CN104450767A - Agrobacterium tumefaciens-mediated tuber melanosporum genetic transformation method - Google Patents
Agrobacterium tumefaciens-mediated tuber melanosporum genetic transformation method Download PDFInfo
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Abstract
The invention discloses an agrobacterium tumefaciens-mediated tuber melanosporum genetic transformation method and belongs to the field of genetic transformation of tuber melanosporum. The agrobacterium tumefaciens-mediated tuber melanosporum genetic transformation method disclosed by the invention comprises the following steps: (1) preparing a tuber melanosporum spore suspension; (2) transforming a binary vector plasmid carrying a target gene to agrobacterium tumefaciens to obtain a positive transformant of agrobacterium tumefaciens so as to prepare a bacterial liquid; (3) mixing the bacterial liquid prepared in the step (2) and the tuber melanosporum spore suspension prepared in the step (1) and co-culturing; and (4) selectively culturing to screen the transformant of agrobacterium tumefaciens and identifying to obtain. By taking spores of tuber melanosporum as a receptor and agrobacterium tumefaciens as a mediator, the method disclosed by the invention has the advantages of being simple and convenient, high and stable in transformation efficiency, high in frequency that the transformant is monoclonal and the like and is suitable for functional genomical and biological studies of tuber melanosporum and the like.
Description
Technical field
The present invention relates to the method for transformation of black truffle, particularly relate to Agrobacterium tumefaciens mediated black truffle genetic transforming method, belong to the field of genetic transformation of black truffle.
Background technology
Black truffle (Tuber melanosporum) is a kind of original from France, with the ectomycorrhiza type edible fungus of the seeds such as Oak Tree, robur Root Symbiont, receives much concern with its inherent unique perfume and " diamond " personal value.Completing of black truffle gene order-checking has promoted researchist from molecular level to the biological understanding of black truffle, but not yet sets up ripe genetic operating system due to black truffle, strongly limit the research of black truffle functional genomics.
The Agrobacterium tumefaciens mediated filamentous fungus method for transformation that development in recent years is got up has following three advantages: first, Agrobacterium can transform complete cell as spore, mycelium, or even the sporophore of macro fungi, this just eliminates the trouble preparing protoplastis, thus do not need consideration to affect factors (the Vianey Olmedo-Monfil of protoplast transformation rate, Carlos Cortes-Penagos, Alfredo Herrera-Estrella.Three decades of fungal transformation:keyconcepts and applications.Methods in Molecular Biology, 2004, 267:297-313.), the second, the transformation efficiency of Agrobacterium-mediated Transformation is very high, research shows, the transformation efficiency of agriculture bacillus mediated filamentous fungus is generally at 300-720 transformant every 10
7individual cell, than other fungal transformation method height 100-1000 doubly (Marcel-J.A.de Groot, Paul Bundock, Paul-J.JHooykaas, Alice-G.M.Beijersbergen.Agrobacterium tumefaciens-mediatedtransformation of filamentous fungi.Nature Biotechnology, 1998,16:839-842.), 3rd, it is most of for singly to copy insertion that T-DNA inserts the mutant produced, the unseparated transformant of offspring can be obtained when adopting the conidium with monokaryon to be transformation receptor, avoid a difficult problem (the ED Mullins of the transformant instability that hypha,hyphae multinuclear causes, X Chen, PRomaine, R Raina, DM Geiser, S Kang.Agrobacterium-MediatedTransformation of Fusarium oxysporum:An Efficient Tool for InsertionalMutagenesis and Gene Transfer.Phytopathology, 2001, 91:173-180.).The shortcoming that Agrobacterium tumefaciens mediated Genetic Transformation System of Filamentous Fungi exists transforms to need a large amount of spores or sporophore as transformation receptor, and it is few or do not produce spore fungi (Olmedo-Monfil et al, 2004) to produce spore under not being suitable for culture conditions.
Black truffle produces spore and seldom or not produces spore on fermentation condition and fungi conventional medium PDA substratum, black truffle not yet has the report successfully preparing protoplastis at present simultaneously, and the genetic transformation system lacking a set of applicable black truffle is the bottleneck of restriction black truffle molecular biology research.
Therefore, urgently build a kind of simple, efficient black truffle genetic transformation system, for the research of promotion black truffle functional genomics, promote that the development and utilization of black truffle resource is significant.
Summary of the invention
Technical problem to be solved by this invention is to provide a kind of Agrobacterium tumefaciens mediated black truffle (Tuber melanosporum) genetic transforming method, and the method is simple and convenient, transformation efficiency is high and stable.
For solving the problems of the technologies described above, the technical solution used in the present invention is:
The invention discloses a kind of Agrobacterium tumefaciens mediated black truffle (Tuber melanosporum) genetic transforming method, comprise the following steps:
(1) black truffle spore suspension is prepared; (2) will the binary vector Plastid transformation agrobacterium tumefaciens of goal gene be carried, obtain agrobacterium tumefaciens positive transformant, preparation bacterium liquid; (3) black truffle spore suspension prepared by bacterium liquid step (2) prepared and step (1) mixes, Dual culture; (4) selectivity is cultivated, and screening black truffle transformant, qualification, to obtain final product.
Wherein, step (1) described black truffle spore suspension miospore concentration is 10
4-10
8individual/mL, is preferably 10
6individual/mL; Described preparation black truffle spore suspension, comprising: black truffle mycelium is inoculated in PDA substratum by (a), cultivates activation in 28 DEG C; Preferably, 3d is activated; B black truffle mycelium after activation is inoculated in VBC substratum by (), cultivate 4-9d for 28 DEG C, be preferably 7d; Wash mycelium with sterilized water, collect the spore of black truffle, dilution, to obtain final product.
The method of the binary vector Plastid transformation agrobacterium tumefaciens carrying goal gene is well known to those skilled in the art by step (2), such as chemical transformation.
Step (2) described preparation bacterium liquid comprises: (a) picking agrobacterium tumefaciens positive transformant, is inoculated into containing in antibiotic LB substratum, and 12-20h is cultivated in 28 DEG C of 200rpm concussions, is preferably 18h, centrifugal, collects thalline; B () is with the resuspended thalline of IM substratum, centrifugal, collect thalline; C () uses the resuspended thalline of IM substratum, by cell concentration OD
600be adjusted to 0.1 ~ 0.6, be preferably 0.15 ~ 0.3,28 DEG C of 200rpm shaking culture 3-8h, be preferably 6h, obtain final product.
Wherein, containing Syringylethanone in described IM substratum, preferably, the concentration of described Syringylethanone is 200mmol/L.
In the black truffle genetic transforming method that the present invention is Agrobacterium tumefaciens mediated, the bacterium liquid that step (2) is prepared by step (3) mixes with black truffle spore suspension equal-volume prepared by step (1); The substratum of described Dual culture is the CM substratum being covered with cellulose nitrate film; Dual culture condition is that 28 DEG C of lucifuges cultivate 24-60h, is preferably 48h.
Wherein, described CM substratum is the IM substratum of interpolation 2% (w/v) agarose, is the Syringylethanone of 200mmol/L containing final concentration.
The described selectivity of step (4) cultivate be by step (3) Dual culture after cellulose nitrate film take off, forward is taped against containing antibiotic PDA substratum, and 28 DEG C are cultured to and occur visible list bacterium colony.Further by selective medium extremely new for single bacterium colony picking, be separated the black truffle bacterial strain having antibiotics resistance.
Microbiotic of the present invention is selected from cephamycin, Totomycin or kantlex.
The black truffle genetic transforming method that the present invention is Agrobacterium tumefaciens mediated, utilizes VBC substratum to promote the large volume production spore of black truffle, Dual culture after being mixed with black truffle spore by the agrobacterium tumefaciens containing binary vector subsequently, by resistance screening, obtains transformant.The transformation efficiency of the method can reach 1-6 × 10
5individual spore, transformation efficiency is high and stable, overcomes the problem that black truffle many cells mycelium is difficult to prepare fast the black truffle genetic transformation difficulty that the reasons such as a large amount of single-celled protozoal plastids cause.
The present invention is the black truffle by pCAMBIA1302-1 Plastid transformation successfully, screens the mutant of a series of integration hygromycin gene.PCR qualification result shows, all fragments that all can amplify expection size for preliminary operation beggar, illustrate that black truffle transformant contains the T-DNA fragment of insertion.Further pcr amplification product is carried out sequencing, Blast analyzes and shows, the consistence of amplified fragments and hygromycin phosphotransferase gene reaches 100%, further illustrates T-DNA and successfully proceeds to black truffle genome, and heredity that can be stable.The present invention is with 10
6spore is the material that sets out, and screen 28,45 and 62 positive transformants respectively by Agrobacterium tumefaciens mediated, positive transformants efficiency reaches 4.53 ± 1.75/10
5individual spore.
The present invention is also successful introduces black truffle by external source green fluorescent protein encoding gene, after expressing, recipient cell shows special green under excitation light, may be used for cytoactive detection, morphological analysis in fermenting process, be also the important tool of research black truffle and host plant interaction relationship simultaneously.The validity of the black truffle genetic transformation system that this experimental result builds from the further proved invention of cell levels, simultaneously also for black truffle biological study provides a kind of important tool.
Technical solution of the present invention compared with prior art, has following beneficial effect:
(1) the inventive method is more simple with compared with protoplast transformation method, and the method is applicable to other and produces the less filamentous fungus of spore simultaneously.
(2) the inventive method utilizes VBC substratum once can prepare the spore of black truffle in a large number, meets the needs building the genetic manipulations such as mutant library.
(3) the present invention with the spore of black truffle for acceptor, take agrobacterium tumefaciens as amboceptor, there is the frequency advantages of higher that transformation efficiency is high, transformant is single copy, be applicable to the structure of black truffle T-DNA radom insertion mutant library, the silence of functional gene or knock out.This is the genetic transformation system of domestic and international reported first black truffle.
the term definition arrived involved in the present invention
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art usually understand identical implication.
Term " genetic transformation " means by certain approach or technology by the genome of Studies of Transfer of Alien Genes Into Receptors cell, and makes it to be expressed in recipient cell.
Term " transformant " means to import foreign DNA thus obtains the single bacterium colony of recipient bacterium of new inherited character.
Term " microbiotic " means the class secondary metabolite with antipathogen or other activity produced in life process by microorganism (comprising bacterium, fungi, actinomyces) or high animals and plants, and other living cells can be disturbed to grow the chemical substance of function; Comprise beta-lactam (as cephalosporins), aminoglycoside (as kantlex), amphenicols (as paraxin) etc. several thousand kinds.
Accompanying drawing explanation
Fig. 1 is that black truffle is producing growing state and the Observations On The Spore Morphology of spore substratum VBC; Wherein, A: black truffle is producing the growing state of spore substratum VBC; B: Observations On The Spore Morphology;
Fig. 2 is the collection of illustrative plates of plasmid pCAMBIA1302-1;
Fig. 3 is part black truffle transformant colonies form; Wherein, A: unconverted black truffle bacterial strain; B: part black truffle transformant colonies;
Fig. 4 is the electrophoretogram of the pcr amplification of the black truffle transformant hygromycin phosphotransferase gene (hph) of random choose; Wherein, 1-10: black truffle transformant; Positive control: the pcr amplification result using pCAMBIA1302 plasmid as template; Negative control (wildtype): using wild-type black truffle genome as template amplification result; Negativecontrol (without template): do not add template in PCR reaction system;
Fig. 5 is the collection of illustrative plates of plasmid pCAMBIA1302;
Fig. 6 is black truffle wild strain and transformant fluoroscopic examination result; Wherein, A: black truffle wild strain; B: black truffle transformant.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.It should be understood that described embodiment is only exemplary, any restriction is not formed to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments or replacement all fall into protection scope of the present invention.
1, experiment material
Black truffle (Mianyang City edible mushrooms institute of Sichuan Province);
Agrobacterium tumefaciens EHA105 (sulphuric acid kanamycin responsive type) (being so kind as to give by Agricultural University Of Nanjing associate professor Wu Jun);
PCAMBIA1302-1 plasmid (the present inventor laboratory builds, after utilizing SpeI and XbaI double digestion pCAMBIA1302, by sticky end from connecting structure pCAMBIA1302-1 plasmid, lacZ α and promoter region deleted);
PCAMBIA1302 plasmid (The small, versatile pPZP family ofAgrobacterium binary vector for plant transformation.Plant MolecularBiology.25 (6), 989-994 (1994)).
The structure of embodiment 1 black truffle mutant library
1, experimental technique
The preparation of 1.1 black truffle spores
(1) black truffle mycelium is inoculated on PDA substratum, cultivates activation 3d in 28 DEG C.PDA substratum: 200g peeled potatoes, adds glucose 20g, MgSO after adding appropriate distilled water boiling 30min
47H
2o 1.5g, KH
2pO
43.0g, V
b150mg, agar 18g, supplementary distilled water is settled to 1000mL, pH nature, 115 DEG C, 30min high-temperature sterilization.
(2) mycelium of the black truffle colony edge after activation is inoculated on VBC substratum, after 28 DEG C of cultivation 7d, with 5mL sterilized water washing mycelium, collects the spore of black truffle.
VBC substratum: potassium primary phosphate 1.0g, saltpetre 1.0g, sucrose 0.5g, V
b1100mg, VC 100mg, agar 20g, distilled water is settled to 1000mL, pH nature, 115 DEG C, 30min high-temperature sterilization.
(3) spore suspension is placed on blood counting chamber, utilizes microscope to count spore, spore suspension is diluted to 10
6individual/mL.
The conversion of 1.2 agrobacterium tumefaciens and cultivation
Adopt chemical transformation by pCAMBIA1302-1 plasmid (Fig. 1) transform Agrobacterium tumefaciens.
1.2.1 prepared by agrobacterium tumefaciens competence
Activated on LB solid medium by the agrobacterium tumefaciens EHA105 (sulphuric acid kanamycin responsive type) preserved, transfer after three times, be inoculated in LB liquid nutrient medium, 28 DEG C, 200rpm, shaking table shaking culture is to OD
600=0.5, collecting cell, utilizes the 0.1M CaCl of precooling
2solution prepares agrobacterium tumefaciens competent cell.
LB Media Components: peptone 10g, yeast powder 5g, NaCl 10g, is settled to 1L.
1.2.2pCAMBIA1302-1 Plastid transformation agrobacterium tumefaciens
Added in 50 μ L agrobacterium tumefaciens competent cells by 10 μ L pCAMBIA1302-1 plasmids, ice bath leaves standstill 30min, is then placed in liquid nitrogen quick-frozen 3min, immediately 37 DEG C of water-bath thermal shock 5min; Be placed in 2min on ice again, add the LB liquid nutrient medium of 650 μ L, 28 DEG C of preheatings, 28 DEG C, 200rpm, shaking table shaking culture 5h, 4000r/min centrifugal bacterium liquid 5min, is resuspended in 100 μ L LB liquid medium, be coated on the LB solid medium containing 50 μ g/mL kantlex, screening agrobacterium tumefaciens positive transformant.
Utilize bacterium colony PCR to identify positive transformant, primers designed is: HPT-5:GACGACACCGTCAGCGCGTC, HPT-3:CTTTGCCCTCGGACGAGTGCTGG.PCR reaction system is 50 μ L, comprises 10 × Buffer 5 μ L, dNTP (20mmolL
-1) 4 μ L, primer (25pmol μ L
-1) each 2 μ L, Mg
2+(25mmolL
-1) 4 μ L, thallus DNA (about 50ng μ L
-1) 1 μ L, Taq archaeal dna polymerase (5U μ L
-1) 0.5 μ L, add H
2o to 50 μ L.Reaction conditions: 94 DEG C of sex change 5min; 94 DEG C of 30sec, 50 DEG C of 30sec, 72 DEG C of 1min, 30 circulations; 72 DEG C extend 10min.
1.2.3 the cultivation of agrobacterium tumefaciens positive transformant
Agrobacterium tumefaciens EHA105 (carrying pCAMBIA1302-1 plasmid) single colony inoculation of picking activation is in LB substratum (containing 50 μ g/mL sulphuric acid kanamycins), 28 DEG C, 200rpm concussion cultivates after 18h, the centrifugal 5min of 5000rpm, supernatant discarded, collects thalline.With the resuspended thalline of IM substratum, the centrifugal 5min of 5000rpm, supernatant discarded, collects thalline, again uses the resuspended thalline of IM substratum, by IM substratum by cell concentration OD
600be adjusted to 0.15 ~ 0.3,28 DEG C of 200rpm shaking table shaking culture 6h.
IM Media Components: KH
2pO
41.45g, K
2hPO
42.05g, (NH
4)
2sO
40.5g, MgSO
47H
2o 0.5g, NaCl 0.15g, CaCl
20.067g, FeSO
47H
2o 0.0025g, 1M MES (pH 5.5) 40mL, 20% glucose (W/V) 10mL, 50% glycerine (V/V) 5mL, micro-5mL, 200mmol Syringylethanone (AS) 1mL, be settled to 1L, 115 DEG C of autoclaving 30min.
Trace element preparation: take ZnSO
47H
2o, CuSO
45H
2o, MnSO
47H
2o, Na
2moO
47H
2o, H
3bO
3each 0.01g is dissolved in 100mL water.
200mmol Syringylethanone is prepared: take 0.039g and be dissolved in 1mL dehydrated alcohol.
20% glucose: take 20g glucose, sterilized water is settled to 100mL.
50% glycerine: 50mL glycerine is dissolved in the sterilized water of 50mL.
1.3 agrobacterium tumefaciens and black truffle spore Dual culture and selectivity are cultivated
By above-mentioned EHA105 (carrying pCAMBIA1302-1 plasmid) the bacterium liquid for preparing and the mixing of black truffle spore suspension equal-volume vortex, draw 200 μ l mixed solutions to be uniformly coated on and to be covered with on the CM substratum of cellulose nitrate film, it is 200mM/L that CM substratum adds AS before use to final concentration.Blown in Bechtop by culture dish to half-dried, 28 DEG C of lucifuges cultivate 48h.Taken off from Dual culture base by cellulose nitrate film, forward is taped against on PDA substratum (containing 200 μ g/mL cephamycins, 250 μ g/mL hygromycin B), and 28 DEG C of cultivations, until occur visible list bacterium colony on PDA substratum.
CM substratum is the IM substratum of interpolation 2% (w/v) agarose.
The screening of 1.4 black truffle transformants and qualification
1.4.1 the screening of black truffle transformant
By on selective medium extremely new for single bacterium colony picking that PDA substratum grows, being separated the black truffle bacterial strain having hygromycin resistance, resistant strain being continued stable to growing containing 50 μ g/mL hygromycin B substratum being cultivated three generations.
1.4.2 the qualification of black truffle transformant
By hygromycin resistance inoculation in the PDA liquid nutrient medium containing 50 μ g/mL hygromycin B, 200 μ g/mL cephamycins, 28 DEG C, 200rpm cultivates the centrifugal 5min of 72h, 13000rpm, abandons supernatant, collects thalline, extracts transformant genome.Genome extracts and adopts fungal gene group to extract test kit (goods number D3390-01, OMEGA biotech company).
PCR qualification adopts the special primer (HPT-F2:GACGACACCGTCAGTGCGTC, HPT-3:CTTTGCCCTCGGACGAGTGCTGG) of target hygromycin gene.PCR reaction system is 50 μ L, comprises 10 × Buffer 5 μ L, dNTP (20mmolL
-1) 4 μ L, primer (25pmol μ L
-1) each 2 μ L, Mg
2+(25mmolL
-1) 4 μ L, thallus DNA (about 50ng μ L
-1) 1 μ L, Taq archaeal dna polymerase (5U μ L
-1) 0.5 μ L, add H
2o to 50 μ L.Reaction conditions: 94 DEG C of sex change 5min; 94 DEG C of 30sec, 50 DEG C of 30sec, 72 DEG C of 1min, 30 circulations; 72 DEG C extend 10min.PCR primer conventionally carries out electrophoresis with 1% sepharose, records electrophoresis result through gel imaging system imaging.
Pcr amplification product is carried out sequencing, the consistence of the analysing amplified fragment of Blast and hygromycin phosphotransferase gene.
2, experimental result
2.1 black truffles are producing growing state and the Observations On The Spore Morphology of spore substratum VBC
Growing state and Observations On The Spore Morphology that black truffle is producing spore substratum VBC are shown in Fig. 2.
The screening of 2.2 black truffle transformants
Part black truffle transformant colonies form is shown in Fig. 3.
The qualification of 2.3 black truffle transformants
PCR qualification result (Fig. 4) shows, the fragment of expection size all can be amplified for preliminary operation beggar and plasmid pCAMBIA1302-1, the wild type strain of unconverted and sterilized water contrast are then increased less than the fragment of corresponding size, illustrate that black truffle transformant contains the T-DNA fragment of insertion.
In order to confirm the reliability of pcr amplification result further, pcr amplification product is carried out sequencing, its nucleotides sequence is classified as shown in SEQ ID NO.1.Blast analyzes and shows, the consistence of this fragment and hygromycin phosphotransferase gene reaches 100%, further illustrates T-DNA and successfully proceeds to black truffle genome, and heredity that can be stable.
The present invention is with 10
6spore is the material that sets out, and screen 28,45 and 62 positive transformants respectively by Agrobacterium tumefaciens mediated, positive transformants efficiency reaches 4.53 ± 1.75/10
5individual spore.
The genetic marker of embodiment 2 black truffle
1, experimental technique
The preparation of 1.1 black truffle spores
Concrete grammar is with embodiment 1.
The conversion of 1.2 agrobacterium tumefaciens and cultivation
Concrete grammar is with embodiment 1, and plasmid used is pCAMBIA1302 (Fig. 5).
1.3 agrobacterium tumefaciens and black truffle spore Dual culture and selectivity are cultivated
Concrete grammar is with embodiment 1.
The screening of 1.4 black truffle transformants and qualification
1.4.1 the screening of black truffle transformant
By on selective medium extremely new for single bacterium colony picking that PDA substratum grows, being separated the black truffle bacterial strain having hygromycin resistance, resistant strain being continued stable to growing containing 50 μ g/mL hygromycin B substratum being cultivated three generations.
1.4.2 black truffle transformant fluorescence intensity detects
The mycelium of picking positive transformant is placed on slide glass, carries out fluoroscopic examination under being placed in inverted fluorescence microscope (IX-51, Japanese Olympus company produces).
2, experimental result
The mycelium of picking positive transformant is placed on slide glass, carries out fluoroscopic examination, adopt blue-light excited light under being placed in inverted fluorescence microscope, can observe positive transformant and produce obvious fluorescence, and wild strain does not produce fluorescence (Fig. 6).
External source green fluorescent protein encoding gene is mainly introduced black truffle by this experiment, after expressing, recipient cell shows special green under excitation light, may be used for cytoactive detection, morphological analysis in fermenting process, be also the important tool of research black truffle and host plant interaction relationship simultaneously.
The validity of the black truffle genetic transformation system that this experimental result builds from the further proved invention of cell levels, simultaneously also for black truffle biological study provides a kind of important tool.
Claims (9)
1. Agrobacterium tumefaciens mediated black truffle (Tuber melanosporum) genetic transforming method, is characterized in that, comprise the following steps:
(1) black truffle spore suspension is prepared; (2) will the binary vector Plastid transformation agrobacterium tumefaciens of goal gene be carried, obtain agrobacterium tumefaciens positive transformant, preparation bacterium liquid; (3) black truffle spore suspension prepared by bacterium liquid step (2) prepared and step (1) mixes, Dual culture; (4) selectivity is cultivated, and screening black truffle transformant, qualification, to obtain final product.
2. according to genetic transforming method according to claim 1, it is characterized in that: step (1) described black truffle spore suspension miospore concentration is 10
4-10
8individual/mL, is preferably 10
6individual/mL.
3. according to the genetic transforming method described in claim 1 or 2, it is characterized in that, step (1) described preparation black truffle spore suspension, comprising: black truffle mycelium is inoculated in PDA substratum by (a), cultivates activation in 28 DEG C; Preferably, 3d is activated; B black truffle mycelium after activation is inoculated in VBC substratum by (), cultivate 4-9d for 28 DEG C, be preferably 7d; Wash mycelium with sterilized water, collect the spore of black truffle, dilution, to obtain final product.
4. according to genetic transforming method according to claim 1, it is characterized in that, step (2) described preparation bacterium liquid comprises: (a) picking agrobacterium tumefaciens positive transformant, be inoculated into containing in antibiotic LB substratum, 12-20h is cultivated in 28 DEG C of 200rpm concussions, centrifugal, collect thalline; B () is with the resuspended thalline of IM substratum, centrifugal, collect thalline; C () uses the resuspended thalline of IM substratum, by cell concentration OD
600be adjusted to 0.1 ~ 0.6,28 DEG C of 200rpm shaking culture 3-8h, to obtain final product.
5. according to genetic transforming method according to claim 4, it is characterized in that, step (2) described preparation bacterium liquid comprises: (a) picking agrobacterium tumefaciens positive transformant, be inoculated into containing in antibiotic LB substratum, 18h is cultivated in 28 DEG C of 200rpm concussions, centrifugal, collect thalline; B () is with the resuspended thalline of IM substratum, centrifugal, collect thalline; C () uses the resuspended thalline of IM substratum, by cell concentration OD
600be adjusted to 0.15 ~ 0.3,28 DEG C of 200rpm shaking culture 6h, to obtain final product.
6. according to genetic transforming method according to claim 1, it is characterized in that: the bacterium liquid that step (2) is prepared by step (3) mixes with black truffle spore suspension equal-volume prepared by step (1); The substratum of described Dual culture is the CM substratum being covered with cellulose nitrate film; Dual culture condition is that 28 DEG C of lucifuges cultivate 24-60h, is preferably 48h.
7. according to the genetic transforming method described in claim 4,5 or 6, it is characterized in that: containing Syringylethanone in the substratum of described IM substratum or Dual culture; Preferably, the concentration of described Syringylethanone is 200mmol/L.
8. according to genetic transforming method according to claim 1, it is characterized in that: the described selectivity of step (4) cultivate be by step (3) Dual culture after cellulose nitrate film take off, forward is taped against containing antibiotic PDA substratum, and 28 DEG C are cultured to and occur visible list bacterium colony.
9., according to the genetic transforming method described in claim 4,5 or 8, it is characterized in that: described microbiotic is selected from cephamycin, Totomycin or kantlex.
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| CN107674881A (en) * | 2016-08-02 | 2018-02-09 | 武汉臻智生物科技有限公司 | Japanese yew diene over-express vector and its application |
| CN107299108A (en) * | 2016-08-31 | 2017-10-27 | 广西壮族自治区农业科学院生物技术研究所 | The genetic transforming method of Agrobacterium tumefaciens mediated ustilago esculenta |
| CN107177622A (en) * | 2017-05-22 | 2017-09-19 | 华中农业大学 | A kind of Agrobacterium tumefaciens mediated Italian mould genetic transforming method |
| CN107177622B (en) * | 2017-05-22 | 2019-09-10 | 华中农业大学 | A kind of Agrobacterium tumefaciens mediated Italian mould genetic transforming method |
| CN108384798A (en) * | 2018-02-13 | 2018-08-10 | 江南大学 | A method of utilizing Agrobacterium tumefaciens transformation Mortierella alpina mycelia |
| CN108949798A (en) * | 2018-07-10 | 2018-12-07 | 中国农业科学院植物保护研究所 | The T contraversa method for transformation of mediated by agriculture bacillus |
| CN108949798B (en) * | 2018-07-10 | 2022-02-18 | 中国农业科学院植物保护研究所 | Agrobacterium-mediated transformation method for Tilletia controversa Kuhn |
| CN111349648A (en) * | 2020-02-28 | 2020-06-30 | 浙江工业大学 | Method for introducing agrobacterium-mediated exogenous gene into cordyceps militaris cells |
| CN112941101A (en) * | 2021-04-16 | 2021-06-11 | 山东大丰园农业有限公司 | Method, system and application for transient expression of blueberry fruits |
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