CN106011141A - Lilium regale inducible promoter and application thereof - Google Patents
Lilium regale inducible promoter and application thereof Download PDFInfo
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Abstract
The invention discloses a lilium regale inducible promoter LrP1 and application thereof. A nucleotide sequence of the LrP1 is as shown in SEQ ID NO: 1. According to the lilium regale inducible promoter LrP1 disclosed by the invention, the technical research related to molecular biology and gene engineering verifies that the lilium regale inducible promoter LrP1 is in response to several plant hormones and biotic and abiotic stress; expression cassettes constructed by connecting the lilium regale inducible promoter LrP1 disclosed by the invention and beta-glucuronidase genes are shifted into tobacco for expression, the activity of glucuronidase of transgene tobacco can be quantitatively detected through a fluorescence method, and a result shows that the activity of the glucuronidase is remarkably enhanced after the transgene tobacco is treated by gibberellin, ethylene, abscisic acid, NaCl, damage factors, fusarium oxysporum, sclerotinia sclerotiorum and botrytis cinerea; therefore, the lilium regale inducible promoter LrP1 is induced by several hormones and biotic and abiotic stress factors, so that the lilium regale inducible promoter LrP1 can be used for plant stress-resistant gene engineering.
Description
Technical field
The present invention relates to molecular biology and genetic engineering Related Research Domain, be specifically related to a kind of inducible promoter LrP1 and application thereof.
Background technology
Promoter is in the DNA sequence of structural gene 5' end upstream, can activate RNA polymerase, is allowed to be combined accurately with template DNA and have the specificity of transcription initiation.Promoter common in plant has constitutive promoter, tissue specific promoter and inducible promoter.Constitutive promoter (constitutive promoter) refers to that the expression somewhat constant of structural gene, on certain level, does not has notable difference at different tissues, position expression under such promoter controls.Tissue specific promoter (tissue-specific promoter), also known as organ specific promoters, is divided into root-specific promoter, stem specific promoter etc..Under this kind of promoter regulation, gene is often only expressed at some specific organ or tissue position, and shows the characteristic of Growth adjustment.Utilize engineered method by Oryza sativa L. (Oryza sativa) sucrose synthase gene promoterRSP1WithRSP2Proceeding to Oryza sativa L., result showsRSP1WithRSP2β-glucuronidase (β-glucuronidase can be driven, GUS) gene efficient specifically expressing in root, stem, leaf and grain husk shell, but in embryo and endosperm, do not express (Li Yongchun, Zhang Xianyin, Xue Qingzhong. specific expressed in sucrose synthase gene promoter clone and transgenic rice plant thereof. Acta Agronomica Sinica, 2002,28 (5): 586-590).
Inducible promoter (inducible promoter) refers to that the promoter of this type can be significantly increased the transcriptional level of gene under the stimulation of some specific physically or chemically signal.Inducible promoter can improve the expression of gene when plant is by extraneous physics or chemical stress, stops the regulation and control to gene after removal is coerced.This characteristic of inducible promoter can ensure to play protection plant, the effect of opposing environmental stimuli when plant is by stress from outside, and ensures that in suitable environment plant energy is not wasted.In genetic engineering, proceed to the exogenous gene of plant if not being controlled by, can in plant great expression, cause albumen accumulate in a large number and waste energy.Carrier adds inducible promoter and can regulate and control the expression of genes of interest when environmental stimuli, well solve the problem that exogenous gene is unrestrictedly expressed.
Inducible promoter includes abiotic inducible promoter and biological factor inducible promoter.Abiotic factor inducible promoter includes the inducible promoters such as the stimulation of arid, salt, temperature；Biological factor inducible promoter i.e. refers to the promoter that pest and disease damage is induced.Utilize genome ends random amplification technology, from Radix sacchari arundinacei (Erianthus arundinaceus) PR10 gene end amplify the promoter of 592bp, and connection proceeds in Nicotiana tabacum L., Oryza sativa L. and Caulis Sacchari sinensis with gus reporter gene, result shows that Radix sacchari arundinacei PR10 promoter can quickly respond process (Chakravarthi M, the Syamaladevi of injury, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate and abscisic acid
DP, Harunipriya P, Augustine SM, Subramonian
N. A novel PR10 promoter from Erianthus arundinaceus directs high constitutive transgene expression and is enhanced upon wounding in heterologous plant systems. Mol Biol
Rep, 2016,43 (1): 17-30).From western kahikatea (Pinus monticolaIn), clone obtainsPmPR10-1.13Promoter and three 5 ' disappearance ends, connect after Reporter gene GUS and proceed to arabidopsis.Result display GUS occurs in plumular axis and the cotyledon of 2-3 days seedling the earliest, after in the top great expression of plant.But in adult plants,PmPR10-1.13Promoter response pathogen infection and wound are coerced, and showPmPR10-1.13Promoter response biology or abiotic stress (Liu JJ, Ekramoddoullah AK, Piggott N, Zamani A. Molecular cloning of a pathogen/wound-inducible
PR10 promoter from Pinus monticola and
characterization in transgenic Arabidopsis plants. Planta,
2005,221 (2): 159-169).Germinatus Phragmitis (Asparagus officinalis) in clone obtain a PR10 gene (AoPR10) promoter, willAoPR10Promoter connects reporter geneGUSProceed to process in arabidopsis and to suitable adverse circumstance,GUSGene can be at injured, pathogen infection and H2O2Process position express and accumulate, showAoPR10Promoter is induced (Mur LA, Sturgess FJ, Farrell GG, Draper J. The AoPR10 by above-mentioned biology and the abiotic stress factor
promoter and certain endogenous PR10 genes respond to oxidative signals in
Arabidopsis. Mol Plant Pathol, 2004,
5 (5): 435-451).
Summary of the invention
It is an object of the present invention to provide a kind of inducible promoter LrP1, derive from Ming River Bulbus Lilii, its nucleotide sequence is as shown in SEQ ID NO:1.
The present invention another object is that and applies in genetic engineering by this promoter, i.e. as abduction delivering promoter regulation exogenous gene specific high-efficiency expression in transgene receptor plant under environment stress.
The present invention relates to separant induction type promoter fragment and identify its expression activity, the present invention clones from Ming River Bulbus Lilii and obtains inducible promoter, and this promoter total length is 1489bp；Bioinformatic analysis shows to comprise a series of different cis acting element in inducible promoter, such as tissue-specific element, the response element of multiple hormone (abscisic acid, gibberellins, ethylene, salicylic acid, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate), abiotic stress (moisture, high salt, injury) response element, the element etc. that Analysis of Defence Genes Involved response pathogenic bacterium inducing is transcribed.WRKY transcription factor plays important regulating and controlling effect in plant disease-resistant defense response, and this inducible promoter sequence has the cis acting element w box(C/TTGACC/T of three WRKY).
By the 35s promoter of the CaMV on the inducible promoter fragment displacement pBI121 carrier of separating clone of the present invention, inducible promoter drive reporter geneGUSExpression cassette, by Agrobacterium tumefaciems (Agrobacterium tumefaciens) mediation will proceed to model plant Nicotiana tabacum L. (Nicotiana tabacumExpress in), and disclosed the expression characterization of inducible promoter by further experiment, lay the foundation for this promoter regulation exogenous gene of later-stage utilization efficient specifically expressing in transfer-gen plant.Inventor is by named for this promoter LrP1.
By LrP1 promoters driven in the present inventionGUSExpression cassette proceed in Nicotiana tabacum L., several plant hormone, biology and abiotic stress is used to process transgenic tobacco plant, and carry out the quantitative fluorescence analysis of GUS activity, testing result shows, LrP1 promoter responds several abiotic, biotic and the process of several plant hormone, gibberellins, ethylene, abscisic acid, NaCl, injury, Fusarium oxysporum, Botrytis cinerea (Botrytis cinerea), sclerotinite (Sclerotinia sclerotiorum) can the activity of obvious evoked promoter LrP1.
Above-mentioned promoter L rP1 can be applied to the abduction delivering of exogenous gene in genetic engineering, and concrete operations are as follows:
(1) special primer of amplification LrP1 is used, genomic DNA is extracted from Ming River Bulbus Lilii tender tissue, by polymerase chain reaction (polymerase chain reaction, PCR) full length sequence of LrP1 is amplified, it is subsequently attached on pGEM-T carrier, obtains through order-checking and there is the clone that sequence is correct；
(2) with digestion with restriction enzyme pGEM-T-LrP1 carrier, promoter fragment is reclaimed；Use suitable digestion with restriction enzyme to remove the composition type expression promoter on plant expression vector simultaneously, reclaimed by glue and obtain carrier large fragment；Again obtained LrP1 fragment is connected with pCAMBIA2300S carrier segments, builds plant inducible expression carrier；Afterwards constructed plant inducible expression carrier is recombinated with genes of interest, and proceed in recipient plant by Agrobacterium tumefaciens mediated, the plant of express transgenic is suffering NaCl, time injury is coerced or Fusarium oxysporum, sclerotinite, Botrytis cinerea are infected, the genes of interest that promoter L rP1 drives can be induced and up-regulated expression level, and the gibberellins of vivo and vitro, ethylene, abscisic acid also can induce genes of interest high level expression in addition.
The present invention is the promoter providing a new abduction delivering in plant genetic engineering application.The conventional 35S promoter from cauliflower mosaic virus of plant overexpression vector in genetic engineering, this promoter is composition type expression promoter, the expression somewhat constant of genes of interest is on certain level, notable difference is not had at different tissues, position expression, so its expression of exogenous gene proceeding to plant is uncontrolled, albumen is caused to accumulate in a large number and waste energy.And inducible promoter can be improved the expression of gene when plant affects by stress from outside or chemical factor; removing the expression coercing or i.e. lowering after chemical treatment genes of interest; can ensure to play protection plant, the effect of opposing environmental stimuli when plant is by environment stress, otherwise in suitable environment, not waste the energy of plant.In genetic engineering application, carrier adds the problem that inducible promoter can overcome genes of interest unrestrictedly to express.The expression activity of inducible promoter LrP1 in several hormones (gibberellins, ethylene, abscisic acid), abiotic stress (NaCl, injury), biotic (Fusarium oxysporum, sclerotinite, Botrytis cinerea) the substantially induction present invention, therefore the present invention has broad application prospects in the genetic engineering of antibiont or abiotic stress.
Accompanying drawing explanation
Fig. 1 is that the glue of promoter L rP1 in the present invention (A figure) and pBI-121 carrier (B figure) reclaims product testing result；
Fig. 2 is pBI121-LrP1-in the present inventionGUSConverting colibacillary positive colony testing result, wherein positive control is the PCR reaction with pGEM-T-LrP1 plasmid as template, and blank is the PCR reaction with sterilized water as template；
Fig. 3 is part pBI121-LrP1-in the present inventionGUSThe PCR the selection result of transgene tobacco, wherein positive control is with plasmid pBI121-LrP1-GUSPCR for template reacts；WT: non-transgenic tobacco (wild type) STb gene is the PCR that template is carried out；
Fig. 4 is the standard curve that in the present invention, GUS enzyme is lived；
Fig. 5 is pBI121-LrP1-in the present inventionGUSTransgene tobacco GUS activity after gibberellins, ethylene, abscisic acid process, wherein CK is the pBI121-LrP1-of normal growthGUSThe GUS activity of transgene tobacco；
Fig. 6 is pBI121-LrP1-in the present inventionGUS transgene tobacco is in NaCl, the activity of the GUS after processing of being injured, and wherein CK is the pBI121-LrP1-of normal growthGUSThe GUS activity of transgene tobacco；
Fig. 7 is pBI121-LrP1-in the present inventionGUSTransgene tobacco is in Fusarium oxysporum, Botrytis cinerea, sclerotinite postvaccinal GUS activity, and wherein CK is the pBI121-LrP1-of normal growthGUSThe GUS activity of transgene tobacco.
Detailed description of the invention
Below by drawings and Examples, the present invention is further described; but scope is not limited to described content; method operating the most according to a conventional method if no special instructions in the present embodiment, agents useful for same if no special instructions use conventional reagent or the reagent configured according to a conventional method.
Detailed description of the invention
Below by drawings and Examples, the present invention is further described; but scope is not limited to described content; method operating the most according to a conventional method if no special instructions in the present embodiment, agents useful for same if no special instructions use conventional reagent or the reagent configured according to a conventional method.
Embodiment 1: the clone of Ming River Bulbus Lilii inducible promoter LrP1 and sequence analysis
With extract Ming River Bulbus Lilii kan gene group DNA as template, with the special primer of amplification promoter L rP1, (forward primer is: 5 ' TCGAGTTTGGTGTTATTGTTATTAG3 ' ', downstream primer is: 5 ' GATGTGTTTGAGAAGGAGGTGT3 ') it is upstream and downstream primer, by the sequence of PCR cloning promoter LrP1.Reaction system (20 μ L) is Ming River Bulbus Lilii genomic DNA 0.5 μ g, 2 μ L
10×Advantage 2 PCR Buffer、1.8 μL dNTP Mix (10mM
each)、0.2 μL
Forward primer (10 μMs), 0.2 μ L
Downstream primer (10 μMs), 0.2 μ L
Advantage 2 PCR Polymerase Mix, 14.6 μ L PCR-Grade water.PCR reaction condition: 94 DEG C of 5 min；94 DEG C of 30 s, 65 DEG C of 30 s, 72 DEG C of 2 min, 32 circulations；72℃ 5 min.After PCR terminates, take 8 μ L and carry out agarose gel electrophoresis, in order to detect specificity and the size of amplified production.
Obtained PCR primer only has a DNA band, therefore directly PCR primer is carried out TA clone, and the test kit of use is pGEM-T
Vector system (Promega), reaction system and operating process be: takes 1.5 μ L
PCR primer, is sequentially added into 1 μ L pGEM-T vector (50 ng/ μ L) and 2.5 μ L 2 × Ligation solution I, and mixing is placed on 16 DEG C of reaction overnight.By heat-shock transformed method, connection product is proceeded in bacillus coli DH 5 alpha competence.With the LB solid medium screening positive clone containing ampicillin (ampicillin, Amp).Select several single bacterium colonies, detect multiple clone site with the special primer of amplification LrP1 after shaking bacterium and insert the clone of LrP1.The positive colony obtained is checked order, the final promoter L rP1 length 1489 obtained
bp.Use PLANTCARE that the cis acting element of promoter is predicted, it was predicted that cis acting element (table 1) relevant to environment stress in promoter sequence.Promoter comprises a series of different cis acting element, such as tissue-specific element, the response element of multiple hormone (abscisic acid, gibberellins, ethylene, salicylic acid, (-)-methyl cis-2-pent-2'-enyl-3-oxocyclopentylacetate), abiotic stress (moisture, high salt, injury) response element, the element etc. that Analysis of Defence Genes Involved response pathogenic bacterium inducing is transcribed；WRKY transcription factor plays important regulating and controlling effect in plant disease-resistant defense response, and promoter sequence has the cis acting element w box(C/TTGACC/T of three WRKY).
Table 1: the cis acting element in promoter sequence predicts the outcome
。
Embodiment 2:LrP1-GUSExpression vector establishment
PBI121 multiple clone site hasHinD III HeBamH I restriction enzyme site, therefore the special primer in amplification promoter adds respectivelyHinD III HeBamThe recognition site of H I.Use SanPrep pillar plasmid DNA in a small amount extraction agent box (the raw work in Shanghai) to extract escherichia coli plasmid pGEM-T-LrP1 and the plant expression vector pBI121 plasmid inserting LrP1, take 1 μ L for agarose gel electrophoresis with detection the integrity of extraction plasmid and concentration level.Use restricted enzymeBamH I HeHinD III carries out double digestion (100 μ L system) respectively to plasmid pGEM-T-LrP1 and pBI121, and reaction system and operating process be: takes 20 μ L pGEM-T-LrP1 and pBI121 plasmid respectively, be sequentially added into 10 μ L 10 × H buffer, 5 μ LBamHⅠ、5 μL HindⅢ、60 μL ddH2O, is centrifuged after mixing in short-term, is placed in 37 DEG C of reaction overnight.All digestion products are carried out agarose gel electrophoresis, then use SanPrep pillar DNA glue to reclaim test kit (the raw work in Shanghai) and promoter fragment and pBI121 carrier large fragment are carried out glue recovery respectively, taking 1 μ L and reclaim the product size by agarose gel electrophoresis detection recovery fragment and concentration, result is as shown in Figure 1.
Utilize T4 DNA Ligase (TaKaRa), promoter dna fragment and the pBI121 carrier segments of recovery being coupled together, reaction system (20 μ L) and operating process be: takes 10 μ L LrP1 DNA fragmentations and is sequentially added into 2 μ L pBI121 carrier DNAs, 2 μ L 10 × T4 DNA Ligase Buffer, 1 μ L T4 DNA Ligase, 5 μ L ddH2O, is centrifuged after mixing in short-term, then 16 DEG C of water-bath reaction overnight.Then heat-shock transformed method is used to proceed in bacillus coli DH 5 alpha by connection product, with containing 50
The solid medium screening positive clone of mg/L kanamycin (kanamycin, Km).Picking individual colonies shakes bacterium, carries out PCR with the special primer that bacterium solution is template amplification promoter L rP1, picks out the clone that LrP1 with pBI121 is successfully connected, and in the positive strain obtained, addition glycerol is placed in-80 DEG C and saves backup.
Use the pBI121-LrP1 in the extraction of SanPrep pillar plasmid extraction test kit the above-mentioned bacillus coli DH 5 alpha of purification-GUSPlasmid.Subsequently by frozen-thawed method by the plant expression vector pBI121-LrP1 of above-mentioned structure-GUSProceed in prepared agrobacterium tumefaciens lba4404 competent cell.Operating procedure is: take 0.2 μ g pBI121-LrP1-GUSPlasmid adds in the centrifuge tube containing 200 μ L competent cells, gently ice bath 5 min after mixing, then continues at freezing 1 in liquid nitrogen
Min, is then immediately placed in 37 DEG C of water-bath 5 min, then ice bath 2 min, adds 500 μ L LB liquid cultures afterwards based on 28 DEG C of shaken cultivation 4
h.Agrobacterium after activation is applied on the LB solid medium containing 50 mg/L Km, is inverted for 28 DEG C and cultivates.Picking individual colonies shakes bacterium, then carries out PCR reaction with the specific primer of amplification LrP1, detects pBI121-LrP1-GUSWhether proceed in Agrobacterium.For the positive colony shown in Fig. 2, addition glycerol is placed on-80 DEG C and saves backup.
Embodiment 3: agriculture bacillus mediated Genetic Transformation in Higher Plants and transgenic plant screening
The transgene receptor of this experiment is Nicotiana tabacum L., by tobacco seed with 75% alcohol-pickled 30 s, sterilized water washing after with 0.1% HgCl2Soak 8 min, wash several times with sterilized water the most again, be seeded in 1/2 MS culture medium, 28 DEG C of light culture 5-8 d, go to illumination box (25 DEG C, 16h/d illumination) after germination, the most monthly with MS culture medium subculture the most once.
By in-80 DEG C of refrigerators preserve containing pBI121-LrP1-GUSThe Agrobacterium LBA4404 bacterium solution of plasmid is taken out, and takes 10 μ L bacterium solution and is inoculated in the LB fluid medium that 1 mL contains 20 mg/L rifampicin and 50 mg/L Km, and 28 DEG C of 200 rpm shaken cultivation is to muddy.Draw 500 μ L bacterium solution to be spread evenly across on the LB solid medium containing 20 mg/L rifampicin and 50 mg/L Km, be inverted for 28 DEG C and cultivate to growing lawn.Being inoculated in 40 mL MGL culture medium containing 25 mg/mL acetosyringones with inoculating loop scraping 3-5 ring lawn, 28 DEG C of 220 rpm shaken cultivation is until OD600It is about 0.6.Aseptic Nicotiana tabacum L. tissue cultured seedling blade is cut into about 1 cm2The leaf dish of size, is soaked in the MGL culture medium containing suspension Agrobacterium, and 15 min are cultivated in 25 DEG C of concussions.Proceed to Nicotiana tabacum L. after the bacterium solution of leaf panel surface being blotted with aseptic filter paper and co-culture culture medium (MS+0.02
Mg/L 6-BA+2.1 mg/L NAA+30 g/L sucrose+6 g/L agar) in, 22 DEG C of light culture 2 days.Leaf dish after co-culturing is transferred in Nicotiana tabacum L. screening culture medium (MS+0.5 mg/L 6-BA+0.1 mg/L NAA+30 g/L sucrose+6 g/L agar+50 mg/L Km+200 mg/L cephamycin), be incubated at illumination box (25 DEG C, 16
H/d illumination).Cultivate about 3 weeks, differentiation tobacco seedling out is cut and subculture is in containing 50 mg/L Km and 300
Root culture is carried out on the root media (MS+30 g/L sucrose+6 g/L agar+50 mg/L Km+300 mg/L cephamycin) of mg/L cephamycin.
Use CTAB method to extract the genomic DNA of transgenic tobacco plant blade, take 1 μ L genomic DNA and carry out agarose gel electrophoresis and detect its integrity and concentration.PCR reaction is carried out with the special primer that the genomic DNA of transfer-gen plant is template amplification promoter L rP1.After PCR terminates, take 8 μ L products for agarose gel electrophoresis to detect positive transgenic plant.The amplification of partial transgenic tobacco plant is as it is shown on figure 3, Ming River Bulbus Lilii inducible promoter LrP1 transgene tobacco screens 22 strain positive transgenic plant altogether.
Embodiment 4: the GUS fluorescent quantitation detection of transgene tobacco
(the Jefferson R. Assaying chimeric such as the quantitative fluorescence analysis reference Jefferson to transgene tobacco root GUS activity
genes in plants: The GUS gene fusion system. Plant Mol Biol
Rep. 1987,5 (4): 387 405) method, its reaction mechanism is: GUS can react with substrate 4-MUG, catalysis produce 4-MU, 4-MU be 365 in excitation wavelength
Producing fluorescence under the conditions of a length of 455 nm of nm, transmitted wave, the fluorescent value of generation can be quantitative determined by spectrofluorophotometer.
The Tobacco Root anticipated is placed in equipped with grind into powder in the mortar of liquid nitrogen, adds 400 μ L GUS Extraction buffers, homogenate is transferred in 1.5 mL centrifuge tubes, in 4 DEG C, 12000 g be centrifuged 10 min.After centrifugal end, collect supernatant in new centrifuge tube.Take 4-MUG solution (1 mmol/L) 37 DEG C of preheating 10 min in 2.0 mL centrifuge tubes of 1 mL in advance.Take 50 μ L of supernatant liquid to add to the GUS reaction buffer of preheating, shake up rapidly, and take 200 μ L reaction mixtures immediately and be placed in the stop buffer of 1.8 mL (operating time be less than 30 s), as enzymatic reaction 0 point sample (as blank during fluoremetry), remaining liq continues 37 DEG C and reacts and start timing.Take 200 μ L reaction mixtures when reacting 15 min, 30 min, 45 min respectively, add to 1.8 mL stop buffers, for fluoremetry.Using spectrofluorophotometer is 365 nm in excitation wavelength, transmitted wave a length of 455
The fluorescent value of each sample is measured under conditions of nm.Make 4-MU standard curve: 1 mM 4-MU mother solution reaction terminating liquid is diluted to 50 nM, 100nM, 200 nM respectively, 400 nM, the different gradient liquid of 500 nM and 1000 nM, it is 365 nm in excitation wavelength, the fluorescent value of each gradient liquid is measured under conditions of a length of 455 nm of transmitted wave, with reaction terminating liquid as blank, draw standard curve (as shown in Figure 4) by the concentration of the fluorescent value recorded and 4-MU.Take 10 μ L of supernatant liquid, use the Coomassie Brilliant Blue of improvement to measure the protein content of sample.Generating the enzyme amount of 1 nmol 4-MU with one minute catalysis 4-MUG is a unit of activity, and GUS enzyme work calculates with the enzyme activity of every mg total protein, is i.e. expressed as 4-MU nmol/min/mg (albumen).By standard curve, calculate the GUS activity of transgene tobacco.
In order to detect Ming River Bulbus Lilii promoter to phytohormone, the response of biotic and abiotic stress, respectively with several plant hormone, biotic and the root of abiotic stress factor treatment transgene tobacco, and the GUS activity before and after being processed by said method mensuration, using normal growth untreated transgene tobacco root GUS activity as comparison (CK).Such as Fig. 5, after gibberellins, abscisic acid and ethylene process, the GUS activity in Ming River Bulbus Lilii promoter L rP1 transgene tobacco root substantially raises, from the point of view of the induction degree of promoter activity, and gibberellins > abscisic acid > ethylene.After NaCl, process of being injured, the GUS activity of transgene tobacco is as shown in Figure 6, injury and NaCl both abiotic stress factors significantly raise the activity of promoter L rP1, compared with coercing with NaCl, the responsiveness that injury is coerced by promoter L rP1 is higher, after injured process, the GUS activity that LrP1 drives is coerced far above NaCl and is compareed.The root of transgene tobacco is processed, also substantially induction of the activity of promoter, from induction degree, Botrytis cinerea with three kinds of pathogen Fusarium oxysporums, sclerotinite and Botrytis cinerea > Fusarium oxysporum > sclerotinite (Fig. 7).Above-mentioned test result indicate that, Ming River Bulbus Lilii promoter L rP1 response several plant hormone, abiotic stress and the process of biotic, gibberellins, abscisic acid, ethylene, NaCl, injured, Botrytis cinerea, Fusarium oxysporum, sclerotinite can substantially raise the GUS activity that LrP1 drives.Obviously, Ming River Bulbus Lilii promoter L rP1 is a Plant Hormone, biotic and abiotic stress factor inducible promoter, can be applicable to plant stress-resistance genetic engineering.
Sequence table
<110>Kunming University of Science and Technology
<120>Ming River Bulbus Lilii inducible promoter and application thereof
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 1489
<212> DNA
<213> Lilium
regale
<400> 1
ttcgagtttg
gtgttattgt tattagtcat tttgacttca tatttgccac caaatacttt 60
gtaactccaa
aacatgttac agtccttaca gatatgtata ctagcatttg tatcaagcta 120
acacccacca
tttactctaa taatattatc ttaagagatc ttcattataa atatctcttc 180
ttcgaatgaa
tttgcttgat ttttttttat attcttcttc tgatgatgac aatcacgatc 240
aaatgattgg
gctttccata atggcagtcg gttccttttg gcttcttctt taggccacta 300
ttcttcttaa
aagaagtgat attcttagcc tttagtttct tgacatcatt actctcattt 360
gtattggcct
tatgggactt aaagttgtca tctttcttct tttgttttct tgaatcctct 420
ccaatctgaa
gatgatgtta aagtgactca agagtccact tattctcttt atgcattatc 480
ttattcttgt
agttattaca agtagaatac tacttagtga taatcattcc tactcgaaag 540
ttatctacaa
ggtcaatccc gacatgcttt atatcatcaa caatcaattg caggtcataa 600
acctgatcga
gggtttactt atcattcacc attttataat taacataatt ggaaattaga 660
aatttctaat
taatgccttt caaagctcct ttgcacttgc tgcttatcga taatggtggt 720
acaaagtctt
tgaaagctcg ttggagggtg tgtctcttgc acatgaattc attttccttt 780
tgcttgtgac
gtttctacct cagaacttca atattgtttg atttggattg aaaatggttt 840
caaaattctt
atccagaatg taacccaact tcatcgtcgt gaggaaaaat ataacattat 900
gttacgacga
gtgtagtttg tcatgctaaa atgcttgagt caaaccagat cttgattcat 960
atctcacaga
gacttgaaaa tggtggagtt catactgtag ccaaaaaaat tgtctaattc 1020
ttttttggaa
tatttgctct aaacttagca atgaaccgtt gcacattcat tactgattta 1080
ttgaacctct
acacataaaa aatcagagag atctactgaa atcatagcaa cctaagacct 1140
cttattgtcc
ttcaaatagc ccaactatta agctaaacaa acattttatt aattttcccg 1200
aaacatctta
tctgactcat tatcattttg aaatattaca ataaaacaca cgtgccaaat 1260
gtcctaaaaa
gattgcagcg tacaagtcaa caagatgatg actgttgttt gaatagcgtg 1320
ttgggcatat
taaactggga ggtgtgatta ttaaaacctc atcataagtc ggcggaactc 1380
ttttatatgg
cggaagacgt ttagatatag cttggcttgc agtgggacta agcacaccaa 1440
cccctccatt
ctcaacccct ataagaatac cccaactctc ttcatcatc 1489
<210> 2
<211> 25
<212> DNA
<213>artificial sequence
<400> 2
tcgagtttgg tgttattgtt
attag 25
<210> 3
<211> 22
<212> DNA
<213>artificial sequence
<400> 3
gatgtgtttg agaaggaggt gt
22
Claims (3)
1. an inducible promoter LrP1, derives from Ming River Bulbus Lilii, and its nucleotide sequence is as shown in SEQ ID NO:1.
2. the application in Genes For Plant Tolerance adverse circumstance stress gene engineering of the inducible promoter LrP1 described in claim 1.
Application the most according to claim 2, it is characterised in that: under environment stress, regulate and control exogenous gene specific high-efficiency expression in transgene receptor plant.
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| CN112608924A (en) * | 2021-01-29 | 2021-04-06 | 昆明理工大学 | Inducible promoter PCHI and application thereof |
| CN112708625A (en) * | 2021-03-16 | 2021-04-27 | 昆明理工大学 | Lilium regale inducible promoter PG1 and application thereof |
| CN112852820A (en) * | 2021-03-16 | 2021-05-28 | 昆明理工大学 | Lilium regale inducible promoter PD1 and application thereof |
| CN113174389A (en) * | 2021-05-27 | 2021-07-27 | 昆明理工大学 | Lilium regale inducible promoter PR4 and application thereof |
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Cited By (10)
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| CN109295080A (en) * | 2018-09-19 | 2019-02-01 | 昆明理工大学 | Panax japonicus majoris β-amyrin synthase gene Pj β-AS purposes |
| CN109295080B (en) * | 2018-09-19 | 2021-08-20 | 昆明理工大学 | Application of rhizoma panacis majoris beta-balsamol synthetase gene Pj beta-AS |
| CN112608924A (en) * | 2021-01-29 | 2021-04-06 | 昆明理工大学 | Inducible promoter PCHI and application thereof |
| CN112608924B (en) * | 2021-01-29 | 2023-06-20 | 昆明理工大学 | Inducible promoter PCHI and application thereof |
| CN112708625A (en) * | 2021-03-16 | 2021-04-27 | 昆明理工大学 | Lilium regale inducible promoter PG1 and application thereof |
| CN112852820A (en) * | 2021-03-16 | 2021-05-28 | 昆明理工大学 | Lilium regale inducible promoter PD1 and application thereof |
| CN112708625B (en) * | 2021-03-16 | 2023-06-16 | 昆明理工大学 | Lilium regale inducible promoter PG1 and application thereof |
| CN112852820B (en) * | 2021-03-16 | 2023-06-20 | 昆明理工大学 | Lilium regale inducible promoter PD1 and application thereof |
| CN113174389A (en) * | 2021-05-27 | 2021-07-27 | 昆明理工大学 | Lilium regale inducible promoter PR4 and application thereof |
| CN113174389B (en) * | 2021-05-27 | 2023-06-16 | 昆明理工大学 | Lilium regale inducible promoter PR4 and application thereof |
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