CN107338248A - Seed specific expression system and its application - Google Patents

Abstract

The invention provides the DNA of separation, and it has SEQ ID NO：Sequence or similar sequence shown in 1.The DNA can instruct to be operatively coupled on nucleic acid specific transcription and/or expression in vegetable seeds downstream.Present invention also offers the expression cassette comprising the DNA and genetically modified plants etc..

Description

Seed specific expression system and its application

Invention field

The invention belongs to plant transgene breeding field, and specifically, the present invention relates to the DNA of separation, and it can instruct to grasp It is connected to nucleic acid specific transcription and/or expression in vegetable seeds downstream with making.In addition, the invention further relates to include the DNA Expression cassette and plant etc., and be related to the application of the DNA.

Background technology

Although the genome sequence of many plants (e.g., rice, especially Oryza sativa.japonica etc.) has been revealed (such as Reference can be made to GenBank accession number AP003843.3, GenBank accession number AP004670.4 etc.), but lots of genes group sequence The function of row is still unknown.In addition, U.S. Patent application US2006075523A1 disclose it is non-from rice and other plant Biological stress response related polypeptide and polynucleotides, wherein polynucleotide sequence SEQ ID NO：15442 are up to 2000 Nucleotides, and the sequence is submerged in numerous sequences disclosed in the document and is not designated specific function；And United States Patent (USP) US7365185B2 discloses the genomic promoter sequence from rice and other plant, but wherein polynucleotide sequence SEQ ID NO：68961 are up to 2760 nucleotides, and the sequence is submerged in numerous sequences disclosed in the document and is not designated Specific function, does not know whether it has the specificity of plant expressive site more.

Exogenous DNA array starts the expression in plant host, the choosing of promoter and enhancer by being connected to specific promoter Select the expression time for determining gene and position.At present in the strong of wide variety of mainly some composing types of agricultural biological technical field Promoter, such as CaMV 35S promoters and corn Ubiquitin-1 promoters, but utilizing these promoters induction purpose The crops such as genetic transformation rice to Crop Improvement quality when, often due to time of destination gene expression, (stage of development is special The opposite sex) or space (tissue and organ specificity) can not well control and cause improved effect unobvious, or due to these groups Constitutive promoter inducible gene expression amount is too high and growing for plant is impacted, and these are all strong currently with composing type Promoter binding function gene carrys out the obstacle run into during crop improvement quality.

In addition, when studying some metabolic processes or regulation approach, it is often necessary to by more than two genetic transformation in same approach Into same strain, another gene, or two are converted again using converting after one of gene obtains transfer-gen plant Individual gene is hybridized again after the completion of converting respectively, is required for waiting longer time, is turned to improve the multiple genes of efficiency reduction The time of change, have been reported that using new carrier while can carry out the conversion of multiple genes recently, but when polygenes converts such as Fruit reuses same promoter, also due to the high homology of promoter sequence may cause gene silencing.

Seed specific expression system can be used for research gene or regulating and controlling sequence to seed grow or the Main Agronomic Characters such as yield Influence, reality production application in, obtain new Seeds oil-body-specific promoter, it is significant to production application.

Therefore, the present inventor is by studying for a long period of time, in fact it has surprisingly been found that a kind of new seed specific promoters, it is being planted Stable specifically expressing in the seed of thing, for agriculture genetic engineering development from now on, there is provided more alternative.

Summary of the invention

The invention provides the DNA of new separation, and it has the function of seed-specific expression promoter.In addition, the present invention is also It is related to the expression cassette comprising the DNA and genetically modified plants etc., and be related to the application of the DNA etc..

Specifically, in a first aspect, the invention provides the DNA of separation, it can instruct to be operatively coupled on downstream Nucleic acid in vegetable seeds specific transcription and/or expression, the DNA of separation sequence is selected from following group of sequence：

(a) there is SEQ ID NO：Sequence shown in 1；

(b) under strict conditions can be with the DNA sequence dna of the DNA hybridization of sequence (a) described；

(c) there is the DNA sequence dna of at least 90% (being preferably at least 95%) sequence identity with (a) described sequence；

(d) SEQ ID NO are included：The DNA sequence dna of (preferably at least 200) continuous nucleotide at least 150 in 1；With

(e) DNA sequence dna complementary with (a)-(d) any sequence.

It is preferred that the DNA of first aspect present invention, its sequence length is less than 2000 nucleotides, preferably smaller than 1500 nucleotides, More preferably less than 1200 nucleotides.

It is preferred that the DNA of first aspect present invention, the nucleic acid that it can instruct to be operatively coupled on downstream is special in vegetable seeds Different transcription and/or expression.

In the specific embodiment of the present invention, the DNA sequence dna such as SEQ ID NO of first aspect present invention：Shown in 1.

In second aspect, the invention provides expression cassette, and it is included：

(a) DNA of first aspect present invention；With

(b) nucleic acid, it is operatively coupled on the DNA of first aspect present invention downstream. It is preferred that the expression cassette of second aspect of the present invention, wherein the nucleic acid can assign the character that plant is selected from following group：

(a) raising of the protein output of the encoded by nucleic acid；

(b) raising of the inhibitory RNA content of the encoded by nucleic acid；

(c) raising of anti-plant pest ability, the raising of pest and disease damage ability of the especially anti-generation in vegetable seeds；

(d) improvement of nutritional quality, including in plant naturally with nutrient composition content raising and plant non-natural with nutrition The imparting of composition；With

(e) raising of the raising of plant products, especially plant economy position such as seed production.

In the third aspect, the invention provides the plant of the transgenosis converted with the expression cassette of second aspect of the present invention, vegetable seeds, Plant tissue or plant cell, preferably wherein, the DNA of first aspect present invention can instruct to be operatively coupled on downstream Nucleic acid in vegetable seeds specific transcription and/or expression.

It is preferred that the plant of third aspect present invention, vegetable seeds, plant tissue or plant cell, wherein the plant is unifacial leaf Plant or dicotyledon, e.g., corn, rice, wheat, barley, sorghum, soybean, rape, cotton, tomato, Ma Ling Potato, sugarcane, beet, tobacco, rape or arabidopsis.

In fourth aspect, the invention provides the production method of the plant of third aspect present invention, and it includes：

(1) expression cassette of second aspect of the present invention is built；

(2) expression cassette for obtaining step (1) imports plant cell；

(3) genetically modified plants are regenerated；With

(4) genetically modified plants are selected, the wherein DNA of first aspect present invention can instruct to be operatively coupled on core downstream Acid is in vegetable seeds specific transcription and/or expression；And

(5) optionally, the plant that amplification step (4) obtains is to obtain offspring.

At the 5th aspect, present invention also offers the method for assigning plant trait, it includes

(1) selection can assign the nucleic acid of plant trait：

(2) expression cassette of the nucleic acid construct second aspect of the present invention obtained using step (1)；

(3) expression cassette for obtaining step (2) imports plant cell；

(4) genetically modified plants are regenerated；With

(5) genetically modified plants for imparting plant trait are selected；And

(6) optionally, amplification step (5) obtain plant to obtain offspring,

Wherein, the character is selected from following group：

(a) raising of the protein output of the encoded by nucleic acid；

(b) raising of the inhibitory RNA content of the encoded by nucleic acid；

(c) raising of anti-plant pest ability, the especially anti-raising occurred in vegetable seeds pest and disease damage ability；

(d) improvement of nutritional quality, including in plant naturally with nutrient composition content raising and plant non-natural with nutrition The imparting of composition；With

(e) raising of the raising of plant products, especially plant economy position yield.

Brief description of the drawings

Fig. 1 is the plant expression vector for the arabidopsis AT4G28520 gene promoters driving GFP genes that the present invention is built, its Middle LB and RB are respectively T-DNA left margin and right margin；Hpt represents hygromycin gene；Cam35SP is represented The promoter of CamV35S genes；NOS represents the terminator of no genes；GFP represents GFP albumen (green fluorescent protein) Gene；3A represents the terminator of 35S genes；520P is the AT4G28520 promoters that embodiment 1 obtains.

Fig. 2 is observation result of the GFP genes of AT4G28520 promoters driving of the present invention in transgenic arabidopsis seed.It is left Figure is images of the T1 for transgenic arabidopsis seed under white transmitted light, and right figure is that T1 swashs for transgenic arabidopsis seed ultraviolet Image under luminous.

Detailed description of the invention

In a first aspect, the invention provides the DNA of separation, its nucleic acid that can instruct to be operatively coupled on downstream is being planted The sub- specific transcription of species and/or expression, the nucleic acid for being especially their ability to instruct to be operatively coupled on downstream are special in vegetable seeds Different transcription and/or expression.

Herein, the DNA of this separation can also be referred to as seed specific promoters, belong to cis-acting elements.Planting Under the driving of sub- specificity promoter, seed this device is often only limited to downstream and with the expression of its gene being operably connected Official or its tissue site, and show the characteristics such as growth adjustment.Seed specific promoters can not only make the expression product of target gene Mainly accumulated in vegetable seeds or its tissue site, such as the nucleic acid mRNA in caused downstream amount is in seed or its tissue 5 times, 10 times, 100 times, 200 times, even 1000 times of other plant body region, increase Zonal expression amount, while also may be used To avoid the unnecessary waste of plant nutrient.It is direct by the observable decoration method of naked eyes in the embodiment of the present invention Differentiate that the seed specific promoters of the present invention instruct transcription and/or expression in vegetable seeds, and the regulation and control continue in development of plants Each stage, and at other positions of plant, the transcription and/or expression that can substantially be differentiated without naked eyes.

Herein, term " being operably connected " refers to a kind of connected mode, and which causes the nucleic acid for being connected to downstream Transcription and/or expression by the present invention seed specific promoters initial and regulate and control.Generally, the nucleic acid being operably connected is to connect Continuous.The nucleic acid in downstream can be spaced a small amount of nucleotides in the case where keeping frame and be connected under seed specific promoters Trip, receives its regulation and control.

Herein, term " separation " DNA refers to DNA from its natural environment (e.g., the genome of plant cell) In be separated, or be synthesized chemically or recombinantly express and come out, and DNA is free of or be substantially free of its source thing Other nucleic acid, cell and the culture medium of kind.The DNA of the separation of the present invention has shorter nucleotide sequence, preferred sequence length Less than 2000 nucleotides, preferably smaller than 1500 nucleotides, more preferably less than 1200 nucleotides.In the specific of the present invention In embodiment, the DNA of separation of the invention is the SEQ ID NO with 1921 nucleotides：Sequence shown in 1.

The DNA of the separation of the present invention also includes having SEQ ID NO：The DNA of sequence shown in 1 function equivalence body, i.e., With SEQ ID NO：The DNA of 1 variant sequence thereof, its nucleic acid for being still able to instruct to be operatively coupled on downstream are being planted The sub- specific transcription of species and/or the SEQ ID NO of expression：Sequence shown in 1.Variant sequence thereof includes under strict conditions can be with With SEQ ID NO：The DNA sequence dna of the DNA hybridization of sequence shown in 1." stringent condition " used herein is public Know, including in 60 DEG C such as in NaCl containing 400mM, 40mM PIPES (pH6.4) and 1mM EDTA hybridization solution Hybridize 12-16 hours, then wash 15-60 minutes with the cleaning solution containing 0.1SDS and 0.1%SSC at 65 DEG C.

Variant sequence thereof also includes and SEQ ID NO：Sequence shown in 1 has at least 90%, 95%, 96%, 97%, 98% or 99% The DNA sequence dna of sequence identity.Wherein, the percentage of sequence identity can be obtained by known bioinformatics , including Myers and Miller algorithms (Bioinformatics, 4 (1)：11-17,1988), Needleman-Wunsch is global Comparison Method (J.Mol.Biol., 48 (3)：443-53,1970), Smith-Waterman Local Alignments method (J.Mol.Biol., 147： 195-197,1981), Pearson and Lipman similarity-searching (PNAS, 85 (8)：2444-2448,1988), Karlin With Altschul algorithm (Altschul etc., J.Mol.Biol., 215 (3)：403-410,1990；PNAS, 90：5873-5877, 1993).This is known to those skilled in the art.

Variant sequence thereof also includes the SEQ ID NO that can keep seed specific promoters function：The fragment of sequence shown in 1, Such as SEQ ID NO：At least 150 in 1,200,300,500,800, until 999 continuous nucleosides The DNA sequence dna of acid.

In second aspect, the invention provides expression cassette, and it is included：

(a) DNA of first aspect present invention；With

(b) nucleic acid, it is operatively coupled on the DNA of first aspect present invention downstream.

The present invention expression cassette on 5 ' -3 ' transcriptional orientation comprising first aspect present invention DNA, be operatively coupled on this hair Terminator (e.g., transcription terminator element or the poly of the nucleic acid in the DNA of bright first aspect downstream, optional transcription and translation Polyadenylation signal).The expression cassette of the present invention can also include, and required replication orgin is replicated in bacterium (for example, coming from PBR322 or P15A ori ORI areas), the element required for agrobacterium T-DNA transfers is (for example, a T-DNA left side Border and/or right margin).The other compositions that expression cassette of the present invention can include include, enhancer, introne, multiple cloning sites, Operator, repressor binding site, Binding site for transcription factor etc..Exemplary enhancer includes opening from CaMV 35S Mover, octopine synthase genes, rice actin I gene, maize alcohol dehydrogenase gene, Maize Dwarf I gene, TMV Ω The enhancer element of the promoter of element and yeast.Foresight with enhancer effectiveness can also be used as using viral leader sequence, Such as come from the targeting sequencing of tobacco mosaic virus (TMV) (TMV), Maize Chlorotic mottle virus (MCMV) and alfalfa mosaic virus (AMV) Deng.Exemplary plant introne include from Adh 1, bronze 1, actin 1, actin 2 introne, with And crose synthase intron.

Herein, the nucleic acid for being operatively coupled on the DNA of first aspect present invention downstream encodes more nucleosides of different first albumen Acid, the polynucleotides of encoding fusion protein, antisense sequences, the polynucleotides, etc. for encoding dsRNA sequences.It is preferably capable of The nucleic acid of the beneficial character of plant (especially vegetable seeds) is assigned, the character includes：

(a) raising of the protein output of the encoded by nucleic acid；

(b) raising of the inhibitory RNA content of the encoded by nucleic acid；

(c) raising of anti-plant pest ability, the especially anti-raising occurred in vegetable seeds pest and disease damage ability；

(d) improvement of nutritional quality, including in plant naturally with nutrient composition content raising and plant non-natural with nutrition The imparting of composition；With

(e) raising of the raising of plant products, especially plant economy position yield.

Correspondingly, the nucleic acid for being operatively coupled on the DNA of first aspect present invention downstream can be with the gene of encoding proteins, such as Insect-resistance gene, bacterial disease resistant gene, fungal disease resistant gene, virus disease resistant gene, nematode diseases resistance Gene, herbicide resistance gene, the gene for influenceing seed constituent or quality, nutrien utilization gene, mycotoxin reduce base Cause, male sterility gene, selectable marker gene, can selection markers thing gene, fluorescent marker gene, negative mark may be selected Remember thing gene, Positive selectable markers gene, the gene for influenceing Plant Agronomic Characteristics (such as yield), tolerance to environmental stress gene (example Such as, assign to arid, heat, cold, freezing, overly moist, salt stress or the resistance of oxidative stress or the gene of patience), improve form sediment Gene that powder characteristic or quantity, oil product quantity or quality, amino acid or protein form, etc..In addition, it is operably connected Nucleic acid in the DNA of first aspect present invention downstream can also encode dsRNA, antisense RNA, siRNA, miRNA, (as, it is necessary to lower the plant promoter of expression, the survival of parasitic plant body biology, growth or breeding institute are necessary with target DNA for it Plant gene) it is complementary and cause the expression of these polynucleotides to be lowered.

The present invention expression cassette can be inserted into plasmid, clay, yeast artificial chromosome, bacterial artificial chromosome or other be adapted to turn Change into any carrier in host cell.Preferable host cell is bacterial cell, in particular for cloning or storing more nucleosides Acid or the bacterial cell for converting plant cell, such as Escherichia coli, Agrobacterium tumdfaciens and Agrobacterium rhizogenes.Work as place When chief cell is plant cell, expression cassette or carrier can be inserted into the genome for the plant cell being converted.Insertion can be fixed Position or random insertion.Preferably, such as homologous recombination is inserted through to realize.In addition, expression cassette or carrier are positively retained at Outside chromosome.The expression cassette or carrier of the present invention may be present in core, chloroplaset, mitochondria and/or the plastid of plant cell.It is excellent Selection of land, expression cassette of the invention or carrier are inserted into the chromosomal DNA of plant nucleolus.

In the third aspect, the invention provides the plant of the transgenosis converted with the expression cassette of second aspect of the present invention, vegetable seeds, Plant tissue or plant cell.

The embodiment of the present invention in two representational monocotyledons and dicotyledon by having effect It can use with the seed specific promoters of the plant use range for the seed specific promoters for demonstrating the present invention, therefore the present invention In any plant species of conversion, including monocotyledon and dicotyledon, such as from the plant of subordinate：Clover category, kind Solanum, Btassica, Cucumis, Solanum, Juglans, Gossypium, Malus, Vitis, antirrhinum, Populus, Fragaria, Arabidopsis, Picea, Capsicum, Chenopodium, Chrysanthemum, ipomoea, Pinus, Pisum, Oryza, Zea, wheat Category, triticale category, Secale, Lolium, Hordeum, Glycine, Pseudotsuga, Bryophyllum, Beta, sunflower Category, Nicotiana, Cucurbita, Rosa, Fragaria, Lotus, the careless category of donkey food, Clover, Trigonella, cowpea Category, tangerine category, linum, Geranium, cassava, Trigonella, Rhaphanus, sinapsis alba category, Atropa, Datura, Hyoscyamus, Nicotiana, petunia juss, Digitalis, Cichorium, Lactuca, Brome, Asparagus, antirrhinum, Hemerocallis, Narcissus, Pelargonium, millet category, Pennisetum, Ranunculus, Senecio, loudspeaker tongue category, Lan Ying flower category, Phaseolus, Avena and allium, preferable plant include corn, rice, wheat, barley, sorghum, soybean, rape, cotton Flower, tomato, potato, sugarcane, beet, tobacco or arabidopsis, more preferably rice.

In fourth aspect, the invention provides the production method of the plant of third aspect present invention, and it includes：

(1) expression cassette of second aspect of the present invention is built；

(2) expression cassette for obtaining step (1) imports plant cell；

(3) genetically modified plants are regenerated；With

(4) genetically modified plants are selected, the wherein DNA of first aspect present invention can instruct to be operatively coupled on core downstream Acid is in vegetable seeds specific transcription and/or expression；And

(5) optionally, the plant that amplification step (4) obtains is to obtain offspring.

The genetically modified plants of the present invention use method for transformation known to plant biotechnology field technical staff to prepare.Any method can It is used to recombinant expression carrier being transformed into plant cell, to produce the genetically modified plants of the present invention.Method for transformation may include directly Connect and indirect method for transformation.Suitable direct method include the DNA intakes of polyethylene glycol induction, liposome-mediated conversion, Imported using particle gun, electroporation and microinjection, etc..In the embodiment of the present invention, present invention uses Transformation technology based on agrobacterium is (reference can be made to Horsch RB etc. (1985) Science 225：1229；White FF, Vectors for Gene Transfer in Higher Plants, Transgenic Plants, volume 1, Engineering and Utilization, Academic Press, 1993, pp.15-38；.Techniques for Gene Transfer, the Transgenic Plants such as Jenes B, Volume 1, Engineering and Utilization, Academic Press, 1993, pp.128-143, etc.).Agrobacterium bacterium Strain (such as Agrobacterium tumdfaciens or Agrobacterium rhizogenes) includes plasmid (Ti or Ri plasmids) and T-DNA elements, the plasmid and member Part is transferred to plant after with Agrobacterium transfection, and T-DNA is integrated into the genome of plant cell.T-DNA can On Ri- plasmids or Ti- plasmids, or it is included separately in so-called binary vector.Agrobacterium-mediated method for transformation is retouched In being set forth in for example.Agrobacterium-mediated conversion is best suitable for dicotyledon, but also is adapted for monocotyledon.Agrobacterium pair During the conversion of plant is described in for example.Conversion can cause instantaneous or stable conversion and expression.Although the nucleotide sequence of the present invention It can be inserted into and fall into any plant and plant cell in these broad varieties, the crop plants cell but it is particularly suitable for use in.

At the 5th aspect, present invention also offers the method for assigning plant trait, it includes

(1) selection can assign the nucleic acid of plant trait：

(2) expression cassette of the nucleic acid construct second aspect of the present invention obtained using step (1)；

(3) expression cassette for obtaining step (2) imports plant cell；

(4) genetically modified plants are regenerated；With

(5) genetically modified plants for imparting plant trait are selected；And

(6) optionally, amplification step (5) obtain plant to obtain offspring,

Wherein, the character is selected from following group：

(a) raising of the protein output of the encoded by nucleic acid；

(b) raising of the inhibitory RNA content of the encoded by nucleic acid；

(c) raising of resistance (including drought resistance, cold resistance, heat-resisting quantity and salt tolerance)；

(d) raising of anti-plant pest ability, the especially anti-raising occurred in vegetable seeds pest and disease damage ability；

(e) improvement of nutritional quality, including in plant naturally with nutrient composition content raising and plant non-natural with nutrition The imparting of composition；With

(f) raising of the raising of plant products, especially plant economy position yield.

Herein, " selection " can be field or greenhouse selection or the heredity selection using genetic marker. Herein, " propagation " can be with generative propagation or vegetative propagation.In generative propagation, it is clear that can carry out extra Hybridization step, and select the offspring for inheriting transgene traits.

The present invention refer to open source literature, and these documents are to more clearly describe the present invention, and their entire contents are included Referred to herein, just look like that repeated description herein has been excessively for their full text.

In order to make it easy to understand, the present invention will be described in detail by specific embodiment below.It is important to note that The description that these descriptions are merely exemplary, and be not meant to limit the scope of the invention.According to the discussion of this specification, this hair Bright many changes, change and will be apparent from for one of ordinary skill in the art.

Embodiment

Method therefor is conventional method unless otherwise instructed in following embodiments, and the primer is public by Shanghai English fine horse biotechnology Department's synthesis, is sequenced and is completed by Beijing Hua Da gene, and the endonuclease during PCR kit, vector construction is purchased from precious biology Engineering Co., Ltd, pEASY-T1 connections kit are purchased from Beijing Quan Shijin biotech companies, and T4DNA ligases are purchased from Promega companies, method are carried out with reference to the method that kit provider is recommended.Carrier pCAMBIA1303 used in experiment It is purchased from CAMBIA companies.

The promoter AT4G28520 of embodiment 1. separation and identification

Design primer needed for following cloning promoter AT4G28520：

Primer 1：5'-AATCTCgAgTGGTCTGCCATGGGACGT-3'(SEQ ID NO:2)

Primer 2：5'-CGCATCGATCATTTTCTTTTTGTTTG-3'(SEQ ID NO:3)

Sequence ctcgag is Xho I restriction enzyme site in primer 1, and sequence atcgat is ClaI restriction enzyme site in primer 2.

Using forward and reverse primer (sequence wherein with underscore part is promoter sequence) of promoter, with plant genome DNA Arabidopsis (Colombia) genomic DNA of extracts kit (TIANGEN Biotech (Beijing) Co., Ltd.) extraction is as mould Plate, expanded, reaction condition is：95 DEG C of pre-degenerations 5 minutes；94 DEG C are denatured 30 seconds；55 DEG C are annealed 30 seconds；72℃ Extension 2 minutes；30 circulations；72 DEG C extend 10 minutes.After reaction terminates, PCR primer is examined through 1% agarose gel electrophoresis Survey time receives, and product is connected into pEASY-T2 carriers, and screening positive clone simultaneously carries out sequence verification.As a result show, institute's extension increasing sequence For it is anticipated that AT4G28520 promoter sequences, its sequence such as SEQ ID NO：Shown in 1.

The structure of the expression vector of embodiment 2.

Sequence verification is already inserted into the pEASY-T2 plasmids Xho I and ClaI double digestions of AT4G28520 promoter sequences, It is connected into equally with the carrier pHPG of Xho I and ClaI double digestions, the pHPG carriers are changed by pCAMBIA1300 carriers Make and form, a GFP gene is connected with behind its multiple cloning sites.Picking colony enters performing PCR detection, chooses PCR results and is Positive bacterium colony is sequenced, and after sequence verification is correct, is extracted corresponding positive colony plasmid, is named as 520GFP.Wherein, bacterium Primer needed for falling PCR detections is primer on pHPG carriers, and positioned at the promoter fragment both sides cloned, amplified fragments about open The length of mover, using bacterium solution as template, augmentation detection, PCR reaction conditions are：95 DEG C of pre-degenerations 5 minutes；94 DEG C of denaturation 30 Second；55 DEG C are annealed 30 seconds；72 DEG C extend 2 minutes；34 circulations；72 DEG C extend 10 minutes.

The collection of illustrative plates in the T-DNA areas of constructed expression vector is as shown in figure 1, wherein：LB and RB is respectively a T-DNA left side Border and right margin；Hpt represents hygromycin gene；Cam35SP represents the promoter of CamV35S genes；NOS tables Show the terminator of no genes；GFP represents GFP albumen (green fluorescent protein) gene；3A represents the termination of 35S genes Son；520P is the AT4G28520 promoters that embodiment 1 obtains.

The Plant Transformation of embodiment 3. and Function Identification

Plasmid 520GFP is transferred to Agrobacterium AGL0 bacterial strains using heat shock method, colored method is dipped in Agrobacterium and infects arabidopsis, in the dark Co-culture 2-3 days, then normal culture to harvest seed.

The plant of transgenic positive is selected, carries out the expression analysis of promoter.As a result find, as shown in the right figure in Fig. 2, Under uv excitation light, the seed of transgenic positive plant sends green fluorescence, and other histoorgans do not detect green fluorescence. Thus illustrate, AT4G28520 promoters provided by the present invention are the promoters of a seed-specific expression.

The present invention also demonstrates expression characterization of the AT4G28520 promoters in the crops such as rape, rice, the results showed that, with it The same in arabidopsis, the promoter also has the characteristic of seed specific expression in other crops.

SEQUENCE LISTING

<110>Shenzhen Crop Molecular Design Breeding Institute

<120>Seed specific expression system and its application

<130>

<160> 3

<170> PatentIn version 3.3

<210> 1

<211> 1921

<212> DNA

<213>Arabidopsis（Arabidopsis thaliana）

<400> 1

gtggtctgcc atgggacgtg tgagaaccct aagcccaatt ttcacctgaa agttttaaaa 60

ccacaaaaaa cttaagagct tatgatgttg aagcgaacga cttcacattc actaaattca 120

caaaacaaaa tgactgaaaa tgggaggagt agaagcagag taccatctga agatcacggc 180

ttccggatgt actctcaaca aggtaaggtc gagcacgaac gtcatagatg accggccttt 240

caaaccaagg aatcataagg tgtgtaccct cagggtaaac ctgcgtaata acaaggttac 300

aagctacata cttgtgaatt aagaagactg tttttacgct caacgagaac aacaaaattt 360

catgactaat cagtctcagg gattaatgaa caacatatga gactttaact aacaaatcaa 420

cctggaaaat ttcatccaaa tctcacaaac agcaaaaaaa ctcattggga aaatacattt 480

actgaaaatc caaaaagacc caatttgaag aaacactgac taaccttatc tttaataccg 540

actaaacgat tgaacatgat ggctcgatgt cctccttcaa cattgtagag actgtgcgta 600

gcaccataga gaccaagccc accaataatc ccaaccttaa gcaacgtcga aatggcacca 660

ccacctggta tctttggaac tttgacgttg ttcatctctt tttctgaaac aatcccaata 720

catgattaat caaaacccat gtgtggtgga aaaactacac aaacatacac tcaaaatcca 780

gactcacatc tactcaatta tgcaacttca tcatgaaaac atcaaaaaca gtcaaagtaa 840

caaaatcaag tcagattcag cacacaaagc cagtaaagat agaaaattta acgaacgctc 900

atgctaagct gcgcaaaata cttcctaatc aaaacagtaa caacgagtaa ttagcaaaat 960

ccgagcagaa aactctcacc cacctccgaa attcacgtct tcactaaaat tttcgaaagg 1020

aatcgatcaa taccaaccca ttacacaaaa tacataatca aaatggcgag aatcgtacct 1080

ggaaactttg cttcaagtcg cagagagagg aaaaggaaga tcgtggagaa aggggtttag 1140

ggtttaagct cagacttcta ttggagtaaa tgggacggtg tcacattttc cgttttggaa 1200

atgaactttg ggctcacgtt atgggctatt agatatttga tgggctttct agtaaataca 1260

atataagtta ttgggcttag tttaaataag cccatgttgg aaatatttga cacatgtctt 1320

ggctactagt gctaaacatg caaccgaaca gttgtcgaga caagtcgcag catatacaat 1380

ggatcaaaca cgcctagtgt cgccgcgtct cgctcatgtg tcaccttgtt tcctcgtttt 1440

tttttaattt ttcataagtt cttttgtttt atcttcaata caaatttttg gctgtatctt 1500

gcaaactctt cgatcatatc gccaatatac gtgaacactg gtgatctaat ttgttgtgtt 1560

aattgttaaa tttagattct attctccggt ttaaaagtga attatatgta tcatggttaa 1620

aacattgtaa gtaagatgat aataaaatga taaatttagt tgatggataa cgtgaagcaa 1680

aaaatgagat agatacattt gattttgtcg tattttgaca tatgcggaga gtgagctacg 1740

cgcatgaaga tcaagagaca cttgctcgag ctcacagagt gacgtgtaaa aagcttagac 1800

tgaagtcccc atgcaaacct aatcctacgt ggctcaaacc acgagctcac ttgacaatat 1860

ataaactcct cctaagtccc gttctcttca tccatctctc acaacaaaca aaaagaaaat 1920

g 1921

<210> 2

<211> 27

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 2

aatctcgagt ggtctgccat gggacgt 27

<210> 3

<211> 26

<212> DNA

<213>It is artificial synthesized（Artificial synthesis）

<400> 3

cgcatcgatc attttctttt tgtttg 26

Claims (10)

1. a kind of promoter, it can instruct to be operatively coupled on nucleic acid specific transcription and/or expression in vegetable seeds downstream, and the DNA sequence dna of the promoter is selected from following group of sequence：

（a）With SEQ ID NO：Sequence shown in 1；

（b）Under strict conditions can be with（a）The DNA sequence dna of the DNA hybridization of the sequence；

（c）With（a）The sequence has at least 90%（Preferably at least 95%）The DNA sequence dna of sequence identity；

（d）Include SEQ ID NO：At least 150 in 1（Preferably at least 200）The DNA sequence dna of continuous nucleotide；With

（e）With（a）-（d）The complementary DNA sequence dna of any sequence.

2. the promoter described in claim 1, its sequence length is less than 2000 nucleotides, preferably smaller than 1500 nucleotides, more preferably less than 1200 nucleotides.

3. the promoter described in claim 1 or 2, it can instruct to be operatively coupled on nucleic acid specific transcription and/or expression in vegetable seeds downstream.

4. claim 1-3 any described promoter, its sequence such as SEQ ID NO：Shown in 1.

5. a kind of expression cassette, it is included：

（a）Claim 1-4 any described promoter；With

（b）Nucleic acid, it is operatively coupled on claim 1-4 any described DNA downstream.

6. the expression cassette described in claim 5, wherein the nucleic acid can assign the character that plant is selected from following group：

（a）The raising of the protein output of the encoded by nucleic acid；

（b）The raising of the inhibitory RNA content of the encoded by nucleic acid；

（c）The raising of anti-plant pest ability；

（d）The improvement of nutritional quality, including in plant naturally with nutrient composition content raising and plant non-natural with nutritional ingredient imparting；With

（e）The raising of the raising of plant products, especially plant economy position yield.

7. plant, vegetable seeds, plant tissue or the plant cell of the transgenosis converted with the expression cassette described in claim 5 or 6, wherein, claim 1-4 any described promoter can instruct to be operatively coupled on nucleic acid specific transcription and/or expression in vegetable seeds downstream.

8. plant, vegetable seeds, plant tissue or plant cell described in claim 7, wherein described plant is monocotyledon or dicotyledon, such as, corn, rice, wheat, barley, sorghum, soybean, rape, cotton, tomato, potato, sugarcane, beet, tobacco or arabidopsis.

9. the production method of the plant described in claim 7 or 8, it includes：

（1）Build the expression cassette described in claim 5 or 6；

（2）By step（1）The expression cassette of acquisition imports plant cell；

（3）Regenerate genetically modified plants；With

（4）Select genetically modified plants, wherein claim 1-4 any described promoter can instruct to be operatively coupled on nucleic acid downstream in vegetable seeds specific transcription and/or expression；And

（5）Optionally, amplification step（4）The plant of acquisition is to obtain offspring.

10.It is a kind ofThe method for assigning plant trait, it includes

（1）Selection can assign the nucleic acid of plant trait：

（2）Use step（1）Expression cassette described in the nucleic acid construct claim 5 or 6 of acquisition；

（3）By step（2）The expression cassette of acquisition imports plant cell；

（4）Regenerate genetically modified plants；With

（5）Select the genetically modified plants for imparting plant trait；And

（6）Optionally, amplification step（5）The plant of acquisition to obtain offspring,

Wherein, the character is selected from following group：

（a）The raising of the protein output of the encoded by nucleic acid；

（b）The raising of the inhibitory RNA content of the encoded by nucleic acid；

（c）The raising of anti-plant pest ability；

（d）The improvement of nutritional quality, including in plant naturally with nutrient composition content raising and plant non-natural with nutritional ingredient imparting；With

（e）The raising of the raising of plant products, especially plant economy position yield.