Application of PP2C.C7 gene in regulation and control of plant stress resistance

Abstract

The invention discloses an application of a PP2C. C7 gene in regulation and control of plant stress resistance, belongs to the technical field of biology, and provides an application of the PP2C. C7 gene in regulation and control of plant stress resistance, and a nucleotide sequence of the PP2C. C7 gene is shown as SEQ ID NO.1 or SEQ ID NO.6. The invention further discloses an application of the PP2C. C7 gene in regulation and control of plant stress resistance. The PP2C.C7 gene is applied to regulation and control of salt stress resistance of plants. The CDS sequence of the PP2C.C7 gene or the protein coded by the PP2C.C7 gene is applied to regulation and control of the salt stress resistance of the plant. The invention verifies that the arabidopsis thaliana mutant and the corn mutant with PP2C.C7 protein function deletion both show significantly higher salt stress tolerance compared with respective wild types of the arabidopsis thaliana mutant and the corn mutant.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of the PP2C.C7 gene in regulating plant stress resistance. Background Art

[0002] The trend of soil salinization is continuously expanding worldwide, severely restricting the growth of crops and becoming an important factor restricting agricultural production. Salt stress not only causes excessive accumulation of Na+ and Cl- ions in the soil, affecting the ion balance of plants, but also triggers osmotic stress and oxidative stress, ultimately inhibiting the growth and development of plants and even leading to death. To cope with salt stress, plants adopt a series of fine molecular regulation mechanisms, such as ion balance regulation, osmotic regulation, antioxidant defense, etc., to maintain cell homeostasis and improve adaptability. With the rapid development of molecular biology, genomics, genetics, biochemistry, and gene editing technologies, the research on the molecular mechanism of plant resistance to salt stress has been continuously deepened.

[0003] Through reversible phosphorylation and dephosphorylation protein modifications, the structure and function of proteins are changed, thereby regulating the activity, stability, subcellular localization, and interaction with other proteins of substrate proteins, etc., so as to precisely regulate cell signal transduction, gene expression, and physiological metabolism to cope with adverse environments. Among them, plant protein phosphatases play a key role in signal transduction, ion transport regulation, and stress resistance mechanisms, and play a crucial role in the process of plants coping with salt stress. Therefore, in-depth study of the function and molecular mechanism of protein phosphatases in plant salt stress responses is of great significance for revealing the regulatory network of plant salt resistance. Summary of the Invention

[0004] To solve the above technical problems, the present 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 thaliana or maize, or knocking out the function of the PP2C.C7 protein in Arabidopsis thaliana or maize, the salt stress resistance performance of Arabidopsis thaliana or maize can be improved.

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

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

[0007] Further 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] The present invention also provides the application of the PP2C.C7 gene in regulating the salt stress resistance performance of plants.

[0009] Preferably, the PP2C.C7 gene negatively regulates the salt stress resistance performance of plants.

[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 performance of *Arabidopsis thaliana*, and the nucleotide sequence of the CDS sequence of the PP2C.C7 gene is as 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 performance of *Arabidopsis thaliana*, and the amino acid sequence of the protein encoded by the PP2C.C7 gene is as 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 performance of maize, and the nucleotide sequence of the CDS sequence of the PP2C.C7 gene is as 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 performance of maize, and the amino acid sequence of the protein encoded by the PP2C.C7 gene is as shown in SEQ ID NO.8.

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

[0016] The present invention verifies that the mutant phenotype with the loss of function of the PP2C.C7 protein shows significant salt stress tolerance compared with the wild-type *Arabidopsis thaliana* or maize before mutation. Thus, it is proved that the PP2C.C7 gene negatively regulates the salt stress resistance performance of *Arabidopsis thaliana* and maize, and the loss of function of the PP2C.C7 protein can improve the stress tolerance of *Arabidopsis thaliana* and maize to high salt. Description of the Drawings

[0017] 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. 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.

[0018] Figure 1 PP2C.C7 negatively regulates the salt tolerance of Arabidopsis thaliana. Among them, A shows the growth phenotypes of Arabidopsis thaliana pp2c.c7 mutant and wild-type (WT) seedlings under control conditions and NaCl treatment, B shows the statistical results of the primary root lengths of Arabidopsis thaliana pp2c.c7 functional knockout mutant and wild-type seedlings under control conditions and NaCl treatment, C shows the statistical results of the fresh weights of individual Arabidopsis thaliana pp2c.c7 mutant and wild-type seedlings under control conditions and NaCl treatment, D shows the growth phenotypes of Arabidopsis thaliana pp2c.c7 mutant and transgenic complementation material seedlings under control conditions and NaCl treatment, E shows the statistical results of the primary root lengths of Arabidopsis thaliana pp2c.c7 mutant and transgenic complementation material seedlings under control conditions and NaCl treatment, F shows the statistical results of the fresh weights of individual Arabidopsis thaliana pp2c.c7 mutant and transgenic complementation material seedlings under control conditions and NaCl treatment, G shows the RT-PCR analysis of the expression levels of PP2C.C7 gene in various Arabidopsis thaliana genetic materials, ACTIN represents the internal reference gene of Arabidopsis thaliana, 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 of Arabidopsis thaliana under salt stress. Among them, A shows the Na + content in the shoots of Arabidopsis thaliana under control conditions and NaCl treatment, B shows the K + content in the shoots of Arabidopsis thaliana under control conditions and NaCl treatment, C shows the Na + content in the roots of Arabidopsis thaliana under control conditions and NaCl treatment, D shows the K + content in the roots of Arabidopsis thaliana under control conditions and NaCl treatment, E shows the K + / Na + ratio analysis in the shoots of Arabidopsis thaliana under control conditions and NaCl treatment, F shows the K + / Na + ratio analysis in the roots of Arabidopsis thaliana under control conditions and NaCl treatment, different letters in A, B, C, D, E, and F indicate significant differences, P < 0.05, n = 3;

[0020] Figure 3PP2C.C7 negatively regulates the oxidation balance in Arabidopsis under salt stress. Among them, A shows the DAB staining results of Arabidopsis cotyledons under control conditions and after NaCl treatment, with a scale bar of 2 mm; B shows the NBT staining results of Arabidopsis cotyledons under control conditions and after NaCl treatment, with a scale bar of 2 mm; C is a statistical graph of the relative DAB staining intensity; D is a statistical graph of the relative NBT staining intensity. Different letters in C and D indicate significant differences, P < 0.05, n = 12;

[0021] Figure 4 It is a phylogenetic relationship analysis diagram of the PP2C C subfamily of different species;

[0022] Figure 5 It is a conserved domain analysis diagram of the PP2C C subfamily of different species;

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

[0024] Figure 7 PP2C.C7 negatively regulates the salt tolerance of maize. Among them, A shows the growth phenotypes of the maize pp2c.c7 functional knockout mutant (zmpp2c.c7-ems) and the wild type (WT) under control conditions and NaCl treatment; B shows the statistical results of the plant heights of the maize pp2c.c7 mutant (zmpp2c.c7-ems) and the wild type (WT) under control conditions and NaCl treatment; C shows the statistical results of the fresh weights of individual seedlings of the maize pp2c.c7 mutant (zmpp2c.c7-ems) and the wild type (WT) under control conditions and NaCl treatment. Different letters in B and C indicate significant differences, P < 0.05, n ≥ 10;

[0025] Figure 8 It is the growth phenotype analysis of the Arabidopsis pp2c43 functional knockout mutant and the wild type (WT) under control conditions and NaCl treatment. Among them, A shows the growth phenotypes of the Arabidopsis pp2c43 mutant and the wild type under control conditions and NaCl treatment; B shows the statistical results of the main root lengths of the Arabidopsis pp2c43 mutant and the wild type under control conditions and NaCl treatment. Different letters in B indicate significant differences, P < 0.05, n ≥ 10;

[0026] Figure 9For the growth phenotype analysis of Arabidopsis PP2C43 overexpression materials under control conditions and NaCl treatment, where A shows the growth phenotypes of Arabidopsis overexpression materials PP2C43-OE 1# and PP2C43-OE 2# and wild-type seedlings, and B shows the statistical results of the primary root lengths of Arabidopsis 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 manners

[0027] 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.

[0028] It should be understood that the terms used in the present invention are only for describing specific 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. Each 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.

[0029] 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 can 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.

[0030] 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 specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are only exemplary. Unless otherwise specified, the technical means used in the examples are conventional means well-known to those skilled in the art, and the raw materials used are all commercially available products.

[0031] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0032] Example 1

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

[0034] 1. Obtaining of PP2C.C7-related mutants

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

[0036] The maize mutant zmpp2c.c7-ems is a function-loss EMS mutagenesis mutant of the protein encoded by the maize ZmPP2C.C7 gene (Zm00001eb421700 gene, whose nucleotide sequence is shown in SEQ ID NO.6; its amino acid sequence is shown in SEQ ID NO.8) with Chang 7-2 (abbreviated as C7-2) as the background, and is named zmpp2c.c7-ems.

[0037] As Figure 1 shown in G, the expression levels of the PP2C.C7 gene in Arabidopsis wild type (WT), Arabidopsis mutants pp2c.c7-1 and pp2c.c7-2 were detected by reverse transcription PCR (RT-PCR) experiments. The results showed that almost no expression of the PP2C.C7 gene was detected in the two mutants pp2c.c7-1 and pp2c.c7-2, indicating that T-DNA insertion led to the loss of function of PP2C.C7.

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

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

[0040] 2. Salt stress phenotype identification of Arabidopsis thaliana pp2c.c7 mutants

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

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

[0043] 3. Salt stress phenotype identification of the Arabidopsis thaliana PP2C.C7 gene complementing the pp2c.c7 mutant

[0044] (1) Preparation of the complementation vector

[0045] The promoter sequence 2 Kb before the start codon of PP2C.C7 was constructed onto 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 onto the pCM1305-Pro:PP2C.C7 vector, and finally the complementation vector pCM1305-ProPP2C.C7:GFP:PP2C.C7 of the PP2C.C7 gene was obtained.

[0046] (2) Preparation of complementation transgenic plants

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

[0048] Forty-eight mutant pp2c.c7-1 materials were transformed with Agrobacterium tumefaciens GV3101 / proPP2C.C7:GFP:PP2C.C7 by the floral dip method, and T0 generation complementation transgenic lines were obtained through cultivation.

[0049] (3) Identification of complementation transgenic plants

[0050] After disinfecting and washing the T0 generation seeds, the seeds were evenly spread on 1 / 2 MS medium containing cefotaxime and hygromycin. After growing for 14 days, the seedlings with normal growth of both roots and crowns were selected. Further, through observation under a fluorescence microscope, the positive seedlings expressing GFP-PP2C.C7 were selected. At this time, the seedlings were the T1 generation of the complementation material. The identified positive lines were numbered and marked, and the seeds harvested from each individual plant were the T2 generation. The T2 generation seeds were screened on 1 / 2 MS medium containing hygromycin, and the plants with normal growth were selected for seed multiplication. The seeds harvested from each individual plant were the T3 generation.

[0051] The T3 generation complementation transgenic seeds were screened on 1 / 2 MS medium containing hygromycin. Those showing no segregation for hygromycin resistance in the T3 generation were homozygous PP2C.C7 complementation transgenic lines: COM-PP2C.C7. At least two independent transgenic complementation material lines were obtained and named COM-1 and COM-2.

[0052] The results were as Figure 1 shown in G. Reverse transcription PCR (RT-PCR) experiments were used to detect the expression levels of the PP2C.C7 gene in the wild type (WT) and the complementation lines. The results showed that the expression levels of PP2C.C7 in two independent PP2C.C7 complementation lines, COM-1 and COM-2, were restored, indicating that PP2C.C7 was expressed in the Arabidopsis transgenic plants.

[0053] (4) Identification of the salt stress phenotypes of complemented Arabidopsis transgenic plants

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

[0055] The results were as Figure 1 shown in D, Figure 1 shown in E andFigure 1 As shown in F, there were no significant growth differences among Col-0 (WT), pp2c.c7-1 mutants, and complemented lines under the condition of 0 mM salt concentration. Under the condition of 100 mM salt concentration, the complemented lines were able to complement the mutant phenotype and showed no significant growth differences from the wild type.

[0056] 4. Physiological mechanism of Arabidopsis PP2C.C7 negatively regulating plant salt tolerance

[0057] To analyze whether Arabidopsis PP2C.C7 regulates plant salt tolerance by affecting ion homeostasis and oxidative homeostasis under salt stress, the Na + and K + contents and reactive oxygen species (ROS) levels of Arabidopsis pp2c.c7 mutants were detected. The results showed that Arabidopsis PP2C.C7 negatively regulated the Na + / K + balance and oxidative balance under salt stress.

[0058] (1) Detection of ion contents in Arabidopsis pp2c.c7 mutants

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

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

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

[0062] (b) Collection of plants treated with salt stress

[0063] Seven days after transplanting the seedlings, the above-ground and underground parts were respectively taken, placed in an oven at 85 °C for drying treatment, and the dry weights were weighed.

[0064] (c) Determination of ion contents in plants treated with salt stress

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

[0066] The results are as Figure 2 shown in A, Figure 2 shown in B, Figure 2 shown in C,Figure 2 In D, Figure 2 in E, and Figure 2 as shown in F, under normal growth conditions, the Na + and K + contents, and the K + / Na + ratio in the crowns and roots of the pp2c.c7 mutant showed no significant difference from those of Col-0 (WT); under salt stress treatment conditions, compared with Col-0 (WT), the mutant accumulated less Na + but more K + in the crowns and roots, so that the K + / Na + ratio in the crowns and roots of the mutant was significantly higher than that of Col-0 (WT). These results indicate that PP2C.C7 negatively regulates the Na + / K + balance under salt stress.

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

[0068] Plants produce excessive reactive oxygen species (ROS) under stress conditions such as salt stress, mainly including superoxide anion (O2 - ), hydrogen peroxide (H2O2), and hydroxyl radical (OH · ). The level of hydrogen peroxide (H2O2) was determined by DAB staining method, and the level of superoxide anion (O2 - ) was determined by NBT staining method.

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

[0070] Wild-type Arabidopsis thaliana and mutant materials pp2c.c7-1 and pp2c.c7-2 of PP2C.C7 grown on normal 1 / 2MS medium for 7 days were respectively transferred to 1 / 2MS medium containing 0 mM and 100 mM NaCl and cultured in a light box for 24 hours.

[0071] (b) Determination of ROS levels in salt-stressed plants

[0072] The treated plants were placed in DAB (or NBT) staining solution, and vacuum was extracted in the dark for 0.5 hour to allow the staining solution to fully penetrate into the plants. After staining in the dark for 6 hours, decolorization was carried out and photos were taken to observe the ROS levels in the plants.

[0073] The results of DAB and NBT staining showed that there was no significant difference in the ROS levels in the leaves of wild-type and pp2c.c7 mutants after mock treatment; 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 (such as Figure 3 in A, Figure 3 in B, Figure 3 in C and Figure 3 in D), indicating that PP2C.C7 negatively regulates the oxidative balance under salt stress.

[0074] The above results indicated that Arabidopsis PP2C.C7 negatively regulates plant ion balance and oxidative balance under salt stress.

[0075] 5. Salt stress phenotype identification of maize zmpp2c.c7-ems mutant

[0076] (1) Analysis of homologous genes of Arabidopsis PP2C.C7 gene in maize

[0077] To search for homologous genes of Arabidopsis PP2C.C7 gene in crops and detect its regulatory function on salt tolerance, phylogenetic analysis was performed on PP2C C family members in crops such as Arabidopsis and maize. The results were as Figure 4 shown that Arabidopsis PP2C.C7 (AtPP2C.C7, AT3G16560) had a closer genetic relationship with maize ZmPP2C19 (Zm00001eb084510) and ZmPP2C98 (Zm00001eb421700). Further conserved domain analysis showed that these three proteins had similar conserved motifs (such as Figure 5 ), indicating that ZmPP2C19 and ZmPP2C98 might have similar functions to Arabidopsis PP2C.C7. Since only a sense mutant of the ZmPP2C98 gene was found in the EMS mutagenesis mutant library of the C7-2 background and there was only one, for unified naming, this mutant was named zmpp2c.c7-ems mutant, and the ZmPP2C98 gene was named ZmPP2C.C7.

[0078] (2) Salt stress phenotype identification of zmpp2c.c7-ems mutant

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

[0080] Furthermore, a pot experiment was conducted to identify the salt stress phenotype of the zmpp2c.c7-ems mutant. The wild type (WT) and the mutant were sown in substrate soil (nutrient soil and vermiculite at a ratio of 1:1) containing 150 mM NaCl or without NaCl. After continuous treatment for 14 days, the salt stress phenotype was recorded, and the plant height and above-ground fresh weight of maize seedlings were statistically analyzed. Different letters indicate significant differences, P < 0.05, n ≥ 10.

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

[0082] Comparative Example 1

[0083] The Arabidopsis PP2C family member PP2C43 is not involved in the regulation of plant salt tolerance

[0084] Some members of the PP2C family have been reported to be involved in the regulation of plant salt tolerance. The present invention first discovers that the Arabidopsis and maize PP2C.C7 genes negatively regulate plant salt tolerance. However, not all members of the PP2C family regulate plant salt tolerance.

[0085] Obtain relevant genetic materials of 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 of the PP2C family, including the loss-of-function mutant pp2c43 (the T-DNA insertion mutant is SALK_094214C) and the overexpression materials PP2C43-OE 1# and PP2C43-OE2# obtained by transferring pro35S:6×Myc:PP2C43 into the wild type (WT).

[0086] Detect the salt stress growth phenotypes of the above-mentioned Arabidopsis PP2C43-related genetic materials. Transfer 6-day-old wild-type Col-0 (WT) and pp2c43 mutant seedlings grown normally to 1 / 2 MS medium with or without 100 mM or 150 mM NaCl, and then culture them vertically for 9 days. Statistically analyze the primary root length. Different letters indicate significant differences, P < 0.05, n ≥ 10. Screen the salt stress phenotypes of the PP2C43 overexpression materials PP2C43-OE 1# and PP2C43-OE 2#. Transfer 6-day-old Arabidopsis seedlings grown normally to 1 / 2 MS medium with or without 100 mM NaCl, and then culture them vertically for 7 days. Statistically analyze the primary root length. Different letters indicate significant differences, P < 0.05, n ≥ 10.

[0087] The results are as Figure 8 shown in Figure 8 A, Figure 9 shown in Figure 9 B,

[0088] The above-described embodiments 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 design spirit of the present invention, various deformations 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 PP2C.C7 gene in regulating plant stress resistance, characterized in that, The nucleotide sequence of the PP2C.C7 gene is as shown in SEQ ID NO.1 or SEQ ID NO.

6.

2. The application according to claim 1, characterized in that, The plant is a cruciferous plant and / or a gramineous plant.

3. The application according to claim 2, characterized in that, The cruciferous plant is Arabidopsis thaliana, and the gramineous plant is Zea mays; 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 Zea mays, the nucleotide sequence of the PP2C.C7 gene is as shown in SEQ ID NO.

6.

4. The application of the PP2C.C7 gene as described in claim 1 in regulating the salt stress resistance performance of plants.

5. The application according to claim 4, wherein The PP2C.C7 gene negatively regulates the salt stress resistance performance of plants.

6. The application according to claim 4, characterized in that The plant is Arabidopsis thaliana and / or Zea mays.

7. Use of the CDS sequence of the PP2C.C7 gene as described in claim 1 in regulating the salt stress resistance performance of Arabidopsis thaliana, characterized in that, The nucleotide sequence of the CDS sequence of the PP2C.C7 gene is as shown in SEQ ID NO.

3.

8. Use of the protein encoded by the PP2C.C7 gene as described in claim 1 in regulating the salt stress resistance performance of Arabidopsis thaliana, characterized in that, The amino acid sequence of the protein encoded by the PP2C.C7 gene is as shown in SEQ ID NO.

2.

9. Use of the CDS sequence of the PP2C.C7 gene as described in claim 1 in regulating the salt stress resistance performance of maize, characterized in that, The nucleotide sequence of the CDS sequence of the PP2C.C7 gene is as shown in SEQ ID NO.

7.

10. Use of the protein encoded by the PP2C.C7 gene as described in claim 1 in regulating the salt stress resistance performance of maize, characterized in that, The amino acid sequence of the protein encoded by the PP2C.C7 gene is as shown in SEQ ID NO.8.

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