CN100567491C - Phytopathogen is induced sequence and the application with tissue specificity expression promoter - Google Patents

Abstract

The invention provides phytopathogen and induce promotor, contain the construction and the carrier of ASR1 promoter element with tissue specificity expression promoter element ASR1, and their purposes.

Description

Phytopathogen is induced sequence and the application with tissue specificity expression promoter

Technical field

The invention belongs to plant biological engineering and plant improvement genetically engineered field.Specifically, the present invention relates to new phytopathogen and induce sequence and application with tissue specificity expression promoter ASR1.

Background technology

Business-like in the world at present transgenic plant are generally adopted the promotor of composing type high expression level promotor such as 35S and ubiquitin (ubiquitin), and the expression of goal gene is not usually organized and development-specific.This class transgenosis is because of being to express in the complete stool property and the time of infertility, and the worry of the biological safety that causes is also maximum.Therefore, separating and utilize the plant promoter of tissue, growth, adverse circumstance (comprising pathogen infection, arid, high temperature etc.) and chemical induction that importance in its theoretical and transgenosis application is arranged, also is the field of competing in the world.This class transgene expression can make target gene express in specific environment or particular organization, replaces in the past the time of infertility and whole plant high expression level, thereby makes plant save metabolisable energy, and effectively reduces ecology and the health risks that transgenosis may have.Especially in plant disease-resistant worm genetically engineered, the transgenosis of disease and pest abduction delivering is expected to eliminate the resistant lose that the gathering because of the disease and pest sudden change/dominant population that continues to select to depress causes, thereby the acquisition durable resistance is for the safe and efficient property of transgenic plant provides a service platform.

Have some the important adjusting growths and the gene of disease resistance response in the plant, the expression of this genoid often has tissue, growth and disease inductive feature.The promotor of separating and using this genoid has important use to be worth in the plant transgene improvement.

Summary of the invention

We are from an Arabidopis thaliana disease resistance response and growth mutant asr1 (auxin and salicylicacid-responsive 1), cloned its functional gene ASR1, analysis revealed, its promotor has showed intensive tissue specificity activity: only high expression level is arranged in meristematic tissue, and be subjected to the abduction delivering of pathogenic bacteria, Whitfield's ointment and derivative thereof, plant hormone.So the ASR1 promotor that this patent relates to has a wide range of applications on plant fixed point and abduction delivering transgenosis.

Therefore, on the one hand, the invention provides a kind of promoter element, it comprises that phytopathogen induces the nucleotide sequence with tissue specificity expression promoter ASR1 or its polynucleotide varient.

In a preferred embodiment, described promoter element comprises the nucleotide sequence shown in the SEQ ID NO:1.

On the other hand, the invention provides a kind of construction specific expressed in the plant meristematic tissue, the promoter element of the present invention that this construction contains foreign gene and is operably connected with this foreign gene.

In a preferred embodiment, described promoter element comprises the nucleotide sequence shown in the SEQ ID NO:1.In another preferred embodiment, described foreign gene is a myb gene.

Another aspect the invention provides a kind of carrier, and it contains promoter element of the present invention.

Another aspect, the invention provides a kind of in the plant meristematic tissue method of specific expressed foreign gene, the method comprising the steps of:

(a) provide a construction, the promoter element of the present invention that described construction contains foreign gene and is operably connected with this foreign gene,

(b) construction in the step (a) is imported vegetable cell, obtain the plant transformed cell;

(c) with the vegetable cell regeneration plant that transforms in the step (b), thus in the meristematic tissue of this plant specific expressed this foreign gene.

In a preferred embodiment, described promoter element comprises the nucleotide sequence shown in the SEQ ID NO:1.In another preferred embodiment, express promotor of the present invention by hormone regulating and controllings such as Whitfield's ointment and derivative, plant hormones, thus specific expressed this foreign gene.

Again on the one hand, the invention still further relates to promoter element of the present invention and be used for purposes at the specific expressed foreign gene of plant meristematic tissue.

The invention still further relates to a kind of host cell that contains carrier of the present invention.

Description of drawings

Fig. 1 demonstration utilizes ASR1 to express gus reporter gene at each vegetative point or meristematic tissue.

Fig. 2 shows with the Northern hybrid experiment and shows that ASR1 is expressed by pathogenic bacterium inducing.Among the figure, " Mock " expression 10mM MgCl ₂Connect blade, in contrast." p.s.m./avrRpm1 " expression contains the p.s.m bacterium inoculation of nontoxic exciton avrRpm1 gene; " p.s.m. " expression is inoculated with the p.s.m bacterium.(bacterium liquid is all used 10mMMgCl ₂The solution preparation) p.s.m is the abbreviation of bacterial classification Pseudomonas syringae maculicola.

Fig. 3 is presented in the wild-type Arabidopis thaliana, and the Whitfield's ointment 12h that executes 0.5mM outward can obviously induce the ASR1 expression of gene.

Embodiment

As used herein, " isolating " is meant that material separates (if natural substance, primal environment promptly is a natural surroundings) from its primal environment.Do not have separation and purification as polynucleotide under the native state in the active somatic cell and polypeptide, but same polynucleotide or polypeptide as from native state with in other materials that exist separately, then for separation and purification.

As used herein, term " ASR1 " is auxin and sAlicylic acid- rThe abbreviation of esponsive 1.

As used herein, term " ASR1 promotor ", " ASR1 promoter region " are used interchangeably, the polynucleotide of refer to have the ASR1 promoter sequence (SEQ ID NO:1) or its active fragments.Last 18 bases are the coding section start among the SEQ ID NO:1.

As used herein, term " promotor " is meant one section sequence of genetic transcription initiation site upstream.

As used herein, term " specifically expressing " is meant that gene is at specific time and specific tissue expression.

As used herein, term " goal gene " is meant gene relevant with the certain quality proterties in the genetically engineered.

The present invention relates to ASR1 promotor and polynucleotide varient thereof.The varient that allelic variant that these polynucleotide varients can be natural generations or non-natural take place.These nucleotide diversity bodies comprise and replace varient, deletion mutation body and insert varient.As known in the art, allelic variant is the replacement form of polynucleotide, replacement, disappearance or the insertion of its (as 1-10,1-8, the 1-5) Nucleotide that may be one or more, but can be from not changing the function of this promotor in fact.

The invention still further relates to nucleic acid fragment with above-mentioned sequence hybridization.As used herein, the length of " nucleic acid fragment " contains 15 Nucleotide at least, better is at least 30 Nucleotide, is more preferably at least 50 Nucleotide, preferably at least 100 above total lengths to the ASR1 promotor of Nucleotide.The amplification technique (as PCR) that nucleic acid fragment can be used for nucleic acid is to determine and/or to separate the nucleotide sequence of promotor of the present invention.

The Nucleotide full length sequence of ASR1 promotor of the present invention or its fragment can obtain with the method for pcr amplification method, recombination method or synthetic usually.For the pcr amplification method, can be disclosed according to the present invention relevant nucleotide sequence design primer, and with cotton genomic dna as template, amplification and must be about sequence.

In case obtained relevant sequence, just can obtain relevant sequence in large quantity with recombination method.This normally is cloned into carrier with it, changes cell again over to, separates obtaining relevant sequence then from the host cell after the propagation by ordinary method.

In addition, also the method for available synthetic is synthesized relevant sequence, especially fragment length more in short-term.Usually, by first synthetic a plurality of small segments, and then connect and to obtain the very long fragment of sequence.At present, can be fully obtain the dna sequence dna of promotor of the present invention (or its fragment, or derivatives thereof) by chemosynthesis.This dna sequence dna can be introduced in various existing dna moleculars as known in the art (or as carrier) and the cell then.

Use method (Saiki, the et al.Science 1985 of round pcr DNA amplification/RNA; 230:1350-1354) be optimized for acquisition promoter element of the present invention.

The present invention also relates to comprise the construction or the carrier of promoter element of the present invention, and transform or the host cell (especially vegetable cell) of transduction, and produce foreign protein and the method that plant transformed cell regeneration is become plant thus with described construction or carrier.

Be applicable to that foreign gene of the present invention is not particularly limited, nearly all foreign gene all can be used for the present invention, especially with.The example of representational foreign gene comprises (but being not limited to): myb gene, GaMYB2 gene, GFP gene etc.These genes are linked to each other with ASR1 promotor of the present invention, form the construction that contains foreign gene, then described construction is used for gene therapy.

The preferred foreign gene of one class is a myb gene.Myb gene is the focus of clone's cotton fiber development key gene, some isolated M YB expression of gene feature and evolutionary relationships in cotton fibre come into one's own (Cedroni etc., 2003, Plant Mol.Biol.51:313-325).

A kind of example of construction is exactly the foreign gene that directly links to each other with the ASR1 promotor.Usually, the ASR1 promotor should be positioned at the upstream of foreign gene.

Among the present invention, the nucleotide sequence that contains the ASR1 promoter element can preferably be inserted into carrier, for example in the expression vector.Term " carrier " refers to bacterial plasmid well known in the art, phage, yeast plasmid, vegetable cell virus or other carriers.The carrier of Shi Yonging includes but not limited in the present invention: expression vector, cloning vector for example are applicable to the carrier in protokaryon (as bacteriums such as intestinal bacteria), eucaryon (as yeast), the vegetable cell.In a word, as long as can duplicate in host and stablize, any plasmid and carrier can be used.In expression vector, except containing replication orgin, ASR1 promoter element, also can contain marker gene and other translation controlling elementss.

Method well-known to those having ordinary skill in the art can be used to make up and contains ASR1 promotor and suitable transcribing/the translate expression vector of control signal.These methods comprise (Sambroook, et al.Molecular Cloning, a Laboratory Manual, Cold SpringHarbor Laboratory.New York, 1989) such as extracorporeal recombinant DNA technology, DNA synthetic technology, the interior recombinant technologys of body.Described dna sequence dna can be connected with treating expressed exogenous gene effectively, and is synthetic to instruct described foreign gene mRNA.

In addition, expression vector preferably comprises one or more selected markers, to be provided for selecting the phenotypic character of transformed host cells, cultivate Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) of usefulness as eukaryotic cell, or be used for colibacillary tsiklomitsin or amicillin resistance.

Comprise the carrier of above-mentioned suitable dna sequence dna and suitable promotor or control sequence, can be used to transform appropriate host cell, so that it can express exogenous protein.

Host cell can be a prokaryotic cell prokaryocyte, as bacterial cell; Or eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.Representative example has: intestinal bacteria, streptomyces; Fungal cell such as yeast; Vegetable cell etc.

When using promotor of the present invention, be enhanced if will make to transcribe when in carrier, inserting enhancer sequence.Enhanser is the cis acting factor of DNA, and nearly 10 to 300 base pairs act on promotor transcribing with enhancing gene usually.Can for example be included in the SV40 enhanser etc. of 100 to 270 base pairs of replication origin side in late period one.

Can carry out with routine techniques well known to those skilled in the art with the recombinant DNA transformed host cell.When the host was prokaryotic organism such as intestinal bacteria, the competent cell that can absorb DNA can be used CaCl in exponential growth after date results ₂Method is handled, and used step is well-known in this area.Another kind method is to use MgCl ₂If desired, transforming also the method for available electroporation carries out.When the host is an eukaryote, can select following DNA transfection method for use: coprecipitation of calcium phosphate method, conventional mechanical method such as microinjection, electroporation, liposome packing etc.Transform plant and also can use methods such as Agrobacterium-mediated Transformation or particle gun conversion, for example leaf dish method.Can use ordinary method regeneration plant for plant transformed cell, tissue or organ, thereby obtain the plant that epidermal hair changes.

The transformant that obtains can be cultivated with ordinary method, expresses the polypeptide of coded by said gene of the present invention.According to used host cell, used substratum can be selected from various conventional substratum in the cultivation.Under the condition that is suitable for the host cell growth, cultivate.After host cell grows into suitable cell density, induce the promotor of selection with suitable method (as temperature transition or chemical induction), cell is cultivated for some time again.

Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, as molecular cloning laboratory manual (Molecular cloning:A laboratory manual, 3rd ed., Sambrook etc., Cold Spring Harbor Laboratory, 2001) and molecular biology of plants laboratory manual (Plant Molecular Biology-A Laboratory Mannual, Clark etc., Springer-Verlag, 1997) condition described in, or the condition of advising according to manufacturer.

The separation of embodiment 1:ASR1 promotor

The separation of ASR1 promotor: according to the sequence that retrieves on the Genbank, design upstream and downstream primer in the 1.6kb scope of asr1 gene translation initiation site upstream, genomic dna with the seedling extraction in 7 days of Arabidopis thaliana Columbia wild-type is that template is carried out PCR, obtains the PCR product.Amplification condition is 94 ℃ of 3min; 94 ℃ then, 30s, 54 ℃, 30s, 72 ℃, 2min circulation 30 are taken turns; Then 72 ℃, 10min.Used primer is as follows:

Reverse primer: 5 ' TTAGTAAGTTTCA GTCGACGTTCTAGA-3 ' (SEQ ID NO:2)

Forward primer: 5 ' TT GGATCCTCAGGCGTGGTTTAAGAG-3 ' (SEQ ID NO:3)

(the underscore place is respectively and introduces restriction enzyme site SalI, BamHI)

Separation can obtain the nucleotide sequence shown in the SEQ ID NO:1.

Embodiment 2: vector construction and Agrobacterium-mediated Transformation

Vector construction and Agrobacterium-mediated Transformation: intermediate carrier is selected SK-for use, is connected into the corresponding site of SK-SalI/BamHI after above-mentioned PCR product S alI/BamHI enzyme is cut.Select enzyme to cut correct cloning and sequencing, after order-checking is correct, downcut asr1 promotor part with SalI/BamHI again, be connected into same binary expression vector pBI101.1, change bacillus coli DH 5 alpha through the SalI/BamHI double digestion.Upgrading grain, enzyme are cut and are identified that correct plasmid changes Agrobacterium GV3101 over to.

Embodiment 3: the screening of Plant Transformation and transgenic progeny

When the major branch of Arabidopis thaliana begins to budding, pinch, to promote collateral generation.Agrobacterium-mediated Transformation was removed blocky kind of pod in preceding 1 day, and overlay film is preserved moisture.Maintain the temperature at 22～24 ℃ between cultivation, relative humidity is 80%, and light source is a luminescent lamp, and intensity of illumination is 85 μ mols ^-1M ^-2, light application time is 16h.With atomizer above-mentioned Agrobacterium bacterium liquid is sprayed at the Arabidopis thaliana plant surface that is in before the full-bloom stage equably, has drop to fall to the blade and be advisable.Preservative film covered 1～2 day, kept relative humidity greater than 95%, infected conversion in order to Agrobacterium.Repeat as stated above to transform 1 time after 7 days (d).Treat behind the seed maturity plant mixing to be gathered in paper bag threshing behind the placement 7d in moisture eliminator.Transformed the seed (T0) sterilization back sowing on the 0.5 * MS substratum that contains 50ug/ml kantlex (Kan), 4 ℃ of vernalization 3 days, normal illumination is cultivated then.After about 10 days, the chooser leaf green, hypocotyl is longer, and grows seedling (T1) transplanting of two normal true leaves.The T1 individual plant carries out same screening after receiving kind, selects the offspring to separate than being under (T2) seed kind of 3: 1 the unseparated homozygous plants that is of T2.Obtain the transgenosis homozygous plants that 20 strain T2 or T3 insert for single copy at last.

Embodiment 4: the molecular biology identification of transgenic plant

Use the transgenosis homozygous plants of embodiment 3 gained to carry out GUS dyeing experiment.Vegetable material is immersed GUS dye liquor vacuum suction, in 37 ℃ of placements, last 70% ethanol decolorization.

The composition of GUS dye liquor is 100mM pH 7.0 sodium phosphate buffers, 10mM EDTA, 0.1% TritonX-100 and 1mM X-Gluc.

The dyeing experimental result is presented among Fig. 1.Among Fig. 1, (a) 7 days Arabidopis thaliana seedling of expression growth, GUS dyeing is at the tip of a root, survey root, plumular axis and root junction, leaf meristematic tissue, newborn leaflet and vein; (b) expression Arabidopis thaliana bud, filigree, the flower pesticide of GUS dyeing in petal, growth; (c) expression Arabidopis thaliana flower, GUS dyes at filigree, flower pesticide, column cap; (d) expression root GUS dyeing forms figure at the tip of a root, survey root, plumular axis and root junction (e-h) survey root.GUS dyeing mainly is distributed in lateral root nidus and the newly-generated lateral root.In sum, the ASR1 promotor is at meristematic tissue such as leaf meristematic tissue, bud, and the lateral root nidus and the tip of a root have high expression level.

Embodiment 5:Northern hybrid experiment shows that ASR1 is expressed by pathogenic bacterium inducing

The Arabidopis thaliana in 5 weeks of growing is used for inoculation experiments.Grow in KB substratum (20gL ^-1Peptone, 10mLL ^-1Glycerine, 1.4gL ^-1Dipotassium hydrogen phosphate and 15gL ^-1Agar, pH 7.2) cultivate pathogenic bacteria P.s.maculicola/avrRpm1 and P.s.maculicola 10mM MgCl through spending the night ₂Be diluted to OD ₆₀₀Be 0.02 (to be equivalent to 1 * 10 ⁷Colony forming unit/mL).Adopt the method for injection inoculation, draw above-mentioned bacterium liquid, enter, note avoiding the middle arteries and veins of blade as far as possible from the back side (abaxial side) injection of lotus throne blade with the 1mL syringe of needle-less.Each blade connects the demifacet leaf.The sampling of inoculation different time sections, liquid nitrogen grinding is extracted RNA.The extraction of RNA is by Shanghai China Shun biotechnology RNAex Reagent ﹠amp of company limited; The method of systems test kit is carried out. and electrophoresis, to change film stand-by.Because the full length cDNA sequence of ASR1 and the gene DFL1 homology of another GH3 family are very high, especially the probe of the higher 3 '-UTR zone of specificity as Northern hybridization, selected in the CDS zone therefore, and its PCR primer is as follows:

ASRf：5’-TAATCAGTATAAGACGCCGAGATGC-3’(SEQ?ID?NO：4)

ASRr：5’-TCGAGAAAGAGTGATGAGAGTTGGTT-3’(SEQ?ID?NO：5)

Amplification condition is 94 ℃ of 3min; 94 ℃ then, 30s, 56 ℃, 30s, 72 ℃, 2min circulation 34 are taken turns; Then 72 ℃, 10min.Genomic dna with Col-0 is a template, goes out the specific band of 353bp with top primer amplification, and this band is reclaimed, and reclaims the test kit purifying with gel.Label probe carries out according to the method in the Random Primer DNA Labeling test kit of TaKaRa company.Radio isotope be (α- ³²P) dCTP, isotopic usage quantity is 2.5 μ L in the reaction system of 25 μ L.

Crossover process is as follows:

(1) will shift good nylon membrane and put into hybrid pipe, add hybridization solution 20mL, 55 ℃ of prehybridization 1h.The composition of hybridization solution is 0.25M phosphoric acid buffer, 1%BSA, 1mM EDTA and 7%SDS.

(2) isotopic labeling is good probe places cooled on ice 3min rapidly then in 100 ℃ of heating 3min, adds in the hybrid pipe 55 ℃ of hybridization 16～24h at last.

(3) after hybridization finishes, take out nylon membrane, 55 ℃ are embathed twice in the solution of 2 * SSC, 0.1%SDS, each 10min.

(4) nylon membrane is wrapped with preservative film, placed the compressing tablet box, in the darkroom, put into X-ray film, place-76 ℃ to spend the night.

(5) in the darkroom, X-ray film is taken out, in developing solution and stop bath, develop a film successively, observe and the scanning results of hybridization.

As shown in Figure 2, when being subjected to pathogen infection, ASR1 is subjected to obviously inducing, and expresses to strengthen, and the pathogenic bacterium inducing ASR1 genetic expression that wherein contains nontoxic gene avrRpm1 is early than its virulent bacteria.

Embodiment 6: the Whitfield's ointment 12h that executes 0.5mM outward can obviously induce the ASR1 expression of gene

Handling vegetative period with Whitfield's ointment (SA) solution spray of 0.5mM is the Arabidopis thaliana wild-type (Col-0) of 30d, handles sampling in back 12 hours, gets untreated 0h simultaneously for contrast, extracts total RNA.Reverse transcription, it is as follows to be with reverse transcription cDNA that template is carried out the PCR.PCR primer:

ASRf：5’-TAATCAGTATAAGACGCCGAGATGC-3’(SEQ?ID?NO：4)

ASRr：5’-TCGAGAAAGAGTGATGAGAGTTGGTT-3’(SEQ?ID?NO：5)

Amplification condition is 94 ℃ of 3min; 94 ℃ then, 30s, 56 ℃, 30s, 72 ℃, 2min circulation 32 are taken turns; Then 72 ℃, 10min.Electrophoresis is taken pictures.

Fig. 3 is presented in the wild-type Arabidopis thaliana, and the Whitfield's ointment 12h that executes 0.5mM outward can obviously induce the ASR1 expression of gene.

All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims institute restricted portion equally.

Sequence table

＜110〉Shanghai Inst. of Life Science, CAS

＜120〉phytopathogen is induced sequence and the application with tissue specificity expression promoter

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Claims (7)

1. a promoter element is characterized in that, the nucleotide sequence of this promoter element is shown in SEQ ID NO:1.

2. a construction specific expressed in the plant meristematic tissue is characterized in that, the described promoter element of claim 1 that it contains foreign gene and is operably connected with this foreign gene.

3. construction as claimed in claim 2 is characterized in that described foreign gene is a myb gene.

4. a carrier is characterized in that, it contains the described promoter element of claim 1.

5. the method for a specific expressed foreign gene in the plant meristematic tissue is characterized in that it comprises step:

(a) provide a construction, the described promoter element of claim 1 that described construction contains foreign gene and is operably connected with this foreign gene;

(b) construction in the step (a) is imported vegetable cell, obtain the plant transformed cell;

(c) with the vegetable cell regeneration plant that transforms in the step (b), thus in the meristematic tissue of this plant specific expressed this foreign gene.

6. the purposes of promoter element as claimed in claim 1 is characterized in that, is used at the specific expressed foreign gene of plant meristematic tissue.

7. a host cell is characterized in that, it contains the described carrier of claim 4.

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