CN108588070A - A kind of Mongolia's Ammopiptanthus mongolicus guard cell specific expression promoter and its application - Google Patents
A kind of Mongolia's Ammopiptanthus mongolicus guard cell specific expression promoter and its application Download PDFInfo
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Abstract
A kind of Mongolia's Ammopiptanthus mongolicus guard cell specific expression promoter and its application, the promoter are nucleotide sequence shown in SEQ ID No.1, can be used for screening and the relevant specific gene of guard cell by the promoter.The present invention provide for the first time one kind can in Ammopiptanthus mongolicus guard cell specifically expressed promoter, the specificity of the promoter is very strong, therefore provides effective tool for the research of Plant Guard Cells function.
Description
Technical field
The present invention relates to plant genetic engineerings and biotechnology, and in particular to a kind of Mongolia Ammopiptanthus mongolicus guard cell is special
Different expression promoter (is named as PAmSLAC1) and its application.
Background technology
The expression of plant gene is had stringent time and spatial ordering by the finely regulating of intraor extracellular environment.Base
Because expression regulation include transcription before, transcription, transcription after, translation, translation after five levels.The regulation and control of wherein transcriptional level are closed the most
Key.Promoter is the critical elements of transcriptional level control.According to the difference of function and the mode of action, promoter can be divided into three
Class：Composing type, induction type and tissue-specific promoter.The target gene of constitutive promoter driving can be in transfer-gen plant
All breeding times and tissue in stablize expression.The most commonly used is containing cauliflower mosaic virus (CaMV) 35S promoter
(Odell, et al.1985), rice actin Actin1 promoters (Mcelroy, et al.1990) and maize ubiquitin
Ubiquitin promoters (Holtorf, et al.1995).But external source target gene continues efficient table in test plant
Up to the waste for not only causing the energy in plant, and in a organized way in expression be possible to that plant itself is caused to poison.
Inducible promoter refers under the stimulation of certain specific physics, chemistry or bio signal, and the type promoter can be substantially
Degree improves the transcriptional level of downstream gene.But inducible promoter also has certain limitation, to the external thorn of test plant
Swash, such as heat shock, pathogen infection, HORMONE TREATMENT may cause a series of biochemical reactions in plant and be unfavorable for plant
Normal growth.Tissue specific promoter can only drive target gene to be expressed at certain specific organ or tissue positions, can
Effectively evade the problem of above two promoter is brought.Therefore, to the research of tissue-specific promoter and application in recent years by
To the attention of more and more researchers.
Under drought environment, plant should preserve internal water, and not influence photosynthesis assimilation CO2, high efficiency regulatory stomata fortune
It is dynamic, it is key that they depend on for existence.Stomata it is open or close, caused by being the reversible expansion or shrinkage of guard cell, therefore, point
From identification and functional study guard cell specific gene and the research of its major regulatory person-promoter is particularly important.
However, species for being limited only to be grown under conventional habitat, such as arabidopsis are studied it at present, for being grown in extreme drought item
Species under part, do not have been reported that also.
Ammopiptanthus mongolicus (Ammopiptanthus mongolicus (Maxim) Cheng f.) is that northwest China Desert Regions are peculiar
The evergreen raw broadleaf shrubs of drought, be grown in annual precipitation throughout the year less than 200mm, year evaporation capacity be more than the extreme drought ring of 3000mm
It is the research good material of plant drought mechanism in border (Cao Ling etc., 2003, Huang Chaoying, 2003).Ammopiptanthus mongolicus long term growth is in pole
It holds under drought environment, has tight demand to the accuracy controlling of stomata.Therefore, Ammopiptanthus mongolicus guard cell specific gene promoter
PAmSLAC1Clone provide theoretical foundation for Ammopiptanthus mongolicus stomatal conductivity mechanism is expanded on further, in genetically modified crops Drought-resistant Breeding
Also there is application value.
Invention content
The technical issues of solution：The present invention provides a kind of Mongolian Ammopiptanthus mongolicus guard cell specific expression promoter and its answers
With, by the promoter can be used for screen with the relevant specific gene of guard cell.
Technical solution：A kind of Mongolia's Ammopiptanthus mongolicus guard cell's specific expression promoter, which is SEQ ID No.1 institutes
The nucleotide sequence shown.
A kind of carrier, the carrier contain above-mentioned promoter.
A kind of bacterial strain, the bacterial strain contain above-mentioned promoter.
The cloning process of above-mentioned Mongolia's Ammopiptanthus mongolicus guard cell's specific expression promoter, step are：Based on known
AmSLAC1 genomic dna sequences design three at its translation initiation codon ATG downstream 249bp, 372bp and 523bp respectively
AmSLAC-SP3, AmSLAC-SP2 and SEQ ID shown in SEQ ID No.3 shown in specific reverse primer SEQ ID No.2
AmSLAC-SP1 shown in No.4 obtains one section by the method for chromosome walking and turns over using Mongolian Ammopiptanthus mongolicus genomic DNA as template
The DNA sequence dna of upstream from start codon is translated, is redesigned on this basis shown in three specific reverse primer SEQ ID No.5
AmSLAC-SP4 shown in AmSLAC-SP5 and SEQ ID No.7 shown in AmSLAC-SP6, SEQ ID No.6, then pass through above-mentioned side
The end of normal direction 5 ' step is moved, this two sections of sequence assemblies are redesigned forward and reverse primer SEQ ID No.8 by section of DNA sequence of getting back
AmSLAC1pro-R shown in shown AmSLAC1pro-F and SEQ ID No.9, the sequence expanded are subcloned to carrier
On pMD19-T, sequencing result shows that the sequence has 1455bp.
Application of the above-mentioned Mongolia's Ammopiptanthus mongolicus guard cell specific expression promoter in screening guard cell's specific gene.
SEQ ID No.1:
Above-mentioned amplified fragments primer sequence is：
SEQ ID No.2：AmSLAC-SP3：5’-CCCCAAACTTGTTCCACTTCTTGTC-3’；
SEQIDNo.3：
AmSLAC-SP2:5’-ACGATGGACTCCAGTCCGGCCCAACTCTCTCTCTTTCCTTGGCAA C-3’；
SEQ ID No.4：AmSLAC-SP1:5’-GCCACTTCTCATCTTTGGGGAGAAG-3’；
SEQ ID No.5：AmSLAC-SP6:5’-CTAAGTATAAGGAAAATATTAAAGC-3’；
SEQ ID No.6：AmSLAC-SP5:5’-GATTTACATCAGAATTAGATTGCAC-3’；
SEQ ID No.7：AmSLAC-SP4:5’-GGAAGGACTATTATGGTAATTC-3’；
SEQ ID No.8：AmSLAC1pro-F:5’-GAGCTCTCTATGTGTTTAAAACAGTGTTTC-3’；
SEQ ID No.9：AmSLAC1pro-R:5’-CCATGGTCTCCTTGTAATAATATGTTGAGC-3’；
SEQ ID No.10：LAD1-1:
5’-ACGATGGACTCCAGAGCGGCCGC(V:G/C/A)N(V:G/C/A)NNNGGAA-3’；
SEQ ID No.11：LAD1-2:
5’-ACGATGGACTCCAGAGCGGCCGC(B:G/C/T)N(B:G/C/T)NNNGGTT-3’；
SEQ ID No.12：LAD1-3:
5’-ACGATGGACTCCAGAGCGGCCGC(V:G/C/A)(V:G/C/A)N(V:G/C/A)NNNCCAA-3’；
SEQ ID No.13：LAD1-4:
5’-ACGATGGACTCCAGAGCGGCCGC(B:G/C/T)(D:G/A/T)N(B:G/C/T)NNNCGGT-3’；
SEQ ID No.14：AC1:5’-ACGATGGACTCCAGAG-3’；
SEQ ID No.15:Vector-R:5’-gtggtgtagagcattacgctgcg-3’
Above-mentioned Ammopiptanthus mongolicus guard cell specific expression promoter PAmSLAC1In regulation and control downstream gene in Ammopiptanthus mongolicus guard cell
In specific expressed application.
Ammopiptanthus mongolicus guard cell's specific expression promoter PAmSLAC1Application in regulating and controlling downstream gene expression, preferably downstream
Gene is the reporter genes such as gus gene, GFP genes.
After the upstream initiation codon (ATG) 1455bp sequence amplifications of Ammopiptanthus mongolicus AmSLAC1 genes, it is cloned into containing report
The plant expression vector pCambia1301 of gene GUS is accused, the carrier arabidopsis thaliana transformation that will be built obtains transgenic line, and
Transfer-gen plant is dyed with GUS display substrates x-Gluc, guard cell's coloring case is observed under microscope.
Advantageous effect：The present invention provide for the first time one kind can in Ammopiptanthus mongolicus guard cell specifically expressed promoter
PAmSLAC1, the specificity of the promoter is very strong, therefore provides effective tool for the research of Plant Guard Cells function.
Description of the drawings
Dyeing step moves third round PCR product electrophoretogram to Fig. 1 AmSLAC1 promoters for the first time；
1,2,3,4 swimming lanes are respectively the product of 4 random primers, and swimming lane 5 is DL2000 Marker (TAKARA).
Fig. 2 AmSLAC1 promoter clone PCR products electrophoretograms；
Swimming lane 1 and 2 is PAmSLAC1Promoter fragment, swimming lane 3 are 1KB Marker (TAKARA).
Fig. 3 is the P of the present inventionAmSLAC1The fusion expression vector collection of illustrative plates of promoter and gus gene；
Fig. 4 is the P of the present inventionAmSLAC1Promoter transfer-gen plant is identified；
Swimming lane 1 is DL2000Marker (TAKARA), and swimming lane 2 is WT lines PCR product, and swimming lane 3-11 is transgenosis
Plant PCR identifies band
Fig. 5 is the P of the present inventionAmSLAC1The histochemical stain result figure of the gus gene of promoter driving；
A：Seedling (blue), B：Blade guard cell (blue), C：Blade table fur (blue), D, hypocotyl guard cell
(blue), E, main root root segment (colourless), F, the tip of a root (colourless), G, flower (blue), H, calyx guard cell (blue), I, pod
(colourless).
Specific implementation mode
According to following embodiment, it can be better understood from the present invention, but following embodiment is not offered as appointing the present invention
What is limited.
Embodiment 1：A kind of Ammopiptanthus mongolicus guard cell different expression gene promoter PAmSLAC1Cloning process
Ammopiptanthus mongolicus Ammopiptanthus mongolicus (Maxim.) Cheng f., material to be tested kind used in the present invention
It plants in growth chamber, incubator condition, 100 μm of ol.m of illumination-2s-1；Photoperiod：16h illumination/8h is dark；Temperature：23℃±
0.5℃；Relative air humidity 70%.
1. Ammopiptanthus mongolicus PAmSLAC1Promoter is cloned：
Ammopiptanthus mongolicus genomic DNA is extracted using cetyl trimethylammonium bromide (CTAB) method, according to Liu et al. people (Liu
And Chen 2007) the carry out chromosome walking, in two steps, often step carries out three-wheel PCR to chromosome walking, and two steps are obtained
PCR product sequence assembly, it is final to obtain Ammopiptanthus mongolicus PAmSLAC1Promoter (1455bp).Step moves the primer such as table 1.
1 chromosome walking of table clones PAmSLAC1Promoter the primer
Ammopiptanthus mongolicus PAmSLAC1The clone of promoter is as follows：
Ammopiptanthus mongolicus genomic DNA is extracted with CTAB methods, the specific steps are：The fresh Ammopiptanthus mongolicus of 1-50g is taken, is ground into liquid nitrogen
Powder；Agar is transferred in the centrifuge tube of precooling, 700 μ L CTAB Extraction buffers are added immediately, 65 DEG C keep the temperature 20min, therebetween
It shakes frequently；700 μ L chloroforms/isoamyl alcohol (v/v=24/1) is added, overturns centrifuge tube mixing, at room temperature, 12000rpm centrifugations
20min；Supernatant is transferred in another centrifuge tube, isometric chloroform/isoamyl alcohol is added, reverse centrifuge tube mixing, room temperature,
12000rpm centrifuges 10min；Upper strata aqueous phase is transferred in the new centrifuge tube through silanization treatment, isometric isopropyl is added
Alcohol, mixing place 30min at room temperature；12000rpm centrifuges 5-10min, goes supernatant, the rinsing of 70% ethyl alcohol, precipitation drying；Wind
40 μ L TE buffer solutions DNA are added after dry, -20 DEG C save backup.
Based on known AmSLAC1 genomic dna sequences, respectively in its translation initiation codon ATG downstream 249bp,
Three specific reverse primer AmSLAC-SP3, AmSLAC-SP2 and AmSLAC-SP1 are separately designed at 372bp and 523bp, it is above
The Ammopiptanthus mongolicus genomic DNA for stating extraction is template, and first round PCR forward primer is respectively four degenerate primers LAD1-1, LAD1-
2, LAD1-3 or LAD1-4, reverse primer AmSLAC-SP1, system are：2 μ L, dNTPs Mix of 10X Ex Taq Buffer
(each 2.5mmol/L) 1.6 μ L, MgCl2(25mmol/L) 1.2 each 0.4 μ L of μ L, LAD1-1/LAD1-2/LAD1-3/LAD1-4,
0.5 units of AmSLAC-SP1 0.4 μ L, genomic DNA 20-50ng, Ex Taq, deionized water polishing to 20 μ L.PCR reacts
Program is：94 DEG C of 3min, 95 DEG C of 1min, 95 DEG C of 30s, 60 DEG C of 1min, 72 DEG C of 3min, 10 cycles, 94 DEG C of 30s, 40 DEG C of 2min,
72 DEG C of 3min, 94 DEG C of 20s, 58 DEG C of 1min, 72 DEG C of 3min, 25 cycles, 72 DEG C of 5min, 4 DEG C of 30s.
First run PCR product is used as second to take turns PCR reaction templates, forward primer AC1 after diluting 40 times, reverse primer is
AmSLAC-SP2, reaction system are：10X Ex Taq Buffer 2 μ L, dNTPs Mix (each 2.5mmol/L) 1.6 μ L, MgCl2
0.4 0.4 μ L of μ L, AmSLAC-SP2 of (25mmol/L) 1.2 μ L, AC1, the first round PCR product after 40 times of dilution, 1 μ L, Ex
0.5 units of Taq, deionized water polishing to 20 μ L.PCR response procedures are：95 DEG C of 30s, 65 DEG C of 1min, 72 DEG C of 3min, 1 is followed
Ring, 95 DEG C of 30s, 68 DEG C of 1min, 72 DEG C of 3min, 94 DEG C of 20s, 58 DEG C of 1min, 72 DEG C
3min, 95 DEG C of 30s, 50 DEG C of 1min, 72 DEG C of 3min, 13 cycles, 72 DEG C of 5min, 4 DEG C of 30s.
Second wheel PCR product is used as third round PCR reaction templates after diluting 10 times, and forward primer AC1, reverse primer is
AmSLAC-SP3, reaction system are：10X Ex Taq Buffer 2 μ L, dNTPs Mix (each 2.5mmol/L) 1.6 μ L, MgCl2
0.4 0.4 μ L of μ L, AmSLAC-SP3 of (25mmol/L) 1.2 μ L, AC1, the second wheel PCR product after 10 times of dilution, 1 μ L, Ex
0.5 units of Taq, deionized water polishing to 20 μ L.PCR response procedures are：95 DEG C of 30s, 68 DEG C of 1min, 72 DEG C of 3min, 95 DEG C
30s, 68 DEG C of 1min, 72 DEG C of 3min, 94 DEG C of 20s, 55 DEG C of 1min, 72 DEG C of 3min, 8 cycles, 72 DEG C of 5min, 4 DEG C of 30s.By
Three-wheel PCR product is detected with the agarose gel electrophoresis of 1% (w/v), and the results are shown in Figure 1.Respectively by 1,2,3 and 4 swimming lane items
Band cuts glue, after gel reclaims kit (purchased from the precious biological Co., Ltd in Dalian, same as below) recycling, is connected to pMD19-T loads
Body (purchased from the precious biological Co., Ltd in Dalian), converts 5 α competent cells of DH, it is solid to be applied to the LB with 50mg/L ampicillins
On body tablet, 37 DEG C are cultivated 16 hours, and picking single bacterium colony carries out bacterium colony PCR, selects positive colony sequencing, obtains AmSLAC1 genes
The DNA sequence dna of translation initiation site upstream 500bp or so,
It is reference to obtain 500bp or so DNA sequence dnas with above-mentioned clone, separately designs three specific reverse primer AmSLAC-
SP6, AmSLAC-SP5 and AmSLAC-SP4, then reacted by above-mentioned three-wheel PCR, we have moved the left sides 1000bp to 5 ' end steps again
This two sections of sequence assemblies are redesigned forward and reverse primer AmSLAC1pro-F and AmSLAC1pro-R, and respectively at this by the right side
5 ' ends of two primers introduce Sac I and I restriction enzyme sites of Nco, PCR system are：2X PrimerSTAR Buffer 10 μ L, dNTP
1.6 0.4 0.4 μ L of μ L, AmSLAC1pro-R (10 μM) of μ L, AmSLAC1pro-F (10 μM) of Mixture (each 2.5mM), Ammopiptanthus mongolicus
2 μ L of genomic DNA, deionized water, 5.6 μ L.PCR response procedures：95 DEG C of 3min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 2min, 30
A cycle, 72 DEG C of 10min, 4 DEG C of 5min.The PCR product agarose gel electrophoresis of 1% (w/v) is detected, as a result such as Fig. 2 institutes
Show.1 and 2 swimming lane bands are cut into glue respectively, after being recycled with gel reclaims kit, are connected to pEasy-blunt zero carriers
(TransGen Biotech) converts Trans1-T1, is applied on the LB solid plates with 50mg/L ampicillins, 37 DEG C
Culture 16 hours, picking single bacterium colony carry out bacterium colony PCR, select positive colony sequencing, obtain AmSLAC1 gene translation initiation sites
Upstream 1455bp DNA sequence dnas,
Embodiment 2：Ammopiptanthus mongolicus PAmSLAC1The conversion of the structure and Agrobacterium tumefaciens strain GV3101 of plant expression vector：
The recombinant vector of structure is by the 35S promoter of the startup gus gene on plasmid pCambia1301 with having cloned
Obtained PAmSLAC1Segment is replaced.To complete this purpose, I double digestion cloning vector pEasy-P of Sac I and Nco is used firstAmSLAC1,
I double digestion pCambia1301 plasmids of Sac I and Nco are used simultaneously.Endonuclease reaction carries out in 37 DEG C of water-baths, after 8 hours, uses
The agarose gel electrophoresis of 1% (w/v) detects.By cloning vector pEasy-PAmSLAC1Small fragment about 1.5kb under digestion and
Large fragment under pCambia1301 digestions is recycled with DNA gel QIAquick Gel Extraction Kit.According to 3:1 (molar concentration rate) ratio will
pEasy-PAmSLAC1Large fragment mixing under small fragment and pCambia1301 digestions under digestion, is added T4DNA ligases
(NEB) 2 unit, 10 X reaction buffer, 1 μ L, 16 DEG C of water-baths connect 16 hours or more.5 α competent cells of DH are converted, are applied
In on the LB solid plates with 50mg/L kanamycins, 37 DEG C are cultivated 16 hours, and picking single bacterium colony carries out bacterium colony PCR, through enzyme
It cuts the correct recombinant plasmid of verification and is named as pCambia1301-PAmSLAC1As shown in Figure 3.
Using electric shocking method by pCambia1301-PAmSLAC1It is transferred to Agrobacterium tumefaciems GV3101, with that is mould containing 50mg/L cards
Element and the Double solid LB plate screenings of 50mg/L rifampins, picking single bacterium colony carry out bacterium colony PCR verifications, are preserved after correct
Bacterial strain is named as pCambia1301-PAmSLAC1/GV3101。
Wherein steps are as follows for electric shocking method：1) 100 μ L Agrobacterium GV3101 receptors are taken, is placed in and slowly melts on ice, 2) plus
Enter 0.5 μ g recombinant plasmids pCambia1301-PAmSLAC1, gently mixing places 30min on ice, 3) and by recombinant plasmid and Agrobacterium
Mixed liquor is placed in 2mm electric shock cups, and voltage 1.8kV, capacitance 25 μ F, 200 Ω of resistance shock by electricity, 4) by the recombination after electric shock
Plasmid and Agrobacterium mixed liquor are sucked out, and are placed in 2mL centrifuge tubes, the LB liquid medium of addition 1mL non-resistants, 28 DEG C,
100rpm cultures 1 hour, 5) 12000rpm, 30s, go supernatant, cell to be resuspended in 200 μ L non-resistant LB liquid mediums；6) will
Cell mixture is spread evenly across the solid LB trainings containing 50mg/L kanamycins, 25mg/L gentamicins and 50mg/L rifampins
Base is supported, 28 DEG C are cultivated 2 days, are verified after bacterium colony occurs in tablet.
Embodiment 3：Plant expression vector pCambia1301-PAmSLAC1Genetic transformation and transgenosis in arabidopsis are planted
Strain screening
Transformation of Arabidopsis thaliana is carried out with reference to the method in arabidopsis laboratory manual, operating procedure is as follows：
1) bud is cut when arabidopsis is bloomed for the first time, promotes the hyperplasia of the more sprays of side shoot, for use；2) picking is verified just
True positive colony first shakes bacterium on a small quantity with 100mL triangular flasks, 28 DEG C, 200rpm overnight incubations；3) it is then transferred to after muddiness
500mL triangular flasks (press 1:100 ratio is inoculated into 200mL kanamycins containing 50mg/L, 25mg/L gentamicins and 50mg/L profits
In the flat LB liquid medium of good fortune) expand and cultivates, 28 DEG C, 200rpm, 8-12 hour, 4) it is surveyed with spectrophotometer (BIO-RAD)
Bacterium solution OD600Value is 0.8-1.2；50mL centrifuge tubes are added in bacterium solution, 12000rpm centrifuges 5min, collects Agrobacterium；5) it uses
It is [dense that 20 μ L Silwet L-77 surfactants are added in sucrose solution in 100mL 10% (m/v) sucrose solution suspension Agrobacterium
Spend 0.02% (v/v)]；6) bacterium solution to suspend is poured into big culture dish, and arabidopsis floral immersion bacterium solution is infected 1 minute, is invaded
The process of dye slowly rotates earthen bowl.It is dried with filter paper after infecting, is put into black plastic bag or film covering, light culture 24 are small
When；7) it after 24 hours, is normally cultivated under illumination condition, keeps moisture sufficient.It disseminates once, is received after seed maturity again after a week
Collection.
It is placed 2 weeks at room ventilation after seed drying, 10min is sterilized in 75% (v/v) ethyl alcohol；Ethyl alcohol is outwelled, is used
10% (v/v) hypochlorite disinfectant 5min；Sodium hypochlorite is outwelled, with sterile water washing 6 times；The seed disinfected is seeded in and is contained
Have on 1/2MS (Sigma) solid medium of 50mg/L hygromycin, 4 DEG C are placed 2 days, and plant incubator, 100 μ of illumination are transferred to
mol.m-2s-1；Photoperiod：16h illumination/8h is dark；Temperature：23℃±0.5℃；Relative air humidity 70%.It is carried according to expression
The distinctive positive seedling of hygromycin resistance screening on body.When blade grows to 3-4 pieces, a piece of blade of clip is put into 1.5mL centrifuge tubes
In, blade is ground with pipette tips；400 μ L extracting solutions (200mM Tris-HCl (pH 7.5), 250mM NaCl, 25mM is added
EDTA, 0.5%SDS.), vortex 5s；12000rpm centrifuges 1min；300 μ L supernatants are drawn in new 1.5mL centrifuge tubes, are added
Enter 300 μ L isopropanols, mixing is stored at room temperature 10min；12000rpm centrifuges 5min；Supernatant is removed, 750 μ L, 70% (v/ are added
V) ethyl alcohol, 12000rpm centrifuge 5min；Supernatant is removed, after air-drying, 30-100 μ L aqua sterilisas are added.
Using the DNA of said extracted as template, PCR amplification is carried out with the primer on carrier and gene order, obtains purpose item
Band as positive transformants plant.The primer when PCR is identified：
Sense primer AmSLAC1pro-F：5’-GAGCTCTCTATGTGTTTAAAACAGTGTTTC-3’
Downstream primer：vector-R:5’-gtggtgtagagcattacgctgcg-3’
PCR response procedures：95 DEG C of 3min, 95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 2min, 35 cycles, 72 DEG C of 10min, 4 DEG C
5min.The PCR product agarose gel electrophoresis of 1% (w/v) is detected, the results are shown in Figure 4, obtains 9 plants of positives altogether and turns base
Because of plant.
Embodiment 4：Ammopiptanthus mongolicus PAmSLAC1The functional analysis and its application of promoter
The present invention clones obtain P for the first timeAmSLAC1Promoter sequence, and functional analysis has been carried out to it.From embodiment 3,
The positive seedling T1 generations screened in transformation of Arabidopsis thaliana and PCR authentication steps, selfing harvest seed, i.e. T2 generations.It is selfed harvest seed again
(i.e. T3 generations).The different times tissue of T3 9 strains of generation is taken to carry out GUS dyeing.Staining procedure is as follows：Vegetable material is soaked in
(0.5mg/mL X-Gluc, 10mM EDTA, 50mM sodium phosphate buffers (pH7.0), 0.5mM iron cyanogen in 1.5mL GUS dyeing liquors
Change potassium, 0.5mM potassium ferrocyanides, 0.001% (v/v) Triton X-100)；Vacuum suction 10min, at 37 DEG C water-bath stay overnight；
The vegetable material that will be impregnated through GUS dyeing liquors is sequentially placed into the ethyl alcohol of 20% (v/v), 35% (v/v) and 50% (v/v),
20min or so is placed per level-one concentration of alcohol.Remove 50% ethyl alcohol, addition fixer (100mM sodium phosphate buffers (pH7.2),
1% (v/v) glutaraldehyde and 4% (v/v) formaldehyde), 40min or more is fixed at room temperature.After the completion of fixation, two are washed with 70% ethyl alcohol
It is secondary, 70% ethyl alcohol of displacement 2-3 times, until negative control is white；NIKON stereomicroscope observations are taken pictures.The arabidopsis seedling taking phase
Whole strain, reproductive stage inflorescence and pod.The position for being dyed to blue is exactly the position of gus gene expression.
Coloration result finds (Fig. 5)：In arabidopsis, blade, hypocotyl, guard cell is dyed to blue at calyx.By
This is as it can be seen that the gus gene of promoter driving is mainly expressed in the guard cell of plant.
The experimental results showed that Ammopiptanthus mongolicus PAmSLAC1With following biological function：The promoter drives gus gene in quasi- south
It is expressed in the guard cell of mustard.This is with the specific expressed promoter of guard cell with answering in terms of plant genetic engineering
With value.
Ammopiptanthus mongolicus PAmSLAC1Application of the promoter in guard cell
It clones to obtain Ammopiptanthus mongolicus P using chromosome walking methodAmSLAC15 ' upstream sequences of the endogenous gene of driving.Structure by
The plant expression vector pCambia1301-P of the Reporter gene GUS of the promoter regulationAmSLAC1.Using agriculture bacillus mediated flower
Sequence dip method arabidopsis thaliana transformation, the T0 of the harvest preliminary screening on the MS culture mediums containing hygromycin for seed, when arabidopsis is long
After going out two panels true leaf, continue to cultivate in transplanting to Nutrition Soil, when plant grows to 4-6 piece leaves, carries out the positive plant of PCR identifications
Strain.Two generation of self propagated.In T3 generations, select 9 transgenic lines to carry out GUS dyeing.
The dyeing of GUS histochemical methods shows that the gus gene of promoter driving is only expressed in the guard cell of plant,
Advantageous tool is provided for research guard cell's specific gene, to improve the drought-resistant ability and photosynthetic efficiency of plant, is improved
The yield and quality of crop provides genetic resources and theoretical foundation.
The invention is not limited in specific implementation modes above-mentioned.The present invention, which expands to, any in the present specification to be disclosed
New feature or any new combination, and disclose any new method or process the step of or any new combination.
Sequence table
<110>Nanjing Soil Inst., Chinese Academy of Sciences
<120>A kind of Mongolia's Ammopiptanthus mongolicus guard cell specific expression promoter and its application
<160> 15
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1455
<212> DNA
<213>Mongolian Ammopiptanthus mongolicus (Ammopiptanthus mongolicus (Maxim.) Cheng f.)
<400> 1
tctatgtgtt taaaacagtg tttcatctct attgatcatt gaaaaattga cagatatcgg 60
actgaaggac cgaatttgca acattgaaaa aataagggga ccaaaagtgc tcaaaaaaat 120
cagagggacc aaaattgaac atgaccaata ataggggatc aaaaatgcaa ttacgccaaa 180
actaaaagaa aataatcaaa caccctttta aaggcaaata atgtttgata cacaacatgt 240
ggatgtatat aattttacat cagctataaa taagtgacaa aatcagagtt aaagtgtaag 300
tgtgaagaaa ccaaaatagg atccgtttta atctaataat gaaaattata taaatgtaag 360
tgatatatgt ataggtgtgt aaataacatt attcttaaaa ggttaacaag ttcaaccttt 420
gtcatcaccc actatgagtt ctcttgcata aattatagga gtggaaacta ttgctacagt 480
gacaaagatg acgaggaaag gagccgaggc cattgctgct gtcggttgaa ctagaggcta 540
gagcagctac tggttatgtt aacaagcttt aatattttcc ttatacttag cagagtgctt 600
gaataaagtt gatcaatcgt attgtattta tagtgcaatc taattctgat gtaaatcaac 660
taagtgttag tgtgatagtt tcaaaatttg atccattgga tgggaattac cataatagtc 720
cttccatgct aaatttatcc ttactctgca aattcctagt ttgcttacct caatcagcat 780
gtaaataatt agagattagc gtgtattgta caataataaa aaatgttcat caattatttg 840
catattgatc attagtatga tagtgccgtg taagaattga tatacctatt tgatttagat 900
tctggtgtcc gcaaacggga ataaacgttt ttcatggtac ttacaatcgg aagcaacaat 960
attgttactt tgatagaagg tacattggga aaagtttttt tttgttgttt gctgccactg 1020
atttaaacca aataggtccc taagtggtgg gaaaggtaga gaagaaaaaa aggaaaatgt 1080
ttcaaaccaa aaaggacctt aatgccactt gttcaaacca tttaggccaa catttctact 1140
tccttaaagt ctgagtttga tccccgtgct cagttgaaaa ctctatcttt tgcatgcaca 1200
ctactttcaa gttgttctta accataacca tatatatatg ccagcatttc gctcacggga 1260
tgttcgtccc atatacctca cttttctctc aaagcagtgg cttccatttc atctaccaaa 1320
caaccatcaa ccaaagaagc ttcactcccc gtgaggtgtc cctcacataa cgcatgtata 1380
aaattaaaaa ctacaccact aagattaaga attcaataga gcatttgttt tgctcaacat 1440
attattacaa ggaga 1455
<210> 2
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ccccaaactt gttccacttc ttgtc 25
<210> 3
<211> 46
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
acgatggact ccagtccggc ccaactctct ctctttcctt ggcaac 46
<210> 4
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gccacttctc atctttgggg agaag 25
<210> 5
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
ctaagtataa ggaaaatatt aaagc 25
<210> 6
<211> 25
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gatttacatc agaattagat tgcac 25
<210> 7
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
ggaaggacta ttatggtaat tc 22
<210> 8
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
gagctctcta tgtgtttaaa acagtgtttc 30
<210> 9
<211> 30
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
ccatggtctc cttgtaataa tatgttgagc 30
<210> 10
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
acgatggact ccagagcggc cgcvnvnnng gaa 33
<210> 11
<211> 33
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
acgatggact ccagagcggc cgcbnbnnng gtt 33
<210> 12
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
acgatggact ccagagcggc cgcvvnvnnn ccaa 34
<210> 13
<211> 34
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
acgatggact ccagagcggc cgcbdnbnnn cggt 34
<210> 14
<211> 16
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 14
acgatggact ccagag 16
<210> 15
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 15
gtggtgtaga gcattacgct gcg 23
Claims (5)
1. a kind of Mongolia's Ammopiptanthus mongolicus guard cell's specific expression promoter, it is characterised in that the promoter is SEQ ID No.1 institutes
The nucleotide sequence shown.
2. a kind of carrier, it is characterised in that the carrier contains promoter according to claim 1.
3. a kind of bacterial strain, it is characterised in that the bacterial strain contains promoter according to claim 1.
4. the cloning process of Mongolian Ammopiptanthus mongolicus guard cell's specific expression promoter described in claim 1, it is characterised in that step
For：Based on known AmSLAC1 genomic dna sequences, respectively in 249 bp of the downstreams its translation initiation codon ATG, 372 bp
With at 523 bp design three specific reverse primer SEQ ID No.2 shown in AmSLAC-SP3, shown in SEQ ID No.3
AmSLAC-SP1 shown in AmSLAC-SP2 and SEQ ID No.4 passes through chromosome using Mongolian Ammopiptanthus mongolicus genomic DNA as template
The method that step is moved obtains the DNA sequence dna of one section of translation initiation codon upstream, and redesigning three on this basis specifically reversely draws
AmSLAC-SP6, AmSLAC- shown in AmSLAC-SP5 and SEQ ID No.7 shown in SEQ ID No.6 shown in object SEQ ID No.5
SP4, then moved to 5 ' end steps by the above method, section of DNA sequence of getting back redesigns this two sections of sequence assemblies positive and negative
To AmSLAC1pro-R, the sequence expanded shown in AmSLAC1pro-F and SEQ ID No.9 shown in primer SEQ ID No.8
On subclone to carrier pMD19-T, sequencing result shows that the sequence has 1455 bp.
5. Mongolian Ammopiptanthus mongolicus guard cell specific expression promoter is in screening guard cell's specific gene described in claim 1
Application.
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Cited By (2)
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| CN111321146A (en) * | 2020-03-19 | 2020-06-23 | 内蒙古自治区农牧业科学院 | Clone of mongolian ammopiptanthus mongolicus inducible promoter and application thereof |
| CN114752597A (en) * | 2022-04-06 | 2022-07-15 | 广东省科学院南繁种业研究所 | Drought-inducible promoter P specifically expressed by plant guard cellsSCBV-CHN2And applications |
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| CN111321146A (en) * | 2020-03-19 | 2020-06-23 | 内蒙古自治区农牧业科学院 | Clone of mongolian ammopiptanthus mongolicus inducible promoter and application thereof |
| CN114752597A (en) * | 2022-04-06 | 2022-07-15 | 广东省科学院南繁种业研究所 | Drought-inducible promoter P specifically expressed by plant guard cellsSCBV-CHN2And applications |
| CN114752597B (en) * | 2022-04-06 | 2023-09-19 | 广东省科学院南繁种业研究所 | Drought-inducible promoter P for plant guard cell specific expression SCBV-CHN2 Application and application thereof |
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