CN110408648A - A kind of method of quick detection plant disease resistance genes - Google Patents
A kind of method of quick detection plant disease resistance genes Download PDFInfo
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Abstract
The present invention provides a kind of methods of quickly detection plant disease resistance genes, comprising the following steps: imports in Agrobacterium disease-resistant gene, obtains recombinant bacterium；The back side that grape leave is infected with the recombinant bacterium, disease-resistant gene is transformed into grape leave, and the front inoculation pathogen of grape leave realizes the detection of disease-resistant gene；The disease-resistant gene is imported in Agrobacterium by recombinant vector；The recombinant vector is the carrier that target gene is cloned into the multiple cloning sites of expression vector and is obtained.The method method of quick detection plant disease resistance genes of the invention is suitable for the detection of expression of the quick instantaneous conversion foreign gene of various genetically modified plants.Have many advantages, such as plant transient expression experiment method simple and quick, expression quantity is high and relatively inexpensive.With the obtained instantaneous conversion plant body of this method can satisfy plant gene function analysis and in terms of application.It haves laid a good foundation further to study the molecular mechanism of grape disease-resistant gene.
Description
Technical field
The invention belongs to agricultural biological technical fields, and in particular to a method of quickly detection plant disease resistance genes
Background technique
Grape (Vitis vinifera L.) is that one of widest fruit is cultivated in China, is always that the whole world is most important
One of fruit, economic value is very high, not only can directly eat raw and also processable be fabricated to raisins, grape wine etc..Mesh
Preceding whole world grape -growing areas is very big, and cultivated area at least occupies the 10% of all types of fruits, by the end of the year 2013,
China's vinegrowing area has reached 71.46 ten thousand hm2, 11,550,000 tons of vintage.
Foreign gene is transferred in plant, is the main means for studying gene function.Genetic Transformation in Higher Plants includes steady
Fixed conversion and instantaneous conversion.It is had been applied in many crop species using the stable conversion technology of mediated by agriculture bacillus, is current
Study the common method of gene in vivo functionality.But the conversion of stable conversion technology and regeneration efficiency are low, and time-consuming long,
If carrying out functional study to multiple genes can take considerable time and energy.Transient expression (transienent gene
Expression) transient expression introduces the exogenous DNA of cell and the chromosomal DNA of host is not integrated, but these DNA are general
It can be expressed in 12 hours after cell can be entered with carrier, and certain time section can be stablized.Instant expression method is more steady
Determining expression has easy to operate, and the period is short, reacts working environment in plant, and expression efficiency is high, safe and efficient etc. many
Outstanding advantages.
The method for realizing transient expression includes that injection osmosis and infestation method, infestation method can infect plant according to different requirements
The different parts of beyond the region of objective existence implant.Such as: the seedling of plant, seed, inflorescence or callus are soaked in containing foreign gene
In bacterium solution, the conversion of foreign gene is realized, while in order to can be shortened the expression time of foreign gene, it is logical at the position for needing to convert
Crossing mechanical external force causes certain mechanical damage that bacterium solution is infected plant damaged part.So whether injection method or passing through
The method of mechanical damage carries out conversion method, and certain damage is all caused to explant, easily causes infection, is carrying out disease-resistant class
When detection, result is formed and is interfered, may not apply to the detection of disease-resistant gene function；And impregnate the portions such as the seedling of plant, seed
Position it cannot be guaranteed that foreign gene being transferred to and expressing, while needing seedling and seed to cultivate one week or so under illumination condition, together
When intensity of illumination it is excessively high, seedling be easy death, intensity of illumination is too low, and the growth conditions of seed and seedling, and illumination is not achieved
The expression translation that influences gene is protected from light that there are contradictions in the light requirement and gene expression of seedling and seed, therefore, by infecting kind
The method for transformation of son and seedling, it is difficult to applied to the application in terms of detection plant exogenous gene expression.
Therefore, a kind of method for finding quickly detection plant exogenous gene expression becomes urgently to be resolved in the art
Problem.
Summary of the invention
A kind of quickly detection plant disease-resistant is provided it is an object of the invention to overcome above-mentioned the deficiencies in the prior art place
The method of gene.
To achieve the above object, the technical scheme adopted by the invention is as follows:
A kind of method of quick detection plant disease resistance genes, comprising the following steps: disease-resistant gene is imported in Agrobacterium, is obtained
To recombinant bacterium；The back side that grape leave is infected with the recombinant bacterium, disease-resistant gene is transformed into grape leave, grape leave
Front inoculation pathogen, realizes the detection of disease-resistant gene；
The disease-resistant gene is imported in Agrobacterium by recombinant vector；
The recombinant vector is the carrier that target gene is cloned into the multiple cloning sites of expression vector and is obtained.
In the above method, the disease-resistant gene is VdWRKY70 gene.
In the above method, the method that the recombinant bacterium infects the back side of grape leave includes the following steps: grape leaf
The back side is floated on downward in the recombination bacterium solution, is placed in vacuum and is handled, and vacuum side of blade cell by after During Agrobacterium, is taken completely
It is placed in dark culturing in incubator out.
In the above method, the vacuum processing parameter are as follows: vacuum degree 20hPa (20mbar), 30 DEG C of processing 5min.
In the above method, the condition of culture are as follows: 24 DEG C of dark culturing 48h of temperature.
In the above method, the method for the front inoculation pathogen of the grape leave includes the following steps: that leaf is positive and connects
Kind of pathogen, 28 DEG C of dark are for 24 hours in incubator.
In the above method, the recombinant bacterium OD600 that the recombinant bacterium infects the grape leave back side is 0.4.
Beneficial effects of the present invention: the method for quick detection plant disease resistance genes of the invention is suitable for the wink of various plants
When convert foreign gene detection of expression.It is observed that within 48 hours the expression of foreign gene in this method, can see within 72 hours
Character mutation is observed, while expression efficiency reaches 100%；Meanwhile appropriate vacuum can help Agrobacterium foreign gene-carrying
Blade is infected from Stoma of Leaves, guarantees the integrality of blade cell, is conducive to the expression of gene and the identification of phenotype relative to it
His method.The characteristics of comprehensive this method, this method have many advantages, such as simple and quick, expression quantity height and relatively inexpensive.Use this method
Obtained instantaneous conversion plant body can satisfy plant gene function analysis and in terms of application, for into one
The molecular mechanism of step research grape disease-resistant gene is had laid a good foundation.
Detailed description of the invention
Fig. 1 is that (A is gus gene expression of results to gus gene of the present invention expression comparing result schematic diagram；B is pBI101 negative
Control).
Fig. 2 is that (it is disease-resistant that a is that the grape leave of transgenosis presents to disease-resistant gene of the present invention expression comparing result schematic diagram
Symptom, the grape leave morbidity without transgenosis is serious, and the blade not processed keeps fresh state；B is to pass through
After western blot detection, the GFP labelled protein expression on VdWRKY70 carrier is higher, and the grape leave of non-transgenosis is not examined
Measure similar expression albumen).
Specific embodiment
For more concise displaying technical solution of the present invention, objects and advantages, combined with specific embodiments below
And its attached drawing is described in further detail the present invention.
Embodiment 1
Step 1. building carries the plant expression vector of gus gene
1.1 E.coli DH5 α competent E.colis, Omega gel extract kit, LB culture medium etc. are purchased from
Henan Bao Sai scientific & technical corporation.
The plant expression vector of 1.2 gus genes uses
The preparation of 1.3 Agrobacterium LBA4404 competence: plate is drawn on the LB solid medium containing 50mg/L rifampin, is chosen
It takes unit cell to shake bacterium 48h in the LB liquid medium containing 50mg/L rifampin to bacterium solution muddiness, is inoculated into 1:100 ratio
Bacterium 5h is shaken in the fresh LB liquid medium containing 50mg/L rifampin of 50ml.Bacterium solution is placed in ice bath 30min on ice, 4 DEG C,
5000g is centrifuged 5min；The calcium chloride suspension that 1ml 0.1% is added in supernatant is abandoned, ice bath 5min, 4 DEG C, 5000g is centrifuged 5min；In abandoning
Reset and add the calcium chloride resuspension into 800 μ L 0.02%, the 8 every 100 μ L ice bath of pipe of pipe of packing is spare.
2 μ g are added in the 1.6 Agrobacterium competence prepared and need the plasmid that converts, mix, liquid nitrogen flash freezer 1min, 37 DEG C
1ml LB liquid medium is added in water-bath 5min, and 28 DEG C, 200rpm shakes bacterium 5h.Centrifugal concentrating is coated onto containing 50mg/L rifampin
On the LB plate of the kanamycins of 50mg/L, 28 DEG C of culture 48h, the verifying of picking single colonie.
1.7 are transferred to Agrobacterium GV1301, and GV3101 plants of Agrobacterium contain PBI121 plasmid (containing 35S-GUS) and PBI101
The carrier of (no 53S promoter, GUS cannot be expressed), under the conditions of 28 DEG C, in the 3ml LB culture medium containing card sodium antibiotic
In, it is shaken up overnight with the speed of 200rpm.Overnight culture is inoculated in 200ml LB culture medium, 28 DEG C are incubated overnight.Agriculture
Bacilli-cell is centrifuged, and cell is collected, and with buffer resuspension, (buffer proportion is 10mM MgCl2,10mM MES (pH value
5.6), 100 μM of acetosyringone), acquisition Agrobacterium bacterial concentration is that OD600nm (OD600) is 0.4.
Step 2. grape leave instantaneous conversion
Thallus is placed in preservation 2-3 hours at room temperature by 2.1.Tissue-cultured seedling ' Rose bella Reid ' grape leave of acquisition 24 days, blade back
Floated in Agrobacterium bacterium solution downward, be placed in Ai Bende concentrating instrument (Concentrator plus), is in vacuum degree: 20hPa
(20mbar), processing 30 DEG C at 5 minutes, after also back side cell by completely by During Agrobacterium after, take out.
After 2.2 wiped cleans, it is placed in culture dish, moistens filter paper moisturizing, after 24 DEG C of dark culturing 48h, to gene protein table
After reaching, the detection of GUS colour developing is carried out, as a result as shown in Figure 1, comparison control group (is carried the leaf that PBI101 Agrobacterium is infected
Piece), after the grape leave (being carried the blade that the Agrobacterium of PBI121 carrier is infected) for having gus gene to express shows GUS expression
Navy blue.
Embodiment 2
Step 1. building carries the carrier of VdWRKY70 disease-resistant gene
1.1 choose the young leaflet tablet of Vitis davidii Foex 0943, E.coli DH5 α competent E.coli, Omega gel
Extract kit, LB culture medium etc. are purchased from Henan Bao Sai scientific & technical corporation；Bioteke plant RNA extraction kit is purchased from Beijing
Hundred Imtech, the super fidelity enzyme of PhusionTM are purchased from Zhengzhou Bo Mei company, pGEMT-easy support agent box, ExTaq enzyme,
DL2000Marker etc. is purchased from TaKaRa (Dalian) company.
The extraction of 1.2 total serum IgEs is extracted using Bioteke plant RNA extraction kit.Use Thermo Scientific
The micro ultra-violet and visible spectrophotometer of Nanodrop 1000 measurement concentration simultaneously determines that RNA's is complete through agarose gel electrophoresis
Property.First chain of cDNA is obtained using the reverse transcription reagent box of Fermentas company.
1.3 VdWRKY70 gene orders clone mRNA using the primer find design primer in Vector NTI 11
Sequence, primer are shown in Table 1.PCR amplification uses the super fidelity enzymatic amplification of PhusionTM of NEB, reaction system are as follows: 10 μ of HF buffer
The 2.5 μ L of dNTPs of L, 2.5mmol/L, 2 μ L of template, each super fidelity enzyme of 2.5 μ L, Phusion of upstream and downstream primer (10mmol/L)
0.5 μ L, distilled water are mended to 50 μ L.PCR response procedures are as follows: 98 DEG C of initial denaturation 3min；98 DEG C of denaturation 10s, 58 DEG C of annealing 10s, 72
DEG C extend 30s, totally 31 circulation；Last 72 DEG C of extensions 10min, 4 DEG C of preservations.PCR product makes through 2% agarose gel electrophoresis
Amplified production is recycled with Omega Gel extract QIAquick Gel Extraction Kit, product after purification is polymerize using the EXTaqTM of TaKaRa
Enzyme adds A, reaction system: the Mg of 10x buffer 2 μ L, 2.5mmol2+The 2 μ L of dNTPs of 2 μ L, 2.5mmol/L, 14 μ L of template.
Response procedures: 72 DEG C of reaction 10min.VdWRKY49-T carrier, conversion are constructed referring to pGEMT-easy TM vector construction specification
E.coli DH5 α Escherichia coli carry out blue hickie screening, choose monoclonal and Sangon Biotech (Shanghai) Co., Ltd. is sent to survey
Sequence, sequencing primer are T7 and T7Terminal universal primer.
Table 1: gene cloning primer
1.4 plant expression vectors choose pCAMBIA1303, and restriction enzyme site chooses NCO I and BstE II, and enzyme chooses the HF of NEB
Super fidelity enzyme, digestion system is referring to specification.PGW serial carrier selects I digestion of NCO, and PW serial carrier removes GUS and GFP sequence
Column, using II double digestion of NCO I and BstE.The agarose gel electrophoresis for passing through 2% after digestion uses Promega gel after cutting glue
The recycling of extract kit.The carrier T of the VdWRKY70 crossed using sequence verification does template, adds connector, adapter-primer respectively
It is shown in Table 2.PW70 carrier uses P70F and PW70R；PGW70 carrier uses P70F and PGW70R.It is super using the PhusionTM of NEB
It is described in fidelity enzyme PCR amplification, system reference material and method 1.3.Target fragment is recycled using Promegagel extract.
According to the Gibson of NEBSpecification constructs plant expression vector PGW70, send raw work sequence verification.It uses
Axygen mini-scale plasmid extracts kit extracts plasmid, spare.
Table 2: vector construction primer connector
The preparation of 1.5 Agrobacterium LBA4404 competence: plate is drawn on the LB solid medium containing 50mg/L rifampin, is chosen
It takes unit cell to shake bacterium 48h in the LB liquid medium containing 50mg/L rifampin to bacterium solution muddiness, is inoculated into 1:100 ratio
Bacterium 5h is shaken in the fresh LB liquid medium containing 50mg/L rifampin of 50ml.Bacterium solution is placed in ice bath 30min on ice, 4 DEG C,
5000g is centrifuged 5min；The calcium chloride suspension that 1ml0.1% is added in supernatant is abandoned, ice bath 5min, 4 DEG C, 5000g is centrifuged 5min；In abandoning
Reset and add the calcium chloride resuspension into 800 μ L 0.02%, the 8 every 100 μ L ice bath of pipe of pipe of packing is spare.
2 μ g are added in the 1.6 Agrobacterium competence prepared and need the plasmid that converts, mix, liquid nitrogen flash freezer 1min, 37 DEG C
1ml LB liquid medium is added in water-bath 5min, and 28 DEG C, 200rpm shakes bacterium 5h.Centrifugal concentrating is coated onto containing 50mg/L rifampin
On the LB plate of the kanamycins of 50mg/L, 28 DEG C of culture 48h, the verifying of picking single colonie.
1.7 are transferred to Agrobacterium GV1301, and GV3101 plants of Agrobacterium contain pCAMBIA1303 (control) and pCAMBIA1303-
The carrier of VdWRKY70 in the 3mlLB culture medium containing card sodium antibiotic, is shaken under the conditions of 28 DEG C with the speed of 200rpm
It is even overnight.Overnight culture is inoculated in 200ml LB culture medium, 28 DEG C are incubated overnight.Agrobatcerium cell is centrifuged, and is collected
Cell, with buffer be resuspended (buffer proportion be 10mM MgCl2,10mM MES (pH value 5.6), 100 μM
Acetosyringone), it is 0.4 that acquisition Agrobacterium bacterial concentration, which is OD600nm (OD600),.
Step 2. grape leave instantaneous conversion
Thallus is placed in preservation 2-3 hours at room temperature by 2.1.Tissue-cultured seedling ' Rose bella Reid ' grape leave of acquisition 24 days, blade back
Floated in Agrobacterium bacterium solution downward, be placed in Ai Bende concentrating instrument (Concentrator plus), is in vacuum degree: 20hPa
(20mbar), processing 30 DEG C at 5 minutes, after also back side cell by completely by During Agrobacterium after, take out.
After 2.2 wiped cleans, it is placed in culture dish, moistens filter paper moisturizing, after 24 DEG C of dark culturing 48h, to gene protein table
After reaching, leaf front is connect bacterium (fruit white rot of grape opportunistic pathogen), in incubator after 28 DEG C of dark 24, as a result as shown in Fig. 2, comparison control group
(being carried the blade that pCAMBIA1303 Agrobacterium is infected), has the grape leave of transgene expression (to be carried pCAMBIA1303-
The blade that the Agrobacterium of VdWRKY70 carrier is infected) the disease-resistant phenotype of performance.
The result shows that detection method of the invention can only quickly detect the exogenous gene expression result of plant with 68h.
Comparative example 1
Use the Agrobacterium-mediated Transformation seedling containing foreign gene in embodiment 1, the specific steps are as follows:
(1) grape seedling (before true leaf does not occur) for taking growth 7 days or so, first washes away the training being stained on seedling with clear water
Nutrient solution and other impurity are put into superclean bench after slightly spraying alcohol (75%, v/v), with the HgCl of 0.1% (w/v)2It carries out
Full scale wash, then seedling is soaked in 5min in ddH2O, weak vibrations wash away remaining HgCl2, ddH is replaced repeatedly2O, cleaning 4
~5 times to ensure to clean up；
(2) seedling after cleaning and sterilizing is placed in high sepage and carries out hypertonic pretreatment 3min, high sepage specifically: contain
165 μM of acetosyringones and 3% sucrose, the 1/2MS solution of pH=5.8, the osmotic pressure of high sepage are 0.7~0.75kg/cm2
Left and right (it should be noted that excessively high osmotic pressure is easy to damage seedling, but it is too low when can not then effectively facilitate genetic transformation).
(3) by middle and high infiltration, treated that seedling is put into instantaneous conversion solution, and 25 DEG C, 120rpm provide suitable illumination, light
According to about 80 μm of ol m-2s-1 of intensity, 5h is cultivated, cultivate under incubation time is too long and non-illuminated conditions, seedling is easy death.
(4) by the seedling ddH after conversion2O is washed twice, and after removing the Agrobacterium being stained on seedling, is placed in containing anti-
Raw element (50mg/L card receive mycin and 50mg/L rifampin) and the 1/2MS of 0.8% (w/v) agar powder cultivate basal growth；Cultivate item
Part are as follows: photoperiod 16h illumination/8h is dark, and light intensity is 80~90 μm of ol m-2s-1, cultivation temperature are as follows: 20 DEG C of 26 DEG C/night of day.
(5) it carries out being inoculated with pathogen to it and cultivating it, condition of culture after cultivating 7 days are as follows: the illumination of photoperiod 16h/
8h is dark, and light intensity is 80~90 μm of ol m-2s-1, cultivation temperature are as follows: 20 DEG C of 26 DEG C/night of day.The results show that comparison control group
(being carried the seedling that pCAMBIA1303 Agrobacterium is infected), has the grape leave of transgene expression (to be carried pCAMBIA1303-
The seedling that the Agrobacterium of VdWRKY70 carrier is infected) the disease-resistant phenotype of performance.
The result shows that the detection method of comparative example 1 needs just detect for 5~7 days the exogenous gene expression result of plant.
It also needs to carry out hypertonic pretreatment to seedling simultaneously, otherwise the effect of genetic transformation is lower.
Comparative example 2
Use the Agrobacterium-mediated Transformation arabidopsis floral containing foreign gene in embodiment 1, the specific steps are as follows:
(1) inflorescence is dipped into 1min in Agrobacterium bacterium solution, taking-up is stained with filter paper and inhales bacterium solution extra on stem.22 DEG C of dark
Under the conditions of place two days, pay attention to moisturizing, converted again every 7 days primary.3 weeks or so after conversion, seed is collected to fruit folder maturation.
(2) dry seed is placed on and 900 μ L are added in 2ml EP pipe are shaken containing 0.2% 70% ethyl alcohol of polysorbas20
9min.The ethyl alcohol for abandoning supernatant addition 90% is washed 3 times, is finally poured on the filter paper of sterilizing with the suspension of 100% ethyl alcohol dry.Configuration 1/
2MS culture medium, addition hygromycin B is to final concentration 25mg/L before a kind of rhyme scheme in Chinese operas serving as the prelude to a complete score for voices.The arabidopsis seed of sterilizing is uniformly sprinkled upon 1/2MS training
It supports on base, normal growth is transgenic arabidopsis positive plant after 7 days.Choose the quasi- south of the good transgenic of upgrowth situation
Mustard connects whiterot fungi using needle point method, observes its disease-resistant phenotype after 7 days respectively.
The result shows that the detection method of comparative example 2 needs just detect for 28 days or so the exogenous gene expression knot of plant
Fruit.
Comparative example 3
Grape leave, specific steps are converted by injecting method using the Agrobacterium containing foreign gene in embodiment 1
It is as follows:
Taking OD600 is that 0.4 pCAMBIA1303-VdWRKY70 bacterium solution is prepared into injection bacterium solution；
It being injected into grape leave with the syringe of 1ml needle-less by bacterium solution is injected, whole process is extremely difficult, and water stain
Local blade can be full of.Culture dish is placed it in after injection, moistens filter paper moisturizing, after 24 DEG C of dark culturing 48h, to gene protein
After expression, leaf front is connect bacterium (fruit white rot of grape pathogen), 28 DEG C dark 24 hours in incubator.The results show that in comparative example 1
Blade fail gene expression and disease-resistant phenotype occur, reason may be to cause mechanical damage, In since blade is injected device
It needs to carry out certain reparation to the damage of itself before expression alien gene, when this extends the expression of external source disease-resistant gene
Between.Therefore, comparative example 3 uses inoculation same as Example 1 and cultural method, and the time of required expression alien gene is more
It is long.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (7)
1. a kind of method of quickly detection plant disease resistance genes, which comprises the following steps: disease-resistant gene is imported agriculture
In bacillus, recombinant bacterium is obtained；The back side that grape leave is infected with the recombinant bacterium, disease-resistant gene is transformed into grape leave,
The front inoculation pathogen of grape leave, realizes the detection of disease-resistant gene；
The disease-resistant gene is imported in Agrobacterium by recombinant vector；
The recombinant vector is the carrier that target gene is cloned into the multiple cloning sites of expression vector and is obtained.
2. the method for quickly detection plant disease resistance genes as described in claim 1, which is characterized in that the disease-resistant gene is
VdWRKY70 gene.
3. the method for quickly detection plant disease resistance genes as described in claim 1, which is characterized in that the recombinant bacterium infects Portugal
The method at the back side of grape blade includes the following steps: to float at the back side of grape leaf downward in the recombination bacterium solution, is placed in vacuum
Middle processing, vacuum side of blade cell is by by after During Agrobacterium, taking-up is placed in dark culturing in incubator completely.
4. the method for quickly detection plant disease resistance genes as claimed in claim 3, which is characterized in that the vacuum processing parameter
Are as follows: vacuum degree 20hPa (20mbar), 30 DEG C of processing 5min.
5. the method for quickly detection plant disease resistance genes as claimed in claim 3, which is characterized in that the condition of culture are as follows:
24 DEG C of dark culturing 48h of temperature.
6. the method for quickly detection plant disease resistance genes as described in claim 1, which is characterized in that the grape leave is just
The method of face inoculation pathogen includes the following steps: the positive inoculation pathogen of leaf, and 28 DEG C of dark are for 24 hours in incubator.
7. the method for quickly detection plant disease resistance genes as described in claim 1, which is characterized in that the recombinant bacterium infects Portugal
The recombinant bacterium OD600 of grape vacuum side of blade is 0.4.
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910603997.2A CN110408648A (en) | 2019-07-05 | 2019-07-05 | A kind of method of quick detection plant disease resistance genes |
| PCT/CN2020/081955 WO2021004098A1 (en) | 2019-07-05 | 2020-03-29 | Method for rapidly detecting plant disease-resistant genes |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
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| CN201910603997.2A CN110408648A (en) | 2019-07-05 | 2019-07-05 | A kind of method of quick detection plant disease resistance genes |
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| CN102121004A (en) * | 2010-12-03 | 2011-07-13 | 西北农林科技大学 | Grape pathogen inducible promoter separating method and application in breeding for disease resistance |
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| WO2021004098A1 (en) * | 2019-07-05 | 2021-01-14 | 中国农业科学院郑州果树研究所 | Method for rapidly detecting plant disease-resistant genes |
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