CN102250892B - Plant endosperm specific expression promoter and use thereof - Google Patents
Plant endosperm specific expression promoter and use thereof Download PDFInfo
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Abstract
The invention discloses a plant endosperm specific expression promoter and a use thereof. A nucleotide sequence of the promoter is one of the following nucleotide sequences of 1) a DNA sequence limited by sequence 1 shown in a sequence list in the patent specification, 2) a DNA sequence which is above 70% homologous to the DNA sequence limited by the sequence 1 shown in the sequence list and has functions the same as that of the DNA sequence limited by the sequence 1 shown in the sequence list, and 3) a nucleotide sequence which can hybridize with the DNA sequence limited by the sequence 1 shown in the sequence list under highly stringent conditions. The promoter can promote specific expression of a foreign gene in plant endosperm, thus is suitable for any plant of which a seed has endosperm. In other words, the promoter is suitable for monocotyledons or endosperm type dicotyledons.
Description
Technical field
The present invention relates to a kind of plant endosperm specificity expression promoter and application thereof.
Background technology
The recent development of Plant Biotechnology has realized that not only the improvement of traditional agronomic shape is (as improving crop yield; Strengthen disease-resistant, pest-resistant, antiweed characteristic; Improvement quality etc.); And make plant become bio-reactor (Daniell et al., Trends Plant Sci.2001, the 6:219-226 of biological medicine and Industrial products; Giddings et al., NatureBiotech.2000,18:1151-1156; Hood and Jilka, Curr.Opin.Biotechnol.1999,10:382-386; Hood and Woodard, Plants as Factories for Protein Production 2002, pp.119-135.Netherlands:Kluwer Academic).For most gramineous crops,, determined it to be the ideal carrier of s-generation transgene product because its output is high, production cost is low, storage endurance and industrial scale characteristics such as control easily.Utilize rice paddy seed production foreign protein with health role and the development of edibility vaccine research very fast in recent years, and obtained immense success.All successfully in paddy rice, express and high level accumulation (Goto et al. like vitamin A, glycinin (Glycinin), soybean ferritin (Ferritin), GLP, Hepatitis B virus vaccine and the pollen hypersensitivity vaccine etc. that have prevention and treatment iron-deficiency anaemia and improve autoimmune function with the prevent diabetes generation function that stimulates insulin secretion with lowering blood glucose effect; Nature Biotech.1999,17:282-286; Katsube et al., Plant Physiol.1999,120:1063-1073; Qu et al., Planta 2005,222:225-233; Takagi et al., Proc Natl.Acad.Sci.2005,102:17525-17530; Ye et al., Science 2000,287:303-305; Zheng et al., PlantPhysiol.1995,109:777-786).Yet lack the limiting factor that corresponding promotor becomes present biotechnology applications (the desirable expressive site of foreign gene, phraseology, expression period and expression level etc.).
The basic goal of plant genetic engineering is to cultivate to make the transfer-gen plant that goal gene is stable and efficiently express.The nucleotide sequence that genetic expression is controlled by the transcription initiation place is the promotor composition, and it mainly comprises the signal that rna polymerase promoter is transcribed, and to instruct synthetic corresponding messenger RNA(mRNA) molecule, further instructs proteinic synthetic.Promotor controlling gene transcriptional level has also determined expression of gene time and phraseology simultaneously.
The promotor (comprising CaMV35S, ubiquitin1, Actin1) of widespread use at present is although its expression efficiency is high; Yet because its expression is not limited by space-time; It can both make exogenous gene expression in nearly all tissue, in carrying out seed, in the external source gene overexpression process, can drive gene and in extraseminal other tissue, express; Both consume bioenergy, and can cause the synthetic of some meta-bolitess that possibly have a negative impact to biological growth and development simultaneously.And the expression intensity of above-mentioned promotor still can't satisfy the needs of transgenic industrialization.If select endosperm-specific HS to express promotor, help regularly the synthetic of directed regulation and control external source useful proteins, both save energy had guaranteed that the plant normal physiological activity was interference-free, can improve the storage level of external source useful proteins in seed simultaneously again.
The rice paddy seed storage protein is main with gluten and protein,alcohol-soluble, accounts for 80% and 10% of seed whole protein respectively.The paddy rice glutenin gene is only expressed in endosperm, and in other tissue, almost detects less than gluten.The paddy rice gluten is by at least 13 Gene Handling, and these genes are divided into GluA, GluB, GluC and four gene subtribes of GluD (Subfamily) according to the homology of its dna sequence dna.Though the paddy rice glutenin gene has higher similarity (80-96% in the subtribe, 60-80% between subtribe) on its base sequence, its promoter region does not almost have similarity, is indicating that the regulatory mechanism of its genetic expression has nothing in common with each other.
The promotor that comes from paddy endosperm storage protein gene has the potential using value for expression of exogenous gene.(Plant Biotechnol.J.2004 2:113-125) utilizes the GUS gene of giving a report, and has studied 3 paddy rice gluten (GluB-1 for Qu and Takaiwa; GluB-2; GluB-4), 3 prolamines (10kD, 13kD; 16kD) with phraseology, tissue expression specificity and the expression intensity of 1 sphaeroprotein (26kD) gene promoter; Obtained the endosperm specificity expression promoter (GluB-4,10kD prolamin, 16kD prolamine and Glb-1) of 4 expression activities than the high several times of Ubiquitin.People such as Qu (J.Exp.Bot.2008,59 (9): 2417-2424) further utilize the GUS gene of giving a report, to 6 paddy rice gluten (GluA-1; GluA-2, GluA-3, GluB-3; GluB-5, GluC) phraseology of gene promoter, tissue expression specificity and expression intensity are studied, and find GluA-1; GluA-2; The GluA-3 promotor is specific expressed in the periphery of endosperm, and GluB-5 and GluC promotor are specific expressed in whole endosperm, and the GluB-3 promotor is then specific expressed in aleurone layer and inferior aleurone layer.
Summary of the invention
The purpose of this invention is to provide a kind of plant endosperm specificity expression promoter and application thereof.
Plant endosperm specificity expression promoter provided by the present invention derives from the upstream sequence of Oryza paddy rice (Oryza sativa) GluB-6 gene, can be one of following nucleotide sequence:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 70% above homology, and dna sequence dna with plant endosperm specificity expression promoter function;
The nucleotide sequence of the dna sequence dna hybridization that 3) under the rigorous condition of height, can limit with sequence in the sequence table 1.
The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ℃, hybridize and wash film.
Wherein, sequence 1 is made up of 2192 deoxynucleotides in the sequence table.
Above-mentioned in sequence table 5 ' end 2123-2129 position Nucleotide of sequence 1 be plant endosperm specificity expression promoter TATA box core area.Therefore promotor other sequences except that TATA box core area are changed through base mutation or disappearance etc., promoter activity is completely lost, also can be as required the controlling element of promotor be changed.
The expression cassette, recombinant expression vector, transgenic cell line and the host bacterium that contain above-mentioned plant endosperm specificity expression promoter all belong to protection scope of the present invention.
In said expression cassette, the downstream syndeton gene of said plant endosperm specificity expression promoter, or regulatory gene; Or the inverted defined gene of structure gene or regulatory gene; The little RNA that perhaps can disturb native gene to express is used for the Drive Structure gene, or regulatory gene; Or the inverted defined gene of structure gene or regulatory gene, the expression of the little RNA of perhaps natural little RNA or synthetic.
Said recombinant expression vector is above-mentioned expression cassette and plasmid, virus or the constructed recombinant vectors of vector expression vector; Said recombinant expression vector is the reorganization plant expression vector; Said recombinant plant expression vector contain above-mentioned expression cassette and can with described expression cassette pass on get into plant host cell, tissue or organ and offspring thereof and can or convenient at least described expression cassette be incorporated in host's the genome, it includes but not limited to binary vector, closes carrier altogether.
Above-mentioned recombinant expression vector can be through using conventional biological method transformed plant cells or tissue or organs such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity led, agriculture bacillus mediated or particle gun, obtain transgenic plant cells or tissue or organ and differentiation, regenerated whole plant and clone thereof or generation thus thereafter.
Above-mentioned plant endosperm specificity expression promoter can be used for cultivating the plant of endosperm specificity expression foreign gene.
The method of utilizing this plant endosperm specificity expression promoter to cultivate the plant of endosperm specificity expression foreign gene also belongs to protection scope of the present invention.
The method of the plant of said cultivation endosperm specificity expression foreign gene can be said foreign gene is connected in said plant endosperm specificity expression promoter downstream; Change in the plant through plant expression vector, screening obtains the transgenic plant of specific expressed said foreign gene in endosperm.
Said plant endosperm specificity expression promoter downstream also can be connected with the controlling element of regulate gene expression.The controlling element of said regulate gene expression, this controlling element comprise the element that can strengthen exogenous gene expression or regulatory gene expressive site in plant, like the controlling element of enhanser, constitutive expression, tissue specific expression, inducible expression.
In the said method, said foreign gene be protein coding gene with (or) non-protein coding gene; Said protein coding gene is preferably the quality-improving gene; Said non-protein coding gene is just rna gene and/or sense-rna gene.
In the said method, said plant is monocotyledons or endosperm type dicotyledons.
Said monocotyledons is paddy rice, wheat, corn, barley, Chinese sorghum, oat etc.; Said endosperm type dicotyledons is buckwheat, the little seeds of a tung oil tree, tung oil tree, castor-oil plant etc.
Plant endosperm specificity expression promoter of the present invention is through the design primer, is template with the oryza sativa genomic dna, the glutenin gene GluB-6 promotor of utilizing PCR method from paddy rice, to separate to obtain.Show that through its expression specificity experiment in paddy rice promotor of the present invention makes beta-glucuronidase (GUS) reporter gene only specific expressed in the rice paddy seed endosperm.Explain that promotor of the present invention can start foreign gene specific expression in the endosperm of plant, be applicable to that any seed has the plant of endosperm, i.e. monocotyledons or endosperm type dicotyledons.
Utilize promotor of the present invention can improve expression and the accumulation level of foreign gene in albumen; But improved seed quality, the albumen that will have physiologically active or small peptide import initiative health function new variety in the seed, utilize seed to produce useful foreign protein or edibility vaccine as bio-reactor, increase agricultural-food science and technology added value etc.Promotor of the present invention and being applied as utilizes researchs such as bio-technology improvement seed quality, molecule medicine farm to lay a good foundation, and has great application prospect.
Description of drawings
Fig. 1 is the structural representation of GluB-6 promotor cloning vector pMD-GluB-6.
Fig. 2 is that the double digestion of pMD-GluB-6 is identified collection of illustrative plates.
Fig. 3 merges the structural representation of gus gene plant expression vector GluB-6-pGPTV-35S-HPT for the GluB-6 promotor.
Fig. 4 identifies collection of illustrative plates for the GluB-6-pGPTV-35S-HPT double digestion.
Fig. 5 is for changeing GluB-6-pGPTV-35S-HPT carrier T
0Electrophoretogram for the rice plant pcr analysis.
Fig. 6 is for changeing GluB-6-pGPTV-35S-HPT carrier T
0For the GUS coloration result in paddy rice leaf, root and the cane.
Fig. 7 is for changeing GluB-6-pGPTV-35S-HPT carrier T
0For the GUS coloration result in the rice paddy seed.
Fig. 8 is for changeing GluB-6-pGPTV-35S-HPT carrier T
0Measure the result for the GUS fluorescence activity in the rice plant seed.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
The acquisition of embodiment 1, plant endosperm specificity expression promoter (GluB-6 promotor)
(J.Exp.Bot.2008,59:2417-2424) report is selected a glutenin gene (gene is called Os02g0248800), names to be GluB-6 according to people such as Qu.From GenBank, search the upstream sequence of GluB-6 gene, design primer amplification GluB-6 promotor.For ease of vector construction, on primer, add two restriction enzyme sites (shown in the underscore) successively.The forward primer of promotor is GluB-6HindF:5 '-TT
AAGCTTTTCGCTGGATTAGAATTC-3 ' (
Hind III), reverse primer is GluB-6SalR:5 ' GC
GTCGACAGCTTTTGTATATACTAATAAAAC-3 ' (
Sal I).
The CTAB method from paddy rice (the wild-type paddy rice platform No. 65, this kind is that China Taiwan Province in 1936 breeds; Qu Leqing etc., Botany Gazette, 2001,43:1167-1171; Institute of Botany, Chinese Academy of Sciences's preservation) extracting genomic dna in a small amount in the blade, is template with it, is primer with GluB-6HindF and GluB-6SalR, carries out pcr amplification GluB-6 promoter sequence.Reaction system is: each 1 μ l of the forward and reverse primer of 10 μ M, and 10 * Ex Buffer, 2 μ l, genomic dna 1 μ l (10ng), ExTaq (TaKaRa) 0.5unit adds ultrapure water to 20 μ l; Response procedures is: 94 ℃ of preparatory sex change 5min, 94 ℃ of 1min then, 55 ℃ of 1min30sec, 72 ℃ of 2min30sec, 30 circulations, last 72 ℃, 10min.Pcr amplification obtains the purpose band of 2.2kb.Reclaim amplified production, be directly connected on the pMD18-T carrier (available from TaKaRa company), check order, the result shows that the GluB-6 promoter sequence size that amplification obtains is 2192bp, the nucleotide sequence with sequence 1 in the sequence table.The order-checking detection is shown the recombinant vectors called after pMD-GluB-6 (its structural representation is as shown in Figure 1) that contains the GluB-6 promoter fragment.The double digestion of pMD-GluB-6 identifies that collection of illustrative plates is as shown in Figure 2, and swimming lane 1 is the fragment of the double digestion acquisition of pMD-GluB-6 among Fig. 2, and swimming lane 2 is a dna molecular amount mark.
1, the GluB-6 promotor merges the plant expression vector construction of gus gene
(2:113-125) described method makes up binary expression vector pGPTV-35S-HPT for Qu and Takaiwa, Plant Biotech J 2004, and Institute of Botany, Chinese Academy of Sciences preserves according to document.With Sal I and Hind III double digestion pMD-GluB-6 plasmid and pGPTV-35S-HPT.Recovery contains the endonuclease bamhi of the 2192bp of GluB-6 promotor, this fragment is connected between the Sal I and Hind III enzyme recognition site of pGPTV-35S-HPT.On the basis that PCR identifies, utilizing Sal I and Hind III to carry out double digestion the recombinant plasmid that obtains identifies, obtains containing the fragment of 2.2kbGluB-6 promotor.The double digestion evaluation is shown the recombinant plasmid called after GluB-6-pGPTV-35S-HPT (its structural representation is as shown in Figure 3) that contains the GluB-6 promotor; Gus gene is positioned at GluB-6 promotor downstream among the GluB-6-pGPTV-35S-HPT, is started by the GluB-6 promotor and expresses.Wherein, the double digestion of GluB-6-pGPTV-35S-HPT is identified collection of illustrative plates, and is as shown in Figure 4.Swimming lane 1 is the double digestion figure of GluB-6-pGPTV-35S-HPT among Fig. 4, and swimming lane 2 is a dna molecular amount mark.
2, change the acquisition of GluB-6-pGPTV-35S-HPT paddy rice
With freeze-thaw method with GluB-6-pGPTV-35S-HPT import Agrobacterium EHA105 (Hood et al., TransgenRes 1993,2:208-218; Institute of Botany, Chinese Academy of Sciences preserves) in, transform wild-type paddy rice Kitaake then.
Utilize hygromycin selection method (according to document Hiei et al., Plant J 1994, the said method of 6:271-282 is carried out), obtain 11 strain T
0In generation, changeed the GluB-6-pGPTV-35S-HPT rice plant.Utilize PCR method that the commentaries on classics GluB-6-pGPTV-35S-HPT paddy rice that above-mentioned screening obtains is carried out the PCR Molecular Detection; The PCR primer is the primer of above-mentioned amplification GluB-6 promotor, that is: forward primer the GluB-6HindF:5 '-GGAAGCTTTTTAAGCTTTTCGCTGGATTAG-3 ' of GluB-6 promotor and reverse primer GluB-6SalR:5 '-AAGTCGACTGCGTCGACAGCTTTTGTATAT-3 '.The PCR reaction system is: each 1 μ l of the forward and reverse primer of 10 μ M, and 10 * Ex Buffer, 2 μ l, genomic dna 1 μ l, ExTaq (TaKaRa) 0.5unit adds ultrapure water to 20 μ l; Response procedures is: 94 ℃ of preparatory sex change 5min, 94 ℃ of 1min then, 55 ℃ of 1min30sec, 72 ℃ of 2min30sec, 30 circulations, last 72 ℃, 10min.The purpose band that pcr amplification obtains 2.2kb is the detection positive.The result shows: obtain 9 strain PCR altogether and detect male commentaries on classics GluB-6-pGPTV-35S-HPT paddy rice T
0For plant.Among Fig. 5, the 1st swimming lane is for being the positive control of template amplification with the pMD-GluB-6 plasmid, and the 13rd swimming lane is the negative control that does not add template in the PCR system, and the 2nd～12 swimming lane is respectively the T that changes the GluB-6-pGPTV-35S-HPT plant expression vector
0For plant.
T
0The seed of tying for transfer-gen plant and be T by the plant that this seed grows up to
1In generation, the rest may be inferred, T
2, T
3Represent the 2nd generation of transfer-gen plant and the 3rd generation respectively.
The functional verification of embodiment 3, plant endosperm specificity expression promoter (GluB-6 promotor)
To changeing GluB-6-pGPTV-35S-HPT paddy rice T
0Carry out histochemical stain for plant.Concrete steps: will change GluB-6-pGPTV-35S-HPT paddy rice T
0Be soaked in GUS reaction solution (0.1M NaPO for the partial blade of plant, root, cane tissue and with the filling stage seed of blooming back 11 days, 15 days, 17 days of scalper longitudinal incision from the middle part
4Damping fluid, pH7.0,10mM EDTA, pH7.0, the 5mM Tripotassium iron hexacyanide, 5mM yellow prussiate of potash, 1.0mMX-Gluc, 0.1%Triton X-100), 37 ℃ of reactions.Being organized in 70% ethanol after the dyeing preserved, observed, and under dissecting microscope, takes a picture.The result shows: change GluB-6-pGPTV-35S-HPT rice root, cane and Ye Zhongjun and do not observe the GUS expression, the result is shown in Figure 6; The seed endosperm of blooming back 11 days is outside to become blue, and endosperm centre and embryo are not dyed blueness; The back 15 days seed of blooming is compared with the back 11 days seed of blooming, and aleurone layer, inferior aleurone layer, endosperm are outside blue high-visible, and embryo is not dyed blueness; Bloom whole endosperm blue high-visible of back 17 days seeds, embryo is not dyed blueness yet, and the result is shown in Figure 7.Coloration result shows that the GluB-6 promotor makes beta-glucuronidase (GUS) reporter gene only specific expressed in the rice paddy seed endosperm.Among Fig. 6: 1,2,3,4 be respectively root, cane, blade, leaf sheath coloration result.11d, 15d, 17d represent to represent respectively to change GluB-6-pGPTV-35S-HPT carrier T respectively among Fig. 7
0For the bloom GUS coloration result of back 11 days, 15 days, 17 day filling stage seed of rice plant.
The GUS fluorescence activity of organizing of embodiment 4, transgenic paddy rice is measured
To changeing GluB-6-pGPTV-35S-HPT carrier T
0The filling stage seed of blooming back 15 days for rice plant carries out the GUS quantitative analysis, method according to the fluorescence detection method of Jefferson etc. (Jefferson, Plant Mol.Biol.Report, 1987,5:387-405) carry out.Be specially: get 1 seed, place 1.5ml eppendorf pipe, seed is pulverized, add 30 μ l extract (50mM NaPO with glass stick
4(pH7.0), 10mM β-Mercaptoethanol, 10mM Na
2EDTA (pH8.0), 0.1%SDS, 0.1%Triton X-100), shake up.4 ℃ 15, the centrifugal 10min of 000rpm gets 10 μ l supernatants in new pipe, adds 90 μ l and is incubated the reaction solution (1mM MUG) to 37 ℃, and 37 ℃ were reacted 60 minutes, and added 900 μ l stop buffer (0.2M Na
2CO
3), the room temperature termination reaction.Under 360nm excitation wavelength and 460nm absorbing wavelength, detect relative 4MU content with F-4500 (Hitachi) type spectrophotofluorometer.With the bovine serum albumin is contrast, utilizes Bio-Rad Protein Assay Kit to measure protein contnt, as shown in Figure 8.The result shows that the activity that the GluB-6 promoter-driven GUS is expressed is 15.65 ± 8.19pmol 4MU/min/ μ g albumen in transgenic paddy rice seed.Among Fig. 8,1～6 is respectively the different GluB-6-pGPTV-35S-HPT of commentaries on classics carrier T
0For GUS expression activity MV in 3 seeds of rice plant: 1 is 11.53pmol 4MU/min/ μ g albumen; 2 is 10.83pmol 4MU/min/ μ g albumen; 3 is 10.67pmol 4MU/min/ μ g albumen; 4 is 29.81pmol 4MU/min/ μ g albumen, and 5 is 21.48pmol 4MU/min/ μ g albumen, and 6 is 9.6pmol 4MU/min/ μ g albumen.
Sequence table
<160>1
<210>1
<211>2192
<212>DNA
< 213>Oryza paddy rice (Oryza sativa)
<400>1
tttaagcttt?tcgctggatt?agaattctaa?tgagatacta?tcaaatgtat?aatgataatt 60
gtaaatacac?ttcgcgcgct?gcttgattgc?taatatatgc?ctatatatgt?atatgtgctg 120
tttgtaacag?ctccaattta?ttctcagatc?caagataaat?ttgaagtttg?tatattaaat 180
tatagttatt?taaaatttaa?tataaaattg?agaacaaaat?gaattattta?aatgttatca 240
agttaatcat?atatttagat?ttataaattt?gtgaagttgt?gcccattcga?aaaaaatgag 300
aacaagatga?tttattcaaa?tgttatcaag?ttgtgcccca?tttcgagcac?attctataaa 360
aatcttaaat?ctaaagttgt?gctaaagagg?tgctaaagag?gagcataatc?tcttccatag 420
aagaaaaaga?agtcacattt?gtgacccagt?cttcgtctag?gatttttaca?tttgcagtca 480
ccatataaga?tgagccgatt?aagctgatca?aatattctta?gattgtgact?aaaagtggta 540
caaaacagtc?ttaatttatt?acaattgacg?cttaatttat?tacaattgac?gagctaaatc 600
tgactcttcg?tgtagataaa?taaatttaaa?gtcttaattt?atcaaaaaaa?gtacaaaaca 660
acacgagacc?tgcttacata?tacttaaaaa?ttcttataga?attcaaataa?tgatcccagt 720
tgtttgaccc?agttgtttga?tttaactaaa?tagaagttat?agataatcta?aatgcactct 780
tataataaca?tatcagtgtt?agtactctct?gcaacccaaa?atgtaagaca?caactacacc 840
attaacacaa?agtccaagaa?ataatttaat?ctagagagca?tcataattaa?cttataataa 900
tgcagctatg?tctgaaagaa?tctttaaatc?aagttagtgg?gttatgtttt?atataagaaa 960
atataaagac?caaattgcaa?tagatggaca?catcaaattt?tgatttgtaa?aattatcaaa 1020
atgaattgtt?tcaaaatagt?tacagaaatt?tgattgacaa?gttataaagt?caagaaaata 1080
taaaaaggct?tttagcatgt?caaaattact?aacacatata?tgttttgtca?acttaactgg 1140
tttattttta?catggcataa?gaggatattg?taccacatac?accagaaaaa?tcagtgttaa 1200
aagaggacca?aaataccctt?cctcgtactt?ctccttctct?aatccttact?catcaacgca 1260
ccctcgttga?gggtcacttg?tcccatctac?tccgatatgc?ttctaatcac?ctccccgcag 1320
ccacaacttc?ataatagact?actactccac?caatgagctt?tagacaaggg?atgcaataag 1380
ctctaatcca?tgtggtgaaa?caaccatcgt?ggccatttct?gtatcaatag?tggtggcgaa 1440
gcagaggcca?acaaatgaag?gtgcgcgagg?gcatttaccc?taagtctcga?tgaactctgg 1500
catcttctca?tatatgttgc?cccctcaatg?tcctcatttg?tcaattgata?gatctgctcc 1560
tcatccaagc?tccatgattg?cgcatgcaaa?aatccgagtt?cagtaaaaga?tggtatagtc 1620
gagcataaga?cagtggtaaa?tttagaagtt?ttaaatgcac?ccagtaatat?tttgtggaga 1680
tgcaaggctc?aggtggtact?acttggtacc?catgaaaata?tagtggtata?gatggaattt 1740
cctccacaaa?atatgccatg?tgaaaatata?ctatgccatg?tgaaaatgac?aacaaaccta 1800
caagtttgct?attttgaaag?aatgtttatc?ttagaaagta?tagtgtgcca?cgcaaaaatg 1860
acaacatgct?tacaagtttg?ttatttaaat?taagacttag?cttgtcaagt?ataggagctt 1920
tatggtgaaa?aacaaatata?ttgacaaggc?aatgacatag?atagctagac?atatcttaat 1980
aagctcccaa?aaacacaaga?agtatgtcat?agctgagtca?tgatatgtat?aactcaattc 2040
caaaagttgc?ttttccctat?atacaatttt?gacaccacaa?gcctgatatt?gtccatagat 2100
gtaacaaact?cattttcttt?gctatataaa?ctagcttgat?ctatccttca?attcacacaa 2160
ttagctttgt?tttattagta?tatacaaaag?ct 2192
Claims (10)
1. plant endosperm specificity expression promoter, its sequence is the dna sequence dna of sequence 1 in the sequence table.
2. the expression cassette that contains the described plant endosperm specificity expression promoter of claim 1.
3. the recombinant expression vector that contains the described plant endosperm specificity expression promoter of claim 1.
4. the host bacterium that contains the described plant endosperm specificity expression promoter of claim 1.
5. the application of the described plant endosperm specificity expression promoter of claim 1 in cultivating transgenic plant, the foreign gene in the said transgenic plant is specific expressed in endosperm; Said plant is a monocotyledons.
6. application according to claim 5; It is characterized in that: the method for said cultivation transgenic plant; Be that said foreign gene is connected in said plant endosperm specificity expression promoter downstream; Change in the plant through plant expression vector, screening obtains the transgenic plant of specific expressed said foreign gene in endosperm.
7. application according to claim 6 is characterized in that: said endosperm specificity expression promoter downstream also are connected with the controlling element of regulate gene expression.
8. application according to claim 7 is characterized in that: said foreign gene is protein coding gene and/or non-protein coding gene.
9. application according to claim 8 is characterized in that: said protein coding gene is the quality-improving gene; Said non-protein coding gene is just rna gene and/or sense-rna gene.
10. according to arbitrary described application among the claim 5-9, it is characterized in that: said monocotyledons is paddy rice, wheat, corn, barley, Chinese sorghum or oat.
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| CN104232654A (en) * | 2013-06-07 | 2014-12-24 | 中国科学院植物研究所 | Expression cassette and application thereof in culturing transgenic plant having seed oil content increased |
| CN104480110B (en) * | 2013-08-30 | 2017-03-29 | 中国农业科学院生物技术研究所 | Corn tissue's specificity promoter and its application |
| CN103667291B (en) * | 2013-11-28 | 2015-09-16 | 中国科学院植物研究所 | Derive from endosperm specificity expression promoter and the application thereof of paddy rice |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US20030135884A1 (en) * | 2002-01-16 | 2003-07-17 | Su-May Yu | Transgenic seeds expressing amylopullulanase and uses therefor |
| CN101063135A (en) * | 2007-04-23 | 2007-10-31 | 中国科学院植物研究所 | Plant endosperm specificity expression promoter and its application |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20030135884A1 (en) * | 2002-01-16 | 2003-07-17 | Su-May Yu | Transgenic seeds expressing amylopullulanase and uses therefor |
| CN101063135A (en) * | 2007-04-23 | 2007-10-31 | 中国科学院植物研究所 | Plant endosperm specificity expression promoter and its application |
Non-Patent Citations (2)
| Title |
|---|
| Le Qing Qu et al..Expression pattern and activity of six glutelin gene promoters in transgenic rice.《Journal of Experimental Botany》.2008,第59卷(第9期),2417-2424. * |
| 王伟权等.水稻谷蛋白GluB-4基因启动子克隆及载体构建.《华北农学报》.2007,第22卷(第2期),22-25. * |
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