Birch m 6 A recognition protein BplCPSF30-L, its encoding gene, and applications.

Abstract

The application discloses a white birch m 6 The application discloses a Bp1 CPSF30-L recognition protein and a coding gene and application thereof, and relates to the technical field of plant genetic engineering. The application discloses a white birch m 6 The Bp1 CPSF30-L recognition protein has an amino acid sequence as shown in SEQ ID NO. 1. The application constructs a white birch m 6 The expression vector of the Bp1 CPSF30-L recognition protein is transformed into mutant Arabidopsis thaliana, and transgenic Arabidopsis thaliana plants with early flowering time are obtained through cultivation and screening. The results show that overexpression of the Bp1 CPSF30-L recognition protein in the Arabidopsis thaliana cpsf30-l mutant can restore the late flowering phenotype of the Arabidopsis thaliana cpsf30-l mutant, and the expression amount of flowering-related genes in the transgenic Arabidopsis thaliana is also restored. The application provides a molecular method and basis for promoting the flowering of white birch and shortening the breeding cycle of white birch.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to birch (Betula sylvestris) m 6 A. Recognition protein BplCPSF30-L, its encoding gene, and applications. Background Technology

[0002] The long generation and breeding cycles of forest trees are major limiting factors in forest tree breeding. Flowering, the process of plant development from vegetative branches, can not only effectively improve the growth cycle of forest trees and enrich the regulatory mechanisms of flower development, but also provide ideal materials for genetic research on forest trees through transgenic lines. However, research on the regulation of plant flowering has mainly focused on the annual model plant Arabidopsis thaliana. Compared to model plants, research on flower development in forest trees is relatively weak. Therefore, it is essential to study the regulation of flower development in forest trees.

[0003] Birch is a globally distributed tree species across Eurasia and an important component of forest ecosystems, playing a vital role in maintaining ecological balance. It provides habitat for numerous wild flora and fauna, promoting biodiversity. Simultaneously, the fallen leaves and root system of birch trees contribute to improved soil fertility, providing a favorable environment for the growth of other plants, thus giving birch significant ecological value. Birch wood is soft, lightweight, and fine-grained, widely used for making agricultural tools and woodenware. Its white, papery bark can be crafted into canoes, fine items, and food baskets, and it is also used as a substitute for ceramic tiles, possessing significant economic value. However, compared to crop breeding, birch grows more slowly, resulting in a longer breeding cycle. Therefore, researching genes that promote flowering in birch is beneficial for shortening the birch breeding cycle and improving breeding efficiency.

[0004] Existing research reports that CPSF30-L acts as m 6 A recognizes the protein, depending on its m 6 The binding activity of A promotes flowering in Arabidopsis thaliana. However, research on the function of the BplCPSF30-L gene in Betula sylvestris is still lacking. Summary of the Invention

[0005] In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide birch m 6 A recognizes the protein BplCPSF30-L. Another technical problem this invention aims to solve is to provide birch m... 6 The gene encoding the recognition protein BplCPSF30-L. Another technical problem this invention aims to solve is to provide birch m 6 The application of the A recognition protein BplCPSF30-L is used to regulate the flowering time of plants.

[0006] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

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

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

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

[0010] Birch m 6 The application of the A recognition protein BplCPSF30-L in regulating the flowering time of mutant plants, wherein the regulation of the flowering time of mutant plants is to promote the early flowering of mutant plants.

[0011] Birch m 6 The application of the A recognition protein BplCPSF30-L in regulating flowering time in mutant plants includes:

[0012] 1) Constructing birch m 6 A vector for the expression of protein A, BplCPSF30-L;

[0013] 2) Construct the birch m 6 The expression vector for the A recognition protein BplCPSF30-L was transformed into the mutant Arabidopsis thaliana.

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

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

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

[0017] Birch m 6 Application of A recognition protein BplCPSF30-L in regulating the number of plant phyllodes.

[0018] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0019] The birch m disclosed in this invention 6The A recognition protein BplCPSF30-L has the amino acid sequence shown in SEQ ID NO.1, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO.2. This invention constructs birch m... 6 A recognition protein BplCPSF30-L expression vector; constructing birch m 6 The expression vector for the recognition protein BplCPSF30-L was transformed into a mutant Arabidopsis thaliana; transgenic Arabidopsis plants with advanced flowering time were bred, screened, and obtained. Results showed that overexpression of BplCPSF30-L in the Arabidopsis thaliana cpsf30-l mutant promoted flowering, while overexpression of BplCPSF30-Lm (W279A, W330A) did not restore flowering in the Arabidopsis thaliana cpsf30-l mutant; the expression levels of flowering-related genes in the transgenic Arabidopsis thaliana were also restored. This invention provides a molecular method and basis for promoting flowering in birch and shortening the birch breeding cycle. Attached Figure Description

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

[0021] Figure 2 Image of EMSA results;

[0022] Figure 3 Screening diagram for plants that are positive for the BplCPSF30-L gene;

[0023] Figure 4 Phenotypic diagrams of Arabidopsis thaliana cpsf30-l mutants transgenic with the BplCPSF30-L gene (A shows the flowering phenotypes of WT, atcpsf30-l, 35S::BplCPSF30-L / atcpsf30-l, and 35S::BplCPSF30-Lm / atcpsf30-l plants; B shows the total number of leaves and flowering time of WT, atcpsf30-l, 35S::BplCPSF30-L / atcpsf30-l, and 35S::BplCPSF30-Lm / atcpsf30-l plants; C shows the average number of leaves per Arabidopsis thaliana plant at flowering).

[0024] Figure 5 This is a graph showing the expression level of the SOC1 gene in 12-day-old Arabidopsis thaliana cpsf30-l mutants transfected with the BplCPSF30-L gene.

[0025] Figure 6The decay curves of SOC1 mRNA in 35S::BplCPSF30-L / cpsf30-1 and cpsf30-1 are shown. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise described in detail, the technical means used in the following embodiments are all conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be performed according to the methods listed in J. Sambrook's *Molecular Cloning: A Laboratory Manual* (3rd Edition) or conventional methods in the art, or according to the kit and product instructions.

[0027] The materials used in this application are two-year-old birch and Arabidopsis thaliana cpsf30-l mutant (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] Total RNA was extracted from 2-year-old birch leaves using a plant total RNA extraction kit (TIANGEN), following the manufacturer's instructions. The results of 1.2% agarose gel electrophoresis are shown below. Figure 1 The bands shown are relatively clear; the absorbance OD of total RNA is [missing value]. 260 / OD 280 The value is 2.32, OD 260 / OD 230 With a value of 2.09, it can be used for gene cloning.

[0031] Using the extracted total RNA as a template, cDNA (Vazyme) was synthesized using a reverse transcription kit, following the instructions.

[0032] 2. Cloning of the target gene

[0033] Based on the BplCPSF30-L gene sequence obtained from the birch genome download, intermediate fragment-specific primers were designed using Primer 5.0. The ORF cloning primer sequences for the BplCPSF30-L gene are shown below:

[0034] BplCPSF30-L-ORF-F:

[0035] 5'-ATGGAGGACACGGAGGGAGTTCTAA-3',

[0036] BplCPSF30-L-ORF-R:

[0037] 5'-TCAGTGATCGGAGGCAGTGGTAG-3'.

[0038] The PCR reaction system consisted of: 2 μL forward primer (10 μM / L), 2 μL reverse primer (10 μM / 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: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 45 s, 35 cycles, and 72℃ extension for 5 min.

[0040] Ligation and Transformation: Add 4 μL of the product to a centrifuge tube, then add 1 μL of Blunt vector, mix, and incubate at room temperature for 15 min. After the reaction, add 50 μL of freshly thawed Trans1-T1 competent cells, mix well, and incubate on ice for 30 min. Place the ligation product in a 42°C metal bath for 40 s, then immediately place it on ice for 2 min. Add 250 μL of LB liquid medium (without Kan) equilibrated to room temperature, and incubate at 37°C for 1 h at 200 rpm. Centrifuge at 10000 rpm for 30 s, discard 150 μL of supernatant, and mix the remaining culture medium thoroughly with a pipette. Spread the mixture onto Kan-resistant medium (pre-prepared at 37°C for 1 h) and incubate overnight at 37°C. Positive clones are then sent to Qingke Biotechnology for sequencing.

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

[0042] Example 2

[0043] 1. Ligation and transformation of target fragments and vectors

[0044] Using pET28a as the prokaryotic expression vector, 4 μL of the BplCPSF30-L gene fragment was gently mixed with 1 μL of pET28a and incubated at room temperature for 5 minutes. After the reaction, the mixture was placed on ice, and the recombinant plasmid was named pET28a-BplCPSF30-L. The plasmid was transformed into *E. coli* DH5α and plated on Amp-resistant LB agar. Sequencing was performed after positive detection. The primer sequences for positive clone detection are shown below:

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

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

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

[0048] The W amino acids at positions 279 and 330 of 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 mutation site to divide the plasmid into two fragments for amplification. The longer fragment is named B, and the shorter fragment is named A. Then, fragments A and B are recombined to obtain the plasmid named pET28a-BplCPSF30-Lm. Transformed into *E. coli* DH5α and plated on Amp-resistant LB agar. Sequencing is performed after positive detection. The primer sequences are shown below:

[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: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 10 s, 58℃ annealing for 10 s, 72℃ extension for 5 min, 35 cycles, and 72℃ extension for 5 min.

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

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

[0062] The PCR reaction procedure was as follows: react at 37℃ for 1 hour.

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

[0064] The PCR reaction system (50 μL) consisted of: 2 μL of Dpn I digestion product (segment A), 2 μL of Dpn I digestion product (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 as follows: 37°C for 30 min; then cool to 4°C or immediately place on ice to cool.

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

[0067] Example 3

[0068] Protein expression vectors pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L were constructed, transformed into *E. coli* for expression, and the proteins were purified. The purified proteins were then combined with m... 6 The A-modified sequence was subjected to EMSA to observe whether binding occurred.

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

[0070] Using pET28a as the prokaryotic expression vector, 4 μL of YTH / BplCPSF30-L and 4 μL of LYTHm / BplCPSF30-L were gently mixed with pET28a and reacted at room temperature for 5 minutes. After the reaction, the mixture was placed on ice to construct the pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L vectors, respectively. These vectors were then transformed into *E. coli* Trans1-T1 cells and plated on Amp-resistant LB agar. Sequencing was performed after positive detection. The primer sequences for positive clone detection are shown below:

[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) following the same steps 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] Single clones of pET28a-YTH / BplCPSF30-L and pET28a-YTHm / BplCPSF30-L were picked and added to 5 mL of LB liquid medium containing 100 mg / L Amp, and incubated at 37°C and 200 rpm for 12 h. The bacterial culture was then inoculated into 50 mL of LB liquid medium containing 100 mg / L Amp and incubated at 37°C and 200 rpm for 3-4 h until OD (Organic Dysfunction) was reached. 600=0.8, take 10mL of bacterial culture as a control and temporarily store it at 4℃. Divide the remaining bacterial culture into two parts and add 0.5mmol / L and 1mmol / L IPTG respectively to induce protein expression. Incubate at 37℃, 200rpm for 6-8h. Centrifuge all bacterial cultures at 6000rpm for 8min to collect the bacteria and discard the supernatant. Add 10mL of 1×PBS, shake and resuspend the bacteria, centrifuge at 6000rpm for 8min to collect the bacteria and discard the supernatant. Repeat twice. Add 10mL of 1×PBS, shake and resuspend the bacteria to prepare for disruption. Use the freeze-thaw method for disruption: first, quickly freeze in liquid nitrogen, then thaw in a 37℃ water bath, shake, and repeat 4-6 times. After disruption, centrifuge at 6000rpm for 8min to collect the bacteria, take the supernatant, and store at 4℃ for later use.

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

[0078] Gel preparation: Place the glass plate into the gel mold, prepare 10 mL of 10% separating gel according to the instructions, immediately pour it into the mold, add distilled water until it is level with the glass, wait 30-40 minutes until the separating gel solidifies, pour off the distilled water and absorb the remaining water with filter paper, then prepare 4 mL of 5% stacking gel, pour it into the mold, insert the comb, let it stand for about 40 minutes, and then put it into the electrophoresis tank to wait for gel running.

[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 the sample, set the voltage to 160V and the electrophoresis time to 50min. Then remove the gel, rinse it clean, stain it with Coomassie Brilliant Blue Ultrafast Staining Solution for 30-40min, and then destain it with water until the protein bands are visible. Take a picture to record the results.

[0081] 3) Protein purification

[0082] Prepare the protein supernatant and purify the protein according to the Ni-NTA affinity chromatography media manufacturer's instructions. Load the media into the column, wash with four column volumes of equilibration buffer, add the clarified protein sample, and maintain a flow rate of 0.5-1 mL / min. Then wash the column with wash buffer at a flow rate of 1 mL / min to remove contaminating proteins until the A280 value of the eluent reaches its minimum and stabilizes. Finally, elute with 5-10 column volumes of elution buffer at a flow rate of 0.5-1 mL / min. Collect the eluent in separate tubes, and stop collecting when the A280 value reaches its minimum and stabilizes. Take the eluent tubes with the highest A280 values ​​for electrophoresis analysis. The buffer formulation is as follows (all dissolved in distilled water):

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

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

[0085] Elution buffer: 50mM Na2HPO4 + 0.3M NaCl + 250mM 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 column B shows the induced protein expression with many impurities. The second to fourth lines show the impurities removed after three washings with running water. The fifth line shows the YTH-6×His / BplCPSF30-L protein collected from the column after elution. Figure 1 The first line in column C shows the induced protein expression, which contains many impurities. The second to fourth lines show the impurities removed after three washings with flow solution. The fifth line shows the YTHm-6×His / BplCPSF30-L protein collected from the column through elution.

[0087] 3. EMSA

[0088] Recombinant proteins YTH-6×His / BplCPSF30-L and YTHm-6×His / BplCPSF30-L were diluted to concentrations of 0–8 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 μL of fluorescein-labeled RNA probe (final concentration 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 using 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, not unmethylated RNA, and YTHm-6×His / BplCPSF30-L loses its ability to recognize m 6 A's ability.

[0090] Example 4

[0091] In this embodiment, after constructing different vectors, transgenic lines (35S::BplCPSF30-Lm / atcpsf30-l, 35S::BplCPSF30-L / atcpsf30-l, and atcpsf30-l) were directly constructed by transforming the Arabidopsis thaliana cpsf30-l mutant for functional verification. The Arabidopsis thaliana cpsf30-l mutant plants exhibit delayed flowering.

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

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

[0094] 1305-BplCPSF30-LF:

[0095] 5′-tttggagagaacacgtctagaATGGAGGACACGGAGGGAGT-3′;

[0096] 1305-BplCPSF30-LR:

[0097] 5′-gtcaagcttgcatgcctgcagGTGATCCGGAGGCAGTGGTAGC-3′.

[0098] After the PCR product is correctly sequenced, the vector is digested with enzymes, ligated, transformed, positively tested, and sent for testing. The plasmid with correct sequencing is transformed into Agrobacterium for Arabidopsis thaliana transformation.

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

[0100] The double enzyme digestion program for the expression vector is: 37℃ for 30 min; 85℃ for 20 s.

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

[0102] 1) Disinfection of Arabidopsis thaliana seeds

[0103] Take an appropriate amount of cpsf30-l mutant seeds and place them in a sterilized EP tube. Add an appropriate amount of 75% ethanol, shake to sterilize for 30 seconds, and then aspirate. Add an equal amount of 0.1% mercuric chloride, sterilize for 2.5 minutes, and then aspirate (discard in a dedicated waste liquid container). Add an equal amount of deionized water, shake to wash, and then aspirate and place the seeds in a new EP tube. Repeat the washing process four times.

[0104] 2) Cultivation of Arabidopsis thaliana

[0105] After sterilization, the seeds were placed in a suspension and evenly sown into 1 / 2 MS medium using a pipette. The medium was sealed and placed in a 4°C refrigerator for vernalization for 3 days. The vernalized medium was then placed in a 23°C constant temperature incubator for 7 days. When the Arabidopsis seedlings had two true leaves, they were transplanted into the prepared substrate and placed in a light incubator with the following settings: 23°C constant temperature, 5 LS light intensity, 75% humidity, and 16 h / d light duration. The seeds were then used for transformation before the Arabidopsis entered its full flowering period.

[0106] 3) Transformation of Agrobacterium

[0107] Different recombinant vectors were transformed into competent Agrobacterium GV3101 cells. Single colonies were picked and inoculated into LB broth, and cultured at 28°C with shaking for 2 days. Full-length primers were used for colony PCR to screen for positive clones, which were then stored at 4°C for later use. Healthy Arabidopsis thaliana plants were allowed to grow until flowering. Positive clones detected by PCR were cultured to OD200. 600 When the concentration was 0.8, Arabidopsis flower organ immersion transformation was performed. The specific steps are as follows:

[0108] Centrifuge the bacterial suspension at 5000 rpm for 5 min, collect the bacterial cells, and suspend them in a 5% sucrose solution. Before soaking, add Silwet L-77 at a concentration of 0.05% (500 μL / L) and shake to remove foam. Soak the aerial parts of Arabidopsis thaliana in the Agrobacterium suspension for 30 seconds, gently shaking during this time. Lay the soaked Arabidopsis thaliana flat on a tray, cover with plastic wrap to retain moisture, and seal with aluminum foil to protect from light for 24 hours. Remove the aluminum foil and culture under normal conditions. Stop watering when the seeds mature. Collect the Arabidopsis thaliana seeds as T1 generation seeds, dry them at 37℃ for one week, and then store them.

[0109] 4) Screening of positive plants

[0110] After disinfection, seeds of Arabidopsis thaliana transformed with Agrobacterium were sown in 1 / 2 MS medium containing kanamycin (50 mg / L). Germination began after 3 days of vernalization, and the seedlings were then transferred to a light-controlled culture room for observation of plant growth. Due to the effects of kanamycin, non-transgenic and control seedlings gradually yellowed and withered, while transgenic seedlings grew normally. After approximately 10 days, all transgenic and control seedlings yellowed and died. Twelve plants were obtained after kanamycin selection and named T1–12. RNA was extracted from the 12 transgenic Arabidopsis lines using a kit, reverse transcribed into cDNA, and then detected by qRT-PCR. The line whose expression level was closest to that of AtCPSF30-L in WT was selected for subsequent experiments. Birch TUB was used as an internal control gene. Following ChamQ… TM The relative expression level of the BplCPSF30-L gene was analyzed using the SYBR qPCR Master Mix kit instructions. The significance of differences between each expression level and the control group was analyzed using the t-test. 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℃ for 1 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 45 s, for 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 thaliana mutant is also used as an experimental control.

[0118] After sterilizing the T1 generation seeds, they were sown in 1 / 2 MS medium containing kanamycin (50 mg / L) and vernalized for 3 days. Then, they were cultured in a suitable environment (culture conditions as above). They were then transferred to substrate soil, and the seeds were harvested as T2 generation transgenic Arabidopsis seeds. The obtained T2 generation transgenic Arabidopsis seeds were cultured using the steps described above. T3 generation homozygous transgenic Arabidopsis seeds and plants were screened, transferred to substrate soil, and then cultured in a light incubator (setting parameters: 23℃ constant temperature and light intensity, 5 LS light intensity, 75% humidity, and 16 h / d light duration).

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

[0120] 1) Phenotype of transgenic Arabidopsis thaliana

[0121] Overexpression lines 35S::BplCPSF30-L / atcpsf30-l, 35S::BplCPSF30-Lm / atcpsf30-l, WT, and atcpsf30-l were cultivated under standardized conditions. Seeds were collected and vernalized, then grown in soil under long-day conditions (16 hours light / 8 hours darkness). Flowering time was defined as the opening of the first flower. The number of leaves and phyllotaxis on the main flower stalk were also counted. 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 flowered earlier and restored the late-flowering phenotype of atcpsf30-l; the 35S::BplCPSF30-L / atcpsf30-l overexpression line could not restore the late-flowering phenotype of atcpsf30-l.

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

[0124] Arabidopsis thaliana Actin2 was selected as the internal control gene. Following ChamQ... TM The relative expression level of the SOC1 gene was analyzed using the SYBR qPCR Master Mix kit instructions. The significance of differences between each expression level and the control group was analyzed using a t-test. The primer sequences are shown below:

[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-l overexpression line restored the AtSOC1 expression level, while the 35S::BplCPSF30-L / atcpsf30-l overexpression line could not restore the AtSOC1 expression level.

[0130] 3) Stability of SOC1 mRNA.

[0131] The experiment was divided into four groups, each consisting of 7-day-old 35S::BplCPSF30-L / cpsf30-l and cpsf30-l seedlings (10 seedlings of each genotype). The seedlings were transferred to 2 mL of incubation buffer (15 mM sucrose, 1 mM potassium chloride, 1 mM PIPES, pH 6.25, and 1 mM sodium citrate). After incubation at 80 rpm for 15 minutes on a shaker, samples were collected at time zero, and timing began. Subsequently, the remaining samples were placed in incubation buffer containing 1 mM cordycepin (Macklin) and subjected to three vacuum soaks (0.6 MPa, 1 minute each, with 1 minute intervals). Samples were collected at 15 min, 30 min, 60 min, and 120 min for further RNA extraction and subsequent experiments. An equal amount of ERCC spike-in was added as a control. Library construction was performed using oligo(dT)25Dynabeads (Thermo Fisher Scientific) to extract poly(A)+ RNA from samples. All degradation curves were fitted with the equation y = exp(-A × x), with the initial expression level set to 1. The relative expression levels and half-lives of each gene at other time points were calculated.

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

[0133] The above description is illustrative only and not restrictive of the present invention. Those skilled in the art will understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all such modifications, variations or equivalents will fall within the protection scope of the present invention.

Claims

1. A type of birch m 6 The A recognition protein BplCPSF30-L has the amino acid sequence shown in SEQ ID NO.

1.

2. The birch m as described in claim 1 6 The gene encoding the A recognition protein BplCPSF30-L has the nucleotide sequence shown in SEQ ID NO.

2.

3. Containing the birch m as described in claim 1 6 A vector and recombinant bacteria that recognizes protein BplCPSF30-L.

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