CN105985957A - Stress inducible expression Ghi10424 promoter and applications thereof - Google Patents
Stress inducible expression Ghi10424 promoter and applications thereof Download PDFInfo
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Abstract
The invention discloses a stress inducible expression Ghi10424 promoter and applications of the tress inducible expression Ghi10424 promoter. The invention discloses DNA molecules shown in any one from (1), (2) and (3) as follows: (1) DNA molecules shown by the nucleotides from the 1st site to the 2079th site in the SEQ ID No.8; (2) DNA molecules shown by the nucleotides from the 15th site to the 2073rd site in the SEQ ID No.11; and (3) DNA molecules shown by the SEQ ID No.11. The promoter disclosed by the invention can be used for influencing the inducible expression of target genes under the stress, and especially has great theoretical and practical significances for cultivating the novel plant varieties capable of resisting compound type stress.
Description
Technical field
The present invention relates to a kind of promoter and application thereof；Particularly to a kind of adverse circumstance abduction delivering Ghi 10424 promoter and application thereof, belong to biological technical field.
Background technology
Low temperature, arid, the abiotic stress serious threat such as saline and alkaline the development of modern agriculture, these abiotic stress cause Cotton Gossypii to produce a series of disadvantageous change in terms of form, physiology, biochemistry and molecule, and then the serious yield affecting Cotton Gossypii and fiber quality.Traditional breeding method is difficult to solve in a short time the degeneration-resistant problem of Cotton Gossypii, engineered method have do not limited by reproduction isolation between kind, the advantage of the multiple merit of rapid polymerization.
The resistance reaction of plant is an extremely complex process, can be completed by a kind of approach, can also through number of ways mutually collaborative or suppression and jointly complete, including the plant plasma membrane serves perception to external source stimulus signal, by second message,second messenger, signal passed to corresponding stress response transcription factor, final transcription factor is specific binding with the cis acting element of stress response, thus adverse circumstance is made response by the expression activating downstream gene.Wherein, promoter sequence comprises many important cis acting element, the expression of plant gene had important function so that it is become the key link of expression regulation, either in gene functional research, still all there is in transgenic technology research highly important status.Therefore, the research of promoter structure, function and mechanism of action be will assist in understanding gene regulation pattern and signaling pathways, the defense mechanism understanding Cotton Gossypii in depth, the resistance that improves crop are had important meaning.
Along with various transgenic crops are in the popularization successively in the whole world, promoter important function in transgenic has been obtained for knowing together widely.People need the expression of different types of promoters driven adversity gene, to provide the solution of different transgene expression pattern.At Resistance Strain of Cotton against in research field, the promoter can expressed by rapid induction under adverse environmental factor exactly that people pay close attention to the most, thus quickly start the Cotton Gossypii resistance to various abiotic stresses, it is ensured that even improve Cotton Gossypii fiber production under adverse environmental factor and quality.
Summary of the invention
The technical problem to be solved is to provide the method cultivating degeneration-resistant border new variety of plant.
In order to solve above technical problem, the present invention provides a kind of DNA molecular, arbitrary 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 above technical problem, the present invention also provides for a kind of raising the method for destination gene expression under Adversity-stressed Plant, comprise the steps: to import above-mentioned DNA molecular to set out in plant, obtain transgenic plant so that the genes of interest in described transgenic plant starts transcribing of described genes of interest using above-mentioned DNA molecular as promoter；Compared with the described plant that sets out, the destination gene expression of described transgenic plant raises；
Described genes of interest sets out the endogenous gene of plant or exogenous gene described in being.
In said method, described environment stress is abiotic stress.
In any of the above-described described method, described genes of interest is adversity gene.
In any of the above-described described method, described adversity gene is the gene with resisting abiotic environment stress ability.
In any of the above-described described method, described abiotic stress be low temperature stress, high-salt stress, abscisic acid coerce, drought stress and/or alkaline stress.
In order to solve above technical problem, the present invention also provides for the application in preparation promoter of the above-mentioned DNA molecular.
In order to solve above technical problem, the present invention also provides for above-mentioned DNA molecular or the application in the plant that preparation resistance strengthens of any of the above-described described method.
In above-mentioned application, described resistance is resisting abiotic environment stress ability.
In above-mentioned application, described abiotic stress be low temperature stress, high-salt stress, abscisic acid coerce, drought stress and/or alkaline stress.
In any of the above-described described method or application, described plant is dicotyledon or monocotyledon, concretely crucifer, such as arabidopsis.
The promoter that the present invention provides can apply to affect the expression of genes of interest under adverse circumstance, especially cultivation degeneration-resistant border new variety of plant is had important theory and realistic meaning.
Accompanying drawing explanation
Fig. 1 be under non-adverse circumstance treatment conditions Ghi 10424 gene expression in the different tissues organ of cotton in seedling stage and under adverse circumstance treatment conditions Ghi 10424 gene expression in the whole young plant of cotton in seedling stage.
Fig. 2 is that the PCR of transgenic arabidopsis identifies.
Fig. 3 is the expression of GUS in variant histoorgan in transgenic arabidopsis.
Fig. 4 is the expression that under different adverse circumstance treatment conditions, promoter is induced rear gene.
Detailed description of the invention
Experimental technique used in following embodiment if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
nullCotton Gossypii C312 (Coker312) (Gossypium hirsutum L.) is at 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/journal.pone.0080218 " disclosed in mistake，The public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.
PZero back/blunt is TIANGEN Biotech's product, and catalog number is VT204-03.
The sequence of pPZP111-GUS-NOS is as shown in SEQ ID No.12, and the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.
Genome Walking Kit is TaKaRa product, and catalog number is 6108.
nullArabidopsis Col-0 environmental (Arabidopsis thaliana L.) is at 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. " disclosed in mistake，The public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.
0.1M phosphate buffer (pH7.0): 1mol/LNa2HPO4Aqueous solution take 5.77mL, 1mol/L NaH2PO4Aqueous solution take 4.23ml, be settled to 100mL with water.
10%SDS solution: somewhat heated by 90ml water, adds 10gSDS, stirring and dissolving, adds several concentrated hydrochloric acid regulation pH to 7.2, then adds water and be settled to 100mL.
0.5M EDTA (pH8.0): add 18.61gNa in 80mL water2EDTA·2H2O, adjusts pH to 8.0 with NaOH, adds water and be settled to 100mL after dissolving.
GUS zyme extract: 0.1M phosphate buffer (pH7.0) takes 50mL, 10%SDS solution and takes 1mL, 0.5M EDTA (pH8.0) and take 2mL, Triton X-100 and take 100 μ L, beta-mercaptoethanol 100 μ L, is settled to 100mL with water.
MUG substrate (4-methyl umbelliferone-D-Glucose aldehydic acid glycosides): claim 8.8mg MUG, be dissolved in 10mL GUS zyme extract, be 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 embodiment is abscisic acid.
Embodiment 1, the screening of Cotton Gossypii adverse circumstance abduction delivering promoter and the qualification of gene expression
One, the process of cotton seeds
The seed of healthy, full Cotton Gossypii C312 (Coker312) (Gossypium hirsutum L.) is processed one minute in the ethanol water of volumn concentration 75%, then solution is poured out, the hydrogen peroxide dipping of addition 10% 2 hours, period rocks several times.Pour out hydrogen peroxide, after the hydrogen peroxide of the surface of the seed being rinsed well with sterile purified water, soaked overnight in sterile purified water, it is ensured that whole process is aseptically carried out.
Two, adverse circumstance processes
The consistent seed that shows money or valuables one carries unintentionally after step one of learning from else's experience process is implanted in 1/2MS culture medium, it is placed in 25 DEG C, the incubator of 12 hour photoperiod is cultivated 5 days so that it is germinate, moving to carry out in corresponding adverse circumstance culture medium adverse circumstance process the most respectively, various adverse circumstance treatment conditions are as shown in table 1.
Treatment conditions respectively organized by table 1
In table 1, the 1/2MS culture medium (pH=5.8) containing 200mM NaCl is made up of solvent and solute；Solute is NaCl, solvent be 1/2MS culture medium (pH=5.8), the NaCl concentration in the 1/2MS culture medium (pH=5.8) containing 200mM NaCl be 200mM；1/2MS culture medium (pH=5.8) containing 1 μM of ABA is made up of solvent and solute；Solute is ABA, solvent be 1/2MS culture medium (pH=5.8), the ABA concentration in the 1/2MS culture medium (pH=5.8) containing 1 μM of ABA be 1 μM；1/2MS culture medium (pH=5.8) containing 200mM mannitol is made up of solvent and solute；Solute is mannitol, and solvent is 1/2MS culture medium (pH=5.8), and mannitol concentration in the 1/2MS culture medium (pH=5.8) containing 200mM mannitol is 200mM.
Three, the LiCl sedimentation method using improvement extract the total serum IgE of the seedling (whole young plant) of each process group in table 1, concentration and the purity of the total serum IgE after dnase digestion are analyzed by NanoDrop ND-1000spectrophotometer and agarose gel electrophoresis, check qualified total serum IgE for following experiment.
Four, the screening of adverse circumstance abduction delivering transcription factor.
nullCompared with matched group，Analyze the cotton seedling of remaining each process group at low temperature、High salt、ABA、Arid、Expression change (reference literature " the Zhu Y-N of the transcript profile under alkali five kinds of abiotic stress conditions of evil,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”)，Obtain a series of candidate gene expressed by five kinds of adverse circumstance coinductions，Found that，Under five kinds of abiotic stress treatment conditions，Up-regulated expression multiple is the highest in the transcript of common up-regulated expression for Ghi 10424 transcript (encoding a vitamin B1 synthetic gene).Therefore, Ghi 10424 gene becomes the crucial candidate gene of adverse circumstance abduction delivering isolation of promoter, and this gene up-regulated expression multiple result in many adverse circumstances chip is as shown in table 2.
Table 2 Ghi 10424 up-regulated expression multiple in many adverse circumstances chip
Table 2 shows, Ghi 10424 can be expressed by ABA, low temperature, arid, high salt, alkali height inducible up regulation, and abduction delivering multiple is all more than 10 times.Therefore, the candidate gene selecting this gene to be abiotic stress abduction delivering isolation of promoter.
Five, the qualification of adverse circumstance abduction delivering transcription factor
Tissue expression specificity and the adverse circumstance abduction delivering characteristic thereof of candidate gene are identified by the method utilizing quantitative fluorescent PCR, further determine that candidate gene.
Take the seedling of matched group, extract the total serum IgE of its root, stem, leaf respectively, extract the total serum IgE of the whole young plant of remaining each process group simultaneously, each total serum IgE reverse transcription is become cDNA.Then Real-Time Fluorescent Quantitative PCR Technique is utilized, use SuperReal PreMix (SYBR Green) (TIANGEN Biotech's product, catalog number is FP204), at quantitative real time PCR Instrument C1000TM Thermal Cycler (CFX96real-time system, Bio-Rad, USA) the upper expression detecting Stress response related gene.With Cotton Gossypii ACTIN1 gene as internal reference, experiment sets three repetitions.
The real-time fluorescence quantitative PCR primer of internal reference ACTIN1:
GhiACTIN1 forward primer: 5'-GTTTATGTGCGGGAAGTTAGG-3'；(SEQ ID No.1)
GhiACTIN1 downstream primer: 5'-TAATGGTGGTGATACGGTGAA-3'.(SEQ ID No.2)
The real-time fluorescence quantitative PCR primer of Cotton Gossypii Ghi 10424 gene:
Ghi 10424 forward primer: 5'-CAGCCTGAGTTTGTTCTA-3'；(SEQ ID No.3)
Ghi 10424 downstream primer: 5'-GTTCACATTCAATCCATCAA-3'.(SEQ ID No.4)
Data process the method using Comparative Ct, and the period that Ct value is experienced when reaching, by fluorescence signal in PCR pipe, the thresholding set, Δ Ct=Ct (Ghi 10424)-Ct (GhiACTIN1), with 2- Δ CtValue weigh gene transcription level, in (matched group) Cotton Gossypii different tissues organ under non-adverse circumstance treatment conditions Ghi 10424 expression and under five kinds of adverse circumstance treatment conditions the expression of (five process groups in addition to matched group) Cotton Gossypii Ghi 10424 be analyzed comparing.
Under non-adverse circumstance treatment conditions, (matched group) Ghi 10424 gene expression in the different tissues organ of cotton in seedling stage is as shown in Figure 1A.
Figure 1A shows, the degeneration-resistant response gene Ghi 10424 expression in blade is the highest, takes second place in stem, and in root, expression is minimum.
Under adverse circumstance treatment conditions (five process groups in addition to matched group) Ghi 10424 gene expression in the whole young plant of cotton in seedling stage is as shown in Figure 1B.
Figure 1B shows, compared with matched group, under five kinds of adverse circumstance treatment conditions, Ghi 10424 gene all presents the up-regulated expression of significance in cotton seedling, wherein up-regulated most under drought condition.
Result above shows, Ghi 10424 gene is a surging abiotic stress expression profile expressed in blade.
Embodiment 2, chromosome walking obtain the promoter sequence of Ghi 10424 upstream region of gene
One, the design of specific primer
Design following three specific primer, design direction is the zone of ignorance direction needing amplification, and the inner side at Ghi 10424SP1 should be designed in the position of Ghi 10424SP2, and Ghi 10424SP3 is positioned at the inner side of Ghi 10424SP2.Carry out chromosome walking, for obtaining the promoter sequence of Ghi 10424 transcriptional domain upstream.
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
CTAB method with improvement extracts the genomic DNA of Cotton Gossypii C312 (Coker312) blade without any process.
Three, chromosome walking
Chromosome walking presses Genome Walking Kit operation, method particularly includes: using step 2 extract genomic DNA as template, using the AP primer in test kit as forward primer, Ghi 10424SP1 is as downstream primer, carry out the reaction of first round PCR, obtain first round pcr amplification product；Then as template after first round pcr amplification product being diluted 1000 times, using AP primer as forward primer, Ghi 10424SP2, as downstream primer, carries out second and takes turns PCR reaction, obtain second and take turns pcr amplification product；Taking turns second after pcr amplification product dilutes 1000 times as template, using AP primer as forward primer, Ghi 10424SP3, as downstream primer, carries out the reaction of third round PCR, obtains third round pcr amplification product again.
nullFour、Take the first round、Second takes turns、The each 20 μ L of third round pcr amplification product carry out the agarose gel electrophoresis of 1%，Cut glue and reclaim electrophoretic band clearly，It is separately recovered each band to be connected with pZero back/blunt carrier and obtain each recombiant plasmid，Carry out the insertion sequence of each recombiant plasmid checking order and splice the DNA fragmentation (as shown in SEQ ID No.8) obtaining an a length of 2.121kb，It is compared with Cotton Gossypii Ghi 10424 gene known array，The DNA fragmentation of this 2.121kb of result contains 33 nucleotide of Ghi 10424 gene cDNA sequence 5 ' end coding region，I.e. 5 ' end coding regions partly overlap，Show the promoter sequence that the DNA fragmentation of remaining a length of 2.088kb comprises Ghi 10424 gene，By named for this promoter Ghi 10424 promoter，Sequence is as shown in the 1st to the 2079th nucleotide from 5 ' ends in SEQ ID No.8.
Five, the sequence analysis of Ghi 10424 promoter
nullBy promoter cis acting element prediction website PLACE (http://www.dna.affrc.go.jp/sigscan) and PlantCARE (http://bioinformatics.psb.ugent.be/webtools/p-lantcare/html/) relevant information software, the cis acting element in Ghi 10424 promoter sequence to obtaining scans for、Prediction and analysis，Result shows，Containing numerous homologous sequences knowing Eukaryotic cis element with oneself in this promoter，In this sequence in addition to containing basic starting element，Also comprise CAAT-box、ABRE、I-box、G-box、W-box、E-box、CACT-box、GATA-box、WRKY、The functional element of the adverse circumstance inducible promoters such as CBF.Result shows, Ghi 10424 promoter is likely to be of the induced activity of Multiple Factors.
Embodiment 3, the clone of Ghi 10424 promoter and the structure of transgene expression vector
One, according to the promoter sequence of the Ghi 10424 obtained, in conjunction with transcriptional start site and the termination site design primer of Bioinformatics Prediction, the primer designed and synthesized is as follows:
Ghi 10424-F:5 '-AACTGCAGGTCGACGGAACTCGAAAAGGCAACTATA-3′；(SEQ ID No.9)
(sequence 5 ' shown in underscore-CTGCAG-3 ' it is PstI enzyme action recognition site, 5 '-GTCGAC-3 ' it is SalI enzyme action recognition site.)
Ghi 10424-R:5 '-GCTCTAGAGCTTGAAGACAGTGAAGTGAGA-3′。(SEQ ID No.10)
(sequence 5 ' shown in underscore-TCTAGA-3 ' it is XbaI enzyme cutting recognition site)
Two, the genomic DNA of the blade of the Cotton Gossypii of matched group in embodiment 1 is extracted, with it as template, with Ghi 10424-F and Ghi 10424-R as primer, carry out PCR amplification, obtain pcr amplification product (as shown in SEQ ID No.11), it is connected with pZero back/blunt carrier, obtain recombiant plasmid, by its named pZero back/blunt-Ghi 10424promoter, recombiant plasmid sending order-checking, result is correct.
In SEQ ID No.11 the 15th to the partial sequence that the 2073rd is Ghi 10424 promoter.
Three, the DNA molecular shown in PstI and XbaI double digestion SEQ ID No.11, obtains genetic fragment；PstI and XbaI double digestion pPZP111-GUS-NOS, obtains carrier large fragment；Genetic fragment is connected with carrier large fragment, obtain recombiant plasmid, by its named PGhi 10424, order-checking is sent by recombiant plasmid PGhi 10424, result is correct, show PGhi 10424 be by PstI and the XbaI recognition site of pPZP111-GUS-NOS between sequence replace with the DNA molecular shown in SEQ ID No.11, remaining sequence of pPZP111-GUS-NOS keeps the constant recombinant vector obtained.
Four, PGhi 10424 is converted Agrobacterium tumefaciems GV3101, obtain recombinational agrobacterium, empty carrier pPZP111-GUS-NOS is converted Agrobacterium GV3101, obtain compareing Agrobacterium, recombinational agrobacterium is environmental with comparison Agrobacterium arabidopsis thaliana transformation Col-0 respectively, respectively obtain T0 for turning PGhi 10424 plasmid arabidopsis and T0 for turning pPZP111-GUS-NOS plasmid control arabidopsis.
Five, in results T0 generation, turns PGhi 10424 plasmid arabidopsis and T0 generation turns the seed of pPZP111-GUS-NOS plasmid control arabidopsis respectively, after drying 3 days, is placed in vernalization 2 days in 4 DEG C of refrigerators in 37 DEG C of couveuses.In T0 generation, is turned PGhi 10424 plasmid arabidopsis and T0 and in the MS screening culture medium containing 50mg/mL kanamycin, carries out selectivity cultivation for the seed turning pPZP111-GUS-NOS plasmid control arabidopsis, respectively obtain T1 for turning PGhi 10424 plasmid arabidopsis positive Seedling and T1 for turning pPZP111-GUS-NOS plasmid control arabidopsis positive Seedling.
Six, in extraction T1 generation, turns the genomic DNA of PGhi 10424 plasmid arabidopsis positive Seedling, with Ghi 10424-F and Ghi 10424-R as primer, carries out PCR amplification, obtains pcr amplification product.And turn the genomic DNA of pPZP111-GUS-NOS plasmid control arabidopsis positive Seedling as template with T1 generation, carry out above-mentioned experiment as negative control, with PGhi 10424 plasmid as template, carry out above-mentioned experiment 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 is that 4 is the pcr amplification product of positive control, and M is DNA marker for pcr amplification product as template of the genomic DNA of PGhi 10424 transgenic arabidopsis positive Seedling with T1.
Fig. 2 shows, T1 has purpose band in the genomic DNA of PGhi 10424 transgenic arabidopsis positive Seedling and the pcr amplification product of PGhi 10424 plasmid, and negative control is without purpose band.Further determine that the T1 generation that step 5 screening obtains turns PGhi 10424 plasmid arabidopsis positive Seedling positive for turning PGhi 10424, be the most all referred to as T1 for transgenic arabidopsis.
GUS expression characterization under Ghi 10424 promoter starts in embodiment 4, transgenic arabidopsis is analyzed
One, dyeing: under the conditions of lucifuge, adds appropriate GUS dyeing liquor in the centrifuge tube of 2mL, is dipped in the GUS dye liquor of centrifuge tube for transgenic arabidopsis by T1, evacuation 30min, is then placed in 37 DEG C of couveuses placement 6h.
Two, rinsing: successively with volumn concentration 30%, the ethanol water immersion rinsing T1 of 50%, 70%, 100%, for transgenic arabidopsis, soaks 30 minutes every time.
Three, decolouring: be eventually adding 100% soak with ethanol T1 for transgenic arabidopsis until decolouring completely.
Four, record: Taking Pictures recording under stereomicroscope.
In transgenic arabidopsis, in variant histoorgan, the expression of GUS is as shown in Figure 3.
In Fig. 3, A is the T1 GUS dyeing for transgenic arabidopsis seedling；B and D is that T1 dyes for transgenic arabidopsis true leaf and cotyledon GUS；C and E is that T1 dyes for transgenic arabidopsis seedling root GUS；F and G is that T1 is for transgenic arabidopsis inflorescence, flower pesticide, stigma GUS dyeing；H and I is that T1 dyes for transgenic arabidopsis angle fruit and seed GUS.
Fig. 3 shows, GUS has expression in T1 is for the root of transgenic arabidopsis, stem, leaf, filigree, sepal, stigma, angle fruit, and wherein Seedling Stage is surging in blade expresses.
PPZP111-GUS-NOS does not exist any original paper that can start GUS expression.
Embodiment 5, GUS quantitative analysis after Ghi 10424 promoter abduction delivering under different adverse circumstance treatment conditions
One, two T1 are carried out each group of process as shown in table 3 respectively for transgenic arabidopsis strain (being respectively designated as PGhi 10424-4 and PGhi 10424-7), obtain each group of seedling.
Table 3 adverse circumstance treatment conditions
Two, the detection by quantitative of gus gene
(1) respectively when processing 16h, 24h and 48h, take step one respectively organizes seedling (quality is about 100mg), with liquid nitrogen by its IQF, then uses the mode of liquid nitrogen grinding to grind the tissue of each process group in mortar respectively.If ground the most immediately, first the plant tissue that liquid nitrogen freezing processes can be stored in-80 DEG C of refrigerators.
(2) forward to grinding broken each tissue respectively in EP pipe, and add the GUS zyme extract of 1mL immediately, fully mix.
(3) 12000rpm, 4 DEG C of centrifugal 5min, go to supernatant, in another EP pipe, place stand-by on ice.
(4) protein concentration during Bradford method measures each supernatant.
(5) quantitative determination of GUS expression
1, take 100 each supernatants of μ L, be separately added into the GUS zyme extract of 400 μ L 37 DEG C preheating, add 500 μ LMUG substrates, 37 DEG C of temperature baths, obtain mixed reactant.Taking mixed reactant 200 μ L respectively at 0min, 15min, 30min, 45min and 60min to join in 800 μ L reaction terminating liquids, obtain each end reaction thing, room temperature keeps in Dark Place.
2, with spectrofluorophotometer under excitation wavelength 365nm, transmitting wavelength 455nm, the fluorescence intensity level of each end reaction thing different time points during slit 10nm, is measured.
3, with fluorescence intensity level, the response time is made curve, obtain the fluorescence intensity change of unit interval.
4, by the fluorescence intensity change of unit interval divided by participating in the protein content reacted, the fluorescence intensity change of the protein units time of 2 transfer-gen plants (PGhi 10424-4 and PGhi 10424-7) unit mass is obtained respectively.Result is as shown in Figure 4.
In Fig. 4, CK represents matched group；Cold represents low temperature group；NaCl represents high salt group；ABA represents ABA group；Mannitol represents arid group；PH represents alkali group.
nullFig. 4 shows，Compare with matched group，PGhi 10424-4 and PGhi 10424-7 transgenic line tie up to ABA、Low temperature (Cold)、High salt (NaCl)、Arid (Mannitol)、After the adverse environmental factors such as alkali (pH) process 24 hours，Gus gene expression is all induced up-regulated expression，And along with prolongation (such as the 48 hours) fluorescence signal intensity of the time of process rises rapidly，Show the abduction delivering by multiple adverse environmental factor of the gene with Ghi 10424 promoter，Consistent with the testing result of gene chip，Thus illustrate，Ghi 10424 promoter is the promoter of typical many adverse circumstances abduction delivering，Therefore，The clone of Ghi 10424 promoter has preferable prospect for plant to improvement and the application of compound stress response，Such as can be applied to affect the expression of genes of interest under adverse circumstance as the promoter of adversity gene，Especially cultivation degeneration-resistant border new variety of plant had important theory and realistic meaning.
Claims (10)
1. a DNA molecular, arbitrary 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. raise a method for destination gene expression under Adversity-stressed Plant, comprise the steps: claim 1
Described DNA molecular imports and sets out in plant, obtains transgenic plant so that the purpose base in described transgenic plant
Because starting transcribing of described genes of interest using the DNA molecular described in claim 1 as promoter；Plant with described setting out
Thing is compared, and the destination gene expression of described transgenic plant raises；
Described genes of interest sets out the endogenous gene of plant or exogenous gene described in being.
Method the most according to claim 2, it is characterised in that: described environment stress is abiotic stress.
The most according to the method in claim 2 or 3, it is characterised in that: described genes of interest is adversity gene.
5. according to the arbitrary described method of claim 2-4, it is characterised in that: described adversity gene is anti-non-for having
Biotic stress coerces the gene of ability.
6. according to the arbitrary described method of claim 2-5, it is characterised in that: described abiotic stress is low
Temperature is coerced, high-salt stress, abscisic acid are coerced, drought stress and/or alkaline stress.
7. the application in preparation promoter of the DNA molecular described in claim 1.
8. DNA molecular or the arbitrary described method of claim 2-6 described in claim 1 increase in preparation resistance
The strong application in plant.
Application the most according to claim 8, it is characterised in that: described resistance is resisting abiotic environment stress
Ability.
Application the most according to claim 9, it is characterised in that: described abiotic stress be low temperature stress,
High-salt stress, abscisic acid are coerced, drought stress and/or alkaline stress.
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| CN112391331A (en) * | 2020-11-12 | 2021-02-23 | 江南大学 | Recombinant escherichia coli for overexpression of GatA gene and application thereof |
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| MUZNA ZAHUR 等: "Functional analysis of cotton small heat shock protein promoter region in response to abiotic stresses in tobacco using Agrobacterium-mediated transient assay", 《MOLECULAR BIOLOGY REPORTS》 * |
| 曾小林 等: "启动子在棉花基因工程中的应用与研发", 《分子植物育种》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107815452A (en) * | 2017-12-06 | 2018-03-20 | 新疆农垦科学院 | A kind of specific expressed promoter of plant leaf blade and its application |
| CN112391331A (en) * | 2020-11-12 | 2021-02-23 | 江南大学 | Recombinant escherichia coli for overexpression of GatA gene and application thereof |
| CN112391331B (en) * | 2020-11-12 | 2022-09-27 | 江南大学 | Recombinant escherichia coli for overexpression of GatA gene and application thereof |
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| Publication number | Publication date |
|---|---|
| CN105985957B (en) | 2018-08-31 |
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