CN101831431B - Promoter afb4 and application thereof - Google Patents
Promoter afb4 and application thereof Download PDFInfo
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- CN101831431B CN101831431B CN2010101832276A CN201010183227A CN101831431B CN 101831431 B CN101831431 B CN 101831431B CN 2010101832276 A CN2010101832276 A CN 2010101832276A CN 201010183227 A CN201010183227 A CN 201010183227A CN 101831431 B CN101831431 B CN 101831431B
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a promoter AFB4 and application thereof. The promoter is a DNA fragment shown in the following 1), 2), 3) or 4): 1) a DNA fragment shown as a sequence 1 in a sequence table; 2) a DNA fragment which hybridizes with the DNA sequence defined in 1) under strict conditions and has the same function; 3) a DNA fragment having 90% or more homology with the DNA sequence defined in 1) and having the same function. Experiments prove that the AFB4DNA fragment is a promoter, and the promoter has strong specific expression activity and is specifically expressed at a vascular column combined by a main root and a lateral root of a plant and at the sawtooth edge of a leaf. The promoter of the invention provides a favorable tool for the research of gene function of specific expression and the transgenic breeding of plants, and has important significance.
Description
Technical field
The present invention relates to promoter AFB 4 and application thereof.
Background technology
Promotor can be divided three classes by its mode of action and function: constitutive promoter, inducible promoter and tissue-specific promoter.Constitutive promoter can both start expression in institute in a organized way, and the persistence of expression is arranged, and does not have the space-time specificity, like the CaMV35S promotor etc.Such promotor has obtained big quantity research and application, in genetically engineered, plays a significant role.But because its expression is regardless of space-time, can cause the excess accumulation of outer rim genetic expression, can influence plant and grow normally.Therefore, seek induction type or tissue-specific promotor, all have vital role for gene functional research and transgenic breeding.
Summary of the invention
An object of the present invention is to provide a kind of dna fragmentation with specific expression promoter function.
Dna fragmentation provided by the present invention is following 1), 2) or 3) shown in:
1) dna fragmentation shown in the sequence 1 in the sequence table;
2) under stringent condition with 1) dna sequence dna hybridization that limits and dna fragmentation with identical function;
3) with 1) dna sequence dna that limits has the homology more than 90% and has the dna fragmentation of identical function.
Increase above-mentioned arbitrary said dna fragmentation total length or its any segmental primer to also belonging to protection scope of the present invention.
Said primer centering, a primer sequence is shown in sequence in the sequence table 2, and another primer sequence is shown in sequence in the sequence table 3.
The recombinant vectors, reorganization bacterium, transgenic cell line or the expression cassette that contain above-mentioned arbitrary said dna fragmentation also belong to protection scope of the present invention.
Above-mentioned arbitrary said dna fragmentation also belongs to protection scope of the present invention in the application that makes goal gene in express at the leaf jagged edges place of lateral root generation place of plant and/or plant.
In the above-mentioned application, generation place of said lateral root is main root and lateral root bonded vascular cylinder place.
In the above-mentioned application, said plant is a dicotyledons.
In the above-mentioned application, said dicotyledons is an Arabidopis thaliana.
The application of above-mentioned arbitrary said dna fragmentation in the genetic breeding of plant also belongs to protection scope of the present invention.
In the above-mentioned application, said plant is a dicotyledons; Said dicotyledons is an Arabidopis thaliana.
Experiment showed, that AFB4 dna fragmentation of the present invention is a promotor, and this promotor there is very strong specifically expressing activity, the jagged edges place expression of special lateral root generation place plant (main root and lateral root bonded vascular cylinder place) and leaf.Promotor of the present invention is that the transgenic breeding of gene functional research on specific expression and plant provides favourable instrument, and is significant.
Description of drawings
Fig. 1 obtains the promotor of AFB4 for pcr amplification.
Fig. 2 cuts evaluation for the enzyme of expression vector.
Fig. 3 is a P-AFB4-1391-GUS carrier synoptic diagram.
Fig. 4 is that the PCR of transgenic arabidopsis identifies.
Fig. 5 is that the GUS of AFB4 promotor is active.
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
The preparation of embodiment 1, promotor and specifically expressing checking
One, the preparation of promotor and affirmation
1, the extraction of arabidopsis thaliana genomic dna
(1) gets Arabidopsis leaf, add liquid nitrogen, after fully grinding, powder is poured in the 1.5ml centrifuge tube;
(2) treat in the centrifuge tube liquid nitrogen volatilization fully after, add 500ul DNA extraction damping fluid (0.1mol/LTrisHCl (pH 8.0), 0.5mol/L NaCl immediately; 0.05mol/L EDTA; 0.5%SDS), fully behind the mixing, 65 ℃ are incubated 30 minutes; The continuous gentleness centrifuge tube that turns upside down makes sample and damping fluid thorough mixing during this time;
(3) with 12, centrifugal 15 minutes of 000rpm room temperature;
(4) supernatant is transferred to another centrifuge tube;
(5) add equal-volume tris-phenol, extracting one time;
(6) use isopyknic chloroform extracting one time again;
(7) get supernatant, add the equal-volume Virahol, softly rock centrifuge tube, make the abundant mixing of Virahol and supernatant ,-20 left standstill 10 minutes, with 12, and centrifugal 10 minutes of 000rpm;
(8), after DNA is deposited in Bechtop and dries up, subsequent use with the dissolving of sterilization distilled water with 70% ethanol lotion.
2, pcr amplification
With the arabidopsis thaliana genomic dna is template, with primer P1 and P2 is carried out pcr amplification.
Primer sequence is following:
P1:ACTGCAGCTGACTCACTAAGTCGTCCAT (sequence 2),
P2:TGGATCCCTCTGACTTTGATCTTATCCA (sequence 3),
Reaction system is following:
The PCR program:
The PCR product is carried out agarose gel electrophoresis.Electrophoresis result (M:Marker as shown in Figure 1;-: the PCR blank; The P:PCR amplified production).Reclaim the purpose band.
The purpose product is connected with carrier EZ-T, connects the product transformed into escherichia coli, resistance screening is cultivated, the picking mono-clonal; Mono-clonal is inoculated in liquid nutrient medium cultivates, extract plasmid, plasmid is checked order.The sequence that records is made P-AFB4-EZ-T with the positive recombinant plasmid note that obtains shown in SEQUENCE ID:1, the note of the dna fragmentation shown in the SEQUENCE ID:1 is made AFB4.
Carrier EZ-T is available from the sincere industry of Beijing Kang Run bio tech ltd, and catalog number is T168-10.
Two, the tissue specificity of promotor checking
PCAMBIA1391Z is available from CAMBIA (Australia).Agrobacterium strains AGL1 ATCC No.BAA-101 is available from ATCC.
(1) structure of transgenic arabidopsis
With Hind III and BamH I digestion with restriction enzyme P-AFB4-EZ-T; Reclaim the purpose segment of about 2000bp; Use Hind III and BamH I digestion with restriction enzyme expression vector pCAMBIA1391Z simultaneously, reclaim the carrier segment, then the purpose segment is connected with the carrier segment; To connect the product transformed into escherichia coli, resistance screening is cultivated, the picking mono-clonal; Mono-clonal is inoculated in liquid nutrient medium cultivates, extract plasmid, plasmid is carried out enzyme cut evaluation and sequence verification.Enzyme is cut result (M:Marker as shown in Figure 2; 1: enzyme is cut product), record the gene order inserted between Hind III and the BamH I restriction enzyme site of pCAMBIA1391Z shown in SEQUENCE ID:1, show that the carrier of structure is correct, the positive recombinant expression vector note that obtains is made P-AFB4-1391Z.GUS is regulated and control by dna fragmentation of AFB4 only in this carrier, does not receive any other promoter regulation (Fig. 3).
The electricity consumption method for transformation imports recombinant expression vector P-AFB4-1391Z among the agrobacterium strains AGL1, and resistance screening obtains positive reorganization Agrobacterium, and note is made AGL1/P-AFB4-1391Z.
Infect the method for inflorescence through Agrobacterium and carry out the Arabidopis thaliana conversion, the seed that conversion obtains is layered on the substratum that contains hygromycin resistance and screens, and the positive young plant that obtains is that T0 is for transgenic arabidopsis.
Simultaneously empty carrier pCAMBIA1391Z is transformed agrobacterium strains AGL1, obtain positive reorganization Agrobacterium, note is made AGL1/pCAMBIA1391Z.With reorganization Agrobacterium AGL1/pCAMBIA1391Z arabidopsis thaliana transformation, obtain T0 for changeing the empty carrier Arabidopis thaliana.
(2) evaluation of transgenic arabidopsis
PCR identifies: is template with T0 for the genomic dna of transgenic arabidopsis, carries out pcr amplification with primer P1/P4; Amplified production carries out agarose gel electrophoresis.With recombinant expression vector P-AFB4-1391Z is the PCR positive control.
P1:ACTGCAGCTGACTCACTAAGTCGTCCAT (sequence 2) is the special primer of AFB4;
P4:GGGTCCTAACCAAGAAAATGA is the special primer of GUS;
The result is as shown in Figure 4.Among the figure, 1,2,3 expression transgenic arabidopsis ,+expression PCR positive control, empty carrier pCAMBIA1391Z Arabidopis thaliana is changeed in-expression.
As a result, obtain 20 strain transgenic arabidopsis young plants.
(3) specifically expressing of promotor in the transgenic arabidopsis
Get T0 and carry out GUS dyeing for 15 days seedling of transgenic arabidopsis growth.Two positions of the active main existence of GUS as a result, the one, lateral root generation place (further observing is the vascular cylinder place in main root and lateral root junction), the 2nd, the jagged edges of blade.
Simultaneously to change empty carrier pCAMBIA1391Z Arabidopis thaliana over to as negative control.
Concrete grammar is: whole Arabidopis thaliana seedling is added staining fluid (50mM phosphoric acid buffer; The 5mM Tripotassium iron hexacyanide; The 5mM yellow prussiate of potash; 10mM EDTA; 1mM X-gluc) in, 37 ℃ of incubated overnight with 70% ethanol decolorization 2-3 time, are white in color to negative control material, microscopic examination, and the blueness under the white background is GUS and expresses the site.
The positive transgenic arabidopsis of 20 strains all carries out GUS dyeing.
The result is as shown in Figure 5.The white arrow indication is GUS dyeing position.The result shows that in the positive transgenic arabidopsis plant, the GUS activity all concentrates on the jagged edges of lateral root generation place (main root and lateral root bonded vascular cylinder place) and leaf; And all do not see painted in the commentaries on classics empty carrier pCAMBIA1391Z Arabidopis thaliana.This presentation of results; AFB4DNA fragment of the present invention is a promotor, has started the expression of GUS, and this promotor has very strong specifically expressing active; Special in main root and lateral root bonded microtubule post place and blade jagged edges high expression level, significant for transgenic breeding in the future.
Sequence table
< 110>Inst. of Genetics and Development Biology, CAS
< 120>promoter AFB 4 and application thereof
<160>3
<210>1
<211>1968
<212>DNA
< 213>Arabidopis thaliana Arabidopsis thaliana
<400>1
ctgactcact?aagtcgtcca?ttacgaacct?atagtagcaa?tgaaacaaat?gaatcattca 60
acaatatctg?acagagtaaa?acgctgtaaa?aatcatcacc?aagagctact?cacaatctta 120
tcaaacagtt?caccaccagt?aatgtactcc?aggatgatat?aaatctttgt?acggcttgct 180
agaacctaaa?aaggaccaaa?tatcaaaatc?ccaaatggaa?agcaagaatc?tttcagtaaa 240
ttataaagca?aagaagaaga?agcaaaacat?tcacttacct?cataaagacg?aacaacacaa 300
ggatgacgaa?caagcttcat?aatcgagatc?tctctcttaa?tctgctaaca?catgtcatca 360
atctaatcat?atctcagcca?aaatgaaatc?gaagagagta?agtagtaaga?agatagagac 420
gggacctgat?ccaccatctt?gcgcttgatg?attgtgctac?gatccacgat?tttcatggcg 480
acgctctcac?cggtctctgt?attctgagcg?aacttgactt?tagcgaaagt?accttcgccg 540
attgttctac?ctaattcata?cttgcccacc?ttccttacca?ccatattctc?ttctgcttct 600
actcaatttc?aatttcactg?catttcttca?agtctagtgt?ttgatagata?gagagagaga 660
aagaggaaag?gcagaaaaaa?agtttcgaat?tttaaaatta?aatttataac?gacaaatata 720
cagagcaaag?cttttaatta?attaggaaag?tctcaaattt?gggaaagaga?taggtgttta 780
aattgcaaat?tggaattaaa?gaagctttaa?tcaatagaaa?gccaaagtaa?ttaagggaaa 840
atatatagat?ttgtgatttg?tgtttgatta?caaatctatg?tagagccaaa?ttaaagtaac 900
aaggggttgg?gggttaaaga?gaagtcagaa?aaggagactt?tgacgaagga?gacgtgttcg 960
gcgactactt?gccacattct?aatatttggt?tgtttacacg?tcattctttg?taatggaagg 1020
tcagtatatt?tataccataa?tttcacacta?atacgactcc?tagcttttga?aaaatatcta 1080
tagacttatg?acccatttat?tagtagttag?tgaccaagac?tatggataca?acgtcgttat 1140
gacttattct?ataaaacttg?acttcgaaat?ttcttctttt?agggaactta?tttgaaattt 1200
atccatattc?taaataattg?ccttttgaaa?attttaagaa?gcatatttga?agatatctat 1260
aacaatttgc?cttgtggaaa?attaagcaaa?cacatttttg?aaaagataat?cgaataaggc 1320
atacagtaca?cttatcttca?aaacccatta?ttggcagtaa?accaaccatt?aatcaccttt 1380
gatcttgttt?tggccaccat?attatgagat?ataataagcc?caaacatttt?tcctttacac 1440
gaaagccttg?aggccttctt?aagagaacac?aatcgaaaag?ttgattgtat?gataaaaatt 1500
aaattcatgt?aaaatgcatt?aacttaaaaa?aggatgaaag?tgactttaga?tattagtatt 1560
attcatttaa?agaaacagta?caaaacactt?ttaaatcctt?ttaattttgg?ttaagattca 1620
acaagtttac?atgattacca?cttacataaa?actctaaatg?tcacctgtca?caccttaaaa 1680
tagaagtaca?aatctttaat?aggattaaaa?atcatgtttg?gtcacaaaat?acttgatgat 1740
tctttttatt?tcttcatcaa?caaaaataat?gtgcaaaacc?aaaaatattt?gttttttttt 1800
cttttacata?gtctcctaaa?taatggaggt?gaaaatggag?agagagagag?agtccaaaca 1860
ccaagaccag?ctcctttttc?acctatctct?cttcttcatc?tgaatcagat?tttgtatcag 1920
aaagagagca?cctgagctca?taaattctgg?ataagatcaa?agtcagag 1968
<210>2
<211>28
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>2
actgcagctg?actcactaag?tcgtccat 28
<210>3
<211>28
<212>DNA
< 213>artificial sequence
<220>
<223>
<400>3
tggatccctc?tgactttgat?cttatcca 28
Claims (9)
1. a dna fragmentation is the dna fragmentation shown in the sequence in the sequence table 1.
2. the amplification said dna fragmentation total length of claim 1 or its any segmental primer are right.
3. primer according to claim 2 is right, it is characterized in that: said primer centering, and a primer sequence is shown in sequence in the sequence table 2, and another primer sequence is shown in sequence in the sequence table 3.
4. the recombinant vectors that contains the said dna fragmentation of claim 1.
5. the reorganization bacterium that contains the said dna fragmentation of claim 1.
6. the transgenic cell line that contains the said dna fragmentation of claim 1.
7. the expression cassette that contains the said dna fragmentation of claim 1.
8. the said dna fragmentation of claim 1 is in the application that makes goal gene in lateral root generation place of plant is expressed;
Generation place of said lateral root is main root and lateral root bonded vascular cylinder place;
Said plant is an Arabidopis thaliana.
9. the application of dna fragmentation described in the claim 1 in the genetic breeding of plant;
Said plant is an Arabidopis thaliana.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101832276A CN101831431B (en) | 2010-05-19 | 2010-05-19 | Promoter afb4 and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010101832276A CN101831431B (en) | 2010-05-19 | 2010-05-19 | Promoter afb4 and application thereof |
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| Publication Number | Publication Date |
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| CN101831431A CN101831431A (en) | 2010-09-15 |
| CN101831431B true CN101831431B (en) | 2012-05-23 |
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| CN110607385A (en) * | 2019-09-23 | 2019-12-24 | 深圳大学 | Functional molecular marker of arabidopsis thaliana leaf jagged edge related gene and application thereof |
| CN111394494B (en) * | 2020-02-12 | 2023-08-25 | 深圳大学 | Application of functional molecular marker of Arabidopsis leaf serrated edge related gene |
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| DE19960843A1 (en) * | 1999-12-16 | 2001-06-28 | Florian Grundler | Root-specific promoter |
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