CN102286484B - Promoter for driving genes to express in flower tissues - Google Patents
Promoter for driving genes to express in flower tissues Download PDFInfo
- Publication number
- CN102286484B CN102286484B CN 201110200465 CN201110200465A CN102286484B CN 102286484 B CN102286484 B CN 102286484B CN 201110200465 CN201110200465 CN 201110200465 CN 201110200465 A CN201110200465 A CN 201110200465A CN 102286484 B CN102286484 B CN 102286484B
- Authority
- CN
- China
- Prior art keywords
- dna
- plant
- promoter
- sequence
- promotor
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a promoter for driving genes to express in flower tissues. The promoter provided by the invention is deoxyribonucleic acid (DNA) molecules shown as 1) or 2) or 3): 1) the DNA molecules shown by the first sequence in a sequence table; 2) the DNA molecules which can realize hybridization with the DNA sequence limited by 1) under the strict condition; and 3) the DNA molecules which have homology being higher than 90 percent with the DNA sequences limited by 1) and 2) and have the promoter functions. The promoter provided by the invention can drive the exogenous gene to express in the flower tissues. The promoter is favorable for promoting people to study the development and the growth of Arabidopsis, in addition, an economic, fast and effect path is provided for the inheritance and the transformation of plants (particularly the vegetable flowers of cruciferae). The promoter has a wide application space and market prospect in the agriculture field.
Description
Technical field
The present invention relates to a kind of promotor that gene is expressed that drives in the flower tissue.
Background technology
Arabidopis thaliana (Arabidopsis thaliana) is a kind of cress, be widely used in plant genetics, developmental biology and molecular biological research, because it has the characteristics such as plant is little, generation time is short, genome is little and makes it become a kind of typical model plant.
The MYB factor is a proteinoid family of containing conservative MYB DNA binding domains.The typical MYB factor is relevant with c-Myb, plays regulating and controlling effect in the cell cycle of animals and plants and some higher eucaryotes.At least contain 100 R2R3-MYB genes in Arabidopis thaliana, these genes are kept the shape of cell at the secondary metabolism of regulating plant, resist disease, and the aspects such as response hormone all play an important role.Before had report Arabidopis thaliana MYB2 1 relevant with the growth of flower with MYB24, and behind Arabidopis thaliana disappearance MYB21, the MYB24 gene, shown as stamen development and be obstructed, filigree can not normally extend, thereby causes male sterile.
Summary of the invention
The purpose of this invention is to provide a kind of promotor that gene is expressed that drives in the flower tissue.
Promotor provided by the invention is following 1) or 2) or 3) dna molecular:
1) dna molecular shown in the sequence 1 in the sequence table;
2) under stringent condition with 1) the dna sequence dna hybridization that limits and the dna molecular with promoter function;
3) with 1) or 2) dna sequence dna that limits has 90% above homology, and have the dna molecular of promoter function.
Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5% SDS, 65 ℃ of lower hybridization, then uses 2 * SSC, 0.1% SDS and 1 * SSC, and 0.1% SDS respectively washes film once.
DNA shown in the sequence 1 in the sequence table is comprised of 1670 deoxyribonucleotides.DNA shown in the sequence 1 can be used as the promoters driven gene and expresses in the flower tissue of plant.
The recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain described promotor all belong to protection scope of the present invention.
Described recombinant expression vector specifically can be the dna fragmentation shown in the sequence 1 is inserted the recombinant plasmid that the multiple clone site of pBI121 carrier obtains.
The present invention also protect a kind of in the flower tissue of plant the method for expression alien gene, be that the dna fragmentation first is imported in the plant, thereby in the flower tissue of plant, express described foreign gene; Described dna fragmentation first contains described promotor and described foreign gene successively to the downstream from the upstream.
Can first described promotor be connected with described foreign gene, form warm DNA, then make up recombinant expression vector with fusion dna, import plant.Also described promotor and described foreign gene can be inserted respectively the multiple clone site that is fit on the carrier that sets out, obtain the recombinant expression vector with the described exogenous gene expression of described promoters driven, then recombinant expression vector be imported plant.
For the ease of transgenic plant cells or plant being identified and screening, can process described recombinant expression vector, as have the antibiotic marker thing of resistance or anti-chemical reagent marker gene etc.Recombinant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, the conventional biological method conversion of plant such as agriculture bacillus mediated by using.
Described method specifically can realize by following recombinant expression vector is imported in the plant: the multiple clone site with described promotor and described foreign gene insertion pBI121 carrier obtains recombinant expression vector respectively; Described promotor is positioned at the upstream of described foreign gene.
Described plant specifically can be cress, such as Arabidopis thaliana (environmental such as Columbia-0) etc.
The invention provides a promotor, this promotor can drive gene and express in the flower tissue of plant.The present invention helps lend some impetus to people to the research of Arabidopis thaliana development growth, and for plant particularly the genetic modification of the vegetable or flower of Cruciferae an economy, approach fast and effectively are provided.The present invention has wide application space and market outlook at agriculture field.
Description of drawings
Fig. 1 is pBI121-MYB24pro-GUS carrier collection of illustrative plates
Fig. 2 is the floral organ GUS dyeing situation of transgenic arabidopsis; A-H: different transfer-gen plants.
Fig. 3 is the floral organ GUS dyeing situation of wild-type Arabidopis thaliana; A-H: different wild-type plant.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.
Used Arabidopis thaliana (Arabidopsis thaliana) is that Columbia-0 is environmental among the embodiment: Arabidopsis Biological Resource Center (ABRC), seed number: CS6673.
Agrobacterium (Agrobacterium tumefaciens) bacterial strain GV3101 is called for short agrobacterium strains GV3101: reference: Floral dip:a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana.Clough SJ, Bent AF.Plant Journal.1998; 16:735-43; The public can obtain from Tsing-Hua University.
The acquisition of embodiment 1, MYB24pro sequence
Take Arabidopis thaliana (Columbia-0) leaf DNA as template, the primer pair that adopts F1 and R1 to form carries out pcr amplification under the effect of the PFU of high-fidelity enzyme, obtain pcr amplification product.
F1：5’-ttaAAGCTT?ttacccaattcctgag-3’；
R1：5’-tttTCTAGA?gagagaaatagaaag-3’。
Pcr amplification product is checked order, shown in the sequence 1 of sequence table.With the DNA called after MYB24 promotor (MYB24pro) shown in the sequence 1 of sequence table.
The acquisition of embodiment 2, transfer-gen plant and evaluation
One, the structure of recombinant expression vector (pBI121-MYB24pro-GUS)
1, the pcr amplification product that obtains with restriction enzyme HindIII and XbaI double digestion embodiment 1 obtains enzyme and cuts product.
2, with restriction enzyme HindIII and XbaI double digestion pBI121 carrier (GenBank:AF485783.1), reclaim the approximately carrier framework of 14kb.
3, the enzyme of step 1 is cut the carrier framework that product is connected with step and under the effect of T4 dna ligase, connected, obtain recombinant plasmid pBI121-MYB24pro-GUS.The structural representation of recombinant plasmid pBI121-MYB24pro-GUS is seen Fig. 1.According to sequencing result, recombinant plasmid pBI121-MYB24pro-GUS is carried out structrual description as follows: the recombinant plasmid that DNA obtains shown in the sequence 1 of insertion sequence table between the HindIII of pBI121 carrier and XbaI enzyme cutting site.
Two, the acquisition of transfer-gen plant
1, recombinant plasmid pBI121-MYB24pro-GUS electric shock is transformed agrobacterium strains GV3101, the Agrobacterium that obtains recombinating (Agrobacterium tumefaciens GV3101/pBI121-MYB24pro-GUS).
2, the restructuring Agrobacterium of 28 ℃ of incubated overnight steps 1 and adjust the bacterium liquid that its concentration is OD600=0.8.
3, by flower infusion method (flower be immersed in the bacterium liquid of step 2 30 seconds) recombinant plasmid pBI121-MYB24pro-GUS is imported respectively 64 strain Arabidopis thalianas (Columbia-0), the seed of results is T0 for the seed of Arabidopis thaliana.
4, with T0 for the planting seed of Arabidopis thaliana in the enterprising row filter of MS substratum that contains kantlex (20mg/L), obtain 24 strains and have the T1 of kalamycin resistance for Arabidopis thaliana (numbering is followed successively by 1-24).
When 5, treating that T1 grows to 4-6 sheet leaf for Arabidopis thaliana it is transplanted on the vermiculite (24 ℃ of growths 45 days; 16 hours/illumination+8 hours/dark), extract respectively 24 strain T1 for the DNA of Arabidopis thaliana leaf, form primer pair with F2 (forward primer of MYB24pro) and R2 (reverse primer of GUS) and carry out the PCR evaluation, if show the approximately pcr amplification product of 1742bp, then plant is transfer-gen plant.
F2：5’-tta?aagctt?ttacccaattcctgag-3’；
R2：5’-cacgggttggggtttctacag-3’。
The result shows, 24 strain T1 are transgenic arabidopsis for Arabidopis thaliana.
Three, the cultivation of wild-type Arabidopis thaliana
The Arabidopis thaliana (Columbia-0) that will grow to 4-6 sheet leaf is transplanted on the vermiculite (24 ℃ of growths 45 days; 16 hours/illumination+8 hours/dark).
Four, GUS staining analysis
To in 45 days transgenic arabidopsis (24 strain) of vermiculite growth and step 3, carry out the GUS staining analysis at 45 days wild-type Arabidopis thaliana (20 strain) of vermiculite growth in the step 2.
24 strain transgenic arabidopsis floral organs all show blueness, and the partial results of floral organ dyeing is seen Fig. 2.20 strain wild-type thaliana flower organs all are not colored, and the partial results of floral organ dyeing is seen Fig. 3.The result shows, the promotor shown in the sequence 1 really can drive gus gene and spend middle expression.
Claims (5)
1. a promotor is the dna molecular shown in the sequence in the sequence table 1.
2. the recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain the described promotor of claim 1.
3. recombinant expression vector as claimed in claim 2 is characterized in that: described recombinant expression vector is that the multiple clone site that the dna fragmentation shown in the sequence 1 inserts the pBI121 carrier is obtained.
4. the method for an expression alien gene in the flower tissue of plant is that the dna fragmentation first is imported in the plant, thereby expresses described foreign gene in the flower tissue of plant; Described dna fragmentation first contains the described promotor of claim 1 and described foreign gene successively to the downstream from the upstream; Described plant is cress; Described cress is Arabidopis thaliana.
5. method as claimed in claim 4, it is characterized in that: described dna fragmentation first imports described plant by recombinant expression vector; The recombinant plasmid that described recombinant expression vector obtains for the multiple clone site with promotor claimed in claim 1 and described foreign gene insertion pBI121 carrier; Described promotor is positioned at the upstream of described foreign gene.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110200465 CN102286484B (en) | 2011-07-18 | 2011-07-18 | Promoter for driving genes to express in flower tissues |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 201110200465 CN102286484B (en) | 2011-07-18 | 2011-07-18 | Promoter for driving genes to express in flower tissues |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN102286484A CN102286484A (en) | 2011-12-21 |
| CN102286484B true CN102286484B (en) | 2013-01-02 |
Family
ID=45333242
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 201110200465 Active CN102286484B (en) | 2011-07-18 | 2011-07-18 | Promoter for driving genes to express in flower tissues |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102286484B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815452A (en) * | 2017-12-06 | 2018-03-20 | 新疆农垦科学院 | A kind of specific expressed promoter of plant leaf blade and its application |
-
2011
- 2011-07-18 CN CN 201110200465 patent/CN102286484B/en active Active
Also Published As
| Publication number | Publication date |
|---|---|
| CN102286484A (en) | 2011-12-21 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106520824A (en) | Multi-target-point editing system and application thereof | |
| CN101186926B (en) | Modified method for transforming gramineous crop by agrobacterium mediated flower-dipping method | |
| CN114752599B (en) | Verticillium dahliae VdNRPS3 gene antipathogen target gene fragment, interference vector and application thereof | |
| CN107012147A (en) | A kind of arid and/or high salt evoked promoter SlWRKY8P and its application from tomato | |
| CN112126707B (en) | Nucleic acid molecules from corn event CA09328 and methods for detecting same | |
| CN105400814B (en) | A method of cultivating insect-resistant transgenic corn | |
| CN110106171B (en) | Long-chain non-coding RNA and application thereof in regulating and controlling low temperature resistance of plants | |
| CN102286484B (en) | Promoter for driving genes to express in flower tissues | |
| CN103172717B (en) | Plant low potassium stress resistant related protein GmWRKY50 as well as encoding gene and application thereof | |
| CN103243096A (en) | Plant tissue specific expression promoter and application of plant tissue specific expression promoter | |
| CN102250898B (en) | Promoter capable of driving expression of gene in tissue of flower | |
| CN103320441B (en) | Plant promoter and application thereof | |
| CN105814208A (en) | Zea mays regulatory elements and uses thereof | |
| CN1995359A (en) | Agrobacterium mediated large size tomato transformation method | |
| CN104628840B (en) | Plant stress tolerance correlative protein VrDREB2A and its encoding gene and application | |
| CN101831454A (en) | Gateway inlet vector pEn-L4*-PrbcS-*T-GFP-L3*, construction method thereof and application thereof | |
| CN107058324A (en) | Rice root specific expression promoter POsRO4 and corresponding rice cultivating method | |
| CN108424911B (en) | Seed-specific bidirectional promoter and application thereof | |
| CN102154320B (en) | Chrysanthemum anti-retroelement DgZFP1, plant expression vector thereof, construction method thereof and application thereof | |
| CN103215271B (en) | Plant promoter and application thereof | |
| CN109486815B (en) | Artificial chimeric promoter and construction method thereof | |
| CN114717255A (en) | Method for improving and cultivating eggplant by RNAi | |
| CN113025621B (en) | Application of CIPK14 gene in improving drought resistance of pigeon pea | |
| CN103305513B (en) | Plant promoter and application thereof | |
| CN106399312A (en) | Inducible promoter NtPCS1P and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant |