CN102757966A - Promoter and application thereof - Google Patents
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- CN102757966A CN102757966A CN2012102619233A CN201210261923A CN102757966A CN 102757966 A CN102757966 A CN 102757966A CN 2012102619233 A CN2012102619233 A CN 2012102619233A CN 201210261923 A CN201210261923 A CN 201210261923A CN 102757966 A CN102757966 A CN 102757966A
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Abstract
The invention discloses a promoter and an application of the promoter. The promoter has 1) a DNA sequence as shown in a sequence 1 in a sequence table, 2) over 90% homology with a DNA sequence limited in the sequence 1 in the sequence table, and a DNA sequence with same functions; and 3) a nucleotide sequence capable of hybridizing with the DNA sequence limited in the sequence 1 in the sequence table under a highly strict condition. The promoter disclosed by the invention can be induced according to specific expression regulation and under various hormones, such as growth hormone, ethephon precursor 1-aminocyclopropane-1-carboxylic acid, cytokinin, salicylic acid, abscisic acid and gibberellin GA; and moreover, the promoter can be subject to stress induction under an abiotic environment, such as drought stress, high-temperature stress (37 DEG C), low-temperature stress (4 DEG C) or mechanical damage.
Description
Technical field
The present invention relates to a kind of promotor and application thereof, particularly a kind of promotor and application thereof that derives from Para rubber tree.
Background technology
In higher plant, sucrose is photosynthetic primary product.By the source in the long-distance transportation process in storehouse; Sucrose is main or even unique form (Williams, L.E., the et al. as carbohydrate transport in the plant materials; Sugar transporters in higher plants-a diversity of roles and complex regulation.Trends Plant Sci; 2000,5,283-290; Sylvie L., et al.The dual function of sugar carriers:transport and sugar sensing.Plant Cell, 1999,11:707-726).Sucrose molecules turnover phloem sieve element carries out through two kinds of different approach; Be synplasm approach and apoplast approach (Buchanan B.B.; Et al.; Biochemistry and Molecular Biology of Plants.Rockville:American Society of Plant Physiologists, 2000,748-776).Because in the crop of many types, the apoplast approach occupies extremely important status, the sucrose transporter that film transports and the distribution in plant need depend on the film of striding of sucrose mediates; Sucrose transporter has the important physical effect as the peculiar one type of carrier proteins of plant in the distribution of sucrose, mainly be at sucrose turnover phloem; The sucrose of storehouse tissue is supplied with; The storage of sucrose, (Gahrtz M., et al. play a significant role in the various physiological processes such as sucrose transhipment regulation and control and other small-molecule substance transhipment; A phloem-specific sucrose-H+symporter from Plantago major L.supports the model of apoplastic phloem loading Plant J; 1994,6 (5), 697-706; Truernit E.; Et al.; The promoter of the Arabidopsis thaliana SUC2sucrose-H+ symporter gene directs expression of β-glucuronidase to the phloem:evidence for phloem loading and unloading by SUC2.Planta; 1995,196:564-570; K ü hn C., Companion cell-specific inhibition of the potato sucrose transporter SUT1.Plant Cell Environ, 1996,19,1115-1123; B ü rkle L., et al., The H+-sucrose cotransporter NtSUT1 is essential for sugar expurt from tobacco leaves.Plant Physiol, 1988,118,59-68; Hackel A, et al., Sucrose transporter LeSUT1 and LeSUT2 inhibition affects tomato fruit development in different ways.The Plant Journal, 2006,45,180 – 192; Gabriel-Neumann E; Et al.; Constitutive overexpression of the sucrose transporter SoSUT1 in potato plants increases arbuscular mycorrhiza fungal root colonization under high, but not under low, soil phosphorus availability.J Plant Physiol; 2011,168 (9): 911-9; Siahpoosha M.R., et al., Modification of OsSUT1 gene expression modulates the salt response of rice Oryza sativa cv.Taipei 309.Plant Science 2011).Some experimental results show that sucrose transporter also participated in abiotic stress and biological response (the Sakr S. that coerces; Et al.; Cutting; Ageing and expression of plant membrane transporters.Biochim.Biophys.Acta, 1997,1330:207-216; Meyer S., AtSUC3, a gene encoding a new Arabidopsis sucrose transporter; Is expressed in cells adjacent to the vascular tissue and in a carpel cell layer.Plant J, 2000,24:869-882); And regulation and control (Barker L., etal., the SUT2 of invertase signal induction and transport efficacy; A putative sucrose sensor in sieve elements.Plant Cell; 2000,12,1153-1164; Schulze W., et al., Function of the cytosolic N-terminus of sucrose transporter AtSUT2 in substrate affinity.FEBS Lett, 2000,485:189-194; Reinders, A., Schulze; W.; Et al., Protein – protein interactions between sucrose transporters of different affinities colocalized in the same enucleate sieve element.Plant Cell, 2002; 14,1567 – 1577).
Para rubber tree is a kind of tropical tree species of in world economy and military developments, all playing an important role, and the tree elastomer of its production is a kind of important industrial raw material and strategic materials.The biosynthesizing of tree elastomer is to be raw material with sucrose, is that carry out the processing site with the latex dust, and the supply capacity of sucrose and rubber output are closely related in the latex dust.In early days; People such as Bouteau prove first on the protoplast membrane of latex dust, to exist through isotopic labeling and electrophysiologic testing and participate in sugar-proton symport system (sugar-H+symport system) (Bouteau F. that latex dust sugar absorbs; Evidence of multiple sugar uptake across the plasma membrane of lacticifer protoplasts from Hevea.Bioelectrochem Bioenerg; 1999,48 (1): 135-139).
Summary of the invention
The purpose of this invention is to provide a kind of plant promoter and application thereof.This promotor has the tissue specific expression characteristic and can be used to cultivate transgenic plant and control expression of exogenous gene specificity and expression amount by multiple factor abduction delivering.
Promotor provided by the present invention derives from the Para rubber tree (Hevea brasiliensis) of Euphorbiaceae genus hevea, and name is called PrHb3, can be one of following nucleotide sequence:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and have the dna sequence dna of promoter function;
The nucleotide sequence of the dna sequence dna hybridization that 3) under the rigorous condition of height, can limit with sequence in the sequence table 1.
The rigorous condition of above-mentioned height can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, under 65 ° of C, hybridize and wash film.
Wherein, sequence 1 is made up of 2096 deoxynucleotides in the sequence table.
5 of sequence 1 ' end 49-99 position Nucleotide in sequence table; 5 of sequence 1 ' end 707-757 position Nucleotide; 5 of sequence 1 ' end 974-1024 position nuclear; There is possible basic promoter region in 5 of sequence 1 ' Nucleotide place, end 2010-2060 position, contains plant promoter TATA box nucleus, and particularly 974-1024 basis promoter region gets score value near 1.0.Also contain hormone in this sequence, coerce, and other functional elements as shown in table 2.
The expression cassette, recombinant expression vector, transgenic cell line and the host bacterium that contain above-mentioned promotor all belong to protection scope of the present invention.
In said expression cassette, the downstream syndeton gene of said plant promoter, or regulatory gene; Or the inverted defined gene of structure gene or regulatory gene; The little RNA that perhaps can disturb native gene to express is used for the Drive Structure gene, or regulatory gene; Or the inverted defined gene of structure gene or regulatory gene, the expression of the little RNA of perhaps natural little RNA or synthetic.
Said recombinant expression vector is above-mentioned expression cassette and plasmid, virus or the constructed recombinant vectors of vector expression vector; Said recombinant expression vector is the reorganization plant expression vector; Said recombinant plant expression vector contain above-mentioned expression cassette and can with described expression cassette pass on get into plant host cell, tissue or organ and offspring thereof and can or convenient at least described expression cassette be incorporated in host's the genome, it includes but not limited to binary vector, closes carrier altogether.
Above-mentioned recombinant expression vector can be through using conventional biological method transformed plant cells or tissue or organs such as Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity led, agriculture bacillus mediated or particle gun, obtain transgenic plant cells or tissue or organ and differentiation, regenerated whole plant and clone thereof or generation thus thereafter.
Promotor of the present invention exist a large amount of maybe be relevant with hormone response, stress response relevant (biology is coerced and abiotic stress), light reply relevant, grow relevant, sugar and reply relevant cis-acting elements, the functional element that wherein the different expression regulation of hormone response and Gent is relevant is the most extensive.Make up the GUS fusion expression vector of this promotor, it is carried out functional verification, find that this promotor activity in root is the highest, find that simultaneously this promoter activity receives multiple hormone and coerces adjusting through transgenic arabidopsis.
In order to verify the function of this promotor, made up the GUS fusion expression vector, and it has been carried out promotor of the present invention carried out the Arabidopis thaliana transgenic analysis, confirmed the activity of this promotor, this promotor is different expression of Gent and inducible promoter.
Experiment showed, that this promotor is different expression regulation of Gent and multiple hormone induction promotor, like ethrel precursor 1-amino-cyclopropane-1-carboxylic acid (ACC), phytokinin, Whitfield's ointment, dormin, Plant hormones regulators,gibberellins; And this promotor can be by the abiotic environment stress-inducing, like high temperature stress (37 ℃), low temperature stress (4 ℃) or mechanical wounding.
In sum, this promotor can instruct foreign gene specific spatial and temporal expression in the development of plants process, and it can not only make the expression product of goal gene accumulate at certain space, increases regional expression amount, avoids the waste of biological nutrition.Therefore, be widely used in other plant and the microbial project of this promotor beyond the rubber tree.
Description of drawings
Fig. 1. the statistical study of hevea brasiliensis transporter gene promotor cis-acting elements.
Fig. 2. transgenic arabidopsis positive plant (changeing pPHb3-GUS Arabidopis thaliana positive plant) tissue staining analysis; A: the coloration result B of Arabidopis thaliana positive plant seedling phase and each tissue thereof: the coloration result of each tissue of transgenic arabidopsis positive plant ripening stage.
Fig. 3. GUS quantitative fluorescence analysis in each tissue of transgenic arabidopsis positive plant, wherein, in the X-coordinate, 1-6 is followed successively by root, lotus throne leaf, stem, stem leaf, seed, the flower of PrHb3 promotor transgenic arabidopsis (changeing pPHb3-GUS Arabidopis thaliana positive plant); 7-12 is followed successively by changes control plasmid PCK Arabidopis thaliana root, lotus throne leaf, stem, stem leaf, seed, flower.
Fig. 4. the ethrel precursor is to PrHb3 promotor transgenic arabidopsis (changeing pPHb3-GUS Arabidopis thaliana positive plant) GUS expression activity quantitative fluorescence analysis;
Fig. 5. phytokinin is to PrHb3 promotor transgenic arabidopsis (changeing pPHb3-GUS Arabidopis thaliana positive plant) GUS expression activity quantitative fluorescence analysis;
Fig. 6. Whitfield's ointment is to PrHb3 promotor transgenic arabidopsis GUS (changeing pPHb3-GUS Arabidopis thaliana positive plant) expression activity quantitative fluorescence analysis;
Fig. 7. dormin is to PrHb3 promotor transgenic arabidopsis GUS (changeing pPHb3-GUS Arabidopis thaliana positive plant) expression activity quantitative fluorescence analysis;
Fig. 8. Plant hormones regulators,gibberellins is to PrHb3 promotor transgenic arabidopsis GUS (changeing pPHb3-GUS Arabidopis thaliana positive plant) expression activity quantitative fluorescence analysis;
Fig. 9. growth hormone is to PrHb3 promotor transgenic arabidopsis GUS (changeing pPHb3-GUS Arabidopis thaliana positive plant) expression activity quantitative fluorescence analysis;
Figure 10. high temperature stress is to PrHb3 promotor transgenic arabidopsis GUS (changeing pPHb3-GUS Arabidopis thaliana positive plant) expression activity quantitative fluorescence analysis;
Figure 11. low temperature stress is to PrHb3 promotor transgenic arabidopsis GUS (changeing pPHb3-GUS Arabidopis thaliana positive plant) expression activity quantitative fluorescence analysis;
Figure 12. injury is coerced PrHb3 promotor transgenic arabidopsis (changeing pPHb3-GUS Arabidopis thaliana positive plant) GUS expression activity quantitative fluorescence analysis;
Figure 13. drought stress is to PrHb3 promotor transgenic arabidopsis (changeing pPHb3-GUS Arabidopis thaliana positive plant) GUS expression activity quantitative fluorescence analysis.
Embodiment
Method among the following embodiment if no special instructions, is ordinary method.
One. make up rubber tree promotor clone library
In order better to make up rubber tree promotor clone library, to the Genome Walker of Clontech
TMJoint long-chain among the Universal Kit is revised, and short chain is not made an amendment, and the short chain of joint and long-chain are dissolved in TE solution respectively; Concentration is 100 μ mol/l; Long-chain and short chain 1:1 mix, sex change annealing (95 ℃ of 5min, 37 ℃ of 10min; Room temperature is placed 30min) form joint, joint concentration is 50 μ mol/l.The blade that grinds 7-33-97 with rubber tree strain heat is the material extraction genomic dna, and method adopts the big improved CTAB method of people that waits in Anze.Choose 25 kinds of restriction enzymes rubber tree heat is ground 7-33-97 (Rubber Institute, Chinese Academy of Agricultural Science's cultivation; Rubber Institute, Chinese Academy of Agricultural Science has seedling to sell for a long time) genomic gene carries out complete degestion, and wherein to cut product be that the restriction enzyme of sticky end comprises BamH I, Bcl I, Bcu I, Bgl II, BspT I, EcoR I, Hind III, Kpn I, Nco I, Nde I, Sty I and Xba I to enzyme; Product is flat terminal BseJ I, Bst1107I, Dra I, Eco105I, Eco147I, Eco72I, EcoR V, HincII, KspA I, Pvu II, Sca I, Ssp I and the Xap I of comprising.Enzyme cuts that system is following, and 5.0 μ l 10x enzymes are cut buffer, 5.0 μ g genomic dnas, and the best enzyme Qie Wendu of 37 ℃ or corresponding restriction endonuclease, enzyme is cut 16h; Enzyme is cut product carry out the extracting of phenol chloroform, ethanol-sodium-acetate deposition reclaims the genomic dna after enzyme is cut.Utilize T4DNA Polymerase that enzyme is cut product and carry out terminal flush end processing, after reaction finishes, the extracting of phenol chloroform, ethanol-sodium-acetate deposition reclaims the flush end enzyme and cuts product, is connected with joint respectively.Linked system is following: 1.0 μ l10xligase buffer, and 1.0 μ l 50%PEG4000,1.0 μ g enzymes are cut product, 1.0 μ l50 μ mol/l joints, 2.5U T4 DNA ligase adds aqua sterilisa to 10.0 μ l, and 16 ℃ connect 24h; 65 ℃, 10min deactivation ligase enzyme promptly can be used as promotor clone's template after diluting 10 times.Rubber tree promotor clone library comprises by the genomic dna enzyme of 25 kinds of restriction enzymes cuts that product adds top connection and the promotor that forms clone template.
Two. the acquisition of latex of panama rubber tree sucrose transporter promoter sequence
Two of the cDNA sequences Design nest-type PRC primers of the hevea brasiliensis translocator that obtains according to this laboratory:
PrHb3-GSP1 5’-GGCGGAGGAGTTGGAAGAGGTTCAGA-3’
PrHb3-GSP2 5’-ATCCAAAATGGTCTACGTCTTCAGTTGC-3’
Utilize two gene-specific primers (GSP1 and GSP2) of design, in conjunction with joint primer (ADP1 and ADP2), the library that obtains with step 1 is a template, carries out the nest-type PRC amplification, to obtain the promoter sequence of gene.First round pcr amplification uses warm start archaeal dna polymerase
the HS DNA Ploymerase of high-fidelity to carry out Touch Down PCR; Reaction system is following: 5.0 μ l, 5 * PrimeSTAR buffer (Mg2+plus); 2.0 μ l 2.5mM dNTP Mixture; 1.0 the library (getting one of 25 templates) that μ l dilution is 10 times; 0.5 μ lADP1 (10 μ M); 0.5 μ l GSP1 (10 μ M), 0.25 μ l PrimeSTAR HS DNA Polymerase (2.5U/ μ l), it is following to add aqua sterilisa to 25.0 μ l. response procedures: 94 ℃ 3 minutes; 98 ℃ 10 seconds, 72 ℃ 5 minutes, 7 circulations; 98 ℃ 10 seconds, 68 ℃ 5 minutes, 32 circulations; 68 ℃ 7 minutes.Second takes turns pcr amplification adopts LA Taq DNA Polymerase to carry out two-step approach PCR; And be template with first round amplified production; Reaction system is following: 2.5 μ l, 5 * LA PCR buffer (Mg2+plus), 2.0 μ l 2.5mM dNTPMixture, the first round pcr amplification product that 1.0 μ l dilution is 100 times; 0.5 μ l ADP2 (10 μ M); 0.5 μ llGSP2 (10 μ M), 0.2 μ l PrimeSTAR HS DNA Polymerase (5.0U/ μ l), it is following to add aqua sterilisa to 25.0 μ l. response procedures: 94 ℃ 3 minutes; 94 ℃ 30 seconds, 68 ℃ of 30 circulations in 5 minutes; 68 ℃ 7 minutes.PCR obtains the nucleotide sequence fragment about big or small 2000bp, and it is cloned into the pMD18-T carrier checks order, and its sequence is analyzed confirmed that this sequence is the promotor of sucrose transporter gene, and promotor length is 2096bp called after PrHb3.
One. latex of panama rubber tree sucrose transporter core promoter site estimation
Utilize promoter Prediction software (http://www.fruitfly.org/seq_tools/promoter.html) that the basic promotor of sucrose transporter (PrHb3) promotor is predicted; The result shows 5 ' end 49-99 position Nucleotide of sequence 1 in sequence table; 5 of sequence 1 ' end 707-757 position Nucleotide; 5 of sequence 1 ' end 974-1024 position Nucleotide; There is possible basic promoter region (table 1) in 5 of sequence 1 ' Nucleotide place, end 2010-2060 position, particularly 974-1024 basis promoter region score value near 1.0, show it is that the safety in core promoter district is very high.With the transcription initiation site of shaded in the time series for prediction.
Table 1BDGP (Berkeley Drosophila Genome Project) DB promotor predicts the outcome
Two. the prediction of rubber tree promotor cis element
Utilize the cis-acting elements of Plantcare software (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) software on-line analysis promotor.Analytical results shows; This promotor except that basal transcription elements T ATA-Box that has promotor and CAAT-Box, exist simultaneously a large amount of maybe be relevant with hormone response, stress response relevant (biology is coerced and abiotic stress), light reply relevant, grow relevant cis-acting elements (table 2).Wherein to reply functional element maximum with light, secondly be the relevant cis-acting elements of stress response and hormone response, grow the cis-acting elements minimum (Fig. 1) of being correlated with.
In the relevant cis-acting elements of hormone response; Contain dormin response element (ABRE); Element responsive to ethylene (ERE); Plant hormones regulators,gibberellins functional element (P-box) and Whitfield's ointment functional element (TCA-element), these hormones are regulated and control growth, growth and the differentiation of plant respectively or each other in phase.In the relevant cis-acting elements of stress response, mainly comprised injury coerce functional element (W-Box, WUN-motift) and drought stress functional element (MBS).In growing relevant cis-acting elements, mainly comprised the cis-acting elements that meristematic tissue (CAT-box) and palisade mesophyll cell (HD-Zip 1) are relevant.
The functional element prediction of taking advantage of a situation of table 2.PrHb3 promoter region
The screening of the plant expression vector construction of embodiment 3, PrHb3 promotor and transgenic arabidopsis sun plant
The promoter sequence that obtains according to order-checking; The Auele Specific Primer of design PrHb3 promotor amplification also grinds 7-33-97 (Rubber Institute, Chinese Academy of Agricultural Science cultivates, and Rubber Institute, Chinese Academy of Agricultural Science has seedling to sell for a long time) to rubber tree heat and carries out genome amplification.Amplimer is: PSUT3-F:5 '-CAAATTAAACATCGAGAATAATCCAAACG-3 ', PSUT3-R:5 '-GGTTGTGGTGGTGGTAGAAGTAGAGAGG-3 '.Amplification obtains the fragment about 2100bp, this fragment is carried out Xcm I enzyme cut, reclaim fragment, and order-checking shows that this fragments sequence is the nucleotide sequence of sequence 1 in the sequence table, and this fragment is promoter fragment of the present invention.To reclaim fragment and cut pCXGUS-P (Chen S.B. through Xcm I enzyme; Et al.; A versatile zero background T-vector system for gene cloning and functional genomics.Plant Physiol.2009; 150:1111-1121) plant expression vector of (available from Arabidopsis Biological Resource Center) recovery connects; Obtain recombinant expression vector, show the recombinant vectors called after pPHb3-GUS that contains above-mentioned promoter fragment through order-checking.The upper reaches of the last gus gene of pPHb3-GUS have only promoter sequence of the present invention, do not have other promotors, and this carrier gus gene only can be expressed under promotor of the present invention starts.
Adopt electric-shocking method to change expression vector over to Agrobacterium (EHA105 above-mentioned pPHb3-GUS; Sky, Beijing bounties Gene Tech. Company Limited); Simultaneously plant expression vector pCXGUS-P behind BamH I single endonuclease digestion from the negative contrast of continuous cropping, with this negative control plasmid called after pCK.Contain gus gene among the pCK, but the gus gene upper reaches has no promoter sequence.With reference to the method for Lu Zhangsen Arabidopis thaliana is carried out Agrobacterium and infect test; Utilize atomizer to change the Agrobacterium of plasmid pPHb3-GUS over to or the agrobacterium suspension of control plasmid pCK is sprayed to respectively on the Arabidopis thaliana titbit with above-mentioned; The Arabidopis thaliana that preservative film will infect (T0 generation) cover gets up to keep humidity; Insert dark culturing 24h in the culturing room, normal cultured is afterwards collected seed to the plant maturation and is placed in the dry environment about a week; With T0 for planting seed to the solid MS culture medium flat plate of the HYG that contains 50 μ g/ml; The screening transformant, the normal Arabidopis thaliana of two week back selection growing ways moves to continued growth in the soil, and T1 is collected for keeping subsequent use in the seed drying environment in ripe back.
Carry out pcr amplification detection with commentaries on classics control plasmid pCK Arabidopis thaliana T1 for seed to changeing the pPHb3-GUS Arabidopis thaliana: the DNA that extracts T1 Arabidopis thaliana positive plant; DNA with T1 Arabidopis thaliana positive plant is a template; With in the plant expression vector gus gene Auele Specific Primer G1 and G2 the amplification (G1:5 '-TCACCATTGGCCACCACCTGCC-3 '; G2:5 '-CCTGTAGAAACCCCAACCCGTG-3 '); All amplified about purpose band 600bp, promptly obtaining male changes control plasmid pCK Arabidopis thaliana or changes the positive T 1 generation seed of pPHb3-GUS Arabidopis thaliana.
For further confirming to change pPHb3-GUS Arabidopis thaliana T1 for promoter fragment contained in the Arabidopis thaliana positive plant; With carrier insert the both sides, site Auele Specific Primer GUS-V and PV (GUS-V:5 '-CCGCATCGAAACGCAGCACG-3 '; PV:5 '-GCCAGGGTTTTCCCAGTCACGA-3 ') increases; All amplifying the purpose stripe size is about 2300bp, and screening changes pPHb3-GUS Arabidopis thaliana positive plant.But above-mentioned primer G1 and G2 amplification obtain purpose band GUS-V and the PV amplification obtains about purpose band 300bp, and this plant is for changeing control plasmid pCK Arabidopis thaliana.
The tissue specificity analysis of PrHb3 promotor in embodiment 4, the transgenic arabidopsis
A. the GUS tissue staining is analyzed in the transgenic arabidopsis
According to Jefferson (Jefferson R.A.; Gus Fusion: β-Glucuronidase as a Sensitive and Versatile Gene Fusion Marker in Higher Plant.EMBO J; 1987; 6:3901-3907) method of classical GUS tissue staining analysis is analyzed the tissue specificity of PrHb3 promotor in transgenic arabidopsis.
Concrete grammar is described below:
Get respectively change the pPHb3-GUS Arabidopis thaliana positive plant seedling phase and the ripening stage plant; Be immersed in the GUS dye liquor; 37 ℃ of insulations several hours or spend the night (decide) according to the GUS expression intensity, get simultaneously transform negative control (above-mentioned commentaries on classics control plasmid pCK Arabidopis thaliana) two period Arabidopis thaliana as contrasting (CK).Change in 70% (volumn concentration) ethanol and decolour 2-3 time, complete to decolouring, naked eyes or electron microscope technique mirror be observation down, and the part of the blueness on the white background is the result that gus gene is expressed.As shown in Figure 2, the PrHb3 promotor has the activity that stronger startup reporter gene gus transcribes.The seedling phase, almost whole plant all was colored, and can find out that vein dyeing is darker after amplifying through blade.Ripening stage mainly dyes and concentrates in root and the lotus throne leaf, also has apparent in view painted during calyx and capsule really press from both sides.No matter and change control plasmid pCK Arabidopis thaliana still is that each of ripening stage organized equal non-coloring in the seedling phase.
B. GUS quantitative fluorescence analysis in the transgenic arabidopsis different tissues
Utilize the GUS 4-MUG that can degrade to generate fluorogenic substrate 4-MU, 4-MU launches the fluorescence of 447nm wavelength under the effect of 365nm wavelength exciting light, and emitting fluorescence intensity is linear with the 4-MU amount, measures the work of GUS enzyme.Mainly with reference to Cervera (Cervera M., Histochemical and Fluorometric Assays for uidA (GUS) Gene Detection.Plant Sci., 2004,286:203-213) method, concrete steps are described below:
Get the PrHb3 promotor transgenic arabidopsis positive plant ripening stage respectively and respectively organize about 100mg, add 800mlGUS and extract damping fluid, be positioned on ice and fully grind; The centrifugation cell fragment; Get supernatant, adopt the Bradford method its albumen to be carried out quantitatively-80 ℃ of preservations.Substrate 4-MUG is dissolved in an amount of GUS extracts buffer solution (sodium phosphate buffer (pH7.0), 50mM; Beta-mercaptoethanol, 10mM; Na2EDTA (pH8.0), 10Mm; Triton X-100,0.1%) middle configuration GUS detection liquid (2mM).Getting the albumen extract is 300ul with body mixing to TVs such as GUS detection liquid, and 37 ° of C incubation appropriate times are got 100 μ l samples at different time and directly are added to 900 μ l Na
2CO
3(reaction solution of getting after the 200 μ l termination reactions is added in the cuvette solution for 0.2mol/L, pH9.5) termination reaction, under excitation wavelength 365nm and emission wavelength 447nm, measures fluorescence, calculates enzyme and lives.The enzymic activity detected result of GUS shows in each tissue; The GUS enzymic activity is the highest in the PrHb3 promotor transgenic arabidopsis positive plant root; Being higher than its hetero-organization far away, is the lotus throne leaf secondly, stem leaf and stem; GUS enzymic activity minimum (Fig. 3) in seed can prove that thus promotor of the present invention is that root-specific is expressed promotor in plant maturity.There is not the GUS enzymic activity in the negative control transfer-gen plant.
The influence of different treatment PrHb3 promoter expression in embodiment 5, the transgenic arabidopsis
A. plant hormone is to the influence of PrHb3 promoter-driven GUS expression activity in the transgenic arabidopsis
Get two weeks of growth on MS (50ug/ml Hyg) flat board, the Arabidopis thaliana seedling carries out plant hormone respectively and handles the transgenic of growing way basically identical (pPHb3-GUS or contrast pCK).4 of following each processing selecting independently transgenic arabidopsis strain system are done repetition.The solid MS substratum of transgenic (pPHb3-GUS or contrast pCK) Arabidopis thaliana growth of seedling is immersed in respectively in the solution that contains 100 μ mol/L dormins (ABA), 100 μ mol/L Whitfield's ointments (SA), 100 μ mol/L Plant hormones regulators,gibberellins (GA), 100 μ mol/L ethrel precursor 1-amino-cyclopropane-1-carboxylic acids (ACC), 100 μ mol/L growth hormone (IAA) or 100 μ mol/L phytokinin (6-BA); 22 ℃ after 0,3,6,9 hour, the detection of drawing materials respectively.Negative control (pCK) transgenic arabidopsis carries out above operation synchronously.Above-mentioned materials is measured enzyme (Cervera M. alive according to the method for different tissues enzyme activity determination; Histochemical and Fluorometric Assays for uidA (GUS) Gene Detection.Plant Sci.; 2004; 286:203-213), analyze albumen (GUS) activity change of reporter gene expression under the different treatment.Detected result shows that after ethrel precursor ACC stimulated the promotor transgenic arabidopsis, GUS was more apparent in view than the fluctuation of living, and integral body presents ascendant trend (Fig. 4).After phytokinin 6-BA, Whitfield's ointment SA and dormin ABA handled, first risings back downward trend appearred in the GUS expression activity at 9 hours, and 9 hours expression activities be higher than handle preceding (Fig. 5, Fig. 6, Fig. 7).Downward trend after appearance raise earlier equally after Plant hormones regulators,gibberellins GA handled, but 9 hours expression activity is lower than processing preceding (Fig. 8).After growth hormone IAA handled, it was not too obvious that the GUS expression activity changes, slightly the trend of some rising (Fig. 9).There is not the GUS enzymic activity in the negative control transfer-gen plant.
A. abiotic stress is to the influence of PrHb3 promoter-driven GUS expression activity in the transgenic arabidopsis
Get two weeks of growth on MS (50ug/ml Hyg) flat board, the Arabidopis thaliana seedling carries out abiotic stress respectively and handles the transgenic of growing way basically identical (pPHb3-GUS or contrast pCK).
Arid is handled, transgenic arabidopsis is transferred to above the exsiccant filter paper, and 22 ℃ after 0,1,2,3 hour, the detection of drawing materials respectively;
High temperature (37 ℃) is handled, and places hot environment to continue illumination cultivation transgenic arabidopsis, and 37 ℃ after 0,3,6,9 hour, the detection of drawing materials respectively;
Low temperature (4 ℃) is handled, and places low temperature environment to continue illumination cultivation transgenic arabidopsis, and 4 ℃ after 0,3,6,9 hour, the detection of drawing materials respectively;
Injury is handled, crushes with the blade of tweezers transgenic arabidopsis, and 22 ℃ after 0,3,6,9 hour, the detection of drawing materials respectively;
4 of above-mentioned each processing selecting independently transgenic arabidopsis strain system are done repetition.Blank: negative control (pCK) transgenic arabidopsis carries out above operation synchronously.
Above-mentioned materials is measured GUS enzyme (Cervera M. alive according to the enzyme activity determination method; Histochemical and Fluorometric Assays for uidA (GUS) Gene Detection.Plant Sci.; 2004; 286:203-213), analyze albumen (GUS) activity change of reporter gene expression under the different treatment.GUS activation analysis result shows, behind high temperature and subzero treatment PrHb3 promotor transgenic arabidopsis, the GUS expression activity presents downtrending in 9 hours, and during subzero treatment, the decline of GUS expression activity more obvious (Figure 10, Figure 11).The back GUS expression activity trend of some rising (Figure 12) is slightly handled in injury, and the arid injury is handled the GUS expression activity and changed not obvious (Figure 13).And do not have the GUS enzymic activity in negative control (pCK) the transgenic arabidopsis plant.
Conclusion
Promotor of the present invention exist a large amount of maybe be relevant with hormone response, stress response relevant (biology is coerced and abiotic stress), light reply relevant, grow relevant, sugar and reply relevant cis-acting elements, the functional element that wherein the different expression regulation of hormone response and Gent is relevant is the most extensive.Make up the GUS fusion expression vector of this promotor, it is carried out functional verification, find that this promotor activity in root is the highest, find that simultaneously this promoter activity receives multiple hormone and coerces adjusting through transgenic arabidopsis.
This promotor is different expression regulation of Gent and multiple hormone induction promotor; This promotor can instruct foreign gene specific spatial and temporal expression in the development of plants process; It can not only make the expression product of goal gene accumulate at certain space, increases regional expression amount, avoids the waste of biological nutrition.Therefore, be widely used in other plant and the microbial project of this promotor beyond the rubber tree.
Claims (10)
1. dna molecular, its sequence is one of following nucleotide sequence:
1) dna sequence dna of sequence 1 in the sequence table;
2) with sequence table in the dna sequence dna that limits of sequence 1 have 90% above homology, and have the dna sequence dna of promoter function;
The nucleotide sequence of the dna sequence dna hybridization that 3) under the rigorous condition of height, can limit with sequence in the sequence table 1.
2. dna molecular according to claim 1 is characterized in that: said dna molecular is a promotor.
3. the expression cassette that contains claim 1 or 2 described dna moleculars.
4. the recombinant expression vector, transgenic cell line or the host bacterium that contain claim 1 or 2 described plant endosperm specificity expression promoters.
5. the described dna molecular of claim 1 is as the application in the plant promoter.
6. the application of the described dna molecular of claim 1 in cultivating transgenic plant.
7. application according to claim 6 is characterized in that: in the said transgenic plant, the downstream of the described dna molecular of said claim 1 connect foreign gene, and said foreign gene is specific expressed in root.
8. application according to claim 6 is characterized in that: said foreign gene is expressed by plant hormone and abiotic environment stress-inducing.
9. application according to claim 8 is characterized in that: said plant hormone is ethrel precursor 1-amino-cyclopropane-1-carboxylic acid and/or phytokinin and/or Whitfield's ointment and/or dormin and/or Plant hormones regulators,gibberellins and/or growth hormone.
10. application according to claim 8 is characterized in that: said abiotic environment is coerced and is high temperature stress, low temperature stress, drought stress or mechanical wounding.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434647A (en) * | 2015-08-11 | 2017-02-22 | 中国农业科学院生物技术研究所 | Phytohormone regulated promoter and application thereof |
| CN108251426A (en) * | 2018-03-23 | 2018-07-06 | 南京林业大学 | A kind of willow promoter pPsTMM and its application by plant hormone induction |
| CN109486819A (en) * | 2018-11-23 | 2019-03-19 | 中国热带农业科学院热带生物技术研究所 | Cassava U6 promoter gene and application |
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2012
- 2012-07-26 CN CN2012102619233A patent/CN102757966A/en active Pending
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| 李凤龙 等: "植物根特异性启动子研究进展", 《安徽农业科学》 * |
| 李和平: "巴西橡胶树蔗糖转运蛋白基因HbSUT5的表达特性研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106434647A (en) * | 2015-08-11 | 2017-02-22 | 中国农业科学院生物技术研究所 | Phytohormone regulated promoter and application thereof |
| CN106434647B (en) * | 2015-08-11 | 2019-02-05 | 中国农业科学院生物技术研究所 | By the promoter and its application of phytohormone Regulation |
| CN108251426A (en) * | 2018-03-23 | 2018-07-06 | 南京林业大学 | A kind of willow promoter pPsTMM and its application by plant hormone induction |
| CN109486819A (en) * | 2018-11-23 | 2019-03-19 | 中国热带农业科学院热带生物技术研究所 | Cassava U6 promoter gene and application |
| CN109486819B (en) * | 2018-11-23 | 2021-04-30 | 中国热带农业科学院三亚研究院 | Cassava U6 promoter gene and application thereof |
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