CN105052733B - Methods for inducing 'Hort 16A' offspring superior plant variation by using EMS and AFLP detection - Google Patents
Methods for inducing 'Hort 16A' offspring superior plant variation by using EMS and AFLP detection Download PDFInfo
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Abstract
The invention relates to methods for inducing 'Hort 16A' offspring superior plant variation by using EMS and AFLP detection, and relates to a kiwifruit breeding and identification method. According to the invention, the method for directly adding the EMS into a culture medium is adopted to induce kiwifruit, and the induction effect of the EMS with the concentration of 0.08 mg/L to an embryonic callus is relatively remarkable. In the AFLP, the influence of the enzyme use quantity, enzyme digestion time and enzyme activity required by PCR amplification to the enzyme digestion is researched; the influence of the enzyme activity and energy to primer connection is analyzed; appropriate primer combinations are screened out in a selective amplification system to be used as selective amplification primers after DNA pre-amplification, so that the selective amplification of polymorphic DNA is facilitated, and the reliability for reflecting the fragment length polymorphism of the polymorphic DNA is improved; therefore, the purpose of detecting the small difference of DNA levels of mutants generated through induction by one-time detection can be achieved.
Description
Technical field
The invention belongs to plant breeding and authentication method, particularly Kiwi berry breeding and authentication method.
Background technology
Kiwi berry (kiwifruit), category Cercopithecidae Actinidia (Actinidiacea,Actinidia spp.), it is many
Nian Sheng defoliation lianas, dioecism.Kiwi berry is the seeds of artificial domesticating cultivation wild fruit tree, by human-induced macaque
Peach variation mutation can produce the kind that nature did not had originally or conventional method is difficult to the new local flavor or new proterties for obtaining.But
It is less for the report of the research that EMS processes Kiwi berry variation breeding.Document 1（Dragon is supported oneself, and Chinese Gooseberry regeneration is lured with variation
The research [D] led.:Zhejiang University, 2008.）It is to process Cultured Actinidia deliciosa Calli with EMS to induce its indefinite shoot mutation different；Document 2
（Zhou Liming, Wang Fei, Wang Jia, EMS mutagenic treatment directed screening Kiwi berrys salt-tolerant mutant research [J]. northwest agriculture
Report, 2009,18 (5):330-335.）It is that Hayward and red kiwi fruit blade are processed with EMS, screens its salt tolerant and dash forward
Variant.But both individually with EMS to the callus immersion long period after, then move into culture medium Kiwi berry lured
Lead, required time is longer.
In the case of the overwhelming majority, variation is nondirectional, and the plant to making a variation detects, contributes to understanding variability
Shape.AFLP (Amplified Fragment Length Polymorphism) is that many will marks of length of amplified fragments are
A kind of molecular marking technique based on DNA polymorphism, few with sample amount with DNA, polymorphic detection sensitivity is high, good stability,
The features such as bands of a spectrum enrich.But because enzyme dosage, digestion time are active related to enzyme needed for PCR amplifications, it is necessary to study these
Impact of the factor to digestion result；Again the activity and energy of enzyme are needed due to connecting primer, therefore, its factor is analyzed to connection effect
The impact of fruit is also necessary.And, AFLP primers include the core sequence CORE complementary with artifical linker sequence at 5 ' ends, limit
The parts of viscosity latter end EXT tri- of the selective base of band at the ends of property restriction endonuclease particular sequence ENZ processed and 3 ', selective amplification primer
Refer to the primer of the viscosity latter end with specific selective base at 3 ' ends.Selective amplification system be the pre- amplification systems of AFLP it
Important step afterwards, in the system, screens appropriate primer combination, can facilitate randomly amplified polymorphic DNA and improve anti-
The reliability of its fragment length polymorphism is reflected, is reached and is once identified small on the mutant DNA level for detecting induction generation
The purpose of difference.
The content of the invention
It is contemplated that with the excellent strain of Chinese gooseberry ' hort16A ' offspring as material, carrying out at EMS mutagenesis to Kiwi berry
Reason, and research and analysis are carried out to the Kiwi berry DNA proterties after mutagenic treatment by improving AFLP systems, there is provided it is adapted to Kiwi berry
The detection method of the molecular labeling of DNA polymorphism.
First, with the EMS inductions ' method of the excellent strain variation of Hort 16A ' offsprings
Comprise the following steps：
（1）The preparation of mutagens culture medium
Kiwi berry mutagens culture medium is：MS+（0.01～0.09mg/L）EMS+0.3mg/L ZT+0.05g/L NAA+
30g/L Sucrose+5g/L Agar, above EMS filtration sterilization add, adjust pH to 5.8, using the callus tissue culture base as
Mutagens culture medium；
（2）Mutagenic treatment
Aseptic Kiwi berry blade is taken, bulk is cut into along the pulse and is inoculated on mutagens culture medium, 24～26 DEG C, lucifuge culture 1
Individual month, after adventitious buds differentiation, it is inoculated in adventitious bud proliferation culture medium：MS+1.5mg/L ZT+0.08mg/L EMS + 30g/L
Sucrose+5g/L Agar, 24～26 DEG C, the lx of intensity of illumination 3000～5000,16 h of photoperiod/d CMC models；
Treat that bud gradually grows up to form seedling, seedling is inoculated in into root media：1/2MS+0.7mg/L IBA+0.5g/L AC+
30g/L Sucrose+5g/L Agar, 24～26 DEG C, the lx of intensity of illumination 3000～5000,16 h of photoperiod/d CMC models.
It is described with EMS inductions ' in the method for the excellent strain variation of Hort 16A ' offsprings, step（1）Mutagens culture medium
EMS concentration is 0.08mg/L.
2nd, with EMS inductions ' the AFLP detection methods of the excellent strain variation of Hort 16A ' offsprings
Comprise the following steps：
（1）The extraction of Kiwi berry STb gene and electrophoresis detection：
Kiwi berry DNA is extracted, the μ L of DNA solution 5 purity and concentration that DNA is detected after 1.0% agarose gel electrophoresis is taken；
（2）In Kiwi berry AFLP endonuclease reactions, using 20 μ l systems：
300ng DNA,
1 U EcoRI/MseI,
4 μ l Buffer,
Remaining is ddH2O；
Response procedures are 37 DEG C of h of digestion 4；65 DEG C, 20min；4 DEG C, preserve；
（3）In Kiwi berry AFLP coupled reactions, using 20ul reaction systems：
5 μ l DNA enzymatics cut product,
2 μ l T4 buffer,
2 U T4 DNA ligases,
1 μ l adapter-primers, primer concentration is 100nM,
2 μ l ATP, primer concentration is 10 mM,
Remaining is ddH2O；
Linker is：22 DEG C, overnight；65 DEG C, 10min；4 DEG C of preservations；
（4）In the pre- amplification systems of Kiwi berry AFLP, using 25 μ l reaction systems：
2 μ l connection products,
0.125 μ l Taq enzymes,
0.5 μ l dNTPs,
2.5 μ l buffer,
The pre- amplimers of 1 μ l, including EcoRI Pre:5'GAC TGC GTA CCA ATT CA 3' and MseI Pre:
5'GAT GAG TCC TGA GTA AC 3', concentration is 50ng/ μ l,
Remaining is ddH2O；
The pre- amplification system for preparing is put in PCR instrument, response parameter is：94 DEG C, 90s, 94 DEG C, 30s, 56 DEG C, 1min,
72 DEG C, 1min, each 30 circulation；72 DEG C, 10min, -20 DEG C save backup；
（5）In Kiwi berry selective amplification system, using 25 μ l reaction systems：
Pre- amplified production after 2 μ l dilutions,
1 μ l EcoR I selective amplification primers,
1 μ l Mse I selective amplification primers,
0.5 μ l dNTPs,
2.5 μ l buffer,
0.25 μ l Taq enzymes,
1:The pre- amplified productions of 2 μ l of 10 dilutions,
Remaining is ddH2O；
The selective amplification primer by E-ACG+M-CTG, E-ACG+M-CAG, E-AAG+M-CAA, E-AAG+M-CTG,
E-ACT+M-CAG, E-ACG+M-CAA, E-AGG+M-CAG and E-AGG+M-CAT are combined by totally 8 pairs of primers；
Response procedures are：94 DEG C, 2min, 94 DEG C, 30s, 65 DEG C, 30s, 72 DEG C, 80s, each 14 circulation, annealing temperature is often followed
Ring successively decreases 0.7 DEG C；94 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 80s, each 23 circulation；72 DEG C, 5min；
（6）Separate and show the amplified production of AFLP with polyacrylamide gel electrophoresis and AgNOR technique respectively.
The marked improvement and essential characteristics that the present invention has be：
The present invention induces the method that EMS is directly added into culture medium to Kiwi berry, it is ensured that Kiwi berry blade embryo is healed
Injured tissue fully can be affected by EMS, wherein, to the Inductive character variable effects of embryo callus during 0.08mg/L concentration
More significantly, ensure for the aberration rate for improving Kiwi berry proterties is provided.
The present invention have studied these factors of the activity of enzyme dosage, digestion time and enzyme needed for PCR amplifications to enzyme in AFLP
Cut the impact of result；Analyze the impact of the activity and energy of enzyme to connection primer effect.And, selective amplification system is made
For important step, in selective amplification system, 64 pairs of primers are screened through repeated multiple times, filter out appropriate primer
Combination：E-ACG+M-CTG、E-ACG+M-CAG、E-AAG+M-CAA、E-AAG+M-CTG、E-ACT+M-CAG、E-ACG+M-CAA、
E-AGG+M-CAG and E-AGG+M-CAT, it is alternatively that property DNA amplification in advance after amplification 3 ' ends of its segment with specific choosing
The primer of the viscosity latter end of selecting property base, thus facilitate randomly amplified polymorphic DNA and raising to reflect its fragment length polymorphism
Reliability, with amplification site it is more, banding pattern quality is good, resolution ratio is higher, band density is suitable, be evenly distributed the features such as can
To reach the purpose that once identification detects the fine difference on the mutant DNA level that induction is produced.In AFLP collection of illustrative plates, variation
The otherness band of Kiwi berry genomic DNA is more, and the band of Normal Rhesus Monkey peach genomic DNA is clear and each band of same primers
More unify.
As shown in fig. 7, the present invention chooses the Kiwi berry genomic DNA and the kiwi fruit leaf piece genome that do not make a variation of character variation
DNA makees same primers contrast：Band variation is more in the AFLP collection of illustrative plates of variation Kiwi berry genomic DNA, has reached the Mi of tissue culture
Monkey peach variation inducing effect；And the AFLP collection of illustrative plates bands of the Kiwi berry genomic DNA that do not make a variation are clear and each bar of same primers takes
Comparison unification.
The invention belongs to the project of the State Administration of Forestry 948（Bullets：2012-6-42）Change with colleges and universities of Yunnan Province forest tree genetics
It is good with breed key lab's Funded Projects.
Description of the drawings
Fig. 1 for culture medium be not added with EMS ' Hort 16A ' superior progeny strains do not make a variation plantlet in vitro.
Fig. 2 is ' Hort 16A ' superior progeny strain character variation seedlings of the invention.In figure, decomposite leaf is obvious.
Fig. 3 is Kiwi berry DNA double digestion effect of the present invention.
Fig. 4 is the comprehensive score figure of Kiwi berry DNA double digestion effect of the present invention.
Fig. 5 is the connection effect of the fragment after Kiwi berry DNA enzymatic of the present invention is cut and joint.
Fig. 6 is the comprehensive score figure that the fragment after Kiwi berry DNA enzymatic of the present invention is cut is connected with joint.
Fig. 7 is the pre- expanding effect figures of Kiwi berry AFLP of the present invention.
Fig. 8 is Kiwi berry AFLP selective amplification design sketch of the present invention.
Fig. 9 is Kiwi berry AFLP collection of illustrative plates of the present invention.
Below in conjunction with specific embodiment, the present invention will be further described.Obviously, specific embodiment include but not
It is limited to pointed content.
Specific embodiment
Material is aseptic Chinese gooseberry ' Hort16A ' superior progeny plantlet in vitro plant.
Experiment main agents used and medicine see the table below 1.
The main agents of table 1 and medicine
The preparation of various solution：
5×TBE：It is standby that 27g Tris-base+13.75g boric acid+10ml0.5M EDTA are settled to 500ml；
Denaturation PAGE glue：Weigh 218g urea to dissolve in large beaker, add the acrylamide/first of 75ml (38% ︰ 2%)
Fork acrylamide, adds 5 × TBE of 100ml and is settled to 500ml；
Fixer：Taking glacial acetic acid 200ml adds mixing in 1.8L distilled water water to be placed in 4 DEG C of pre- cold standbies；
Silver staining liquid：4g silver nitrates are completely dissolved in 2L distilled water, add 37% formaldehyde 3ml, are prepared with first 5 minutes；
Developer solution：120g natrium carbonicum calcinatums are configured to 2L solution, add 37% formaldehyde 3ml, 400 μ L sodium thiosulfate
（10mg/ml）.
（1）The preparation of mutagens：
Cultured Actinidia deliciosa Calli culture medium：MS+0.3mg/L ZT+0.05g/L NAA+30g/L Sucrose+5g/L
It is placed in after Agar autoclavings on superclean bench, the EMS after filtration sterilization is rapidly added into callus tissue culture base.EMS ends
Concentration be respectively 0.01mg/L, 0.02mg/L, 0.03mg/L, 0.04mg/L, 0.05mg/L, 0.06mg/L, 0.07mg/L,
0.08mg/L, 0.09mg/L, 0.10mg/L, adjust pH5.8, are used to be inoculated with after culture medium solidifying.
（2）Mutagenic treatment：
Take aseptic Kiwi berry blade, be inoculated in bud inducement cultivation base, 24～26 DEG C, intensity of illumination 3000-5000 lx,
Under the conditions of 16 h of photoperiod/d cultivate 1 month, by bud proceed to proliferated culture medium MS+0.08mg/L EMS+1.5 mg/L ZT+
30g/L Sucrose+5g/L Agar are cultivated.Treat that bud gradually grows up to form seedling, then seedling is inoculated in into root media：1/2MS+
0.7mg/L IBA+0.5g/L AC+30g/L Sucrose+5g/L Agar are cultivated.
Variable concentrations EMS processes the impact to Kiwi berry blade embryo callus：EMS concentration be 0.01～
In 0.08mg/L is interval, Kiwi berry blade increases with EMS concentration, and the ratio of embryo callus differentiation rises；Exceed
0.08mg/L numerical value, the ratio of embryo callus differentiation declines.Wherein, when EMS concentration be 0.08mg/L when inducing effect most
Good, when EMS concentration reaches 0.10mg/L, the survival rate and differentiation rate of Kiwi berry is all close to 0.
2nd, with EMS inductions ' the AFLP detection methods of the excellent strain variation of Hort 16A ' offsprings
（1）The extraction of Kiwi berry STb gene and electrophoresis detection
Kiwi berry DNA is extracted using CTAB methods, the μ L of DNA solution 5 that then will be extracted, 1.0% 20 points of agarose gel electrophoresis
Clock, with fluorescent quantitative detector the purity and concentration of DNA are detected.
（2）Aflp analysis
It is prepared by template DNA
Kiwi berry genomic DNA is carried out into digestion with restriction enzyme ECoRI and MseI, then uses T4DNA ligases, will
DNA enzymatic is cut and is completed with manual splice one step of connection, and system is 20 μ L.
Due to enzyme dosage and digestion time it is active related to enzyme, therefore, by L16(45) orthogonal and synthesis
Point system research digestion result.The result shows：By 300ngDNA+1U EcoRI/MseI+2 μ l buffer solution+ddH2O=20μl
20 μ l reaction systems of composition, 60 DEG C of child care 20min after 37 DEG C of h of water-bath 4,4 DEG C of preservation effects are optimal.This is because, by Fig. 3,
The 4 factor level variation tendencies for being given, digestion effect is first raised and declined afterwards with being incremented by for DNA consumptions, is reached most at 300ng
High level；Digestion effect is highest at 1U in inscribe enzyme dosage, minimum at 4U, overall on a declining curve；Digestion effect is with digestion
Being incremented by for time and rise after falling before, digestion 4h effects are best.
Meanwhile, the extreme difference of DNA consumptions, inscribe enzyme dosage and digestion time is respectively 5,1.5 and 1.25, shows DNA consumptions
It is the key factor for affecting digestion effect.
Because connection needs energy, and the activity of enzyme is the key factor for affecting connection effect, therefore, we adopt L9
(34) orthogonal and comprehensive scoring method have studied connection experiment.As a result show：Using 20 μ l reaction systems, i.e. 2U T4
The μ l digestion products of+2 μ l ATP (10 mM) of+1.0 μ l adapter-primers (100nM) of μ l T4 buffer solutions of DNA ligase+2+5+
ddH2O=20 μ l, 65 DEG C of child care 10min after 22 DEG C of overnight process, 4 DEG C of preservation connection effects are optimal.This is because, give by Fig. 5,6
The factor level result of variations for going out：Connection effect is first raised and declined afterwards with being incremented by for T4 DNA ligase consumptions, at 2U
Peak is obtained, minimum is obtained at 1U；Connection effect is first raised and declined afterwards with being incremented by for adapter-primer consumption,
Peak is obtained at 1.0 μ l, minimum is obtained at 0.5 μ l；Connection effect increases with being incremented by for ATP consumptions, using 2 μ l
ATP connection effects are best.
Pre- amplified reaction
The pre- amplified reaction total systems of AFLP are 25 μ L, are respectively shown in Table 2 into subassembly.
The AFLP of table 2 expands in advance PCR reaction systems
| Component | Volume (μ L) |
| Template DNA (from digestion connection) | 2 |
| Pre-ampmix | 1 |
| dNTPs | 0.5 |
| 10×PCR buffer | 2.5 |
| TaqDNA polymease(2 U/μL) | 0.5 |
| Primer(50ng/μl) | 1 |
| AFLP-Water | 17.5 |
Primer is EcoRI Pre:5'GAC TGC GTA CCA ATT CA 3' and MseI Pre: 5' GAT GAG
TCC TGA GTA AC 3'。
The pre- amplification system for preparing is put in PCR instrument, response parameter is：94 DEG C, 90s；94 DEG C, 30s, 56 DEG C, 1min,
72 DEG C, 1min, 30 circulations；72 DEG C, 10min.The detection of 1.0% agarose gel electrophoresis is carried out after the completion of PCR, -20 DEG C of preservations are standby
With.
See Fig. 7.In pre- amplification experiment, with being incremented by for primer consumption, band is brighter, and expanding effect is better.When leading
And the consumption of rear guide chain primer reaches 1 μ l（Concentration is 50ng/ μ l）Afterwards, amplified band no longer blast；With passing for Taq enzyme consumption
Increase, band is more and more brighter, as TaqDNA polymease（2 U/μL）Consumption is reached after 1 μ L, and band brightness is not further added by.
Selective amplification reacts
It is selective to expand for the template DNA of restrictive amplification by pre- amplified production with TE buffer according to 1: 10 dilution
Increase PCR per part of 25 μ L of reactant liquor, system composition is as shown in table 3.
The AFLP selective amplification PCR reaction systems of table 3
| Component | Volume (μ L) |
| Pre- amplification dilute sample | 2 |
| 10×PCR buffer | 2.5 |
| TaqDNA polymease(2 U/μL) | 1 |
| dNTPs | 0.5 |
| The primers of EcoR I | 1 |
| The primers of Mse I | 1 |
| AFLP-Water | 17 |
Mix, be centrifuged, PCR is expanded, response parameter is：94℃90s；94 DEG C of 30s, 65 DEG C of 1min, 72 DEG C of 1min, 13 follow
Ring, per 0.7 DEG C of cycle down；94 DEG C of 30s, 56 DEG C of 1min, 72 DEG C of 1min, 25 are circulated；72 DEG C of 5min, -20 DEG C of preservations.Such as Fig. 8
Shown in the right-hand component of Kiwi berry AFLP selective amplification design sketch, with being incremented by for Taq enzyme consumption, band is more and more brighter, expands
Synergy fruit is better.
The present invention is screened through repeated multiple times to 64 pairs of AFLP primers, selects with amplification that site is more, banding pattern matter
Measure, resolution ratio is higher, band density is suitable, the features such as be evenly distributed and be adapted to 8 pairs of primers combination of Kiwi berry AFLP reactions,
It is as follows：E-ACG+M-CTG、E-ACG+M-CAG、E-AAG+M-CAA、E-AAG+M-CTG、E-ACT+M-CAG、E-ACG+M-CAA、
E-AGG+M-CAG and E-AGG+M-CAT.Sequence is as follows：
E-ACG+M-CTG: 5' GAC TGC GTA CCA ATT CACG 3’+5’ GAT GAG TCC TGA GTA
ACTG 3’
E-ACG+M-CAG: 5' GAC TGC GTA CCA ATT CACG 3’+5’ GAT GAG TCC TGA GTA
ACAG 3’
E-AAG+M-CAA: 5' GAC TGC GTA CCA ATT CAAG 3’+5’ GAT GAG TCC TGA GTA
ACAA 3’
E-AAG+M-CTG: 5' GAC TGC GTA CCA ATT CAAG 3’+5’ GAT GAG TCC TGA GTA
ACTG 3’
E-ACT+M-CAG: 5' GAC TGC GTA CCA ACT CAAG 3’+5’ GAT GAG TCC TGA GTA
ACAG 3’
E-ACG+M-CAA: 5' GAC TGC GTA CCA ATT CACG 3’+5’ GAT GAG TCC TGA GTA
ACAA 3’
E-AGG+M-CAG: 5' GAC TGC GTA CCA ATT CAGG 3’+5’ GAT GAG TCC TGA GTA
ACAG 3’
E-AAG+M-CAT: 5' GAC TGC GTA CCA ATT CAAG 3’+5’ GAT GAG TCC TGA GTA
ACAT 3’
Polyacrylamide gel electrophoresis
Retreaded by drying with distilled water flushing after glass plate and ear plate are fully cleaned, wiped anyhow with absolute ethyl alcohol respectively
Wipe using face 3 times, sweep net for the last time.By the affine silane for preparing（The μ L of affine silane 30, the μ L of glacial acetic acid 30, absolute ethyl alcohol
4.5ml）Embrocate on a glass, gently wiped once with absolute ethyl alcohol after drying.Gloves are changed, with stripping silane ear is wiped
After piece plate, then gently wiped once with absolute ethyl alcohol.Then, under, both sides of the edge bleed off pressure bar to glass plate, and ear plate is at upper, bottom
Portion aligns, and plate is assembled, and both sides are clamped with clip.Finally by the new 6%PAGE glue prepared（The μ L of 50ml PAGE glues+60
TEMED+160μL 10%APS）In pouring into gap, note there can not be bubble, place more than 4 hours, it is to be solidified rear for electrophoresis.
10 μ L Loading Buffer are added in selective amplification product, centrifugation, mixing are stood after 95 DEG C of min of denaturation 5
It is placed in ice bath.Using 1 × TBE as electrophoretic buffer, after the min of constant voltage 50V/cm prerunning 30,5 μ L denaturation are taken
Liquid is added in polyacrylamide gel carries out electrophoresis, and when blue bands reach offset plate bottom electrophoresis is stopped.
Silver staining
In being put into equipped with pre-prepd fixer after carefully glass plate is separated, shaking table is placed in（50r/min）On shake up
20min, to be instructed dose of decolouring, fixer retains.With distillation washing twice, each 3min is carried out on shaking table, and glass plate is put into
In the silver staining liquid for having prepared, it is placed on shaking table and shakes 30min.After with distilled water flushing 5s, the developer solution of precooling is immediately placed in
In, it is placed in shaking table（50r/min）On shake to band and occur, glass plate is put in above-mentioned 10% glacial acetic acid, on shaking table it is fixed about
5min, offset plate is put and is dried naturally at room temperature.
3rd, Kiwi berry variation interpretation of result
Paired observation is carried out to the Kiwi berry present situation after EMS process, it is found that Part Traits are more special, occur in that various changes
Different phenomenon（Meristic variation proterties such as Fig. 2）.
The present invention is with the Kiwi berry genomic DNA of character variation after EMS inductions and without the Normal Rhesus Monkey peach gene for inducing
Group DNA makees same primers to Polyacrylamide Gel Electrophoresis.After dyed, paired observation finds, make a variation Kiwi berry gene
Band variation is more in the AFLP collection of illustrative plates of group DNA, and the AFLP collection of illustrative plates bands of the Kiwi berry genomic DNA that do not make a variation are clear and identical
The each band of primer is relatively unified.This illustrates that EMS is fine to the Kiwi berry variation inducing effect of tissue culture（Such as Fig. 9）.Further grind
Study carefully the variation for showing that AFLP can also disclose the plant DNA level that phenotype does not change, and the technology can be used for Kiwifruit Tissue Culture
Hereditary variation dynamic monitoring in seedling industrialized production.
Claims (1)
1. with AFLP detection EMS inductions, ' method of Hort 16A ' progeny variations, comprises the following steps one kind：
（1）The extraction of Kiwi berry STb gene and electrophoresis detection：
Kiwi berry DNA is extracted, the μ L of DNA solution 5 purity and concentration that DNA is detected after 1.0% agarose gel electrophoresis is taken；
（2）In Kiwi berry AFLP endonuclease reactions, using 20 μ l systems：
300ng DNA,
1 U EcoRI/MseI,
4 μ l Buffer,
Remaining is ddH2O；
Response procedures are 37 DEG C of h of digestion 4；65 DEG C, 20min；4 DEG C, preserve；
（3）In Kiwi berry AFLP coupled reactions, using 20ul reaction systems：
5 μ l DNA enzymatics cut product,
2 μ l T4 buffer,
2 U T4 DNA ligases,
1 μ l adapter-primers,
2 μ l ATP,
Remaining is ddH2O；
Linker is：22 DEG C, overnight；65 DEG C, 10min；4 DEG C of preservations；
（4）In the pre- amplification systems of Kiwi berry AFLP, using 25 μ l reaction systems：
2 μ l connection products,
0.125 μ l Taq enzymes,
0.5 μ l dNTPs,
2.5 μ l buffer,
The pre- amplimers of 1 μ l, including EcoRI Pre:5'GAC TGC GTA CCA ATT CA 3' and MseI Pre: 5'
GAT GAG TCC TGA GTA AC 3', concentration is 50ng/ μ l,
Remaining is ddH2O；
The pre- amplification system for preparing is put in PCR instrument, response parameter is：94 DEG C, 90s, 94 DEG C, 30s, 56 DEG C, 1min, 72
DEG C, 1min, it is each 30 circulation；72 DEG C, 10min, -20 DEG C save backup；
（5）In Kiwi berry selective amplification system, using 25 μ l reaction systems：
1:The pre- amplified productions of 2 μ l of 10 dilutions,
1 μ l EcoR I selective amplification primers,
1 μ l Mse I selective amplification primers,
0.5 μ l dNTPs,
2.5 μ l buffer,
0.25 μ l Taq enzymes,
Remaining is ddH2O；
The selective amplification primer is by E-ACG+M-CTG, E-ACG+M-CAG, E-AAG+M-CAA, E-AAG+M-CTG, E-ACT
+ M-CAG, E-ACG+M-CAA, E-AGG+M-CAG and E-AGG+M-CAT are combined by totally 8 pairs of primers；
Response procedures are：94 DEG C, 2min, 94 DEG C, 30s, 65 DEG C, 30s, 72 DEG C, 80s, each 14 circulation, annealing temperature is often circulated passs
Subtract 0.7 DEG C；94 DEG C, 30s, 55 DEG C, 30s, 72 DEG C, 80s, each 23 circulation；72 DEG C, 5min；
（6）Separate and show the amplified production of AFLP with polyacrylamide gel electrophoresis and AgNOR technique respectively.
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| CN107446924B (en) * | 2017-08-16 | 2020-01-14 | 中国科学院华南植物园 | Kiwi fruit gene AcPDS editing vector based on CRISPR-Cas9 and construction method and application thereof |
| CN110122335B (en) * | 2019-06-27 | 2022-03-22 | 西南林业大学 | Induction method of salt-tolerant mutant of slow-fragrant kiwi fruit tissue culture seedling |
| CN113818237A (en) * | 2021-07-30 | 2021-12-21 | 百事基材料（青岛）股份有限公司 | Silk large biological fiber containing peach blossom active ingredients and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US7695901B2 (en) * | 2000-07-26 | 2010-04-13 | North Carolina State University | Identification of poinsettia cultivars |
| CN103548680A (en) * | 2013-10-30 | 2014-02-05 | 西南林业大学 | Tissue culture and rapid propagation method for actinidia chinensis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US7695901B2 (en) * | 2000-07-26 | 2010-04-13 | North Carolina State University | Identification of poinsettia cultivars |
| CN103548680A (en) * | 2013-10-30 | 2014-02-05 | 西南林业大学 | Tissue culture and rapid propagation method for actinidia chinensis |
Non-Patent Citations (3)
| Title |
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| Adventitious plant regeneration on leaf explants from adult male kiwifruit and AFLP analysis of genetic variation;M.J.Prado等;《Plant Cell Tiss Organ Cult》;20070131;第88卷(第1期);第1-10页 * |
| EMS诱变处理定向筛选猕猴桃耐盐突变体研究;周立名等;《西北农业学报》;20091031;第18卷(第5期);第1.1、1.2.1至1.2.4节 * |
| 猕猴桃AFLP分析体系的建立;陈华等;《西北植物学报》;20050831;第25卷(第8期);1528-1535 * |
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