CN102712929B - Identification and use of plant root-specific expression promoter - Google Patents
Identification and use of plant root-specific expression promoter Download PDFInfo
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- CN102712929B CN102712929B CN201180005657.1A CN201180005657A CN102712929B CN 102712929 B CN102712929 B CN 102712929B CN 201180005657 A CN201180005657 A CN 201180005657A CN 102712929 B CN102712929 B CN 102712929B
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
- C12N15/8227—Root-specific
Abstract
An isolated DNA, which has a sequence as shown in SEQ ID NO: 1 or a similar sequence thereof, can direct the nucleic acid which is operably linked to the downstream thereof to be transcribed and/or expressed specifically in plant roots. Also provided is an expression cassette and a transgenic plant comprising the DNA.
Description
Technical field
The invention belongs to plant transgene breeding field, particularly, the present invention relates to the DNA separated, it can instruct the nucleic acid that is operatively coupled on its downstream to transcribe and/or express plant root is special.In addition, the invention still further relates to the expression cassette that comprises this DNA and plant etc., and relate to the application of this DNA.
Background technology
Although many plants (as, paddy rice, especially
oryza sativa.japonicadeng) genome sequence revealed (as can be referring to GenBank accession number AP003843.3, GenBank accession number AP004670.4 etc.), but the function of lots of genes group sequence is still unknown.In addition, U.S. Patent application US2006075523A1 discloses polypeptide and the polynucleotide relevant from the inanimate stress response of paddy rice and other plant, wherein polynucleotide sequence SEQ ID NO:15442 reaches 2000 Nucleotide, and this sequence is submerged in the disclosed numerous sequences of the document and not designated specific function; And U.S. Pat 7365185B2 discloses the genomic promoter sequence from paddy rice and other plant, but wherein polynucleotide sequence SEQ ID NO:68961 reaches 2760 Nucleotide, and this sequence is submerged in the disclosed numerous sequences of the document and not designated specific function, more do not know whether it has the specificity that plant materials is expressed position.
Thereby exogenous DNA array is enabled in the expression in plant host by being connected to specific promotor, the selection of promotor type determines expression time and the position of gene.In the agricultural biological technical field widespread use, be at present mainly the strong promoter of some composing types, such as CaMV 35S promoter and corn Ubiquitin-1 promotor, yet when utilizing these promotors to induce the crops such as goal gene rice transformation to the quality of Crop Improvement, tending to time (etap specificity) due to destination gene expression or space (tissue and organ specificity) can not control well and cause improved effect not obvious, perhaps because these constitutive promoter inducible gene expression amounts are too high, growing of plant impacted, these are all the obstacles run into while utilizing at present composing type strong promoter combined function gene to carry out the Crop Improvement quality.
In addition, when some metabolic process of research or the approach of adjusting, usually need the plural gene transformation on same approach in same strain, adopt one of them gene of conversion to obtain transforming again the another one gene after transfer-gen plant, perhaps two genes are hybridized after having transformed respectively again, all need to wait for the longer time, in order to raise the efficiency the time that shortens a plurality of gene transformation, there is recently report can utilize new carrier to carry out the conversion of a plurality of genes simultaneously, if but reuse same promotor when polygene transforms, also can may cause gene silencing due to the high homology of promoter sequence.
Root is the vitals that plant materials absorbs moisture and nutritive substance, and the height that the different expression system of Gent can be used for studying plant oozes stress-tolerance, phytoremediation and rhizosphere secretion etc.Although the documents such as Chinese patent application CN1196753A, CN1791677A, CN101389762A and CN101589144A disclose a series of root-specific promoters, these promoter sequence configurations, can't expect and obtain new root-specific promoter.
For this reason, the inventor is through studying for a long period of time, found surprisingly a kind of new root-specific promoter, it is not only shorter with respect to disclosed sequences of document such as U.S. Pat 7365185B2 and confirmed to have the function of root-specific promoter, and each etap in dicotyledonous and monocotyledons can keep this function, greatly widened the scope of its use.
Summary of the invention
summary of the invention
The invention provides the DNA of new separation, it has the function of root-specific promoter.In addition, the invention still further relates to the expression cassette that comprises this DNA and transgenic plant etc., and relate to application of this DNA etc.
Particularly, in first aspect, the invention provides the DNA of separation, it can instruct the nucleic acid that is operatively coupled on its downstream to transcribe and/or express plant root is special, and the sequence of the DNA of described separation is selected from the sequence of following group:
(a) there is the sequence shown in SEQ ID NO:1;
(b) DNA sequence dna that can hybridize with the DNA of (a) described sequence under stringent condition;
(c) with (a) described sequence, have at least 90%(to be preferably at least 95%) DNA sequence dna of sequence identity;
(d) DNA sequence dna that comprises at least 150 (being preferably at least 200) continuous nucleotides in SEQ ID NO:1; With
(e) with the DNA sequence dna of arbitrary described sequence complementation (a)-(d).
The DNA of preferred first aspect present invention, its sequence length is less than 2000 Nucleotide, preferably is less than 1500 Nucleotide, is more preferably less than 1200 Nucleotide.
The DNA of preferred first aspect present invention, it can instruct, and the nucleic acid that is operatively coupled on its downstream is special in the plant root pericycle transcribes and/or expresses.
In a specific embodiment of the present invention, the DNA sequence dna of first aspect present invention is as shown in SEQ ID NO:1.
In second aspect, the invention provides expression cassette, it comprises:
(a) DNA of first aspect present invention; With
(b) nucleic acid, it is operatively coupled on the downstream of the DNA of first aspect present invention.
The expression cassette of preferred second aspect present invention, wherein said nucleic acid can be given plant and be selected from the proterties of following group:
(a) raising of the coded protein output of described nucleic acid;
(b) raising of the coded inhibitory RNA content of described nucleic acid;
(c) raising of resistance (comprising drought resistance, winter hardiness, high thermal resistance and salt tolerance);
(d) raising of anti-plant pest ability, the especially anti-raising that occurs in plant root disease and pest ability;
(e) improvement of nutritional quality, comprise giving of the raising of the natural nutrient composition content had in plant and nutritive ingredient that the plant non-natural has; With
(f) raising of plant biomass, the especially raising of plant economy position output.
In the third aspect, the invention provides genetically modified plant, plant seed, plant tissue or vegetable cell with the expression cassette conversion of second aspect present invention, preferably wherein, the DNA of first aspect present invention can instruct the nucleic acid that is operatively coupled on its downstream to transcribe and/or express plant root is special.
Plant, plant seed, plant tissue or the vegetable cell of preferred third aspect present invention, wherein said plant is monocotyledons or dicotyledons, as, corn, paddy rice, wheat, barley, Chinese sorghum, soybean, rape, cotton, tomato, potato, sugarcane, beet, tobacco or Arabidopis thaliana, preferably paddy rice.
In fourth aspect, the invention provides the production method of the plant of third aspect present invention, it comprises:
(1) build the expression cassette of second aspect present invention;
(2) expression cassette step (1) obtained imports vegetable cell;
(3) transgenic plant that regenerate; With
(4) select transgenic plant, wherein the DNA of first aspect present invention can instruct the nucleic acid that is operatively coupled on its downstream to transcribe and/or express plant root is special; And
(5) plant that optionally, propagation step (4) obtains is to obtain the offspring.
Aspect the 5th, the present invention also provides the method for giving plant trait, and it comprises
(1) select to give the nucleic acid of plant trait:
(2) expression cassette of the nucleic acid construct second aspect present invention of using step (1) to obtain;
(3) expression cassette step (2) obtained imports vegetable cell;
(4) transgenic plant that regenerate; With
(5) select the transgenic plant of having given plant trait; And
(6) optionally, breed the plant of step (5) acquisition to obtain the offspring,
Wherein, described proterties is selected from following group:
(a) raising of the coded protein output of described nucleic acid;
(b) raising of the coded inhibitory RNA content of described nucleic acid;
(c) raising of resistance (comprising drought resistance, winter hardiness, high thermal resistance and salt tolerance);
(d) raising of anti-plant pest ability, the especially anti-raising that occurs in plant root disease and pest ability;
(e) improvement of nutritional quality, comprise giving of the raising of the natural nutrient composition content had in plant and nutritive ingredient that the plant non-natural has; With
(f) raising of plant biomass, the especially raising of plant economy position output.
The accompanying drawing explanation
Fig. 1 is the T-DNA district collection of illustrative plates of expression vector pHPG.LB and RB are respectively left margin and the right margin of T-DNA; Hpt means hygromycin gene; Pnos means the promotor of no gene; Tnos means the terminator of no gene; GUS means gus albumen (beta-Glucuronidase (β-glucuronidase)) gene; T35s means the terminator of 35s gene; HindIII and BamHI mean respectively the restriction enzyme site of restriction enzyme HindIII and BamHI; Root-specific promoter is the root specific expression promoter of separation of the present invention.
Fig. 2 is the tissue of KT630P transgenic paddy rice, the GUS dyeing of organ.Wherein, A is rice seedling phase plant body; B is the transgenosis resistant calli; C is seedling leaf; The stem that D is seedling stage; The root that E is seedling stage; F is rice flower organ.
Fig. 3 is the RT-PCR detected result of the expression of gus gene in each histoorgan of transgenic paddy rice of KT630P promoters driven of the present invention.
Fig. 4 is the GUS dyeing of KT630P transgenic arabidopsis plant.Wherein, A is Arabidopsis thaliana Seedlings phase plant body; B is plant body before the Arabidopis thaliana bolting; C is Arabidopis thaliana plant in flowering period body; The root GUS dyeing situation that D is body formula sem observation.
detailed Description Of The Invention
In first aspect, the invention provides the DNA of separation, it can instruct the nucleic acid that is operatively coupled on its downstream to transcribe and/or express plant root is special, and especially it can instruct that the nucleic acid that is operatively coupled on its downstream is special in the plant root pericycle transcribes and/or express.
In this article, the DNA of this separation also can be called as root-specific promoter, belongs to cis-acting elements.Under the driving of root-specific promoter, the expression of its downstream the gene that is operably connected with it often is only limited to this organ of root or its tissue site, and the characteristics such as adjusting are grown in performance.Root-specific promoter can not only make the expression product of goal gene mainly in plant root or the accumulation of its tissue site, the amount of the nucleic acid mRNA in the downstream for example produced in root or its tissue is 5 times, 10 times, 100 times, 200 times, even 1000 times of other plant body region, increase the Zonal expression amount, also can avoid the unnecessary waste of plant nutrition simultaneously.In the specific embodiment of the present invention, directly differentiating root-specific promoter of the present invention by the observable staining of naked eyes instructs and transcribes and/or express at plant root, and these regulation and control continue the stages at development of plants, and, at other positions of plant, what there is no that naked eyes can obviously differentiate transcribing and/or expressing.
In this article, term " is operably connected " and refers to a kind of mode of connection, and this mode makes transcribing and/or express by root-specific promoter of the present invention and opening and begin and regulate and control of the nucleic acid that is connected to downstream.Usually, the nucleic acid be operably connected is continuous.The nucleic acid in downstream in the situation that keep the frame can a small amount of Nucleotide in interval and be connected to the downstream of root-specific promoter, be accepted its regulation and control.
In this article, term " separation " DNA refer to the DNA environment natural from it (as, the genome of vegetable cell) in, be separated, or by chemosynthesis or out recombinant expressed, and DNA does not contain or substantially contains other nucleic acid, cell and the substratum of its source species.The DNA of separation of the present invention has shorter nucleotide sequence, and preferred sequence length is less than 2000 Nucleotide, preferably is less than 1500 Nucleotide, is more preferably less than 1200 Nucleotide.In the specific embodiment of the present invention, the DNA of separation of the present invention has the sequence shown in the SEQ ID NO:1 of 1000 Nucleotide.
The DNA of separation of the present invention also comprises the function equivalence body of the DNA with the sequence shown in SEQ ID NO:1, the DNA that has the variant sequence of SEQ ID NO:1, it still can instruct the nucleic acid that is operatively coupled on its downstream in the sequence shown in the special SEQ ID NO:1 that transcribes and/or express of plant root.The variant sequence is included in the DNA sequence dna that can hybridize with the DNA with the sequence shown in SEQ ID NO:1 under stringent condition." stringent condition " used herein is known, comprise such as containing 400mM NaCl, 40mM PIPES(pH6.4) and the hybridization solution of 1mM EDTA in 60 ℃ of hybridization 12-16 hour, then under 65 ℃, with the washings containing 0.1SDS and 0.1%SSC, wash 15-60 minute.
The variant sequence also comprises the DNA sequence dna that at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity is arranged with sequence shown in SEQ ID NO:1.Wherein, the per-cent of sequence identity can obtain by known information biology algorithm, comprise Myers and Miller algorithm (Bioinformatics, 4 (1): 11-17, 1988), Needleman-Wunsch overall comparison method (J.Mol.Biol., 48 (3): 443-53, 1970), Smith-Waterman Local Alignment method (J.Mol.Biol., 147:195-197, 1981), Pearson and Lipman similarity searching method (PNAS, 85 (8): 2444-2448, 1988), algorithm (the Altschul etc. of Karlin and Altschul, J.Mol.Biol., 215 (3): 403-410, 1990, PNAS, 90:5873-5877,1993).This is familiar with to those skilled in the art.
The variant sequence also comprises the fragment of the sequence shown in the SEQ ID NO:1 that can keep the root-specific promoter function, for example in SEQ ID NO:1 at least 150,200,300,500,800 until the DNA sequence dna of 999 continuous nucleotides.
In second aspect, the invention provides expression cassette, it comprises:
(a) DNA of first aspect present invention; With
(b) nucleic acid, it is operatively coupled on the downstream of the DNA of first aspect present invention.
The DNA that expression cassette of the present invention comprises first aspect present invention on 5 '-3 ' transcriptional orientation, be operatively coupled on first aspect present invention DNA downstream nucleic acid, the optional terminator of transcribing and translating (as, Transcription Termination element or polyadenylation signal).Expression cassette of the present invention can also comprise, copies required replication orgin (for example, from pBR322 or P15A ori ORI district) in bacterium, and edaphic bacillus T-DNA shifts needed element (for example, the left margin of T-DNA and/or right margin).Other compositions that expression cassette of the present invention can comprise comprise, enhanser, intron, multiple clone site, operator gene, repressor binding site, transcription factor binding site point etc.Exemplary enhanser comprises from the stunt enhancer element of promotor of I gene, TMV Ω element and yeast of CaMV 35S promoter, octopine synthase gene, rice Actin muscle I gene, corn alcohol dehydrogenase gene, corn.Also can adopt the virus leader sequence to be used as having the foresight of enhanser effectiveness, as leader sequence from tobacco mosaic virus (TMV) (TMV), corn chlorisis mottle virus (MCMV) and alfalfa mosaic virus (AMV) etc.Exemplary plant introne comprises from the intron of Adh 1, bronze 1, Actin muscle 1, Actin muscle 2 and sucrose synthase intron.
In this article, be operatively coupled on polynucleotide, antisense sequences, the polynucleotide of coding dsRNA sequence of polynucleotide, encoding fusion protein of the different first albumen of nucleic acid encoding in downstream of the DNA of first aspect present invention, etc.Preferably can give the nucleic acid of the useful proterties of plant (especially plant root), described proterties comprises:
(a) raising of the coded protein output of described nucleic acid;
(b) raising of the coded inhibitory RNA content of described nucleic acid;
(c) raising of resistance (comprising drought resistance, winter hardiness, high thermal resistance and salt tolerance);
(d) raising of anti-plant pest ability, the especially anti-raising that occurs in plant root disease and pest ability;
(e) improvement of nutritional quality, comprise giving of the raising of the natural nutrient composition content had in plant and nutritive ingredient that the plant non-natural has; With
(f) raising of plant biomass, the especially raising of plant economy position output.
Correspondingly, be operatively coupled on the gene that the nucleic acid in downstream of the DNA of first aspect present invention can proteins encoded, as the insect-resistant gene, the bacterial disease resistant gene, the fungal disease resistant gene, the virus disease resistant gene, nematode disease resistance gene, herbicide resistance gene, affect the gene of grain composition or quality, the nutrien utilization gene, mycotoxins reduces gene, male sterility gene, the selectable marker gene, but selection markers thing gene, negative selectable marker gene, positive selectable marker gene, affect the gene of plant agronomy feature (as productive rate), the tolerance to environmental stress gene (for example, give arid, heat, cold, freezing, cross wet, the resistance of salt stress or oxidative stress or the gene of patience), improve starch property or quantity, oil product quantity or quality, the gene that amino acid or protein form, etc..In addition, the nucleic acid in downstream that is operatively coupled on the DNA of first aspect present invention also can encode dsRNA, sense-rna, siRNA, miRNA, itself and disadvantageous polynucleotide (as, the plant promoter of need to lower expressing, the biological survival of phytoparasite body, grow or breed necessary plant gene) complementary and cause the downward of these polynucleotide.
Expression cassette of the present invention can be inserted into plasmid, clay, yeast artificial chromosome, bacterial artificial chromosome or other are applicable to being transformed in any carrier in host cell.Preferred host cell is bacterial cell, in particular for the clone or store polynucleotide or for example, for the bacterial cell of transformed plant cells, intestinal bacteria, Agrobacterium tumefaciems and Agrobacterium rhizogenes.When host cell is vegetable cell, expression cassette or carrier can be inserted in the genome of the vegetable cell be converted.Insertion can be location or random insertion.Preferably, be inserted through such as homologous recombination and realize.In addition, expression cassette or carrier can remain on outside karyomit(e).Expression cassette of the present invention or carrier can be present in core, chloroplast(id), plastosome and/or the plastid of vegetable cell.Preferably, expression cassette of the present invention or carrier are inserted in the chromosomal DNA of plant nucleolus.
In the third aspect, the invention provides genetically modified plant, plant seed, plant tissue or vegetable cell with the expression cassette conversion of second aspect present invention.
The specific embodiment of the present invention the effective application attestation by two representational monocotyledonss and dicotyledons the plant use range of root-specific promoter of the present invention, therefore root-specific promoter of the present invention can be used for transforming any plant species, comprise monocotyledons and dicotyledons, for example, from the plant with the subordinate: Medicago, tomato belongs to, Btassica, Cucumis, Solanum, Juglans, Gossypium, Malus, Vitis, antirrhinum, Populus, Fragaria, Arabidopsis, Picea, Capsicum, Chenopodium, Chrysanthemum, ipomoea, Pinus, Pisum, Oryza, Zea, Triticum, triticale belongs to, Secale, lolium, Hordeum, Glycine, Pseudotsuga, Bryophyllum, Beta, Helianthus, Nicotiana, Cucurbita, rose, Fragaria, Lotus, donkey food grass belongs to, Clover, Trigonella, Vigna, tangerine belongs to, linum, Geranium, cassava, Trigonella, Rhaphanus, sinapsis alba belongs to, Atropa, Datura, poison tobacco, Nicotiana, green winter Solanum, Digitalis, Cichorium, Lactuca, Brome, Asparagus, antirrhinum, hemerocallis, Narcissus, Pelargonium, millet belongs to, Pennisetum, Ranunculus, Senecio, the loudspeaker tongue belongs to, blue English Pittosporum, Phaseolus, Avena and allium, preferred plant comprises corn, paddy rice, wheat, barley, Chinese sorghum, soybean, rape, cotton, tomato, potato, sugarcane, beet, tobacco or Arabidopis thaliana, be more preferably paddy rice.
In fourth aspect, the invention provides the production method of the plant of third aspect present invention, it comprises:
(1) build the expression cassette of second aspect present invention;
(2) expression cassette step (1) obtained imports vegetable cell;
(3) transgenic plant that regenerate; With
(4) select transgenic plant, wherein the DNA of first aspect present invention can instruct the nucleic acid that is operatively coupled on its downstream to transcribe and/or express plant root is special; And
(5) plant that optionally, propagation step (4) obtains is to obtain the offspring.
Transgenic plant of the present invention are used plant biotechnology field method for transformation preparation known to the skilled.Any method can be used to recombinant expression vector is transformed in vegetable cell, to produce transgenic plant of the present invention.Method for transformation can comprise directly and method for transformation indirectly.Suitable direct method comprises that the DNA that polyoxyethylene glycol induces takes in, liposome-mediated conversion, use particle gun importing, electroporation and microinjection, etc.In the specific embodiment of the present invention, the present invention has used the transformation technology based on edaphic bacillus (can be referring to (1985) Science 225:1229 such as Horsch RB; White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, the 1st volume, Engineering and Utilization, Academic Press, 1993, pp.15-38; The .Techniques for Gene Transfer such as Jenes B, Transgenic Plants, the 1st volume, Engineering and Utilization, Academic Press, 1993, pp.128-143, etc.).Edaphic bacillus bacterial strain (for example Agrobacterium tumefaciems or Agrobacterium rhizogenes) comprises plasmid (Ti or Ri plasmid) and T-DNA element, described plasmid and element are with after the edaphic bacillus transfection, being transferred to plant, and T-DNA is integrated in the genome of vegetable cell.T-DNA can be positioned on Ri-plasmid or Ti-plasmid, or is included in independently in so-called binary vector.During agrobacterium-mediated method for transformation for example is described in.The agrobacterium-mediated the most applicable dicotyledons of conversion, but also be applicable to monocotyledons.During edaphic bacillus for example is described in to the conversion of plant.Conversion can cause instantaneous or stable conversion and expression.Although nucleotide sequence of the present invention can be inserted into any plant and the vegetable cell that falls into these broad variety, it is particularly useful for the crop plants cell.
Aspect the 5th, the present invention also provides the method for giving plant trait, and it comprises
(1) select to give the nucleic acid of plant trait:
(2) expression cassette of the nucleic acid construct second aspect present invention of using step (1) to obtain;
(3) expression cassette step (2) obtained imports vegetable cell;
(4) transgenic plant that regenerate; With
(5) select the transgenic plant of having given plant trait; And
(6) optionally, breed the plant of step (5) acquisition to obtain the offspring,
Wherein, described proterties is selected from following group:
(a) raising of the coded protein output of described nucleic acid;
(b) raising of the coded inhibitory RNA content of described nucleic acid;
(c) raising of resistance (comprising drought resistance, winter hardiness, high thermal resistance and salt tolerance);
(d) raising of anti-plant pest ability, the especially anti-raising that occurs in plant root disease and pest ability;
(e) improvement of nutritional quality, comprise giving of the raising of the natural nutrient composition content had in plant and nutritive ingredient that the plant non-natural has; With
(f) raising of plant biomass, the especially raising of plant economy position output.
In this article, " selection " can be that field or greenhouse are selected, and can be also to use the heredity of genetic marker to select.In this article, " propagation " can sexual propagation, can be also vegetative propagation.In sexual propagation, obviously can carry out extra hybridization step, and select the offspring who has inherited the transgenosis proterties.
The present invention has quoted open source literature, and these documents are in order more clearly to describe the present invention, and their full text content is all included this paper in and carried out reference, just looks like that repeated description is excessively the same in this article for their full text.
For the ease of understanding, below will to the present invention, be described in detail by specific embodiment.It needs to be noted, these descriptions are only exemplary descriptions, do not form limitation of the scope of the invention.According to the discussion of this specification sheets, many variations of the present invention, change are all apparent concerning one of ordinary skill in the art.
Embodiment
In following embodiment, method therefor is ordinary method if no special instructions, the primer is synthetic by Shanghai Ying Jun biotech company, order-checking is completed by Beijing Hua Da gene, endonuclease in PCR test kit, vector construction process is purchased from precious biotechnology company limited, pEASY-T1 connects test kit purchased from Beijing Quan Shijin biotech company, the T4 DNA ligase is purchased from Promega company, and the method that the equal reference reagent box of method provider is recommended is carried out.In experiment, carrier pCAMBIA1303 used can be purchased from CAMBIA company.
separation and the evaluation of embodiment 1. promotor KT630P
Design the required primer of following cloning promoter KT630P:
Primer 1:5'-CCCaagctt
gCTATATGTGTACGTGATAGTATAT-3'
Primer 2: 5'-CGggatcc
tTAATTTGCTCTTGTATTAGCTCTA-3'
The restriction enzyme site that in primer 1, sequence aagctt is HindIII, the restriction enzyme site that in primer 2, sequence ggatcc is BamHI.
Utilize forward and reverse primer (wherein the sequence with the underscore part is promoter sequence) of promotor, the plant genome DNA of usining extracts the paddy rice that test kit (TIANGEN Biotech (Beijing) Co., Ltd.) extracts (in spend 11) genomic dna as template, increased, reaction conditions is: 95 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds; Anneal 30 seconds for 55 ℃; 72 ℃ are extended 1 minute; 30 circulations; 72 ℃ are extended 10 minutes.After reaction finishes, the PCR product detects and reclaims through 1% agarose gel electrophoresis, and product is connected into the pEASY-T1 carrier, and screening positive clone also carries out sequence verification.Result shows, the KT630P promoter sequence that institute's extension increasing sequence is our expection, and its sequence is as shown in SEQ ID NO:1.
the structure of embodiment 2. expression vectors
Sequence verification has been inserted to HindIII and the BamHI double digestion for the pEASY-T1 plasmid of KT630P promoter sequence, be connected in the same carrier pHPG with HindIII and BamHI double digestion, picking colony carries out the PCR detection, choosing the positive bacterium colony of PCR result is checked order, after sequence verification is correct, extract corresponding positive colony plasmid, called after pHPG-KT630P.Wherein, it is primer on the pHPG carrier that bacterium colony PCR detects required primer, is positioned at cloned promoter fragment both sides, and amplified fragments is about the length of promotor, take bacterium liquid as template, augmentation detection, and the PCR reaction conditions is: 95 ℃ of denaturations 5 minutes; 94 ℃ of sex change 30 seconds; Anneal 30 seconds for 55 ℃; 72 ℃ are extended 1 minute; 34 circulations; 72 ℃ are extended 10 minutes.
As shown in Figure 1, wherein: LB and RB are respectively left margin and the right margin of T-DNA to the collection of illustrative plates in the T-DNA district of constructed expression vector; Hpt means hygromycin gene; Pnos means the promotor of no gene; Tnos means the terminator of no gene; GUS means gus albumen (beta-Glucuronidase (β-glucuronidase)) gene; T35S means the terminator of 35S gene; HindIII and BamHI mean respectively the restriction enzyme site of inscribe HindIII and BamHI; Root-specific promoter is the KT630P promotor that embodiment 1 obtains.
embodiment 3. Plant Transformation and Function Identification
Utilize the heat shock method that plasmid pHPG-KT630P is proceeded to Agrobacterium AGL0 bacterial strain, utilize agrobacterium-mediated transformation to carry out cotransformation to paddy rice, utilize Agrobacterium inflorescence infestation method arabidopsis thaliana transformation plant simultaneously.Separate each histoorgan from transfer-gen plant, carry out the active detection of GUS, each histoorgan is placed in to the centrifuge tube that contains GUS dyeing damping fluid, be put in 37 ℃ of incubators and be incubated overnight, then under room temperature condition, in dehydrated alcohol, decolour and preserve.
3.1 the histoorgan of transgenic paddy rice seedling dyeing
The Rice Callus with hygromycin resistance that conversion KT630P promotor is obtained, carry out the GUS Coloration experiment, result, as shown in Fig. 2 B, does not demonstrate the blueness that naked eyes can observe in resistant calli, show substantially not have the expression of gus gene in this tissue.
To transform the histoorgan of the transgenic paddy rice of KT630P promotor, blade, stem, root and flower carry out respectively GUS dyeing.Result is respectively as shown in Fig. 2 C, Fig. 2 D, Fig. 2 E and Fig. 2 F, and gus gene only has very strong expression in the root system of transgenic paddy rice seedling, shows the very strong blueness that naked eyes can obviously observe; And, in other each organ (blade, stem and flower), the expression of gus gene substantially do not detected.In addition, take whole transgenic paddy rice seedling and carry out GUS dyeing, result as shown in Figure 2 A, can be found by obvious colour contrast, only have root to demonstrate the very strong blueness that naked eyes can obviously observe, and other organ sites all do not have the discernible obvious blue of naked eyes.This shows, promotor of the present invention can instruct the gus protein in its downstream to express in the transfer-gen plant root, and this expression has the specificity of root; And, no matter at seedling stage or florescence, a plurality of etap all show the specificity that this expression has root.
3.2 the RT-PCR expression analysis of transgenic paddy rice
Get root, stem, leaf and the floral organ of KT630P transgenic rice plant pure lines, extract RNA, reverse transcription be cDNA as template, using paddy rice ACTIN gene as internal reference, analyze the expression of gus reporter gene in transgenic paddy rice of KT630P promoters driven.
The detection primer of RT-PCR is:
Primer 3:5'-TAATGTTCTGCGACGCTCAC-3'
Primer 4:5'-CGGCGAAATTCCATACCTG-3'
Primer 5:5'-TGTTCCTGCCATGTATGT-3'
Primer 6:5'-ATGTCCCTCACAATTTCC-3'
Wherein, primer 3 and primer 4 are
gUSthe detection primer of gene, its amplified fragments size is 321bp; Primer 5 and primer 6 are paddy rice reference genes
aCTINthe analysis primer, its amplified fragments size is 252bp.PCR detection system and program are:
10×buffer 2μL
10mM dNTP 0.4μL
10 mM primer 3(check analysiss are used 10 mM primers 5) 0.4 μ L
10 mM primer 4(check analysiss are used 10 mM primers 6) 0.4 μ L
Taq polymerase 0.4μL
cDNA 1μL
ddH2O 15.4μL
The PCR reaction conditions: 95 ℃, denaturation 5 minutes; 94 ℃, sex change 30 seconds; 55 ℃, anneal 30 seconds; 72 ℃, extend 25 seconds; 28 circulations, 72 ℃, 10 minutes.
After reaction finishes, the PCR product is carried out to 1.5% agarose gel electrophoresis detection.As shown in Figure 3, in check analysis, the expression of all ACTIN internal references all has been detected the PCR detected result in the organs such as root, stem, leaf and flower, there is no notable difference; And, for the expression of gus gene, in the organs such as root, stem, leaf and flower, only in root tissue, detected
gUSthe expression of gene.This shows again, and promotor of the present invention can instruct the heterologous protein expression in its downstream in the transfer-gen plant root, and this expression has the specificity of root; And, no matter at seedling stage or florescence, a plurality of etap all show the specificity that this expression has root.
3.3 the histoorgan of transgenic arabidopsis dyeing
By the positive seedling of KT630P promotor arabidopsis thaliana transformation gained and the plant body of each etap thereof, whole is carried out GUS dyeing.Result is as shown in Fig. 4 A, Fig. 4 B and Fig. 4 C, and in each etap of transgenic arabidopsis, reporter gene GUS only shows very strong expression at plant root, and the dye levels in other histoorgan is all in the invisible level of naked eyes.This shows again, and for various plants, even the allos plant, promotor of the present invention also can instruct the heterologous protein expression in its downstream in the transfer-gen plant root of each etap, and this expression has the specificity of root.
By the root system dyeing situation of body formula sem observation KT630P transgenic arabidopsis, find result as shown in Figure 4 D, gus gene is only expressed in the pericycle of root system.This shows, the expression that promotor of the present invention instructs has root pericycle expression specificity.
SEQUENCE LISTING
<110 > Beijing Weiming Kaituo Crops Design Center Ltd
<120 > evaluation of roots of plants specific expression promoter and application
<130>
<150> CN201010547550.7
<151> 2010-11-16
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 1001
<212> DNA
<213 > paddy rice (Oryza sativa L.ssp.japonica)
<400> 1
gctatatgtg tacgtgatag tatatttaac aatgaatcaa atgatatgaa aataataaat 60
aattacttaa atattttgaa taagacgaat ggtcaaacac gtactaaaaa gtcaacggtg 120
tcaaacattt tgaaacggaa ggagtatatt cttttaactt tgtaatagtt atatataatt 180
gttcgtacct cgagtagtta tcataaaact attcaactat tcagaaaaaa aagagtacat 240
cttacgggag aaggccagcc aaaattagta catctcatgg tggatgccaa caatcaacaa 300
aacctaccaa tccacacatt attacacctg aaaacgatct ctagctctga ttaatttcaa 360
acttcgatta tggactagtc aacacttcct aatagcagtg aactttgatt tggccatatg 420
aatactccca actttcatac cgaaaatttt atcttcaaac tatgaattct aaggatattc 480
ccttttaggc cgatcactag ttcccctttt tatttgcaga gaggaaacat gcaaatttga 540
tatagaaata tacaaggggg aagcatacat atgatgccta tctgccatcc caggtacatg 600
cctatctgcc aaaacaatca actaacctac caagtaccaa tcctcccata attgaacagc 660
tggaccagga aaaatcatca tctgtgtaca tcttaattga ccatattcat gcaagctctg 720
atcaagttgt gcaatgtcag tattatatta ctaagttagt agctagttag ttaaatatca 780
gtctaataaa tgtctaatca gagctgttag taggaaagag taggtcacca atctctcaaa 840
gacttataca agctagattc catcaccaca ctatataaac acacacacac ctgaacacca 900
gtcacacaac caaatcaagc tcagcttaat caatcacctc atcacacact cttagctaag 960
ctaagctaag ctaagctaga gctaatacaa gagcaaatta a 1001
<210> 2
<211> 20
<212> DNA
<213 > synthetic primer
<400> 2
taatgttctg cgacgctcac 20
<210> 3
<211> 19
<212> DNA
<213 > synthetic primer
<400> 3
cggcgaaatt ccatacctg 19
<210> 4
<211> 18
<212> DNA
<213 > synthetic primer
<400> 4
tgttcctgcc atgtatgt 18
<210> 5
<211> 18
<212> DNA
<213 > synthetic primer
<400> 5
atgtccctca caatttcc 18
Claims (3)
1. the DNA separated, it can instruct the nucleic acid that is operatively coupled on its downstream to transcribe and/or express plant root is special, and the sequence of the DNA of described separation is the sequence shown in SEQ ID NO:1.
2. expression cassette, it comprises:
(a) DNA claimed in claim 1; With
(b) nucleic acid, it is operatively coupled on the downstream of DNA claimed in claim 1.
3. the production method of the transgenic paddy rice transformed with expression cassette claimed in claim 2, it comprises:
(1) build expression cassette claimed in claim 2;
(2) expression cassette Introduced into Rice cell step (1) obtained;
(3) transgenic paddy rice of regenerating; With
(4) select transgenic paddy rice, wherein DNA claimed in claim 1 can instruct the nucleic acid that is operatively coupled on its downstream to transcribe and/or express plant root is special; And
(5) paddy rice that optionally, propagation step (4) obtains is to obtain the offspring.
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| CN2010105475507A CN102465114A (en) | 2010-11-16 | 2010-11-16 | Identification and application of specific expression promoter of plant root |
| CN201010547550.7 | 2010-11-16 | ||
| PCT/CN2011/082126 WO2012065528A1 (en) | 2010-11-16 | 2011-11-14 | Identification and use of plant root-specific expression promoter |
| CN201180005657.1A CN102712929B (en) | 2010-11-16 | 2011-11-14 | Identification and use of plant root-specific expression promoter |
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| CN102876710B (en) * | 2012-10-25 | 2014-03-19 | 北京未名凯拓作物设计中心有限公司 | Identification and applications of ground tissue specificity expression promoter |
| CN103233040B (en) * | 2013-04-01 | 2015-04-01 | 北京兴科创控股有限公司 | Method for cultivating antiviral momordica grosvenori |
| CN103305513B (en) * | 2013-06-08 | 2015-03-04 | 清华大学 | Plant promoter and application thereof |
| WO2016000236A1 (en) * | 2014-07-03 | 2016-01-07 | Pioneer Overseas Corporation | Plants having altered agronomic characteristics under nitrogen limiting conditions and related constructs and methods involving genes encoding nac3/onac067 polypeptides |
| CN113416735B (en) * | 2021-03-17 | 2023-01-31 | 云南中烟工业有限责任公司 | Tobacco germ cell specific high expression gene and application thereof |
| CN113308468B (en) * | 2021-04-12 | 2023-03-10 | 上海交通大学 | Plant root specific promoter and application thereof |
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|---|---|---|---|---|
| CN1487084A (en) * | 2003-08-13 | 2004-04-07 | 浙江大学 | Rice root system phosphorus starvation induction specific expression promoter and its plant culture method |
| CN1492925A (en) * | 2001-01-05 | 2004-04-28 | �ձ��̲ݲ�ҵ��ʽ���� | Rice sucrose transporter gene promoter |
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| CN1042565A (en) * | 1988-11-08 | 1990-05-30 | 复旦大学 | The cloning process of DNA initiator sequence for paddy rice |
| GB9524395D0 (en) * | 1995-11-29 | 1996-01-31 | Nickerson Biocem Ltd | Promoters |
| FR2772787B1 (en) * | 1997-12-24 | 2001-12-07 | Rhone Poulenc Agrochimie | H3C4 PROMOTER OF BUT ASSOCIATED WITH THE FIRST INTRON OF RICE ACTINE, CHIMERIC GENE INCLUDING IT AND TRANSFORMED PLANT |
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|---|---|---|---|---|
| CN1492925A (en) * | 2001-01-05 | 2004-04-28 | �ձ��̲ݲ�ҵ��ʽ���� | Rice sucrose transporter gene promoter |
| CN1487084A (en) * | 2003-08-13 | 2004-04-07 | 浙江大学 | Rice root system phosphorus starvation induction specific expression promoter and its plant culture method |
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