CN104195138A - Low-temperature inducible promoter and application thereof - Google Patents
Low-temperature inducible promoter and application thereof Download PDFInfo
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- CN104195138A CN104195138A CN201410442189.XA CN201410442189A CN104195138A CN 104195138 A CN104195138 A CN 104195138A CN 201410442189 A CN201410442189 A CN 201410442189A CN 104195138 A CN104195138 A CN 104195138A
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Abstract
The invention discloses a low-temperature inducible promoter and application thereof, belongs to the field of genetic engineering, and in particular relates to non-coded nucleic acid for adjusting the genetic expression in DNA recombination techniques. The nucleotide sequence of the promoter is as shown in SEQIDNO:1. By adopting the promoter disclosed by the invention, experimental and technical support can be provided for research and development of plant biological reactors. Vaccines, antibodies and other medical protein can be expressed by utilizing the low-temperature inducible promoter and taking plants as the biological reactors, the conventional agriculture scope is broken through and the medical field is achieved.
Description
Technical field
The invention belongs to genetically engineered field, be specifically related to the non-coding nucleic acid that in DNA recombinant technology, a kind of regulatory gene is expressed.
Background technology
In plant genetic engineering research, external source goal gene is expressed in acceptor, to improve the resistance of crop to environment stresses such as arid, low temperature, saline and alkaline and disease and pests, or raising crop quality and Crop Improvement economical character etc., become a kind of important means of current crop breeding.Eukaryotic gene expression regulation comprises pretranscriptional control, transcriptional control, post-transcriptional control, translational control and post-translational control.Wherein, the regulation and control of transcriptional level are subject to cis-acting elements and transcription factor coordinative role, are the key links of expression regulation.Therefore, separation and the research about promotor becomes one of focus of domestic and international research.
Use constitutive promoter to order about exogenous gene expression, not only cause the wasting of resources, also directly affect growing and normal physiological metabolism of plant, and use inducible promoter, not only can not cause the waste of various resources, can strengthen again plant materials resistance to external world, reduce the impact on growth and development of plants and other pathways metabolisms.Therefore, how controlling foreign gene high efficient expression at regular time and quantity, is the key point that genetic engineering technique is effectively applied aspect crop genetic improvement.Except finding effective goal gene, find that the promotor of effectively controlling exogenous gene expression becomes the major issue that needs solution in genetically engineered improvement.Promotor is roughly divided into three kinds of constitutive promoter, tissue-specific promoter and inducible promoters according to its function and efficacy mode.The genetic expression of constitutive promoter regulation and control is not subject to the impact of external environmental condition, almost in all plant different tissues, can express.Be typically cauliflower mosaic virus (cauli-flower mosaic virus, CaMV) 35S promotor, (Benfey etc., 1990, Science 250,959-966) to be widely used in dicotyledons transgenic engineering.Other unifacial leaf have corn ubiquitin Ubiquitin promotor (Streatfield etc., 2004, Transgenic Res 13,299-312) and rice actin (Actin) promotor.Such promoter activity is higher, but because causing foreign gene, it at the different sites of the whole growth period of transgenic plant, expresses, affect growing and normal physiological metabolism of plant, some product may cause toxic action, be unfavorable for the raising of quality and yield, even can cause the death of plant.Kasuga etc. utilize induction type rd29A promotor to replace CaMV35S promotor arabidopsis thaliana transformation, improved the drought resistance of transgenic arabidopsis, reduced composing type crosses and expresses that the plant strain growth causing is stagnated or the generation (Kasuga of dwarfism simultaneously, 2004, Plant and Cell Physiology 45,346-350).Therefore,, for the expression of regulating plant gene better, the research of organizing specific type promotor and inducible promoter and application are more and more subject to people and pay attention to.
Organizing specific type promotor is that regulation and control goal gene is only expressed in certain organs or tissue.Generally there are some special motifs relevant to tissue specificity in such promotor.Nitz etc. are the separated myrosin gene Pyk10 promoter sequence that obtains being coded in specifically expressing in root and hypocotyl from Arabidopis thaliana, this promotor can regulate and control goal gene at transgenic arabidopsis root high level specifically expressing (Nitz etc., 2001, Plant Science, 161,337-346).The promotor that Bate etc. analyze potato specific expression gene LAT25 by deletion mutantion finds, regulation and control pollen-specific Expression element be positioned at these gene promoter-492 to-52 sections (Bate etc., 1998,37,859-869).Maize leaf specificity PPCA1 promotor (Gowik etc., 2004, Plant Cell 16,1077-1090), strawberry fruit specificity GalUR promotor (Agius etc., 2005, J Exp Bot 56,37-46) etc.The further investigation of such promotor is contributed to illustrate to the basic theory of growth and development of plants and physiological metabolism, and be with a wide range of applications.Inducible promoter refers under standard state and does not express or low expression, is subject to the stimulation of some physics or chemical signal, can improve significantly the expression level one class promotor of gene.According to induction factor, inducible promoter can be divided into photoinduction type promotor, temperature-induced type promotor, wound inducement type promotor, hormone induction type promotor and fungal induction type promotor etc.Be characterized in: be subject to the induction of physics or chemical factor; Contain several functions element, the collaborative activity that strengthens or reduce promotor; Part inducible promoter has the feature of tissue-specific promoter simultaneously.Other inducible promoters are subject to the induction (Huda etc., 2013, PLoS One 8) of arid, cold, methyl jasmonate and dormin as paddy rice plasma membrane CaATPase promotor.Arabidopis thaliana RBCS-1A promotor is photoinduction type promotor, have simultaneously tissue specificity (practise rain beautiful jade etc., 2012, Acta Agronomica Sinica 38,1561-1569); For inducible promoter, goal gene only just can be expressed after accepting inducement signal, not only can not cause the waste of various resources, can strengthen again plant materials resistance to external world, reduces the impact on growth and development of plants and other pathways metabolisms.Therefore, study and utilize inducible promoter, for the adversity gene engineering of studying the various physiological Mechanism of coercing of plant responding and research farm crop, have important meaning, aspect fundamental research and Plant Biotechnology, also having broad application prospects.Yet up to the present, can be applied to the inducible promoter of transgenic research still seldom.Therefore, for the interaction between the determining of new degeneration-resistant promoter related clone, the concrete sequence of cis-acting elements, each element, and the research of the transcription factor of doing mutually with these elements remains the emphasis of promotor research from now on.
Summary of the invention
The object of this invention is to provide a kind of promotor and application thereof that can start destination gene expression when low temperature induction.One aspect of the present invention provides a kind of promotor, and the nucleotides sequence of this promotor is classified SEQ ID NO:1 as.
The present invention also provides recombinant vectors, expression cassette, transgenic cell or the recombinant bacterium that contains above-mentioned promotor.
Described recombinant vectors is to insert at the multiple clone site place of carrier the recombinant plasmid obtaining after described promotor.
Described expression cassette is by described promotor, the goal gene of this promoter expression, and transcription termination sequence forms; Described promotor is connected with described goal gene in functional mode, and described goal gene is connected with described transcription termination sequence.
The present invention has an aspect to be to provide the application in expression of plants at startup goal gene of described promotor., when temperature-induced, be preferably particularly preferably when low temperature induction.
Described low temperature induction, refers to that some plant genes are under cold environmental conditions (referring generally to be greater than 0 ℃, lower than 15 ℃), starts the mode of expressing.
A further aspect of the invention has been to provide a kind of method of utilizing described promotor to cultivate transgenic plant, comprises the steps:
1) build goal gene recombinant expression vector: goal gene is inserted in the recombinant vectors that contains described promotor, low temperature induction makes described promotor start described destination gene expression, obtains goal gene recombinant expression vector;
2) goal gene recombinant expression vector step 1) being built imports in object plant, and low temperature induction makes described promotor start described destination gene expression, obtains expressing the transgenic plant of described goal gene.
A further aspect of the invention has been to provide and has a kind ofly made plant when low temperature induction, express the method for goal gene, it is that goal gene is inserted in the recombinant vectors that contains described promotor, and this recombinant vectors is imported in object plant, then low temperature induction goal gene is expressed in plant.
Promotor of the present invention can provide for the research and development of plant bioreactor experiment and technical support.Utilize described low temperature induction type promotor, using plant as bio-reactor, express vaccine, antibody and other pharmaceutical proteins etc., can break through traditional agriculture category, extend to medical field.
Accompanying drawing explanation
Fig. 1: promotor of the present invention
pCD1 build PCR electrophorogram.
Fig. 2: promotor of the present invention
pCD1 t vector plasmid enzyme is cut evaluation electrophorogram.
Fig. 3: promotor of the present invention
pCD1 connect pC0390GUS enzyme and cut evaluation electrophorogram.
Fig. 4: under low temperature induction condition, promotor
pCD1 the gus gene histochemical stain of transformation of tobacco blade.
Fig. 5: under low temperature induction condition, promotor
pCD1 with the GUS specific activity contrasting.
Embodiment
Below in conjunction with accompanying drawing, method of the present invention is described in detail, but object described below is exemplary, rather than limits the scope of the invention.
Embodiment 1: low temperature induction type promotor of the present invention
pCD1separation
The preparation of 1.1 sweet buckwheat genomic dnas
By the sweet buckwheat seedling with culture medium, at illumination condition, be 8000LUX, temperature is 25
ounder C condition, grow 3 weeks.Then proceeding to illumination condition is 8000LUX, then adopts the plant genes group DNA rapid extraction test kit (catalog number (Cat.No.) DN15) of Ai Delai bio tech ltd, Beijing to extract DNA.
1.2 promotor
pCD1 clone and sequential analysis
The DNA extracting above of take is template, with PCR method amplification FeDREB1 upstream region of gene promoter sequence.Its amplimer is as follows, introduces Hind III restriction enzyme site in its upstream primer, introduces Bamh I restriction enzyme site in downstream primer:
Upstream primer: 5'-CCCAAGCTTATAGACCTAGTCAGGATCA-3'
Downstream primer: 5'-GCGGATCCGATCGATCCTGAATATAT-3'
PCR reaction system
PCR response procedures: 94
oc sex change 5 min; 94
oc sex change 30 s, 60
oc 30 s that anneal, 72
oc extends 2 min, 29 circulations; 72
oc 10 min, 4
oc insulation.
With the agarose gel electrophoresis of 1.0 %, detect PCR result, see Fig. 1.
The recovery Yong Tiangen biochemical technology company limited DNA of goal gene DNA fragment from sepharose reclaims test kit and reclaims, and operation is carried out according to test kit explanation.
PMD19-T Simple Vector) ligation
Ligation operates according to the explanation of the PMD19-T Simple Vector of takara company support agent box, and ligation system is as follows:
Mix reactant, of short duration centrifugal, 4 ℃ are spent the night.
Evaluation with positive colony
The intestinal bacteria that are used for transforming are that DH5a(is purchased from Tian Gen biochemical technology company limited).The preparation of competent cell all operates with the conversion that is connected product under aseptic condition.Picking transforms the white colony on flat board, adopt plasmid to extract on a small quantity test kit and extract plasmid (being purchased from Tian Gen biochemical technology company limited) as template, according to foregoing PCR primer and amplification condition, carry out pcr amplification, through agarose gel electrophoresis, detect the positive colony that produces 270bp gene, positive colony called after
pCD1 .
4. sequence verification
Positive colony through identifying, send Sangon (Shanghai Sheng Gong Bioisystech Co., Ltd) to carry out DNA sequencing, and its nucleotide sequence is as shown in sequence table 1.
embodiment 2: promotor
pCD1the structure of plant transient expression carrier
Utilize Bam HI and
hind IIIendonuclease enzyme is cut recombinant cloning vector (T+
pCD1 ) and
pC0390::GUScarrier, then will
promotor
pCD1 with
pC0390::GUSthe gus reporter gene of carrier connects, and builds plant transient expression carrier (Fig. 2, Fig. 3).The plant transient expression carrier called after building
pCD1::GUS.
pC0390::GUScarrier and
pC35S::GUScarrier is provided by xuwei doctor Rong of Ningxia University, concrete building process reference literature (Xu etc., 2010, Planta, 231:475 – 487).Adopt electric shocking method, by what build
pCD1::GUScarrier,
pC0390::GUScarrier (negative control) and
pC35S::GUScarrier (positive control) imports respectively in agrobacterium tumefaciens competence bacterial strain GV3101 with standby.
embodiment 3: identify under cold condition promotor
pCD1active
3.1
3.1.1 tobacco is cultivated and instantaneous conversion
Ben Shi tobacco seed, is seeded in the sterilization matrix of nutrition pot after 1 week through 4 ℃ of subzero treatment, and covers preservative film, in seeding room, grows after within 3 weeks, emerging and moves into artificial intelligence weather incubator.In incubator, cultivate the tobacco plant of 5 weeks for agriculture bacillus mediated instantaneous conversion analysis.Concrete method for transformation is as follows: 1. inoculation comprises Agrobacterium 20 μ l that promotor and GUS construct in the fresh LB liquid nutrient medium of 20mL, adds Rifampin 50mg L
-1, Kan 50mg L
-1, in 28 ℃ of constant-temperature table 240rpm, incubated overnight; 2. the Agrobacterium bacterium liquid of incubated overnight is transferred in the sterilizing centrifuge tube of 50mL, at room temperature 6000rpm, centrifugal 10min; 3. abandon supernatant, and with infiltration damping fluid (10 mM MES, the 10 mM MgCl of 10mL
2, 100 μ M AS pH, 5.7) and resuspended thalline; 4. get the bacterium liquid 2mL after resuspended, measure OD600 value, with permeating bacterium liquid that damping fluid is diluted to OD600 value approximately 0.6 for conversion; 5. before the Agrobacterium bacterium liquid injection tobacco leaf epidermic cell with after dilution, tobacco is taken out from climate box, and be placed in 1h under incandescent light, pore is opened completely and be beneficial to injection infiltration.6. with the syringe of the needle-less of 1mL, draw ready Agrobacterium bacterium liquid, the injection point choosing, props up syringe needle, slowly bacterium liquid is injected to the intercellular substance of tobacco leaf, makes bacterium liquid be penetrated into as far as possible whole blade.7. the tobacco plant after infiltration covers preservative film, puts back to weather incubator, renewal cultivation 1d; 8. cultivate the low temperature incubator that transformation of tobacco plant after 1 d is put into 4 ℃ and carry out low temperature induction processing; 9. above-mentioned each transformation of tobacco plant is cultivated after 1 d in low temperature stress processing and normal condition respectively, and the blade that clip adjoining tree and low temperature stress are processed plant is immediately further to analyze use.
3.1.2 transformation of tobacco plant leaf GUS expression activity is analyzed
GUS histochemical stain is with reference to the scheme of Jefferson (1987).Tobacco leaf material is placed in to sterilizing plate, adds GUS staining fluid (10 mM Na
2eDTA, 100 mM NaH
2pO
4, 0.5 Mm K
4fe (CN)
63H
2o, 0.1% Triton X-100 and 0.5 mg L
-1x-Gluc, pH 8.0) covering material; The constant incubator that plate is placed in to 37 ℃ is cultivated 24h; Reclaim staining fluid; Give in plate and add 70% ethanol, in 37 ℃ of incubation 5-6h; Outwell the ethanol in plate, add 90% ethanol, at 37 ℃, continue incubation 10h; Outwell ethanol, carry out GUS dyeing and observe and take a picture.Contrast and the processing Tissues of Tobacco of getting 1g left and right are placed in mortar, powdered by liquid nitrogen grinding, add Extraction buffer (containing 50 mM L
-1sodium phosphate, 10 mM L
-1eDTA, 0.1% TritonX-100,1% Sarcosyl, 10 mmol L
-1beta-mercaptoethanol), mix, 4 ℃, after the centrifugal 10min of 5000 rpm, get supernatant liquor and obtain GUS soluble proteins crude extract.Take out part and add 1 mM L
-1gUS reaction substrate 4-MUG, 37 ℃ insulation 30 min after, add 0.2 m M L
-1na
2cO
3reaction terminating liquid termination reaction.Utilize Hitachi's 850 type fluorescent spectrophotometer assay fluorescent values, exciting light 365 nm, utilizing emitted light 455 nm.Soluble protein content is with reference to Bradford(1976) method mensuration, with UV-2102C ultraviolet spectrophotometer, measure GUS protein content, excitation wavelength 595 nm.The required enzyme amount that the 1 min hydrolysis 4-MUG of take generates 1 pM 4-MU is an enzyme activity unit, with the enzyme activity of every milligram of albumen, represents that GUS is active.Above-mentioned experiment in triplicate.
Fig. 4 shows, GUS activity is very strong demonstrates very dark blueness, and light blue expression activity is lower, without blue, represents that GUS activity is lower.Wild-type (WT) and conversion
pC0390::GUSthe tobacco leaf of plant expression vector (negative control) can not be dyed blueness by X-Gluc solution under normal growth, low temperature induction condition, but transforms
pC35S::GUSthe tobacco leaf of plant expression vector all can by X-Gluc solution, be dyed blueness, explanation under various conditions
pC0390::GUSplant expression vector can be used for the activation analysis of the promoter fragment of this experiment.Compare normal growth condition in contrast, transformed
pCD1::GUStobacco leaf under low temperature induction condition, by X-Gluc, dyed blueness, illustrated
pCD1(270bp) is low temperature induction type promotor.The fluorescent quantitation result of GUS in Fig. 5 shows,
pCD1(270bp) promotor section activity is the activity 32.6% of positive control, illustrates that this promotor is a kind of promotor of high efficient cryogenic induction type.
3.2
Select respectively Arabidopis thaliana, paddy rice and sweet buckwheat, use and 3.1 similar approach, analyze the activity of promotor pCD1 activity under cold condition.Found that, compare normal growth condition in contrast, transformed
pCD1::GUSarabidopis thaliana, paddy rice and sweet buckwheat blade under low temperature induction condition, all by X-Gluc, dyed blueness, illustrated
pCD1(270bp) is low temperature induction type promotor.The fluorescent quantitation of GUS shows, in three kind of plant
pCD1(270bp) promotor section activity is respectively activity 36.2 %, 32.5% and 37.8% of positive control, illustrates that this promotor is a kind of promotor of high efficient cryogenic induction type.
Sequence table
<110> Changjiang University
<120> low temperature induction type promotor and application thereof
<160> 3
<170> PatentIn version 3.5
<210>1
<211> 270
<212> DNA
The sweet buckwheat of <213>
<220>
<223> promotor pCD4
<400> 1
atagacctag tcaggatcac gactgtactc ccttacccag aaacctcctt acgaacactt 60
gcgccagctc cctgaatatc caatccgcca gaccgccacc agctgtctcc tccgtgttaa 120
ccaccaccac actcttccca cactctattt cctcgtcccc aattcaacca acactatata 180
aaatcacacc atccatctta caacttaatt agattaaatt cttaaatctt aatcaactca 240
aaattacttc atatatattc aggatcgatc 270
<210>2
<211> 28
<212> DNA
<213> artificial sequence
<220>
<223> primer 1
<400> 2
cccaagctta tagacctagt caggatca 28
<210>3
<211> 26
<212> DNA
<213> artificial sequence
<220>
<223> primer 2
<400> 3
gcggatccga tcgatcctga atatat 26
Claims (7)
1. a promotor, is characterized in that the nucleotides sequence of this promotor is classified SEQ ID NO:1 as.
2. contain recombinant vectors, expression cassette, transgenic cell or the recombinant bacterium of promotor as claimed in claim 1.
3. promotor as claimed in claim 1 is starting the application of goal gene in expression of plants.
4. promotor as claimed in claim 3 starts the application of goal gene in expression of plants when temperature-induced.
5. promotor as claimed in claim 4 starts the application of goal gene in expression of plants when low temperature induction.
6. the method for utilizing the promotor described in claim 1 to cultivate transgenic plant, comprises the steps:
Build goal gene recombinant expression vector: goal gene is inserted in the recombinant vectors that contains described promotor, low temperature induction makes described promotor start described destination gene expression, obtains goal gene recombinant expression vector;
The goal gene recombinant expression vector that step 1) is built imports in object plant, and low temperature induction makes described promotor start described destination gene expression, obtains expressing the transgenic plant of described goal gene.
7. one kind makes plant when low temperature induction, express the method for goal gene, it is characterized in that the method is that goal gene is inserted in the recombinant vectors that contains promotor as claimed in claim 1, and this recombinant vectors is imported in object plant, then low temperature induction goal gene is expressed in plant.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845973A (en) * | 2015-06-11 | 2015-08-19 | 安徽省农业科学院水稻研究所 | Cloning and applications of low-temperature strong inducible promoter POscold6 of rice |
| CN114107305A (en) * | 2021-12-14 | 2022-03-01 | 朱博 | Low-temperature inducible enhancer and application thereof in enhancing gene expression during low-temperature induction of plants |
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| CN103483436A (en) * | 2013-08-27 | 2014-01-01 | 西北农林科技大学 | Common buckwheat DREB (Dehydration Responsive Element Binding protein) transcription factor |
| CN104250647A (en) * | 2014-09-02 | 2014-12-31 | 长江大学 | Drought induced type promoter and application thereof |
-
2014
- 2014-09-02 CN CN201410442189.XA patent/CN104195138B/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103483436A (en) * | 2013-08-27 | 2014-01-01 | 西北农林科技大学 | Common buckwheat DREB (Dehydration Responsive Element Binding protein) transcription factor |
| CN104250647A (en) * | 2014-09-02 | 2014-12-31 | 长江大学 | Drought induced type promoter and application thereof |
Non-Patent Citations (4)
| Title |
|---|
| Z.W. FANG ET AL.: "Characterization of FeDREB1 promoter involved in cold- and drought-inducible expression from common buckwheat (Fagopyrum esculentum)", 《GENETICS AND MOLECULAR RESEARCH》, vol. 14, no. 3, 17 July 2015 (2015-07-17), pages 7990 - 8000 * |
| ZHENGWU FANG ET AL.: "A Buckwheat (Fagopyrum esculentum) DRE-Binding Transcription Factor Gene, FeDREB1, Enhances Freezing and Drought Tolerance of Transgenic Arabidopsis", 《PLANT MOL BIOL REP》, vol. 33, 31 January 2015 (2015-01-31), pages 1510 - 1525 * |
| 方正武: "甜荞FeDREB1基因及其启动子的功能分析", 《WWW.CNKI.NET》, 30 June 2015 (2015-06-30) * |
| 李凤艳 等: "小麦转录因子TaDREB6 基因启动子的克隆及活性分析", 《麦类作物学报》, vol. 31, no. 5, 15 September 2011 (2011-09-15), pages 793 - 798 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104845973A (en) * | 2015-06-11 | 2015-08-19 | 安徽省农业科学院水稻研究所 | Cloning and applications of low-temperature strong inducible promoter POscold6 of rice |
| CN104845973B (en) * | 2015-06-11 | 2017-10-13 | 安徽省农业科学院水稻研究所 | The strong evoked promoter POscold6 of paddy rice low clone and application |
| CN114107305A (en) * | 2021-12-14 | 2022-03-01 | 朱博 | Low-temperature inducible enhancer and application thereof in enhancing gene expression during low-temperature induction of plants |
| CN114107305B (en) * | 2021-12-14 | 2023-11-28 | 朱博 | Low-temperature induction type enhancer and application thereof in enhancing gene expression during low-temperature induction of plants |
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