CN107904238A - Thick boisiana high salt, drought-inducible promoter IpLEA PRO and its application - Google Patents

Abstract

The present invention relates to a kind of promoter (being abbreviated as IpLEA PRO) of thick boisiana LEA protein IpLEA genes, its nucleotide sequence shown in SEQ ID No.1.Expression of results the present invention provides through detection GUS proves that the polynucleotides have the activity of promoter, and are strengthened by high salt and drought stress induced activity, this show the effect that the LEA protein gene responds high salt/Drought Stress in thick boisiana.The present invention is further to study the expression of resistant gene of salt related to applied to thick boisiana is regulated and controled, and improves and improve thick boisiana and the salt tolerance of other plant, and salt/relevant target protein of drought stress is expressed using plant as bioreactor and provides theoretical foundation.

Description

Thick boisiana high salt, drought-inducible promoter IpLEA-PRO and its application

Technical field

The invention belongs to plant genetic engineering field, and in particular to thick boisiana high salt, a drought-inducible promoter IpLEA-PRO and its application.

Background technology

Thick boisiana (Ipomoea pes-caprae L.), also known as saddle rattan (Fujian, Guangdong, Guangxi), husky lampwick (Guangdong), Membranaceous marshmarigold herb, king crab rattan (Fujian), extra large potato, laciniate blumea herb, Ma Liuteng, trachelospermum jasminoides (Hainan), Sha Teng (Zhejiang), is that Convolvulaceae Ipomoea is more Year gives birth to the sprawling herbs plant that crawls, and in the whole world, the sand beach of tropical and subtropical zone or island area have widely distributed.Thick boisiana resists Inverse property is extremely strong, and salt tolerant and drought resistance are especially pronounced.Meanwhile thick boisiana has higher gardens as a kind of evergreen liana Greening value.In addition, multiple areas, such as Australia, Brazil, Southeast Asia and China's Lingnan area, thick boisiana are also in the world A kind of common traditional herbal medicine.It can be seen from the above that thick boisiana has larger potentiality to be exploited as a kind of excellent wild plant resource And application value, while also can deeply be ground as the research object of a kind of plant stress-resistance molecule mechanism, excavation adversity gene Study carefully.

LEA protein for late embryo occur Abundant protein family (Late Embryogensis Abundant proteins II family of II, LEA-), it is that one kind in plant has high heat stability, hydrophilic small molecular protein, can be in plant Great expression under Late Embryogenesis and dehydration environment stress, is widely present in plant kingdom.Plant LEA protein has extensive Biological function, such as prevents cell dehydration, stabilizing cell membrane, bind metal ion, removing hydroxyl radical free radical from preventing film fat peroxide Change, protect chill sensitive enzymatic activity, as molecular chaperones and the characteristic for combining DNA/RNA etc..Research shows, many plants The expression of LEA protein gene is all influenced be subject to Water deficits such as high salt/arid and cryogenic freezings, and by improving LEA eggs White gene is expressed to improve resistance of the plant to Water deficit.And this improved gene expression amount by environment stress and showed Induced expression as being referred to as gene, is determined by the startup subcharacter of gene.

Promoter (promoter) is one section of noncoding DNA sequence positioned at structural gene 5'- ends upstream, can be by RNA Polymerase identification, combine, and the accurately initial time and expression intensity of control transcription (gene expression), is gene transcription regulation Center.The expression of gene and the structure of promoter are closely related, and the species of cis-acting elements determines gene in gene promoter Expression characteristic.When in promoter containing certain special cis-acting elements, the expression of the gene may be by associated The influence of factor.

The content of the invention

There is plant high salt, the promoter of drought stress induction type it is an object of the invention to provide a kind of.

Another object of the present invention is to provide the promoter, prepare transgenosis plant and for improve plant to height The application in the negative effect of foreign gene overexpression is reduced when salt and arid resistance.

In order to complete above-mentioned purpose, technical scheme is as follows：

A kind of high salt, drought-inducible promoter DNA sequence dna, the promoter DNA sequence are to contain SEQ ID No.1 Shown nucleotide sequence；Or for containing having replacing for one or several nucleotide in the sequence shown in SEQ ID No.1 and sequence Change, lack or increase, but the nucleotide sequence with identical function；Or it is the nucleosides with the pairing of SEQ ID NO.1 complete complementaries Acid sequence；Or to pass through what chromosome walking clonal expansion obtained by SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 Nucleotide sequence.

Present invention also offers a kind of above-mentioned high salt, the preparation method of drought-inducible promoter DNA sequence dna, with wild thickness The genomic DNA of rattan is template, is cloned by SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 by chromosome walking Amplification obtains.

Present invention also offers a kind of plant expression vector, contains above-mentioned high salt, drought-inducible promoter DNA sequences Row.

Preferably, the plant expression vector lures to connect high salt, arid described in claim 1 after BamHI digestions The pBI101.2 carriers of conductivity type promoter DNA sequence.

Present invention also offers above-mentioned high salt, drought-inducible promoter DNA sequence dna, or above-mentioned plant expression vector Application in gene expression in plants is regulated and controled.

Present invention also offers above-mentioned high salt, drought-inducible promoter DNA sequence dna, or the plant described in claim 3 Thing expression vector high salt, drought stress plant transgene breeding in application.

Preferably, the plant is arabidopsis or crops.

The method of arabidopsis or crops to high salt, Drought Stress adaptability is improved present invention also offers a kind of, including Arabidopsis or agriculture will be transformed into containing above-mentioned high salt, drought-inducible promoter DNA sequence dna, or above-mentioned plant expression vector In crop.

Preferably, by GV3101 agrobacterium mediation converteds arabidopsis or crops, arabidopsis or crops are obtained Transgenic seed.

The advantages of the present invention are as follows：

The present invention has found thick boisiana LEA protein IpLEA gene promoters IpLEA- by long-term experience and substantial amounts of experiment PRO, it is in addition to containing core promoter element TATA-BOX, the cis acting member also containing multiple response environment stresses Part, including abscisic acid response element ABRE, the drought-induced and relevant Myb factor binding sites MBS of stress, and stress are related TC-rich repeats cis-acting elements.In addition, cis work necessary to endosperm expression is also found in the promoter sequence With element Skn-1_Motif.The promoter can be lured in thick boisiana and arabidopsis with controlling gene be subject to high salt/drought stress Expression is led, which can be applied to the plant transgene breeding for high salt/drought stress, for culture and improvement high salt/dry The genetically modified plants of non-irrigated anti-adversity ability, while the expression of foreign gene under regular culture conditions is reduced, to reduce foreign gene To the negative effect of growth of transgenic plants and development.

IpLEA-PRO promoters of the present invention are high salt and drought stress inducible promoter, available for plant gene Change plant in engineering to foreign gene in the response mode of high salt and arid and adjustment genetically modified plants in normal growing conditions Lower expression way, overcomes common using composition type expression promoter (such as 35S promoter, maize ubiquitin promoter, rice flesh Filamentous actin promoter etc.) carry out transgenosis work in negative effect, with obtain meet improve Plant Tolerance high salt and arid coerce Compel desired genetically modified plants new varieties.Particularly, the IpLEA-PRO promoter fragments are in heterologous plant, such as arabidopsis In, it may have the feature that induction exogenous gene is expressed under high salt and drought stress conditions, while reduce under regular culture conditions The expression of foreign gene, to reduce negative effect of the foreign gene to growth of transgenic plants and development.

Brief description of the drawings

Fig. 1 is according to the method for Genome Walking Kit, the promoter of electrophoresis detection thick boisiana LEA protein IpLEA genes The PCR fragment of IpLEA-PRO amplifications.M shows DNA molecular amount standard, and No. 1 swimming lane shows first round nested PCR electrophoresis as a result, No. 2 swimming Road shows the second wheel nested PCR electrophoresis as a result, No. 3 swimming lanes show third round nested PCR electrophoresis result.

The promoter IpLEA-PRO sequential structure figures of Fig. 2 thick boisiana LEA protein genes.ATG shows that the translation of IpLEA genes rises Beginning codon, remaining special marking are different cis-acting elements.

Fig. 3 Real time RT-PCR detect expression of the thick boisiana LEA protein IpLEA genes in thick boisiana plant different parts Situation.

Expression during Fig. 4 Real time RT-PCR detect thick boisiana LEA protein IpLEA genes in thick boisiana body is subject to height The induction of salt and drought stress.

Fig. 5 thick boisiana promoters IpLEA-PRO is inserted into the physical map of plant transgene binary expression vector pBI101.2, The carrier contains kalamycin resistance screening-gene, and promoter is fused to the end regions of gus gene 5 '.

Fig. 6 thick boisiana promoters IpLEA-PRO starts gus gene expression schematic diagram in transgenic arabidopsis.

Embodiment

For the ease of understanding the present invention, the present invention will be described more fully below.The present invention can many differences Form realize, however it is not limited to implementation example described herein.On the contrary, it is to make pair to provide these purposes for implementing examples The understanding of the disclosure more thorough and comprehensive.

The experimental method of actual conditions is not specified in the following example, usually according to normal condition, such as Sambrook etc. People, molecular cloning：Laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989) institute in The condition stated, or according to the condition proposed by manufacturer.Used various common chemical reagent, are commercially available in embodiment Product.

Unless otherwise defined, technical field of all technical and scientific terms used in the present invention with belonging to the present invention The normally understood implication of technical staff it is identical.The term used in the description of the present invention is intended merely to describe specific reality The purpose of example is applied, is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed The arbitrary and all combination of project.

The present invention finds the DNA sequence dna of one section of 1495 base first, and position of this section of sequence on thick boisiana genome abuts The upstream of the reading frame translation initiation codon ATG of thick boisiana LEA genes IpLEA, is the startup of thick boisiana LEA protein gene IpLEA Son, is named as IpLEA-PRO.

The nucleotide sequence of IpLEA-PRO promoters of the present invention is as shown in SEQ.ID.NO1,1495 bases of sequence. According to plant cis-regulating element database (PlantCARE, http://bioinformatics.psb.ugent.be/ Webtools/plantcare/html/ search), promoter region provided by the present invention are removed containing necessary to promoter Outside TATA Box, CAAT Box, also containing multiple environment stresses induction cis-acting elements, which is that the promoter is Environment stress inducible promoter.

The present invention have detected the expression of IpLEA genes in thick boisiana plant, Real time RT-PCR analyses first The result shows that under conditions of normal growth, IpLEA genes have wide expression in thick boisiana different parts and developmental stage, but For its expression quantity for reference gene IpUBQ, expression quantity is very low.Under high salt and drought stress treatment conditions, thick boisiana The expression of IpLEA genes is induced, and is shown in thick boisiana body, and the promoter of IpLEA genes is a high salt/drought stress Promoter.

Afterwards, using the DNA fragmentation of chromosome walking method PCR amplification IpLEA-PRO, then with In-Fusion technologies (In-Fusion HD Cloning Kit,TaKaRa Code:PT5162-1), which is inserted into plant transgene On binary expression vector pBI101.2, by agriculture bacillus mediated transgenic method, transgenosis work, detection are carried out to arabidopsis IpLEA-PRO regulates and controls the expression of gus gene in heterologous plant arabidopsis, shows that the promoter can open in arabidopsis Dynamic foreign gene gus gene expression.High salt and drought stress processing are carried out to transgenic arabidopsis, detects and intends south in transgenosis In mustard plant, the expression of gus gene is also induced, and shows the promoter fragment in heterologous plant arabidopsis, it may have The feature that induction exogenous gene is expressed under high salt and drought stress conditions.

Invention technician should be understood the nucleotide sequence according to SEQ ID No.1, it is replaced, lack or Increase one or several nucleotide, obtain the nucleotide sequence with identical function, for example, in response element or functional element, Replace one or several bases.Therefore, the nucleotide sequence of the present invention further included shown in SEQ ID No.1 through replace, Lack or increase one or several nucleotide, and the nucleotide sequence with identical function, this segment DNA sequence can be can be by Induced to high salt and drought stress, strengthen gene expression.

Embodiment 1：The Cloning and sequence analysis of the promoter IpLEA-PRO of thick boisiana LEA protein IpLEA genes

In the present invention thick boisiana plant culture used in South China Botanical Garden greenhouse group (23 ° 18 ' 75.91 " N, 113 ° 37 ' 02.38 " E) in；Thick boisiana seed collection is in Zhuhai seabeach (22 ° 16 ' 25.37 " N, 113 ° 34 ' 18.00 " E).Thick boisiana tissue sampling Afterwards, be immediately placed in it is quick-frozen in liquid nitrogen after to be stored in -80 DEG C of refrigerators spare.The full thick boisiana seed of 100 or so is taken, using 10% When sulfuric acid immersion 12 is small, clean 20 times with tap water afterwards, using vermiculite sprouting thick boisiana seed (28 DEG C, it is daily 16 it is small when illumination/ 8 is dark when small), grow up to seedling after about one month.The small seedling leaf 0.1g of thick boisiana of healthy growth is taken, is put into mortar and adds liquid nitrogen Powder is ground to, using the plant genome DNA extracts kit One-Tube of Beijing day bounties Gene Tech. Company Limited Plant DNAOUT (article No.s：60705) genomic DNA of thick boisiana blade is extracted.Using electrophoresis detection and and uv-spectrophotometric The purity and concentration of the method detection thick boisiana genomic DNA of meter, and use ddH₂O adjusts the concentration of DNA to 100ng/ μ L.

Design three specific primer SP1:5 '-GGGATCAGCCCCGAGATCAG-3 ' (SEQ ID NO.2), SP2:5′- ACAGGATTTGGATTCTTCAC-3 ' (SEQ ID NO.3) and SP3:5′-AATCTCGGACACATCTGCAG-3′(SEQ ID NO.4)。

Using above-mentioned thick boisiana genomic DNA as template, the 1st, 2,3 wheels moved with SP1, SP2 and SP3 homologue step are random Primer (AP1) carries out chromosome walking cloned promoter.After three-wheel PCR, examined with 1% agarose gel electrophoresis Survey, bright band PCR product (as shown in Figure 1) is selected, according to Magen companies HiPure Gel Pure DNA Kits explanations Book is recycled into row agarose gel electrophoresis, and is connected in the pGEM carrier Ts of Promega companies.Method to specifications will Reaction product converts e. coli jm109 competence bacterial strain.Picking monoclonal, extracts plasmid, send biotech firm to be sequenced, and It is spare to preserve correct plasmid (being named as IpLEA-PRO-pGEMT).The thick boisiana LEA protein gene promoter that sequencing display is cloned into IpLEA-PRO full length sequences are 1495 bases, labeled as SEQ ID NO.1.

Promoter IpLEA-PRO sequences are analyzed, find TATA-box, CAAT-box and other are that may be present suitable Formula functional element (as shown in Figure 2).

Embodiment 2：The expression of thick boisiana LEA protein IpLEA genes is induced be subject to high salt/drought stress

The present invention disclose first by thick boisiana LEA protein IpLEA genes its own promoter regulation and control under in thick boisiana body Expression, the detection method used is Real time RT-PCR technology.The IpLEA bases obtained by this laboratory clone The cDNA sequence and website NCBI (http of cause://www.dtd.nlm.nih.gov/) Photographing On-line Real time RT-PCR draw Thing.Primer for detecting IpLEA gene expression patterns is IpLEA-RTF:5′-AAGGAGGGTGGTACCACCCG-3′(SEQ ID NO.5) and IpLEA-RTR:5′-CTTTAAGTCTGAAACACCAAACGC-3′(SEQ ID NO.6).Reference gene is thickness Rattan ubiquitin protein gene IpUBQ, primer IpUBQ-RTF:5 '-TCGACAATGTGAAGGCAAAG-3 ' (SEQ ID NO.7) and IpUBQ-RTR:5’-CTTGATCTTCTTCGGCTTGG-3’(SEQ ID NO.8).With reference to BIO-RAD companies iTaq^TM The specification of Universal SYBR Green Supermix prepares real time RT-PCR reaction systems (operating on ice). It is detected using Roche quantitative fluorescent PCR LightCycler480 application methods.

All detections are repeated using two biological samples, and each biological sample carries out repeating detection reaction three times. Program Stage3 detection solubility curves are added when primer uses for the first time, confirm the specificity of primer.

Thick boisiana seedling is chosen respectively from South China Botanical Garden greenhouse and garden and adult is bloomed the different tissues position of plant, inspection Expression of the thickness measuring rattan LEA protein IpLEA genes under the conditions of stress from outside is not affected by under its own promoter regulates and controls.Such as Fig. 3 Shown, under conditions of normal growth, IpLEA genes have expression in the different parts of thick boisiana, but expression quantity is relative to composition Under type promoter regulation for IpUBQ genes, its expression quantity is relatively low, and maximum expression quantity is only IpUBQ (in adult plants root) 0.04 times of gene expression amount, shows under regular culture conditions, and thick boisiana LEA protein IpLEA promoters open for low expression level Mover.

Take seed sprout after the healthy growth thick boisiana seedling of one month, by thick boisiana seedling transfer 1/2MS fluid nutrient mediums after Continuous culture 3 days.Then with 1/ containing NaCl (300mM, simulates high-salt stress) and mannitol (300mM, Drought stress simulation) 2MS fluid nutrient mediums handle thick boisiana seedling, collect Stress treatment 0h (control), 2 it is small when and 24 it is small when after thick boisiana spire and children Each 0.5g of root, for extracting total serum IgE.The extraction of RNA is said according to Magen companies HiPure Plant RNA Kits (R4151's) Bright book carries out.Two-step method is used using total serum IgE as template reverse transcription cDNA.The synthesis of cDNA chains is according to Quan Shi King Companies The specification of TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix carries out.

As shown in figure 4, under 300mM NaCl Stress treatments, no matter in the root of thick boisiana seedling, rattan or leaf, IpLEA bases The expression of cause is all subject to the induced strong of salt stress, its maximum inducing amount can reach more than 7 times.And in the arid (sweet dew of 300mM Alcohol processing can result in plant dehydration) under Stress treatment, no matter in the root of thick boisiana seedling, rattan or leaf, the expression of IpLEA genes Also more than 8 times be can reach by induced strong, its maximum inducing amount.It is one that disclosure above, which indicates IpLEA genes, High salt and drought stress induced gene, gene promoter IpLEA-PRO, which has, starts target gene thick boisiana LEA genes in salt drought The lower induced expression of stress, improves ability of the thick boisiana to high salt drought stress adaptability.

Embodiment 3：The GUS transgenosis built under the promoter IpLEA-PRO regulation and control of thick boisiana LEA protein IpLEA genes is intended The structure of southern mustard material

Using the IpLEA-PRO-pGEMT Plasmid DNA inserted with thick boisiana IpLEA gene promoters as template, design is following to draw Thing IpLEAProF:5 '-CGACTCTAGAGGATCCAATCTCGGACACATCTGCAG-3 ' (SEQ ID NO.9) and IpLEAProR:5 '-ACCTACCCGGGGATCCACCTTCTCACAAGCTGAGAT-3 ' (SE Q ID NO.10) are to thick boisiana LEA The promoter IpLEA-PRO of protein I pLEA genes carries out PCR amplification.PCR product is according to Magen companies HiPure Gel Pure DNA Kits specifications are recycled into row agarose gel electrophoresis.At the same time using B amHI single endonuclease digestions processing arabidopsis transgenosis double base Expression vector pBI101.2, recycles linearization plasmid.IpLEA-P RO promoters PCR fragment and linearisation pBI101.2 after recycling Plasmid is through Nanodrop companies ultraviolet specrophotometer measured concentration, using TaKaRa companies HD Cloning Kit carry out DNA fragmentation and are connected with the homologous recombination of carrier.Reaction product is converted large intestine by method to specifications Bacillus JM109 competence bacterial strains.Picking monoclonal, extracts plasmid, after sequencing identification is correct positive colony, is named as IpLEA-PRO-pBI101.2 (structure diagram is as shown in Figure 5), it is spare to preserve plasmid.After sequencing analysis are correct, IpLEA- PRO-pBI101.2 recombinant plasmids are transferred in GV3101 Agrobacteriums using freeze-thaw method, are added 50% glycerine to put -80 DEG C and are saved backup.

Using Clombia wildtype Arabidopsis thalianas as transgenic line, the method that is infected using inflorescence, by what is built IpLEA-PRO-pBI101.2 plant expression vectors pass through GV3101 agrobacterium mediation converted arabidopsis.The arabidopsis that will be obtained Transgenosis T3 is put on the MS culture mediums containing 50ug/mL kanamycins for seed and is screened.

The transgenic arabidopsis T3 of growth 10 days is taken for plant, when being placed in that processing 3 is small in GUS staining reaction liquid, using group Weave chemistry method is detected, afterwards with 95% ethanol fade 48 it is small when after taken pictures using LEICA DM2500 Stereo microscopes. As shown in fig. 6, there is blue spot in the true leaf of transgenic arabidopsis seedling, it is positive to show that GUS staining reactions occur, it was demonstrated that Gus gene begins transcription under the regulation and control of thick boisiana LEA gene promoters IpLEA-PRO, shows thick boisiana LEA of the present invention Gene promoter sequence (SEQ ID NO.1) possesses the function that promotor gene is transcribed in heterologous plant (arabidopsis), is one The promoter that can be applied in plant transgene work.

Embodiment 4：Expression of the gus gene in arabidopsis under IpLEA-PRO regulation and control is lured by high salt and drought stress Lead

After the seed of the transgenic positive plant arabidopsis of acquisition is sprouted, it is placed in 1/2MS culture medium flat plates in 22 DEG C (16 it is small when illumination/8 it is small when it is dark) condition of culture under culture obtain Arabidopsis thaliana Seedlings fortnight, carry out high salt afterwards (NaCl of 300mM) and simulating drought (mannitol of 300mM) Stress treatment, processing method is with example 2 is implemented, in processing time To harvest Arabidopsis thaliana Seedlings during 24h, control is used as using untreated Arabidopsis thaliana Seedlings.Real time RT-PCR skills are carried out afterwards Art detects expression of the gus gene in arabidopsis body under by the regulation and control of thick boisiana promoter IpLEA-PRO.The extraction of RNA and The program of reverse transcription is referring to embodiment 2.Reference gene in arabidopsis uses arabidopsis ubiquitin gene AtUBQ10 (At4g05320), the primer sequence of design is respectively：AtUBQF:5′-GATCTTTGCCGGAAAACAATTGGAGGATGGT-3′ (SEQ ID NO.11) and AtUBQR:5′-CGACTTGTCATTAGAAAGAAAGAGATAACAGG-3′(SEQ ID NO.12). The special primer of gus gene detection is as follows：GUS-F:5′-ATGTTACGTCCTGTAGAAAGGAAG-3′(SEQ ID NO.13) And GUS-R:5′-TCATTGTTTGCCTCCCTGCTGC-3′(SEQ ID NO.14).With reference to BIO-RAD companies iTaq^TM The specification of Universal SYBR Green Supermix prepares real time RT-PCR reaction systems (operating on ice). It is detected using Roche quantitative fluorescent PCR LightCycler480 application methods.

All detections are repeated using two biological samples, and each biological sample carries out repeating detection reaction three times. Program Stage3 detection solubility curves are added when primer uses for the first time, confirm the specificity of primer.

Find after testing, the gus gene of arabidopsis is under the regulation and control of thick boisiana LEA protein gene promoter IpLEA-PRO, nothing By be salt stress (NaCl of 300mM) processing under, or simulating drought (mannitol of 300mM) stress under, gus gene exists Expression in arabidopsis, which receives, consumingly to be induced, and shows the thick boisiana LEA protein gene promoter IpLEA- that the present invention announces PRO is really high salt drought-inducible promoter in heterologous plant (arabidopsis) Transgenic studies.

The present embodiment only discloses in arabidopsis external source gus gene by promoter IpLEA-PRO in high salt drought stress Lower induced expression, the present invention also may extend to other functional genes and other plant or crops, and be applied to plant gene work The induced expression of target gene under high salt drought stress conditions is tackled in journey.Since the promoter is under the conditions of plant normal growth The expression of promotor gene is relatively low, therefore, may have potential poison to plant growth and development under normal operation for some Property gene for, can reduce transgenosis work the potential side effect of foreign gene so that cultivate specificity adapt to high salt do The modified form genetically modified plants of non-irrigated adverse circumstance.It is can also be applied to transformation is adapted to the plant biological reaction of high salt Drought Stress Device, to obtain the genetically modified plants bioreactor of the target protein high yield under high salt drought stress conditions.

Embodiment described above only expresses the several embodiments of the present invention, its description is more specific and detailed, but simultaneously Therefore the limitation to the scope of the claims of the present invention cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Sequence table

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tcattgtttg cctccctgct gc 22

Claims (9)

1. a kind of high salt, drought-inducible promoter DNA sequence dna, it is characterised in that：The promoter DNA sequence be containing Nucleotide sequence shown in SEQ ID No.1；Or it is one or several containing having in the sequence shown in SEQ ID No.1 and sequence Replacement, missing or the increase of nucleotide, but the nucleotide sequence with identical function；Or it is and SEQ ID NO.1 complete complementaries The nucleotide sequence of pairing；Or to be cloned by SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 by chromosome walking Expand obtained nucleotide sequence.

2. a kind of high salt, the preparation method of drought-inducible promoter DNA sequence dna described in claim 1, it is characterised in that：With The genomic DNA of wild thick boisiana is template, is walked by SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 by chromosome Clonal expansion is moved to obtain.

A kind of 3. plant expression vector, it is characterised in that：Contain the high salt described in claim 1, drought-inducible promoter DNA Sequence.

4. plant expression vector according to claim 3, it is characterised in that：The plant expression vector is BamHI enzymes High salt, the pBI101.2 carriers of drought-inducible promoter DNA sequence dna after cutting described in connection claim 1.

5. high salt, drought-inducible promoter DNA sequence dna described in claim 1, or the plant expression load described in claim 3 Application of the body in gene expression in plants is regulated and controled.

6. high salt, drought-inducible promoter DNA sequence dna described in claim 1, or the plant expression load described in claim 3 Body high salt, drought stress plant transgene breeding in application.

7. the application according to claim 5 or 6, the plant is arabidopsis or crops.

8. a kind of improve the method for arabidopsis or crops to high salt, Drought Stress adaptability, it is characterised in that including by right It is required that high salt, drought-inducible promoter DNA sequence dna described in 1, or plant expression vector claim 3 obtained described in 4 turns Change into arabidopsis or crops.

9. according to the method described in claim 8, it is characterized in that, pass through GV3101 agrobacterium mediation converteds arabidopsis or farming In thing, arabidopsis or the transgenic seed of crops are obtained.