CN100999736A - Rice high efficient expression starter and application thereof - Google Patents
Rice high efficient expression starter and application thereof Download PDFInfo
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- CN100999736A CN100999736A CN 200610117929 CN200610117929A CN100999736A CN 100999736 A CN100999736 A CN 100999736A CN 200610117929 CN200610117929 CN 200610117929 CN 200610117929 A CN200610117929 A CN 200610117929A CN 100999736 A CN100999736 A CN 100999736A
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Abstract
The present invention relates to one kind of high efficiency expression promoter of rice and its application. The promoter has the nucleotide sequence as shown in SEQ ID No.1. It is shown by means of cloning one Os252 promoter to be predicted to express in stem in high efficiency, constituting GUS fused expression vector transferred to rice and performing PCR detection and chemical GUS tissue staining, that Os252 promoter has expression in the leaf, stem and seed albumen of rice. It is shown by means of GUS enzyme activity measurement that Os252 promoter has enzyme activity in leaf and seed albumen of rice about twice that of 35S promoter and enzyme activity in stem slighter lower than that of 35S promoter, and is one high efficiency expressed promoter. The present invention may be used in the genetic improvement and production research of rice.
Description
Technical field
The invention belongs to biological technical field, particularly relate to a kind of rice starter and cloning process and application thereof.
Background technology
The expression of plant gene is subjected to the control of promotor, and the separation of promotor and functional analysis are the important contents of gene expression in plants study on regulation.Promotor is meant that one section can combine the initial dna sequence dna of transcribing of realization accurate and effective with the trans factor that RNA polymerase and some other influences are transcribed in the gene.The important content of gene expression in plants study on regulation is not only in the separation of promotor and functional analysis, also is an importance of plant genetic engineering research.
The expression of plant gene is subjected to the control of promotor and is arranged and the spatial specificity its time, in recent years, in higher plant to seed protein gene, light regulatory gene, hormone induction gene, environmental injury (as anoxic, outside injury, pathogenic agent injury) etc. the promotor of induced gene and some other genes involveds carried out comparatively deep research, confirm that promotor playing the part of crucial role (Plant Cell such as document Chen W as a kind of cis-acting elements of genetic expression in the plant gene regulation and control, 2002,14 (3): 559-574; He Y and Gan S, Plant Mol Biol, 2001,47 (5): Plant Cell such as 595-605:YamaguchiS, 1998,10:2115-2126; Li Y K and Wang J F, Chinese Bull Bot, 1998,15 (suppl): 1-6).
Grass in the monocotyledons comprises many important cash crop such as paddy rice, Chinese sorghum, corn, sugarcane, wheat and barley, and wherein paddy rice is most important in the world food crop.Rice genome is little and extensively elected as model plant, separates the promotor that efficiently expresses and have important economic worth from cereal plants.
Paddy rice is the model plant of development of plants biology and molecular biology research, the research of finishing to the paddy gene function of rice genome examining order provides a great convenience, but at present about the isolating work sutdy of rice high efficient expression starter and few, at present be separated to promotor such as 35S promoter (the Odell JT etc. 1985 that efficiently express from plant virus and plant itself, Nature 313:810-812), tissue specific promoter (Van Tunen A J etc. 1990, The Plant Cell 2:393-401) and inducible promoter (Zuo J and Chua NH 2000, Curren Opinion in Biotechnology11:146-151).These promotors have become the important tool of plant genetic engineering.The 35S promoter of separating from cauliflower mosaic virus is a most frequently used promotor the present plant genetic operation, the expression of promotor gene in dicotyledons strongly of this promotor, but a little less than this promotor is expressed relatively in monocotyledons.
Summary of the invention
Technical problem to be solved
Technical problem to be solved by this invention provides a kind of rice high efficient expression starter and application thereof, expresses relative more weak or contain the defective of more copy at cereal plants itself in monocotyledons to overcome existing promotor.
Technical scheme
One of technical scheme of the present invention provides a kind of rice high efficient expression starter, has the nucleotide sequence shown in the SEQ IDNo.1.
Two of technical scheme of the present invention provides a kind of paddy rice host cell, and this cell has been imported into above-mentioned promoter sequence.
Three of technical scheme of the present invention provides a kind of expression vector that contains described rice high efficient expression starter.
Four of technical scheme of the present invention provides a kind of bacterial host cell, and this cell contains described expression vector sequence.
Five of technical scheme of the present invention provides a kind of Agrobacterium host cell, and this cell contains described expression vector sequence.
Six of technical scheme of the present invention provides a kind of transgenic paddy rice, and this paddy rice contains described promoter sequence.
One of optimal technical scheme of above-mentioned transgenic paddy rice is that the expression of the gene that said promotor guided in the leaf of paddy rice surpasses the homologous genes that 35S promoter guided and express.
Two of the optimal technical scheme of above-mentioned transgenic paddy rice is that the expression of the gene that said promotor guided in the endosperm of paddy rice mature seed surpasses the homologous genes that 35S promoter guided and express.
The further optimal technical scheme of above-mentioned transgenic paddy rice is that the gene that said promotor guided is a gus gene.
Seven of technical scheme of the present invention provides a kind of method that obtains transgenic paddy rice, it is characterized in that, utilizes the expression vector rice transformation cell that contains described promotor and target gene, then by the tissue culture regeneration plant.
The present invention realizes in the following manner:
In brief, be by from Zhejiang University's est database, selecting and cloned a promotor Os252 that prediction efficiently expresses in rice stem, sequence is: 5 '-3 '
AGTTTATGTGCTTATACAGATGAGTGCACATGTTATTTGAGAGTTTGCGTTTGTAGTGT
ATTTCGATTTTTTTAAAAAAAAGAAATCAATAAGGGAGTCTACCGGTAGAGAGGCGA
AAACATTGATTTTTCACCAGCGAGGAGATAAACCTTGTAAACCTTTTTCTCTAATCCA
AAAACAAACTTGAATTATGTGTACTTACTTGCGCCCTGCGAAACCTCCCGAGACTTAA
ACGTAACGCAAACGTTTGAACAGGCTGACCGCAGGATTAACATCGAACGATCAATAC
TGCAACGCAACAACAACCATCAGACAAAGATCGGACGGCTAGGAAGGAACCCTAGC
ACCCGTGAGTGCCTATATAAGAGACAATCCTCTCGCCCTAATCCCTCCCCTCTCCCATC
TCACCCCGCCGCCGCCGCCGCCGCAGCCTCCTCAAGGCTGCTCCCATCCTCTCCTTCG
AGGTCAGCTGCAGATCTTCTCTCTTCTCCTTGTTTGCGCCGGTTCATGGTAGTTCGTAG
CCGTAGATCTGATTCGATGGAGCGAGGTTTGGGTGATTTGATGCCTGGGCATGTTGTT
TTTGTCCATTATTAGTAACTTTTTCTGTGTATTCGTCTCACTGCTATGATCTTAGTTTGCT
GTTCGTTGACGCGATGATTTATTGACTTGTCGGTGAATCATTTATGTACGTTGAAAATT
ATCGGTAGAATCGGACTATTAACTTGATGTGCTGCAGAATCATGTACATAAGTTGAAA
ATCATCGGTAGAATCAGTAGAAGTATTTCTATCTAGAATCCGTTCGAATATCTCTGTTTT
TATGTTCGAATAGATGGTGTTATCGTCTATGCCTGGTTCGGTTTGGTCGATTACTGCGC
GTTAGCACTTATAATTGATGTCGAAGTTCATCTGATCTGTGAGCTTGCCTGTAATATTAT
TTGGAGTTAGAAATTATGCACGCCTGTTGTTTCTAATCTTTGTTTCGTTCTGTTTTGCA
GTTAGCTTCCTCCTTATTCAACC
This promotor is one of them promotor of not cloned among 10 TUT of the highly redundant degree in stem library of Zhejiang University's est database (http://www.estarray.org), change paddy rice over to so selected GenBank ID and be the promotor (being Os252) of BI807252 and made up the GUS fusion expression vector, studied the activity of this promotor by rice transformation.Transgenic paddy rice carried out PCR detects and the GUS histochemical stain shows that experiment is incorporated into 252 promotors in the rice genome.The mensuration of the GUS enzymic activity of carrying out subsequently, and the result that the enzymic activity of 252 promotors and 35S promoter compare shown: the enzymic activity of 252 promotors in paddy rice leaf and seed endosperm is the twice of 35S approximately, enzymic activity in stem is slightly low than 35S, proves and successfully separated a promotor that efficiently expresses from paddy rice.
Particularly, at first need to design and synthesize the primer sequence that is used for cloning this promotor: F:5 ' AGTTTATGTGCTTATACAGATGAG 3 '; R:5 ' GGTTGAATAAGGAGGAAGC 3 '.The total DNA of pcr amplification rice leaf, obtaining the product clip size is 1018bp, has comprised technical scheme one said promoter sequence SEQ ID NO.1.
Then, the pcr amplification product that has promoter sequence that obtains is cloned into T-Vector.With binary vector pCAMBIA 1301 usefulness Hindlll and Ncol double digestion, separate obtaining big fragment, after filling and leading up with T4DNA polymerase end, connect with the T4 ligase enzyme, be built into the pCAM-Gus carrier.The T carrier that will contain promoter sequence reclaims small segment and is connected with the big fragment of pCAM-Gus plasmid double digestion with BamH I and Sal I double digestion, constitutes technical scheme three said paddy rice expression vector pCAMBIA 1301.(Fig. 5)
With the intestinal bacteria of above-mentioned paddy rice expression vector pCAMBIA 1301 transformed competence colibacillus, resistance screening obtains the technical scheme four said bacterial host cells that contain expression vector.The intestinal bacteria transformant extracts the expression vector plasmid and transforms Agrobacterium in 37 ℃ of overnight incubation of shaking table, obtains the technical scheme five said Agrobacterium host cells that contain the expression vector sequence.
Agrobacterium-mediated Transformation of picking-70 ℃ preservation is gone up line at YEP substratum (contain card receive mycin 50mg/L), 28 ℃ of dark 2d. picking mono-clonals of cultivating in 5mL YEP liquid nutrient medium, 28 ℃ in a small amount the shaking table overnight incubation cultivate.Next day, in a small amount the bacterium (1-3mL) cultivated of shaking table changed a large amount of shaking tables cultivations of 100mL AB (YEP) substratum (contain card receive mycin 50mg/L) over to.When being cultured to the OD value for 0.3-0.5,4000rpm gets precipitation and suspends with equal-volume AAM substratum after centrifugal 10 minutes, leave standstill 1-2h.The picking color is fresh faint yellow, and the fine and close particulate state callus of growth immerses bacterium liquid, is placed on 28 ℃ of shaking tables less than 100rpm, 20 minutes, transfers to ND after blotting with aseptic filter paper
2Cultivate 3d altogether under (fill up one sterilization filter paper) 22 ℃, the dark condition on-As (As200ug/L) substratum.
Callus after cultivating is altogether taken out, wash 5 times to remove the Agrobacterium of surface adsorption, soak 2h with the sterilized water that contains cephamycin 100mg/L then with sterilized water.After blotting with aseptic filter paper, be transferred to N
6D
2The SI substratum, screening 14d.Change ND over to
2The SII substratum continues screening 14d, obtains the two said paddy rice host cells that are imported into the Os252 promoter sequence of technical scheme.
Resistant calli (color is fresh, partially white) is met the people break up substratum in advance, 26 ℃, secretly cultivate 8d.Change over to again on the division culture medium, illumination cultivation, every 12-13d subculture is once, until seedling differentiation. the seedling that will grow to 3-5cm moves on the root media, is cultured to (30-50d) behind the 10cm flush away substratum, one week of hardening moves in the soil in sterilized water, is cultured to maturation.Transfer-gen plant is the six described transgenic paddy rices that contain promoter sequence of technical scheme.
And, utilize the expression vector rice transformation cell that contains described promotor and target gene, then by the process of tissue culture regeneration plant, be the method for seven said acquisition transgenic paddy rices of technical scheme.
Further the PCR that transgenic paddy rice is carried out detects and the specific implementation process of the mensuration of GUS histochemical stain and GUS enzymic activity is seen embodiment 1.The result shows that the Os252 promotor all has expression in the endosperm of leaf, stem and the seed of paddy rice; Measurement result by the GUS enzymic activity shows that the enzymic activity of Os252 promotor in paddy rice leaf and seed endosperm is the twice of 35S promoter approximately, and the enzymic activity in stem is slightly low than 35S.
Beneficial effect
Itself being separated to the promotor such as 35S promoter, tissue specific promoter and the inducible promoter that efficiently express from plant virus and plant compares, Os252 promotor of the present invention is expressed in monocotyledons apparently higher than the 35S promoter of separating from cauliflower mosaic virus, the latter's expression of promotor gene in dicotyledons strongly; This Os252 promotor is expected to become promotor commonly used in the plant genetic operation, becomes the important tool of plant genetic engineering.
Description of drawings
Fig. 1 be the activity ratio of different promoters in the transfer-gen plant leaf.
Fig. 2 be the activity ratio of different promoters in the transfer-gen plant stem.
Fig. 3 be the activity ratio of different promoters in the transfer-gen plant endosperm.
Fig. 4 is the comparison of Os252 promotor enzymic activity in the transfer-gen plant different tissues.
Fig. 5 is paddy rice expression vector pCAMBIA 1301 collection of illustrative plates.
Fig. 6 is the PCR Molecular Identification result of transgenosis seedling.M is a molecular weight marker, with 1-12 swimming lane totally 12 Os252 transgenic paddy rice blades, 13 swimming lanes are that 35S transgenic paddy rice blade is the PCR product electrophoresis result demonstration of template, 12 transfer-gen plants of Os252 and 35S contain the band about hygromycin gene 500bp, and wild-type paddy rice and water (14 and 15 swimming lane) do not have corresponding amplified production as negative control.
Embodiment
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.Should be understood that in addition those skilled in the art can make various changes or modifications the present invention after the content of having read the present invention's instruction, these equivalent form of values fall within the application's appended claims institute restricted portion equally.
The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, as: plasmid extracts, dna fragmentation is cloned, recombinant plasmid is identified with reference to molecular cloning experiment guide (Sambrook, J.and Russell, D. (Huang Peitang etc. translate), 2002, molecular cloning experiment guide (the 3rd edition). (Beijing: Science Press), pp:27-30.), the perhaps condition of advising according to manufacturer, as: dna fragmentation reclaims and adopts vast Imtech to reclaim test kit, and operates by its specification sheets.Callus induction and leaf dish transform: " Japan is fine " callus induce and agriculture bacillus mediated rice conversion with reference to Liu Qiaoquan (Liu Qiaoquan, Zhang Jingliu, Wang Chongyang, Hong Mengmin, Gu Minghong, 1998, the foundation of Agrobacterium tumefaciens mediated paddy rice colleges and universities conversion system, the plant physiology journal, 24 (3): 259-271.) method of Denging is carried out.
All inorganic chemical reagents and organic solvent are available from Shanghai chemical reagent factory, and reagent is homemade analytical pure.Primer is given birth to worker company by Shanghai and is synthesized.Eppendorf gradient type pcr amplification instrument is available from German Eppendorf company.Vegetable material: japonica rice variety Japan is fine to be preserved by Shanghai Normal University plant function genome laboratory.Agrobacterium EHA105 bacterial strain and e.colistraindh5 are preserved by Shanghai Normal University plant function genome laboratory; Plasmid vector: pMD18-Tvector, (available from TAKARA company), pCAMBIA-1301 is preserved by Shanghai Normal University's laboratory plant function genome.Restriction enzyme and T4DNA ligase enzyme are all available from TAKARA company.
The clone of promotor and vector construction: according to 10 TUT data of the highly redundant degree in stem library of Zhejiang University's est database (http://www.estarray.org), selected one of them promotor of not cloned, its GenBank ID is BI807252 (being Os252), designs and synthesizes primer: F:5 ' AGTTTATGTGCTTATACAGATGAG 3 '; R:5 ' GGTTGAATAAGGAGGAAGC 3 '.The total DNA of pcr amplification rice leaf, obtaining the product clip size is 1018bp, be cloned into T-Vector, binary vector pCAMBIA 1301 usefulness Hindlll and Ncol double digestion separate big fragment, after filling and leading up with T4 polymerase end, the T4 ligase enzyme is built into the pCAM-Gus carrier after connecting, the T carrier that will contain promoter sequence reclaims small segment and is connected with the big fragment of pCAM-Gus plasmid double digestion with BamH I and Sal I double digestion, constitutes paddy rice expression vector pCAMBIA 1301.(Fig. 5)
With the intestinal bacteria of above-mentioned paddy rice expression vector pCAMBIA 1301 transformed competence colibacillus, resistance screening obtains to contain the bacterial host cell of expression vector.The intestinal bacteria transformant extracts the expression vector plasmid and transforms Agrobacterium in 37 ℃ of overnight incubation of shaking table, obtains to contain the Agrobacterium host cell of expression vector sequence.
Agriculture bacillus mediated rice conversion and the regeneration of transformed plant, screening and differentiation are taken root: Agrobacterium-mediated Transformation of picking-70 ℃ preservation is gone up line at YEP substratum (contain card receive mycin 50mg/L), 28 ℃ of dark 2d. picking mono-clonals of cultivating are in 5mL YEP liquid nutrient medium, and 28 ℃ of a small amount of shaking table overnight incubation are cultivated.Next day, in a small amount the bacterium (1-3mL) cultivated of shaking table changed a large amount of shaking tables cultivations of 100mL AB (YEP) substratum (contain card receive mycin 50mg/L) over to.When being cultured to the OD value for 0.3-0.5,4000rpm gets precipitation and suspends with equal-volume AAM substratum after centrifugal 10 minutes, leave standstill 1-2h.The picking color is fresh faint yellow, and the fine and close particulate state callus of growth immerses bacterium liquid, is placed on 28 ℃ of shaking tables less than 100rpm, 20 minutes, transfers to ND after blotting with aseptic filter paper
2Cultivate 3d altogether under (fill up one sterilization filter paper) 22 ℃, the dark condition on-As (As200ug/L) substratum.
Callus after cultivating is altogether taken out, wash 5 times to remove the Agrobacterium of surface adsorption, soak 2h with the sterilized water that contains cephamycin 100mg/L then with sterilized water.After blotting with aseptic filter paper, be transferred to N
6D
2The SI substratum, screening 14d.Change the ND2SII substratum over to, continue screening 14d.
Resistant calli (color is fresh, partially white) is met the people break up substratum in advance, 26 ℃, secretly cultivate 8d.Change over to again on the division culture medium, illumination cultivation, every 12-13d subculture is once, until seedling differentiation. the seedling that will grow to 3-5cm moves on the root media, is cultured to (30-50d) behind the 10cm flush away substratum, one week of hardening moves in the soil in sterilized water, is cultured to maturation.
The PCR Molecular Identification of transgenosis seedling: the CTAB method is extracted total DNA, carries out pcr amplification with the primer that designs on the hygromycin gene, and primer is: HF, 5 '-GATCGTTATGTTTATCGGCACT-3 '; HR, 5 '-TTGGCGACCTCGTATTGG-3 ', the PCR program is: 94 ℃ of 30s, 54 ℃ of 30s, 72 ℃ of 30s, 40 circulations, the product size is 500bp.Result such as Fig. 6 can see that the PCR product electrophoresis result that 12 transgenic paddy rice blades are template shows 12 bands that all contain about hygromycin gene 500bp, and wild-type and water do not have band as negative control.
The GUS histochemical stain of transfer-gen plant: the different tissues to 12 transgenic paddy rices carries out GUS dyeing, dyeing process: with reference to (Jefferson such as Jefferson, R.A., Burgess, S.M., andHirsh, D, beta-Glucuronidase from Escherichia coli as a gene-fusion marker.Proc.Natl.Acad.Sci.USA, 1986, method 83:8447-8451) is made into the 20mM mother liquor to X-Gluc with dimethyl formamide, contains the Tripotassium iron hexacyanide of 1mM/L and yellow prussiate of potash and the 10mM NA of 1mM/L in the phosphoric acid buffer of 50mM/L (pH7.0)
2EDTA; In phosphoric acid buffer: the ratio of methyl alcohol: X-Gluc (40: 10: 1) is made into staining fluid.Get a small amount of blade of transgenic paddy rice regeneration plant, the mature seed of stem tissue and incision epidermis adds staining fluid in the pipe in 96 orifice plates, and the amount that adds is as the criterion with the submergence material.37 ℃ of dyeing of good seal two days with the ethanol decolorization several of massfraction 70%, are observed coloration result.
Detect the GUS activity in the transfer-gen plant.The measuring method of GUS enzymic activity: with reference to Jefferson method (Jefferson, R.A, Assay Chimeric Genes in Plants:The GUS GeneFusion System.Plant Molecular Biology Reporter, 1987,4 (5): the 397-405) total protein in extraction transgenic paddy rice leaf, stem and the endosperm tissue measures A with spectrophotometric
280Value also calculates protein concentration by typical curve, reacts with protein solution with substrate MUG, thereby draws the speed of response of GUS enzyme with the fluorescence intensity that spectrophotofluorometer is measured the differential responses time, calculates enzyme activity by formula.The result shows that the Os252 promotor has the leaf of 6 plant, and GUS dyeing all is positive in stem and the endosperm tissue, and 35S promoter is at leaf, and stem and endosperm all are positive, and do not detect the expression of gus gene in the unconverted seedling.
From PCR and the insertion that the Os252 promotor is successful as can be seen of GUS qualification result the rice genome.
Typical curve by the BSA formulation, calculate leaf, stem and the endosperm tissue total protein content of the individual plant of transfer-gen plant, 0.5mL the MUG solution of 37 ℃ of preheatings adds V=100 μ L protein extract, reacts every interval 10min and gets 100 μ L reaction solutions and add and to contain 900 μ L Na
2CO
3Termination reaction in the termination reaction liquid, with the emission light of spectrophotofluorometer at 455nm, the exciting light of 365nm, measure the fluorescence intensity of each time period under the condition of slit width 5mm, obtain slope by reaction times and fluorescence intensity relation curve, then every μ L protein extract speed of response is: 10 * 0.8 * slope * [(V+500) ÷ V] * [1000 ÷ V], and enzyme activity promptly is speed of response and proteic ratio, the enzyme activity of same promotor different tissues is averaged data such as following table:
| Sample | Protein concentration (mg/mL) | Slope | Speed of response (nmMUG/minmL) | Enzyme activity (nmMUG/min/mg protein) |
| 35S-L | 45.760 | 6.356 | 3050.640 | 66.617 |
| 252-L | 32.140 | 8.023 | 3850.920 | 128.507 |
| 35S-S | 37.380 | 6.629 | 3181.680 | 92.606 |
| 252-S | 24.860 | 3.921 | 1881.920 | 79.003 |
| 35S-E | 27.600 | 2.646 | 1270.080 | 50.032 |
| 252-E | 21.780 | 4.694 | 2253.080 | 124.907 |
Illustrate: L-Leaf leaf, S-Stem stem, E-EndOsperm endosperm
SEQUENCE LISTING
The nucleotides sequence tabulation
＜110〉Shanghai Normal University
＜120〉a kind of rice high efficient expression starter and application thereof
<130>Patent
<160>1
<170>Patentln version 3.3
<210>1
<211>1018
<212>DNA
<213>Oryza sativa
<220>
<221>promoter
<222>(1)..(1018)
<400>1
agtttatgtg cttatacaga tgagtgcaca tgttatttga gagtttgcgt ttgtagtgta 60
tttcgatttt tttaaaaaaa agaaatcaat aagggagtct accggtagag aggcgaaaac 120
attgattttt caccagcgag gagataaacc ttgtaaacct ttttctctaa tccaaaaaca 180
aacttgaatt atgtgtactt acttgcgccc tgcgaaacct cccgagactt aaacgtaacg 240
caaacgtttg aacaggctga ccgcaggatt aacatcgaac gatcaatact gcaacgcaac 300
aacaaccatc agacaaagat cggacggcta ggaaggaacc ctagcacccg tgagtgccta 360
tataagagac aatcctctcg ccctaatccc tcccctctcc catctcaccc cgccgccgcc 420
gccgccgcag cctcctcaag gctgctccca tcctctcctt cgaggtcagc tgcagatctt 480
ctctcttctc cttgtttgcg ccggttcatg gtagttcgta gccgtagatc tgattcgatg 540
gagcgaggtt tgggtgattt gatgcctggg catgttgttt ttgtccatta ttagtaactt 600
tttctgtgta ttcgtctcac tgctatgatc ttagtttgct gttcgttgac gcgatgattt 660
attgacttgt cggtgaatca tttatgtacg ttgaaaatta tcggtagaat cggactatta 720
acttgatgtg ctgcagaatc atgtacataa gttgaaaatc atcggtagaa tcagtagaag 780
tatttctatc tagaatccgt tcgaatatct ctgtttttat gttcgaatag atggtgttat 840
cgtctatgcc tggttcggtt tggtcgatta ctgcgcgtta gcacttataa ttgatgtcga 900
agttcatctg atctgtgagc ttgcctgtaa tattatttgg agttagaaat tatgcacgcc 960
tgttgtttct aatctttgtt tcgttctgtt ttgcagttag cttcctcctt attcaacc 1018
Claims (10)
1. a rice high efficient expression starter has the nucleotide sequence shown in the SEQ ID No.1.
2. paddy rice host cell, this cell has been imported into the promoter sequence described in the claim 1.
3. expression vector that contains the described rice high efficient expression starter of claim 1.
4. bacterial host cell, this cell contains the expression vector sequence described in the claim 3.
5. Agrobacterium host cell, this cell contains the expression vector sequence described in the claim 3.
6. transgenic paddy rice, this paddy rice contains the promoter sequence described in the claim 1.
7. transgenic paddy rice according to claim 6 is characterized in that, the expression of the gene that said promotor guided in the leaf of paddy rice surpasses the homologous genes that 35S promoter guided and express.
8. transgenic paddy rice according to claim 6 is characterized in that, the expression of the gene that said promotor guided in the endosperm of paddy rice mature seed surpasses the homologous genes that 35S promoter guided and express.
9. according to claim 7 or 8 described transgenic paddy rices, it is characterized in that the gene that said promotor guided is a gus gene.
10. a method that obtains transgenic paddy rice is characterized in that, utilizes the expression vector rice transformation cell that contains described promotor of claim 1 and target gene, then by the tissue culture regeneration plant.
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| CN102206636A (en) * | 2010-03-29 | 2011-10-05 | 中国科学院上海生命科学研究院 | Element for driving gene to express in seed and application thereof |
| CN102206637A (en) * | 2010-03-29 | 2011-10-05 | 中国科学院上海生命科学研究院 | Element for driving gene to be expressed in embryo and application thereof |
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|---|---|---|---|---|
| CN102206636A (en) * | 2010-03-29 | 2011-10-05 | 中国科学院上海生命科学研究院 | Element for driving gene to express in seed and application thereof |
| CN102206637B (en) * | 2010-03-29 | 2014-03-05 | 中国科学院上海生命科学研究院 | Element for driving gene to be expressed in embryo and application thereof |
| CN102206636B (en) * | 2010-03-29 | 2013-12-25 | 中国科学院上海生命科学研究院 | Element for driving gene to express in seed and application thereof |
| CN102206637A (en) * | 2010-03-29 | 2011-10-05 | 中国科学院上海生命科学研究院 | Element for driving gene to be expressed in embryo and application thereof |
| CN101831428A (en) * | 2010-04-07 | 2010-09-15 | 华中农业大学 | Separation clone and expression mode identification of promotor region of rice endosperm special expression gene |
| CN101831428B (en) * | 2010-04-07 | 2011-11-30 | 华中农业大学 | Separation clone and expression mode identification of promotor region of rice endosperm special expression gene |
| CN101831429A (en) * | 2010-04-15 | 2010-09-15 | 华中农业大学 | Promoter and expression mode identification of rice endosperm specific expression gene |
| CN102206640A (en) * | 2010-04-23 | 2011-10-05 | 深圳华大基因科技有限公司 | Promoter SbUbi2, its preparation method and use |
| CN102206641A (en) * | 2010-04-23 | 2011-10-05 | 深圳华大基因科技有限公司 | Promoter SbUbi1, its preparation method and use |
| CN102206641B (en) * | 2010-04-23 | 2013-02-27 | 深圳华大基因科技有限公司 | Promoter SbUbi1, its preparation method and use |
| CN101979547A (en) * | 2010-09-02 | 2011-02-23 | 华中农业大学 | Identification of isolation cloning and core region of promoters suitable for gene expression of skeletal muscles in pigs |
| CN102121004A (en) * | 2010-12-03 | 2011-07-13 | 西北农林科技大学 | Grape pathogen inducible promoter separating method and application in breeding for disease resistance |
| CN102199602A (en) * | 2011-02-16 | 2011-09-28 | 中国科学院植物研究所 | DNA (deoxyribonucleic acid) fragment and application thereof |
| CN102199602B (en) * | 2011-02-16 | 2012-10-24 | 中国科学院植物研究所 | DNA (deoxyribonucleic acid) fragment and application thereof |
| CN105340731A (en) * | 2015-11-13 | 2016-02-24 | 上海师范大学 | Photo-thermo-sensitive genic male-sterile rice creation method |
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