CN100395337C - Plant ethylene response component binding protein and its coding gene - Google Patents

Abstract

The present invention discloses a binding protein of a plate ethylene response element and a coded gene thereof. The conjugated protein of a plate ethylene response element of the present invention is a protein with an amino acid residue sequence of SEQ ID No. 2 in a sequence list, or a protein which is used for substituting, deleting or adding one or a plurality of amino acid residues of the amino acid residue sequence of SEQ ID No. 2, has the same activity with the amino acid residue sequence of SEQ ID No. 2 and is derived from SEQ ID No. 2. By experimental verification, TaEREB2 of the present invention is used for expression under the induction of drought, high salt content and pathogenic bacteria, and can especially regulate and control the expression of a gene containing GCC-cassette cis elements (a core sequence is GCCGCC.). The TaEREB2 of the present invention lays the foundation for artificially controlling the expression of a stress resistant gene, and plays an important role in breeding plant varieties with strengthened stress resistance.

Description

Conjugated protein and the encoding gene of one kind of plant element responsive to ethylene

Technical field

The present invention relates in the plant a kind of with coerce relevant transcription factor and encoding gene thereof, the particularly conjugated protein and encoding gene of a kind of plant element responsive to ethylene.

Background technology

Environment stresses such as arid, high salt and low temperature are the obstruction factors that influences wheat growth, growth.Therefore, the understanding wheat is replied and signal transduction mechanism adverse environmental factor, improves the resistance of wheat breed, becomes one of vital task of wheat genetic research and wheat breed improvement.

Under environment stress, can produce a series of responsing reactions in the plant materials, the variation that is accompanied by many Physiology and biochemistries and grows.Clear and definite plant is to the reaction mechanism of adverse circumstance, will provide the science argument for adversity gene engineering research and application.At present, the plant stress-resistance Journal of Sex Research is deep into cell, molecular level gradually, and combines with genetics and genetic engineering research, explores and improves plant growth characteristics with biotechnology, its objective is and improves the adaptive faculty of plant to adverse circumstance.

Under the adverse environmental factor of environment-stress such as arid, high salt and low temperature, plant can be made corresponding adjustment on molecule, cell and integral level, with the injury that reduces environment to the full extent and caused and survived.Many genes are expressed by stress-inducing, the product of these genes not only can be participated in the stress response of plant directly, and can regulate other Expression of Related Genes or participate in signal transduction path, thereby plant is avoided or reduce injury, strengthen coercing the resistance of environment.With coerce relevant gene product and can be divided into two big classes: the product of first kind genes encoding comprises that ionophorous protein, aquaporin, the osmoregulation 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 second genoid coding comprises the protein factor that participates in coercing relevant signal transmission and genetic expression adjusting, as protein kinase, transcription factor etc.Wherein, transcription factor plays an important role in the gene expression regulation that plant stress is replied.

Transcription factor is also referred to as trans-acting factor, is can be conjugated protein with the DNA of cis-acting elements generation specific effect in the eukaryotic gene promoter region, by between them and and other associated protein between interaction, activate or suppress and transcribe.The DNA land of transcription factor has determined it and cis-acting elements bonded specificity, and transcription regulatory region has determined it that genetic expression is risen to activate or restraining effect.In addition, himself activity also is subjected to appraising and deciding the influence of effects such as position and oligomerization.

At present known in plant with coerce relevant transcription factor and mainly contain: AP2 (APETALA2)/EREBP (element responsive to ethylene is conjugated protein, the ethylene responsive element bindingprotein) transcription factor family with AP2 structural domain, bZIP (basic region/leucine zipper motif transcription factors) the class transcription factor that contains alkalescence zone and leucine zipper, the WRKY transcription factor family that contains conservative WRKY aminoacid sequence, the MYC family and MYB family of containing alkaline helix-loop-helix (bHLH) and leucine zipper with tryptophane bunch (Trp cluster).These five transcription factor families, except that WRKY family not the water of involved in plant coerce the reaction, other four families all participate in regulating the environment stress reaction of plant to arid, high salt and low temperature etc.Wherein, AP2/EREBP class transcription factor extensively exists in higher plant, it is the peculiar class transcription factor of plant, in recent years, report is all arranged in Arabidopis thaliana, tobacco, corn, paddy rice, soybean and rape, and this shows AP2/EREBP class transcription factor ubiquity and have vital role in higher plant.

DREB (DRE-binding protein) class transcription factor is a member in the EREBP-like subfamily in the AP2 family.They do not have significant homogeny DREB and EREBP class transcription factor on aminoacid sequence, but all contain one section very conservative DNA calmodulin binding domain CaM (EREBP/AP2 structural domain) of being made up of 58 left and right sides amino acid.The protein three dimensional analysis shows that 3 beta sheets are contained in this zone, plays a crucial role to discerning all kinds of cis-acting elements.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 class transcription factor and different cis-acting elements.DREB class transcription factor the 14th amino acids is Xie Ansuan (V14), the 19th amino acids is L-glutamic acid (E19), wherein the 19th amino acid is not conservative, for example the 19th amino acids of the OsDREB1 transcription factor of paddy rice is exactly Xie Ansuan (Dubouzet J.G., Sakuma Y., Ito Y., Kasuga M., Dubouzet E.G., Miura S., Seki M., Shinozaki K., Yamaguchi-Shinozaki K., OsDREB genes in RICE, Oryza sativa L., encodetranscription activators that function in drought-, high-salt-andcold-responsive gene expression.The Plant Journal, 2003,33:751-763).In the DREB associated protein, determine aspect the DNA bonded specificity, the effect of V14 is obviously than important (the Sakuma Y. of E19, Liu Q., Dubouzet J.G., Abe H., Shinozaki K.andYamaguchi-Shinozaki K., DNA-Binding specificity of the ERF/AP2 domain ofArabidopsis DREBs, transcription factors involved in dehydration-andcold-Inducible gene expression.Biochemical and Biophysical ResearchCommunications, 2002,290:998-1009); And ERF class transcription factor the 14th amino acids is a glycine, and the 19th is aspartic acid, thereby DREB specific combination DRE/CRT cis element, ERF specific combination GCC-box.The C-petiolarea of AP2/EREBP structural domain also comprises 1 core sequence of being made up of 18 amino-acid residues, and this sequence forms amphiphatic alpha-helix, and this alpha-helix may participate in the interaction between other transcription factor and DNA.

At present, in many plants, all find the transcription factor of this EREBP/AP2 of containing structural domain, and transmit relevant (Liu Qiang with signal such as disease-resistant, degeneration-resistant respectively, Zhao Nanming, Yamaguchi-Shinozaki K., ShinozakiK., the effect of DREB transcription factor in improving stress resistance of plant. Science Bulletin, 2000, the 45 volume 1:11-16).Liu Qiang etc. think, a dreb gene can be regulated and control a plurality of and plant arid, high salt and low temperature patience function associated expression of gene (Liu Qiang, Zhao Nanming, Yamaguchi-Shinozaki K., Shinozaki K., the effect of DREB transcription factor in improving stress resistance of plant. Science Bulletin, 2000, the 45 volume 1:11-16).Kasuga etc. studies confirm that, the DREB1A gene that imports to Arabidopis thaliana can promote the gene rd29 relevant with environment stress patience simultaneously, rd17, kin1, cor6.6, the expression of cor15a and erd10, the resistance of transfer-gen plant strengthens (Kasuga M. greatly, Liu Q., Miura S., Yamaguchi-Shinozaki K., Shinozaki K., Improving plant drought, salt, and freezing tolerance by gene transfer of asingle stress-inducible transcription factor.Nature Biotechnology, 1999,17:287-292).Equally, the low temperature tolerance ability of the transfer-gen plant of low temperature patience transcription factor CBF1 (the Jaglo-Ottosen K.R. that is significantly increased, Gilmour S.J., Zarka D.G., Schabenberger O., Thomashow M.F., Arabidopsis CBF1 overexpression induces COR genes andenhances freezing tolerance.Science, 1,998 280 (5360): 104-106).Because the stress tolerance of plant is the complex character by the polygene regulation and control, rely on to import the comprehensive raising that the individual feature protein gene is difficult to realize stress resistance of plant.Therefore, utilize a key transcription factor to promote the expression of a plurality of functional genes, thereby strengthen the resistance of plant, become the engineered research focus of plant stress-resistance.

According to the number that contains the DNA land, the AP2/EREBP transcription factor is divided into AP2 (APETALA2) and the conjugated protein EREBP of element responsive to ethylene (ethylene-responsive element binding protein) and three major types of RAV.AP2 type transcription factor comprises AP2, the ANT of Arabidopis thaliana, the Glossy of corn, idsl etc.Such transcription factor contains two AP2/EREBP structural domains, regulates growing of cell, has found 14 AP2 type transcription factor genes in Arabidopis thaliana; EREBP type transcription factor only contains 1 AP2/EREBP structural domain, regulates plant to ground molecule responsing reactions such as hormone (ethene), cause of disease, low temperature, arid and high salt.In the EREBP type transcription factor, found many members such as tobacco EREBP1-4, tomato Pti4-6, Arabidopis thaliana RAV1-2, AtEBP, AtERF1-5, DREB1A-C (CBF1-3) and DREB2A-B, transmitted relevant with signals such as cell development, hormone, disease-resistant, low temperature and arid, high salt respectively.These EREBP type transcription factors can be further divided into again: EREBP (ethylene-responsive element binding protein, be ERF) subgroup, comprise tobacco EREBP1-4, tomato Pti4-6, Arabidopis thaliana AtEBP, AtERF1-5, this class transcription factor and the GCC-box specific combination that contains core sequence AGCCGCC, therefore, its DNA land is called the GCC-box again in conjunction with territory (GCC-boxbinding domain, GBD), the 2nd G wherein, the 5th G, the 7th C be proteic identification (the Hao D. that plays an important role to ERF, Ohme-Takagi M., Sarai A., Unique mode of GCC box recognitionby the DNA-binding domain of ethylene responsive element-binding factor (ERFdomain) in plant.The Journal of Biological Chemistry, 1998,273:26857-26861).With nucleus magnetic resonance its three-D space structure be studies show that the GBD of AtERF1 combines with the major groove of its target sequence GCC-box by forming 3 reverse β-lamellas; The DREBP subgroup comprises Arabidopis thaliana DREB1A-C (CBF1-3) and DREB2A-B, and this class transcription factor is specific combination arid response element DRE/CRT under arid, high salt, low temperature, finds 124 DREBP type transcription factor genes in the arabidopsis gene group; RAV type transcription factor comprises Arabidopis thaliana RAV1, RAV2, contains two different DNA land-ERF/AP2 and B3, has found 6 RAY type transcription factor genes in Arabidopis thaliana.Also have the special transcription factor AL079349 of a class, it is all different with above transcription factor, constitutes a class by itself.

Recent findings EREBPs albumen has participated in the signal conduction and the gene expression regulation of arid, high salt and low temperature stress.People such as Mine have been separated to EREBP transcription factor CIP353 from the potato stem tuber of cryopreservation, be subjected to low temperature stress to induce strong expression (Mine T., Hiyoshi T., Kasaoka K., Ohyama A., 2003.CIP353 Encodesan AP2/ERF-Domain Protein in Potato (Solanum tuberosum L.) and RespondsSlowly to Cold Stress.Plant Cell Physiol., 44:10-15), illustrate to have the gene expression regulation that EREBP albumen has participated in being subjected to low temperature stress.Park etc. utilize tomato to be material, obtained being subjected to high salt, the EREBP transcription factor Tsi gene of ethene or jasmonic abduction delivering, EMSA (Electrophoretic mobilityshift assays) analysis of experiments is found, Tsi albumen can both be in conjunction with (Park J.M. with GCC-box and DRE/CRT cis element, Park C.J., Lee S.B., Ham B.K., Shin R., and Paek K.H., Overexpression of the Tobacco Tsil gene encoding an EREBP/AP2-Type transcriptionfactor enhances resistance against pathogen attack and osmotic stress inTobacco.The Plant Cell, 2001,13:1035-1046), although the former binding ability is greater than the latter, but illustrate that some EREBP albumen can activate the gene that is subjected to the osmotic stress abduction delivering.Under the normal growth condition, the overexpression of Tsi gene has improved the salt tolerance of transfer-gen plant (35S::Tsil), strengthened disease resistance (ParkJ.M., Park C.J., Lee S.B., Ham B.K., Shin R., and Paek K.H., Over expressionof the Tobacco Tsil gene encoding an EREBP/AP2-Type transcription factorenhances resistance against pathogen attack and osmotic stress in Tobacco.The Plant Cell, 2001,13:1035-1046), illustrated that more than the Tsi gene may participate in biology and coerce and abiotic stress two bars pathways.By a class MAPK of high-salt stress activated signal transfer mode (comprising SIMKK and SIMK), to coerce signal and pass to EIN2 (in the CTR1 downstream of Ethylene Signal pipeline) (Guo H.W.and Ecker J., The ethylene signaling pathway:newinsights.CurrentOpinion in Plant Biology, 2004,7:40-49), activate some EREBPs transcription factor at last, regulation and control osmotic stress Expression of Related Genes improves the salt tolerance of plant.Contain the GCC-box element for whether existing, and expression product participates in the gene of abiotic stress response directly, be still waiting to do further confirmation.

Comprehensive present result of study, the signal pipeline of plant under the environment stress condition has following six approach at least: the signal pipeline that (1) depends on ABA has three: induced by arid, high salt, activate MYB, MYC class transcription factor gene, regulation and control have the target gene of MYBR or MYCR cis-acting elements; Induced by arid, high salt, activate ABF/AREB class transcription factor gene, regulation and control have the target gene of ABRE cis-acting elements; Induced by arid, high salt, activate CBF4, DREB1 class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements.(2) the signal pipeline that does not rely on ABA has three: induced by arid, high salt, activate DREB2 class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements; Be subjected to low temperature induction, activate CBF1-3/DREB1A-C class transcription factor gene, regulation and control have the target gene of DRE/CRT cis-acting elements; Induced by arid, high salt or ethene, activate ERF class transcription factor gene, regulation and control have the target gene of DRE/CRT or GCC cis-acting elements.

With the cardinal principle of the activation characteristic of yeast-one-hybrid system proof transcription factor as shown in Figure 2, GCC-box cis-acting elements and mutant GCC-box cis-acting elements are building up to basic promotor Pmin (minimal promoter) upstream of pHISi-1 carrier and pLacZi carrier respectively, and Pmin promotor downstream connects reporter gene (His3, LacZ and URA3).After being transformed into the yeast cell that is connected with GCC-box cis-acting elements and mutant GCC-box cis-acting elements respectively as the expression vector YEP-GAP (not containing mobilizing function) of the goal gene that is connected with the encoding transcription factor, if the reporter gene that is connected with in the yeast cell of mutant GCC-box cis-acting elements can not be expressed, and the reporter gene that is connected with in the yeast cell of specific GCC-box cis-acting elements can be expressed, illustrate that this transcription factor can combine with GCC-box cis-acting elements, and has mobilizing function, activate the Pmin promotor, impelled reporter gene to express.Thereby interior binding specificity of the body that has proved the purpose transcription factor and mobilizing function.

The innovation and creation content

The purpose of this invention is to provide the conjugated protein and encoding gene of a kind of plant element responsive to ethylene.

Plant element responsive to ethylene provided by the present invention is conjugated protein, name is called TaEREB2, derive from Triticum wheat (Triticum aestivum L.), be to have SEQ ID № in the sequence table: the protein of 2 amino acid residue sequences, or with SEQ ID №: 2 amino acid residue sequence is through replacement, disappearance or the interpolation of one or several amino-acid residue and have the № with SEQ ID: 2 amino acid residue sequence is identical active by SEQ ID №: 2 deutero-protein.

The protein that the amino acid residue sequence of sequence 2 is made up of 296 amino-acid residues in the sequence table, from the 39th-40 amino acids residue sequence of aminoterminal and the 216th-218 amino acids residue sequence is two possible nuclear localization signals, is conservative AP2/EREBP structural domain from the 76th-133 amino acids residue sequence of aminoterminal.

The conjugated protein encoding gene of plant element responsive to ethylene, name is called TaEREB2, derives from Triticum wheat (Triticum aestivum L.), is one of following nucleotide sequences:

1) SEQ ID № in the sequence table: 1 dna sequence dna;

2) SEQ ID № in the code sequence tabulation: the polynucleotide of 2 protein sequences;

3) with sequence table in SEQ ID №: 1 dna sequence dna that limits has 90% above homology, and the identical function protein DNA sequence of encoding.

CDNA sequence in the sequence 1 is by 1794 based compositions, and the open reading frame of this gene is from the 415th-1305 bit base of 5 ' end, 296 amino acid of encoding altogether (sequence 2 in the sequence table).

Contain expression carrier of the present invention and clone and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification TaEREB2.

Experimental results show that TaEREB2 of the present invention expresses under the inducing of arid, high salt and pathogenic bacteria, and can special regulation and control contain GCC-box cis element (core sequence: expression of gene GCCGCC), TaEREB2 of the present invention will play an important role in cultivating resistance and the plant breeding of resistance of reverse enhanced for the degeneration-resistant and anti-retrocorrelation expression of gene of artificial control provides the foundation.

Description of drawings

Fig. 1 a-1c is the Nothern hybridization collection of illustrative plates that TaEREB2 is expressed by stress-inducing

Fig. 2 is the principle schematic with binding specificity in the body of yeast-one-hybrid system proof transcription factor and activation characteristic

Embodiment

The clone of embodiment 1, TaEREB2

One, the separation of mRNA

The wheat tri-leaf period seedling of hydroponics growing about 10 days carried out arid as follows handled 2 hours: seedling is carefully taken out, note not hindering root, blot with the moisture of clean thieving paper, again seedling is placed on the clean thieving paper In Shade 2 hours blade and root.Use liquid nitrogen flash freezer then ,-80 ℃ of preservations are standby.Adopt Quikprep Micro mRNA Purification Kit (Pharmacia) to carry out the separation of mRNA.

Two, the structure in cDNA library and titer determination

1, the structure in cDNA library

Adopt Timesaver ^TMCDNA Synthesis Kit (Pharmacia) will separate the synthetic cDNA two strands of the mRNA that obtains in the step 1, and adds EcoRI/No tI adaptor; Adopt ZAP

Predigested

III Gold Cloning Kit (Stratagene) carries out the structure in cDNA library, obtains 500ul storehouse liquid altogether.

2, the mensuration of titre

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

(2) get 1ul respectively, 10ul, 100ul diluent go in three 10ml centrifuge tubes, add 100ul competence host bacterium XL1-Blue MRF ' (OD respectively ₆₀₀Be 1.0), bathe 20min in 37 ℃ of temperature;

(3) add 3ml top glue (50 ℃) mixing respectively, be laid on immediately on the solid NZY flat board, solidify the back and be inverted 37 ℃ of overnight incubation;

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

Calculation formula:

As calculated, the titre in this cDNA library is 3.0 * 10 ⁶Individual plaque.

Three, cDNA library screening

1, the preparation of probe

According to the AP2 conserved regions sequences Design primer WAPF and the WAPR of the dreb gene of having cloned, be that template is carried out pcr amplification with the cDNA of common wheat, program and system are 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) 1μl

Primer (25 μ M) 1 μ l

10×buffer 5μl

ddh ₂O 42.1μl

Taq(5U/μl) 0.4μl

Amplification condition (PTC-200):

Get amplified production 2ul electrophoresis detection in 1.2% sepharose, bromination second pyridine dyeing, whether ultraviolet gel imaging instrument scanning has a bright band in the position of 180bp.

2, the recovery of probe

Adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV805A) to reclaim the purifying probe.

(1) under ultraviolet lamp, downcut the band that contains target DNA fragment rapidly, general in the position of 180bp, exhaust gel surface liquid and chopping with paper handkerchief.Calculated for gel weight (writing down 1.5ml centrifuge tube weight in advance), this weight is as a gel volume (as 100mg=100ul); The DR-I solution that adds 3 gel volumes mixes the back in 75 ℃ of heating, is interrupted and mixes, and melts (about 6-8min) fully until gel piece;

(2) add the DR-II solution of 0.5 DR-I volume, adding Virahol to final concentration again is 20%, mixes;

(3) mixed solution in the absorption step (2) is transferred to DNA preparation pipe (placing the 2ml centrifuge tube), and the centrifugal 1min of 3600rpm abandons filtrate;

(4) will prepare pipe and put back centrifuge tube, and add 0.5ml RinseA solution, the centrifugal 30s of 3600rpm abandons filtrate;

(5) will prepare pipe and put back centrifuge tube, and add 0.7ml RinseB solution, the centrifugal 30s of 3600rpm abandons filtrate.Use 0.7ml RinseB solution washing more once with same method;

(6) will prepare pipe and place centrifuge tube, the centrifugal 1min of top speed;

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

(8) wash-out goes out is target DNA, preserves standby.

Wherein, DR-I solution: gel fusing agent (contain the DNA protective material, prevent that DNA from high temperature degrading).The airtight storage of room temperature (TaKaRa company, Code No.:DV805A).

DR-II solution: high from liquid sequence solution (impelling dna fragmentation greater than 100bp to be selectively bound to DNA prepares on the film).The airtight storage of room temperature.If precipitation occurs, should after dissolving and be cooled to room temperature, 70 ℃ of incubations re-use (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, change film

(1) gets 1ul storehouse liquid (seeing the structure part in cDNA library), in the culture dish of diameter 150mm, cultivate phage, general 6.0 * 10 ³Pfu;

(2) need to be placed on 4 ℃ of coolings after plaque is cultivated, face the time spent taking-up and place super clean bench to dry up, the top gelatinous envelope glues when preventing the spline film;

(3) with 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 the film both sides with tweezers, have up literally, middle contact earlier is dull and stereotyped, comes up, and moves, and bubble is not arranged, and film is shakeout naturally, after shakeouing fully, and timing;

(5) on film, prick three asymmetric holes with syringe needle, carry out mark with marking pen in the position of culture dish back side correspondence;

(6) behind the 3min, Yi Bian with tweezers from beginning to uncover gently film, do not take up top glue;

(7) film is put into culture dish (putting one deck filter paper and 15ml sex change liquid) the sex change 7min that fills sex change liquid rapidly, had down literally, note avoiding solution to arrive the upper surface of film;

(8) film is transferred to (put one deck filter paper and 15ml neutralizer) in the culture dish that fills neutralizer neutralization twice, each 3min;

(9) film is changed over to wash 30min in the rinsing liquid then, can shake gently;

(10) take out film, on clean filter paper, blot, have down literal;

(11) with preservative film film is wrapped, crosslinked 1min on UV-crosslinked instrument deposits standby for 4 ℃;

Wherein,

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 the 700-800ml water dissolution,, be settled to 1000ml at last with HCl adjust pH to 8.0.

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

* need sex change before ssDNA adds: put 10min on ice immediately after boiling 10min.

Wherein, 5 * HSB (pH6.8)Molecular weight (MW) g/200ml

NaCl(3M) 58.44 35.064

PIPES(0.1M) 302.4 6.048

EDTA(20mM) 372.24 1.488

Denhardt’sIII g/100ml

2％Gelatin 2

2％Ficoll-400 2

2％PVP-360 2

10％SDS 10

5％Na ₄P ₂O·10H ₂O 5

With the prehybridization solution mixing, put into nylon membrane after being preheated to clarification, 65 ℃ of prehybridization 5-6h (new film) in 65 ℃; After probe mark is intact, add isopyknic 0.4N NaOH solution, leave standstill 10min in room temperature behind the mixing and make the probe sex change, join then in the prehybridization solution, 65 ℃ of hybridization are spent the night;

5, wash-out

Under 55 ℃ of-65 ℃ of conditions, wash film.

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

II washes: 0.1 * SSC/0.1%SDS, wash detection signal strength in the membrane process, and determine elution time.

Blot washed film with filter paper, preservative film is wrapped, and presses the X-mating plate.

6, two of positive colony take turns screening

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

(2) on projection reading set, put X-ray sheet and corresponding culture dish, make the culture dish location according to asymmetric point;

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

(4) vibration is 30 seconds, and room temperature was placed 1 hour, and is centrifugal, gets supernatant;

(5) get 10-50ul supernatant bed board once more, cultivate phage and carry out postsearch screening;

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

Four, obtain TaEREB2

1, deletion (Mass excision) in the cluster

(1) preparation XL1-Blue MRF ' and XLOLR bacterium.With liquid LB culture medium culturing XL1-Blue MRF ' and XLOLR bacterium, 30 ℃ are spent the night, and add 0.2% (w/v) maltose, 10mM MgSO in the 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) in the aseptic centrifuge tube of a 10ml, add:

1 μ l storehouse liquid (seeing the structure part in cDNA library) (contains 6.0 * 10 approximately ³Phage particle)

XL1-Blue MRF ' 200 μ l (OD ₆₀₀Be 1.0)

ExAssist assistant phage 2 μ l (＞1 * 10 ¹⁰Pfu/ml)

(3) 37 ℃ of incubation 15min;

(4) add 20ml liquid NZY substratum, 37 ℃ of shaking culture 2.5-3h;

(5) 65-70 ℃ of heating 20min;

The centrifugal 10min of (6) 1000 * g, supernatant move in the new pipe;

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

(8) 37 ℃ of incubation 15min;

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

2, segmental inspection is inserted in the cDNA library

(1) single bacterium colony of deletion in the cluster in the picking step 1 at random extracts their plasmid DNA;

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

10 * damping fluid H, 1 μ l

EcoR?I(12U/μl) 0.5μl

Plasmid DNA 2 μ l

ddH ₂O 6.5μl

(3) 37 ℃ of digestion 2h, 0.8% agarose gel electrophoresis finds that the carrier more than 95% has the insertion fragment, illustrate that the phage more than 95% contains recon, so the actual recon that contains in library is 2.85 * 10 ⁶(titre in cDNA library is 3.0 * 10 ⁶).The insertion fragment of the recon more than 50% illustrates that the library that makes up is more complete between 800bp-4Kb.

3, deletion (Single-clone excision) in the mono-clonal

(1) will obtain single positive plaque (see three, the step 6 postsearch screening cDNA library of cDNA library screening part) scratches down from flat board, put into one aseptic, be added with in the centrifuge tube of 500 μ l SM damping fluids and 20 μ l chloroforms, vortex oscillation 10sec, 4 ℃ of storages;

(2) with liquid LB culture medium culturing XL1-Blue MRF ' and XLOLR bacterium, 30 ℃ are spent the night, and add 0.2% (w/v) maltose, 10mM MgSO in the 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) in the aseptic centrifuge tube of a 10ml, add:

XL1-Blue MRF ' 200 μ l (OD ₆₀₀Be 1.0)

Phage stock solution 250 μ l (contain 1 * 10 at least ⁵Phage particle)

ExAssist assistant phage 1 μ l (＞1 * 10 ¹⁰Pfu/ml)

(5) 37 ℃ of incubation 15min;

(6) add 3ml liquid NZY substratum, 37 ℃ of shaking culture 2.5-3h;

(7) in 65-70 ℃ of water-bath centrifuge tube 20min, the centrifugal 15min of 1000 * g then;

(8) supernatant is moved in the new centrifuge tube, be phagemid suspension;

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

(10) get 50 μ l bacterium liquid and be applied on the LB solid medium (containing ammonia benzyl 50 μ g/ml) 37 ℃ of overnight incubation;

(11) second days picking positive colonies spend the night with liquid LB culture medium culturing, extract plasmid, cut with the EcoRI enzyme, and electrophoresis detection is inserted fragment length.

(12) choose and insert fragment and check order greater than the clone of 800bp, on ABI733 sequenator (GenecoreBiological Company), adopt the dideoxy nucleotide chain cessation method to measure sequence, the complete sequence and the Nucleotide databases such as EMBL Bank and GENEBANK that obtain are compared, analyze with DNASIS software.Find that No. 17 clones have a conservative AP2/EREBP structural domain.And gene structure is complete.

(13) No. 17 clones' of analysis nucleotide sequence and corresponding amino acid sequence, the result obtains having the TaEREB2 of the nucleotide sequence shown in the sequence 1 in the sequence table, its open reading frame is from the 415th-1305 bit base of 5 ' end, 296 amino acid (sequence 2 in the sequence table) of encoding altogether, in the sequence 2 is two possible nuclear localization signals from the 39th-40 amino acids residue sequence of aminoterminal and the 216th-218 amino acids residue sequence, is conservative AP2/EREBP structural domain from the 76th-133 amino acids residue sequence of aminoterminal.

The expression characterization of embodiment 2, Northern hybridization analysis TaEREB2

1, the preparation of vegetable material

Seedling age is 10 days a wheat seedling, carries out following processing:

(1) arid is handled: the wheat seedling of water planting is taken out the moisture that blots on the root, place on the exsiccant filter paper, arid is cultivated after 2 hours, 5 hours, 10 hours and is taken out material, uses liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.

(2) salt marsh is handled: with wheat seedling place 2% by NaCl and Na ₂SO ₄(NaCl and Na in the sodium salt solution of forming ₂SO ₄Mass percent be 3: 2) in, illumination cultivation is taken out material respectively after 2 hours, 5 hours, 10 hours, use liquid nitrogen flash freezer ,-80 ℃ of preservations are standby.

(3) ABA handles: wheat seedling is placed the ABA solution of 200 μ M, and illumination cultivation takes out and uses liquid nitrogen flash freezer respectively after 2 hours, 5 hours, 10 hours, and-80 ℃ of preservations are standby.

(4) Powdery Mildew is handled: wheat seedling is inoculated the Powdery Mildew bacterial strain, and illumination cultivation 24 and after 48 hours is taken out also and is used liquid nitrogen flash freezer, and-80 ℃ of preservations are standby.

(5) damage to plants caused by sudden drop in temperature processing: wheat seedling is placed 4 ℃ of incubators, and illumination cultivation takes out and uses liquid nitrogen flash freezer after 6 hours, 10 hours, 24 hours, and-80 ℃ of preservations are standby.

(6) Dui Zhao processing: the wheat seedling of directly getting without any processing-80 is ℃ in contrast frozen.

2, change film

(1) processing of electrophoresis and commentaries on classics film apparatus: 0.1N NaOH/1mM EDTA soaks more than the 1h, and is totally standby with flushing with clean water;

(2) prepare the denaturing formaldehyde gel: in an Erlenmeyer flask, add 10ml 10 * MOPs, water and 1g agarose that 73ml DEPC handles, heating and melting; Add 17ml formaldehyde when being cooled to 55 ℃ of left and right sides, mixing is poured in the glue groove of RNA special use;

(3) in the 0.5ml centrifuge tube that DEPC handles, add:

RNA+DEPC treating water 4 μ l (RNA is about 30 μ g, utilizes total RNA of the above-mentioned seedling that the RNAgents Total RNA Isolation System kit of Promega company extracts)

Methane amide 12.5 μ l

Formaldehyde 4.0 μ l

10×MOPs 2.5μl

Bromjophenol blue (10 *) 2.5 μ l

(4) sample is at 65 ℃ of water bath processing 5min, and point sample begins electrophoresis then, and damping fluid is 1 * MOPs.When bromjophenol blue arrives gel 1/3-1/2 position, stop electrophoresis;

(5) use the distilled water flushing gel, change film with 20 * SSC.To be placed on the filter paper bridge after the gel upset, put one and the sizable nylon membrane (Hybond-N of gel ⁺, Amarsharm), catch up with except that bubble, on film, put 3 metafiltration paper, identical with the nylon membrane size, catch up with except that bubble, seal with film around the gel, put sizable thieving paper, press the weight of about 500g, change film 16-24h, change thieving paper therebetween.After the commentaries on classics film is finished, wash film 10min with 2 * SSC, blot with filter paper, preservative film is wrapped, and ultra violet lamp 3min deposits to hybridization for 4 ℃;

3, the preparation of probe

At 3 ' design special primer EREB2F and EREB2R (avoiding the AP2/EREBP zone) of TaEREB2 gene, be that template is carried out pcr amplification with the TaEREB2 gene of cloning, program and system are as follows:

Primer sequence: EREB2F:5 '-TCC ACG AGC GCA CCC AAG-3 '

EREB2R：5’-ACA?CTT?GCG?AGT?GTC?CTA?TCC?TAT?CC-3’

Reaction system (50 μ l):

Template (60ng/ul) 0.5 μ l

dNTP(10mM) 1μl

Primer (25 μ M) 1 μ l

10×buffer 5μl

ddh ₂O 42.1μl

Taq(5U/μl) 0.4μl

Amplification condition (PTC-200):

Get amplified production 2ul electrophoresis detection in 1.2% sepharose, bromination second pyridine dyeing, the scanning of ultraviolet gel imaging instrument, whether the position of observing about 540bp has a bright band.

4, the recovery of probe

Adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) to reclaim the purifying probe recovery of the probe of cDNA library screening (with three).

5, probe mark

Probe mark is a random hexamer primer method, and every 1-2 opens the dna probe that film adds step 4, about 50-100ng with, add water to 14.5 μ l, 100 ℃ place ice bath 5min after boiling sex change 5min immediately, instantaneous centrifugal, add following ingredients then, final volume 25 μ l are in 37 ℃ of reaction 5h-8h.

5 * Oligo damping fluid, 5 μ l

Klenow fragment (5U/ μ l) 0.7 μ l

10 * damping fluid 2.5ul

BSA(10mg/ml) 1μl

α- ³²P-dCTP(10mCi/ml) 1.5μl

Wherein, contain in 1ml 5 * Oligo damping fluid:

160μl H ₂O

250μl 1M?Tris-HCl

25μl 1M?MgCl ₂

5 μ l 1M mercaptoethanols

500μl 2M?HEPES

20μl 100mM?dATP

20μl 100mM?dGTP

20μl 100mM?dTTP

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

* need sex change before ssDNA adds: put 10min on ice immediately after boiling 10min.

Wherein, 5 * HSB (pH6.8)Molecular weight (MW) g/200ml

NaCl(3M) 58.44 35.064

PIPES(0.1M) 302.4 6.048

EDTA(20mM) 372.24 1.488

Denhardt’sIII g/100ml

2% gelatin 2

2％Ficoll-400 2

2％PVP-360 2

10％SDS 10

5％Na ₄P ₂O·10H ₂O 5

With the prehybridization solution mixing, put into nylon membrane after being preheated to clarification, 65 ℃ of prehybridization 5-6h (new film) in 65 ℃; After probe mark is intact, add isopyknic 0.4N NaOH solution, leave standstill 10min in room temperature behind the mixing and make the probe sex change, join then in the prehybridization solution, 65 ℃ of hybridization are spent the night;

5, wash-out

The film of having hybridized taken out from hybrid pipe put into the wash-out box, (excessive hybridization solution is removed in 2 * SSC/0.1%SDS) rinsings, washes twice, each 20min with washing lotion I in 45 ℃ then to add a small amount of washing lotion I; (0.2 * SSC/0.2%SDS) washes twice, each 15min in 45 ℃ to use washing lotion II again.After washing lotion I washes, detect hybridization signal and background, whether decision continues wash-out again.Blot washed film with filter paper, preservative film is wrapped (bubble is removed in attention);

6, radioautograph

In X-radiography magazine, Hybond membrane is placed on the intensifying screen, in the darkroom, the X-mating plate is placed on the Hybond membrane, put another intensifying screen again, compress X-radiography magazine ,-70 ℃ of refrigerators exposed 7-15 days.

The Northern analytical results shows at drought stress after 2 hours shown in Fig. 1 a-1c, and at high salt (2% (NaCl+NaSO ₄) coerce after 2 hours the TaEREB1 gene and begin to express.Equally, under white powder germ inductive condition, TaEREB1 genetic expression strengthens, and illustrates that the TaEREB1 gene is expressed by arid, high salt and pathogenic bacterium inducing respectively.But, to coerce under the condition of handling with ABA cold, the TaEREB1 gene is not expressed.Illustrating that this gene does not catch a cold to coerce with ABA induces.Among Fig. 1 a-1c, CK is contrast.

The activation characteristic of embodiment 3, TaEREB2

One, TaEREB2 is gene constructed to expression vector YEP-GAP

1, obtains the complete sequence of TaEREB2 gene coding amino acid part

According to the sequences Design primer of the TaEREB2 gene of having cloned, the primer end adds EcoRI and XhoI restriction enzyme site respectively, and pcr amplification obtains the complete sequence of coding nitrogen base acid moieties, and program and system are as follows:

Primer sequence:

TaEREB2-EI：5’-TTATGAATTCGCAAAGATGTGCGGCGGTG-3’

TaEREB2-XI：5’-TTTTCTCGAGTTGCGAGTGTCCTATCCTATCCTGTTG-3’

Reaction system (50 μ l):

Template (60ng/ul) 0.5 μ l

dNTP(10mM) 1μl

Primer (25 μ M) 1 μ l

10×buffer 5μl

ddh ₂O 42.1μl

Taq(5U/μl) 0.4μl

Amplification condition (PTC-200):

Get amplified production 2ul electrophoresis detection in 1.2% sepharose, bromination second pyridine dyeing, the scanning of ultraviolet gel imaging instrument, whether the position of observing about 1.1Kb has a bright band.

Adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) reclaim the purified pcr product method with embodiment 1 three, the step 2 in the cDNA library screening, the recovery of probe).

2, TaEREB2 is gene constructed to expression vector YEP-GAP

With the PCR product and the expression vector YEP-GAP Liu Q of purifying in the step 1, Kasuga M, Sakuma Y, AbeH, Miura S, Yamaguchi-Shinozaki K, Shinozaki K., 1998), with EcoRI (Takara) and XhoI (Takara) respectively enzyme cut 4-6hr, reaction system is as follows:

10 * damping fluid H, 5 μ l

EcoRI(12U/μl) 2μl

XhoI(12U/μl) 2μl

PCR product/YEP-GAP 20 μ l

ddH ₂O 21μl

Adopt Agarose Gel DNA Purification Kit Ver.2.0 (TaKaRa company, Code No.:DV807A) reclaim purifying enzyme cut product (method with embodiment 1 three, the step 2 in the cDNA library screening, the recovery of probe).

The enzyme of purifying is cut the PCR product cut carrier YEP-GAP with enzyme and be connected 4-8hr, reaction system is as follows:

10×Ligase?buffer 1μl

Enzyme is cut PCR product 4 μ l

Enzyme is cut carrier YEP-GAP 4 μ l

T4DNA?Ligase 1μl

Get 0.5 μ l and connect product, electric shock transforms the JM109 bacterial strain, 37 ℃ of incubated overnight, and the picking positive colony, whether the sequencing analysis sequence is correct, obtains containing the recombinant expression vector YEP-GAP-TaEREB2 of TaEREB2 gene.

Two, the checking of binding specificity and activation characteristic in the body of TaEREB2

1, the structure of yeast reporter

The tandem sequence 5 '-GAATTC-GAGCCGCCAC-GAGCCGCCAC-GAGCCGCCAC-GAGCCGCCAC-GTCGA C-3 ' (core sequence of GCC-box is GCCGCC) that will contain 4 GCC-box elements is building up to the Pmin of pHis-1 carrier (MATCHMAKER One-Hybrid System, Clontech company) respectively _HIS3Promotor and pLacZi carrier (MATCHMAKER One-Hybrid System, Clontech company) P _CYCIThe promotor upstream obtains recombinant vectors pHis-1-GC and pLacZi-GC respectively, respectively pHis-1-GC and pLacZi-GC is cut into wire with Xho I and Nco I restriction endonuclease.Earlier wire pHis-1-GC is transformed in the yeast cell (YM4271 strain system, MATCHMAKEROne-Hybrid System, Clontech company), acquisition can be at SD/His ^-The yeast transformant of normal growth on the substratum (Yeast transformant).Be host cell then, continue to transform the pLacZi-GC carriers that contain 4 repetition GCC-box elements with this yeast transformant.The SD/His that lacks Histidine and uridylic so at the same time ^-/ Ura ^-On the substratum, select to obtain to contain the normal dual yeast reporter of pHis-1-GC and pLacZi-GC; The core sequence GCCGCC of 4 GCC-box elements is mutated into TTTTTT (MGCC box), i.e. 5 '-GAATTC-GATTTTTTAC-GATTTTTTAC-GATTTTTTAC-GATTTTTTAC-GTCGA C-3 ', by normal dual yeast reporter construction process, make up a dual yeast reporter of mutant that contains 4 MGCC boxes again.

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

(1) inoculation yeast bacterial strain (YM4271 strain system, MATCHMAKER One-Hybrid System, Clontech company) in 1ml YPD liquid nutrient medium, concuss 2 minutes, disperse behind the agglomerate suspension to be gone in the triangular flask that contains 50ml YPD liquid nutrient medium, 30 ℃/250rpm shakes and spends the night, and surveys OD600=1.7-1.8 and (counts about 4 * 10 ⁷Individual/mL);

(2) get 30ml step (1) overnight culture and receive in the fresh YPD substratum of 300ml, 30 ℃/250rpm cultivates, and about 3 hours to 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, suspends with the freshly prepared 1 * TE/LiAc solution of 1.5ml, and the vibration mixing is standby;

(4) taking out 0.1ml yeast competence transforms, add following solution successively: 0.1 μ g expression vector YEP-GAP-TaEREB2,0.1mg ssDNA (salmon sperm dna, Sigma), 0.6mlPEG/LiAc vibrated 30 ℃/200rpm shaking culture 30 minutes at a high speed 1 minute;

(5) add 70ul DMSO (sigma=D8779), be inverted mixing gently, 42 ℃ of heat shocks 30 minutes, vibration gently therebetween, 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) dip in transfering loop and get suspension, containing 0 respectively, the SD/His of 15mmol/L 3-AT ^-/ Ura ^-/ Trp ^-Setting-out is cultivated on the selective medium.

(8) the normal dual yeast reporter of half dull and stereotyped culturing step 1 structure, the dual yeast reporter of mutant that second half culturing step 1 makes up is so that do check analysis.

(9) be placed upside down in incubator, cultivated 3-4 days for 30 ℃.

(10) found that SD/His at 0mmol/L 3-AT ^-/ Ura ^-/ Trp ^-Culture medium flat plate on the yeast reporter of normal yeast reporter and sudden change growth is all arranged, but the diameter of the yeast reporter of sudden change is obviously little; And at the SD/His of 15mmol/L 3-AT ^-/ Ura ^-/ Trp ^-Culture medium flat plate on normal yeast reporter can normal growth, but the yeast reporter of sudden change is not restrained 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 bacterium colony of the normal yeast reporter of picking and sudden change respectively.Go in the YPD liquid nutrient medium,, wait to grow to the logarithmic growth later stage, get 1.5ml bacterium liquid, the centrifugal 30s of 3000rpm in 30 ℃ of shaking culture;

(2) abandon supernatant, liquid in the control main places liquid nitrogen quick-frozen 10min with centrifuge tube, taking-up is melted it naturally, adds 50ul Z/X-gal solution, 30 ℃ of incubations, found that normal yeast reporter becomes blue in 6-8h, and the yeast reporter of sudden change changes not in 12h, still is white.Illustrate that transcription factor TaEREB2 can combine with GCC-box cis-acting elements, and have mobilizing function, activated the Pmin promotor and (comprised Pmin _HIS3Promotor and P _CYCIPromotor), impel reporter gene to express.Thereby interior binding specificity of the body that has proved TaEREB2 and mobilizing function.

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 5.8,60 ℃ of Glucose of adding 40% are later on reduced in 121 ℃/15min sterilization, and making its final concentration is 20g/L.

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

Do not contain amino acid whose yeast nitrogen (Yeast nitrogen base) 6.7g/L

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

Agar powder (Bacteriological agar) 20g/L

Regulate pH to 5.8,121 ℃/15min sterilization adds 40%Glucose after reducing to 60 ℃, and making its final concentration is 20g/L.

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

L-Isoleucine (Isoleucine) 300mg/L

L-Valine (Xie Ansuan) 1500mg/L

L-Adenine (VITAMIN B4) 200mg/L

L-Arginine (arginine) 200mg/L

L-Histidine Hcl monohydrate (Histidine) 200mg/L

L-Leucine (leucine) 1000mg/L

L-Lysine Hcl (Methionin) 300mg/L

L-Methionine (methionine(Met)) 200mg/L

L-Phenylalanine (phenylalanine) 500mg/L

L-Threonine (Threonine) 2000mg/L

L-Tyrosine (tyrosine) 300mg/L

(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 ℃ of autoclavings, room temperature preservation.

(6)1×TE/LiAc：

10×TE?buffer 1ml

10×LiAc 1ml

ddH ₂O 8ml

(7)Z?Buffer：

Na ₂HPO ₄·7H ₂O 16.1g/L

NaH ₂PO ₄·H ₂O 5.5g/L

KCl 0.75g/L

MgSO ₄·7H ₂O 0.246g/L

Regulate pH to 7.0,121 ℃/15min sterilization, 4 ℃ of preservations.

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

Use N, N-dimethyl-formamide (DMF) dissolves X-gal, and making its final concentration is 20mg/ml ,-20 ℃ of storages.

(9) contain the Z buffer damping fluid 100ml (Z buffer with X-gal) of X-gal, note matching while using:

Z?buffer 98ml

Beta-mercaptoethanol (the 0.27ml of β-mercaptoethanol)

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

<160>2

<210>1

<211>1794

<212>DNA

＜213〉Triticum wheat (Triticum aestivum L.)

<400>1

tttttttttt?tgactaataa?tgtttgctac?aggttgcgct?taaaacactg?atagtgatac 60

ataggttact?gagagaaggc?gacgggtcat?tcaaagatga?cttcctgagc?tattcgtaca 120

gaggaaacat?tttgcagctt?ccaaatttca?gggatgactc?gagcccatta?ggtttgttct 180

atcgcgcatc?attctgaact?gcagcaacca?acggaagctc?ccgctcttct?gtttcggtac 240

ctgacgcgac?gctctcctcc?accccctttt?gtcagcatgg?gattccgcac?caccttgatc 300

agcctgccct?gagttcatca?ctcaaagtaa?gaaagagggt?agcgacggac?aagcagaggg 360

acgcaggcac?gtactatacc?ttagtcacgt?acacacagcg?agaagtgagc?aaagatgtgc 420

ggcggtgcca?tcctcgccga?gctcatgccg?agcgcgccgc?ccaggagcgt?cacggcggtc 480

cacctcttgc?ccaagcggca?gagggtcgac?gacttcgagg?ttgctttcaa?gcgcttcgac 540

gaggaccccg?aggaggagga?ggaggtgcgc?cagtgcgcgg?gcttcgagtt?tggcgcccgc 600

gggcagccgg?cagcgaggcg?cggcggcggt?aggccggcaa?agtacagagg?cgtccggcgc 660

aggccttggg?gcaaatgggc?ggcagagatc?cgcgaccccg?tcaagggcgt?ccgggtctgg 720

ctcggaacct?tcccctccgc?cgaggccgcc?gcgcacgcct?acgacgacgc?cgcacgcggc 780

ttccgcggcg?ccaacgccag?gctcaacttc?ccctcctcgt?ccacgagcgc?acccaagtgc 840

cgcgtagccg?aaaaaccgac?accgttcgtt?gttattgacc?tcgacgacga?caaagagggt 900

gatgccggag?cagcggatgc?cggcgggata?agctccgaat?ccagcggcgc?cctgccggac 960

ttctcgtggc?agggcatgtc?cgcgtccgac?gaggtcacgg?cgcaatctgt?ccatgctgaa 1020

gtggagtccg?gccagtccgt?cgtcgacctg?ggcagcgcca?agaagcggcc?ccggatcgaa 1080

gccgacgagg?ttctgccggc?agcgtccgac?gactccgcca?accaactgtt?agatcctttc 1140

atgttcgatg?acgaactcgg?tttcttggac?agcagctcgt?acgagtggct?ggatggcctg 1200

ttcggcgccg?atgctgagaa?gatcgacggc?agtcagctgg?ggctctggag?ctttggcgac 1260

gatgactgtc?tcgccgagga?cagcgcgtgc?gtgcaagtag?agtagcagta?ttgtagtatc 1320

atcaaagaca?acaggatagg?ataggacact?cgcaagtgtt?tgacgccctc?ccacaccttg 1380

tttttctttt?gaggataccc?ccacaccttg?ttgatgatat?ctctttcatc?aaagaaataa 1440

aaaaacaccc?tgttaatgat?gtaaaaaaaa?aaaaaaaagt?ttgcgccgcc?gccgcctcga 1500

ggacaatggg?acgcacggtg?gtttccgacg?aggaagagga?cgacttcatc?gaggccgagg 1560

aagaggacga?gccgcggccg?tcgcggaggg?ccagggatga?cgacgacgag?caggacgacg 1620

atgaggagga?ggaggatgat?gaagatgagg?gacagaatga?gtacgagaag?gatggcttca 1680

tagtggatga?tgcggatgaa?gacgaggagg?aggaggagga?ggaggcgaga?gctagtgatg 1740

atgaaagacg?caagaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaaaaaaaa?aaaa 1794

<210>2

<211>296

<212>PRT

＜213〉Triticum wheat (Triticum aestivum L.)

<400>2

Met?Cys?Gly?Gly?Ala?Ile?Leu?Ala?Glu?Leu?Met?Pro?Ser?Ala?Pro?Pro

1 5 10 15

Arg?Ser?Val?Thr?Ala?Val?His?Leu?Leu?Pro?Lys?Arg?Gln?Arg?Val?Asp

20 25 30

Asp?Phe?Glu?Val?Ala?Phe?Lys?Arg?Phe?Asp?Glu?Asp?Pro?Glu?Glu?Glu

35 40 45

Glu?Glu?Val?Arg?Gln?Cys?Ala?Gly?Phe?Glu?Phe?Gly?Ala?Arg?Gly?Gln

50 55 60

Pro?Ala?Ala?Arg?Arg?Gly?Gly?Gly?Arg?Pro?Ala?Lys?Tyr?Arg?Gly?Val

65 70 75 80

Arg?Arg?Arg?Pro?Trp?Gly?Lys?Trp?Ala?Ala?Glu?Ile?Arg?Asp?Pro?Val

85 90 95

Lys?Gly?Val?Arg?Val?Trp?Leu?Gly?Thr?Phe?Pro?Ser?Ala?Glu?Ala?Ala

100 105 110

Ala?His?Ala?Tyr?Asp?Asp?Ala?Ala?Arg?Gly?Phe?Arg?Gly?Ala?Asn?Ala

115 120 125

Arg?Leu?Asn?Phe?Pro?Ser?Ser?Ser?Thr?Ser?Ala?Pro?Lys?Cys?Arg?Val

130 135 140

Ala?Glu?Lys?Pro?Thr?Pro?Phe?Val?Val?Ile?Asp?Leu?Asp?Asp?Asp?Lys

145 150 155 160

Glu?Gly?Asp?Ala?Gly?Ala?Ala?Asp?Ala?Gly?Gly?Ile?Ser?Ser?Glu?Ser

165 170 175

Ser?Gly?Ala?Leu?Pro?Asp?Phe?Ser?Trp?Gln?Gly?Met?Ser?Ala?Ser?Asp

180 185 190

Glu?Val?Thr?Ala?Gln?Ser?Val?His?Ala?Glu?Val?Glu?Ser?Gly?Gln?Ser

195 200 205

Val?Val?Asp?Leu?Gly?Ser?Ala?Lys?Lys?Arg?Pro?Arg?Ile?Glu?Ala?Asp

210 215 220

Glu?Val?Leu?Pro?Ala?Ala?Ser?Asp?Asp?Ser?Ala?Asn?Gln?Leu?Leu?Asp

225 230 235 240

Pro?Phe?Met?Phe?Asp?Asp?Glu?Leu?Gly?Phe?Leu?Asp?Ser?Ser?Ser?Tyr

245 250 255

Glu?Trp?Leu?Asp?Gly?Leu?Phe?Gly?Ala?Asp?Ala?Glu?Lys?Ile?Asp?Gly

260 265 270

Ser?Gln?Leu?Gly?Leu?Trp?Ser?Phe?Gly?Asp?Asp?Asp?Cys?Leu?Ala?Glu

275 280 285

Asp?Ser?Ala?Cys?Val?Gln?Val?Glu

290 295

Claims (6)

1. a kind of plant element responsive to ethylene is conjugated protein, and amino acid residue sequence is shown in SEQ ID NO:2.

2. the plant element responsive to ethylene protein-bonded encoding gene of amino acid residue sequence shown in SEQ ID NO:2.

3. gene according to claim 2 is characterized in that: the base sequence of this gene is shown in SEQ ID NO:1.

4. contain claim 2 or 3 described expression carrier.

5. the clone that contains claim 2 or 3 described genes.

6. the primer of amplification claim 2 or 3 described genes is right.

Images