CN106754916B - A kind of ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans - Google Patents

Abstract

The invention discloses a kind of ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans, the nucleotide sequence such as SEQ ID NO of the evoked promoter:Shown in 1, the invention also discloses application of the ABA evoked promoters of the gene in being started target gene expression by ABA inducement efficients.Experiments have shown that, under ABA inductions, promoter of the present invention can raise expression of the driving reporter gene in transgenic arabidopsis and soybean protoplast, this helps to carry out the drought-enduring regulatory mechanism research of GmNAC15 genes, ABA inducible promoters to obtain universal provide sequence material, and being also used to cultivate drought-resistant crops kind for it provides the foundation.

Description

A kind of ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans

Technical field

The present invention relates to a kind of a kind of ABA of promoter more particularly to No. 9 GmNAC15 genes of soybean sage beans inductions to start Son.

Background technology

Soybean, pulse family (Fabaceae), Glycine (Glycine), annual herb is originating in China, has all over China Cultivation is also cultivated in all over the world extensively.Soybean (Glycine max (L.) Merr) is important cereal crops and economic work Object is rich in protein, fat and a variety of active materials beneficial to human body of high quality, has very high nutritive value.In addition, Soybean is also the quality raw materials for being a variety of processing industry such as food, feed, and consequence is in national economy.With warp The development of Ji also will constantly increase Soybean demand amount, however soybean yield-increasing limited by environmental degradation it is increasingly apparent.Arid, Salt, extreme temperature, chemistry poison and oxidative stress be influence crop yield main abiotic stress factor, wherein arid and Salt damage limits the development of soybean planting industry.

Plant will maintain normal growth in complicated natural environment, and the internal environmental stimuli factor is needed to make corresponding sound It answers, accurate regulation and control is made to plant itself various functions gene.It is known that plant is resisting arid, with high salt and low temperature etc. There are 4 bars pathways when environment-stress：2 therein belong to and rely on ABA signal transduction systems, and other 2 then not Rely on ABA signal transduction systems.The biosynthesis of MYC and myb transcription factor is needed in relying on ABA signal pathways I to swash Adversity gene expression in downstream living.It is activation bZIP class transcription factors that another, which relies on ABA signal pathways II then, is transcribed by bZIP classes The factor reacts regulating element (ABRE PyCGTGGC) combination with the ABA in target gene promoter, to regulate and control the degeneration-resistant base in downstream Because of expression.For not depending on ABA signal transduction paths III then by activating DREB class transcription factors, downstream base is identified by it Because of the DRE cis-acting elements of promoter region to regulate and control downstream destination gene expression.

It is to cultivate degeneration-resistant crop new product to be expressed in transgenic plants using adverse circumstance inducible promoter driving adversity gene The effective ways of kind.The inducible promoter that can apply to transgenic research at present is still seldom, for new degeneration-resistant phase start or stop The discovery of mover, clone, cis-acting elements are analyzed and are still with the research of the transcription factor of cis element interaction The degeneration-resistant promoter of definite functions is successfully applied to regulate and control the expression of adversity gene in genetically modified plants by the emphasis studied from now on It is the research direction of plant stress-resistance genetic engineering.Adverse circumstance related gene often responds a variety of environment stresses in NAC families, this is big Part give the credit to on gene promoter containing there are many relevant cis-acting elements of environment stress, by the relevant NAC family of adverse circumstance Race's gene promoter research can obtain a variety of and coerce relevant crucial DNA fragmentation and cis-acting elements, effective to building Adverse circumstance inducible promoter it is significant.Research is more deep at present has：Abscisic acid response element (ABA-responsive Element, ABRE), ethylene response element (ethylene-responsive element, ERE), jasmonate response Element (jasmonate-responsive element, JRE), low temperature response element (low-temprature reponsive Element, LTRE) and arid response element (dehydration-responsive element, DRE) etc..Plant is started The research of son helps to understand gene transcription regulation expression pattern and its regulatory mechanism, and to specificity and inducible promoter work( The research of energy, helps to obtain specific expressed genetically modified plants normally and efficiently.

It is verified in this laboratory previous work to derive from salt-enduring cultivars --- the GmNAC15 genes of holy beans 9 are salt With ABA responsive genes, and to salt stress be positive control (patent ZL2012101285907).It is holy by comparing salt tolerant soybean Beans 9 salt and it is non-salt under the conditions of gene expression profile variation, it was found that GmNAC15 genes up-regulated expression under salt treatment, will The over-express vector of the gene is transferred in model plant arabidopsis, and render transgenic arabidopsis obtains non-transgenic arabidopsis institute not The ability for the salt resistance/drought tolerance having.Simultaneously under ABA processing, transgenic arabidopsis seed germination rate and leaf stoma degree of leading height In nontransgenic plants (control), show that GmNAC15 genes participate in arabidopsis drought resisting/salt tolerance by ABA Dependents.Pass through Soybean Germinating Embryo vacuum infiltration auxiliary exogenous gene transforming method the over-express vector of the gene is transferred in soybean, by than Compared with analytical proof, Transgenic soybean plants are significantly increased than salt resistance/drought tolerance of nontransgenic plants.Simultaneously in ABA processing Under, genetically engineered soybean germination rate and stomatal conductance are above nontransgenic plants (control), show that GmNAC15 genes pass through ABA Dependent participates in soybean drought resisting/salt tolerance.Currently, GmNAC15 genes participate in the mechanism of ABA signal paths and indefinite, grind Studying carefully the ABA evoked promoters of GmNAC15 genes can help preferably to illustrate the function of GmNAC15 genes, study simultaneously The ABA inducing properties of GmNAC15 promoters can provide important references to obtain universal ABA inducible promoters, in the future may be used Cultivation for transgenic salt-tolerant wheat crop.But retrieval is found, yet there are no about GmNAC15 Gene A BA evoked promoters Relevant report.

Invention content

The object of the present invention is to provide a kind of ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans and its applications.

The ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans of the present invention, it is characterised in that：The ABA The nucleotide sequence of evoked promoter is one of following nucleotide sequences：

A) SEQ ID No in sequence table:1 DNA sequence dna；

With SEQ ID No in sequence table:1 DNA sequence dna has 90% or more homology, and DNA with the same function Sequence.

The present invention also provides a kind of reporter genes of No. 9 GmNAC15 Gene A BA evoked promoters containing above-mentioned soybean sage beans GUS expression vectors pGmNAC15::PKGWFS7, the nucleotide sequence such as SEQ ID No of the expression vector:Shown in 2；And it is a kind of The reporter gene LUC expression vectors pGmNAC15 of No. 9 GmNAC15 Gene A BA evoked promoters containing above-mentioned soybean sage beans:: PGreenII-0800LUC, the nucleotide sequence such as SEQ ID No of the expression vector:Shown in 3.

The ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans of the present invention are being started mesh by ABA inducement efficients Mark the application in gene expression.

Applicant is cloned into GmNAC15 gene promoters in No. 9 plant of soybean sage beans first；Utilize Gateway systems System reacts by BP, LR, GmNAC15 gene promoters is connected to gus reporter gene expression vector pKGWFS7 (see Fig. 2) On；By pGmNAC15::PKGWFS7 carriers are transferred in agrobacterium strains GV3101, and pattern is transferred to by the agrobacterium strains of conversion In plant Arabidopsis thaliana, to verify GmNAC15 gene promoter subfunctions.

Applicant is also connected by digestion and GmNAC15 gene promoters is connected in pGreenII-0800LUC carriers, By arabidopsis and soybean mesophyll protoplast transient transfection systems, verification GmNAC15 gene promoters are induced by ABA Function.

Experiment confirms：The ABA evoked promoters of sage's No. 9 GmNAC15 genes of beans of the present invention can be in ABA inductive conditions The expression of lower up-regulation target gene, it is expected to be used for the cultivation of transgenosis adversity resistant plant.The wherein described preferred soybean of plant or quasi- south Mustard.

The present invention has the beneficial effect that：Using PCR clone technologies, the present invention clones for the first time has obtained soybean sage beans 9 GmNAC15 gene promoters, and gus reporter gene is driven to be expressed in arabidopsis, render transgenic arabidopsis obtains non- The ability of the not available expression gus gene of transgenic arabidopsis, and can higher be expressed under ABA inductive conditions.Indicate this hair Bright described No. 9 GmNAC15 genes of soybean sage beans and its ABA evoked promoters can be widely used for cultivating degeneration-resistant plant variety.

Description of the drawings

Fig. 1 is PCR clone's soybean sage No. 9 GmNAC15 gene promoter electrophoretograms of beans, wherein：M is Marker, swimming lane 1,2 For promoter DNA.

Fig. 2 is pKGWFS7.0 carrier schematic diagrames.

Fig. 3 is GmNAC15::GUS transgenic arabidopsis GUS coloration results

Wherein：A is the coloration result of complete stool after transgenic arabidopsis culture 2 days, in apical meristem, the dimension of hypocotyl There are GUS signal representations at tubing and tip of a root position；B is 7 days after seed is sprouted, and gus gene is in cotyledon vein, true leaf, true leaf base High expression in portion, apical meristem, main root tip of a root position and the lateral root tip of a root；C is blade；D is true leaf base portion, growing point；E is The tip of a root；F is inflorescence；G is silique.

Fig. 4 is GmNAC15::GUS transgenic arabidopsis GUS staining versus in the case where having ABA (B) and being handled without ABA (A) schemes. (B is shown：ABA processing under in transgenic arabidopsis cotyledon GUS signal enhancings).

Fig. 5 is the insert districts pGreenII-0800LUC carrier T-DNA schematic diagram.

Fig. 6 is GmNAC15 promoters relative fluorescence element enzymatic determination result under the conditions of ABA and non-ABA, wherein MOCK is The protoplast normally relative luciferase activity of culture for 24 hours is converted, experimental group (ABA) is conversion protoplast 50mM ABA The relative luciferase activity of processing for 24 hours.

Specific implementation mode

The acquisition of 1 soybean GmNAC15 gene promoters of embodiment

According to the upstreams the GmNAC15 gene transcription start site ATG 2026kb sequences found on the websites NCBI, design starts Sub- primer (F：5’-CGCGTCGACCATGATTGCGAATTATTTATC-3’：5’-ACTGCAGGGGGGAAGACAGAGGGA-3’) No. 9 genomic DNAs of holy beans are extracted using CTAB methods, the genomic DNA of extraction is diluted to 100ug/ul, are used as amplification promoter Template.Using high fidelity enzyme HIFI (Takara), GmNAC15 gene promoters are expanded.

1.1 No. 9 extracting genome DNAs of holy beans

(1) CTAB extracting solutions are put into 65 DEG C of water-baths in advance and are preheated；

(2) No. 9 vegetable materials of holy beans are put into mortar, powder is ground under the protection of liquid nitrogen；

(3) etc. after liquid nitrogen volatilization, 100-200mg plant powders are transferred in 1.5ml centrifuge tubes immediately, then promptly The 650 μ l of 2%CTAB extracting solutions of 65 DEG C of preheatings are added, overturns mixing rapidly, is placed in 65 DEG C of water-bath 30min, during which run per 5min Mixing is primary；

(4) it takes out material to be cooled to room temperature, isometric phenol/chloroform/isoamyl alcohol, mixing to milky, room temperature is added 12000rpm centrifuges 10min；

(5) supernatant is transferred in new sterile centrifugation tube, isometric chloroform/isoamyl alcohol is added, gently overturns mixing, Room temperature, 12000rpm centrifuge 10min；

(6) supernatant is transferred in new sterile centrifugation tube again, isometric isopropanol is added, gently overturn mixing, -20 DEG C place 20min；

(7) 4 DEG C, 12000rpm, 10min is centrifuged, abandons supernatant, 70% ethyl alcohol is washed twice, dried up in super-clean bench；

(8) precipitation is dissolved in 40 μ l distilled waters (A containing 0.1%RNase), and -20 DEG C save backup.

1.2 GmNAC15 gene promoters are cloned

The reaction system of high fidelity enzyme HIFI amplifications is following (50 μ l systems)：

Amplification condition is as follows：

After PCR, clone products are detected in 0.8%TAE agarose gel electrophoresis.Electrophoresis result is as shown in Figure 1.

The purifying recycling (Tiangeng kit) of 1.3 clone gene segments

1) 500 μ l equilibrium liquids (BL) are taken, are added in recovery column (CB2), 12000rpm, 1min is centrifuged；

2) gel with target fragment will be cut to be put into 1.5ml centrifuge tubes, the sol solutions of 3 times of volumes of addition, 50 DEG C Colloidal sol 10min, during which overturns every now and then；

3) it after gel melts completely, is transferred in equilibrated recovery column (CB2), 12000rpm, centrifuges 1min；

4) rinsing liquid (PW) of 600 μ l, room temperature 2-5min, 12000rpm is added to centrifuge 1min into adsorption column (CB2)；

5) step 4 is repeated；

6) void column, 12000rpm centrifuge 2min；

7) recycling pillar is uncapped, after several minutes of dryings are blown in super-clean bench, is put into the 1.5ml centrifuge tubes of sterilizing, be added 40 μ l are preheating to 60 DEG C of EB buffer solutions, place 1min；

8) 12000rpm centrifuges 1min, and acquired solution is i.e. containing recycling segment.

1.4 connection GmNAC15 promoters and pMD19-T carriers

Reaction system is following (20 μ l systems)：

16 DEG C of connections overnight.(pMD19-T carriers are bought from TAKARA companies).

The plasmid conversion (sterile working) of 1.5 Escherichia coli

(1) 1-5 μ l connection products are added in the competent escherichia coli cell of 50 μ l, gently mixing, is placed on ice 30min；

(2) 42 DEG C of water-bath heat shock 90sec, place 2-3min on ice immediately；

(3) 1ml LB culture mediums are added in super-clean bench, 40-50min is cultivated on 37 DEG C of shaking tables；

(4) room temperature, 5000rpm centrifuge 3min；

(5) LB culture mediums are outwelled, bacterium is coated on the culture dish containing corresponding antibiotic, 37 DEG C of inversions were cultivated Night.

1.6 colibacillus PCRs are verified

Reaction system is following (20 μ l systems)：

Amplification condition is as follows：

After PCR, reaction solution is detected in 0.8%TAE agarose gel electrophoresis.

1.7 DNA sequencing

The single bacterium colony of PCR test positive is shaken with the liquid LB containing Amp (50mg/L), 37 DEG C are shaken overnight, are then served Hai Boshang Bioisystech Co., Ltd is sequenced, and obtains sequencing result, promoter DNA sequence is as shown in sequence table SEQ ID No.1.

By sequence alignment analysis, above-mentioned DNA sequence dna (SEQ ID No.1) is same with the nucleotide of soybean Williams 82 Source 99.69%, three times biology repetition prove that the DNA sequence dna (SEQ ID No.1) is exactly No. 9 GmNAC15 bases of soybean sage beans Because of promoter, it is named as the ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans.

PGmNAC15 in 1.8 extraction Positive E. colis::PMD19-T plasmids (Tiangeng kit)

(1) correct bacterium will be sequenced to be inoculated in the LB liquid medium that 10ml contains Amp (50mg/L), 37 DEG C were shaken Night；

(2) bacterium solution is added in the 1.5ml centrifuge tubes of sterilizing, 12000rpm, centrifuges 1min, collect thalline；

(3) supernatant is abandoned, the P1 solution of 4 DEG C of 250 μ l is added, with the soft suspension thalline of the liquid-transfering gun of 1ml；

(4) the P2 solution gentle inversion mixings of 250ul are added, 5min is stood；

(5) the P3 solution of 350 μ l is added after solution clarification, gently places 5min after mixing；

(6) room temperature, 12,000rpm, 10min is centrifuged, while 600 μ l BL equilibrium liquids being added in adsorption column, is placed at room temperature for 2min, 12000rpm centrifuge 1min, outwell the liquid in collecting pipe；

(7) supernatant after centrifugation is transferred in equilibrated adsorption column, is placed at room temperature for 2min, 12000rpm, centrifuged 1min outwells the liquid in collecting pipe；

(8) 600 μ l rinsing liquid PW are added in adsorption column, are placed at room temperature for 2min, 12000rpm, centrifuges 1min, outwells receipts Liquid in collector repeats primary；

(9) adsorption column blank pipe 12000rpm centrifuges 5min；

(10) adsorption column is placed in a sterile 1.5ml centrifuge tube, it is pre- that 55 μ l are added dropwise to the intermediate position of adsorption column Heat is placed at room temperature for 2min, 12000rpm to 65 DEG C of EB eluents, centrifuges 2min, plasmid solution is collected into centrifuge tube ,- 20 DEG C save backup.

2. No. 9 GmNAC15 gene promoter sequences analyses of soybean sage beans

Utilize online promoter Analysis software PLACE (http://www.dna.affrc.go.jp/PLACE/) and PLANTCARE(http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) to clone Obtained GmNAC15 gene promoters carry out sequence analysis, find the cis- of in GmNAC15 gene promoter regions middle prediction Element have 34 may be related to stress (as shown in the table), including (2) 4 GT1GMSCAM4,6 ARE, 1 DRE, 3 GRE and some MYB, MYC show related containing abundant stress in the promoter to WRKY families Binding site for transcription factor Cis-acting elements may be regulated and controled by a variety of transcription factors, participate in plant and responded to a variety of biologies and abiotic stress.

Embodiment 2 stablizes transgenic technology and analyzes GmNAC15 promoter activities

2.1. Reporter gene GUS expression vector pGmNAC15::GUS is built

Utilize Gateway systems structure promoter pGmNAC15::The flows of GUS expression vectors as shown in Fig. 2, according to GmNAC15 promoter sequences design the primer (primer pair 5 '-containing attB connectors respectivelyAAA AAG CAG GAC CGT CGT GTT CAC AAA TC-3 ', R：5’-AGA AAG CTG GGTTTT TTC A AA AAC ACA ATT TCG-3 ', under It is respectively attB1 connectors and attB2 connectors at scribing line).

2.1.1 PCR amplification contains the GmNAC15 promoter gene fragments of attB connectors

(1) high fidelity enzyme HIFI carries out the first round amplification (20 μ l systems) of Gateway systems：

Amplification condition is as follows：

(2) high fidelity enzyme HIFI carries out the second step amplification of Gateway systems.

Attb primer sequences：

attb-F：5’-G GGG ACA AGT TTG TAC AAA AAA GCA GGC T-3’

attb-R：5’-GGG GAC CAC TTT GTA CAA GAA AGC TGG GT-3’

Reaction system is following (50 μ l systems)：

Amplification condition is as follows：

2.1.2 the purifying recycling (Tiangeng kit) of clone gene segment (with 1.3)

2.1.3 the BP reactions of clone gene segment

It is as follows that BP reacts (Gateway systems) system：

25 DEG C react 8 hours-overnight.

2.1.4 E. coli competent plasmid conversion (with 1.5)

2.1.5 colibacillus PCR verification (with 1.6)

2.1.6 DNA sequencing (with 1.8)

Verification is connected to the GmNAC15 promoter cloning regions nucleotide sequence such as SEQ ID No.1 institutes in pDONR221 Show.

2.1.7 the extraction of e. coli plasmid dna obtains pGmNAC15::PDONR221 plasmids (with 1.7)

2.1.8 the LR reactions of clone gene segment

It is as follows that LR reacts (Gateway systems) system：

25 DEG C react 8 hours-overnight.(pKGWFS7 is bought from Invitrogen pKGWFS7 carriers schematic diagram as shown in Figure 2 Company).

2.1.9 the preparation of E. coli competent and plasmid conversion (with 1.5)

2.1.10 colibacillus PCR verification (with 1.6)

2.1.11 the extraction of e. coli plasmid dna (with 1.8)

Verification is transferred to the pGmNAC15 of the positive plasmid of pKGWFS7 carriers::PKGWFS7, that is, pGmNAC15::GUS clones area Domain nucleotide sequence is as shown in SEQ ID No.1.

2.1.12 the preparation (sterile working) of Agrobacterium competence

(1) Agrobacterium GV3101 strains are taken to be inoculated in 10ml YEP fluid nutrient mediums, 28 DEG C of shaking table cultures are stayed overnight；

(2) 1 is pressed：50 are inoculated in 50ml YEP fluid nutrient mediums, 28 DEG C of shaken cultivations 3-4 hours, until OD₆₀₀Value is 0.4-0.6；

(3) 4 DEG C, 4200rpm, 10min is centrifuged, collects thalline；

(4) supernatant is abandoned, the NaCl suspension thallines of the 0.15M of 10ml precoolings are added；

(5) step 3 is repeated；

(6) supernatant is abandoned, the CaCl of the 20mM of 2ml precoolings is added₂Suspension thalline is sub-packed in 1.5ml centrifuge tubes, it is current or Final volume 7%DMSO is added to save backup for -80 DEG C after liquid nitrogen flash freezer.

2.1.13 the plasmid conversion (sterile working) of Agrobacterium

(1) 10 μ l Plasmid DNA are added in the competent cell of 50 μ l, flick centrifuge tube mixing, ice bath 30min；

(2) liquid nitrogen flash freezer 1min；Then 37 DEG C of water-bath 5min, immediately ice bath 2-3min；

(3) 1ml YEP culture mediums, 28 DEG C of culture 2-4h are added；

(4) room temperature, 4000rpm centrifuge 3min, collect thalline；

(5) bacterium is coated on the YEP culture plates containing corresponding antibiotic, 28 DEG C are inverted culture 48h.

2.1.14 the PCR verifications of conversion Agrobacterium

Reaction system is following (20 μ l systems)：

Amplification condition is as follows：

After reaction, reaction solution is detected in 0.8%TAE agarose gel electrophoresis.

2.2 verify the functional verification of No. 9 GmNAC15 gene promoters of holy beans in arabidopsis

2.2.1 flower infestation method arabidopsis thaliana transformation

(1) when arabidopsis (Col-0 wild types) grows to bolting 1cm, top is cut to the generation to induce side to give birth to inflorescence；

(2) in conversion the previous day, the Agrobacterium GV3101 containing expression vector plasmid that 1ml was activated is taken to be added to containing corresponding In the 40ml YEP culture mediums of antibiotic and 50 μ g/ml rifampins, 28 DEG C of shake cultures to OD600 are about 1.0-1.2；

(3) room temperature, 4200rpm centrifuge 10min, thalline are collected, with dip dyeing liquid for shell (5% sucrose, 0.05%Silwet L- 77) thalline is resuspended, it is about 0.8 to make OD600；

(4) Agrobacterium is dripped on inflorescence with pipettor and is disseminated, after all inflorescences are all infected, arabidopsis is put Enter and vacuumizes 1min in vacuum desiccator；

(5) inflorescence is covered with freshness protection package, being protected from light culture as 20-22 DEG C cuts off top exposing inflorescence for one day, is further cultured for one Freshness protection package, culture to seed maturity are thrown off after it.

2.2.2 the surface sterilization of arabidopsis seed

Appropriate arabidopsis neutron subject to sterilization is put into 1.5ml centrifuge tubes, the ethyl alcohol that 1ml 75% is added (contains The TritonX-100 of 0.03% volume ratio) concussion disinfection 1min, then 1min (twice) is sterilized with 70% ethyl alcohol concussion, finally Seed is drawn onto on aseptic filter paper with suction nozzle and is dried up, is then clicked and entered in culture medium with sterile toothpick.

2.2.3 the screening of transfer-gen plant

Using flower infestation method by Agrobacterium by pGmNAC15::GUS carriers are transferred in arabidopsis.By the T0 generation kinds of harvest Son is broadcast to be screened on the 1/2MS solid mediums containing 50mg/L card sodium mycins, is green by blade after seed is sprouted 10 days Color, the longer seedling of root move into soil, take a small amount of blade slightly to carry genomic DNA when seedling fast bolting, and PCR method verification is taken to turn Gene plant, primer are att B1/2.Wait for that positive plant is ripe and harvests T1 for seed.T1 is used for seed contains 50mg/L card sodium The 1/2MS solid mediums of mycin carry out resistance screening, choose resistance：

Non-resistance ratio is 3:1 single copy is inserted into strain, plants positive plant, and i.e. harvest T2 is for seed after maturation.T2 generations Seed screens to obtain transgenic homozygous system by card sodium chloramphenicol resistance.

2.2.4 transgenic arabidopsis analyzes GmNAC15 promoters

In order to analyze the activity and tissue specificity of GmNAC15 promoters, to pGmNAC15::GUS transgenic arabidopsis into Row GUS tissue stainings.The results show that pGmNAC15::For GUS when sprouting two days, gus gene is in apical meristem, hypocotyl Vascular tissue and the tip of a root position expression (Fig. 3-A)；7 days after seed sprouting, gus gene is in cotyledon vein, true leaf, true leaf base High expression (Fig. 3-B/C/E/G) in portion, apical meristem, main root tip of a root position and the lateral root tip of a root,.The floral organ of transfer-gen plant There is strong gus gene to express in official：Stamen part, GUS expressions are higher in petal, filigree and sepal, do not detected in anther To GUS signals (Fig. 3-G)；Gynoecium part, GUS signals are concentrated mainly in style and column cap；In ripe silique, pericarp and Significant GUS signals (Fig. 3-H) are all detected at carpopodium.The experimental results showed that the obtained GmNAC15 promoters of clone have compared with Strong promoter activity, also have Space-time speciality, can start foreign gene the specific developmental stage of arabidopsis specific organization Middle high expression.

Embodiment 3. is induced using protoplasts of Arabidopsis thaliana broken by ultrasonic instantaneous conversion technical identification GmNAC15 promoters by ABA.

The structure of 3.1 GmNAC15 promoter pGreen II-0800LUC carriers

3.1.1 the GmNAC15 promoter fragments containing restriction enzyme site are cloned

Double fluorescence report carrier pGreen II-0800LUC of GmNAC15 promoters are built using double digestion-connection method. The restriction enzyme site in No. 9 GmNAC15 promoters of holy beans is analyzed using Primer 5, with pGreen II-0800LUC Multiple cloning sites (insert districts pGreen II-0800LUC carrier T-DNA are as shown in Figure 5) be compared, filter out available enzyme Enzyme site is Sal I and Pst I.

Contain the primer (F of restriction enzyme site using GmNAC15：5’-ACG CGT CGA CAC CGT CGT GTT CAC AAA TC-3 ', R：5’-AAC TGC AGT TTT TCA AAA ACA CAA TTT CG-3 ') PCR amplification GmNAC15 startups Son,

The reaction system of high fidelity enzyme HIFI amplifications is following (20 μ l systems)：

Amplification condition is as follows：

After reaction, reaction solution is detected in 0.8%TAE agarose gel electrophoresis.

3.1.2 glue recycling PCR product (with 1.3)

3.1.3 Sal I and Pst I difference double digestion PCR products and pGreen II-0800LUC carriers are stayed overnight.

Reaction system is as follows：

37 DEG C of overnight abundant digestions.

3.1.4 second day glue recycling digestion products and pGreen II-0800LUC carriers.(with 1.3)

3.1.5 the digestion products of recycling and carrier are attached reaction overnight.(with 1.4)

3.1.6 E. coli competent plasmid converts.(with 1.5)

3.1.7 colibacillus PCR is verified.(with 1.6)

3.1.8 DNA sequencing (with 1.7).

Verification is connected to the GmNAC15 promoter cloning regions nucleotide sequence such as SEQ ID in pGreenII-0800LUC Shown in No.1.

3.1.9 the extraction of e. coli plasmid dna obtains pGmNAC15::PGreenII-0800LUC plasmids：Endotoxin-free Plasmid purification DNA purification systems (NucleoBond Xtra Midi EF) (MACHEREY-NAGEL)

(1) culture of thalline is collected：4500g, 15min, 4 DEG C

(2) cellular lysates：Thalline is thoroughly resuspended in 8ml Buffer RES-EF, and 8ml Buffer LYS-EF cracking is added Thalline, soft mixing, room temperature crack 5min；

(3) balances pillar and filter：15ml Buffer EQU-EF

(4) is neutralized:8ml NEU-EF, soft mixing 10-15 times, place 5min on ice

(5) purifications and dress column lysate：Reverse test tube 3 times, lysate is poured into pillar

(6) .1st is washed：5ml Buffer FIL-EF

(7) removes Filter column：Remove Nucleobond Xtra Column Filter

(8) .2ed is washed：35ml Buffer ENDO-EF

(9) dissolves：5ml ELU-EF

(14) is precipitated：3.5ml isopropanols, 15000g, 4 DEG C, 30min

(15) cleanings and dry DNA precipitation：70% alcohol at normal temperature of 2ml, 5min, 15000g, room temperature, 5min

(16) dissolving DNAs：The H2O-EF of appropriate volume is added, DNA concentration is made to reach 1000ug/ml, -20 DEG C of preservations.

Response condition of the 3.2 GmNAC15 promoters to ABA

In order to analyze response condition of the GmNAC15 gene promoters to stress, to pGmNAC15::GUS arabidopsis is coerced Compel processing, the expression that detection gus gene is dyed by GUS changes.It will be cultivated in 1/2MS solid mediums 2 weeks pGmNAC15::GUS transgenic seedlings are transplanted in the MS fluid nutrient mediums containing various abiotic stress molecule and carry out Stress treatment, point ABA the and MOCK processing for not carried out various concentration gradient, in 3h, 6h and for 24 hours when materials carry out GUS dyeing.The result shows that GUS staining powers, which only have when ABA is handled for 24 hours, to be significantly increased, and enhances region mainly in cotyledon, tip of a root GUS dye levels Enhancing unobvious (Fig. 4).The experimental results showed that GmNAC15 promoters have the characteristic of ABA processing induced expressions, and it is lured Expression is led to occur mainly in cotyledon.

3.3 quantitatively detect the activity of 5 ' deletion fragment of GmNAC15 promoters difference using LUC reporter genes

Utilize PromegaThe original of Luciferase Assay System detection promoter L UC plasmids conversions Raw plastid relative luciferase activity, operation is with reference to specification.- 80 DEG C of protoplast is taken out to be placed on and is melted on ice, is taken 20ul protoplast solutions+20ulFirefly hair is measured after Luciferase Reagent, 25 DEG C of reaction 10min 20ul is added in light valueStop&Reagent is measured, and 25 DEG C of reaction 10min measure sea pansy luminous value.It calculates Relative luciferase activity value, relative activity value=fire fly luminescence value/sea pansy luminous value.GmNAC15 promoters are lived relatively Property value and by A BA induction situation as shown in fig. 6, the results show that under ABA inductions, GmNAC15 promoters can raise driving report Expression of the gene in transgenic arabidopsis and soybean protoplast is accused, GmNAC15 promoters have ABA inducing functions, can For GmNAC15 genes salt tolerant from now on/drought regulatory mechanism research, imply that being widely used in cultivation salt tolerant/drought turns base in the future Because of plant.

Sequence table

<110>Shandong University

<120>A kind of ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans

<141> 2016-11-30

<160> 3

<210> 1

<211> 2004

<212> DNA

<213>Glycine soybean（Glycine max.）

<221>The ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans

<222>（1）…（2004）

<400>1

catgattgcg aattatttat catttaaaca aattgcctag aatgagttac aactttaaat 60

tgaccgaatt gtatgggtta aattgattaa aaaaattatt aactaactat ttaagttgaa 120

agtattcaat aaagttatct aactttcttt tgataaaata aaattaacta actagccaat 180

gcatataaat taatgtaaaa aggtaaaacc atgtgtacgc gtatataata attttaaaaa 240

aatggaaaaa aaaactgtaa agttcccagt ttataaaaaa tacattattt ttattaactt 300

atgaaaaaaa attaaaatta aaattttctt taaaaaaatc tctgtaatta aactcccctg 360

ataacttatt ttttgtcaaa taaagtaatt aattaactta taattttccc aacacacaca 420

taaacgtaag acacagaata ctaaataata atttgagctg agtaatgtgg tgtttgacta 480

gtgcttaatt taatttaaaa acacttgttc cacactccca agtaacttag acgtttccat 540

tattttgaga ataaactggg aaaataaaca acaaattagg cccacataga aatataatat 600

gactggaaaa cacacgatca tgtctgatat agaatagatt ttctcattgg ttttatatgg 660

ctatagtttt tatgcatgta gctttaataa gaatatatgc caatttgtat ggttagtcga 720

tttcagcgta atttagaccc ttaagttatt gaaaagctaa cagaatattt taattgatct 780

gaaaatgatt tcgcaagtgt atgtttacgt tcacggctcc ttaaatcaat gtggtacaat 840

aaattaattt ttcatttgag tctcagaaac ataattaatt atttgtgatc taaaaaaaat 900

cttgagcaag cgtaaacctt aaaaatgaaa agcttactca aagctaaaaa gtaaatggta 960

aagtagaaac atatggagta gtaaatcttt gttaaaataa ataatattat tttcaggaga 1020

ctttttataa gaaaaaatga aagagaagtg aggaaataaa aatttacatc tataattatt 1080

aataacaaat aattatccta tttggtgtat acatcttatt agacaattta atccttaaat 1140

tatttctttt aacttagatt aacttttata cttttacatt taacttctat tatagtatta 1200

atcttgtaaa tgtagaagct aacagaagag agagagagag agagagagag gggaaggggt 1260

aagtaaaggt aataccgaca gaaaggggtg tgtctgaaga agttacgtta ggagcaccaa 1320

aacgaaacgg cagaggagag actagtggtt gggtaagggt tgagtttccg tacaaattaa 1380

ctgttgcttc ttttaagggg ccagggtgag ttgcgttgca aaatctgata gtattcatct 1440

catttcattt gttccccgtt aagggagaga cagggttact tgtgtacttt cactcactca 1500

ctcactcact cactctccag ctgtatccaa tcacataagc tgacttcatt gcattctctc 1560

gcctcatttt tattttaact aacaaaaatc cttacttttt ttacatttca taaaacatac 1620

tctacttttc tttctttttg ttttgttcaa ggaaggaaca ctgtgacagt agaagcaagc 1680

aagaattgga attggaattg gaattggaat gaattctcct cctcctccaa gaccaagagt 1740

ggagaggtcg agcggaacta ggctagcagc agtttccaca caagccaaaa ccaaaaccaa 1800

aaccaaaacc aaaataaaaa ctgtagttgt caaagtaaaa gaaaggttga gtctagttta 1860

cgccaaatgt gtcttcttat tcttctcctt cttgtcagac ccattactat aaaagaagcc 1920

acactgcatc cttctccata cccttttact ttctttatcc aataataata actccatttt 1980

ccctctgtct tccccccctc ctct 2004

<210> 2

<211> 13046

<212> DNA

<213>Artificial sequence

<221>Reporter gene GUS expression vector pGmNAC15::pKGWFS7

<222>（1）…（13046）

<400>2

tgatcacagg cagcaacgct ctgtcatcgt tacaatcaac atgctaccct ccgcgagatc 60

atccgtgttt caaacccggc agcttagttg ccgttcttcc gaatagcatc ggtaacatga 120

gcaaagtctg ccgccttaca acggctctcc cgctgacgcc gtcccggact gatgggctgc 180

ctgtatcgag tggtgatttt gtgccgagct gccggtcggg gagctgttgg ctggctggtg 240

gcaggatata ttgtggtgta aacaaattga cgcttagaca acttaataac acattgcgga 300

cgtttttaat gtactgaatt aacgccgaat tgaattatca gcttgcatgc cggtcgatct 360

agtaacatag atgacaccgc gcgcgataat ttatcctagt ttgcgcgcta tattttgttt 420

tctatcgcgt attaaatgta taattgcggg actctaatca taaaaaccca tctcataaat 480

aacgtcatgc attacatgtt aattattaca tgcttaacgt aattcaacag aaattatatg 540

ataatcatcg caagaccggc aacaggattc aatcttaaga aactttattg ccaaatgttt 600

gaacgatctg cttgactcta gctagagtcc gaaccccaga gtcccgctca gaagaactcg 660

tcaagaaggc gatagaaggc gatgcgctgc gaatcgggag cggcgatacc gtaaagcacg 720

aggaagcggt cagcccattc gccgccaagc tcttcagcaa tatcacgggt agccaacgct 780

atgtcctgat agcggtccgc cacacccagc cggccacagt cgatgaatcc agaaaagcgg 840

ccattttcca ccatgatatt cggcaagcag gcatcgccgt gggtcacgac gagatcctcg 900

ccgtcgggca tccgcgcctt gagcctggcg aacagttcgg ctggcgcgag cccctgatgc 960

tcttcgtcca gatcatcctg atcgacaaga ccggcttcca tccgagtacg tgctcgctcg 1020

atgcgatgtt tcgcttggtg gtcgaatggg caggtagccg gatcaagcgt atgcagccgc 1080

cgcattgcat cagccatgat ggatactttc tcggcaggag caaggtgaga tgacaggaga 1140

tcctgccccg gcacttcgcc caatagcagc cagtcccttc ccgcttcagt gacaacgtcg 1200

agcacagctg cgcaaggaac gcccgtcgtg gccagccacg atagccgcgc tgcctcgtct 1260

tggagttcat tcagggcacc ggacaggtcg gtcttgacaa aaagaaccgg gcgcccctgc 1320

gctgacagcc ggaacacggc ggcatcagag cagccgattg tctgttgtgc ccagtcatag 1380

ccgaatagcc tctccaccca agcggccgga gaacctgcgt gcaatccatc ttgttcaatc 1440

atgcctcgat cgagttgaga gtgaatatga gactctaatt ggataccgag gggaatttat 1500

ggaacgtcag tggagcattt ttgacaagaa atatttgcta gctgatagtg accttaggcg 1560

acttttgaac gcgcaataat ggtttctgac gtatgtgctt agctcattaa actccagaaa 1620

cccgcggctg agtggctcct tcaacgttgc ggttctgtca gttccaaacg taaaacggct 1680

tgtcccgcgt catcggcggg ggtcataacg tgactccctt aattctcatg tataattcga 1740

gctcggtacc cggggatcct ctagagtcga cctgcaggca tgcaagctct cccatatggt 1800

cgacctgcag gcggccgcac tagtgatatc acaagtttgt acaaaaaagc tgaaccatga 1860

ttgcgaatta tttatcattt aaacaaattg cctagaatga gttacaactt taaattgacc 1920

gaattgtatg ggttaaattg attaaaaaaa ttattaacta actatttaag ttgaaagtat 1980

tcaataaagt tatctaactt tcttttgata aaataaaatt aactaactag ccaatgcata 2040

taaattaatg taaaaaggta aaaccatgtg tacgcgtata taataatttt aaaaaaatgg 2100

aaaaaaaaac tgtaaagttc ccagtttata aaaaatacat tatttttatt aacttatgaa 2160

aaaaaattaa aattaaaatt ttctttaaaa aaatctctgt aattaaactc ccctgataac 2220

ttattttttg tcaaataaag taattaatta acttataatt ttcccaacac acacataaac 2280

gtaagacaca gaatactaaa taataatttg agctgagtaa tgtggtgttt gactagtgct 2340

taatttaatt taaaaacact tgttccacac tcccaagtaa cttagacgtt tccattattt 2400

tgagaataaa ctgggaaaat aaacaacaaa ttaggcccac atagaaatat aatatgactg 2460

gaaaacacac gatcatgtct gatatagaat agattttctc attggtttta tatggctata 2520

gtttttatgc atgtagcttt aataagaata tatgccaatt tgtatggtta gtcgatttca 2580

gcgtaattta gacccttaag ttattgaaaa gctaacagaa tattttaatt gatctgaaaa 2640

tgatttcgca agtgtatgtt tacgttcacg gctccttaaa tcaatgtggt acaataaatt 2700

aatttttcat ttgagtctca gaaacataat taattatttg tgatctaaaa aaaatcttga 2760

gcaagcgtaa accttaaaaa tgaaaagctt actcaaagct aaaaagtaaa tggtaaagta 2820

gaaacatatg gagtagtaaa tctttgttaa aataaataat attattttca ggagactttt 2880

tataagaaaa aatgaaagag aagtgaggaa ataaaaattt acatctataa ttattaataa 2940

caaataatta tcctatttgg tgtatacatc ttattagaca atttaatcct taaattattt 3000

cttttaactt agattaactt ttatactttt acatttaact tctattatag tattaatctt 3060

gtaaatgtag aagctaacag aagagagaga gagagagaga gagaggggaa ggggtaagta 3120

aaggtaatac cgacagaaag gggtgtgtct gaagaagtta cgttaggagc accaaaacga 3180

aacggcagag gagagactag tggttgggta agggttgagt ttccgtacaa attaactgtt 3240

gcttctttta aggggccagg gtgagttgcg ttgcaaaatc tgatagtatt catctcattt 3300

catttgttcc ccgttaaggg agagacaggg ttacttgtgt actttcactc actcactcac 3360

tcactcactc tccagctgta tccaatcaca taagctgact tcattgcatt ctctcgcctc 3420

atttttattt taactaacaa aaatccttac tttttttaca tttcataaaa catactctac 3480

ttttctttct ttttgttttg ttcaaggaag gaacactgtg acagtagaag caagcaagaa 3540

ttggaattgg aattggaatt ggaatgaatt ctcctcctcc tccaagacca agagtggaga 3600

ggtcgagcgg aactaggcta gcagcagttt ccacacaagc caaaaccaaa accaaaacca 3660

aaaccaaaat aaaaactgta gttgtcaaag taaaagaaag gttgagtcta gtttacgcca 3720

aatgtgtctt cttattcttc tccttcttgt cagacccatt actataaaag aagccacact 3780

gcatccttct ccataccctt ttactttctt tatccaataa taataactcc attttccctc 3840

tgtcttcccc ccctcctctc agctttcttg tacaaagtgg tgatatcccg cggatggtga 3900

gcaagggcga ggagctgttc accggggtgg tgcccatcct ggtcgagctg gacggcgacg 3960

taaacggcca caagttcagc gtgtccggcg agggcgaggg cgatgccacc tacggcaagc 4020

tgaccctgaa gttcatctgc accaccggca agctgcccgt gccctggccc accctcgtga 4080

ccaccctgac ctacggcgtg cagtgcttca gccgctaccc cgaccacatg aagcagcacg 4140

acttcttcaa gtccgccatg cccgaaggct acgtccagga gcgcaccatc ttcttcaagg 4200

acgacggcaa ctacaagacc cgcgccgagg tgaagttcga gggcgacacc ctggtgaacc 4260

gcatcgagct gaagggcatc gacttcaagg aggacggcaa catcctgggg cacaagctgg 4320

agtacaacta caacagccac aacgtctata tcatggccga caagcagaag aacggcatca 4380

aggtgaactt caagatccgc cacaacatcg aggacggcag cgtgcagctc gccgaccact 4440

accagcagaa cacccccatc ggcgacggcc ccgtgctgct gcccgacaac cactacctga 4500

gcacccagtc cgccctgagc aaagacccca acgagaagcg cgatcacatg gtcctgctgg 4560

agttcgtgac cgccgccggg atcactctcg gcatggacga gctgtacaag cccggcatgt 4620

tacgtcctgt agaaacccca acccgtgaaa tcaaaaaact cgacggcctg tgggcattca 4680

gtctggatcg cgaaaactgt ggaattgatc agcgttggtg ggaaagcgcg ttacaagaaa 4740

gccgggcaat tgctgtgcca ggcagtttta acgatcagtt cgccgatgca gatattcgta 4800

attatgcggg caacgtctgg tatcagcgcg aagtctttat accgaaaggt tgggcaggcc 4860

agcgtatcgt gctgcgtttc gatgcggtca ctcattacgg caaagtgtgg gtcaataatc 4920

aggaagtgat ggagcatcag ggcggctata cgccatttga agccgatgtc acgccgtatg 4980

ttattgccgg gaaaagtgta cgtatcaccg tttgtgtgaa caacgaactg aactggcaga 5040

ctatcccgcc gggaatggtg attaccgacg aaaacggcaa gaaaaagcag tcttacttcc 5100

atgatttctt taactatgcc ggaatccatc gcagcgtaat gctctacacc acgccgaaca 5160

cctgggtgga cgatatcacc gtggtgacgc atgtcgcgca agactgtaac cacgcgtctg 5220

ttgactggca ggtggtggcc aatggtgatg tcagcgttga actgcgtgat gcggatcaac 5280

aggtggttgc aactggacaa ggcactagcg ggactttgca agtggtgaat ccgcacctct 5340

ggcaaccggg tgaaggttat ctctatgaac tgtgcgtcac agccaaaagc cagacagagt 5400

gtgatatcta cccgcttcgc gtcggcatcc ggtcagtggc agtgaagggc caacagttcc 5460

tgattaacca caaaccgttc tactttactg gctttggtcg tcatgaagat gcggacttac 5520

gtggcaaagg attcgataac gtgctgatgg tgcacgacca cgcattaatg gactggattg 5580

gggccaactc ctaccgtacc tcgcattacc cttacgctga agagatgctc gactgggcag 5640

atgaacatgg catcgtggtg attgatgaaa ctgctgctgt cggctttaac ctctctttag 5700

gcattggttt cgaagcgggc aacaagccga aagaactgta cagcgaagag gcagtcaacg 5760

gggaaactca gcaagcgcac ttacaggcga ttaaagagct gatagcgcgt gacaaaaacc 5820

acccaagcgt ggtgatgtgg agtattgcca acgaaccgga tacccgtccg caagtgcacg 5880

ggaatatttc gccactggcg gaagcaacgc gtaaactcga cccgacgcgt ccgatcacct 5940

gcgtcaatgt aatgttctgc gacgctcaca ccgataccat cagcgatctc tttgatgtgc 6000

tgtgcctgaa ccgttattac ggatggtatg tccaaagcgg cgatttggaa acggcagaga 6060

aggtactgga aaaagaactt ctggcctggc aggagaaact gcatcagccg attatcatca 6120

ccgaatacgg cgtggatacg ttagccgggc tgcactcaat gtacaccgac atgtggagtg 6180

aagagtatca gtgtgcatgg ctggatatgt atcaccgcgt ctttgatcgc gtcagcgccg 6240

tcgtcggtga acaggtatgg aatttcgccg attttgcgac ctcgcaaggc atattgcgcg 6300

ttggcggtaa caagaaaggg atcttcactc gcgaccgcaa accgaagtcg gcggcttttc 6360

tgctgcaaaa acgctggact ggcatgaact tcggtgaaaa accgcagcag ggaggcaaac 6420

aatgaccatg gcggccggga gcatgcggcc atgctagagt ccgcaaaaat caccagtctc 6480

tctctacaaa tctatctctc tctatttttc tccagaataa tgtgtgagta gttcccagat 6540

aagggaatta gggttcttat agggtttcgc tcatgtgttg agcatataag aaacccttag 6600

tatgtatttg tatttgtaaa atacttctat caataaaatt tctaattcct aaaaccaaaa 6660

tccagtgacc tgcaggcatg cgacgtcgag cttagcttga gcttggatca gattgtcgtt 6720

tcccgccttc agtttaaact atcagtgttt gacaggatat attggcgggt aaacctaaga 6780

gaaaagagcg tttattagaa taacggatat ttaaaagggc gtgaaaaggt ttatccgttc 6840

gtccatttgt atgtgcatgc caaccacagg gttcccctcg ggatcaaagt actttgatcc 6900

aacccctccg ctgctatagt gcagtcggct tctgacgttc agtgcagccg tcttctgaaa 6960

acgacatgtc gcacaagtcc taagttacgc gacaggctgc cgccctgccc ttttcctggc 7020

gttttcttgt cgcgtgtttt agtcgcataa agtagaatac ttgcgactag aaccggagac 7080

attacgccat gaacaagagc gccgccgctg gcctgctggg ctatgcccgc gtcagcaccg 7140

acgaccagga cttgaccaac caacgggccg aactgcacgc ggccggctgc accaagctgt 7200

tttccgagaa gatcaccggc accaggcgcg accgcccgga gctggccagg atgcttgacc 7260

acctacgccc tggcgacgtt gtgacagtga ccaggctaga ccgcctggcc cgcagcaccc 7320

gcgacctact ggacattgcc gagcgcatcc aggaggccgg cgcgggcctg cgtagcctgg 7380

cagagccgtg ggccgacacc accacgccgg ccggccgcat ggtgttgacc gtgttcgccg 7440

gcattgccga gttcgagcgt tccctaatca tcgaccgcac ccggagcggg cgcgaggccg 7500

ccaaggcccg aggcgtgaag tttggccccc gccctaccct caccccggca cagatcgcgc 7560

acgcccgcga gctgatcgac caggaaggcc gcaccgtgaa agaggcggct gcactgcttg 7620

gcgtgcatcg ctcgaccctg taccgcgcac ttgagcgcag cgaggaagtg acgcccaccg 7680

aggccaggcg gcgcggtgcc ttccgtgagg acgcattgac cgaggccgac gccctggcgg 7740

ccgccgagaa tgaacgccaa gaggaacaag catgaaaccg caccaggacg gccaggacga 7800

accgtttttc attaccgaag agatcgaggc ggagatgatc gcggccgggt acgtgttcga 7860

gccgcccgcg cacgtctcaa ccgtgcggct gcatgaaatc ctggccggtt tgtctgatgc 7920

caagctggcg gcctggccgg ccagcttggc cgctgaagaa accgagcgcc gccgtctaaa 7980

aaggtgatgt gtatttgagt aaaacagctt gcgtcatgcg gtcgctgcgt atatgatgcg 8040

atgagtaaat aaacaaatac gcaaggggaa cgcatgaagg ttatcgctgt acttaaccag 8100

aaaggcgggt caggcaagac gaccatcgca acccatctag cccgcgccct gcaactcgcc 8160

ggggccgatg ttctgttagt cgattccgat ccccagggca gtgcccgcga ttgggcggcc 8220

gtgcgggaag atcaaccgct aaccgttgtc ggcatcgacc gcccgacgat tgaccgcgac 8280

gtgaaggcca tcggccggcg cgacttcgta gtgatcgacg gagcgcccca ggcggcggac 8340

ttggctgtgt ccgcgatcaa ggcagccgac ttcgtgctga ttccggtgca gccaagccct 8400

tacgacatat gggccaccgc cgacctggtg gagctggtta agcagcgcat tgaggtcacg 8460

gatggaaggc tacaagcggc ctttgtcgtg tcgcgggcga tcaaaggcac gcgcatcggc 8520

ggtgaggttg ccgaggcgct ggccgggtac gagctgccca ttcttgagtc ccgtatcacg 8580

cagcgcgtga gctacccagg cactgccgcc gccggcacaa ccgttcttga atcagaaccc 8640

gagggcgacg ctgcccgcga ggtccaggcg ctggccgctg aaattaaatc aaaactcatt 8700

tgagttaatg aggtaaagag aaaatgagca aaagcacaaa cacgctaagt gccggccgtc 8760

cgagcgcacg cagcagcaag gctgcaacgt tggccagcct ggcagacacg ccagccatga 8820

agcgggtcaa ctttcagttg ccggcggagg atcacaccaa gctgaagatg tacgcggtac 8880

gccaaggcaa gaccattacc gagctgctat ctgaatacat cgcgcagcta ccagagtaaa 8940

tgagcaaatg aataaatgag tagatgaatt ttagcggcta aaggaggcgg catggaaaat 9000

caagaacaac caggcaccga cgccgtggaa tgccccatgt gtggaggaac gggcggttgg 9060

ccaggcgtaa gcggctgggt tgtctgccgg ccctgcaatg gcactggaac ccccaagccc 9120

gaggaatcgg cgtgacggtc gcaaaccatc cggcccggta caaatcggcg cggcgctggg 9180

tgatgacctg gtggagaagt tgaaggccgc gcaggccgcc cagcggcaac gcatcgaggc 9240

agaagcacgc cccggtgaat cgtggcaagc ggccgctgat cgaatccgca aagaatcccg 9300

gcaaccgccg gcagccggtg cgccgtcgat taggaagccg cccaagggcg acgagcaacc 9360

agattttttc gttccgatgc tctatgacgt gggcacccgc gatagtcgca gcatcatgga 9420

cgtggccgtt ttccgtctgt cgaagcgtga ccgacgagct ggcgaggtga tccgctacga 9480

gcttccagac gggcacgtag aggtttccgc agggccggcc ggcatggcca gtgtgtggga 9540

ttacgacctg gtactgatgg cggtttccca tctaaccgaa tccatgaacc gataccggga 9600

agggaaggga gacaagcccg gccgcgtgtt ccgtccacac gttgcggacg tactcaagtt 9660

ctgccggcga gccgatggcg gaaagcagaa agacgacctg gtagaaacct gcattcggtt 9720

aaacaccacg cacgttgcca tgcagcgtac gaagaaggcc aagaacggcc gcctggtgac 9780

ggtatccgag ggtgaagcct tgattagccg ctacaagatc gtaaagagcg aaaccgggcg 9840

gccggagtac atcgagatcg agctagctga ttggatgtac cgcgagatca cagaaggcaa 9900

gaacccggac gtgctgacgg ttcaccccga ttactttttg atcgatcccg gcatcggccg 9960

ttttctctac cgcctggcac gccgcgccgc aggcaaggca gaagccagat ggttgttcaa 10020

gacgatctac gaacgcagtg gcagcgccgg agagttcaag aagttctgtt tcaccgtgcg 10080

caagctgatc gggtcaaatg acctgccgga gtacgatttg aaggaggagg cggggcaggc 10140

tggcccgatc ctagtcatgc gctaccgcaa cctgatcgag ggcgaagcat ccgccggttc 10200

ctaatgtacg gagcagatgc tagggcaaat tgccctagca ggggaaaaag gtcgaaaagg 10260

tctctttcct gtggatagca cgtacattgg gaacccaaag ccgtacattg ggaaccggaa 10320

cccgtacatt gggaacccaa agccgtacat tgggaaccgg tcacacatgt aagtgactga 10380

tataaaagag aaaaaaggcg atttttccgc ctaaaactct ttaaaactta ttaaaactct 10440

taaaacccgc ctggcctgtg cataactgtc tggccagcgc acagccgaag agctgcaaaa 10500

agcgcctacc cttcggtcgc tgcgctccct acgccccgcc gcttcgcgtc ggcctatcgc 10560

ggccgctggc cgctcaaaaa tggctggcct acggccaggc aatctaccag ggcgcggaca 10620

agccgcgccg tcgccactcg accgccggcg cccacatcaa ggcaccctgc ctcgcgcgtt 10680

tcggtgatga cggtgaaaac ctctgacaca tgcagctccc ggagacggtc acagcttgtc 10740

tgtaagcgga tgccgggagc agacaagccc gtcagggcgc gtcagcgggt gttggcgggt 10800

gtcggggcgc agccatgacc cagtcacgta gcgatagcgg agtgtatact ggcttaacta 10860

tgcggcatca gagcagattg tactgagagt gcaccatatg cggtgtgaaa taccgcacag 10920

atgcgtaagg agaaaatacc gcatcaggcg ctcttccgct tcctcgctca ctgactcgct 10980

gcgctcggtc gttcggctgc ggcgagcggt atcagctcac tcaaaggcgg taatacggtt 11040

atccacagaa tcaggggata acgcaggaaa gaacatgtga gcaaaaggcc agcaaaaggc 11100

caggaaccgt aaaaaggccg cgttgctggc gtttttccat aggctccgcc cccctgacga 11160

gcatcacaaa aatcgacgct caagtcagag gtggcgaaac ccgacaggac tataaagata 11220

ccaggcgttt ccccctggaa gctccctcgt gcgctctcct gttccgaccc tgccgcttac 11280

cggatacctg tccgcctttc tcccttcggg aagcgtggcg ctttctcata gctcacgctg 11340

taggtatctc agttcggtgt aggtcgttcg ctccaagctg ggctgtgtgc acgaaccccc 11400

cgttcagccc gaccgctgcg ccttatccgg taactatcgt cttgagtcca acccggtaag 11460

acacgactta tcgccactgg cagcagccac tggtaacagg attagcagag cgaggtatgt 11520

aggcggtgct acagagttct tgaagtggtg gcctaactac ggctacacta gaaggacagt 11580

atttggtatc tgcgctctgc tgaagccagt taccttcgga aaaagagttg gtagctcttg 11640

atccggcaaa caaaccaccg ctggtagcgg tggttttttt gtttgcaagc agcagattac 11700

gcgcagaaaa aaaggatctc aagaagatcc tttgatcttt tctacggggt ctgacgctca 11760

gtggaacgaa aactcacgtt aagggatttt ggtcatgcat gatatatctc ccaatttgtg 11820

tagggcttat tatgcacgct taaaaataat aaaagcagac ttgacctgat agtttggctg 11880

tgagcaatta tgtgcttagt gcatctaatc gcttgagtta acgccggcga agcggcgtcg 11940

gcttgaacga atttctagct agacattatt tgccgactac cttggtgatc tcgcctttca 12000

cgtagtggac aaattcttcc aactgatctg cgcgcgaggc caagcgatct tcttcttgtc 12060

caagataagc ctgtctagct tcaagtatga cgggctgata ctgggccggc aggcgctcca 12120

ttgcccagtc ggcagcgaca tccttcggcg cgattttgcc ggttactgcg ctgtaccaaa 12180

tgcgggacaa cgtaagcact acatttcgct catcgccagc ccagtcgggc ggcgagttcc 12240

atagcgttaa ggtttcattt agcgcctcaa atagatcctg ttcaggaacc ggatcaaaga 12300

gttcctccgc cgctggacct accaaggcaa cgctatgttc tcttgctttt gtcagcaaga 12360

tagccagatc aatgtcgatc gtggctggct cgaagatacc tgcaagaatg tcattgcgct 12420

gccattctcc aaattgcagt tcgcgcttag ctggataacg ccacggaatg atgtcgtcgt 12480

gcacaacaat ggtgacttct acagcgcgga gaatctcgct ctctccaggg gaagccgaag 12540

tttccaaaag gtcgttgatc aaagctcgcc gcgttgtttc atcaagcctt acggtcaccg 12600

taaccagcaa atcaatatca ctgtgtggct tcaggccgcc atccactgcg gagccgtaca 12660

aatgtacggc cagcaacgtc ggttcgagat ggcgctcgat gacgccaact acctctgata 12720

gttgagtcga tacttcggcg atcaccgctt cccccatgat gtttaacttt gttttagggc 12780

gactgccctg ctgcgtaaca tcgttgctgc tccataacat caaacatcga cccacggcgt 12840

aacgcgcttg ctgcttggat gcccgaggca tagactgtac cccaaaaaaa catgtcataa 12900

caagaagcca tgaaaaccgc cactgcgccg ttaccaccgc tgcgttcggt caaggttctg 12960

gaccagttgc gtgacggcag ttacgctact tgcattacag cttacgaacc gaacgaggct 13020

tatgtccact gggttcgtgc ccgaat 13046

<210> 3

<211> 5871

<212> DNA

<213>Artificial sequence

<221>Reporter gene LUC expression vectors pGmNAC15::pGreenII-0800LUC

<222>（1）…（5871）

<400>3

agatcttggc aggatatatt gtggtgtaac gttatcgtac ccctactcca aaaatgtcaa 60

agatacagtc tcagaagacc aaagggctat tgagactttt caacaaaggg taatttcggg 120

aaacctcctc ggattccatt gcccagctat ctgtcacttc atcgaaagga cagtagaaaa 180

ggaaggtggc tcctacaaat gccatcattg cgataaagga aaggctatca ttcaagatgc 240

ctctgccgac agtggtccca aagatggacc cccacccacg aggagcatcg tggaaaaaga 300

agacgttcca accacgtctt caaagcaagt ggattgatgt gacatctcca ctgacgtaag 360

ggatgacgca caatcccact atccttcgca agacccttcc tctatataag gaagttcatt 420

tcatttggag aggacagccc accaccatga cttcgaaagt ttatgatcca gaacaaagga 480

aacggatgat aactggtccg cagtggtggg ccagatgtaa acaaatgaat gttcttgatt 540

catttattaa ttattatgat tcagaaaaac atgcagaaaa tgctgttatt tttttacatg 600

gtaacgcggc ctcttcttat ttatggcgac atgttgtgcc acatattgag ccagtagcgc 660

ggtgtattat accagacctt attggtatgg gcaaatcagg caaatctggt aatggttctt 720

ataggttact tgatcattac aaatatctta ctgcatggtt tgaacttctt aatttaccaa 780

agaagatcat ttttgtcggc catgattggg gtgcttgttt ggcatttcat tatagctatg 840

agcatcaaga taagatcaaa gcaatagttc acgctgaaag tgtagtagat gtgattgaat 900

catgggatga atggcctgat attgaagaag atattgcgtt gatcaaatct gaagaaggag 960

aaaaaatggt tttggagaat aacttcttcg tggaaaccat gttgccatca aaaatcatga 1020

gaaagttaga accagaagaa tttgcagcat atcttgaacc attcaaagag aaaggtgaag 1080

ttcgtcgtcc aacattatca tggcctcgtg aaatcccgtt agtaaaaggt ggtaaacctg 1140

acgttgtaca aattgttagg aattataatg cttatctacg tgcaagtgat gatttaccaa 1200

aaatgtttat tgaatcggac ccaggattct tttccaatgc tattgttgaa ggtgccaaga 1260

agtttcctaa tactgaattt gtcaaagtaa aaggtcttca tttttcgcaa gaagatgcac 1320

ctgatgaaat gggaaaatat atcaaatcgt tcgttgagcg agttctcaaa aatgaacaat 1380

aattctagcc ggtacgctga aatcaccagt ctctctctac aaatctatct ctctctattt 1440

tctccataaa taatgtgtga gtagtttccc gataagggaa attagggttc ttatagggtt 1500

tcgctcatgt gttgagcata taagaaaccc ttagtatgta tttgtatttg taaaatactt 1560

ctatcaataa aatttctaat tcctaaaacc aaaatccagt actaaaatcc agatcgataa 1620

cattaacgtt tacaatttcc attcgccatt caggctgcgc aactgttggg aagggcgatc 1680

ggtgcgggcc tcttcgctat tacgccagct ggcgaaaggg ggatgtgctg caaggcgatt 1740

aagttgggta acgccagggt tttcccagtc acgacgttgt aaaacgacgg ccagtgaatt 1800

gtaatacgac tcactatagg gcgaattggg taccgggccc cccctcgagg tcgaccatga 1860

ttgcgaatta tttatcattt aaacaaattg cctagaatga gttacaactt taaattgacc 1920

gaattgtatg ggttaaattg attaaaaaaa ttattaacta actatttaag ttgaaagtat 1980

tcaataaagt tatctaactt tcttttgata aaataaaatt aactaactag ccaatgcata 2040

taaattaatg taaaaaggta aaaccatgtg tacgcgtata taataatttt aaaaaaatgg 2100

aaaaaaaaac tgtaaagttc ccagtttata aaaaatacat tatttttatt aacttatgaa 2160

aaaaaattaa aattaaaatt ttctttaaaa aaatctctgt aattaaactc ccctgataac 2220

ttattttttg tcaaataaag taattaatta acttataatt ttcccaacac acacataaac 2280

gtaagacaca gaatactaaa taataatttg agctgagtaa tgtggtgttt gactagtgct 2340

taatttaatt taaaaacact tgttccacac tcccaagtaa cttagacgtt tccattattt 2400

tgagaataaa ctgggaaaat aaacaacaaa ttaggcccac atagaaatat aatatgactg 2460

gaaaacacac gatcatgtct gatatagaat agattttctc attggtttta tatggctata 2520

gtttttatgc atgtagcttt aataagaata tatgccaatt tgtatggtta gtcgatttca 2580

gcgtaattta gacccttaag ttattgaaaa gctaacagaa tattttaatt gatctgaaaa 2640

tgatttcgca agtgtatgtt tacgttcacg gctccttaaa tcaatgtggt acaataaatt 2700

aatttttcat ttgagtctca gaaacataat taattatttg tgatctaaaa aaaatcttga 2760

gcaagcgtaa accttaaaaa tgaaaagctt actcaaagct aaaaagtaaa tggtaaagta 2820

gaaacatatg gagtagtaaa tctttgttaa aataaataat attattttca ggagactttt 2880

tataagaaaa aatgaaagag aagtgaggaa ataaaaattt acatctataa ttattaataa 2940

caaataatta tcctatttgg tgtatacatc ttattagaca atttaatcct taaattattt 3000

cttttaactt agattaactt ttatactttt acatttaact tctattatag tattaatctt 3060

gtaaatgtag aagctaacag aagagagaga gagagagaga gagaggggaa ggggtaagta 3120

aaggtaatac cgacagaaag gggtgtgtct gaagaagtta cgttaggagc accaaaacga 3180

aacggcagag gagagactag tggttgggta agggttgagt ttccgtacaa attaactgtt 3240

gcttctttta aggggccagg gtgagttgcg ttgcaaaatc tgatagtatt catctcattt 3300

catttgttcc ccgttaaggg agagacaggg ttacttgtgt actttcactc actcactcac 3360

tcactcactc tccagctgta tccaatcaca taagctgact tcattgcatt ctctcgcctc 3420

atttttattt taactaacaa aaatccttac tttttttaca tttcataaaa catactctac 3480

ttttctttct ttttgttttg ttcaaggaag gaacactgtg acagtagaag caagcaagaa 3540

ttggaattgg aattggaatt ggaatgaatt ctcctcctcc tccaagacca agagtggaga 3600

ggtcgagcgg aactaggcta gcagcagttt ccacacaagc caaaaccaaa accaaaacca 3660

aaaccaaaat aaaaactgta gttgtcaaag taaaagaaag gttgagtcta gtttacgcca 3720

aatgtgtctt cttattcttc tccttcttgt cagacccatt actataaaag aagccacact 3780

gcatccttct ccataccctt ttactttctt tatccaataa taataactcc attttccctc 3840

tgtcttcccc ccctcctctc tgcagcccgg gggatccact agttctagag cggccgccac 3900

cgcggtggag atcgaattcc catggaagac gccaaaaaca taaagaaagg cccggcgcca 3960

ttctatccgc tggaagatgg aaccgctgga gagcaactgc ataaggctat gaagagatac 4020

gccctggttc ctggaacaat tgcttttaca gatgcacata tcgaggtgga catcacttac 4080

gctgagtact tcgaaatgtc cgttcggttg gcagaagcta tgaaacgata tgggctgaat 4140

acaaatcaca gaatcgtcgt atgcagtgaa aactctcttc aattctttat gccggtgttg 4200

ggcgcgttat ttatcggagt tgcagttgcg cccgcgaacg acatttataa tgaacgtgaa 4260

ttgctcaaca gtatgggcat ttcgcagcct accgtggtgt tcgtttccaa aaaggggttg 4320

caaaaaattt tgaacgtgca aaaaaagctc ccaatcatcc aaaaaattat tatcatggat 4380

tctaaaacgg attaccaggg atttcagtcg atgtacacgt tcgtcacatc tcatctacct 4440

cccggtttta atgaatacga ttttgtgcca gagtccttcg atagggacaa gacaattgca 4500

ctgatcatga actcctctgg atctactggt ctgcctaaag gtgtcgctct gcctcataga 4560

actgcctgcg tgagattctc gcatgccaga gatcctattt ttggcaatca aatcattccg 4620

gatactgcga ttttaagtgt tgttccattc catcacggtt ttggaatgtt tactacactc 4680

ggatatttga tatgtggatt tcgagtcgtc ttaatgtata gatttgaaga agagctgttt 4740

ctgaggagcc ttcaggatta caagattcaa agtgcgctgc tggtgccaac cctattctcc 4800

ttcttcgcca aaagcactct gattgacaaa tacgatttat ctaatttaca cgaaattgct 4860

tctggtggcg ctcccctctc taaggaagtc ggggaagcgg ttgccaagag gttccatctg 4920

ccaggtatca ggcaaggata tgggctcact gagactacat cagctattct gattacaccc 4980

gagggggatg ataaaccggg cgcggtcggt aaagttgttc cattttttga agcgaaggtt 5040

gtggatctgg ataccgggaa aacgctgggc gttaatcaaa gaggcgaact gtgtgtgaga 5100

ggtcctatga ttatgtccgg ttatgtaaac aatccggaag cgaccaacgc cttgattgac 5160

aaggatggat ggctacattc tggagacata gcttactggg acgaagacga acacttcttc 5220

atcgttgacc gcctgaagtc tctgattaag tacaaaggct atcaggtggc tcccgctgaa 5280

ttggaatcca tcttgctcca acaccccaac atcttcgacg caggtgtcgc aggtcttccc 5340

gacgatgacg ccggtgaact tcccgccgcc gttgttgttt tggagcacgg aaagacgatg 5400

acggaaaaag agatcgtgga ttacgtcgcc agtcaagtaa caaccgcgaa aaagttgcgc 5460

ggaggagttg tgtttgtgga cgaagtaccg aaaggtctta ccggaaaact cgacgcaaga 5520

aaaatcagag agatcctcat aaaggccaag aagggcggaa agatcgccgt gtaattctag 5580

agaattcgct gaaatcacca gtctctctct acaaatctat ctctctctat tttctccata 5640

aataatgtgt gagtagtttc ccgataaggg aaattagggt tcttataggg tttcgctcat 5700

gtgttgagca tataagaaac ccttagtatg tatttgtatt tgtaaaatac ttctatcaat 5760

aaaatttcta attcctaaaa ccaaaatcca gtactaaaat ccagatccac tagccttgac 5820

aggatatatt ggcgggtaaa ctaagtcgct gtatgtgttt gtttgagatc t 5871

Claims (4)

1. a kind of ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans, it is characterised in that：The ABA evoked promoters Nucleotide sequence be SEQ ID No in sequence table:DNA sequence dna shown in 1.

2. the Reporter gene GUS expression containing No. 9 GmNAC15 Gene A BA evoked promoters of soybean sage beans described in claim 1 carries Body pGmNAC15::PKGWFS7, the nucleotide sequence such as SEQ ID No of the expression vector:Shown in 2.

3. the reporter gene LUC expression containing No. 9 GmNAC15 Gene A BA evoked promoters of soybean sage beans described in claim 1 carries Body pGmNAC15::PGreenII-0800LUC, the nucleotide sequence such as SEQ ID No of the expression vector:Shown in 3.

4. the ABA evoked promoters of No. 9 GmNAC15 genes of soybean sage beans described in claim 1 are being started mesh by ABA inducement efficients Mark the application in gene expression.