Application of pp2c.c7 gene in regulating plant stress resistance

Abstract

The application discloses application of a PP2C.C7 gene in regulating plant stress resistance, belongs to the technical field of biology, and provides application of the PP2C.C7 gene in regulating plant stress resistance, wherein the nucleotide sequence of the PP2C.C7 gene is shown in SEQ ID NO. 1 or SEQ ID NO. 6. The application provides application of the PP2C.C7 gene in regulating plant salt stress resistance. The application provides application of a CDS sequence of the PP2C.C7 gene or a protein encoded by the PP2C.C7 gene in regulating plant salt stress resistance. The application verifies that, compared with respective wild types, a PP2C.C7 protein function loss Arabidopsis mutant and a corn mutant show significantly higher salt stress tolerance.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and in particular relates to the application of the PP2C.C7 gene in regulating plant stress resistance. Background Technology

[0002] Soil salinization is expanding globally, severely limiting crop growth and becoming a major factor restricting agricultural production. Salt stress not only leads to excessive accumulation of Na+ and Cl- ions in the soil, affecting plant ion balance, but also triggers osmotic and oxidative stress, ultimately inhibiting plant growth and development, and even causing death. To cope with salt stress, plants employ a series of sophisticated molecular regulatory mechanisms, such as ion balance regulation, osmotic regulation, and antioxidant defense, to maintain cellular homeostasis and enhance adaptability. With the rapid development of molecular biology, genomics, genetics, biochemistry, and gene editing technologies, research into the molecular mechanisms by which plants resist salt stress has been continuously deepened.

[0003] Through reversible phosphorylation and dephosphorylation, protein structure and function are altered, thereby regulating the activity, stability, subcellular localization, and interactions with other proteins of substrate proteins. This precisely regulates cell signaling, gene expression, and physiological metabolism to cope with adverse environments. Plant protein phosphatases play a crucial role in signal transduction, ion transport regulation, and stress resistance mechanisms, and are particularly important in plant responses to salt stress. Therefore, in-depth research into the function and molecular mechanisms of protein phosphatases in plant salt stress responses is of great significance for elucidating the regulatory network of plant salt tolerance. Summary of the Invention

[0004] To address the aforementioned technical problems, this invention proposes the application of the PP2C.C7 gene in regulating plant stress resistance. By reducing the expression of the PP2C.C7 gene in Arabidopsis or maize, or by knocking out the function of the PP2C.C7 protein in Arabidopsis or maize, the salt stress resistance of Arabidopsis or maize can be improved.

[0005] To achieve the above objectives, the present invention provides the application of the PP2C.C7 gene in regulating plant stress resistance, wherein the nucleotide sequence of the PP2C.C7 gene is shown in SEQ ID NO.1 or SEQ ID NO.6.

[0006] Preferably, the plant is a cruciferous plant and / or a grass plant.

[0007] More preferably, the cruciferous plant is Arabidopsis thaliana, and the gramineous plant is maize; when the PP2C.C7 gene regulates the stress resistance of Arabidopsis thaliana, the nucleotide sequence of the PP2C.C7 gene is as shown in SEQ ID NO.1, and when the PP2C.C7 gene regulates the stress resistance of maize, the nucleotide sequence of the PP2C.C7 gene is as shown in SEQ ID NO.6.

[0008] This invention also provides the application of the PP2C.C7 gene in regulating the salt stress resistance of plants.

[0009] Preferably, the PP2C.C7 gene negatively regulates the plant's salt stress resistance.

[0010] Preferably, the plant is Arabidopsis thaliana and / or maize.

[0011] The present invention also provides the application of the CDS sequence of the PP2C.C7 gene in regulating the salt stress resistance of Arabidopsis thaliana, the nucleotide sequence of the CDS sequence of the PP2C.C7 gene is shown in SEQ ID NO.3.

[0012] The present invention also provides the application of the protein encoded by the PP2C.C7 gene in regulating the salt stress resistance of Arabidopsis thaliana, and the amino acid sequence of the protein encoded by the PP2C.C7 gene is shown in SEQ ID NO.2.

[0013] The present invention also provides the application of the CDS sequence of the PP2C.C7 gene in regulating the salt stress resistance of maize, and the nucleotide sequence of the CDS sequence of the PP2C.C7 gene is shown in SEQ ID NO.7.

[0014] The present invention also provides the application of the protein encoded by the PP2C.C7 gene in regulating the salt stress resistance of maize, and the amino acid sequence of the protein encoded by the PP2C.C7 gene is shown in SEQ ID NO.8.

[0015] Compared with the prior art, the present invention has the following advantages and technical effects:

[0016] This invention verified that the mutant phenotype with loss of function of the PP2C.C7 protein, compared with the unmutated wild-type Arabidopsis or maize, exhibited significant salt stress tolerance. This demonstrates that the PP2C.C7 gene negatively regulates the salt stress resistance of Arabidopsis and maize, and that loss of function of the PP2C.C7 protein can improve the high salt tolerance of Arabidopsis and maize. Attached Figure Description

[0017] 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.

[0018] Figure 1 The negative regulation of salt tolerance in Arabidopsis thaliana by PP2C.C7 is shown in the following figures: A represents the growth phenotypes of Arabidopsis thaliana pp2c.C7 mutant and wild-type (WT) seedlings under control and NaCl treatments; B represents the taproot length statistics of Arabidopsis thaliana pp2c.C7 functional knockout mutant and wild-type seedlings under control and NaCl treatments; C represents the fresh weight statistics of single seedlings of Arabidopsis thaliana pp2c.C7 mutant and wild-type under control and NaCl treatments; and D represents the salt tolerance statistics of Arabidopsis thaliana pp2c.C7 mutant and transgenic seedlings under control and NaCl treatments. The growth phenotypes of supplementary material seedlings are shown in Figure E, which represents the main root length of Arabidopsis pp2c.c7 mutant and transgenic supplementary material seedlings under control and NaCl treatment conditions; Figure F represents the fresh weight of single seedlings of Arabidopsis pp2c.c7 mutant and transgenic supplementary material under control and NaCl treatment conditions; Figure G represents the expression level of PP2C.C7 gene in each Arabidopsis genetic material analyzed by RT-PCR; ACTIN represents the Arabidopsis internal reference gene; different letters in B, C, D and F indicate significant differences; P < 0.05, n ≥ 10.

[0019] Figure 2 PP2C.C7 negatively regulates the sodium-potassium balance in Arabidopsis thaliana under salt stress, where A represents the Na+ content in the crown of Arabidopsis thaliana under control and NaCl treatment conditions. + Content, B represents the K content in the crown of Arabidopsis thaliana under control conditions and NaCl treatment. + Content, C represents the Na content in Arabidopsis roots under control and NaCl treatment conditions. + Content, D represents the potassium content in Arabidopsis roots under control conditions and NaCl treatment. + Content, E represents the K content in the crown of Arabidopsis thaliana under control conditions and NaCl treatment. + / Na + Proportional analysis, F represents the control condition and the K content of Arabidopsis roots under NaCl treatment. + / Na + Proportional analysis, where different letters in A, B, C, D, E, and F represent significant differences, P < 0.05, n = 3;

[0020] Figure 3To demonstrate the negative regulation of oxidative equilibrium in Arabidopsis thaliana by PP2C.C7 under salt stress, A represents the DAB staining results of Arabidopsis thaliana cotyledons under control and NaCl treatment (scale bar: 2 mm), B represents the NBT staining results of Arabidopsis thaliana cotyledons under control and NaCl treatment (scale bar: 2 mm), C represents the statistical graph of relative DAB staining intensity, and D represents the statistical graph of relative NBT staining intensity. Different letters in C and D indicate significant differences (P < 0.05, n = 12).

[0021] Figure 4 A phylogenetic analysis diagram of the PP2C C subfamily of different species;

[0022] Figure 5 Diagram illustrating the conserved structural domains of the PP2C C subfamily in different species;

[0023] Figure 6 Genotyping of the maize pp2c.c7 functional knockout mutant (zmpp2c.c7-ems) is performed. In the figure, A is a schematic diagram of the zmpp2c.c7-ems mutation, and B is the base alignment result of the ZmPP2C.C7 gene in wild-type maize Chang 7-2 (abbreviated as C7-2) and the zmpp2c.c7-ems mutant.

[0024] Figure 7 The negative regulation of salt tolerance in maize by PP2C.C7 is shown in Figure 1. A represents the growth phenotype of maize pp2c.c7 functional knockout mutant (zmpp2c.c7-ems) and wild-type (WT) seedlings under control and NaCl treatment conditions. B represents the plant height of maize pp2c.c7 mutant (zmpp2c.c7-ems) and wild-type (WT) seedlings under control and NaCl treatment conditions. C represents the fresh weight of single seedlings of maize pp2c.c7 mutant (zmpp2c.c7-ems) and wild-type (WT) seedlings under control and NaCl treatment conditions. Different letters in B and C indicate significant differences, P < 0.05, n ≥ 10.

[0025] Figure 8 To analyze the growth phenotypes of Arabidopsis pp2c43 functional knockout mutants and wild-type (WT) plants under control and NaCl treatment conditions, A represents the growth phenotypes of Arabidopsis pp2c43 mutant and wild-type seedlings under control and NaCl treatment conditions, and B represents the statistical analysis of the taproot length of Arabidopsis pp2c43 mutant and wild-type seedlings under control and NaCl treatment conditions. Different letters in B indicate significant differences, P<0.05, n≥10;

[0026] Figure 9To analyze the growth phenotypes of Arabidopsis thaliana PP2C43 overexpression materials under control and NaCl treatment conditions, A represents the growth phenotypes of Arabidopsis thaliana overexpression materials PP2C43-OE 1# and PP2C43-OE 2# and wild-type seedlings, and B represents the statistical analysis of the taproot length of Arabidopsis thaliana overexpression materials PP2C43-OE 1# and PP2C43-OE 2# and wild-type seedlings. Different letters indicate significant differences, P<0.05, n≥10. Detailed Implementation

[0027] 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.

[0028] 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. Every smaller range between any stated value or intermediate value within a stated range, and 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.

[0029] 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.

[0030] 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, which 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. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] 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.

[0032] Example 1

[0033] I. PP2C.C7 negatively regulates salt tolerance in Arabidopsis and maize

[0034] 1. Obtaining PP2C.C7 related mutants

[0035] The Arabidopsis mutants SALK_023206 and SALK_204106 (obtained from TAIR) are mutations caused by T-DNA insertion into the PP2C.C7 gene (AT3G16560 gene, whose nucleotide sequence is shown in SEQ ID NO.1) of the Arabidopsis PP2C family C subfamily member, and are named pp2c.c7-1 and pp2c.c7-2, respectively.

[0036] The maize mutant zmpp2c.c7-ems is an EMS-induced mutant with a loss of function of the protein encoded by the maize ZmPP2C.C7 gene (Zm00001eb421700 gene, whose nucleotide sequence is shown in SEQ ID NO.6) in maize with a background of Chang 7-2 (abbreviated as C7-2), whose amino acid sequence is shown in SEQ ID NO.6. It is named zmpp2c.c7-ems.

[0037] like Figure 1 As shown in Figure G, the expression level of the PP2C.C7 gene in wild-type Arabidopsis thaliana (WT) and the Arabidopsis thaliana mutants pp2c.c7-1 and pp2c.c7-2 was detected by reverse transcription PCR (RT-PCR). The results showed that the expression of the PP2C.C7 gene was almost undetectable in the 2 mutants pp2c.c7-1 and pp2c.c7-2, indicating that T-DNA insertion leads to the loss of PP2C.C7 function.

[0038] like Figure 6 China A and Figure 6 As shown in Figure B, genomic DNA was extracted from maize C7-2 and the mutant zmpp2c.c7-ems. The ZmPP2C.C7 gene fragment was amplified by PCR and sequenced. It was found that in the zmpp2c.c7-ems mutant, the 1645th base was mutated from guanine (G) to adenine (A), and subsequently, the glutamine (Q, codon CAA) at position 131 encoded by the ZmPP2C.C7 gene was mutated to a stop codon (TAA), leading to premature termination of translation of the ZmPP2C.C7 transcript and loss of protein function. To rule out possible interference from other potential mutation sites, this mutant was backcrossed with C7-2 for two generations, resulting in the isolation of the mutant zmpp2c.c7-ems and the corresponding wild-type maize material, which were used for salt stress phenotypic analysis.

[0039] The nucleotide sequence of the CDS sequence of the Arabidopsis PP2C.C7 gene is shown in SEQ ID NO.3, the amino acid sequence of the protein encoded by the Arabidopsis PP2C.C7 gene is shown in SEQ ID NO.2, and the nucleotide sequence of the promoter of the Arabidopsis PP2C.C7 gene is shown in SEQ ID NO.4. The nucleotide sequence of the CDS sequence of the maize PP2C.C7 gene is shown in SEQ ID NO.7, and the amino acid sequence of the protein encoded by the maize PP2C.C7 gene is shown in SEQ ID NO.8.

[0040] 2. Identification of salt stress phenotype in Arabidopsis thaliana pp2c.c7 mutant

[0041] The mutant materials pp2c.c7-1 and pp2c.c7-2 of wild-type Arabidopsis thaliana Col-0(WT) and PP2C.C7 were sown on 1 / 2 MS medium and grown vertically in a full-sunlight chamber for 5 days. Wild-type and mutant materials with consistent growth were selected and transferred to 1 / 2 MS medium with NaCl concentrations of 0 mM and 100 mM, respectively. After culturing in a light chamber for 7 days, the phenotype was observed.

[0042] The results are as follows Figure 1 China A Figure 1 China B and Figure 1 As shown in Figure C, under a salt concentration of 0 mM, there was no significant difference between the Arabidopsis functional knockout mutants pp2c.c7-1 and pp2c.c7-2 and the wild-type Arabidopsis Col-0 (WT). Under a salt concentration of 100 mM, the mutants showed stronger salt tolerance than the wild-type Arabidopsis. The main root length of the mutants was significantly longer than that of the wild-type, and the whole plant fresh weight of the mutants was significantly greater than that of the wild-type.

[0043] 3. Identification of salt stress phenotype in Arabidopsis thaliana PP2C.C7 gene complementation pp2c.c7 mutant

[0044] (1) Preparation of replenishment carrier

[0045] The promoter sequence of the first 2 kb of the start codon of PP2C.C7 was constructed into the pCM1305 vector to obtain pCM1305-Pro:PP2C.C7; then the GFP tag sequence and the CDS sequence (SEQ ID NO.3) of the PP2C.C7 gene were constructed into the pCM1305-Pro:PP2C.C7 vector to finally obtain the complementation vector of the PP2C.C7 gene pCM1305-ProPP2C.C7:GFP:PP2C.C7.

[0046] (2) Preparation of replacement transgenic plants

[0047] The complementation vector ProPP2C.C7:GFP:PP2C.C7 was transformed into Agrobacterium GV3101 to obtain Agrobacterium GV3101 / proPP2C.C7:GFP:PP2C.C7.

[0048] Forty-eight mutant strains of pp2c.c7-1 were transformed with Agrobacterium GV3101 / proPP2C.C7:GFP:PP2C.C7 using the inflorescence soaking method, and T0 generation replacement transgenic lines were obtained.

[0049] (3) Identification of reintroduced transgenic plants

[0050] After disinfecting and washing the T0 generation seeds, they were evenly spread on 1 / 2 MS medium containing cephalosporins and hygromycin. After 14 days of growth, seedlings with normal root and crown growth were selected. Further observation under a fluorescence microscope revealed positive seedlings expressing GFP-PP2C.C7; these seedlings were designated as the supplementary material, T1 generation. The identified positive lines were numbered and marked, and the seeds collected from each plant were designated as T2 generation. T2 generation seeds were then screened on 1 / 2 MS medium containing hygromycin, and normally growing plants were selected for propagation; the seeds collected from each plant were designated as T3 generation.

[0051] Seeds from the T3 generation of reintroduced transgenic plants were screened on 1 / 2 MS medium containing hygromycin. A homozygous reintroduced transgenic line, COM-PP2C.C7, was obtained in the T3 generation, exhibiting no segregation of hygromycin resistance. At least two independent reintroduced transgenic lines were obtained and named COM-1 and COM-2.

[0052] The results are as follows Figure 1 As shown in Figure G, the expression level of the PP2C.C7 gene in wild-type (WT) and replenishment lines was detected by reverse transcription PCR (RT-PCR). The results showed that the expression level of PP2C.C7 was restored in the two independent PP2C.C7 replenishment lines COM-1 and COM-2, indicating that PP2C.C7 is expressed in transgenic Arabidopsis plants.

[0053] (4) Identification of salt stress phenotypes in recombinant Arabidopsis transgenic plants

[0054] Wild-type Arabidopsis thaliana Col-0(WT), pp2c.c7-1 mutants, and T3 generation homozygous reintroduced lines COM-1 and COM-2 were sown on 1 / 2 MS medium and grown vertically for 5 days. Materials with consistent growth were selected and transferred to 1 / 2 MS medium containing 0 mM and 100 mM NaCl, respectively, and cultured in a light box to observe phenotypes.

[0055] The results are as follows Figure 1 D, Figure 1 China E and Figure 1 As shown in Figure F, under a salt concentration of 0 mM, there was no significant growth difference between the Col-0(WT), pp2c.c7-1 mutants and the replacement line. Under a salt concentration of 100 mM, the replacement line was able to correct the mutant phenotype and showed no significant growth difference from the wild type.

[0056] 4. Physiological mechanisms by which Arabidopsis thaliana PP2C.C7 negatively regulates salt tolerance.

[0057] To investigate whether Arabidopsis thaliana PP2C.C7 regulates salt tolerance under salt stress by affecting ion and oxidative homeostasis, the Na+ content of the Arabidopsis thaliana pp2c.C7 mutant was measured. + and K + The results showed that PP2C.C7 in Arabidopsis thaliana negatively regulated Na+ content and reactive oxygen species (ROS) levels under salt stress. + / K + Balance and oxidation balance.

[0058] (1) Detection of ion content in Arabidopsis thaliana pp2c.c7 mutant

[0059] (a) Salt stress treatment of Arabidopsis mutant plants

[0060] In plants under salt stress, the regulation of cellular ion homeostasis, especially Na+, is crucial. + and K + The dynamic changes.

[0061] Wild-type Arabidopsis thaliana Col-0 (WT) and mutant materials pp2c.c7-1 and pp2c.c7-2, which had been grown on ordinary 1 / 2 MS medium for 5 days, were transferred to 1 / 2 MS medium containing 0 mM and 100 mM NaCl, respectively, and cultured in a light box for 7 days.

[0062] (b) Collection of plants subjected to salt stress

[0063] Seven days after transplanting, the above-ground and underground parts were taken separately, dried at 85℃, and weighed.

[0064] (c) Determination of ion content in plants subjected to salt stress

[0065] The sample is excited in high-temperature plasma, and the characteristic emission spectra of the elements can be used to quantify Na. + K + This method, which involves detecting metal ions, is called inductively coupled plasma optical emission spectrometry (ICP-OES).

[0066] The results are as follows Figure 2 China A Figure 2 B, Figure 2 C, Figure 2 D, Figure 2 China E and Figure 2 As shown in Figure F, under normal growth conditions, the Na+ content in the crown and roots of the pp2c.c7 mutant is... + and K + Content, K + / Na + The proportions were not significantly different from those of Col-0(WT); under salt stress treatment, the mutant accumulated less Na in the crown and roots compared to Col-0(WT). + But more K + Thus, the K in the crown and root of the mutant + / Na + The proportion was significantly higher than that of Col-0 (WT). These results indicate that PP2C.C7 negatively regulates Na+ under salt stress. + / K + balance.

[0067] (2) Detection of ROS levels in Arabidopsis thaliana pp2c.c7 mutant

[0068] Plants produce excessive reactive oxygen species (ROS) under adverse conditions such as salt stress, mainly including superoxide anions (O2). - ), hydrogen peroxide (H2O2) and hydroxyl radicals (OH) · Hydrogen peroxide (H2O2) levels were determined using DAB staining, and superoxide anion (O2) levels were determined using NBT staining. - )level.

[0069] (a) Salt stress treatment of mutant plants

[0070] Wild-type Arabidopsis thaliana and mutant materials pp2c.c7-1 and pp2c.c7-2, which had been grown on ordinary 1 / 2 MS medium for 7 days, were transferred to 1 / 2 MS medium containing 0 mM and 100 mM NaCl, respectively, and cultured in a light box for 24 hours.

[0071] (b) Measurement of ROS levels in plants under salt stress

[0072] The treated plants were placed in DAB (or NBT) staining solution and vacuumed for 0.5 hours in the dark to allow the staining solution to fully penetrate the plants. After staining for 6 hours in the dark, the stain was removed, and the ROS levels in the plants were observed by photographing.

[0073] DAB and NBT staining results showed no significant difference in ROS levels in leaves between wild-type and pp2c.c7 mutants after the control treatment (Mock); however, after salt stress treatment, ROS accumulated significantly in all materials, but the ROS level in the pp2c.c7 mutant was significantly lower than that in the wild type (e.g., Figure 3 China A Figure 3 B, Figure 3 C and Figure 3 The result (D) indicates that PP2C.C7 negatively regulates the oxidation balance under salt stress.

[0074] The above results indicate that under salt stress, Arabidopsis thaliana PP2C.C7 negatively regulates plant ion and oxidation balance.

[0075] 5. Identification of salt stress phenotype in maize zmpp2c.c7-ems mutant

[0076] (1) Homologous gene analysis of Arabidopsis PP2C.C7 gene in maize

[0077] To identify homologs of the Arabidopsis PP2C.C7 gene in crops and to examine its regulatory function on salt tolerance, a phylogenetic analysis of PP2C C family members in Arabidopsis and maize was performed. The results are as follows: Figure 4 As shown, Arabidopsis PP2C.C7 (AtPP2C.C7, AT3G16560) is closely related to maize ZmPP2C19 (Zm00001eb084510) and ZmPP2C98 (Zm00001eb421700). Further analysis of conserved domains revealed that these three proteins share similar conserved motifs (such as...). Figure 5 This indicates that ZmPP2C19 and ZmPP2C98 may have similar functions to Arabidopsis PP2C.C7. Since only one sense mutant of the ZmPP2C98 gene was found in the EMS mutagenesis mutant library with a C7-2 background, for the sake of consistent nomenclature, this mutant was named zmpp2c.c7-ems mutant, and the ZmPP2C98 gene was named ZmPP2C.C7.

[0078] (2) Identification of salt stress phenotype of zmpp2c.c7-ems mutant

[0079] like Figure 6As shown in Figure A, the 1645th base in the zmpp2c.c7-ems mutant is mutated from guanine (G) to adenine (A), which in turn causes the glutamine (Q, codon CAA) at position 131 encoded by the ZmPP2C.C7 gene to mutate into a stop codon (TAA), resulting in premature termination of translation of the ZmPP2C.C7 transcript and loss of protein function. To rule out possible interference from other potential mutation sites, this mutant was backcrossed with C7-2 for two generations, and the mutant zmpp2c.c7-ems and the corresponding wild-type maize material were isolated.

[0080] Further, the salt stress phenotype of the zmpp2c.c7-ems mutant was identified through pot experiments. Wild-type (WT) and mutant were sown in substrate soil containing 150 mM NaCl or without NaCl (nutrient soil to vermiculite ratio of 1:1) for 14 days. After continuous treatment, the salt stress phenotype was recorded, and the seedling height and aboveground fresh weight of maize were statistically analyzed. Different letters indicate significant differences, P<0.05, n≥10.

[0081] The results are as follows Figure 7 As shown, under normal conditions (Mock), there was no significant difference between the zmpp2c.c7-ems mutant and the wild-type material; under a salt concentration of 150 mM, the mutant showed stronger salt tolerance than the wild-type maize, the plant height of the mutant was significantly greater than that of the wild-type, and the aboveground fresh weight of the mutant was significantly greater than that of the wild-type, indicating that ZmPP2C.C7 negatively regulates the salt tolerance of maize.

[0082] Comparative Example 1

[0083] PP2C43, a member of the Arabidopsis PP2C family, does not participate in the regulation of plant salt tolerance.

[0084] Some members of the PP2C family have been reported to participate in the regulation of plant salt tolerance. This invention is the first to discover that the PP2C.C7 gene in Arabidopsis thaliana and maize negatively regulates plant salt tolerance. However, not all members of the PP2C family regulate plant salt tolerance.

[0085] Genetic materials related to the PP2C43 gene (AT3G17250 gene, whose nucleotide sequence is shown in SEQ ID NO.5, and the CDS sequence of the Arabidopsis PP2C43 gene is shown in SEQ ID NO.9) of the G subfamily member of the PP2C family were obtained, including the loss-of-function mutant pp2c43 (T-DNA insertion mutant SALK_094214C) and the overexpression materials PP2C43-OE 1# and PP2C43-OE 2# of pro35S:6×Myc:PP2C43 transformed into wild-type (WT).

[0086] Salt stress growth phenotypes of the above Arabidopsis thaliana PP2C43-related genetic materials were detected. Wild-type Col-0(WT) and pp2c43 mutant seedlings that had grown normally for 6 days were transferred to 1 / 2 MS medium containing or without 100 mM or 150 mM NaCl and cultured vertically for 9 days. Taproot length was statistically analyzed; different letters indicate significant differences (P < 0.05, n ≥ 10). Salt stress phenotype screening of PP2C43 overexpression materials PP2C43-OE 1# and PP2C43-OE 2# was conducted. Arabidopsis thaliana seedlings that had grown normally for 6 days were transferred to 1 / 2 MS medium containing or without 100 mM NaCl and cultured vertically for 7 days. Taproot length was statistically analyzed; different letters indicate significant differences (P < 0.05, n ≥ 10).

[0087] The results are as follows Figure 8 China A Figure 8 B, Figure 9 China A and Figure 9 As shown in Figure B, there was no significant growth difference between the Arabidopsis WT and pp2c43 mutants under both normal and salt stress conditions, nor was there a significant growth difference between the Arabidopsis WT and PP2C43-OE materials. These results indicate that PP2C43 does not regulate salt tolerance in Arabidopsis, suggesting that not all members of the PP2C family of plant protein phosphatases regulate salt tolerance.

[0088] 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. PP2C.C7 The application of genes in negatively regulating the salt stress resistance of plants is characterized by, The PP2C.C7 The nucleotide sequence of the gene is shown in SEQ ID NO.1 or SEQ ID NO.

6.

2. As described in claim 1 PP2C.C7 The application of genes in negatively regulating the salt stress resistance of plants is characterized by, The plants mentioned are Arabidopsis thaliana and / or maize.

3. As described in claim 1 PP2C.C7 The application of genes in negatively regulating the salt stress resistance of plants is characterized by, The PP2C.C7 The nucleotide sequence of the gene's CDS sequence is shown in SEQ ID NO.

3.

4. As described in claim 1 PP2C.C7 The application of genes in negatively regulating the salt stress resistance of plants is characterized by, The PP2C.C7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

5. As described in claim 1 PP2C.C7 The application of genes in negatively regulating the salt stress resistance of plants is characterized by, The PP2C.C7 The nucleotide sequence of the gene's CDS sequence is shown in SEQ ID NO.

7.

6. As described in claim 1 PP2C.C7 The application of genes in negatively regulating the salt stress resistance of plants is characterized by, The PP2C.C7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.8.

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