CN106636125A - Method for improving basic stress resistance of plants and application of method - Google Patents
Method for improving basic stress resistance of plants and application of method Download PDFInfo
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
Abstract
The invention discloses a method for improving the basic stress resistance of plants and application of the method, and belongs to the field of stress resistance of plants. The expression quantities of in-vivo ERF-VII (ethylene response factor VII) members of the plants are subjected to up-regulation by the aid of biotechnologies, and the basic resistance of the plants can be improved after the in-vivo expression quantity of an ERF-VII in each plant is subjected to up-regulation. Expression of reactive oxygen species synthesis key genes RbohD (respiratory burst oxidase homologue D) of the plants is subjected to up-regulation by the aid of the ERF-VII under abiotic stress such as drought, strong-light, high-temperature and heavy-metal-ion stress after expression of the ERF-VII members is subjected to up-regulation, adversity signals are transferred via reactive oxygen species, the plants can sense the stress early, related stress response genes can be subjected to up-regulation, and accordingly the adversity tolerance of the plants can be enhanced. The method and the application have the advantages that the fact that the basic stress resistance of the plants can be obviously improved by ERF-VII family gene members AtERF74 of over-expression Arabidopsis thaliana is discovered, the signals can be quickly burst and transferred by the plants via the ROS (reactive oxygen species), and the downstream stress response genes can be activated and respond to the environmental stress.
Description
Technical field
The invention belongs to plant stress-resistance field, more particularly to a kind of to improve the degeneration-resistant method in plant basis and its application.
Background technology
As climate warming and greenhouse effects are increasingly serious, plant and crop are faced with increasing environment-stress,
Such as arid, water logging, high temperature, salt stress, environment-stress becomes one of important factor in order for limiting crop yield.
Active oxygen (Reactive oxygen species, ROS) is the middle product that oxygen molecule is produced in reduction reaction
Thing.Wherein, H2O2It is the most important members of ROS, the ROS of other forms can be converted into H2O2, H2O2Can readily penetrate through
Cell membrane and plasma membrane so as to can flow freely with iuntercellular in the cell.On the one hand, ROS is to DNA, protein, unsaturated lipid etc.
Macromolecular has obvious destruction, so that protein degradation, Lipid peroxidation metabolism, DNA break；The also conduct of another aspect ROS
One important signaling molecule, participates in regulation and control plant and grows and various Stress responses, such as the defence of fungi invasion, non-
Synthesis regulation of response, the regulation and control of pore folding and lignin of biotic etc..In plant normal growth, ROS mainly makees
For the metabolite of mitochondria, chloroplaset and peroxisome, and in environment stress, ROS can pass through in cytoplasm
Three type peroxidating of nadph oxidase (Respiratory burst oxidase homologue, Rboh) and cell wall-bound
Thing enzyme (cell wall bound type III peroxidases) is produced.It is this rapid to produce in plant stress-resistance early stage
ROS is called ROS and bursts out, and bursting out for this ROS signals can be perceived by plant and transmitted toward downstream as alarm signal,
So as to start various stress response mechanism.
The content of the invention
In order to overcome the shortcoming and deficiency of prior art, it is an object of the invention to provide a kind of raising plant basis is degeneration-resistant
Method.Subfamily (the ethylene response of the ethylene responses factor the 7th in plant body are raised using biotechnology
Factor VII, ERF-VII) member expression, after expressions of the ERF-VII in plant body is raised, the basis of plant resists
Inversion adds.The present invention relates to after ERF-VII member's up-regulated, under various abiotic stress, such as arid, high light, high temperature, weight
Under metal ion stress, plant synthesizes the expression of key gene RbohD by ERF-VII upregulated activities oxygen, and by active oxygen
Transmission adverse circumstance signal, makes plant sense stress earlier, related stress response gene is raised, so that tolerance of the plant to adverse circumstance
Property strengthen.
Another object of the present invention is to provide the application of the degeneration-resistant method in above-mentioned raising plant basis.
The purpose of the present invention is achieved through the following technical solutions：
It is a kind of to improve the degeneration-resistant method in plant basis, improve plant by raising the ERF-VII family genes of plant
It is basic degeneration-resistant.
ERF74 gene upregulations are adjusted on described ERF-VII family genes.
It is adjusted on described ERF-VII family genes and ERF-VII family gene members is raised by transgenic technology, reaches
The basic degeneration-resistant purpose of plant.
Described plant is arabidopsis, willow, eucalyptus or paddy rice.
Further, described plant is arabidopsis.
The plant that a kind of ERF-VII family genes are raised, is obtained by said method.
The plant that described ERF-VII family genes are raised normally is being planted, and salt-soda soil is difficult to planting plants with other
Plant in soil.
Described improves application of the degeneration-resistant method in plant basis in raising plant basis is degeneration-resistant.
During the current physiology in plant stress-resistance is studied with molecular regulation, existing many researchs show that adverse circumstance can cause H2O2's
Accumulation, and have found H2O2Signal can be transmitted as signaling molecule, make plant responding adverse circumstance.But for H2O2In adverse circumstance how
Produced rapidly, how to carry out conduction and the research of the molecular mechanism how to be eliminated as signal and still extremely lack.The present invention
Meaning be propose H2O2How to be regulated and controled by ERF-VII family members in adverse circumstance early stage, bursting out and transmit signal rapidly makes plant
Response adverse circumstance.The expression that the present invention passes through raising ERF-VII family members, improves the basic degeneration-resistant of plant.
First by using chimeric suppressor silent technology, (2004) Hiratsu et al. suppress arabidopsis to the present invention
The transcriptional activity of ERF-VII family members ERF71～ERF75, studies function of the ERF-VII families in plant.Research is sent out
Existing, ERF74dominant repressor (ERF74-DR) transfer-gen plants drop significantly to the tolerance of high light and drought stress
It is low.Based on the working experience of inventor, inventor thinks that this phenotype is caused by ROS balances are destroyed in plant body
's.By the analysis to ERF-VII family members in arabidopsis gene group, in arabidopsis gene group, ERF-VII families are total to
There are 5 members, be respectively ERF71～ERF75, based on now to the progress of ERF-VII families, inventor is thought in ERF-
ERF74 in VII families, ERF75 play Main Function.And then inventor constructs 35S::ERF74 overexpression plant, and from
ABRC (Arabidopsis Biological Resource Center) has been subscribed to erf74, the mutant of erf75, and right
They are hybridized, and finally give erf74erf75 double-mutant plant.
By 35S::ERF74, WT, erf74, erf75, erf74erf75 plant carries out drought stress and processes discovery,
Erf74erf75 mutant plants are seriously withered after arid 10 days, and erf74 also has slight wilting symptoms, and erf75 is then without bright
Aobvious wilting symptoms, and 35S::ERF74 then grows more more healthy than wild type.Above plant is watered after recovering 3 days, 35S::
ERF74, WT, erf75 plant phenotype is restored, and the plant that erf74erf75 mutant plants then have half can not recover table
Type and it is dead, other continue survive plant also grow less than wild type.Although major part erf74 mutant plants water after simultaneously
Without death, but observe and find that its some leaf is withered blackening because of dehydration.
Then to 35S::ERF74, WT, erf74, erf75, erf74erf75 plant carries out high light and processes and observe phenotype,
Research finds that erf74erf75 blades have turned into atropurpureus, and overexpression and WT lines blade are still green, to the above
Plant leaf carries out anthocyanidin content and determines discovery erf75, and the anthocyanidin content of erf74, erf74erf75 plant compares wild type
5～15 times are risen, this shows erf75, erf74, erf74erf75 plant is subject to different degrees of strong ligh stress.
In order to whether further study ERF74 and ERF75 in the stress that other are not studied, such as high temperature and heavy metal adverse circumstance rises
Effect, inventor is to 35S::The protoplast of ERF74, WT, erf74, erf75, erf74erf75 carries out 42 DEG C of high-temperature process,
After high-temperature process 0S, 180S, 600S, 900S, diacetic acid fluorescein (fluorescein diacetate, FDA) is added to carry out
Dyeing, and determine the survival condition of protoplast using Flow cytometry.It is found that 35 from streaming figure::ERF74 crosses table
There is higher tolerance to high temperature up to protoplast, still there is 50% cell survival after high-temperature process 900S, and
Erf74erf75 mutant plants are decreased obviously to the tolerance of high temperature, just only 37.8% cell after high-temperature process 180S
Survival, and arrived the cell survived after 900S and be only left 20.9%.This shows ERF74 and ERF75 in the response of high temperature stress
Play an important role.
Inventor also carries out aluminium chloride process to above protoplast, and using Flow cytometry cell survival rate is determined.Grind
Study carefully and find erf74, the different degrees of decline of survival rate appearance under aluminium chloride process of erf74erf75 mutant, 35::ERF74
Overexpression protoplast then has higher tolerance to aluminium chloride process.This shows ERF74 and ERF75 equally in heavy metal ion
Play an important role in the response of stress.
The above adverse circumstance process test result indicate that, overexpression ERF-VII family members ERF74 can improve plant in arabidopsis
Tolerance of the strain to arid, high light, high temperature and heavy metal ion, and erf75, erf74, erf74erf75 plant then to it is arid,
The tolerance of high light, high temperature and heavy metal ion weakens.This shows that ERF74 and ERF75 play an important role in plant stress-resistance.
Hereinafter act on played in plant stress-resistance for ERF74 and ERF75 and being illustrated.
The change of the expression in Stress treatment and recovery with qRT-PCR and trans-activation method analysis ERF71～ERF75
Change.Research finds that the expression of ERF74 and ERF75 is significantly gone up by high light, arid, high temperature and aluminium chloride induction, expression
Rise, and after stress terminates, the expression of ERF74 and ERF75 is lowered to quickly original level.And the expression of ERF71～ERF73
Although also can be induced by certain stress, inducing amount is totally be not as obvious as ERF74 and ERF75.ERF74 and ERF75 exist
The rising of expression in adverse circumstance shows that it plays a significant role in plant stress.
In order to study in water logging, under arid and high light process, the change that AtERF74 is positioned in plant, by AtERF74
Proceed to pEarleyGate101 (p35S::AtERF74-GFP) fusion protein is formed.Agrobacterium C58 is proceeded to again, infects Tobacco Leaf
Piece.Observe that under normal circumstances major part is positioned in cell membrane AtERF74 under laser confocal microscope, and in water logging
(conversion stops watering for first 10 days, and lasts till observation, altogether for (observation under light Laser Scanning Confocal Microscope at once after water logging 3h), arid
Arid 13 days) and high light (observation 300 μm of olm of front 36h placements-2·s-1In the incubator of light intensity) under treatment, AtERF74
Will be transferred in nucleus from cell membrane.This shows ERF74 under the conditions of normal plants, in being positioned at cell membrane, no
Transcriptional activation is played, and when plant is forced, moment is reoriented in nucleus.
To 35S::ERF74, WT, erf74, erf75, erf74erf75 protoplast or plant carry out high temperature, ABA,
AlCl3, water logging, arid and high light process, and using 6- carboxyl -2,7- dichlorofluorescein ethyl acetate (H2DCFDA, 2,7-
Dichlorodihydrofluorescein diacetate, a kind of nonpolar chemicals, can free diffusing enter cell, its quilt
The DCF with high fluorescent can be generated after ROS oxidations) cell is dyeed, then by Flow cytometry to cell body
Interior ROS amounts are detected.Research finds that the ROS early stages of erf75, erf74, erf74erf75 protoplast burst out by difference
The suppression of degree, and 35S::The ROS of ERF74 protoplast early stages bursts out, and appears earlier higher.This show ERF74 and
ERF75 bursts out to the ROS of plant degeneration-resistant early stage and plays indispensable effect.
RbohD (NADPH-dependent oxidase D) is the important gene that plant produces ROS, and inventor utilizes
The increase of qPCR and trans-activation method detection ERF74 and ERF75 lacks tables of the RbohD under adverse environmental factor whether can be affected
Up to amount.Research shows, the expression of RbohD is subject to arid, high light, water logging, the induction of high temperature and chlorination Acid-Al stress, and this lures
Lead in 35S::Strengthen in ERF74 plant, and had weakened at different degrees in erf75, erf74, erf74erf75 plant.
Before this have plurality of articles report, ERF transcription combined with GCC elements (Fujimoto et al., 2000).
By promoter Analysis, RbohD upstreams 1 are found, 709bp has a GCC element.Inventor utilizes Gateway system constructings
The RbohDpro2-LUC (lacking GCC elements) two of the RbohDpro1-LUC (containing GCC elements) and 1.676kb of 1.876kb
Carrier and core 35S::ERF71-75 (ERF71-75-PUGW2) carrier is transferred to arabidopsis wild-type protoplasts cell jointly
In.Research shows that ERF71, ERF73, ERF75 have weaker activation to RbohD promoters, and ERF74 is then opened RbohD
Mover has stronger activation.After RbohD promoters lose GCC elements, the activation of ERF74 have dropped 60%.
In order to obtain the positive evidence that AtERF74 is combined with RbohD, inventor has carried out gel shift experiment
(electrophoretic mobility shift assay, EMSA).ERF74 albumen is mixed with the GCC elements of RbohD promoters
Substantially occur being detained band after conjunction, it was confirmed that ERF74 and RbohD directly in conjunction with.After GGCGGC series jumps into GGAGGA,
It is detained band to disappear；Add 20 times and 100 times not containing after the RbohD probe of biotin labeling, be detained band brightness obvious
Weaken, mutating experiment and competitive assay show ERF74 and RbohD probe specific bonds.In sum.Inventor is by anti-
Formula activation experiment and gel shift experiment confirm AtERF74 by GCC elements and RbohD directly in conjunction with.
Inventor has found water logging responsive genes SUS4, LDH, responses of drought stress gene by qPCR and trans-activation detection
RD20, aluminium chloride responsive genes AOX1a, the induction of high-temperature response gene HSP18.2 all relies on ERF74-Rbohd signals and leads to
Road, when WT lines or protoplasm somatocyte are after 10 μM of DPI (RbohD inhibitor) pretreatments of 30 minutes, the above is rung
The induction for answering gene is suppressed, and carries out similar experiment with rbohd mutant and same situation also occurs.And
Erf74erf75 mutant is also that the induction of stress response gene is pressed down because the expression of RbohD can not be raised in stress
System.
In addition, there is also ERF-VII families in the plants such as willow, eucalyptus, paddy rice, their overexpression plant also has can
The result of expected basic degeneration-resistant raising.The overexpression plant of ERF-VII family genes is in plants such as willow, eucalyptus, paddy rice
In can also be obtained by transgenosis.
The present invention has the following advantages and effect relative to prior art：
With climate warming and greenhouse effects it is increasingly serious, environment-stress become limit crop yield material impact because
One of element.And with expanding economy, China can be fewer and feweri with arable land.Trees, the base of crops are increased by transgenosis
Plinth resistance then in a disguised form increased the yield of trees and crop, reduce planting cost, make the soil of more arids and heavy metal pollution
Ground is adapted to plant trees and plant.To improving air quality, pressure important role is alleviated in soil erosion.The present invention
It is found that overexpression arabidopsis ERF-VII family gene member AtERF74 can significantly improve plant basis resistance, leads to plant
Cross faster that promptly ROS bursts out transmission signal, activate downstream stress response gene, response environment stress.
Description of the drawings
Fig. 1 is the phenotype of ERF74-DR transfer-gen plants.
Fig. 2 is 35S::ERF74, WT, erf74, erf75, erf74erf75 in normal growing conditions, drought condition and strong
The phenotype of optical condition.
Fig. 3 is 35S::ERF74, WT, erf74, erf75, erf74erf75 protoplast is processed in heat treatment and aluminium chloride
Period, cell survival rate is schemed over time.
Fig. 4 is AtERF74 in normal growing conditions, water logging, the Subcellular Localization figure under arid and high light process.
Fig. 5 is 35S::ERF74, WT, erf74, erf75, erf74erf75 plant or protoplast under various process,
Average ROS is measured with the curve map for processing time change in cell body.
Fig. 6 is 35S::ERF74, WT, erf74, erf75, erf74erf75 plant under the conditions of different disposal, RbohD tables
Up to amount variation diagram.
Fig. 7 is 35S::ERF74, WT, erf74, erf75, erf74erf75 protoplasm somatocyte is in different condition, RbohD
Gene expression amount variation diagram.
Fig. 8 is that trans-activation checking ERF74 is combined by GCC elements with RbohD.
Fig. 9 is that EMSA checking ERF74 is combined by GCC elements with RbohD.
Figure 10 is the change of stress response gene expression under different adverse environmental factors.
Specific embodiment
With reference to embodiment and accompanying drawing, the present invention is described in further detail, but embodiments of the present invention are not limited
In this.
The experimental technique of unreceipted specific experiment condition in the following example, generally according to conventional laboratory conditions or according to system
Make the experiment condition proposed by manufacturer.
Embodiment 1
PERF74-ERF74 is proceeded in PHGEAR carriers.Agrobacterium C58 is proceeded to again, in infecting arabidopsis WT, T1 generation tides
Mycin screening obtains the transgenosis of the positive and obtains ERF74dominant repressor (ERF74-DR) transfer-gen plant, observation
There is blade nigrescence phenomenon in phenotype, the transfer-gen plant (ERF74-DR) for finding 56.8%, sees Fig. 1 (a).Further carry out arid
(Drought) adverse circumstance test, after ERF74-DR and WT plant are normally watered 15 days, stopping is watered 15 days, finds ERF74-DR
Plant is withered, and WT plant strain growths are also more normal, see Fig. 1 (b).
Embodiment 2
By 35S::ERF74, WT, erf74, erf75, erf74erf75 seed after planting, normal growth.Drought tests are
Recover to water 3 days and observe phenotype after the plant of 15 days normal growths is stopped watering 15 days, as a result as shown in Fig. 2 finding
Erf74, erf74erf75 weaken to drought tolerance, and 35S::ERF74 then strengthens drought tolerance.High light (HL) is tested
It is that 20 days normal growth plant are transferred under intense light conditions to grow 10 days and observe phenotype.The another plant by sowing simultaneously is just
Growth is used as control under the conditions of being frequently grown.As a result as shown in Fig. 2 erf75, erf74, erf74erf75 subtract to high light tolerance
It is weak, 35S::ERF74 strengthens high light tolerance.
Embodiment 3
The just normal growth 35S of 20～25 days::ERF74, WT, erf74, erf75, erf74erf75 plant is extracted former
After raw plastid, 42 degree of high temperature (Heat) are carried out and after 0.5mM aluminium chloride (Al) the process scheduled time, add FDA to be dyeed, lead to
Overflow-type cell instrument carries out detecting the life or death of protoplasm somatocyte.Experiment finds, as a result as shown in figure 3,35::ERF74 overexpression
Plant pair high temperature has higher tolerance, the cell survival for still having 50% after high-temperature process 900S, and erf74erf75 is prominent
The tolerance of variant plant pair high temperature is decreased obviously, just only 37.8% cell survival after high-temperature process 180S, and arrives
The cell survived after 900S is only left 20.9%.And aluminium chloride result is also close with high-temperature process result.
Embodiment 4
In order to study at water logging (Flooding), under arid and high light process, the change that AtERF74 is positioned in plant
Change, AtERF74 is proceeded into pEarleyGate101 (p35S::AtERF74-GFP) fusion protein is formed.Agriculture bar is proceeded to again
Bacterium C58, infects tobacco leaf.Observe that under normal circumstances major part is positioned at AtERF74 under laser confocal microscope
In cell membrane, and (convert and stop within first 10 days pouring in water logging (observation under light Laser Scanning Confocal Microscope at once after water logging 3h), arid
Water, and last till observation, it is arid 13 days altogether) and high light (observation 300 μm of olm of front 36h placements-2·s-1The incubator of light intensity
In) under treatment, AtERF74 will be transferred in nucleus from cell membrane, as a result as shown in Figure 4.
Embodiment 5
To 35S::ERF74, WT, erf74, erf75, erf74erf75 protoplast or plant carry out high temperature, ABA and (come off
Acid), AlCl3, water logging, arid and high light process, and using 6- carboxyl -2,7- dichlorofluorescein ethyl acetate (H2DCFDA, 2,7-
Dichlorodihydrofluorescein diacetate) cell is dyeed, then by Flow cytometry to cell
In vivo ROS amounts are detected.
First, to 35S::ERF74, WT, erf74, erf75, erf74erf75 protoplast carries out 20 μM of ABA process,
As a result as shown in Fig. 5 (b).A kind of ABA not environment stresses at last, simply a kind of transduction signal related to ROS generations.In ABA
When processing protoplast only 7 minutes, 35S::ERF74, WT, erf75 protoplast just produces obviously ROS and bursts out, and
Erf74 only produces a small amount of ROS and bursts out, and erf74erf75 is barely perceivable the generation that ROS bursts out, until ABA processes 15
During minute, just observe that obvious ROS bursts out in erf74erf75 protoplasts.And arrived ABA process 60 minutes when, send out
Existing erf74, the ROS total amounts of erf74erf75 plant are apparently higher than 35S::ERF74 and WT plant.
Then to 35S::ERF74, WT, erf74, erf75, erf74erf75 protoplasm somatocyte has carried out 42 DEG C of high temperature
Process, as a result as shown in Fig. 5 (a).In high-temperature process 45s, all of protoplasm somatocyte has different degrees of ROS to burst out
Occur, occur the peak that ROS bursts out during to 180s.Now, 35S::The ROS of the ERF74 protoplasm somatocytes intensity that bursts out is substantially high
In WT.And erf74 and erf74erf75 protoplasm somatocytes only have weaker ROS to burst out appearance.420s, all plasms are arrived
Somatic ROS bursts out and disappear substantially, and held stationary.ROS has in erf74 and erf74erf75 protoplasm somatocytes after 420s
It is a small amount of to rise, and 35S::ROS has but been reduced in ERF74 protoplasm somatocytes
Then to 35S::ERF74, WT, erf74, erf75, erf74erf75 protoplasm somatocyte carries out 0.5mM aluminium chloride
Process, as a result as shown in Fig. 5 (c).Research found when aluminium chloride processes protoplast 5～10 minutes, 35S::ERF74, WT are former
Raw plastid cell just produces obviously ROS and bursts out, and erf74, erf75 and erf74erf75 protoplasm somatocytes are only produced
A small amount of ROS bursts out.Arrived aluminium chloride process 60 minutes when, the ROS total amounts of erf74, erf74erf75 protoplasm somatocyte are much
Higher than 35S::ERF74 and WT protoplasm somatocytes.Inventor carries out water logging to above plant, arid, highlight test, as a result such as
Shown in Fig. 5 (d)～(f), similar result is also obtained.
Embodiment 6
The increase of ERF74 and ERF75 is detected using qPCR or lack expression of the RbohD under adverse environmental factor whether can be affected
Amount.By extracting 35S::ERF74, WT, erf74, erf75, erf74erf75 plant throughout manages the blade RNA of time period, and
Reverse transcription is analyzed discovery into cDNA finally by qPCR, and the expression of RbohD is subject to arid, high light and water logging condition
Induction, and this induction is in 35S::Strengthen in ERF74 plant, and had in erf75, erf74, erf74erf75 plant
Weakened at different degrees, as a result as shown in Figure 6.
Then RbohD about 1.8kb promoters are connected into (pRbohD-LUC) in Luciferase reporter genes, and this is carried
Body (pRbohD-PUGW35) proceeds to respectively 35S::In ERF74, WT, erf74, erf75, erf74erf75 protoplasm somatocyte,
Then 42 DEG C of high-temperature process are carried out to above protoplasm somatocyte 5 minutes, 20 μM of ABA and 0.5mM aluminium chloride are processed 15 minutes,
Then the fluorescence intensity of LUC reporter genes is detected, as a result as shown in Figure 8.Study and find under aluminium chloride, ABA and high-temperature process,
The fluorescence intensity of pRbohD-LUC reporter genes has all increased, but RbohD luring in erf74, erf74erf75 mutant
Directive/guide receives different degrees of suppression.
Embodiment 7
By promoter Analysis, RbohD upstreams 1 are found, 709bp has a GCC element, before this existing plurality of articles report
Road, ERF transcription combined with GCC elements (Fujimoto et al., 2000).So inventor utilizes Gateway system structures
The RbohDpro2-LUC (lacking GCC elements) of the RbohDpro1-LUC (containing GCC elements) and 1.676kb of 1.876kb is built
Two carriers.Inventor is by the two carriers and 35S::ERF71-75 (ERF71-75-PUGW2) carrier proceeds to respectively wild type
In protoplasm somatocyte.Research finds, as a result as shown in fig. 7, ERF71, ERF73, ERF75 have weaker to RbohD promoters
Activation, and ERF74 then has stronger activation to RbohD promoters.After RbohD promoters lose GCC elements,
The activation of ERF74 have dropped 60%.
In order to obtain the positive evidence that AtERF74 is combined with RbohD, gel shift experiment has been carried out
(electrophoretic mobility shift assay, EMSA).First by Escherichia coli, prokaryotic expression goes out GST-
ERF74 fusion proteins.As a result as shown in figure 9, gel shift experiment shows that ERF74 albumen is mixed with the GCC elements of RbohD promoters
Substantially occur being detained band after conjunction, it was confirmed that ERF74 and RbohD directly in conjunction with.And work as inventor GGCGGC series jumps into
After GGAGGA, it is detained band and disappears；Add 20 times and 100 times not containing after the RbohD probe of biotin labeling, be detained band
Brightness substantially weakens, and mutating experiment and competitive assay show ERF74 and RbohD probe specific bonds.
Water logging responsive genes SUS4, LDH, responses of drought stress gene RD20, chlorination are found by qPCR and trans-activation detection
Aluminium responsive genes AOX1a, the induction of high-temperature response gene HSP18.2 all relies on ERF74-Rbohd signal paths, works as wild type
After 10 μM of DPI (RbohD inhibitor) pretreatments of 30 minutes, above responsive genes are lured for plant or protoplasm somatocyte
Lead and be suppressed, carry out similar experiment with rbohd mutant and same situation also occur.And erf74erf75 mutant
It is that the induction of stress response gene is suppressed, as a result as shown in Figure 10 because the expression of RbohD can not be raised in stress.
Above-described embodiment is the present invention preferably embodiment, but embodiments of the present invention not by above-described embodiment
Limit, other any Spirit Essences without departing from the present invention and the change, modification, replacement made under principle, combine, simplification,
Equivalent substitute mode is should be, is included within protection scope of the present invention.
Claims (8)
- It is 1. a kind of to improve the degeneration-resistant method in plant basis, it is characterised in that：Raised by making the ERF-VII family genes of plant, To improve the basic degeneration-resistant of plant.
- It is 2. according to claim 1 to improve the degeneration-resistant method in plant basis, it is characterised in that：ERF74 gene upregulations are adjusted on described ERF-VII family genes.
- It is 3. according to claim 1 to improve the degeneration-resistant method in plant basis, it is characterised in that：It is adjusted on described ERF-VII family genes and ERF-VII family gene members is raised by transgenic technology, reaches plant Basic degeneration-resistant purpose.
- 4. the degeneration-resistant method in plant basis is improved according to any one of claims 1 to 3, it is characterised in that：Described plant Thing is arabidopsis, willow, eucalyptus or paddy rice.
- 5. the degeneration-resistant method in plant basis is improved according to any one of claims 1 to 3, it is characterised in that：Described plant Thing is arabidopsis.
- 6. the plant that a kind of ERF-VII family genes are raised, it is characterised in that by the side described in any one of Claims 1 to 5 Method is obtained.
- 7. the plant that ERF-VII family genes according to claim 6 are raised, it is characterised in that：The plant that described ERF-VII family genes are raised normally is being planted, and salt-soda soil is difficult to the soil of planting plants with other Plantation.
- 8. the degeneration-resistant method in plant basis answering in raising plant basis is degeneration-resistant is improved described in any one of Claims 1 to 5 With.
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| CN110272911A (en) * | 2019-07-05 | 2019-09-24 | 四川大学 | Application of the AOX1a gene in terms of improving drought resistance in plants |
| CN112501182A (en) * | 2020-12-07 | 2021-03-16 | 山西农业大学 | Poplar ERF transcription factor gene and application thereof |
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| CN108070027A (en) * | 2018-02-12 | 2018-05-25 | 中国农业科学院作物科学研究所 | The breeding method and its relevant biological material of waterlogging and volume increase transgenic wheat |
| CN110128516A (en) * | 2019-04-16 | 2019-08-16 | 扬州大学 | Barley moisture-proof controlling gene HvERF2.11, albumen and its application in breeding |
| CN110128516B (en) * | 2019-04-16 | 2022-04-22 | 扬州大学 | Barley moisture-resistant regulatory gene HvERF2.11, protein and application thereof in breeding |
| CN110272911A (en) * | 2019-07-05 | 2019-09-24 | 四川大学 | Application of the AOX1a gene in terms of improving drought resistance in plants |
| CN112501182A (en) * | 2020-12-07 | 2021-03-16 | 山西农业大学 | Poplar ERF transcription factor gene and application thereof |
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| Publication number | Publication date |
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| CN106636125B (en) | 2019-11-15 |
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