Pinus massoniana PmYTH5 gene as well as encoding protein and application thereof

Abstract

The invention discloses a pinus massoniana PmYTH5 gene as well as an encoding protein and application thereof, and relates to the technical field of plant genetic engineering. The invention discloses a pinus massoniana PmYTH5 gene, and the nucleotide sequence of the pinus massoniana PmYTH5 gene is shown as SEQ ID NO. 1. The PmYTH5 gene is used for constructing a protein expression vector, a receptor is introduced for protein expression, and the result shows that PmYTH5-HIS is specifically combined with m6A modified RNA (Ribonucleic Acid) instead of unmethylated RNA, and PmYTH5m-HIS loses the capability of recognizing m6A. A transgenic arabidopsis thaliana plant of the pinus massoniana PmYTH5 gene is constructed, and the result shows that the salt tolerance of arabidopsis thaliana can be improved by overexpressing the PmYTH5 in the arabidopsis thaliana. The PmYTH5 gene disclosed by the invention is beneficial to deep research on functions of the PmYTH5 gene of the pinus massoniana, and a molecular means and basis are provided for cultivating saline-alkaline tolerant improved varieties of the pinus massoniana.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a Masson pine PmYTH5 gene and its encoded protein and application. Background Art

[0002] Masson pine (Pinus massoniana Lamb.) is the main native afforestation tree species in southern China and a pioneer tree species for improving the environment and greening barren hills. It is widely distributed in the eastern subtropical humid zone of my country and extends southward to the northern tropics. Due to its wide distribution, multiple uses, high economic value, easy reproduction and strong adaptability, it has become one of the most important artificial afforestation tree species in southern China. As a typical tree species for the development of comprehensive utilization, Masson pine is widely used in papermaking and construction. It plays a very important role in medical care, feed processing and forest chemical industry. It has high economic, ecological and social benefits. Breeding salt-alkali, drought-resistant, disease-resistant and insect-resistant Masson pine varieties is an important support for maintaining ecological balance, improving forest functions, improving forest quality and ensuring national ecological security.

[0003] Salt stress response is an important mechanism for plants to cope with adverse environments and improve their survival ability. When the salt content in the soil exceeds the normal range, it will cause adverse effects such as osmotic stress and ionic stress on the plants, thereby affecting the normal growth and development of the plants. In order to adapt to this adverse environment, plants will activate the salt stress response mechanism to reduce the damage caused by salt stress by adjusting the ion balance in the cells, synthesizing osmotic regulating substances, and increasing the activity of antioxidant enzymes. Salt stress response involves the regulation of multiple signal transduction pathways and gene expression, and is a complex and delicate process. Studying salt stress response not only helps us to have a deeper understanding of the plant's stress resistance mechanism, but also provides important theoretical basis and practical guidance for the breeding of salt-tolerant plant varieties.

[0004] Studies have reported that m 6 A reader protein ECT8 plays a vital role in plant response to various stresses. Therefore, the cloning and functional study of the homologous gene PmYTH5 of ECT8 in Masson pine will provide an effective molecular means and scientific basis for improving the adaptability of Masson pine under different environmental conditions. 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 Masson pine PmYTH5 gene. Another technical problem to be solved by the present invention is to provide a protein encoded by the Masson pine PmYTH5 gene. Another technical problem to be solved by the present invention is to provide an application of the Masson pine PmYTH5 gene for regulating the stress resistance of forest trees.

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

[0007] A Masson pine PmYTH5 gene, whose nucleotide sequence is shown in SEQ ID NO. 1.

[0008] The amino acid sequence of the protein encoded by the Masson pine PmYTH5 gene is shown in SEQ ID NO. 2.

[0009] Vectors and recombinant bacteria containing the Masson pine PmYTH5 gene.

[0010] Application of the Masson pine PmYTH5 gene in regulating plant growth under NaCl stress, wherein regulating plant growth is promoting plant growth, including:

[0011] 1) Constructing the expression vector of Masson pine PmYTH5 gene;

[0012] 2) Transforming the constructed expression vector of the Masson pine PmYTH5 gene into the Arabidopsis mutant;

[0013] 3) Cultivate, screen and obtain transgenic Arabidopsis plants with significantly increased salt tolerance.

[0014] Application of PmYTH5 gene in regulating seed germination under NaCl stress.

[0015] Application of Masson pine PmYTH5 gene in improving plant salt tolerance.

[0016] Application of the Masson pine PmYTH5 gene in regulating the expression of genes in a salt stress response pathway, wherein the genes in the salt stress response pathway are PPRT1, MSI1, BIL1, and ENGD-1.

[0017] Compared with the prior art, the present invention has the following beneficial effects:

[0018] 1) The present invention is the first to discover and successfully isolate the PmYTH5 gene in Masson pine. Its nucleotide sequence is shown in SEQ ID NO. 1, and its amino acid sequence of the encoded protein is shown in SEQ ID NO. 2. The PmYTH5 gene was constructed into a protein expression vector, introduced into the receptor, and protein expression was performed. The results showed that PmYTH5-HIS specifically binds to m 6 A modified RNA, but not unmethylated RNA, and PmYTH5m-HIS lost recognition of m 6 A's ability.

[0019] 2) The present invention constructs an expression vector of the Masson pine PmYTH5 gene; transforms the constructed expression vector of the Masson pine PmYTH5 gene into an Arabidopsis mutant; and cultivates, screens, and obtains transgenic Arabidopsis plants with significantly increased salt tolerance. The results show that overexpression of PmYTH5 in Arabidopsis can improve the salt tolerance of Arabidopsis. The present invention contributes to the in-depth study of the function of the Masson pine PmYTH5 gene and provides a molecular means and basis for cultivating salt-alkali tolerant Masson pine varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The expression and purification diagram of PmYTH5-HIS and PmYTH5m-HIS proteins (M is a marker; lanes 1 and 3 are before induction; lanes 2 and 4 are purified recombinant proteins);

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

[0022] Figure 3 This is the qRT-PCR analysis result of the relative expression of the target gene in Arabidopsis transgenic plants (OE1 is PmYTH5 transgenic Arabidopsis, and OE2 is PmYTH5m transgenic Arabidopsis);

[0023] Figure 4 The phenotype diagram of PmYTH5 transgenic Arabidopsis (A is the phenotype diagram of seed germination of transgenic Arabidopsis ect8 mutant under 0mM, 100mM, and 150mM NaCl stress; B is the statistical diagram of seed germination rate of transgenic Arabidopsis ect8 mutant under 0mM, 100mM, and 150mM NaCl stress; C is the statistical diagram of green cotyledons of transgenic Arabidopsis ect8 mutant seeds after germination under 0mM, 100mM, and 150mM NaCl stress);

[0024] Figure 5 The expression level diagram of salt stress response pathway genes PPRT1, MSI1, BIL1, and ENGD-1 in 12-day-old transgenic Arabidopsis mutants;

[0025] Figure 6 This is the decay curve of mRNA of salt stress response pathway genes PPRT1, MSI1, BIL1, and ENGD-1 in transgenic Arabidopsis mutants. 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 by referring to the methods listed in the book "Molecular Cloning Laboratory Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0027] The plant materials used in this application are Pinus massoniana needles and Arabidopsis thaliana ect8 mutants, both of which are preserved in the National Key Laboratory of Tree Genetics and Breeding (60503 Pine Genetic Improvement Laboratory) of Nanjing Forestry University.

[0028] Example 1

[0029] 1. Total RNA Extraction

[0030] The total RNA of one-year-old Masson pine leaves was extracted using the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. Before extraction, the mortar and pestle were wrapped with tin foil and baked in an oven at 180°C for about 4 hours. After the sample was ground into powder in liquid nitrogen, RNA was extracted according to the instructions of the kit and stored at -80°C. The 1.2% agarose gel electrophoresis bands of the total RNA of Masson pine needles were clear; the absorbance OD of the total RNA was 260 / OD 280 The value is 2.17, OD 260 / OD 230 The value is 2.06, which can be used for gene cloning.

[0031] 2. Obtaining cDNA

[0032] The HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme) was used to obtain cDNA by reverse transcription using the extracted RNA as a template. The specific process is as follows:

[0033] 1) Prepare the following mixture in an RNase-free centrifuge tube: 5 μg Total RNA, RNase-free ddH2O to 8 μL. Heat at 60°C for 5 min, quickly cool on ice, and leave on ice for 2 min.

[0034] 2) Add 2 μL of 5×g DNA wiper mix to 8 μL of the previous mixture, mix gently with a pipette, and incubate at 42°C for 2 min.

[0035] 3. Cloning of target gene

[0036] According to the PmYTH5 gene sequence downloaded from the Masson pine genome, intermediate fragment-specific primers were designed using Primer 5.0 to clone the ORF fragment of the PmYTH5 gene. The PmYTH5 cloning primers are:

[0037] PmYTH5-ORF-F: 5'-ATGGCTGTTGCAGTCCCGG-3';

[0038] PmYTH5-ORF-R: 5'-CTAGCAGGCTACAGATCCAACA-3'.

[0039] The PCR reaction system (50 μL) is: 2 μL Forward primer (10 μM / L), 2 μL Reverse primer (10 μM / L), 2 μL Template cDNA (100ng / μL), 25 μL 2×Taq PCR MASTER Mix, and 19 μL ddH2O.

[0040] 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 45 s, 35 cycles, and extension at 72°C for another 5 min.

[0041] Add 4 μL of product to a centrifuge tube, then add 1 μL of Blunt vector, mix and react at room temperature for 15 minutes. After the reaction, add 50 μL of freshly thawed Trans1-T1 competent cells, mix and place on ice for 30 minutes. Place the ligation product in a 42°C metal bath for 40 seconds, then immediately place it in ice for 2 minutes. Add 250 μL of LB liquid culture medium (without Kan) equilibrated to room temperature, and culture at 200 rpm and 37°C for 1 hour. Centrifuge at 10000 rpm for 30 seconds, discard 150 μL of supernatant, and use a pipette to blow and mix the remaining culture medium evenly, then spread it on Kan-resistant culture medium (the culture medium was placed in a 37°C incubator for 1 hour in advance), and culture it in a 37°C incubator overnight. After positive clone detection, it was sent to Qingke Bio for sequencing. The final sequencing obtained the ORF nucleotide sequence of the PmYTH5 gene as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2.

[0042] Example 2

[0043] The 435th and 448th W amino acids in the PmYTH5 amino acid sequence were mutated to A using a point mutation kit (Vazyme), and the amino acid sequence after site mutation was named PmYTH5m. The specific steps are as follows:

[0044] 1. Ligation and transformation of target fragment and vector

[0045] Use pET28a vector as prokaryotic expression vector, gently mix 4 μL PmYTH5 and 1 μL pET28a, react at room temperature for 5 minutes, and place on ice after the reaction. Transform E. coli DH5α and apply Amp-resistant LB solid medium. Sequencing is performed after positive detection. The vector pET28a-PmYTH5 is constructed. The positive clone detection primers are:

[0046] pET28a-F: 5'-TAATGCCGCTGCGCAACTCATG-3';

[0047] pET28a-R: 5'-CTTTGTTAGCAGCCGGATCGC-3'.

[0048] 2. Mutations in key sites of the PmYTH5 gene

[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-PmYTH5m, that is, the vector pET28a-PmYTH5m is constructed. Transform Escherichia coli DH5α and apply Amp-resistant LB solid culture medium. Sequencing is performed after positive detection. The primer sequences are as follows:

[0050] PmYTH5-AF:

[0051] 5'-GTAAAGGCGCACATGATAAAGGATATTCCAAATTCACAGTTTCGCC-3',

[0052] PmYTH5-AR:

[0053] 5'-TCTTGCTGCgcGAAATCCATGCTTTTGTCAAAATCTACACTACC-3';

[0054] PmYTH5-BF:

[0055] 5'-TCATGTGCGCCTTTACAGGGAAAAAACCATTCCAC-3',

[0056] PmYTH5-BR:

[0057] 5'-ATGGATTTCgcGCAGCAAGATAAGTGGAATGGT-3'.

[0058] The PCR reaction system (50 μL) is: 2 μL Forward primer (10 μM / L), 2 μL Reverse primer (10 μM / L), 2 μL Template cDNA (100ng / μL), 25 μL 2×Taq PCR MASTER Mix, and 19 μL 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) The amplified product contains the original template plasmid. To prevent it from forming false positive transformants after transformation, Dpn I digestion must be 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 fragments A and B 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 program 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 pET28a-PmYTH5 and pET28a-PmYTH5m protein expression vectors 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. The specific steps are:

[0069] 1. Construction of pET28a-PmYTH5 and pET28a-PmYTH5m protein expression vectors

[0070] Use pET28a vector as a prokaryotic expression vector, gently mix 4 μL PmYTH5 and 4 μL PmYTH5m with pET28a, react at room temperature for 5 minutes, and place on ice after the reaction. Transform E. coli Trans1-T1, the transformation steps are the same as Example 1 (4), and apply Amp-resistant LB solid medium. After positive detection, sequencing is performed. The positive clone detection primers are:

[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) to obtain the pET28a-PmYTH5 and pET28a-PmYTH5m recombinant strains.

[0074] 2. Prokaryotic expression analysis and protein purification

[0075] 1) IPTG-induced protein expression

[0076] Pick the pET28a-PmYTH5 and pET28a-PmYTH5m recombinant strains and place them in 5 mL of 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 of 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, 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 10mL 1×PBS and shake to resuspend the bacteria, centrifuge at 6000rpm for 8min to collect the bacteria, discard the supernatant, repeat twice, add 10mL 1×PBS and shake to resuspend the bacteria, and prepare for crushing. Use the freeze-thaw method to crush: first put it in liquid nitrogen for quick freezing, then thaw it in a 37℃ water bath, shake, and repeat 4-6 times. After the crushing 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 same level as 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, 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.

[0083] The buffer method is as follows (all dissolved in distilled water):

[0084] Lysis equilibrium buffer (LE Buffer): 50mM Na2HPO4+0.3M NaCl;

[0085] Washing buffer: 50 mM Na2HPO4 + 0.3 M NaCl + 10 mM imidazole;

[0086] Elution buffer: 50 mM Na2HPO4 + 0.3 M NaCl + 250 mM imidazole.

[0087] The results are as follows Figure 1 As shown, the recombinant protein "PmYTH5-HIS" was purified from the "pET28a-PmYTH5" strain; and the recombinant protein "PmYTH5m-HIS" was purified from the "pET28a-PmYTH5m" strain.

[0088] 4) EMSA

[0089] Recombinant proteins PmYTH5-HIS and PmYTH5m-HIS were diluted to concentrations 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). One 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 min. 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).

[0090] The results are as follows Figure 2 As shown, PmYTH5-HIS specifically binds to m 6 A modified RNA, but not unmethylated RNA, and PmYTH5m-HIS lost recognition of m 6 A's ability.

[0091] Example 4

[0092] The pCAMBIA1305-PmYTH5 overexpression vector was constructed and transformed into the Arabidopsis ect8 mutant. The phenotypic differences between the T2 generation transgenic Arabidopsis and wild-type Arabidopsis were compared to analyze the function of the Masson pine PmYTH5 gene.

[0093] 1. Construction of pCAMBIA1305-PmYTH5 and pCAMBIA1305-PmYTH5m vectors

[0094] The Escherichia coli strain used in the present invention is Trans1-T1 (TransGen); the expression vector is pCAMBIA1305 (preserved in the laboratory of Nanjing Forestry University); the restriction endonuclease and ligase are purchased from Vazyme.

[0095] The specific steps are as follows:

[0096] The pCAMBIA-1305 expression vector was treated with XbaI and PstI endonucleases for restriction digestion. The double restriction digestion system (50 μL) of the expression vector is: 1 μg pCAMBIA1305 plasmid, 1 μL QuickCut XbaⅠ, 1 μL QuickCut Pst I, 5 μL 10×QuickCut Buffer, ddH2O up to 50 μL. The double restriction digestion program of the expression vector is: 37℃ 30min; 85℃ 20s. Xba I and Pst I restriction sites were added to the upstream and downstream of the target fragment by PCR, and separated by 1.2% agarose gel electrophoresis; the restriction digestion products were recovered and purified using Gel DNA Extraction Mini Kit and dissolved in 20 μL Elution Buffer. The primer sequences used are as follows:

[0097] 1305-PmYTH5-F:

[0098] 5′-tttggagagaacacgtctagaATGGCTGTTGCAGTCCCGG-3′,

[0099] 1305-PmYTH5-R:

[0100] 5′-cttgctcaccatgtagtcgacGCAGGCTACAGATCCAACAATTAA-3′;

[0101] 1305-PmYTH5m-F:

[0102] 5′-tttggagagaacacgtctagaATGGCTGTTGCAGTCCCGG-3′,

[0103] 1305-PmYTH5m-R:

[0104] 5′-cttgctcaccatgtagtcgacGCAGGCTACAGATCCAACAATTAA-3′.

[0105] 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.

[0106] 2. Screening of positive Arabidopsis T1 plants transfected with PmYTH5 / PmYTH5m genes

[0107] Disinfection of Arabidopsis seeds: Take an appropriate amount of Arabidopsis ect8 mutant seeds and put them into a sterilized EP tube, add an appropriate amount of 75% ethanol, shake and disinfect for 30 seconds, and then suck them out; add the same amount of 0.1% mercuric chloride, disinfect for 2.5 minutes, and then suck them out (discard them in a special waste liquid tank); add the same amount of deionized water, shake and wash, then suck them out and put the seeds in a new EP tube; repeat the water washing four times.

[0108] Cultivation of Arabidopsis: Place the sterilized seeds in a suspension and sow them evenly into 1 / 2MS culture medium using a pipette; seal the culture medium and place it in a 4°C refrigerator for vernalization for 3 days; place the vernalized culture medium in a 23°C constant temperature culture room for 7 days; when the Arabidopsis seedlings grow two true leaves, transplant the Arabidopsis into the pre-prepared substrate culture soil and place it in a light incubator for cultivation. The setting parameters of the light incubator are: 23°C constant temperature, light intensity 5LS, humidity 75%, and light time 16h / d; use it for transformation before the Arabidopsis enters the flowering period.

[0109] Transform the pCAMBIA1305-PmYTH5 and pCAMBIA1305-PmYTH5m vectors into competent Agrobacterium GV3101, pick a single colony and inoculate it into LB liquid medium, shake and culture at 28℃ for 2 days; use full-length primers to perform bacterial liquid PCR to screen positive clones, and store positive clones at 4℃ for later use. Grow healthy Arabidopsis to bloom. Shake the positive clones detected by PCR to OD 600 When the concentration was 0.8, Arabidopsis flower organ immersion transformation was performed.

[0110] The specific steps are as follows:

[0111] The bacterial solution was centrifuged at 5000 rpm for 5 min, the cells were collected and suspended in 5% sucrose solution;

[0112] 1) Before soaking, add Silwet L-77 at a concentration of 0.05% (500 μL / L) and shake out the foam;

[0113] 2) Soak the unpollinated inflorescence in the Agrobacterium suspension solution for 30 seconds, shaking gently during the process;

[0114] 3) Lay the soaked Arabidopsis flat on a tray, cover with plastic wrap to keep it moist, and seal with tin foil to avoid light for 24 hours;

[0115] 4) Remove the tin foil, culture under normal conditions, and stop watering when the seeds are mature;

[0116] 5) Arabidopsis seeds were collected as T1 generation seeds and dried at 37°C for one week before storage.

[0117] After disinfection, the collected seeds were 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 plants gradually turned yellow and withered, while the transgenic seedlings grew normally. After about 10 days, all the transgenic plants and control plants turned yellow and died. After kanamycin screening, 9 plants were obtained, named T1-1 to T1-9. The RNA of these 9 transgenic Arabidopsis strains was extracted by the kit, reverse transcribed into cDNA, and then qRT-PCR was performed to select the strain with the highest expression level for subsequent experiments.

[0118] The RNA extraction and the synthesis of the first strand of cDNA were the same as in Example 1. After reverse transcription, the cDNA was diluted 10 times. Masson pine TUA was selected as the internal reference gene. The relative expression of the PmYTHDF1 gene was analyzed according to the instructions of the ChamQ™ SYBR qPCR Master Mix kit, and the t-test method was used to analyze the significance of the difference between each expression and the control group. The primer sequences are as follows:

[0119] qTUA-F: 5'-CAAACTTGGTCCCGTATCCTC-3';

[0120] qTUA-R: 5'-CACAGAAAGCTGCTCATGGTAA-3';

[0121] qPmYTH5-F: 5'-TAGATGGAGCATACCAAGAA-3';

[0122] qPmYTH5-R: 5'-TGTGCCACTTTACAGGGA-3'.

[0123] 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.

[0124] 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.

[0125] 3. Obtaining T2 transgenic Arabidopsis

[0126] After the T1 generation seeds are disinfected (the method is the same as that of Example 3 (2) 1), they are sown in 1 / 2MS culture 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 transplanted into matrix soil, and the seeds are collected as T2 generation transgenic Arabidopsis seeds. After transplanting into 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 for cultivation).

[0127] The results are as follows Figure 3 As shown, compared with the wild type, the relative expression levels of the target genes in the transgenic plants were significantly increased.

[0128] 4. Phenotypic Observation of Transgenic Arabidopsis

[0129] Plants of different genotypes were cultivated under standardized conditions, and their seeds were collected and stored for phenotypic experiments. All experiments were repeated at least three times. To determine NaCl tolerance, WT, ect8, 35S::PmYTH5 / ect8, and 35S::PmYTH5m / ect8 were sown on 1 / 2 MS medium supplemented with 0mM, 100mM, and 150mM NaCl in the NaCl treatment experiment, with 30 seeds of each type. Germination (radicle emergence) and post-germination growth (cotyledon greenness) were measured on the 4th and 12th days after vernalization, respectively.

[0130] The results are as follows Figure 4 As shown in the results, overexpression of PmYTH5 in the Arabidopsis ect8 mutant improved the tolerance of the Arabidopsis ect8 mutant to NaCl, and the number of green cotyledons increased after germination; however, overexpression of PmYTH5m could not restore the tolerance of the Arabidopsis ect8 mutant to NaCl. 6 A binding activity improves tolerance of Arabidopsis ect8 mutant to salt stress

[0131] 5. Relative expression of PPRT1, MSI1, BIL1, and ENGD-1

[0132] Arabidopsis thaliana TUB8 was selected as the internal reference gene, and specific primers for each gene were designed by Primer 5.0. The amplification length of each target fragment was 150-200bp. The relative expression levels of salt stress response pathway genes PPRT1, MSI1, BIL1, and ENGD-1 were analyzed according to the instructions of the ChamQ™ SYBR qPCR Master Mix kit, 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:

[0133] qTUB8-F: 5'-CAAAGATGCGAGAGATTCTTCA-3';

[0134] qTUB8-R: 5'-CTTAGCTCCGATCTGGTTTCC-3';

[0135] qPPRT1-F: 5'-TCCTGTAATCAAAGCTCGACA-3';

[0136] qPPRT1-R: 5'-AGCACTCTGCTATTCCGAAGTT-3';

[0137] qMSI1-F: 5'-ATCTCGTTATTACTCACGTCTC-3';

[0138] qMSI1-R: 5'-ATCTGCTGGATAATTTGTACCTTTC-3';

[0139] qBIL1-F: 5'-CCACCCTTGGCATAAGGTTT-3';

[0140] qBIL1-R: 5'-GCCTGTTGATAAGCTCCGGT-3';

[0141] qENGD-1-F:5'-GCCAAAGATTCATGCCTGGG-3';

[0142] qENGD-1-R: 5'-GCCAGCACTTCACCTCATCA-3'.

[0143] The results are as follows Figure 5 As shown in the figure, the expression levels of salt stress response pathway genes PPRT1, MSI1, BIL1, and ENGD-1 were significantly increased.

[0144] 6. Cordycepin inhibits transcription and determines the stability of PPRT1, MSI1, BIL1, and ENGD-1 mRNA

[0145] The experiment was divided into 4 groups, each representing a different time point. Each group consisted of 7-day-old 35S::PmYTH5 / ect8 and ect8 seedlings (10 plants for each genotype). They were transferred into 2 mL of incubation buffer (15 mm sucrose, 1 mm KCl, 1 mm PIPES, pH 6.25, and 1 mm sodium citrate). After incubation on a shaker at 80 rpm for 15 min, samples were collected at time zero and the timer was started. Subsequently, the remaining samples were placed in incubation buffer containing 1 mM cordycepin (Macklin) and vacuum infiltrated three times (0.6 MPa, each lasting 1 min, with 1 min interval). Samples were collected at 15 min, 30 min, 60 min, and 120 min for further RNA extraction and subsequent experiments. The same amount of ERCC spike-in was added as a control. Oligo(dT)25 Dynabeads (Thermo Fisher Scientific) were used to extract poly(A) + RNA from the samples and construct RNA libraries. All degradation curves were fitted using 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.

[0146] The results are as follows Figure 6 As shown, the mRNAs of PPRT1, MSI1, BIL1, and ENGD-1 were more unstable in the 35S::PmYTH5 / ect8 line.

[0147] 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 Masson pine PmYTH5 gene, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The protein encoded by the Masson pine PmYTH5 gene of claim 1, whose amino acid sequence is shown in SEQ ID NO.

2.

3. A vector and a recombinant bacterium containing the Masson pine PmYTH5 gene according to claim 1.

4. Use of the Masson pine PmYTH5 gene according to claim 1 in regulating plant growth under NaCl stress.

5. The use according to claim 4, characterized in that: The regulating plant growth is promoting plant growth.

6. The use according to claim 5, characterized in that: include: 1) Constructing the expression vector of Masson pine PmYTH5 gene; 2) Transforming the constructed expression vector of the Masson pine PmYTH5 gene into the Arabidopsis mutant; 3) Cultivate, screen and obtain transgenic Arabidopsis plants with significantly increased salt tolerance.

7. Use of the Masson pine PmYTH5 gene according to claim 1 in regulating seed germination under NaCl stress.

8. Use of the Masson pine PmYTH5 gene according to claim 1 in promoting the improvement of plant salt tolerance.

9. Use of the Masson pine PmYTH5 gene according to claim 1 in regulating the expression of genes in a salt stress response pathway.

10. The use according to claim 9, characterized in that: The salt stress response pathway genes are PPRT1, MSI1, BIL1, and ENGD-1.

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