CN105985957B - A kind of adverse circumstance induced expression Ghi10424 promoters and its application - Google Patents
A kind of adverse circumstance induced expression Ghi10424 promoters and its application Download PDFInfo
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Abstract
The invention discloses a kind of 10424 promoters of adverse circumstance induced expression Ghi and its applications.The present invention discloses a kind of DNA molecular, any shown for following (1) (3)：(1) DNA molecular shown in the 1st to the 2079th nucleotide in SEQ ID No.8；(2) DNA molecular shown in the 15th to the 2073rd nucleotide in SEQ ID No.11；(3) DNA molecular shown in SEQ ID No.11.Promoter disclosed by the invention can be applied to influence the induced expression of target gene under adverse circumstance, especially resist compound adversity plant new varieties to have important theory and realistic meaning to cultivating.
Description
Technical field
The present invention relates to a kind of promoter and its applications；More particularly to a kind of 10424 promoters of adverse circumstance induced expression Ghi
And its application, belong to biotechnology.
Background technology
The abiotic stress such as low temperature, arid, saline and alkaline seriously threaten the development of modern agriculture, these abiotic stress cause
Cotton generates a series of unfavorable changes, and then the serious yield for affecting cotton in terms of form, physiology, biochemistry and molecule
And fiber quality.Traditional breeding method is difficult solve the problems, such as cotton in a short time degeneration-resistant, and the method for genetic engineering has not
Limited by inter-species reproduction isolation, rapid polymerization multiple merits the advantages of.
The resistance reaction of plant is an extremely complex process, can be completed, can also be passed through by a kind of approach
Number of ways mutually cooperates with or inhibits and complete jointly, the perception including plant plasma membrane serves to external source stimulus signal, leads to
It crosses second messenger and signal is passed into corresponding stress response transcription factor, the cis acting of final transcription factor and stress response
Element is specifically bound, to activate the expression of downstream gene to make response to adverse circumstance.Wherein, many is included in promoter sequence
Important cis-acting elements plays an important roll the expression of plant gene, becomes the crucial ring of expression regulation
Section either still all has highly important status in gene functional research in transgenic technology research.Therefore, to starting
The research of minor structure, function and mechanism of action will be helpful to understand gene regulation pattern and signaling pathways, to understanding in depth
The defense mechanism of cotton, the resistance for improving crop have important meaning.
Popularization successively with various transgenic crops in the whole world, important function of the promoter in transgenosis have obtained
Extensive common recognition is arrived.People need the expression of different types of promoter driving adversity gene, to provide different transgenosis tables
The solution of expression patterns.In Resistance Strain of Cotton against in research field, what people paid close attention to the most be exactly can be fast under adverse environmental factor
The promoter of fast induced expression ensures that even improving cotton exists to quickly start resistance of the cotton to various abiotic stresses
Fiber production under adverse environmental factor and quality.
Invention content
Technical problem to be solved by the invention is to provide the methods for cultivating degeneration-resistant border new variety of plant.
In order to solve the above technical problems, the present invention provides a kind of DNA molecular, it is any shown for following (1)-(3)：
(1) DNA molecular shown in the 1st to the 2079th nucleotide in SEQ ID No.8；
(2) DNA molecular shown in the 15th to the 2073rd nucleotide in SEQ ID No.11；
(3) DNA molecular shown in SEQ ID No.11.
In order to solve the above technical problems, the present invention also provides destination gene expressions under a kind of up-regulation Adversity-stressed Plant
Method includes the following steps：Above-mentioned DNA molecular is imported in the plant that sets out, obtains genetically modified plants so that the transgenosis
Target gene in plant starts the transcription of the target gene using above-mentioned DNA molecular as promoter；With the plant that sets out
It compares, the destination gene expression up-regulation of the genetically modified plants；
The target gene is the endogenous gene or foreign gene of the plant that sets out.
In the above method, the environment stress is abiotic stress.
In any of the above-described method, the target gene is adversity gene.
In any of the above-described method, the adversity gene is the gene with resisting abiotic environment stress ability.
In any of the above-described method, the abiotic stress is low temperature stress, high-salt stress, the abscisic acid side of body
Urgent, drought stress and/or alkaline stress.
In order to solve the above technical problems, the application the present invention also provides above-mentioned DNA molecular in preparing promoter.
In order to solve the above technical problems, the present invention also provides above-mentioned DNA moleculars or any of the above-described method to make
Application in the plant of standby resistance enhancing.
In above application, the resistance is resisting abiotic environment stress ability.
In above application, the abiotic stress is low temperature stress, high-salt stress, the acid stress that falls off, drought stress
And/or alkaline stress.
In any of the above-described method or application, the plant is dicotyledon or monocotyledon, concretely
Crucifer, such as arabidopsis.
Promoter provided by the invention can be applied to influence the expression of target gene under adverse circumstance, especially to cultivating degeneration-resistant border
New variety of plant has important theory and realistic meaning.
Description of the drawings
Fig. 1 is expression water of 10424 genes of Ghi in the different tissues organ of cotton in seedling stage under non-adverse circumstance treatment conditions
Flat and expression of 10424 genes of Ghi in the whole young plant of cotton in seedling stage under adverse circumstance treatment conditions.
Fig. 2 is that the PCR of transgenic arabidopsis is identified.
The expression that Fig. 3 is GUS in variant histoorgan in transgenic arabidopsis.
Fig. 4 is the expression that promoter is induced rear gene under different adverse circumstance treatment conditions.
Specific implementation mode
Experimental method used in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Cotton C312 (Coker312) (Gossypium hirsutum L.) is in document " Zhu Y-N, Shi D-Q, Ruan
M-B,Zhang L-L,Meng Z-H,Liu J,Yang W-C(2013)Transcriptome Analysis Reveals
Crosstalk of Responsive Genes to Multiple Abiotic Stresses in Cotton
(Gossypium hirsutum L.).PLoS ONE 8(11):e80218.doi:10.1371/
It is disclosed in journal.pone.0080218 ", the public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research.
PZero back/blunt are TIANGEN Biotech's product, catalog number VT204-
03。
For the sequence of pPZP111-GUS-NOS as shown in SEQ ID No.12, the public can be from Chinese Academy of Sciences's heredity and development
Biological study is obtained.
Genome Walking Kit are TaKaRa products, catalog number 6108.
Arabidopsis Col-0 environmental (Arabidopsis thaliana L.) is in document " Xin-Ran Li, Hong-Ju
Li,Li Yuan,Man Liu,Dong-Qiao Shi,Jie Liu and Wei-Cai Yang.(2014)Arabidopsis
DAYU/ABERRANT PEROXISOME MORPHOLOGY9Is a Key Regulator of Peroxisome
Biogenesis and Plays Critical Roles during Pollen Maturation and Germination
in Planta.The Plant Cell,26:619-635. " it is disclosed in, the public can give birth to from Chinese Academy of Sciences's heredity and development
Wu Xue research institutes obtain.
0.1M phosphate buffers (pH7.0)：1mol/LNa2HPO4Aqueous solution take 5.77mL, 1mol/L NaH2PO4Water
Solution takes 4.23ml, and 100mL is settled to water.
10%SDS solution：90ml water is slightly heated, adds 10gSDS, stirring and dissolving, be added several drop concentrated hydrochloric acids adjusting pH to
7.2, then plus water is settled to 100mL.
0.5M EDTA(pH8.0)：18.61gNa is added in 80mL water2EDTA·2H2O, it is molten with NaOH tune pH to 8.0
After solution plus water is settled to 100mL.
GUS zyme extracts：0.1M phosphate buffers (pH7.0) take 50mL, 10%SDS solution to take 1mL, 0.5M EDTA
(pH8.0) take 2mL, Triton X-100 that 100 μ L, 100 μ L of beta -mercaptoethanol is taken to be settled to 100mL with water.
MUG substrates (4-methyl umbelliferone-D-Glucose aldehydic acid glycosides)：Claim 8.8mg MUG, is dissolved in 10mL GUS zyme extracts
In, it is configured to the working concentration of 2mmol/L.
Reaction terminating liquid (0.2mol/L Na2CO3Aqueous solution)：Claim 2.12g Na2CO3, with water constant volume to 100mL.
ABA in following embodiments is abscisic acid.
The identification of screening and the gene expression of embodiment 1, cotton adverse circumstance induced expression promoter
One, the processing of cotton seeds
By healthy, full cotton C312 (Coker312) (Gossypium hirsutum L.) seed in volume hundred
Divide in the ethanol water of content 75% and handle one minute, then pours out solution, 10% hydrogen peroxide dipping is added 2 hours, the phase
Between shake several times.Hydrogen peroxide is poured out, after being rinsed well the hydrogen peroxide of the surface of the seed with sterile purified water, in sterile purified water
Middle soaked overnight, it is ensured that whole process aseptically carries out.
Two, adverse circumstance is handled
The consistent seed that shows money or valuables one carries unintentionally after step 1 of learning from else's experience processing is planted on 1/2MS culture mediums, is placed in 25 DEG C, 12 hours
It is cultivated 5 days in the incubator of photoperiod, makes its germination, then move to progress adverse circumstance processing on corresponding adverse circumstance culture medium respectively,
Various adverse circumstance treatment conditions are as shown in table 1.
1 each group treatment conditions of table
In table 1, the 1/2MS culture mediums (pH=5.8) of the NaCl containing 200mM are made of solvent and solute；Solute is NaCl,
Solvent is 1/2MS culture mediums (pH=5.8), and NaCl is a concentration of in the 1/2MS culture mediums (pH=5.8) of the NaCl containing 200mM
200mM；1/2MS culture mediums (pH=5.8) containing 1 μM of ABA are made of solvent and solute；Solute is ABA, solvent 1/2MS
Culture medium (pH=5.8), a concentration of 1 μM in the 1/2MS culture mediums (pH=5.8) containing 1 μM of ABA of ABA；Contain 200mM
The 1/2MS culture mediums (pH=5.8) of mannitol are made of solvent and solute；Solute is mannitol, and solvent is 1/2MS culture mediums
(pH=5.8), a concentration of 200mM of the mannitol in the 1/2MS culture mediums (pH=5.8) containing 200mM mannitol.
Three, the total serum IgE of the seedling (whole young plant) of each processing group in table 1 is extracted using the LiCl precipitation method of improvement, DNA enzymatic disappears
The concentration and purity of total serum IgE after change pass through NanoDrop ND-1000spectrophotometer and agarose gel electrophoresis
It is analyzed, qualified total serum IgE is examined to be used for following experiments.
Four, the screening of adverse circumstance induced expression transcription factor.
Compared with the control group, the cotton seedling for analyzing remaining each processing group does harm to five kinds in low temperature, with high salt, ABA, arid, alkali
The expression of transcript profile under abiotic stress conditions changes (reference literature " Zhu Y-N, Shi D-Q, Ruan M-B, Zhang L-
L,Meng Z-H,Liu J,Yang W-C(2013).Transcriptome Analysis Reveals Crosstalk of
Responsive Genes to Multiple Abiotic Stresses in Cotton(Gossypium hirsutum
L.).PLoS ONE 8(11):e80218.doi:10.1371/journal.pone.0080218 "), it obtains a series of by five kinds
The candidate gene of adverse circumstance coinduction expression, as a result, it has been found that, under five kinds of abiotic stress treatment conditions, 10424 transcripts of Ghi (are compiled
One vitamin B1 synthetic gene of code) the up-regulated expression multiple highest in the transcript of common up-regulated expression.Therefore, Ghi
10424 genes become the crucial candidate gene of adverse circumstance induced expression isolation of promoter, up-regulation of the gene in more adverse circumstance chips
Expressing multiple, the results are shown in Table 2.
Up-regulated expression multiples of 2 Ghi 10424 of table in more adverse circumstance chips
Table 2 shows that Ghi 10424 can be expressed by ABA, low temperature, arid, with high salt, alkali height inducible up regulation, and be induced
Multiple is expressed at 10 times or more.Therefore, select the gene for the candidate gene of abiotic stress induced expression isolation of promoter.
Five, the identification of adverse circumstance induced expression transcription factor
Using quantitative fluorescent PCR method to the tissue expression specificity of candidate gene and its adverse circumstance induced expression characteristic into
Row identification, further determines that candidate gene.
The seedling of control group is taken, extracts the total serum IgE of its root, stem, leaf respectively, while extracting the whole strain of remaining each processing group
The total serum IgE of seedling, by each total serum IgE reverse transcription at cDNA.Then Real-Time Fluorescent Quantitative PCR Technique is utilized, using SuperReal
PreMix (SYBR Green) (TIANGEN Biotech's product, catalog number FP204), it is fixed in fluorescence
Detection adverse circumstance is answered on amount PCR instrument C1000TM Thermal Cycler (CFX96real-time system, Bio-Rad, USA)
Answer the expression of related gene.Using cotton ACTIN1 genes as internal reference, experiment is set to be repeated three times.
The real-time fluorescence quantitative PCR primer of internal reference ACTIN1：
GhiACTIN1 sense primers：5'-GTTTATGTGCGGGAAGTTAGG-3'；(SEQ ID No.1)
GhiACTIN1 downstream primers：5'-TAATGGTGGTGATACGGTGAA-3'.(SEQ ID No.2)
The real-time fluorescence quantitative PCR primer of 10424 genes of cotton Ghi：
10424 sense primers of Ghi：5'-CAGCCTGAGTTTGTTCTA-3'；(SEQ ID No.3)
10424 downstream primers of Ghi：5'-GTTCACATTCAATCCATCAA-3'.(SEQ ID No.4)
The method that data processing uses Comparative Ct, Ct values are the thresholding that fluorescence signal reaches setting in PCR pipe
When the recurring number that is undergone, Δ Ct=Ct (Ghi 10424)-Ct (GhiACTIN1), with 2-ΔCtValue weighs gene transcription level,
Under non-adverse circumstance treatment conditions in (control group) cotton different tissues organ at the expression of Ghi 10424 and five kinds of adverse circumstances
The expression of (five processing groups in addition to control group) cotton Ghi 10424 carries out analysis comparison under the conditions of reason.
Expression of 10424 genes of (control group) Ghi in the different tissues organ of cotton in seedling stage under non-adverse circumstance treatment conditions
It is horizontal as shown in Figure 1A.
Figure 1A shows expression quantity highests of the degeneration-resistant response gene Ghi 10424 in blade, takes second place in stem, the table in root
It is minimum up to measuring.
10424 genes of (five processing groups in addition to control group) Ghi are in the whole young plant of cotton in seedling stage under adverse circumstance treatment conditions
Expression it is as shown in Figure 1B.
Figure 1B shows compared with the control group, and under five kinds of adverse circumstance treatment conditions, 10424 genes of Ghi are in cotton seedling
Present conspicuousness up-regulated expression, wherein under drought condition up-regulated expression it is most.
The above result shows that 10424 genes of Ghi are the abiotic stress induced expressions of a surging expression in blade
Gene.
Embodiment 2, chromosome walking obtain the promoter sequence of 10424 upstream region of gene of Ghi
One, the design of specific primer
Following three specific primer is designed, design direction is the zone of ignorance direction for needing to expand, Ghi 10424SP2
Position should design in the inside of Ghi 10424SP1, Ghi 10424SP3 are located at the inside of Ghi 10424SP2.It is dyed
Body step is moved, the promoter sequence for obtaining the 10424 transcriptional domain upstreams Ghi.
Chromosome walking specific primer：
Ghi 10424SP1：5′-CCGGAGCCATATATCCACTAATGC-3′；(SEQ ID No.5)
Ghi 10424SP2：5′-TGAACTGAAACTAGAAGAGCGGTGC-3′；(SEQ ID No.6)
Ghi 10424SP3：5′-ACTGTCCGCTCCAGCAAAGATTC-3′.(SEQ ID No.7)
Two, the extraction of cotton genomic dna
The genomic DNA of cotton C312 (Coker312) blade without any processing is extracted with the CTAB methods of improvement.
Three, chromosome walking
Chromosome walking is operated by Genome Walking Kit, and specific method is：The genomic DNA extracted with step 2
As template, using the AP primer in kit as sense primer, Ghi 10424SP1 carry out first as downstream primer
PCR reactions are taken turns, first round pcr amplification product is obtained；Then template is used as after first round pcr amplification product being diluted 1000 times,
Using AP primer as sense primer, Ghi 10424SP2 carry out the second wheel PCR reactions, obtain the second wheel as downstream primer
Pcr amplification product；It is used as template after the second wheel pcr amplification product is diluted 1000 times again, is drawn using AP primer as upstream
Object, Ghi 10424SP3 carry out third round PCR reactions, obtain third round pcr amplification product as downstream primer.
Four, it takes the first round, the second wheel, each 20 μ L of third round pcr amplification product to carry out 1% agarose gel electrophoresis, cuts
Glue recycles clearly electrophoretic band, and each band is separately recovered and connect to obtain each recombinant plasmid with pZero back/blunt carriers,
The insetion sequence of each recombinant plasmid is sequenced and splices DNA fragmentation (such as SEQ ID for obtaining that a length is 2.121kb
Shown in No.8), it is compared with 10424 gene known arrays of cotton Ghi, as a result the DNA fragmentation of the 2.121kb contains
10424 gene cDNA sequences 5 ' of Ghi hold 33 nucleotide of code area, i.e., 5 ' end code areas to partly overlap, show remaining
Length be 2.088kb DNA fragmentation include the promoter sequences of 10424 genes of Ghi, which is named as Ghi
10424 promoters, in sequence such as SEQ ID No.8 from 5 ' ends shown in the 1st to the 2079th nucleotide.
Five, the sequence analysis of 10424 promoters of Ghi
Website PLACE (http are predicted by promoter cis-acting elements://www.dna.affrc.go.jp/
) and PlantCARE (http sigscan://bioinformatics.psb.ugent.be/webtools/p-lantcare/
Html/) relevant information software scans for the cis-acting elements in 10424 promoter sequences of Ghi of acquisition, predicts
And analysis, the results showed that, numerous homologous sequences that Eukaryotic cis element is known with oneself are contained in the promoter, in the sequence
Also include CAAT-box, ABRE, I-box, G-box, W-box, E-box, CACT- other than containing basic starting element
The functional element of the adverse circumstances inducible promoter such as box, GATA-box, WRKY, CBF.The result shows that 10424 promoters of Ghi can
There can be the induced activity of Multiple Factors.
The structure of embodiment 3, the clone of 10424 promoters of Ghi and transgene expression vector
One, according to the promoter sequence of the Ghi 10424 obtained, in conjunction with the transcription initiation site of Bioinformatics Prediction
And termination site design primer, the primer designed and synthesized are as follows：
Ghi 10424-F：5′-AACTGCAGGTCGACGGAACTCGAAAAGGCAACTATA-3′；(SEQ ID No.9)
(sequence 5 '-shown in underscoreCTGCAG- 3 ' be PstI digestion recognition sites, 5 '-GTCGAC- 3 ' be SalI digestions
Recognition site.)
Ghi 10424-R：5′-GCTCTAGAGCTTGAAGACAGTGAAGTGAGA-3′。(SEQ ID No.10)
(sequence 5 '-shown in underscoreTCTAGA- 3 ' be XbaI enzyme cutting recognition site)
Two, the genomic DNA for extracting the blade of the cotton of control group in embodiment 1, using it as template, with Ghi 10424-
F and Ghi 10424-R be primer, carry out PCR amplification, obtain pcr amplification product (as shown in SEQ ID No.11), by its with
PZero back/blunt carriers connect, and obtain recombinant plasmid, are named as pZero back/blunt-Ghi
10424promoter send recombinant plasmid to sequencing, as a result correctly.
15th to the 2073rd partial sequence for 10424 promoters of Ghi in SEQ ID No.11.
Three, DNA molecular shown in PstI and XbaI double digestions SEQ ID No.11, obtains genetic fragment；PstI and XbaI
Double digestion pPZP111-GUS-NOS obtains carrier large fragment；Genetic fragment is connect with carrier large fragment, obtains recombinant plasmid,
Be named as PGhi 10424, send recombinant plasmid PGhi 10424 to sequencing, as a result correctly, show PGhi 10424 be by
Sequence between PstI the and XbaI recognition sites of pPZP111-GUS-NOS replaces with DNA molecular shown in SEQ ID No.11,
The recombinant vector that remaining sequence of pPZP111-GUS-NOS remains unchanged.
Four, PGhi 10424 is converted into Agrobacterium tumefaciems GV3101, recombinational agrobacterium is obtained, by empty carrier pPZP111-
GUS-NOS converts Agrobacterium GV3101, obtains control Agrobacterium, and recombinational agrobacterium and control Agrobacterium are distinguished arabidopsis thaliana transformation
Col-0 is environmental, respectively obtains that T0 generations turn 10424 plasmid arabidopsis of PGhi and T0 generations turn pPZP111-GUS-NOS plasmid controls
Arabidopsis.
Five, in harvest T0 generations, turn 10424 plasmid arabidopsis of PGhi respectively and T0 generations turn pPZP111-GUS-NOS plasmid controls
The seed of arabidopsis after being dried 3 days in 37 DEG C of incubators, is placed in vernalization 2 days in 4 DEG C of refrigerators.In T0 generations, are turned into PGhi 10424
In plasmid arabidopsis and T0 generations, turn the seed of pPZP111-GUS-NOS plasmid control arabidopsis containing 50mg/mL kanamycins
Selective culture is carried out on MS screening and culturing mediums, respectively obtains that T1 generations turn 10424 plasmid arabidopsis positive seedlings of PGhi and T1 generations turn
PPZP111-GUS-NOS plasmid control arabidopsis positive seedlings.
Six, in extraction T1 generations, turn the genomic DNA of 10424 plasmid arabidopsis positive seedlings of PGhi, with Ghi 10424-F and Ghi
10424-R is primer, carries out PCR amplification, obtains pcr amplification product.And pPZP111-GUS-NOS plasmid controls are turned with T1 generations and are intended
The genomic DNA of southern mustard positive seedling is template, carries out above-mentioned experiment as negative control, using 10424 plasmids of PGhi as template,
Above-mentioned experiment is carried out as positive control.The electrophoresis result of pcr amplification product is as shown in Figure 2.
In Fig. 2,1 is the pcr amplification product of negative control, and 2 and 3 be positive for 10424 transgenic arabidopsis of PGhi with T1
The genomic DNA of seedling is the pcr amplification product of template, and 4 be the pcr amplification product of positive control, and M is DNA marker.
Fig. 2 shows genomic DNA and PGhi 10424 plasmids of the T1 for PGhi 10424 transgenic arabidopsis positive seedlings
There is purpose band, negative control is without purpose band in pcr amplification product.Further determine that the T1 generations that step 5 is screened turn
10424 plasmid arabidopsis positive seedlings of PGhi are to turn 10424 positives of PGhi, are referred to as T1 generations turn in the following embodiments
Gene arabidopsis.
GUS expression characterizations analysis under 10424 promoters of Ghi start in embodiment 4, transgenic arabidopsis
One, it dyes：Under the conditions of being protected from light, appropriate GUS dyeing liquors are added in the centrifuge tube of 2mL, T1 is intended for transgenosis
Southern mustard is dipped into the GUS dye liquors of centrifuge tube, vacuumizes 30min, then places it in 37 DEG C of incubators and places 6h.
Two, it rinses：Volumn concentration 30%, 50%, 70%, 100% ethanol water is used to impregnate rinsing T1 successively
For transgenic arabidopsis, impregnate 30 minutes every time.
Three, it decolourizes：It is eventually adding 100% ethyl alcohol and impregnates T1 for transgenic arabidopsis until decoloration completely.
Four, it records：It is photographed to record under stereomicroscope.
The expression of GUS is as shown in Figure 3 in variant histoorgan in transgenic arabidopsis.
In Fig. 3, A is that T1 is dyed for the GUS of transgenic arabidopsis seedling；B and D be T1 for transgenic arabidopsis true leaf and
Cotyledon GUS dyeing；C and E is that T1 is dyed for transgenic arabidopsis seedling root GUS；F and G is T1 for transgenic arabidopsis inflorescence, flower
Medicine, column cap GUS dyeing；H and I is T1 for transgenic arabidopsis silique and seed GUS dyeing.
Fig. 3 shows that GUS has expression in T1 is for the root of transgenic arabidopsis, stem, leaf, filigree, sepal, column cap, silique,
Wherein Seedling Stage surging expression in blade.
There is no any original papers that can start GUS expression in pPZP111-GUS-NOS.
Embodiment 5, the GUS quantitative analyses under different adverse circumstance treatment conditions after 10424 promoter induced expressions of Ghi
One, by two T1 for transgenic arabidopsis strain (being respectively designated as PGhi 10424-4 and PGhi 10424-7) point
Each group processing as shown in table 3 is not carried out, obtains each group seedling.
3 adverse circumstance treatment conditions of table
Two, the quantitative detection of gus gene
(1) respectively handle 16h, for 24 hours with 48h when, take each group seedling (quality be 100mg or so) of step 1, use liquid
Then its rapid freezing is ground the tissue of each processing group by nitrogen respectively by the way of liquid nitrogen grinding in mortar.If do not stood
It grinds, plant tissue that can be first by liquid nitrogen frozen processing is stored in -80 DEG C of refrigerators.
(2) the broken each tissue of grinding is gone to respectively in EP pipes, and the GUS zyme extracts of 1mL is added immediately, fully
Mixing.
(3) 12000rpm, 4 DEG C of centrifugation 5min, supernatant is gone in another EP pipes, is placed on ice for use.
(4) Bradford methods measure the albumen concentration in each supernatant.
(5) quantitative determination of GUS expressions
1, the 100 each supernatants of μ L are taken, the GUS zyme extracts of 400 μ L, 37 DEG C of preheatings is separately added into, adds 500 μ LMUG
Substrate, 37 DEG C of warm bath, obtains mixed reactant.Mixed reactant is taken respectively in 0min, 15min, 30min, 45min and 60min
200 μ L are added in 800 μ L reaction terminating liquids, obtain each end reaction object, and room temperature is kept in dark place.
2, measured with sepectrophotofluorometer at excitation wavelength 365nm, launch wavelength 455nm, when slit 10nm it is each most
The fluorescence intensity level of end reaction object different time points.
3, curve is made to the reaction time with fluorescence intensity level, finds out the fluorescence intensity change of unit interval.
4, with the fluorescence intensity change of unit interval divided by the protein content of participation reaction, 2 transfer-gen plants are found out respectively
The fluorescence intensity change of the protein units time of (PGhi 10424-4 and PGhi 10424-7) unit mass.As a result such as Fig. 4 institutes
Show.
In Fig. 4, CK represents control group；Cold represents low temperature group；NaCl represents with high salt group；ABA represents ABA groups；
Mannitol represents arid group；PH represents alkali group.
Fig. 4 shows to compare with control group, and PGhi 10424-4 and PGhi 10424-7 transgenic lines tie up to ABA, low temperature
(Cold), after the adverse environmental factors such as (NaCl) with high salt, arid (Mannitol), alkali (pH) are handled 24 hours, gus gene expression quantity is equal
It is induced up-regulated expression, and as the extension of processing time (such as 48 hours) fluorescence signal intensity rises rapidly, shows to carry
The gene of 10424 promoters of Ghi by a variety of adverse environmental factors induced expression, it is consistent with the testing result of genetic chip, thus
Illustrate, 10424 promoters of Ghi are the promoter of a typical more adverse circumstance induced expressions, therefore, 10424 promoters of Ghi
Clone has preferable foreground for plant to the improvement of compound stress response and application, such as can be as degeneration-resistant base
The promoter of cause is applied to influence the expression of target gene under adverse circumstance, especially to cultivating degeneration-resistant border new variety of plant with important
Theoretical and realistic meaning.
Claims (6)
1. a kind of DNA molecular, any shown for following (1)-(3)：
(1) DNA molecular shown in the 1st to the 2079th nucleotide in SEQ ID No.8；
(2) DNA molecular shown in the 15th to the 2073rd nucleotide in SEQ ID No.11；
(3) DNA molecular shown in SEQ ID No.11.
2. the method for destination gene expression, includes the following steps under a kind of up-regulation Adversity-stressed Plant：It will be described in claim 1
DNA molecular importing is set out in plant, obtains genetically modified plants so that the target gene in the genetically modified plants is with claim
DNA molecular described in 1 starts the transcription of the target gene as promoter；Compared with the plant that sets out, the transgenosis
The destination gene expression of plant raises；
The target gene is the endogenous gene or foreign gene of the plant that sets out；
The plant is arabidopsis or cotton；
The environment stress is abiotic stress；
It is low temperature stress, high-salt stress, the acid stress that falls off, drought stress and/or alkaline stress to state abiotic stress.
3. according to the method described in claim 2, it is characterized in that：The target gene is adversity gene.
4. according to the method described in claim 3, it is characterized in that：The adversity gene is with resisting abiotic environment stress energy
The gene of power.
5. application of the DNA molecular described in claim 1 in preparing promoter.
6. DNA molecular described in claim 1 or any methods of claim 2-4 are in the plant for preparing resistance enhancing
In application；
The plant is arabidopsis or cotton；
The resistance is resisting abiotic environment stress ability；
The abiotic stress is low temperature stress, high-salt stress, the acid stress that falls off, drought stress and/or alkaline stress.
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