CN106893780A - A kind of method for screening cotton gene related to drought tolerance - Google Patents
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Abstract
The invention discloses a kind of method for screening cotton gene related to drought tolerance.Method provided by the present invention includes：(1) following experimental group and control group are set：Experimental group：Drought stress treatment is carried out to cotton to be measured；Control group：Drought stress treatment is not carried out to the cotton to be measured；(2) detect and relatively described in the experimental group and the control group genomic DNA of cotton to be measured methylation level and/or pattern, the gene that methylation level and/or pattern have differences in two groups is found, as or candidate is and cotton drought resistance related gene.The present invention utilizes MSAP technical Analysis upland cotton seedling methylating under drought stress and/or patterns of change difference, the change of CCGG sites cytosine methylation level and/or pattern before and after analysis stress, gene related to drought tolerance is searched for using methylation differential fragment, can be that Resistance Strain of Cotton drought epigenetic regulation Mechanism Study and gene related to drought tolerance screening provide foundation.
Description
Technical field
The invention belongs to biological technical field, it is related to a kind of method for screening cotton gene related to drought tolerance, more particularly to one
Plant by detecting through related to screen cotton drought resisting to the change of the cotton gene group methylation level processed without drought stress
The method of gene.
Background technology
Global climate change is aggravated, and water resource lacks, and greenhouse effects aggravation, the soil salinization is serious, and me is seriously restricted
State's agricultural production.From the point of view of in world wide, arid is most wide influence area, causes economic loss the most serious, it is considered to be
One of natural calamity of most serious in the world.There is 2/3 Dry land in the arid of whole world soil 28%, China.With China's grain and cotton
The production situation fluctuating for striving the industrial crops such as the contradiction on ground, cotton, potato is indefinite, allows of no optimist.For cotton
Study on drought resistance, always be the focus of domestic and foreign scholars research, even more increase cotton yield per unit area, improve cotton product
Matter, solves the not enough key point of cotton production capacity.According to statistics, China nonirrigated farmland gross area is about 7.58 × 107hm2, account for China and always plough
The 73.70% of ground area, cut-off China in 2010 has 5.5 × 105hm2Arable land is suffered from drought, and the economic loss for causing every year is accounted for always
More than the 70% of natural calamity.Therefore, influence research of the drought stress to China industrial crops, rational exploitation and utilization drought are carried out
Ground, has its own strategic significance to my agricultural production and economic development.
Cotton is described as the pioneer crop in salt-soda soil because having stronger resistance.The drought resistance of cotton is studied, is excavated
Gene related to drought tolerance, improves cotton resistance, significant.DNA methylation is the important content of epigenetics phenomenon
One of, there are close ties with many vital movements such as normal growth development, plant defense, the environment stress of animals and plants, whole
All played an important role in life process.Under normal circumstances, blooming has the cytimidine of 20-30% in angiosperm genome
Residue can methylate, recent research indicate that, this ratio can also be higher, the overwhelming majority all occur in CG dinucleotides and
In CNG trinucleotides, other sites are relatively fewer.The change of DNA methylation site and pattern is a dynamic change, can be with
The life processes such as growth and development of plants, stress response change.The change of methylation level and state can cause base accordingly
Because the change of expression, research show that genetic transcription is methylated to be influenceed, methylation gene higher can suppress table
Reach, and the gene that demethylation occurs in itself will Enhanced expressing expression.
Methylation-sensitive amplified polymorphism (Methylation-sensitive amplification
Polymorphism, MSAP) a kind of technology technology for growing up on the basis of the AFLP of improvement, using different restricted
I pair of 5 '-CCGG-3 ' site of restriction endonuclease Hpa II and Msp carries out the digestion that methylates, and two kinds of enzymes are different to methylation sensitive, but two
Person can be recognized and cut CCGG sequences, can produce different cutting fragments, and methyl is analyzed by expanding these cleaved products
Change level and patterns of change.This method has been widely used in many other crops such as arabidopsis, paddy rice and cotton,
It is considered as a kind of classical DNA methylation detection method.But on drought stress induction upland cotton DNA methylation change
Report is also rarely reported.
The content of the invention
It is an object of the invention to provide a kind of new screening and the method for cotton drought resistance related gene.
Screening provided by the present invention and the method for cotton drought resistance related gene, specifically may include following steps：
(1) following experimental group and control group are set：
Experimental group：Drought stress treatment is carried out to cotton to be measured；
Control group：Drought stress treatment is not carried out to the cotton to be measured；
Whether the difference of the experimental group and the control group is only in that processed by drought stress, remaining all conditions
It is consistent.
(2) detect and the genomic DNA of cotton to be measured methylates relatively described in the experimental group and the control group
There is the gene of significant difference in level and/or pattern, methylation level and/or the pattern in two groups of finding, as or candidate is
With cotton drought resistance related gene.
In the present invention, the cotton is upland cotton, specially upland cotton " middle H177 ".
The step of methods described in (1), the cotton to be measured is carried out to be selected in cotton seedling when drought stress is processed
Long to three leaves, wholeheartedly period is carried out, and drought stress treatment is completed when soil relative water content drops to 7.0%.
The step of methods described in (2), the present invention is using MSAP technology for detection and the comparing experimental group and described
The methylation level of the genomic DNA of cotton to be measured described in control group.
The present invention uses the base of cotton to be measured described in MSAP technology for detection and the relatively experimental group and the control group
Because group DNA methylation level and/or pattern when, it is used as follows：
(a1) double digestion is combined as EcoR I/Hpa II and EcoR I/Msp I.
(a2) two single strand dnas of the joint of EcoR I as shown in sequence in sequence table 1 and sequence 2 are directed to mutually to recruit
To being formed；For the two single strand dna complementations of the joint as shown in sequence in sequence table 3 and sequence 4 of Hpa II and Msp I
Pairing is formed.
(a3) the pre- amplimer for EcoR I is single strand dna shown in sequence 5 in sequence table；For Hpa II and
The pre- amplimer of Msp I is single strand dna shown in sequence 6 in sequence table.
(a4) for EcoR I selective amplification primer shown in sequence 7, sequence 8, sequence 9 or sequence 10 in sequence table
Single strand dna；For Hpa II and Msp I selective amplification primer be sequence 11 in sequence table, sequence 12, sequence 13 or
Single strand dna shown in sequence 14.
Accordingly, pre- amplification PCR system uses 50 μ L reaction systems, containing 5 μ L connections cut back, two pre- amplimers
Respectively 1 μ L (primer concentration is 10 μM), 2.5U Taq enzymes, 1 × PCR buffer, 5 μ L 2.5mM dNTP.Pre- amplification PCR is anti-
The program is answered to be：94℃2min；94 DEG C of 30s, 56 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72℃10min.
Selective PCR amplification system uses 20 μ L reaction systems, containing the 2 pre- amplified productions of μ L, 1 μ L selective amplification primers
(each 1 μ L of upstream and downstream primer, primer concentration is 10 μM), 1 × PCR buffer, 0.5U Taq enzyme, 2 μ L2.5mM dNTP.Selection
Property PCR response procedures are：94 DEG C of 30s (denaturation)；65 DEG C of 1min (annealing), 72 DEG C of 1min (extension)；The 13rd is recycled to from the 2nd
Individual circulation, annealing temperature is gradually successively decreased, and each circulation reduces by 0.7 DEG C；35 cycle annealing temperature are recycled to from the 14th to keep
At 56 DEG C, and other programs are constant；Last 72 DEG C of extensions 10min.
It is described to find the gene that methylation level and/or pattern have differences in two groups in methods described, concretely：
According to MSAP electrophoresis results, the differential band in the experimental group and the control group is found, the differential band is cut back
Sequencing is received, according to sequencing result, homology search is carried out in ncbi database, find out corresponding gene, as or candidate is and cotton
Flower drought resistance related gene.
Application of the product containing following combination in screening and cotton drought resistance related gene falls within guarantor of the invention
Shield scope：
(1) restriction enzyme EcoR I, Hpa II and Msp I；
(2) for the joint of EcoR I, and for the joint of Hpa II and Msp I；
Two single strand dnas of the joint for EcoR I as shown in sequence 1 in sequence table and sequence 2 are mutually recruited
To being formed；Two single strand dnas of the joint for Hpa II and Msp I as shown in sequence in sequence table 3 and sequence 4
Complementary pairing is formed；
(3) pre- amplimer for EcoR I and the pre- amplimer for Hpa II and Msp I；
The pre- amplimer for EcoR I is single strand dna shown in sequence 5 in sequence table；It is described for Hpa
The pre- amplimer of II and Msp I is single strand dna shown in sequence 6 in sequence table；
(4) selective amplification primer for EcoR I and the selective amplification primer for Hpa II and Msp I；
The selective amplification primer for EcoR I is in sequence table shown in sequence 7, sequence 8, sequence 9 or sequence 10
Single strand dna；The selective amplification primer for Hpa II and Msp I is sequence 11, sequence 12, sequence in sequence table
13 or single strand dna shown in sequence 14.
The present invention is also claimed a kind of product for screening with cotton drought resistance related gene.
Product for screening with cotton drought resistance related gene provided by the present invention, containing as follows：
(1) restriction enzyme EcoR I, Hpa II and Msp I；
(2) for the joint of EcoR I, and for the joint of Hpa II and Msp I；
Two single strand dnas of the joint for EcoR I as shown in sequence 1 in sequence table and sequence 2 are mutually recruited
To being formed；Two single strand dnas of the joint for Hpa II and Msp I as shown in sequence in sequence table 3 and sequence 4
Complementary pairing is formed；
(3) pre- amplimer for EcoR I and the pre- amplimer for Hpa II and Msp I；
The pre- amplimer for EcoR I is single strand dna shown in sequence 5 in sequence table；It is described for Hpa
The pre- amplimer of II and Msp I is single strand dna shown in sequence 6 in sequence table；
(4) selective amplification primer for EcoR I and the selective amplification primer for Hpa II and Msp I；
The selective amplification primer for EcoR I is in sequence table shown in sequence 7, sequence 8, sequence 9 or sequence 10
Single strand dna；The selective amplification primer for Hpa II and Msp I is sequence 11, sequence 12, sequence in sequence table
13 or single strand dna shown in sequence 14.
In the application and the product, the cotton can be upland cotton, concretely upland cotton " middle H177 ".
The present invention is poor using MSAP technical Analysis upland cotton seedling methylating under drought stress and/or patterns of change
Different, the change of CCGG sites cytosine methylation level and/or pattern, is searched using methylation differential fragment before and after analysis stress
Rope gene related to drought tolerance, can be that Resistance Strain of Cotton drought epigenetic regulation Mechanism Study and gene related to drought tolerance screening provide foundation.
Brief description of the drawings
Fig. 1 is metamorphosis before and after cotton drought stress.
Fig. 2 is H177 genome dna electrophoresis results in cotton material.
Fig. 3 is DNA methylation MSAP AFLP systems.Wherein, H：HpaⅡ/EcoRⅠ；M：MspⅠ/EcoR.Type I：Non- first
Base band；Type II：Hemimethylation band；Type-iii：Permethylated band.
Fig. 4 be drought stress with compare between cotton gene group DNA methylation pattern analysis.Wherein H0And M0It is control
MSAP banding patterns, H and M be drought stress treatment MSAP banding patterns；H0It is the digestions of II/EcoR of Hpa I, M with H0With M be Msp I/
The digestions of EcoR I.
Fig. 5 is the relative expression quantity of M1-M5 these homologous genes under drought stress.
Specific embodiment
Experimental technique used in following embodiments is conventional method unless otherwise specified.
Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.
H177 in upland cotton drought resisting material：From the Chinese Academy of Agriculture Science and Technologys Cotton Research Institute.It is recorded in " Song Guifang land
Cotton gene related to drought tolerance (GhTrx, GhGR) is cloned and its functional analysis He'nan University, 2012, Master's thesis " text, Gong Zhongke
Obtained from applicant, can only be used to repetition present invention experiment and use.
Embodiment 1, using methylation-sensitive amplified polymorphism technology (MSAP) screen upland cotton gene related to drought tolerance
First, test material
The present invention using H177 in cotton drought resisting material, using sand culture method, nursery (the illumination 14h, 30 DEG C in incubator；
Dark 10h, 25 DEG C).Select full seed, the seed of uniformity, be through concentration 5% hypochlorite disinfectant after, use sterilized water
Clean, the vernalization 36h in sterile petri dish is rinsed, the consistent seed of germination is selected and is planted.It is long extremely in cotton seedling
Wholeheartedly period carries out drought stress to three leaves, and monitoring sand water content change treats that soil relative water content drops to 7.0% or so
When, carry out blade sampling, liquid nitrogen flash freezer, -80 DEG C of preservations.Wherein, metamorphosis such as Fig. 1 institutes before and after H177 drought stresses in cotton
Show.
Experiment sets the middle H177 without drought stress treatment as control simultaneously.
2nd, the extraction of genomic DNA, purifying and measure
With CTAB methods (Porebski S, Grant-Bailey L, the Baum B R.Modification fof a for improveing
CTAB extraction protocol for plants containing high polysaccharide and
polyphenol components.Plant Molecular Biology Reporter,1997,15(1):8-15.) extract
Genomic DNA, is processed with RNase (10mgmL-1), removes RNA, DNA mass after purification with 1.0% Ago-Gel
Electrophoresis (Fig. 2) and nucleic acid determination instrument (Nanodrop_2000) are detected, are dispensed that 4 DEG C and -20 DEG C save backup.
3rd, methylation-sensitive amplified polymorphism analysis (MSAP)
1st, test method
With reference to (Zhao Y L, Yu S X, Ye WW, et al.Study on DNA cytosine such as Zhao
methylation of cotton(Gossypium hirsutum L.)genome and its implication for
salt tolerance.Agricultural Science in China,2010,9(6):Method 783-791.) is carried out
MSAP is analyzed, and double digestion is combined as I/Hpa of EcoR II and I/Msp of EcoR I, and joint, pre- amplification and selective amplification primer are shown in Table
1。
The joint of table 1, pre- amplification and selective amplification primer
Digestion system (20 μ L)：400ngDNA, EcoR I and HpaII (or MspI) each 10U, digestion 3h.
Linked system (20 μ l)：200ng digestion products, 5pmol EcoRI joints, 50pmol HpaII-MspI joints,
10U T4 ligases, 16 DEG C overnight.10 times of connection product dilution, it is standby.
Pre- amplification PCR system (50 μ L)：5 μ L connect cut back, and pre- amplimer E1 and HM1 is respectively 1 μ L, and (primer is dense
Spend is 10 μM), 2.5U Taq enzymes, 1 × PCR buffer, 5 μ L 2.5mM dNTP.Pre- amplification PCR response procedures：94℃2min；
94 DEG C of 30s, 56 DEG C of 1min, 72 DEG C of 1min, 30 circulations；72℃10min.10 times of product dilution is standby.
Selective PCR amplification system (20 μ)：2 μ L expand cut back, 1 μ L selective amplification primer (upstream and downstream primers in advance
Each 1 μ L, primer concentration be 10 μM), 1 × PCR buffer, 0.5U Taq enzyme, 2 μ L 2.5mM dNTP.Selective PCR reaction interval
Sequence is：94 DEG C of 30s (denaturation)；65 DEG C of 1min (annealing), 72 DEG C of 1min (extension)；The 13rd circulation is recycled to from the 2nd, is annealed
Temperature is gradually successively decreased, and each circulation reduces by 0.7 DEG C；35 cycle annealing temperature are recycled to from the 14th and are maintained at 56 DEG C, and its
Its program is constant；Last 72 DEG C of extensions 10min.
After reaction terminates, polyacrylamide gel electrophoresis is carried out, genomic DNA is by II/EcoR of enzymes combinations Hpa I (H)
4 kinds of banding patterns are generally produced with after I/EcoR of Msp I (M) digestion, but 3 kinds can only be detected on polyacrylamide gel electrophoresis
Banding pattern (Fig. 3).Type I, 2 swimming lanes have band, show without the generation that methylates；Type II, H swimming lanes have band, and M swimming lanes do not have
Band, shows that CCGG sites occur hemimethylation；Type III, H swimming lanes are without band, and M swimming lanes have band, show that CCGG sites occur full first
Base.The banding pattern situation of the H/M swimming lanes presented according to electrophoresis result, carries out statistical analysis.
2nd, result of the test
(1) the cotton genomic dna methylation level change that drought stress causes
MSAP analyses are carried out using the 16 pairs of primers (selective amplification primer amounts to 16 kinds of combinations) in table 1, without dry
Band sum amplifiable in H177 in the cotton material of non-irrigated Stress treatment is 324, and the band ratio that methylates is 26.85%,
Permethylated band ratio is 20.06%.And band number amplifiable after being processed through drought stress is 354, methylate band
Ratio is 29.94%, and permethylated band ratio is 24.57%, is specifically shown in Table 2.I.e. after drought stress, cotton gene group
Methylation level is raised.
The drought stress of table 2 influences on cotton leaf DNA methylation level
Note：Amplified band sum=I+II+III；Methylate band sum=II+III；Permethylated band sum=
III。
(2) the cotton genomic dna methylation patterns change that drought stress causes
Analyzed by MSAP, understand specific CCGG sites methylation patterns situation of change after being processed through drought stress.As a result
Show that the MSAP analysis coamplifications of test material go out 11 kinds of banding patterns (Fig. 4,11 kinds of banding patterns are corresponding with the type in table 3), divide many
2 kinds of state property and monomorphism.Polymorphism refers to that control is different on methylation patterns from drought stress treatment group, there is three state again
Methylate (Type B), demethylation (c-type) and indefinite type (D types)；Monomorphism is to compare and have identical banding pattern between processing
(A types), shows that the methylation state after treatment does not change.Methylation level increases after Type B represents stress, and c-type represents the side of body
Methylation level declines after compeling, and D types represent that control cannot determine with the methylation state of DNA in treatment group.By to banding pattern
Statistical analysis, find cotton after drought stress, methylation state of DNA has a very large change, and is specifically shown in Table 3.
Influence of the drought stress of table 3 to cotton leaf methylation state of DNA
Note：H is the digestions of II/EcoR of Hpa I, and M is the digestions of I/EcoR of Msp I；+：There is band ,-：Without band；CThe expression born of the same parents that methylate are phonetic
Pyridine.
4th, the recovery of MSAP differential fragments, sequencing and homologous comparison
The MSAP differential fragments of control group and experimental group are reclaimed, is expanded and sequence analysis.It is specific as follows：Using going out
The knife blade cutting MSAP differential fragments of bacterium, are then crushed with the band that slide will cut.Differential band uses Poly-
Gel (Omega BioTek, USA) is reclaimed, and recovery product is used as DNA cloning template, is then sequenced.According to sequencing result,
Homology search is carried out in ncbi database, gene related to drought tolerance (table 4) is found out.
The homology analysis of the diversity sequence of table 4
According to this problem early stage drought resisting dielectrophoresis experiment analysis results (bibliography：Under the drought stresses such as land license not
With the leaf protein group comparative studies northwests Botany Gazette of drought resisting horizontal land cotton, 2013,33 (12):2401-2409),
Show that the gene in table 4 is related to drought resistance.
5th, homologous gene expression quantity detection (qRT-PCR)
The mRNA expressions of homologous gene are detected using quantitative fluorescent PCR (qRT-PCR) method.Extract experimental group and right
According to the total serum IgE of group sample, the reverse transcription synthesis chains of cDNA first, -20 DEG C preserve for qRT-PCR analyses.With cDNA as template, with
Actin (Actin) is reference gene, and homologous gene primer is as follows：M1：5 '-GATCTGCGAGTCCCTACTGC-3 ', 5 '-
GGGGCGACCATACTTGTTCA-3′；M2：5 '-AAAACAGCGGTAGCCACAGA-3 ', 5 '-
TACCTGCGCCACAGAAGATG-3′；M3：5 '-CAGCTACAGCGCGTTCTTTG-3 ', 5 '-
TGCCAGGCCAGACAATTAGG-3′；M4：5 '-GAGGTGATGCAACTGGGACA-3 ', 5 '-
GTTGATTCCGATGGCCCTGA-3′；M5：5 '-ACCCAGCAAACCAAGTAGGG-3 ', 5 '-
CACTCAGCACGATGGACAGT-3′.Quantitative fluorescent PCR system is 20 μ L, and amplification program is 94 DEG C of 30s；94 DEG C of 5s, 55 DEG C
15s, 72 DEG C of 10s, 40-45 circular response collect CT values after terminating, using 2-ΔΔCTMethod calculates the relative expression quantity of gene.
Result is as shown in Figure 5, it is seen that the relative expression quantity of M1-M5 these homologous genes under drought stress is relatively to photograph
Than having different degrees of rising, and significant difference.
<110>The Chinese Academy of Agriculture Science and Technologys Cotton Research Institute
<120>A kind of method for screening cotton gene related to drought tolerance
<130> GNCLN170535
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<170> PatentIn version 3.5
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Claims (10)
1. a kind of method screened with cotton drought resistance related gene, comprises the following steps：
(1) following experimental group and control group are set：
Experimental group：Drought stress treatment is carried out to cotton to be measured；
Control group：Drought stress treatment is not carried out to the cotton to be measured；
(2) detect and relatively described in the experimental group and the control group genomic DNA of cotton to be measured methylation level
And/or pattern, the gene that methylation level and/or pattern have differences in two groups is found, as or candidate is and cotton drought resisting
Property related gene.
2. method according to claim 1, it is characterised in that：The cotton is upland cotton.
3. method according to claim 1 and 2, it is characterised in that：In step (2), using MSAP technology for detection and compare
The methylation level and/or pattern of the genomic DNA of cotton to be measured described in the experimental group and the control group.
4. method according to claim 3, it is characterised in that：Using MSAP technology for detection and relatively more described experimental group and institute
When stating the methylation level and/or pattern of the genomic DNA of cotton to be measured described in control group, double digestion is combined as EcoR I/
Hpa II and EcoR I/Msp I.
5. method according to claim 4, it is characterised in that：Using MSAP technology for detection and relatively more described experimental group and institute
When stating the methylation level and/or pattern of the genomic DNA of cotton to be measured described in control group, for EcoR I joint by sequence
Sequence 1 and two single strand dna complementary pairings shown in sequence 2 are formed in list, for Hpa II and Msp I joint by
Sequence 3 and two single strand dna complementary pairings shown in sequence 4 are formed in sequence table.
6. method according to claim 5, it is characterised in that：Using MSAP technology for detection and relatively more described experimental group and institute
When stating the methylation level and/or pattern of the genomic DNA of cotton to be measured described in control group, the pre- amplification for EcoR I is drawn
Thing is single strand dna shown in sequence 5 in sequence table, and the pre- amplimer for Hpa II and Msp I is sequence 6 in sequence table
Shown single strand dna.
7. method according to claim 6, it is characterised in that：Using MSAP technology for detection and relatively more described experimental group and institute
When stating the methylation level and/or pattern of the genomic DNA of cotton to be measured described in control group, the selectivity for EcoR I expands
It is sequence 7, sequence 8, sequence 9 or single strand dna shown in sequence 10 in sequence table to increase primer, for Hpa II and Msp I's
Selective amplification primer is sequence 11, sequence 12, sequence 13 or single strand dna shown in sequence 14 in sequence table.
8. the application containing the following product for combining in screening and cotton drought resistance related gene：
(1) restriction enzyme EcoR I, Hpa II and Msp I；
(2) for the joint of EcoR I, and for the joint of Hpa II and Msp I；
Two single strand dna complementary pairing shapes of the joint for EcoR I as shown in sequence 1 in sequence table and sequence 2
Into；Two single strand dna complementations of the joint for Hpa II and Msp I as shown in sequence in sequence table 3 and sequence 4
Pairing is formed；
(3) pre- amplimer for EcoR I and the pre- amplimer for Hpa II and Msp I；
The pre- amplimer for EcoR I is single strand dna shown in sequence 5 in sequence table；It is described for Hpa II and
The pre- amplimer of Msp I is single strand dna shown in sequence 6 in sequence table；
(4) selective amplification primer for EcoR I and the selective amplification primer for Hpa II and Msp I；
The selective amplification primer for EcoR I is single-stranded shown in sequence 7, sequence 8, sequence 9 or sequence 10 in sequence table
DNA molecular；The selective amplification primer for Hpa II and Msp I be sequence table in sequence 11, sequence 12, sequence 13 or
Single strand dna shown in sequence 14.
9. it is a kind of for screen with the product of cotton drought resistance related gene, containing as follows：
(1) restriction enzyme EcoR I, Hpa II and Msp I；
(2) for the joint of EcoR I, and for the joint of Hpa II and Msp I；
Two single strand dna complementary pairing shapes of the joint for EcoR I as shown in sequence 1 in sequence table and sequence 2
Into；Two single strand dna complementations of the joint for Hpa II and Msp I as shown in sequence in sequence table 3 and sequence 4
Pairing is formed；
(3) pre- amplimer for EcoR I and the pre- amplimer for Hpa II and Msp I；
The pre- amplimer for EcoR I is single strand dna shown in sequence 5 in sequence table；It is described for Hpa II and
The pre- amplimer of Msp I is single strand dna shown in sequence 6 in sequence table；
(4) selective amplification primer for EcoR I and the selective amplification primer for Hpa II and Msp I；
The selective amplification primer for EcoR I is single-stranded shown in sequence 7, sequence 8, sequence 9 or sequence 10 in sequence table
DNA molecular；The selective amplification primer for Hpa II and Msp I be sequence table in sequence 11, sequence 12, sequence 13 or
Single strand dna shown in sequence 14.
10. it is according to claim 8 application or claim 9 described in product, it is characterised in that：The cotton is land
Cotton.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112655502A (en) * | 2020-12-31 | 2021-04-16 | 新疆农垦科学院 | Method for screening cotton drought-resistant related gene |
| CN113981136A (en) * | 2021-12-20 | 2022-01-28 | 宜宾学院 | Method for detecting DNA methylation pattern variation of plants after physiological stress treatment |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102080128A (en) * | 2010-11-29 | 2011-06-01 | 山东农业大学 | Application of methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants |
| CN102965367A (en) * | 2012-12-04 | 2013-03-13 | 中国农业科学院棉花研究所 | Method for acquiring plant candidate anti-disease gene sequence |
| CN104087656A (en) * | 2014-05-16 | 2014-10-08 | 安徽农业大学 | Method for screening white cotton cotyledon ageing related gene by using methylation-sensitive amplification polymorphism technology |
| CN104293775A (en) * | 2013-07-16 | 2015-01-21 | 北京林业大学 | Molecule marker, screening method, kit and application for stress-resistant poplar |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102080128A (en) * | 2010-11-29 | 2011-06-01 | 山东农业大学 | Application of methylation sensitive amplification polymorphism technique in toxicity analysis of transgenic plants |
| CN102965367A (en) * | 2012-12-04 | 2013-03-13 | 中国农业科学院棉花研究所 | Method for acquiring plant candidate anti-disease gene sequence |
| CN104293775A (en) * | 2013-07-16 | 2015-01-21 | 北京林业大学 | Molecule marker, screening method, kit and application for stress-resistant poplar |
| CN104087656A (en) * | 2014-05-16 | 2014-10-08 | 安徽农业大学 | Method for screening white cotton cotyledon ageing related gene by using methylation-sensitive amplification polymorphism technology |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN112655502A (en) * | 2020-12-31 | 2021-04-16 | 新疆农垦科学院 | Method for screening cotton drought-resistant related gene |
| CN113981136A (en) * | 2021-12-20 | 2022-01-28 | 宜宾学院 | Method for detecting DNA methylation pattern variation of plants after physiological stress treatment |
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