Phyllostachys edulis sourced PeDHN2 gene and application of encoding protein thereof

Abstract

The invention discloses a phyllostachys edulis sourced PeDHN2 gene and application of a coding protein of the phyllostachys edulis sourced PeDHN2 gene, and belongs to the technical field of plant biological breeding. The nucleotide sequence of the PeDHN2 gene is as shown in SEQ ID NO. 11; the sequence of the PeDHN2 gene coding protein is as shown in SEQ ID NO. 12. According to the PeDHN2 gene cloned from moso bamboos, experiments show that expression of the gene is remarkably up-regulated under salt stress, drought and abscisic acid (ABA) stress, and after the gene is introduced into rice through a transgenic technology, the germination rate and root growth ability of the rice under salt stress are remarkably improved. The invention provides key gene resources and technical support for molecular design and breeding of saline-alkaline tolerant crops.

Description

Technical Field

[0001] The present invention relates to the technical field of plant biological breeding, and in particular to an application of a PeDHN2 gene derived from moso bamboo and a protein encoded therein. Background Art

[0002] Salt stress is one of the most serious abiotic stresses currently, and as secondary salinization caused by agricultural irrigation and improper use of chemical fertilizers is showing an intensifying trend, breeding new 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-yield, adaptable, high-quality tree with a wide range of uses. It has strong vitality and adaptability, can grow in a variety of environments, and grows very fast, averaging 2-3 meters per year. It reproduces primarily through the expansion of underground bamboo rhizomes and produces shoots, making it an ideal tree species for ecological restoration. However, most current bamboo varieties are not salt-tolerant, and the increasing severity of soil salinization has seriously threatened bamboo production. Therefore, it is of great significance to explore key salt-tolerant species and cultivate salt-tolerant varieties through molecular design breeding. Summary of the Invention

[0004] The present invention aims to provide a PeDHN2 gene derived from moso bamboo and its encoded protein for use in order to address the aforementioned problems in the prior art. The present invention isolates the PeDHN2 gene from moso bamboo, reveals its key role in salt stress response, and demonstrates its application value in significantly improving salt tolerance through transformation of rice.

[0005] To achieve the above object, the present invention provides the following solutions:

[0006] Technical solution 1: Application of the PeDHN2 gene or its encoded protein in improving plant stress resistance, 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 as SEQ ID NO.12.

[0008] Technical solution 2: A plant expression vector comprising the PeDHN2 gene.

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

[0010] Technical solution three: A primer set for detecting the expression of the PeDHN2 gene, the primer set sequence is shown in SEQ ID NO.5 and SEQ ID NO.6.

[0011] Technical solution four: A method for detecting the expression of PeDHN2 gene in plants, including performing real-time fluorescence quantitative PCR analysis using the primer set described above.

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

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

[0014] The present invention isolates the PeDHN2 gene from Phyllostachys edulis for the first time and clarifies its key role under salt stress. The results show that the germination rate of transgenic rice OE-PeDHN2 can reach 55%, significantly higher than that of WT. Under 8 μM ABA stress, the germination rate of OE-PeDHN2 remains at 95%, much higher than that of the control group. After continuous treatment with NaCl for 50 days, the overexpression line OE-PeDHN2 shows phenotypes of longer roots and vigorous leaf growth. After continuous treatment with ABA for 50 days, the roots of the transgenic line OE-PeDHN2 are significantly longer and thicker than those of the control, indicating that the overexpression of PeDHN2 endows rice with stronger resistance to NaCl and ABA. The present invention reveals the key role of the PeDHN2 gene in salt stress response and verifies its application value of significantly improving salt tolerance through transforming rice, filling the technical gap in this field. Description of the Drawings

[0015] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0016] Figure 1 It is a schematic diagram of the subcellular localization of PeDHN2 in tobacco leaf cells; among them, the positive control is the 35S::EGFP empty vector; its fusion protein is expressed in both the nucleus and the cytoplasm; the staining of tobacco leaf cells shows the position of the fusion protein, and the green fluorescence indicates the position of the fusion protein; the blue fluorescence represents the nuclear signal marker; the red fluorescence represents the cytoplasmic signal marker;

[0017] Figure 2 It is the expression pattern of PeDHN2 in Phyllostachys edulis after treatment with ABA, NaCl, and PEG; in the figure, green represents ABA treatment; yellow represents NaCl treatment; orange represents PEG treatment;

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

[0019] Figure 4 Heat map of germination rate statistics of PeDHN2 overexpressing rice under untreated, 80 mM NaCl treatment and 8 mM ABA treatment;

[0020] Figure 5 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) of transgenic rice. Detailed implementation manners

[0021] The various exemplary implementation manners 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, characteristics, and implementation schemes of the present invention.

[0022] It should be understood that the terms used in the present invention are only for describing particular implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0023] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0024] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the present invention specification are obvious to those skilled in the art. The present invention specification and examples are only exemplary.

[0025] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0026] Example 1

[0027] 1. Materials

[0028] The invention uses Chinese Guilin bamboo progeny seedlings as materials, cultivates bamboo seedlings in an artificial climate chamber (temperature 25°C, relative humidity 80%, 16 hours of light, 8 hours of darkness) until they are 6 months old, selects 60 bamboo seedlings with consistent growth, cleans the root system, and transfers them to Kimura nutrient solution for hydroponic culture for 4 weeks. Stress treatment is performed using Kimura nutrient solution (Huogelan, Hunan, China) containing 20% PEG-6000 (simulated drought, polyethylene glycol (PEG)), 300mM NaCl (salt stress), and 100μM ABA (hormone treatment). Leaf samples are collected at 0, 1, 3, 6, 9, 12, and 24 hours, quickly frozen in liquid nitrogen, and stored in a -80°C ultra-low temperature refrigerator for later use.

[0029] The transgenic rice experiment was carried out simultaneously, and the plant culture conditions were 24±1℃, 75% relative humidity, and a light intensity of 40μmol·m - 2 s - 1. Photoperiod: 16h / 8h (light / dark). Positive T1 rice seeds were tested for stress sensitivity in germination bags by preparing Kimura nutrient solution containing 80mM NaCl or 8μM ABA.

[0030] 2. Gene cloning and vector construction

[0031] RNA was extracted from leaves of Phyllostachys pubescens using the Quick RNA Isolation Kit (Hua Yueyang, Beijing, China). RNA concentration was determined using a microvolume 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 using a reverse transcription kit (TaKaRa, Kusatsu, Japan).

[0032] Using cDNA as a template, Snap Gene software (San Diego, CA, USA) was used to design specific primers F and R for PeDHN2.

[0033] PeDHN2-specific primer F:

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

[0035] PeDHN2 specific primer R:

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

[0037] Using 2× Max Master Mix kit (Vazyme, Nanjing, China), amplify the target sequence. Then use the DNA purification and recovery kit (Tiangen, Beijing, China) for purification. Insert the target sequence into the pCE2TA / Blunt-Zero vector (Vazyme, Nanjing, China) using a cloning kit in a metal bath at 25°C. Then transform the ligation product into Escherichia coli DH5α competent cells (Coolaber, Beijing, China) for colony PCR. Pick positive colonies from the colony PCR for sampling. Send them for Sanger DNA sequencing to Suzhou Azenta. Use SnapGene software to align and analyze the sequence results. Finally, extract the plasmid using a plasmid extraction kit (Tiangen, Beijing, China) and store it at -20°C for later use.

[0038] Design PeDHN2 seamless cloning specific primers using SnapGene software, and the sequences are shown in Table 1. Using the pCE2TA / Blunt-Zero vector plasmid containing the PeDHN2 fragment as a template, fuse the CDS sequence of the PeDHN2 gene with the enhanced green fluorescent protein (EGFP) tag into the pHG vector using the 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 tumefaciens competent cell GV3101 (Weidibio, Shanghai, China) by heat shock method and cultured at 28 °C for 2 days on LB solid medium containing kanamycin (Kan, 50 μg / mL) and rifampicin (Rif, 50 μg / mL). Single colonies identified as positive by PCR were picked and inoculated into 25 mL LB liquid medium, and cultured with shaking at 28 °C and 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 with the suspension (10 mM MES-KOH, 10 mM MgCl2, 200 μM acetosyringone, pH 5.7) to prepare an infection solution with an optical density (OD 600 ) reaching approximately 0.6. After standing for 3 h, 1 mL of the infection solution was injected into the back of tobacco using a 1 mL syringe. After infection, the plants were watered thoroughly, cultured in the dark at 25 °C for 1 day, and then cultured under light for 2 days. The infected tobacco leaves were cut into 1 cm 2 square pieces, 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 at 465 and 509 nm respectively.

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

[0044] Quantitative PCR primers for the PeDHN2 gene were designed using Primer Premier v.6, and the sequences are as follows:

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

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

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

[0048] 3. Transformation and stress treatment of rice

[0049] (1) Transforming Agrobacterium

[0050] Take 1 μL of plasmid and add it to 50 μL of Agrobacterium tumefaciens EHA105 competent cells. After thorough mixing, transfer it to an electroporation cuvette. After electroporation, add 1 mL of LB liquid medium, mix well, and transfer it to a 1.5 mL centrifuge tube. Incubate it on a shaker at 30 °C and 180 rpm for 30 min. Pipette 50 μL of the activated Agrobacterium liquid and inoculate it on an LB solid medium, and incubate it in the dark at 30 °C for 48 h.

[0051] 1) Detection of Agrobacterium

[0052] Prepare a PCR amplification system. After completion, mix well and use a PCR instrument for amplification. The amplification program is set according to primer information, etc.; forward primer: ATGGAGTACCAGGGGCAGCACG (SEQ ID NO.9), reverse primer: AGCGGCTCAGTGCTGGCC (SEQ ID NO.10); reaction system: forward primer (10 μM), 1 μL; reverse primer (10 μM), 1 μL; 2×Taq PCR Mix, 10 μL; ddH2O, 7 μL; template, 1 μL; total 20 μL.

[0053] Gel electrophoresis detection: Prepare 1% agarose gel (weigh 1.5 g of agarose powder, dissolve it in 150 mL of 1×TAE buffer, heat it in a microwave oven for 3 minutes until the liquid becomes transparent. Add EB to the gel-making plate, pour the dissolved agarose liquid into the gel-making plate, mix well, insert the comb, and let it stand for 40 minutes until the gel turns milky white), load the samples, and complete the electrophoresis process; Check the results of PCR amplification. When the electrophoresis bands of the positive control and the samples are clear and of the correct size, and there are no bands in the negative control, it indicates that the sample can proceed to the next step.

[0054] 2) Rice genetic transformation

[0055] A. Induce rice callus: Select rice grains without mildew spots and normal germ pores, disinfect them with 75% alcohol for 1 min, wash them with sterile water, 1 min / time; disinfect them with 15% sodium hypochlorite for 20 min, wash them with sterile water 3 times, 1 min / time; Inoculate the disinfected rice grains on the induction medium and culture them under light at 26°C for 20 days to induce callus;

[0056] B. Agrobacterium infection: Pick Agrobacterium into the infection solution to prepare an Agrobacterium resuspension with OD 600 = 0.2. Pick the callus into a triangular flask, add the Agrobacterium resuspension, after infecting for 10 - 15 min, discard the bacterial liquid, and inoculate the callus on the co-culture medium, and co-culture at 20°C for 48 - 72 h;

[0057] C. Screening of positive callus: Inoculate the co-cultured callus on the screening medium and culture it in the dark at 26°C for 20 - 30 days; Inoculate the positive callus into the secondary screening medium. When picking the callus, be sure to pick monoclonal callus and culture it in the dark at 26°C for 7 - 10 days;

[0058] D. Differentiation and rooting: Inoculate the positive callus on the differentiation medium and culture it under light at 25 - 27°C for 15 - 20 days. After the buds grow to 2 - 5 cm, inoculate them on the rooting medium and culture them under light at 30°C for 7 - 10 days;

[0059] E. Identification of positive seedlings: Extract rice genomic DNA using the CTAB method and perform PCR detection. The detection method is the same as that for Agrobacterium detection.

[0060] The components of each medium used above are detailed in Lei Shi. Optimization of the rapid transformation system of rice [D]. Hubei: Huazhong Agricultural University, 2023.

[0061] 4. Experimental results

[0062] (1) The CDS sequence and protein sequence of the PeDHN2 gene were cloned

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

[0064] ATGGAGTACCAGGGGCAGCACGGCGACGCCACCAGCCGCGTCGACGAGTACGGCAACCCGGTGGCCGCACACGGCGCCGCCGGCGGGCAGTTCCAACCGGCGAGGGAGGAGCACAAGACCGGAGGCATCCTGCAGCGCTCCGGCAGCTCCAGCTCCAGCTCGTCTTCTGAGGACGACGGCATGGGCGGGAGGAGGAAGAAGGGCATCAAGGAGAAGATCAAGGAGAAGCTCCCCGGCGGCCACAAGGACGAGCAGCACCAGACTACGGCGACAGGCGGCGCCTACGGGCAGCAAGGGCACGCTGGCATGACCAGCGGCACCGGGGCGCACGGGACCCAGGGCACCGGCGAGAAGAAGGGCCTCATGGACAAGATCAAGGAGAAGCTGCCCGGCCAGCACTGA;

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

[0066] MEYQGQHGDATSRVDEYGNPVAAHGAAGGQFQPAREEHKTGGILQRSGSSSSSSSSEDDGMGGRRKKGIKEKIKEKLPGGHKDEQHQTTATGGAYGQQGHAGMTSGTGAHGTQGTGEKKGLMDKIKEKLPGQH。

[0067] (2) Subcellular localization

[0068] The subcellular localization result of PeDHN2 is shown in Figure 1 , and the result shows that PeDHN2 is localized in the plasma membrane.

[0069] (3) Expression trend of PeDHN2 gene in Phyllostachys edulis under stress

[0070] The results of the expression pattern of PeDHN2 in Phyllostachys edulis after ABA, NaCl and PEG treatments are shown in Figure 2It can be seen that under the treatment of 100 μM ABA, after 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h, PeDHN2 shows an oscillatory response type. A rapid response occurred at 1 h after the stress treatment, and the expression level was significantly up-regulated compared with the control group (p<0.05). However, with the prolongation of the treatment time, its expression level decreased at 3 h. Under continuous stress conditions, a secondary activation trend was presented during 6 - 9 h, and the expression level gradually recovered to the peak, but it decreased again at 12 h. After 24 h of treatment, the gene expression showed an obvious rebound and finally stabilized at a high expression level trend.

[0071] Under the treatment of 300 mM NaCl, after 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h, PeDHN2 rapidly responded to the stress from 1 h to 12 h, and the expression level gradually increased, reaching the peak at 12 h, but the expression level began to show a downward trend at 24 h.

[0072] Under the treatment of 20% PEG, after 0 h, 1 h, 3 h, 6 h, 9 h, 12 h, and 24 h, all 5 PeDHNs genes showed a similar expression pattern of first increasing, then decreasing, and then increasing again. Under PEG treatment, PeDHN2 increased at 1 h, and its expression decreased relatively at 3 h and 6 h, and gradually increased from 9 h to 24 h.

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

[0074] The identification results of rice overexpressing PeDHN2 are shown in Figure 3 To evaluate the seed germination and early seedling growth of transgenic lines under different stress conditions, the present invention used double germination bag hydroponics to group-culture transgenic rice, and set 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 detection results of the germination rate of OE-PeDHN2 transgenic rice seeds treated with NaCl and ABA are shown in Figure 4 By observing and counting the germination rates on the 7th day and the 15th day, the results showed that under normal conditions, the germination rates of WT and transgenic rice seeds were both close to 100%, indicating that the experimental materials had good germination ability. Under salt stress conditions, the germination rate of WT decreased significantly to 25%, showing obvious salt stress sensitivity. While the germination rate of OE-PeDHN2 could reach 55%, which was significantly higher than that of the wild type (WT). Under the stress of 8 μM ABA, the germination rate of WT decreased to 55% on the 7th day, also showing sensitivity to ABA stress, while the germination rate of PeDHN2 remained at 95%, much higher than that of the control group.

[0076] The growth conditions of transgenic rice after 50 days of no treatment, NaCl stress, and ABA treatment are shown in Figure 5 . It can be seen that after 50 days of continuous NaCl treatment, the above-ground parts of WT showed wilting and death, while several overexpression lines showed phenotypes of longer root length and vigorous leaf growth. Similarly, after 50 days of continuous ABA treatment, there were no significant phenotypic differences in the above-ground parts between 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 conferred stronger resistance to NaCl and ABA in rice.

[0077] The embodiments described above are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of the PeDHN2 gene or its encoded protein in enhancing the stress resistance of plants, characterized in that, The nucleotide sequence of the PeDHN2 gene is shown in SEQ ID NO.

11.

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

12.

3. A plant expression vector, characterized in that, Comprising the PeDHN2 gene according to claim 1.

4. The plant expression vector according to claim 3, characterized in that, The plant expression vector is constructed by fusing the pHG vector with the EGFP and PeDHN2 genes.

5. A primer set for detecting the expression of the PeDHN2 gene described in claim 1, characterized in that, The primer set sequences are shown in SEQ ID NO.5 and SEQ ID NO.

6.

6. A method for detecting the expression of the PeDHN2 gene in plants, characterized in that, The method comprises using the primer set according to claim 5 to perform real-time fluorescence quantitative PCR analysis.

7. A method for improving the salt stress tolerance of plants, characterized in that, The method comprises the steps of introducing the PeDHN2 gene or its encoded protein into the 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.

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