CN106497970A - A kind of method that agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System is set up - Google Patents
A kind of method that agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System is set up Download PDFInfo
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Abstract
The invention discloses a kind of method of the foundation of agriculture bacillus mediated Bulbus Allii Cepae Transient Expression System.The method infects onion epidermis cell in infecting in culture medium IM containing Agrobacterium, and 20～22 DEG C are infected 10～20 minutes, cultivates 24 hours under light.The inventive method is quick, low cost, transformation efficiency up to more than 90%, can accurately be used for the Subcellular Localization of gene and the research of albumen and interactions between protein.
Description
Technical field
The invention belongs to field of plant genetic, is that a kind of agriculture bacillus mediated efficient Study of Exogenous gene exists
The Subcellular Localization of Bulbus Allii Cepae endepidermis and the transient transformation methods of albumen and interactions between protein.
Background technology
The transient expression of target gene is a simple and useful method (Zhou et for analyzing biological function
al.2009).The transient expression of gene is in research protein function such as protein active analysis, Subcellular Localization and albumen and egg
Highly useful information (Lee et al.2008 are provided in terms of white interaction；Ueki et al.2009；Hollender and
Liu 2010).(Scott et al.1999), instantaneous conversion compared with complex operation, costly and time-consuming stable conversion system
Method i.e. quick flexibly do not affect the position of chromosome and can be used for well differentiated plant tissue (Fischer et again
al.1999).Instantaneous conversion has more and more been used as the supplement of stable conversion, accelerates the process of further investigation
(Wroblewski et al.2005).
On research gene function, instantaneous conversion can implement instantaneous conversion at present as the good supplement of stable conversion
Main vegetable material is Nicotiana tabacum L. or Bulbus Allii Cepae.Bulbus Allii Cepae as monocotyledon, for the gene that clones from monocotyledon
Subcellular Localization credibility may be higher, therefore we select Bulbus Allii Cepae as the object of our instantaneous conversion Establishings.Ocean
Herba Alii fistulosi epidermis cell have be readily available, cell monolayer, transparent and disturb without chloroplast, be postgraduate's activity of science, egg
The ideal experiment material of white activity, cyto-dynamics, gene transient expression, protein subcellular positioning and albumen and interactions between protein
Material (Scott et al.1999；Walter et al.2004；Cheng et al.2009；Hollender and Liu
2010；Zhang et al.2011).
Particle bombardment and Agrobacterium-medialed transformation method are the main methods of current instantaneous conversion.Particle bombardment receives carrier
Restriction less and can be used for monocotyledon and dicot plant tissue, in Bulbus Allii Cepae, conventional method for transformation is mainly
Application particle bombardment (Christou 1995；Eady et al.1996；Scott et al.1999；Arnim 2007；Cheng
et al.2009；Hollender and Liu 2010).But, using particle bombardment carry out conversion have transformation efficiency low, conversion
Procedure expensive and limited by instrument and equipment and auxiliary material.
In recent years, also it has been reported that carrying out Bulbus Allii Cepae instantaneous conversion (Sun et al.2007 with agriculture bacillus mediated method；Xu
et al.2014).Its advantage is to only need to conventional bacterial strain, carrier and culture medium not need expensive instrument and equipment and gold
Powder etc., and simple to operate, and low cost, copy number are low.But current transformation efficiency is not also very high, in the application of system also
Without successfully reporting in terms of albumen and interactions between protein.
Content of the invention
The present invention is aiming at above-mentioned defect and provides a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae transient expression body
The method that system sets up.The technical problem to be solved in the present invention is to optimize agriculture bacillus mediated Bulbus Allii Cepae instantaneous conversion system, improves and turns
Change efficiency.Utilize system of the present invention quickly can judge first new structure for stable conversion with fluorescent protein tag
Carrier whether active, it is to avoid wasting manpower and material resources during stable conversion；The Asia of target protein can also be carried out simultaneously
Cellular localization and albumen and the work such as the interaction of albumen, are the analysis provided auxiliary means of gene function.
The method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System of the present invention is set up passes through following technical side
Case is realizing：Onion epidermis cell is infected in infecting in culture medium containing Agrobacterium, 20～22 DEG C are infected 10～20 minutes,
Cultivate 24 hours under light.
The described culture medium that infects is based on MS culture medium, by adjusting the content of dextrose and saccharose, increased
Agrobacterium infects the osmotic potential of liquid；Add 0.01% Silwet L-77 simultaneously, increased richness of the Agrobacterium on onion epidermis
Collection, improves transformation efficiency.
The method that described a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System is set up, comprises the following steps：
(1) Bulbus Allii Cepae endepidermis block is taken；
(2) Agrobacterium containing target gene infects the configuration of culture medium：Agriculture by activation carrying binary vector once
Bacillus is inoculated in 10ml YEB fluid mediums, and 28 DEG C of 200rpm wave and culture are overnight；Bacterium solution is transferred to again fresh
In 10ml YEB fluid mediums, 28 DEG C of 200rpm shake 45 hours, OD600=0.5～0.8；By 4 DEG C of low temperature 4000rpm of bacterium solution
Supernatant is outwelled after centrifugation 6min, concentration OD is caused with culture medium is infected by resuspended for thalline600=0.3, add Silwet L-77；
(3) onion epidermis infect：Aseptically, Bulbus Allii Cepae endepidermis block is put into and with the addition of Silwet containing thalline
L-77 is infected in culture medium, is gently rocked, is infected 10～20min；After infecting by onion epidermis near the side of mesophyll down
It is put in co-cultivation culture medium；Co-culture culture dish and be put into 20 ± 2 DEG C of 24 hours under light.
Step (1) is specially：By onion bulb stem, 1～2 layer of scale leaf of outer layer is removed, in ultra-clean work after onion bulb stem is sterilized
Make the interior sterilized water of platform by bulb wash clean, longitudinal incision onion bulb stem, take the inner more plump scale leaf in middle level, in these scale leafs
1cm gently drawn by inside dissecting knife2Cross square some, these Bulbus Allii Cepae endepidermis blocks of tearing.
The onion bulb stem sterilization is the ethanol disinfection of 70～75% (volumetric concentrations).
Agrobacterium strains described in step (2) are EHA105 or GV3101 is (from the extra large spring problem of plant institute of Chinese Academy of Sciences scape
Group).Carry the exogenous gene importing onion epidermis cell with reporter gene fusions such as GUS, GFP or YFP.
In step (2) by the Agrobacterium inoculation of the carrying binary vector for activating once in 10ml YEB fluid mediums,
Volume ratio is 1:It is grand that 100, YEB fluid mediums contain 100 μ g/mL rifampicin, 50 μ g/ml kanamycin or 100 μ g/ml
Mycin, 100 μM of acetosyringones；
Bacterium solution is transferred to again in fresh 10ml YEB fluid mediums, volume ratio 1:10；YEB fluid mediums contain
There are 100 μM of acetosyringones.
Concentration OD is caused by resuspended for thalline with infecting culture medium in step (2)600=0.3, used infects culture medium for Sorghum vulgare Pers.
Culture medium is contaminated, the Silwet L-77 for adding 0.01% in culture medium are infected.
The Sorghum vulgare Pers. that with the addition of Silwet L-77 is infected culture medium and includes following component per L：MS culture medium 4.43g, glucose
36g, sucrose 68.5g, MES culture medium 0.5g, Silwet L-770.01% (percent by volume), 100 μM of acetosyringone, pH
5.8.
It is that Sorghum vulgare Pers. co-cultures culture medium to co-culture culture medium in step (3), includes following component per L：MS culture medium
4.43g, glucose 10g, sucrose 20g, MES culture medium 0.5g, agar 8g, 100 μM of acetosyringone, pH 5.8
In order to reduce infringement of the Agrobacterium to onion epidermis cell, make transformation efficiency more stable, optimize in step (2)
The concentration of thalline in culture medium is infected, it is OD to select the concentration for infecting culture medium thalline600=0.3.
The present invention has advantages below compared with prior art：The culture medium that infects of the present invention is with MS culture medium as base
Plinth, by adjusting the content of dextrose and saccharose, increased the osmotic potential that Agrobacterium infects liquid；Add 0.01% simultaneously
Silwet L-77, increased enrichment of the Agrobacterium on onion epidermis, improve transformation efficiency.
Utilize system of the present invention quickly can judge first new structure for stable conversion with fluorescin mark
Whether the carrier of label is active, it is to avoid wasting manpower and material resources during stable conversion.
The present invention does not rely on the expensive experiment material such as particle gun, low cost；Conversion process simple and fast, whole process are only needed
2～3 days；The vegetable material that could be converted after not needed plantation by such as Nicotiana tabacum L., arabidopsiss etc. is limited；Transformation efficiency is up to
More than 90%；The research of accurate Subcellular Localization research and albumen and interactions between protein can be carried out, is gene function
Analysis provided auxiliary means.
Description of the drawings
Fig. 1 show used carrier framework in embodiment 1 and embodiment 2；
Fig. 2 show used carrier framework in embodiment 3；
Fig. 3 show agriculture bacillus mediated onion epidermis instantaneous conversion program；Wherein, a：Fresh Bulbs, b：Bulb is indulged
Cut, c：Bulbus Allii Cepae endepidermis cut 1cm2 fritters, d：Onion epidermis are infected in centrifuge tube, e：Co-culture, f：To train altogether
After supporting, onion epidermis are placed on preparation microexamination, scale=1cm on microscope slide；
Fig. 4 show the expression of embodiment 1p3300-GFP green fluorescence；Wherein, a：P3300-GFP green fluorescence figures, b：
Light field, c：A and b stacking charts, d：Blank (unconverted onion epidermis cell), e：Light field, f：D and e stacking charts, scale=
100 microns；
Fig. 5 show impact of the bacterial concentration to transformation efficiency；
Fig. 6 show Subcellular Localization of the CWIN10B-mCherry fusion protein in onion epidermis；a：p1300-
CWIN10B-mCherry red fluorescent proteins (plasmolysis figure), b：Light field, c：A and b stacking charts, d blanks are (unconverted
Onion epidermis cell), e light fields, f：D and e stacking charts；Scale=100 micron；
Fig. 7 show and detects CYCH and CDKD using onion epidermis cell；2 interaction；a：pSPYNE-CYCH-YCHA/
CDKD；2-YNEE albumen and interactions between protein yellow fluorescence, b：Light field, c：A and b stacking charts, d：PSPYNE-YCHA/YNEE is negative
Control, without interaction yellow fluorescence, e：Light field, f：D is superimposed with e.
Specific embodiment：
For a better understanding of the present invention, technical scheme is described in detail with instantiation below, but this
Invention is not limited thereto.
Example 1
The optimization of agriculture bacillus mediated onion epidermis cell Subcellular Localization system
(1) choose and grow vigorous, energetic onion bulb stem, remove 1～2 layer of scale leaf (Fig. 3 a) of outermost layer, by Bulbus Allii Cepae squama
Stem soaks sterilization in 10 minutes in 70～75% ethanol, then washs bulb 3-5 time with sterilized water in superclean bench, uses
Sterile scalpel longitudinal incision onion bulb stem (Fig. 3 b), takes the inner more plump scale leaf in middle level, (recessed in these scale leaf endepidermis
Face) about 1cm is gently drawn with dissecting knife2Cross square some, these Bulbus Allii Cepaes endepidermis block (Fig. 3 c) of tearing；
(2) with GFP as reporter gene, optimize transformation system.GFP reporter genes are connected in pCAMBIA3300 carriers, structure
Build expression vector pCAMBIA3300-ubi-gfp binary expression vectors (Fig. 1).By in vector introduction Agrobacterium EHA105, incite somebody to action
Be identified as positive colony and activate once carrying binary vector Agrobacterium inoculation in 10ml YEB fluid mediums (body
Product compares 1:100；Contain 100 μ g/mL rifampicin, 50 μ g/ml kanamycin, 100 μM of acetosyringones), 28 DEG C of 200rpm shook
Night；Bacterium solution is transferred to (volume ratio 1 again in fresh 10ml YEB fluid mediums:10；100 μM of acetosyringones), 28
DEG C 200rpm shakes 4-5 hours, OD600=0.5～0.8；Supernatant will be outwelled after 4 DEG C of low temperature 4000rpm of bacterium solution centrifugation 6min, with invading
Dye culture medium SbIM, AtIM and TnIM (add or without 0.01% Silwet L-77) (contaminate culture medium ask for an interview table 1) will
Thalline is resuspended to cause concentration OD600=0.1,0.3 and 0.5.
(3) onion epidermis infect：Aseptically, 5-10 block Bulbus Allii Cepae endepidermis blocks are put into containing the different of thalline
Infect in culture medium and (infect liquid to be placed in 2ml centrifuge tubes) (Fig. 3 d), gently rock, infect 10～20min.By Bulbus Allii Cepae after infecting
Epidermis is transferred in co-cultivation culture medium (SbCOM) (Fig. 3 e) near the side court of mesophyll.Co-culture culture dish to be put into 20 under light
± 2 DEG C of 24 hours, onion epidermis are cleaned in sterilized water several times, are then put on microscope slide, covered (Fig. 3 f),
Observation result under laser confocal microscope is as shown in figure 4, onion epidermis cell has obvious green fluorescence to express (see explanation
Book accompanying drawing Fig. 4).By Fig. 5 block diagrams it will be seen that when bacterial concentration is OD600When=0.3, transformation efficiency up to 92% with
On, it is significantly higher than OD600When=0.1, with OD600When=0.3, difference is notable (see Figure of description Fig. 5).For improving conversion effect
Rate, present invention optimizes infecting the composition of culture medium in step (2), the Agrobacterium is infected culture medium and co-cultures culture medium and sees
Table 1；It is based on MS culture medium that IM infects culture medium, by adjusting the content of dextrose and saccharose, increased Agrobacterium and invades
The osmotic potential of dye liquor；Add 0.01% Silwet L-77 simultaneously, increased enrichment of the Agrobacterium on onion epidermis, improve
Transformation efficiency (as shown in table 2).
In order to reduce infringement of the Agrobacterium to onion epidermis cell, make transformation efficiency more stable, optimize in step (2)
The concentration of thalline in culture medium is infected, it is OD to select the concentration for infecting culture medium thalline600=0.3.
1 difference of table infects culture medium and co-cultures culture medium (SbCOM)
Note：SbIM (Sorghum vulgare Pers. infects culture medium), SbIM+s (Sorghum vulgare Pers. is infected culture medium and with the addition of Silwet L-77), SbCOM
(Sorghum vulgare Pers. co-cultures culture medium), (Nicotiana tabacum L. is infected culture medium and with the addition of Silwet L- for TnIM (Nicotiana tabacum L. infects culture medium), TnIM+s
77), AtIM (arabidopsiss infect culture medium), AtIM+s (arabidopsiss infect culture medium and with the addition of Silwet L-77).
2 difference of table infects culture medium and different impacts of the time of infection to Bulbus Allii Cepae transformation efficiency
Note：Capitalization A-D represents that transformation efficiency difference in 0.01 significant level is extremely notable, and lower case a-e is represented
Transformation efficiency significant difference in 0.05 level, using SPSS13 statistical analysis softwares and Duncan methods to turning between different disposal
Changing efficiency carries out significant difference analysis；+s：Add Silwet L-77 in culture medium.
From form, Sorghum vulgare Pers. is infected culture medium and with the addition of the transformation efficiency of Silwet L-77 and is up to 90%, far above which
He infects culture medium, while also infecting the transformation efficiency that culture medium is not added with the 50～60% of Silwet L-77 far above Sorghum vulgare Pers..
Embodiment 2
The method for transformation of agriculture bacillus mediated onion epidermis cell is applied to the research of protein subcellular positioning
(1) choose and grow vigorous, energetic onion bulb stem, remove 1～2 layer of scale leaf (Fig. 3 a) of outermost layer, by Bulbus Allii Cepae squama
Stem soaks sterilization in 10 minutes in 70～75% ethanol, then washs bulb 3～5 times with sterilized water in superclean bench,
With sterile scalpel longitudinal incision onion bulb stem (Fig. 3 b), the inner more plump scale leaf in middle level is taken, (recessed in these scale leaf endepidermis
Face) about 1cm is gently drawn with dissecting knife2Cross square some, these Bulbus Allii Cepaes endepidermis block (Fig. 3 c) of tearing；
(2) with mCherry as reporter gene, CWIN10B is connected in pCAMBIA1300-mCherry carriers, builds table
Reach carrier pCAMBIA1300-CWIN10B-mCherry binary expression vectors (Fig. 1).By vector introduction Agrobacterium GV3101
In, by be identified as positive colony and activate once carrying binary vector Agrobacterium inoculation to 10ml YEB fluid mediums
In (volume ratio 1:100；Contain 100 μ g/mL rifampicin, 50 μ g/ml kanamycin, 100 μM of acetosyringones), 28 DEG C of 200rpm
Shake overnight；Bacterium solution is transferred to (volume ratio 1 again in fresh 10ml YEB fluid mediums:10；100 μM of acetyl Flos Caryophyllis
Ketone), 28 DEG C of 200rpm shake 4～5 hours, OD600=0.5～0.8；To outwell after 4 DEG C of low temperature 4000rpm centrifugation 6min of bacterium solution
Clearly, use culture medium SbIM (adding 0.01% Silwet L-77) (table 1) is infected by resuspended for thalline cause concentration OD600=0.3.
(3) onion epidermis infect：Aseptically, 5～10 pieces of Bulbus Allii Cepae endepidermis blocks are put into the difference containing thalline
Infect in culture medium and (infect liquid to be placed in 2ml centrifuge tubes) (Fig. 3 d), gently rock, infect 10-20min.By ocean after infecting
Herba Alii fistulosi epidermis is transferred in co-cultivation culture medium (SbCOM) (Fig. 3 e) near the side court of mesophyll.Co-culture culture dish to be put under light
20+2 DEG C of 24 hours, onion epidermis are cleaned in sterilized water several times, are then put on microscope slide, covered (figure
3f), observe under laser confocal microscope, as a result show that CWIN10B albumen is positioned on cell wall (Fig. 6).
This embodiment illustrates the method and can be rapidly performed by accurate gene Subcellular Localization research
Embodiment 3
The method for transformation of agriculture bacillus mediated onion epidermis cell is applied to the research of albumen and interactions between protein
(1) choose and grow vigorous, energetic onion bulb stem, remove 1～2 layer of scale leaf (Fig. 3 a) of outermost layer, by Bulbus Allii Cepae squama
Stem soaks sterilization in 10 minutes in 70～75% ethanol, then washs bulb 3～5 times with sterilized water in superclean bench,
With sterile scalpel longitudinal incision onion bulb stem (Fig. 3 b), the inner more plump scale leaf in middle level is taken, (recessed in these scale leaf endepidermis
Face) about 1cm is gently drawn with dissecting knife2Cross square some, these Bulbus Allii Cepaes endepidermis block (Fig. 3 c) of tearing；
(2) by CYCH and CDKD；2 are connected into composition pSPYNE- in pSPYNE-YCHA and pSPYNE-YNEE carriers respectively
CYCH-YCHA/CDKD；2-YNEE carriers, pSPYNE-YCHA and pSPYNE-YNEE zero loads are used as negative control (Fig. 2).By institute
Have in vector introduction Agrobacterium EHA105, the Agrobacterium for being identified as positive colony and activate carrying binary vector once is connect
Plant (the volume ratio 1 in 10ml YEB fluid mediums:100；Contain 100 μ g/mL rifampicin, 50 μ g/ml kanamycin, 100 μM
Acetosyringone), 28 DEG C of 200rpm shake overnight；Bacterium solution is transferred to (volume again in fresh 10ml YEB fluid mediums
Than 1:10；100 μM of acetosyringones), 28 DEG C of 200rpm shake 4～5 hours, OD600=0.5～0.8；By 4 DEG C of low temperature of bacterium solution
Supernatant is outwelled after 4000rpm centrifugation 6min, is used and culture medium SbIM (adding 0.01% Silwet L-77) (table 1) is infected by bacterium
Body weight is outstanding to cause concentration OD600=0.3.
(3) onion epidermis infect：Aseptically, 5～10 pieces of Bulbus Allii Cepae endepidermis blocks are put into the difference containing thalline
Infect in culture medium and (infect liquid to be placed in 2ml centrifuge tubes) (Fig. 3 d), gently rock, infect 10～20min.By ocean after infecting
Herba Alii fistulosi epidermis is transferred in co-cultivation culture medium (SbCOM) (Fig. 3 e) near the side court of mesophyll.Co-culture culture dish to be put under light
20 ± 2 DEG C of 24 hours, onion epidermis are cleaned in sterilized water several times, are then put on microscope slide, covered (figure
3f), observe under laser confocal microscope, as a result show to have turned pSPYNE-CYCH-YCHA/CDKD；The Bulbus Allii Cepae table of 2-YNEE
Chrotoplast occurs in that strong yellow fluorescence in nucleus and Cytoplasm, (Fig. 7) occurs without fluorescence in negative control.
This example demonstrates the research that technical solution of the present invention can be applicable to accurate albumen and interactions between protein.
Claims (10)
1. a kind of method that agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System is set up, it is characterised in that onion epidermis are thin
Born of the same parents are infected in infecting in culture medium containing Agrobacterium, and 20～22 DEG C are infected 10～20 minutes, are cultivated 24 hours under light.
2. the method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System according to claim 1 is set up, which is special
Levy and be, comprise the following steps：
(1) Bulbus Allii Cepae endepidermis block is taken；
(2) Agrobacterium containing target gene infects the configuration of culture medium：Agrobacterium by activation carrying binary vector once
It is inoculated in 10ml YEB fluid mediums, 28 DEG C of 200rpm wave and culture are overnight；Bacterium solution is transferred to fresh 10ml again
In YEB fluid mediums, 28 DEG C of 200rpm shake 4～5 hours, OD600=0.5～0.8；By 4 DEG C of low temperature 4000rpm of bacterium solution from
Supernatant is outwelled after heart 6min, concentration OD is caused with culture medium is infected by resuspended for thalline600=0.3, add Silwet L-77；
(3) onion epidermis infect：Aseptically, Bulbus Allii Cepae endepidermis block is put into and with the addition of Silwet L- containing thalline
77 infect in culture medium, gently rocks, infects 10～20min；After infecting by onion epidermis near the side of mesophyll towards decentralization
To in co-cultivation culture medium；Co-culture culture dish and be put into 20 ± 2 DEG C of 24 hours under light.
3. the method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System according to claim 1 is set up, which is special
Levy and be, step (1) is specially：By onion bulb stem, 1～2 layer of scale leaf of outer layer is removed, in ultra-clean work after onion bulb stem is sterilized
Bulb wash clean, longitudinal incision onion bulb stem are taken the inner more plump scale leaf in middle level, in these scale leafs by the interior sterilized water of platform
1cm gently drawn by portion's dissecting knife2Cross square some, these Bulbus Allii Cepae endepidermis blocks of tearing.
4. the method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System according to claim 3 is set up, which is special
Levy and be, the onion bulb stem sterilization is 70～75% ethanol disinfection.
5. the method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System according to claim 2 is set up, which is special
Levy and be, agrobacterium strains described in step (2) are EHA105 or GV3101.
6. the method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System according to claim 2 is set up, which is special
Levy and be, in step (2) by the Agrobacterium inoculation of the carrying binary vector for activating once in 10ml YEB fluid mediums, body
Product is than being 1:It is grand mould that 100, YEB fluid mediums contain 100 μ g/mL rifampicin, 50 μ g/ml kanamycin or 100 μ g/ml
Element, 100 μM of acetosyringones；
Bacterium solution is transferred to again in fresh 10ml YEB fluid mediums, volume ratio 1:10；YEB fluid mediums contain
100 μM of acetosyringones.
7. the method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System according to claim 2 is set up, which is special
Levy and be, concentration OD is caused by resuspended for thalline with infecting culture medium in step (2)600=0.3, used infects culture medium for Sorghum vulgare Pers.
Culture medium is contaminated, the Silwet L-77 for adding 0.01% in culture medium are infected.
8. the method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System according to claim 7 is set up, which is special
Levy and be, the Sorghum vulgare Pers. that with the addition of Silwet L-77 is infected culture medium and includes following component per L：MS culture medium 4.43g, glucose
36g, sucrose 68.5g, MES culture medium 0.5g, Silwet L-77 0.01%, 100 μM of acetosyringone, pH 5.8.
9. the method that a kind of agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System according to claim 2 is set up, which is special
Levying and being, it is that Sorghum vulgare Pers. co-cultures culture medium that culture medium is co-cultured in step (3), includes following component per L：MS culture medium 4.43g,
Glucose 10g, sucrose 20g, MES culture medium 0.5g, agar 8g, 100 μM of acetosyringone, pH 5.8.
10. a kind of method that agriculture bacillus mediated efficient Bulbus Allii Cepae Transient Expression System is set up as claimed in claim 1 is in target
Application in terms of the interaction of the Subcellular Localization of albumen and albumen and albumen.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN106916848A (en) * | 2017-04-11 | 2017-07-04 | 浙江大学 | A kind of method that gene transient expression is realized in Peach fruits |
| CN107014792A (en) * | 2017-04-20 | 2017-08-04 | 南京农业大学 | A kind of method that utilization Chinese rose transient expression system identifies interactions between protein |
| CN109722447A (en) * | 2019-03-10 | 2019-05-07 | 华中农业大学 | A kind of method of the citrusfruit instantaneous conversion of mediated by agriculture bacillus |
| CN113106121A (en) * | 2021-05-25 | 2021-07-13 | 赣南师范大学 | Method for establishing genetic transformation system of tillered onion |
| CN113462716A (en) * | 2021-06-24 | 2021-10-01 | 天津师范大学 | Duckweed instantaneous transformation method |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106916848A (en) * | 2017-04-11 | 2017-07-04 | 浙江大学 | A kind of method that gene transient expression is realized in Peach fruits |
| CN106916848B (en) * | 2017-04-11 | 2020-04-10 | 浙江大学 | Method for realizing gene transient expression in peach fruit |
| CN107014792A (en) * | 2017-04-20 | 2017-08-04 | 南京农业大学 | A kind of method that utilization Chinese rose transient expression system identifies interactions between protein |
| CN109722447A (en) * | 2019-03-10 | 2019-05-07 | 华中农业大学 | A kind of method of the citrusfruit instantaneous conversion of mediated by agriculture bacillus |
| CN113106121A (en) * | 2021-05-25 | 2021-07-13 | 赣南师范大学 | Method for establishing genetic transformation system of tillered onion |
| CN113462716A (en) * | 2021-06-24 | 2021-10-01 | 天津师范大学 | Duckweed instantaneous transformation method |
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