CN105647963A - Target gene apple conversion method and application thereof to transient expression of target gene in apples - Google Patents
Target gene apple conversion method and application thereof to transient expression of target gene in apples Download PDFInfo
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Abstract
The invention discloses a target gene apple conversion method and application thereof to transient expression of a target gene in apples. The apple conversion method includes: using infection liquid containing the target gene to infect apple embryo protoplast to complete apple conversion. An apple embryo protoplast preparation method includes: A1) subjecting apple embryonic calluses to suspension subculture in a suspension subculture medium to obtain suspension cells; A2) using CPW13M for treating the suspension cells to obtain the suspension cells subjected to CPW13M treatment; A3) using cellulose and pectinase for enzymolysis of the suspension cells subjected to CPW13M treatment to obtain the apple embryo protoplast. According to experiments, the apple conversion method can realize transient expression of the target gene in the apples, can be used for functional verification of the target gene and protein and organelle positioning and can also be used for detection of protein interaction and cell metabolism.
Description
Technical field
The present invention relates to the method for genes of interest Transformation of Apple in biological technical field and the application in transient expression genes of interest in Fructus Mali pumilae thereof.
Background technology
Malus sieversii (MalussieversiiRoem.) is mainly distributed on Central Asia Tianshan Mountains mountain range, it it is considered as ancestors' kind of modern Cultivated apple, and there is abundant genetic diversity, it it is the one that conventional apple rootstock medium drought resistant ability is the strongest, Separation Research to its anti-drought gene, contributes to disclosing the fruit tree response mechanism to adverse circumstance. embryo callus, as a kind of good test examination material, is widely used in the researchs such as the genetic transformation of plant, regeneration plant, be free protoplast well try material, the metabolic process of the protoplast of acellular wall, signal transduction pathway, reaction etc. to environmental factors and external irritant, all with complete plant cell or organize essentially identical, by the DNA to import feature gene in protoplast Yu reporter gene fusion, utilize reporter gene transient expression in plant protoplast can carry out protein and organelle location rapidly and accurately, related gene functional study, protein is studied and cellular process research etc. mutually, for studying the physiological change of plant on a cellular level, gene expression, signal transmission etc. provides simple and effective experimental system.
Current Malus sieversii is stablized genetic conversion system and is not yet successfully constructed, and related gene functional study in this platymiscium cannot be carried out smoothly.
Summary of the invention
The technical problem to be solved is how to carry out the transient expression of genes of interest in wilding.
For solving above-mentioned technical problem, present invention firstly provides Fructus Mali pumilae method for transformation.
Fructus Mali pumilae method for transformation provided by the present invention, carries out Fructus Mali pumilae conversion including with Fructus Mali pumilae embryo protoplast for receptor;
The preparation method of described Fructus Mali pumilae embryo protoplast includes following A 1) and A2):
A1) Fructus Mali pumilae embryo callus is carried out suspension successive transfer culture, obtain suspension cell;
A2) described Fructus Mali pumilae embryo protoplast is obtained with suspension cell described in cellulase and pectinase enzymatic hydrolysis.
In said method, described Fructus Mali pumilae embryo callus is carried out suspension successive transfer culture include: described Fructus Mali pumilae embryo callus is carried out suspension successive transfer culture in suspension system subculture medium, obtains suspension cell;
Described suspension system subculture medium is add the culture medium that BA, 2,4-D, vitamin C and sucrose obtain in MS culture medium; The concentration of BA described in described suspension system subculture medium, described 2,4-D, described vitamin C and described sucrose can respectively 0.5mg/L, 3mg/L, 2mg/L and mass percent concentration 3%, pH is 5.8.
In said method, described A1) specifically comprise the steps that the described Fructus Mali pumilae embryo callus of grinding, obtain abrasive material; Described abrasive material is carried out successive transfer culture in described suspension system subculture medium, obtains described suspension cell.
In the method for above-mentioned purpose gene transformation Fructus Mali pumilae, described grinding Fructus Mali pumilae embryo callus can be grind described Fructus Mali pumilae embryo callus with mesh screen. Described mesh screen can be rustless steel mesh screen, such as 40 order rustless steel mesh screens.
In said method, described with Fructus Mali pumilae embryo protoplast for receptor carry out Fructus Mali pumilae convert can for infect described Fructus Mali pumilae embryo protoplast with the liquid that infects containing genes of interest. Described use contain genes of interest infect liquid infect described Fructus Mali pumilae embryo protoplast can for the protoplast instant expression method utilizing PEG to mediate complete described in infect.
In said method, described time of infection can be 10-30min, such as 15min or 20min.
In said method, the number of times of described suspension successive transfer culture can be more than or equal to 2 times, and the time of each suspension successive transfer culture can be 7-10d, such as 8d. Described suspension successive transfer culture can carry out under dark, 150r/min. The temperature of described suspension successive transfer culture can be 22-24 DEG C.
In said method, also include described suspension cell CPW13M process before suspension cell described in described cellulase and pectinase enzymatic hydrolysis; Described CPW13M adds the solution that mannitol obtains in cell-protoplast cleanout fluid (cellprotoplastwashmedium, hereinafter referred to as CPW solution); In described CPW13M, the mass percent concentration of mannitol can be 11%-15% (such as 13%).
Wherein, described CPW solution is made up of water and solute, the solute of described CPW solution and concentration respectively KH thereof2PO427.2mg/L��KNO3101.0mg/L��CaCl2��2H2O1480.0mg/L��MgSO4��7H2O246.0mg/L, KI0.16mg/L and CuSO4��5H2O0.025mg/L��
In said method, the time processing described suspension cell with described CPW13M can be 0.5-2h. The described CPW13M of described use processes the soak time concretely 1h of described suspension cell.
In said method, the ratio of the suspension cell that described cellulase, described pectase and described CPW13M process can be 100U:15U:10mL.
In said method, described A2) A21 can be included) and A22):
A21) steep described suspension cell by the enzyme immersion containing described cellulase and described pectase enzyme, obtain enzyme-protoplast mixed liquor;
A22) described enzyme-protoplast mixed liquor is added on CPW25S, obtain lower floor to be CPW25S upper strata be the stratified liquid 1 of enzyme-protoplast mixed liquor; Described stratified liquid 1 is centrifuged, makes protoplast be positioned at the interface of described stratified liquid 1, separate protoplast, obtain described Fructus Mali pumilae embryo protoplast;
Described CPW25S adds the solution that sucrose obtains in described CPW solution; Sucrose mass percent concentration concretely 23%-27% (such as 25%) in described CPW25S.
In said method, described enzyme liquid is made up of solute and solvent; Described solvent is described CPW solution, described in cellulase, 15000U/L described in described solute and concentration thereof respectively 100000U/L, pectase, 0.65mol/LD-mannitol, 1.0% (mass percent concentration) polyvinylpyrrolidone (PVP), 0.1% (mass percent concentration) MES and 0.1% (mass percent concentration) BSA, pH are 5.6.
In said method, with described enzyme immersion steep described suspension cell can the low light level (such as below 500lux), 22-24 DEG C, carry out under 40r/min. The time steeping the described CPW13M suspension cell processed by described enzyme immersion can be 15-25h, such as 20h.
In said method, described cellulase can be CellulaseR-10. Described pectase can be PectolaseY-23. Excellent Nikon bio tech ltd, described CellulaseR-10 concretely Beijing product, article No. is C8260-100. Described PectolaseY-23 is Beijing Mei Laibo medical science and technology company limited product concretely, and article No. is MMF-1069.
In said method, the preparation method of described Fructus Mali pumilae embryo callus comprises the steps that Fructus Mali pumilae embryo carries out inducing culture on inducing culture obtains Fructus Mali pumilae embryo callus;
Described inducing culture is the solid medium that addition BA, 2,4-D and sucrose obtain in MS culture medium;
Wherein, BA in described inducing culture, 2,4-D and the concentration of sucrose can be 3mg/L, 1mg/L and 3% (mass percent concentration) respectively.
In said method, described inducing culture can 22-24 DEG C, carry out under dark. The incubation time of described inducing culture can be 7-21d, such as 14d.
In said method, described Fructus Mali pumilae embryo is the Fructus Mali pumilae embryo contained and spend rear 60-80d.
In said method, described Sheng is spent rear 60-80d concretely to contain and is spent rear 70d.
In said method, the preparation method of described Fructus Mali pumilae embryo callus also includes described Fructus Mali pumilae embryo callus is carried out successive transfer culture on subculture medium;
Described subculture medium is the solid medium that addition BA, 2,4-D and sucrose obtain in MS culture medium;
In wherein said subculture medium, the concentration of BA, 2,4-D and sucrose can be 4mg/L, 2mg/L and 3% (mass percent concentration) respectively.
In said method, described successive transfer culture can 22-24 DEG C, carry out under dark. The incubation time of described successive transfer culture can be 7-21d, such as 14d.
For solving above-mentioned technical problem, the method that present invention also offers following P1 or P2:
P1, described Fructus Mali pumilae embryo protoplast preparation method;
P2, described Fructus Mali pumilae embryo callus preparation method.
For solving above-mentioned technical problem, present invention also offers the product of following M1 or M2:
M1, for preparing the reagent set of Fructus Mali pumilae embryo protoplast, at least two in described suspension system subculture medium, described CPW13M, described CPW25S, described CPW solution, described cellulase, described pectase, described inducing culture and described subculture medium;
M2, for inducing the reagent of Fructus Mali pumilae embryo callus, for described inducing culture and/or described subculture medium.
For solving above-mentioned technical problem, present invention also offers following arbitrary application:
X1, the application in transient expression genes of interest in Fructus Mali pumilae of described Fructus Mali pumilae method for transformation;
X2, described Fructus Mali pumilae embryo protoplast preparation method application in transient expression genes of interest in Fructus Mali pumilae;
X3, described Fructus Mali pumilae embryo callus preparation method application in transient expression genes of interest in Fructus Mali pumilae;
The application in transient expression genes of interest in Fructus Mali pumilae of X4, described product;
The application in plant gene function is verified of X5, described Fructus Mali pumilae method for transformation;
X6, described Fructus Mali pumilae embryo protoplast preparation method plant gene function verify in application;
X7, described Fructus Mali pumilae embryo callus preparation method plant gene function verify in application;
The application in plant gene function is verified of X8, described product.
X9, described Fructus Mali pumilae embryo callus preparation method application in preparing Fructus Mali pumilae embryo protoplast;
X10, described Fructus Mali pumilae embryo callus preparation method Fructus Mali pumilae convert in application;
X11, described Fructus Mali pumilae embryo protoplast preparation method application in preparing Fructus Mali pumilae embryo protoplast.
In the present invention, described Fructus Mali pumilae can be the Fructus Mali pumilae kind that can be used for apple rootstock, such as Malus sieversii (MalussieversiiRoem).
In the present invention, described suspension system subculture medium can be aseptic culture medium. Described inducing culture can be aseptic culture medium. Described subculture medium can be aseptic culture medium. Described CPW13M can be sterile liquid. Described enzyme liquid can be sterile liquid. Described CPW25S can be sterile liquid.
Experiment proves, genes of interest can be carried out transient expression by the Fructus Mali pumilae method for transformation of the present invention in wilding, and making genes of interest be expressed: comparison Green fluorescence signal is distributed in whole protoplasm somatocyte, and the Fructus Mali pumilae embryo protoplast Green fluorescence signal converting genes of interest MsDREB2C gene concentrates on nuclear area, show that MsDREB2C gene has expression at nuclear area, consistent with MsDREB2C gene expression characteristic in natural situation. The preparation method of the Fructus Mali pumilae embryo callus of the present invention can prepare Fructus Mali pumilae embryo callus: utilize that the callus color that the preparation method of the Fructus Mali pumilae embryo callus of the present invention obtains is yellowish, quality is crisp, surface has graininess protruding, microscopy observes that cell is round cell, and form stable after repeatedly successive transfer culture, multiplication capacity is strong. The preparation method of the Fructus Mali pumilae embryo protoplast of the present invention can prepare the high-quality spherical shape Fructus Mali pumilae embryo protoplast being wrapped in by cell membrane.
It is demonstrated experimentally that genes of interest can be carried out transient expression by the Fructus Mali pumilae method for transformation of the present invention in Fructus Mali pumilae, it is possible to be used for carrying out the functional verification of genes of interest and the location of protein and organelle, can also be used with and detect protein and make mutually and cellular metabolism.
Accompanying drawing explanation
Fig. 1 is the outward appearance of Fructus Mali pumilae embryo callus. Wherein, A is 1d after the inoculation of Fructus Mali pumilae rataria; B is 7d after the inoculation of Fructus Mali pumilae rataria; C is 21d after the inoculation of Fructus Mali pumilae rataria; D is successive transfer culture Fructus Mali pumilae embryo callus once; E is the Fructus Mali pumilae embryo callus of successive transfer culture three times; F is the Fructus Mali pumilae embryo callus of successive transfer culture five times.
Fig. 2 is suspension cell.
Fig. 3 is the enzymolysis result of Fructus Mali pumilae embryo callus. Wherein A is before enzymolysis, and B is after enzymolysis.
Fig. 4 is purification and the microscopy of Fructus Mali pumilae embryo protoplast. Wherein A is the delamination in purge process, and 1 is enzymolysis solution, and 2 is protoplast layer, and 3 is residue and CPW25S; B is not purified protoplast microscopy result; C is the protoplast microscopy result after purification.
Gene expression in Fructus Mali pumilae embryo protoplast for the purpose of Fig. 5. Wherein, A is the Fructus Mali pumilae embryo protoplast of the Transformed E 3025-MsDREB2C under light field;B is the Fructus Mali pumilae embryo protoplast of the Transformed E 3025-MsDREB2C under details in a play not acted out on stage, but told through dialogues; C is the Fructus Mali pumilae embryo protoplast of the Transformed E 3025-MsDREB2C under light and shade superimposed field; D is the Fructus Mali pumilae embryo protoplast of the Transformed E 3025 under light field; The Fructus Mali pumilae embryo protoplast of the Transformed E 3025 under E details in a play not acted out on stage, but told through dialogues; F is the Fructus Mali pumilae embryo protoplast of the Transformed E 3025 under light and shade superimposed field. Bar=10 ��m.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention being further described in detail, the embodiment provided is only for illustrating the present invention, rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, all commercially obtain.
Experimental technique in following embodiment, if no special instructions, is and carries out under room temperature (23 �� 1 DEG C). Malus sieversii (MalussieversiiRoem) in following embodiment (Li Yu agriculture .2001. Malus research of fruit germplasm resource. Beijing: Chinese agriculture publishing house: 20-134.), the public can obtain from applicant, this biomaterial only attach most importance to duplicate invention related experiment used by, can not use as other purposes.
RFP gene in pSAT6 RFP N1 carrier is replaced with GFP gene by the carrier E3025 in following embodiment, keeps the constant carrier obtained of other sequences, carrier E3025 expressing green fluorescent protein GFP. Wherein pSAT6 RFP N1 carrier is documented in document (Sang-MinChungetal.Aversatilevectorsystemformultiplegenee xpressioninplants.TRENDSinPlantScienceVol.10No.8August20 05). The public can obtain carrier E3025 from applicant, this biomaterial only attach most importance to duplicate invention related experiment used by, can not use as other purposes.
Inducing culture in following embodiment is the sterile solid culture medium that addition BA, 2,4-D and sucrose obtain in MS culture medium; Wherein the concentration of BA, 2,4-D and sucrose respectively 3mg/L, 1mg/L and 3% (mass percent concentration).
Subculture medium in following embodiment is the sterile solid culture medium that addition BA, 2,4-D and sucrose obtain in MS culture medium; Wherein the concentration of BA, 2,4-D and sucrose respectively 4mg/L, 2mg/L and 3% (mass percent concentration).
Suspension system subculture medium in following embodiment be in MS culture medium add BA, 2, the aseptic culture medium that 4-D, vitamin C and sucrose obtain, wherein BA, 2, the concentration of 4-D, vitamin C and sucrose respectively 0.5mg/L, 3mg/L, 2mg/L and 3% (mass percent concentration), pH is 5.8.
CPW13M in following embodiment adds the sterile solution that mannitol mass percent concentration is 13% that mannitol obtains in CPW solution. CPW solution is made up of water and solute, the solute of CPW solution and concentration thereof respectively KH2PO427.2mg/L��KNO3101.0mg/L��CaCl2��2H2O1480.0mg/L��MgSO4��7H2O246.0mg/L, KI0.16mg/L and CuSO4��5H2O0.025mg/L��
Enzyme liquid in following embodiment is by solute and solution; Solvent is CPW13M, solute and concentration thereof respectively 100000U/LCellulaseR-10,15000U/LPectolaseY-23,0.65mol/LD-mannitol, 1.0% (mass percent concentration) polyvinylpyrrolidone (PVP), 0.1% (mass percent concentration) MES and 0.1% (mass percent concentration) BSA, pH are 5.6. CellulaseR-10 is excellent Nikon bio tech ltd, Beijing product, and article No. is C8260-100;PectolaseY-23 is Beijing Mei Laibo medical science and technology company limited product, and article No. is MMF-1069.
W5 solution in following embodiment is the sterile solution being made up of water and solute, the solute of W5 solution and concentration thereof respectively CaCl2��2H2O125mmol/L, NaCl154.0mmol/L, KCl5.0mmol/L, glucose 5.0mmol/L, one water morpholino b acid (MES) 5.0mmol/L.
CPW25S in following embodiment adds the sterile solution that sucrose mass percent concentration is 25% that sucrose obtains in CPW solution.
MMg in following embodiment is the sterile solution being made up of solute and solvent; Solvent is water, solute and concentration thereof respectively MgCl215mmol/L, MES0.1% (mass percent concentration) and mannitol (mannitol) 0.4mol/L.
PEG6000 aqueous solution in following embodiment is the sterile solution being made up of solute and solvent; Solvent is water, solute and concentration thereof respectively PEG600040% (mass percent concentration).
The transient expression of genes of interest in embodiment 1, Fructus Mali pumilae
The transient expression of genes of interest in Fructus Mali pumilae, including utilizing Fructus Mali pumilae method for transformation to go in Fructus Mali pumilae by genes of interest, is specially and infects Fructus Mali pumilae embryo protoplast with the liquid that infects containing genes of interest, completes Fructus Mali pumilae and converts. In triplicate, the concrete operation step every time repeating experiment is as follows in experiment:
One, the preparation of Fructus Mali pumilae embryo protoplast
1, the preparation of Fructus Mali pumilae embryo callus
1.1 outer implant sterilizations
By 70DAFB, (Sheng spends rear natural law, Dayafterfullbloom) young fruit of Malus sieversii (MalussieversiiRoem) cleans up, and with the ethanol water surface sterilization 3min of 75% (concentration of volume percent), then with the aqueous sodium hypochlorite solution sterilization 15min that effective chlorine mass percent is 5%, finally use aseptic water washing 2-3 time, obtain aseptic young fruit.
The induction of 1.2 Fructus Mali pumilae embryo callus
Take out the seed of the aseptic young fruit of step 1.1, aseptically carefully remove seed coat with tweezers, obtain Fructus Mali pumilae rataria, be inoculated on inducing culture after Fructus Mali pumilae rataria is cut into the square fritter of 2mm, then at 23 �� 1 DEG C, lucifuge cultivates 21d, obtains Fructus Mali pumilae embryo callus.
The successive transfer culture of 1.3 Fructus Mali pumilae embryo callus
The Fructus Mali pumilae embryo callus of step 1.2 is inoculated on subculture medium, then at 23 �� 1 DEG C, lucifuge cultivates 18d, obtain the Fructus Mali pumilae embryo callus of successive transfer culture, by the Fructus Mali pumilae embryo callus of this successive transfer culture at 23 �� 1 DEG C, dark lower repetition successive transfer culture 5 times.
Observe the Fructus Mali pumilae embryo callus of the successive transfer culture of the Fructus Mali pumilae embryo callus that obtains of step 1.2 and step 1.3, the Fructus Mali pumilae embryo callus color that after the inoculation of Fructus Mali pumilae rataria, 7-21d obtains is yellowish, quality is crisp, there is graininess protruding (in Fig. 1 B and C) on surface, microscopy observes that cell is round cell, and form stable after repeatedly successive transfer culture, multiplication capacity strong (in Fig. 1 D-F).
2, the preparation of Fructus Mali pumilae embryo protoplast
The preparation of 2.1 suspension cells
The Fructus Mali pumilae embryo callus obtained with 40 order rustless steel mesh screen grinding steps 1, obtains abrasive material, this abrasive material (particle diameter about 40 ��m); By abrasive material suspension successive transfer culture 8d in suspension system subculture medium, the condition of suspension successive transfer culture is that under dark, 150r/min, temperature is 23 �� 1 DEG C, repeats successive transfer culture 2 times, obtains suspension cell (Fig. 2).
The preparation of 2.2 Fructus Mali pumilae embryo protoplasts
The suspension cell 1h obtained with CPW13M soaking step 2.1, abandons supernatant liquid, obtains the CPW13M suspension cell processed;In the CPW13M suspension cell processed, enzyme liquid is added according to the ratio that volume ratio is 1:10 of the CPW13M suspension cell processed and enzyme liquid, obtain enzyme-suspension cell mixed liquor, by enzyme-suspension cell mixed liquor the low light level (500lux), 23 �� 1 DEG C, hatch 20h under 40r/min, obtain the enzyme after enzymolysis-suspension cell mixed liquor (Fig. 3); Enzyme after enzymolysis-suspension cell mixed liquor is lightly ground filtration with 300 order nylon mesh screens, discharges protoplast, obtain enzyme-protoplast mixed liquor;
Protoplast in purifying enzyme-protoplast mixed liquor by the following method: enzyme-protoplast mixed liquor is added on CPW25S gently, process is softly slow, avoid colliding separating surface, as far as possible make it remain stable for, obtain lower floor to be CPW25S upper strata be the stratified liquid 1 of enzyme-protoplast mixed liquor; By stratified liquid 1 centrifugal 15min at 800 rpm, make protoplast be arranged in the interface (Fig. 4 A) of described stratified liquid 1, carefully by the protoplast layer sucking-off of interface, obtain Fructus Mali pumilae embryo protoplast; Being placed in new centrifuge tube by Fructus Mali pumilae embryo protoplast, with W5 solution washing 2-3 time, washing centrifugal 6min all at 800 rpm, finally with the resuspended Fructus Mali pumilae embryo protoplast of W5 solution, obtains Fructus Mali pumilae embryo Protoplast suspension every time.
Fructus Mali pumilae embryo protoplast is carried out microscopy, and in result such as Fig. 4 shown in B and C, result shows, can obtain the high-quality spherical shape Fructus Mali pumilae embryo protoplast being wrapped in by cell membrane after purification.
Two, the transient expression of genes of interest in Fructus Mali pumilae
1, the preparation of genes of interest
DNA fragmentation between EcoRI and the SalI recognition sequence of carrier E3025 is replaced with the MsDREB2C gene (MsDREB2C gene is apple gene) shown in SEQIDNo.1, obtain recombinant vector E3025-MsDREB2C, E3025-MsDREB2C and express the protein shown in SEQIDNo.2.
Wherein, SEQIDNo.1 is the sequence of MsDREB2C gene, encodes the MsDREB2C albumen shown in SEQIDNo.2. MsDREB2C gene is expressed in nucleus.
2, the transient expression of genes of interest in Fructus Mali pumilae
Complete genes of interest Transformation of Apple embryo protoplast by the following method, and using carrier E3025 as comparison:
1) by the Fructus Mali pumilae embryo Protoplast suspension of the 2.2 of step one after placing 30min on ice, under 800rpm, centrifugal 6min, abandons supernatant, obtains Fructus Mali pumilae embryo protoplast; In Fructus Mali pumilae embryo protoplast, add the 500 resuspended protoplasts of �� LMMg, obtain Fructus Mali pumilae embryo protoplast MMg suspension;
2) to step 1) 100 �� L Fructus Mali pumilae embryo protoplast MMg suspensions in add the recombinant vector E3025-MsDREB2C-GFP of 10 �� g steps 1, mix gently, obtain Fructus Mali pumilae embryo protoplast-E3025-MsDREB2C-GFP mixed liquor; In Fructus Mali pumilae embryo protoplast-E3025-MsDREB2C-GFP mixed liquor, add 110 �� LPEG6000 aqueous solutions, after mixing, obtain liquid to be transformed; Liquid to be transformed is hatched at 23 DEG C 10min and carries out the conversion of protoplast, obtain the conversional solution of protoplast;
3) to step 2) protoplast conversional solution in add 440 �� LCPW13M, overturn gently, liquid after being converted; After converting, liquid centrifugal 6min under 800rpm, abandons supernatant, the protoplast after being converted;
4) to step 3) conversion after protoplast in add after 100 �� LCPW13M solution softly mix, add 900 �� LCPW13M, then on Tissue Culture Plate, 23 �� 1 DEG C, hatch 18h under the low light level (500lux), obtain converting the Fructus Mali pumilae embryo protoplast of 10min.
According to step 1)-4) method, 10min will be hatched replace with respectively and hatch 15min, hatch 20min and hatch 30min, other steps are all constant, the Fructus Mali pumilae embryo protoplast of the Fructus Mali pumilae embryo protoplast respectively obtaining conversion 15min, the Fructus Mali pumilae embryo protoplast converting 20min and conversion 30min.
The Fructus Mali pumilae embryo protoplast observe, under laser confocal microscope, the Fructus Mali pumilae embryo protoplast converting 10min, converting 15min, the Fructus Mali pumilae embryo protoplast converting 20min and the basic zero difference of transient expression situation of genes of interest in converting the Fructus Mali pumilae embryo protoplast of 30min, wherein convert in the Fructus Mali pumilae embryo protoplast of 20min the transient expression situation of genes of interest as shown in Figure 5.
Result shows, comparison Green fluorescence signal is distributed in whole protoplasm somatocyte, and the Fructus Mali pumilae embryo protoplast Green fluorescence signal converting the Fructus Mali pumilae embryo protoplast of 10min, the Fructus Mali pumilae embryo protoplast of conversion 15min, the Fructus Mali pumilae embryo protoplast converting 20min and conversion 30min all concentrates on nuclear area, show that MsDREB2C gene has expression at nuclear area, consistent with MsDREB2C gene expression characteristic in natural situation. The above results shows, genes of interest can be carried out transient expression by the method for the present invention in Fructus Mali pumilae, and can carry out the functional verification of genes of interest.
Claims (10)
1. Fructus Mali pumilae method for transformation, carries out Fructus Mali pumilae conversion including with Fructus Mali pumilae embryo protoplast for receptor;
The preparation method of described Fructus Mali pumilae embryo protoplast includes following A 1) and A2):
A1) Fructus Mali pumilae embryo callus is carried out suspension successive transfer culture, obtain suspension cell;
A2) described Fructus Mali pumilae embryo protoplast is obtained with suspension cell described in cellulase and pectinase enzymatic hydrolysis.
2. method according to claim 1, it is characterised in that: also include described suspension cell CPW13M process before suspension cell described in described cellulase and pectinase enzymatic hydrolysis; Described CPW13M is the solution that mass percent concentration is 11%-15% adding the mannitol that mannitol obtains in cell-protoplast cleanout fluid.
3. method according to claim 2, it is characterised in that: the time processing described suspension cell with described CPW13M is 0.5-2h.
4. according to described method arbitrary in claim 1-3, it is characterised in that: described A2) include A21) and A22):
A21) steep described suspension cell by the enzyme immersion containing described cellulase and described pectase enzyme, obtain enzyme-protoplast mixed liquor;
A22) described enzyme-protoplast mixed liquor is added on CPW25S, obtain lower floor to be CPW25S upper strata be the stratified liquid 1 of enzyme-protoplast mixed liquor; Described stratified liquid 1 is centrifuged, makes protoplast be positioned at the interface of described stratified liquid 1, separate protoplast, obtain described Fructus Mali pumilae embryo protoplast;
Described CPW25S adds the solution that sucrose obtains in described CPW solution; In described CPW25S, sucrose mass percent concentration is 23%-27%.
5. according to described method arbitrary in claim 1-4, it is characterised in that: the preparation method of described Fructus Mali pumilae embryo callus includes: Fructus Mali pumilae embryo carries out inducing culture on inducing culture and obtains Fructus Mali pumilae embryo callus;
Described inducing culture is the solid medium that addition BA, 2,4-D and sucrose obtain in MS culture medium.
6. method according to claim 4 or 5, it is characterised in that: the preparation method of described Fructus Mali pumilae embryo callus also includes described Fructus Mali pumilae embryo callus is carried out successive transfer culture on subculture medium;
Described subculture medium is the solid medium that addition BA, 2,4-D and sucrose obtain in MS culture medium.
7. according to described method arbitrary in claim 1-6, it is characterised in that: described Fructus Mali pumilae is the Fructus Mali pumilae kind for apple rootstock.
8. the method for following P1 or P2:
The preparation method of arbitrary described Fructus Mali pumilae embryo protoplast in P1, claim 1-7;
The preparation method of arbitrary described Fructus Mali pumilae embryo callus in P2, claim 5-7.
9. the product of following M1 or M2:
M1, for preparing the reagent set of Fructus Mali pumilae embryo protoplast, at least two in suspension system subculture medium described in claim 1-6, described CPW13M, described CPW25S, described cell-protoplast cleanout fluid, described cellulase, described pectase enzyme, described inducing culture, described subculture medium;
M2, for inducing the reagent set of Fructus Mali pumilae embryo callus, for inducing culture described in claim 5 and/or subculture medium described in claim 6.
10. following arbitrary application:
Arbitrary described Fructus Mali pumilae method for transformation application in transient expression genes of interest in Fructus Mali pumilae in X1, claim 1-7;
Preparation method application in transient expression genes of interest in Fructus Mali pumilae of arbitrary described Fructus Mali pumilae embryo protoplast in X2, claim 1-7;
Preparation method application in transient expression genes of interest in Fructus Mali pumilae of arbitrary described Fructus Mali pumilae embryo callus in X3, claim 5-7;
Product described in X4, claim 9 is the application in transient expression genes of interest in Fructus Mali pumilae;
Arbitrary described Fructus Mali pumilae method for transformation application in plant gene function is verified in X5, claim 1-7;
The preparation method of arbitrary described Fructus Mali pumilae embryo protoplast application in plant gene function is verified in X6, claim 1-7;
The preparation method of arbitrary described Fructus Mali pumilae embryo callus application in plant gene function is verified in X7, claim 5-7;
The application in plant gene function is verified of the product described in X8, claim 9;
The preparation method of arbitrary described Fructus Mali pumilae embryo callus application in preparing Fructus Mali pumilae embryo protoplast in X9, claim 5-7;
The preparation method of arbitrary described Fructus Mali pumilae embryo callus application in Fructus Mali pumilae converts in X10, claim 5-7;
The preparation method of arbitrary described Fructus Mali pumilae embryo protoplast application in preparing Fructus Mali pumilae embryo protoplast in X11, claim 1-7.
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| CN115715524A (en) * | 2022-11-30 | 2023-02-28 | 广东省科学院生物与医学工程研究所 | Tissue culture method for improving embryogenic callus induction rate of apple leaves |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106480163A (en) * | 2016-10-19 | 2017-03-08 | 山东农业大学 | A kind of joint Fructus Mali pumilae callus cell culture and the method for genetic transformation identification Fructus Mali pumilae disease-resistant gene |
| CN106480163B (en) * | 2016-10-19 | 2019-11-01 | 山东农业大学 | A method of joint apple callus cell culture and genetic transformation identify apple disease-resistant gene |
| CN115715524A (en) * | 2022-11-30 | 2023-02-28 | 广东省科学院生物与医学工程研究所 | Tissue culture method for improving embryogenic callus induction rate of apple leaves |
| CN115715524B (en) * | 2022-11-30 | 2023-10-03 | 广东省科学院生物与医学工程研究所 | Tissue culture method for improving embryogenic callus induction rate of apple leaves |
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