CN101280313B - Root specific promoter and recombinant expression vector thereof - Google Patents
Root specific promoter and recombinant expression vector thereof Download PDFInfo
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- CN101280313B CN101280313B CN2008101122176A CN200810112217A CN101280313B CN 101280313 B CN101280313 B CN 101280313B CN 2008101122176 A CN2008101122176 A CN 2008101122176A CN 200810112217 A CN200810112217 A CN 200810112217A CN 101280313 B CN101280313 B CN 101280313B
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Abstract
The invention discloses a specific promoter of a root and a recombinant expression vector. The promoter adopts DNA molecular: a of following a) or b) or c) DNA molecular: a) is a DNA molecular which is composed of the deoxyribonucleotide shown in the sequence 1 in the sequence table; b) is a DNA molecular hybridized with a) defined DNA sequence under the strict condition; and c) is a specific expression DNA molecular in the root of the plant and has over 90 percents of homology and the gene with a adjusting and controlling purpose with a) defined DNA sequence. The invention also discloses a recombinant expression vector with the promoter, and the recombinant expression vector can be applied for cultivating the transgenic plant with a specific expressing target gene in the plant root.
Description
Technical field
The present invention relates to plant genetic engineering field, specifically, relate to a kind of root-specific promoter and recombinant expression vector thereof.
Background technology
That the expression of eukaryotic gene is subjected to transcribing is preceding, transcriptional level, transcribe the regulation and control of post-treatment, transhipment, translation skill and protein-active, wherein the regulation and control of transcriptional level are links the most basic in the genetic expression, promotor is a necessary for gene expression, having determined the intensity of space, time and the expression of exogenous gene expression, is the critical limitation factor that people utilize genetically engineered and transgenic technology directional transformation plant.
Encoding sequence only accounts for the 1%-10% of its genomic dna in most of eukaryotic gene groups, and a big chunk sequence plays the gene expression regulation effect in the non-coding sequence, and promoter sequence occupies very big ratio in non-coding sequence.The mode of action according to promotor can be divided into it 3 classes, i.e. constitutive promoter, inducible promoter and tissue-specific promoter, and wherein, tissue-specific promoter is called organ specific promoters again.
Currently obtain genetic modified organism by the transgenosis means (genomic modified organism GMO), thereby changes the plant especially growth characteristics, the important component part that fruit quality has become modern agriculture of crop.In transgenic plant, the most frequently used promotor is a constitutive promoter, promptly utilizes CaMV 35S promoter driving purposes gene high level expression in each tissue.This may cause some adverse influences for growth and development of plant under some situation, is unfavorable for realizing the purpose of people's plant modification.For example, after utilizing the CaMV 35S promoter to drive DREB1A gene transformation Arabidopis thaliana plant, drought resistance, the salt tolerance of transfer-gen plant improve greatly, but because the excessive ectopic expression of this gene, make the plant strain growth under the normal condition seriously be suppressed, show as that plant is downgraded, blade is short and small.
Tissue-specific promoter can the driving purposes gene at root, stem, leaf, flower or even pore specifically expressing.Root is plant absorbs moisture and nutrition from soil a major organs, and root-specific promoter can drive the goal gene specifically expressing in its downstream at root.By the transgenosis means, can be with separated and K
+The transporter that absorption is correlated with overexpression specifically will improve fertilising efficient effectively in root.Successfully identified at present and isolated multiple S, P, K transporter,, utilized root-specific promoter to drive these genes and express, can improve the bioavailability of nutrient at root as AKT1 and SULTR etc.Simultaneously, many cash crop be with root as storage organ, as potato, can be with the gene relevant at special its expression level of enhancing of root with starch and sugar metabolism, thereby improve its utility value.
Along with the development of functional genomics research, the more function gene is cloned, but will obtain better transfer-gen plant, and selecting suitable promotor that it is efficiently expressed at special position is the problem that at first will consider.
Summary of the invention
The purpose of this invention is to provide a kind of roots of plants specificity promoter.
Roots of plants specificity promoter provided by the invention, name is called SP, be following a) or b) or dna molecular c):
A) dna molecular of forming by the deoxyribonucleotide sequence shown in the sequence in the sequence table 1;
B) under stringent condition with dna sequence dna hybridization that a) limits and the dna molecular that can regulate and control goal gene specifically expressing in the root of plant;
C) and a) dna sequence dna that limits has the homology more than 90% and can regulate and control the dna molecular of goal gene specifically expressing in the root of plant.
Promotor in the described step c) preferably has homology more than 95% with a) promotor.
Above-mentioned roots of plants specificity promoter can be regulated and control goal gene and express in the vascular bundle tissue of plant root.
The expression cassette, recombinant vectors, transgenic cell line and the reorganization bacterium that contain promotor of the present invention also belong to protection scope of the present invention.
Described expression cassette is followed successively by promotor of the present invention, goal gene and transcription terminator to the downstream from the upstream.
The described SP promotor total length that increases and any segmental primer thereof are to also belonging to protection scope of the present invention.
Described recombinant expression vector specifically can be expression vector as shown in Figure 1 or the gus gene of expression vector shown in Figure 1 is replaced with the carrier that required goal gene obtains.
SP promotor of the present invention and described recombinant expression vector can be used for cultivating the transgenic plant of specifically expressing goal gene in roots of plants.
When the recombinant expression vector that will contain described SP promotor changes in monocotyledons or the dicotyledons, can in the root of monocotyledons or dicotyledons, efficiently express goal gene; Described plant optimization is an Arabidopis thaliana.
Experiment showed, promoters driven goal gene of the present invention specifically expressing in root.By the online database compare of analysis, found that, contain the cis-acting elements that two known root-specifics are expressed in the dna sequence dna of this 845bp.ATATT is the relevant element of a kind of root-specific, is called rootmotif, is the fragment of rolA gene promoter in the agrobacterium tumefaciens.In the AtSBP3 promotor, contain 13 ATATT cis-acting elements.The cis-acting elements that in this promoter sequence, also comprises an xylem specifically expressing: ACAAAGAA.
Root-specific promoter provided by the invention can substitute 35S promoter and be applied to transfer-gen plant, make goal gene be positioned root, perhaps as the part of promotor, obtain other characteristic after connecting other cis-acting elements, as strengthen expression activity or increase the abduction delivering ability, thereby be widely used in transgenosis process with this end in view.
Description of drawings
Fig. 1 is the physical map of recombinant expression vector pCAMBIA1391-SP
Fig. 2 is SP promoters driven gus gene specifically expressing in the Arabidopis thaliana root
Fig. 3 is the Photomicrograph that SP promoters driven gus gene is expressed at root
Embodiment
The acquisition of embodiment 1, SP promoter sequence
With 1 Colombia in age in week environmental wild-type Arabidopis thaliana plant is experiment material, extracts its genomic dna and is template with it, designs primer, pcr amplification SP promoter sequence from the arabidopsis gene group database of announcing.Introduce BamHI and EcoRI restriction enzyme site respectively at the primer two ends, primer sequence is as follows: the upstream: 5 '-ACGGGATCCACCATTCACGGTAGGTTCTTTTAT-3 ', introduce the BamHI restriction enzyme site; The downstream: 5 '-ACGGAATTCTCTTGCGTTTACTGTTTTTGC-3 ', introduce the EcoRI restriction enzyme site.
Pcr amplification product is carried out agarose gel electrophoresis to be detected, downcutting purpose bar zone purification reclaims, enzyme is cut product to be connected on the pGEM-T Easy carrier and transformed into escherichia coli DH10 β competent cell, the mono-clonal that picking grows on the Amp resistant panel, extraction plasmid, enzyme are cut evaluation and are checked order, and will change the plasmid called after p-SP of SP promoter sequence over to.Sequencing result shows that the deoxyribonucleotide sequence of the SP promotor that amplification obtains is shown in sequence in the sequence table 1.
Embodiment 2, carry the structure of the recombinant expression vector of SP promotor and gus gene
To use BamHI and EcoRI double digestion 3h respectively under plasmid p-SP and 37 ℃ of conditions of plasmid pCAMBIA1391 (available from pCAMBIA company), enzyme is cut product and is carried out 1% agarose gel electrophoresis analysis respectively, big fragment after the SP promoter sequence of recovery 845bp and the plasmid pCAMBIA1391 enzyme of 12kb are cut is spent the night with 4 ℃ of connections of T4DNA ligase enzyme.Connect product transformed into escherichia coli DH10 β competent cell, the mono-clonal that picking grows on the Kan resistant panel extracts plasmid, carries out PCR evaluation and enzyme and cuts evaluation.With the recombinant expression vector called after pCAMBIA1391-SP that obtains, the physical map of recombinant expression vector pCAMBIA1391-SP as shown in Figure 1.
Embodiment 3, SP promoters driven gus gene specifically expressing in the Arabidopis thaliana root
One, recombinant expression vector pCAMBIA1391-SP transforms the wild-type Arabidopis thaliana
The recombinant expression vector pCAMBIA1391-SP that embodiment 2 is made up transforms agrobacterium tumefaciens GV3101 competent cell, and single bacterium colony that picking is grown on Rif and kantlex (Kan) resistant panel carries out PCR to be identified.
The Agrobacterium that will contain recombinant expression vector pCAMBIA1391-SP transforms the environmental wild-type Arabidopis thaliana of Colombia with Floral dip method, gathers in the crops the seed (T of transgenic arabidopsis plant in this in present age
1Generation), the positive seedling of screening on the MS substratum that adds 25mg/L Totomycin (Hyg).Gather in the crops this T
1Seed (T for transgenic arabidopsis
2Generation), with T
2For the transgenic arabidopsis planting seed in above-mentioned substratum, to the T that grows
2Carry out the detection of gus gene for the 10d seedling in age of transgenic arabidopsis and each organ of ripe plant.
Two, GUS staining analysis
The preparation of GUS dye liquor (1mL): the X-Gluc that takes by weighing 1mg, add 1-2 and drip N, dinethylformamide dissolves fully to X-Gluc, add the Tripotassium iron hexacyanide 10 μ L of 0.1mol/L phosphoric acid buffer 980 μ L, 5mmol/L of pH 7.0 and the yellow prussiate of potash 10 μ L of 5mmol/L then, mixture is stirred, add 1 μ LTriton X-100 at last.
Choose the T that changes the SP promoter sequence over to
2For the 10d seedling in age of transgenic arabidopsis, the different tissues of ripe plant, put into the EP pipe that 100 μ L GUS dye liquors are housed respectively, 37 ℃ of temperature are bathed 4h, alcohol with 50% and 70% is wash-out 12h successively, observe the dyeing situation of each organ of transgenic arabidopsis afterwards at stereoscope and microscopically, and by the CCD pickup image.Change the T of SP promoter sequence over to
2For observations under the stereoscope after 10d seedling in the age dyeing of transgenic arabidopsis as shown in Figure 2, its microscopically observations as shown in Figure 3.Fig. 2 shows, changes the T of SP promoter sequence over to
2Root for transgenic arabidopsis has the painted of blueness, SP promoters driven gus gene is described only in the root expression, and promptly the SP promotor is a root-specific promoter.Fig. 3 shows that SP promoters driven gus gene expresses in the vascular bundle tissue of root.
Sequence table
<160>1
<210>1
<211>845
<212>DNA
＜213〉Arabidopis thaliana (Arabidopsis thaliana)
<400>1
cattcacggt?aggttctttt?attcgctctc?actgataatc?ctttaatatg?caactcaaag 60
atttgaacct?catgtgaacg?ttctatgaat?atcgttacag?atagacaacg?gtttaatgac 120
accgacaatg?aaaataagac?gggacaaggt?ggttgatcaa?tacaagaatg?aaatagaaag 180
actctacaag?tagactagaa?attttagcat?cctctttatt?ttacaattat?cttgttaagg 240
attagcaaat?gtgaaccagt?tcttactcca?ttgttaatat?aagctgcata?tatctttgtt 300
catcaactag?ggatttccaa?gttctgcagc?actatggata?cttattgagt?ctaatagaat 360
caataattgg?aagaagcagt?taaaccggtc?aaaagaccta?agtatgttgg?agggtttgag 420
atatcttgac?attctgtgtt?tagaggaagg?ctagatgttg?atacaatatt?gtataatttg 480
cataattata?ttatccgttc?aatccaaaag?catttaagtg?cgatgtaaag?tgatggtttt 540
ttctagttta?tgttaaaaca?aatgattagt?ctcactctct?agtcgataga?atattgcaac 600
ttgtttataa?atttgattag?gtgagctatg?attagtagtg?cataaataca?ttatctatga 660
gacaaagaaa?cgtgataggt?tattgttttt?tcttgtttgc?gtctggcata?tgtatgtata 720
aaaccaaata?tcaagcagaa?catatgcgat?tttcaaaatg?tgggcggtgc?tgaaaacata 780
tattagttta?ctccaacttg?cagaatattt?gagtatatta?atgcaaaaac?agtaaacgca 840
agagc 845
Claims (9)
1. promotor, the dna molecular of forming by the deoxyribonucleotide sequence shown in the sequence in the sequence table 1.
2. the expression cassette that contains the external source goal gene of the described promotor of claim 1.
3. the recombinant expression vector that contains the expression cassette of described promotor of claim 1 or the described external source goal gene of claim 2.
4. according to the described recombinant expression vector of claim 3, it is characterized in that: the carrier that sets out of described recombinant expression vector is plasmid pCAMBIA1391.
5. according to claim 3 or 4 described recombinant expression vectors, it is characterized in that: described recombinant expression vector is following 1) or 2) expression vector:
1) expression vector as shown in Figure 1;
2) gus gene with expression vector shown in Figure 1 replaces with the expression vector that required goal gene obtains.
6. the transgenic cell line or the reorganization bacterium that contain arbitrary described recombinant expression vector among the described promotor of claim 1 or described expression cassette of claim 2 or the claim 3-5.
7. the application of arbitrary described recombinant expression vector in the transgenic plant of cultivating specifically expressing goal gene in roots of plants among the described promotor of claim 1 or described expression cassette of claim 2 or the claim 3-5.
8. application according to claim 7 is characterized in that: described plant is monocotyledons or dicotyledons.
9. application according to claim 8 is characterized in that: described plant is an Arabidopis thaliana.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102367451B (en) * | 2011-11-04 | 2013-04-10 | 合肥工业大学 | Eukaryotic recombinant plasmid of gene capable of improving content of fragrant substances in rice and application thereof |
| CN102618543B (en) * | 2012-03-13 | 2015-01-21 | 青岛农业大学 | Root-specific and harm inducible promoter from glycine max |
| CN103320441B (en) * | 2013-06-17 | 2015-04-22 | 清华大学 | Plant promoter and application thereof |
| CN104673793B (en) * | 2013-11-27 | 2017-12-08 | 中国科学院上海生命科学研究院 | Legume root system tissue-specific promoter and its application |
| CN103740720B (en) * | 2013-12-30 | 2015-07-08 | 安徽省农业科学院水稻研究所 | Identification and application of rice root specific strong promoter POsRo2 |
| MX2017017161A (en) * | 2015-06-23 | 2018-03-09 | Iowa Corn Promotion Board | Plants having enhanced yield-related traits and methods of making them. |
| CN106047878B (en) * | 2016-08-12 | 2020-03-24 | 湖南农业大学 | Rice root specific expression promoter POsr1 and application thereof |
| CN112877330B (en) * | 2020-03-02 | 2022-06-21 | 山东农业大学 | Method for efficiently expressing target gene in protoplast cell |
| CN113913430B (en) * | 2021-11-11 | 2023-04-25 | 中国热带农业科学院橡胶研究所 | Promoter for specific expression of plant overground tissues and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001044454A2 (en) * | 1999-12-16 | 2001-06-21 | Planton Gmbh | Root-specific promoter |
| CN1954075A (en) * | 2004-03-12 | 2007-04-25 | 辛根塔参与股份公司 | Inducible promoters |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2001044454A2 (en) * | 1999-12-16 | 2001-06-21 | Planton Gmbh | Root-specific promoter |
| CN1954075A (en) * | 2004-03-12 | 2007-04-25 | 辛根塔参与股份公司 | Inducible promoters |
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