Application of PpALKBH2 gene in improving abiotic stress resistance of plants

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a PpALKBH2 gene to improvement of abiotic stress resistance of plants, and the ID number of the PpALKBH2 gene is Pp3c211520. According to the phytoneum parvum PpALKBH2 gene knockout mutant constructed by the invention, the PpALKBH2 is subjected to frame shift mutation, so that the drought stress resistance is reduced, and the methylation damage resistance is reduced; the constructed PpALKBH2 gene is used for overexpressing the erencigia parviflora, the gene expression quantity is increased, the protein content is accumulated, the methylation damage resistance is enhanced, and the salt stress tolerance is also enhanced, which indicates that the PpALKBH2 gene can regulate and control the abiotic stress resistance of plants. Experiments show that when the PpALKBH2 gene is used for changing the expression level of the PpALKBH2 and improving the stress resistance of plants, the PpALKBH2 gene has relatively high practical application value.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of the PpALKBH2 gene in improving the abiotic stress resistance of plants. Background Art

[0002] Bryophytes are higher plants that landed early. They require relatively simple nutrients for growth, have a short life cycle, and can complete the entire process of "sporophyte - protonema - gametophyte - sporophyte" in 8 weeks. Bryophytes are in a special evolutionary position between aquatic plants and terrestrial plants, possessing the strong growth and reproduction ability of aquatic plants, the multicellular traits of algae plants, and the complete plant morphology of higher plants. In addition, bryophytes are widely used in environmental monitoring. They can be used as indicator species to evaluate the pollution degree and ecosystem health, and can also be used in the research of vegetation succession and ecosystem restoration, etc., with broad application prospects. Currently, abiotic stress is one of the important factors affecting the growth of bryophytes. Therefore, it is very important to explore bryophyte genes resistant to abiotic stress, analyze the mechanism of bryophyte resistance to abiotic stress, and improve the abiotic stress resistance of bryophytes for their growth and development. Summary of the Invention

[0003] The purpose of the present invention is to provide the application of the PpALKBH2 gene in improving the abiotic stress resistance of plants, construct Physcomitrella patens with overexpression of the PpALKBH2 gene, whose ability to resist methylation damage is enhanced and whose tolerance to salt stress is also enhanced, indicating that the PpALKBH2 gene can regulate the abiotic stress resistance of plants.

[0004] The present invention provides the application of the PpALKBH2 gene in regulating the abiotic stress resistance of plants, and the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0005] As a preferred embodiment, the regulation includes overexpressing the PpALKBH2 gene to improve the abiotic stress resistance of plants; knocking out or silencing the PpALKBH2 gene to reduce the abiotic stress resistance of plants.

[0006] As a preferred embodiment, the abiotic stress includes one or more of drought stress, salt stress, and methylation damage stress.

[0007] The present invention also provides a primer pair for amplifying the PpALKBH2 gene, and the primer pair includes a forward primer and a reverse primer; the nucleotide sequence of the forward primer is as shown in SEQ ID NO:1; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:2; the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0008] The present invention also provides a biological material capable of regulating the expression level of the PpALKBH2 gene, including a biological material overexpressing the PpALKBH2 gene and a biological material knocking out or silencing the PpALKBH2 gene; the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0009] As a preferred embodiment, the biological material overexpressing the PpALKBH2 gene includes one or more of a recombinant expression vector containing the PpALKBH2 gene, a recombinant microorganism containing the PpALKBH2 gene, and a transgenic cell line containing the PpALKBH2 gene.

[0010] As a preferred embodiment, the biological material knocking out or silencing the PpALKBH2 gene includes one or more of a gene editing target site of the PpALKBH2 gene, a gene editing vector of the PpALKBH2 gene, a recombinant vector knocking out or silencing the PpALKBH2 gene, a recombinant microorganism containing the recombinant vector knocking out or silencing the PpALKBH2 gene, and a transgenic cell line containing the recombinant vector knocking out or silencing the PpALKBH2 gene;

[0011] The gene editing target sites of the PpALKBH2 gene include target site 1 and target site 2; the nucleotide sequence of target site 1 is as shown in SEQ ID NO:3; the nucleotide sequence of target site 2 is as shown in SEQ ID NO:4.

[0012] The present invention also provides the use of the primer pair or the biological material in regulating the abiotic stress resistance of plants and / or creating new germplasms.

[0013] The present invention also provides a method for improving the abiotic stress resistance of plants, including the following steps: overexpressing the PpALKBH2 gene in the genome of a target plant to obtain a plant with improved abiotic stress resistance, and the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0014] The present invention also provides a method for creating a plant model with reduced abiotic stress resistance, including the following steps: knocking down the expression level of the PpALKBH2 gene in the genome of a target plant or knocking out the PpALKBH2 gene in the genome of a target plant to obtain the plant with reduced abiotic stress resistance, and the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0015] Beneficial effects: The present invention provides the application of the PpALKBH2 gene in improving the abiotic stress resistance of plants. The ID number of the PpALKBH2 gene is Pp3c2_11520. The knockout mutant of the Physcomitrella patens PpALKBH2 gene constructed in the present invention has a frameshift mutation in PpALKBH2, resulting in reduced drought stress resistance and reduced resistance to methylation damage. The overexpressed Physcomitrella patens with the PpALKBH2 gene constructed has enhanced resistance to methylation damage and also enhanced tolerance to salt stress. The experiments of the present invention show that the PpALKBH2 gene can regulate the abiotic stress resistance of plants. It is shown that using the PpALKBH2 gene to change the expression level of PpALKBH2 and improve the stress resistance of plants has high practical application value. Description of the drawings

[0016] 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 for use in the embodiments.

[0017] Figure 1 For targeted knockout of PpALKBH2 by gene editing means, where a is a schematic diagram of the PpALKBH2 knockout target site; b is the amplification of the target site DNA fragment of the PpALKBH2 knockout line; c is the sequencing of the PpALKBH2 knockout site; CRISPR-8, CRISPR-15, CRISPR-25, and CRISPR-28 are 4 obtained knockout mutants;

[0018] Figure 2 It is a schematic diagram and identification diagram of the pPOG1(ubi)-PpALKBH2 vector, where a is a schematic diagram of the pPOG1(ubi)-PpALKBH2 vector structure; b is the DNA identification diagram of the OE-PpALKBH2 positive material, c is the RNA level identification of the OE-PpALKBH2 material; d is the protein level identification of the OE-PpALKBH2 positive material; OE-PpALKBH2-1, OE-PpALKBH2-4, OE-PpALKBH2-5, and OE-PpALKBH2-6 are 4 obtained overexpressed materials;

[0019] Figure 3 It is the phenotypes and relative water content of the wild type and mutants during repeated drought and rewatering treatments. Among them, a is the phenotypic changes of the wild type and Ppalkbh2 mutants before and after repeated drought and rewatering treatments; b is the change in the relative water content in plants of the wild type and Ppalkbh2 mutants during multiple drought and rewatering treatments;

[0020] Figure 4is the maximum photochemical efficiency of Physcomitrium patens during repeated drought and rehydration treatments. Among them, a-b shows the changes in the maximum photochemical efficiency of the wild type and Ppalkbh2 mutants during multiple drought and rehydration treatments. The F v / F m of the wild type (WT), and the F v / F m of Ppalkbh2. Statistical significance was analyzed by t-test (ns indicates no significant difference, ****p < 0.001);

[0021] Figure 5 are the protonema survival rates of wild type and mutants of Physcomitrium patens under different concentrations of MMS treatment; 11520-CRISPR-15, 11520-CRISPR-25, and 11520-CRISPR-28 are mutant materials; the scale bar is 1 cm;

[0022] Figure 6 are the phenotypic characteristics of wild type and mutants of Physcomitrium patens under different concentrations of MMS treatment. Among them, a shows the observation and statistics of the protonema area of mutants and wild type when recovered for 14 D after MMS treatment; b shows the observation and statistics of the number of new gametophytes of mutants and wild type when recovered for 21 D after MMS treatment; Ppalkbh2-15, Ppalkbh2-25, and Ppalkbh2-28 are mutant materials; the scale bar is 2 mm;

[0023] Figure 7 are the protonema survival rates of wild type and overexpression of Physcomitrium patens under different concentrations of MMS treatment; PU-11520-1, PU-11520-4, and PU-11520-6 are overexpression materials; the scale bar is 1 cm;

[0024] Figure 8 are the phenotypic characteristics of wild type and overexpression of Physcomitrium patens under different concentrations of MMS treatment. Among them, a shows the observation and statistics of the protonema area of overexpression and wild type when recovered for 14 D after MMS treatment; b shows the observation and statistics of the number of new gametophytes of overexpression and wild type when recovered for 21 D after MMS treatment; OE-PpALKBH2-1, OE-PpALKBH2-4, and OE-PpALKBH2-6 are overexpression materials; the scale bar is 2 mm;

[0025] Figure 9Phenotypic characteristics of wild-type Physcomitrella patens and Ppalkbh2 mutants under 0 M NaCl treatment, where a is the phenotypic observation of mutants and wild-type under 0 M NaCl treatment; b is the statistical result of photosynthetic parameters of mutants and wild-type under 0 M NaCl treatment; Ppalkbh2-15, Ppalkbh2-25, and Ppalkbh2-28 are mutant materials; the scale bar is 2 mm;

[0026] Figure 10 Phenotypic characteristics of wild-type Physcomitrella patens and Ppalkbh2 mutants under 0.4 M NaCl treatment, where a is the phenotypic observation of mutants and wild-type under 0.4 M NaCl treatment; b is the statistical result of photosynthetic parameters of mutants and wild-type under 0.4 M NaCl treatment; Ppalkbh2-15, Ppalkbh2-25, and Ppalkbh2-28 are mutant materials; the scale bar is 2 mm;

[0027] Figure 11 Phenotypic characteristics of wild-type Physcomitrella patens and overexpressed OE-PpALKBH2 under 0 M NaCl treatment, where a is the phenotypic observation of overexpression and wild-type under 0 M NaCl treatment; b is the statistical result of photosynthetic parameters of overexpression and wild-type under 0 M NaCl treatment; OE-PpALKBH2-1, OE-PpALKBH2-4, and OE-PpALKBH2-6 are overexpression materials; the scale bar is 2 mm;

[0028] Figure 12 Phenotypic characteristics of wild-type Physcomitrella patens and overexpressed OE-PpALKBH2 under 0.4 M NaCl treatment, where a is the phenotypic observation of overexpression and wild-type under 0.4 M NaCl treatment; b is the statistical result of photosynthetic parameters of overexpression and wild-type under 0.4 M NaCl treatment; OE-PpALKBH2-1, OE-PpALKBH2-4, and OE-PpALKBH2-6 are overexpression materials; the scale bar is 2 mm;

[0029] Figure 5 、 Figure 6 、 Figure 9 and Figure 10 In, 15 refers to the Ppalkbh2-15 mutant material, 25 refers to the Ppalkbh2-25 mutant material, and 28 refers to the Ppalkbh2-28 mutant material; Figure 7 、 Figure 8 、 Figure 11 and Figure 12 In, 1 refers to the OE-PpALKBH2-1 overexpression material, 4 refers to the OE-PpALKBH2-4 overexpression material, and 6 refers to the OE-PpALKBH2-6 overexpression material;

[0030] Figures 1 - 12 Among them, the CRISPR-15 mutant material, namely the 11520-CRISPR-15 mutant material, namely the Ppalkbh2-15 mutant material; the CRISPR-25 mutant material, namely the 11520-CRISPR-25 mutant material, namely the Ppalkbh2-25 mutant material; the CRISPR-28 mutant material, namely the 11520-CRISPR-28 mutant material, namely the Ppalkbh2-28 mutant material; the OE-PpALKBH2-1 overexpression material, namely the PU-11520-1 overexpression material; the OE-PpALKBH2-4 overexpression material, namely the PU-11520-4 overexpression material; the OE-PpALKBH2-6 overexpression material, namely the PU-11520-6 overexpression material. Specific implementation manners

[0031]

[0032] As a specific embodiment, the regulation includes overexpressing the PpALKBH2 gene to improve the abiotic stress resistance of plants; knocking out or silencing the PpALKBH2 gene to reduce the abiotic stress resistance of plants. As a specific embodiment, the abiotic stress includes one or more of drought stress, salt stress, and methylation damage stress.

[0033] The present invention also provides a primer pair for amplifying the PpALKBH2 gene, the primer pair including a forward primer and a reverse primer; the nucleotide sequence of the forward primer is as shown in SEQ ID NO:1; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:2; the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0034] The sequences of the primer pair of the present invention are shown as follows:

[0035] Forward primer (SEQ ID NO:1): 5'-TGTCGTGAGTAGTAGGGGTT-3',

[0036] Reverse primer (SEQ ID NO:2): 5'-ATCACCTTTCCTGCCGATGT-3'.

[0037] The present invention also provides a biological material capable of regulating the expression level of the PpALKBH2 gene, including a biological material overexpressing the PpALKBH2 gene and a biological material knocking out or silencing the PpALKBH2 gene; the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0038] The biological material overexpressing the PpALKBH2 gene of the present invention includes one or more of a recombinant expression vector containing the PpALKBH2 gene, a recombinant microorganism containing the PpALKBH2 gene, and a transgenic cell line containing the PpALKBH2 gene. The basic backbone of the recombinant expression vector containing the PpALKBH2 gene of the present invention includes the pPOG1(ubi) vector. As a specific embodiment, the recombinant expression vector containing the PpALKBH2 gene includes inserting the PpALKBH2 gene into the pPOG1(ubi) vector driven by the Ubiquitin strong promoter to obtain an overexpression recombinant vector of PpALKBH2.

[0039] The biomaterials for knocking out or silencing the PpALKBH2 gene of the present invention include one or more of a gene editing target site for knocking out or silencing the PpALKBH2 gene, a gene editing vector of the PpALKBH2 gene, a recombinant microorganism containing a recombinant vector for knocking out or silencing the PpALKBH2 gene, and a transgenic cell line containing a recombinant vector for knocking out or silencing the PpALKBH2 gene. As a specific embodiment, the gene editing target sites of the PpALKBH2 gene include target site 1 and target site 2; the nucleotide sequence of target site 1 is as shown in SEQ ID NO:3: 5'-GTAACAAACACGTCTTTATGATG-3'; the nucleotide sequence of target site 2 is as shown in SEQ ID NO:4: 5'-TGCCGTAGGAAAGCAAAATCTAG-3'. In the embodiments of the present invention, the basic backbone of the recombinant vector for knocking out the PpALKBH2 gene includes a pCR-gRNA vector, and the knockout target site of the PpALKBH2 gene is connected to the pCR-gRNA vector to obtain a gene editing vector for knocking out the PpALKBH2 gene.

[0040] The present invention also provides the use of the primer pair or the biomaterial in regulating the abiotic stress resistance of plants and / or creating new germplasms. By constructing a knockout mutant of the PpALKBH2 gene and an overexpression vector of the PpALKBH2 gene, the present invention found that after treatment with the methylation reagent MMS (alkylating reagent), the survival rate of protonemata of the mutant was significantly lower than that of the wild type, and the number of newly generated gametophytes on the protonemata of the mutant was significantly lower than that of the wild type. The survival rate of protonemata of the overexpression material was higher than that of the wild type, and the area of newly generated protonemata of the overexpression was higher than that of the wild type, indicating that PpALKBH2 is involved in responding to the stress of the methylation reagent MMS on the protonemata of Physcomitrium patens. After treatment with NaCl, the mutant showed more severe phenotypic abnormalities, with the gametophyte leaves showing large areas of chlorosis and whitening. The tips of the wild-type plants had more leaves starting to turn chlorotic and white, while the gametophyte leaves of the overexpression lines showed less chlorosis and whitening and had greener tissues overall, indicating that the PpALKBH2 gene can regulate the abiotic stress resistance of plants.

[0041] The present invention also provides a method for improving the abiotic stress resistance of plants, including the following steps: overexpressing the PpALKBH2 gene in the genome of a target plant to obtain a plant with improved abiotic stress resistance, and the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0042] The present invention also provides a method for creating a plant model with reduced abiotic stress resistance, comprising the following steps: knocking down the expression level of the PpALKBH2 gene in the genome of a target plant or knocking out the PpALKBH2 gene in the genome of the target plant to obtain the plant with reduced abiotic stress resistance, and the ID number of the PpALKBH2 gene is Pp3c2_11520.

[0043] In the PpALKBH2 gene knockout mutant of Physcomitrella patens constructed in the present invention, PpALKBH2 is frameshift mutated, and its drought stress resistance and anti-methylation damage ability are reduced; in the Physcomitrella patens with overexpression of the PpALKBH2 gene constructed, the gene expression level increases, the protein content accumulates, the anti-methylation damage ability is enhanced, and the salt stress tolerance is also enhanced, indicating that the PpALKBH2 gene can regulate the abiotic stress resistance of plants. The experiments of the present invention show that using the PpALKBH2 gene to change the PpALKBH2 expression level and improve the stress resistance of plants has high practical application value.

[0044] To further illustrate the present invention, the following describes in detail the application of the PpALKBH2 gene provided by the present invention in improving the abiotic stress resistance of plants in conjunction with embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0045] Unless otherwise specified, the present invention has no special requirements for the preparation raw materials, and commercially available products well-known to those skilled in the art can be used.

[0046] The formula of the BCDAT medium used in the present invention is shown in Table 1, and the formula of the BCD medium is shown in Table 2.

[0047] Table 1 BCDAT medium

[0048] Mother liquor Addition amount StockB 10 mL StockC 10 mL <![CDATA[KNO3(SIGMAP8291)]]> 1.01g <![CDATA[FeSO4·7H2O]]> 0.012g Alternative TES 1 mL Ammonium tartrate (Sigma - Aldrich V900149 - 100G) 0.92g <![CDATA[1MCaCl2·2H2O(VETECV900269-500G)]]> 1 mL Agar (BIOFROX 8211KG001) 9g Fill up to 1000 mL

[0049] Table 2 BCD medium

[0050]

[0051]

[0052] Mother liquor preparation: StockB(100X)Autoclave: MgSO4·7H2O 25g(100mM)H2O Fill up to1000mLStockC(100X)Autoclave: KH2PO425 g(1.84mM)H2O Fill up to 1000mL

[0053] Stock D(100X)Do not autoclave:KNO3 101 g(1M)FeSO4·7H2O 1.25g(4.5mM)H2O Fill up to 1000mL

[0054] Alternative TES(1000X)Autoclave:CuSO4·5H2O 55mg(0.22mM)H3BO3 614 mg(10mM)CoCl2·6H2O 55mg(0.23mM)Na2MoO4·2H2O 25mg(0.1mM)ZnSO4·7H2O 55mg(0.19mM)MnCl2·4H2O 389mg(2mM)KI 28mg(0.17mM)H2O Fill up to 1000mL

[0055] Example

[0056] 1. Method for culturing moss

[0057] Use BCDAT medium to culture the protonema of Physcomitrella patens. After grinding and subculturing the protonema material cultured for 6 days, culture it under a photoperiod of 16 h light / 8 h dark, 500 μmol m -2 s -1 light intensity at 25 °C for 40 days to enter the uniform gametophyte generation.

[0058] For gametophyte culture, use BCD medium. Pick uniform-growing small gametophytes from the uniformly growing protonema dish after grinding and culture them on BCD medium under a light intensity of 500 μmol m -2 s -1 light intensity at 25 °C for 40 days for drought re-treatment.

[0059] 2. Construction of recombinant vector

[0060] The sequences of the guide RNAs for the target sites of the mutants (target site 1 and target site 2) were synthesized by the company. Target site 1 and target site 2 were ligated onto the co - vector of pU6 - sgRNA, pActCas12a, and pBNRf to obtain the gene - editing vector plasmid for the PpALKBH2 gene (Pu, X., Liu, L., Li, P., Huo, H., Dong, X., Xie, K., Liu, L. (2019). A CRISPR / LbCas12a - based method for highly efficient multiplex gene editing in Physcomitrella patens. Plant J, 100(4), 863 - 872. doi:10.1111 / tpj.14478).

[0061] Target site 1: 5'-GTAACAAACACGTCTTTATGATG-3' (shown as SEQ ID NO:3);

[0062] Target site 2: 5'-TGCCGTAGGAAAGCAAAATCTAG-3' (shown as SEQ ID NO:4).

[0063] Construction of Pp3c2_11520 gene overexpression vector: The ALKBH gene of Physcomitrella patens (gene ID: Pp3c2_11520) was amplified by PCR in vitro, and an overexpression vector was constructed. The vector backbone used for constructing the expression vector was pPOG1(ubi) (Yang, Z., Duan, L., Li, H., Tang, T., Chen, L., Hu, K., Liu, L. (2022). Regulation of Heat Stress in Physcomitrium (Physcomitrella) patens Provides Novel Insight into the Functions of Plant RNase H1s. Int J Mol Sci, 23(16). doi:10.3390 / ijms23169270). Using the primer sequences [forward primer, F: 5'-GGTGTTACTTCTGCAGAAGCTTATGCCAGTTCATTCTTGTCG-3' (as shown in SEQ ID NO:6); reverse primer, R: 5'-GCTGCCGCTACCTCCAAGCTTCTCAGTGATGGTCTGCTCCA-3' (as shown in SEQ ID NO:7)] and the genomic DNA of Physcomitrella patens as a template for PCR amplification. After recovering the PCR product, it was ligated to the linearized vector pPOG1(ubi) (HindIII multiple cloning site), and then transformed into Escherichia coli DH5α (Shanghai Weidi - DL1001M). For the grown monoclonal colonies, positive identification was carried out. For the bacterial liquid identified as positive, the bacteria were shaken and the plasmid was extracted to obtain the plasmid of pPOG1(ubi)-PpALKBH2.

[0064] 3. Transformation of moss

[0065] The method of introducing DNA into protoplasts mediated by polyethylene glycol (PEG) (Sigma - 88276-1KG-F) was selected. 30 μL of the plasmid (the gene editing vector plasmid of PpALKBH2 gene or the plasmid of pPOG1(ubi)-PpALKBH2) was transferred into 300 μL of the protoplasts of Physcomitrella patens. The materials used for transformation were young protonemata.

[0066] 4. Identification of Ppalkbh2 mutants and overexpression materials

[0067] Total DNA of moss was extracted by CTAB method, and the fragment containing the target site was amplified by PCR using 2×Taq Master Mix from novoprotein company. The reaction system (50 μL): 25 μL of 2×Taq Master Mix, 1 μL of Forward primer (10 μM), 1 μL of Reverse primer (10 μM), 2 μL of genomic DNA, and 21 μL of ddH2O;

[0068] The identification of mutant materials and overexpression materials is described as follows, and the identification primers are shown in Table 3.

[0069] Table 3 Identification primers

[0070]

[0071] Using an eppendorf PCR instrument, the PCR reaction procedure: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 30 s, annealing at 52°C for 15 s, extension at 72°C for 60 s (amplification efficiency 1 kb / min), 34 cycles; extension at 72°C for 10 min; preservation at 4°C. Identification by electrophoresis and sequencing.

[0072] The results are as Figure 1 shown. The first and second target sites of the Ppalkbh2 mutant gene (Ppalkbh2 shown in the figure) were edited. Some bases were added or deleted in this mutant, resulting in a frameshift mutation. It is a mutant with loss of function of the PpALKB gene. The obtained mutants are CRISPR-8, CRISPR-15, CRISPR-25, and CRISPR-28. Figure 2 It represents the identification of overexpression materials. After overexpression, the expression level of PpALKBH2 was significantly increased, indicating that the overexpression materials were successfully constructed. The obtained overexpression materials are OE-PpALKBH2-11520-1, OE-PpALKBH2-11520-4, and OE-1PpALKBH2-1520-6.

[0073] 5. Drought rewater treatment of moss

[0074] Select the Physcomitrella patens materials (wild type and Ppalkbh2 mutant materials) that have been cultured for 40 days and entered the gametophyte stage of uniform growth. Take the plant materials out of the culture medium and place them in ddH2O for 2 h to reach a uniform water content state. Then take the materials to filter paper to remove the water on the surface of the materials, and perform air drying under constant temperature and humidity conditions (temperature 25 °C, humidity about 40%) for 2 h. Then add sterilized distilled water for rehydration treatment for 22 h to restore to the growth state before treatment. Repeat this process 5 times to obtain R0 (referring to the materials placed in ddH2O for 2 h), S1 (referring to the materials of the first drought treatment), R1 (referring to the materials of the first rehydration treatment), S2 (referring to the materials of the second drought treatment), R2 (referring to the materials of the second rehydration treatment), S3 (referring to the materials of the third drought treatment), R3 (referring to the materials of the third rehydration treatment), S4 (referring to the materials of the fourth drought treatment), R4 (referring to the materials of the fourth rehydration treatment), S5 (referring to the materials of the fifth drought treatment) materials, and calculate their relative water content.

[0075] The results are as Figure 3 shown. The relative water content of the wild type and Ppalkbh2 mutant after drought rehydration treatment was measured. It was found that the mutant lost the ability of drought memory, while the wild type still maintained drought memory. After the first round of drought treatment, the relative water content (RWC) of the wild type plants dropped sharply to about 40%, which was in line with the response characteristics of typical dehydration-sensitive plants. However, in subsequent drought treatments (S2 - S4), its water loss rate showed a significant slowdown, and the water retention ability was enhanced. In the drought treatment of S4, it could be seen that the RWC of the wild type was significantly higher than the results of the previous three days. The Ppalkbh2 mutant did not show the "memory" ability during repeated drought treatments, and its RWC showed a continuous downward trend from the S1 stage to the S4 stage. Combining the observations of the phenotypic changes of the wild type and mutant during the drought rehydration process, it was found that the phenomenon of gametophyte whitening in the mutant was significantly higher than that in the wild type plants' gametophytes. This indicates that Physcomitrella patens established drought memory through repeated "drought training" and formed a water retention adaptation mechanism. Moreover, this memory effect has obvious gene specificity.

[0076] 6. Measurement of Chlorophyll Fluorescence

[0077] Use FluorCam 800MF of Waltz Company in Germany (Photon Systems Instruments, Drasow, Czech Republic) to measure the chlorophyll fluorescence of moss plants before and after drought stress treatment and during the recovery process (that is, detect the samples of the drought rehydration treatment of the moss in step 4). The samples need to be dark adapted for 30 min before measurement.

[0078] 7. Salt Stress Treatment and MMS Treatment of Moss

[0079] Take the protonemata that have grown uniformly for 40 days after polishing (wild type, Ppalkbh2 mutants, and overexpression materials), transfer them to BCD plates containing different concentrations of NaCl (0.4 M) for treatment, and observe their phenotypes after 5 days of treatment.

[0080] Take the protonemata (wild type, Ppalkbh2 mutants, and overexpression materials) that have been uniformly polished for 14 days, soak them in different concentrations of MMS (methyl methanesulfonate) solutions (0 mM, 10 mM, 30 mM) (Sigma - Aldrich 200 - 625 - 0) for 1 h, then polish them into uniform protonemata, aspirate 5 μL and culture them on ordinary BCDAT for 21 days for observation.

[0081] The results are as Figure 4 shown. Further analysis by chlorophyll fluorescence values found that the change trend of the photosynthesis parameter (F v / F m ) of wild - type Physcomitrella patens during repeated drought - rewatering treatment: it decreased significantly at S1, but increased to some extent at subsequent S2 and S3, and the photosynthetic efficiency parameter was higher than that at S1; while the F v / F m value of the mutant showed a significant decreasing trend from S1 to S4. This indicates that the wild type maintains better photosynthetic activity compared to the Ppalkbh2 mutant.

[0082] The results are as Figure 5 shown. When the MMS concentration is 0, it is observed that there is no difference in the growth of the wild type, Ppalkbh2 - 15 / 25 / 28 mutants. After the protonemata of the wild type, Ppalkbh2 - 15 / 25 / 28 mutant materials are treated with 30 mM MMS respectively and cultured normally for 14 D, the growth (protonemata) of the materials is observed. The survival rate of the mutant protonemata is significantly lower than that of the wild type. Figure 6 In a, it is found that the area of the newly - formed protonemata of the mutant is lower than that of the wild type. As Figure 6 shown in b, after culturing for 21 D, the number of newly - formed gametophytes on the mutant protonemata is significantly lower than that of the wild type; the above experiments show that PpALKBH2 is involved in responding to the stress of the methylation reagent MMS on the protonemata materials of Physcomitrella patens, and the Ppalkbh2 mutant is more sensitive to the methylation reagent.

[0083] When the MMS concentration is 0, it is observed that there is no difference in the growth of the wild type, OE - PpALKBH2 - 1 / 4 / 6 overexpression materials ( Figure 7) After the protonemata of wild-type, OE-PpALKBH2-1 / 4 / 6 overexpression materials were treated with 50 mM MMS respectively for 14 days, it was found that compared with the wild-type, the survival rate of the protonemata of the overexpression materials was higher than that of the wild-type( Figure 7 )

[0084] After treatment with 50 mM MMS and normal culture for 14 days, the area of the protonemata was counted, and it was found that after treatment with different concentrations of MMS, the area of the newly formed protonemata of OE-PpALKBH2-1 / 4 / 6 overexpression was higher than that of the wild-type( Figure 8 in a); after treatment with 50 mM MMS and culture for 21 days, the number of newly formed gametophytes on the MMS-treated protonemata was counted, and it was found that the number of newly formed gametophytes of the overexpression materials was significantly more than that of the wild-type( Figure 8 in b).

[0085] The results are as Figure 9 shown. Through phenotypic observation, it was found that for the plants without salt stress treatment, there were no obvious differences in the growth phenotypes and photosynthetic parameters of plants with different genotypes. After the wild-type (WT), Ppalkbh2 mutants grew on the BCD medium with 0.4 M NaCl for 5 days, the whole wild-type plants still maintained relatively intact green tissues, while the mutants Ppalkbh2-15 / 25 / 28 showed more serious phenotypic abnormalities, and the gametophyte leaves showed large areas of chlorosis and whitening (such as Figure 10 in a). At the same time, by detecting chlorophyll fluorescence, after treatment with 0.4 M NaCl, the maximum photosynthetic efficiency F v / F m and NPQ of the mutants were significantly smaller than those of the wild-type (such as Figure 10 in b).

[0086] As Figure 11 , through phenotypic observation, it was found that when the NaCl concentration was 0, there were no obvious differences in the growth phenotypes of the gametophytes of the wild-type (WT) and overexpression lines (OE-PpALKBH2-1 / 4 / 6), and after chlorophyll fluorescence detection, their photosynthetic parameters were relatively consistent. After the wild-type (WT), overexpression lines OE-PpALKBH2-1 / 4 / 6 grew on the BCD medium with 0.4 M NaCl for 7 days, more leaves at the tips of the wild-type plants began to show chlorosis and whitening, while the gametophyte leaves of the overexpression lines OE-PpALKBH2-1 / 4 / 6 showed less chlorosis and whitening, and the whole had greener tissues (such as Figure 12 in a). At the same time, by detecting chlorophyll fluorescence, after treatment with 0.4 M NaCl, the maximum photosynthetic efficiency F v / F m and NPQ of the overexpression were significantly higher than those of the wild-type (such as Figure 12 in b).

[0087] It can be seen that in the PpALKBH2 gene knockout mutant of Physcomitrella patens constructed in the present invention, PpALKBH2 undergoes frameshift mutation, resulting in reduced drought stress resistance and reduced resistance to methylation damage. In the overexpressed Physcomitrella patens of the PpALKBH2 gene, the gene expression level increases, protein content accumulates, its resistance to methylation damage is enhanced, and its tolerance to salt stress is also enhanced, indicating that the PpALKBH2 gene can regulate the abiotic stress resistance of plants. It is shown that using the PpALKBH2 gene to change the PpALKBH2 expression level and improve plant stress resistance has high practical application value.

[0088] Although the above embodiments have made a detailed description of the present invention, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on these embodiments without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of PpALKBH2 gene in regulating abiotic stress resistance of plants, characterized in that, The ID number of the PpALKBH2 gene is Pp3c2_11520.

2. The application according to claim 1, characterized in that, The regulation includes overexpressing the PpALKBH2 gene to improve the abiotic stress resistance of plants; knocking out or silencing the PpALKBH2 gene to reduce the abiotic stress resistance of plants.

3. The application according to claim 1, characterized in that The abiotic stress includes one or more of drought stress, salt stress, and methylation damage stress.

4. Primer pair for amplifying PpALKBH2 gene, characterized in that, The primer pair includes a forward primer and a reverse primer; the nucleotide sequence of the forward primer is as shown in SEQ ID NO:1; the nucleotide sequence of the reverse primer is as shown in SEQ ID NO:2; the ID number of the PpALKBH2 gene is Pp3c2_11520.

5. A biological material capable of regulating the expression level of PpALKBH2 gene, characterized in that, Biological materials including overexpressing the PpALKBH2 gene and biological materials for knocking out or silencing the PpALKBH2 gene; the ID number of the PpALKBH2 gene is Pp3c2_11520.

6. The biological material according to claim 5, wherein Biological materials for overexpressing the PpALKBH2 gene include one or more of a recombinant expression vector containing the PpALKBH2 gene, a recombinant microorganism containing the PpALKBH2 gene, and a transgenic cell line containing the PpALKBH2 gene.

7. The biomaterial according to claim 5, characterized in that, Biological materials for knocking out or silencing the PpALKBH2 gene include a gene editing target site of the PpALKBH2 gene, a gene editing vector of the PpALKBH2 gene, a recombinant vector for knocking out or silencing the PpALKBH2 gene, a recombinant microorganism containing the recombinant vector for knocking out or silencing the PpALKBH2 gene, and a transgenic cell line containing the recombinant vector for knocking out or silencing the PpALKBH2 gene; The gene editing target sites of the PpALKBH2 gene include target site 1 and target site 2; the nucleotide sequence of target site 1 is as shown in SEQ ID NO:3; the nucleotide sequence of target site 2 is as shown in SEQ ID NO:

4.

8. Use of the primer pair according to claim 4 or the biological material according to any one of claims 5 to 7 in regulating the abiotic stress resistance of plants and / or creating new germplasms.

9. A method for improving the abiotic stress resistance of plants, characterized in that, It includes the following steps: overexpressing the PpALKBH2 gene in the genome of the target plant to obtain a plant with improved abiotic stress resistance, and the ID number of the PpALKBH2 gene is Pp3c2_11520.

10. A method for creating a plant model with reduced abiotic stress resistance, characterized in that, It includes the following steps: Knock down the expression level of the PpALKBH2 gene in the genome of the target plant or knock out the PpALKBH2 gene in the genome of the target plant to obtain the plant with reduced abiotic stress resistance, and the ID number of the PpALKBH2 gene is Pp3c2_11520.

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