CN107904238B - Thick boisiana is with high salt, drought-inducible promoter IpLEA-PRO and its application - Google Patents
Thick boisiana is with high salt, drought-inducible promoter IpLEA-PRO and its application Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
- C12N15/8223—Vegetative tissue-specific promoters
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- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
Abstract
The present invention relates to a kind of promoters (being abbreviated as IpLEA-PRO) of thick boisiana LEA protein IpLEA gene, with nucleotide sequence shown in SEQ ID No.1.The present invention provides through the expressions of results of detection GUS to prove, which has the activity of promoter, and is enhanced by with high salt and drought stress induced activity, and this show the effects that the LEA protein gene responds with 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 the salt tolerance of raising and improvement thick boisiana and other plant is expressed salt/relevant target protein of drought stress using plant as bioreactor and provides theoretical basis.
Description
Technical field
The invention belongs to plant genetic engineering fields, and in particular to a thick boisiana is with high salt, drought-inducible promoter
IpLEA-PRO and its application.
Background technique
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) are 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 is anti-
Inverse property is extremely strong, and salt tolerant and drought resistance are especially pronounced.Meanwhile thick boisiana is as a kind of evergreen liana, gardens with higher
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 that thick boisiana has biggish potentiality to be exploited as a kind of excellent wild plant resource
And application value, while also can be used as a kind of plant stress-resistance molecule mechanism, the research object of excavating adversity gene is deeply ground
Study carefully.
LEA protein be late embryo occur Abundant protein family (Late Embryogensis Abundant proteins
II family of II, LEA-), it is that plant is intracorporal a kind of with high heat stability, hydrophilic small molecular protein, it 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 rouge peroxide
Change, protect chill sensitive enzymatic activity, as molecular chaperones and the characteristic for combining DNA/RNA etc..Studies have shown that many plants
The expression of LEA protein gene is all influenced by with high salt/Water deficits such as arid and cryogenic freezing, and by improving LEA egg
The expression of white gene is to improve plant to the resistance of Water deficit.And it this improved gene expression amount by environment stress and is showed
Inducing expression as being referred to as gene is determined by the starting subcharacter of gene.
Promoter (promoter) is one section of noncoding DNA sequence positioned at the end structural gene 5'- upstream, can be by RNA
Polymerase identification combines, and accurately controls the initial time and expression intensity of transcription (gene expression), is gene transcription regulation
Center.The expression of gene and the structure of promoter are closely related, and the type 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.
Summary of the invention
With high salt with plant, drought stress induction type promoter that the purpose of the present invention is to provide a kind of.
Another object of the present invention is to provide the promoters, in prepare transgenosis plant and are directed to raising 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 with high salt, drought-inducible promoter DNA sequence dna, the promoter DNA sequence are to contain SEQ ID No.1
Shown in nucleotide sequence；Or for containing having replacing for one or several nucleotide in sequence shown in SEQ ID No.1 and sequence
It changes, lack or increases, but nucleotide sequence with the same function；Or the nucleosides to be matched with SEQ ID NO.1 complete complementary
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.
Preparation method with high salt, drought-inducible promoter DNA sequence dna that the present invention also provides above-mentioned one kind, 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.
The present invention also provides a kind of plant expression vectors, contain above-mentioned with high salt, drought-inducible promoter DNA sequence
Column.
Preferably, the plant expression vector is that described in claim 1 with high salt, arid lure is connected after BamHI digestion
The pBI101.2 carrier of conductivity type promoter DNA sequence.
The present invention also provides above-mentioned with high salt, drought-inducible promoter DNA sequence dna or above-mentioned plant expression vectors
Application in regulation gene expression in plants.
The present invention also provides above-mentioned with high salt, drought-inducible promoter DNA sequence dna or plants as claimed in claim 3
Application of the object expression vector in plant transgene breeding with high salt, drought stress.
Preferably, the plant is arabidopsis or crops.
Arabidopsis or crops are improved to the method for Drought Stress adaptability with high salt the present invention also provides a kind of, including
Arabidopsis or agriculture will be transformed into containing above-mentioned with high salt, drought-inducible promoter DNA sequence dna or above-mentioned plant expression vector
In crop.
Preferably, by GV3101 agrobacterium mediation converted 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 promoter IpLEA- by long-term experience and a large amount of experiment
PRO, the cis acting member other than containing core promoter element TATA-BOX, also containing multiple response environment stresses
Part, including abscisic acid response element ABRE, the drought-induced and relevant Myb factor binding site MBS of stress, and stress are related
TC-rich repeats cis-acting elements.In addition, endosperm is also found in the promoter sequence expresses required cis- work
With element Skn-1_Motif.The promoter can be lured with controlling gene by with high salt/drought stress in thick boisiana and arabidopsis
Expression is led, which can be applied to the plant transgene breeding for high salt/drought stress, it is with high salt/dry for culture and improvement
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 promoter of the present invention is with high salt and drought stress inducible promoter, can be used for plant gene
Change plant in engineering to foreign gene in response mode with high salt and arid and adjustment genetically modified plants in normal growing conditions
Lower expression way overcomes commonly 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 the Plant Tolerance side of body with high salt and arid
Compel desired genetically modified plants new varieties.In particular, the IpLEA-PRO promoter fragment is in heterologous plant, such as arabidopsis
In, it may have the feature that induction exogenous gene is expressed under with high salt and drought stress conditions, while reducing under regular culture conditions
The expression of foreign gene, to reduce foreign gene to the negative effect of growth of transgenic plants and development.
Detailed description of the invention
Method of the Fig. 1 according to Genome Walking Kit, the promoter of electrophoresis detection thick boisiana LEA protein IpLEA gene
The PCR fragment of IpLEA-PRO amplification.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 figure of Fig. 2 thick boisiana LEA protein gene.ATG shows that the translation of IpLEA gene rises
Beginning codon, remaining special marking are different cis-acting elements.
Fig. 3 Real time RT-PCR detects thick boisiana LEA protein IpLEA gene in the expression of thick boisiana plant different parts
Situation.
Fig. 4 Real time RT-PCR detect thick boisiana LEA protein IpLEA gene in thick boisiana body in expression by height
The induction of salt and drought stress.
Fig. 5 thick boisiana promoter IpLEA-PRO is inserted into the physical map of plant transgene binary expression vector pBI101.2,
Carrier resistance screening gene containing kanamycin, promoter are fused to 5 ' end regions of gus gene.
Fig. 6 thick boisiana promoter IpLEA-PRO starts gus gene in transgenic arabidopsis and expresses schematic diagram.
Specific embodiment
It to facilitate the understanding of the present invention, below will be to invention is more fully described.The present invention can many differences
Form realize, however it is not limited to implementation example described herein.On the contrary, providing the purpose that these implement examples is to make pair
The understanding of the disclosure is more thorough and comprehensive.
In the following examples, the experimental methods for specific conditions are not specified, usually according to normal condition, such as Sambrook etc.
People, molecular cloning: institute in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989)
The condition stated, or according to the normal condition proposed by manufacturer.Used various common chemical reagent, are commercially available in embodiment
Product.
Unless otherwise defined, all technical and scientific terms used in the present invention and belong to technical field of the invention
The normally understood meaning of technical staff it is identical.Term used in the description of the invention is intended merely to describe specific reality
The purpose for applying example is not used in the limitation present invention.Term "and/or" used in the present invention includes one or more relevant listed
Any and all combinations of project.
The present invention finds the DNA sequence dna of one section of 1495 base for the first time, 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 gene IpLEA is the starting of thick boisiana LEA protein gene IpLEA
Son is named as IpLEA-PRO.
The nucleotide sequence of IpLEA-PRO promoter 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
Except TATA Box, CAAT Box, the cis-acting elements also induced containing multiple environment stresses, which is that the promoter is
Environment stress inducible promoter.
The present invention has detected the expression of IpLEA gene in thick boisiana plant, Real time RT-PCR analysis first
The result shows that IpLEA gene has wide expression in thick boisiana different parts and developmental stage under conditions of normal growth, but
For its expression quantity for reference gene IpUBQ, expression quantity is very low.Under with high salt and drought stress treatment conditions, thick boisiana
The expression of IpLEA gene is induced, and is shown in thick boisiana body, and the promoter of IpLEA gene is a with high salt/drought stress
Promoter.
Later, using the DNA fragmentation of chromosome walking method PCR amplification IpLEA-PRO, In-Fusion technology is then used
(In-Fusion HD Cloning Kit, TaKaRa Code:PT5162-1), is inserted into plant transgene for the segment DNA sequence
On binary expression vector pBI101.2, by the transgenic method of mediated by agriculture bacillus, 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.The processing of with high salt and drought stress is carried out to transgenic arabidopsis, is detected in the quasi- south of 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 with high salt and drought stress conditions.
Invention technician should understand that the nucleotide sequence according to shown in SEQ ID No.1, it is replaced, lack or
Increase one or several nucleotide, obtains nucleotide sequence with the same function, for example, in response element or functional element,
Replace one or several bases.Therefore, it is of the present invention further include nucleotide sequence shown in SEQ ID No.1 through replacement,
Lack or increase one or several nucleotide, and nucleotide sequence with the same function, this segment DNA sequence can be can be by
To the induction of with high salt and drought stress, enhance gene expression.
Embodiment 1: the Cloning and sequence analysis of the promoter IpLEA-PRO of thick boisiana LEA protein IpLEA gene
Thick boisiana plant culture used in the present invention 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 for taking 100 or so, using 10%
Sulfuric acid impregnate 12 hours, clean 20 times with tap water later, using vermiculite sprouting thick boisiana seed (28 DEG C, daily illumination in 16 hours/
8 hours dark), grow up to seedling after about one month.The small seedling leaf 0.1g of the thick boisiana of healthy growth is taken, is put into mortar and liquid nitrogen is added
It is ground to powder, using the plant genome DNA extracts kit One-Tube of Beijing day bounties Gene Tech. Company Limited
Plant DNAOUT (article No.: 60705) extracts the genomic DNA of thick boisiana blade.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
It surveys, bright band PCR product (as shown in Figure 1) is selected, according to Magen company HiPure Gel Pure DNA Kits explanation
Book carries out agarose gel electrophoresis recycling, and is connected in the pGEM carrier T of Promega company.Method to specifications will
Reaction product converts e. coli jm109 competence bacterial strain.Picking monoclonal extracts plasmid, biotech firm is sent to be sequenced, and
It is spare to save 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 sequence is 1495 bases, is labeled as SEQ ID NO.1.
Promoter IpLEA-PRO sequence is analyzed, finds TATA-box, CAAT-box and other are that may be present suitable
Formula functional element (as shown in Figure 2).
Embodiment 2: induction of the expression of thick boisiana LEA protein IpLEA gene by with high salt/drought stress
The present invention disclose first by thick boisiana LEA protein IpLEA gene own promoter regulation under in thick boisiana body
Expression, the detection method used is Real time RT-PCR technology.The IpLEA base obtained by this laboratory clone
The cDNA sequence and website NCBI (http://www.dtd.nlm.nih.gov/) Photographing On-line Real time RT-PCR of cause are drawn
Object.Primer for detecting IpLEA gene expression pattern 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 company iTaqTM
The specification of Universal SYBR Green Supermix prepares real time RT-PCR reaction system (operating on ice).
It is detected using Roche quantitative fluorescent PCR LightCycler480 application method.
All detections are all made of two biological samples and repeat, and each biological sample carries out repeating detection reaction three times.
Addition program Stage3 detects solubility curve when primer uses for the first time, confirms 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 under the conditions of thickness measuring rattan LEA protein IpLEA gene is not affected by stress from outside under own promoter regulation.Such as Fig. 3
Shown, under conditions of normal growth, IpLEA gene has expression in the different parts of thick boisiana, but expression quantity is relative to composition
Under type promoter regulation for IpUBQ gene, expression quantity is lower, and maximum expression quantity (in adult plants root) is only IpUBQ
0.04 times of gene expression amount, shows under regular culture conditions, and thick boisiana LEA protein IpLEA promoter opens for low expression level
Mover.
Healthy growth one month thick boisiana seedling after taking seed to sprout, by thick boisiana seedling transfer 1/2MS fluid nutrient medium after
Continuous culture 3 days.Then with 1/ containing NaCl (300mM simulates high-salt stress) and mannitol (300mM, Drought stress simulation)
2MS fluid nutrient medium handles thick boisiana seedling, collects Stress treatment 0h (control), 2 hours and thick boisiana spire and children after 24 hours
Each 0.5g of root, for extracting total serum IgE.The extraction of RNA is said according to Magen company HiPure Plant RNA Kits (R4151's)
Bright book carries out.Use two-step method using total serum IgE as template reverse transcription cDNA.The synthesis of cDNA chain is according to Quan Shi King Company
The specification of TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix carries out.
As shown in figure 4, under 300mM NaCl Stress treatment, no matter in the root of thick boisiana seedling, rattan or leaf, IpLEA base
All by the induced strong of salt stress, maximum inducing amount can reach 7 times or more for the expression of cause.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 gene
Also by induced strong, maximum inducing amount can reach 8 times or more.It is one that disclosure above, which shows IpLEA gene,
With high salt and drought stress induced gene, gene promoter IpLEA-PRO have starting target gene thick boisiana LEA gene in salt drought
Lower inducing expression is coerced, improves thick boisiana to the ability of drought stress adaptability with high salt.
Embodiment 3: the GUS transgenosis under the promoter IpLEA-PRO regulation of building thick boisiana LEA protein IpLEA gene is quasi-
The building of southern mustard material
Using the IpLEA-PRO-pGEMT Plasmid DNA inserted with thick boisiana IpLEA gene promoter as template, following draw is designed
Object IpLEAProF:5 '-CGACTCTAGAGGATCCAATCTCGGACACATCTGCAG-3 ' (SEQ ID NO.9) and
IpLEAProR:5 '-ACCTACCCGGGGATCCACCTTCTCACAAGCTGAGAT-3 ' (SE Q ID NO.10) is to thick boisiana LEA
The promoter IpLEA-PRO of protein I pLEA gene carries out PCR amplification.PCR product is according to Magen company HiPure Gel Pure
DNA Kits specification carries out agarose gel electrophoresis recycling.Arabidopsis transgenosis double base is handled using B amHI single endonuclease digestion simultaneously
Expression vector pBI101.2 recycles linearization plasmid.IpLEA-P RO promoter PCR fragment and linearisation pBI101.2 after recycling
Plasmid measures concentration through Nanodrop company ultraviolet specrophotometer, using TaKaRa companyHD Cloning
Kit carries out DNA fragmentation and connects with the homologous recombination of carrier.Reaction product is converted Escherichia coli by method to specifications
JM109 competence bacterial strain.Picking monoclonal extracts plasmid, after sequencing is accredited as correct positive colony, is named as IpLEA-
It is spare to save plasmid by PRO-pBI101.2 (structural schematic diagram is as shown in Figure 5).After sequencing analysis is correct, IpLEA-PRO-
PBI101.2 recombinant plasmid is transferred in GV3101 Agrobacterium using freeze-thaw method, is added 50% glycerol to put -80 DEG C and is saved backup.
Using Clombia wildtype Arabidopsis thaliana as transgenic line, the method infected using inflorescence, by what is built
IpLEA-PRO-pBI101.2 plant expression vector passes through GV3101 agrobacterium mediation converted arabidopsis.By arabidopsis obtained
Transgenosis T3 is put on the MS culture medium containing 50ug/mL kanamycins for seed and is screened.
It takes the transgenic arabidopsis T3 of growth 10 days for plant, is placed in GUS staining reaction liquid and handles 3 hours, using group
Weave chemistry method is detected, and is taken pictures after being faded 48 hours with 95% ethyl alcohol later using LEICA DM2500 Stereo microscope.
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 reaction occurs, it was demonstrated that
Gus gene begins transcription under the regulation of thick boisiana LEA gene promoter IpLEA-PRO, shows thick boisiana LEA of the present invention
Gene promoter sequence (SEQ ID NO.1) has the function that promotor gene is transcribed in heterologous plant (arabidopsis), is one
The promoter that can be applied in plant transgene work.
Expression of the gus gene in arabidopsis under embodiment 4:IpLEA-PRO regulation is lured by with high salt and drought stress
It leads
After the seed of the transgenic positive plant arabidopsis of acquisition is sprouted, it is placed in 1/2MS culture medium flat plate in 22
Culture obtains Arabidopsis thaliana Seedlings fortnight under the condition of culture of DEG C (16 hours illumination/8 hour dark), carries out later with high salt
(NaCl of 300mM) and simulating drought (mannitol of 300mM) Stress treatment, processing method is with example 2 is implemented, in the processing time
For for 24 hours when harvest Arabidopsis thaliana Seedlings, using untreated Arabidopsis thaliana Seedlings as control.Real time RT-PCR skill is carried out later
Art detects expression of the gus gene in arabidopsis body under by the regulation 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 as follows: 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)
With GUS-R:5 '-TCATTGTTTGCCTCCCTGCTGC-3 ' (SEQ ID NO.14).With reference to BIO-RAD company iTaqTM
The specification of Universal SYBR Green Supermix prepares real time RT-PCR reaction system (operating on ice).
It is detected using Roche quantitative fluorescent PCR LightCycler480 application method.
All detections are all made of two biological samples and repeat, and each biological sample carries out repeating detection reaction three times.
Addition program Stage3 detects solubility curve when primer uses for the first time, confirms the specificity of primer.
Find that the gus gene of arabidopsis is under the regulation of thick boisiana LEA protein gene promoter IpLEA-PRO, nothing through detection
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 drought-inducible promoter with high salt in heterologous plant (arabidopsis) Transgenic studies really.
The present embodiment only discloses in arabidopsis external source gus gene by promoter IpLEA-PRO in drought stress with high salt
Lower inducing expression, the present invention also may extend to other function gene and other plant or crops, and be applied to plant gene work
The inducing expression of target gene under drought stress conditions with high salt is coped in journey.Since the promoter is under the conditions of plant normal growth
The expression of promotor gene is lower, therefore, may have potential poison to plant growth and development under normal operation for certain
Property gene for, can reduce the transgenosis work potential side effect of foreign gene, thus cultivate specificity adapt to it is with high salt dry
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 Drought Stress with high salt
Device, to obtain the genetically modified plants bioreactor of the target protein high yield under drought stress conditions with high salt.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
Limitations on the scope of the patent of the present invention therefore 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 guarantor of the invention
Protect range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Sequence table
<110>South China Botanical Garden Chinese Academy of Sciences
<120>thick boisiana is with high salt, drought-inducible promoter IpLEA-PRO and its application
<160> 14
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1495
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aatctcggac acatctgcag ttggttttcc aaacccgatt gtctcctcta tcttctcccc 60
tacatcatgg ataaaatctt tcaccttgtc caagaaccca cccttttctt cctccttgtc 120
ttccttgtcc ttgatacccc tttccactat ctctggatta tcagacgatg ccataccttc 180
tcacaagctg agatcaataa atattgaact aattactcac tcaatattgc aagcaaaatc 240
ttacttacta ttcatgaaca cagatttatt tttttttttt atcaatgcat agaaatgaca 300
cattgatttg tttaatttaa attttaaaaa gttcatacgt ttaattagct tgtagtataa 360
agttcaggtg tgtatttaaa cttttatttc aaataaaaaa aagttatggg ccaaaatcta 420
atgaggtatt tacacaggac aaaaatttgt gtgagatatt tcacggatct taatttgtaa 480
gacggatcaa atatttgatt aataagatca aaatttctac tcattaataa atattcgact 540
tgtcttactg attgaaactc gtaaaccagt gttaccctta cacgggtggt ttccgttaga 600
tttttgaagt gtttgagatg aaatcaaatg ataatttgga ccacaaatta ggcgacacgt 660
acaaaaacag ctgaataaac taccccgtat taaaatgatg atgatgattg ggccgtgtgt 720
ggtcggctgg tcggtgcatc aaataatgag tcgggtccgc gtgaaccgat aaccaacccg 780
aacccactta cgcaccagtc accccagtca tcgactcgat catttcttct gtatttatta 840
caccaaaaat ttatagcgtc tatcgctctg tgtccggaca acctcttcgt cttcaccatt 900
tgcagccgat tatctctcgt taggtaaatc ttcattgttt cgtttttgat tgatcatccc 960
ccatattatt ggagtttgga gattctgatc gtttagcttt gctttttgaa taatgatgat 1020
gcgatctgat ctaggttgac ttgatcggtt gattttttta attttctgaa atgaatggtg 1080
gtctcttgta gtcttgtcag tcactctcac tccactcaaa ccgctaattc ctccattgat 1140
gattgatgat taccgctttt gaacctagct tagtactact tctagtaatc taggcaaatg 1200
ctataattcc gatcgtgtgt gtgttttgta ttcacttgtt ttaagttcat tgtatatgtg 1260
ggaattgatg ttcttgagat aattacacag gcattaattg cttcttaggt ctagtgaatt 1320
gctccttcta atactagtag actagtaatt agtgtgatca cgaaagaaat ggaaaataaa 1380
tgctttttaa aaaaaaaaaa ataaatctgt gttcatgaat agtaagtaag attttgcttg 1440
caatattgag tgagtaatta gttcaatatt tattgatctc agcttgtgag aaggt 1495
<210> 2
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
gggatcagcc ccgagatcag 20
<210> 3
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 3
acaggatttg gattcttcac 20
<210> 4
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 4
aatctcggac acatctgcag 20
<210> 5
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 5
aaggagggtg gtaccacccg 20
<210> 6
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 6
ctttaagtct gaaacaccaa acgc 24
<210> 7
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 7
tcgacaatgt gaaggcaaag 20
<210> 8
<211> 20
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 8
cttgatcttc ttcggcttgg 20
<210> 9
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 9
cgactctaga ggatccaatc tcggacacat ctgcag 36
<210> 10
<211> 36
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 10
acctacccgg ggatccacct tctcacaagc tgagat 36
<210> 11
<211> 31
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 11
gatctttgcc ggaaaacaat tggaggatgg t 31
<210> 12
<211> 32
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 12
cgacttgtca ttagaaagaa agagataaca gg 32
<210> 13
<211> 24
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 13
atgttacgtc ctgtagaaag gaag 24
<210> 14
<211> 22
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 14
tcattgtttg cctccctgct gc 22
Claims (8)
1. a kind of with 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.
2. a kind of plant expression vector, it is characterised in that: contain with high salt, drought-inducible promoter DNA described in claim 1
Sequence.
3. plant expression vector according to claim 2, it is characterised in that: the plant expression vector is BamHI enzyme
With high salt, drought-inducible promoter DNA sequence dna pBI101.2 carrier described in claim 1 is connected after cutting.
4. with high salt, drought-inducible promoter DNA sequence dna described in claim 1 or plant as claimed in claim 2 expression carry
Application of the body in high salt, drought stress control of plant gene expression.
5. with high salt, drought-inducible promoter DNA sequence dna described in claim 1 or plant as claimed in claim 2 expression carry
Application of the body in plant transgene breeding with high salt, drought stress.
6. application according to claim 4 or 5, the plant is arabidopsis or crops.
7. a kind of improve arabidopsis or crops to the method for Drought Stress adaptability with high salt, which is characterized in that including by right
It is required that with high salt described in 1, drought-inducible promoter DNA sequence dna, or plant expression vector described in claim 2 or 3 is turned
Change into arabidopsis or crops.
8. the method according to the description of claim 7 is characterized in that passing through GV3101 agrobacterium mediation converted arabidopsis or farming
In object, the transgenic seed of arabidopsis or crops is obtained.
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|---|---|---|---|
| CN201711192230.2A CN107904238B (en) | 2017-11-24 | 2017-11-24 | Thick boisiana is with high salt, drought-inducible promoter IpLEA-PRO and its application |
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| CN201711192230.2A CN107904238B (en) | 2017-11-24 | 2017-11-24 | Thick boisiana is with high salt, drought-inducible promoter IpLEA-PRO and its application |
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| CN107904238B true CN107904238B (en) | 2019-05-17 |
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| CN114875025B (en) * | 2022-03-25 | 2023-09-19 | 广东省科学院南繁种业研究所 | Drought and ABA inducible promoter P SCBV-YZ2060 And applications thereof |
| CN114752597B (en) * | 2022-04-06 | 2023-09-19 | 广东省科学院南繁种业研究所 | Drought-inducible promoter P for plant guard cell specific expression SCBV-CHN2 Application and application thereof |
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