CN107881172A - A kind of adverse circumstance inducible promoter, adverse circumstance inducible promoter plant expression vector and induction target gene expression - Google Patents
A kind of adverse circumstance inducible promoter, adverse circumstance inducible promoter plant expression vector and induction target gene expression Download PDFInfo
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Abstract
The present invention relates to a kind of adverse circumstance inducible promoter sequence, includes SEQ ID NO:1 1388bp DNA nucleotide sequence, belongs to field of plant genetic.Present invention also offers a kind of adverse circumstance inducible promoter plant expression vector and the method for making plant express target gene under low temperature, arid and Salt treatment, target gene can be replacedGUSGene is inserted into the plant expression vector containing promoter of the present invention, and the recombinant vector is imported into target plant；SEQ ID NO:2 and SEQ ID NO:3 be PCR primer, is included respectivelyEcoThe Hes of R IHinThe restriction enzyme sites of d III, the primer are applied to amplification ID containing SEQ NO:1 DNA sequence dna；The present invention can be used for the high efficient expression for starting foreign gene under cold, arid and salt stress, suitable for plant genetics and breeding Upgrading.
Description
Technical field
The present invention relates to gene engineering technology field, and in particular to a kind of adverse circumstance inducible promoter, adverse circumstance induction type open
Promoter expression vector and induction target gene expression.
Background technology
Chilling injury, arid and the soil salinization are to limit agricultural production, threaten the important disaster of grain security, especially
The large area that the cold wave and the arid of plant growth key period of season in spring and autumn can result in crops is damaged, and causes serious subtract
Production.Therefore, in addition to strengthening to the Defensive Technology Study of low temperature, arid and salt damage, by plant gene engineering technology means,
Adversity gene is expressed in crop, to improve resistance of the crop to environment stress, reduce low temperature, arid and high salt to making
Grievous injury caused by thing a, it has also become important means of crop breeding.
The gene expression regulation in plant is mainly adjusted comprising DNA level regulation and control, transcriptional level control, post-transcriptional level at present
Level modulation after control, translation skill regulation and control and translation.Promoter is to be located at the section of DNA sequence before transcription initiation site, energy
RNA polymerase is enough activated, it is accurately combined with template DNA, so as to originate related gene transcription, and has many in promoter
Cis-acting elements, by being interacted with trans-acting factor, the expression of downstream target gene can be regulated and controled, be that transcriptional level is adjusted
Committed step in control.
Research finds that plant promoter mainly has constitutive promoter, tissue-specific promoter and inducible promoter.
Wherein constitutive promoter can stablize promotor gene expression in the whole stage of development of plant, do not induced and sent out by external environment
Raw acute variation；Tissue-specific promoter can in specific period, specific tissue promotor gene expression；Induction type
Promoter then can be according to the expression of the induced genes such as different environment-stress or hormonal stimulation, and then strengthens plant to unfavorable ring
The defence capability of border condition, the normal physiological of plant is protected to be metabolized and grow.It is most normal in plant transgene research at present
Constitutive promoter is used, common are cauliflower mosaic virus CaMV 35S promoters, rice actin Actin is opened
Mover and maize ubiquitin Ubiquitin promoters, wherein CaMV 35S promoters are mainly used in the transgenosis of dicotyledon
In research, Actin and Ubiquitin promoters are used in monocotyledon, are especially studied in rice and corn gene
Middle Ubiquitin promoters are widely used in driving exogenous gene expression.Although constitutive promoter is starting foreign gene table
Up to when have the advantages that it is stable, efficiently and wide spectrum, but due in the whole growth and development process of plant efficiently, continuous expression mesh
Gene is marked, easily causes the overexpression of albumen, not only causes the waste on energy, while also easily influence plant itself
Physiological equilibrium, and then yield may be had influence on.Therefore, increasing researcher has turned to research emphasis under given conditions
The inducible promoter of induced expression and the tissue-specific promoter expressed in privileged site, specific period, to alleviate by different
The overexpression of source protein and to caused by plant growth injury.
Existing much researchs on plant tissue specific promoter are reported at present：Such as the specifically expressing in blade
ArabidopsisatslAGene and cornC4PdKGene promoter；The specifically expressing in fruitE4、E8、ACCSynthase gene starts
Son；The Kidney bean of specifically expressing in vascular bundlePAL、GRP118Gene promoter；The arabidopsis of specifically expressing in rootPyk10Base
Because of promoter etc..Research report on inducible promoter mainly lures comprising abiotic stress induction type, biotic
Conductivity type, Light-inducible and hormone inducible etc..This inducible promoter is not expressed under normal operation or expression quantity is very low,
Start great expression GAP-associated protein GAP when corresponding inductive condition occurs, injured so as to reduce poor environment to caused by plant.Grind
Study carefully and show that some inducible promoters can while be induced by many factors, be referred to as polynary inducible promoter, such as：Intend south
MustardCor15aGene promoter has low temperature and drought-induced characteristic；Arabidopsisrd29APromoter has low temperature, arid and high salt
Induced activity；Rice plasma membraneCaATPasePromoter has arid, cold, MeJA and ABA inducing properties.
Drive the high efficient expression of adversity gene under given conditions using inducible promoter, enhancing plant is to poor environment
The resistance of condition, the focus of research has been increasingly becoming in recent years.But the induction type that can be widely applied for transgenic research opens
Current still fewer, the particularly polynary inducible promoter of mover.
The content of the invention
The technical problem to be solved in the present invention（One）It is：It is outer to propose that one kind can induce under low temperature, arid and salt stress
The adverse circumstance inducible promoter sequence of source gene expression.
The present invention is to solve the technical scheme that above-mentioned technical problem proposes to be：A kind of adverse circumstance inducible promoter, it is described inverse
Border inducible promoter includes SEQ ID NO:1 1388bp DNA nucleotide sequence.
Further, the sequence of the adverse circumstance inducible promoter comes from two fringe false bromegrass AP2/EREBP family genesBdDREB-47。
The technical problem to be solved in the present invention（Two）It is：It is proposed that a kind of plant adverse circumstance that can be applied to genetic transformation induces
Type promoter plant expression vector.
The present invention is to solve the technical scheme that above-mentioned technical problem proposes to be：The plant expression vector includes above-mentioned inverse
Border inducible promoter sequence.
The technical problem to be solved in the present invention（Three）It is：It is proposed that one kind makes plant be induced under low temperature, arid and salt stress
The method for expressing target gene.
The present invention is to solve the technical scheme that above-mentioned technical problem proposes to be：First target gene is inserted into and opened containing above-mentioned
In the plant recombinant vector of mover, then the recombinant vector imported in target plant, and under low temperature, arid and condition of salt stress
Induction target gene is expressed in plant.
The beneficial effects of the invention are as follows：
Inducible promoter provided by the invention belongs to polynary inducible promoter, can be under cold, arid and condition of salt stress
Start the expression of foreign gene.According to technical scheme provided by the invention, foreign gene is replacedGUSGene is inserted into the present invention
Adverse circumstance inducible promoter plant expression vector in, artificial control environmental condition can be passed through（Temperature, humidity and salt contain
Amount）, the expression of foreign gene is controlled, avoids energy dissipation and possibility injury caused by continuous expression target gene.Separately
Outside,, can be under cold, arid, salt stress using inducible promoter provided by the invention using adversity gene as foreign gene
Efficiently start adversity gene expression, strengthen the resistance of plant.
Brief description of the drawings
Fig. 1 is that promoter PCR expands electrophoretogram in the present invention, and wherein M is DL2000 Marker, stripe size it is supreme and
It is 2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp down；1 is promoter pBdDREB-47 of the present invention.
Fig. 2 is passed through after pCAMBIA1381-GUS carriers connect carrier T with promoter of the present inventionEcoThe Hes of R IHinIII pair of enzyme of d
Cut electrophoretogram, wherein M is DNA Marker III, stripe size is from top to down 4500bp, 3000bp, 2000bp, 1200bp,
800bp、500bp、200bp；1 is pBdDREB-47-T plasmids；2 be pCAMBIA1381-GUS plasmids.
Fig. 3 is bacterium solution PCR electrophoretograms after promoter connection pCAMBIA1381-GUS plant expression vectors, and wherein M is
DL2000 Marker；1-10 is monoclonal.
Fig. 4 is after promoter connects pCAMBIA1381-GUS plant expression vectorsEcoThe Hes of R IHinThe double digestions of d III identification electricity
Swimming figure, wherein M is DNA Marker III；1 is pC1381-pBdDREB-47-GUS plasmids.
Fig. 5 is Agrobacterium bacterium solution PCR detection electrophoretograms, and wherein M is DL2000 Marker；1-3 is Agrobacterium monoclonal；4
For feminine gender；5 be pC1381-pBdDREB-47-GUS plasmids.
Fig. 6 be part T0 for transgene tobacco positive detection electrophoretogram, wherein M is DL2000 Marker；1-7 is to turn base
Because tobacco T0 is for individual plant；8 be wild-type tobacco；9 be pC1381-pBdDREB-47-GUS plasmids.
Fig. 7 is promoter plant expression vector construction schematic diagram of the present invention.
Fig. 8 is cold, arid and salt stress transgene tobacco GUS colored graphs before and after the processing.
Embodiment
Embodiment one
The present embodiment is on two fringe false bromegrassBdDREB-47The clone of gene promoter.
The preparation of 1.1 two fringe false bromegrass genomic DNAs
Using the blade of the two fringe false bromegrass of CTAB methods extraction normal growth, examined by ultramicron nucleic acid-protein analyzer Q5000
Extracted DNA concentration and quality is surveyed, it is standby that DNA concentration is diluted to 100ng/ μ l.
The clone of 1.2 promoters
Using the two fringe false bromegrass DNA of said extracted as template, SEQ ID NO are utilized:2 and SEQ ID NO:3 primers, enter performing PCR
Amplification obtains SEQ ID NO:1 sequence.
PCR reaction systems are as follows：
| Reagent | Volume（μl） |
| ddH2O | 5.8 |
| 2×Prime Star GC Buffer | 10 |
| dNTP mix（2.5mM） | 2 |
| Sense primer（10μM） | 0.5 |
| Anti-sense primer（10μM） | 0.5 |
| Takara Prime Star Taq enzymes | 0.2 |
| DNA profiling | 1 |
| Total system | 20 |
PCR response procedures：98 DEG C of pre-degeneration 5min；98 DEG C of denaturation 30s, 55 DEG C of annealing 20s（Each cycle annealing temperature increase
0.5℃）, 72 DEG C of extension 90s, expand 20 circulations；98 DEG C of denaturation 30s, 65 DEG C of annealing 20s, 72 DEG C of extension 90s, expand 15
Circulation；72 DEG C of extension 10min；4 DEG C of insulations.
Above PCR primer to be drawn into 10 μ l and carries out 1% agarose gel electrophoresis detection, concrete outcome is shown in Fig. 1, wherein
Marker is DL2000, and target stripe size is 1388bp.
By above-mentioned remaining PCR primer connection carrier T, the present invention uses Takara Prime Star Taq high-fidelities
Enzyme, amplified production are flat end, and carrier T is pEASY-Blunt Cloning Vector（Purchase the full formula gold biotechnology in Beijing
Co., Ltd）, operated according to kit specification, linked system is 5 μ l（The μ l carrier Ts of 4 μ l PCR primers+1）.
Above-mentioned connection product is transferred to competent escherichia coli cell Trans-T1 by heat shock method（Purchase the full Shi Jinsheng in Beijing
Thing Technology Co., Ltd.）, concrete operation method is shown in kit specification.Picking monoclonal carries out bacterium colony PCR detections, chooses positive
Clone is sequenced, and sequencing result shows extension increasing sequence and SEQ ID NO:1 fits like a glove.
Will sequencing correctly clone expand it is numerous, and with the small extraction reagent kit of plasmid（It is purchased from Tiangeng biochemical technology Co., Ltd）
Plasmid is extracted, plasmid concentration is carried by ultramicron nucleic acid-protein analyzer Q5000 detections.
Embodiment two
The present embodiment is on adverse circumstance inducible promoter（pBdDREB-47）Plant expression vector construction.
WithEcoThe Hes of R IHindⅢ（It is purchased from NEB companies）Double digestion recombinant plasmid pBdDREB-47-T and carrier
PCAMBIA1381-GUS, digestion result are shown in Fig. 2, purpose fragment are separately recovered（1388bp）With pCAMBIA1381-GUS carrier-pellets
Section, and with T4 DNA ligases（It is purchased from NEB companies）Connection, competent escherichia coli cell Trans- is transferred to by heat shock method
In T1, result is shown in Fig. 3 after picking monoclonal carries out positive detection, expands numerous extraction plasmid, and the further digestion of positive plasmid is identified
（ EcoRI HeHinThe double digestions of d III）As a result Fig. 4 is seen, and it is correct through sequencing result, show plant recombination expression vector
PC1381-pBdDREB-47-GUS is successfully constructed, and pC1381-pBdDREB-47-GUS further can be recombinated into matter by heat shock method
Grain is transferred to Agrobacterium competent cell EHA105（Purchase in Shanghai Shi Feng bio tech ltd）, positive detection, as a result such as
Shown in Fig. 5, it can be used for follow-up plant transgene after correct.
Build the promoter containing pBdDREB-47 plant expression vector schematic diagram as shown in fig. 7, wherein 35S Pro and
PBdDREB-47 is promoter, and 35S Ter and Nos Ter are terminator, and selected marker is hygromycin resistance, and gus gene is report
Gene is accused, the activity of pBdDREB-47 promoters can be judged by the expression of gus gene.
Embodiment three
The present embodiment is to pass through Agrobacterium-mediated transformation tobacco on pC1381-pBdDREB-47-GUS recombinant plasmids.
The preparation of 3.1 transgenic acceptors
The seed broadcasting of wild-type tobacco is put in 25 degree of culturing room in flowerpot（12h illumination/12h is dark）, while cover fresh-keeping
Film moisturizing, progressively removes preservative film after coming up.Transplanted after two weeks, blade can use when tobacco grows big to 2 months
In transgenic experiments.
3.2 tobacco leafs sterilize and preculture
Choose the preferable tobacco of upgrowth situation, its leaflet tablet is cut with scissors, on superclean bench first with 75% ethanol immersion
Sterilize tobacco leaf 50s.Again with 2.5% liquor natrii hypochloritis soaking disinfection 10min, during which constantly rock so as to sufficiently sterilised,
Finally by tobacco leaf sterile water wash 5 times.
The tobacco leaf disinfected is placed on aseptic filter paper and is sufficiently absorbed through surface moisture, with sterile razor blade by tobacco leaf
It is cut into about 0.5 × 0.5 cm2Fritter（Pay attention to avoiding vein）, the blade that cuts is laid in tobacco and co-cultures culture medium（MS+
6-BA（2 mg/L）+ NAA（0.2 mg/L）+ Agar（8 g/L）+ sucrose（30g/L）pH5.8）On, under 25 DEG C of dark conditions
Preculture 3 days.
The preparation of 3.3 Agrobacterium bacterium solutions
Agrobacterium bacterium solution containing pC1381-pBdDREB-47-GUS recombinant plasmids is put down in the LB solids containing 50mg/L kan
Rule on plate, 28 DEG C of light cultures are resuspended in 50ml MS fluid nutrient mediums after 1 day, 28 DEG C of shaking table 200r/min concussion trainings
30min is supported, and carries out next step experiment immediately.
3.4 Agrobacteriums are infected
Tobacco leaf after preculture 3 days is put in the Agrobacterium bacterium solution of fully shaking, soaks 10 minutes, during which constantly shakes
It is dynamic.Blade is put on aseptic filter paper after the completion of infecting, to be sufficiently absorbed through the bacterium solution remained on blade, then by the leaf after dip-dye
Piece is scattered to be put on the co-cultivation base for being covered with aseptic filter paper, 25 DEG C of light cultures 3 days.
The differentiation of 3.5 tobacco leafs is with taking root
Tobacco leaf after light culture 3 days is transferred to differential medium（MS+ 6-BA（2 mg/L）+ NAA（0.2 mg/L）+
Cb（500 mg/L）+ Kan （100 mg/L）+ Agar（8 g/L）+ sucrose（30 g/L）pH5.8）On, in 25 DEG C, 12h light
According to being cultivated under/12h dark conditions, until differentiation budding.
When budlet grows to 2 ~ 3 cm, sprout is cut, is inserted into root media（1/2MS+ Kan（100 mg/L）+
Cef（250 mg/L）+ Agar（8 g/L）+ sucrose（30 g/L）pH5.8）On, under 25 DEG C, 12h illumination/12h dark conditions
Culture.Explant can take root after general two weeks, be transplanted to when its root growth is more flourishing in flowerpot, continue to train in greenhouse
Support.Extraction transgenosis T0 treats the genomic DNA of tobacco, and positive detection, the testing result of plant part are carried out with gus gene primer
See Fig. 6, SEQ ID NO:4 and SEQ ID NO:5 be gus gene PCR detection primers.
Example IV
The present embodiment is the activity identification on adverse circumstance inducible promoter
The present invention is mainly cold by being carried out to T1 for transgene tobacco seedling（4℃）, arid and salt（300mM NaCl）Stress
Processing, then detect by GUS dyeing the expression of gus reporter gene, identify the present invention promoter have it is cold, arid and
Salt treatment characteristic.
The preparation of 4.1 experiment materials
The seed in appropriate wild type and transgene tobacco T0 generations is taken to soak 1-2h in 2ml centrifuge tubes, make seed complete respectively
Portion is sunken to ttom of pipe, washs 2-3 times to remove the impurity in seed.After on super-clean bench by immersion seed be transferred to sterile 2ml from
In heart pipe, add sterilized water and wash twice, then sterilized 8-10min with 70% ethanol disinfection 50s, 1% liquor natrii hypochloritis, finally used
Sterile water washing 3-5 times（Centrifuge tube is constantly rocked in whole sterilization and washing process）.T0 after sterilization is seeded in for seed
On the MS culture mediums of the hygromycin containing 50mg/L, wild-type tobacco seed is seeded on MS culture mediums, be put in 25 DEG C, 12h illumination/
Grown under conditions of 12h dark, germination after about 5-7 days, after two weeks by seedling replanting into small alms bowl it is standby.
4.2 cold, the arid and lower promoter activity identifications of salt stress processing
1 month big wild type and transgenosis T1 are put into 4 DEG C of growth cabinets for tobacco seedling, clip cold treatment respectively
0h, 12h and 24h blade add appropriate non-diffusing type GUS dye liquors in 10ml centrifuge tubes（It is purchased from that Beijing is extensive and profound in meaning scientific and technological to be had
Limit responsible company）, the amount of GUS dye liquors will fully submerge blade, and vacuumizing 10min makes GUS dye liquors fully permeate blade, and 37 DEG C are kept away
Light dyes 12h, and absolute ethyl alcohol decolourizes.Drought stress and salt stress processing are the wild type big to 1 month and transgenosis T1 respectively
Carry out controlling water arid and the NaCl solution for pouring 300mM daily once for tobacco seedling, progress is sampled in the 0d and 6d of processing
GUS is dyed.Black position is the expressive site of gus gene, and black is deeper, and expression quantity is higher.As shown in figure 8, before cold treatment
（0h）Gus gene expression quantity is very low in transgenic tobacco leaf（Black is very faint）, gus gene table after cold treatment 12h and 24h
Up to amount significantly rise（Blade black is deepened）, absolutely proved that promoter of the present invention does not start substantially at a normal temperature
Gus gene is expressed or expression quantity is very low, starts target gene expression rapidly if low temperature stress is run into.Drought stress simultaneously
With salt stress processing, before processing GUS expression quantity is very low, and GUS expression quantity dramatically increases after 6d is handled.Therefore it is provided by the present invention
Promoter have efficient cold, arid and Salt treatment active, available in plant genetics and breeding Upgrading.
The present invention's is not limited to above-described embodiment, and the technical scheme of above-mentioned each embodiment of the invention can be handed over each other
Fork combination forms new technical scheme, in addition all technical schemes formed using equivalent substitution, all falls within the guarantor of application claims
In the range of shield.
Claims (4)
- A kind of 1. adverse circumstance inducible promoter, it is characterised in that：The adverse circumstance inducible promoter includes SEQ ID NO:1 1388bp DNA nucleotide sequence.
- 2. adverse circumstance inducible promoter according to claim 1, it is characterised in that：The sequence of the adverse circumstance inducible promoter From two fringe false bromegrass AP2/EREBP family genesBdDREB-47。
- A kind of 3. adverse circumstance inducible promoter plant expression vector, it is characterised in that：The plant expression vector will comprising right Seek the adverse circumstance inducible promoter sequence described in 1.
- 4. one kind induction target gene expression, it is characterised in that：First target gene is inserted into containing institute in claim 1 In the plant recombinant vector for stating promoter, then the recombinant vector imported in target plant, and in low temperature, arid and salt stress bar Target gene is induced to be expressed in plant under part.
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| CN111763672A (en) * | 2020-06-30 | 2020-10-13 | 安徽省农业科学院水稻研究所 | Rice low-temperature inducible expression promoter Poscold10 and application thereof |
| CN113151273A (en) * | 2021-04-14 | 2021-07-23 | 新疆农业大学 | Abiotic stress inducible promoter and application thereof |
| CN114717259A (en) * | 2022-05-26 | 2022-07-08 | 山西大学 | Temperature control inducible gene editing system and application thereof |
| CN114854746A (en) * | 2022-04-20 | 2022-08-05 | 浙江省农业科学院 | Adversity stress inducible promoter TIP1-6-pro and expression vector construction method and application thereof |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111763672A (en) * | 2020-06-30 | 2020-10-13 | 安徽省农业科学院水稻研究所 | Rice low-temperature inducible expression promoter Poscold10 and application thereof |
| CN111763672B (en) * | 2020-06-30 | 2022-03-25 | 安徽省农业科学院水稻研究所 | Rice low-temperature inducible expression promoter Poscold10 and application thereof |
| CN113151273A (en) * | 2021-04-14 | 2021-07-23 | 新疆农业大学 | Abiotic stress inducible promoter and application thereof |
| CN114854746A (en) * | 2022-04-20 | 2022-08-05 | 浙江省农业科学院 | Adversity stress inducible promoter TIP1-6-pro and expression vector construction method and application thereof |
| CN114717259A (en) * | 2022-05-26 | 2022-07-08 | 山西大学 | Temperature control inducible gene editing system and application thereof |
| CN114717259B (en) * | 2022-05-26 | 2023-06-20 | 山西大学 | Temperature control inducible gene editing system and application thereof |
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