CN102533766B - Seed-specific promoter and application thereof - Google Patents
Seed-specific promoter and application thereof Download PDFInfo
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- CN102533766B CN102533766B CN 201210007246 CN201210007246A CN102533766B CN 102533766 B CN102533766 B CN 102533766B CN 201210007246 CN201210007246 CN 201210007246 CN 201210007246 A CN201210007246 A CN 201210007246A CN 102533766 B CN102533766 B CN 102533766B
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Abstract
The invention discloses a seed-specific promoter and application thereof. The promoter is a DNA molecule shown as a sequence 1 in a sequence table. The invention also provides a method for specifically expressing genes in plant seeds. The method comprises the following steps of: transferring a recombinant expression vector containing the DNA molecule into a plant, and promoting downstream genes to be specifically expressed in the plant seeds by using the DNA molecule. The promoter has high-efficiency promoting activity and high tissue-specific expression, and provides a good base material for improving traits of wheat seeds by using a transgenic technology.
Description
Technical field
The present invention relates to a kind of seed specificity promoter and application thereof.
Background technology
Promotor is that RNA polymerase can be identified and combination with it, thereby the section of DNA sequence that initial gene is transcribed is usually located at upstream region of gene.A typical promotor comprises CAAT-box and TATA-box, and they are respectively identification and the binding site of RNA polymerase, generally is positioned at tens base places, transcription initiation site upstream.Usually have some special dna sequence dnas in the core promoter upstream, i.e. cis-acting elements, thus transcription factor is with it in conjunction with activating or the transcribing of suppressor gene.In case the RNA polymerase location also is combined on the promotor and can transcribes by promotor gene, therefore promotor is the critical elements of gene expression regulation, and the interaction of it and trans-acting factors such as RNA polymerase and other albumen cofactors is the essence of promoter regulation genetic transcription.Transcriptional profile according to promotor can be divided three classes it: constitutive promoter, specificity promoter (tissue or organ specific promoters) and inducible promoter.
Under the constitutive promoter regulation and control, the genetic expression of different tissues organ and etap does not have notable difference, thereby is referred to as constitutive promoter.In the dicotyledons the constitutive promoter of normal use be cauliflower mosaic virus (CaMV) 35S promoter, constitutive promoter commonly used in the monocotyledons is ubiquitin protein (ubiquitin) promotor of corn, the Actin muscle of paddy rice (actin) promotor etc.Express because the gene that constitutive promoter drives all has in various degree in each tissue of plant, in application, expose some problems gradually.For example foreign gene is expressed in whole strain plant, the a large amount of heterologous proteins that produce or meta-bolites have been broken the original metabolic balance of plant at plant interior accumulation, and some product is to plant and nonessential even poisonous, thereby hindered the normal growth of plant, even cause death.In addition, repeated use may cause gene silencing or suppress phenomenon altogether with the two or more foreign genes of a kind of promoters driven.Therefore, people seek more efficiently specificity promoter and replace constitutive promoter, in order to regulate and control gene expression in plants better.
Genetic transcription under the specificity promoter regulation and control generally only occurs in some certain organs or the tissue, as root, stem, leaf, fruit and seed specific promoters.At present, found generally to exist simultaneously in this class promotor the element of several control tissue specific expressions, its expression specificity determines by kind, number and the relative position etc. of these elements are common.
Cereal crop is human main food source, and seed is its edible part.Along with engineered development, people have cloned many seed specific promoters from cereal crop, and they are applied among the improvement of cereal class grain quality.Using more is hmw glutenin subunit 1Dx5, the Bx17 that derives from common wheat, the G1t of paddy rice etc.But the startup exogenous gene expression activity of these promotors there are differences, and its expression activity can not satisfy engineered needs, and when the needs polygene is connected, if utilize the different genetic expression of same promoters driven, is easy to cause gene silencing.Therefore, multipath is sought and is transcribed the higher seed specific promoters of efficient, and for the expression that improves target gene, processing and the nutritional quality of improvement cereal plants are significant.
Summary of the invention
The purpose of this invention is to provide a kind of seed specificity promoter and application thereof.
Promotor provided by the invention is opened swallow No. 1 available from the oat varieties Ji, is the dna molecular shown in the sequence 1 in the sequence table.
The recombinant expression vector, expression cassette, transgenic cell line or the reorganization bacterium that contain described promotor all belong to protection scope of the present invention.
Available existing expression vector establishment contains the recombinant expression vector of described promotor.For the ease of identifying and screening, can process used expression vector, can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change as adding to encode.
Described recombinant expression vector specifically can be described dna molecular is inserted the recombinant plasmid that the multiple clone site of plasmid pCAMBIA3301 obtains.
The present invention also protect a kind of in plant seed the method for specifically expressing foreign gene, be that the dna fragmentation first is imported in the plant, thus in plant seed the described foreign gene of specifically expressing; Start described expression of exogenous gene by described promotor in the described dna fragmentation first.Described dna fragmentation first specifically can import described plant by recombinant expression vector; Described recombinant expression vector can be described dna molecular and described foreign gene are inserted the recombinant plasmid that the multiple clone site of plasmid pCAMBIA3301 obtains.Described recombinant expression vector can be by using conventional biological method transformed plant cells or tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, agriculture bacillus mediated, particle gun, and the plant transformed tissue cultivating become plant.
The present invention also protect a kind of in plant seed the method for specific expression gene, be that the recombinant expression vector that will contain described promotor imports in the plant, start its downstream gene specifically expressing in plant seed by described promotor.Described recombinant expression vector specifically can be described dna molecular is inserted the recombinant plasmid that the multiple clone site of plasmid pCAMBIA3301 obtains.Described gene specifically can be the Gus gene.Described recombinant expression vector can be by using conventional biological method transformed plant cells or tissues such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, agriculture bacillus mediated, particle gun, and the plant transformed tissue cultivating become plant.
More than in arbitrary described method, described plant can be monocotyledons or dicotyledons.Described monocotyledons specifically can be wheat, as wheat K35.
Promotor provided by the invention has the tissue specific expression that starts activity and height efficiently, for the grain characters of utilizing transgenic technology improvement wheat provides the good basis material.
Description of drawings
Fig. 1 is the comparison chart of new clone promotor (sequence 1) and existing sequence (sequence 2).
Fig. 2 is the PCR electrophorogram in the promotor discovery procedure; 1-6 is the PCR product, and M is molecular weight marker.
Fig. 3 is that the PCR in the construction recombination plasmid first process identifies electrophorogram; M is molecular weight marker, and other swimming lane is the connection product.
Fig. 4 cuts the evaluation electrophorogram for the enzyme in the construction recombination plasmid first process; M is molecular weight marker, and other swimming lane is the connection product.
Fig. 5 is the PCR evaluation electrophorogram with the regrowth of recombinant plasmid first preparation; M is molecular weight marker; CK+ is the recombinant plasmid first, and CK-is wheat K35; Other swimming lane is regrowth.
Fig. 6 is the root of three kinds of transfer-gen plants and the photo after the leaf Gus dyeing; 1: be the transfer-gen plant first; 2 is transfer-gen plant second; 3 is transfer-gen plant third.
Fig. 7 is the photo after the seed Gus of three kinds of transfer-gen plants dyes.
Embodiment
Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique among the following embodiment if no special instructions, is ordinary method.Used test materials among the following embodiment if no special instructions, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all arranges repeated experiments three times, results averaged.
Plasmid pGEM-Teasy, plasmid pCAMBIA3301 and plasmid pCAMBIA1381xb are all available from Changsha Yingrun Biological Technology Co., Ltd. (http://www.yrbio.com).Be used for the test kit of plasmid DNA and extracting genome DNA available from sky root biochemical technology company limited (http://www.tiangen.com/).In bronze and the particle gun conversion process required consumptive material all available from industry Bole development in science and technology company limited of Beijing unit (
Http:// www.ybr.com.cn).Various inorganic salt, VITAMIN, microbiotic, hormone, and the required reagent of Gus dyeing all available from Sigma-Aldrich China company (
Http:// www.sigmaaldrich.com/china-mainland.html).
Swallow is opened No. 1 in the oat varieties Ji: the public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science and Crop Inst. of shandong Prov. Agriculture science Academy; Reference: utilize the sterile naked oats ZY gene of nuclear to breed high-quality high protein oat new variety. poplar, Zhang Xinjun, Yang Xiaohong, Li Tianliang. Hebei North institute journal (natural science edition), 2009,25 (1) 39-41.
Wheat K35: be academy of agricultural sciences, Shandong Province crop voluntarily seed selection have a new germ plasm that good rataria cultivates one's ability; The public can obtain from Institute of Crop Science, Chinese Academy of Agricultural Science and Crop Inst. of shandong Prov. Agriculture science Academy; Reference: Sui Xinxia, Huang Chengyan, Chu Xiusheng, Li Genying, Fan Qingqi. easy anther cultural precocious new wheat germplasm K35. Shandong agricultural sciences, the first phase in 2007,116-117 page or leaf.
The dedifferentiation substratum is prepared as follows: 2,4-D, Ticarcillin/Clavulanate Acid and MS substratum are mixed, obtain the dedifferentiation substratum; The concentration of 2,4-D in described dedifferentiation substratum is 2mg/L, and the concentration of Ticarcillin/Clavulanate Acid in described dedifferentiation substratum is 150mg/L.
The infiltration substratum is prepared as follows: 2,4-D, Ticarcillin/Clavulanate Acid, sorbyl alcohol, N.F,USP MANNITOL and MS substratum are mixed, obtain permeating substratum; The concentration of 2,4-D in described infiltration substratum is 2mg/L, and the concentration of Ticarcillin/Clavulanate Acid in described infiltration substratum is 150mg/L, and the concentration of sorbyl alcohol in described infiltration substratum is 0.2M, and the concentration of N.F,USP MANNITOL in described infiltration substratum is 0.2M.
Induce screening culture medium to be prepared as follows: 2,4-D, Biolaphos and MS substratum to be mixed, obtain inducing screening culture medium; The concentration of 2,4-D in inducing screening culture medium is 2mg/L, and the concentration of Biolaphos in inducing screening culture medium is 5mg/L.
The regeneration screening culture medium is prepared as follows: KT, Biolaphos and MS substratum are mixed, obtain the screening culture medium of regenerating; The concentration of KT in described regeneration screening culture medium is 2mg/L, and the concentration of Biolaphos in described regeneration screening culture medium is 5mg/L.
The later stage screening culture medium: Biolaphos and MS substratum are mixed, obtain the later stage screening culture medium, the concentration of described Biolaphos in described later stage screening culture medium is 5mg/L.
The discovery of experimental example 1, specificity promoter and sequential analysis
One, the discovery of promoter sequence
1, opens the seedling that swallow sprouts for No. 1 in the dark from the oat varieties Ji with DNA extraction test kit (day root) and extract genomic dna.
2, as follows according to a pair of primer of conserved regions design of existing promotor (GENBANKACCESSION NO.AY795082, the sequence 2 of seeing sequence table):
F1 (upstream primer): 5 '-cg
AagcttTggaaagtcattttgcctc-3 ';
R1 (downstream primer): 5 '-gc
CcatggGagattgtagaaggtggattgg-3 '.
3, the genomic dna with step 1 is template, to carrying out pcr amplification, obtains pcr amplification product with the primer of step 2 design.
PCR system (20 μ l): contain 50ng genomic dna, 10pmol upstream primer, 10pmol downstream primer, each 250 μ M of dNTPs, 2 μ l, 10 * buffer (50mM KCl, 10mM Tris-HCl, 1.5mM MgCl
2, pH 8.3) and 2.0U pfu polysaccharase (Dalian is precious biological), all the other are water.
The PCR program: 94 ℃ 3 minutes; 94 ℃ 45 seconds, 60 ℃ 45 seconds, 72 ℃ 1 minute, 36 circulations; 72 ℃ 10 minutes.
4, pcr amplification product is carried out 1.2% agarose gel electrophoresis, carry out ultraviolet visualization after the EB dyeing and take a picture, electrophorogram is seen Fig. 2.
5, from the running gel of step 4, reclaim the purpose fragment of about 961bp, with check order after plasmid pGEM-Teasy is connected, sequencing result shows, the purpose fragment shown in the sequence 1 of sequence table, will be in plasmid pGEM-Teasy the recombinant plasmid called after recombinant plasmid pTglo that obtains of DNA shown in the sequence 1 of insertion sequence table (but also DNA shown in the artificial synthesized sequence 1 and be connected with plasmid pGEM-Teasy).
Two, the sequential analysis of promotor
Experimental example 2, construction of recombinant plasmid
One, the structure of recombinant plasmid first
(1) be template with recombinant plasmid pTglo, the primer of forming with F1 and R1 obtains pcr amplification product to carrying out pcr amplification.
(2) with the pcr amplification product of restriction enzyme HindIII and NcoI double digestion step (1), reclaim enzyme and cut product.
(3) with restriction enzyme HindIII and NcoI double digestion plasmid pCAMBIA3301, reclaim carrier framework (about 11076bp).
(4) carrier framework of the enzyme of step (2) being cut product and step (3) is connected, and obtains connecting product.
(5) the connection product of step (4) is carried out successively PCR and identify that (adopt primer that F1 and R1 form to), enzyme cut evaluations (adopting HindIII and NcoI double digestion) and the evaluation of checking order, obtain the recombinant plasmid first.
PCR identifies that electrophorogram sees Fig. 3, has purpose band positive of about 961bp.
Enzyme is cut and is identified that electrophorogram sees Fig. 4, has purpose band positive of about 961bp.
According to sequencing result, it is as follows that the recombinant plasmid first is carried out structrual description: HindIII and the small segment between the NcoI restriction enzyme site (CaMV35S promotor) with the DNA shown in the sequence 1 of sequence table has replaced plasmid pCAMBIA3301 start the Gus expression of gene by the DNA shown in the sequence 1.
Two, the structure of recombinant plasmid second
(1) dna molecular shown in the sequence 2 of composition sequence table.
(2) be template with the synthetic dna molecular of step 1, to carrying out pcr amplification, obtain pcr amplification product with the primer of F1 and R1 composition.
(3) with the pcr amplification product of restriction enzyme HindIII and NcoI double digestion step (2), reclaim enzyme and cut product.
(4) with restriction enzyme HindIII and NcoI double digestion plasmid pCAMBIA3301, reclaim carrier framework (about 11076bp).
(5) carrier framework of the enzyme of step (3) being cut product and step (4) is connected, and obtains recombinant plasmid second.According to sequencing result, it is as follows that recombinant plasmid second is carried out structrual description: HindIII and the small segment between the NcoI restriction enzyme site (CaMV35S promotor) with the DNA shown in the sequence 2 of sequence table has replaced plasmid pCAMBIA3301 start the Gus expression of gene by the DNA shown in the sequence 2.
Three, the structure of recombinant plasmid third
With plasmid pCAMBIA1381xb (a kind of plant expression vector contains the Gus gene, but is not activated the promotor of Gus genetic expression) as negative control (recombinant plasmid third).
The acquisition of embodiment 3, transgenic plant
Respectively with recombinant plasmid first, recombinant plasmid second and the recombinant plasmid third preparation transfer-gen plant (every kind of plasmid transforms 2000 scultellums):
One, the separation of wheat scultellum and pre-treatment
Gather 14 days prematurity wheat K35 seed of pollination (scultellum size 1mm), handled 30 seconds 0.1% (g/100mL) mercuric chloride (HgCl with 75% (volume ratio) aqueous ethanolic solution
2) aqueous solution handled 10 minutes, took off rataria with dissecting needle in the aseptic technique platform, plumular axis is inoculated in the dedifferentiation substratum down.Next day, excise plumular axis with No. 15 blades from plumular axis and the scultellum contact position of seed at microscopically, obtain scultellum.The scultellum of removing plumular axis is put back to respectively on the dedifferentiation substratum, kept identical direction (tangent plane contact substratum), 26 ℃ of dark cultivations 3 days.There are in the scope of the about 2.5cm in culture dish center of infiltration substratum (about 40 of every ware) 26 ℃, secretly cultivated 4 hours in then scultellum being concentrated on.
Two, the preparation of bullet
1, the preparation of bronze mother liquor
Get the bronze 10mg of 1.0 μ m, with 70% (volume ratio) ethanol aqueous wash 3 times (centrifugal 1 minute of each 10000rpm), add 1ml aqua sterilisa vibration 2 minutes, obtain the bronze storage solution.
2, the preparation of bullet
With the abundant vortex of bronze storage solution, get the 1.5ml centrifuge tube that 100 μ l put into silication, centrifugal 30 seconds of 14000rpm abandons supernatant.Adding 50 μ l water suspends bronze, the plasmid (recombinant plasmid first, recombinant plasmid second or recombinant plasmid third) that the limit adds 10 μ l 1ug/ μ l is inward slightly shaken in earthquake device top, add the 20 μ l 0.1M spermidine aqueous solution of new preparation again, add 50 μ l 2.5M CaCl again
2The aqueous solution, high speed spiral 3 minutes, with centrifuge tube be placed on allowed in about 2 minutes on ice bronze automatically precipitation a little while, centrifugal 10 seconds of 10000rpm abandons supernatant.Add 750 μ l dehydrated alcohols, the high speed spiral is placed on centrifuge tube then on ice and allowed bronze precipitate automatically in about 2 minutes, and centrifugal 10 seconds of 10000rpm abandons supernatant, suspends with 100 μ l dehydrated alcohols, is inserted on ice or puts into-20 ℃ of refrigerators and temporarily preserve, and prepares to shoot.
3, the scultellum after the bullet implant steps one that adopts particle bombardment that step 2 is prepared is handled, parameter is as follows: the bronze consumption is that 100ug/ rifle, plasmid consumption are that 1ug/ μ l, target distance are 9cm, and particle gun pressure is 28, and the bronze granularity is 1.0 μ m.
Three, the acquisition of transgenic wheat
Scultellum after particle gun transformed places induces on the screening culture medium 26 ℃ of dark cultivations 10 days.At microscopically callus is cut into about the fritter about 1mm, the fritter that derives from same callus comes together, is placed on and induces on the screening culture medium, 26 ℃ of dark cultivations 20 days.Select callus to select glossy shinny callus, transfer on the regeneration screening culture medium, under 26 ℃, the condition of intensity of illumination 1000Lux, cultivated 7 days.Select the seedling about 5cm to transfer on the later stage screening culture medium, under 26 ℃, the condition of intensity of illumination 1000Lux, cultivated 20 days, obtain regrowth (T0 generation).Regrowth is transferred in the peat composed of rotten mosses (KLASMAN), in the controlled environment chamber middle the cultivation (20 ℃ of daytimes, 16 ℃ of nights; Illumination 16 hours/dark 8 hours) until results.
The Gus activity of gene expression of experimental example 4, transfer-gen plant different sites is analyzed
One, the PCR of regrowth identifies
Each regrowth that embodiment 3 is obtained carries out following evaluation respectively: the long blade tip of the about 3cm of clip, extract genomic dna, and the primer of forming with F2 and R2 carries out the PCR evaluation to (target sequence is the Gus gene), and target fragment is about 617bp.
F2 (upstream primer): 5 '-GGTCACTCATTACGGCAAAGT-3 ';
R2 (downstream primer): 5 '-GACGACCAAAGCCAGTAAAGT-3 '.
PCR system (20 μ l): contain 50ng genomic dna, 10pmol upstream primer, 10pmol downstream primer, each 250 μ M of dNTPs, 2 μ l, 10 * buffer (50mM KCl, 10mM Tris-HCl, 1.5mM MgCl
2, pH 8.3) and 2.0U rTaq polysaccharase (Dalian is precious biological), all the other are water.
The PCR program: 94 ℃ 3 minutes; 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 45 seconds, 36 circulations; 72 ℃ 8 minutes.
Partial results with the regrowth of recombinant plasmid first preparation is seen Fig. 5, obtains 12 strain positive plants (transfer-gen plant first).Adopt recombinant plasmid second to obtain 8 strain positive plants (transfer-gen plant second).Adopt recombinant plasmid third to obtain 5 strain positive plants (transfer-gen plant third).
2, Gus dyeing
PCR is accredited as positive plant carries out following Gus dyeing respectively:
The GUS staining fluid uses liquid by solute and solvent composition; Described solvent is the sodium phosphate buffer of pH 7.0,0.2M; Concentration in described solute and the GUS staining fluid thereof is as follows: the 0.5M Tripotassium iron hexacyanide, 0.5M yellow prussiate of potash, 10 μ M EDTA, 0.6mg/L Triton, 0.5mg/ml X-gluc.
Plant blossom was got its part seed, leaf and root and is respectively charged in the 5ml centrifuge tube after two weeks, and with the complete submergence of Gus staining fluid, 37 ℃ of concussions are spent the night, and Gus genetic expression intensity is observed in inferior daily 70% aqueous ethanolic solution flushing 3 times.
The root of three kinds of transfer-gen plants and Ye Zhongjun do not have blue signal to occur, and the photo of part plant root and leaf is seen Fig. 6.
There is blue signal to occur in the seed of all transfer-gen plant first, has blue signal to occur in the seed of all transfer-gen plant second, and the color of the seed of transgenosis first saturate many than the seed of transfer-gen plant second.All there is not blue signal to occur in the seed of all transfer-gen plants third.The photo of part plant seed is seen Fig. 7.The result shows that promotor provided by the invention has good seed tissue specificity, and the startup activity is better than existing promotor.
Claims (10)
1. the dna molecular shown in the sequence 1 in the sequence table.
2. the recombinant expression vector, expression cassette or the reorganization bacterium that contain the described dna molecular of claim 1.
3. recombinant expression vector as claimed in claim 2 is characterized in that: described recombinant expression vector is that described dna molecular is inserted the recombinant plasmid that the multiple clone site of plasmid pCAMBIA3301 obtains.
4. the method for a specifically expressing foreign gene in plant seed is that the dna fragmentation first is imported in the plant, thus in plant seed the described foreign gene of specifically expressing; Start described expression of exogenous gene by the described dna molecular of claim 1 in the described dna fragmentation first.
5. method as claimed in claim 4, it is characterized in that: described dna fragmentation first imports described plant by recombinant expression vector; The recombinant plasmid that described recombinant expression vector obtains for the multiple clone site with described dna molecular and described foreign gene insertion plasmid pCAMBIA3301.
6. the method for a specific expression gene in plant seed is that the recombinant expression vector that will contain the described dna molecular of claim 1 imports in the plant, starts its downstream gene specifically expressing in plant seed by described dna molecular.
7. method as claimed in claim 6 is characterized in that: described recombinant expression vector is that described dna molecular is inserted the recombinant plasmid that the multiple clone site of plasmid pCAMBIA3301 obtains.
8. method as claimed in claim 7, it is characterized in that: described gene is the Gus gene.
9. as arbitrary described method in claim 4 or 8, it is characterized in that: described plant is monocotyledons or dicotyledons.
10. method as claimed in claim 9, it is characterized in that: described monocotyledons is wheat.
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Non-Patent Citations (4)
| Title |
|---|
| A novel cis-acting element, ESP, contributes to high-level endosperm-specific expression in an oat globulin promoter;Claudia E. Vickers等;《Plant Mol Biol》;20060817;第62卷;195-214 * |
| Avena sativa globulin (Glo1) gene, promoter region;Vickers,C.E.等;《GenBank》;20060912;1-2 * |
| Claudia E. Vickers等.A novel cis-acting element, ESP, contributes to high-level endosperm-specific expression in an oat globulin promoter.《Plant Mol Biol》.2006,第62卷195-214. |
| Vickers,C.E.等.Avena sativa globulin (Glo1) gene, promoter region.《GenBank》.2006,1-2. |
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