Phyllostachys edulis-derived pedhn2 gene and application of the encoded protein thereof

Abstract

The application discloses a Phyllostachys edulis-derived PeDHN2 gene and application of a coded protein thereof, and belongs to the technical field of plant biological breeding. A nucleotide sequence of the PeDHN2 gene is shown as SEQ ID NO. 11; and a sequence of the coded protein of the PeDHN2 gene is shown as SEQ ID NO. 12. The PeDHN2 gene is cloned from the Phyllostachys edulis, and experiments show that the gene is significantly up-regulated under salt stress, drought and abscisic acid (ABA) stress, and after being introduced into rice through a transgenic technology, the germination rate and root growth ability of the rice under salt stress are significantly improved. The application provides a key gene resource and technical support for molecular design breeding of salt-tolerant crops.

Description

Technical Field

[0001] This invention relates to the field of plant biobreeding technology, and in particular to the application of a PeDHN2 gene derived from moso bamboo and its encoded protein. Background Technology

[0002] Salt stress is one of the most serious abiotic stresses, and secondary salinization caused by improper agricultural irrigation and fertilizer use is intensifying. Therefore, cultivating salt-tolerant varieties and developing and utilizing saline-alkali land are important strategic needs to ensure food security.

[0003] Moso bamboo (Phyllostachy edulis) is a high-yielding, highly adaptable, high-quality, and versatile species. It possesses strong vitality and adaptability, capable of growing in diverse environments, and grows very rapidly, averaging 2-3 meters in height annually. It primarily reproduces through the expansion of underground rhizomes and the production of new shoots, making it an ideal species for ecological restoration. However, most current moso bamboo varieties are not salt-tolerant, and with the increasing severity of soil salinization, moso bamboo yields are seriously threatened. Therefore, identifying key salt-tolerant varieties and employing molecular design breeding to cultivate salt-tolerant varieties is of paramount importance. Summary of the Invention

[0004] The purpose of this invention is to provide an application of the PeDHN2 gene derived from moso bamboo and its encoded protein, thereby addressing the problems existing in the prior art. This invention isolates the PeDHN2 gene from moso bamboo, reveals its crucial role in salt stress response, and verifies its application value in significantly enhancing salt tolerance through transformation of rice.

[0005] To achieve the above objectives, the present invention provides the following solution:

[0006] Technical Solution 1: Application of the PeDHN2 gene or its encoded protein in enhancing plant stress resistance, wherein the nucleotide sequence of the PeDHN2 gene is shown in SEQ ID NO.11.

[0007] Furthermore, the sequence of the protein encoded by the PeDHN2 gene is shown in SEQ ID NO.12.

[0008] Technical Solution 2: A plant expression vector containing the PeDHN2 gene.

[0009] Furthermore, the plant expression vector was constructed by fusing the pHG vector with the EGFP and PeDHN2 genes.

[0010] Technical Solution 3: A primer set for detecting the expression of the PeDHN2 gene, the primer set sequences of which are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0011] Technical Solution 4: A method for detecting PeDHN2 gene expression in plants, comprising real-time quantitative PCR analysis using the primer set described above.

[0012] Technical Solution 5: A method for improving the salt stress tolerance of plants, comprising the step of introducing the PeDHN2 gene or its encoded protein into a target plant; the nucleotide sequence of the PeDHN2 gene is shown in SEQ ID NO.11; the sequence of the protein encoded by the PeDHN2 gene is shown in SEQ ID NO.12.

[0013] The present invention discloses the following technical effects:

[0014] This invention is the first to isolate the PeDHN2 gene from moso bamboo and clarify its crucial role under salt stress. Results showed that the germination rate of the transgenic rice OE-PeDHN2 reached 55%, significantly higher than that of the control group (WT). Under 8 μM ABA stress, the germination rate of OE-PeDHN2 remained at 95%, far exceeding that of the control group. After 50 days of continuous NaCl treatment, the overexpressing line OE-PeDHN2 exhibited a phenotype with longer roots and vigorous leaf growth. After 50 days of continuous ABA treatment, the roots of the transgenic line OE-PeDHN2 were significantly longer and thicker than the control, indicating that overexpression of PeDHN2 endowed rice with strong resistance to NaCl and ABA. This invention reveals the crucial role of the PeDHN2 gene in the salt stress response and verifies its application value in significantly enhancing salt tolerance through rice transformation, filling a technological gap in this field. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0016] Figure 1 This diagram illustrates the subcellular localization of PeDHN2 in tobacco leaf cells; the positive control is the empty 35S::EGFP vector; its fusion protein is expressed in both the nucleus and cytoplasm; staining of tobacco leaf cells shows the location of the fusion protein, with green fluorescence indicating the location of the fusion protein; blue fluorescence represents nuclear signal markers; and red fluorescence represents cytoplasmic signal markers.

[0017] Figure 2 The expression patterns of PeDHN2 in moso bamboo after treatment with ABA, NaCl, and PEG are shown in the figure; green represents ABA treatment; yellow represents NaCl treatment; and orange represents PEG treatment.

[0018] Figure 3 Identification of positive strains of pHG-PeDHN2 transgenic rice; Figure caption: +, positive control; M, marker; -O, negative control; 1-11 are transgenic lines;

[0019] Figure 4 Heatmap of germination rate in PeDHN2 overexpressing rice under untreated, 80 mM NaCl treated, and 8 mM MABA treated conditions;

[0020] Figure 5 The growth status of wild-type rice (WT) and transgenic rice (OE-PeDHN2) after 50 days of no treatment (A), NaCl stress (B), and ABA treatment (C). Detailed Implementation

[0021] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0022] It should be understood that the terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any stated value or intermediate value within a stated range, as well as each smaller range between any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0024] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0025] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0026] Example 1

[0027] 1. Materials

[0028] This invention uses bamboo seedlings from Guilin, China as material. Bamboo seedlings were cultivated to 6 months of age in an artificial climate chamber (temperature 25℃, relative humidity 80%, 16h light, 8h darkness). Sixty uniformly growing bamboo seedlings were selected, and after root cleaning, they were transferred to Kimura nutrient solution for 4 weeks of hydroponic cultivation. Stress treatments were applied using Kimura nutrient solution (Huogelan, Hunan, China) containing 20% ​​PEG-6000 (simulating drought, polyethylene glycol (PEG)), 300mM NaCl (salt stress), and 100μM ABA (hormone treatment). Leaf samples were collected at 0, 1, 3, 6, 9, 12, and 24h, flash-frozen in liquid nitrogen, and stored at -80℃ for later use.

[0029] The transgenic rice experiment was conducted simultaneously, with plant culture conditions of 24±1℃, 75% relative humidity, and a light intensity of 40 μmol·m⁻². - 2·s - 1. Photoperiod 16h / 8h (light / dark). The germination bag stress sensitivity of positive T1 generation rice seeds was tested by preparing Kimura nutrient solution containing 80mM NaCl or 8μMABA.

[0030] 2. Gene cloning and vector construction

[0031] RNA was extracted from bamboo leaves using the Quick RNA Isolation Kit (Hua Yueyang, Beijing, China). RNA concentration was determined using a micro-volume spectrophotometer (Thermo Scientific, Waltham, MA, USA), and RNA integrity and quality were assessed by 1% agarose gel electrophoresis (Bio-Rad, Hercules, CA, USA). First-strand cDNA was synthesized using the extracted RNA as a template via a reverse transcription kit (TaKaRa, Kusatsu, Japan).

[0032] Using cDNA as a template, specific primers F and R for PeDHN2 were designed using Snap Gene software (San Diego, CA, USA).

[0033] PeDHN2 specific primer F:

[0034] ATGGAGTACCAGGGGCAGCACG(SEQ ID NO.1);

[0035] PeDHN2 specific primer R:

[0036] AGCGGCTCAGTGCTGGCC (SEQ ID NO. 2).

[0037] Use 2× The target sequence was amplified using the Max Master Mix kit (Vazyme, Nanjing, China). Then, it was purified using a DNA purification and recovery kit (Tiangen, Beijing, China). The target sequence was inserted into the pCE2TA / Blunt-Zero vector (Vazyme, Nanjing, China) using a cloning kit in a 25°C metal bath. The ligation product was then transformed into *E. coli* DH5α competent cells (Coolaber, Beijing, China) for colony PCR. Positive colonies were picked from the colony PCR results and sent for Sanger DNA sequencing to Suzhou Azenta. Sequence alignment was performed using SnapGene software. Finally, plasmids were extracted using a plasmid extraction kit (Tiangen, Beijing, China) and stored at -20°C for later use.

[0038] PeDHN2 seamless cloning-specific primers were designed using SnapGene software; their sequences are shown in Table 1. Using the pCE2TA / Blunt-Zero vector plasmid containing the PeDHN2 fragment as a template, the CDS sequence of the PeDHN2 gene was fused with an enhanced green fluorescent protein (EGFP) tag into the pHG vector using a 2×Phanta Max Master Mix high-fidelity enzyme kit (Vazyme, Nanjing, China).

[0039] Table 1 Seamless Cloning Specific Primers

[0040]

[0041] (1) Subcellular localization

[0042] The constructed plant expression vector plasmid was transformed into Agrobacterium competent cells GV3101 (Weidibio, Shanghai, China) using the heat shock method. The cells were cultured for 2 days at 28°C on LB solid medium containing kanamycin (Kan, 50 μg / mL) and rifampin (Rif, 50 μg / mL). Single colonies that tested positive by PCR were picked and inoculated into 25 mL of LB liquid medium, and cultured at 28°C with shaking at 200 rpm / min for 16 h. The samples were centrifuged at 4000 rpm for 10 min at room temperature to collect bacterial cells. The supernatant was discarded, and the cells were resuspended in a suspension (10 mM MES-KOH, 10 mM MgCl2, 200 μM acetosyringone, pH 5.7) to prepare an optical density (OD) reading. 600 The infection solution reached a concentration of approximately 0.6. After standing for 3 hours, the infection solution was injected into the back of the tobacco plant using a 1 mL syringe. After infection, the plants were thoroughly watered and incubated in the dark at 25°C for 1 day, followed by 2 days of light incubation. A 1 cm section of the infected tobacco leaf was then cut off. 2 Small square pieces were stained with 4′,6-diamidino-2-phenylindole (DAPI) solution (10 mg / mL) at room temperature for 10–15 min, and then rinsed 5 times with sterile water. The fluorescence signals of DAPI and EGFP were observed and photographed using an Axio Imager M2 microscope (Zeiss, Oberkochen, Germany) under light excitation of 465 and 509 nm, respectively.

[0043] (2) Real-time quantitative PCR under stress treatment

[0044] Primers for quantitative PCR of the PeDHN2 gene were designed using Primer Premier v6, and the sequences are as follows:

[0045] PeDHN2-F:CTCCAGCTCCAGCTCGTCTTCT (SEQ ID NO.5);

[0046] PeDHN2-R: TCTGGTGCTGCTCGTCCTTGT (SEQ ID NO. 6).

[0047] Seven-month-old bamboo seedlings were used as material and treated with Kimura nutrient solution, nutrient solution containing 80mM MABA, nutrient solution containing 100mM NaCl, and nutrient solution containing 20% ​​PEG for 0h, 3h, 6h, 9h, 12h, and 24h, respectively. The second leaf from the top was collected at each time point for qRT-PCR. Real-time quantitative PCR (qRT-PCR) was performed using TB Green & Premix Ex TaqTMII (TaKaRa, Kusatsu, Japan) on a qTOWER RT-PCR system (Analytik, Jena, Germany). GAPDH was used as an internal reference gene (GAPDH forward primer: CAAGGCTGTTGGCAAGGTTC (SEQ ID NO.7), reverse primer R: CATATGAGGCAGACTTCTCGATTC (SEQ ID NO.8)). A 2 -ΔΔCT The relative gene expression levels in moso bamboo tissues after different treatments were analyzed, with the expression level in moso bamboo leaf tissues at 0d serving as the control group.

[0048] 3. Transformation and stress treatment of rice

[0049] (1) Transformation of Agrobacterium tumefaciens

[0050] Add 1 μL of plasmid to 50 μL of EHA105 Agrobacterium competent cells, mix thoroughly, and then transfer to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix thoroughly, and then transfer to a 1.5 mL centrifuge tube. Incubate at 30°C and 180 rpm for 30 min on a shaker. Then, inoculate 50 μL of the activated Agrobacterium culture onto LB solid medium and incubate in the dark at 30°C for 48 h.

[0051] 1) Agrobacterium detection

[0052] Prepare the PCR amplification system, mix thoroughly after preparation, and perform amplification using a PCR instrument. The amplification program is set according to the primer information, etc.; Forward primer: ATGGAGTACCAGGGGCAGCACG (SEQ ID NO.9), reverse primer: AGCGGCTCAGTGCTGGCC (SEQ ID NO.10); Reaction system: forward primer (10μM), 1μL; reverse primer (10μM), 1μL; 2×Taq PCR Mix, 10μL; ddH2O, 7μL; template, 1μL; total 20μL.

[0053] For gel electrophoresis detection, prepare a 1% agarose gel (weigh 1.5g of agarose powder and dissolve it in 150mL of 1×TAE buffer, microwave for 3 minutes until the liquid becomes transparent. Add EB to the gel casting plate, pour the dissolved agarose liquid into the plate, mix well, insert a comb, and let stand for 40 minutes until the gel turns milky white), spot the sample, and complete the electrophoresis process; check the PCR amplification results. If the electrophoretic bands of the positive control and the sample are clear and of the correct size, and the negative control has no band, the sample can proceed to the next step.

[0054] 2) Rice genetic transformation

[0055] A. Induction of rice callus: Select rice grains without mold spots and with normal bud openings, disinfect with 75% alcohol for 1 min, rinse with sterile water for 1 min / time; disinfect with 15% sodium hypochlorite for 20 min, rinse with sterile water 3 times, 1 min / time; inoculate the disinfected rice grains into the induction medium, and culture at 26℃ under light for 20 days to induce callus.

[0056] B. Agrobacterium infection: Agrobacterium was picked up and placed in the infection solution to prepare OD. 600 =0.2% Agrobacterium resuspension, pick the callus into an Erlenmeyer flask, add Agrobacterium resuspension, infect for 10-15 min and discard the bacterial solution, inoculate the callus into co-culture medium and co-culture at 20℃ for 48-72 h;

[0057] C. Positive callus screening: Inoculate the co-cultured callus into the screening medium and incubate in the dark at 26°C for 20-30 days; inoculate the positive callus into the secondary screening medium, and be sure to select single-clonal callus during the callus picking process, and incubate in the dark at 26°C for 7-10 days.

[0058] D. Differentiation and rooting: Inoculate positive callus into differentiation medium and culture at 25-27℃ under light for 15-20 days. After the shoots of 2-5cm have differentiated, inoculate them into rooting medium and culture at 30℃ under light for 7-10 days.

[0059] E. Identification of positive seedlings: Rice genomic DNA was extracted using the CTAB method and then detected by PCR. The detection method was the same as that for Agrobacterium detection.

[0060] For details of the components of each culture medium used above, please refer to Shi Lei, Optimization of Rapid Conversion System for Rice [D]. Hubei: Huazhong Agricultural University, 2023.

[0061] 4. Experimental Results

[0062] (1) The CDS sequence and protein sequence of the PeDHN2 gene were obtained by cloning.

[0063] The CDS sequence of PeDHN2 (SEQ ID NO.11):

[0064] ATGGAGTACCAGGGGCAGCACGGCGACGCCACCAGCCGCGTCGACGAGTACGGCAACCCGGTGGCCGCACACGGCGCCGCCGGCGGGCAGTTCCAACCGGCGAGGGAGGAGCACAAGACCGGAGGCATCCTGCAGCGCTCCGGCAGCTCCAGCTCCAGCTCGTCTTCTGAGGACGACGGCATGGGCGGGAGGAGGAAGAAG GGCATCAAGGAGAAGATCAAGGAGAAGCTCCCCGGCGGCCACAAGGACGAGCAGCACCAGACTACGGCGACAGGCGGCGCCTACGGGCAGCAAGGGCACCGCTGGCATGACCAGCGGCACCGGGGCGCACGGGACCCAGGGCACCGGCGAGAAGAAGGGCCTCATGGACAAGATCAAGGAGAAGCTGCCCGGCCAGCACTGA;

[0065] The protein sequence of PeDHN2 (SEQ ID NO.12):

[0066] MEYQGQHGDATSRVDEYGNPVAAHGAAGGQFQPAREEHKTGGILQRSGSSSSSSSEDDGMGGRRKKGIKEKIKEKLPGGHKDEQHQTTATGGAYGQQGHAGMTSGTGAHGTQGTGEKKGLMDKIKEKLPGQH.

[0067] (2) Subcellular localization

[0068] The subcellular localization results of PeDHN2 are shown in […]. Figure 1 The results showed that PeDHN2 was located in the cell membrane.

[0069] (3) Expression trend of PeDHN2 gene in the stress response of moso bamboo

[0070] The expression patterns of PeDHN2 in moso bamboo after treatment with ABA, NaCl, and PEG are shown in the figure. Figure 2It is evident that under 100 μMABA treatment, PeDHN2 exhibits an oscillating response at 0h, 1h, 3h, 6h, 9h, 12h, and 24h. A rapid response is observed at 1h of stress treatment, with expression levels significantly upregulated compared to the control group (p<0.05). However, with prolonged treatment, expression levels decrease after 3h. Under continuous stress, a secondary activation trend is observed between 6-9h, with expression levels gradually recovering to peak levels, only to decline again at 12h. After 24h of treatment, gene expression shows a significant rebound, eventually stabilizing at a high expression level.

[0071] Under 300mM NaCl treatment, PeDHN2 rapidly responded to stress from 1h to 12h, with expression gradually increasing and reaching a peak at 12h. However, the expression level began to decline at 24h.

[0072] Under 20% PEG treatment, the expression of the five PeDHN genes showed a similar pattern of first increasing, then decreasing, and then increasing again after 0h, 1h, 3h, 6h, 9h, 12h, and 24h. Under PEG treatment, PeDHN2 expression increased at 1h, decreased relatively at 3h and 6h, and gradually increased from 9h to 24h.

[0073] (4) Rice overexpressing PeDHN2 acquires strong salt tolerance

[0074] The results of rice overexpression of PeDHN2 are shown in the figure. Figure 3 To evaluate the seed germination and early seedling growth of transgenic lines under different stress conditions, this invention employed double-germination bag hydroponics to culture transgenic rice in groups, setting NaCl (80 mM) and ABA (8 μM) as stress conditions. Three biological replicates were set up, named OE-PeDHN2 and WT1 / 2 / 3 (Wild-Type), respectively.

[0075] The germination rate of OE-PeDHN2 transgenic rice seeds treated with NaCl and ABA was detected as follows: Figure 4 By observing and statistically analyzing the germination rates on days 7 and 15, the results showed that under normal conditions, the germination rates of both WT and transgenic rice seeds were close to 100%, indicating that the experimental materials had good germination capabilities. However, under salt stress, the germination rate of WT significantly decreased to 25%, demonstrating obvious sensitivity to salt stress. In contrast, the germination rate of OE-PeDHN2 reached 55%, significantly higher than that of wild-type (WT). Under 8 μMABA stress, the germination rate of WT decreased to 55% on day 7, also showing sensitivity to ABA stress, while the germination rate of PeDHN2 remained at 95%, far higher than the control group.

[0076] The growth status of transgenic rice after 50 days under no treatment, NaCl stress, and ABA treatment is shown in the figure. Figure 5 It is evident that after 50 days of continuous NaCl treatment, the aboveground parts of the WT plants wilted and died, while several overexpression lines exhibited phenotypes with longer roots and vigorous leaf growth. Similarly, after 50 days of continuous ABA treatment, there were no significant phenotypic differences in the aboveground parts of the control and transgenic lines, but the roots of the transgenic lines were significantly longer and thicker than those of the control, indicating that overexpression of PeDHN2 endowed rice with strong resistance to NaCl and ABA.

[0077] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. Application of PeDHN2 gene or its coded protein in improving salt resistance of plants, characterized in that, The nucleotide sequence of the PeDHN2 gene is shown in SEQ ID NO.11; the plant is rice or moso bamboo.

2. Use according to claim 1, characterized in that, The sequence of the protein encoded by the PeDHN2 gene is shown in SEQ ID NO.

12.

3. A method for improving the salt stress tolerance of a plant, characterized in that, The method includes the step of introducing the PeDHN2 gene or its encoded protein into a target plant; the nucleotide sequence of the PeDHN2 gene is shown in SEQ ID NO.11; the sequence of the protein encoded by the PeDHN2 gene is shown in SEQ ID NO.12; and the plant is rice or moso bamboo.

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