CN107287195A - One kind is in the single-minded expression promoter of guard cell and its creation method - Google Patents
One kind is in the single-minded expression promoter of guard cell and its creation method Download PDFInfo
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- CN107287195A CN107287195A CN201710350170.6A CN201710350170A CN107287195A CN 107287195 A CN107287195 A CN 107287195A CN 201710350170 A CN201710350170 A CN 201710350170A CN 107287195 A CN107287195 A CN 107287195A
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- C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8237—Externally regulated expression systems
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
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Abstract
The invention discloses one kind in the single-minded expression promoter of guard cell and its creation method, create one by environment stress induce can guard cell specificity expression promoter, pass through the method for gene expression regulation, make plant when moisture supply is sufficient, stomatal opening, and when adverse circumstance such as low temperature, arid come interim, stomata is closed rapidly, to reduce moisture loss, strengthen the resistance of reverse and WUEL of plant.
Description
Technical field
It is specifically a kind of abiotic inverse by low temperature, arid and high salt etc. the present invention relates to biotechnology research field
The induction of border stress factors can in guard cell specificity expression's promoter establishment process, can be used for the gene work of plant
Journey.
Background technology
Plant is among its all one's life, unavoidably by the stress damage of the abiotic stresses such as low temperature, arid, high salt.Stomata is not
The only change of sensing external environment signal, but also be plant and the extraneous door for carrying out gas exchanges.Global plant is annual
About consume 65% freshwater resources, these moisture are lost by the transpiration of stomata.Therefore, from regulation and control
Air vent switch movement into hand, strengthens the saving water, resisting drought ability of plant, it has also become the focus paid close attention to both at home and abroad.
The content of the invention
For the above-mentioned state of the art, the present invention is intended to provide a kind of in the single-minded expression promoter of guard cell and its establishment side
Method, realize create one by environment stress induce can guard cell specificity expression promoter, pass through gene expression
The method of regulation and control, makes plant when moisture supply is sufficient, stomatal opening, and when adverse circumstance such as low temperature, arid come interim, stomata is rapid
Close, to reduce moisture loss, strengthen the resistance of reverse and WUEL of plant.
To achieve these goals, the present invention is adopted the following technical scheme that：
One kind is in the single-minded expression promoter of guard cell, and its nucleotide sequence is SEQ NO ID.1.
The creation method of the above-mentioned single-minded expression promoter of guard cell, comprises the following steps：
S1 designs a pair of specific primers：
Sense primer：5′-CACAAGCTTCACCCACTTTTCTTCCTCT-3′；
Anti-sense primer：5′-GTCTCTAGAAGTGGCGAGTTGTGGTT-3′；
S2 using round pcr by ABA response elements amplified from Dc3 promoters come；
S3 designs another pair primer：
Sense primer：5′-CACTCTAGAAGTAGGCAAGTAGCAATGTC-3′；
Anti-sense primer：5′-TTCCCCCGGGTATTATATATTGCTGCTTC-3′；
S4 using round pcr by guard cell specificity expression element amplified from kst1 promoters come；
The pcr amplification product obtained in step S2 and step S4 is first coupled on T-easy carriers and is sequenced by S5, after confirmation
The pcr amplification product HindIII and XbaI enzyme cutting that will first be obtained in plant expression vector pBI121 and step S2, are separately recovered
PBI121 large fragment and purpose fragment, is attached with ligase again afterwards, then by obtained connection product and step S4
In obtained pcr amplification product XbaI and SmaI digestions, the large fragment and purpose fragment of the connection product is separately recovered, it
It is attached again with ligase afterwards, the promoter of the guard cell specificity expression induced by environment stress so created is with regard to structure
It is built on the plant expression vector pBI121 containing gus reporter gene.
The beneficial effects of the present invention are：Create one by environment stress induce can be in guard cell's selectivity table
The promoter reached, by the method for gene expression regulation, makes plant when moisture supply is sufficient, stomatal opening, and works as adverse circumstance such as
Low temperature, arid come interim, and stomata is closed rapidly, to reduce moisture loss, strengthen the resistance of reverse and WUEL of plant.
Brief description of the drawings
Fig. 1 observes schematic diagram for the histochemical stain of promoter of the present invention.
Fig. 2 a- Fig. 2 d for the present invention promoter ABA, high salt, damage to plants caused by sudden drop in temperature and arid under GUS determination of activity experimental results show
It is intended to.
Embodiment
Below with reference to accompanying drawing, the invention will be further described, it is necessary to which explanation, the present embodiment is with this technology side
Premised on case, detailed embodiment and specific operating process are given, but protection scope of the present invention is not limited to this reality
Apply example.
One kind is in the single-minded expression promoter of guard cell, and its nucleotide sequence is SEQ NO ID.1.
The structure of the promoter, by ABA response elements, guard cell specificity expression element and basal promoter element structure
Into.
ABA response elements, are made up of the TCGT motifs of 7 series connection.
Guard cell's specificity expression's element, is made up of 3 guard cell's specificity T AAAG motifs.
Basal promoter element includes 1 TATA box.
A kind of creation method in the single-minded expression promoter of guard cell, comprises the following steps：
S1 designs a pair of specific primers：
Sense primer：5′-CACAAGCTTCACCCACTTTTCTTCCTCT-3′(Hind111)；
Anti-sense primer：5′-GTCTCTAGAAGTGGCGAGTTGTGGTT-3′)(Xba1)；
S2 using round pcr by ABA response elements amplified from Dc3 promoters come；
S3 designs another pair primer：
Sense primer：5′-CACTCTAGAAGTAGGCAAGTAGCAATGTC-3′(Xba1)；
Anti-sense primer：5′-TTCCCCCGGGTATTATATATTGCTGCTTC-3′(Sma1)；
S4 using round pcr by guard cell specificity expression element amplified from kst1 promoters come；
The pcr amplification product obtained in step S2 and step S4 is first coupled on T-easy carriers and is sequenced by S5, after confirmation
The pcr amplification product HindIII and XbaI enzyme cutting that will first be obtained in plant expression vector pBI121 and step S2, are separately recovered
PBI121 large fragment and purpose fragment, is attached with ligase again afterwards, then by obtained connection product and step S4
In obtained pcr amplification product XbaI and SmaI digestions, the large fragment and purpose fragment of the connection product is separately recovered, it
It is attached again with ligase afterwards, the promoter of the guard cell specificity expression induced by environment stress so created is with regard to structure
It is built on the plant expression vector pBI121 containing gus reporter gene.
In this way, new chimeric promoters just instead of the 35S promoter of constitutive expression completely.
Performance test
Using agriculture bacillus mediated genetic plant transformations method, the above-mentioned plant expression vector built is transferred to tobacco
In, using the method for histochemical stain, observed, such as Fig. 1, a refers to control；B, c, d, e refer to the low temperature (4 of 1 hour respectively
DEG C), arid (PEG6000), salt (250mM NaCl), (100 μM) processing of ABA；F refers to the observation result of control epidermal cell, and g refers to
Epidermal cell by the low temperature of 1 hour (4 DEG C), arid (PEG6000), salt (250mM NaCl), (100 μM) processing of ABA is seen
Examine result.GUS activity highest in epidermis is confirmed using the method for enzyme activity determination simultaneously, and by arid, ABA, high salt and cold
Harmful induction, as shown in Fig. 2 a- Fig. 2 d, the position of the wherein tobacco that each cylinders of Fig. 2 b- Fig. 2 d are referred to is identical with Fig. 2 a.
For those skilled in the art, technical scheme that can be more than and design, make various corresponding
Change and deform, and all these change and deformation should be construed as being included within the protection domain of the claims in the present invention.
SEQUENCE LISTING (sequence tables）
<110>Li Jun, Song Xiyun, Liu Ligong
<120>One kind is in the single-minded expression promoter of guard cell and its creation method
<160>1
<210> 1
<211>746
<212> DNA
<213>Artificial sequence
<400> 1
CACAAGCTTC ACCCACTTTT CTTCCTCTTT TCCACTACTT TATACATATT TCTTAACCTC 60
CGTGCCCAAC CCATTTGATA AGAAATCGGA GGCACGGAGG GAGTATATCA TTACACATAT 120
ATAATAAATA TAATTTACAA ATATTTTATG TACTTTCGTG TATTTCGTGT ACCCTAAATG 180
AAAACCCATA TCCGACACGA AATCTATCGT GTACTTTCGT GTTTCGTGTA TCGAAAATCA 240
AAAACCGTAT CATTTTTTTC GTGTCGTTTC GTTTCGTATT AGCGTGTTAC GTGTCAAATG 300
GTCAGGTCTA GTTGAAAACG AAACCGTGAG ACGTGTATGG CATTGGTCAA ACGTGTTGGA 360
ACTTGAAAGC GAGACATCTA TATAAATTAT AAACTTGACT TGTCATGCGT CACCGTTAGT 420
CTTTATCCAC AGACATCAAC CATAAAGTTG TGTCGATTAT CCATGCACAC CCGAGCTAAC 480
CACAACTCGC CACTTCTAGA GTAGGCAAGT AGCAATGTCA CGTTCTTAAA GCTAAATGCT 540
TTTTCAAAAG AATCACAATA AAGAAACACT TGACCCGTGT ATCACCCCAA CTACTTCTTC 600
ATCTACATCC TCTATATATA AACACGCTAA AAATAACTAG TTAGTATTTT TAAATATTAC 660
ACATTGCCTT TCCAAGAAAC TCGAAAAAAA AAAAAAAAAA AAAAACCACA TCAACAAAAA 720
AGAAGCAGCA ATATATAATA CCCGGG 746
Claims (2)
1. one kind is in the single-minded expression promoter of guard cell, it is characterised in that its nucleotide sequence is SEQ NO ID.1.
2. the creation method as claimed in claim 1 in the single-minded expression promoter of guard cell, it is characterised in that including as follows
Step：
S1 designs a pair of specific primers：
Sense primer：5′-CACAAGCTTCACCCACTTTTCTTCCTCT-3′；
Anti-sense primer：5′-GTCTCTAGAAGTGGCGAGTTGTGGTT-3′；
S2 using round pcr by ABA response elements amplified from Dc3 promoters come；
S3 designs another pair primer：
Sense primer：5′-CACTCTAGAAGTAGGCAAGTAGCAATGTC-3′；
Anti-sense primer：5′-TTCCCCCGGGTATTATATATTGCTGCTTC-3′；
S4 using round pcr by guard cell specificity expression element amplified from kst1 promoters come；
The pcr amplification product obtained in step S2 and step S4 is first coupled on T-easy carriers and is sequenced by S5, first will after confirmation
The pcr amplification product HindIII and XbaI enzyme cutting obtained in plant expression vector pBI121 and step S2, is separately recovered
PBI121 large fragment and purpose fragment, is attached with ligase again afterwards, then by obtained connection product and step S4
In obtained pcr amplification product XbaI and SmaI digestions, the large fragment and purpose fragment of the connection product is separately recovered, it
It is attached again with ligase afterwards, the promoter of the guard cell specificity expression induced by environment stress so created is with regard to structure
It is built on the plant expression vector pBI121 containing gus reporter gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710350170.6A CN107287195A (en) | 2017-05-18 | 2017-05-18 | One kind is in the single-minded expression promoter of guard cell and its creation method |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201710350170.6A CN107287195A (en) | 2017-05-18 | 2017-05-18 | One kind is in the single-minded expression promoter of guard cell and its creation method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1922327A (en) * | 2004-02-27 | 2007-02-28 | 米兰大学 | Stomacal guard cell specific promoter |
| CN102367439A (en) * | 2011-10-31 | 2012-03-07 | 中国农业大学 | Plant stomata specific promoter and application thereof |
| CN102433338A (en) * | 2012-01-04 | 2012-05-02 | 中国农业大学 | Plant stomata specific promoter and application thereof |
-
2017
- 2017-05-18 CN CN201710350170.6A patent/CN107287195A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1922327A (en) * | 2004-02-27 | 2007-02-28 | 米兰大学 | Stomacal guard cell specific promoter |
| CN102367439A (en) * | 2011-10-31 | 2012-03-07 | 中国农业大学 | Plant stomata specific promoter and application thereof |
| CN102433338A (en) * | 2012-01-04 | 2012-05-02 | 中国农业大学 | Plant stomata specific promoter and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| GUNNAR PLESCH ET AL.: "Involvement of TAAAG elements suggests a role for Dof transcription factors in guard cell-speci®c gene expression", 《THE PLANT JOURNAL》 * |
| HWA-JEE CHUNG ET AL.: "Abscisic acid-inducible nuclear proteins bind to bipartite promoter elements required for ABA response and embryo-regulated expression of the carrot Dc3 gene", 《PLANTA》 * |
| 李军: "诱导型保卫细胞特异性启动子的构建及Atdof1.7基因超表达对气孔运动的影响", 《中国博士学位论文全文数据库基础科学辑》 * |
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