CN103589725A - Promoter with both plant overground tissue organ specificity and photoinduced specificity and application thereof - Google Patents
Promoter with both plant overground tissue organ specificity and photoinduced specificity and application thereof Download PDFInfo
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- CN103589725A CN103589725A CN201310540693.9A CN201310540693A CN103589725A CN 103589725 A CN103589725 A CN 103589725A CN 201310540693 A CN201310540693 A CN 201310540693A CN 103589725 A CN103589725 A CN 103589725A
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Abstract
The invention relates to a newly discovered DNA sequence which can be used as a promoter for regulating and controlling specific expression of a target gene in tissue organs of a plant overground part in a photoinduced form. A promoter sequence of an AtcpSecY2 gene is cloned from a model plant arabidopsis thaliana; and subsequently in a transgenic arabidopsis thaliana, the promoter is confirmed to be capable of driving a GUS reporter gene to be specifically expressed in the tissue organs of the plant overground part in the photoinduced form. With application of the promoter, a 'promoter-target gene to be expressed' fusion gene is obtained by construction, a plant is transformed, and the transgenic plant having both plant overground tissue organ specificity and photoinduced specificity expression of the target gene can be obtained. The promoter not only contributes to research on a transcription regulation expression mode of plant genes under a condition of illumination, but also is applied in plant gene engineering to allow exogenous genes to be moderately expressed along with plant physiological statuses under the condition of illumination, at the same time, accurately and effectively changes photosynthetic characteristics and agronomic characters of the plant overground part, realizes crop strain improvement, and has wide application value.
Description
Technical field
The invention belongs to plant genetic engineering field, is to utilize Protocols in Molecular Biology to obtain a kind of have concurrently plant histoorgan and the specific promotor of photoinduction and the application in transgenic plant thereof on the ground specifically.
Background technology
Promotor be positioned at structure gene upstream, to RNA polymerase identify, the section of DNA sequence of combination and transcriptional start, promotor, just as " switch ", can be controlled initial time and the intensity of its downstream configurations genetic transcription.The expression regulation of higher plant gene is one of centre point of plant genetic engineering research, as a kind of important molecular regulation element on transcriptional level, the research of promotor is become to molecular biological focus (Wang Ying etc., 2003; Li Jie etc., 2006).
According to promotor in plant, starting pattern and the function of transcribing can be divided three classes: constitutive promoter, tissue and organ specificity promotor and inducible promoter (Wang Ying etc., 2003).Constitutive promoter can promotor gene in a organized way expression, there is persistence, do not show Space-time speciality; Genetic transcription under tissue and organ specificity promoter regulation generally only occurs in some certain organs or tissue; Inducible promoter is under the induction of some physics or chemical signal, can promote goal gene strong expression (Wang Ying etc., 2003; Wait quietly on road, and 2004).
In constitutive promoter, at present most popular is cauliflower mosaic virus
caMV 35Spromotor, from the rouge alkali synthetase gene in agrobacterium tumefaciens Ti-plasmids T-DNA region
nospromotor and octopine synthase gene
ocs(Wang Ying etc., 2003 such as promotor; Li Jie etc., 2006).As in monocotyledons, Tsuchiya etc. (1996) find
caMV 35Spromotor and paddy rice
actlpromotor has efficient transcriptional regulatory activity in the gene transformation of lily.In dicotyledons, Sunilkumar etc. (2002) with
caMV 35Spromoters driven green fluorescent protein (GFP) gene is expressed in transgene cotton.Although utilize expression that constitutive promoter drives goal gene to make some progress for the functional study of gene, but due to constitutive promoter can cause goal gene plant institute in a organized way in great expression, consume intracellular matter and energy excessively, therefore in application, there is a lot of drawback (Gittins et al., 2000).As use constitutive promoter can cause goal gene to be expressed in whole plant, and produce a large amount of meta-bolitess, hindered the normal growth of plant and grown, be unfavorable for the raising of yield and quality; Some expression product is even poisonous, causes death (Robinson, 1996 of plant; Gilmour et al., 2004; Qin et al., 2004; Savitch et al., 2005; Thomashow, 2010).In addition, reuse the expression that constitutive promoter drives a plurality of foreign genes and may cause gene silencing (Kumpatla et al., 1998).Therefore, urgently need to find more efficiently tissue and organ specificity or inducible promoter and replace constitutive promoter, with the expression of regulating plant gene better.
Tissue and organ specificity or inducible promoter are because it drives the expression of goal gene to occur in some specific organ-tissue position or its expression is subject to the induction of external stimulus signal, so can overcome to a great extent the many disadvantages that constitutive promoter highlights in theoretical and applied research.In many plants, had been found that at present this two classes specificity promoter.If (2002) such as Mao Zichao are by tomato fruit-specific promoter
2A12with
iptthe fusion gene that goal gene forms proceeds to tomato, has obtained seedless tomatoes fruit.Mariani etc. (1990; 1992) by the promotor of tobacco anther tapetum specific expression gene
tA29with
barnaseconversion of plant nuclease gene specifically expressing in flower pesticide after gene fusion, has obtained the male sterile material of tobacco and rape, and has carried out commercial applications.According to the difference of exogenous stimulation or inductor, inducible promoter is divided into again the inducible promoters such as light, heat, injury, hormone and pathogenic bacteria.In plant, found at present multiple inducible promoter, as Sunflower Receptacle heat shock protein
sHSPSgene promoter, potato
pin2(Xu et al., 1993 such as hormone induction type promotor and P69B, P69C pseudomonas inducible promoter; Coca et al., 1996; Jorda and Vera, 2000).
Arabidopsis is in Cruciferae, due to features such as its individual morphology are little, growth cycle is short, fecundity is strong, genome is little and Biological Information Resources is abundant, become the model plant (Zhang Zhen's hardwood etc., 2006) of plant genetics, developmental biology and molecular biology research.The over-ground part of plant and underground part, in different environment, are connected by vascular bundle between the two, exist a large amount of exchanges of nutritive substance and information.The activity of underground part root tissue depends on photosynthate, hormone and the VITAMIN etc. that over-ground part provides; The vegetative activity of over-ground part blade, branch and flowers and fruits etc. needs root system that moisture, mineral, nitrogen, hormone and amino acid etc. are provided.Only affect one of most important environmental factor of growth and development of plants, especially to plant shoot, divide the physiological property of histoorgan and functional status to have the greatest impact.The seed of plant germination and growth in soil, be accompanied by the process coming up, over-ground part adapts to illumination gradually, and under the induction of light, involved in plant is growing of histoorgan on the ground, particularly participate in the photosynthetic gene strong expression of chlorenchyma, the growth that is follow-up plant by photosynthesis, bloom and solidly provide topmost matter and energy to originate.Therefore, plant shoot divides the vital movement of histoorgan and illumination to exist closely to contact.
Also do not have at present a kind ofly to have plant on the ground histoorgan and the specific promotor of photoinduction concurrently, so this area is in the urgent need to screening and the exploitation plant promotor of tissue and organ specificity on the ground under the induction of light.Acquisition effectively utilization have plant ground histoorgan concurrently and the specific promotor of photoinduction not only contributes to study transcriptional control expression pattern and the regulatory mechanism of plant gene under illumination condition, and be applied to make foreign gene under illumination condition, be accompanied by the expression of plant physiology state appropriateness in plant genetic engineering, change accurately and effectively photosynthesis characteristics and the economical character that plant shoot divides simultaneously, realize the strain improvement of farm crop, with growing of regulating plant better, be with a wide range of applications.
Summary of the invention
The present invention seeks to solve in existing plant gene engineering technology technology, because using constitutive promoter, make foreign gene overexpression heterologous protein or meta-bolites in transgenic plant, make the original metabolic imbalance of plant, having hindered the normal growth of plant grows, even cause death, and reuse the problem that expression that same constitutive promoter drives a plurality of foreign genes may cause gene silencing.Be the transcriptional control expression pattern of further investigation plant gene under illumination condition simultaneously, and be applied to make foreign gene under illumination condition, be accompanied by the expression of plant physiology state appropriateness in plant genetic engineering, change accurately and effectively photosynthesis characteristics and the economical character that plant shoot divides simultaneously, realize the strain improvement of farm crop, with growing of regulating plant better, provide a kind of have concurrently plant histoorgan and the specific promotor of photoinduction and the application in transgenic plant thereof on the ground.
What the invention provides a kind of new, 1851bp has plant histoorgan and the specific promotor of photoinduction on the ground concurrently, and this nucleic acid sequence of promoter is selected from:
(a) nucleotide sequence as shown in sequence table 1;
(b) nucleotide sequence at least in the nucleotide sequence limiting at (a) with more than 80% homology.
As the example of concrete application, the invention provides a kind of cloning process that has plant ground histoorgan and the specific promotor of photoinduction concurrently, concrete operation step is as follows:
The first, take arabidopsis thaliana genomic dna as template, with PCR method amplification, have plant histoorgan and photoinduction specificity promoter on the ground concurrently;
The second, reclaim pcr amplification product;
Three, the amplified production above-mentioned second step being reclaimed carries out ligation with pMD-18 carrier (purchased from TaKaRa company) under ligase enzyme catalysis;
Four, will connect product and transform bacillus coli DH 5 alpha competent cell;
Five,, by ammonia benzyl mycin resistance screening, obtain the positive TA clone of this promotor.
The present invention provides the application that has plant ground histoorgan and the specific promotor of photoinduction concurrently simultaneously, that is: what utilize to obtain has plant histoorgan and the specific promotor of photoinduction on the ground concurrently, build the fusion gene of " promotor-goal gene ", carry out Plant Transformation, thus obtain in genome, contain the above nucleic acid sequence of promoter, can photoinduced form the plant transgenic plant of histoorgan specifically expressing goal gene on the ground.Goal gene in wherein said fusion gene can be the goal gene of any photosynthesis characteristics dividing for fundamental research, transformation technology, plant shoot and economical character improvement needs.
As the example of concrete application, the invention provides a kind of " promotor-
gUSreporter gene " fusion gene structure and in transgenic arabidopsis, with photoinduced form, part is specific expressed on the ground.Specific operation process is as follows:
The first, use
xbai/
ncoi double digestion contains the TA cloned plasmids that has plant ground histoorgan and the specific promotor of photoinduction concurrently, reclaims this specificity promoter fragment;
The second, use
xbai/
ncoi double digestion pCAMBIA 1301 (purchased from CAMBIA company) plasmid, reclaims large carrier segments and (wherein contains
gUSthe encoding sequence of reporter gene);
That three, mixes that the above-mentioned the first step obtains has the plant pCAMBIA 1301 carrier large fragments that histoorgan and the specific promoter fragment of photoinduction and second step obtain concurrently on the ground, under ligase enzyme catalysis, carry out ligation, complete on pCAMBIA 1301 carriers " have concurrently plant on the ground histoorgan and the specific promotor of photoinduction-
gUS" structure of fusion gene.
Wherein the designed PCR amplimer that has plant ground histoorgan and photoinduction specificity promoter concurrently is as follows, and wherein upstream primer has been introduced
xbai restriction enzyme site, downstream primer has been introduced
ncoi restriction enzyme site:
Upstream primer: 5 '-
tCTAGAcTTGTCGACAACTACTGATCCG-3 '
Downstream primer: 5 '-
cCATGGcGAGAGGATGAGGAGAGAAA-3 '
In described application " promotor-
gUSreporter gene " in transgenic arabidopsis with photoinduced form part specifically expressing on the ground, operating process is as follows:
The concrete grammar of transformation of Arabidopsis thaliana, adopts the method (Clough and Bent, 1998) of agriculture bacillus mediated Floral dip, and the seed of acquisition is through 50 mg l
-1hygromycin resistance screening, the normal resistant plant of growing turns earth culture supports, and utilizes the method (Blume and Grierson, 1997) of histochemical stain, detects under transgenic arabidopsis photoinduction condition and in whole growth and development process in each histoorgan
gUSthe specificity of reporter gene expression.
Advantage of the present invention and positively effect:
The present invention utilize effectively have concurrently plant on the ground histoorgan and the specific promotor of photoinduction replace constitutive promoter, can be for being structured in the ground histoorgan of plant the fusion gene with the specific expressed goal gene of photoinduced form.Utilize genetic transfoumation to be proceeded in the genome of plant, can realize the directional operation to goal gene, obtain the transgenic plant that have plant ground histoorgan and the specific expressed goal gene of photoinduction concurrently.This not only contributes to study transcriptional control expression pattern and the regulatory mechanism of plant gene under illumination condition, and be applied to make foreign gene under illumination condition, be accompanied by the expression of plant physiology state appropriateness in plant genetic engineering, change accurately and effectively photosynthesis characteristics and the economical character that plant shoot divides simultaneously, realize the strain improvement of farm crop, with growing of regulating plant better, be with a wide range of applications.
Accompanying drawing explanation
Fig. 1 is the electrophoresis detection figure that has plant ground histoorgan and photoinduction specificity promoter clone concurrently.
Fig. 2 be Arabidopis thaliana " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" PCR of transgenic arabidopsis plant identifies.
Fig. 3 be " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" the GUS coloration result of transgenic arabidopsis plant under photoinduction condition.
Fig. 4 be " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" GUS coloration result in each organ growth course of transgenic arabidopsis plant.
Embodiment
Embodiment 1: the clone who has plant ground histoorgan and photoinduction specificity promoter concurrently
The first, take arabidopsis thaliana genomic dna as template, that utilizes PCR method amplification 1851bp has plant histoorgan and photoinduction specificity promoter on the ground concurrently, reclaims amplified production and also carries out TA clone.
(1) pcr amplification object fragment:
1. according to known Arabidopis thaliana
atcpSecY2the sequences Design special primer of gene promoter area is introduced in upstream primer
xbai restriction enzyme site is introduced in downstream primer
ncoi restriction enzyme site.
Upstream primer: 5 '-
tCTAGAcTTGTCGACAACTACTGATCCG-3 ' (introduces
xbai restriction enzyme site)
Downstream primer: 5 '-
cCATGGcGAGAGGATGAGGAGAGAAA-3 ' (introduces
ncoi restriction enzyme site)
2. extract according to a conventional method arabidopsis thaliana genomic dna, take genomic dna as template, utilize above-mentioned primer to carry out pcr amplification, preparation has plant histoorgan and photoinduction specificity promoter fragment on the ground concurrently.
PCR response procedures:
94 ℃ 3 minutes;
94 ℃ 30 seconds, 56 ℃ 30 seconds, 72 ℃ 2 minutes, 32 circulations;
72 ℃ 10 minutes;
4 ℃ of insulations.
PCR reaction system:
(2) clone of object fragment and the evaluation of positive colony:
1. the recovery of object fragment:
By agarose gel electrophoresis, reclaim target DNA fragment, recovery method adopts the DNA sepharose of the precious biotech firm in Dalian to reclaim test kit, and concrete operation step is shown in catalogue.
2. connect:
The reagent that adds following reaction system, 16 ℃ of reactions are spent the night, and realize being connected of object fragment and pMD18-T (purchased from TaKaRa company) carrier.
Linked system:
| Reagent | Add-on (μ l) |
| PCR product (50 ng μ l after purifying reclaims -1) | 4.5 |
| PMD18-T carrier (50 ng μ l -1) | 0.5 |
| Solution I ligase enzyme | 5.0 |
3. the evaluation of conversion and positive colony:
CaCl routinely
2induction and method for transformation, prepare bacillus coli DH 5 alpha competent cell, with 10 μ l, connects product transformed competence colibacillus cell, is then evenly applied on the flat board that contains Amp, X-gal and IPTG, is inverted for 37 ℃ and cultivates 12-14 hour.Select the white colony transforming on flat board, extract according to a conventional method plasmid, use
xbai and
ncoi double digestion, produces and contains the 1861 bp fragments that have plant ground histoorgan and photoinduction specificity promoter concurrently.By foregoing PCR primer and amplification condition, take plasmid extraction thing as template, carry out PCR amplification, what through agarose gel electrophoresis, detect to produce 1863bp has plant histoorgan and photoinduction specificity promoter fragment on the ground concurrently, is the positive colony that contains this promoter sequence.As shown in Figure 1, swimming lane M:DL2000 DNA Marker; Swimming lane 1: with H
2o is the negative contrast of the PCR system of template; Swimming lane 2: the PCR product that has plant ground histoorgan organ and photoinduction specificity promoter clone concurrently.
4. sequence verification
Through the positive colony of identifying, deliver bacterium stab culture to Shanghai Sheng Gong Bioisystech Co., Ltd) carry out DNA sequencing, its nucleotide sequence is as shown in sequence table 1.
Embodiment 2: utilize pCAMBIA1301 carrier (to contain
gUSreporter gene) build " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" fusion gene
(1) from intestinal bacteria, extract carrier pCAMBIA 1301 plasmids (purchased from CAMBIA company), use
xbai/
ncoafter I double digestion, reclaiming large carrier segments (wherein includes
gUSreporter gene sequence).
(2) from the prepared TA of embodiment 1 clones, extract plasmid, use
xbai/
ncoi double digestion, reclaims (with embodiment 1) Arabidopis thaliana by agarose gel electrophoresis and has histoorgan and photoinduction specificity promoter fragment on the ground concurrently.
(3) above-mentioned 2 fragments are spent the night in 16 ℃ of connections under ligase enzyme catalysis, complete on pCAMBIA 1301 carriers " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" Fusion gene construction.
Linked system:
| Reagent | Add-on (μ l) |
| Have plant histoorgan and photoinduction specificity promoter fragment (50 ng μ l on the ground concurrently -1) | 2.0 |
| PCAMBIA1301 carrier large fragment (50 ng μ l -1) | 3.0 |
| Solution I ligase enzyme | 5.0 |
(4) with connecting mixture, transform bacillus coli DH 5 alpha competent cell, method is with embodiment 1.
(5) select the white colony transforming on dull and stereotyped (kalamycin resistance), extract according to a conventional method plasmid, use
xbai and
bste II double digestion plasmid DNA produces two fragments, and one is the pCAMBIA1301 carrier segments of 9002 bp, another be 3901 bp " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" fusion gene.
(6) take plasmid carries out PCR reaction as template, identify in plasmid " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" fusion gene, the size of amplified fragments is 2656 bp.The primer is as follows:
Upstream primer: 5 '-TCTAGACTTGTCGACAACTACTGATCCG-3 '
Downstream primer: 5 '-TACAGTCTTGCGCGACATGCG-3 '
(7) through enzyme, cut the positive colony of identifying with PCR, deliver the order-checking of order-checking company.
(8) from positive colony, extract plasmid, by ordinary method, transform Agrobacterium GV3101, obtain through engineering approaches Agrobacterium, for Plant Transformation.
Embodiment 3:
The preparation of transgenic arabidopsis plant
(1) with embodiment 2, build " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" fusion gene arabidopsis thaliana transformation, concrete method for transformation adopts the method (Clough and Bent, 1998) of agriculture bacillus mediated Floral dip, and the seed of acquisition is through 50 mg l
-1hygromycin resistance screening, the normal plant that grows turns earth culture and supports.
(2) PCR of transfer-gen plant detects: the blade of difference clip transfer-gen plant and wild-type plant, with reference to < < molecular cloning experiment guide (third edition) > > (Huang Peitang etc., 2002) method is extracted leaves genomic DNA, with following primer, carry out PCR reaction, reaction system is as embodiment 1:
Upstream primer: 5 '-TCTAGACTTGTCGACAACTACTGATCCG-3 '
Downstream primer: 5 '-TACAGTCTTGCGCGACATGCG-3 '
PCR product carries out agarose gel electrophoresis, transfer-gen plant occur 2656 bp " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" fusion gene band, there is not fusion gene band in non-transgenic plant, proves that object fragment is incorporated in Plant Genome.As shown in Figure 2, swimming lane M:DL2000 DNA Marker; Swimming lane 1: with have concurrently plant on the ground histoorgan and the specific promotor of photoinduction-
gUSplasmid is the PCR positive control of template; Swimming lane 2-3: with arabidopsis thaliana transformation have concurrently plant on the ground histoorgan and the specific promotor of photoinduction-
gUSthe PCR product that the transformed plant DNA of fusion gene is template; Swimming lane 4: the negative contrast of the PCR that the genomic dna of wild-type Arabidopis thaliana of take is template.
Embodiment 4:
Transgenic arabidopsis
gUSthe expression of reporter gene is subject to the induction of light
Utilize the method for histochemical stain (Blume and Grierson, 1997), under detecting and having plant at T1 in for transgenic plant concurrently histoorgan and photoinduction specificity promoter drive on the ground
gUSthe photoinduction specificity of expressing.Blueness in transgenic plant in each organ, tissue or cell is reporter gene
gUShistochemical stain, representing to have plant concurrently on the ground histoorgan and photoinduction specificity promoter have expression activity at these positions.Dyeing time is 10 h, preserves after the coloration result of material is scanned into picture.
Coloration result is as shown in Figure 3: a: the Arabidopis thaliana etiolated seedling of growing under dark condition 4 days; The Arabidopis thaliana etiolated seedling of b:4 days was through photoinduction 12 hours; The Arabidopis thaliana etiolated seedling of c:4 days was through photoinduction 24 hours.Under photoperiods 16 h light/8 h dark condition, " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" activity of GUS is very weak in 4 days etiolated seedlings of transgenic arabidopsis; but when the etiolated seedling of 4 days is transferred under illumination condition and be cultivated; find that the GUS activity of transgenic arabidopsis is stronger after illumination 12 h, the activity of promotor very strong (Fig. 3) after prolonging light time to 24 h.Result proves: have plant ground histoorgan and photoinduction specificity promoter concurrently and can drive under illumination condition
gUSreporter gene is specific expressed in transgenic arabidopsis, illustrates that this promotor has good photoinduction characteristic in transgenic arabidopsis.
Embodiment 5:
The GUS chemical staining of each organ-tissue in transgenic arabidopsis
Utilize the method for histochemical stain (Blume and Grierson, 1997), under detecting and having plant at T1 in for transgenic plant concurrently histoorgan and photoinduction specificity promoter drive on the ground
gUSorgan specificity and the development-specific of expressing.As embodiment 4, the blueness in each organ of transgenic plant, tissue or cell is reporter gene
gUShistochemical stain, representing to have plant concurrently on the ground histoorgan and photoinduction specificity promoter have expression activity at these positions.Dyeing time is 10 h, preserves after the coloration result of material is scanned into picture.
Coloration result is as shown in Figure 4: a: seedling; B: ripe seedling; C: stem (comprising stem leaf) d: perfect flower; E: ripe kind pod.Under photoperiods 16 h light/8 h dark condition, " have concurrently plant on the ground histoorgan and photoinduction specificity promoter-
gUS" have very strong GUS active in the over-ground part histoorgan of transgenic arabidopsis, comprise cotyledon, lotus throne leaf, stem, stem leaf, flower and ripe kind pod.In underground part root tissue without GUS active (Fig. 4).Result proves: having plant ground histoorgan and photoinduction specificity promoter concurrently can drive
gUSreporter gene is expressed at the over-ground part tissue and organ specificity of transgenic arabidopsis, illustrates that this promotor has good over-ground part tissue and organ specificity in transgenic arabidopsis.
The above embodiments result shows and confirms, Arabidopis thaliana provided by the present invention
atcpSecY2it is active that gene promoter not only has strong photoinduction, divide tissue and organ specificity, and the activity of this promotor can stably entail offspring but also possess good plant shoot.Therefore, Arabidopis thaliana provided by the present invention
atcpSecY2gene promoter is that one stable, activity is strong and has plant histoorgan and the specific promotor of photoinduction on the ground concurrently.Obtain and effectively utilize this promotor, not only contribute to study transcriptional control expression pattern and the regulatory mechanism of plant gene under illumination condition, and be applied to make foreign gene under illumination condition, be accompanied by the expression of plant physiology state appropriateness in plant genetic engineering, change accurately and effectively photosynthesis characteristics and the economical character that plant shoot divides simultaneously, realize the strain improvement of farm crop, with growing of regulating plant better, be with a wide range of applications.
Claims (5)
1. have plant histoorgan and the specific promotor of photoinduction on the ground concurrently, its nucleotide sequence is selected from:
(a) nucleotide sequence as shown in sequence table 1;
(b) nucleotide sequence at least in the nucleotide sequence limiting at (a) with more than 80% homology.
2. plant on the ground histoorgan and the specific promotor of photoinduction of having concurrently according to claim 1, it is characterized in that, designed Arabidopis thaliana has plant concurrently, and on the ground the pcr amplification primer of histoorgan and photoinduction specificity promoter is as follows, wherein in upstream primer, has introduced
xbai restriction enzyme site, has introduced in downstream primer
ncoi restriction enzyme site:
Upstream primer: 5 '-
tCTAGAcTTGTCGACAACTACTGATCCG-3 '
Downstream primer: 5 '-
cCATGGcGAGAGGATGAGGAGAGAAA-3 '.
3. plant on the ground histoorgan and the specific promotor of photoinduction of having concurrently according to claim 1, it is characterized in that, from arabidopsis thaliana genomic dna, clone obtains and has plant histoorgan and photoinduction specificity promoter on the ground concurrently, and concrete operation step is as follows:
(1) take arabidopsis thaliana genomic dna as template, with PCR method amplification, have plant histoorgan and photoinduction specificity promoter on the ground concurrently;
(2) reclaim pcr amplification product;
(3) amplified production above-mentioned (2) step being reclaimed carries out ligation under ligase enzyme catalysis with pMD-18T;
(4) will connect product and transform bacillus coli DH 5
αcompetent cell;
(5), by resistance screening, obtain the positive TA clone of this promotor.
4. an application that has plant ground histoorgan and photoinduction specificity promoter concurrently claimed in claim 1, it is characterized in that, utilize this promotor to build the fusion gene that obtains promotor-goal gene, by its arabidopsis thaliana transformation or other plant, thus obtain in genome, contain nucleic acid sequence of promoter described in claim 1, can be in the over-ground part histoorgan of plant with the transgenic plant of the specific expressed corresponding goal gene of photoinduced form.
5. claimed in claim 1ly have a plant application for histoorgan and photoinduction specificity promoter on the ground concurrently, it is characterized in that, utilize this promotor to build to obtain promotor-
gUSfusion gene, drives in histoorgan on the ground plant
gUSreporter gene is expressed corresponding goal gene specifically with photoinduced form.
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