CN100562575C - A kind of composition type expression promoter of virus satellite - Google Patents
A kind of composition type expression promoter of virus satellite Download PDFInfo
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Abstract
The invention discloses a kind of composition type expression promoter of virus satellite.Specifically, this promotor comes from the satellite molecule DNA β that accompanies with tobacco curly shoot virus.Isolating promotor is total length promotor and the excalation promoter sequence of the β C1 ORF of this DNA beta molecule.Experiment shows that the promotor of the β C1 ORF that comes from tobacco curly shoot virus DNA β is the promotor of constitutive expression.The present invention relates to tobacco curly shoot virus DNA β β C1 ORF the total length promotor and the disappearance promoter sequence and contain this β C1 ORF total length promotor and the disappearance promoter sequence expression cassette.Promotor among the present invention derives from dicotyledons virus satellite molecule, develops this promotor and is applied to the various crop gene transformation of (comprising the cereal class), and application prospect will be very wide.
Description
Technical field
The present invention relates to a kind of composition type expression promoter of virus satellite.Specifically, the present invention relates to the separation of β C1 ORF total length promoter sequence and the disappearance promoter sequence thereof of tobacco curly shoot virus DNA β.The present invention also relates to contain the β C1 ORF total length promotor of the tobacco curly shoot virus DNA β that connects heterologous gene and the structure of the carrier box of disappearance promotor.In addition, the present invention also relates to carry out the method for moment expression and stably express with the expression vector of the various promotors of β C1 ORF that contain tobacco curly shoot virus DNA β.
Background technology
The purpose of plant genetic engineering is after foreign gene is imported recipient plant it correctly and effectively to be expressed.Yet this process is subjected to the influence and the restriction of a lot of factors, and suitable promotor is the key factor of foreign gene effective expression.
So far, the promotor of existing many different sourcess is used to transformed plant cells.Wherein a big class is separated from agrobacterium tumefaciens, another big class is the promotor in plant source itself, because the target gene expression amount of above-mentioned promotor control is often lower, and have certain homology with recipient plant cellular genome sequence, so people select plant virus promoters usually for use in transgenic plant.The constitutive promoter of widespread use has in gene expression in plants at present, cauliflower mosaic virus (CaMV) 35S promoter that uses in most of dicotyledonous transgenic plant, unifacial leaf transgenic plant mainly use from the Ubiquitin promotor of corn with from the Actinl promotor of paddy rice.Constitutive promoter can both drive expression of exogenous gene in dicotyledons, monocotyledons and fungi, not limited by space-time, and expression amount is constant.
The DNA beta molecule is the satellite molecule of following with some monad geminivirus infection.The about 1.3kb of this molecular size is that its helper virus is genomic about half.The DNA beta molecule depends on its helper virus and carries out dna replication dna, and the mechanism of transcribing that depends on host plant is carried out transcript and expression.The β C1 ORF that has a high conservative in its genome, there are some researches show that this coding region expressed proteins is a kind of pathogenic correlation factor, its disappearance does not influence duplicating of DNA beta molecule, and existing experiment shows that the DNA beta molecule can carry out genetic manipulation as expression vector.The activity that determines the promotor of pathogenic associated protein β C1 on the one hand by understanding of carrying out of the present invention can be understood this proteic expression intensity in depth, and can be reflected the expression efficiency of this expression vector; On the other hand, owing to identified that the promotor that derives from the DNA beta molecule that tomato yellow leaf curl china virus accompanies is the promotor of phloem specific expressing, and DNA beta molecule that tobacco curly shoot virus accompanies and above-mentioned DNA beta molecule have bigger difference on the phenotype of causing a disease, expect also to be different from the promoter expression type in this DNA beta molecule the promotor of above-mentioned DNA beta molecule, for gene expression in plants provides some valuable Expression elements, and be the basis that good molecular biology aspect is laid in the research of the different pathogenic mechanism of two class DNA beta molecules.β C1 ORF promotor is the important factor that influences this virus satellite vector expression efficient, and the expression type of this promotor has also determined its using value in transgenosis.Based on this, the present invention has identified total length promotor active of β C1ORF and the expression activity of expressing type and disappearance promotor.
Summary of the invention
The composition type expression promoter that the purpose of this invention is to provide a kind of virus satellite.
The composition type expression promoter sequence of virus satellite: sequence shown in the total length promotor SEQ ID NO:1 of the β C1 ORF of tobacco curly shoot virus DNA β and disappearance promotor SEQ ID NO:2 thereof.
The effective gene that DNA construct comprises the described separated DNA of claim 1 and carries out with it being operatively connected, wherein said effective gene is selected from the gene of following composition: sterilant gene, herbicide resistance gene, anti-fungal gene, antiviral gene, antimicrobial gene.
Utilize vegetable cell to express the method for sterilant gene or herbicide resistance gene or anti-fungal gene or antiviral gene or antimicrobial gene, comprise the following steps:
(a) in effable mode sterilant gene or anti-herbicide gene or anti-fungal gene or antiviral gene or antimicrobial gene are connected to the separated DNA downstream of claim 1 to obtain recombinant dna fragment;
(b) this recombinant dna fragment is inserted in the carrier;
(c) with this carrier transformed plant cells;
(d) cultivate this vegetable cell and express sterilant gene or anti-herbicide gene or anti-fungal gene or antiviral gene or antimicrobial gene.
Utilize vegetable cell to prepare method of protein, comprise the following steps:
(a) be connected to the downstream of separated DNA of claim 1 with the acquisition recombinant dna fragment according to will the encode DNA of desired protein of the mode that can be expressed of DNA of coding desired protein;
(b) recombinant dna fragment is inserted in the carrier;
(c) recombinant dna fragment is cultivated the required protein of plant transformed cell expressing through Agrobacterium or particle bombardment transformed plant cells;
(d) from culture, reclaim required protein.
Expression vector contains the DNA of claim 1, wherein said carrier is plasmid vector or the expression vector that is applicable to vegetable cell, described carrier can insert effective gene, wherein said effective gene is selected from the gene of following composition: the sterilant gene, herbicide resistance gene, anti-fungal gene, antiviral gene, antimicrobial gene.
The beneficial effect that the present invention has:
1) promotor among application the present invention can realize effective disease-resistant purpose in the plant virus resistance gene engineering.Though the direct infringement target of many plant pests, particularly some piercing sucking insects mainly is at phloem, it is systemic infection plant other tissues except phloem that some disease and pests are also arranged.The employing transgenic method is prevented and treated these virus diseases and disease and pest, if the application organizes specificity promoter, its action effect can only be confined to some position of plant so, control that can not disease and pest is in full force and effect.So prevent and treat in the process at this type of and to use constitutive promoter and just seem particularly important.Constitutive promoter is compared with tissue-specific promoter, and it can continue to efficiently express goal gene, can effectively control disease and pest and spread in whole plant, and all kinds of histocytes of plant all are protected.The result of histochemical staining method shows that total length promotor among the present invention and minimum disappearance promotor are at the tegumental cell of transfer-gen plant root, stem, leaf, and the phloem of mesophyll cell and vascular tissue can both be expressed (as Fig. 6, Fig. 7) in the part xylem.The strong promoter of constitutive expression can cause foreign gene at the intravital overexpression of plant usually, thereby vine growth and development is had a negative impact.The total length promoter activity slightly is better than the activity of minimum disappearance promotor among the present invention, compare with strong composition type expression promoter CaMV 35S promoter activity, the total length promoter activity is its 41.80%, so the expression of exogenous gene amount of this promoters driven can't have a negative impact to growing of plant.The constitutive expression of cumulated volume invention promotor and the advantage of non-overexpression foreign gene are applied to can reach in the genetic engineering of plant for disease resistance cost-effective disease-resistant purpose.
2) promotor of using among the present invention is the promotor of constitutive expression, can efficiently expressing exogenous gene, increase the output of foreign protein.The promotor of constitutive expression in a organized way in and all stages of plant-growth all promotor gene express, do not show the space-time specificity; RNA also is relative constant with the protein expression amount.If produce certain target protein, set of applications moulding expression promoter expressing quantity is apparently higher than tissue-specific promoter.
3) promotor among application the present invention can effectively be applied to transgenic plant genetic engineering.The plant promoter that uses in the plant genetic engineering is owing to have certain homology with the recipient plant genome, tends to cause the gene silencing phenomenon in the host plant body and causes the foreign gene inactivation.This phenomenon is more general when polygene transforms.And the promotor among the present invention, owing to do not have homology with the Plant Genome sequence, so can avoid the generation of gene silencing phenomenon.In addition, studies show that in a large number that the promotor of dicotyledons viral source can be effectively applied to monocotyledonous genetic transformation.Promotor among the present invention derives from dicotyledons virus satellite molecule, develops this promotor and is applied to the various crop gene transformation of (comprising the cereal class), and application prospect will be very wide.
Description of drawings
Fig. 1 is the series disappearance figure of the β C1 ORF promotor of tobacco curly shoot virus DNA beta molecule.Rotaring intertranslating start site (ATG) with β C1 ORF is+1.
Fig. 2 is the promoter expression vector design of graphics.Promoter fragment behind the clone inserts the HindIII/BamH I site of expression vector pINT121 behind restriction enzyme HindIII/BamH I double digestion, be built into promoter expression vector.
Fig. 3 is expression vector establishment figure.The GUS-NOS fragment of expression vector pINT121 through producing behind the restriction enzyme BamH I/EcoRI double digestion is connected with pBINPLUS carrier segments behind the BamH I/EcoR I double digestion, be built into expression vector pBINGUS, wherein pINT121 is used for positive control, and pBINGUS is used for negative control.
Fig. 4 expresses the dyeing picture GUS moment of each promotor.A, b represent promotor 997,997 Δs 797 respectively, the negative contrast of c pBINGUS, the positive contrast of d pINT121, bar=25 μ m..
Fig. 5 is the transient expression GUS active A of each promotor, the repeatedly repetition GUS activity of each promotor; B, the average relatively GUS activity of each promotor.A, the negative contrast of pBINGUS among the B, the positive contrast of pINT121, n represents the independent experiment multiplicity of each promotor.By the check of the LSD among the multiple comparisons method ANOVA among the SAS of statistical software 8.0 experimental data is carried out significance of difference analysis (P＜0.05or P＜0.01), error line is represented standard deviation.
Fig. 6 is the transgenosis dyeing picture of total length promotor 997 (a-e) and positive control pINT121 (f-j).A, f, the square section of root; B, g, the square section of stem; C, h, the back of blade; D, i, the square section of blade; E, j, the profile of root.Rc, root cap; Svb, secondary vascular bundle; X, xylem; Ip, endophloem; Ep, outer phloem; Pm, palisade tissue; Sm, spongy tissue; Bar=25 μ m.
Fig. 7 is the transgenosis dyeing picture (a-e) of disappearance promotor 997 Δs 797.A, the square section of root; B, the square section of stem; C, the back of blade; D, the square section of blade; E, the profile of root.Rc, root cap; Svb, secondary vascular bundle; X, xylem; Ip, endophloem; Ep, outer phloem; Pm, palisade tissue; Sm, spongy tissue; Bar=25 μ m.
Embodiment
The detection that the present invention proposes to transformation of tobacco plant gene expression product, to estimate the expression intensity of promotor, and analyze the Histochemical localization of promotor in the intravital expression of plant, many technology of knowing have very much to those skilled in the art been used, comprising the spectrophotofluorimetry that is not limited to be used for the GUS enzyme activity assay, be used for the technology such as free-hand slicing method of tissue chemical analysis.
The invention describes can constitutive expression the virus satellite promotor, it is the β C1 ORF promotor of the DNA beta molecule (AJ421484) that accompanies with tobacco curly shoot virus.With infection the total DNA of tobacco plant of tobacco curly shoot virus being arranged among the present invention is template, has cloned and identified the β C1ORF total length promotor and the disappearance promotor of tobacco curly shoot virus DNA beta molecule, and sequence table has shown the sequence of each promotor.The all natural sequence of the β C1 ORF total length promotor of tobacco curly shoot virus DNA β has 997 base pairs, according to the genomic constitutional features of DNA beta molecule, the total length promotor is lacked.The feature of disappearance promotor 997 Δs 797 is fragments (sequence of promotor is SEQ ID NO:2) of-201 to-997 in the disappearance total length promotor, finds that in the activation analysis of promotor the disappearance promotor has still kept active (Fig. 4).
The invention describes the method for gene product expression in the vegetable cell.The promotor and the GRD beta-glucuronidase gene (gus) of having cloned are constructed plant expression vector, make gus gene in the tobacco plant cell, carry out moment by the Agrobacterium soaking method and express; Plant expression vector after making up is changed in the tobacco cell by Ye Panfa, make the gus gene in tobacco cell, carry out stably express.
The present invention has also described active evaluation of GUS and detection method in the vegetable cell.In the moment of vegetable cell expression system, plant tissue is carried out whether can identifying by the appearance in blue site whether its gus gene expresses after the histochemical stain; Detect its expression activity by fluorophotometric analysis.Experimental results show that β C1 ORF total length promotor and disappearance promotor thereof among the present invention all have promoter activity, the former activity is strong slightly by contrast, be 41.80% of CaMV 35S promoter, the minmal sequence of expression activity is positioned at disappearance promotor 997 Δs 797; The expression vector of total length promotor and disappearance promotor is changed in the tobacco plant by Ye Panfa, further identify the expression type of its upstream promoter after the GUS histochemical stain by the expressive site in blue site.The result shows, β C1 ORF total length promotor and disappearance promotor thereof can drive gus gene constitutive expression in organs such as transfer-gen plant root, stem, leaf, and has promoter activity disappearance promotor 997 Δs 797 and also have same expression pattern.
But the β C1 ORF total length promotor of the tobacco curly shoot virus DNA β among the present invention or the needed any heterologous gene of its disappearance promotor constitutive expression.The example of heterologous gene comprises: insecticidal toxin gene, herbicide resistance gene, antimicrobial gene, anti-fungal gene, antiviral gene.
To contain the β C1 ORF promotor of tobacco curly shoot virus DNA β and the construct of heterologous gene among the present invention is inserted in a kind of carrier, with this carrier transformed plant cells, preferred carrier is can be a kind of agrobacterium tumefaciens (agrobacterium) Ti-plasmids derivative for plant, this plasmid derivative thing imports vegetable cell by agriculture bacillus mediated binary vector technology, and this technology is known in this area.Transformed plant cells is incubated in a kind of suitable culture medium, and the plant that regenerates.
The present invention includes β C1 ORF total length promotor that comes from tobacco curly shoot virus DNA β and the DNA construct that lacks the promoter DNA sequence and contain above sequence.Simultaneously, the present invention also comprises the dna sequence dna of " function equivalent " of the β C1 ORF promotor that is tobacco curly shoot virus DNA β, and contains the construct that effectively is connected to the such dna sequence dna on the heterologous gene.
The present invention comprises that also " function equivalent " that use β C1 ORF promotor as described herein give the constitutive expression that is in any gene under their control.
The term that uses among the present invention " promoter activity " but be meant when effective gene being connected to the downstream of promotor with the phraseology of this gene, and import among host's (vegetable cell), this host shows the function that has in the host or produce this effective gene product outside the host.Normally the protein coding gene (reporter gene) of easy qualitative and quantitative analysis is connected to the downstream of promotor, gene after will making up then imports in the host, detect the expressing protein situation, with this activity that determines whether to exist specific promotor with and how render a service.
" disappearance promotor " is meant anyly disappearance is arranged and still have the β C1 ORF promotor of the tobacco curly shoot virus DNA β of promoter activity.
" function equivalent " is meant under stringent hybridization condition and an any dna sequence dna of dna sequence dna complementary, this sequence and the hybridization of this object of reference and the activity of the β C1 ORF promotor that is similar to tobacco curly shoot virus DNA β is arranged.
Non-limiting example below adopting is further described the present invention.
The clone and the sequencing of the β C1 ORF promoter fragment of embodiment 1 tobacco curly shoot virus DNA β
Basically implement molecular engineering according to described methods such as Sambrook (Molecular Cloning:A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor Laboratory, NY, 1989).
Tobacco leaf with the infection tobacco curly shoot virus is a material, extracts the total DNA[Murray MG of plant et al., 1987, EMBO J, 6:3901～3907 with the CTAB method].Each promoter fragment of pre-amplification is seen Fig. 1.With the total DNA of plant is template, each promoter fragment of the β C1ORF by primer amplified tobacco curly shoot virus DNA β.Primers F L (5 ' GAAGCTTATACGTATTTTAATCCGTATG3 ', corresponding to the 1-21 position among the SEQ ID NO1, introduced the HindIII site) and RL (5 ' CGGATCCATTTGTTCTTGTGACCAAAAC 3 ', corresponding to the 997-977 position among the SEQ ID NO 1, introduced the BamHI site) amplification total length promotor (SEQ ID NO:1).Primers F 1 (5 ' CAAGCTTAGTACTAACGTCTAACTAC 3 ', corresponding to the 1-19 position among the SEQ ID NO:2, introduced the HindIII site) and RL (5 ' GGATCCATTTGTTCTTGTGACCAAAAC 3 ', corresponding to the 200-180 position among the SEQ ID NO:2, introduced the BamHI site) amplification disappearance promotor 997 Δs 797 (SEQ ID NO:2).PCR product after the amplification through HindIII and BamHI direct enzyme cutting after QiaGen gel-purified test kit reclaims the purpose fragment, with being used for vector construction after the Sanger dideoxy chain termination sequence verification.
The structure of embodiment 2 promoter expression vectors and GUS fusion expression vector
With Restriction Enzyme HindIII and BamHI digestion binary expression vector pINT121 (Liu et al, Planta, 2003,216:824-833), total length promotor and various disappearance promoter fragment after enzyme cut insert this site, be configured to promoter expression vector (Fig. 2): the total length promoter construct is p β-FL, and the construct of disappearance promotor 997 Δs 797 is a p β Δ 797.
With BamHI and EcoR I double digestion expression vector pINT121, GUS-NOS fragment under the cutting is inserted into expression vector pBINPLUS, and (structure of this carrier is seen Transgenic Research, 1995,4, in BamHI/EcoR I site 288-290), carrier behind the structure is pBINGUS, and this carrier is as negative control, and carrier pINT121 is as positive control (Fig. 3).
Embodiment 3 Agrobacterium-mediated Transformation
With each promotor construction by the triparental mating method import the Agrobacterium host cell [Wang Guanlin, plant genetic engineering philosophy and technique. Beijing: education of science press, 1998].Concrete grammar is as follows: the promoter expression vector behind the structure need just can enter Agrobacterium EHA105 by the mediation of helper plasmid pRK2013.Promoter expression vector inoculation 5ml behind the structure contains Km (50 μ g/ml) LB liquid nutrient medium, 200rpm incubated overnight under 37 ℃ of conditions; EHA105 inoculation 5ml contains Sm (50 μ g/ml) YEP liquid nutrient medium, and 28 ℃ of following 200rpm cultivated 48 hours; Helper plasmid pRK2013 inoculation 5ml contains Km (50 μ g/ml) LB liquid nutrient medium, 200rpm incubated overnight under 37 ℃ of conditions.Three kinds of bacterium liquid are respectively got 400 μ l, put upside down mixing after, 6000rpm 30 seconds collects bacterium liquid, the thalline of collection is with 200 μ l ddH
2O suspends, being coated with rod with glass coats and does not have the YEP of resistance solid medium flat board to carry out triparental mating, cultivate after 24 hours for 28 ℃, dip on two YEP solid medium flat boards that the continuous coating of the bacterial plaque that takes a morsel contains Km (50 μ g/ml) and Sm (50 μ g/ml) respectively, obtain containing single bacterium colony of promoter construct with this.With Auele Specific Primer it is carried out PCR and identify screening positive clone.
The moment of embodiment 4 promotors expresses
1) cultivation of Agrobacterium and inducing: reference literature [Jyoti Kapila et al., 1997.Plant Science122:101-108] the Agrobacterium bacterium liquid of each promoter expression vector is inoculated in contains corresponding antibiotic liquid nutrient medium YEB (5g/L beef extract, the 1g/L yeast powder, the 5g/L Tryptones, 5g/L sucrose, 2mM sal epsom, pH5.8) in, be cultured to logarithmic phase, centrifugal collection thalline, be resuspended in MMA solution (10mmol/L2-(N-morpholine)-ethylsulfonic acid (MES, pH 5.6), 100 μ mol/L Syringylethanones, and adjust bacterial concentration and make its OD the 10mmol/L magnesium chloride),
600=1.5, room temperature leaves standstill places 3h.
2) moment expresses: reference literature [Liu Zhaoming etc., 2002, biotechnology journal 18 volume 4 phases: 411-414; Yinong Yang et al.2000.The Plant Journal, 22 (6), 543-551] plant leaf is taked the Agrobacterium soaking method.Concrete grammar: choose age in 6-7 week Ben Shi cigarette plant middle part and do not launch blade fully, get the disposable syringe of a 1ml, remove syringe needle, the resuspended bacterium liquid of 500 μ l is injected fully cell between the arteries and veins of every blade with the needle tubing depended on pressure.Base of the plant is adopted the Agrobacterium injection, be about to the resuspended bacterium liquid of 500 μ l injects plants stems with the disposable syringe of 1ml middle part.Plant after Agrobacterium infiltration and the Agrobacterium injection is cultivated in 25 ℃ of thermostatic chambers, and periodicity of illumination is 16/8 hour.GUS histochemical stain and the analysis of GUS fluorescence activity are carried out in sampling behind the 60-72h.
Embodiment 5GUS detects
Histochemical staining method: the base of the plant after reference literature [Jefferson RA et al.EMBO J, 1987,6:3901～3907.] will be injected is done free-hand section.Place contain 1mmol/L X-Gluc (5-bromo-4-chloro-3-indoles-β-D-glucuronic acid) staining fluid in 37 ℃ of incubations 3 hours to spending the night.Sample to colourless, is observed photograph through anatomical lens through 75% ethanol dehydration.As Fig. 4, a among the figure, b represent promotor 997,997 Δs 797 respectively, the positive contrast of d pINT121, the negative contrast of c pBINGUS.As can be seen from the figure, total length promotor 997 and disappearance promotor 997 Δs 797 thereof all can drive the moment expression in axis of GRD beta-glucuronidase gene, wherein the expression activity of total length promotor 997 slightly is better than disappearance promotor 997 Δs 797, positive control pINT121 has presented tangible blue expression site, and the blue site of expressing does not appear in negative control pBINGUS.From expression intensity, the gus gene expression intensity of the total length promotor of the β C1 ORF of tobacco curly shoot virus DNA β is approximately half of positive control pINT121.
Fluorometry: reference literature [Jefferson RA et al.EMBO J, 1987,6:3901～3907.] plant leaf after will sampling is through liquid nitrogen grinding, GUS extracting solution (the 50mmol/L phosphoric acid buffer pH 7.0 that adds 3 times of volumes, the 10mmol/L disodium ethylene diamine tetraacetate, 0.1% (w/v) Triton X-100,0.1% (w/v) sarcosyl, the 10mmol/L beta-mercaptoethanol), get supernatant 10ul and 4-MUG (4-methyl umbelliferone-β-D-glucuronic acid) reaction after centrifugal, the fluorophotometric instrument is measured the GUS activity.As Fig. 5, A repeatedly repeats the GUS activity that back (multiplicity is not less than 6) records for each promotor construction, and B is the average expression activity of each promotor construction.Experimental data is analyzed by the LSD method that repeatedly repeats comparison ANOVA of SAS8.0 software, 35S promoter activity with positive control pINT121 is reference, the result shows that the activity of total length promotor 997 is 41.80% of positive controls, and the activity of disappearance promotor 997 Δs 797 is 39.24% of 35S promoters.And total length promotor 997 does not have significant difference (P＞0.05) with disappearance promotor 997 Δs 797; Have utmost point significant difference (P＜0.01) between both and the positive control pINT121, concrete data see Table 1.
Table 1
Annotate :-representative does not have the disappearance fragment; Relative reactivity is reference with the CaMV 35S promoter in the table.
Gather blade from the common cigarette tobacco plant of health, reference [Horsch RB et al., Science, 1985,227:1229～1231].Specific as follows: the tobacco leaf fritter that will cut after will sterilizing places pre-the cultivation 2 days on the division culture medium of nonreactive.Transform with the Agrobacterium bacterium liquid that is cultured to logarithmic phase then, place dark the cultivation 2 days on the division culture medium of nonreactive, go to again that (kantlex 100mg/L and Pyocianil 500mg/L) cultivates on the resistance substratum.Treat to downcut when the resistance budlet grows to about 1 centimetre, move to (kantlex 100mg/L and Pyocianil 100mg/L) in the root media, the resistance seedling that grows to a month size is used for tissue chemical analysis.
Cut bud after one month, the positive transgenosis seedling that picked at random 4 strain PCR screen, respectively its root, stem are made free-hand section and fritter, blade is cut into small pieces, and places 1mmol/L X-Gluc (5-bromo-4-chloro-3-indoles-β-D-glucuronic acid) staining fluid in 37 ℃ of incubations 3 to 12 hours.The free-hand section of root, stem and blade fritter are colourless to negative contrast with the dehydration of 75% ethanolic soln.The fritter of the root after the dyeing, stem and blade fixedly spends the night for 4 ℃ through 2.5% glutaraldehyde.Ethanolic soln (50%, 70%, 80%, 90%, 95%, 100%) with gradient concentration carries out processed to sample, spends the night with the Resins, epoxy embedding at last.Each embedded sample is cut into slices with the semithin section machine, and microscopically is observed and taken a picture.Find total length promotor 997 and lack promotor 997 Δs 797 can express at root, stem and the Ye Zhongjun of transfer-gen plant.Wherein root cap and tip of a root meristematic zone all have tangible GUS express (Fig. 6, e); In the square section of root tissue, can observe to have in primary structure of root mediopellis and the phloem cell and significantly blue express the site, and in the xylem without any express the site occur (Fig. 6, a); In the section of stem, cortex parenchyma cell and vascular tissue all observe GUS express site, especially vascular tissue have significantly GUS dyeing (Fig. 6, b); Observe tangible blue site (Fig. 6 of expression at the rotaring gene plant blade back, c), the cross section observations of blade is consistent therewith, at all kinds of tissues of blade, comprise all observe in palisade tissue, spongy tissue and the secondary vascular tissue blue expression site (Fig. 6, d).In the transfer-gen plant of positive control, observed identical expression pattern (Fig. 6, e-j), promptly at the cortex parenchyma cell of its root, stem, leaf, phloem cell in the vascular tissue, can both express the gus gene in part tracheid, the mesophyll cell, but strength ratio promotor of the present invention is eager to excel.(Fig. 7 a-e), promptly also can both express the gus gene in its root, stem, leaf, but intensity descends slightly than the total length promotor also to have observed identical expression pattern in the transfer-gen plant of disappearance promotor 997 Δs 797.Above result shows that β C1 ORF total length promotor 997 has the constitutive expression characteristic in vegetable cell, and disappearance promotor 997 Δs 797 can keep its expression pattern, and the controlling element that has constitutive expression in-1～-200 zone is described.
Above example is to be used for describing the present invention, rather than restriction the present invention.By the description of front, except this paper illustrated and describe, various modifications of the present invention are clear and easy to understand for a person skilled in the art.
Sequence table
(1) physical data
(i) applicant: the Zhou Xueping of Zhejiang University, Ding Chenjun, Wu Jianxiang
(ii) subject matter: a kind of composition type expression promoter
(iii) sequence number: 3
(2) data of SEQ ID NO:1:
(i) sequence signature:
(A) length: 997 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) geometry: linearity
(ii) molecule type: genomic dna
(iii) infer: non-
(iv) antisense: non-
(v) sequence description: SEQ ID NO:1
atacgtattt?taatccgtat?gtaatgcata?acataagtat?taaataataa?aataaatatt?60
tatattatca?acgtagtact?gtgtagaccc?aaattgaaag?agcccaacaa?gagaccaaat?120
tgcataccca?gtaatctttt?ttggcccatt?taaggcccaa?tttcatatgt?ggggcccacc?180
atcagcacca?aagaccccgc?tcgccaccgg?taatattata?acggtggcga?gctaagctcc?240
ggcgtagcta?aggctgctgc?gtagcgtagt?ggtttctacc?ctcccagggg?tacacaacgc?300
cgcgcgtgtc?gcaaattggt?gaacggaagt?gcctgaatcg?gcggtcggag?gagttttagg?360
agagagaaac?tctacttttt?cagtccactc?tcacgcgcct?accagacaca?gggacaaggc?420
tattttggga?aagtaaatta?atagtcccca?attgagtccc?cgatgtatcg?ggtcctcatt?480
tacctatcag?gtcctcatgt?ttttacccat?tcacctcagt?aattattaat?tactgattta?540
ccgtgcagta?aatcatttta?ttctttttat?ttattctttt?aattaggtgg?ggtccactga?600
aatttcttca?aggtttattg?ttctgagtgt?atgagttttt?cttttgtttt?tatttttttt?660
tcttgttttc?ttgttcgtca?ttggggtttt?cgtttcgctg?cgctcccatt?ttaggataat?720
ggatgttatg?agttttattg?tatttaattt?cagttttttt?cttttttctt?ttttcctttc?780
ctaatcatgt?tatgattagt?actaacgtct?aactacacat?ttaattctaa?ttacacatta?840
accacaccat?taacaacagc?gtcacatccg?atacaaagat?ctccactacc?aaacatatga?900
acaagcaaga?aacacgtaaa?tcatttcaga?ccatgaatag?catatgaaaa?tgattattta?960
aagggacaga?actaacgttt?tggtcacaag?aacaaat 997
(3) data of SEQ ID NO:2:
(i) sequence signature:
(A) length: 200 base pairs
(B) type: nucleic acid
(C) chain: two strands
(D) geometry: linearity
(ii) molecule type: genomic dna
(iii) infer: non-
(iv) antisense: non-
(v) sequence description: SEQ ID NO:2
agtactaacg?tctaactaca?catttaattc?taattacaca?ttaaccacac?cattaacaac?60
agcgtcacat?ccgatacaaa?gatctccact?accaaacata?tgaacaagca?agaaacacgt?120
aaatcatttc?agaccatgaa?tagcatatga?aaatgattat?ttaaagggac?agaactaacg?180
ttttggtcac?aagaacaaat
Claims (8)
1. the composition type expression promoter sequence of a virus satellite is characterized in that: described promoter sequence is dna sequence dna shown in the total length promotor SEQ ID NO:1 of the β Cl ORF of tobacco curly shoot virus DNA β or its disappearance promotor SEQ ID NO:2.
2. DNA construct, it is characterized in that the effective gene that comprises the described promoter DNA sequence of claim 1 and carry out with it being operatively connected, wherein said effective gene is selected from the gene of following composition: sterilant gene, herbicide resistance gene or antimicrobial gene.
3. DNA construct according to claim 2, wherein said antimicrobial gene is anti-fungal gene or antiviral gene.
4. utilize vegetable cell to express the method for sterilant gene or herbicide resistance gene or antimicrobial gene, comprise the following steps:
(a) in effable mode sterilant gene or herbicide resistance gene or antimicrobial gene are connected to the promoter DNA sequence downstream of claim 1 to obtain recombinant dna fragment;
(b) this recombinant dna fragment is inserted in the carrier;
(c) with this carrier transformed plant cells;
(d) cultivate this vegetable cell and express sterilant gene or anti-herbicide gene or antimicrobial gene.
5. method according to claim 4, wherein said antimicrobial gene is anti-fungal gene or antiviral gene.
6. utilize vegetable cell to prepare method of protein, comprise the following steps:
(a) be connected to the promoter DNA sequence downstream of claim 1 with the acquisition recombinant dna fragment according to will the encode DNA of desired protein of the mode that can be expressed of DNA of coding desired protein;
(b) recombinant dna fragment is inserted in the carrier;
(c) recombinant dna fragment is cultivated the required protein of plant transformed cell expressing through Agrobacterium or particle bombardment transformed plant cells;
(d) from culture, reclaim required protein.
7. expression vector, it is characterized in that containing the promoter DNA sequence of claim 1, wherein said carrier is the expression vector that is applicable to vegetable cell, described carrier inserts effective gene, and wherein said effective gene is selected from the gene of following composition: sterilant gene, herbicide resistance gene or antimicrobial gene.
8. expression vector according to claim 7, wherein said antimicrobial gene is anti-fungal gene or antiviral gene.
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