CN114181291A

CN114181291A - Poplar RNAm6A methyltransferase marker PtrMTA protein, polyclonal antibody and application

Info

Publication number: CN114181291A
Application number: CN202111446272.0A
Authority: CN
Inventors: 李瑞丽; 鲁良; 葛颜锐
Original assignee: Beijing Forestry University
Current assignee: Beijing Forestry University
Priority date: 2021-11-30
Filing date: 2021-11-30
Publication date: 2022-03-15

Abstract

The invention discloses a poplar RNA m⁶A methyltransferase marker PtrMTA protein, polyclonal antibody and application thereof, belonging to the technical field of epigenetics. The PtrMTA protein is derived from populus trichocarpa, has extremely strong specificity and is RNA m⁶A methyltransferase tagged protein; the invention clones the full length and prepares the polyclonal antibody of the anti-PtrMTA protein, and has high specificity and high sensitivity of immunoreaction.

Description

Poplar RNAm6A methyltransferase marker PtrMTA protein, polyclonal antibody and application

Technical Field

The invention relates to the technical field of epigenetics, in particular to populus tomentosa RNAm6A methyltransferase marker PtrMTA protein and application thereof.

Background

Epigenetics, also known as "pseudogenetics," is the study of reversible and heritable changes in gene function due to other mechanisms, including DNA modifications, histone modifications, and RNA modifications, without changes in nuclear DNA sequence. In recent years, more RNA modification functions are discovered, for example, 6-methyladenine modification can be involved in regulation of expression after gene transcription of organisms by regulating the shearing, positioning, transport and stability of mRNA.

m⁶A is a prevalent RNA epigenetic modification, catalyzed by the m6A methyltransferase complex. m is⁶The A methyltransferase complex methylates the N hydrogen at position 6 of adenine to form m with S-adenosyl methionine (SAM) as methyl donor⁶A. The complex is composed of 3 parts, including METTL3 (methytranferase like 3), METTL14 (methytranferase like 14) and WTAP (metals' promoter 1-associating protein). Wherein the METTL3 and METTL14 subunits can be present in a ratio of 1: 1 form a stable heterodimer, but the two are differentially split, METTL3 acts as a catalytic center, METTL14 binds primarily to RNA substrates. The WTAP subunit acts as a regulatory subunit, regulating the intracellular activity of the heterodimer. Deletion of any subunit of the complex results in m⁶A significant decrease in a level.

Current RNA m⁶The study of a modifications has become one of the popular areas of epigenetics. However, RNA m in plants⁶The study of A methyltransferases has lagged far behind animals, so molecular biological methods have been used to mine specific RNA m⁶A methyltransferase marker and further on RNA m⁶The intensive study of the modification A is a problem which needs to be solved by those skilled in the art.

Disclosure of Invention

In view of the above, the invention provides a populus tomentosa RNAm⁶A methyltransferase marker PtrMTA protein and its application in detecting and researching RNAm by prepared polyclonal antibody⁶A methyltransferase is important to reveal the RNA modification of populus tomentosa.

In order to achieve the purpose, the invention adopts the following technical scheme:

poplar RNAm⁶A methyltransferase marker PtrMTA protein, the amino acid sequence of which is set forth in SEQ ID NO: 1 is shown.

The method specifically comprises the following steps:

MEIHPNKNQNNNGEDKTVATIKNLRSRLESRIETQHKTQLDLLASLQDLVPNIVSSLDLSLQIVSSFNNKPFTPTPPLPASTSRAHLEIGSNSRNPNDPRANIDSRPEILGESSKLERKDGNFDGDGAESGSPLAVVRVMVAECLLQRVPFNPIDSSTVLRKLENDQNATEAEKAAIREVGGESGAILAVEMALRSMAEENRGIELEEFVVSGKSRVMVLNIDRNRLVKELPESAQYTQNLELSGSSDFNQNQSSGIGNNVNSNGGVDVNGNGVYGIGGPVVMQRPLMGDMWMGGGGDMWPRGGMMGPRGMMMGPRGMMQRPPMPLPMQSQQQKQRSEEDEMKDLEALLNKKSFREMQKSKTGEELLDLIHRPTARETAVAAKFKTKGGSQLKEYCSALTKEDCRRQCGSFIACEKVHFRRIIAPHTDVSQGDCSFLDTCRHMKTCKYVHYELDPTPDVSPMAMGAAALPPPKALKPQRAEYCSEVELGEPQWINCDIRNFKMEILGQFGVIMADPPWDIHMELPYGTMADDEMRNLNVPVLQTDGLIFLWVTGRAMELGRECLELWGYKRVEEIIWVKTNQLQRIIRTGRTGHWLNHSKEHCLVGIKGNPEVNRNIDTDVIVAEVRETSRKPDEMYPLLERISPRTRKLELFARMHNTQAGWMSLGNQLEGVRLVDEGLRARFKAAYPDVVVQPSSPPRASAMEIDSTASQMRSPFSVTESKSMATQFADRAAPETVYTSEEKPMALDAVMAG。

another object of the present invention is to provide a polyclonal antibody obtained by immunizing an animal with the above-mentioned PtrMTA protein as an antigen.

The PtrMTA protein obtained by the invention has good immunogenicity, the polyclonal antibody has good specificity and high titer, the titer is 1:51200, and the background value is low.

Still another object of the present invention is to provide the above method for preparing polyclonal antibody, wherein the PtrMTA protein is used to immunize an animal, the blood of the immunized animal is collected to prepare antiserum, and the antiserum is separated and purified to obtain the polyclonal antibody.

The invention firstly designs and synthesizes PtrMTA full-length protein, and then uses the PtrMTA protein as immunogen to successfully immunize animals to obtain purified polyclonal antibody, wherein the mode of immunizing the animals is selected from subcutaneous injection, intradermal injection or intraperitoneal injection, and the immunization dose is determined by the type of the immunized animals.

Still another object of the present invention is to provide the use of the above polyclonal antibody in the preparation of a test RNAm⁶Application in A preparation.

Compared with the prior art, the invention discloses and provides an RNAm⁶A methyltransferase marker PtrMTA protein. In order to further research the function of PtrMTA, the inventor clones and expresses the PtrMTA full-length protein, and obtains the PtrMTA antigen protein with high purity after purification. High-purity antigen is adopted to immunize rabbits and mice,high-titer polyclonal antibodies were obtained, and the purity and antibody titer were confirmed by SDS-PAGE and ELISA:

(1) the PtrMTA protein of the invention has extremely strong specificity and is RNA m⁶A methyltransferase tagged protein.

(2) The invention clones the whole length of the monoclonal antibody and prepares a corresponding polyclonal antibody, and the polyclonal antibody has high specificity and high sensitivity of immunoreaction.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below, it is obvious that the drawings in the following description are only embodiments of the present invention, and for those skilled in the art, other drawings can be obtained according to the provided drawings without creative efforts.

FIG. 1 is a graph of protein expression of engineering bacteria according to an embodiment of the present invention, where M is protein marker, lane 1 is non-induced, lane 2 is whole bacteria after sonication, lane 3 is supernatant after sonication, and lane 4 is precipitation after sonication;

FIG. 2 is a diagram showing a purification pattern of PtrMTA expressed protein according to an embodiment of the present invention, wherein M is protein marker, lane 1 is a result obtained by diluting purified protein 5 times, lane 2 is a result obtained by diluting purified protein 10 times, and lane 3 is a BSA control of 0.5 mg/ml;

FIG. 3 is a diagram showing the purity profile of a polyclonal antibody against PtrMTA detected by SDS-PAGE according to an embodiment of the present invention, wherein M is protein marker, and lane 1 is Anti-MTA;

FIG. 4 is a map of a polyclonal antibody against PtrMTA detected by ELISA according to an embodiment of the present invention;

FIG. 5 is a graph showing the results of Western blotting test according to an embodiment of the present invention.

Detailed Description

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The embodiment of the invention discloses a Chinese white poplar RNAm⁶A methyltransferase marker PtrMTA protein, polyclonal antibody and application, the experimental methods described in the following examples are all conventional methods unless otherwise specified; the reagents and biomaterials, if not specifically indicated, are commercially available and are not described in detail herein.

Example 1

Preparation of PtrMTA protein

(1) Designing specific primers according to PtrMTA gene sequence by taking a cDNA library of the Chinese white poplar as a template, connecting BamH I and EcoR I enzyme cutting sites at two ends of the primers, carrying out PCR amplification on the full length of the PtrMTA gene, recombining with pET30a through double enzyme cutting sites to obtain a recombinant plasmid pET30a-PtrMTA, transforming to a competent cell TOP10, carrying out amplification culture on TOP10, coating a plate, selecting a single clone to extract a plasmid for sequencing, and verifying that the sequence is correct.

PCR primers:

5'-CGGGATCCATGGAGATTCATCCAAACAAG-3', shown as SEQ ID NO: 2 is shown in the specification;

5'-TAAGAATGCGGCCGCTCAGCCAGCCATAACAGCA-3' as shown in SEQ ID NO: 3, respectively.

And (3) PCR system:

PCR procedure: 5min at 95 ℃; 35 cycles of 95 ℃ 30s 60 ℃ 30s 72 ℃ 2.5 min; stretching for 5min at 72 ℃.

(2) A pET30a-PtrMTA Plasmid (TIANGEN BIOTECH company TIANPrep Mini Plasmid Kit) was extracted from the positive bacterium which had been correctly sequenced, Codon plus competent cells were transformed, and then screened with kanamycin to obtain positive clones, which were then added with 0.5mmol/L IPTG and induced overnight at 16 ℃. The strains after induction are subjected to ultrasonic treatment (500W,180 times, 5s each time and 5s interval) and then centrifuged (12000rpm,15min), and the obtained supernatant and precipitate are subjected to SDS-PAGE electrophoresis respectively, so that a target band appears in the supernatant, and an expression product is soluble protein (shown in figure 1).

(3) Carrying out amplification culture on the positive clone strain in the step (2) and carrying out induced expression

1) 1-2. mu.l of activated bacterial suspension (Codon plus strain) was inoculated into 5mL of the corresponding resistant LB liquid medium and cultured at 37 ℃ at 200 rpm.

2) The cultured bacterial suspension was transferred to 250mL of the corresponding resistant LB liquid medium and cultured at 37 ℃ and 200rpm until OD was 0.6-0.8, and IPTG (0.5mM) was induced overnight at 16 ℃.

3) Collecting bacteria: 8000rpm, and 6 min. The supernatant was discarded. 4) Ultrasonic bacteria breaking: the bacteria are blown off by 20-30ml of 10mM Tris-HCl (pH8.0), ultrasonic crushing is carried out (500W,180 times, 5s each time and 5s interval), extraction is carried out (the bacteria suspension after ultrasonic crushing is taken, 12000rpm is carried out, centrifugation is carried out for 10min, the supernatant is carried out to another EP tube, the supernatant is removed and the precipitate is blown off by the equal volume of 10mM Tris-HCl (pH8.0) solution), soluble PtrMTA protein is combined by a Ni-NAT affinity chromatographic column, 100mM imidazole elutes PtrMTA protein, SDS-PAGE (shown in figure 2) is carried out, a strip is cut to carry out MALDI-TOF/TOF identification, and the expression product is confirmed to be the PtrMTA protein.

Example 2

Preparation of PtrMTA protein polyclonal antibody

Test animals: new Zealand female white rabbit (2kg)

Serum was isolated from rabbit leg vein blood before primary immunization as a pre-immune serum control. 400 mu g of PtrMTA full-length protein prepared by the method of example 1 and equal volume of complete Freund's adjuvant are mixed uniformly, fully emulsified and injected into the back of a New Zealand white rabbit at multiple points subcutaneously; two weeks later, performing primary boosting immunization, uniformly mixing 200 mu g of PtrMTA full-length protein stored at (-80 ℃) with an incomplete Freund's adjuvant with the same volume, fully emulsifying, and performing subcutaneous multi-point injection on the back of a New Zealand white rabbit; then, every 2 weeks of the boost immunity, and in the second boost immunity after 7 days from the rabbit leg vein blood, using ELISA method to determine antibody titer; and (3) bleeding carotid artery on the 7 th day after the fourth boosting immunization, standing at the room temperature of 20-25 ℃ for 4-5 hours, sucking serum, centrifuging at the temperature of 4 ℃ and 5000rpm for 5 minutes, collecting supernatant, subpackaging and freezing at the temperature of-80 ℃ for later use.

The supernatant was purified from Ig G using protein a: 1) 20-30ml of 10mM Tris-HCl (pH8.0) solution is used for resuspending the precipitate obtained by ultrasonic centrifugation and standing for 10 min. 2)12000rpm, centrifugal 10min, supernatant transfer to another tube for preservation. 3) 20-30ml of 10mM Tris-HCl (pH8.0) solution is used for resuspension of the pellet, and the pellet is allowed to stand for 10 min. 4)12000rpm, centrifugation for 10min, and discarding the supernatant. 5) Repeating the steps (3) and (4) once. 6) The pellet was resuspended by adding a small amount of 10mM Tris-HCl (pH8.0) solution, and then the protein was solubilized by adding 5-10 ml of 10mM Tris-HCl (pH8.0) solution containing 8M urea. 7)12000rpm, centrifugation for 10min, collecting supernatant, and taking 50. mu.l electrophoresis.

Polyclonal antibodies against PtrMTA were obtained and the purity of the polyclonal antibodies was determined by SDS-PAGE (shown in FIG. 3).

Example 3

Validation of polyclonal antibodies

The specificity and sensitivity of the antibody were verified by ELISA.

(1) Antigen (PtrMTA protein obtained in example 1) was diluted with the coating solution to a final concentration of 2. mu.g/mL, 100. mu.L/well, 4 ℃ overnight; then washing with washing liquor for 2 times;

(2) sealing with sealing solution, 200 μ L/well, incubating at 37 deg.C for 2h, and washing with lotion for 1 time;

(3) the polyclonal antibody obtained in example 2 is diluted by a gradient of 2 times from 200 times (PBS is used), a blank control is PBS, and a negative control is negative serum diluted by 200 times (PBS is used); 100 mu L/hole, incubating at 37 ℃ for 1h, and washing with washing solution for 3 times;

(4) adding PBS to dilute antibody 20000 times, 100 μ L/hole, incubating at 37 deg.C for 1h, taking out, and washing with washing solution for 3 times;

(5) developing with developing solution 100 μ L/hole for 5-15 min;

(6) adding 50 mu L of stop solution into each hole to stop;

(7) absorbance values were measured at two wavelengths (450, 630).

The ELISA experiment result (as shown in figure 4) shows that the titer of the polyclonal antibody of the invention is 1:51200, and the polyclonal antibody has extremely strong specificity and high sensitivity with PtrMTA protein,background values were low for identification and further study of PtrMTA protein and RNA m⁶A methyltransferase is of great significance.

Example 4

Western blot

(1) 50mg of 84K overexpressed poplar leaves were weighed, 200. mu.L of a protein extract (protein extract: 0.125M (pH 8.8)) was added, and 1g of SDS, 10mL of glycerol, and 0.05M of Na were weighed₂SO₅Then add ddH₂O is added to 100mL to be fully dissolved), ground into homogenate, centrifuged at 12000rpm at normal temperature for 10min, and carefully absorbed into a new centrifuge tube;

(2) sucking 20 μ L of supernatant, measuring total protein content with enzyme-labeling instrument, adding the rest supernatant into 1/10 volume sample buffer solution, boiling in water bath for 10min, 2 times, and storing in-20 deg.C refrigerator;

(3) thoroughly cleaning the glass plate, aligning the glass plate, putting the glass plate into a clamp for clamping, and vertically clamping the glass plate on a frame for preparing glue pouring;

(4) 15% separation gel (10mL) was prepared by adding 5mL of 30% acrylamide, 2.5mL of 1.5M Tris-HCl (pH 8.8), 0.1mL of 10% SDS, 30. mu.L of 15% APS, 5. mu.L of TEMED (tetramethylethylenediamine), and ddH₂O to 10mL), shaking up immediately after adding TEMED, sucking 5mL of separation gel with a 10mL gun along the glass until the gel surface rises to the height of the middle line of the green band. Then adding absolute ethyl alcohol to the glue for liquid sealing. After the gel is solidified, the absolute ethyl alcohol on the upper layer of the gel is poured out and is sucked dry by absorbent paper. The above procedure was followed to prepare 5% concentrated gel (5mL) by adding 30% acrylamide 0.83mL, 1.5M Tris-HCl (pH 8.8)0.63mL, 10% SDS 50. mu.L, 15% APS 30. mu.L, TEMED (tetramethylethylenediamine) 5. mu.L, and ddH₂O to 5mL), adding TEMED, shaking up immediately, filling the rest space of the rubber plate with concentrated glue, and then inserting a comb;

(5) washing the concentrated gel with water, placing into an electrophoresis tank, adding sufficient electrophoresis solution, sucking with a gun head, and discharging air bubbles. Adding the denatured protein sample according to the concentration of the protein to be measured; note that: the sample is added slowly during the sample adding, the sample can rush out the sample adding hole due to too fast sample adding, and the generation of bubbles is avoided during the sample adding process. Before adding the next sample, the sample injector needs to be washed in the electrophoresis buffer solution for 3 times, so that cross contamination is avoided;

(6) and after the sample adding is finished, carrying out electrophoresis for 4-5h, wherein during the electrophoresis, carrying out electrophoresis at 60V for 30min, then carrying out electrophoresis at 120V continuously until bromophenol blue just runs out of the lowest edge of the separation gel, stopping the electrophoresis, and carrying out membrane conversion.

(7) 6 filter papers and 1 nitrocellulose membrane were prepared and soaked in the membrane transfer buffer before use. The clamp is opened to keep the black side horizontal, a spongy cushion is arranged on the black side, and air bubbles in the spongy cushion are rolled away by a glass rod. Then three layers of filter paper are padded on the sponge cushion, and air bubbles are rolled out by a glass rod.

(8) Prying off the glass plate, carefully taking down the separation gel, covering the separation gel on filter paper, properly adjusting to align the separation gel with the filter paper, slightly rolling the separation gel with a glass rod to remove bubbles, then covering 3 pieces of filter paper on the membrane, continuously removing the bubbles with the glass rod, finally covering another sponge pad, rolling the bubbles, closing the clamp, and enabling the whole clamp to be in a sandwich type of a blackboard, a sponge, 3 layers of filter paper, gel, a nitrocellulose membrane, filter paper, a sponge and a whiteboard; note that: the whole operation process is carried out in the transfer liquid, and bubbles are continuously rolled out;

(9) placing the clamp in a transfer groove, wherein the black surface of the clamp faces the black surface of the groove, the white surface of the clamp faces the red surface of the groove, and rotating the film for 90min at 180 mA;

(10) transferring the membrane to a plate containing a sealing solution (5% skimmed milk powder dissolved in TTBS solution), and sealing for 1h on a decoloring shaker at room temperature or sealing overnight at 4 ℃;

(11) diluting the blocking solution to a concentration of 1% by using TTBS, then diluting the primary antibody to a proper concentration, putting the blocked nitrocellulose membrane and the diluted primary antibody (PtrMTA protein antibody) into a hybridization bag together, and incubating for 2h at normal temperature (or incubating overnight at 4 ℃); then rinsing the nitrocellulose membrane with TTBS for 5min each time 3 times on a decoloring shaker at room temperature;

(12) diluting the secondary antibody with 1% skimmed milk powder, putting the nitrocellulose membrane and the diluted secondary antibody into a hybridization bag together, and incubating for 1h at room temperature; then rinsing the nitrocellulose membrane with TTBS for 5min each time for 3 times on a decoloring shaker at the temperature;

(13) mixing the solution A and the solution B of the Western Blot luminescent solution in equal amount, paving a layer of preservative film on a stage of a chemiluminescence imager, then placing a nitrocellulose membrane on the preservative film, uniformly dripping the prepared Western Blot luminescent solution on the nitrocellulose membrane, setting exposure time in a chemiluminescence imaging system, and taking a picture.

Results of western blot experiments (as shown in figure 5). The polyclonal antibody of the invention is combined with the PtrMTA protein in the plant, which shows that the PtrMTA protein antibody has extremely strong specificity and high sensitivity, and plays an important role in the deep research of methyltransferase in plants.

The embodiments in the present description are described in a progressive manner, each embodiment focuses on differences from other embodiments, and the same and similar parts among the embodiments are referred to each other.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Beijing university of forestry

<120> poplar RNAm6A methyltransferase marker PtrMTA protein, polyclonal antibody and application

<160> 3

<170> SIPOSequenceListing 1.0

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<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Glu Ile His Pro Asn Lys Asn Gln Asn Asn Asn Gly Glu Asp Lys

1 5 10 15

Thr Val Ala Thr Ile Lys Asn Leu Arg Ser Arg Leu Glu Ser Arg Ile

20 25 30

Glu Thr Gln His Lys Thr Gln Leu Asp Leu Leu Ala Ser Leu Gln Asp

35 40 45

Leu Val Pro Asn Ile Val Ser Ser Leu Asp Leu Ser Leu Gln Ile Val

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Ser Ser Phe Asn Asn Lys Pro Phe Thr Pro Thr Pro Pro Leu Pro Ala

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Ser Thr Ser Arg Ala His Leu Glu Ile Gly Ser Asn Ser Arg Asn Pro

85 90 95

Asn Asp Pro Arg Ala Asn Ile Asp Ser Arg Pro Glu Ile Leu Gly Glu

100 105 110

Ser Ser Lys Leu Glu Arg Lys Asp Gly Asn Phe Asp Gly Asp Gly Ala

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Glu Ser Gly Ser Pro Leu Ala Val Val Arg Val Met Val Ala Glu Cys

130 135 140

Leu Leu Gln Arg Val Pro Phe Asn Pro Ile Asp Ser Ser Thr Val Leu

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Arg Lys Leu Glu Asn Asp Gln Asn Ala Thr Glu Ala Glu Lys Ala Ala

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Ile Arg Glu Val Gly Gly Glu Ser Gly Ala Ile Leu Ala Val Glu Met

180 185 190

Ala Leu Arg Ser Met Ala Glu Glu Asn Arg Gly Ile Glu Leu Glu Glu

195 200 205

Phe Val Val Ser Gly Lys Ser Arg Val Met Val Leu Asn Ile Asp Arg

210 215 220

Asn Arg Leu Val Lys Glu Leu Pro Glu Ser Ala Gln Tyr Thr Gln Asn

225 230 235 240

Leu Glu Leu Ser Gly Ser Ser Asp Phe Asn Gln Asn Gln Ser Ser Gly

245 250 255

Ile Gly Asn Asn Val Asn Ser Asn Gly Gly Val Asp Val Asn Gly Asn

260 265 270

Gly Val Tyr Gly Ile Gly Gly Pro Val Val Met Gln Arg Pro Leu Met

275 280 285

Gly Asp Met Trp Met Gly Gly Gly Gly Asp Met Trp Pro Arg Gly Gly

290 295 300

Met Met Gly Pro Arg Gly Met Met Met Gly Pro Arg Gly Met Met Gln

305 310 315 320

Arg Pro Pro Met Pro Leu Pro Met Gln Ser Gln Gln Gln Lys Gln Arg

325 330 335

Ser Glu Glu Asp Glu Met Lys Asp Leu Glu Ala Leu Leu Asn Lys Lys

340 345 350

Ser Phe Arg Glu Met Gln Lys Ser Lys Thr Gly Glu Glu Leu Leu Asp

355 360 365

Leu Ile His Arg Pro Thr Ala Arg Glu Thr Ala Val Ala Ala Lys Phe

370 375 380

Lys Thr Lys Gly Gly Ser Gln Leu Lys Glu Tyr Cys Ser Ala Leu Thr

385 390 395 400

Lys Glu Asp Cys Arg Arg Gln Cys Gly Ser Phe Ile Ala Cys Glu Lys

405 410 415

Val His Phe Arg Arg Ile Ile Ala Pro His Thr Asp Val Ser Gln Gly

420 425 430

Asp Cys Ser Phe Leu Asp Thr Cys Arg His Met Lys Thr Cys Lys Tyr

435 440 445

Val His Tyr Glu Leu Asp Pro Thr Pro Asp Val Ser Pro Met Ala Met

450 455 460

Gly Ala Ala Ala Leu Pro Pro Pro Lys Ala Leu Lys Pro Gln Arg Ala

465 470 475 480

Glu Tyr Cys Ser Glu Val Glu Leu Gly Glu Pro Gln Trp Ile Asn Cys

485 490 495

Asp Ile Arg Asn Phe Lys Met Glu Ile Leu Gly Gln Phe Gly Val Ile

500 505 510

Met Ala Asp Pro Pro Trp Asp Ile His Met Glu Leu Pro Tyr Gly Thr

515 520 525

Met Ala Asp Asp Glu Met Arg Asn Leu Asn Val Pro Val Leu Gln Thr

530 535 540

Asp Gly Leu Ile Phe Leu Trp Val Thr Gly Arg Ala Met Glu Leu Gly

545 550 555 560

Arg Glu Cys Leu Glu Leu Trp Gly Tyr Lys Arg Val Glu Glu Ile Ile

565 570 575

Trp Val Lys Thr Asn Gln Leu Gln Arg Ile Ile Arg Thr Gly Arg Thr

580 585 590

Gly His Trp Leu Asn His Ser Lys Glu His Cys Leu Val Gly Ile Lys

595 600 605

Gly Asn Pro Glu Val Asn Arg Asn Ile Asp Thr Asp Val Ile Val Ala

610 615 620

Glu Val Arg Glu Thr Ser Arg Lys Pro Asp Glu Met Tyr Pro Leu Leu

625 630 635 640

Glu Arg Ile Ser Pro Arg Thr Arg Lys Leu Glu Leu Phe Ala Arg Met

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His Asn Thr Gln Ala Gly Trp Met Ser Leu Gly Asn Gln Leu Glu Gly

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Val Arg Leu Val Asp Glu Gly Leu Arg Ala Arg Phe Lys Ala Ala Tyr

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Pro Asp Val Val Val Gln Pro Ser Ser Pro Pro Arg Ala Ser Ala Met

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Glu Ile Asp Ser Thr Ala Ser Gln Met Arg Ser Pro Phe Ser Val Thr

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taagaatgcg gccgctcagc cagccataac agca 34

Claims

1. Poplar RNAm⁶A methyltransferase marker PtrMTA protein, wherein the amino acid sequence of the PtrMTA protein is as set forth in SEQ ID NO: 1 is shown.

2. A polyclonal antibody obtained by immunizing an animal with the PtrMTA protein of claim 1 as an antigen.

3. The method for producing a polyclonal antibody according to claim 2, wherein the PtrMTA protein is used to immunize an animal, the blood of the immunized animal is collected to produce an antiserum, and the antiserum is isolated and purified to obtain the polyclonal antibody.

4. Use of polyclonal antibodies according to claim 2 for the detection of RNAm⁶Application in A preparation.