CN106893780B - Method for screening cotton drought-resistant related gene - Google Patents
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Abstract
The invention discloses a method for screening genes related to cotton drought resistance. The method provided by the invention comprises the following steps: (1) the following experimental and control groups were set up: experimental groups: carrying out drought stress treatment on cotton to be detected; control group: carrying out no drought stress treatment on the cotton to be detected; (2) and detecting and comparing the methylation level and/or pattern of the genome DNA of the cotton to be detected in the experimental group and the control group, and finding out the genes with difference in methylation level and/or pattern in the two groups, namely the genes are or are candidate genes related to the cotton drought resistance. The method utilizes an MSAP technology to analyze the methylation and/or mode change difference of upland cotton seedlings under drought stress, analyzes the change of the cytosine methylation level and/or mode of CCGG loci before and after the stress, utilizes methylation difference fragments to search drought-resistant related genes, and can provide a basis for the research of cotton drought-resistant epigenetic regulation mechanism and the screening of the drought-resistant related genes.
Description
Technical Field
The invention belongs to the technical field of biology, relates to a method for screening cotton drought-resistant related genes, and particularly relates to a method for screening cotton drought-resistant related genes by detecting changes of methylation levels of cotton genomes subjected to and without drought stress treatment.
Background
Global climateThe change is aggravated, the water resource is lacked, the greenhouse effect is aggravated, the soil salinization is serious, and the agricultural production of China is severely restricted. From the world, drought is one of the most serious natural disasters in the world, because the drought affects the most area and causes the most serious economic loss. The land is 28% dry all over the world, and China has 2/3 dry land. With the increasingly outstanding contradiction of land competition between grains and cotton in China, the production situation of economic crops such as cotton, potato and the like is irregular and is not optimistic. The research aiming at the drought resistance of cotton is always a hot point of research of scholars at home and abroad, and is the key point for increasing the unit area yield of cotton, improving the quality of cotton and solving the problem of insufficient cotton yield. According to statistics, the total dry land area of China is about 7.58 multiplied by 107hm2Accounts for 73.70 percent of the total cultivated land area of China, and the total cultivated land area of China is 5.5 multiplied by 10 by 20105hm2The economic loss caused by dry farmland accounts for more than 70% of the total natural disasters each year. Therefore, the research on the influence of drought stress on economic crops in China is developed, the dry land is reasonably developed and utilized, and the method has important strategic significance on agricultural production and economic development.
Cotton is known as a pioneer crop in saline-alkali soil because of its strong stress resistance. The method has important significance in researching the drought resistance of cotton, excavating genes related to the drought resistance and improving the stress resistance of the cotton. DNA methylation is one of important contents of epigenetic phenomena, is closely related to many life activities such as normal growth and development of animals and plants, plant defense, adversity stress and the like, and plays an important role in the whole life process. Under normal conditions, 20-30% of cytosine residues in the genome of flowering angiosperms are methylated, and recent studies have shown that this ratio is also higher, mostly occurring in CG dinucleotides and CNG trinucleotides, with relatively few other sites. The change of DNA methylation sites and patterns is a dynamic change and can change along with the life processes of plant growth and development, stress response and the like. The change of the methylation level and the state correspondingly causes the change of the gene expression level, and researches show that the gene transcription is influenced by methylation, the gene with higher methylation degree can inhibit the expression, and the gene which is subjected to demethylation per se can enhance the expression.
Methylation-sensitive amplification polymorphism (MSAP) technology is developed on the basis of improved AFLP, methylase cleavage is carried out on 5 '-CCGG-3' sites by adopting different restriction endonucleases HpaII and mspI, the two enzymes have different Methylation sensitivities, but both can recognize and cleave CCGG sequences, different cleavage fragments can be generated, and Methylation level and pattern change are analyzed by amplifying cleavage products. This method has been widely used in many other crops, such as Arabidopsis, rice and cotton, and has been considered as a classical DNA methylation detection method. However, there are few reports on drought stress induced DNA methylation changes in upland cotton.
Disclosure of Invention
The invention aims to provide a novel method for screening genes related to cotton drought resistance.
The method for screening the genes related to the drought resistance of the cotton, provided by the invention, specifically comprises the following steps:
(1) the following experimental and control groups were set up:
experimental groups: carrying out drought stress treatment on cotton to be detected;
control group: carrying out no drought stress treatment on the cotton to be detected;
the experimental group and the control group differed only in whether they were subjected to drought stress treatment, and all other conditions remained the same.
(2) And detecting and comparing the methylation level and/or pattern of the genome DNA of the cotton to be detected in the experimental group and the control group, and finding out the genes with significant difference in methylation level and/or pattern in the two groups, namely the genes are or are candidate genes related to the cotton drought resistance.
In the present invention, the cotton is upland cotton, specifically "medium H177".
In the step (1) of the method, when the cotton to be tested is subjected to drought stress treatment, the drought stress treatment is completed when the cotton seedlings grow to three leaves and one heart and the relative water content of soil is reduced to 7.0%.
In the step (2) of the method, the invention adopts MSAP technology to detect and compare the methylation level of the genomic DNA of the cotton to be detected in the experimental group and the control group.
The invention adopts MSAP technology to detect and compare the methylation level and/or the pattern of the genome DNA of the cotton to be detected in the experimental group and the control group, and the method comprises the following steps:
(a1) the double enzyme cutting combination is EcoR I/Hpa II and EcoR I/Msp I.
(a2) The adaptor aiming at the EcoR I is formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table; the linker aiming at Hpa II and Msp I is formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table.
(a3) The pre-amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 5 in a sequence table; the pre-amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown in a sequence 6 in a sequence table.
(a4) The selective amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 7, a sequence 8, a sequence 9 or a sequence 10 in a sequence table; the selective amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 11, a sequence 12, a sequence 13 or a sequence 14 in a sequence table.
Correspondingly, the pre-amplification PCR system adopts a 50 mu L reaction system, and contains 5 mu L of connecting dilution product, 1 mu L of two pre-amplification primers (the concentration of the primers is 10 mu M), 2.5U of Taq enzyme, 1 XPCR buffer and 5 mu L of 2.5mM dNTP. The pre-amplification PCR reaction procedure is as follows: 94 ℃ for 2 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 1min, 72 ℃ for 1 min; 10min at 72 ℃.
The selective PCR amplification system used 20. mu.L reaction system, containing 2. mu.L pre-amplification product, 1. mu.L selective amplification primer (1. mu.L each of upstream and downstream primers, primer concentration 10. mu.M), 1 XPCR buffer, 0.5U Taq enzyme, 2. mu.L 2.5mM dNTP. The selective PCR reaction procedure was: 30s at 94 ℃ (denaturation); annealing at 65 deg.C for 1min, and extending at 72 deg.C for 1 min; from cycle 2 to cycle 13, the annealing temperature is gradually reduced, and each cycle is reduced by 0.7 ℃; the annealing temperature was maintained at 56 ℃ from cycle 14 to cycle 35, while the other procedures were unchanged; finally, extension is carried out for 10min at 72 ℃.
In the method, the finding of the genes with different methylation levels and/or patterns in the two groups may specifically be: and finding out a difference band in the experimental group and the control group according to the MSAP electrophoresis result, cutting, recovering and sequencing the difference band, and performing homology search in an NCBI database according to the sequencing result to find out a corresponding gene, namely or candidate genes related to the cotton drought resistance.
The application of the product containing the following combination in screening genes related to the drought resistance of cotton also belongs to the protection scope of the invention:
(1) restriction enzymes EcoR I, Hpa II and Msp I;
(2) a linker for EcoR I, and linkers for Hpa II and Msp I;
the adaptor aiming at the EcoR I is formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table; the joints aiming at Hpa II and Msp I are formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table;
(3) pre-amplification primers for EcoR I and pre-amplification primers for Hpa II and Msp I;
the pre-amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 5 in a sequence table; the pre-amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 6 in a sequence table;
(4) a selective amplification primer for EcoR I and a selective amplification primer for Hpa II and Msp I;
the selective amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 7, a sequence 8, a sequence 9 or a sequence 10 in a sequence table; the selective amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 11, a sequence 12, a sequence 13 or a sequence 14 in a sequence table.
The invention also requests to protect a product for screening genes related to the drought resistance of cotton.
The product for screening the genes related to the drought resistance of the cotton comprises the following components:
(1) restriction enzymes EcoR I, Hpa II and Msp I;
(2) a linker for EcoR I, and linkers for Hpa II and Msp I;
the adaptor aiming at the EcoR I is formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table; the joints aiming at Hpa II and Msp I are formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table;
(3) pre-amplification primers for EcoR I and pre-amplification primers for Hpa II and Msp I;
the pre-amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 5 in a sequence table; the pre-amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 6 in a sequence table;
(4) a selective amplification primer for EcoR I and a selective amplification primer for Hpa II and Msp I;
the selective amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 7, a sequence 8, a sequence 9 or a sequence 10 in a sequence table; the selective amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 11, a sequence 12, a sequence 13 or a sequence 14 in a sequence table.
In the application and the product, the cotton may be upland cotton, and may be, in particular, upland cotton "medium H177".
The method utilizes an MSAP technology to analyze the methylation and/or mode change difference of upland cotton seedlings under drought stress, analyzes the change of the cytosine methylation level and/or mode of CCGG loci before and after the stress, utilizes methylation difference fragments to search drought-resistant related genes, and can provide a basis for the research of cotton drought-resistant epigenetic regulation mechanism and the screening of the drought-resistant related genes.
Drawings
FIG. 1 shows morphological changes of cotton before and after drought stress.
FIG. 2 shows the result of electrophoresis of H177 genomic DNA in cotton material.
FIG. 3 is a map of DNA methylation MSAP amplification. Wherein, H: hpa II/EcoRI; m: mspI/EcoR. Type I: an unmethylated band; type II: hemimethylated bands; type III: fully methylated bands.
FIG. 4 is a DNA methylation pattern analysis of cotton genomes between drought stress and control. Wherein H0And M0MSAP banding pattern as control, H and M are MSAP banding pattern of drought stress treatment; h0And H is an HpaII/EcoRI enzyme, M0And M is mspI/EcoRI enzyme.
FIG. 5 shows the relative expression amounts of several homologous genes M1-M5 under drought stress.
Detailed Description
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
H177 in upland cotton drought-resistant materials: from the cotton institute of the Chinese academy of agricultural sciences. The invention is described in the research of the clone of drought-resistant related genes (GhTrx, GhGR) of Gossypium hirsutum and the functional analysis thereof, university of Henan, 2012, Master thesis, which is publicly available from the applicant and can only be used for repeated experiments.
Example 1 screening of drought-resistant related genes in upland cotton by Methylation Sensitive Amplification Polymorphism (MSAP)
First, test materials
The invention adopts H177 in cotton drought-resistant material, adopts sand culture method, and raises seedlings in incubator (illumination 14H, 30 deg.C; dark 10H, 25 deg.C). Selecting full and uniform seeds, disinfecting the seeds by sodium hypochlorite with the concentration of 5%, washing the seeds by sterile water, accelerating germination in a sterile culture dish for 36 hours, and selecting the seeds with uniform germination for planting. Carrying out drought stress when the cotton seedlings grow to be three-leaf one-heart, monitoring the change of the water content of sandy soil, sampling leaves when the relative water content of the soil is reduced to about 7.0%, quickly freezing by using liquid nitrogen, and storing at-80 ℃. Wherein, the morphological changes of H177 in cotton before and after drought stress are shown in figure 1.
Experiment H177 without drought stress treatment was also set as a control.
Secondly, extracting, purifying and measuring genome DNA
Genomic DNA was extracted by the modified CTAB method (Porebski S, Grant-Bailey L, Baum B R. modification of fon a CTAB extraction protocol for plants containing high levels of polysaccharide and heterophenol components. plant Molecular Biology Reporter,1997,15(1):8-15.), treated with RNase (10 mg. mL-1), RNA was removed, the quality of the purified DNA was checked by 1.0% agarose gel electrophoresis (FIG. 2) and a nucleic acid analyzer (Nanodrop-2000), dispensed, and stored at 4 ℃ and-20 ℃ for further use.
Methylation sensitive amplification polymorphism analysis (MSAP)
1. Test method
MSAP analysis was performed by reference to the method of Zhao et al (Zhao Y L, Yu S X, Ye WW, et al. study on DNA methylation of cotton (Gossypium hirsutum L.), genome and its implementation for analysis of genomic DNA, agricultural Science in China,2010,9(6): 783-791), the two-enzyme combination being EcoRI/Hpa II and EcoRI/mspI, and the linker, pre-amplification and selective amplification primers are shown in Table 1.
TABLE 1 adaptor, Pre-amplification and Selective amplification primers
Enzyme digestion system (20 μ L): 400ng of DNA, 10U each of EcoR I and HpaII (or MspI), was digested for 3 h.
Ligation system (20 μ l): 200ng of the cleavage product, 5pmol of EcoRI linker, 50pmol of HpaII-MspI linker, 10U T4 ligase, 16 ℃ overnight. The ligation product was diluted 10-fold and ready for use.
Pre-amplification PCR System (50. mu.L): mu.L of the ligation dilution product, 1. mu.L of pre-amplification primers E1 and HM1 (primer concentration 10. mu.M), 2.5U of Taq enzyme, 1 XPCR buffer, and 5. mu.L of 2.5mM dNTP, respectively. Pre-amplification PCR reaction procedure: 94 ℃ for 2 min; 30 cycles of 94 ℃ for 30s, 56 ℃ for 1min, 72 ℃ for 1 min; 10min at 72 ℃. The product was diluted 10-fold for use.
Selective PCR amplification system (20 μ): mu.L of the pre-amplified dilution, 1. mu.L of selective amplification primers (1. mu.L of each of the upstream and downstream primers, 10. mu.M primer concentration), 1 XPCR buffer, 0.5U of Taq enzyme, 2. mu.L of 2.5mM dNTP. The selective PCR reaction procedure was: 30s at 94 ℃ (denaturation); annealing at 65 deg.C for 1min, and extending at 72 deg.C for 1 min; from cycle 2 to cycle 13, the annealing temperature is gradually reduced, and each cycle is reduced by 0.7 ℃; the annealing temperature was maintained at 56 ℃ from cycle 14 to cycle 35, while the other procedures were unchanged; finally, extension is carried out for 10min at 72 ℃.
After the reaction, polyacrylamide gel electrophoresis was performed, and the genomic DNA was cleaved with the combination of HpaII/EcoRI (H) and mspI/EcoRI (M) to generate 4 band patterns, but only 3 band patterns were detected by polyacrylamide gel electrophoresis (FIG. 3). Type I, 2 lanes all have bands, indicating no methylation occurred; type II, with bands in lane H and no band in lane M, indicating that the CCGG site is hemimethylated; type III, no band in lane H, and a band in lane M, indicates complete methylation of the CCGG site. Statistical analysis was performed based on the band pattern of H/M lanes shown by the electrophoresis results.
2. Test results
(1) Cotton genome DNA methylation level change caused by drought stress
MSAP analysis was performed using 16 pairs of primers (16 combinations of selective amplification primers) in Table 1, resulting in 324 amplified bands in total, 26.85% methylated bands and 20.06% fully methylated bands in H177 in cotton material that was not treated with drought stress. The number of bands amplified after drought stress treatment is 354, the methylation band ratio is 29.94%, and the total methylation band ratio is 24.57%, which is shown in Table 2. That is, the methylation level of cotton genome is increased after drought stress.
TABLE 2 Effect of drought stress on DNA methylation levels in Cotton leaves
Note: the total number of amplified bands is I + II + III; total number of methylated bands ═ II + III; total number of fully methylated bands III.
(2) Cotton genome DNA methylation pattern changes caused by drought stress
Through MSAP analysis, the methylation mode change condition of a specific CCGG locus after drought stress treatment is known. The results show that MSAP analysis of the test material co-amplified 11 band patterns (fig. 4, 11 band patterns correspond to the patterns in table 3), with 2 polymorphic and monomorphic. Polymorphism means that the control and drought stress treated groups differ in methylation pattern, with 3 states, methylation (type B), demethylation (type C) and indeterminate (type D); the singlet state, i.e., the same band pattern between control and treatment (type A), indicates no change in methylation status after treatment. Type B indicates increased methylation levels after stress, type C indicates decreased methylation levels after stress, and type D indicates that DNA methylation status in control and treatment groups was not determinable. Statistical analysis of the banding patterns shows that the DNA methylation state of cotton is changed greatly after drought stress, and the specific table is shown in Table 3.
TABLE 3 Effect of drought stress on DNA methylation status of Cotton leaves
Note: h is Hpa II/EcoR I enzyme digestion, M is Msp I/EcoR I enzyme digestion; +: with the band, -: no belt;Crepresents methylated cytosine.
Fourth, recovery, sequencing and homologous comparison of MSAP differential fragments
And (3) recovering, amplifying and analyzing the sequence of the MSAP differential fragments of the control group and the experimental group. The method comprises the following specific steps: MSAP differential fragments were cut with a sterile surgical blade and the cut strips were then crushed with a glass slide. The differential band was recovered by Poly-Gel (Omega BioTek, USA), and the recovered product was used as a DNA amplification template, followed by sequencing. According to the sequencing result, homology search is carried out in NCBI database to find out the drought-resistant related gene (Table 4).
TABLE 4 homology analysis of the different sequences
According to the early stage drought resistance dielectrophoresis experiment analysis results (reference documents, such as permission of land, comparative research on leaf proteomics of upland cotton at different drought resistance levels under drought stress, northwest plant science, 2013,33(12):2401-2409), the genes in the table 4 are related to drought resistance.
Fifthly, detecting the expression quantity of homologous genes (qRT-PCR)
And detecting the mRNA expression level of the homologous gene by adopting a fluorescent quantitative PCR (qRT-PCR) method. Total RNA was extracted from the experimental and control samples and reverse transcribed to synthesize the first strand of cDNA, which was stored at-20 ℃ for qRT-PCR analysis. cDNA is taken as a template, Actin (Actin) is taken as an internal reference gene, and homologous gene primers are as follows: m1: 5'-GATCTGCGAGTCCCTACTGC-3', 5'-GGGGCGACCATACTTGTTCA-3', respectively; m2: 5'-AAAACAGCGGTAGCCACAGA-3', 5'-TACCTGCGCCACAGAAGATG-3', respectively; m3: 5'-CAGCTACAGCGCGTTCTTTG-3', 5'-TGCCAGGCCAGACAATTAGG-3', respectively; m4: 5'-GAGGTGATGCAACTGGGACA-3', 5'-GTTGATTCCGATGGCCCTGA-3', respectively; m5: 5'-ACCCAGCAAACCAAGTAGGG-3', 5'-CACTCAGCACGATGGACAGT-3' are provided. The fluorescent quantitative PCR system is 20 mu L, and the amplification program is 94 ℃ for 30 s; collecting CT values after 40-45 cyclic reactions at 94 ℃ for 5s, 55 ℃ for 15s and 72 ℃ for 10s, and adopting 2-ΔΔCTThe relative expression level of the gene is calculated.
The results are shown in fig. 5, and it can be seen that the relative expression amounts of several homologous genes M1-M5 under drought stress are increased to different degrees compared with the control, and the difference is significant.
<110> Cotton research institute of Chinese academy of agricultural sciences
<120> method for screening cotton drought-resistant related gene
<130> GNCLN170535
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<170> PatentIn version 3.5
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Claims (6)
1. A method for screening genes related to cotton drought resistance comprises the following steps:
(1) the following experimental and control groups were set up:
experimental groups: carrying out drought stress treatment on cotton to be detected;
control group: carrying out no drought stress treatment on the cotton to be detected;
(2) detecting and comparing the methylation level and/or pattern of the genome DNA of the cotton to be detected in the experimental group and the control group, and finding out the genes with difference in methylation level and/or pattern in the two groups, wherein the genes are or are candidate genes related to the drought resistance of the cotton;
in the step (2), detecting and comparing the methylation level and/or the methylation mode of the genomic DNA of the cotton to be detected in the experimental group and the control group by adopting an MSAP technology;
detecting and comparing methylation levels and/or patterns of the genomic DNA of the cotton to be detected in the experimental group and the control group by adopting an MSAP technology, wherein the double enzyme digestion combination is EcoR I/Hpa II and EcoR I/Msp I;
when detecting and comparing the methylation level and/or pattern of the genomic DNA of the cotton to be detected in the experimental group and the control group by adopting an MSAP technology, a joint aiming at EcoR I is formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table, and a joint aiming at Hpa II and Msp I is formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in the sequence table;
when the methylation level and/or the methylation mode of the genomic DNA of the cotton to be detected in the experimental group and the control group are detected and compared by adopting an MSAP (multiple-site amplification) technology, a pre-amplification primer aiming at EcoR I is a single-chain DNA molecule shown as a sequence 5 in a sequence table, and pre-amplification primers aiming at Hpa II and Msp I are single-chain DNA molecules shown as a sequence 6 in the sequence table;
and when the methylation level and/or the methylation mode of the genomic DNA of the cotton to be detected in the experimental group and the control group are detected and compared by adopting an MSAP technology, the selective amplification primers aiming at the EcoR I are single-stranded DNA molecules shown as a sequence 7, a sequence 8, a sequence 9 and a sequence 10 in a sequence table, and the selective amplification primers aiming at the Hpa II and the Msp I are single-stranded DNA molecules shown as a sequence 11, a sequence 12, a sequence 13 and a sequence 14 in the sequence table.
2. The method of claim 1, wherein: the cotton is upland cotton.
3. The application of a product containing the following composition in screening genes related to cotton drought resistance is as follows:
(1) restriction enzymes EcoR I, Hpa II and Msp I;
(2) a linker for EcoR I, and linkers for Hpa II and Msp I;
the adaptor aiming at the EcoR I is formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table; the joints aiming at Hpa II and Msp I are formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table;
(3) pre-amplification primers for EcoR I and pre-amplification primers for Hpa II and Msp I;
the pre-amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 5 in a sequence table; the pre-amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 6 in a sequence table;
(4) a selective amplification primer for EcoR I and a selective amplification primer for Hpa II and Msp I;
the selective amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 7, a sequence 8, a sequence 9 and a sequence 10 in a sequence table; the selective amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 11, a sequence 12, a sequence 13 and a sequence 14 in a sequence table.
4. Use according to claim 3, characterized in that: the cotton is upland cotton.
5. A product for screening genes related to cotton drought resistance comprises the following components:
(1) restriction enzymes EcoR I, Hpa II and Msp I;
(2) a linker for EcoR I, and linkers for Hpa II and Msp I;
the adaptor aiming at the EcoR I is formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 1 and a sequence 2 in a sequence table; the joints aiming at Hpa II and Msp I are formed by complementary pairing of two single-stranded DNA molecules shown in a sequence 3 and a sequence 4 in a sequence table;
(3) pre-amplification primers for EcoR I and pre-amplification primers for Hpa II and Msp I;
the pre-amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 5 in a sequence table; the pre-amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 6 in a sequence table;
(4) a selective amplification primer for EcoR I and a selective amplification primer for Hpa II and Msp I;
the selective amplification primer aiming at the EcoR I is a single-stranded DNA molecule shown as a sequence 7, a sequence 8, a sequence 9 and a sequence 10 in a sequence table; the selective amplification primers aiming at Hpa II and Msp I are single-stranded DNA molecules shown as a sequence 11, a sequence 12, a sequence 13 and a sequence 14 in a sequence table.
6. The product of claim 5, wherein: the cotton is upland cotton.
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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 |
| 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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| 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 |
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