CN102250894B

CN102250894B - Specific expression promoter for plant embryo and application thereof

Info

Publication number: CN102250894B
Application number: CN 201010184251
Authority: CN
Inventors: 曲乐庆; 代玲玲
Original assignee: Institute of Botany of CAS
Current assignee: Institute of Botany of CAS
Priority date: 2010-05-20
Filing date: 2010-05-20
Publication date: 2012-12-19
Anticipated expiration: 2030-05-20
Also published as: CN102250894A

Abstract

The invention discloses a specific expression promoter for a plant embryo and application thereof. The promoter is derived from (1) a DNA (Deoxyribonucleic Acid) sequence shown as a sequence 1 in a sequence table, (2) a DNA sequence having over 70 percent of homology with the DNA sequence defined by the sequence 1 in the sequence table and having the function of a specific expression promoter for the plant embryo, and (3) a nucleotide sequence which can be hybridized with the DNA sequence defined by the sequence 1 in the sequence table under a highly serious condition. The promoter can be used for promoting specific expression of an exogenous gene in the plant embryo, and is suitable for any seed plant.

Description

A kind of plant embryo-specific is expressed promotor and application thereof

Technical field

The present invention relates to kind of plant breast specific expressing promoter and application thereof.

Background technology

The recent development of Plant Biotechnology has realized that not only the improvement of traditional economical character 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 embryo-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 growth of plant seed comprises two important processes: the embryo is taken place and seed maturity.Along with the progressively maturation of embryo, the storage in bulk product, as: protein, fat, glucide begin synthetic and accumulation, and seed is progressively ripe.The form stage of growth of fetal development has determined the development program and the one-piece construction pattern of plant individual.At present obtained certain progress for the morphology and the Physiologic Studies of seed; But because clone's seed embryo-specific gene has difficulties technically; For example; The high level expression of seed storage protein gene be easy to cover some low abundance genes information (Goldberg et al., cell 1989,56:149-160); Lack the reasons such as some particular molecule or cell marking in the embryo development procedure, at present on the molecular biology level to the understanding of seed fetal development also seldom.

Thereby; Separate, obtain seed embryo-specific expression promotor; Both helped to understand more carefully plant embryos grow in form the molecule mechanism with cytodifferentiation takes place, can be widely used in again in the plant genetic engineering research external source goal gene being efficiently expressed in specific tissue; To realize the three-dimensional accuracy controlling of timing, site-directed quantitative, in the improvement crop quality, can reduce disadvantageous effect again to plant to exogenous gene expression.

Lipid is the most effectively form of plant seed stored energy, and the lipid material in nearly all plant is mainly with triglyceride (triacylglycerols, form storage AG).Different with the TAG in the white adipose tissue, not polymeric each other between the TAG molecule of plant seed, but be dispersed into many little metastable ubcellular droplets that these little ubcellular droplets are called oil body.Half unit membrane that the oil body periphery is made up of individual layer phosphoric acid adipose membrane and oil body protein (oleosin) covers.According to different on length and sequence of the both sexes N-terminal of oil body protein and C-terminal, can with its be divided into two types of lower molecular weight (L) and HMWs (H) (Hsieh K, Huang AHC.Plant Physiol.2004,136:3427-34).18KDa that contains in the rice paddy seed embryo and 16KDa oil body protein belong to H type and L type (Tzen et al., Plant Physiol.1998,94:1282-1289 respectively; Wu et al., J.Biochem.1998,123 (3): 386-391).(Transgenic Res.2006 such as Junko; 15:95-100) utilize EMBRYO IN RICE specific expressing promoter Ole18 (Qu and Takaiwa, Plant Biotech.J., 2004; 2:113-125) driving linoleate isomerase gene expresses in transgenic paddy rice; Not only improve the linoleic acid content in the rice paddy seed, and strengthened the anti-cancer and the arteriosclerosis function of rice, embodied the great application potential of EMBRYO IN RICE specific expressing promoter.

Summary of the invention

The purpose of this invention is to provide a kind of plant embryo-specific and express promotor and application thereof.

Plant embryo-specific provided by the present invention is expressed promotor, derives from the upstream sequence of paddy rice 16 kDa oleosin genes, 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 have the dna sequence dna of plant embryo-specific 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 at 0.1 * SSPE (or in the solution of 0.1 * SSC), 0.1% SDS, hybridizes under 65 ℃ and washes film.

Wherein, sequence 1 is made up of 1438 deoxynucleotides in the sequence table.

Above-mentioned in sequence table the 1295th～1300 Nucleotide of 5 ' end of sequence 1 be that plant embryo-specific is expressed promotor 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 embryo-specific expression promotor all belong to protection scope of the present invention.

In said expression cassette, said plant embryo-specific is expressed the downstream syndeton gene of promotor, 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 that the constructed said recombinant expression vector of recombinant vectors of above-mentioned expression cassette and plasmid, virus or vector 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 embryo-specific is expressed the plant that promotor can be used for cultivating the embryo-specific expression foreign gene.

The method of utilizing this plant embryo-specific to express the plant of promotor cultivation embryo-specific expression foreign gene also belongs to protection scope of the present invention.

The method of the plant of said cultivation embryo-specific expression foreign gene can be said foreign gene is connected in said plant embryo-specific expression promotor downstream; Change in the plant through plant expression vector, screening obtains the transgenic plant of specific expressed said foreign gene in embryo.

Said plant embryo-specific is expressed the controlling element that the promotor downstream also can be connected with 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 is protein coding gene and/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 dicotyledons.

Said monocotyledons is paddy rice, wheat, corn, barley, Chinese sorghum, oat etc.; Said dicotyledons is soybean, rape, Sunflower Receptacle etc.

It is through the design primer that plant embryo-specific of the present invention is expressed promotor, is template with the oryza sativa genomic dna, the oil body protein gene 16 kDa oleosin promotors 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 embryo.Explain that promotor of the present invention can start foreign gene specific expression in the embryo of plant, is applicable to that any seed has the plant of embryo, i.e. monocotyledons or dicotyledons.Utilize promotor of the present invention can improve expression and the accumulation level of foreign gene in plant embryos; 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 be applied as the process and the research of mechanism that be to disclose the whole plants fetal development utilizes researchs such as bio-technology improvement seed quality, the medical farm of molecule to lay a good foundation, and has great application prospect.

Description of drawings

Fig. 1 is the electrophoretogram of pcr amplification 16 kDa oleosin promotors from oryza sativa genomic dna.

Fig. 2 is the structural representation that 16 kDa oleosin promotors merge gus gene expression vector oleP-pGPTV-35S-HPT.

Fig. 3 is that oleP-pGPTV-35S-HPT carrier double digestion is identified collection of illustrative plates.

Fig. 4 is for changeing oleP-pGPTV-35S-HPT carrier T ₀For rice plant chimeric primers pcr amplification electrophoretogram.

Fig. 5 is for changeing oleP-pGPTV-35S-HPT carrier T ₀For rice plant Southern results of hybridization.

Fig. 6 is for changeing oleP-pGPTV-35S-HPT carrier T ₀For rice plant root, stem, leaf sheath and blade GUS coloration result.

Fig. 7 is for changeing oleP-pGPTV-35S-HPT carrier T ₀Blooming back 7 days for rice plant, 12 days, the GUS coloration result of 17 days seeds.

Fig. 8 is for changeing oleP-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.

Embodiment 1, plant embryo-specific are expressed the acquisition of promotor (16 kDa oleosin promotor)

According to paddy rice 16 kDa oleosin gene cDNA sequences (GenBank number is L76464), from GenBank, search the upstream sequence of 16 kDa oleosin genes, design primer amplification 16 kDa oleosin promotors.For ease of vector construction, on every pair of primer, add two restriction enzyme sites (shown in the underscore) successively.The forward primer of 16kDa oleosin promotor is F1:5 '-ACC AAGCTTCGCTGATGTGGTATCATAG-3 ' (Hind III) and reverse primer R1:5 ' AA GTCGACGGGGATCGAGCACACCTGCAG-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 F1 and R1, carries out pcr amplification 16 kDa oleosin promoter sequences.Reaction system is: each 1 μ l of the forward and reverse primer of 10 μ M, and 2 * GC Buffer, 10 μ l, genomic dna 1 μ l (10ng), LATaq (TAKARA) 0.5 unit 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 1min30sec, 30 circulations, last 72 ℃, 10min.Pcr amplification obtains the purpose band of 1.5kb, and is as shown in Figure 1.The 1st swimming lane is a molecular weight standard among Fig. 1, and the 2nd swimming lane is 16 kDa oleosin promotors.Reclaim amplified production, be directly connected on the pT7Blue carrier (available from Novagen company) and check order.The result shows that the 16 kDa oleosin promoter sequences size that amplification obtains is 1438bp, the nucleotide sequence with sequence 1 in the sequence table.The order-checking detection is shown the recombinant vectors called after pT7-oleP that contains 16 kDa oleosin promoter fragments.

Embodiment 2, plant embryo-specific are expressed promotor (16 kDa oleosin promotor) expression vector establishment and genetic transformation

1,16KDa oleosin promotor merges the plant expression vector construction of gus gene

(2:113-125) described method makes up the pGPTV-35S-HPT reference literature for Qu and Takaiwa, Plant Biotech J 2004, and Institute of Botany, Chinese Academy of Sciences preserves.With Hind III and Sal I double digestion pT7-oleP plasmid and binary expression vector pGPTV-35S-HPT.Recovery contains the endonuclease bamhi of the 16 kDa oleosin promotors of 1438bp, this fragment is connected between the Hind III and Sal I enzyme recognition site of pGPTV-35S-HPT.On the basis that PCR identifies, utilizing Hind III and Sal I to carry out double digestion the recombinant plasmid that obtains identifies, obtains the fragment of the 16 kDa oleosin promotors of 1.4kb.Enzyme is cut evaluation show the recombinant plasmid called after oelP-pGPTV-35S-HPT (its structural representation is as shown in Figure 2) that contains 16 kDa oleosin promotors.Wherein, the double digestion of oelP-pGPTV-35S-HPT is identified collection of illustrative plates, and is as shown in Figure 3.Swimming lane 1 is a dna molecular amount mark among Fig. 3, and

swimming lane

2 and 3 is the fragment that oelP-pGPTV-35S-HPT Hind III and Sal I double digestion obtain.

2, change the acquisition of oleP-pGPTV-35S-HPT paddy rice

With freeze-thaw method oelP-pGPTV-35S-HPT is imported Agrobacterium EHA105 (Hood et al., Transgen Res1993,2:208-218; 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 9 strain T ₀In generation, changeed the oelP-pGPTV-35S-HPT rice plant.

Utilize PCR method that the commentaries on classics oelP-pGPTV-35S-HPT paddy rice that above-mentioned screening obtains is carried out the PCR Molecular Detection; The PCR primer is the chimeric primers of 16 kDa oleosin promoter sequences and gus gene sequence; That is: the inner forward primer P1:5 ' of 16 kDaoleosin promoter sequences-gatgaacttgttccttcagttacacg-3 ' and inner reverse primer the p2:5 '-cgtgacatcggcttcaaatggcgta-3 ' of GUS sequence.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) 0.5 unit 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 1min30sec, 30 circulations, last 72 ℃, 10min.The purpose band that pcr amplification obtains 0.8kb is the detection positive, and the result shows that obtaining 9 strain PCR detects male commentaries on classics oelP-pGPTV-35S-HPT paddy rice T ₀For plant (as shown in Figure 4).The 1st swimming lane is a molecular weight standard among Fig. 4, and the 2nd swimming lane is the positive control of template with the plasmid, and the 3rd swimming lane is that the 4th～11 swimming lane is respectively the T that changes the oelP-pGPTV-35S-HPT plant expression vector with the negative control of unconverted strain system as template 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 Southern hybridization of embodiment 3, transgenic rice plant detects

Extract PCR with the CTAB method and identify the T that is positive ₀Genomic dna for the paddy rice rotaring gene plant blade; About 40 μ g DNA add after the 50 unit abundant enzymes of EcoR I cut; After 0.8% agarose gel electrophoresis separates, DNA is transferred on the nylon membrane (available from Amersham pharmacia company) that positively charged examines with the NaOH of 0.4N.Film after the transfer 65 ℃ of prehybridizations 5 hours in the 0.5M sodium phosphate buffer that contains 7% (W/V) SDS carry out probe mark (available from Chinese Fu Rui company) with (α-32P) dCTP random priming.Employed hybridization probe is to be positioned at the inner 441bp dna fragmentation of GUS (sequence is for to be the 1344-1784 position nucleotide sequence of S69414.1 from GENBANK number; With the pGPTV-35S-HPT plasmid is masterplate, and 5 '-CTGCGACGCTCACACCGATACC-3 ' and 5 '-TCACCGAAGTTCATGCCAGTCCAG-3 ' is the pcr amplification product of primer) be probe.65 ℃ of hybridization were washed film twice in 65 ℃ after 20 hours in containing 2 * SSC of 0.1%SDS, each 15 minutes, in 1 * SSC of 0.1%SDS, wash film once, 15 minutes in 65 ℃ again.Press the X-ray sheet after 24～48 hours, radioactive automatic developing.The Southern results of hybridization shows; Can both detect the hybridization band in each transgenic line; And in different strain systems; The number of hybridization band and the position incomplete same (as shown in Figure 5) of appearance, this shows that the oelP-pGPTV-35S-HPT expression structure successfully is incorporated in the rice genome, and is separate conversion individuality between each transgenic line.Among Fig. 5, the 1st swimming lane is the contrast of non-transgenic plant, and 2～10 swimming lanes are for changeing the oelP-pGPTV-35S-HPT plant.

Embodiment 4, plant embryo-specific are expressed the functional verification of promotor (16 kDa oleosin promotor)

To changeing oleP-pGPTV-35S-HPT paddy rice T ₀Carry out histochemical stain for plant.Concrete steps: will change oleP-pGPTV-35S-HPT carrier T ₀Partial blade, root, cane tissue for rice plant and contrast wild-type paddy rice (Kitaake) plant are cut into small pieces; The filling stage seed that to bloom back 7 days, 12 days, 17 days is with scalper longitudinal incision from the middle part.The sample of handling well is soaked in GUS reaction solution (0.1M NaPO ₄Damping fluid, pH 7.0,10mMEDTA, pH7.0, the 5mM Tripotassium iron hexacyanide, 5mM yellow prussiate of potash, 1.0mM X-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, changes root, blade, leaf sheath, the cane of oelP-pGPTV-35S-HPT carrier rice plant and does not all observe GUS expression (as shown in Figure 6); The seed aleurone layer of blooming back 7 days, inferior aleurone layer become blue, and endosperm and embryo are not dyed blueness; The back 12 days seed of blooming is compared with the back 7 days seed of blooming, and aleurone layer, inferior aleurone layer blueness are more obvious, embryo becomes blue, and endosperm is not dyed blueness; The seed aleurone layer of blooming back 17 days, inferior aleurone layer and whole embryo blue high-visible compared with the back 12 days seed of blooming, and the blueness of embryo is darker; And contrast wild-type paddy rice (Kitaake) root, blade, leaf sheath, cane and the filling stage seed of blooming back 7 days, 12 days, 17 days are not all observed GUS and are expressed (as shown in Figure 7).The result shows that 16 kDa oleosin promotors make beta-glucuronidase (GUS) reporter gene only specific expressed in the embryo of rice paddy seed and aleurone layer.Among Fig. 6,1,2,3 are respectively the coloration result of transfer-gen plant root, stem, leaf sheath and blade.Among Fig. 7, the filling stage seed coloration result that ck is bloomed back 17 days for contrast wild-type paddy rice (Kitaake); 7d, 12d, 17d represent to change oelP-pGPTV-35S-HPT carrier T respectively ₀For the bloom GUS coloration result of back 7 days, 12 days, 17 day filling stage seed of rice plant.

The GUS fluorescence activity of organizing of embodiment 5, transgenic paddy rice is measured

To changeing oleP-pGPTV-35S-HPT carrier T ₀The filling stage seed of blooming back 17 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, 10mMNa ₂EDTA (pH 8.0), 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.The result shows that the activity that 16 KDa oleosin promoter-driven GUS are expressed is 7.7 ± 3.5pmol 4MU/min/ μ g albumen (as shown in Figure 8) in transgenic paddy rice seed.Among Fig. 8,1-8 is respectively the different oleP-pGPTV-35S-HPT of commentaries on classics carrier T ₀For GUS expression activity in the rice plant seed: 1 is 6.4pmol 4MU/min/ μ g albumen; 2 is 11.8pmol 4MU/min/ μ g albumen, and 3 is 11.7pmol 4MU/min/ μ g albumen, and 4 is 4.2pmol 4MU/min/ μ g albumen; 5 is 1.7pmol4MU/min/ μ g albumen; 6 is 7.3pmol 4MU/min/ μ g albumen, and 7 is 8.8pmol 4MU/min/ μ g albumen, and 8 is 9.6pmol 4MU/min/ μ g albumen.

Sequence table

<160>1

<210>1

<211>1438

<212>DNA

< 213>Oryza paddy rice (Oryza sativa)

<400>1

ttcgctgatg?tggtatcata?gtaagcaaaa?taaaaaataa?aaaaatatga?gacccacatg 60

taagtctctt?ctttcttctc?tattcttctc?tccgctattc?tcttttcttc?ttctctacta 120

ttttcttcag?cgatggcata?gaggagatag?gccggagtgg?cagagagggg?agaggcaagt 180

gggcgagcat?ggctgagctc?tccaccgcct?caagaggctc?cgcacccgtt?gccgctcgcg 240

accgaggagg?aggagaagag?gggtgggggt?gggggggggg?ggggaggctg?ccggcgggaa 300

atgagggagg?ggagaggcgc?gggacggcac?catagctagc?cttgccgcat?gtgggttcac 360

cgcggaaggg?cgcagccgcc?gccgccgtct?ccgggtgacg?acttggacca?tccacaacga 420

ggtgccttcc?gacgccctcg?ccttccttgt?cgtgccgtgc?gaggtcccct?tccgtgaagt 480

aagggtacgg?ctagcagaga?atgtcggtgc?ctctgctgcc?accatcactg?cagtcaggta 540

cagcctcgcg?cgacgaggac?ctgttggtgt?cggtctcctt?cgatgaggag?ctcgcccaca 600

tgcgcgacga?gtacgaccgt?ctcagggcaa?tgcatccgtt?cgcgagcttc?cagctgttcg 660

tctccaccgc?tgttgcttat?acctcctccc?gtcgcgaggc?gctcgaggtg?gcggagttcg 720

gccgggctcg?acggcggcct?ctcccctctc?tgccgctgcc?ggcctatccc?ctctttgcta 780

tcgctgaaga?gaagagaaga?gaagaagaaa?agagaagaga?ggagaaaaga?aagaagagac 840

ttatatgtgg?gttccataat?ttattttttt?aattttgctg?actagaataa?catgtcagcg 900

aaacccgaaa?ccagagatct?atactgccgt?ggaatcgaaa?ttgcacggtt?ttatatagtt 960

tagggataaa?gatttctggt?tttggggttt?cgggatgtca?tacaaacttg?tcgtaaagtt 1020

gagggatgac?tgatgaactt?gttccttcag?ttacacgcgc?acgcatggtg?atacggccca 1080

cgagagatcg?ggcccaatac?aggcagcggc?ggccgaatgc?agcagcgccc?gtaacctcgt 1140

acgtatcgcc?tcgtgggaag?cggccgaggc?cgcgcgcggc?ggcaaccaca?gcaaaccaca 1200

ggtgtcgctc?ggcgtccgac?acgagtcccg?catgcgcccc?acgcggcggc?tccagcaccc 1260

gcctccacgc?cgccccatgc?acggcgcctc?cccctataaa?ccccaccctc?ttctccctcc 1320

tcaccgtcgt?cagtccactt?ctcactagct?cgtagacagt?gctgcacgtg?ggttagctac 1380

ttagctcttt?ctctgcattg?ctggcttaat?tttgcagctg?caggtgtgct?cgatcccc 1438

Claims

1. a plant embryo-specific is expressed promotor, is the dna sequence dna of sequence 1 in the sequence table.

2. contain the expression cassette that the described plant embryo-specific of claim 1 is expressed promotor.

3. contain the recombinant expression vector that the described plant embryo-specific of claim 1 is expressed promotor.

4. contain the host bacterium that the described plant embryo-specific of claim 1 is expressed promotor.

5. the described plant embryo-specific of claim 1 is expressed the application of promotor in cultivating transgenic plant, and the foreign gene in the said transgenic plant is specific expressed in embryo;

Said plant is a monocotyledons.

6. application according to claim 5; It is characterized in that: said cultivation transgenic plant; Be said foreign gene to be connected in said plant embryo-specific express the promotor downstream; Change in the plant through plant expression vector, screening obtains the transgenic plant of specific expressed said foreign gene in embryo.

7. application according to claim 6 is characterized in that: said embryo-specific expression promotor 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; Said protein coding gene is the quality-improving gene; Said non-protein coding gene is just rna gene and/or sense-rna gene.

9. application according to claim 5 is characterized in that: said monocotyledons is paddy rice, wheat, corn, barley, Chinese sorghum or oat.