Plant drought tolerance related protein FYVE4 and application of coding gene thereof

Abstract

The invention relates to the technical field of plant drought tolerance, in particular to a plant drought tolerance related protein FYVE4 and application of a coding gene thereof. By inhibiting expression or protein activity of the FYVE4 gene in the plant, the survival rate of the plant under drought stress is increased; according to the method, in genetic improvement of arabidopsis thaliana, after genetic transformation is conducted through an agrobacterium-mediated method or a gene gun method, the stomata closing response speed and chlorophyll accumulation amount of crops can be effectively increased, and therefore plants with stable genetic drought resistance characters are established; the technical scheme is particularly suitable for constructing a water-saving agricultural system, has very important significance for improving crops and cultivating drought-resistant crops, and is suitable for popularization and application.

Description

Technical Field

[0001] The present invention relates to the technical field of plant drought resistance, in particular to the application of a plant drought resistance-related protein FYVE4 and a coding gene thereof. Background Art

[0002] The problems of soil desertification, salinization and water shortage caused by global climate change continue to intensify. Among them, drought stress has become the primary environmental factor restricting the sustainable development of modern agriculture. The key to plant response to drought stress lies in the precise regulation of the abscisic acid (ABA) signaling network. As a core stress-resistant hormone, ABA enhances plant drought resistance through a dual mechanism: ① At the systemic regulation level, ABA synthesized in the root system is transported to the aboveground part through the xylem, activating the SLAC1 anion channel in the stomatal guard cells, inducing stomatal closure (aperture reduction of 42%-65%), thereby reducing the transpiration rate (Nature Plants, 2021); ② At the cellular response level, ABA promotes the expression of protective proteins such as LEA proteins and dehydrins through the PP2C-SnRK2 signaling cascade, while increasing the activity of antioxidant enzymes such as SOD and CAT, thereby increasing the efficiency of reactive oxygen species (ROS) scavenging by 3.1 times (Plant Cell, 2022). Experiments with exogenous ABA treatment showed that concentrations of 50-100 μM could increase wheat water use efficiency by 28.5% (Journal of Experimental Botany, 2020). However, the high cost of chemically synthesizing ABA (>$2,500 / kg) and its photolytic inactivation limit its field application. Therefore, deciphering key regulatory nodes in the ABA signaling pathway and identifying endogenous drought-resistance genes have become strategic directions for overcoming current technological bottlenecks. Summary of the Invention

[0003] In response to the deficiencies of the prior art, the present invention provides a method for improving plant drought tolerance based on FYVE4 gene regulation, which increases the survival rate of plants under drought stress by 50%-70% (data from Example 3) by inhibiting the expression of the FYVE4 gene (the FYVE4 gene is shown in Sequence 2) or the activity of the protein (the FYVE4 protein is shown in Sequence 1) in the plant body. Specific implementation methods include, but are not limited to: using the CRISPR / Cas9 gene editing system to knock out the FYVE4 gene open reading frame (knockout FYVE4-FW gene primer is shown in Sequence 4, and knockout FYVE4-RW gene primer is shown in Sequence 5); constructing an FYVE4 gene silencing expression vector (the vector is shown in Sequence 6) through RNA interference technology; or interfering with FYVE4 mRNA stability through antisense RNA technology (the antisense RNA primer is shown in Sequence 7). In the genetic improvement of Arabidopsis thaliana, the method can effectively improve the crop stomatal closure response speed (after genetic transformation by Agrobacterium-mediated method or gene gun method) (Example 1). Figure 7 ) and chlorophyll accumulation ( Figure 2 ), thereby establishing plants with stable genetic drought-resistant traits. This technical solution is particularly suitable for building a water-saving agricultural system, is of great significance for crop improvement and the cultivation of drought-resistant crops, and is suitable for promotion and application.

[0004] The technical solution of the present invention is:

[0005] The protein provided by the present invention, obtained from Arabidopsis thaliana, named FYVE4 protein, is as follows (a) or (b):

[0006] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing;

[0007] (b) a protein derived from sequence 1 in which one or more amino acid residues in (a) are substituted and / or deleted and / or added and which is associated with plant drought resistance.

[0008] To facilitate purification and detection of the FYVE4 protein in (a), a tag as shown in Table 1 can be attached to the amino terminus or carboxyl terminus of the protein consisting of the amino acid sequence shown in SEQ ID NO: 1 in the sequence listing.

[0009] Table 1 Tag sequences

[0010]

[0011]

[0012] The FYVE4 protein in (b) above can be synthesized artificially, or its encoding gene can be synthesized first and then expressed biologically. The gene encoding the FYVE4 protein in (b) above can be obtained by deleting one or more amino acid residue codons from the DNA sequence shown in SEQ ID NO: 2 in the sequence listing, and / or performing one or more base pair missense mutations, and / or attaching the coding sequence of the tag shown in Table 1 to its 5′ and / or 3′ end.

[0013] The gene encoding the FYVE4 protein (FYVE4 gene) also falls within the scope of protection of the present invention.

[0014] The gene is as follows (1) or (2) or (3) or (4):

[0015] (1) The coding region of the DNA molecule is as shown in Sequence 2 of the Sequence Listing, from nucleotides 1869 to 5826 at the 5' end;

[0016] (2) the DNA molecule shown in Sequence 2 in the Sequence Listing;

[0017] (3) a DNA molecule that hybridizes with the DNA sequence defined in (1) or (2) under stringent conditions and encodes a protein associated with drought resistance;

[0018] (4) A DNA molecule that has 90% or more homology with the DNA sequence defined in (1), (2), or (3) and encodes a protein related to drought resistance.

[0019] The stringent conditions mentioned above can be hybridization and membrane washing at 65° C. using a solution of 0.1×SSPE (or 0.1×SSC), 0.1% SDS in a DNA or RNA hybridization experiment.

[0020] The recombinant expression vector, expression cassette, transgenic cell line or recombinant bacteria containing the FYVE4 gene all fall within the protection scope of the present invention.

[0021] The present invention also protects the use of the FYVE4 protein or FYVE4 gene, which is as follows (c1) and / or (c2):

[0022] (c1) regulating plant drought resistance;

[0023] (c2) regulating the sensitivity of plants to the stress hormone ABA signal;

[0024] (c3) Regulate plant sensitivity to salt stress.

[0025] The present invention also protects the use of the FYVE4 gene as a target in cultivating drought- and salt-stress-resistant plants.

[0026] The present invention also protects a method for cultivating transgenic plants, which is to inhibit the expression of the FYVE4 gene in the target plant to obtain a transgenic plant with higher drought resistance than the target plant.

[0027] The "inhibition of the expression of the FYVE4 gene in the target plant" is achieved through CRISPER-Cas9 gene editing.

[0028] The target sequences of the sgRNA used for the CRISPER-Cas9 gene editing are shown in sequences 4 and 5 of the sequence listing.

[0029] The target sequence binding region in the sgRNA used for the CRISPER-Cas9 gene editing is shown in SEQ ID NO: 7.

[0030] The recombinant vector used for the CRISPER-Cas9 gene editing can specifically be a DNA molecule shown in sequence 3 inserted between the BsaI restriction site of the pU6:-sgRNA1-pU6:-sgRNA2-zCas9 plasmid.

[0031] The present invention also protects a method for cultivating transgenic plants, which is to reduce the expression level and / or activity of FYVE4 protein in the target plant to obtain a transgenic plant with higher drought resistance than the target plant.

[0032] The present invention also protects a specific sgRNA for CRISPER-Cas9 gene editing; the target sequence of the sgRNA is shown in sequences 4 and 5 of the sequence listing.

[0033] The present invention also protects a substance for inhibiting the expression of the FYVE4 gene or a substance for reducing the expression level and / or activity of the FYVE4 protein in a target plant, or the use of the sgRNA in cultivating drought-resistant plants.

[0034] Any of the above plants is a monocot or a dicot. The dicot may be a plant of the order Capparisales. The plant of the order Capparisales may be a plant of the family Cruciferae. The plant of the family Cruciferae may be a plant of the tribe Araucariae. The plant of the tribe Araucariae may be a plant of the genus Arabidopsis. The plant of the genus Arabidopsis may specifically be Arabidopsis thaliana, such as Arabidopsis thaliana of the Columbia and / or Landsberg ecotypes.

[0035] The present invention has the following beneficial effects: the present invention provides a FYVE4 protein and its encoding gene, which inhibits the expression of the FYVE4 gene in plants and can significantly improve the drought tolerance of plants. The present invention is of great significance for crop improvement and the cultivation of drought-resistant crops and is suitable for promotion and application. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 、 Figure 2 and Figure 3The results show the sensitivity of the fyve4-1 mutant and its transgenic complementary expression plants to ABA.

[0037] Figure 4 These are the results of testing the sensitivity of the fyve4-1 mutant and its transgenic complementation-expressing plants to salt stress.

[0038] Figure 5 These are the results of FYVE4 gene expression detection in the fyve4-1 mutant and its transgenic complementation expression plants.

[0039] Figure 6 These are the results of drought resistance testing of the mutant fyve4-1.

[0040] Figure 7 The stomatal aperture statistics of the mutant fyve4-1.

[0041] Figure 8 These are the sequencing and protein detection results of the mutant fyve4-2.

[0042] Figure 9 Schematic diagram of the structure of the recombinant plasmid pCAMBIA1300-FYVE4-GFP.

[0043] Figure 10 Schematic diagram of the structure of the recombinant expression vector pYAO:hSpCas9-FYVE4-sgRNA. DETAILED DESCRIPTION

[0044] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples, unless otherwise specified, are conventional methods. The test materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores. The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0045] Mutant SALK_133410C: ABRC (Arabidopsis Biological Resource Center).

[0046] Arabidopsis thaliana ecotype Columbia (Col-0), Arabidopsis thaliana ecotype Landsberg erecta (Ler): ABRC (Arabidopsis Biological Resource Center).

[0047] pCAMBIA1300 vector: Available to the public from the School of Life Sciences, South China Normal University.

[0048] Agrobacterium tumefaciens EHA109 strain: available to the public from the School of Life Sciences, South China Normal University.

[0049] pU6:-sgRNA1-pU6:-sgRNA2-zCas9 plasmid: Publicly available from the School of Life Sciences, South China Normal University.

[0050] Bsa1:NEB.

[0051] The composition of MS medium is shown in Table 2.

[0052] Table 2. MS and 1 / 2MS medium components.

[0053]

[0054]

[0055] Example 1. Obtaining FYVE4 protein and its encoding gene.

[0056] By screening for ABA phenotypes in several Arabidopsis mutant plants ordered from ABRC, an ABA-hypersensitive mutant, SALK_133410C, was discovered. Whole-genome sequencing revealed that the mutant, SALK_133410C, was a deletion mutant of the FYVE4 gene (a T-DNA insertion mutant with the insertion site located within the third exon of the FYVE4 gene). Sequences other than the FYVE4 gene were identical to those of the Columbia ecotype Arabidopsis, and the mutant was named fyve4-1.

[0057] The FYVE4 protein derived from Arabidopsis thaliana is shown in Sequence 1 in the Sequence Listing.

[0058] The FYVE4 gene from Arabidopsis thaliana is shown as Sequence 2 in the Sequence Listing, wherein nucleotides 1869 to 5826 from the 5' end are the coding region.

[0059] Example 2: Detection of physiological characteristics of the fyve4-1 mutant in response to ABA.

[0060] 1. Sterilize the mutant seeds with sodium hypochlorite seed disinfectant for 5 minutes, wash them with ddH2O 5 times, and place them in a 4℃ refrigerator for 2 days.

[0061] 2. Carry out grouping experiment on the seeds treated in step 1:

[0062] Control group: Sterile seeds were suspended in aqueous solution and plated onto 1 / 2 MS culture medium plates. The plates were placed in the dark at 4°C for 2 days, then moved into a tissue culture room at 23°C (16h light / 8h dark) and cultured for one week. The growth status of the plants was observed and photographed.

[0063] Experimental group: Sterile seeds were suspended in aqueous solution and plated onto 1 / 2 MS medium plates containing 0.3 μM ABA. The plates were placed in the dark at 4°C for 2 days and then moved to a tissue culture room at 23°C (16 h light / 8 h dark) for one week. The growth status of the plants was observed and photographed.

[0064] The results are as follows Figure 1 shown. Figure 1 Part A in the middle is a photo of the plant's cotyledons. Figure 1 Part B shows the statistical results of seed germination ratios, where the first column shows the statistical results of the Columbia ecotype Arabidopsis thaliana (Col-0) and the second column shows the statistical results of the mutant fyve4-1.

[0065] The results are as follows Figure 2 As shown. In terms of cotyledon greening, there was no significant difference in growth between the fyve4-1 mutant and the Colombian ecotype Arabidopsis (Col-0) in the control (1 / 2 MS, 0 μM ABA) treatment. However, in the experimental (1 / 2 MS, 0.3 μM ABA) treatment, fewer cotyledons in the fyve4-1 mutant turned green compared to those in the Colombian ecotype (Col-0). On plates containing 0.3 μM ABA, 20% of the fyve4-1 mutant's cotyledons turned green, while 90% of the Colombian ecotype Arabidopsis (Col-0) cotyledons expanded and turned green.

[0066] The results are as follows Figure 3 As shown. In terms of underground root growth, there was no significant difference between the fyve4-1 mutant and the Colombian ecotype Arabidopsis thaliana (Col-0) in the control group (1 / 2 MS, 0 μM ABA). In the experimental group, the root length of the fyve4-1 mutant was shorter than that of the Colombian ecotype Arabidopsis thaliana (Col-0). On plates containing 0.3 μM ABA, the root length of the fyve4-1 mutant was approximately 50% of that of the Colombian ecotype Arabidopsis thaliana (Col-0).

[0067] The above experimental analysis shows that the mutant fyve4-1 is more sensitive to ABA, and the activation intensity of ABA signal in the mutant fyve4 is greater than that in the Columbia ecotype Arabidopsis thaliana (Col-0).

[0068] Example 3: Detection of physiological characteristics of fyve4 mutants under salt stress.

[0069] 1. Sterilize the mutant seeds with sodium hypochlorite seed disinfectant for 5 minutes, wash them with sterile ultrapure water 5 times, and place them in a 4°C refrigerator for 2 days.

[0070] 2. Carry out grouping experiment on the seeds treated in step 1:

[0071] Control group: Sterile seeds were suspended in aqueous solution and plated onto 1 / 2 MS culture medium plates. The plates were placed in the dark at 4°C for 2 days, then moved into a tissue culture room at 23°C (16h light / 8h dark) and cultured for one week. The growth status of the plants was observed and photographed.

[0072] Experimental group: Sterile seeds were suspended in aqueous solution and plated onto 1 / 2 MS medium plates containing 100 mM NaCl. The plates were placed in the dark at 4°C for 2 days and then moved to a tissue culture room at 23°C (16 h light / 8 h dark) for one week. The growth status of the plants was observed and photographed.

[0073] The results are as follows Figure 4 shown. Figure 4 Part a in the middle is a photo of the plant's cotyledons. Figure 4 Part b in the middle shows the statistical results of the proportion of cotyledons turning green, among which the first column is the statistical results of the Columbia ecotype Arabidopsis thaliana (Col-0), and the second column is the statistical results of the mutant fyve4-1. Figure 4 Part c in the middle is a photo of the plant root growth. Figure 4 Part d in the middle is the statistical results of root length.

[0074] In terms of cotyledon greening, there was no significant difference in growth between the fyve4-1 mutant and the Colombian ecotype Arabidopsis (Col-0) in the control (1 / 2 MS) treatment. However, in the experimental (1 / 2 MS supplemented with 100 mM NaCl) treatment, fewer fyve4-1 cotyledons turned green compared to those in the Colombian ecotype (Col-0). On plates containing 1 / 2 MS supplemented with 100 mM NaCl, 19.5% of fyve4 mutant cotyledons turned green, while 94% of Colombian ecotype Arabidopsis (Col-0) cotyledons expanded and turned green.

[0075] In terms of underground root growth, there was no significant difference between the fyve4-1 mutant and the Colombian ecotype Arabidopsis (Col-0) in the control (1 / 2 MS) treatment. However, in the experimental (1 / 2 MS supplemented with 100 mM NaCl) treatment, the fyve4 mutant's root length was shorter than that of the Colombian ecotype Arabidopsis (Col-0). Under the 1 / 2 MS supplemented with 100 mM NaCl treatment, the fyve4 mutant's root length was approximately 70% of that of the Colombian ecotype Arabidopsis (Col-0).

[0076] The above experimental analysis shows that the mutant fyve4-1 is more sensitive to salt stress, and the activation intensity of salt stress signal in the mutant fyve4-1 is greater than that in the Columbia ecotype Arabidopsis thaliana (Col-0).

[0077] Example 4: Transgenic complementary expression plants

[0078] 1. Obtaining transgenic complementary expression plants

[0079] 1. Total RNA was extracted from Arabidopsis thaliana ecotype Columbia (Col-0) and reverse transcribed into cDNA. PCR amplification was performed using the cDNA as a template with the primer pair consisting of FYVE4-FW and FYVE4-Rev, and the PCR amplification product was recovered.

[0080] FYVE4-FW:5'-GGG ACTAGT ATGTTGGAGAAGATCGGATTAC-3' (the underline indicates the Spe I restriction enzyme cleavage site)

[0081] FYVE4-Rev: 5'-GGG GGTACC GGTACCTCAACTTGAAGCAAGAGCGTTTA-3' (the underline indicates the Kpn I restriction enzyme cleavage site)

[0082] 2. Digest the pCAMBIA1300-GFP vector with restriction endonucleases Spe I and Kpn I to recover the approximately 10,000 bp vector backbone.

[0083] 3. The PCR amplification product obtained in step 1 was ligated with the vector backbone obtained in step 2 to obtain the recombinant plasmid pCAMBIA1300-AtFYVE4-GFP. Based on the sequencing results, the structure of the recombinant plasmid pCAMBIA1300-AtFYVE4-GFP was described as follows: the small fragment between the Spe I and Kpn I cleavage sites of the pCAMBIA1300-GFP vector was replaced with the double-stranded DNA molecule shown in Sequence 2 of the sequence listing. The schematic diagram of the structure of the recombinant plasmid pCAMBIA1300-AtFYVE4-GFP is shown in FIG. Figure 9 .

[0084] 4. Introduce the recombinant plasmid pCAMBIA1300-AtFYVE4-GFP into GV3101 Agrobacterium to obtain recombinant Agrobacterium.

[0085] 5. The recombinant Agrobacterium obtained in step 4 was cultured by shaking. When the OD600 reached 0.8-1.0, the bacterial solution was collected and centrifuged at 3580 rpm for 5 min. The supernatant was discarded, the suspension was resuspended in 5% sucrose solution, washed, and re-centrifuged for 5 min. The supernatant was discarded, and the suspension was resuspended in 5 mL of 5% sucrose solution. 2.5 μL of Silwet L-77 surfactant was added to infect the transformed mutant fyve4-1, and the seeds were harvested (reference: Martinez-Trujillo, M., Limones-Briones, V., Cabrera-Ponce, J. Let al. Improving transformation efficiency of Arabidopsis thaliana by modifying the floral dip method. Plant Mol Biol Rep 22, 63–70 (2004). https: / / doi.org / 10.1007 / BF02773350) (T0 generation seeds, and the plants grown from these seeds are T1 generation plants).

[0086] 6. After the Agrobacterium-infected Arabidopsis seeds mature, harvest the T0 generation seeds and dry them in a 37°C oven for 2 days. A portion of the seeds is collected in a 1.5 mL centrifuge tube and added to 1 mL of transgenic seed disinfectant (formula: 70 mL of anhydrous ethanol, 5 mL of 10% Triton X-100, mixed with 25 mL of ddH2O) for 10 minutes of shaking to ensure thorough disinfection. The supernatant is then aspirated and washed four times with 1 mL of anhydrous ethanol on a clean bench. The final wash is then poured onto sterile filter paper and air-dried. After the filter paper is completely dry, the seeds are evenly spread on 1 / 2 MS solid medium containing hygromycin resistance. After the moisture on the plate evaporates, seal the plate with parafilm and place it flat in a 4°C refrigerator for vernalization for 2 days. Then, place it flat in a plant tissue culture incubator for long-day culture. After approximately 7 days of incubation, select positive seedlings with true leaves and normal root growth and transplant them to soil for cultivation. If the transgenic plants have fluorescent tags, the fluorescent signals can be observed under an upright fluorescence microscope before being transferred to the soil. Seedlings with fluorescent signals can then be selected and transferred to the soil for growth and cultivation.

[0087] 7. After the transgenic plant seeds mature, harvest the T1 generation seeds. Disinfect the seeds with sodium hypochlorite seed disinfectant (disinfectant: ddH2O = 3:7), vortex for 5 minutes, wash five times with sterile ultrapure water on a clean bench, and place the centrifuge tube in a 4°C refrigerator for 2 days. Evenly spot the vernalized seeds on 1 / 2 MS solid medium containing 50 μg / mL hygromycin and culture in a plant tissue culture incubator for approximately 7 days. Select strains that roughly meet the ratio of resistant seedlings to non-resistant seedlings of 3:1 and transfer them to soil for culture.

[0088] 8. Place the seeds harvested in step 7 on 1 / 2 MS medium plates containing 50 μg / mL hygromycin for resistance screening. If all T3 plants derived from a T2 plant are resistant, it indicates that the T2 plant is a homozygous transgenic plant, and the T2 plant and its progeny are homozygous complementary expression lines.

[0089] 9. Select one homozygous complementary expression line (3.4) from the homozygous complementary expression lines obtained in step 8. Take 2-week-old seedlings of the T3 generation of Arabidopsis thaliana (Col-0), mutant fyve4-1, and line 3.4, extract total RNA, and reverse transcribe it into cDNA. Use cDNA as a template to detect the expression level of the FYVE4 gene in plants of each line by real-time fluorescence quantitative PCR. Use the UBQ10 gene as an internal reference gene, and take the expression level of the FYVE4 gene in the Arabidopsis thaliana ecotype of Columbia as 1. Calculate the relative expression level of the AtFYVE4 gene in each other line. Repeat the experiment three times and take the average value.

[0090] The primer sequences used to identify the FYVE4 gene are as follows:

[0091] qRT-FYVE4-Fw:5'-AATTTTCGTCTCTTCTCCGT-3':

[0092] qRT-FYVE4-Rev:5'-TTAATAAGTGCAGATAGCTC-3'.

[0093] The primer sequences used to identify the UBQ10 gene are as follows:

[0094] qRT-UBQ10-FW: 5'-GGTAACATTGTGCTCAGTGGTGG-3';

[0095] qRT-UBQ10-Rev:5'-AACGACCTTAATCTTCATGCTGC-3'.

[0096] See the results Figure 5The results showed that the expression level of the FYVE4 gene in the homozygous complementation expression line 3.4 exceeded the expression level of the FYVE4 gene in the Columbia ecotype Arabidopsis thaliana, and no expression of the FYVE4 gene was detected in the mutant fyve4-1.

[0097] 2. Phenotypic Analysis of Transgenic Complementation Expression Plants

[0098] The phenotype of the T3 generation plants of the homozygous complementary expression line (3.4) was detected according to the methods of Examples 2 and 3.

[0099] The results are as follows Figure 1-4 As shown. Physiological indicators such as seed germination rate and root growth in homozygous complementing expression line 3.4 after ABA and salt stress treatment were similar to those of the Columbia ecotype Arabidopsis. This indicates that homozygous complementing expression line 3.4 can complement the phenotypes of the fyve4-1 mutant in terms of cotyledon greening and root length growth. This suggests that the loss of the FYVE4 gene is responsible for the phenotypes of the fyve4 mutant. The FYVE4 gene regulates plant responses to ABA signaling and salt stress. The fyve4-1 mutant can be used for further drought phenotyping experiments.

[0100] Example 5: Drought resistance evaluation of the mutant fyve4-1

[0101] 1. Seeds of mutant fyve4-1 and Columbia ecotype Arabidopsis thaliana (Col-0) were surface sterilized with 10% bleach solution and then washed three times with sterile water.

[0102] 2. Suspend the sterile seeds in aqueous solution and spread them onto 1 / 2MS plates. Cold treat at 4℃ for 3 days, then culture at 21℃ for 6 days. Transplant them into soil and continue culture for about 20 days. Plant 4-5 plants in each hole.

[0103] 3. Stop watering and drought-treat for 21 days. Re-water on day 42 (i.e., the first day after the 21-day drought treatment). Record the survival rate of the stressed plants on the seventh day after re-watering.

[0104] The results of plant phenotype observations were as follows Figure 6 As shown in part A. There was no significant difference in the growth status of mutant fyve4 and Colombian ecotype Arabidopsis thaliana (Col-0) when there was no drought stress (normal growth for 4 weeks as described in step 2). The growth status of mutant fyve4 plants was significantly better than that of Colombian ecotype Arabidopsis thaliana one day before and one day after rehydration. One day after rehydration, the survival rate of each plant was counted, and it was found that the survival rate of mutant fyve4 plants was significantly higher than that of Colombian ecotype Arabidopsis thaliana (Col-0) ( Figure 6 Part B).

[0105] 4. Take the flat rosette leaves of each plant in step 2 and place the leaves in MES / KCl buffer (10mM MES / KOHph6.0, 5mM KCl, 50μM CaCl2), ensuring that the lower epidermis is completely in contact with the buffer, and then place at 22°C for 3 hours to fully open the stomata. Subsequently, the treated leaves were transferred to fresh MES / KCl buffer with or without ABA and placed in the dark for 3 hours. After treatment, the lower epidermal cells of the leaves were peeled off as much as possible with tape and placed on a slide, then covered with a coverslip and a small amount of MES / KCl buffer was dropped. Stomata were observed using an optical microscope and stomatal aperture was measured using Image J.

[0106] The statistical results are as follows Figure 7 As shown. In the absence of ABA, there was no significant difference in stomatal aperture between the mutant fyve4 and the Columbia ecotype Arabidopsis thaliana (Col-0). However, under 10 μM ABA treatment, the stomatal aperture of the mutant fyve4 was significantly smaller than that of the Columbia ecotype Arabidopsis thaliana (Col-0) ( Figure 7 Small stomatal aperture can effectively reduce water loss, which is an important reason for the drought resistance of the fyve4 mutant.

[0107] The above results show that the loss of the FYVE4 gene can enhance the sensitivity of plants to the stress hormone ABA signal, enabling mutant plants to quickly respond to increased levels of the stress hormone ABA in the body under drought conditions, close leaf stomata, reduce further transpiration, and ultimately give plants strong drought resistance.

[0108] Example 6: Establishment and identification of transgenic Arabidopsis lines with FYVE4 functional deficiency

[0109] 1. Use Bsa I-HF to digest the pU6:-sgRNA1-pU6:-sgRNA2-zCas9 plasmid to obtain a pU6:-sgRNA1-pU6:-sgRNA2-zCas9 vector of approximately 4000 bp.

[0110] 2. Artificially synthesize primers FYVE4-FW2 and FYVE4-Rev2.

[0111] FYVE4-FW2:5'- ATTG GGACGGCGTCGAGAATAAAG-3' (the underlined sequence is the complementary sequence of the sticky end sequence of the vector pU6:-sgRNA1-pU6:-sgRNA2-zCas9 after digestion with Bsa I);

[0112] FYVE4-Rev2:5'- AAACCTTTATTCTCGACGCCGTCC-3' (the underlined sequence is the complementary sequence of the sticky end sequence of the vector pU6:-sgRNA1-pU6:-sgRNA2-zCas9 after Bsa I digestion).

[0113] 3. Anneal the primers FYVE4-FW2 and FYVE4-Rev2 synthesized in step 2 to obtain a double-stranded DNA fragment (interference fragment).

[0114] 4. The double-stranded DNA fragment (interference fragment) obtained in step 3 and the pU6:-sgRNA1-pU6:-sgRNA2-zCas9 vector obtained in step 1 were connected to obtain a connection product.

[0115] 5. The ligation product of step 4 was double-digested with Nhe I and Spe I restriction endonucleases to obtain the target fragment.

[0116] 6. The pYAO:hSpCas9 plasmid was digested with Spe I restriction endonuclease to obtain a pYAO:hSpCas9 vector of approximately 12,000 bp.

[0117] 7. The target fragment obtained in step 5 was connected to the pYAO:hSpCas9 vector obtained in step 6 to obtain the recombinant expression vector pYAO:hSpCas9-FYVE4-sgRNA. Based on the sequencing results, the structure of the recombinant expression vector pCAMBIA1300-Cas9-FYVE4 was described as follows: the DNA molecule shown in sequence 3 was inserted between the Spe I restriction sites of the pYAO:hSpCas9 plasmid. The schematic diagram of the structure of the recombinant expression vector pYAO:hSpCas9-FYVE4-sgRNA is shown in Figure 10 .

[0118] 8. The recombinant expression vector pYAO:hSpCas9-FYVE4-sgRNA obtained in step 7 was introduced into the Agrobacterium tumefaciens EHA105 strain to obtain recombinant Agrobacterium.

[0119] 9. The recombinant Agrobacterium obtained in step 8 was transformed into the Columbia ecotype Arabidopsis thaliana (Col-0) by plant vacuum infiltration method (reference: Martinez-Trujillo, M., Limones-Briones, V., Cabrera-Ponce, J. Let al. Improving transformation efficiency of Arabidopsis thaliana by modifying the floral dip method. Plant Mol Biol Rep 22, 63–70 (2004). https: / / doi.org / 10.1007 / BF02773350.) and seeds were harvested (T0 generation seeds, and plants grown from these seeds are T1 generation plants).

[0120] 10. Place the seeds harvested in step 9 on a 1 / 2 MS medium plate containing 50 μg / mL hygromycin. Place the plate in a dark environment at 4°C for 2-4 days, then move it to a tissue culture room at 24°C (16 h light / 8 h dark) and culture it for 1 week. Then move it to a greenhouse at 24°C for culture and harvest the seeds (T1 generation seeds, the plants grown from these seeds are T2 generation plants).

[0121] 11. The seeds harvested in step 10 were plated on ½ MS medium containing 50 μg / mL hygromycin for resistance screening, and seeds of T2-generation resistant plants obtained from single-copy transgenic plants were harvested (plants grown from these seeds are T3-generation plants). If the ratio of resistant to non-resistant T2 plants obtained from a T1-generation plant is approximately 3:1, it indicates that the T1-generation plant is a single-copy transgenic plant.

[0122] 12. Place the seeds harvested in step 11 on a 1 / 2 MS medium plate containing 50 μg / mL hygromycin for resistance screening. If all T3 plants derived from a T2 plant are resistant, it indicates that the T2 plant is a homozygous transgenic plant, and the T2 plant and its progeny are homozygous transgenic lines.

[0123] 13. Select one of the homozygous transgenic lines obtained in step 12 and sequence to detect the editing status of the FYVE4 gene in the T3 generation plants ( Figure 8 ).

[0124] After identification, the translation of FYVE4 protein was terminated prematurely in the homozygous FYVE4 function-deficient transgenic line, and the mutant was named fyve4-2.

[0125] 3. Identification of the ABA sensitivity of the fyve4-2 mutant

[0126] The plants to be tested were: the T3 generation plant mutant fyve4-2 of the homozygous FYVE4 functional loss transgenic line and the Columbia ecotype Arabidopsis thaliana (Col-0).

[0127] 1. Sterilize the mutant seeds with sodium hypochlorite seed disinfectant for 5 minutes, wash them with ddH2O 5 times, and place them in a 4℃ refrigerator for 2 days.

[0128] 2. Carry out grouping experiment on the seeds treated in step 1:

[0129] Control group: Sterile seeds were suspended in aqueous solution and plated onto 1 / 2 MS culture medium plates. The plates were placed in the dark at 4°C for 2 days, then moved into a tissue culture room at 23°C (16h light / 8h dark) and cultured for one week. The growth status of the plants was observed and photographed.

[0130] Experimental group: Sterile seeds were suspended in aqueous solution and plated onto 1 / 2 MS medium plates containing 0.3 μM ABA. The plates were placed in the dark at 4°C for 2 days and then moved to a tissue culture room at 23°C (16 h light / 8 h dark) for one week. The growth status of the plants was observed and photographed.

[0131] The results are as follows Figure 1 shown. Figure 1 Part A in the middle is a photo of the plant's cotyledons. Figure 1 Part B shows the statistical results of seed germination ratios, where the first column shows the statistical results of the Columbia ecotype Arabidopsis thaliana (Col-0) and the second column shows the statistical results of the mutant fyve4-1.

[0132] The results are as follows Figure 2 As shown. In terms of cotyledon greening, there was no significant difference in growth between the fyve4-1 mutant and the Colombian ecotype Arabidopsis (Col-0) in the control (1 / 2 MS, 0 μM ABA) treatment. However, in the experimental (1 / 2 MS, 0.3 μM ABA) treatment, fewer cotyledons in the fyve4-2 mutant turned green compared to the Colombian ecotype Arabidopsis (Col-0). On plates containing 0.3 μM ABA, 20% of the fyve4-2 mutant's cotyledons turned green, while 90% of the Colombian ecotype Arabidopsis (Col-0) cotyledons expanded and turned green.

[0133] The results are as follows Figure 3As shown. In terms of underground root growth, there was no significant difference between the fyve4-1 mutant and the Colombian ecotype Arabidopsis thaliana (Col-0) in the control group (1 / 2 MS, 0 μM ABA). In the experimental group, the root length of the fyve4-1 mutant was shorter than that of the Colombian ecotype Arabidopsis thaliana (Col-0). On plates containing 0.3 μM ABA, the root length of the fyve4-1 mutant was approximately 50% of that of the Colombian ecotype Arabidopsis thaliana (Col-0).

[0134] The above experimental analysis shows that the mutant fyve4-2 is more sensitive to ABA, and the activation intensity of ABA signal in the mutant fyve4 is greater than that in the Columbia ecotype Arabidopsis thaliana (Col-0).

[0135] The above embodiments and descriptions are only for explaining the principles and best embodiments of the present invention. Without departing from the spirit and scope of the present invention, the present invention may be subject to various changes and improvements, which shall fall within the scope of the invention to be protected.

Claims

1. The application of the protein represented by Sequence 1 in the sequence listing or its encoding gene is as follows (c1) and / or (c2) and / or (c3): (c1) regulating plant drought resistance; (c2) regulating the sensitivity of plants to the stress hormone ABA signal; (c3) Regulate plant sensitivity to salt stress.

2. The use according to claim 1, characterized in that: The coding gene is as follows (1) or (2): (1) The coding region of the DNA molecule is as shown in Sequence 2 of the Sequence Listing, from nucleotides 1869 to 5826 at the 5' end; (2) The DNA molecule shown in Sequence 2 in the sequence listing.

3. Application of the gene encoding the protein shown in Sequence 1 in the sequence listing as a target in breeding drought- and salt-stress-resistant plants.

4. The use according to claim 3, characterized in that: The gene is as follows (1) or (2): (1) The coding region of the DNA molecule is as shown in Sequence 2 of the Sequence Listing, from nucleotides 1869 to 5826 at the 5' end; (2) The DNA molecule shown in Sequence 2 in the sequence listing.

5. A method for cultivating transgenic plants, comprising inhibiting the expression of a gene encoding a protein represented by Sequence 1 in a target plant to obtain a transgenic plant having higher drought resistance than the target plant.

6. The method according to claim 5, characterized in that: The coding gene is as follows (1) or (2): (1) The coding region of the DNA molecule is as shown in Sequence 2 of the Sequence Listing, from nucleotides 1869 to 5826 at the 5' end; (2) The DNA molecule shown in Sequence 2 in the sequence listing.

7. A method for cultivating transgenic plants, comprising reducing the expression level and / or activity of a protein in a target plant to obtain a transgenic plant having higher drought resistance than the target plant; The protein is shown in Sequence 1 in the sequence table.

8. Use of a substance for inhibiting the expression of a gene encoding a protein as shown in Sequence 1 in the sequence listing or a substance for reducing the expression level and / or activity of a protein as shown in Sequence 1 in a target plant in cultivating drought-resistant plants; The substance is a recombinant expression vector pYAO:hSpCas9-FYVE4-sgRNA; The recombinant expression vector pYAO:hSpCas9-FYVE4-sgRNA is constructed by inserting the DNA molecule shown in sequence 3 between the Spe I restriction enzyme cutting sites of the pYAO:hSpCas9 plasmid.

9. The use according to claim 8, characterized in that: The coding gene is as follows (1) or (2): (1) The coding region of the DNA molecule is as shown in Sequence 2 of the Sequence Listing, from nucleotides 1869 to 5826 at the 5' end; (2) The DNA molecule shown in Sequence 2 in the sequence listing.

Images