Betula platyphylla m6A recognition protein BplCPSF30-L and coding gene and application thereof

Abstract

The invention discloses a white birch m6A recognition protein BplCPSF30-L as well as a coding gene and application thereof, and relates to the technical field of plant genetic engineering. The white birch m6A recognition protein BplCPSF30-L disclosed by the invention has an amino acid sequence as shown in SEQ ID NO. 1. The method comprises the following steps: constructing an expression vector of the white birch m6A recognition protein BplCPSF30-L, and transforming the expression vector into a mutant arabidopsis thaliana; and cultivating and screening to obtain a transgenic arabidopsis thaliana plant with advanced flowering time. The result shows that the late flower phenotype of the arabidopsis thaliana cpsf30-1 mutant can be recovered by overexpressing the BplCPSF30-L in the arabidopsis thaliana cpsf30-1 mutant; and the expression quantity of the flowering related gene in the transgenic arabidopsis thaliana is also recovered. The invention provides a molecular means and basis for promoting the flowering of the white birch and shortening the breeding period of the white birch.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to birch m 6 A recognition protein BplCPSF30-L, its encoding gene and application. Background Art

[0002] The long generation and breeding cycle of forest trees is the main constraint on forest tree breeding. Flowering is the development of flowers from nutrient trees. It can not only effectively improve the growth cycle of forest trees and enrich the floral development regulation mechanism of forest trees, but also the transgenic lines obtained are expected to provide ideal materials for genetic research of forest trees. However, the research on plant floral regulation is mainly concentrated in the annual model plant Arabidopsis thaliana. Compared with model plants, the research on floral development of forest trees is relatively weak. Therefore, it is necessary to study the floral development regulation of forest trees.

[0003] Birch is a world-wide tree species widely distributed in Eurasia. It is an important component of forest ecosystems and plays an important role in maintaining ecological balance. It can provide habitats for many wild animals and plants and promote biodiversity. At the same time, the fallen leaves and roots of birch trees help improve soil fertility and provide a good environment for the growth of other plants. Therefore, birch has important ecological value. The wood of birch is soft and light, with fine texture. It is mostly used to make agricultural tools and wooden utensils. Its white papery bark can be made into canoes, exquisite items and food baskets, etc. It is also used as a substitute for tiles and has important economic value. However, compared with crop breeding, birch grows slower and has a longer breeding cycle. Therefore, studying genes that promote flowering of birch is conducive to shortening the breeding cycle of birch and improving the breeding efficiency of birch.

[0004] Existing studies have reported that CPSF30-L is a 6 A recognition protein, dependent on its m 6 A binding activity, promoting flowering of Arabidopsis. However, research on the function of the BplCPSF30-L gene in birch is still blank. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a 6 A recognition protein BplCPSF30-L. Another technical problem to be solved by the present invention is to provide birch m 6 A recognition protein BplCPSF30-L encoding gene. The technical problem to be solved by the present invention is to provide birch m 6 Application of A recognition protein BplCPSF30-L for regulating plant flowering time.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A kind of birch 6 A recognition protein BplCPSF30-L, whose amino acid sequence is shown in SEQ ID NO.1.

[0008] Birch 6 The nucleotide sequence of the gene encoding the A recognition protein BplCPSF30-L is shown in SEQ ID NO.2.

[0009] Contains birch m 6 A. Vector and recombinant bacteria that recognize protein BplCPSF30-L.

[0010] Birch 6 Application of the A recognition protein BplCPSF30-L in regulating the flowering time of mutant plants, wherein the regulating the flowering time of mutant plants is to promote the mutant plants to bloom earlier.

[0011] Birch 6 Application of A recognition protein BplCPSF30-L in regulating flowering time of mutant plants, including:

[0012] 1) Build Birch m 6 A expression vector for the recognition protein BplCPSF30-L;

[0013] 2) The constructed birch m 6 The expression vector of A recognition protein BplCPSF30-L was transformed into mutant Arabidopsis thaliana;

[0014] 3) Cultivate, screen and obtain transgenic Arabidopsis plants with advanced flowering time.

[0015] Birch 6 Application of the A recognition protein BplCPSF30-L in regulating the expression level of flowering-related genes, wherein the regulation of the expression level of flowering-related genes is to promote the expression of the flowering-related gene SOC1.

[0016] Birch 6 Application of A recognition protein BplCPSF30-L in regulating plant leaf number.

[0017] Birch 6 Application of A recognition protein BplCPSF30-L in regulating plant leaf arrangement number.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] The birch m disclosed in the present invention 6A recognition protein BplCPSF30-L, the amino acid sequence of which is shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2. 6 A recognition protein BplCPSF30-L expression vector; the constructed birch m 6 The expression vector of the A recognition protein BplCPSF30-L was transformed into a mutant Arabidopsis; transgenic Arabidopsis plants with early flowering time were cultivated and screened. The results showed that overexpression of BplCPSF30-L in the Arabidopsis cpsf30-l mutant could promote flowering of the Arabidopsis cpsf30-l mutant, while overexpression of BplCPSF30-Lm (W279A, W330A) could not restore flowering of the Arabidopsis cpsf30-l mutant; the expression of flowering-related genes in the transgenic Arabidopsis was also restored. The present invention provides a molecular means and basis for promoting flowering of birch and shortening the breeding cycle of birch. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The expression and purification diagram of YTH-6×His / BplCPSF30-L and YTHm-6×His / BplCPSF30-L proteins (M is a marker; 1 and 3 are proteins before induction; 2 and 4 are induced proteins; 5 and 6 are purified recombinant proteins);

[0021] Figure 2 Figure 1 is the result of EMSA;

[0022] Figure 3 This is a screening diagram for BplCPSF30-L gene-positive plants;

[0023] Figure 4 The phenotype diagram of the BplCPSF30-L gene-transfected Arabidopsis cpsf30-1 mutant (A is the flowering phenotype diagram of WT, atcpsf30-1, 35S::BplCPSF30-L / atcpsf30-1 and 35S::BplCPSF30-Lm / atcpsf30-1 plants; B is the statistics of the total number of leaves and flowering time of WT, atcpsf30-1, 35S::BplCPSF30-L / atcpsf30-1 and 35S::BplCPSF30-Lm / atcpsf30-1 plants when flowering; C is a schematic diagram of the average number of leaves per Arabidopsis plant when flowering);

[0024] Figure 5 The expression level of SOC1 gene in 12-day-old Arabidopsis cpsf30-l mutant transformed with BplCPSF30-L gene;

[0025] Figure 6This is the decay curve of SOC1 mRNA in 35S::BplCPSF30-L / cpsf30-1 and cpsf30-1. DETAILED DESCRIPTION

[0026] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be carried out with reference to the methods listed in the book "Molecular Cloning Experiment Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0027] The materials used in this application are 2-year-old birch and Arabidopsis thaliana cpsf30-l mutants (characterized by delayed flowering) (published at https: / / doi.org / 10.1016 / j.molp.2021.01.014), both of which are deposited in the National Key Laboratory of Forest Genetics and Breeding, Nanjing Forestry University.

[0028] Example 1

[0029] 1. Total RNA extraction and cDNA acquisition

[0030] The total RNA of 2-year-old birch leaves was extracted using a plant total RNA extraction kit (TIANGEN), and the specific operation was carried out according to the instructions. The results of 1.2% agarose gel electrophoresis are shown in Figure 1 The bands shown are clearer; the absorbance OD of total RNA 260 / OD 280 The value is 2.32, OD 260 / OD 230 The value is 2.09, which can be used for gene cloning.

[0031] The extracted total RNA was used as a template to synthesize cDNA using a reverse transcription kit (Vazyme), and the specific operations were performed according to the instructions.

[0032] 2. Cloning of target gene

[0033] According to the BplCPSF30-L gene sequence downloaded from the Betula platyphylla genome, intermediate fragment-specific primers were designed using Primer 5.0. The ORF cloning primer sequence of the BplCPSF30-L gene is as follows:

[0034] BplCPSF30-L-ORF-F:

[0035] 5'-ATGGAGGACACGGAGGGAGTTCTAA-3',

[0036] BplCPSF30-L-ORF-R:

[0037] 5'-TCAGTGATCGGAGGCAGTGGTAG-3'.

[0038] The PCR reaction system was: 2 μL Forward primer (10 uM / L), 2 μL Reverse primer (10 uM / L), 2 μL Template cDNA (100 ng / μL), 25 μL 2×Taq PCR MASTER Mix, and 19 μL ddH2O.

[0039] The PCR reaction conditions were as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 58°C for 10 s, extension at 72°C for 45 s, 35 cycles, and extension at 72°C for another 5 min.

[0040] Connect vector and transform: add 4μL product to a centrifuge tube, then add 1μL Blunt vector, mix and react at room temperature for 15min. After the reaction, add 50μL of freshly thawed Trans1-T1 competent cells, mix and place on ice for 30min. Place the connected product in a 42℃ metal bath for 40s, then immediately place it in ice for 2min. Add 250μL LB liquid culture medium (without Kan) equilibrated to room temperature, and culture at 200rpm and 37℃ for 1h. Centrifuge at 10000rpm for 30s, discard 150μL of supernatant, mix the remaining culture medium with a pipette, and spread it on Kan-resistant culture medium (the culture medium was placed in a 37℃ incubator for 1h in advance), and culture it in a 37℃ incubator overnight. After positive clone detection, send it to Qingke Bio for sequencing.

[0041] The final ORF sequence of the BplCPSF30-L gene obtained by sequencing is shown in SEQ ID NO.1, and the coding region of BplCPSF30-L was translated into an amino acid sequence shown in SEQ ID NO.2 using ORFfinder.

[0042] Example 2

[0043] 1. Connection and transformation of target fragment and vector

[0044] Use pET28a vector as prokaryotic expression vector, gently mix 4 μL BplCPSF30-L gene fragment with 1 μL pET28a, react at room temperature for 5 minutes, place on ice after the reaction, and the recombinant plasmid is named pET28a-BplCPSF30-L. Transform Escherichia coli DH5α and apply Amp-resistant LB solid medium. Sequencing is performed after positive detection. The primer sequence for positive clone detection is as follows:

[0045] pET28a-F: 5'-TAATGCCGCTGCGCAACTCATG-3';

[0046] pET28a-R: 5′-CTTTGTTAGCAGCCGGATCGC-3′.

[0047] 2. Mutations in key sites of the BplCPSF30-L gene

[0048] The 279th and 330th W amino acids in the BplCPSF30-L amino acid sequence were mutated to A using a point mutation kit (Vazyme).

[0049] 1) Design a pair of reverse complementary or partially reverse complementary primers at each site to be mutated to divide the plasmid into two segments for amplification. The long segment is named B and the short segment is named A. Then the A segment and the B segment are recombined to obtain a plasmid named pET28a-BplCPSF30-Lm. Transform Escherichia coli DH5α and apply Amp-resistant LB solid medium. Sequencing is performed after positive detection. The primer sequences are as follows:

[0050] BplCPSF30-L-AF:

[0051] 5'-AAGGAGTAGCGGCAACTCAAAGGAGCAACGAAGCT-3',

[0052] BplCPSF30-L-AR:

[0053] 5'-GGGCAACGCGAAATATGCACATGGAACTGCACATTAT-3';

[0054] BplCPSF30-L-BF:

[0055] 5'-GAGTTGCCGCTACTCCTTGTTGTACAGATAATTCCA-3',

[0056] BplCPSF30-L-BR:

[0057] 5'-GTGCATATTTCGCGTTGCCCCCACCTACAGAACC-3'.

[0058] The PCR reaction system (50μL) is: 2μL Forward primer (10uM / L), 2μL Reverse primer (10uM / L), 2μL Template cDNA (100ng / μL), 25μL 2×Taq PCR MASTER Mix, 19μL ddH2O.

[0059] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, denaturation at 95°C for 10 s, annealing at 58°C for 10 s, extension at 72°C for 5 min, 35 cycles, and extension at 72°C for another 5 min.

[0060] 2) To prevent the original template plasmid contained in the amplified product from forming false positive transformants after transformation, Dpn I digestion was performed before recombination circularization to remove the methylated template plasmid.

[0061] The PCR reaction system (50 μL) was: 1 μL Dpn I, 49 μL fragment A or 49 μL fragment B.

[0062] The PCR reaction program was: constant temperature reaction at 37°C for 1 h.

[0063] 3) The digested A and B fragments are recombined using a recombinase.

[0064] The PCR reaction system (50 μL) was: 2 μL of Dpn I digestion product of segment A, 2 μL of Dpn I digestion product of segment B, 4 μL of 5×CE II Buffer, 2 μL of Exnase II, and 9 μL of ddH2O.

[0065] The PCR reaction procedure is: 37°C, 30 min; cool to 4°C or immediately cool on ice.

[0066] The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) according to the same steps as in Example 1.

[0067] Example 3

[0068] The protein expression vectors pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L were constructed and transformed into Escherichia coli for expression and protein purification. 6 A modified sequence was subjected to EMSA to observe whether binding occurred.

[0069] 1. Construction of pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L vectors

[0070] Using pET28a vector as a prokaryotic expression vector, 4 μL YTH / BplCPSF30-L and 4 μL YTHm / BplCPSF30-L were gently mixed with pET28a, reacted at room temperature for 5 minutes, and placed on ice after the reaction to construct pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L vectors. The pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L vectors were transformed into Escherichia coli Trans1-T1, and coated with Amp-resistant LB solid medium. Sequencing was performed after positive detection. The primer sequences for positive clone detection are as follows:

[0071] T7 promoter primer: 5′-TAATACGACTCACTATAGGG-3′;

[0072] T7 terminator primer: 5′-TGCTAGTTATTGCTCAGCGG-3′.

[0073] The recombinant plasmid was transformed into Escherichia coli BL21 (DE3) in the same manner as in Example 1. pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L strains were constructed.

[0074] 2. Prokaryotic expression analysis and protein purification

[0075] 1) IPTG induced protein expression

[0076] Pick the pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L monoclonal strains respectively and put them into 5 mL LB liquid medium containing 100 mg / L Amp, and shake them at 200 rpm at 37°C for 12 hours. Inoculate the bacterial liquid into 50 mL LB liquid medium containing 100 mg / L Amp, and shake them at 37°C and 200 rpm for 3-4 hours until the OD 600=0.8, take out 10mL of bacterial solution as a control and temporarily store it at 4℃. Divide the remaining bacterial solution into two parts and add 0.5mmol / L and 1mmol / L IPTG to induce protein expression respectively, and shake the bacteria at 37℃ and 200rpm for 6-8h. Centrifuge all the bacterial solution at 6000rpm for 8min to collect the bacteria and discard the supernatant. Add 10mL1×PBS to shake and resuspend the bacteria, centrifuge at 6000rpm for 8min to collect the bacteria, discard the supernatant, repeat twice, add 10mL 1×PBS to shake and resuspend the bacteria, and prepare to break. Use the freeze-thaw method to break: first put it in liquid nitrogen for quick freezing, then thaw it in a 37℃ water bath, shake it, and repeat 4-6 times. After the breaking is completed, centrifuge at 6000rpm for 8min to collect the bacteria, take the supernatant, and store it at 4℃ for later use.

[0077] 2) Detection of target protein by polyacrylamide gel electrophoresis (SDS-PAGE)

[0078] Prepare gel: install the glass plate into the gel mold, prepare 10mL of 10% separation gel according to the instructions, immediately inject it into the mold, inject distilled water to the level of the glass, wait for 30-40 minutes, after the separation gel solidifies, pour out the distilled water and absorb the remaining water with filter paper, then prepare 4mL of 5% concentrated gel, inject it into the mold, insert the comb, let it stand for about 40 minutes, then put it into the electrophoresis tank and wait for the gel to run.

[0079] Prepare 5× Tris-glycine electrophoresis buffer: add 0.125 mol / L Tris, 1.25 mol / L glycine, and 0.5% SDS to deionized water and store at room temperature.

[0080] After spotting, set the voltage to 160V and the electrophoresis time to 50 minutes. Then take out the gel, rinse it, stain it with Coomassie Brilliant Blue Ultrafast Staining Solution for 30-40 minutes, decolorize it with clean water until the protein bands are clearly visible, and take pictures to record.

[0081] 3) Protein purification

[0082] Prepare the protein supernatant and perform protein purification according to the instructions of Ni-NTA affinity chromatography medium. Load the medium into the column, wash with four column volumes of equilibrium buffer, add the protein clarification sample, control the flow rate to 0.5-1mL / min, and then wash the column with washing buffer at a flow rate of 1mL / min to remove impurities until the A280 value of the effluent reaches the lowest and stable. Finally, use 5-10 column volumes of elution buffer at a flow rate of 0.5-1mL / min to elute, collect the eluate in separate tubes, measure the A280 value to the lowest and stable, stop collecting, and take the eluate with the highest A280 value for electrophoresis detection. The buffer formula is as follows (all dissolved in distilled water):

[0083] Lysis equilibrium buffer (LE Buffer): 50mM Na2HPO4+0.3M NaCl;

[0084] Washing buffer: 50 mM Na2HPO4 + 0.3 M NaCl + 10 mM imidazole;

[0085] Elution buffer: 50 mM Na2HPO4 + 0.3 M NaCl + 250 mM imidazole.

[0086] The results are as follows Figure 1 As shown, the recombinant protein "YTH-6×His / BplCPSF30-L" was purified from the "pET28a-YTH / BplCPSF30-L" strain; the recombinant protein "YTHm-6×His / BplCPSF30-L" was purified from the "pET28a-YTHm / BplCPSF30-L" strain. Figure 1 The first line in B is the protein induced to be expressed, with many impurities. The second to fourth lines are washed with three times of flow cytometry, and the impurities are washed away. The fifth line is the elution to collect the YTH-6×His / BplCPSF30-L protein on the column. Figure 1 The first line in C is the induced expressed protein with many impurity bands. The second to fourth lines are washed with flow cytometry three times to remove the impurities. The fifth line is the elution to collect the YTHm-6×His / BplCPSF30-L protein on the column.

[0087] 3. EMSA

[0088] The recombinant proteins YTH-6×His / BplCPSF30-L and YTHm-6×His / BplCPSF30-L were diluted to a concentration of 0 to 8 mM in binding buffer (10 mM HEPES [pH 8.0], 50 mM KCl, 1 mM EDTA, 0.05% Triton X-100, 5% glycerol, 10 mg / mL salmon DNA, 1 mM DTT, and 40 U / mL RNase inhibitor). 1 microliter of fluorescein amide (FAM)-labeled RNA probe (final concentration of 4 nM) and 1 mL of protein were mixed in 10 mL of binding buffer and incubated on ice for 30 minutes. The entire RNA-protein mixture was loaded into a Novex 4%-20% TBE gel (Thermo Fisher Scientific) and separated by electrophoresis at 4°C. The gel was visualized by ChemiDoc (Bio-Rad).

[0089] The results are as follows Figure 2As shown, YTH-6×His / BplCPSF30-L specifically binds to m 6 A-modified RNA, but not unmethylated RNA, and YTHm-6×His / BplCPSF30-L lost recognition of m 6 A's ability.

[0090] Example 4

[0091] After constructing different vectors in this example, Arabidopsis cpsf30-1 mutants were directly transformed to construct transgenic lines (35S::BplCPSF30-Lm / atcpsf30-1, 35S::BplCPSF30-L / atcpsf30-1, atcpsf30-1) for functional verification. Arabidopsis cpsf30-1 mutant plants have the characteristic of delayed flowering.

[0092] 1. Construction of pCAMBIA1305-BplCPSF30-L vector

[0093] The pCAMBIA-1305 expression vector was treated with XbaI and PstI endonucleases for restriction digestion. XbaI and PstI restriction sites were added to the upstream and downstream of BplCPSF30-L and BplCPSF30-Lm fragments by PCR to construct pCAMBIA1305-BplCPSF30-L and pCAMBIA1305-BplCPSF30-Lm overexpression vectors. Separation was performed by 1.2% agarose gel electrophoresis; the digestion products were recovered and purified using Gel DNA Extraction Mini Kit and dissolved in 20 μL Elution Buffer. The primer sequences are as follows:

[0094] 1305-BplCPSF30-LF:

[0095] 5′-tttggagagaacacgtctagaATGGAGGACACGGAGGGAGT-3′;

[0096] 1305-BplCPSF30-LR:

[0097] 5′-gtcaagcttgcatgcctgcagGTGATCCGGAGGCAGTGGTAGC-3′.

[0098] After the PCR product is sequenced correctly, the vector is digested with enzymes, connected, transformed, tested positive, and sent for testing. The correctly sequenced plasmid is transferred into Agrobacterium for transformation of Arabidopsis thaliana.

[0099] The double enzyme digestion system (50μL) of the expression vector is: 1μg pCAMBIA1305 plasmid, 1μL QuickCut XbaⅠ, 1μL QuickCut Pst I, 5μL 10×QuickCut Buffer, ddH2O up to 50μL.

[0100] The double enzyme digestion procedure of the expression vector was: 37°C for 30 min; 85°C for 20 s.

[0101] 2. Genetic transformation of BplCPSF30-L gene into Arabidopsis thaliana

[0102] 1) Disinfection of Arabidopsis seeds

[0103] Take an appropriate amount of cpsf30-l mutant seeds and put them into a sterilized EP tube, add an appropriate amount of 75% ethanol, shake and disinfect for 30 seconds, then suck out; add an equal amount of 0.1% mercuric chloride, sterilize for 2.5 minutes, then suck out (discard in a dedicated waste liquid tank); add an equal amount of deionized water, shake and wash, then suck out and put the seeds in a new EP tube; repeat the water washing four times.

[0104] 2) Cultivation of Arabidopsis thaliana

[0105] The sterilized seeds were placed in a suspension and sown evenly into 1 / 2MS culture medium using a pipette; the culture medium was sealed and placed in a 4°C refrigerator for vernalization for 3 days; the vernalized culture medium was placed in a 23°C constant temperature culture room for 7 days; when the Arabidopsis seedlings grew two true leaves, the Arabidopsis was transplanted into the pre-prepared substrate culture soil and placed in a light incubator for culture. The light incubator was set with the following parameters: 23°C constant temperature, 5LS light intensity, 75% humidity, and 16h / d light duration; the Arabidopsis was used for transformation before it entered the flowering period.

[0106] 3) Transformation of Agrobacterium

[0107] Different recombinant vectors were transformed into Agrobacterium GV3101 competent cells, and single colonies were picked and inoculated into LB liquid culture medium, and cultured at 28℃ for 2 days; full-length primers were used for bacterial liquid PCR to screen positive clones, and positive clones were stored at 4℃ for later use. Healthy Arabidopsis to be planted grew until flowering. Positive clones detected by PCR were shaken to OD 600 When the pH is 0.8, Arabidopsis flower organ immersion transformation is performed. The specific steps are as follows:

[0108] The bacterial solution was centrifuged at 5000rpm for 5min, the bacteria were collected and suspended with 5% sucrose solution; before soaking, SilwetL-77 was added at a concentration of 0.05% (500μL / L) and the foam was shaken out; the aerial part of Arabidopsis was soaked in the Agrobacterium suspension solution for 30sec, with gentle shaking during the period; the soaked Arabidopsis was laid flat on a tray, covered with plastic wrap to keep moisture, and sealed with tin foil to avoid light for 24h; the tin foil was removed and cultured under normal conditions, and watering was stopped when the seeds matured; Arabidopsis seeds were collected as T1 generation seeds, and stored after drying at 37℃ for one week.

[0109] 4) Positive plant screening

[0110] The seeds of Arabidopsis thaliana transformed by Agrobacterium infection were disinfected and sown in 1 / 2MS medium containing kanamycin (50 mg / L). After germination began 3 days after vernalization, they were transferred to the light culture room to observe the growth changes of the plants. Due to the influence of kanamycin, the seedlings of non-transgenic plants and control group plants gradually turned yellow and withered, while the transgenic seedlings grew normally. After about 10 days, all the transgenic plants and control group plants turned yellow and died. After kanamycin screening, 12 plants were obtained and named T1 to 12. The RNA of the 12 transgenic Arabidopsis lines was extracted using a kit, reverse transcribed into cDNA, and then qRT-PCR was performed. The line with the expression level closest to that of AtCPSF30-L in WT was selected for subsequent experiments. Birch TUB was selected as the internal reference gene. According to ChamQ TM The relative expression of BplCPSF30-L gene was analyzed according to the SYBR qPCR Master Mix kit instructions, and the t-test method was used to analyze the significance of the difference between each expression level and the control group. The primer sequences are as follows:

[0111] qTUB-F: 5'-GCTCTCCTTTTGTTGCTGTT-3';

[0112] qTUB-R: 5'-GAATCTCTCAGCACCAATCG-3';

[0113] qBplCPSF30-LF: 5'-AGATTCCACCGCCGTTCC-3';

[0114] qBplCPSF30-LR: 5'-TCGCCCTTCATGCACAGG-3'.

[0115] The qRT-PCR reaction system (10 μL) is: 0.4 μL Forward primer (10 μM / L), 0.4 μL Reverseprimer (10 μM / L), 1 μL Template cDNA, 5 μL SYBR Green Mix, and 3.2 μL ddH2O.

[0116] The qRT-PCR reaction program was: 95°C for 1 min; 95°C for 15 s, 58°C for 15 s, 72°C for 45 s, and 40 cycles.

[0117] The results are as follows Figure 3 As shown, 35S::BplCPSF30-L / atcpsf30-l is the overexpression line of BplCPSF30-L in the cpsf30-l mutant, 35S::BplCPSF30-Lm / atcpsf30-l is the overexpression line of BplCPSF30-Lm in the cpsf30-l mutant, WT is the wild-type control, and the atcpsf30-l Arabidopsis mutant is also used as an experimental control.

[0118] After the above T1 generation seeds are disinfected, they are sown in 1 / 2MS medium containing kanamycin (50 mg / L), and after 3 days of vernalization, they are placed in a suitable environment for cultivation (culture conditions are the same as above). Then they are transferred to the matrix soil, and the seeds are collected as T2 generation transgenic Arabidopsis seeds. The obtained T2 generation transgenic Arabidopsis seeds are cultured as in the above steps, and T3 generation homozygous transgenic Arabidopsis seeds and plants are screened. After being transferred to the matrix soil, they are placed in a light incubator for cultivation (the setting parameters are: 23°C constant temperature and light, light intensity 5LS, humidity 75%, and light time 16h / d cultivation).

[0119] 3. Statistics and quantitative analysis of transgenic plant phenotypes

[0120] 1) Phenotype of transgenic Arabidopsis

[0121] The 35S::BplCPSF30-L / atcpsf30-l overexpression line, 35S::BplCPSF30-Lm / atcpsf30-l overexpression line, WT, and atcpsf30-l were cultivated under standardized conditions, and seeds were collected for vernalization and then grown in soil under long-day conditions (16 hours of light / 8 hours of darkness). The flowering time was defined as the opening of the first flower. The number of leaves and the number of leaf orders on the main flower stem were counted at the same time. All experiments were repeated at least three times.

[0122] The results are as follows Figure 4As shown, the 35S::BplCPSF30-L / atcpsf30-l overexpression line blooms early and restores the late flowering phenotype of atcpsf30-l; the 35S::BplCPSF30-L / atcpsf30-l overexpression line cannot restore the late flowering phenotype of atcpsf30-l.

[0123] 2) Relative expression of SOC1 in transgenic Arabidopsis

[0124] Arabidopsis Actin2 was selected as the internal reference gene. TM The relative expression of SOC1 gene was analyzed according to the SYBR qPCR Master Mix kit instructions, and the t-test method was used to analyze the significance of the difference between each expression level and the control group. The primer sequences are as follows:

[0125] qActin2-F: 5'-GCTCTCCTTTTGTTGCTGTT-3';

[0126] qActin2-R: 5'-GAATCTCTCAGCACCAATCG-3';

[0127] qSOC1-F: 5'-GGATCGAGTCAGCACCAAACC-3';

[0128] qSOC1-R: 5'-CCCAATGAACAATTGCGTCTC-3'.

[0129] The results are as follows Figure 5 As shown, the 35S::BplCPSF30-L / atcpsf30-1 overexpression line restored the expression level of AtSOC1, while the 35S::BplCPSF30-L / atcpsf30-1 overexpression line could not restore the expression level of AtSOC1.

[0130] 3) Stability of SOC1 mRNA.

[0131] The experiment was divided into 4 groups, each group was 7d old 35S::BplCPSF30-L / cpsf30-l and cpsf30-l seedlings (10 plants for each genotype). They were transferred to 2mL incubation buffer (15mm sucrose, 1mm potassium chloride, 1mm PIPES, pH 6.25 and 1mm sodium citrate). After incubation on a shaker at 80 rpm for 15 minutes, samples were collected at time zero and timing was started. Subsequently, the remaining samples were placed in an incubation buffer containing 1mM cordycepin (Macklin) and vacuum infiltrated 3 times (0.6Mpa, each lasting 1 minute, with an interval of 1 minute). Samples were collected at 15min, 30min, 60min and 120min, and RNA was further extracted and subsequent experiments were performed. The same amount of ERCC spike-in was added as a control. Oligo(dT)25Dynabeads (Thermo Fisher Scientific) were used to extract poly(A)+RNA from the samples and construct RNA libraries. All degradation curves were fitted with the equation y=exp(-A×x), the expression level at the initial time was set to 1, and the relative expression level and half-life of each gene at other time points were calculated.

[0132] The results are as follows Figure 6 As shown, the mRNA of SOC1 was more stable in the 35S::BplCPSF30-L / cpsf30-l overexpression line.

[0133] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A kind of birch 6 A recognition protein BplCPSF30-L, whose amino acid sequence is shown in SEQ ID NO.

1.

2. The birch tree of claim 1 6 The nucleotide sequence of the gene encoding the A recognition protein BplCPSF30-L is shown in SEQ ID NO.

2.

3. Containing the birch m according to claim 1 6 A. Vector and recombinant bacteria that recognize protein BplCPSF30-L.

4. The birch tree of claim 1 6 Application of A recognition protein BplCPSF30-L in regulating flowering time of mutant plants.

5. The use according to claim 4, characterized in that: The method of regulating the flowering time of mutant plants is to promote the mutant plants to bloom earlier.

6. The use according to claim 5, characterized in that: include: 1) Build Birch m 6 A expression vector for the recognition protein BplCPSF30-L; 2) The constructed birch m 6 The expression vector of A recognition protein BplCPSF30-L was transformed into mutant Arabidopsis thaliana; 3) Cultivate, screen and obtain transgenic Arabidopsis plants with advanced flowering time.

7. The birch tree of claim 1 6 Application of A recognition protein BplCPSF30-L in regulating the expression of flowering-related genes.

8. The use according to claim 7, characterized in that: The regulation of the expression of flowering-related genes is to promote the expression of flowering-related gene SOC1.

9. The birch tree of claim 1 6 Application of A recognition protein BplCPSF30-L in regulating plant leaf number.

10. The birch tree of claim 1 6 Application of A recognition protein BplCPSF30-L in regulating plant leaf arrangement number.

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