CN101921773A - Capsella bursa-pastoris CBF pathway key gene and application in cultivation of cold resistant plant thereof - Google Patents

Abstract

The invention belongs to the technical field of molecular biology and genetic engineering, in particular to a capsella bursa-pastoris CBF path key gene and application in cultivation of a cold resistant plant thereof. In the invention, capsella bursa-pastoris is used as an experimental material, a nucleotide sequence and a promoter of a COR gene (a key gene in the CBF pathway of cloning the capsella bursa-pastoris by a PCR technique) and the nucleotide sequence of the CBF gene, which are represented by SEQ ID No. 1, SEQ ID No. 2 and SEQ ID No.3 respectively, are used to construct a plant express vector, and the cold resistant transgenic plant is obtained by agrobacterium-mediated genetic transformation. By fully exploring potential gene resources of the capsella bursa-pastori and using the gene resources for improving crop variety, the invention has great economic value.

Description

Shepherd's purse CBF pathway key gene and the application in cultivating cold resistant plant thereof

Technical field

The invention belongs to molecular biology, gene engineering technology field.Particularly, the present invention relates to a kind of key gene CBF (CRT/DRE binding factor) gene that in shepherd's purse (Capsellabursa-pastoris), has a CBF approach and COR ( CoLd- rEgulated) gene and promotor, behind these gene transferred plants, the CBF albumen of expressing in plant can activate the promotor of COR gene, and then improves COR expression of gene amount, the winter hardiness of regulating plant at last.

Background technology

Plant has enough adaptability and resistivity to the change of circumstances and poor environment, and this resistance was controlled by the genetic type of phyletic evolution both, restricted by the physiological ecological factor in the ontogeny.Temperature is as one of important environmental factor, distribution and growth and the output of restriction plant, growth and development of plants is played decisive role (Viswanathan C, ZhuJK.Molecular genetic analysis of cold-regulated gene transcription.Philos Trans R Soc Lond BBiol Sci.2002,357 (1423): 877-886.).Therefore improve plant particularly those winter hardiness or suitable to cold as the plant of useful agricultural crops be very important.

Former study shows that the raising of plant winter hardiness is subjected to controlled by multiple genes under the low temperature stress, and polygenic expression is induced by two approach: one is the activity expression that relies on ABA (dormin) approach; Another does not rely on the approach of ABA, in this approach, transcriptional activity factor CBF is attached on the CRT/DRE gene order, induce COR genetic expression and improved the winter resistance (Zhong Keya of plant, Ye Miaoshui, Hu Xinwen, Guo Jianchun. the vital role heredity of transcription factor CBF in plant cold resistance, 2006,28 (2): 249-254.).

The CBF transcription factor is that a class is subjected to low temperature induction, is present in the trans-acting factor in the plant.This class transcription factor combines with CRT/DRE (C-repeat dehydration-responsive-element) DNA controlling element specifically, activate that have this controlling element in the promotor cold induced and (or) expression of dehydration induced gene, thereby cause that the intravital multiple Physiology and biochemistry of plant changes, and produce certain cold tolerance.The COR gene ( CoLd- rEgulated) be the general designation of a series of cold induced genes, expression product is the protein of a class highly-hydrophilic, contains amphipathic polypeptide, influences the bendability of phosphatide bilayer capsule interior molecules, reduce the possibility of fatal phase change, improve the stability of film under low-temperature condition thus.Its promoter region all contain the CRT/DRE cis-acting elements ( C- rEpea t/ dEhydration rEsponsive eLement), its core area is 5 base pair conserved sequence CCGAC.The encoding gene of transcription factor CBF can be regulated cor (cold-regulated) expression of gene during the confirmation domestication by low temperature in Arabidopis thaliana, finishes a series of Physiology and biochemistry adaptations in the body thereby make to plant, and finally improves the tolerance of plant to environment-stress.

Find through literature search prior art, at present relevant COR gene also mainly is at salt mustard (Thellungiellasalsuginea) (GenBank Accession No.:ABX79675), Arabidopis thaliana (A.thaliana), high mountain Roripa montana (Drabaalpina) (GenBank Accession No.:ABW71518), rape (B.napus) waits in the cress to be separated to.The research that the COR gene is used for the improvement of transgenosis cold resistant plant lays particular emphasis on the cDNA sequence that imports single COR gene more, and more employing is the CaMV35S promotor, and recipient plant adopts the model plant Arabidopis thaliana more.In the Arabidopis thaliana of overexpression CBF1, also can express even without low temperature stimulation cor gene.The electrolyte leakage measuring finds that the plant frost resistance of overexpression CBF1 is higher 3.3 ℃ than the frost resistance of only expressing the cor15a plant, the CBF1 abduction delivering can also improve the freezing survival rate of whole plant, and only express not all right (the Jagglo-Ottosen KR of cor15a, Gilmour SJ, Zarka DG, SchabenbergerO, Thomashow MF.Arabidopsis cbf1overexpression induces COR genes and enhances freezingtolerance.Science, 1998,280:104-106.).Therefore think that the winter resistance of plant is the accumulation proterties of the anti-cold gene regulating of the many little effects of audient, the co expression of many genes just can reach the purpose that strengthens cold resistance of plant.This has brought very big difficulty to the cold resistance that improves plant by transgenic technology.Therefore in the cold acclimatization process, more gene is participated in, and has become the engineered preferred object of plant cold resistance.

Though utilize transgenic technology to improve the existing multiple trial of winter hardiness of plant, up to the present, successful illustration is also few.To resist cold gene (as Cor15a) and anti-cold generegulation factor CBF1 to transform the target plant and the coordinate expression of resistance to cold difference simultaneously, be expected to significantly improve its cold resistance, introduce intron simultaneously in the effect that strengthens in expressing, the expansion of many important hot belt type economic plants distributed areas will be helped lend some impetus to, fully excavate its market potential, have industrial application prospect widely.

Shepherd's purse (Capsella bursa-pastoris) is a kind of 1 year or 2 years wild herbaceous plant, tiling ground, happiness is cloudy, is a kind of edible vegetables that are seen everywhere in south, belong to Cruciferae, shepherd's purse belongs to Capsella Medik, can normal growth under cold condition grow and solid.Studies confirm that in the past has the degeneration-resistant acknowledgement mechanism of CBF in the shepherd's purse, comprise CBF and COR gene in the shepherd's purse.(the GenBank accession number: full length cDNA sequence 1034bp AY391121) comprises a two-way nuclear localization sequence (NLS) structural domain, the AP2 structural domain of 60 amino acid primitives of inferring and an ALA-Rich structural domain to CBF gene in the shepherd's purse.CBF expression of gene in the acclimatization to cold analysis revealed shepherd's purse is caught a cold and is induced adjusting (Wang X, Liu S, Liu X, Chen Z, Liu X, Pang Y, Sun X, Tang K.Molecular Cloning andCharacterization of a CBF Gene from Capsella bursa-pastoris.DNA sequence.2004,15 (3): 180-187).(the GenBank accession number: AY437888) full-length cDNA is 588bp to COR15b gene in the shepherd's purse, comprises a potential chloroplast(id) signal shearing site and two protein kinase phosphorylation action sites that depend on adenylic acid (AMP) and guanylic acid.The acclimatization to cold analysis revealed, COR15b expression of gene in the shepherd's purse is subjected to low temperature induction (Liu S, Wang X, FanZ, Pang Y, Sun X, Wang X, Tang K.Molecular cloning and characterization of a novelcold-regulated gene from Capsella bursa-pastoris.DNA sequence.2004,15 (4): 262-268.).

CBF pathway key gene involved in the present invention is to be cloned into from total DNA of shepherd's purse blade, comprises nucleotide sequence and its promoter sequence of COR gene; The nucleotide sequence of CBF gene.Find to utilize the relevant report of shepherd's purse CBF pathway key gene CBF and COR gene cotransformation cultivation hardy plant at present as yet.

Summary of the invention

An object of the present invention is provides cold-resistant gene C OR of valuable shepherd's purse and promotor thereof for the plant cold resistance breeding, another object of the present invention is the overexpression by said gene, the anti-low temperature stress performance of plant is greatly enhanced, the anti-obvious enhanced good plant of (anti-) low temperature ability kind of final acquisition.

The present invention clones cold induced gene CbCOR from shepherd's purse, its sequence is SEQ ID NO.1.

The present invention clones the promotor CbCORP of cold induced gene from shepherd's purse, its sequence is SEQ ID NO.2.

The present invention clones the transcription factor CbCBF that is subjected to low temperature induction from shepherd's purse, its sequence is SEQ ID NO.3.

Cold induced gene CbCOR of the present invention realizes by following process:

Extract the DNA of shepherd's purse, utilize the full length sequence of the cold induced gene CbCOR of pcr clone, its sequence is SEQ IDNO.1.

The promotor of above-mentioned cold induced gene realizes by following process:

Extract the DNA of shepherd's purse, utilize the chromosome walking technology to clone the promotor of cold-resistant gene C bCOR, its sequence is SEQID NO.2.

The transcription factor CbCBF of low temperature induction that is subjected to of the present invention realizes by following process:

Extract the DNA of shepherd's purse, utilize pcr clone to be subjected to the full length sequence of the transcription factor CbCBF of low temperature induction, sequence is shown in SEQ ID NO.3.

The transcription factor CbCBF that is subjected to low temperature induction in the shepherd's purse is a trans-acting factor, comprise a CRT/DRE DNA controlling element binding member in this transcription factor, can combine with the CRT/DRE cis acting factor of cold induced gene promoter, thereby activate the expression of cold induced gene, and then improve the winter resistance of plant.The effect of CbCOR gene promoter is vital.The function of CbCOR gene promoter is analyzed by following process:

The activation analysis of cold induced gene promoter in the shepherd's purse.

Make up CbCOR gene plant expression vector pCAMBA2301-CbCORP-CbCOR; Obtain cold-resistant transgenic plant by the agrobacterium-mediated transformation genetic transformation.

The bonded functional analysis of the promotor of cold induced gene and CbCBF gene in the shepherd's purse.

Make up polynary plant expression vector pCAMBA2301-35S-CbCBF-CbCORP-CbCOR.Obtain cold-resistant transgenic plant by agriculture bacillus mediated genetic transformation, carry out the promotor of cold-resistant gene C OR gene in the shepherd's purse and the binding analysis of CbCBF gene.

CbCOR gene plant expression vector establishment of the present invention is as follows:

Make up two kind of plant expression vectors:

The one, only comprise CbCOR gene a: pCAMBA2301-CbCORP-CbCOR;

The 2nd, comprise two genes, a CbCOR gene, CbCBF gene a: pCAMBA2301-35S-CbCBF-CbCORP-CbCOR.

The application of above-mentioned CbCOR gene etc.:

Utilize said gene to make up plant expression vector, obtain cold-resistant transgenic plant by agriculture bacillus mediated genetic transformation.

This gene integration of detections such as PCR, Southern blot and Western blot analysis proofs in the transfer-gen plant genome and prove this gene can be in plant normal expression, the mensuration that the simulation low temperature stress is handled experiment and physical signs proves that all the transgenic line comparison according to having significantly improved the plant cold resistance performance, has proved that commentaries on classics CbCBF and CbCOR gene synergism increase substantially the plant cold tolerance.

The present invention is an experiment material with the shepherd's purse seedling, cDNA sequences Design primer according to shepherd's purse CbCOR and CbCBF, adopt the homologous clone technical point not clone the dna sequence dna of shepherd's purse CbCOR and CbCBF, make up the chromosome walker kit of shepherd's purse simultaneously, adopt the chromosome walking technology to clone the promotor of shepherd's purse CbCOR, they are built into plant expression vector transformation mode plant tobacco, detect the cold-resistant physical signs of transgene tobacco.

The tangible effect that the present invention has:

1, the transfer-gen plant of the cold-resistant gene C bCOR of energy overexpression shepherd's purse among the present invention, its winter resistance is compared with wild plant and is significantly improved.

2, the present invention change the cold-resistant gene C bCOR of shepherd's purse genome sequence (comprising an intron) to intermediate carrier, forward plant expression vector again to, transformed plant is improved significantly the winter resistance of plant then;

3, the present invention uses is that the promoter sequence of CbCOR is linked intermediate carrier, and transformed plant is improved significantly the winter resistance of plant then;

4, the cold-resistant gene C bCOR of shepherd's purse and the CbCBF gene of the present invention's use are connected on the identical carrier, and transformed plant is improved significantly the winter resistance of plant then.

Shepherd's purse is the stronger wild plant of freezing tolerance, is a kind of fabulous cold-resistant genetic resources, so far, the genome sequence of the cold-resistant gene of shepherd's purse and the research of its promoter sequence be yet there are no bibliographical information both at home and abroad.Utilize this characteristic wild plant, therefrom filter out and cold-resistant directly related gene, disclose the cold-resistant mechanism of shepherd's purse, study its hereditary basis, fully excavate its potential genetic resources, and use it for the variety of crops improvement, have huge science and economic worth.

Embodiment

Below in conjunction with the concrete testing data in laboratory and in conjunction with specific embodiments, further set forth the present invention.These embodiment only are used to the present invention is described and are not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, for example the Sambrook equimolecular is cloned: laboratory manual (New York:Cold SpringHarbor Laboratory Press, 1989) condition described in, or the condition of advising according to manufacturer.

The key gene CbCBF of embodiment 1 shepherd's purse CBF approach and the clone of CbCOR gene

1. the cultivation of shepherd's purse seedling

The shepherd's purse seed source is in Shanghai City seeds company, and the shepherd's purse seed is seeded in and contains MS through after disinfecting ₀In the culture tank of substratum, place 25 ℃ to cultivate for 4 weeks down, wherein periodicity of illumination is 16h illumination, the 8h dark.

2.DNA separation

Get the shepherd's purse blade, behind the adding liquid nitrogen, pulverize, use the CTAB method to carry out the extraction of DNA.With the quality of agarose gel electrophoresis identification of dna, on spectrophotometer, measure the content of DNA then.

3. the clone of gene

Amino acid whose coding region (GenBank Accession No.:AY391121 and GenBank AccessionNo.:AY437888) according to the CBF and the COR gene of known shepherd's purse, design two pairs of primers, wherein, a pair of (5 '-ATGTCTTTCTCAGGAGCTGT-3 ' and 5 '-CTACTTGGTGGCATCC TTAG-3 ', be designated as SEQ ID NO.4) be used to clone the nucleotide sequence of the CbCOR gene of shepherd's purse; A pair of (5 '-ATGAACTCATCTTTCTCTGCT-3 ' and 5 '-TTAATAACTCCATAGCGAGAC-3 ', be designated as SEQ ID NO.5) be used to clone the nucleotide sequence of CbCBF gene.Detailed process is as follows: be template with DNA, carry out pcr amplification, the PCR condition be 94 ℃ 5 minutes, carried out 35 circulations in 2 minutes with 94 ℃ 1 minute, 58 ℃ 1 minute and 72 ℃ thereupon, extended 10 minutes with 72 ℃ at last.The electrophoresis detection pcr amplification product obtains expanding fragment length and is respectively 837bp (CbCOR gene) and 660bp (CbCBF gene).Clone, check order with pcr amplification product according to a conventional method then, obtain the sequence shown in SEQ ID NO.1 and the SEQ ID NO.3 respectively.

The clone of the promoter sequence of the key gene CbCOR gene of embodiment 2 shepherd's purse CBF approach

Adopt the promoter sequence of chromosome walking technology amplification shepherd's purse CbCOR gene.Experimental implementation is according to UniversalGenomeWalker ^TMThe service manual of Kit (CLONTECH) carries out.After build up in the chromosome walking storehouse of shepherd's purse, according to 2 primers of SEQ ID NO.1 sequences Design (5 '-TACTCCGCTGTGGAAAGAAGAACCC-3 ' and 5 '-GTCATCGAGGATGTTGCCGTCACCT-3 ', be designated as SEQ ID NO.6), cooperate with 2 joint primers in the test kit, carry out two-wheeled PCR reaction, simultaneously electrophoresis detection two-wheeled pcr amplification product.Selecting second the specific band in taking turns clones, checks order.Sequencing result shows the promoter sequence total length 1180bp of the CbCOR gene of being cloned into.See SEQ ID NO.2.

The promoter sequence and the SEQ ID NO.1 sequence of the shepherd's purse of gained are compared, the promoter sequence that discovery is cloned into has overlapping of 57bp with SEQ ID NO.1 sequence, first initiator codon of shepherd's purse CbCOR genes encoding frame is located at (1124-1126bp), 1123 bases before first initiator codon ATG are 5 ' flanking sequence of CbCOR gene, called after CbCORP.Compare among the promoter sequence submission GenBank with 1123bp, wherein 5 ' the flanking sequence of the cold-regulated protein cor15b precursor of 412bp (666-1070) sequence of 3 ' end and Arabidopis thaliana (Arabidopsis thaliana) has 82% homology, all the other sections are not found any homologous sequence, illustrate that resulting 1123bp sequence is defined as 5 ' flanking sequence of CbCOR gene, and be newfound sequence.Table 1 shows that AtCOR5 ' the flanking sequence sequence of shepherd's purse CbCOR 5 ' flanking sequence sequence and Arabidopis thaliana compares (GAP) table.

The key gene CbCBF of embodiment 3 shepherd's purse CBF approach and the analysis of CbCOR gene

The length of the shepherd's purse CbCBF sequence that the present invention is cloned into is 660bp, and detailed sequence is seen SEQ ID NO.3, and wherein open reading frame is positioned at 1-660 position Nucleotide.By comparing with shepherd's purse CbCBF cDNA sequence, similarity can reach 100%, and illustrating in the nucleotide sequence of shepherd's purse CbCBF does not have intron.The length of the shepherd's purse CbCOR sequence that the present invention is cloned into is 837bp, and detailed sequence is seen SEQ ID NO.1, and wherein open reading frame is positioned at 1-191 and 612-837 position Nucleotide.By comparing with shepherd's purse CbCOR cDNA sequence, the sequence similarity degree of coding region can reach 100%, but the intron of a 420bp is arranged in the CbCOR sequence, and shepherd's purse CbCOR dna sequence dna and shepherd's purse CbCOR cDNA sequence relatively see Table 2.

The activation analysis of the promotor of embodiment 4 shepherd's purse CbCOR genes

In this embodiment, the shepherd's purse CbCOR gene promoter sequence (SEQ ID NO.2) of being cloned into is connected to pCAMBIA1301 (by Australian CAMBIA[the Center ofthe Application of Molecular Biology toInternational Agriculture, Australia] be so kind as to give) on the carrier, make up one and drive the plant expression vector that gus gene is expressed.The promotor transient expression experiment shows, in unifacial leaf and dicotyledons, shepherd's purse CbCOR gene promoter is subjected to all that low temperature is cold induces, and the expression of gus gene is strengthened.Therefore, shepherd's purse CbCOR gene promoter has application promise in clinical practice in the cold-resistant genetically engineered of crop.

The structure of transient expression carrier

In the present embodiment, make up transient expression carrier pCAMBA1301-CbCORP-GUS.

Excise the CaMV35S promotor that plant expression vector pCAMBA1301 carries, be connected into the promotor of CbCOR gene, drive the expression of gus gene.Use plant expression vector pCAMBA1301, as the contrast of promotor transient expression experiment in unifacial leaf and dicotyledons of CbCOR gene.

Particle bombardment maize transformation embryo callus and tobacco spire

With 1300psi system pressure and 28 o'clock mercury column vacuum tightness, on the DuPont PDS1000/He of Bio-Rad company type particle gun, difference maize transformation embryo callus and tobacco spire, every rifle 1 μ g plasmid DNA.The maize callus is at N ₆Inducing culture on the substratum, the tobacco spire is cultivated on the 1/2MS substratum.The inducible expression vector pCAMBA1301-CbCORP-GUS that starts with the CbCOR gene promoter transforms, and is contrast with constitutive expression carrier pCAMBA1301,90 of 150 of each maize transformation embryo callus and tobacco spires.

The low temperature adverse circumstance is handled and the GUS histochemical stain

After the conversion 1/2 maize calli is transferred to N ₆On the substratum, 27 ℃ of dark culturing; 1/2 tobacco spire is transferred on the 1/2MS substratum, under 24 ℃ of conditions, every day the 16h illumination cultivation, as the contrast of normal growth condition.Simultaneously other 1/2 maize calli or tobacco spire are cultivated in 4 ℃ of low temperature on former substratum.

Handle after the 3d, and usefulness GUS reaction solution (the 0.1mol/LNaH2PO4 damping fluid, pH 7.0; 10mmol/L EDTA, pH 7.0; The 5mmol/L Tripotassium iron hexacyanide; The 5mmol/L yellow prussiate of potash; 1.0mmol/L X-gluc; 0.1%Triton X-100) soak above-mentioned each maize calli or tobacco spire of handling, vacuumize (200Mbar repeats 2 times, each 30s), 37 ℃ of incubation 16h clean with distilled water.The maize calli non-pigment disturbs, and directly examines under a microscope and takes pictures.The tobacco spire has pigment to disturb, with stationary liquid (dehydrated alcohol: fixing 1h glacial acetic acid=3: 1), the dehydration of decolouring step by step of 25%-95% ethanol, the distilled water cleaning, observation is taken pictures then.

The expression product of gus gene can be hydrolyzed into blue material with the X-Gluc in the reaction solution, makes tissue present blueness, and the blue depth and amount of speckle can be reacted the expression level of gus gene to a certain extent.Under low temperature and collating condition, maize calli and tobacco spire with constitutive expression carrier pCAMBA1301 transforms normally manifest blue spot.Maize calli and tobacco leaf with cold abduction delivering plasmid pCAMBA1301-COR-GUS conversion, under normal culture condition, detect less than blue spot, can't differentiate positive colony, but under high salt and cold condition, maize calli and tobacco spire all manifest blue spot, and the area of spot and tinctorial strength all are higher than the alignment processing that transforms with constitutive expression carrier.Explanation thus, the CbCOR gene promoter can be subjected to all that low temperature is cold induces in unifacial leaf and dicotyledons, start the expression of structure gene.

Embodiment 5 yeast expressions and functional evaluation

In this embodiment, the cDNA sequence of the CbCOR of total length and CbCBF is built into respectively among the commercial Yeast expression carrier,, its frost resistance is made functional evaluation by its overexpression in yeast.

The structure of Yeast expression carrier, and zymic transforms.

According to the nucleotide sequence of CbCOR and CbCBF gene, the primer of design protein-coding region, and on positive anti-primer, introduce restriction endonuclease sites (this decides according to the pYES2 carrier of selecting for use) respectively, so that construction of expression vector.Total RNA is a template with shepherd's purse, behind pcr amplification, CbCOR and CbCBF is being guaranteed to be cloned into pYES2 carrier (Invitrogen) under the correct prerequisite of reading frame.Identify that good expression vector utilizes the Lithium Acetate method to change yeast saccharomyces cerevisiae (Saccharomycescerevisiae) W303 (ura3-1 over to, can-1-100, leu2-3,112trp1-1, his3-11,15), Screening and Identification obtains containing the engineering bacteria W303-pYES2-CbCOR or the W303-pYES2-CbCBF of pYES2-CbCOR or pYES2-CbCBF protein expression vector.

The cultivation of wild yeast W303, and subculture YPD substratum (1% yeast vat liquor, 2% peptone, 2% glucose, pH5.8).Transformed the cultivation of the W303 of W303-pYES2-CbCOR or W303-pYES2-CbCBF and unloaded pYES2, subculture SD-ura substratum (Difco, uridylic defective type substratum).

Yeast rna extracts and the Northern hybridization analysis

1.W303-pYES2-CbCOR or W303-pYES2-CbCBF cultivates up to OD in the SD-ura substratum ₆₀₀Reach 0.7, the part culture forwards in YPD or the YPGAL substratum and cultivates.

2.24h after, OD ₆₀₀Reach 2.0, the centrifugal 5min of 8000rpm collects thalline.With cell suspension in 400 μ l AE damping fluids (50mM Sodium Acetate, 10mM EDTA adjust pH value to 5.2).

3. the 10%SDS that adds 1/10 volume, thorough mixing.

4. add the acidic phenol (pH4.5) of equal-volume, mix 65 ℃ of preheatings.

5.65 ℃ heating 5min places 10min on ice.

6. the centrifugal 10min of 8000rpm/min at room temperature.

7. water intaking phase adds isopyknic phenol/chloroform/primary isoamyl alcohol (25: 24: 1), the centrifugal 8min of 10000rpm/min.

8. water intaking phase adds the centrifugal 8min of equal-volume chloroform/primary isoamyl alcohol (49: 1) 10000rpm/min.

9. the sodium-acetate that adds 1/10 volume 3M pH5.2, the straight alcohol of 2.5 times of volumes ,-20 ℃ of precipitations of spending the night.

10. deposit sample is removed liquid in 4 ℃ of centrifugal 5min.

11., use an amount of DEPC water dissolution RNA at last dry RNA on ice (placing 15min).

12.30 the total RNA of μ g forwards Hybond-N to behind 112% agarose sex change gel electrophoresis ⁺Nylon membrane is used for the Northern hybridization analysis.The cDNA of total length CbCOR or CbCBF α- ³²Making probe behind the P-dCTP mark hybridizes.Hybond membrane is containing 2 * SSC the 1st time, and in the 0.1%SDS film washing liquid 45 ℃, rinsing 15min; Change 1 * SSC then over to, 45 ℃ of rinsing 5min of the film washing liquid of 0.1%SDS.After raffinate blots with filter paper on the Hybond membrane, wrap up with preservative film.Place the phosphorus screen to carry out radioautograph film.

Northern hybridization shows that CbCOR or CbCBF albumen can be by abduction deliverings in yeast.The expression amount of YPGal substratum inducible protein that contains semi-lactosi is than the YPD substratum abduction delivering amount height that does not contain semi-lactosi, the yeast of the unloaded pYES2 of wild commentaries on classics then all detects less than hybridization signal in two kinds of substratum, show the pYES2-CbCOR of structure or pYES2-CbCBF expression vector can be in yeast fine expression CbCOR or CbCBF albumen, and do not have the interference of the albuminoid of yeast self.

The frost resistance test

Get bacterial classification one ring to be measured, be inoculated in the YPD substratum, 30 ℃ are cultured to lag period, and centrifugal (3000rpm) washed twice is suspended in thalline in the sucrose solution of 3mL different concns, puts into refrigerator and cooled and freezes, and chilling rate is controlled at about 1 ℃.20min thaws in 30 ℃ of incubators.With viable bacteria counting method measure freezing before and freezing after viable count, calculate the thalline survival rate.

With the yeast that changes unloaded pYES2 is contrast, with the sucrose concentration gradient analysis change pYES2-CbCOR or pYES2-CbCBF zymic survival rate behind the freezing treatment 3h.The result shows, the yeast that changes pYES2-CbCOR or pYES2-CbCBF can significantly improve the frost resistance of cell, show as in the solution of sucrose concentration 0%, 2%, 5%, 10%, 15%, change pYES2-CbCOR or pYES2-CbCBF zymic survival rate and all be significantly higher than control group, survival rate is higher than high sugar soln in the low sugar soln.CbCOR or CbCBF overexpression in yeast is described, has improved the growth conditions of yeast under cold coercing, improved survival rate.

The leaf dish method Plant Transformation that embodiment 6 is agriculture bacillus mediated

The structure of plant expression vector

Make up two kind of plant expression vectors:

The one, only comprise CbCOR gene a: pCAMBA2301-CbCORP-CbCOR,

The 2nd, comprise two genes, a CbCOR gene, CbCBF gene a: pCAMBA2301-35S-CbCBF-CbCORP-CbCOR.

In the present embodiment, the CbCOR gene is placed in after the promotor of COR gene, and the promotor of COR gene is subjected to again to be connected to pCAMBA2301 again under the regulation and control of CbCBF, makes up a plant expression vector, is used for the conversion of plant.

Agrobacterium-mediated transformation is to the conversion of tobacco

1. the cultivation picking list bacterium colony of Agrobacterium, in 2ml Agrobacterium liquid substratum, 28 ℃ of overnight incubation; Get the above culture of 1mL, add in the 50mL Agrobacterium liquid substratum, 28 ℃ are cultured to OD ₆₀₀=0.6-1.0; The centrifugal 10min of 8000rpm collects thalline, uses MS ₀Resuspended, make OD ₆₀₀=1.0.

2. cultivate altogether and get young tender, the healthy and strong blade of tobacco aseptic seedling, remove master pulse, blade is cut into leaf dish about 0.8cm * 0.8cm, be placed on MS ₁On the substratum, anti-uppermost leaf dish dehydration; The leaf dish is put into the Agrobacterium nutrient solution, and 190rpm contaminates 10min on 28 ℃ of shaking tables; The medication spoon is pulled the leaf dish out, is placed on the thieving paper of sterilization, blots the unnecessary bacterium liquid of Ye Panshang; The leaf dish that blots is lain in the MS that adds lid layer filter paper ₁On the substratum, about 25 ℃, cultivate about 2d altogether in the dark place.

3. after induced bundle is sprouted common cultivation, according to the following steps the Agrobacterium of leaf panel surface is washed off, shaken frequently therebetween, make the leaf dish fully contact following solution: sterilized water, 15min; Sterilized water+Pyocianil (500mg/L), 15min; MS ₀+ Pyocianil (500mg/L), 20min.The medication spoon is pulled the leaf dish out, is placed on the thieving paper, blots excessive moisture; The leaf dish is placed on MS ₂On the substratum, the leaf plate edge gently is pressed in the substratum; About 25 ℃, the 16h illumination cultivation.

4. root induction is at MS ₂After inducing about 4 weeks on the substratum, the young shoot that leaf margin is grown cuts from base portion and leaf dish, and young shoot is inserted MS ₃Root induction in the substratum, about 25 ℃, the 16h illumination cultivation.

The Molecular Detection of embodiment 7 transgene tobaccos

The transgene tobacco minim DNA extracts and PCR detects

1. the rotaring gene plant blade that takes a morsel shreds, and puts in the mortar, adds 1mL and extracts damping fluid (500mmol/LNaCl 1.5%SDS), grinds pulping for 100mmol/L TrisClpH 8.0,20mmol/L EDTA.

2. suck in the 1.5mL EP pipe, acutely shake mixing.

3.60 ℃ water bath heat preservation 30-60min puts upside down mixing frequently.

4. the centrifugal 5min of 10000rpm under the room temperature.

5. carefully draw supernatant in new centrifuge tube, add the equal-volume chloroform, strenuous vibration.

6. the centrifugal 5min of 10000rpm under the room temperature.

7. carefully supernatant is sucked in the new centrifuge tube.

8. add 1 times of volume Virahol, cotton-shaped DNA precipitation promptly appears for a moment in placement under the room temperature.The centrifugal 5min of 8000rpm discards supernatant; 75% alcohol is washed once, dries precipitation.

9. add 5 μ L RNaseA (10 μ g/ μ L), 37 ℃ of 10min remove RNA.

10. add 50-100 μ L aqueous fusion and separate-20 ℃ of storages.

11. the DNA that extracts is a template, carries HYG gene primer and CbCOR or CbCBF gene-specific primer with expression vector and carries out the PCR reaction respectively, identifies the expression situation of goal gene in the transgene tobacco genome.

The DNA extraction of transgene tobacco and Southern blot detect

Tobacco leaf 100mg is got in the extraction of tobacco leaf DNA, adds 500 μ l and extracts damping fluid (63.77g/L Sorbitol Powder, 12.1g/LTris-HCl pH8.2,1.861g/LEDTA-Na ₂Salt); The nucleic acid lysis buffer of 1 times of volume (200ml1M Tris-HCl pH 7.5,200ml 0.25M EDTA, 400ml 5M NaCl, 20gCTAB, 200ml MQ Water); 0.4 the 5%SDS of volume doubly; Add bisulfite before using and receive (final concentration is 0.02M)], after 30min are placed in 65 ℃ of water-baths, add 750 μ l chloroforms: Virahol (24: 1), centrifugal (12000rpm) 5min pipettes in the new centrifuge tube of supernatant liquor to, adds the cold primary isoamyl alcohol with volume, shake, occur up to the DNA precipitation.Centrifugal (12000rpm) 10min, abandoning supernatant.Wash precipitation with 70% ethanol, be dissolved in after the drying among an amount of TE.

Use spectrophotometric quantitative analysis DNA or RNA, the concrete operations step is as follows: with TE or distilled water testing sample is done an amount of dilution.As blank, is 260nm at wavelength with TE or distilled water, and 280nm and 310nm place regulate uv-spectrophotometric instrument reading to zero, adds sample solution and reads OD value in wavelength place, three places, record OD value, and concentration and purity by definite DNA of calculating or RNA.

We have designed two pairs of primers the preparation of probe, the a pair of nucleotide sequence that is used to clone the CBF of shepherd's purse (5 '-ATGTCTTTCTCAGGAGCTGT-3 ' and 5 '-CTACTTGGTGGCATCCTTAG-3 ') (seeing SEQ IDNO.4), a pair of nucleotide sequence that is used to clone the COR gene (5 '-ATGAACTCATCTTTCTCTGCT-3 ' and 5 '-TTAATAACTCCATAGCGAGAC-3 ') (seeing SEQ ID NO.5).Obtain expanding fragment length and be respectively 660bp (CbCBF gene) and 837bp (CbCOR) template as hybridization probe, adopt the PCR method that probe is carried out digoxin-dUTP (digoxigenin (DIG)-dUTP) (PCR DIG Probe Synthesis Kit, Roche) mark.Add respectively in 500 μ L Eppendorf pipes at 2 labels on the frozen water: the glue of CbCBF or CbCOR reclaims product 0.1 μ L, 10 * PCR buffer (with MgCl ₂) 5 μ L, 10 * PCR Dig Mix, 5 μ L, 10 * dNTP stock solution, 5 μ L, 10 μ mol/L primers, 11 μ L, 10 μ mol/L primer 2s, 1 μ L, Enzyme mix, Expand High Fidelity 0.75 μ L (2.6 unit), ddH ₂O 32.15 μ L, cumulative volume 50 μ L.Forward to behind the mixing on the PCR instrument that has been preheating to 94 ℃ and increase, amplification program is: 94 ℃, and 2min → (94 ℃, 30sec → 60 ℃, 30sec → 72 ℃, 40sec) * and 30cycles → 72 ℃, 10min.Every pipe is got 5 μ L PCR products behind agarose/ethidium bromide/1 * TAE gel electrophoresis and ultraviolet detection, judges that probe is whether on the mark and concentration.

Enzyme is cut and is chosen the restriction enzyme EcoRI that do not have recognition site in the probe sequence and HindIII and respectively the shepherd's purse genomic dna is carried out enzyme and cut, and adds following composition in 2 500 μ LEppendorf pipes respectively:

Composition EcoRI group HindIII group

10×buffer 15μL?NEBuffer?2 15μL?NEBuffer?2

DNA 40μL(30μg) 40μL(30μg)

Enzyme (10units/ μ L) EcoRI 12 μ L HindIII 12 μ L

Deionization H ₂O 83 μ L 83 μ L

Cumulative volume 150 μ L 150 μ L

Gently behind the mixing more than 37 ℃ of incubation 24h, enzyme is cut behind the 3h gently mixing once, every pipe was got 5 μ L in 0.5% agarose/ethidium bromide/1 * TAE gel electrophoresis after enzyme was cut 24h, changed next step until confirming that enzyme is cut over to after thorough substantially.

The Southern gel electrophoresis is cut to every pipe enzyme and is added 355 μ L ddH in the product ₂O and 500 μ L phenol: chloroform: primary isoamyl alcohol, put upside down mixing 10min;

1.12000rpm centrifugal 10min;

2. suct clearly, add the dehydrated alcohol of 1000 μ L-20 ℃ precoolings, mixing;

3.12000rpm centrifugal 10min;

4. abandon supernatant, dry slightly back with 30 μ L TE (pH 8.0) dissolution precipitations;

5. prepare 0.8% agarose/1 * TAE gel;

6. cold back the cutting to every pipe DNA enzyme of gel adds 5 μ L 6 * last sample buffer, last sample in the product;

Under the normal temperature in 1.0vcm ^-1Electrophoresis migrates to 3/5ths distances of gel to bromjophenol blue about 12 hours.

The Southern gel changes film and fixing

1. take out gel after electrophoresis finishes, cut unnecessary colloid, downcut the upper left corner and serve as a mark, vibration sex change 45min in sex change liquid;

2. discard sex change liquid, use ddH ₂O pauses to clean the back in neutralizer in the vibration and 2 * 15min.

3. the good kapillary of frame changes the platform of film, pours 500mL 20 * SSC in the aluminium box, completes double-deck 3MM filter paper bridge and wetting;

4. gel aperture one side is placed on the platform bubble of rushing downwards;

5. cut one and gel Hybond-N of a size ⁺Nylon membrane is laid on the gel bubble of rushing after wetting;

6. surround four limits of gel with Parafilm, in case short circuit;

7. cut with nylon membrane two-layer 3MM filter paper of a size and be laid on the nylon membrane bubble of rushing;

8. press a folded and nylon membrane thieving paper of the same area, evenly press the weights box about a 500g on the top, change more than the film 24h;

9. after the commentaries on classics film is finished, take off gel ethidium bromide staining 10min, and carry out ultraviolet detection, check and change membrane efficiency;

10. use 6 * SSC rinsing nylon membrane, 2 * 5min simultaneously;

11. nylon membrane placed on the thieving paper dries 30min in room temperature;

12. wrap nylon membrane in 80 ℃ of fixing 2h with 3MM filter paper;

13. nylon membrane is wrapped in the tin pool paper, and room temperature preservation is standby.

The Southern hybridization and the detection of probe and nylon membrane

1. nylon membrane take out is immersed 5min among 2 * SSC, DNA towards on be laid in the hybrid pipe;

2. add 20mL DIG Easy Hyb (Roche), in Shake ' n ' Stack hybrid heater (Hybaid), rotate prehybridization 30min in 42 ℃;

3. pour out prehybridization solution, add the fresh DIG Easy Hyb of 12mL, continue at 42 ℃ and rotate prehybridization;

4. the probe liquid that the detection of proper amt has been marked water-bath sex change 5min in boiling water bath, chilling 3min in the frozen water;

5. probe is joined rapidly among the DIG Easy Hyb of heat, 42 ℃ are rotated hybridization 16h;

6. hybridization time is taken out nylon membrane to the back, washes 2 * 5min in 2 * SSC, 0.1%SDS under the greenhouse;

7.68 in 0.1 * SSC, 0.1%SDS, wash nylon membrane 2 * 15min under ℃;

8. rinsing film 2min vibrates in 100mL Washing Buffer (Roche) under the room temperature;

9. blocking-up 1h vibrates film under the room temperature in 100mL Blocking Solution;

Under the room temperature with film in 20mL AP Solution (DIG Luminescent Detection Kit, Roche) in vibration balance 30min;

Rinsing nylon membrane 2 * 15min 11. in 100mL Washing Buffer, vibrate under the room temperature;

12. under the room temperature with nylon membrane and 20mL Detection Buffer (DIG Luminescent Detection Kit, Roche) balance 3min;

13. in hybridization bag, DNA simultaneously upwards also evenly spreads 1mL CSPD, and (DIG LuminescentDetection Kit Roche), seals evenly, in 37 ℃ of preheating 10min with film;

14. in the darkroom, press a Fuji X-ray sheet, about 37 ℃ of insulation 10min;

15. develop, stop shadow and photographic fixing.Develop photographic film with a large amount of tap water after photographic fixing is finished, dry the back, cut the film of every film correspondence by the corresponding position that on film, marks numbering and three gauge points of numbering.

Total RNA of transgene tobacco extracts and Nouthern blot detects

Total RNA of transgene tobacco extracts

Two pairs of primers of southernblot design are adopted in the preparation of probe, with the total RNA of shepherd's purse is template, clone the cDNA sequence (5 '-ATGTCTTTCTCAGGAGCTGT-3 ' and 5 '-CTACTTGGTGGCATCCTTAG-3 ') (seeing SEQ ID NO.4) of the CBF of shepherd's purse and the nucleotide sequence of CBF gene (5 '-ATGAACTCATCTTTCTCTGCT-3 ' and 5 '-TTAATAACTCCATAGCGAGAC-3 ') (seeing SEQ ID NO.5) respectively.Obtain length and be respectively the template of the amplified fragments of 660bp (CbCBF gene) and 417bp (CbCOR gene) as hybridization probe, preparation agarose denaturing formaldehyde gel is done in the triangular flask of baking 2h in one 180 ℃ and is added: DEPC treated water 37.5mL; 10 * MOPS 7.5mL; The low melting-point agarose 0.55g of RNase-free; Cumulative volume 45mL.Change glue in microwave oven, add 10mL formaldehyde when being chilled to 60 ℃, wait behind the mixing to be chilled to and fall glue about 45 ℃, plug the 3mm comb, the cold back of gel is standby.

The preparation of RNA sex change sample and electrophoresis are got the blade of 30 strain transgenic tobacco plants and total RNA of 4 ℃ of inductive transgenic tobacco plants.Adopt ethanol precipitation with more than RNA sample concentration to the 3 μ g/ μ L in advance, and calculate the μ L number of the required RNA of each sample, prepare the 500 μ L Eppendorf pipe of the RNase-free of respective numbers, add respectively after the numbering: total RNA 30 μ g; Formaldehyde 7 μ L; Deionized formamide 20 μ L; 10 * MOPS, 2 μ L; The DEPC treated water complements to behind the 40 μ L mixings in 65 ℃ of sex change 10min, and chilling on ice adds the last sample buffer of micro-ethidium bromide and 6 μ L RNase-free, goes up sample immediately, in 1 * MOPS damping fluid in 1vcm ^-1Electrophoresis treats to change when bromjophenol blue migrates to a half-distance of gel film.

The Northern gel changes film, fixing and hybridization and detects the electrophoresis back that finishes and take out gel, cuts unnecessary colloid, downcuts the upper left corner and serves as a mark; Gel is paused to clean with the DEPC treated water; 20 * the SSC that adopts DEPC to handle changes film;

Prehybridization, hybridization (all at 50 ℃) and develop a film under the environment of RNase-free.Crossover process is all with Southern hybridization and detection.

The freezing test of embodiment 8 transgene tobaccos

The transgenic seedling and the similar wild-type tobacco seedling of contemporaneously cultivating and growing situation in two weeks of will taking root places 4 ℃ of illumination cultivation, and acclimatization to cold 3d places-15 ℃ to handle dark 1-2h afterwards, record survival seedling quantity.Experiment is provided with three groups of repetitions, by the statistical significance of t-test evaluation transgenosis group and wild-type control group difference.

Detect tobacco seedling survival rate as seen by subzero treatment, behind-15 ℃ of processing 1h, Cbcbf transfer-gen plant survival rate is significantly higher than the wild-type contrast; The Cbcbf survival rate reduces behind the 2h, still apparently higher than the wild-type plant.The three groups of data based t-testing identity of repeated experiments P＜0.05, the coincidence statistics meaning.Prompting shepherd's purse gene C bcbf encoded protein may play an important role aspect plant winter hardiness and the frost resistance raising.

The mensuration of the cold-resistant physical signs of embodiment 9 transgene tobaccos

Anthrone colorimetric method for determining soluble sugar content

The making of typical curve: preparation 0,0.2,0.4,0.6,0.8, the sucrose standardized solution of 1.0ug/mL, be settled to 20mL, add the 0.5mL anthrone acetic acid ethyl ester reagent and the 5mL vitriol oil respectively, fully vibration, immediately test tube is put into boiling water bath, all accurately be incubated 1min, so be cooled to room temperature after the taking-up in vain, from comparing, measuring absorbancy at 630nm wavelength F with sky, is ordinate zou with the absorbancy, with sugared content is X-coordinate, the drawing standard curve.

The mensuration of soluble sugar content: take by weighing the transgenic positive seedling leaf (avoiding master pulse) about 0.5g, add 5-10mL distilled water, the boiling water bath 30min that jumps a queue (extracting 2 times), extracting solution changes in the 25mL scale test tube, and it is stand-by to add water to 25mL scale place.Draw extracting solution..5mL in the 20mL test tube, adding distil water 1.5mL, add the 0.5mL anthrone ethyl acetate reagent and the 5mL vitriol oil respectively, fully vibration, immediately test tube is put into boiling water bath and all accurately be incubated 1min, naturally cool to room temperature after the taking-up, compare with blank, under the 630nm wavelength, measure absorbancy, calculate the content of soluble sugar according to typical curve.

Ninhydrin colorimetry is measured proline content

The preparation of PA reagent: take by weighing the 2.5g triketohydrindene hydrate, add Glacial acetic acid 60mL and 6mol/L phosphatase 24 0mL, 80 ℃ are stirred to dissolving fully, cooling rapidly, and 4 ℃ of following refrigerators are preserved standby (existing RE is usefulness now, is no more than 48h).

The making of typical curve: preparation 1.0,2.5,5.0,7.5,10,15,20,25, the L-proline(Pro) standardized solution of 30ug/mL adds respectively in each 2mLr test tube of standardized solution, Glacial acetic acid and mensuration reagent, boiling water bath 15min jumps a queue, measuring its absorbance value (transferring 0 with measuring reagent) after cooling under 515nm, is X-coordinate with the absorbance value, and concentration of proline is an ordinate zou production standard curve.Obtaining the typical curve equation is: y=1.0276+34.8401x (coincidence coefficient: 0.9992).

PA: take by weighing the transgenic positive seedling leaf (avoiding master pulse) about 0.5g, branch adds 5mL ethanol altogether three times and is ground to homogenate, change in the 25mL scale test tube, add water to 25mL scale place, in 80 ℃ of water-baths, heat 20min then, cold slightly permutite and the gac that promptly adds respectively about 0.2g, vibration is evenly left standstill the 10min after-filtration.Get filtrate, Glacial acetic acid and each 2mL of mensuration reagent in test tube, boiling water bath 15min measures its absorbance value (transferring 0 with measuring reagent) after cooling under 515nm.Calculate concentration of proline according to typical curve, represent its proline content (unit: ppm).

The mensuration of membrane permeability

Getting 10 sequins (avoiding master pulse) on the transgenic positive tobacco leaf in the special-purpose scale test tube of 20mL with punch tool, add ultrapure water to the scale place, place moisture eliminator with the vacuum pump 60min that bleeds altogether at twice then, to extract the air in the intercellular substance out, 20min is left standstill in the test tube taking-up of taking out gas, sheet when stirring gently with glass rod then, under 2025 ℃ of constant temperature, measure its specific conductivity with conductivity meter, place 100 ℃ of boiling water bath 10min then, with the tissue of killing the plant, add ultrapure water behind the naturally cooling to scale, under 20-25 ℃ of constant temperature, measure it with conductivity meter and boil specific conductivity, injure rate=processing specific conductivity/boil specific conductivity * 100% with cytolemma, the size of expression tenuigenin membrane permeability.

Table 1 is that AtCOR5 ' the flanking sequence sequence of shepherd's purse CbCOR 5 ' flanking sequence sequence of the present invention and Arabidopis thaliana compares (GAP) table.

gb|AC007087.7|Arabidopsis?thaliana?chromosome?2?clone?F14N22?map?mi551，complete

Sequence?Length＝96685，Score＝318?bits(172)，Expect＝3e-83，Identities＝338/412(82％)，Gaps＝36/412(8％)，Strand＝Plus/Plus

Query 666 ACATTTTCAGCTTAACGACTAATACAACTATCCTTTATGTATACCTCAATGATC-AGTCT?724

|||||||||||||||||||||||||| |?||||||||?|||| || |?|||?|||||

Sbjct?75524?ACATTTTCAGCTTAACGACTAATACATTTTTCCTTTATATATATATCTCT-ATCGAGTCT?75582

Query?725 ACTAATCGTTAGTGTTGAAAGTTGCAAATCAAATAAAAATGCTAACATGTAAAGTTTGGT?784

|?| | |||?|||||||||||||||||?|||?|?|||||||||||||||||?|?|?||

Sbjct?75583?AGT--T-ATTAATGTTGAAAGTTGCAAATAAAACAGAAATGCTAACATGTAAA-TATCGT?75638

Query?785 TGCCAAAAATGCTAACATGTATATAACGGTTATAACCACAACTTGATGGCCGACCTCttt?844

||||||||||||||||||| |||||||||||||||||||||||||||||||||||||||

Sbjct?75639?AGCCAAAAATGCTAACATGTGTATAACGGTTATAACCACAACTTGATGGCCGACCTCTTT?75698

Query?845 ttttGTTGGTGCTAAGTTAGTAACCATAGAAATGATTACACGTAGCTAAAAATACGAACG?904

||| || | | | |||||||||||||||?|||||||||?||| |||||||

Sbjct?75699?TTTC-TT--T--T--G---GTAACCATAGAAATGGTTACACGTAACTAG---TACGAACC?75745

Query?905 AACAAAAACTCTTCTTAGTTAACGATACT-A--GCTAGTA--A-TGCGCAAAAATATCTT?958

|||?|||||||||||||?|| |||||?|?| |?||?|| |?|||||||||||||||

Sbjct?75746?AACGAAAACTCTTCTTA-TT--CGATAGTTAAAGATAATAGCAATGCGCAAAAATATCTA?75802

Query?959 GAACTGACACGTGTACATAAATTTGGATTCTCTCATTGGCCGAGAGGTCTATAAGACGAT?1018

|?|||?||||||||| | ||||||||||| |||||?||||||?|||||||?|||||

Sbjct?75803?GCACTCACACGTGTAG-T---TTTGGATTCTC--ATTGGTCGAGAGATCTATAAAACGAT?75856

Query?1019 ACTATTGGAGATTAGATGCTTCTCATCTCACTTTCTCCATATGTACAAACTC?1070

||||||||||?|||||| |||||||||||||||||||||?|?||?||||||

Sbjct?75857?ACTATTGGAGGTTAGATTTTTCTCATCTCACTTTCTCCATCT-TA-AAACTC?75906

Table 2 is comparison (GAP) table of shepherd's purse CbCOR dna sequence dna of the present invention and CbCOR cDNA sequence.

1 50

CbCOR15b-DNA (1)?ATGTCTTTCTCAGGAGCTGTTCTCAGTGGCATGGGTTCTTCTTTCCACAG

CbCOR15b-mRNA (1)?ATGTCTTTCTCAGGAGCTGTTCTCAGTGGCATGGGTTCTTCTTTCCACAG

51 100

CbCOR15b-DNA (51)CGGAGTAACCAAGCAGAGTAGCGTTGGCGCCGTCGGAGTTGGCCGGAAGA

CbCOR15b-mRNA (51)CGGAGTAACCAAGCAGAGTAGCGTTGGCGCCGTCGGAGTTGGCCGGAAGA

101 150

CbCOR15b-DNA (101)GTGAACTCGTCGTCGTTGCTCAGCGCAAGAAGTCGTTGATATACGCCGTC

CbCOR15b-mRNA (101)GTGAACTCGTCGTCGTTGCTCAGCGCAAGAAGTCGTTGATATACGCCGTC

151 200

CbCOR15b-DNA (151)AAAGGTGACGGCAACATCCTCGATGACCTTAACGAAGCCAC

gagtcta

CbCOR15b-mRNA (151)AAAGGTGACGGCAACATCCTCGATGACCTTAACGAAGCCAC

201 250

CbCOR15b-DNA (201)tgttatatatctttgaacaatcttcctcgtgtgactagtttaagtctatc

CbCOR15b-mRNA (193)--------------------------------------------------

251 300

CbCOR15b-DNA (251)tagatatttttactcaaaaaactctaatgtatattacgtacgtagtactc

CbCOR15b-mRNA (193)--------------------------------------------------

301 350

CbCOR15b-DNA (301)cacatgaatttttgtttttggttaattccaccatattagtttgataaaga

CbCOR15b-mRNA (193)--------------------------------------------------

351 400

CbCOR15b-DNA (351)ctcactgtaatataattaatggtctttgttgaggattgattttatccact

CbCOR15b-mRNA (193)--------------------------------------------------

401 450

CbCOR15b-DNA (401)gagccatttggaaaaaatatacatatataccgatctacttttataaaaaa

CbCOR15b-mRNA (193)--------------------------------------------------

451 500

CbCOR15b-DNA (451)aaagatgtttactttaataagtatatgataaaagtaaatatttgtttttg

CbCOR15b-mRNA (193)--------------------------------------------------

501 550

CbCOR15b-DNA (501)gaaatatataatatataaaagatatatttgagtactctttagaacaaacg

CbCOR15b-mRNA (193)--------------------------------------------------

551 600

CbCOR15b-DNA (551)actcaaattcatgaccaaatgttttaaaaccgtttactcaataatctgaa

CbCOR15b-mRNA (193)--------------------------------------------------

601 650

CbCOR15b-DNA (601)taaacgtgc AAAGAAAGCTTCGGATTTCATGACGGAGAAGACAAAGGA

CbCOR15b-mRNA (194)-----------AAAGAAAGCTTCGGATTTCATGACGGAGAAGACAAAGGA

651 700

CbCOR15b-DNA (651)AGCTTTGGTGGATGGTGAGAAAGCAAAAGACTACGTTGTTGAGAAAACCA

CbCOR15b-mRNA (233)AGCTTTGGTGGATGGTGAGAAAGCAAAAGACTACGTTGTTGAGAAAACCA

701 750

CbCOR15b-DNA (701)TCGAAGCCAATGATACGGCGACAGAGGAAGCAAAGAAAGCTTTTGATTAT

CbCOR15b-mRNA (283)TCGAAGCCAATGATACGGCGACAGAGGAAGCAAAGAAAGCTTTTGATTAT

751 800

CbCOR15b-DNA (751)GTGACGGAGAAAGGCAAAGAAGCCGGAAACAAGGCGGCTGAGTTCGTAGA

CbCOR15b-mRNA (333)GTGACGGAGAAAGGCAAAGAAGCCGGAAACAAGGCGGCTGAGTTCGTAGA

801 837

CbCOR15b-DNA (801)GGGTAAAGCTGGAGAGGCTAAGGATGCCACCAAGTAG

CbCOR15b-mRNA (383)GGGTAAAGCTGGAGAGGCTAAGGATGCCACCAAGTAG

Sequence that the present invention relates to and mark apportion are as follows:

SEQ?ID?NO.1

＜110〉Fudan University

＜120〉shepherd's purse CbCOR gene coded sequence

<160>2

<170>PatentIn?version?3.1

<210>1

<211>837

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

<220>

<221>CDS

<222>(1)...(191)、(612)...(837)

<223>

<400>1

ATG?TCT?TTC?TCA?GGA?GCT?GTT?CTC?AGT?GGC?ATG?GGT?TCT?TCT?TTC?CAC

AGC?GGA?GTA?ACC?AAG?CAG?AGT?AGC?GTT?GGC?GCC?GTC?GGA?GTT?GGC?CGG

AAG?AGT?GAA?CTC?GTC?GTC?GTT?GCT?CAG?CGC?AAG?AAG?TCG?TTG?ATA?TAC

GCC?GTC?AAA?GGT?GAC?GGC?AAC?ATC?CTC?GAT?GAC?CTT?AAC?GAA?GCC?AC

tgagtctatg?ttatatatct?ttgaacaatc?ttcctcgtgt?gactagttta?agtctatcta

gatattttta?ctcaaaaaac?tctaatgtat?attacgtacg?tagtactcca?catgaatttt

tgtttttggt?taattccacc?atattagttt?gataaagact?cactgtaata?taattaatgg

tctttgttga?ggattgattt?tatccactga?gccatttgga?aaaaatatac?atatataccg

atctactttt?ataaaaaaaa?agatgtttac?tttaataagt?atatgataaa?agtaaatatt

tgtttttgga?aatatataat?atataaaaga?tatatttgag?tactctttag?aacaaacgac

tcaaattcat?gaccaaatgt?tttaaaacc?gtttactcaa?taatctgaat?aaacgtgc

A

AAG?AAA?GCT?TCG?GAT?TTC?ATG?ACG?GAG?AAG?ACA?AAG?GAA?GCT?TTG?GTG

GAT?GGT?GAG?AAA?GCA?AAA?GAC?TAC?GTT?GTT?GAG?AAA?ACC?ATC?GAA?GCC

AAT?GAT?ACG?GCG?ACA?GAG?GAA?GCA?AAG?AAA?GCT?TTT?GAT?TAT?GTG?ACG

GAG?AAA?GGC?AAA?GAA?GCC?GGA?AAC?AAG?GCG?GCT?GAG?TTC?GTA?GAG?GGT

AAA?GCT?GGA?GAG?GCT?AAG?GAT?GCC?ACC?AAG?TAG

<211>138

<212>PRT

＜213〉shepherd's purse (Capsella bursa-pastoris)

Met?Ser?Phe?Ser?Gly?Ala?Val?Leu?Ser?Gly?Met?Gly?Ser?Ser?Phe?His

1 5 10 15

Ser?Gly?Val?Thr?Lys?Gln?Ser?Ser?Val?Gly?Ala?Val?Gly?Val?Gly?Arg

20 25 30

Lys?Ser?Glu?Leu?Val?Val?Val?Ala?Gln?Arg?Lys?Lys?Ser?Leu?Ile?Tyr

35 40 45

Ala?Val?Lys?Gly?Asp?Gly?Asn?Ile?Leu?Asp?Asp?Leu?Asn?Glu?Ala?Thr

50 55 60

Lys?Lys?Ala?Ser?Asp?Phe?Met?Thr?Glu?Lys?Thr?Lys?Glu?Ala?Leu?Val

65 70 75 80

Asp?Gly?Glu?Lys?Ala?Lys?Asp?Tyr?Val?Val?Glu?Lys?Thr?Ile?Glu?Ala

85 90 95

Asn?Asp?Thr?Ala?Thr?Glu?Glu?Ala?Lys?Lys?Ala?Phe?Asp?Tyr?Val?Thr

100 105 110

Glu?Lys?Gly?Lys?Glu?Ala?Gly?Asn?Lys?Ala?Ala?Glu?Phe?Val?Glu?Gly

115 120 125

Lys?Ala?Gly?Glu?Ala?Lys?Asp?Ala?Thr?Lys

130 135

SEQ?ID?NO.2

＜110〉Fudan University

＜120〉promoter sequence of shepherd's purse CbCOR gene

<160>2

<170>PatentIn?version?3.1

<210>2

<211>1180

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

<400>1

gatcagtctg?ttgtaatgat?ttctattaaa?ttcacatcaa?ccctcgaaat?aagagacatg 60

gctcgcagtt?aaactaattg?tactcttaca?accagcttgt?gctaggacga?gcatatgtaa 120

ttaagctcgt?tcgttaggct?cttgtctctt?ttgtgacgaa?aataaatggg?atggcacaaa 180

agaaaaataa?ttaattatca?ccacatctcg?gcactccccg?tcacacacat?catcatcaag 240

tagtctatcc?ataattagat?tgtctctgcc?cgataatatg?actctctccc?aaatgtgaag 300

gctctattac?tattagtagt?ggaacttaaa?tcacatccca?aactagcgaa?gtatgtcatt 360

tggcattgcc?acaacagcac?actgttcaat?gttcatatac?ctaattaacc?aatgtgagac 420

gacatatctt?ataggctcct?ttgttgttag?tttcaacttt?caagcaagag?aggaatacac 480

atgctcattt?cacataattt?ttgtgtcctt?tctagcttat?tgtaaataac?gttttgtgct 540

gttgagcaat?ataggtttta?gagtaacatt?gacatttata?agtacaaact?gacccctaag 600

gcactgaaga?taaaccaaaa?taatgttaaa?tagttgttct?taactcaatt?gctaatcact 660

agtacacatt?ttcagcttaa?cgactaatac?aactatcctt?tatgtatacc?tcaatgatca 720

gtctactaat?cgttagtgtt?gaaagttgca?aatcaaataa?aaatgctaac?atgtaaagtt 780

tggttgccaa?aaatgctaac?atgtatataa?cggttataac?cacaacttga?tggccgacct 840

ctttttttgt?tggtgctaag?ttagtaacca?tagaaatgat?tacacgtagc?taaaaatacg 900

aacgaacaaa?aactcttctt?agttaacgat?actagctagt?aatgcgcaaa?aatatcttga 960

actgacacgt?gtacataaat?ttggattctc?tcattggccg?agaggtctat?aagacgatac 1020

tattggagat?tagatgcttc?tcatctcact?ttctccatat?gtacaaactc?agttcctttt 1080

ctattttatt?tatttccttc?taagaaacat?atctctcatg?gcgatgtctt?tctcaggagc 1140

tgttctcagt?ggcatgggtt?cttctttcca?cagcggagta 1180

SEQ?ID?NO.3

＜110〉Fudan University

＜120〉encoding sequence of shepherd's purse CbCBF gene

<160>2

<170>PatentIn?version?3.1

<210>3

<211>660

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

<400>1

ATG?AAC?TCA?TCT?TTC?TCT?GCT?TTC?TCT?GAA?ATG?TTT?GGT?TCC?GAG?TAC

GAG?TCT?CCG?GTT?TCT?TCA?GGC?GGC?GGA?GAT?TAT?TGT?CCG?ACG?CTG?GCG

ACG?AGC?TGT?CCC?AAG?AAA?CCA?GCG?GGT?AGG?AAG?AAG?TTT?CGT?GAG?ACC

CGT?CAC?CCA?GTT?TAC?AGA?GGA?GTT?CGT?CGG?AGA?AAC?TCC?GGT?AAG?TGG

GTT?TGT?GAG?GTT?AGA?GAG?CCA?AAC?AAG?AAA?TCT?AGG?ATT?TGG?CTC?GGA

ACT?TTC?CCT?ACG?GCC?GAG?ATG?GCT?GCT?CGT?GCT?CAC?GAC?GTC?GCC?GCT

ATA?GCC?CTC?CGT?GGC?AGG?TCA?GCC?TGT?CTC?AAT?TTC?GCT?GAC?TCT?GCT

TGG?CGG?CTA?CGG?ATC?CCC?GAG?TCA?ACA?GGC?GCC?AAG?GAA?ATC?CAG?AAG

GCG?GCG?GCT?GAA?GCT?GCG?CTG?GCC?TTT?CAG?GAT?GAG?ATG?ATG?ATG?AGC

GAT?ACC?ACG?ACG?ACG?GAT?CAT?GGC?TTT?GAC?ATG?GAG?GAA?ACG?TTT?GTG

GAA?GCA?ATT?GTG?ACG?GCG?GAA?CAG?AGC?GCT?TCG?TTA?TAT?ATA?GAC?GAA

GAG?GAC?ATG?TTC?GGT?ATG?CCG?AGT?TTG?ATG?GCT?AGT?ATG?GCC?GAA?GGT

ATG?CTT?TTG?CCT?CTG?CCG?TCC?GTA?CAA?TGG?AAC?CAC?AAC?TAT?GAC?ATC

GAC?GGC?GAT?GAT?GAC?GTC?TCG?CTA?TGG?AGT?TAT?TAA

<211>219

<212>PRT

＜213〉shepherd's purse (Capsella bursa-pastoris)

Met?Asn?Ser?Ser?Phe?Ser?Ala?Phe?Ser?Glu?Met?Phe?Gly?Ser?Glu?Tyr

1 5 10 15

Glu?Ser?Pro?Val?Ser?Ser?Gly?Gly?Gly?Asp?Tyr?Cys?Pro?Thr?Leu?Ala

20 25 30

Thr?Ser?Cys?Pro?Lys?Lys?Pro?Ala?Gly?Arg?Lys?Lys?Phe?Arg?Glu?Thr

35 40 45

Arg?His?Pro?Val?Tyr?Arg?Gly?Val?Arg?Arg?Arg?Asn?Ser?Gly?Lys?Trp

50 55 60

Val?Cys?Glu?Val?Arg?Glu?Pro?Asn?Lys?Lys?Ser?Arg?Ile?Trp?Leu?Gly

65 70 75 80

Thr?Phe?Pro?Thr?Ala?Glu?Met?Ala?Ala?Arg?Ala?His?Asp?Val?Ala?Ala

85 90 95

Ile?Ala?Leu?Arg?Gly?Arg?Ser?Ala?Cys?Leu?Asn?Phe?Ala?Asp?Ser?Ala

100 105 110

Trp?Arg?Leu?Arg?Ile?Pro?Glu?Ser?Thr?Gly?Ala?Lys?Glu?Ile?Gln?Lys

115 120 125

Ala?Ala?Ala?Glu?Ala?Ala?Leu?Ala?Phe?Gln?Asp?Glu?Met?Met?Met?Ser

130 135 140

Asp?Thr?Thr?Thr?Thr?Asp?His?Gly?Phe?Asp?Met?Glu?Glu?Thr?Phe?Val

145 150 155 160

Glu?Ala?Ile?Val?Thr?Ala?Glu?Gln?Ser?Ala?Ser?Leu?Tyr?Ile?Asp?Glu

165 170 175

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

180 185 190

Met?Leu?Leu?Pro?Leu?Pro?Ser?Val?Gln?Trp?Asn?His?Asn?Tyr?Asp?Ile

195 200 205

Asp?Gly?Asp?Asp?Asp?Val?Ser?Leu?Trp?Ser?Tyr

210 215

SEQ?ID?NO.4

<211>20

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

ATGTCTTTCTCAGGAGCTGT

<211>20

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

CTACTTGGTGGCATCC?TTAG

SEQ?ID?NO.5

<211>21

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

ATGAACTCATCTTTCTCTGCT

<211>21

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

TTAATAACTCCATAGCGAGAC

SEQ?ID?NO.6

<211>25

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

TACTCCGCTGTGGAAAGAAGAACCC

<211>25

<212>DNA

＜213〉shepherd's purse (Capsella bursa-pastoris)

GTCATCGAGGATGTTGCCGTCACCT

Claims (5)

1. a cold induced gene CbCOR gene and promotor CbCORP thereof of being cloned into from shepherd's purse, its sequence is respectively SEQ ID NO.1 and SEQ ID NO.2.

2. transcription factor CbCBF gene that is subjected to low temperature induction of being cloned into from shepherd's purse, its sequence is SEQ ID NO.3.

3. the plant expression vector of a structure is characterized in that comprising the described CbCOR gene of claim 1: pCAMBA2301-CbCORP-CbCOR.

4. the plant expression vector of a structure is characterized in that comprising described CbCOR gene of claim 1 and claim 2 described CbCBF gene: pCAMBA2301-35S-CbCBF-CbCORP-CbCOR.

5. CbCOR gene as claimed in claim 1 and promotor CbCORP thereof, and the application of CbCBF gene as claimed in claim 2 in cultivating cold resistant plant.