth.

3, galactosidase activity detects

(1) from the SD/His of 0mmol/L 3-AT ^-/ Ura ^-/ Trp ^-culture medium flat plate on the yeast reporter daughter colony of respectively normal yeast reporter of picking and sudden change.Go in YPD liquid nutrient medium, in 30 DEG C of shaking culture, in the logarithmic growth later stage to be grown to, get 1.5ml bacterium liquid, the centrifugal 30s of 3000rpm;

(2) supernatant is abandoned, liquid in control main, centrifuge tube is placed in liquid nitrogen quick-frozen 10min, taking-up makes it naturally melt, add 50ul Z/X-gal solution, 30 DEG C of incubations, found that normal yeast reporter becomes blue in 6-8h, and the not change in 12h of yeast reporter of sudden change, be still white.Illustrate that transcription factor TaDREB6 can be combined with DRE cis-acting elements, and there is mobilizing function, have activated Pmin promotor (or Pmin _hIS3promotor or P _cYCIpromotor?), impel reporter gene to express.Thus demonstrate Binding in vivo specificity and the mobilizing function of TaDREB6.

Three, medicament preparation

(1) YPD liquid nutrient medium

Microbial culture yeast extract (Bacto-Yeast Extract) 10g/L

Microbial culture tryptone (Bacto-Peptone) 20g/L

Regulate pH to 5.8,121 DEG C/15min sterilizing, be down to the Glucose that 60 DEG C add 40% later, make its final concentration be 20g/L.

(2) SD/His ^-/ Ura ^-/ Trp ^-selective medium

Not containing amino acid whose yeast nitrogen (Yeast nitrogen base) 6.7g/L

Auxotroph mixture (drop-out media without His/Ura/Trp) 100ml

Agar powder (Bacteriological agar) 20g/L

Regulate pH to 5.8,121 DEG C/15min sterilizing, add 40%Glucose after being down to 60 DEG C, make its final concentration be 20g/L.

(3) auxotroph mixture (Drop-out mix): (10X)

(4)1×PEG/LiAc：

50％(w/v)PEG3350 8ml

10×TE buffer 1ml

10×LiAc 1ml

(5)10×TE Buffer：

100mM Tris-Hcl

10mM EDTA，pH＝7.5

121 DEG C of autoclavings, room temperature preservation.

(6)1×TE/LiAc：

10×TE buffer 1ml

10×LiAc 1ml

ddH ₂O 8ml

(7)Z Buffer：

Regulate pH to 7.0,121 DEG C/15min sterilizing, 4 DEG C of preservations.

(8) X-gal storage liquid (X-gal Stock Solution):

Dissolve X-gal with N, N-dimethyl-formamide (DMF), make its final concentration be 20mg/ml ,-20 DEG C of storages.

(9) the Z buffer damping fluid 100ml (Z buffer with X-gal) containing X-gal, notes matching while using:

Z buffer 98ml

β-dredge base ethanol (β-mercaptoethanol) 0.27ml

X-gal storage liquid (X-gal stock solution) 1.67ml

Embodiment 5, TaDREB6 Subcellular Localization

1, material prepares

At 9cm culture dish upper berth skim MS substratum, after tearing, internal surface is upward, is laid in MS substratum center, diameter within the scope of 3cm, 25 DEG C of preculture 4h.

2, the process of bronze

Getting 100mg diameter is that 1.5ml centrifuge tube put into by the bronze of 1.0 μMs, and add 1ml dehydrated alcohol, fully vibrate 3min, with the centrifugal 1min of 12000rpm, removes supernatant, then adds after 1ml sterilized water fully mixes, centrifugal with 12000rpm, repeats above-mentioned steps 3 times.Finally, be suspended in by bronze in 1ml ultrapure water ,-20 DEG C save backup.

3, particulate bullet is prepared

3 μ g add bronze suspension 6 μ l (50mg/ml) that diameter is 1.0 μMs, 0.1M spermidine (spermidine) 4 μ l, 2.5M CaCl containing the recombinant plasmid dna of TaDREB6 gene ₂6 μ l, mixing of bronze, DNA, spermidine and calcium chloride first being vibrated respectively, then after mixing vibration mixing 3min, leaves standstill 15min on ice.The centrifugal 10s of 12000rpm (referring to that rotating speed reaches 10s after 12000rpm), abandons supernatant.Add 140 μ l dehydrated alcohols, the centrifugal 10s of (breaing up bronze) 12000rpm after thick vibration, collect bronze precipitation.20 μ l dehydrated alcohols suspend and precipitate, some film.

Note: the required product of experiment must sterilizing, and wherein Spermidine and plasmid DNA are suction filtration sterilizing.

4, biolistic bombardment acceptor material

(1) select the split film (this experiment 1100psi) of certain pressure, together with bombardment film, in the alcohol of 70%, soak 1 ~ 2h, taking-up is dried;

(2) metal baffle is with alcohol-pickled, sterilizing on spirit lamp, the Bechtop ultraviolet sterilization of particle gun;

(3) get the above-mentioned bronze-plasmid complex prepared of 20 μ l, be spread evenly across on the mid-way of bombardment film, be not applied on whole film, size is consistent with the pore diameter range on carrier fixed ring, dries, and is then fixed on carrier fixed ring;

(4) above-mentioned carrier fixed ring is installed on launching device;

(5) film can be split and be installed to gas acceleration tube lower end;

(6) onion epidermis culture dish is put into vacuum chamber, take off culture dish lid;

(7) pointer is vacuumized to 26In/Hg;

(8) put helium in gas acceleration tube, until pressure reaches when can split pressure that film can bear in pipe, film can be split and break;

(9) gas is flushed on bombardment film, and carrier moves downward, and is blocked by metal baffle, and bronze-plasmid complex is below through the mesh directive target cell of metal baffle;

(10) onion epidermis cell bombarded is put into 25 DEG C of incubators, observe under laser confocal microscope after light culture 16 ~ 24h.

5, onion epidermis cell microscopy

By the onion epidermis compressing tablet after biolistic bombardment, light culture 16-24h, then at laser scanning co-focusing microscope (Bio-Rad MicroRadiance) (Laser scanning confocal microscopy, LSMC) observe GFP (green fluorescent protein) fluorescence, and carry out scanning and take pictures.The working parameter of LSCM is: Ex=488nm, Em=525 ± 15nm, Power=10%, Zoom7, medium sweep, Frame512 × 512.Software is TIME-COURSE and PHOTOSHOP5.0.See Fig. 6.

Claims (5)

1. The encoding gene of 1.TaDREB6 albumen is cultivating the application in high temperature resistant transgenic dicots;

   Be that the encoding gene of TaDREB6 albumen is imported in object dicotyledons, obtain the transgenic dicots of heat-resisting ability higher than described object dicotyledons;

   The protein that described TaDREB6 albumen is made up of the aminoacid sequence shown in sequence in sequence table 1.
2. 2.2 .application as claimed in claim 1, is characterized in that: the encoding gene of described TaDREB6 albumen is following 1) to 3) in arbitrary described DNA molecular:

   1) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end the 19th to 783 Nucleotide;

   2) sequence 2 of sequence table is from the DNA molecular shown in 5 ' end the 19th to 814 Nucleotide;

   3) DNA molecular shown in sequence 2 of sequence table.
3. 3.3 .application as claimed in claim 1, is characterized in that: the encoding gene of described TaDREB6 albumen imports in described object dicotyledons by recombinant vectors; Described recombinant vectors is the recombinant plasmid multiple clone site that the encoding gene of described TaDREB6 albumen inserts plant expression vector obtained.
4. 4.4 .application as claimed in claim 3, is characterized in that: described plant expression vector is pBI121 plasmid.
5. 5.5 .the application of TaDREB6 albumen in regulation and control dicotyledons heat-resisting ability;

   The protein that described TaDREB6 albumen is made up of the aminoacid sequence shown in sequence in sequence table 1.