Pinus massoniana PmYTH1 gene as well as encoding protein and application thereof

Abstract

The invention discloses a pinus massoniana PmYTH1 gene as well as an encoding protein and application thereof, and relates to the technical field of plant genetic engineering. The nucleotide sequence of the pinus massoniana PmYTH1 gene disclosed by the invention is as shown in SEQ ID NO. 1. The method comprises the following steps: constructing an expression vector of the Pinus massoniana PmYTH1 gene, and transforming the expression vector into an arabidopsis thaliana mutant and a wild type; and cultivating and screening to obtain a transgenic arabidopsis thaliana plant of which the tolerance to ABA and NaCl is obviously increased. The results show that the tolerance of the arabidopsis ect2 / 3 / 4 mutant to ABA is improved by overexpressing the PmYTH1 in the arabidopsis ect2 / 3 / 4 mutant, and the tolerance of the wild arabidopsis Col-0 to NaCl is improved by overexpressing the PmYTH1 in the wild arabidopsis Col-0; the expression quantity of ABA signal channel genes is obviously increased. The invention provides a molecular means and basis for cultivating a new pinus massoniana variety which is efficiently tolerant to ABA, NaCl and adversity stress.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the PmYTH1 gene of Masson pine, its encoded protein, and its applications. Background Technology

[0002] Masson pine (Pinus massoniana L.) is a native tree species endemic to the subtropical regions of my country. It has a wide range of uses, high economic value, is easy to propagate, and is highly adaptable, making it one of the most important artificial afforestation tree species in southern my country. Masson pine is widely used in land greening, pulp and paper making, and the board manufacturing industry. Its resin and pollen are important raw materials for the chemical industry and health products; it can be used as a culture medium for Poria cocos, and its needles can be used as a feed additive. Masson pine has a high calorific value, making it an important fuelwood and a key tree species for recreational and ecological forests.

[0003] Abscisic acid (ABA) is an endogenous plant hormone that plays a crucial role in plant growth, development, and stress responses. Studies have found that ABA regulates seed dormancy and germination, cell division and elongation, photoperiod response, leaf senescence, and abscission in plant growth and development. Simultaneously, ABA regulates plant responses to abiotic stresses such as drought, high salinity, low temperature, and salinity, thereby mitigating the harmful effects of these stresses on plants. Furthermore, ABA regulates downstream gene expression through specific signal transduction pathways, thus controlling plant physiological and developmental processes. The ABA signaling pathway plays a vital role in plant economic production and environmental adaptability. Therefore, studying the genes in *Pinus massoniana* that respond to ABA can optimize its biomass production and improve its adaptability under different environmental conditions, which is of great significance for the cultivation and utilization of *Pinus massoniana*.

[0004] Previous studies have shown that Arabidopsis thaliana ECT2 acts as an m 6 A recognizes the protein, depending on its m 6 The binding activity of A can enhance its own ABA tolerance. PmYTH1 in Masson pine is a homolog of AtECT2 in Arabidopsis thaliana. Research on the sequence and function of PmYTH1 in Masson pine provides a molecular approach and basis for improving the adaptability of Masson pine under different environmental conditions. Summary of the Invention

[0005] To address the aforementioned problems in existing technologies, the technical problem this invention aims to solve is to provide the *Pinus massoniana* PmYTH1 gene. Another technical problem this invention aims to solve is to provide the protein encoded by the *Pinus massoniana* PmYTH1 gene. A further technical problem this invention aims to solve is to provide applications for the *Pinus massoniana* PmYTH1 gene in regulating the stress resistance of forest trees.

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

[0007] Application of the PmYTH1 gene from Pinus massoniana, with nucleotide sequence as shown in SEQ ID NO. 1, in promoting the growth of Arabidopsis thaliana mutants under ABA stress.

[0008] The applications include:

[0009] 1) Construct an expression vector for the PmYTH1 gene of Masson pine;

[0010] 2) The constructed expression vector of the Masson pine PmYTH1 gene was transformed into Arabidopsis mutants;

[0011] 3) Breed, screen and obtain transgenic Arabidopsis plants with significantly increased tolerance to ABA.

[0012] Application of the PmYTH1 gene from Masson pine, with nucleotide sequence as shown in SEQ ID NO. 1, in increasing the expression of genes in the ABA signaling pathway.

[0013] In the aforementioned application, the ABA signaling pathway genes are DWA1, DWA2, SDIRIP1, and CPN20 genes.

[0014] Application of the PmYTH1 gene from Pinus massoniana, with nucleotide sequence as shown in SEQ ID NO. 1, in promoting seed germination of Arabidopsis thaliana under NaCl stress.

[0015] Application of the PmYTH1 gene from *Pinus massoniana*, with nucleotide sequence as shown in SEQ ID NO. 1, in promoting an increase in the number of green cotyledons after germination of *Arabidopsis thaliana* seeds under NaCl stress.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention constructs an expression vector for the PmYTH1 gene of *Pinus massoniana* and transforms it into *Arabidopsis thaliana* mutants and wild-type plants; transgenic *Arabidopsis thaliana* plants with significantly increased tolerance to ABA and NaCl are then bred, screened, and obtained. Results show that overexpression of PmYTH1 in the *Arabidopsis thaliana* ect2 / 3 / 4 mutant improves the ect2 / 3 / 4 mutant's tolerance to ABA, and overexpression of PmYTH1 in the wild-type *Arabidopsis thaliana* Col-0 improves the wild-type *Arabidopsis thaliana* Col-0's tolerance to NaCl; the expression levels of genes in the ABA signaling pathway are all significantly increased. This invention provides a molecular method and basis for breeding new *Pinus massoniana* varieties with high tolerance to ABA, NaCl, and abiotic stress. Attached Figure Description

[0018] Figure 1The graph shows the expression and purification of MBP-PmYTHDF1 / YTH-His and MBP-PmYTHDF1 / YTHm-His proteins (M is the marker; lanes 1 and 3 are the proteins before induction; lanes 2 and 4 are the purified recombinant proteins).

[0019] Figure 2 The image shows the qRT-PCR detection results of the T1 generation of transgenic Arabidopsis thaliana. In the image, A represents the identification results of transgenic seedlings of PmYTH1 in Arabidopsis thaliana ect2 / 3 / 4 mutants, and B represents the identification results of transgenic seedlings of PmYTH1 in wild-type Arabidopsis thaliana.

[0020] Figure 3 Figure 1 shows the results of PmYTH1 enhancing the tolerance of Arabidopsis ect2 / 3 / 4 mutants to ABA. In the figure, A represents the seed germination phenotypes of transgenic Arabidopsis ect2 / 3 / 4 mutants under ABA stress of 0, 0.4 μM, and 0.8 μM; B represents the statistical data on germination rate and green cotyledon rate of transgenic Arabidopsis ect2 / 3 / 4 mutants under ABA stress of 0, 0.4 μM, and 0.8 μM; WT represents wild-type Arabidopsis lines, and Arabidopsis ect2 / 3 / 4 mutants are the control lines; 35S::PmYTH1 / ect2 / 3 / 4 represents the PmYTH1 transgenic Arabidopsis lines.

[0021] Figure 4 Figure 1 shows the results of PmYTH1 improving the tolerance of wild-type Arabidopsis thaliana to NaCl. In the figure, A is the seed germination phenotype of transgenic Arabidopsis thaliana Col-0 under NaCl stress of 0 mM and 150 mM; B is the statistical data of germination rate and green cotyledon rate of transgenic Arabidopsis thaliana Col-0 under NaCl stress of 0 mM and 150 mM; WT is the wild-type Arabidopsis thaliana line; 35S::PmYTH1-L1 and 35S::PmYTH1-L2 are transgenic Arabidopsis thaliana lines of PmYTH1.

[0022] Figure 5 This figure shows the expression levels of the DWA1, DWA2, SDIRIP1, and CPN20 genes in 12-day-old transgenic Arabidopsis mutants. Detailed Implementation

[0023] 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.

[0024] The plant materials used in this application are pine needles, Arabidopsis thaliana ect2 / 3 / 4 mutants, and wild-type Arabidopsis thaliana Col-0, all of which are preserved in the National Key Laboratory of Forest Genetics and Breeding (60503 Pine Genetic Improvement Research Laboratory) of Nanjing Forestry University.

[0025] Example 1

[0026] 1. Total RNA extraction

[0027] Total RNA was extracted from Pinus massoniana needles using the Tiangen Polysaccharide and Polyphenol Plant Total RNA Extraction Kit. Before extraction, the mortar and pestle were wrapped in aluminum foil and baked in an oven at 180℃ for about 4 hours. The sample was then ground into powder in liquid nitrogen, and RNA was extracted according to the kit instructions and stored at -80℃.

[0028] The results are as follows Figure 1 As shown, the 1.2% agarose gel electrophoresis bands of total RNA from pine needles are clear; the absorbance OD of total RNA is [data missing]. 260 / OD 280 The value is 2.12, OD 260 / OD 230 With a value of 2.05, it can be used for gene cloning.

[0029] 2. Obtaining cDNA

[0030] Using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme), cDNA was obtained by reverse transcription using extracted RNA as a template. The specific process is as follows:

[0031] 1) Prepare the following mixture in an RNase-free centrifuge tube: 5 μg Total RNA, 8 μL RNase-free ddH2O. Heat at 60°C for 5 min, then rapidly cool on ice and let stand on ice for 2 min.

[0032] 2) Add 2 μL of 5×gDNA wiper Mix to 8 μL of the mixture from the previous step, gently pipette to mix, and incubate at 42℃ for 2 min.

[0033] 3. Cloning the target gene

[0034] Based on the PmYTH1 gene sequence obtained from the Masson pine genome download, intermediate fragment-specific primers were designed using Primer 5.0 to clone the ORF fragment of the PmYTH1 gene. The PmYTH1 cloning primers are:

[0035] PmYTH1-ORF-F: 5'-ATGGCAGCCGCTTCGGCACAA-3';

[0036] PmYTH1-ORF-R: 5'-CTAGCAACCATTTGTTTGCACTGG-3'.

[0037] 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.

[0038] 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 45 s, 35 cycles, and 72℃ extension for 5 min.

[0039] 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℃ 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℃ for 1 h at 200 rpm. Centrifuge at 10000 rpm for 30 s, discard 150 μL of the supernatant, and mix the remaining culture medium by pipetting. Spread the mixture onto Kan-resistant medium (pre-prepared at 37℃ for 1 h) and incubate overnight at 37℃. Positive clones were detected and sent to Qingke Biotechnology for sequencing. The final sequencing yielded the ORF nucleotide sequence of the PmYTH1 gene as shown in SEQ ID NO.1, and the amino acid sequence of the encoded protein as shown in SEQ ID NO.2.

[0040] Example 2

[0041] The pCAMBIA1305-PmYTH1 overexpression vector was constructed and transformed into Arabidopsis thaliana ect2 / 3 / 4 mutants and wild-type Arabidopsis thaliana Col-0. The phenotypic differences between T2 generation transgenic Arabidopsis thaliana and wild-type Arabidopsis thaliana were compared, and the function of the PmYTH1 gene in Pinus massoniana was analyzed.

[0042] 1. Construction of the pCAMBIA1305-PmYTH1 vector

[0043] The Escherichia coli strain used in this invention is Trans1-T1 (TransGen); the expression vector is pCAMBIA1305 (preserved in the laboratory of Nanjing Forestry University); the restriction endonuclease and ligase were purchased from Vazyme.

[0044] The specific steps are as follows:

[0045] The pCAMBIA-1305 expression vector was digested with XbaI and PstI restriction enzymes. The typical double digestion system (50 μL) for the expression vector consisted of: 1 µg pCAMBIA1305 plasmid, 1 μL QuickCut XbaI, 1 μL QuickCut PstI, 5 μL 10×QuickCut Buffer, and ddH2O up to 50 μL. The double digestion program was: 37℃ for 30 min; 85℃ for 20 s. XbaI and PstI restriction sites were added upstream and downstream of the target fragment via PCR, and the fragments were separated 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. The primer sequences used are shown below:

[0046] 1305-PmYTH1-F:

[0047] 5′-tttggagagaacacgtctagaATGGCAGCCGCTTCGGCA-3′,

[0048] 1305-PmYTH1-R:

[0049] 5′-gtcaagcttgcatgcctgcagCTAGCAACCATTTGTTTGCACTG-3′;

[0050] 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.

[0051] 2. Screening of positive Arabidopsis T1 generation plants transgenic with the PmYTH1 gene

[0052] Disinfection of Arabidopsis thaliana seeds: Take an appropriate amount of wild-type Arabidopsis thaliana seeds and put them into a sterilized EP tube. Add an appropriate amount of 75% ethanol, shake to disinfect for 30 seconds, and then aspirate. Add an equal amount of 0.1% mercuric chloride, disinfect 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 put the seeds into a new EP tube. Repeat the washing process four times.

[0053] Arabidopsis thaliana cultivation: 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℃ refrigerator for vernalization for 3 days; the vernalized medium was then placed in a 23℃ constant temperature incubator for 7 days; when the Arabidopsis thaliana seedlings had two true leaves, they were transplanted into the prepared substrate and placed in a light incubator with the following settings: 23℃ constant temperature, light intensity 5LS, humidity 75%, and light duration 16h / d; the seeds were used for transformation before the Arabidopsis thaliana entered its full flowering period.

[0054] The pCAMBIA1305-PmYTH1 vector was 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 value was 0.8, Arabidopsis flower organ immersion transformation was performed.

[0055] The specific steps are as follows:

[0056] Centrifuge the bacterial culture at 5000 rpm for 5 min, collect the bacterial cells, and suspend them in a 5% sucrose solution;

[0057] 1) Before soaking, add Silwet L-77 at a concentration of 0.05% (500 μL / L) and shake to remove foam;

[0058] 2) Soak the unpollinated inflorescences in Agrobacterium suspension for 30 seconds, gently shaking them during this time;

[0059] 3) Lay the soaked Arabidopsis thaliana flat on a tray, cover with plastic wrap to keep it moist, and seal with aluminum foil to protect it from light for 24 hours;

[0060] 4) Remove the aluminum foil and cultivate under normal conditions. Stop watering when the seeds are mature.

[0061] 5) Collect Arabidopsis thaliana seeds as T1 generation seeds, dry them at 37℃ for one week, and then store them.

[0062] After sterilization, the collected seeds were sown in 1 / 2 MS medium containing kanamycin (50 mg / L). Germination began after 3 days of vernalization, and the seeds 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. Nine plants were obtained after kanamycin screening and named T1-1 to T1-9. RNA was extracted from these nine transgenic Arabidopsis lines using a kit, reverse transcribed into cDNA, and then detected by qRT-PCR. The line with the highest expression level was selected for subsequent experiments.

[0063] RNA extraction and cDNA first-strand synthesis were performed using the same methods as in Example 1. The cDNA was diluted 10-fold after reverse transcription. *Pinus massoniana* TUA was selected as the internal control gene. The relative expression level of the *PmYTHDF1* gene was analyzed according to the instructions of the ChamQ™ SYBR qPCR Master Mix kit, and the significance of each expression level compared to the control group was analyzed using the t-test. Primer sequences are as follows:

[0064] qTUA-F: 5'-CAAACTTGGTCCCGTATCCTC-3';

[0065] qTUA-R: 5'-CACAGAAAGCTGCTCATGGTAA-3';

[0066] qPmYTH1-F: 5'-GTGCCTTCAACTGATAGGA-3';

[0067] qPmYTH1-R: 5'-CCAACAGGACCATCGTAA-3'.

[0068] 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.

[0069] The qRT-PCR reaction program was as follows: 95℃ for 1 min; 95℃ for 15 s, 58℃ for 15 s, 72℃ for 45 s, for 40 cycles.

[0070] 3. Obtaining T2 generation transgenic Arabidopsis thaliana

[0071] 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). Next, they were transferred to substrate soil, and the seeds were harvested as T2 generation transgenic Arabidopsis seeds. After being transferred to substrate soil, they were placed in a light incubator for cultivation (setting parameters: constant temperature and light intensity 23℃, light intensity 5 LS, humidity 75%, and light duration 16 h / d).

[0072] The results are as follows Figure 2 As shown, compared with the wild type, the relative expression levels of the target gene in the transgenic plants were significantly increased.

[0073] 4. Phenotypic observation of transgenic Arabidopsis thaliana

[0074] 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.

[0075] To determine ABA tolerance, in the ABA treatment experiment, WT, ect2 / 3 / 4, and 35S::PmYTH1 / ect2 / 3 / 4 seeds were sown on 1 / 2 MS medium supplemented with 0 μM, 0.4 μM, and 0.8 μM ABA, respectively, with 12 seeds of each type.

[0076] To determine NaCl tolerance, in the NaCl treatment experiment, WT, 35S::PmYTH1-L1, and 35S::PmYTH1-L2 seeds were sown on 1 / 2 MS medium supplemented with 0 mM and 150 mM NaCl, respectively, with 18 seeds of each type.

[0077] Germination (radicle sprouting) and post-germination growth (cotyledons turning green) were measured on the 4th and 12th day after vernalization, respectively.

[0078] The results are as follows Figure 3 As shown, A is the seed germination phenotype of transgenic Arabidopsis ect2 / 3 / 4 mutants under 0, 0.4 μM, and 0.8 μM ABA stress; B is a statistical graph of the germination rate and green cotyledon rate of transgenic Arabidopsis ect2 / 3 / 4 mutants under 0, 0.4 μM, and 0.8 μM ABA stress. The results show that overexpression of PmYTH1 in Arabidopsis ect2 / 3 / 4 mutants improves the tolerance of Arabidopsis ect2 / 3 / 4 mutants to ABA and increases the number of green cotyledons after germination.

[0079] like Figure 4As shown in the figure, A is the seed germination phenotype of transgenic Arabidopsis thaliana Col-0 under NaCl stress of 0 mM and 150 mM; B is the statistical data of germination rate and green cotyledon rate of transgenic Arabidopsis thaliana Col-0 under NaCl stress of 0 mM and 150 mM; WT is the wild-type Arabidopsis thaliana line; 35S::PmYTH1-L1 and 35S::PmYTH1-L2 are Arabidopsis thaliana lines transgenic with the PmYTH1 gene. The results confirmed that in wild-type Arabidopsis thaliana Col-0, overexpression of PmYTH1 improved the tolerance of wild-type Arabidopsis thaliana Col-0 to NaCl and increased the number of green cotyledons after germination.

[0080] 5. Relative expression levels of DWA1, DWA2, SDIRIP1, and CPN20

[0081] Arabidopsis thaliana TUB8 was selected as the internal control gene. The relative expression levels of DWA1, DWA2, SDIRIP1, and CPN20 genes were analyzed according to the instructions of the ChamQ™ SYBR qPCR Master Mix kit. The significance of differences between each expression level and the control group was analyzed using the t-test. Primer sequences are as follows:

[0082] qTUB8-F: 5'-CAAAGATGCGAGAGATTCTTCA-3';

[0083] qTUB8-R: 5'-CTTAGCTCCGATCTGGTTTCC-3';

[0084] qDWA1-F: 5'-ACGGAGACGCTACAAACTGG-3';

[0085] qDWA1-R: 5'-TTCGTGTGCCCTAACCACTC-3';

[0086] qDWA2-F: 5'-GTGTCTACGGCCTTGCTTG-3';

[0087] qDWA2-R: 5'-CCATCGGGACCTATCTTCTTG-3';

[0088] qSDIRIP1-F: 5'-AAGGTCTGCCACCACTTTAGG-3';

[0089] qSDIRIP1-R: 5'-AGAAGTTCCTCCTTCCTGATCATT-3';

[0090] qCPN20-F: 5'-GCCTTCTATTGGCACGGTGA-3';

[0091] qCPN20-R: 5'-TTCCTCGTCTAGGGAACCCG-3'.

[0092] The results are as follows Figure 5 As shown, the expression levels of ABA signaling pathway genes DWA1, DWA2, SDIRIP1, and CPN20 were all significantly increased.

[0093] 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. Application of the PmYTH1 gene of Pinus massoniana, with nucleotide sequence as shown in SEQ ID NO. 1, in promoting the growth of Arabidopsis thaliana mutants under ABA stress.

2. The application according to claim 1, characterized in that, include: 1) Construct an expression vector for the PmYTH1 gene of Masson pine; 2) The constructed expression vector of the Masson pine PmYTH1 gene was transformed into Arabidopsis mutants; 3) Breed, screen and obtain transgenic Arabidopsis plants with significantly increased tolerance to ABA.

3. Application of the PmYTH1 gene of Pinus massoniana, with nucleotide sequence as shown in SEQ ID NO. 1, in increasing the expression level of genes in the ABA signaling pathway.

4. The application according to claim 3, characterized in that, The ABA signaling pathway genes are DWA1, DWA2, SDIRIP1, and CPN20 genes.

5. Application of the PmYTH1 gene of Pinus massoniana, with nucleotide sequence as shown in SEQ ID NO. 1, in promoting the germination of Arabidopsis thaliana seeds under NaCl stress.

6. Application of the PmYTH1 gene of Pinus massoniana, whose nucleotide sequence is shown in SEQ ID NO. 1, in promoting an increase in the number of green cotyledons after germination of Arabidopsis thaliana seeds under NaCl stress.

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