CN117783354A - Use of compositions for detecting N6-adenylate methylation modification in mRNA binding to plant proteins - Google Patents
Use of compositions for detecting N6-adenylate methylation modification in mRNA binding to plant proteins Download PDFInfo
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Abstract
The invention relates to the field of biotechnology, and discloses application of a composition in detecting N6-adenylate methylation modification in mRNA combined with plant protein, wherein the composition consists of ethanol, sodium acetate and glycogen, and the ratio of the ethanol to the sodium acetate to the glycogen in the composition is 500uL:3mol sodium acetate: 0.1g glycogen. The composition of the invention can enrich more RNA modification, reduce the use of harmful reagents and improve the binding of plant protein to RNA m 6 Accuracy of the A modification detection.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of a composition in detecting N6-adenylate methylation modification in mRNA combined with protein.
Background
RNA Immunoprecipitation (RIP), a key technology used in the field of epigenetic science in recent years, is mainly used for the interaction of target protein binding RNA, and RIP mainly focuses on a class of RNA Binding Proteins (RBPs) and identifies different types of RNA such as binding mRNA, lncRNA and the like by immunoprecipitation together with the RNA. RNA-protein interactions can regulate the functions of mRNA and non-coding RNA, and research into analyzing chemical modifications near target protein-binding RNA will help us understand the biological functions of RNA.
Methylation modification of N6-adenylate (N6-methylaadenylate, m is obtained after modification) 6 A, CAS number 1867-73-8) is the most abundant modification in eukaryotic mRNA and is also the first endogenous modification detected in mRNA. m is m 6 A plays an important role in regulating gene expression and mRNA stability. In animals, m 6 A affects almost all aspects of RNA metabolism, however m in plants 6 The mechanism by which A regulates plant development is still unclear. m is m 6 A is essential in embryonic development, which is conserved in both mammals and plants. M in mammals 6 Loss of function of the a-methylase complex (including METTL3, METTL14 and WTAP) results in embryonic lethality. In Arabidopsis thaliana, m was knocked out 6 The a methylase (including MTA, MTB, FIP37 and VIR) genes lead to embryonic lethality, with embryonic development stopped at the globular stage. Recovery mutations of MTA or FIP37 further confirm m 6 A function in the developmental stage of plant embryo.
The existing method for detecting N6-adenylate methylation near RNA in target protein conjugate is mainly reported in animal experiments, but the technical method is not reported in plants. The RIP method in animal experiment has less RNA modification, and the research aims at developing and optimizing a quantitative detection method for m near the plant protein binding RNA 6 A method of abundance of a.
Disclosure of Invention
One technical problem to be solved by the invention is how to detect the methylation modified abundance of protein-bound N6-adenylate.
In order to solve the technical problems, the invention firstly provides application of a composition in detecting N6-adenylate methylation modification in mRNA combined with protein, wherein the composition consists of ethanol, sodium acetate and glycogen, and the ratio of the ethanol to the sodium acetate to the glycogen in the composition is 500ul:3mol sodium acetate: 0.1g glycogen.
The invention also provides the composition.
The present invention also provides a method for detecting the abundance of an N6-adenylate methylation modification in mRNA bound by a protein (i.e., a method for detecting the abundance of an N6-adenylate methylation modification near an RNA in a protein-bound body of interest), in particular a method for detecting the abundance of an N6-adenylate methylation modification bound by a RIP (RNA immunoprecipitation) bound by LC-MS (liquid chromatography-mass spectrometry) or MS (liquid chromatography), wherein the method for detecting the abundance of an N6-adenylate methylation modification bound by a protein by a RIP (RNA immunoprecipitation) bound by LC-MS (liquid chromatography-mass spectrometry) comprises the steps of:
s1, extracting mRNA of a plant;
s2, fragmenting the mRNA by metal ions to obtain a fragmented mRNA solution, and adding the composition to promote mRNA precipitation to obtain purified fragmented mRNA;
S3, expressing target proteins of the plants in vitro;
s4, incubating and combining the target protein of S3 and the purified fragmented mRNA of S2 in vitro to obtain a protein-RNA complex;
s5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;
s6, adding the composition into the target mRNA solution to promote mRNA precipitation, so as to obtain purified target mRNA;
s7, preparing the purified target mRNA of the S6 into mononucleotide, further dephosphorylating, and purifying the mononucleotide to obtain an on-line sample;
s8, detecting by liquid chromatography-mass spectrometry, and adopting an external standard method to detect N6-methyl adenylate (m) in the sample of the machine in S7 6 A) And adenine ribonucleoside (rA) content, divided by N6-methyladenosine contentObtaining the protein binding N6-adenylate methylation modified abundance of the biological sample by the sugar nucleoside content;
the N6-methyl adenosine acid content is quantified according to a chromatographic peak corresponding to an ion peak with collision energy of 16V and m/z of 282.1-150.1;
the adenine ribonucleoside content was quantified according to the chromatographic peak corresponding to the ion peak at collision energy of 10V and m/z of 268.1-136.1.
The invention also provides a method for detecting the methylation modified abundance of protein-bound N6-adenylate by combining RIP (RNA immunoprecipitation) with MS (liquid chromatography), which comprises the following steps:
S1, extracting mRNA of a plant;
s2, fragmenting the mRNA by metal ions to obtain a fragmented mRNA solution, and adding the composition to promote mRNA precipitation to obtain purified fragmented mRNA;
s3, expressing target proteins of the plants in vitro;
s4, incubating and combining the target protein of S3 and the purified fragmented mRNA of S2 in vitro to obtain a protein-RNA complex;
s5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;
s6, adding the composition into the target mRNA solution to promote mRNA precipitation, so as to obtain purified target mRNA;
s7, preparing the purified target mRNA of the S6 into mononucleotide, further dephosphorylating, and purifying the mononucleotide to obtain an on-line sample;
s8', detecting by liquid chromatography, and adopting an external standard method to detect N6-methyl adenylate (m) in the sample of the machine in S7 6 A) And quantifying the adenine ribonucleoside (rA) content, dividing the N6-methyladenosine content by the adenine ribonucleoside content to obtain the protein-bound N6-adenylate methylation modified abundance of the biological sample; the mobile phase adopted by the liquid chromatography consists of a phase A and a phase B, wherein the phase A is formic acid aqueous solution with the volume percentage content of 0.1 percent (the solvent is water and the solute is formic acid), the phase B is acetonitrile solution with the volume percentage content of 0.1 percent of formic acid (the solvent is acetonitrile, The solute is formic acid); the elution procedure used for liquid chromatography was as follows: from 0min to 8min, the volume ratio of the phase A to the mobile phase is linearly reduced from 100% to 99%; from more than 8min to 10min, the volume ratio of the A phase to the mobile phase is linearly reduced from less than 99% to 94%; from more than 10min to 15min, the volume ratio of the A phase to the mobile phase is linearly reduced from less than 94% to 0%; from more than 15min to 20min, the volume fraction of the A phase in the mobile phase is linearly increased from 0% to 100%; the chromatographic column adopted by the liquid chromatography is Thermo Scientific Hypersil GOLD aQ reverse phase column, the column length is 100mm, the column inner diameter is 2.1mm, and the particle size of the filler is 1.9um; the column temperature of the chromatographic column is 25 ℃, and the flow rate of the mobile phase is 0.3ml/min; the retention time of the chromatographic peak of the N6-methyl adenylate is 10.5+/-1 min; the retention time of the chromatographic peak of the adenine ribonucleotide is 5.5+ -1 min.
In the above method, the addition of the above composition to S2 or S6 promotes mRNA precipitation, and the composition of claim 1 is added to the fragmented mRNA solution or the target mRNA solution to obtain a reaction system in which ethanol is contained in an amount of 2.5L, sodium acetate is contained in an amount of 0.3M, and glycogen is contained in an amount of 100. Mu.g ml -1 。
In the above method, the addition of the above composition as described in S2 or S6 promotes mRNA precipitation, which is carried out at-80 ℃.
In the above method, both the purified fragmented mRNA and the purified mRNA of interest are prepared as solutions in sterile water without enzyme prior to use.
In the above method, the metal ion in S2 is a zinc ion.
In the above method, the fragmentation of S2 is carried out by fragmenting the mRNA to a size of 100 nt.
In the above method, the fragmenting of S2 is performed in a reaction system a, which is a liquid composed of: mRNA, tris-HCl, zinc chloride and water; the mRNA content was 20. Mu.g, the Tris-HCl content was 100mM, the zinc chloride content was 100mM, and the balance was water.
In the above method, the fragmentation of S2 was performed at 94℃for 5min and then stopped by adding 2. Mu.l of 0.5M EDTA to obtain a fragmented mRNA solution.
In the method, the in-vitro incubation and combination of S4 is that a reaction system B is incubated on ice for 1 hour and then is incubated with the magnetic beads with His labels for 2 hours in a rotating way at the temperature of 4 ℃; the reaction system B is a liquid composed of the following substances: the protein of interest, the purified fragmented mRNA, tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA, CAS number 60-00-4), ethylphenyl polyethylene glycol (NP-40, cat. Number N8032), dithiothreitol (DTT, CAS number 27565-41-9), ribonuclease inhibitors (RNasin, promega company product, cat. Number N2511) and water; the content of the target protein is 20 mug/μl, the content of the purified fragmented mRNA is 1 mug/μl, the content of Tris-HCl is 50mM, the content of sodium chloride (NaCl) is 250mM, the content of ethylenediamine tetraacetic acid (EDTA) is 0.4mM, the mass percent of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of Dithiothreitol (DTT) is 1mM, the content of ribonuclease inhibitor (RNasin) is 0.4U/μl, and the balance is water.
In the method, the protein-RNA complex is eluted and separated, and the protein-RNA complex is subjected to rotary elution at 4 ℃ by using a buffer solution X to remove nonspecific fragments, then is digested for 20min by using a PK buffer solution at 37 ℃, and finally is incubated for 20min by using 200 μl PK-urea buffer solution at 37 ℃ to obtain a target mRNA solution.
In the above method, the buffer solution X is a liquid composed of: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA, CAS number 60-00-4), ethylphenyl polyethylene glycol (NP-40, cat. Number N8032-4), dithiothreitol (DTT, CAS number 27565-41-9), ribonuclease inhibitors (RNasin, promega company, cat. Number N2511) and water; the content of sodium chloride (NaCl) is 250mM, the content of ethylenediamine tetraacetic acid (EDTA) is 0.4mM, the mass percentage of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of Dithiothreitol (DTT) is 1mM, the content of ribonuclease inhibitor (RNasin) is 0.4U/. Mu.l, and the balance is water.
In the above method, the PK buffer is a liquid consisting of: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA), proteinase K (protein K, CAS number 39450-01-6) and water; the content of Tris-HCl is 100mM, the content of sodium chloride (NaCl) is 50mM, the content of ethylenediamine tetraacetic acid (EDTA) is 10mM EDTA, the content of proteinase K is 4 mug/mul, and the balance is water.
In the above method, the PK-urea buffer is a liquid composed of: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA), urea and water; the content of Tris-HCl is 100mM, the content of sodium chloride (NaCl) is 50mM, the content of ethylenediamine tetraacetic acid (EDTA) is 10mM EDTA, the content of urea is 7M, and the balance is water.
In the above method, the preparation of the mononucleotide as described in S7 is performed in a reaction system C which is a liquid composed of: RNA, adenosine deaminase inhibition, tetrahydrouridine, antioxidant, 2, 6-di-tert-butyl-P-methylphenol, nuclease P1, phosphodiesterase I, sodium acetate, znCl 2 And water; the RNA content is 500ng/300 μl, the adenosine deaminase inhibition content is 0.005 μg/μl, the tetrahydrouridine content is 0.05 μg/μl, the antioxidant content is 0.1mM, the 2, 6-di-tert-butyl-P-methylphenol content is 0.1mM, the nuclease P1 content is 1U, the phosphodiesterase I content is 0.01U, the sodium acetate content is 0.1mM, the ZnCl content is high 2 The content of (C) was 0.0067mM, the balance being water.
In the above method, the preparation of S7 as a single nucleotide was carried out at 37℃for 3 hours.
In the above method, the dephosphorylation of S7 is performed using alkaline phosphatase.
In the above method, the dephosphorylation of S7 is carried out at 37℃for 30 minutes.
In the above method, the purification of the mononucleotide of S7 is performed by using an ultrafiltration tube having a molecular weight cut-off of 10 kDa.
In the above method, in the detection by the liquid chromatography-mass spectrometry of S8, the mobile phase adopted by the liquid chromatography is composed of a phase a and a phase B, wherein the phase a is an aqueous solution of formic acid (the solvent is water, the solute is formic acid) with a volume percentage of 0.1%, and the phase B is an acetonitrile solution of formic acid (the solvent is acetonitrile, the solute is formic acid) with a volume percentage of 0.1%; the elution procedure used for liquid chromatography was as follows: from 0min to 8min, the volume ratio of the phase A to the mobile phase is linearly reduced from 100% to 99%; from more than 8min to 10min, the volume ratio of the A phase to the mobile phase is linearly reduced from less than 99% to 94%; from more than 10min to 15min, the volume ratio of the A phase to the mobile phase is linearly reduced from less than 94% to 0%; the volume fraction of the phase A in the mobile phase is linearly increased from 0% to 100% from more than 15min to 20min, a chromatographic column used in the liquid chromatography is Thermo Scientific Hypersil GOLD aQ reverse phase column, the column length is 100mm, the inner diameter of the column is 2.1mm, and the particle size of the filler is 1.9um; the column temperature of the chromatographic column is 25 ℃, and the flow rate of the mobile phase is 0.3ml/min; the retention time of the chromatographic peak of the N6-methyl adenylate is 10.5+/-1 min; the retention time of the chromatographic peak of the adenine ribonucleotide is 5.5+ -1 min.
LC-MS/MS is considered to be an accurate and rapid means for detecting RNA chemical modification, and the technology has been widely used in the field of apparent biology. The technology compares the molecular weight of mononucleotidated base and the peak time in a detection sample through a standard substance, so as to judge the related chemical modification. Compared with specific chemical modification antibody detection, LC-MS/MS has the characteristics of quick time, low cost, high safety coefficient and the like. The invention discloses the detection of m on mRNA in plant body bound with plant protein through RIP binding LC-MS for the first time 6 A is modified and optimized on the existing in vitro RIP technology, compared with the existing animal protein detection technology, the detection method provided by the invention can enrich more RNA modification, greatly optimize the existing method for detecting the RNA modification nearby the target protein and reduce the use of harmful reagents, provide an excellent detection method for accurately detecting the modification nearby the plant protein binding RNA fragment, and further improve the detection of the plant protein binding RNA m 6 Accuracy of the A modification detection. m is m 6 A modification is reported to play an important role in participating in plant growth and development and responding to stress, and target protein is developed to bind mRNA m 6 The detection method of the A modification has important value for researching plant RNA epigenetic science and RNA apparent transcriptome breeding.
Drawings
FIG. 1 is an electrophoretogram of mRNA after fragmentation in examples 1 and 2, with nucleic acids maker being 5000bp,3000bp,2500bp,1500bp,1000bp,750bp,500bp,200bp,100bp from top to bottom, respectively.
FIG. 2 is a graph of in vitro purified proteins of examples 1 and 2, with protein sizes between 30-35 kd.
FIG. 3 is a graph of the detection of m in example 1 6 The peak shape of the A output is the retention time (min), i.e. m 6 The peak time of A was 10.5min.
FIG. 4 is a graph of the peak shape of the rA output of example 1 with retention time (min) on the abscissa, i.e., the peak time of rA is 5.5min.
FIG. 5 is m in example 2 6 The peak shape of the A output is shown as retention time (min) on the abscissa, namely, the peak emergence time of rA is 10.5min.
FIG. 6 is a graph of the peak shape of the rA output of example 2 with retention time (min) on the abscissa, i.e., the peak time of rA is 5.5min.
FIG. 7 is a comparison of changes in m6A enrichment in examples 1 and 2, with the pre-optimized example 1 as a control, and the m6A enrichment in example 2 after optimization increased 40-fold (P= 2.44495E) -06 )。
FIG. 8 shows the comparison of the rA enrichment changes in examples 1 and 2, with the comparison of example 1 before optimization, and the increase in rA enrichment in example 2 after optimization by a factor of 51 (P= 1.57716E) -07 )。
Detailed Description
The following detailed description of the invention is provided in connection with the accompanying drawings that illustrate the invention and are not to be construed as limiting the scope of the invention. The examples provided below serve as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.
The experimental methods in the following examples, unless specifically indicated, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified. The quantitative tests in the following examples were all set up in triplicate and the results averaged.
N6-methyl adenylate (m) in the following examples 6 A) Is a product of Shanghai Yuan Ye Biotechnology Co., ltd, and the product number is 1867-73-8. Adenine ribonucleotide (rA) is a product of Shanghai Yes Biotechnology Co., ltd, product number 58-61-7. The expression strain BL21 is a product of Nanjinopran biotechnology limited Co., ltd, and the product number is C504-02.
The mass spectrum multi-reaction monitoring (multiple reaction monitoring, MRM) technology is adopted, is a technology for acquiring data in a targeted manner based on known information or assumed information and collecting mass spectrum signals, and has the advantages of sensitivity, accuracy, specificity and the like.
The detection is carried out by adopting a liquid chromatograph-mass spectrometer, and the following example specifically adopts an Agilent 1260 liquid chromatograph-G6400 series three-stage quadrupole mass spectrometer. The target detection object is m 6 A and rA.
In mass spectrometry, N6-methyladenosine (m 6 A) The m/z parameter is set to 282.1-150.1,Collision Energy parameter is set to 16; adenine ribonucleotide (rA) m/z parameter was set to 268.1-136.1,Collision Energy parameter was set to 10; the "m/z" and "CID" parameters in the mass spectrum were specifically set as in Table 1.
Wash table 1 mass spectral parameters
In Table 1, m 6 A represents N6-methyladenosine and rA represents adenine ribonucleotide.
The column for liquid chromatography was TThermo Scientific Hypersil GOLD aQ reverse phase column (100x2.1 mm,1.9 um). The column temperature of the liquid chromatography was 25 ℃. In the mobile phase of liquid chromatography, phase A was 0.1% (volume ratio) aqueous formic acid solution, and phase B was acetonitrile (0.1% formic acid). The mobile phase flow rate of the liquid chromatograph was 0.3ml/min.
The elution process of liquid chromatography is shown in table 2: from 0min to 8min, the volume ratio of the phase A to the mobile phase is linearly reduced from 100% to 99%; from 8min to 10min, the volume ratio of the phase A to the mobile phase is linearly reduced from 99% to 94%; from 10min to 15min, the volume ratio of the phase A to the mobile phase is linearly reduced from 94% to 0%; from 15min to 20min, the volume fraction of the A phase in the mobile phase is linearly increased from 0% to 100%.
TABLE 2 elution procedure
| Retention time | The A phase accounts for the volume ratio (%) |
| 0.00min | 100.00 |
| 8.00min | 99.00 |
| 10.00min | 94.00 |
| 15.00min | 0.00 |
| 20.00min | 100.00 |
The "m/z" and "CID" parameters in the mass spectrum and the elution procedure of the liquid chromatograph in Table 2 were set according to Table 1, m 6 The retention time corresponding to the peak value of the A standard substance is 10.5min, and if the sample has the peak value of 10.5+/-1 min, the compound corresponding to the peak value can be judged to be m 6 A. The retention time corresponding to the peak value of rA standard is 5.5min, and if the sample has a peak value of 5.5+ -1 min, the peak value can be judgedThe corresponding compound is rA.
The relative abundance (relative abundance) represents the intensity of an ion, and the relative intensity is determined based on the strongest peak (base peak) as 100 and the other peaks as the standard.
m 6 Relative abundance of a = m in peak shape plot 6 Peak area corresponding to a/peak pattern diagram corresponding to rA.
Preparation of concentration gradient N6-methyladenosine (m) 6 A) Concentrations of 1ng/ml, 10ng/ml, 50ng/ml, 100ng/ml, respectively, m established 6 A standard curve, linear distribution between 1ng/ml and 100ng/ml, wherein the correlation coefficient R 2 Above 0.96, the prepared standard curve is proved to have strong feasibility and linear relation. Preparing concentration gradient adenine ribonucleoside (rA) with concentrations of 1ng/ml, 10ng/ml, 50ng/ml and 100ng/ml respectively, and establishing rA standard curve with linear distribution between 1ng/ml and 100ng/ml, wherein the correlation coefficient R 2 Above 0.95, the prepared standard curve has strong feasibility and linear relation.
Example 1 optimization of ex-precursor RIP in combination with LC-MS/MS detection of protein of interest binding mRNA m 6 A modification
Z1, extracting plant mRNA;
the total RNA in the corn leaves is enriched by a commercial plant RNA extraction kit (Nanjinouzan Biotechnology Co., ltd., product number: rA 101) to obtain the total RNA of the corn leaves, and then mRNA purification enrichment is carried out from the total RNA of the corn leaves by a commercial purification mRNA kit (Nanjinouzan Biotechnology Co., ltd., product number: N403-01) to obtain the purified mRNA.
Z2, fragmenting the purified mRNA by metal ions to obtain a fragmented mRNA solution;
the fragmentation is carried out in a reaction system A which is a liquid consisting of: mRNA, tris-HCl, zinc chloride and water; the mRNA content was 20. Mu.g, the Tris-HCl content was 100mM, the zinc chloride content was 100mM, and the balance was water.
The fragmentation was stopped by adding 2. Mu.l of 0.5M EDTA after 5min at 94℃to give a fragmented mRNA solution. The nucleic acid electrophoresis pattern of the fragmented mRNA is shown in FIG. 1.
Z3. expressing a protein of interest of said plant in vitro;
The constructed fusion protein coding gene of the target protein zc1 (zc 1 in the example is corn Zm00001D028655 gene coding protein) with His tag (5071-5973 of SEQ ID No.1 is a coding sequence of the fusion protein, SEQ ID No.1 is a carrier sequence for expressing the fusion protein, SEQ ID No.2 is an amino acid sequence of the fusion protein), the fusion protein coding gene is transferred into an expression strain BL21, 1M isopropyl-beta-D-thiogalactoside IPTG (Beijing Soy Co., ltd., product, cat# I8070) is utilized for carrying out overnight induction at 16 ℃, after induction, a supernatant is obtained through ultrasound, a commercial His tag magnetic bead (Beijing Soy Bay Co., ltd., cat# M2300) is utilized for carrying out incubation on supernatant, the zc1 protein is eluted through imidazole, and the target protein after in vitro purification is obtained, the protein is shown in FIG. 2, and the protein size is between 30 kd and 35 kd.
Z4, incubating and combining the target protein of Z3 and the fragmented mRNA of Z2 in vitro to obtain a protein-RNA complex;
the in vitro purified target protein (see Z3) and the fragmented mRNA solution (see Z2) were added to a buffer solution (50 mM Tris-HCl pH 7.5, 250mM NaCl,0.4mM EDTA,0.1% NP-40,1mM DTT, 0.4U/. Mu.l RNasin (Promega, cat. No.: N2511)) to constitute a reaction system B (100. Mu.l system) which was a liquid composed of: protein of interest, fragmented mRNA, tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA, CAS number 60-00-4), ethylphenyl polyethylene glycol (NP-40, cat# N8032-4), dithiothreitol (DTT, CAS number 27565-41-9), ribonuclease inhibitors (RNasin, promega company product, cat# N2511) and water. The content of the target protein is 20 mug/ul, the content of the fragmented mRNA is 1 mug/ul, the content of Tris-HCl is 50mM, the content of sodium chloride (NaCl) is 250mM, the content of ethylenediamine tetraacetic acid (EDTA) is 0.4mM, the mass percent of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of Dithiothreitol (DTT) is 1mM, the content of ribonuclease inhibitor (RNasin) is 0.4U/ul, and the balance is water.
The reaction system B is incubated for 1 hour on ice, and then is rotated and incubated with commercial His-tagged magnetic beads for 2 hours at 4 ℃ to obtain the protein-RNA complex.
Z5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;
the protein-RNA complex is eluted and separated, and the protein-RNA complex is subjected to rotary elution at 4 ℃ by using a buffer solution X to remove nonspecific fragments, then is digested for 20min by using a PK buffer solution at 37 ℃, and finally is incubated for 20min by using 200 μl PK-urea buffer solution at 37 ℃ to obtain a target mRNA solution.
The buffer solution X is a liquid composed of the following substances: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA, CAS number 60-00-4), ethylphenyl polyethylene glycol (NP-40, cat# N80324), dithiothreitol (DTT, CAS number 27565-41-9), ribonuclease inhibitors (RNasin, promega company, cat# N2511) and water; the content of sodium chloride (NaCl) is 250mM, the content of ethylenediamine tetraacetic acid (EDTA) is 0.4mM, the mass percentage of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of Dithiothreitol (DTT) is 1mM, the content of ribonuclease inhibitor (RNasin) is 0.4U/. Mu.l, and the balance is water.
The PK buffer is a liquid consisting of: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA), proteinase K (protein K, CAS number 39450-01-6) and water; the content of Tris-HCl is 100mM, the content of sodium chloride (NaCl) is 50mM, the content of ethylenediamine tetraacetic acid (EDTA) is 10mM EDTA, the content of proteinase K is 4 mug/mul, and the balance is water.
The PK-urea buffer is a liquid consisting of the following substances: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA), urea and water; the content of Tris-HCl is 100mM, the content of sodium chloride (NaCl) is 50mM, the content of ethylenediamine tetraacetic acid (EDTA) is 10mM EDTA, the content of urea is 7M, and the balance is water.
mRNA enrichment
To the target mRNA solution was added 1ml of phenol: extracting chloroform mixed solution (product of Mock company, product number: P3803), centrifuging at 12000rpm at 4deg.C for 15min to remove supernatant, adding 1ml anhydrous ethanol and final concentration of 100 μg ml -1 The supernatant was removed by centrifugation again at 12000rpm at 4℃for 15min, and the excess absolute ethanol was dried to give a precipitate enriched in the target mRNA.
Z7, preparing the enriched target mRNA of the Z6 into mononucleotide, further dephosphorylating, and purifying the mononucleotide to obtain an on-machine sample;
The enriched target mRNA obtained in Z6 was taken and prepared into a solution by adding 15. Mu.l of sterile water, followed by single nucleotide preparation.
The preparation of the mononucleotide is carried out in a reaction system C (300. Mu.l system) which is a liquid composed of: RNA (provided by nucleic acid sample), adenosine deaminase inhibition, tetrahydrouridine, antioxidant, 2, 6-di-tert-butyl-P-methylphenol, nuclease P1, phosphodiesterase I, sodium acetate, znCl 2 And water; the RNA content is 500ng/300 μl, the adenosine deaminase inhibition content is 0.005 μg/μl, the tetrahydrouridine content is 0.05 μg/μl, the antioxidant content is 0.1mM, the 2, 6-di-tert-butyl-P-methylphenol content is 0.1mM, the nuclease P1 content is 1U, the phosphodiesterase I content is 0.01U, the sodium acetate content is 0.1mM, the ZnCl content is high 2 The content of (C) was 0.0067mM, the balance being water.
A specific adenosine deaminase inhibitor used in this example is erythro-9- (2-Hydroxy-3-onyl) adenine (EHMA) hydrochloride, product of Sigma company, cat. Number E114.
The specific antioxidant used in this example was deferoxamine, product of sigma aldrich (Shanghai) trade company, cat No. Y0001937.
The specific phosphodiesterase I used in this example is snake venom phosphodiesterase I, product of Shanghai Yuan Yes Biotechnology Co., ltd., product number S10225.
Reaction conditions when the nucleic acid sample is an RNA sample: 37℃for 3 hours.
10U alkaline phosphatase was added to the system to carry out the reaction.
The specific alkaline phosphatase used in this example is calf intestinal alkaline phosphatase (CIP), NEB Inc., cat# MO290S.
Reaction conditions: 37℃for 30 minutes.
The system was added to a 10KD ultrafilter tube and centrifuged at 13000rpm for 10min, and the filtrate was collected as an on-machine sample from the corn leaf.
Z8, detecting by liquid chromatography-mass spectrometry, and adopting an external standard method to detect N6-methyl adenylate (m) in the sample of the machine in Z7 6 A) And adenine ribonucleoside (rA) content.
The on-machine samples obtained from corn leaves as described in S7 were tested according to the settings of the "m/z" and "CID" parameters in the mass spectrum of Table 1 and the elution procedure of the liquid chromatograph in Table 2, to detect m 6 The peak shape of the A output is shown in FIG. 5. In FIG. 5, the retention time (min), i.e., m, is plotted on the abscissa 6 The peak time of A was 10.5min. The mass spectrum of rA output is shown in FIG. 6, and the abscissa is the retention time (min), i.e. the peak time of rA is 5.5min.
Calculating the m of the corn leaf 6 The relative abundance of a is 0.00249.
According to m established 6 A standard curve and rA standard curve to obtain m contained in corn leaf 6 The absolute content of A is 0.00249ng/ml, and the absolute content of rA is 0.2848ng/ml.
Example 2 in vitro RIP-combined LC-MS/MS detection of mRNA m near the target protein after optimization 6 A modification
S1, extracting mRNA of a plant;
the total RNA in the corn leaves was enriched by a commercial plant RNA extraction kit (Nanjinouzan Biotechnology Co., ltd., cat# rA 101), and then mRNA was purified and enriched from the total RNA in the corn leaves by a commercial purification mRNA kit (Nanjinouzan Biotechnology Co., ltd., cat# N403-01), to obtain purified mRNA.
S2, fragmenting the mRNA by metal ions to obtain a fragmented mRNA solution, and adding a composition to promote mRNA precipitation to obtain purified fragmented mRNA;
the fragmentation is carried out in a reaction system A which is a liquid consisting of: mRNA, tris-HCl, zinc chloride and water; the mRNA content was 20. Mu.g, the Tris-HCl content was 100mM, the zinc chloride content was 100mM, and the balance was water.
The fragmentation was stopped by adding 2. Mu.l of 0.5M EDTA after 5min at 94℃to give a fragmented mRNA solution.
At this time, mRNA was fragmented to a size of 100nt, and the nucleic acid electrophoresis pattern of the fragmented mRNA was shown in FIG. 1.
The optimization here compared to example 1 is as follows: the addition of the composition (in which the ratio of ethanol, sodium acetate and glycogen is 500ul:3mol sodium acetate: 0.1g glycogen) promotes mRNA precipitation.
Adding the composition to promote mRNA precipitation, namely adding ethanol (volume percentage content is 100%), 3M sodium acetate aqueous solution (pH value is 5.2) (Beijing Soy Bao technology Co., ltd., product number: A1070) and glycogen (product number: AM 9510) into the fragmented mRNA solution to obtain a reaction system, wherein the ethanol content in the reaction system is 500L, the sodium acetate content is 3M, and the glycogen content is 100 mug ml -1 。
After overnight at-80℃the pellet was washed by further centrifugation at 15000g for 25min at 4℃with 1ml of 75% ethanol by volume and the supernatant was discarded, and the pellet obtained was purified fragmented mRNA by centrifugation at 15000g for 15min at 4 ℃.
S3, expressing target proteins of the plants in vitro;
the constructed fusion protein zc1 (zc 1 in the example is corn Zm00001D028655 gene encoding protein) with His tag is encoded by a fusion protein encoding gene (5071-5973 of SEQ ID No.1 is the encoding sequence of the fusion protein, SEQ ID No.1 is the carrier sequence for expressing the fusion protein, SEQ ID No.2 is the amino acid sequence of the fusion protein), the fusion protein encoding gene is transferred into an expression strain BL21, 1M isopropyl-beta-D-thiogalactosyl IPTG (Beijing Soy Bao technology Co., ltd., product number: I8070) is utilized for overnight induction at 16 ℃, after induction, a protein is obtained by taking supernatant through ultrasound, a commercial His tag magnetic bead (Beijing Soy Bao, product number: M2300) is utilized for incubating the supernatant, and the zc1 protein is eluted through imidazole to obtain the target protein after in vitro purification, the protein is shown in FIG. 2, and the protein size is between 30 and 35 kd.
S4, incubating and combining the target protein of S3 and the purified fragmented mRNA of S2 in vitro to obtain a protein-RNA complex;
the purified fragmented mRNA was used by adding 15. Mu.l of enzyme-free sterile water to obtain a purified fragmented mRNA solution.
The in vitro purified target protein (see S3) and the purified fragmented mRNA solution were added to a buffer solution (50 mM Tris-HCl pH 7.5, 250mM NaCl,0.4mM EDTA,0.1% NP-40,1mM DTT, 0.4U/. Mu.l RNasin (Promega, cat. No.: N2511)) to constitute a reaction system B (1000. Mu.l system) which was a liquid composed of: the protein of interest, purified fragmented mRNA, tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA, CAS number 60-00-4), ethylphenyl polyethylene glycol (NP-40, cat# N8032-4), dithiothreitol (DTT, CAS number 27565-41-9), ribonuclease inhibitors (RNasin, promega company product, cat# N2511) and water. The content of the target protein is 20 mug/ul, the content of the purified fragmented mRNA is 1 mug/ul, the content of Tris-HCl is 50mM, the content of sodium chloride (NaCl) is 250mM, the content of ethylenediamine tetraacetic acid (EDTA) is 0.4mM, the mass percent of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of Dithiothreitol (DTT) is 1mM, the content of ribonuclease inhibitor (RNasin) is 0.4U/ul, and the balance is water.
The reaction system B is incubated for 1 hour on ice, and then is rotated and incubated with commercial His-tagged magnetic beads for 2 hours at 4 ℃ to obtain the protein-RNA complex.
S5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;
the protein-RNA complex is eluted and separated, and the protein-RNA complex is subjected to rotary elution at 4 ℃ by using a buffer solution X to remove nonspecific fragments, then is digested for 20min by using a PK buffer solution at 37 ℃, and finally is incubated for 20min by using 200 μl PK-urea buffer solution at 37 ℃ to obtain a target mRNA solution.
The buffer solution X is a liquid composed of the following substances: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA, CAS number 60-00-4), ethylphenyl polyethylene glycol (NP-40, cat# N8032), dithiothreitol (DTT, CAS number 27565-41-9), ribonuclease inhibitors (RNasin, promega company, cat# N2511) and water; the content of sodium chloride (NaCl) is 250mM, the content of ethylenediamine tetraacetic acid (EDTA) is 0.4mM, the mass percentage of ethylphenyl polyethylene glycol (NP-40) is 0.1%, the content of Dithiothreitol (DTT) is 1mM, the content of ribonuclease inhibitor (RNasin) is 0.4U/. Mu.l, and the balance is water.
The PK buffer is a liquid consisting of: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA), proteinase K (protein K, CAS number 39450-01-6) and water; the content of Tris-HCl is 100mM, the content of sodium chloride (NaCl) is 50mM, the content of ethylenediamine tetraacetic acid (EDTA) is 10mM EDTA, the content of proteinase K is 4 mug/mul, and the balance is water.
The PK-urea buffer is a liquid consisting of the following substances: tris-HCl (pH 7.5), sodium chloride (NaCl), ethylenediamine tetraacetic acid (EDTA), urea and water; the content of Tris-HCl is 100mM, the content of sodium chloride (NaCl) is 50mM, the content of ethylenediamine tetraacetic acid (EDTA) is 10mM EDTA, the content of urea is 7M, and the balance is water.
S6, adding the composition into the target mRNA solution to promote mRNA precipitation, so as to obtain purified target mRNA;
the optimization here compared to example 1 is as follows: the addition of the composition (in which the ratio of ethanol, sodium acetate and glycogen is 500ul:3mol sodium acetate: 0.1g glycogen) promotes mRNA precipitation.
Adding the composition to promote mRNA precipitation comprises adding ethanol (volume percentage 100%), 3M sodium acetate aqueous solution (pH 5.2) (Beijing Soy Bao technology Co., ltd., product number: A1070) and glycogen (Saiemerer) Airline product, goods number: AM 9510); the composition consisted of 2.5L ethanol, 0.3M sodium acetate and 100. Mu.g/ml glycogen -1 。
S7, preparing the purified target mRNA of the S6 into mononucleotide, further dephosphorylating, and purifying the mononucleotide to obtain an on-line sample;
the purified target mRNA obtained in S6 was prepared by adding 15. Mu.l of sterile water to prepare a solution, and then single nucleotide preparation was performed.
The preparation of the mononucleotide is carried out in a reaction system C (300. Mu.l system) which is a liquid composed of: RNA (provided by nucleic acid sample), adenosine deaminase inhibition, tetrahydrouridine, antioxidant, 2, 6-di-tert-butyl-P-methylphenol, nuclease P1, phosphodiesterase I, sodium acetate, znCl 2 And water; the RNA content is 500ng/300 μl, the adenosine deaminase inhibition content is 0.005 μg/μl, the tetrahydrouridine content is 0.05 μg/μl, the antioxidant content is 0.1mM, the 2, 6-di-tert-butyl-P-methylphenol content is 0.1mM, the nuclease P1 content is 1U, the phosphodiesterase I content is 0.01U, the sodium acetate content is 0.1mM, the ZnCl content is high 2 The content of (C) was 0.0067mM, the balance being water.
A specific adenosine deaminase inhibitor used in this example is erythro-9- (2-Hydroxy-3-onyl) adenine (EHMA) hydrochloride, product of Sigma company, cat. Number E114.
The specific antioxidant used in this example was deferoxamine, product of sigma aldrich (Shanghai) trade company, cat No. Y0001937.
The specific phosphodiesterase I used in this example is snake venom phosphodiesterase I, product of Shanghai Yuan Yes Biotechnology Co., ltd., product number S10225.
Reaction conditions when the nucleic acid sample is an RNA sample: 37℃for 3 hours.
10U alkaline phosphatase was added to the system to carry out the reaction.
The specific alkaline phosphatase used in this example is calf intestinal alkaline phosphatase (CIP), NEB Inc., cat# MO290S.
Reaction conditions: 37℃for 30 minutes.
The system was added to a 10KD ultrafilter tube and centrifuged at 13000rpm for 10min, and the filtrate was collected as an on-machine sample from the corn leaf.
S8, detecting by liquid chromatography-mass spectrometry, and adopting an external standard method to detect N6-methyl adenylate (m) in the sample of the machine in S7 6 A) And adenine ribonucleoside (rA) content.
The on-machine samples obtained from corn leaves as described in S7 were tested according to the settings of the "m/z" and "CID" parameters in the mass spectrum of Table 1 and the elution procedure of the liquid chromatograph in Table 2, to detect m 6 The peak shape of the A output is shown in FIG. 5. In FIG. 5, the retention time (min), i.e., m, is plotted on the abscissa 6 The peak time of A was 10.5min. The mass spectrum of rA output is shown in FIG. 6, and the abscissa is the retention time (min), i.e. the peak time of rA is 5.5min.
Calculating the m of the corn leaf 6 The relative abundance of a is 0.1050.
According to m established 6 A standard curve and rA standard curve to obtain m contained in corn leaf 6 The absolute content of A is 0.1050ng/ml, and the absolute content of rA is 13.5550ng/ml.
Optimization of enriched m before and after by comparison of example 1 and example 2 6 The relative abundance of A (1:0.00249 ng/ml, 2:0.1050 ng/ml) and the absolute rA content (1:0.2848 ng/ml, 2:13.5550 ng/ml), m after optimization (example 2) 6 The A modification is 40 times more than before the optimization (see FIG. 7), the absolute rA content is 51 times more after the optimization (see FIG. 8), and the optimized experimental method replaces phenol and chloroform with sodium acetate and glycogen, which greatly reduces the risk during the experimental operation.
The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific examples, it will be understood that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.
SEQ ID No.1：
TGGCGAATGGGACGCGCCCTGTAGCGGCGCATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCGCCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTCTAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACTTGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACGTTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCTCGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCTGATTTAACAAAAATTTAACGCGAATTTTAACAAAATATTAACGTTTACAATTTCAGGTGGCACTTTTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCCGCTCATGAATTAATTCTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTAATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGACGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAATGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAATGCTGTTTTCCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGAGGCATAAATTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAATCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTAGAGCAAGACGTTTCCCGTTGAATATGGCTCATAACACCCCTTGTATTACTGTTTATGTAAGCAGACAGTTTTATTGTTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTCCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGAGCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAGAGCGCCTGATGCGGTATTTTCTCCTTACGCATCTGTGCGGTATTTCACACCGCATATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAGTTAAGCCAGTATACACTCCGCTATCGCTACGTGACTGGGTCATGGCTGCGCCCCGACACCCGCCAACACCCGCTGACGCGCCCTGACGGGCTTGTCTGCTCCCGGCATCCGCTTACAGACAAGCTGTGACCGTCTCCGGGAGCTGCATGTGTCAGAGGTTTTCACCGTCATCACCGAAACGCGCGAGGCAGCTGCGGTAAAGCTCATCAGCGTGGTCGTGAAGCGATTCACAGATGTCTGCCTGTTCATCCGCGTCCAGCTCGTTGAGTTTCTCCAGAAGCGTTAATGTCTGGCTTCTGATAAAGCGGGCCATGTTAAGGGCGGTTTTTTCCTGTTTGGTCACTGATGCCTCCGTGTAAGGGGGATTTCTGTTCATGGGGGTAATGATACCGATGAAACGAGAGAGGATGCTCACGATACGGGTTACTGATGATGAACATGCCCGGTTACTGGAACGTTGTGAGGGTAAACAACTGGCGGTATGGATGCGGCGGGACCAGAGAAAAATCACTCAGGGTCAATGCCAGCGCTTCGTTAATACAGATGTAGGTGTTCCACAGGGTAGCCAGCAGCATCCTGCGATGCAGATCCGGAACATAATGGTGCAGGGCGCTGACTTCCGCGTTTCCAGACTTTACGAAACACGGAAACCGAAGACCATTCATGTTGTTGCTCAGGTCGCAGACGTTTTGCAGCAGCAGTCGCTTCACGTTCGCTCGCGTATCGGTGATTCATTCTGCTAACCAGTAAGGCAACCCCGCCAGCCTAGCCGGGTCCTCAACGACAGGAGCACGATCATGCGCACCCGTGGGGCCGCCATGCCGGCGATAATGGCCTGCTTCTCGCCGAAACGTTTGGTGGCGGGACCAGTGACGAAGGCTTGAGCGAGGGCGTGCAAGATTCCGAATACCGCAAGCGACAGGCCGATCATCGTCGCGCTCCAGCGAAAGCGGTCCTCGCCGAAAATGACCCAGAGCGCTGCCGGCACCTGTCCTACGAGTTGCATGATAAAGAAGACAGTCATAAGTGCGGCGACGATAGTCATGCCCCGCGCCCACCGGAAGGAGCTGACTGGGTTGAAGGCTCTCAAGGGCATCGGTCGAGATCCCGGTGCCTAATGAGTGAGCTAACTTACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCCAGGGTGGTTTTTCTTTTCACCAGTGAGACGGGCAACAGCTGATTGCCCTTCACCGCCTGGCCCTGAGAGAGTTGCAGCAAGCGGTCCACGCTGGTTTGCCCCAGCAGGCGAAAATCCTGTTTGATGGTGGTTAACGGCGGGATATAACATGAGCTGTCTTCGGTATCGTCGTATCCCACTACCGAGATATCCGCACCAACGCGCAGCCCGGACTCGGTAATGGCGCGCATTGCGCCCAGCGCCATCTGATCGTTGGCAACCAGCATCGCAGTGGGAACGATGCCCTCATTCAGCATTTGCATGGTTTGTTGAAAACCGGACATGGCACTCCAGTCGCCTTCCCGTTCCGCTATCGGCTGAATTTGATTGCGAGTGAGATATTTATGCCAGCCAGCCAGACGCAGACGCGCCGAGACAGAACTTAATGGGCCCGCTAACAGCGCGATTTGCTGGTGACCCAATGCGACCAGATGCTCCACGCCCAGTCGCGTACCGTCTTCATGGGAGAAAATAATACTGTTGATGGGTGTCTGGTCAGAGACATCAAGAAATAACGCCGGAACATTAGTGCAGGCAGCTTCCACAGCAATGGCATCCTGGTCATCCAGCGGATAGTTAATGATCAGCCCACTGACGCGTTGCGCGAGAAGATTGTGCACCGCCGCTTTACAGGCTTCGACGCCGCTTCGTTCTACCATCGACACCACCACGCTGGCACCCAGTTGATCGGCGCGAGATTTAATCGCCGCGACAATTTGCGACGGCGCGTGCAGGGCCAGACTGGAGGTGGCAACGCCAATCAGCAACGACTGTTTGCCCGCCAGTTGTTGTGCCACGCGGTTGGGAATGTAATTCAGCTCCGCCATCGCCGCTTCCACTTTTTCCCGCGTTTTCGCAGAAACGTGGCTGGCCTGGTTCACCACGCGGGAAACGGTCTGATAAGAGACACCGGCATACTCTGCGACATCGTATAACGTTACTGGTTTCACATTCACCACCCTGAATTGACTCTCTTCCGGGCGCTATCATGCCATACCGCGAAAGGTTTTGCGCCATTCGATGGTGTCCGGGATCTCGACGCTCTCCCTTATGCGACTCCTGCATTAGGAAGCAGCCCAGTAGTAGGTTGAGGCCGTTGAGCACCGCCGCCGCAAGGAATGGTGCATGCAAGGAGATGGCGCCCAACAGTCCCCCGGCCACGGGGCCTGCCACCATACCCACGCCGAAACAAGCGCTCATGAGCCCGAAGTGGCGAGCCCGATCTTCCCCATCGGTGATGTCGGCGATATAGGCGCCAGCAACCGCACCTGTGGCGCCGGTGATGCCGGCCACGATGCGTCCGGCGTAGAGGATCGAGATCTCGATCCCGCGAAATTAATACGACTCACTATAGGGGAATTGTGAGCGGATAACAATTCCCCTCTAGAAATAATTTTGTTTAACTTTAAGAAGGAGATATACCATGGGCAGCAGCCATCATCATCATCATCACAGCAGCGGCCTGGTGCCGCGCGGCAGCCATATGGCTAGCATGACTGGTGGACAGCAAATGGGTCGCGGATCCGAATTCatggcggagacgctcgacatgaccctcgatgacatcatcaagaacaacaagaagagtaacccctcctccggcggggcccgccgcagccgcggcggatccgcccctggcggcggcagcggcggggtcgggccgaccaggcgctccttcaagagggctgggaacaggcaggcgccctaccagccgccgaaggcccctgacgctgcgtggcagcacgacatgtaccctgcggtcgccgcaggaggcggcggcggcgggagggtctcggcgctcgagacgggcaccaagctctacatctccaacctggactttggggtttcgaacgacgatatcaaggagctgttctctgagctaggtgatctgaagcgtttttcgataatatatgatcgaagtgggaggtctaagggaacagctgaagttgtatttgcaaggcgttctgatgctgtagcggcggtgaagaaatataacaatgtccaacttgatggtaagcccatgaagatagagatagttgggaccaatactccaactgcatctgctgctcttccagtctccaatggtggccatgctaggaatgctgtgaggagtgcaccgaggggtgctgccccagcaggtgtgccgcagcgaagacctcatcagaggggtgggaggcgtagtggtggatctgggggtggtcgtcgtggcaaggagcgcagcaagccaaagtcggctgaagaactcgacgctgacttggagaagtatcatgctgatgcgatgcagaccaacGCGGCCGCACTCGAGCACCACCACCACCACCACTGAGATCCGGCTGCTAACAAAGCCCGAAAGGAAGCTGAGTTGGCTGCTGCCACCGCTGAGCAATAACTAGCATAACCCCTTGGGGCCTCTAAACGGGTCTTGAGGGGTTTTTTGCTGAAAGGAGGAACTATATCCGGAT
SEQ ID No.2：
MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEFMAETLDMTLDDIIKNNKKSNPSSGGARRSRGGSAPGGGSGGVGPTRRSFKRAGNRQAPYQPPKAPDAAWQHDMYPAVAAGGGGGGRVSALETGTKLYISNLDFGVSNDDIKELFSELGDLKRFSIIYDRSGRSKGTAEVVFARRSDAVAAVKKYNNVQLDGKPMKIEIVGTNTPTASAALPVSNGGHARNAVRSAPRGAAPAGVPQRRPHQRGGRRSGGSGGGRRGKERSKPKSAEELDADLEKYHADAMQTNAAALEHHHHHH
Claims (10)
1. Use of a composition consisting of ethanol, sodium acetate and glycogen in a ratio of 500uL:3mol sodium acetate: 0.1g glycogen.
2. The composition of claim 1.
3. A method for detecting the abundance of N6-adenylate methylation modifications in mRNA bound by a protein, comprising: the method comprises the following steps:
s1, extracting mRNA of a plant;
s2, fragmenting the mRNA by metal ions to obtain a fragmented mRNA solution, and adding the composition of claim 1 to promote mRNA precipitation to obtain purified fragmented mRNA;
s3, expressing target proteins of the plants in vitro;
s4, incubating and combining the target protein of S3 and the purified fragmented mRNA of S2 in vitro to obtain a protein-RNA complex;
s5, eluting and separating the protein-RNA complex to obtain a target mRNA solution;
s6, adding the composition in claim 1 into the target mRNA solution to promote mRNA precipitation, so as to obtain purified target mRNA;
s7, preparing the purified target mRNA of the S6 into mononucleotide, further dephosphorylating, and purifying the mononucleotide to obtain an on-line sample;
S8, detecting by a liquid chromatography-mass spectrometry, quantifying the content of N6-methyl adenylate and adenine ribonucleoside in the sample of the machine according to S7 by adopting an external standard method, and dividing the content of N6-methyl adenylate by the content of adenine ribonucleoside to obtain the methylation modified abundance of protein binding N6-adenylate of the biological sample;
the N6-methyl adenosine acid content is quantified according to a chromatographic peak corresponding to an ion peak with collision energy of 16V and m/z of 282.1-150.1;
the adenine ribonucleoside content was quantified according to the chromatographic peak corresponding to the ion peak at collision energy of 10V and m/z of 268.1-136.1.
A method for detecting the methylation modified abundance of protein-bound N6-adenylate by rip-bound MS, characterized by: comprising the S1, the S2, the S3, the S4, the S5, the S6, and the S7 of claim 1, further comprising S8':
s8', detecting by a liquid chromatography, quantifying the content of N6-methyl adenylate and adenine ribonucleoside in the sample of the machine according to S7 by adopting an external standard method, and dividing the content of N6-methyl adenylate by the content of adenine ribonucleoside to obtain the methylation modified abundance of protein combined with N6-adenylate of the biological sample; the mobile phase adopted by the liquid chromatography consists of a phase A and a phase B, wherein the phase A is a formic acid aqueous solution with the volume percentage content of 0.1%, and the phase B is an acetonitrile solution of formic acid with the volume percentage content of 0.1%; the elution procedure used for liquid chromatography was as follows: from 0min to 8min, the volume ratio of the phase A to the mobile phase is linearly reduced from 100% to 99%; from more than 8min to 10min, the volume ratio of the A phase to the mobile phase is linearly reduced from less than 99% to 94%; from more than 10min to 15min, the volume ratio of the A phase to the mobile phase is linearly reduced from less than 94% to 0%; from more than 15min to 20min, the volume fraction of the A phase in the mobile phase is linearly increased from 0% to 100%; the chromatographic column adopted by the liquid chromatography is Thermo Scientific Hypersil GOLD aQ reverse phase column, the column length is 100mm, the column inner diameter is 2.1mm, and the particle size of the filler is 1.9um; the column temperature of the chromatographic column is 25 ℃, and the flow rate of the mobile phase is 0.3ml/min; the retention time of the chromatographic peak of the N6-methyl adenylate is 10.5+/-1 min; the retention time of the chromatographic peak of the adenine ribonucleotide is 5.5+ -1 min.
5. A method according to claim 3 or 4, characterized in that: the addition of the composition of claim 1 to promote mRNA precipitation as described in S2 or S6, the composition of claim 1 is added to the fragmented mRNA solution or the target mRNA solution to obtain a reaction system in which ethanol is contained in an amount of 2.5L, sodium acetate is contained in an amount of 0.3M, and glycogen is contained in an amount of 100. Mu.g ml -1 。
6. A method according to any one of claims 3-5, characterized in that: the addition of the composition of claim 1 to S2 or S6 promotes mRNA precipitation, which is carried out at-80 ℃.
7. The method according to any one of claims 3-6, wherein: and S2, the metal ions are zinc ions.
8. The method according to any one of claims 3-7, wherein: the fragmentation in S2 is performed in a reaction system a, which is a liquid consisting of: mRNA, tris-HCl, zinc chloride and water; the mRNA content was 20. Mu.g, the Tris-HCl content was 100mM, the zinc chloride content was 100mM, and the balance was water.
9. The method according to any one of claims 3-8, wherein: s4, in-vitro incubation and combination are that the reaction system B is incubated on ice for 1 hour, and then is incubated with the magnetic beads with His labels for 2 hours at the temperature of 4 ℃; the reaction system B is a liquid composed of the following substances: the target protein, the purified fragmented mRNA, tris-HCl with pH value of 7.5, sodium chloride, ethylenediamine tetraacetic acid, ethylphenyl polyethylene glycol, dithiothreitol, ribonuclease inhibitor and water; the content of the target protein is 20 mug/… mu l, the content of the purified fragmented mRNA is … mug/… mu l, the content of Tris-HCl is 50mM, the content of sodium chloride is 250mM, the content of ethylenediamine tetraacetic acid is 0.4mM, the mass percentage of ethylphenyl polyethylene glycol is 0.1%, the content of dithiothreitol is 1mM, the content of ribonuclease inhibitor is 0.4U/mu l, and the balance is water.
10. The method according to any one of claims 3-9, characterized in that: s5, eluting and separating the protein-RNA complex, namely performing rotary elution on the protein-RNA complex at 4 ℃ by using a buffer solution X to remove non-specific fragments, then performing digestion for 20min at 37 ℃ by using a PK buffer solution, and finally performing incubation for 20min at 37 ℃ by using 200 μl of PK-urea buffer solution to obtain a target mRNA solution;
the buffer solution X is a liquid composed of the following substances: tris-HCl with pH value of 7.5, sodium chloride, ethylenediamine tetraacetic acid, ethylphenyl polyethylene glycol, dithiothreitol, ribonuclease inhibitor and water; the content of sodium chloride is 250mM, the content of ethylenediamine tetraacetic acid is 0.4mM, the mass percentage of ethylphenyl polyethylene glycol is 0.1%, the content of dithiothreitol is 1mM, the content of ribonuclease inhibitor is 0.4U/μl, and the balance is water;
the PK buffer is a liquid consisting of: tris-HCl with pH value of 7.5, sodium chloride, ethylenediamine tetraacetic acid, proteinase K and water; the content of Tris-HCl is 100mM, the content of sodium chloride is 50mM, the content of ethylenediamine tetraacetic acid is 10mM EDTA, the content of proteinase K is 4 mug/μl, and the balance is water;
The PK-urea buffer is a liquid consisting of the following substances: tris-HCl with pH value of 7.5, sodium chloride, ethylenediamine tetraacetic acid, urea and water; the content of Tris-HCl is 100mM, the content of sodium chloride is 50mM, the content of ethylenediamine tetraacetic acid is 10mM EDTA, the content of urea is 7M, and the balance is water.
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