CN110079549A - A method of induce potato quickly to tie potato by being overexpressed potato StSN2 gene - Google Patents
A method of induce potato quickly to tie potato by being overexpressed potato StSN2 gene Download PDFInfo
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- A—HUMAN NECESSITIES
- A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
- A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
- A01H4/00—Plant reproduction by tissue culture techniques ; Tissue culture techniques therefor
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/66—General methods for inserting a gene into a vector to form a recombinant vector using cleavage and ligation; Use of non-functional linkers or adaptors, e.g. linkers containing the sequence for a restriction endonuclease
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Abstract
The method that the invention discloses a kind of to induce potato knot potato by being overexpressed potato StSN2 gene, comprising: (1) construct over-express vector；(2) transgenic plant: the 1. induction of potato set is constructed by mediated by agriculture bacillus；2. the activation and resuspension of Agrobacterium；3. preculture and co-cultivation；4. bud induction and root induction；(3) transgenic line is determined；(4) potato knot potato is induced.StSN2 gene participates in potato tubers sleep procedure in the present invention, increase the dormancy time of stem tuber by being overexpressed StSN2 gene, to maladjustment environment, simultaneously by being overexpressed StSN2 gene, can Effective Regulation potato tubers dormant state, and the release to potato tubers sleep procedure can be achieved with by RNAi, makes its rudiment process mutually be suitable for environment, promote its fast-growth, improves its yield and quality.
Description
Technical field
The invention belongs to gene engineering technology fields, and in particular to one kind is by being overexpressed the induction of potato StSN2 gene
The method of potato knot potato.
Background technique
Potato (Solanum tuberosum L.) is Solanaceae nightshade, adaptable, yield is high, full of nutrition,
It is the important cereal crops in China.Potato tubers application is wide, to make based on grain vegetable, also makees potato seed, to the control bud of the two in production
Demand is different, and as fresh food or the stem tuber of processing, rudiment can make it reduce or lose commodity value, need to completely inhibit；It is right
In potato seed, especially in the southwest that can be produced in more seasons, then needs to adjust its sprouting time and sowing season matches, otherwise can
There is stem tuber rudiment too early, too late or the improper problem of potato seed physiological age.
Although having some methods for adjusting stem tuber rudiments at present, due to a lack of to suspend mode and rudiment mechanism deeply
There is the problems such as blindly random in solution, these methods, cannot reach the requirement of accurate and flexible regulation.Such as: (1) low temperature controls: 2-4 DEG C
The stem tuber storage time can be appropriately extended in low temperature, but different cultivars responds different and makes that effect is delayed to have differences to low temperature, and low
Temperature control bud Expenses Cost is high, improves effect with greater need for combination chemical regulation.(2) permanence Suckering agents: chlorpropham
(Chlorpropham, product name CIPC) is currently used potato bud inhibition agent, and handling potato tubers with it will lead to
Potato, which cannot germinate, does not have control bud effect, and residual harm is also required to pay attention to.(3) it interim Suckering agents: is developed from plant
The sprout inhibition within a certain period of time of volatility sprout inhibition substance out, stem tuber can still restore to germinate after removal, can be used for potato seed storage.Such as 1,
4- dimethylnaphthalene (Isosorbide-5-Nitrae-dimethylnaphthalene DMN), coriander, Peppermint essential oil (Teper-Bamnolker et al,
2010), but concentration for the treatment of and time are not easy to control, and the time too long can damage potato seed rudiment and growth, and the time not enough reaches
Less than expected storage time.
In conclusion there is the problems such as expending high, medicament harm, effect is not significant, root in potato tubers dormancy control
This is the reason is that the internal mechanism converted from suspend mode to rudiment of potato tubers is indefinite.Therefore, it studies in potato tubers and stops
Sleep relevant gene expression and regulation mechanism and how to induce potato knot potato to be a problem to be solved.
Summary of the invention
For above-mentioned deficiency in the prior art, lured the present invention provides a kind of by being overexpressed potato StSN2 gene
The method for leading potato knot potato, the dormant state of potato can be effectively controlled in this method, and then can effectively induce potato knot potato.
To achieve the above object, the technical solution adopted by the present invention to solve the technical problems is:
A method of potato knot potato is induced by being overexpressed potato StSN2 gene, comprising the following steps:
(1) over-express vector of the gene containing StSN2 is constructed, and is converted into Agrobacterium competent cell；
(2) transgenic plant is constructed by mediated by agriculture bacillus
1. the induction of potato set
It is accessed in minimal medium using the stem section of potato set as explant, being placed in temperature is 18 ± 1 DEG C, light application time
It is 35-45 μm of ol m for 8h/16h day/night, intensity of illumination-2s-1Under conditions of cultivate 15-20 days；
2. the activation and resuspension of Agrobacterium
3. preculture and co-cultivation
Preculture: taking the potato plant cultivated 15-20 days, aseptically takes interstitial growth healthy and strong and without bud eye
Stem section, be placed on pre-culture medium, in 20-24 DEG C dark culture 2-3 days；
It co-cultures: by the stem section 2-3min after the During Agrobacterium preculture of resuspension, then using sterile water wash 2 times, go
After falling excessive moisture, access is lined in the co-cultivation base of one layer of filter paper, in 24-26 DEG C of dark culture 36h；
4. bud induction and root induction
Bud induction: the stem section after co-cultivation is put into the sterile water of the Cef containing 100mg/L and is cleaned 3-4 times, it is extra to remove
It is transferred in bud screening and culturing medium and is cultivated after moisture, cultivation temperature is 23 ± 1 DEG C, and intensity of illumination is 35-45 μm of ol m-2s-1,
Light application time was 16h/8h day/night, every 10-14 days one subcultures of replacement；
Root induction: cutting access for the long regeneration bud to 0.5-1cm and take root and cultivate in screening and culturing medium, cultivation temperature
It is 20 ± 1 DEG C, intensity of illumination is 55-65 μm of ol m-2s-1, light application time is 16h/8h day/night；
(3) transgenic line is determined
To the strain that can normally send out roots, its genomic DNA is extracted, carries out target gene PCR by template of genomic DNA
Amplification and sequencing, determine transgenic line；
(4) potato knot potato is induced
Part more than the transgenic plant root system of growth 15-30 days is inoculated into and is lured in potato culture medium, in 23 ± 1 DEG C
Under the conditions of dark culture 35-45 days.
Further, detailed process in step (1) are as follows: using restriction enzyme to gram containing StSN2 genetic fragment
Grand carrier and over-express vector carry out double digestion, by target fragment and carrier recovery that digestion obtains and connect, by connection product
Into competent escherichia coli cell, picking single colonie expands numerous, extraction plasmid for conversion, after PCR, digestion and sequencing identification, will
The plasmid for being connected with target fragment is transformed into the competent cell of Agrobacterium tumefaciems, after PCR is identified, cultivates Agrobacterium.
Further, 1. middle minimal medium is MS culture medium+0.6wt% agar+3wt% sucrose to step, and pH value is
5.8-5.9。
Further, step 2. in Agrobacterium activation and be resuspended detailed process are as follows: take conversion after Agrobacterium original bacteria liquid
It crosses in solid medium, is subsequently placed in 28 DEG C of culture 48h；Picking single colonie is seeded to Kan containing 50mg/L and 50mg/L
In the liquid YEB of Rif, at 28 DEG C, 48h is cultivated under the conditions of 200rpm, and finally bacterium solution is diluted and continues culture to OD600=
0.4-0.6, centrifugation, removes supernatant, isometric outstanding bacterium solution is added, thallus is resuspended.
Further, 2. middle re-suspension liquid is MS culture medium+3wt% sucrose, pH value 5.9-6.0 to step.
Further, step 3. in pre-culture medium ingredient are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+2mg/
L 6-BA+0.25mg/L TDZ+0.1mg/L GA3+ 0.03mg/L 2,4-D, pH value 5.9-6.0.
Further, step 3. in co-culture base ingredient are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+2mg/
L 6-BA+0.25mg/L TDZ+0.1mg/L GA3+ 0.03mg/L 2,4-D, pH value 5.9-6.0.
Further, step 4. in bud screening and culturing medium ingredient are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+
2mg/L 6-BA+0.25mg/L TDZ+0.1mg/L GA3+0.03mg/L 2,4-D+50mg/L Kan+100mg/L Cef+
250mg/L Car, pH value 5.9-6.0.
Further, step 4. in take root the ingredient of screening and culturing medium are as follows: MS culture medium+0.6wt% agar+3wt% sugarcane
Sugar+50mg/L Kan, pH value 5.9-6.0.
Further, the ingredient of potato culture medium is lured in step (4) are as follows: MS+0.6wt% agar+8wt% sucrose+
0.05wt% active carbon+3wt% paclobutrazol, pH value 5.8.
It is provided by the invention it is a kind of by be overexpressed potato StSN2 gene induce potato knot potato method, have with
It is lower the utility model has the advantages that
StSN2 gene participates in potato tubers sleep procedure in the present invention, increases stem tuber by being overexpressed StSN2 gene
Dormancy time, to maladjustment environment, at the same by be overexpressed StSN2 gene, can Effective Regulation potato tubers stop
Dormancy state, and can be achieved with the release to potato tubers sleep procedure by RNAi, makes its rudiment process mutually be suitable for environment,
Promote its fast-growth, improves its yield and quality.
In addition, the expression of StSN2 gene can also influence the mechanism of action of hormone such as GA and ABA in potato tubers, energy
It is enough effectively to control its suspend mode and budding state, to influence the knot potato situation of potato.
Detailed description of the invention
Fig. 1 is different overexpression StSN2 strain blade StSN2 gene expression results.
Fig. 2 is different overexpression StSN2 strain potato wedge StSN2 gene expression results.
Fig. 3 is that different materials induce the knot potato quantity after potato 7d, 10d, 13d.
Fig. 4 is that different materials induce the knot potato yield after potato 7d, 10d, 13d.
Fig. 5 is the potato knot potato yield and size picture of different materials.
Specific embodiment
Embodiment 1
A method of potato knot potato is induced by being overexpressed potato StSN2 gene, comprising the following steps:
(1) over-express vector is constructed
Double digestion (StSN2 gene, CDS nucleosides containing target gene fragment is distinguished using restriction enzyme BamH1 and SmaI
Acid sequence is shown in that SEQ ID NO:5, amino acid sequence are shown in SEQ ID NO:6) cloning vector and over-express vector pBI121 it is (unloaded
Body), the pBI121 carrier that digestion obtains is recycled with target fragment and is connected with T4 ligase.Endonuclease reaction is carried out at 37 DEG C,
Connection reacts on 4 DEG C overnight.
Wherein, digestion system (50 μ l) are as follows: 10 × Buffer, 5 μ l, 10 × BSA5 μ l, Restriction
endonucleases 2.5μl、PCR products or Vectors 10μl、ddH2O 27.5μl。
Linked system (10 μ l) are as follows: 1 μ l of T4DNA ligase, 10 × Buffer, 1 μ l, 2 Vector μ l, PCR
products 6μl。
Connection product is converted into competent escherichia coli cell, picking single colonie expands numerous, extraction plasmid, through PCR, enzyme
It is transformed into the competent cell GV3101 of Agrobacterium tumefaciems after cutting and being sequenced identification.
(2) transgenic plant is constructed
1. the induction of test tube seedling
Using No. 10 test tube seedling stem sections of river taro as explant, accessed in minimal medium, be placed in temperature be 18 ± 1 DEG C,
Light application time is 8h/16h day/night, intensity of illumination is 40 μm of ol m-2s-1Under conditions of cultivate 15-20 days；Wherein, base
Basal culture medium includes MS culture medium+0.6wt% agar+3wt% sucrose, pH value 5.8-5.9.
2. the activation and resuspension of Agrobacterium
Agrobacterium original bacteria liquid scribing line after taking conversion, is placed in 27 DEG C of culture 48h, picking single colonie is seeded to 5mL liquid YEB
In (Kan containing 50mg/L and 50mg/L Rif), in 28 DEG C, 48h is cultivated under the conditions of 200rpm, then press 1:200 dilution proportion
Bacterium solution is cultivated under similarity condition to OD in fresh liquid YEB600=0.4-0.6, then 4000rpm is centrifuged 10min, goes
Clearly, isometric outstanding bacterium solution is added, thallus is resuspended.
Wherein, 1L Agrobacterium YEB culture medium includes following component: tryptose 5.0g, sucrose 5.0g, MgSO4·7H2O
0.5g, yeast powder 10.0g, beef extract 5.0g add water 1L, and with triss adjusting PH with base to 7.0,15g agar powder is added if solid,
121 DEG C of sterilizing 20min.
Outstanding bacterium solution includes following component: MS culture medium+3wt% sucrose, pH value 5.9-6.0.
3. preculture and co-cultivation
Preculture: taking the potato plant cultivated 15-20 days, aseptically cuts off terminal bud and plant lower part
Stem section takes interstitial growth healthy and strong and the stem section without bud eye, is placed on pre-culture medium, in 20-24 DEG C dark culture 2-3 days.
Wherein, the ingredient of pre-culture medium are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+2mg/L 6-BA+
0.25mg/L TDZ+0.1mg/L GA3+ 0.03mg/L 2,4-D, pH value 5.9-6.0.
It co-cultures: then the stem section 2-3min after the During Agrobacterium preculture of resuspension being used sterile water wash 2 times, used
After filter paper removes excessive moisture, access is lined in the co-cultivation base of one layer of filter paper, in 24 DEG C of dark culture 36h.
Wherein, the ingredient of base is co-cultured are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+2mg/L 6-BA+
0.25mg/L TDZ+0.1mg/L GA3+ 0.03mg/L 2,4-D, pH value 5.9-6.0.
4. bud induction and root induction
Bud induction: the stem section after co-cultivation is put into the sterile water of the Cef containing 100mg/L and is cleaned 3-4 times, with sterile filter
Paper is sopped up to be transferred in bud screening and culturing medium after excessive moisture and be cultivated, and cultivation temperature is 23 ± 1 DEG C, and intensity of illumination is 40 μm of ol
m-2s-1, light application time is 16h/8h day/night, every 10-14 days one subcultures of replacement and is removed because Agrobacterium is excessive
The lethal of growth, pollution and complete albefaction plant.
Wherein, the ingredient of bud screening and culturing medium are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+2mg/L 6-BA+
0.25mg/L TDZ+0.1mg/L GA3+ 0.03mg/L 2,4-D+50mg/L Kan+100mg/L Cef+250mg/L Car, pH
Value is 5.9-6.0.
Root induction: cutting access for the long regeneration bud to 0.5-1cm and take root and cultivate in screening and culturing medium, cultivation temperature
It is 20 ± 1 DEG C, intensity of illumination is 60 μm of ol m-2s-1, light application time is 16h/8h day/night.
Wherein, it takes root the ingredient of screening and culturing medium are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+50mg/L
Kan, pH value 5.9-6.0.
All culture mediums of the present invention are basal mediums after 121 DEG C of high pressure sterilization 20min, are cooled to 60 DEG C or so again
Hormone and antibiotic after adding filtration sterilization.
(3) transgenic line is determined
To the strain (general 10d or so starts to grow) that can normally send out roots, can be initially believed that it is transgenic line, into one
Step extracts its genomic DNA, using genomic DNA as template, carries out target gene PCR amplification and sequencing, it is determined whether to turn base
Because of material；RNA is extracted, the transgenic line that destination gene expression amount is higher than 5 times of control is filtered out, for later experiments.
(4) potato knot potato is induced
Part more than the transgenic plant root system of growth 15-30 days is inoculated into and is lured in potato culture medium, in 23 ± 1 DEG C
Under the conditions of carry out dark culture, dark culture 40 days, wait tie potato quantity it is more when, observe the potato shape and size of transgenic line tying potato.
It is above-mentioned to lure potato culture medium are as follows: MS+0.6wt% agar+8wt% sucrose+0.05wt% active carbon+3wt% paclobutrazol,
PH value is 5.8.
As a result it detects:
1, resistant gene Preliminary detection
(1) potato test tube seedling leaves genomic DNA, using the DNA of extraction as template, resistance base are extracted using CTAB method
Because of anti-kanamycins npt II, then design primer carries out PCR reaction.
The primer of design are as follows:
Upstream primer: 5'-CTCCTTGCTCCTTCTCG-3'(SEQ ID NO:1)；
Downstream primer: 5'-TAGCAAGGGCAAGTCTCAG-3'(SEQ ID NO:2).
PCR reaction system: ddH212.5 μ l of O, 10 × PCR buffer, 2.0 μ l, upstream primer (10 μM) 0.5 μ l, under
Swim primer (10 μM) 0.5 μ l, dNTP Mixture (2.5mM each) 2.0 μ l, 0.5 μ l of DNA profiling, TaKaRa Ex Taq
0.5μl、MgCl2 1.5μl。
PCR response procedures are as follows: 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 30s, 58 DEG C of annealing/extension 1.5min, totally 30 are followed
Ring, last 58 DEG C of extensions 10min.
It takes 4 μ l amplified productions to carry out agarose gel electrophoresis, has detected whether purpose band, using plasmid as positive control,
It is negative control with the unconverted aseptic seedling total DNA of corresponding kind.
By testing result it is found that potato test tube seedling leaves genomic DNA can amplify purpose band, illustrate transgenosis
Success.
2, StSN2 expression quantity detects in blade
(1) potato test tube miaoye reverse transcription: is extracted according to the product description of Invitrogen TRIzol Reagent
Then piece total serum IgE synthesizes first chain of cDNA with RevertAidTM First Strand cDNA Synthesis kit,
Sequentially add following component: 1 μ L of oligodT, 3 RNA μ L, DEPC processing 8 μ L of water slightly mix and are centrifuged 30s, 65 DEG C of changes
Property 5min, chilling 2min, sequentially adds 5 × Reaction Buffer, 4 μ L, RNase Inhibitor (20U μ L on ice-1)1
μL、10mM dNTP Mix 2μL、Reverse Transcriptase(200UμL-1) 1 μ L, it slightly mixes and is slightly centrifuged, 42 DEG C of water
Bath, 60min, 72 DEG C of 10min terminate reaction, -20 DEG C of preservations.
(2) qRT-PCR:
Reaction system (25 μ L): RNase-Free ddH2O 10.5μL、2×SGExcel FastSYBR Mixture
12.5μL、Forward Primer(10μM)0.5μL、Reverse Primer(10μM)0.5μL、cDNA 1.0μL。
Response procedures: 95 DEG C of initial denaturation 20s, 95 DEG C of denaturation 5s, 60 DEG C of annealing/extension 30s, totally 40 recycle.
Using quantitative fluorescence analysis transgenic line and control group StSN2 expression conditions, difference is overexpressed StSN2 plants
It is that blade StSN2 gene expression results are shown in Fig. 1；Wherein, overexpression StSN2 material strain number: 68,108,110, it marks respectively
Are as follows: OE-StSN2-#68, OE-StSN2-#108, OE-StSN2-#110.As shown in Figure 1, control group StSN2 gene expression amount is aobvious
It writes and is lower than transgenic line StSN2 gene expression amount.
3, StSN2 gene expression amount detects in potato wedge
It chooses 500 plants of transgenic lines and carries out potting, be control with river taro No. 10, it is total to extract each tissue of the fresh sample of potato
RNA, with Oligo (dT) for primer, according to First Strand cDNA Synthesis Kit Revertra Ace- α-reverse
Recording method synthesizes cDNA, and designs internal reference EF1 α L primer, i.e. EF1 α L F:5'-CTTGTACACCACGCTAAGGAG-3'(SEQ
ID NO:3), EF1 α L R:5'-GTCAATGCAAACCATTCCTTG-3'(SEQ ID NO:4) and StSN2 quantify primer
StSN2-F:5'-CTCCTTGCTCCTTCTCG-3'(SEQ ID NO:1), StSN2-R:5'-TAGCAAGGGCAAGTCTCAG-
3'(SEQ ID NO:2).
Quantitative PCR reaction system (15 μ L): RNase-Free ddH2O 5μL、2×SGExcel FastSYBR
Mixture 5.0μL、Forward Primer(10μM)0.25μL、Reverse Primer(10μM)0.25μL、cDNA 4.5μ
L。
Response procedures: 95 DEG C of initial denaturation 30s, 95 DEG C of denaturation 5s, 55 DEG C of annealing/extension 5s, 95 DEG C of 10s, 65 DEG C of 5s, 95 DEG C
5s, totally 40 recycle.
Using quantitative fluorescence analysis transgenic line and control group StSN2 expression conditions, difference is overexpressed StSN2 plants
It is that potato wedge StSN2 gene expression results are shown in Fig. 2；Wherein, overexpression StSN2 material strain number: 68,108,110, it marks respectively
Are as follows: OE-StSN2-#68, OE-StSN2-#108, OE-StSN2-#110.
Is carried out by induction knot potato and is overexpressed StSN2 as shown in Figure 2 for 68,108,110 and control group material for strain number
The gene expression amount of potato wedge is apparently higher than control group in material.
4, potato quantity and yield are tied
Record induction potato 7d, 10d, 13d, the knot potato quantity and knot potato yield of different materials.Knot potato quantity result is shown in
Fig. 3 induces knot potato yield result after potato 13d to see that Fig. 4, potato knot potato yield and size picture are shown in Fig. 5；Wherein it is overexpressed
StSN2 material strain number: 68,108,110, it is respectively labeled as: OE-StSN2-#68, OE-StSN2-#108, OE-StSN2-#
110。
From the figure 3, it may be seen that transgenosis StSN2 accelerates the small potato knot potato time, control group is inducing 13 talentes to start to tie potato, and
Transgenic line, which lures, begins to knot potato for potato 7 days, shows that potato knot potato can be accelerated by being overexpressed StSN2.
By Fig. 4 and Fig. 5 it is found that transgenic line can dramatically increase potato knot potato yield, and with Fig. 1 and Fig. 2 result knot
Conjunction is analyzed, discovery StSN2 expression quantity it is higher, knot potato amount it is more, show StSN2 can promote Potatoes knot potato to
Increase its yield.
5, the comparison of transgenic plant and WT lines
By being overexpressed StSN2 gene, obtained transgenic potato plant is compared with WT lines, plant height and leaf
Area is obviously higher than WT lines and big, illustrates that the gene can promote the growth and development of potato haulm.
Claims (10)
1. a kind of method for inducing potato knot potato by being overexpressed potato StSN2 gene, which is characterized in that including following step
It is rapid:
(1) over-express vector of the gene containing StSN2 is constructed, and is converted into Agrobacterium competent cell；
(2) transgenic plant is constructed by mediated by agriculture bacillus
1. the induction of potato set
It is accessed in minimal medium using the stem section of potato set as explant, being placed in temperature as 18 ± 1 DEG C, light application time is
8h/16h day/night, intensity of illumination are 35-45 μm of ol m-2s-1Under conditions of cultivate 15-20 days；
2. the activation and resuspension of Agrobacterium
3. preculture and co-cultivation
Preculture: taking the potato plant cultivated 15-20 days, aseptically takes interstitial growth healthy and strong and the stem without bud eye
Section, be placed on pre-culture medium, in 20-24 DEG C dark culture 2-3 days；
It co-cultures: by the stem section 2-3min after the During Agrobacterium preculture of resuspension, then using sterile water wash 2 times, remove more
After remaining moisture, access is lined in the co-cultivation base of one layer of filter paper, in 24-26 DEG C of dark culture 36h；
4. bud induction and root induction
Bud induction: the stem section after co-cultivation is put into the sterile water of the Cef containing 100mg/L and is cleaned 3-4 times, remove excessive moisture
After be transferred in bud screening and culturing medium and cultivated, cultivation temperature is 23 ± 1 DEG C, and intensity of illumination is 35-45 μm of ol m-2s-1, illumination
Time was 16h/8h day/night, every 10-14 days one subcultures of replacement；
Root induction: cutting access for the long regeneration bud to 0.5-1cm and take root and cultivate in screening and culturing medium, cultivation temperature 20
± 1 DEG C, intensity of illumination is 55-65 μm of ol m-2s-1, light application time is 16h/8h day/night；
(3) transgenic line is determined
To the strain that can normally send out roots, its genomic DNA is extracted, carries out target gene PCR amplification by template of genomic DNA
And sequencing, determine transgenic line；
(4) potato knot potato is induced
Part more than the transgenic plant root system of growth 15-30 days is inoculated into and is lured in potato culture medium, in 23 ± 1 DEG C of condition
Lower dark culture 35-45 days.
2. the method according to claim 1 for inducing potato knot potato by being overexpressed potato StSN2 gene, feature
It is, detailed process in step (1) are as follows: table to the cloning vector containing StSN2 genetic fragment and is crossed using restriction enzyme
Double digestion is carried out up to carrier, by target fragment and carrier recovery that digestion obtains and connects, connection product is converted to large intestine bar
In bacterium competence cell, picking single colonie expands numerous, extraction plasmid, after PCR, digestion and sequencing identification, will be connected with target fragment
Plasmid be transformed into the competent cell of Agrobacterium tumefaciems, after PCR is identified, cultivate Agrobacterium.
3. the method according to claim 1 for inducing potato knot potato by being overexpressed potato StSN2 gene, feature
It is, 1. middle minimal medium is MS culture medium+0.6wt% agar+3wt% sucrose, pH value 5.8-5.9 to step.
4. the method according to claim 1 or 2 for inducing potato knot potato by being overexpressed potato StSN2 gene,
Be characterized in that, step 2. in Agrobacterium activation and be resuspended detailed process are as follows: take conversion after Agrobacterium original bacteria liquid solid train
It supports and crosses in base, be subsequently placed in 28 DEG C of culture 48h；Picking single colonie is seeded to the liquid of Kan containing 50mg/L and 50mg/L Rif
In YEB, at 28 DEG C, 48h is cultivated under the conditions of 200rpm, and finally bacterium solution is diluted and continues culture to OD600=0.4-0.6, from
The heart removes supernatant, isometric outstanding bacterium solution is added, thallus is resuspended.
5. the method according to claim 4 for inducing potato knot potato by being overexpressed potato StSN2 gene, feature
It is, 2. middle re-suspension liquid is MS culture medium+3wt% sucrose, pH value 5.9-6.0 to step.
6. the method according to claim 1 for inducing potato knot potato by being overexpressed potato StSN2 gene, feature
Be, step 3. in pre-culture medium ingredient are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+2mg/L 6-BA+
0.25mg/L TDZ+0.1mg/L GA3+ 0.03mg/L 2,4-D, pH value 5.9-6.0.
7. the method according to claim 1 for inducing potato knot potato by being overexpressed potato StSN2 gene, feature
Be, step 3. in co-culture base ingredient are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+2mg/L 6-BA+
0.25mg/L TDZ+0.1mg/L GA3+ 0.03mg/L 2,4-D, pH value 5.9-6.0.
8. the method according to claim 1 for inducing potato knot potato by being overexpressed potato StSN2 gene, feature
Be, step 4. in bud screening and culturing medium ingredient are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+2mg/L 6-BA+
0.25mg/L TDZ+0.1mg/L GA3+ 0.03mg/L 2,4-D+50mg/L Kan+100mg/L Cef+250mg/L Car, pH
Value is 5.9-6.0.
9. the method according to claim 1 for inducing potato knot potato by being overexpressed potato StSN2 gene, feature
Be, step 4. in take root the ingredient of screening and culturing medium are as follows: MS culture medium+0.6wt% agar+3wt% sucrose+50mg/L
Kan, pH value 5.9-6.0.
10. the method according to claim 1 for inducing potato knot potato by being overexpressed potato StSN2 gene, special
Sign is, the ingredient of potato culture medium is lured in step (4) are as follows: MS+0.6wt% agar+8wt% sucrose+0.05wt% active carbon+
3wt% paclobutrazol, pH value 5.8.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113637686A (en) * | 2021-08-30 | 2021-11-12 | 华中农业大学 | Application of potato StABL1 gene in regulation and control of potato maturity |
| CN114085838A (en) * | 2021-12-02 | 2022-02-25 | 甘肃农业大学 | Potato stu-miRn220 and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421813A (en) * | 2013-07-09 | 2013-12-04 | 华中农业大学 | Application of SN1 gene in controlling high temperature resistance and drought resistance of rice |
| CN103436538A (en) * | 2013-08-30 | 2013-12-11 | 中国农业科学院蔬菜花卉研究所 | Antimicrobial peptide as well as preparation method and application thereof |
-
2019
- 2019-05-23 CN CN201910436150.XA patent/CN110079549B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103421813A (en) * | 2013-07-09 | 2013-12-04 | 华中农业大学 | Application of SN1 gene in controlling high temperature resistance and drought resistance of rice |
| CN103436538A (en) * | 2013-08-30 | 2013-12-11 | 中国农业科学院蔬菜花卉研究所 | Antimicrobial peptide as well as preparation method and application thereof |
Non-Patent Citations (3)
| Title |
|---|
| MARTA BERROCAL-LOBO 等: "Snakin-2, an Antimicrobial Peptide from Potato Whose Gene Is Locally Induced by Wounding and Responds to Pathogen Infection", 《PLANT PHYSIOLOGY》 * |
| MEIYALAGHAN S 等: "Towards disease resistance in potatoes using intragenic approaches", 《COMMUNICATIONS IN AGRICULTURAL AND APPLIED BIOLOGICAL SCIENCES》 * |
| 任玉霞: "马铃薯抗晚疫病转基因植株的获得及其抗病鉴定", 《万方学位论文》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113637686A (en) * | 2021-08-30 | 2021-11-12 | 华中农业大学 | Application of potato StABL1 gene in regulation and control of potato maturity |
| CN114085838A (en) * | 2021-12-02 | 2022-02-25 | 甘肃农业大学 | Potato stu-miRn220 and application thereof |
| CN114085838B (en) * | 2021-12-02 | 2023-08-15 | 甘肃农业大学 | Potato stu-miRn220 and application thereof |
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