CN101684149B - Guinea grass drought-induced transcription factor and coding gene and application thereof - Google Patents
Guinea grass drought-induced transcription factor and coding gene and application thereof Download PDFInfo
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Abstract
The invention discloses a guinea grass drought-induced transcription factor and a coding gene and an application thereof. The transcription factor is a protein as (1) or (2) as follows: (1) a protein formed by an amino acid sequence shown in a sequence 1 in a sequence table; (2) a protein which is obtained by replacing and/or deleting and/or adding one or a plurality of amino acid residues to an amino acid residue sequence of the sequence 1 in the sequence table, has a transcription activation function, regulates plant resistance, and is derived from (1). LcDREB3a and a coding gene thereof have important theoretical and practical meanings for cultivating the guinea grass with improved resistance and other new species of plant, can be applied to the cultivation and authentication of resistant plant species required by agriculture and animal husbandry and ecological environment improvement, and have higher practical application value. The invention has wide application prospects in the fields of agriculture and animal husbandry.
Description
Technical field
The present invention relates to a kind of with coerce relevant transcription factor and encoding sox thereof and application, the application of the drought-induced transcription factor of sheep's hay and encoding sox thereof and its that particularly relates to a kind of EREBP/AP2 of deriving from family in cultivating the plant that resistance improves.
Background technology
Bad physical environment; Act on plant like low temperature, high temperature, arid and salt marsh etc. and can cause that a series of physiological metabolism takes place in the plant materials reacts; The reversibility that shows as metabolism and growth suppresses, and when serious even cause irreversible injury, thereby causes whole plant dead.Wherein arid disaster is particularly frequent, is the topmost environmental factor that influences plant-growth and crop yield.Utilize the drought tolerance of genetic engineering means improvement crop, the harm of avoiding as far as possible or alleviating the poor environment factor is current biology and modern agricultural technology hot research fields, also is the key subjects that China and world agriculture are badly in need of solution.Plant is called resistance to opposing of coercing and the ability of restraining oneself, and resistance is the flexibility to poor environment that plant forms in the long-term evolution process.For many years; People have carried out big quantity research from physiology, biochemistry, metabolism, ecology and heredity, evolution equal angles; Accumulated abundant data; Particularly along with development of molecular biology, people can be familiar with the resistance of reverse mechanism of plant to coercing on genomic constitution, expression regulation and signal conduction equimolecular level, for the anti-performance of coercing of utilizing genetic engineering means improvement plant has been opened up new approach.Because the complicacy of plant stress-resistance proterties; Adopt traditional breeding method to improve the resistance ten minutes difficulty of plant; Along with development of molecular biology; Genetic engineering means has been opened up the new way of plant stress-resistance breeding, but being separated into of efficient adversity gene limits the engineered principal element of plant stress-resistance.In the past, the clone of adversity gene and application mainly are single functional genes, and like trimethyl-glycine synthase gene, proline(Pro) synthase gene etc., though obtained certain effect, the resistance of plant is not comprehensively improved.
Stress resistance of plant receives controlled by multiple genes, numerous functional genes that the plant stress-resistance proterties is exerted an influence are given full expression to and plays a role, resistance that could more effective improvement plant.Along with the continuous development of biotechnology, research emphasis has turned to the regulatory factor (like promotor and transcription factor) of each species specificity or high efficiency from general functional gene.Because a transcription factor can be regulated and control a plurality of and resistance Expression of Related Genes in the plant, strengthens the effect of a transcription factor, just can make the degeneration-resistant proterties of plant obtain comprehensive improvement.
Summary of the invention
The purpose of this invention is to provide a kind of transcription factor that receives the sheep's hay EREBP/AP2 family of drought-induced expression.
Drought-induced transcription factor provided by the present invention, name is called LcDREB3a, derive from sheep's hay (Leymuschinensis (Trin.) Tzvel), is following 1) or 2) protein:
1) protein of forming by the amino acid residue sequence shown in the sequence in the sequence table 1;
2) with the amino acid residue sequence of sequence in the sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have transcriptional activation function the regulation and control stress resistance of plant by 1) deutero-protein.
For the ease of the purifying of LcDREB3a, label as shown in table 1 on proteinic N-terminal that can the amino acid residue sequence of sequence 1 is formed in by sequence table or C-terminal connect.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | |
| FLAG | ||
| 8 | DYKDDDDK | |
| Strep-tag?II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned 2) but in the LcDREB3a synthetic, also can synthesize its encoding sox earlier, carry out biology again and express and to obtain.Above-mentioned 2) encoding sox of the LcDREB3a in can be through the codon that in the dna sequence dna shown in the 5 ' terminal 119-1225 bit base, lacks one or several amino-acid residue with sequence in the sequence table 2; And/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The cDNA gene of the drought-induced transcription factor of above-mentioned sheep's hay also belongs to protection scope of the present invention.
The cDNA gene of the drought-induced transcription factor of above-mentioned sheep's hay specifically can be following 1)-4) in arbitrary described gene:
1) its encoding sequence is the deoxyribonucleotide from 5 ' terminal 119-1225 position of sequence 2 in the sequence table;
2) its nucleotide sequence is the sequence 2 in the sequence table;
The dna sequence dna hybridization that 3) under stringent condition, can limit with sequence in the sequence table 2 and the dna molecular of the drought-induced transcription factor of above-mentioned sheep's hay of encoding;
4) with 1) gene have the homology more than 90%, and the dna molecular of the drought-induced transcription factor of above-mentioned sheep's hay of encoding.
Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.
Increase above-mentioned LcDREB3a full length gene or its arbitrary segmental primer to also belonging to protection scope of the present invention.
The recombinant vectors, transgenic cell line and the reorganization bacterium that contain the drought-induced transcription factor encoding gene of above-mentioned sheep's hay also belong to protection scope of the present invention.
Available existing plant expression vector construction contains the recombinant expression vector of LcDREB3a gene.Said plant expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for the plant micropellet bombardment, like pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries the drought-induced transcription factor encoding gene LcDREB3a of sheep's hay of the present invention can lead through Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.By the plant transformed host both can be monocotyledonss such as wheat, paddy rice, corn, also can be dicotyledonss such as Arabidopis thaliana, tobacco, soybean, cucumber, tomato, cotton, willow, lucerne place.
When using the gene constructed recombinant expression vector of LcDREB3a; Can before its transcription initiation Nucleotide, add any enhancement type, composing type, organizing specific type or inducible promoter; Like cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., they can use separately or be used in combination with other plant promoter; In addition; When using gene constructed plant expression vector of the present invention; Also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc.; But must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of said translation wave and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening; Can process used plant expression vector, can in plant, express enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker thing (qingfengmeisu qiong affinity tag, kantlex affinity tag etc.) or the anti-chemical reagent marker gene (like anti-weedkiller gene) etc. that can produce colour-change with resistance as adding.
Said recombinant expression vector specifically can be between the MCS of carrier p1302 the recombinant expression vector that the encoding sox that inserts the drought-induced transcription factor of above-mentioned sheep's hay obtains, like p1302-BD-LcDREB3a.
Another object of the present invention provides a kind of method of cultivating the transgenic plant of resistance of reverse raising.
The method of the transgenic plant that cultivation resistance of reverse provided by the present invention improves is in the encoding sox LcDREB3a importing plant with the drought-induced transcription factor of above-mentioned sheep's hay, obtains the transgenic plant that drought resistance improves.
The encoding sox LcDREB3a of the drought-induced transcription factor of said sheep's hay imports in the plant through said recombinant expression vector.
Said plant both can be monocotyledonss such as wheat, paddy rice, corn, also can be dicotyledonss such as Arabidopis thaliana, tobacco, soybean, cucumber, tomato, cotton, willow, lucerne place.
Said plant specifically can be sheep's hay.
In order to prove the Subcellular Localization of LcDREB3a gene; Be inserted on the carrier that contains the GFP green fluorescent protein and obtain recombinant expression vector; Utilize particle bombardment that recombinant expression vector is imported onion epidermis cell, the fluorescence co-focusing microscopically is observed its location situation.The result shows that the LcDREB3a assignment of genes gene mapping is in the nucleus of onion epidermis cell, and is consistent with the prediction of positioning sequence.
Chinense seedlings is carried out arid, cold, high salt, ABA or adverse circumstance such as cradles inducing LcDREB3a expression of gene situation before and after utilizing quantifying PCR method to detect to coerce.
The invention provides a drought-induced transcription factor LcDREB3a of sheep's hay and an encoding sox thereof that derives from EREBP/AP2 family.Experiment showed, LcDREB3a can with the DRE combination of elements, be positioned in the nucleus; Adverse circumstance is induced to receive arid, cold, high salt, ABA or cradle etc., can participate in the response of sheep's hay to multiple environment stress, improves the resistance of plant.LcDREB3a and encoding sox thereof have important theory and practical significance for sheep's hay and other neies variety of plant of cultivating the resistance raising; Can be used for the cultivation and the evaluation of the required resistance plant kind of husbandry and ecological environment treatment, have higher actual application value.The present invention has broad application prospects in agricultural, livestock industry field.
Description of drawings
Fig. 1 is the agarose gel electrophoresis detected result through the total RNA of chinense seedlings of 20%PEG processing
Fig. 2 is the agarose gel electrophoresis detected result of 3 ' RACE product
Fig. 3 is the agarose gel electrophoresis detected result of 5 ' RACE product
Fig. 4 is the agarose gel electrophoresis detected result of pcr amplification LcDREB3a full-length cDNA
Fig. 5 is the structural representation of LcDREB3a full-length cDNA
Fig. 6 is the homology analysis result of other DREB proteinoid aminoacid sequence of having cloned in LcDREB3a and the plant
Fig. 7 is the systematic evolution tree analytical results of other DREB proteinoid aminoacid sequence of having cloned in LcDREB3a and the plant
Fig. 8 is the tissue specific expression analytical results of LcDREB3a gene
Expression of results under the stress conditions such as Fig. 9 cradles in difference for the LcDREB3a gene, arid, low temperature and high salt
Figure 10 is the Subcellular Localization result of LcDREB3a gene
Figure 11 is the transcriptional activation activity analytical results of LcDREB3a
Embodiment
Method is ordinary method if no special instructions described in the following embodiment, and the primer is given birth to worker biotech firm by Shanghai and synthesized.
The acquisition of embodiment 1, the drought-induced transcription factor gene LcDREB3a of sheep's hay full length cDNA sequence
One, the clone of the drought-induced transcription factor gene LcDREB3a3 ' of sheep's hay terminal sequence
1, the extraction of vegetable material processing and total RNA
With sheep's hay (lucky living No., Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province) seedling is material, and PEG handles and extracts its total RNA after 6 hours, carries out 1% agarose gel electrophoresis and detects, and the result is as shown in Figure 1.The result shows that the RNA that is extracted has two tangible electrophoretic bands, is followed successively by 28S RNA and 18S RNA from top to bottom, shows to have obtained higher, the more complete total RNA of purity.
2, the clone of the drought-induced transcription factor gene LcDREB3a3 ' of sheep's hay terminal sequence
Amino acid residue sequence according to disclosed plant DREB is sought conservative region, and according to conservative region sequences Design primer, concrete primer sequence is following:
LC1：5′-AGGCATTACGGCCAACAAGAGA-3′
The total RNA through arid sheep's hay of handling (Ji is given birth to No., Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province) seedling that extracts with above-mentioned steps 1 is a template, uses PrimeScript
TMThe requirement of 1st Strand cDNA Synthesized Kit test kit (Takara company) and reference reagent box specification sheets, synthetic its first chain cDNA of counter-rotating.Reaction system and reaction conditions are following: Oligo-dT (10pmol/ μ l) 1 μ l; Total RNA (≤1 μ g) 2 μ l; DNTP Mixture (10mmol/l each) 1.0 μ l, 5 * Buffer4.0 μ l, RNase Inhibitor (40U/ μ l) 0.5 μ l; PrimeScriptRTase (200U/ μ l) 0.5 μ l, RNase-free distilled water11 μ l; 65 ℃ of 5min, 42 ℃ of 45min, 70 ℃ of 15min.With the synthetic first chain cDNA be stored in-20 ℃ subsequent use.
Be template with the first chain cDNA that obtains again, pcr amplification is carried out in primer LC1 and primer OligodT-adaptor5 '-GATTTCTGTCCGACGACTTTTTTTTTTTTTTTTTT-3 ' pairing; The PCR reaction system is: each 1 μ l of cDNA template, LC1 primer and OligodT-adaptor, 10 * Buffer2.5 μ l, dNTP Mixture (10mmol/l each) 2 μ l, Taq enzyme 0.25 μ l, ddH
2O12.25 μ l; Reaction conditions is: 94 ℃ of preparatory sex change 5min of elder generation; 94 ℃ of 30s then, 55 ℃ of 30s, 72 ℃ of 60s, totally 36 circulations; Last 72 ℃ are extended 10min.
After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, the result is as shown in Figure 2.Wherein, swimming lane M is the dna molecular amount standard of DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is 3 ' RACE pcr amplification product, the result shows, has obtained the purpose fragment that length is about 750bp through pcr amplification.Reclaim and purifying 3 ' RACE product; Be connected on the PMD-18T carrier, connect product transformed into escherichia coli DH5 α competent cell, screening positive clone carries out bacterium liquid PCR to be identified; The plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.The result shows; This segmental length is 744bp; Its deoxyribonucleotide sequence has higher homology with 3 ' terminal sequence of known DREB genoid in the plant shown in sequence in the sequence table 3, show that this fragment possibly be 3 ' terminal sequence of the conjugated protein encoding sox of sheep's hay AP2 class.
Two, the clone of sheep's hay drought stress induced transcription factor gene LcDREB3a5 ' terminal sequence
LcDREB3a gene 3 ' according to above-mentioned steps one obtains is held cDNA sequences Design primer: R:5 '-TTCGTTCCAGCAGCCCCCAGTCGTCG-3 '; The sheep's hay of handling through arid of extracting with step 1 (give birth to No. one by Ji; Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province) total RNA of seedling is a template; Adopt 5 ' the RACE test kit and the reference reagent box specification sheets of Promega company, its first chain cDNA is synthesized in reverse transcription.Reaction system and condition are following: 1 μ l RNA, 1 μ l5 '-CDS primer A, 1 μ l SMART II A oligo; 1 μ l DTT (20mM), l μ l dNTP Mix (10mM), 1 μ l MMLV ReverseTranscriptase; 2 μ l5X First-StrandBuffer, 2 μ l sterile H
2O; 70 ℃ of 2min, 2min on ice, 42 ℃ of 1.5h, 72 ℃ of 7min.
The first chain cDNA to obtain is a template; Primer R and primer UPM (Promega company: Long (0.4 μ M): 5 '-CTAATACGACTCACTATAGGGCAAGCAGTGGTATCAACGCAGAGT-3 ', Short (2 μ M): 5 '-CTAATACGACTCACTATAGGGC-3 ') match and carry out pcr amplification; The PCR reaction system is: 1 μ l50X Advantage2Polymerase Mix; 34.5 μ l PCR-Grade Water, 5 μ l10XAdvantage2PCR Buffer, l μ l dNTP Mix (10mM); 1 μ l50X Advantage2PolymeraseMix; 5 μ lUPM, 1 μ l primer R, 2.5 μ lcDNA templates; Reaction conditions is: first 94 ℃ of 30s, 68 ℃ of 30s then, 70 ℃ of 60s, totally 40 circulations; Last 70 ℃ are extended 10min.
After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, the result is as shown in Figure 3.Wherein, swimming lane M is the dna molecular amount standard of DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is 5 ' RACE pcr amplification product, the result shows, has obtained the purpose fragment that length is about 1000bp through pcr amplification.Reclaim and purifying 5 ' RACE product; Be connected on the PMD-18T carrier, connect product transformed into escherichia coli DH5 α competent cell, screening positive clone carries out bacterium liquid PCR to be identified; The plasmid that extracts positive colony checks order, and sequencing result is carried out BLAST analyze.The result shows; This segmental length is 1006bp; Its deoxyribonucleotide sequence has higher homology with 5 ' terminal sequence of known DREB2 genoid in the plant shown in sequence in the sequence table 4, show that this fragment possibly be 5 ' terminal sequence of the conjugated protein encoding sox of sheep's hay AP2 class.
Three, the acquisition of the drought-induced transcription factor gene LcDREB3a of sheep's hay full length cDNA sequence and PCR detect
The length of utilizing above-mentioned steps one and step 2 to obtain is the overlap between 750bp and the 1000bp fragment, the full length cDNA sequence of the LcDREB3a gene that splicing obtains by Contig software, and its deoxyribonucleotide sequence is shown in sequence in the sequence table 2.Sequence 2 is its encoding sequence by 1543 based compositions from 5 ' the 119th the-the 1225th at end in the sequence table, and coding has the protein of the amino acid residue sequence shown in the sequence 1 in the sequence table.Sequence 1 is made up of 369 amino-acid residues in the sequence table.According to the following primer of LcDREB3a full length gene cDNA sequences Design: 1:5 '-GGATCCCCTTTTCTTTCTCCCCTC-3 ',
2：5′-TTGCCACGGAATTGCATTTTCA-3′。
The total RNA through arid sheep's hay of handling (Ji is given birth to No., Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province) seedling that extracts with above-mentioned step 1 is a template through the reverse transcription synthetic first chain cDNA, carries out pcr amplification.Pcr amplification product is carried out 1% agarose gel electrophoresis detect, the result is as shown in Figure 4.Wherein, swimming lane M is the dna molecular amount standard of DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane 1 is a pcr amplification product.The result shows, obtained the fragment that length is about 1500bp through pcr amplification.Reclaim and this product of purifying, be connected on the PMD-18T carrier, connect product transformed into escherichia coli DH5 α competent cell, screening positive clone carries out bacterium liquid PCR identifies that the plasmid that extracts positive colony checks order.Sequencing result shows; The sequence that this pcr amplification product and above-mentioned splicing obtain is identical in the amplification part; The sequence total length is 1505bp; Showing that 3 ' the RACE fragment and 5 ' the RACE fragment of being cloned belong to same gene, is LcDREB3a with this unnamed gene, with its encoded protein called after LcDREB3a.
The bioinformatic analysis of embodiment 2, LcDREB3a and proteins encoded thereof
One, the structure function of LcDREB3a Gene Sequence Analysis and proteins encoded thereof prediction
Utilize DNAMAN and OMIGA software that the full length cDNA sequence of the LcDREB3a of embodiment 1 acquisition is carried out bioinformatic analysis; This sequence total length 1543bp; From 5 ' the 119th the-the 1226th at end is ORF; The protein LcDREB3a that coding is made up of 369 amino-acid residues, the structural representation of LcDREB3a is as shown in Figure 5.Infer that its molecular weight is 40kDa, iso-electric point pI value 4.72.Analyze the structural domain of LcDREB3a with SMART (http://coot.embl-heidelberg.de/SMART/) server; The result shows; This albumen contains a typical EREBP/AP2 structural domain; Be that sequence 1 is from the 91st the-the 154th amino acids residue of aminoterminal in the sequence table, this structural domain is made up of 64 amino-acid residues, shows that this albumen is a member in the EREBP/AP2 family.Analytical results shows that also there is a typical acid activatable zone in the proteic carboxyl terminal of LcDREB3a.Above-mentioned analytical results shows that LcDREB3a is a kind of transcription factor, belongs to the AP2 protein family.
The homology and the systematic evolution tree analysis of the DREB2 proteinoid encoding amino acid sequence that two, other has been cloned in LcDREB3a and the plant
(the GeneBank number of landing is: AtDREB2A (NP_196160) to the aminoacid sequence of the DREB class transcription factor that other has been cloned in LcDREB3a and the plant to utilize DNAMAN software; GhDBP2 (AY619718); GmDREB2 (ABB36645); HvDRF1 (AAO38209), TaDREB4B (AAX13283) and ZmDREB2A (BAE96012)) carry out homology analysis and systematic evolution tree analysis, the homology analysis result is as shown in Figure 6.The result shows; The homology of LcDREB3a and monocotyledons wheat, barley and DREB transcription factor of corn is respectively 90%, 87% and 61%; And be respectively 36%, 27% and 38% with the homology of dicotyledons Arabidopis thaliana, soybean and cotton DREB class transcription factor; The homology that shows LcDREB3a and monocotyledons DREB class transcription factor is higher, and lower with the homology of dicotyledons DREB class transcription factor.The systematic evolution tree analytical results is as shown in Figure 7, and the result shows that bigger difference has appearred in DREB proteinoid during evolution, but this albumen is relatively conservative in each type plant.
The tissue specific expression analysis of embodiment 3, LcDREB3a gene
Extract sheep's hay (lucky the giving birth to No. one of handling through drought stress among the embodiment 1 respectively; Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province) total RNA of the root of seedling, rhizome, leaf sheath and Ye Si kind tissue; Utilize primer 5 '-CAACATCCAACCACTCCG-3 ' and 5 '-AGGCTCCAATGGCTCAAAG-3 ' carries out RT-PCR and analyzes, simultaneously with the Actin gene as interior mark.After reaction finishes, the RT-PCR product is carried out 1% agarose gel electrophoresis detect, the result is as shown in Figure 8.Wherein, Swimming lane M is the dna molecular amount standard of DL2000DNA molecular weight standard (Beijing Quanshijin Biotechnology Co., Ltd), and swimming lane R is a LcDREB3a expression of gene situation in the root, and swimming lane RH is a LcDREB3a expression of gene situation in the rhizome; Swimming lane S is a LcDREB3a expression of gene situation in the leaf sheath; Swimming lane L is a LcDREB3a expression of gene situation in the leaf, and the result shows that the expression of LcDREB3a gene in four kinds of tissues there are differences; Minimum with expression amount in the leaf sheath, expression amount is higher in leaf and the rhizome.
Embodiment 4, the expression pattern analysis of LcDREB3a under different abiotic stress factor conditions
With the normal growth sheep's hay in 8 weeks (lucky giving birth to No. one; Jisheng Improved Variety Station of Aneurolepidium Chinense, Jilin Province) seedling carries out the low temperature (4 ℃) of different time (0,0.5,3,6,12,24 or 48 hour), high salt (400mM NaCl), arid (20% polyethylene glycol 6000), dormin (ABA, 100 μ M) respectively and cradles coercing processing.Extract total RNA of the chinense seedlings of above-mentioned different treatment respectively, quantifying PCR method is analyzed the expression pattern of LcDREB3a gene under different abiotic stress factor conditions, and the result is as shown in Figure 9.The result shows that LcDREB3a gene transcription level obviously receives inducing of drought stress, and along with the prolongation of the time of coercing, the relative expression quantity of LcDREB3a gene increases sharply, and reaches peak to 12h, and expression amount afterwards reduces gradually; LcDREB3a expression of gene amount is low slightly under low temperature induction, when 12h, reaches peak, and at ABA, cradle and high salt is induced down, LcDREB3a expression of gene amount increases sharply, and reaches peak at 6h.
Embodiment 5, the checking of LcDREB3a functional transcription factor
One, the Subcellular Localization of LcDREB3a
The LcDREB3a gene that the foregoing description 1 amplification is obtained is inserted into through between the NcoI and SpeI restriction enzyme site of the carrier p1302 (available from CAMBIA company) that same enzyme is cut, with the recombinant expression vector called after p1302-BD-LcDREB3a that obtains after NcoI and SpeI enzyme are cut.With the above-mentioned recombinant expression vector p1302-BD-LcDREB3a of 5 μ g with 360 μ g goldc grains embeddings after, adopt particle bombardment to change onion epidermis cell over to, simultaneously with the onion epidermis cell that changes carrier p1302 over to as contrast.The onion epidermis cell that transforms is observed down and photograph at laser confocal scanning microscope (Bio-Rad MRC1024) after cultivating 24h down at 25 ℃, dark condition on MS (Murashige-Skoog) substratum, and the result is shown in figure 10.Wherein, Figure 10 a and Figure 10 d are the form of onion epidermis cell under the bright field.As can be seen from Figure 10, change that expressed proteins is distributed in the whole cell in the transgenic cell of carrier p1302 over to, like Figure 10 b and Figure 10 c; Change that expressed proteins then is positioned in the nucleus in the transgenic cell of LcDREB3a recombinant expression vector p1302-BD-LcDREB3a over to, like Figure 10 e and Figure 10 f.The result shows that LcDREB3a is positioned in the nucleus.
Two, the transcriptional activation activity analysis of LcDREB3a
The LcDREB3a gene that the foregoing description 1 amplification is obtained is inserted into through between the BamHI and SalI restriction enzyme site of the Yeast expression carrier pBridge (available from CAMBIA company) that same enzyme is cut, with the recombinant vectors called after pBridge-BD-LcDREB3a that obtains after BamHI and SalI enzyme are cut.It is (available from U.S. Clontech company that recombinant vectors pBridge-BD-LcDREB3a is imported to the yeast AH109 strain that contains His3 and LacZ reporter; Cat. no K1612-1) in; Change Yeast expression carrier pBridge over to yeast AH109 strain system as negative contrast simultaneously, changing pGAL4 (available from U.S. Clontech company) over to yeast AH109 strain is as over against photograph.The transgenic yeast bacterium is cultivated not containing on the SD substratum (SD/-His-Trp) of His and Trp, and the result is shown in figure 11.Figure 11 a representes the position of various transgenic yeasts on flat board, and wherein, 1 for containing the yeast strain of LcDREB3a gene, and 2 for changing pGAL4 over to as the yeast strain over against photograph, and 3 for changing the yeast strain of Yeast expression carrier pBridge as negative contrast over to; Figure 11 b representes yeast strain at the upgrowth situation that does not contain on the SD substratum of His and Trp, and Figure 11 c representes the zymic betagalactosidase activity.The result shows; The transgenic yeast bacterium that changes Yeast expression carrier pBridge over to is not containing on the SD substratum of His and Trp and can not grow, and the transgenic yeast bacterium that changes recombinant vectors pBridge-BD-LcDREB3a over to can not contain on the SD substratum of His and Trp growth and show blue.The result shows that LcDREB3a has transcriptional activation activity.
Sequence table
<160>4
<210>1
<211>369
<212>PRT
< 213>sheep's hay (Leymus chinensis)
<400>1
<210>2
<211>1543
<212>DNA
< 213>sheep's hay (Leymus chinensis)
<400>2
<210>3
<211>744
<212>DNA
< 213>sheep's hay (Leymus chinensis)
<400>3
<210>4
<211>1006
<212>DNA
< 213>sheep's hay (Leymus chinensis)
<400>4
Claims (9)
1. transcription factor, the protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1.
2. the encoding sox of the said transcription factor of claim 1.
3. encoding sox according to claim 2 is characterized in that: the cDNA gene of said transcription factor is that nucleotide sequence is the sequence 2 described genes in the sequence table.
4. the recombinant expression vector or the reorganization bacterium that contain claim 2 or 3 said genes.
5. recombinant expression vector according to claim 4 is characterized in that: said recombinant expression vector is for inserting the recombinant expression vector that claim 2 or 3 said genes obtain between the MCS of carrier pBridge or p1302.
6. a method of cultivating the transgenic plant of resistance raising is that claim 2 or 3 described encoding soxs are changed in the plant, obtains the transgenic plant that resistance improves.
7. method according to claim 6 is characterized in that, claim 2 or 3 described encoding soxs are to import in the plant through claim 4 or 5 described recombinant expression vectors.
8. method according to claim 6 is characterized in that, said plant is a wheat.
9. according to arbitrary described method among the claim 6-8, it is characterized in that: said resistance is the resistance to low temperature, high salt, arid or dormin.
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Non-Patent Citations (5)
| Title |
|---|
| GS Liu et al.Isolation and characterization of dehydration responsive element binding protein from Leymus chinensis.《GenBank: EU999998.1》.2008, * |
| GS Liu et al.Isolation and characterization of dehydration responsive element binding protein from Leymus chinensis.《GenBank:ACH73204.1》.2008, * |
| 李丽娟等.植物中的DREB类转录因子.《首都师范大学学报(自然科学版)》.2006,第27卷(第05期), * |
| 赵利锋等.AP2/EREBP转录因子在植物发育和胁迫应答中的作用.《植物学通报》.2008,第25卷(第01期), * |
| 阳文龙等.高羊茅中一个DREB类转录因子基因的分离及鉴定分析.《应用与环境生物学报》.2006,第12卷(第02期), * |
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