CN109750039A - A kind of plant touches response promoter and its application - Google Patents

Abstract

The invention discloses a kind of plants to touch response promoter and its application.Specific DNA molecular provided by the present invention, nucleotide sequence is as shown in sequence 4 in sequence table.It is demonstrated experimentally that specific DNA molecular provided by the invention can illustrate that the specific DNA molecular is one and touches response promoter in the expression for touching (such as mechanical pressure) Shi Qidong target gene (encoding gene of such as luciferase).The present invention has important application value.

Description

A kind of plant touches response promoter and its application

Technical field

The invention belongs to field of biotechnology, and in particular to a kind of plant touches response promoter and its application.

Background technique

Promoter is the section of DNA sequence positioned at the upstream gene start codon (ATG), can be in conjunction with RNA polymerase And the transcription of promotor gene, it is equivalent to " switch " of genetic transcription.Promoter mainly includes two regions: core promoter region and non- Core promoter region.Core promoter region forms generality transcription structure, usually by some short conserved sequence (such as transcription initiation positions Point, TATA-box, CAAT-box etc.) composition.The structure feature of these sequences decide promoter with RNA polymerase identification, In conjunction with and starting transcription.Non-core promoter region includes proximal end control region and distal end control region, often there are some specificity Transcription factor combine site, controlling gene transcription time and Region-specificity.It is different according to the transcriptional profile of promoter It is classified as composing type, induction type and tissue-specific promoter's three classes.Constitutive promoter can in different types of cell and The different phase of cell development continues efficiently to drive genetic transcription, and transcriptional activity is relative constant, by constitutive promoter The gene of driving is often referred to as house-keeping gene (housekeeping gene).Tissue-specific promoter is only in certain spies The a certain moment of the cell (tissue) or cell (tissue) development of determining type is efficiently transcribed, and is contained and is determined promoter The cis-regulating element of specificity, will combine with transcription factor existing for the tissue specificity, just show promoter activity, The gene of tissue-specific promoter's driving is often closely related with the cell of this type (tissue) function.Inducible promoter Then inducement signal is needed to stimulate, the transcription of ability promotor gene, inducible promoter is more common in plant, what it was driven Gene usually plays a significant role during plant growth and development is adjusted and poor environment is coped with.

Plant is faced with many environmental factors complicated and changeable in its life cycle, such as maintains plant normal growth development The primary conditions variation such as temperature, illumination and moisture, in addition to this touch the institute (such as gravity, wind, the touching of sleet, barrier) Caused stimulation is also a signal of interest in external environment, affects the growth and development process of plant extensively.Plant responding The process that mechanical stimulus changes in morphological development in turn is known as triggering form, is mainly reflected in mechanical stimulus meeting Reduce plant blade area, the radial extent of stem is suppressed, to form short and small sturdy plant.People ring plant Answer the interest of mechanical stimulus long-standing, most for known to people as the emotivity of sensitive plant leaf is closed up, climbing plant tendril Haptotropism curling etc..However, being mainly reflected in morphogenesis, development models for the research of plant responding mechanical stimulus at present With the research in terms of physiological status, in addition to organizing and the research of organ level, the mechanical response of plant is also manifested in cell and thin Born of the same parents' device is horizontal.However, about the molecule that mechanical stimulation signal and plant responding mechanical stimulus how are responded and transmitted to plant Mechanism Study is less.

Luciferase (Luciferase) is the general designation that the enzyme of bioluminescence can be generated in nature, it can be in biology Internal produces chemiluminescence (luciferin) or fatty aldehyde (firefly aldehyde) oxyluminescence.Fluorescence existing for nature Plain enzyme is from firefly, photobacteria, luminous starfish, luminescent segment worm, luminous fish, luminous beetles etc., wherein most representative It is a kind of intracorporal luciferase of firefly that scientific name is Photinus pyralis, 550 amino acid of the gene codified Luciferase protein is the monomeric enzyme of a 61kDa, without modifying after expression, directly has complete enzyme activity.Luciferase can be with It is generated with the method for genetic engineering and has been used for different experiments.The gene order of coding fluorescence element enzyme is identified, and It is widely applied to the numerous areas of biology, for studying the regulation of gene and the detection of expression.As a kind of fluorescent marker Object, the gene of coding fluorescence element enzyme can be synthesized and be inserted into organism or be transfected into cell, with the target protein that makes marks, Track positioning and the dynamic process of albumen.Common is that the gene of coding fluorescence element enzyme is inserted under the promoter of gene to be studied Trip, and under specific space-time condition detect coding fluorescence element enzyme gene expression, with the work of this goal in research albumen Use mode.Luciferase label has very strong specificity, is shone with enzyme and substrate specificity effect.In addition, luciferase Also have the characteristics that high sensitivity and stability are good.Therefore, luciferase has great importance in field of biology research.

Summary of the invention

The purpose of the present invention is start the expression of target gene by touching (such as mechanical pressure).

The present invention protects a kind of specific DNA molecular first, can be following (a1) or (a2) or (a3):

(a1) DNA molecular shown in sequence 4；

(a2) nucleotide sequence limited with (a1) has 75% or 75% or more identity, and with promoter function DNA molecular；

(a3) nucleotide sequence hybridization limited under strict conditions with (a1) or (a2), and with promoter function DNA molecular.

Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation Method is mutated the nucleotide sequence of specific DNA molecular of the invention.Those have and the present invention by manually modified The nucleotide sequence 75% of the specific DNA molecular of offer or the nucleotide of higher identity, as long as there is promoter function, It is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.

Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair The nucleotide sequence of bright specific DNA molecular has 75% or higher, 80% or higher or 85% or higher or 90% or more The nucleotide sequence of height or 95% or higher identity.Identity can with the naked eye or computer software is evaluated.Use meter Calculation machine software, the identity between two or more sequences can be indicated with percentage (%), can be used to evaluate related sequence Identity between column.

Expression cassette containing any of the above-described specific DNA molecular also belongs to protection scope of the present invention.

The expression cassette (from 5 ' to 3 ') may include promoter region (being made of the specific DNA molecular), transcription initiation region, Target gene area, transcription termination region and optional end of translation.The promoter region and target gene area to host cell and Yan Kewei it is natural/it is similar, alternatively, the promoter region and target gene area can be natural/similar between each other, or Person, the promoter region and/or target gene area are for host or it is heterologous between each other." heterologous " refers to sequence For the sequence from alien species, or, if coming from same species, by human intervention deliberately in component and/or gene Group has carried out substantive sex modification to native form in terms of site.The transcription termination region optionally contained can be homologous with transcription initiation region, It is homologous with the target gene area being operably connected, it is homologous with host；Or；Target gene area, host are external source or heterologous.

The expression cassette may also include 5 ' boot sequences.5 ' boot sequences can enhance translation.

When preparing expression cassette, using adapter or linker connection DNA fragmentation or other operations can be can be related to mention For the extra DNA of restriction site appropriate, removal, removal restriction site etc..To reach this purpose, body can be carried out Outer mutation, restricted digestion, annealing, is replaced at primer reparation again, such as conversion and transversion.

The expression cassette may also include the selected marker for screening transformed cells.Selected marker can For screening transformed cells or tissue.Marker gene includes the gene for encoding antibiotic resistance.Other selected markers include Phenotypic markers such as fluorescin.Selected marker listed above does not have restricted.Any selectivity can be used in the present invention Marker gene.

Recombinant plasmid containing any of the above-described specific DNA molecular also belongs to protection scope of the present invention.

The recombinant plasmid can be plasmid that any of the above-described specific DNA molecular insertion is set out, obtained recombinant plasmid. Any of the above-described specific DNA molecular is concretely inserted into the multiple cloning sites for the plasmid that sets out by the recombinant plasmid, is obtained Recombinant plasmid.

The recombinant plasmid may include any of the above-described expression cassette for containing the specific DNA molecular.

The recombinant plasmid recombinant plasmid pLUC-TCH3p that concretely embodiment refers to.The recombinant plasmid pLUC- TCH3p is that the DNA small fragment between the restriction enzyme HindIII and BamHI of recombinant plasmid pLUC is replaced with sequence table DNA molecular shown in middle sequence 4, obtained recombinant plasmid.

Transgenic cell line containing any of the above-described specific DNA molecular also belongs to protection scope of the present invention.

The transgenic cell line can be transgenic plant cells system.

The transgenic plant cells system does not include propagation material.The genetically modified plants are interpreted as not only including by institute The first generation genetically modified plants that specific DNA molecular transformation receptor plant obtains are stated, also include its filial generation.For genetically modified plants, The specific DNA molecular can be bred in the species, it is also possible to which the specific DNA molecular is transferred by traditional breeding techniques Other kinds of same species, particularly including in commercial variety.The genetically modified plants include seed, callus, complete plant Strain and cell.

In one embodiment of the invention, the plant can be crucifer.The crucifer is specific It can be arabidopsis.Concretely wildtype Arabidopsis thaliana (Arabidopsis thaliana) Columbia-0 is sub- for the arabidopsis Type.

Any of the above-described specific DNA molecular is as promoter or touches the of the invention using also belonging to of response promoter Protection scope.

Any of the above-described specific DNA molecular, any of the above-described expression cassette or any of the above-described recombinant plasmid are opening Application in the expression of dynamic target gene also belongs to protection scope of the present invention.

The method that the present invention also protects expression target gene.

The method for the expression target gene that the present invention is protected, concretely method one, the method one can be for above-mentioned Any specific DNA molecular is as promoter or touches the expression for responding promoter to start target gene.

The method for the expression target gene that the present invention is protected, concretely method two, the method two may include as follows Any of the above-described specific DNA molecular: being inserted into the upstream of any target gene or enhancer by step, starts purpose with this The expression of gene.

The method for the expression target gene that the present invention is protected, concretely method three, the method three may include as follows Step: target gene is inserted into the downstream of the specific DNA molecular in any of the above-described expression cassette, by the specific DNA Molecule starts the expression of the target gene.

The method for the expression target gene that the present invention is protected, concretely method four, the method four may include as follows Target gene: being inserted into the downstream of the specific DNA molecular in any of the above-described recombinant plasmid by step, by described special DNA molecular starts the expression of the target gene.

The expression of target gene when (such as mechanical pressure) is concretely touched in the expression of any of the above-described target gene.

Any of the above-described specific DNA molecular can be used as promoter (concretely touching response promoter) can be in plant Middle expressing gene (such as foreign gene).The plant can be crucifer.The crucifer concretely intends south Mustard.The arabidopsis concretely wildtype Arabidopsis thaliana (Arabidopsis thaliana) Columbia-0 hypotype.

Any of the above-described target gene can be the encoding gene of luciferase.The amino acid sequence of the luciferase can As shown in sequence 2 in sequence table.The encoding gene of the luciferase can be as shown in sequence 1 in sequence table.

Any of the above-described described touch can be mechanical pressure.In one embodiment of the invention, the mechanical pressure can be by It is formed in the quartz sand of plant (concretely arabidopsis) surface covering certain thickness (such as 5mm).In another reality of the invention It applies in example, the mechanical pressure can be by the glass plate in plant (concretely arabidopsis) surface covering certain thickness (such as 5mm) It is formed.Cover time can be 20min or more (such as 30min).

It is demonstrated experimentally that specific DNA molecular provided by the invention can touch (such as mechanical pressure) Shi Qidong target gene The expression of (encoding gene of such as luciferase) illustrates that the specific DNA molecular is one and touches response promoter.The present invention has Important application value.

Detailed description of the invention

Fig. 1 is the luminous testing result that TCH1p and TCH3p drives luciferase.

Fig. 2 is the luminous testing result that CaMV 35S promoter drives luciferase.

Fig. 3 is the testing result for the transcriptional level that wildtype Arabidopsis thaliana touches stimulation front and back TCH1 gene and TCH3 gene.

Specific embodiment

Embodiment below facilitates a better understanding of the present invention, but does not limit the present invention.

Experimental method in following embodiments is unless otherwise specified conventional method.

Test material as used in the following examples is unless otherwise specified to buy from routine biochemistry reagent shop It arrives.

Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.

Wildtype Arabidopsis thaliana (Arabidopsis thaliana) Columbia-0 hypotype is recorded in following document: Kim H, Hyun Y, Park J, Park M, Kim M, Kim H, Lee M, Moon J, Lee I, Kim J.A genetic link between cold responses and flowering time through FVE in Arabidopsis Thaliana.Nature Genetics.2004,36:167-171.Wildtype Arabidopsis thaliana (Arabidopsis thaliana) Columbia-0 hypotype hereinafter abbreviation wildtype Arabidopsis thaliana or arabidopsis.

In following embodiments, alternation of light and darkness culture (i.e. light culture and dark culture alternating) condition are as follows: 22 ± 2 DEG C；16h illumination Culture/8h dark culturing；Intensity of illumination when illumination cultivation is 80~100 μ E/m²/s。

In following embodiments, the sand grains diameter of quartz sand is 1-2mm.

Embodiment 1, arabidopsis touch the screening of responsive genes

The present inventor is touching post-stimulatory difference expression gene using RNA-seq method detection arabidopsis, into And it screens and obtains arabidopsis and touch responsive genes.Specific step is as follows:

1, arabidopsis seed is taken, is sterilized with 75% (v/v) ethanol water containing 0.05% (v/v) triton X-100 Then 15min is cleaned 3-5 times with aqua sterilisa.

2, after completing step 1, arabidopsis seed is seeded in 1/2MS solid medium, 4 DEG C of vernalization 3d.

3, the 1/2MS solid medium for completing step 2 is placed in incubator, alternation of light and darkness culture 5d obtains arabidopsis children Seedling.

Part Arabidopsis thaliana Seedlings (i.e. untreated Arabidopsis thaliana Seedlings) (complete stool) are rapidly frozen with liquid nitrogen, -80 DEG C of preservations.

4, after completing step 3, Xiang Qiyu Arabidopsis thaliana Seedlings surface covers (with a thickness of 5mm) quartz sand 30min, obtains machinery The Arabidopsis thaliana Seedlings of stimulation.The quartz sand on the Arabidopsis thaliana Seedlings of mechanical stimulus is removed, then by the arabidopsis children of mechanical stimulus Seedling (complete stool) is rapidly frozen with liquid nitrogen, -80 DEG C of preservations.

5, the total serum IgE of the Arabidopsis thaliana Seedlings of untreated Arabidopsis thaliana Seedlings or mechanical stimulus is extracted, then with coupling oligo (dT) column (the peace excellent product up to company of promise) purifies and separates contain the mRNA of poly (A).

6, after completing step 5, the mRNA for containing poly (A) is taken, mRNA fragmentation buffer is added, and (peace promise is excellent up to public affairs The product of department), 94 DEG C of processing 2min obtain the RNA segment of fragmentation.

7, after completing step 6, using the RNA segment of the fragmentation as template, first using random primed reverse transcription synthesis the One chain cDNA；Then DNA polymerase i synthetic dsdna is used, while RNaseH digestion RNA template is added.The cDNA of reverse transcription is produced Object QiaQuick PCR purification kit (product of Qiagen company) recovery purifying.Finally cDNA segment is drawn plus connector Object carries out PCR amplification, is sequenced with the HiSeq2500 of Illumina company.

8, after completing step 7, RNA-seq points are carried out to the Arabidopsis thaliana Seedlings of untreated Arabidopsis thaliana Seedlings or mechanical stimulus Analysis obtains the difference expression gene obviously raised after mechanical stimulus processing (i.e. arabidopsis touches responsive genes).

The result shows that TCH1 gene (gene ID is AT5G37780) and TCH3 gene (gene ID is AT2G41100) are Arabidopsis touches responsive genes.

9, the expression of responsive genes (TCH1 gene or TCH3 gene) is touched with arabidopsis in untreated Arabidopsis thaliana Seedlings Amount is used as 1, detects the relative expression quantity that arabidopsis in the Arabidopsis thaliana Seedlings of mechanical stimulus touches responsive genes.

The result shows that arabidopsis touches sound in the Arabidopsis thaliana Seedlings of mechanical stimulus compared with untreated Arabidopsis thaliana Seedlings The expression quantity of gene is answered to be significantly increased.

Embodiment 2, arabidopsis touch the clone of the promoter of responsive genes and the detection that shines

One, the building of recombinant plasmid

1, the building of recombinant plasmid pLUC

(1) with pGreenII 0800-LUC plasmid (product of BioVector company) for template, using primers F: 5 '-C GGGATCCATGGAAGACGCCAAAAACATAAAG-3 ' (recognition site that underscore is restriction enzyme BamHI) and primer R:5 '-GGGGTACCTTACAATTTGGACTTTCCGCC-3 ' (recognition site that underscore is restriction enzyme KpnI) composition Primer pair carry out PCR amplification, recycle size about 1700bp pcr amplification product.

Response procedures: 94 DEG C of 3min；94 DEG C 15 seconds, 57 DEG C 30 seconds, 68 DEG C of 2min, 35 recycle；68℃10min.

(2) after completing step (1), pcr amplification product is sequenced.

Sequencing result shows in pcr amplification product containing DNA fragmentation shown in the sequence 1 in ordered list.In sequence table Protein shown in sequence 2 (i.e. luciferase) in DNA fragmentation polynucleotide shown in sequence 1.

(3) after completing step (1), pcr amplification product is taken, with restriction enzyme BamHI and KpnI digestion, recycling is about The digestion products of 1.7kb.

(4) plasmid pBI121 (product of Beijing Baeyer enlightening Bioisystech Co., Ltd, catalog number MP-091) is taken, With restriction enzyme SacI and EcoRI digestion, the DNA fragmentation of about 277bp is recycled.The DNA fragmentation is Noster poly A Termination sequence.

(5) carrier pUC19 (product of hundred Tyke Bioisystech Co., Ltd of Beijing, catalog number DP7801) is taken, With restriction enzyme SacI and EcoRI digestion, the carrier framework of about 2686bp is recycled.

(6) DNA fragmentation that step (4) obtains and the carrier framework that step (5) obtains are attached, obtain recombinant plasmid pUC19-Noster。

(7) recombinant plasmid pUC19-Noster is taken, with restriction enzyme BamHI and KpnI digestion, recycles about 2950bp Digestion products.

(8) digestion products that step (3) obtains and the digestion products that step (7) obtains are attached, obtain middle interstitial Grain.

(9) middle interstitial granules are taken, with restriction enzyme BamHI and EcoRI digestion, recycle the DNA fragmentation of about 2kb.

(10) plasmid pCAMBIA1301 (product of Biovector Co.) is taken, with restriction enzyme BamHI and EcoRI The carrier framework of about 10kb is recycled in digestion.

(11) DNA fragmentation that step (9) obtains and the carrier framework that step (10) obtains are attached, obtain recombination matter Grain pLUC.

2, arabidopsis touches the clone of the promoter of responsive genes

(1) clone of the promoter (hereinafter referred to as TCH1p) of TCH1 gene

A, extract arabidopsis genomic DNA and using it as template, using upstream primer: 5 '-CCAAGCTTagctta Ttactctcttccttggttttg-3 ' (recognition site that underscore is restriction enzyme HindIII) and downstream primer: 5 '- CGGGATCCAgcttcttcgagaaatcgtctttc-3 ' (recognition site that underscore is restriction enzyme BamHI) composition Primer pair carry out PCR amplification, recycle size about 1374bp pcr amplification product.The pcr amplification product contains TCH1p.

Reaction system is 30 μ L, by 0.6 μ L KOD (concentration is 5U/ μ L) (product that KOD is TOYOBO company), 3 μ L10 (concentration of dATP, dTTP, dGTP and dCTP is by × the buffer component of KOD (10 × buffer be), 3 μ L dNTPs 2.0mM), 2.4 μ L MgSO4 solution (concentration 25mM), 0.9 μ L upstream primer (concentration is 10 μM), 0.9 μ L downstream primer are (dense Degree is 10 μM), the genomic DNA and 16.2 μ LddH of 3 μ L arabidopsis₂O composition.

Response procedures: 94 DEG C of 2min；94 DEG C 15 seconds, 56 DEG C 30 seconds, 68 DEG C of 2min, 35 recycle；68℃10min.

B, after completing step a, the pcr amplification product is taken, is sequenced.

Sequencing result shows the nucleotide sequence of TCH1p as shown in sequence 3 in sequence table.

(2) clone of the promoter (hereinafter referred to as TCH3p) of TCH3 gene

A, extract arabidopsis genomic DNA and using it as template, using upstream primer: 5 '- CCAAGCTTCATTAGGGTCTGGCTGGTATG-3 ' (recognition site that underscore is restriction enzyme HindIII) and downstream Primer: 5 '-CGGGATCC(underscore is the identification position of restriction enzyme BamHI to ACCCGAATTATTTGAAATGACG-3 ' Point) composition primer pair carry out PCR amplification, recycle size about 1400bp pcr amplification product.The pcr amplification product contains TCH3p。

The reaction system of a in the same step of reaction system (1).

The response procedures of a in the same step of response procedures (1).

B, after completing step a, the pcr amplification product is taken, is sequenced.

Sequencing result shows the nucleotide sequence of TCH3p as shown in sequence 4 in sequence table.

3, the clone of CaMV 35S promoter (hereinafter referred to as 35Sp)

A, using pGreenII 0800-LUC plasmid as template, using upstream primer: 5 '-CCAAGCTTTGAGACTTTTCA ACAAAGGGTAATTTC-3 ' (recognition site that underscore is restriction enzyme HindIII) and downstream primer: 5 '-CGGGA TCCTGTCCTCTCCAAATGAAATGAACTTC-3 ' (recognition site that underscore is restriction enzyme BamHI) composition draws Object recycles the pcr amplification product of size about 346bp to PCR amplification is carried out.The pcr amplification product contains 35Sp.

The reaction system of a in the same step of reaction system (1).

The response procedures of a in the same step of response procedures (1).

B, after completing step a, the pcr amplification product is taken, is sequenced.

Sequencing result shows the nucleotide sequence of 35Sp as shown in sequence 5 in sequence table.

4, the building of recombinant plasmid pLUC-TCH1p, recombinant plasmid pLUC-TCH3p and recombinant plasmid pLUC-35Sp

(1) building of recombinant plasmid pLUC-TCH1p

The recombinant plasmid pLUC that (1-1) takes step 1 to construct, with restriction enzyme HindIII and BamHI digestion, recycling The carrier framework of about 10kb.

The pcr amplification product that (1-2) takes in step 2 (1) to expand, with restriction enzyme HindIII and BamHI digestion, Recycle the endonuclease bamhi of about 1374bp.

The carrier framework that step (1-1) obtains and the endonuclease bamhi that step (1-2) obtains are attached by (1-3), are obtained Recombinant plasmid pLUC-TCH1p.

Recombinant plasmid pLUC-TCH1p is sequenced.According to sequencing result, recombinant plasmid pLUC-TCH1p is tied Structure is described as follows: the DNA small fragment between the restriction enzyme HindIII and BamHI of recombinant plasmid pLUC is replaced with sequence DNA molecular shown in sequence 3 in list, obtained recombinant plasmid.In recombinant plasmid pLUC-TCH1p, fluorescence is started by TCH1p The expression of plain enzyme.

(2) building of recombinant plasmid pLUC-TCH3p

According to the method for step (1), " pcr amplification product that (1) expands in step 2 " in step (1-2) is replaced with into " step The pcr amplification product that (2) expand in rapid 2 ", other steps are constant, obtain recombinant plasmid pLUC-TCH3p.

Recombinant plasmid pLUC-TCH3p is sequenced.According to sequencing result, recombinant plasmid pLUC-TCH3p is tied Structure is described as follows: the DNA small fragment between the restriction enzyme HindIII and BamHI of recombinant plasmid pLUC is replaced with sequence DNA molecular shown in sequence 4 in list, obtained recombinant plasmid.In recombinant plasmid pLUC-TCH3p, fluorescence is started by TCH3p The expression of plain enzyme.

(3) building of recombinant plasmid pLUC-35Sp

According to the method for step (1), " pcr amplification product that (1) expands in step 2 " in step (1-2) is replaced with into " step The pcr amplification product of rapid 3 amplification ", other steps are constant, obtain recombinant plasmid pLUC-35Sp.

Recombinant plasmid pLUC-35Sp is sequenced.According to sequencing result, structure is carried out to recombinant plasmid pLUC-35Sp It is described as follows: the DNA small fragment between the restriction enzyme HindIII and BamHI of recombinant plasmid pLUC is replaced with into sequence DNA molecular shown in sequence 5 in table, obtained recombinant plasmid.In recombinant plasmid pLUC-35Sp, luciferase is started by 35Sp Expression.

Two, the acquisition of recombinational agrobacterium

Recombinant plasmid pLUC-TCH1p is imported into Agrobacterium tumefaciems GV3101, recombinational agrobacterium is obtained, is named as GV3101/ pLUC-TCH1p。

Recombinant plasmid pLUC-TCH3p is imported into Agrobacterium tumefaciems GV3101, recombinational agrobacterium is obtained, is named as GV3101/ pLUC-TCH3p。

Recombinant plasmid pLUC-35Sp is imported into Agrobacterium tumefaciems GV3101, recombinational agrobacterium is obtained, is named as GV3101/ pLUC-35Sp。

Three, the acquisition of transgenic arabidopsis

1, using arabidopsis floral dip-flower conversion method (be recorded in Clough in following document, S.J., and Bent, A.F..Floraldip:asimplified method for Agrobacterium-mediated transformation Of Arabidopsis thaliana.Plant J. (1998) 16,735-743.), GV3101/pLUC-TCH1p is gone into open country In raw type arabidopsis, T is obtained₁In generation, turns TCH1p arabidopsis seed.

2, the T for obtaining step 1₁In generation, turns the 1/2MS training that TCH1p arabidopsis seed is seeded in the hygromycin containing 30mg/L It supports on base, the arabidopsis (resistance seedling) for capableing of normal growth is T₁In generation, turns TCH1p positive seedling, T₁In generation, turns TCH1p positive seedling and receives Seed be T₂In generation, turns TCH1p arabidopsis seed.

3, the T for the different strains for filtering out step 2₂In generation, turns TCH1p arabidopsis seed and is seeded in the tide containing 30mg/L Screened on the 1/2MS culture medium of mycin, if be capable of in certain strain the number of the arabidopsis (resistance seedling) of normal growth with Can not normal growth arabidopsis (non-resistance seedling) number ratio be 3:1, then the strain be TCH1p be inserted into one copy Strain, the seed that the resistance seedling in the strain receives is T₃In generation, turns TCH1p arabidopsis seed.

4, the T for filtering out step 3₃In generation, turns TCH1p arabidopsis seed and is seeded in the hygromycin containing 30mg/L again It is screened on 1/2MS culture medium, is the as T of resistance seedling₃Turn TCH1p arabidopsis for homozygosis.To wherein 2 T₃In generation, is homozygous Turn TCH1p arabidopsis strain and be respectively designated as TCH1p-1 and TCH1p-2, and carries out subsequent experimental.

According to the method described above, GV3101/pLUC-TCH1p is replaced with into GV3101/pLUC-TCH3p, other steps are homogeneous Together, T is obtained₃Turn TCH3p arabidopsis for homozygosis.To wherein 2 T₃Turn TCH3p arabidopsis strain for homozygosis to be respectively designated as TCH3p-1 and TCH3p-2, and carry out subsequent experimental.

According to the method described above, GV3101/pLUC-TCH1p is replaced with into GV3101/pLUC-35Sp, other steps are homogeneous Together, T is obtained₃Turn 35Sp arabidopsis for homozygosis.To wherein 1 T₃Turn 35Sp arabidopsis strain for homozygosis and is named as 35S:LUC, and Carry out subsequent experimental.

Four, shine detection

D-Luciferin mother liquor: 1g D-Luciferin powder (MW=318.42g/mol) is dissolved in 62.8mL water, The D-Luciferin mother liquor that concentration is 50mM is obtained, -80 DEG C of packing are kept in dark place.

D-Luciferin working solution: it is 1mM that D-Luciferin mother liquor to concentration, which is diluted with water,.

Arabidopsis seed to be measured is the T of TCH1p-1₃For seed, the T of TCH1p-2₃For seed, the T of TCH3p-1₃For seed, The T of TCH3p-2₃For seed, the T of 35S:LUC₃For seed or wildtype Arabidopsis thaliana seed.

1, arabidopsis seed to be measured is taken, is gone out with 75% (v/v) ethanol water containing 0.05% (v/v) triton X-100 Bacterium 15min, is then cleaned 3-5 times with aqua sterilisa.

2, after completing step 1, arabidopsis seed to be measured is seeded in 1/2MS solid medium, 4 DEG C of vernalization 3d.

3, the 1/2MS solid medium for completing step 2 is placed in incubator, alternation of light and darkness culture 5d obtains quasi- south to be measured Mustard seedling.

4, after completing step 3, the Arabidopsis thaliana Seedlings to be measured is taken, are sprayed with D-Luciferin working solution, place is then allowed to stand Reason for 24 hours, takes pictures (Berthold LB985) with plant living body imager.

5, after completing step 4, the piece of surface covering glass plate (with a thickness of 5mm) of part Arabidopsis thaliana Seedlings to be measured is gone after 30min Fall glass plate, takes pictures (Berthold LB985) with plant living body imager.

6, after completing step 4, part Arabidopsis thaliana Seedlings to be measured are without any processing, and plant living body imager is used after 30min Take pictures (Berthold LB985).As control.

Part of test results is shown in that (A is control to Fig. 1, and wherein the 1# of upper left is the T of TCH1p-1₃For seed, the 1# of upper right is The T of TCH1p-2₃For seed, the 2# of lower-left is the T of TCH3p-1₃For seed, the 2# of bottom right is the T of TCH3p-2₃For seed；B is Cover glass plate handles 30min, and wherein the 1# of upper left is the T of TCH1p-1₃For seed, the 1# of upper right is the T of TCH1p-2₃Generation kind Son, the 2# of lower-left are the T of TCH3p-1₃For seed, the 2# of bottom right is the T of TCH3p-2₃For seed) and Fig. 2 (left figure is control, right Figure is that the processing of cover glass plate 30min, Col are the seed of wildtype Arabidopsis thaliana, and 35S:LUC is the T of 35S:LUC₃For seed). The result shows that touching before stimulation (i.e. non-cover glass plate), TCH1p-1, TCH1p-2, TCH3p-1 and TCH3p-2 do not have substantially Fluorescence；After touching stimulation (cover glass plate handles 30min), the fluorescence intensity of TCH1p-1 and TCH1p-2 are weaker, TCH3p-1 It is then relatively strong with the fluorescence intensity of TCH3p-2；It touches before stimulation and after touching stimulation, the fluorescence intensity of 35S:LUC is relatively strong and nothing Significant changes；It touches before stimulation and after touching stimulation, the equal unstressed configuration signal of wildtype Arabidopsis thaliana.

It can be seen that TCH1p and TCH3p, which are able to respond, touches stimulation (such as mechanical pressure).Stimulation is being touched (such as machinery Pressure) under, TCH3p and TCH1p can drive luciferase to generate fluorescence.

Five, the transcriptional level of TCH1 gene and TCH3 gene is detected

Arabidopsis thaliana Seedlings to be measured are the wildtype Arabidopsis thaliana seedling or complete for completing the non-cover glass plate in surface in 6 in step 4 At the wildtype Arabidopsis thaliana seedling of piece of surface covering glass plate processing in step 45.

1, Arabidopsis thaliana Seedlings to be measured are taken, are extracted using plant total serum IgE kit (product of SIGMA-ALDRICH company) total RNA obtains arabidopsis total serum IgE to be measured.

2, arabidopsis total serum IgE to be measured is taken, referring to the RT-PCR kit (production of TIANGEN Biotech (Beijing) Co., Ltd. Product) specification operating procedure, synthesize the cDNA of arabidopsis to be measured.

(1) 2 μ g arabidopsis total serum IgE to be measured is taken, 2 μ L 5 × gDNA Buffer are added, then uses RNase-Free ddH₂O Polishing is to 10 μ L, after mixing of short duration centrifugation, 42 DEG C of standing 3min.

(2) take into the system of step (1), be added 10 μ L reverse transcription mix (by 2 μ 10 × King of L RT Buffer, 1 μ LFastKing RT Enzyme Mix, 2 μ L FQ-RT Primer Mix and RNase-Free ddH2O composition), it mixes；First 42 DEG C of incubation 15min, then 95 DEG C of incubation 3min are placed on the cDNA for obtaining arabidopsis to be measured on ice.

5×gDNA Buffer、RNase-Free ddH₂O、10×King RT Buffer、FastKing RT Enzyme Mix and FQ-RT Primer Mix is the component in RT-PCR kit.

3, after completing step 2, using TB Green^TMFast qPCR Mix kit (product of Takara company) carries out Real Time PCR (qPCR) detection.

(1) cDNA for taking arabidopsis to be measured, uses ddH₂O dilutes 20 times, obtains template solution.

(2) reaction system is prepared.Reaction system is 20 μ L, by 5 μ L template solutions, 10 μ L TB Green Fast qPCR Mix (2 ×), 0.8 μ L upstream primer (concentration is 10 μM), 0.8 μ L downstream primer (concentration is 10 μM), 0.4 μ L ROX Reference Dye (50 ×) and ddH₂O composition.

TB Green Fast qPCR Mix (2 ×) and ROX Reference Dye (50 ×) is TB Green^TMFast Component in qPCR Mix kit.

The nucleotides sequence for detecting the upstream primer of TCH1 gene is classified as 5 '-CCCGAGTTCCTGAACCTGAT-3 ', and downstream is drawn The nucleotides sequence of object is classified as 5 '-CAGCCTCACGGATCATCTCT-3 '.

The nucleotides sequence for detecting the upstream primer of TCH3 gene is classified as 5 '-ATGGTGACGGAACCATCAGT-3 ', and downstream is drawn The nucleotides sequence of object is classified as 5 '-CGCTATGTCCCTCTCCCAAT-3 '.

(3) after completing step (2), the reaction system is taken, carries out Real-time PCR detection.

Response procedures are as follows: 95 DEG C of 20s；95 DEG C of 3s, 60 DEG C of 30s, 40 circulations；95℃15s；60℃1min；95℃15s； 60℃15s。

Real-time PCR testing result is shown in that (0h is the non-cover glass plate in surface to Fig. 3, and 0.5h is piece of surface covering glass plate 30min).The result shows that touching before stimulation (i.e. non-cover glass plate), with the expression quantity of TCH1 gene and TCH3 gene for 1；? After touching stimulation, the expression quantity of TCH1 gene and TCH3 gene significantly raises (2 times of the expression quantity up-regulation of TCH1 gene, TCH3 base The expression quantity of cause raises 31 times).It can be seen that TCH1 gene and TCH3 gene pairs, which touch stimulation (such as mechanical pressure), to be had obviously Response, further relate to TCH1 gene promoter and TCH3 gene promoter to touch stimulation (such as mechanical pressure) have it is bright Aobvious response.

<110>Peking University

<120>a kind of plant touches response promoter and its application

<160> 5

<170> PatentIn version 3.5

<210> 1

<211> 1653

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 1

atggaagacg ccaaaaacat aaagaaaggc ccggcgccat tctatcctct agaggatgga 60

accgctggag agcaactgca taaggctatg aagagatacg ccctggttcc tggaacaatt 120

gcttttacag atgcacatat cgaggtgaac atcacgtacg cggaatactt cgaaatgtcc 180

gttcggttgg cagaagctat gaaacgatat gggctgaata caaatcacag aatcgtcgta 240

tgcagtgaaa actctcttca attctttatg ccggtgttgg gcgcgttatt tatcggagtt 300

gcagttgcgc ccgcgaacga catttataat gaacgtgaat tgctcaacag tatgaacatt 360

tcgcagccta ccgtagtgtt tgtttccaaa aaggggttgc aaaaaatttt gaacgtgcaa 420

aaaaaattac caataatcca gaaaattatt atcatggatt ctaaaacgga ttaccaggga 480

tttcagtcga tgtacacgtt cgtcacatct catctacctc ccggttttaa tgaatacgat 540

tttgtaccag agtcctttga tcgtgacaaa acaattgcac tgataatgaa ttcctctgga 600

tctactgggt tacctaaggg tgtggccctt ccgcatagaa ctgcctgcgt cagattctcg 660

catgccagag atcctatttt tggcaatcaa atcattccgg atactgcgat tttaagtgtt 720

gttccattcc atcacggttt tggaatgttt actacactcg gatatttgat atgtggattt 780

cgagtcgtct taatgtatag atttgaagaa gagctgtttt tacgatccct tcaggattac 840

aaaattcaaa gtgcgttgct agtaccaacc ctattttcat tcttcgccaa aagcactctg 900

attgacaaat acgatttatc taatttacac gaaattgctt ctgggggcgc acctctttcg 960

aaagaagtcg gggaagcggt tgcaaaacgc ttccatcttc cagggatacg acaaggatat 1020

gggctcactg agactacatc agctattctg attacacccg agggggatga taaaccgggc 1080

gcggtcggta aagttgttcc attttttgaa gcgaaggttg tggatctgga taccgggaaa 1140

acgctgggcg ttaatcagag aggcgaatta tgtgtcagag gacctatgat tatgtccggt 1200

tatgtaaaca atccggaagc gaccaacgcc ttgattgaca aggatggatg gctacattct 1260

ggagacatag cttactggga cgaagacgaa cacttcttca tagttgaccg cttgaagtct 1320

ttaattaaat acaaaggata tcaggtggcc cccgctgaat tggaatcgat attgttacaa 1380

caccccaaca tcttcgacgc gggcgtggca ggtcttcccg acgatgacgc cggtgaactt 1440

cccgccgccg ttgttgtttt ggagcacgga aagacgatga cggaaaaaga gatcgtggat 1500

tacgtcgcca gtcaagtaac aaccgcgaaa aagttgcgcg gaggagttgt gtttgtggac 1560

gaagtaccga aaggtcttac cggaaaactc gacgcaagaa aaatcagaga gatcctcata 1620

aaggccaaga agggcggaaa gtccaaattg taa 1653

<210> 2

<211> 550

<212> PRT

<213>artificial sequence

<220>

<223>

<400> 2

Met Glu Asp Ala Lys Asn Ile Lys Lys Gly Pro Ala Pro Phe Tyr Pro

1 5 10 15

Leu Glu Asp Gly Thr Ala Gly Glu Gln Leu His Lys Ala Met Lys Arg

20 25 30

Tyr Ala Leu Val Pro Gly Thr Ile Ala Phe Thr Asp Ala His Ile Glu

35 40 45

Val Asn Ile Thr Tyr Ala Glu Tyr Phe Glu Met Ser Val Arg Leu Ala

50 55 60

Glu Ala Met Lys Arg Tyr Gly Leu Asn Thr Asn His Arg Ile Val Val

65 70 75 80

Cys Ser Glu Asn Ser Leu Gln Phe Phe Met Pro Val Leu Gly Ala Leu

85 90 95

Phe Ile Gly Val Ala Val Ala Pro Ala Asn Asp Ile Tyr Asn Glu Arg

100 105 110

Glu Leu Leu Asn Ser Met Asn Ile Ser Gln Pro Thr Val Val Phe Val

115 120 125

Ser Lys Lys Gly Leu Gln Lys Ile Leu Asn Val Gln Lys Lys Leu Pro

130 135 140

Ile Ile Gln Lys Ile Ile Ile Met Asp Ser Lys Thr Asp Tyr Gln Gly

145 150 155 160

Phe Gln Ser Met Tyr Thr Phe Val Thr Ser His Leu Pro Pro Gly Phe

165 170 175

Asn Glu Tyr Asp Phe Val Pro Glu Ser Phe Asp Arg Asp Lys Thr Ile

180 185 190

Ala Leu Ile Met Asn Ser Ser Gly Ser Thr Gly Leu Pro Lys Gly Val

195 200 205

Ala Leu Pro His Arg Thr Ala Cys Val Arg Phe Ser His Ala Arg Asp

210 215 220

Pro Ile Phe Gly Asn Gln Ile Ile Pro Asp Thr Ala Ile Leu Ser Val

225 230 235 240

Val Pro Phe His His Gly Phe Gly Met Phe Thr Thr Leu Gly Tyr Leu

245 250 255

Ile Cys Gly Phe Arg Val Val Leu Met Tyr Arg Phe Glu Glu Glu Leu

260 265 270

Phe Leu Arg Ser Leu Gln Asp Tyr Lys Ile Gln Ser Ala Leu Leu Val

275 280 285

Pro Thr Leu Phe Ser Phe Phe Ala Lys Ser Thr Leu Ile Asp Lys Tyr

290 295 300

Asp Leu Ser Asn Leu His Glu Ile Ala Ser Gly Gly Ala Pro Leu Ser

305 310 315 320

Lys Glu Val Gly Glu Ala Val Ala Lys Arg Phe His Leu Pro Gly Ile

325 330 335

Arg Gln Gly Tyr Gly Leu Thr Glu Thr Thr Ser Ala Ile Leu Ile Thr

340 345 350

Pro Glu Gly Asp Asp Lys Pro Gly Ala Val Gly Lys Val Val Pro Phe

355 360 365

Phe Glu Ala Lys Val Val Asp Leu Asp Thr Gly Lys Thr Leu Gly Val

370 375 380

Asn Gln Arg Gly Glu Leu Cys Val Arg Gly Pro Met Ile Met Ser Gly

385 390 395 400

Tyr Val Asn Asn Pro Glu Ala Thr Asn Ala Leu Ile Asp Lys Asp Gly

405 410 415

Trp Leu His Ser Gly Asp Ile Ala Tyr Trp Asp Glu Asp Glu His Phe

420 425 430

Phe Ile Val Asp Arg Leu Lys Ser Leu Ile Lys Tyr Lys Gly Tyr Gln

435 440 445

Val Ala Pro Ala Glu Leu Glu Ser Ile Leu Leu Gln His Pro Asn Ile

450 455 460

Phe Asp Ala Gly Val Ala Gly Leu Pro Asp Asp Asp Ala Gly Glu Leu

465 470 475 480

Pro Ala Ala Val Val Val Leu Glu His Gly Lys Thr Met Thr Glu Lys

485 490 495

Glu Ile Val Asp Tyr Val Ala Ser Gln Val Thr Thr Ala Lys Lys Leu

500 505 510

Arg Gly Gly Val Val Phe Val Asp Glu Val Pro Lys Gly Leu Thr Gly

515 520 525

Lys Leu Asp Ala Arg Lys Ile Arg Glu Ile Leu Ile Lys Ala Lys Lys

530 535 540

Gly Gly Lys Ser Lys Leu

545 550

<210> 3

<211> 1374

<212> DNA

<213>arabidopsis (Arabidopsis thaliana)

<400> 3

agcttattac tctcttcctt ggttttgttt tatttttatc tagttttagt tttgaaattt 60

atttttttta ttatgaattt taaaaattta atttttattt gtgttggaat ttttttattt 120

tctttgtaac ctatatgaac ttggatattt tttttctttc ttacattaat taagtacgta 180

agtttcgaca atcaagaata gggttttgtt gattccaatc gtgttaccca aaaattagaa 240

taaggtttgt ccaacaattt attaaatttg gtcttagagt tgtatacgag cctacgaggt 300

atcttttaga tatgccgaag agatggtagt gcgaagaaga aaagaaacct cattttccat 360

ggtattaagg atttcaagct attcaaactt ttgtagtgac ccaaatacgt gtagaactgg 420

cccaaagaca aatagcttaa ttaagattat cacctaatct taggtattgg tgataggtga 480

ttcgctcaag ccttatgatc aatattagct ccatattatg gagagtgagc agcacatact 540

cgtagataga aaagtattgt tgctcgagta atatttttct gatgataatt gtgtttcgac 600

cactgttggc atctttcaca catgaataca tgatgtacat gtacgacatg caatctttct 660

cttaacgctc gactgctgtc tgctgtgatg gatcatcaca agtaaacttt gtgaagcata 720

tttcgtgagc caaatgatgt tagaaaacaa tcgtgtgagc caaaggcgtg agagcgcttg 780

tagaaagtgg tttatgttgt agttaaaata atccgaatat tcatcttccg gaaaaaagtg 840

aagttcaacc tctcaacggt acgtttttat acctcaacta agttacattg tgtatatcaa 900

catcagctaa ctttttatta atatttcggt tcacaatatc attatctttt tcaaaattta 960

ttaagatttc ttttgctcat tttatttatg tgaattttaa aaataaaata atttttataa 1020

tatagaaaca aaatattact aaaagtaatt tttaaaaaat aaataaaatt aatgtcatat 1080

caacgaaaaa ttcatagttt attaaagtag taaatataaa agtaataata ttttaattat 1140

tttggtatta aattatacac ttttattaat atatgtattt tcttctaaaa attaaaaatt 1200

aattgggttt ttgtttggaa tttttccgac ttaccttttc tccttctttt tgtcgttttg 1260

cttagatatt tgtttgcatc attttcaaag agagacgact ctgaatccaa aaaaaaaaaa 1320

aactcattaa gtaaagacaa aggaagagaa gaaagacgat ttctcgaaga agct 1374

<210> 4

<211> 1400

<212> DNA

<213>arabidopsis (Arabidopsis thaliana)

<400> 4

cattagggtc tggctggtat gttagatctc tctaaaaagg cgtttgatct ttgaaaataa 60

tttctatgta ataaatttat tgtgttacca tcttttcttg catgcaattt attaaaatga 120

gtttgtaata tggtttcagt atcgaatata attggggtat caatgtatct tttgagaaaa 180

acatttaaaa cgtagcagaa ctaattattg aaaactggta gcattactaa aaattgatct 240

aaatcggtta atgatttggc acatacagtg atcaaatcag ctaagcctca tatctcaccg 300

tcccagagtt cttcaagact cttataagga ctcatctaat gaataatgac atgcctcgca 360

ccaacctgcc ctgcaacatt gaaaacacac gtctacaatt tcgataaccc aagtgttttg 420

agcaaaacaa aggacgccct ttgacccatt tggactacaa tgtgtggaaa attactgcat 480

gttcaatatc agaaaacttg gataacatgt cttcgatatg ggcttttctt attttgtacc 540

cgtgtaattt cgctggccca aaatccaaag caacaagggt aaatttgtat gcagtacatg 600

tggcatgttg ggtcatatgg aaacaggtca caagactcag attctttaca ttgcggcggg 660

tagtagtagc tttctagtct accttgagat tgcccctgcc cgtgattctt gagtcgggca 720

caggaagtgg ttttctgtca actttacttg ctcaagccat ccccgcaagg tagaaacttc 780

tgtggtcttt ctcgttattt cttatgtacc atgtttttgg tcgtttgtgt agtttcttct 840

aaccttccac acacttgtct tgcgaattcc ttccacagca gagatgttaa atcaggaata 900

ttcaggatgt agaaacttct ctgctctttc tcaagtagaa tgcacttgtg gaatccctca 960

gatcagattt cattggtaac gttcgttaac ttcaacaaca acactttact aaacatgtgc 1020

taatcaattg tcatacgctt gtatttcttc gattcatttt tatcttatgc aagtgttttc 1080

tctattatga tttatttagt tttgcataaa taaggacact ccttcggagt cagtataaga 1140

aaactataac ctaaaatata aattaggcaa atgtccactc acccatccaa aattccattg 1200

taaataaatg tttctttcaa ataaatgaaa aaaaaaaaaa aaaacaagtt ggaagaagga 1260

accaaccaag taattgccag tcaaaggagg cgactcacac ttgaaaggat aagctgacat 1320

ggcagacaac gatcctgcgt agaaattgcg tgaacgtgga aaagtttgag gatatagccg 1380

tcatttcaaa taattcgggt 1400

<210> 5

<211> 346

<212> DNA

<213>artificial sequence

<220>

<223>

<400> 5

tgagactttt caacaaaggg taatttcggg aaacctcctc ggattccatt gcccagctat 60

ctgtcacttc atcgaaagga cagtagaaaa ggaaggtggc tcctacaaat gccatcattg 120

cgataaagga aaggctatca ttcaagatgc ctctgccgac agtggtccca aagatggacc 180

cccacccacg aggagcatcg tggaaaaaga agacgttcca accacgtctt caaagcaagt 240

ggattgatgt gacatctcca ctgacgtaag ggatgacgca caatcccact atccttcgca 300

agacccttcc tctatataag gaagttcatt tcatttggag aggaca 346

Claims (10)

1. specific DNA molecular, for as follows (a1) or (a2) or (a3):

(a1) DNA molecular shown in sequence 4；

(a2) nucleotide sequence limited with (a1) has 75% or 75% or more identity, and the DNA with promoter function Molecule；

(a3) nucleotide sequence hybridization limited under strict conditions with (a1) or (a2), and the DNA with promoter function points Son.

2. the expression cassette containing specific DNA molecular described in claim 1.

3. the recombinant plasmid containing specific DNA molecular described in claim 1.

4. the transgenic cell line containing specific DNA molecular described in claim 1.

5. specific DNA molecular described in claim 1 as promoter or touch response promoter application.

6. recombinant plasmid described in expression cassette described in specific DNA molecular, claim 2 described in claim 1 or claim 3 is opening Application in the expression of dynamic target gene.

7. a kind of method for expressing target gene, using specific DNA molecular described in claim 1 as promoter or to touch response Promoter starts the expression of target gene.

8. a kind of method for expressing target gene includes the following steps: for be inserted into specific DNA molecular described in claim 1 and take office The upstream of what target gene or enhancer, starts the expression of target gene with this.

9. a kind of method for expressing target gene includes the following steps: for be inserted into expression cassette described in claim 2 target gene The specific DNA molecular downstream, start the expression of the target gene by the specific DNA molecular.

10. a kind of method for expressing target gene includes the following steps: that matter will be recombinated described in target gene insertion claim 3 The downstream of the specific DNA molecular in grain, is started the expression of the target gene by the specific DNA molecular.