CN104293890A - Method and kit for detecting specific DNA methylation modification site in plant flower organ - Google Patents
Method and kit for detecting specific DNA methylation modification site in plant flower organ Download PDFInfo
- Publication number
- CN104293890A CN104293890A CN201310298633.0A CN201310298633A CN104293890A CN 104293890 A CN104293890 A CN 104293890A CN 201310298633 A CN201310298633 A CN 201310298633A CN 104293890 A CN104293890 A CN 104293890A
- Authority
- CN
- China
- Prior art keywords
- seq
- ecori
- hpaii
- mspi
- joint
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6858—Allele-specific amplification
Abstract
The invention discloses a method and a kit for detecting a specific DNA methylation modification site in a plant flower organ. The method includes following steps of: S1) extracting genome DNA of the plant flower organ; S2) subjecting the genome DNA of the plant flower organ to enzyme digestion; S3) respectively connecting restriction enzyme adaptors to the products of the enzyme digestion; S4) subjecting pre-amplification primers which are designed by adopting the adaptors as templates to PCR pre-amplification to obtain PCR pre-amplification products; S5) diluting the PCR pre-amplification products, adding screening primers with selective bases and performing PCR amplification; S6) detecting PCR amplification products by electrophoresis, and counting and analyzing DNA bands; and S7) recovering, cloning DNA methylation differentially expressed bands in the S6) step and performing sequence measurement. By adoption of the adaptors, the pre-amplification primers and the screening primers, the specific DNA methylation modification site can be detected stable and efficiently.
Description
Technical field
The present invention relates to technical field of molecular biology, to methylate the method for decorating site and test kit in particular to a kind of plant flower organ specific DNA that detects.
Background technology
DNA methylation play the part of in plant development process and genome defence environmental factor stresses etc. important effect (
et al.2004; Ruiz-Garcia et al.2005; Teyssier et al.2008; Verhoeven et al.2010).In plant, DNA methylation usually occurs in CpG or CpNpG sequence, and can remain unchanged (Wassenegger2000) in the circulation of DNA reparation.All plant development process are all based upon on the basis of space-time specifically expressing and gene expression regulation accurately.Increasing evidence shows that the Space-time speciality of genetic expression is not only by special DNA sequence dna (cis and transacting element control) regulation and control, also by some apparent modifying factors, wherein topmost is exactly that DNA methylation (Chan et al.2005) regulates and controls.
At present, carrying out the most frequently used technology of research to genomic methylation is methylation sensitive amplified polymorphism technology (Methylation sensitive amplified polymorphism, MSAP), the method utilizes the responsive enzyme (Hpa II and Msp I) of the two kinds of DNA methylations identifying CpG or CpNpG, the DNA methylation decorating site of full-length genome is identified, recycling PCR increases to endonuclease bamhi, modifies differential fragment in conjunction with polyacrylamide gel electrophoresis display DNA methylation.MSAP technology can detect the complete genome DNA decorating site that methylates, and has advantage that is efficient, the genome Given information that saves time, accurately and not relies on.Therefore, the method is used widely and is developed rapidly, is with a wide range of applications at life science.
Because MSAP technology needs to utilize the responsive enzyme of two kinds of DNA methylations to carry out endonuclease reaction, therefore for obtaining in the general reaction system of plant different tissues, need to be optimized for enzyme, DNA profiling, reaction times and temperature.But traditional MSAP method can only obtain DNA methylation difference site, cannot obtain the nucleotide sequence in this site, is unfavorable for carrying out of follow-up study.
Summary of the invention
The present invention aims to provide a kind of plant flower organ specific DNA that detects and to methylate the method for decorating site and test kit, to solve traditional MSAP method effect difference and cannot obtain the technical problem of nucleotide sequence in difference site in plant flower organ.
To achieve these goals, according to an aspect of the present invention, provide a kind of plant flower organ specific DNA that detects to methylate the method for decorating site.The method adopts MSAP technology for detection DNA methylation, comprises the following steps: S1, extracts plant flower organ genomic dna; S2, carries out enzyme with Msp I/EcoRI and Hpa II/EcoRI two groups of enzymes to plant flower organ genomic dna respectively and cuts; S3, connects the joint of upper restriction enzyme respectively to digestion products; S4, is that the pre-amplimer of stencil design carries out PCR and increases in advance with joint, obtains the pre-amplified production of PCR; S5, after pre-for PCR amplified production dilution, adds and is with the screening primer of selective base to carry out pcr amplification; S6, electrophoresis detection pcr amplification product, statistics is analyzing DNA band also, S7, the DNA methylation differential band obtained in recovery, clone S6 also checks order, and wherein, joint comprises EcoRI joint and HpaII/MspI joint, the sequence of EcoRI joint is SEQ ID NO:1 and SEQ ID NO:2, and the sequence of HpaII/MspI joint is SEQ ID NO:3 and SEQ ID NO:4; Pre-amplimer comprises the pre-amplimer of EcoRI and the pre-amplimer of HpaII/MspI, and the sequence of the pre-amplimer of EcoRI is the sequence of the pre-amplimer of SEQ ID NO:5, HpaII/MspI is SEQ ID NO:6; Screening primer comprises EcoRI and screens primer and HpaII/MspI screening primer, and it is SEQ ID NO:7 ~ 16 that EcoRI screens primer sequence, and it is SEQ ID NO:17 ~ 27 that HpaII/MspI screens primer sequence.
Further, the enzyme system of cutting in S2 is that 20 μ l enzymes cut system, comprises 10 × amplification buffer 2.0 μ l, DNA4.0 μ l, EcoRI/HpaII3U/3.2U or EcoRI/MspI3U/4U, EcoRI joint 0.5 μ l, HpaII/MspI joint 0.5 μ l, T4 ligase enzyme 0.5 μ l, ddH
2o11.85 μ l.
Further, the pre-amplification system in S4 is 25 μ l systems, comprises the digestion products 2.5 μ l of dilution 100 times, and concentration is the pre-amplimer 19.5 μ l of 10 μm of ol/L, 10 × amplification buffer 2.5 μ l.
Further, the pre-amplification program in S4 is 94 DEG C of sex change 2min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, and 72 DEG C extend 80s, 30 circulations, and last 72 DEG C extend 5min.
Further, the selection amplification system in S5 is 20 μ l amplification systems, comprises pcr amplification mixed solution 10 μ l, dilutes the PCR pre-amplified production 3 μ l of 100 times, HpaII/MspI primer 1 μ l, EcoRI primer 1 μ l, ddH
2o5 μ l, wherein, pcr amplification mixed solution comprises ddH
2o155.0 μ l, 10 × amplification buffer 40.0 μ l, 5U Taq archaeal dna polymerase 5 μ l.
Further, the selection amplification program in S5 is 94 DEG C of sex change 5min, 94 DEG C of sex change 30s, and 0.7 DEG C of annealing 30s falls in 65 DEG C and each cycle annealing temperature, and 72 DEG C extend 1min, after 13 circulations; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 80s, 23 circulations, and 72 DEG C extend 5min.
Further, the combination of screening primer is respectively: SEQ ID NO:9+SEQ ID NO:21, SEQ ID NO:9+SEQ ID NO:22, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:12+SEQ ID NO:17, SEQ ID NO:12+SEQ ID NO:18, SEQ ID NO:12+SEQ ID NO:23, SEQ ID NO:12+SEQ ID NO:26, SEQ ID NO:14+SEQ ID NO:21, SEQ ID NO:14+SEQ ID NO:23, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:14+SEQ ID NO:26, SEQ ID NO:15+SEQ ID NO:17, SEQ ID NO:15+SEQ ID NO:19, SEQ ID NO:15+SEQ ID NO:21, SEQ ID NO:15+SEQ ID NO:23, SEQ ID NO:15+SEQ ID NO:26, SEQ ID NO:16+SEQ ID NO:17, SEQ ID NO:16+SEQ ID NO:19, SEQ ID NO:16+SEQ ID NO:21, SEQ ID NO:16+SEQ ID NO:23, SEQ ID NO:16+SEQ ID NO:24, SEQ ID NO:16+SEQ ID NO:26.
Further, S6 comprises: utilize polyacrylamide gel electrophoresis to detect pcr amplification product.
According to another aspect of the present invention, a kind of plant flower organ specific DNA that detects is provided to methylate the test kit of decorating site.This test kit comprises MSAP technology joint used, pre-amplimer and screening primer sequence, wherein, joint comprises EcoRI joint and HpaII/MspI joint, the sequence of EcoRI joint is SEQ ID NO:1 and SEQ ID NO:2, and the sequence of HpaII/MspI joint is SEQ ID NO:3 and SEQ ID NO:4; Pre-amplimer comprises the pre-amplimer of EcoRI and the pre-amplimer of HpaII/MspI, and the sequence of the pre-amplimer of EcoRI is the sequence of the pre-amplimer of SEQ ID NO:5, HpaII/MspI is SEQ ID NO:6; Screening primer comprises EcoRI and screens primer and HpaII/MspI screening primer, and it is SEQ ID NO:7 ~ 16 that EcoRI screens primer sequence, and it is SEQ ID NO:17 ~ 27 that HpaII/MspI screens primer sequence.
Further, the combination of screening primer is respectively: SEQ ID NO:9+SEQ ID NO:21, SEQ ID NO:9+SEQ ID NO:22, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:12+SEQ ID NO:17, SEQ ID NO:12+SEQ ID NO:18, SEQ ID NO:12+SEQ ID NO:23, SEQ ID NO:12+SEQ ID NO:26, SEQ ID NO:14+SEQ ID NO:21, SEQ ID NO:14+SEQ ID NO:23, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:14+SEQ ID NO:26, SEQ ID NO:15+SEQ ID NO:17, SEQ ID NO:15+SEQ ID NO:19, SEQ ID NO:15+SEQ ID NO:21, SEQ ID NO:15+SEQ ID NO:23, SEQ ID NO:15+SEQ ID NO:26, SEQ ID NO:16+SEQ ID NO:17, SEQ ID NO:16+SEQ ID NO:19, SEQ ID NO:16+SEQ ID NO:21, SEQ ID NO:16+SEQ ID NO:23, SEQ ID NO:16+SEQ ID NO:24, SEQ ID NO:16+SEQ ID NO:26.
Adopt joint provided by the invention, pre-amplimer and screening primer, can stablize, detect plant flower organ specific DNA efficiently and to methylate decorating site; In addition, adopt technical scheme of the present invention, also achieve plant flower organ specific DNA methylation site screening and candidate gene acquisition, for follow-up gene expression regulation analysis and epigenetic marker development provide technical support.
Accompanying drawing explanation
The Figure of description forming a application's part is used to provide a further understanding of the present invention, and schematic description and description of the present invention, for explaining the present invention, does not form inappropriate limitation of the present invention.In the accompanying drawings:
Fig. 1 shows in the embodiment of the present invention 1 schematic diagram utilizing polyacrylamide gel electrophoresis to show DNA difference section decorating site, wherein, H, M represent the primer pair of H/E and M/E combination of primers respectively, arrow instruction DNA methylation difference site, A-G represents that different primers combination is followed successively by (A:H/M-3+EcoR I-5, B:H/M-3+Eco R I-6, C:H/M-3+EcoR I-8, D:H/M-6+EcoR I-1, E:H/M-6+EcoR I-2, F:H/M-6+EcoR I-7, G:H/M-6+EcoR I-10)
represent instaminate flower organ,
represent male floral organs; And show this site female flower in the black box of accompanying drawing E combination of primers and have permethylated modification relative to male flower, and the data in the black box of G combination of primers represent male flower has permethylated modification relative to female flower.
Fig. 2 shows the differential DNA methylation sites filtered out by H/M-6+EcoR I-2 combination of primers, through reclaiming the DNA difference site nucleotide sequence of cloning and obtaining, its position in gene is demonstrated after the comparison of willow JGI database, the differential band that red representative is obtained by double digestion, the yellow CCGG site representing the modification that methylates, green represents 5 ' UTR, and pink colour represents 3 ' UTR, blueness represents the exon of gene, and colourless part represents intron.The methylating modification that this figure shows on a CCGG site is positioned on the First Exon of this gene.
Embodiment
It should be noted that, when not conflicting, the embodiment in the application and the feature in embodiment can combine mutually.Below with reference to the accompanying drawings and describe the present invention in detail in conjunction with the embodiments.
According to a kind of typical embodiment of the present invention, a kind of plant flower organ specific DNA that detects is provided to methylate the method for decorating site.The method adopts MSAP technology for detection DNA methylation, comprises the following steps: S1, extract plant flower organ genomic dna, S2, carries out enzyme with Msp I/EcoRI and Hpa II/EcoRI two groups of enzymes to plant flower organ genomic dna respectively and cuts, S3, connects the joint of upper restriction enzyme respectively to digestion products, S4, is that the pre-amplimer of stencil design carries out PCR and increases in advance with joint, obtains the pre-amplified production of PCR, S5, after pre-for PCR amplified production dilution, adds and is with the screening primer of selective base to carry out pcr amplification, S6, electrophoresis detection pcr amplification product, statistics is analyzing DNA band also, S7, reclaim, the DNA methylation differential band obtained in clone S6 also checks order, wherein, wherein, the sequence of EcoRI joint is SEQ ID NO:1 and SEQ ID NO:2, the sequence of HpaII/MspI joint is SEQ ID NO:3 and SEQ ID NO:4, the sequence of the pre-amplimer of EcoRI is SEQ ID NO:5, the sequence of the pre-amplimer of HpaII/MspI is SEQ ID NO:6, it is SEQ ID NO:7 ~ 16 that HpaII/MspI screens primer sequence, it is SEQ ID NO:17 ~ 27 that EcoRI screens primer sequence, as shown in table 1.
Table 1
Adopt joint provided by the invention, pre-amplimer and screening primer, can stablize, detect plant flower organ specific DNA efficiently and to methylate decorating site; In addition, adopt technical scheme of the present invention, also achieve plant flower organ specific DNA methylation site screening and candidate gene acquisition, for follow-up gene expression regulation analysis and epigenetic marker development provide technical support.
For determining the nucleotide sequence of DNA methylation decorating site, present method incorporates recovery clone technology on the basis of MSAP.In order to obtain better effect, needing that system, amplification system etc. are cut to enzyme and carrying out further integrated optimization.
Preferably, the enzyme system of cutting in S2 is that 20 μ l enzymes cut system, comprise 10 × amplification buffer 2.0 μ l, DNA4.0 μ l, EcoRI/HpaII3U/3.2U(is EcoRI (12U/ μ l)/HpaII(8U/ μ l such as) 0.25/0.4 μ l) or EcoRI/MspI3U/4U(EcoRI (12U/ μ l)/MspI(10U/ μ l) 0.25/0.4 μ l), EcoRI joint 0.5 μ l, HpaII/MspI joint 0.5 μ l, T4 ligase enzyme 0.5 μ l, ddH2O11.85 μ l.What this system adopted is that single stage method enzyme cuts linked system, enzyme can be cut connection one step and complete, time saving and energy saving.
Preferably, the pre-amplification system in S4 is 25 μ l systems, comprises 100 times of digestion products 2.5 μ L of dilution, pre-amplimer 10 μm of ol/L19.5 μ L, 10 × amplification buffer 2.5 μ L.This system can obtain pre-amplified production to greatest extent on the basis utilizing minimum digestion products.
Preferably, the pre-amplification program in S4 is 94 DEG C of sex change 2min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, and 72 DEG C extend 80s, 30 circulations, and last 72 DEG C extend 5min.This program, according to pre-amplified production clip size setting sex change and annealing time, comparatively time savingly can complete pcr amplification.
Preferably, the selection amplification system in S5 is 20 μ l amplification systems, comprises amplification work mixed solution 10 μ L, dilutes the PCR pre-amplified production 3 μ l of 100 times, Hpa II/Msp I primer 1 μ l, EcoRI primer 1 μ l, ddH
2o5 μ l, wherein, amplification work mixed solution comprises ddH
2o155.0 μ l, 10 × amplification buffer 40.0 μ l, 5U Taq archaeal dna polymerase 5 μ l.This system has used pcr amplification damping fluid, can ensure when pcr amplification in enormous quantities, the stability of PCR system and consistence, is conducive to the consistence ensureing population sample amplification.
Preferably, the selection amplification program in S5 is 94 DEG C of sex change 5min, 94 DEG C of sex change 30s, and 0.7 DEG C of annealing 30s falls in 65 DEG C and each cycle annealing temperature, and 72 DEG C extend 1min, after 13 circulations; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 80s, 23 circulations, and 72 DEG C extend 5min.Equally, this program sets sex change and annealing time according to pre-expansion product clip size, comparatively time savingly can complete pcr amplification.
Preferably, S6 comprises: utilize polyacrylamide gel electrophoresis to detect pcr amplification product, polyacrylamide gel gel electrophoresis has higher resolving power, can reach about 1bp, therefore have employed this technology as electrophoresis detection technology.
Preferably, the combination of described screening primer is respectively: SEQ ID NO:9+SEQ ID NO:21, SEQ ID NO:9+SEQ ID NO:22, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:12+SEQ ID NO:17, SEQ ID NO:12+SEQ ID NO:18, SEQ ID NO:12+SEQ ID NO:23, SEQ ID NO:12+SEQ ID NO:26, SEQ ID NO:14+SEQ ID NO:21, SEQ ID NO:14+SEQ ID NO:23, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:14+SEQ ID NO:26, SEQ ID NO:15+SEQ ID NO:17, SEQ ID NO:15+SEQ ID NO:19, SEQ ID NO:15+SEQ ID NO:21, SEQ ID NO:15+SEQ ID NO:23, SEQ ID NO:15+SEQ ID NO:26, SEQ ID NO:16+SEQ ID NO:17, SEQ ID NO:16+SEQ ID NO:19, SEQ ID NO:16+SEQ ID NO:21, SEQ ID NO:16+SEQ ID NO:23, SEQ ID NO:16+SEQ ID NO:24, SEQ ID NO:16+SEQ ID NO:26, i.e. H/M-3+EcoR I-5, H/M-3+Eco R I-6, H/M-3+EcoR I-8, H/M-6+EcoR I-1, H/M-6+EcoR I-2, H/M-6+EcoR I-7, H/M-6+EcoR I-10, H/M-8+EcoR I-5, H/M-8+EcoR I-7, H/M-3+EcoR I-8, H/M-8+EcoR I-10, H/M-9+EcoR I-1, H/M-9+EcoR I-3, H/M-9+EcoR I-5, H/M-9+EcoR I-7, H/M-9+EcoR I-10, H/M-10+EcoR I-1, H/M-10+EcoR I-3, H/M-10+EcoR I-5, H/M-10+EcoR I-7, H/M-10+EcoR I-8, H/M-10+EcoR I-10.The screening primer amplification of aforesaid combination goes out that the amplification of effective band is stable, specific fragment is more, amplification efficiency is high.
According to a kind of typical embodiment of the present invention, a kind of plant flower organ specific DNA that detects is provided to methylate the test kit of decorating site.It is as shown in table 1 that this test kit comprises MSAP technology joint used, pre-amplimer and screening primer sequence.
The present invention to reaction times of MSAP double digestion system, temperature of reaction, damping fluid selects and DNA profiling usage quantity is optimized, and thereby is achieved the MSAP double digestion reaction system that poplar flower organ is suitable for.The present invention also gropes the optimum condition of PCR, especially to annealing temperature, cycle number and the optimization of extension time.The present invention is optimized the condition such as reaction times, transformation efficiency, carrier selection reclaiming clone's system.In a kind of typical embodiment of the present invention, the methylate method of decorating site of rapid detection plant flower organ specific DNA comprises the steps:
(1) CTAB method or utilize DNA extraction kit to extract plant flower organ genomic dna, and diluted for use;
(2) utilize Msp I and Hpa II to combine respectively at EcoR I, build double digestion reaction system;
(3) round pcr and Adapter a, Adapter b is utilized; H/M NO:1-10 and E NO:1-11 primer sets carry out pre-amplified reaction and selective amplification successively;
(4) polyacrylamide gel electrophoresis and silver dye;
(5) utilize small-molecular-weight DNA fragmentation to reclaim test kit, differential DNA band is reclaimed;
(6) to object fragment Cloning Transformation.
Wherein, what extract in step (1) is willow genomic dna, is diluted to 200ng/ μ l, and stand-by-20 DEG C of preservations.
Wherein, in step (2), the cumulative volume of double digestion ligation system is 20 μ l, specific as follows:
Wherein, in described step (3), pre-expansion reaction system is 20 μ l, specific as follows:
Mix gently, centrifugal.
Amplification program: 94 DEG C of sex change 2min, 30 circulations (94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 80s), last 72 DEG C extend 5min, 4 DEG C of preservations.Agarose gel electrophoresis detects expanding effect, and get appropriate pre-expansion product 100 × dilution, be used as choosing and expand template, resultant product saves backup with-20 DEG C.
Wherein, in described step (4), it is 20 μ l that reaction system is expanded in choosing, specific as follows:
Selective amplification Working mix prepares
Selective amplification:
Mix gently, centrifugal at the bottom of pipe.
Amplification program: 94 DEG C, 5min; 13 circulations (94 DEG C of 30s, 65 DEG C of 30s(-0.7 DEG C/Cycle), 30s; 72 DEG C, 1min); 23 circulations (94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 80s); 72 DEG C of 5min.4 DEG C of preservations.Before electrophoresis, add 5ul sample-loading buffer in 10ul amplified production, 94 DEG C of 3min to make after denaturing samples loading immediately.
Wherein, in described step (5), select amplified production, 94 DEG C of 5min, adopt 1900V constant voltage to carry out 7% polyacrylamide gel electrophoresis 1.5h, silver dye.The formula of formamide denaturation liquid has its detailed configuration method in " molecular cloning texts guide ".
Wherein, in step (6), glue is cut to polymorphic DNA fragment on polyacrylamide gel and reclaims, carry out selective PCR amplification as template, then obtain object band by agarose gel electrophoresis.Use Beijing vast Tai Heng biotechnology limited liability company small-molecular-weight DNA fragmentation high-performance flash purification to reclaim test kit (High Yield Nucleic Acid Purification Kit For Small DNA Fragments) and carry out the recovery of DNA purifying.
Wherein, in step (7), concrete reaction system is as follows:
(1) connection of object fragment and carrier
Use the PMD-18T carrier of Takara company, method reference Manual also slightly changes (it, between 30s-60s, is improve transformation efficiency that its specification sheets is recommended in heat shock in recombinant conversion process, and it is 90s that present method is changed)..
1, melt carrier on ice and be connected damping fluid, gentle centrifugation.
2, in 0.5ml centrifuge tube, following solution is added:
3, mix, gentle centrifugation.16 DEG C of connections are spent the night.
(2) conversion of recombinant plasmid
1, getting the above-mentioned connection product of 5ul joins in 50ul intestinal bacteria TOP10 recipient cell, mixing, ice bath 30min.
2,42 DEG C of water-bath heat shock 90s, ice bath 3min.
3, LB liquid nutrient medium 200ul is added, mixing.In 37 DEG C of incubators, 150rpm cultivates 30 ~ 60min.
4, get 100ul nutrient solution, coat on the LB solid medium containing Amp50mg/L, be inverted overnight incubation for 37 DEG C.Get single bacterium colony and carry out PCR qualification.
(3) the pcr amplification qualification of recombinant clone
1, picking 10 single bacterium colonies are distinguished in 20ul LB liquid medium from flat board.
2, PCR amplification system is as follows:
Amplification program: 94 DEG C of 30s, 94 DEG C of 30s, 65 DEG C of (often circulation reduction by 0.7 DEG C) 30s, 72 DEG C of 1min, 13 circulations, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 1min, 30 circulations, 72 DEG C of 5min.
The present invention is further illustrated below in conjunction with embodiment.
Embodiment 1
The following experiment parameter do not mentioned, specifically operates according to above-mentioned test parameter.
1) select the ripe female plant of Cortex Populi Tomentosae and staminiferous plant (taking from national Cortex Populi Tomentosae germplasm resource bank), take instaminate flower organ and male floral organs respectively.
2) DNA extraction kit is utilized to extract the DNA of floral organ, as MSAP template.
3) utilize the DNA sample (200ng/ μ l) of previous reaction system to dilution to carry out enzyme to cut.
4) utilize polyacrylamide gel electrophoresis to show DNA difference and modify band.
Fig. 1 shows in the present embodiment the schematic diagram utilizing polyacrylamide gel electrophoresis to show DNA difference section decorating site, wherein, H, M represent the primer pair of H/E and M/E combination of primers respectively, arrow instruction DNA methylation difference site, A-G represents that different primers combination is followed successively by (A:H/M-3+EcoR I-5, B:H/M-3+Eco R I-6, C:H/M-3+EcoR I-8, D:H/M-6+EcoR I-1, E:H/M-6+EcoR I-2, F:H/M-6+EcoR I-7, G:H/M-6+EcoR I-10)
represent instaminate flower organ,
represent male floral organs; And show this site female flower in the black box of accompanying drawing E combination of primers and have permethylated modification relative to male flower, and the data in the black box of G combination of primers represent male flower has permethylated modification relative to female flower.
Fig. 2 shows the differential DNA methylation sites filtered out by H/M-6+EcoR I-2 combination of primers, through reclaiming the DNA difference site nucleotide sequence of cloning and obtaining, its position in gene is demonstrated after the comparison of willow JGI database, the differential band that red representative is obtained by double digestion, the yellow CCGG site representing the modification that methylates, green represents 5 ' UTR, and pink colour represents 3 ' UTR, blueness represents the exon of gene, and colourless part represents intron.The methylating modification that this figure shows on a CCGG site is positioned on the First Exon of this gene.
Clear readable DNA differential band is reclaimed, after Cloning Transformation, measures the nucleotide sequence in this site.Concrete outcome is as table 2:
Table 2
For Cortex Populi Tomentosae male and female flowering organ, 110 pairs of primer screenings are utilized to obtain the DNA modification site of 212 differences, have selected 37 sites to reclaim, obtain 28 sequences altogether, warp and the comparison of willow genome database obtain 24 genes (as table 2) relevant with flower development, confirm reliability and the high efficiency of the method.
Embodiments of the invention obtain following beneficial effect:
1) optimize MSAP reaction system, this system is applicable to the screening of poplar flower organ DNA methylation site, expands the use range of this technology.
2) acquisition that the nucleotides sequence that present method can obtain DNA methylation difference site is classified as candidate gene provides the foundation, and is conducive to carrying out of follow-up study.
3) present method is simply effective, is conducive to the exploitation of epigenetic mark.
To sum up, the invention discloses a kind of rapid detection plant flower organ specific DNA to methylate the method for decorating site.The present invention utilizes Msp I and the responsive enzyme of Hpa II two kinds of DNA methylations and EcoR I to carry out enzyme to genomic dna and cuts, utilize polyacrylamide gel electrophoresis to show difference decorating site to after endonuclease bamhi amplification, utilize cloning and sequencing technology to reclaim subsequently and obtain DNA methylation difference modification fragment sequence.The present invention relates to by sequence is Adapter a, Adapter b, and the primer sets of the primer composition of H/M NO:1-10 and E NO:1-11, comprises the test kit of described primer sets and the purposes of described primer sets.The present invention has screened the reaction system of Msp I and Hpa II double digestion, and by reclaiming the nucleotide sequence that can obtain DNA methylation decorating site to the clone of differential fragment.Method of the present invention is fast and reliable, does not rely on genomic known array, easy handling.At the specific DNA methylation decorating site of plant flower organ, the aspects such as epigenetic marker development and important candidate gene screening are with a wide range of applications.
The foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, for a person skilled in the art, the present invention can have various modifications and variations.Within the spirit and principles in the present invention all, any amendment done, equivalent replacement, improvement etc., all should be included within protection scope of the present invention.
Sequence table
<110> Beijing Forestry University
<120> detects plant flower organ specific DNA and to methylate the method for decorating site and test kit
<130> P73604
<160> 27
<170> PatentIn version 3.2
<210> 1
<211> 17
<212> DNA
<213> artificial
<220>
<223> EcoRI joint
<400> 1
ctcgtagact gcgtacc 17
<210> 2
<211> 15
<212> DNA
<213> artificial
<220>
<223> EcoRI joint
<400> 2
aattggtacg cagtc 15
<210> 3
<211> 16
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI joint
<400> 3
gatcatgagt cctgct 16
<210> 4
<211> 16
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI joint
<400> 4
cgagcaggac tcatga 16
<210> 5
<211> 17
<212> DNA
<213> artificial
<220>
The pre-amplimer of <223> EcoRI
<400> 5
gactgcgtac caattca 17
<210> 6
<211> 18
<212> DNA
<213> artificial
<220>
The pre-amplimer of <223> HpaII/MspI
<400> 6
atcatgagtc ctgctcgg 18
<210> 7
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 7
gactgcgtac caattcaac 19
<210> 8
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 8
gactgcgtac caattcaag 19
<210> 9
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 9
gactgcgtac caattcaca 19
<210> 10
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 10
gactgcgtac caattcact 19
<210> 11
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 11
gactgcgtac caattcacc 19
<210> 12
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 12
gactgcgtac caattcacg 19
<210> 13
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 13
gactgcgtac caattcagc 19
<210> 14
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 14
gactgcgtac caattcagg 19
<210> 15
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 15
gactgcgtac caattcaga 19
<210> 16
<211> 19
<212> DNA
<213> artificial
<220>
<223> HpaII/MspI screens primer
<400> 16
gactgcgtac caattcatc 19
<210> 17
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 17
atcatgagtc ctgctcggtc t 21
<210> 18
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 18
atcatgagtc ctgctcggtc g 21
<210> 19
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 19
atcatgagtc ctgctcggtc c 21
<210> 20
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 20
atcatgagtc ctgctcggtt c 21
<210> 21
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 21
atcatgagtc ctgctcggtt g 21
<210> 22
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 22
atcatgagtc ctgctcggtt a 21
<210> 23
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 23
atcatgagtc ctgctcggtg a 21
<210> 24
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 24
atcatgagtc ctgctcggtg t 21
<210> 25
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 25
atcatgagtc ctgctcggtg c 21
<210> 26
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 26
atcatgagtc ctgctcggta t 21
<210> 27
<211> 21
<212> DNA
<213> artificial
<220>
<223> EcoRI screens primer
<400> 27
atcatgagtc ctgctcggta c 21。
Claims (10)
1. detect plant flower organ specific DNA to methylate the method for decorating site, it is characterized in that, adopt MSAP technology for detection DNA methylation, comprise the following steps:
S1, extracts plant flower organ genomic dna;
S2, carries out enzyme with Msp I/EcoRI and Hpa II/EcoRI two groups of enzymes to described plant flower organ genomic dna respectively and cuts;
S3, connects the joint of upper restriction enzyme respectively to digestion products;
S4, the pre-amplimer being stencil design with described joint carries out PCR and increases in advance, obtains the pre-amplified production of PCR;
S5, after pre-for described PCR amplified production dilution, adds and is with the screening primer of selective base to carry out pcr amplification;
S6, electrophoresis detection pcr amplification product, statistics is analyzing DNA band also,
S7, reclaims, clones the DNA methylation differential band that obtains in described S6 and check order,
Described joint comprises EcoRI joint and HpaII/MspI joint, and the sequence of described EcoRI joint is SEQ ID NO:1 and SEQ ID NO:2, and the sequence of described HpaII/MspI joint is SEQ ID NO:3 and SEQ ID NO:4; Described pre-amplimer comprises the pre-amplimer of EcoRI and the pre-amplimer of HpaII/MspI, and the sequence of the pre-amplimer of described EcoRI is the sequence of the pre-amplimer of SEQ ID NO:5, described HpaII/MspI is SEQ ID NO:6; Described screening primer comprises EcoRI and screens primer and HpaII/MspI screening primer, and it is SEQ IDNO:7 ~ 16 that described EcoRI screens primer sequence, and it is SEQ ID NO:17 ~ 27 that described HpaII/MspI screens primer sequence.
2. method according to claim 1, it is characterized in that, the enzyme system of cutting adopted in described S2 is that 20 μ l enzymes cut system, comprise 10 × amplification buffer 2.0 μ l, DNA4.0 μ l, EcoRI/HpaII3U/3.2U or EcoRI/MspI3U/4U, EcoRI joint 0.5 μ l, HpaII/MspI joint 0.5 μ l, T4 ligase enzyme 0.5 μ l, ddH
2o11.85 μ l.
3. method according to claim 1, it is characterized in that, the pre-amplification system in described S4 is 25 μ l systems, comprises the described digestion products 2.5 μ l of dilution 100 times, concentration is described pre-amplimer 19.5 μ l, the 10 × amplification buffer 2.5 μ l of 10 μm of ol/L.
4. method according to claim 1, is characterized in that, the pre-amplification program in described S4 is 94 DEG C of sex change 2min, 94 DEG C of sex change 30s, 56 DEG C of annealing 30s, and 72 DEG C extend 80s, 30 circulations, and last 72 DEG C extend 5min.
5. method according to claim 1, is characterized in that, the selection amplification system in described S5 is 20 μ l amplification systems, comprise pcr amplification mixed solution 10 μ l, dilute the described PCR pre-amplified production 3 μ l of 100 times, HpaII/MspI primer 1 μ l, EcoRI primer 1 μ l, ddH
2o5 μ l, wherein, described pcr amplification mixed solution comprises ddH
2o155.0 μ l, 10 × amplification buffer 40.0 μ l, 5U Taq archaeal dna polymerase 5 μ l.
6. method according to claim 1, is characterized in that, the selection amplification program in described S5 is 94 DEG C of sex change 5min, 94 DEG C of sex change 30s, and 0.7 DEG C of annealing 30s falls in 65 DEG C and each cycle annealing temperature, and 72 DEG C extend 1min, after 13 circulations; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C extend 80s, 23 circulations, and 72 DEG C extend 5min.
7. method according to claim 1, is characterized in that, the combination of described screening primer is respectively: SEQ ID NO:9+SEQ ID NO:21, SEQ ID NO:9+SEQ ID NO:22, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:12+SEQ ID NO:17, SEQ ID NO:12+SEQ ID NO:18, SEQ ID NO:12+SEQ ID NO:23, SEQ ID NO:12+SEQ ID NO:26, SEQ ID NO:14+SEQ ID NO:21, SEQ ID NO:14+SEQ ID NO:23, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:14+SEQ ID NO:26, SEQ ID NO:15+SEQ ID NO:17, SEQ ID NO:15+SEQ ID NO:19, SEQ ID NO:15+SEQ ID NO:21, SEQ ID NO:15+SEQ ID NO:23, SEQ ID NO:15+SEQ ID NO:26, SEQ ID NO:16+SEQ ID NO:17, SEQ ID NO:16+SEQ ID NO:19, SEQ ID NO:16+SEQ ID NO:21, SEQ ID NO:16+SEQ ID NO:23, SEQ ID NO:16+SEQ ID NO:24, SEQ ID NO:16+SEQ ID NO:26.
8. method according to claim 1, is characterized in that, described S6 comprises: utilize polyacrylamide gel electrophoresis to detect described pcr amplification product.
9. one kind is detected plant flower organ specific DNA and to methylate the test kit of decorating site, it is characterized in that, comprise MSAP technology joint used, pre-amplimer and screening primer sequence, wherein, described joint comprises EcoRI joint and HpaII/MspI joint, the sequence of described EcoRI joint is SEQ ID NO:1 and SEQ ID NO:2, and the sequence of described HpaII/MspI joint is SEQ ID NO:3 and SEQ ID NO:4; Described pre-amplimer comprises the pre-amplimer of EcoRI and the pre-amplimer of HpaII/MspI, and the sequence of the pre-amplimer of described EcoRI is the sequence of the pre-amplimer of SEQ ID NO:5, described HpaII/MspI is SEQ ID NO:6; Described screening primer comprises EcoRI and screens primer and HpaII/MspI screening primer, and it is SEQ ID NO:7 ~ 16 that described EcoRI screens primer sequence, and it is SEQ ID NO:17 ~ 27 that described HpaII/MspI screens primer sequence.
10. test kit according to claim 9, is characterized in that, the combination of described screening primer is respectively: SEQ ID NO:9+SEQ ID NO:21, SEQ ID NO:9+SEQ ID NO:22, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:12+SEQ ID NO:17, SEQ ID NO:12+SEQ ID NO:18, SEQ ID NO:12+SEQ ID NO:23, SEQ ID NO:12+SEQ ID NO:26, SEQ ID NO:14+SEQ ID NO:21, SEQ ID NO:14+SEQ ID NO:23, SEQ ID NO:9+SEQ ID NO:24, SEQ ID NO:14+SEQ ID NO:26, SEQ ID NO:15+SEQ ID NO:17, SEQ ID NO:15+SEQ ID NO:19, SEQ ID NO:15+SEQ ID NO:21, SEQ ID NO:15+SEQ ID NO:23, SEQ ID NO:15+SEQ ID NO:26, SEQ ID NO:16+SEQ ID NO:17, SEQ ID NO:16+SEQ ID NO:19, SEQ ID NO:16+SEQ ID NO:21, SEQ ID NO:16+SEQ ID NO:23, SEQ ID NO:16+SEQ ID NO:24, SEQ ID NO:16+SEQ ID NO:26.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310298633.0A CN104293890A (en) | 2013-07-16 | 2013-07-16 | Method and kit for detecting specific DNA methylation modification site in plant flower organ |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310298633.0A CN104293890A (en) | 2013-07-16 | 2013-07-16 | Method and kit for detecting specific DNA methylation modification site in plant flower organ |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104293890A true CN104293890A (en) | 2015-01-21 |
Family
ID=52313838
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310298633.0A Pending CN104293890A (en) | 2013-07-16 | 2013-07-16 | Method and kit for detecting specific DNA methylation modification site in plant flower organ |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104293890A (en) |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105256379A (en) * | 2015-11-23 | 2016-01-20 | 武汉大学 | Method for preparing novel genome simplified methylation sequencing library |
| CN106701939A (en) * | 2016-12-22 | 2017-05-24 | 中国热带农业科学院热带生物技术研究所 | Cytosine methylation excavation method |
| CN106755534A (en) * | 2017-03-01 | 2017-05-31 | 北京林业大学 | A kind of DNA Methylation in Plants tissue specificity acquisition methods |
| CN106967806A (en) * | 2017-04-06 | 2017-07-21 | 西北农林科技大学 | A kind of method that utilization MSAP methods differentiate cherry dwarfing rootstock |
| CN109593839A (en) * | 2017-09-29 | 2019-04-09 | 上海交通大学 | A kind of DNA mutation and methylation status detection method |
Citations (1)
| 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 |
-
2013
- 2013-07-16 CN CN201310298633.0A patent/CN104293890A/en active Pending
Patent Citations (1)
| 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 |
Non-Patent Citations (3)
| Title |
|---|
| XIANG GAO等: "In Vitro Micropropagation of Freesia hybrida and the Assessment of Genetic and Epigenetic Stability in Regenerated Plantlets", 《J PLANT GROWTH REGUL》, vol. 29, 30 December 2009 (2009-12-30), pages 257 - 267, XP019845156 * |
| YUEPENG SONG等: "Sex-specific DNA methylation and gene expression in andromonoecious poplar", 《PLANT CELL REP》, vol. 31, 4 April 2012 (2012-04-04), pages 1393 - 1405, XP035085290, DOI: doi:10.1007/s00299-012-1255-7 * |
| 高寰等: "三叶木通MSAP 反应体系的优化及引物筛选", 《中草药》, vol. 43, no. 3, 31 December 2012 (2012-12-31), pages 572 - 576 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105256379A (en) * | 2015-11-23 | 2016-01-20 | 武汉大学 | Method for preparing novel genome simplified methylation sequencing library |
| CN106701939A (en) * | 2016-12-22 | 2017-05-24 | 中国热带农业科学院热带生物技术研究所 | Cytosine methylation excavation method |
| CN106755534A (en) * | 2017-03-01 | 2017-05-31 | 北京林业大学 | A kind of DNA Methylation in Plants tissue specificity acquisition methods |
| CN106755534B (en) * | 2017-03-01 | 2020-06-16 | 北京林业大学 | Method for acquiring specificity of plant DNA methylated tissue |
| CN106967806A (en) * | 2017-04-06 | 2017-07-21 | 西北农林科技大学 | A kind of method that utilization MSAP methods differentiate cherry dwarfing rootstock |
| CN109593839A (en) * | 2017-09-29 | 2019-04-09 | 上海交通大学 | A kind of DNA mutation and methylation status detection method |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018024082A1 (en) | Method for constructing serially-connected rad tag sequencing libraries | |
| CN104293890A (en) | Method and kit for detecting specific DNA methylation modification site in plant flower organ | |
| CN107201408B (en) | Method for developing sisal hemp SSR primer based on transcriptome sequencing | |
| CN104032007A (en) | Method for detecting exogenous gene copy number in transgenic tobacco | |
| CN104480202B (en) | A kind of Fructus Luffae reference gene and application thereof | |
| KR102121570B1 (en) | KASP primer set based on SNP for discriminating or classifying Panax ginseng cultivar or resource and uses thereof | |
| CN102433379B (en) | Method for rapidly detecting expression of ANS (Anthocyanidin Synthetase) genes from different sources in rape seed capsule and application thereof | |
| CN103468672A (en) | Method for rapidly discriminating species of cotton | |
| CN105463093A (en) | Primer, kit and amplification method for fish mitochondria whole genome amplification | |
| CN107988334B (en) | Method for SNP typing by direct PCR of oral swab | |
| CN112442547A (en) | Development and application of SNP molecular marker of rice blast resistance gene Pita | |
| CN108642209B (en) | Wheat plant thousand grain weight judgment marker and application thereof | |
| CN101550449B (en) | Method for analyzing diversity of biological enzyme genes in compost | |
| CN112592995B (en) | Universal primer for detecting target gene expression in transgenic plant and detection method | |
| CN104099423B (en) | For the molecular labeling of cutter long-tailed anchovy different ecological type population identification | |
| CN113512608B (en) | LAMP (Loop-mediated isothermal amplification) detection primer system, kit and method for transgenic tobacco | |
| CN106636065B (en) | Whole-genome efficient gene region enrichment sequencing method | |
| KR101435446B1 (en) | Novel marker and primer to identify and discriminate cultivars discrimination in Brassica napus L. and use thereof | |
| CN104293775B (en) | The molecular marker of degeneration-resistant willow, screening technique, test kit and application | |
| CN101760555A (en) | Method for identifying single-copy transgenic tobacco based on PCR technology | |
| CN104073562B (en) | A kind of molecular marker for cutter long-tailed anchovy different ecological type population identification | |
| CN102965367B (en) | Method for acquiring plant candidate anti-disease gene sequence | |
| CN106701927B (en) | Method and kit for rapidly detecting chrysophyceae through loop-mediated isothermal amplification | |
| CN101824410A (en) | Simple method for establishing plant artificial microRNA | |
| CN104087583A (en) | Moschus anhuiensis microsatellite loci, primers and application of primers |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150121 |