CN109576299A - A kind of Agrobacterium tumefaciens mediated how main stick spore genetic transforming method - Google Patents
A kind of Agrobacterium tumefaciens mediated how main stick spore genetic transforming method Download PDFInfo
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Abstract
The present invention provides a kind of Agrobacterium tumefaciens mediated how main stick spore genetic transforming methods, include the following steps: (1) how main stick spore to the sensitivity testing of hygromycin；(2) antibiotic inhibits the concentration screening of Agrobacterium normal growth；(3) how main stick spore conidial suspension preparation；(4) prepared by the bacterium solution of Agrobacterium tumefaciems containing binary vector；(5) Agrobacterium tumefaciems and how main stick spore conidium co-culture；(6) screening of transformant；(7) Molecular Identification of transformant is inserted into copy number and genetic stability analysis.The present invention does not need removal fungal cell wall and prepares protoplast, easy to operate, high conversion efficiency, and obtained transformant list copy ratio is big, can stablize heredity.The present invention provides the Agrobacterium tumefaciens mediated how main stick spore genetic transforming method of a set of stability and high efficiency, provides potentially possible to obtain pathogenic mutation body, lay the foundation for further investigation pathogenic related gene and pathogenic mechanism.
Description
Technical field
The present invention relates to a kind of Agrobacterium tumefaciens mediated how main stick spore genetic transforming methods, belong to genetic engineering field.
Background technique
Mostly main stick spore is a kind of important phytopathogen, and host range is extensive.The pathogen usually endangers plant leaf portion
Target shape scab is formed, flower, fruit, stem and the root of host can also be infected.However, related how main stick spore causes a disease, genetic mechanism is still unclear
Chu develops new strategy, new method is provided, in molecular water in view of Economic Importance of the how main stick spore leaf spot on crop
Flat upper further investigation pathogenic related gene and pathogenic mechanism are imperative.
Fungal transformation technology generates mutant with random or targeting failure modes gene, this is to go deep on a molecular scale
Research fungal attack related gene provides mutant material.Most commonly used genetic transforming method has original in filamentous fungi at present
The genetic transformation (PEG) that raw plastid mediates, genetic transformation (REMI) that restriction enzyme mediates and Agrobacterium tumefaciens mediated
Genetic transformation (ATMT).In these method for transformation, because ATMT technology avoids some of PEG and REMI transformation technology and lacks
Point and gradually become fungi in study gene function important method.Firstly, ATMT technology is different from PEG technology, do not need
Except fungal cell wall prepares protoplast, and the preparation of protoplast is a complicated process.ATMT technology is better than other turns
Second outstanding feature of change method is that T-DNA can be random is inserted into genome and usually with single shape for copying insertion
Formula is integrated into genome, so, the mutant of any one phenotypic alternation is all likely due to caused by insertion.Pass through sieve
T-DNA insertional mutagenesis library is selected, phenotypic mutation body is obtained, T-DNA insertion point flanking sequence is then expanded, distinguishes impacted
Gene.
There are much examples about ATMT successful conversion in different fungies at present, and also confirms that the transformation technology exists
The deletion of target gene and destruction aspect are highly effective.However, condition needed for different genetic of fungi conversions is different, Transformation Parameters
It has differences, influence factor is different.Up to the present, not about Agrobacterium tumefaciens mediated how main stick spore genetic transformation
The report of research can not be successfully and be converted referring to the method for transformation of other filamentous fungis.
Summary of the invention
The object of the present invention is to provide a kind of Agrobacterium tumefaciens mediated how main stick spore genetic transforming methods, pass through this method
T-DNA insertion bacterial strain can be obtained, phenotypic mutation body can be obtained, then cause to it by screening T-DNA insertional mutagenesis library
Characteristic of disease test, evaluates its pathogenecity.Its T-DNA insertion copy number, amplification insertion are studied to the mutant of pathogenecity variation
Site flanking sequence, then impacted gene is distinguished by Relational database and molecular biology software.The present invention is to obtain
The offer of pathogenic mutation body is potentially possible, lays the foundation for further investigation Disease-causing gene and pathogenic mechanism.
In order to solve the above technical problems, the technical solution adopted by the present invention are as follows:
A kind of Agrobacterium tumefaciens mediated how main stick spore genetic transforming method, comprising the following steps:
(1) sensitivity testing of the how main stick spore to hygromycin: how main stick is inoculated on the PDA plate of the hygromycin containing various concentration
Spore, culture observation bacterium colony growing state, determines hygromycin (hph) optimal use concentration；
(2) antibiotic inhibits the concentration screening of Agrobacterium tumefaciems normal growth: after the activation of picking Agrobacterium monoclonal expands culture,
It collects mycelium dilution to be spread evenly across on the plate of cephalosporin containing various concentration and Ticarcillin/Clavulanate Acid, culture observation determines antibiotic
Optimal use concentration；
(3) it how main stick spore conidial suspension preparation: with the how main stick spore conidium on induced medium elution plate, adjusts
Arthrospore concentration is 1 × 105-6A/mL；
(4) prepared by the bacterium solution of Agrobacterium tumefaciems containing binary vector: dilute with induced medium after picking Agrobacterium monoclonal activation culture
It releases, is further cultured for 5~6h and makes OD600Reach 0.5~0.6；
(5) Agrobacterium tumefaciems and how main stick spore co-culture: by ready Agrobacterium tumefaciems and how main stick spore sectional growing spore suspension
Liquid is uniformly mixed is coated on the nitrocellulose filter of the CM culture medium containing acetosyringone (AS) in equal volume, 22-25 DEG C of dark
Culture is no less than 48h；
(6) screening of transformant: by the cellulose nitrate on CM plate after Agrobacterium tumefaciems and the co-cultivation of how main stick spore conidium
Plain film is cut into 1cm × 4cm size, and overturns in tiling to the plate of the pressure containing selection, 25 DEG C of dark culturings, until bacterium colony goes out
Existing, the bacterium colony that can be grown primarily determines as transformant；The single colonie grown is transferred to the plate of the new pressure containing selection respectively
Upper culture to mycelia is paved with plate, is used for Molecular Detection；
(7) the PCR identification of transformant is inserted into copy number and genetic stability analysis: above-mentioned transformant is carried out hygromycin phosphorus
The PCR of sour transferase gene is verified, and the transformant for determining that these assume is real transformant, then carries out Southern
Blot test analyzes it and is inserted into copy number, finally, detecting the genetic stability of these transformants.
Preferably: hygromycin concentration is 100 μ g/ mL in the step (1).
Preferably: mycelium dilution is to OD in the step (1)600Even spread when being 0.5.
Preferably: antibiotic concentration described in the step (2) is 400 μ g/mL Cef+200 μ g/mL Tim.
Preferably: conidium concentration described in the step (3) is 1 × 106A/mL.
Preferably: Agrobacterium tumefaciems concentration OD used in the step (4)600It is 5.6 for 0.5, IM Medium's PH Value.
Preferably: acetosyringone concentration used in the step (5) is 200 μM, and CM Medium's PH Value is 5.6, is trained altogether
Supporting temperature is 23 DEG C, and the co-cultivation time is 54-72h.
Preferably: the induced medium is to contain 50 μ g/mL kanamycins (Kan) and 25 μ g/mL rifampins (Rif)
LB culture medium.
Beneficial effects of the present invention:
The present invention for the first time studies Agrobacterium tumefaciens mediated how main stick spore genetic transformation, provides a set of stability and high efficiency
Agrobacterium tumefaciens mediated how main stick spore genetic transforming method, with stochastic model destroy gene and generate mutant, for point
Further investigation fungal attack related gene provides mutant material in sub- level, lays the foundation for further investigation pathogenic mechanism.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
Some embodiments of invention for those of ordinary skill in the art without creative efforts, can be with
It obtains other drawings based on these drawings.
Fig. 1 various concentration hygromycin stick spore how main to muskmelonCc-The inhibiting effect of GX bacterial strain；
Transformant on Fig. 2 screening and culturing medium；
Fig. 3 PCR detects transformant；
The T-DNA that Fig. 4 Southern blot detects transformant is inserted into copy number；
The detection of Fig. 5 transformant genetic stability.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
The Agrobacterium tumefaciens strain that following tests is selected is that AGL-1(is bought in Zhengzhou Shang Yi Biotechnology Co., Ltd),
The resistance screening that conversion carrier contains is labeled as hygromix phosphotransferase resistant gene and Kan resistant gene, the termination contained
Son is trpC.
Following tests select transformation receptor bacterium be how main stick spore bacterium (Corynespora cassiicola) muskmelon separation
Object Guangxi bacterial strain (Cc-GX), separated by this laboratory and identify and save.
The culture medium prescription that following tests is selected is as follows:
PDA: 200 g of peeling potatoes shave, add boiling to boil 30 min, double gauze filtering adds 20g glucose, 15g fine jade
Rouge, distilled water are settled to 1 L.Under 0.1MPa pressure, 121 °C of moist heat sterilization 15min.
LB: tryptone 10g, sodium chloride 10g, yeast extract 5g, NaOH adjust pH to 7.0 or so.Solid LB culture
Base, another plus 15g/L agar powder.Distilled water is settled to 1 L.Under 0.1MPa pressure, 121 °C of moist heat sterilization 20min.
IM:K2HPO42.05g KH2PO41.45g, NaCl 0.15g, MgSO4·7H2O 0.5g, CaCl2·6H2O
0.1g, FeSO4·7H2O 0.0025g, (NH4)2SO40.5g, glucose 2g, glycerine 5mL, MES 7.8g.PH is 5.5-
5.8, distilled water is settled to 1 L.Under 0.1MPa pressure, 121 °C of moist heat sterilization 20min.
CM:K2HPO42.05g KH2PO41.45g, NaCl 0.15g, MgSO4·7H2O 0.5g, CaCl2·6H2O
0.1g, FeSO4·7H2O 0.0025g, (NH4)2SO40.5g, glucose 1g, glycerine 5mL, MES 7.8g, agar powder 15g.
PH is 5.5-5.8, and distilled water is settled to 1 L.Under 0.1MPa pressure, 121 °C of moist heat sterilization 20min.
Embodiment 1
Agrobacterium tumefaciens mediated how main stick spore genetic transformation test, the specific steps are as follows:
(1)Cc-Sensitivity testing of the GX bacterial strain to hygromycin.The how main stick spore of Agrobacterium tumefaciens mediated muskmelonCc-GX bacterial strain is lost
It passes in transformation system, selects suitable hygromycin concentration, it is most important to selection mutant.The bacterium colony that will be saved on inclined-plane turns
It is connected in PDA plate, in 25 °C of dark activation cultures.Then, being beaten with the punch of sterilizing from colony edge and taking diameter is 5mm's
Bacteria cake, then bacteria cake is successively placed into new PDA plate center, each one ferfas cake of plating sets the plate after inoculation
In constant incubator, 25 °C of dark culturing 7d.The bacteria cake for taking diameter to be 5mm is beaten from colony edge with the punch of sterilizing again,
By bacteria cake be placed into new hygromycin containing various concentration (0,50,55,60,65,70,75,80,85,90,95,
100,105,110,115, and 120 μ g/ mL) PDA plate center, 25 °C of dark culturing 7d, observation bacterium colony growth
Situation, 3 repetitions of every processing, determines hygromycin optimal use concentration.
(2) Cef inhibits the concentration screening of Agrobacterium tumefaciems normal growth.Agriculture bar is inhibited using the method screening Cef of coated plate
The concentration of bacterium growth.By the Agrobacterium tumefaciems AGL-1 streak inoculation containing binary vector in contain 50 μ g/mL Kan and 25 μ g/
On the solid LB plate of mL Rif, 28 DEG C of dark culturing 2d.AGL-1 Agrobacterium single bacterium on picking LB solid plate falls on 10mL
LB liquid medium containing 50 μ g/mL Kan and 25 μ g/mL Rif, 200rpm shaken cultivation.Setting contains different concentration of Ce f
The plate of (0,25,50,100,150,200,250,300,350,400,450,500 μ g/mL).Work as Agrobacterium
OD600Even spread plate when value is 0.5,200 μ L bacterium solutions/plate, 28 DEG C of culture 3d, the growing state of record observation Agrobacterium,
3 repetitions of every processing, determine the optimal use concentration of Cef.
(3) Tim inhibits the concentration screening of Agrobacterium normal growth.Inhibit Agrobacterium raw using the method screening Tim of coated plate
Long concentration.By the Agrobacterium tumefaciems AGL-1 streak inoculation containing binary vector in contain 50 μ g/mL Kan and 25 μ g/mL
On the solid LB plate of Rif, 28 DEG C of dark culturing 2d.AGL-1 Agrobacterium single bacterium on picking LB solid plate falls on 10mL and contains
The LB liquid medium of 50 μ g/mL Kan and 25 μ g/mL Rif, 200rpm shaken cultivation.Setting contains various concentration Tim(0,
25,50,75,100,150,200,250,300 μ g/mL) plate.As the OD of Agrobacterium600Value is uniform when being 0.5
Spread plate, 200 μ L/ plates, 28 DEG C of culture 3d, the growing state of record observation Agrobacterium, 3 repetitions of every processing determine Tim's
Optimal use concentration.
(4) the how main stick spore conidial suspension preparation of muskmelon.By what is saved on inclined-planeCc-GX bacterial strain, switching are flat in PDA
Then activation culture in plate beats the bacteria cake for taking diameter to be 5mm from colony edge with the punch of sterilizing, then successively put bacteria cake
Set new PDA plate center, each one ferfas cake of plating.The plate after inoculation is placed in constant incubator again, in
25 °C of dark culturing 20d its produce spore naturally.Add 5mL IM on cultured flat-plate bacterial colony, then is swept with the writing brush of sterilizing and fall spore
Son, then three layers of lens wiping paper filtering obtain spore suspension, and 12,000rpm, 10min is concentrated spore, removes supernatant.It is trained with IM liquid
It supports base and dilutes spore, blood counting chamber counts, and adjusting spore concentration is 1 × 106A/mL is spare.
(5) preparation of the bacterium solution of Agrobacterium tumefaciems containing binary vector.By the Agrobacterium AGL-1 streak inoculation containing binary vector
In on the solid LB plate containing 50 μ g/mL Kan and 25 μ g/mL Rif, 28 DEG C of dark culturing 2d.On picking LB solid plate
AGL-1 Agrobacterium single bacterium fall on 5mL contain 50 μ g/mL Kan and 25 μ g/mL Rif LB liquid medium, 28 DEG C, 200rpm
Shaken cultivation is stayed overnight.1.5mL bacterium solution is taken, 1,000rpm, 1min remove supernatant, are cleaned 2 times with IM culture medium, then outstanding with 1mL IM
Floating thallus, the IM fluid nutrient medium for taking bacterium solution to contain 200 μM of AS in 5mL make the OD of AGL-1600Control is between 0.2~0.3, and 28
DEG C, 5~6h of 200rpm shaken cultivation makes OD600Reach 0.5, it is spare.
(6) Agrobacterium tumefaciems and how main stick spore bacterium co-culture.Ready Agrobacterium and spore suspension are mixed in equal volume
It closes uniform.It is 0.45 μm by aperture, the sterile nitrocellulose filter tiling that size is 4cm × 5cm is flat to the CM containing 200 μM of AS
On plate.200 μ L mixed liquors are taken to be uniformly coated on nitrocellulose filter, 23 DEG C of dark culturing 65h.
(7) screening of transformant.Agrobacterium tumefaciems and how main stick spore conidium co-culture the nitre on CM plate after 65h
Acid cellulose film is cut into 1cm × 4cm size, and overturns tiling to (PDA+100 μ g/mL hph+400 μ g/mL in screening flat board
Cef+200 μ g/mL Tim).25 DEG C of 5-7 d of dark culturing, until bacterium colony occurs, the bacterium colony that can be grown is primarily determined as conversion
Son.The single colonie grown is transferred on new PDA+100 μ g/mL hph plate respectively, carries out postsearch screening, positive bacteria is fallen within
4 DEG C of PDA plate save backup.
(8)CcThe PCR of-GX transformant is identified.Because of the binary vector carried in test Agrobacterium tumefaciems AGL-1 used
On contain hygromycin phosphotransferase gene, it is possible to contain hygromycin (hph) culture medium preliminary screening transformant, with
This measurement how main stick spore of muskmelonCcWhether the insertion of T-DNA is had on the genomic DNA of-GX conversion bacterial strain.In order to make result of study
It is more accurate, PCR amplification is carried out to transformant according to hygromycin phosphotransferase gene sequence design specific primer, further
Identification.
(9) template marked using the hygromycin phosphotransferase gene on binary vector as probe, prepares hybridization probe.
20 conversion bacterial strains are randomly choosed, its genomic DNA is extracted, are usedEcoRI andHinDIII carries out double digestion.It will after complete degestion
Digestion sample spot is added on 0.75% gel, is separated by electrophoresis, for Southern blot detect, finally with the spy of label
Needle hybridization, analysis T-DNA are inserted into copy number.
(10)CcThe genetic stability of-GX transformant is analyzed.Randomly select 10 transformants, while withCc- GX wild type
Bacterial strain does control and exists to measure hygromycin phosphotransferase geneCc- GX converts the genetic stability in subgenom.Strain is first
Then activation culture beats the bacteria cake for taking diameter to be 5mm from colony edge with the punch of sterilizing again, then is successively placed into bacteria cake
It continuously cultivated for 5 generations in the new PDA culture medium without hygromycin, finally transfers in the PDA culture containing 100 μ g/mL hygromycin
On base, the growth conditions of observation conversion bacterial strain determine whether transformant still has hygromycin resistance, to detect the something lost of transformant
Pass stability.
Test result:
(1) as shown in Figure 1, as hygromycin concentration >=50 μ g/mL,Cc-The growth of GX bacterium colony is obvious suppressed, when hygromycin is dense
When spending >=100 μ g/mL,Cc-GX bacterium colony is not grown completely, and therefore, the hygromycin concentration that 100 μ g/mL are chosen during test is made
For the screening concentration of positive transformant.
(2) with the raising of Cef concentration, reduced trend is presented in AGL-1 increment.When Cef concentration reaches 250 μ g/mL
When, the growth of AGL-1 can be completely inhibited.Test process chooses the Cef concentration of 400 μ g/mL as the examination for inhibiting AGL-1 growth
Test concentration.
(3) with the raising of Tim concentration, reduced trend is presented in AGL-1 increment.When Tim concentration reaches 150 μ g/mL
When, the growth of AGL-1 can be completely inhibited.Test process chooses the Tim concentration of 200 μ g/mL as the examination for inhibiting AGL-1 growth
Test concentration.
(4) as shown in Fig. 2, by the nitric acid on CM plate after Agrobacterium tumefaciems and how main stick spore conidium co-cultivation 65h
Cellulose membrane is cut into strip and overturns tiling to screening flat board.25 DEG C of 5-7 d of dark culturing, that adds AS has bacterium colony to grow,
Growing without bacterium colony for AS is not added.
(5) as shown in figure 3, binary vector and all hygromycin resistant transformed sons can amplify the tide of a treaty 1355bp
Neomycin phosphotransferase genetic fragment particular target band, and unconverted wild-type strain shows T- without this specific band
Hygromycin phosphotransferase gene on DNA has been integrated into the how main stick spore of muskmelonCcIn-GX strain gene group.
(6) in randomly selected 20 plants of T-DNA insertion mutation body, each mutant strain has a unique hybridization item
Band, this illustrates that T-DNA is that random integration arrivesCcIn-GX genome.Wherein 18 plants of transformants present single hybrid belt, table
This bright 18 plants of transformant T-DNA are inserted into the form singly copied, and single copy insertion ratio is 90%.Other two plants, there is one plant
There are 2 hybrid belts, in addition there are 3 hybrid belts for one plant, shows that the two transformants T-DNA is inserted into the form of multicopy,
Account for 10%(Fig. 4).So carrying out the how main stick spore of muskmelon using ATMT technologyCcWhen the genetic transformation of-GX bacterial strain, the mono- copy of T-DNA
Insertion ratio is big, this is convenient for transformant convenient for the determination of mutant character gene loci and the amplification of insertion point flanking fragment
Genetic analysis, the genetic conversion system established is suitable for constructing how main stick spore mutant library.
(7) 10 transformants randomly selected after continuously cultivating for 5 generations in the PDA culture medium without hygromycin, then are transferred
Onto the PDA culture medium containing 100 μ g/mL hygromycin, transformant can still be grown, while compare wild typeCc- GX bacterial strain
It cannot grow.And PCR authenticates to the presence of T-DNA Insert Fragment, illustrates that these conversion bacterial strains still have hygromycin resistance.
So the T-DNA that Agrobacterium tumefaciems carries in plasmid can successfully be integrated into the how main stick spore of muskmelon by ATMT method for transformationCcIn the genome of-GX bacterial strain, and being capable of normal expression.These transformants have genetic stability, as shown in Figure 5.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Within mind and principle, any modification, equivalent replacement, improvement and so on be should all be included in the protection scope of the present invention.
Claims (8)
1. a kind of Agrobacterium tumefaciens mediated how main stick spore genetic transforming method, which comprises the following steps:
(1) sensitivity testing of the how main stick spore to hygromycin: how main stick is inoculated on the PDA plate of the hygromycin containing various concentration
Spore, culture observation bacterium colony growing state, determines hygromycin optimal use concentration；
(2) antibiotic inhibits the concentration screening of Agrobacterium tumefaciems normal growth: after the activation of picking Agrobacterium monoclonal expands culture,
It collects mycelium dilution to be spread evenly across on the plate of cephalosporin containing various concentration and Ticarcillin/Clavulanate Acid, culture observation determines antibiotic
Optimal use concentration；
(3) how main stick spore conidial suspension preparation: on cultured flat-plate bacterial colony plus 5mL IM fluid nutrient medium elutes
How main stick spore conidium on plate, then swept with the writing brush of sterilizing and fall spore, then it is outstanding to obtain spore for three layers of lens wiping paper filtering
Supernatant liquid, adjusting spore concentration is 1 × 105-6A/mL；
(4) bacterium solution of Agrobacterium tumefaciems containing binary vector prepare: by the Agrobacterium streak inoculation containing binary vector in contain 50 μ g/
On the solid LB plate of mL kanamycins and 25 μ g/mL rifampins, dark culturing 2d, picking single bacterium falls on 5mL containing 50 μ g/mL
The LB liquid medium of Kan and 25 μ g/mL Rif, shaken cultivation is overnight, takes 1.5mL bacterium solution, supernatant is removed in centrifugation, with IM culture medium
Cleaning 2 times, then with 1mL IM suspension thalline, take a small amount of bacterium solution in IM fluid nutrient medium of the 5mL containing AS, 5~6h of shaken cultivation makes
It obtains OD600 and reaches 0.5~0.6；
(5) Agrobacterium tumefaciems and how main stick spore co-culture: being 0.45 μm by aperture, size is the sterile cellulose nitrate of 4cm × 5cm
In plain film tiling to the CM culture medium containing AS；Ready Agrobacterium and spore suspension are uniformly mixed in equal volume, take 200 μ L
Mixed liquor is uniformly coated on nitrocellulose filter, and 22-25 DEG C of dark culturing is no less than 48h；
(6) screening of transformant: by the cellulose nitrate on CM plate after Agrobacterium tumefaciems and the co-cultivation of how main stick spore conidium
Plain film is cut into fine strip shape, and overturns and tile onto screening flat board, 25 DEG C of dark culturings, until bacterium colony occurs, the bacterium colony that can be grown
It primarily determines as transformant；The single colonie grown is transferred to culture to mycelia on the plate of the new pressure containing selection respectively to be paved with
Plate is used for Molecular Detection；
(7) the PCR identification of transformant is inserted into copy number and genetic stability analysis: above-mentioned transformant is carried out hygromycin phosphorus
The PCR of sour transferase gene is verified, and the transformant for determining that these assume is real transformant, then carries out Southern
Blot test analyzes it and is inserted into copy number, finally, detecting the genetic stability of these transformants.
2. the Agrobacterium tumefaciens mediated how main stick spore genetic transforming method of one kind according to claim 1, it is characterised in that:
Hygromycin concentration is 100 μ g/ mL in the step (1).
3. the Agrobacterium tumefaciens mediated how main stick spore genetic transforming method of one kind according to claim 1, it is characterised in that:
Mycelium dilution is to OD in the step (1)600Even spread when being 0.5.
4. the Agrobacterium tumefaciens mediated how main stick spore genetic transforming method of one kind according to claim 1, it is characterised in that:
Step (2) antibiotic concentration is 400 μ g/mL Cef+200 μ g/mL Tim.
5. the Agrobacterium tumefaciens mediated how main stick spore genetic transforming method of one kind according to claim 1, it is characterised in that:
The conidium concentration of the step (3) is 1 × 106A/mL.
6. the Agrobacterium tumefaciens mediated how main stick spore genetic transforming method of one kind according to claim 1, it is characterised in that:
Agrobacterium tumefaciems concentration OD used in the step (4)600It is 5.6 for 0.5, IM Medium's PH Value.
7. the Agrobacterium tumefaciens mediated how main stick spore genetic transforming method of one kind according to claim 1, it is characterised in that:
Acetosyringone concentration used in the step (5) is 200 μM, and CM Medium's PH Value is 5.6, and co-culturing temperature is 23 DEG C, altogether
Incubation time is 54-72h.
8. the Agrobacterium tumefaciens mediated how main stick spore genetic transforming method of one kind according to claim 1, it is characterised in that:
Nitrocellulose filter is cut into 1cm × 4cm size, incubation time 5-7d, screening and culturing medium PDA+ in the step (6)
100μg/mL hph+400μg/mL Cef+200μg/mL Tim。
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| CN110257416A (en) * | 2019-06-26 | 2019-09-20 | 上海润庄农业科技有限公司 | A kind of how main stick spore high-efficiency genetic transforming method of mediated by agriculture bacillus |
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| CN105296526A (en) * | 2015-09-02 | 2016-02-03 | 中国农业科学院郑州果树研究所 | Infectious clone vector of melon necrotic spot virus and construction method of infectious clone vector |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN110257416A (en) * | 2019-06-26 | 2019-09-20 | 上海润庄农业科技有限公司 | A kind of how main stick spore high-efficiency genetic transforming method of mediated by agriculture bacillus |
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