SPX4 gene for negative regulation of plant drought tolerance and application thereof

Abstract

The invention relates to the technical field of biological genes, in particular to an SPX4 gene for negative regulation of plant drought tolerance and application of the SPX4 gene. The negative regulation effect of the SPX4 in plant drought stress response is disclosed, and specifically, the SPX4 can regulate the nucleation process of a downstream core transcription factor; under the treatment of ABA, compared with a wild type, the overexpression of the SPX4 can inhibit the expression of a plurality of stress response genes such as OsNCED4, OsPSY3, OsPM1, DREB1C and OsbHLH120, which suggests that the SPX4 plays a negative regulation role in the stress response process; the SPX4-OE strain shows the insensitivity of ABA treatment in a root growth experiment; under PEG 6000 simulated drought treatment, the SPX4 overexpression plant shows drought sensitivity, and the negative regulation ability of the SPX4 in the drought response process is further proved; in a large field, under the low-nitrogen or high-nitrogen drought condition, the yield of SPX4-OE plants is remarkably reduced, particularly under the low-nitrogen drought condition, the yield loss is particularly remarkable, and it is proved that the SPX4-OE serves as a negative regulation component to participate in the plant stress tolerance process. The invention provides a new target and an effective strategy for crop stress resistance breeding.

Description

Technical Field

[0001] This invention relates to the field of biogenetics, specifically to an SPX4 gene that negatively regulates plant drought tolerance and its applications. Background Technology

[0002] SPX domain proteins are widely distributed in eukaryotes, including animals, plants, and fungi. These proteins are named according to the first letters of the names of the three proteins: Syg1, Pho81, and XPRI. In rice, SPX4 is an important repressor protein known to play a role in the transduction pathways of phosphorus and nitrate signals, regulating nucleoplasmic shuttling of the core transcription factor PHR2 for phosphorus starvation and the core transcription factor NLP3 for nitrate signaling.

[0003] Abiotic stresses, such as drought, salinity, high temperature, and low temperature, are abiotic environmental factors that negatively impact plant growth and development and are major environmental factors limiting crop growth and yield. However, current technologies have not yet revealed the specific function and mechanism of action of SPX4 in plant responses to abiotic stresses, particularly drought stress. Therefore, there is an urgent need in this field to discover new regulatory targets that can coordinate the balance between plant stress resistance and growth, providing new strategies and gene resources for crop genetic improvement. Summary of the Invention

[0004] This invention overcomes the shortcomings of existing technologies by providing an SPX4 gene that negatively regulates plant drought resistance and its applications. SPX4 can regulate the nuclear translocation of downstream core transcription factors. By obtaining SPX4-overexpressing plants, these plants exhibit the following characteristics: suppression of stress-response gene expression under ABA treatment; significantly reduced survival rate under PEG 6000 simulated drought treatment; and significantly reduced yield under low-nitrogen or high-nitrogen drought conditions. This demonstrates the negative regulatory capacity of SPX4 in drought response, providing a new target and effective strategy for crop stress-resistance breeding.

[0005] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, an SPX4 gene is provided, the CDS sequence of which is shown in SEQ ID NO.1, and the sequence of the protein encoded by which SPX4 is shown in SEQ ID NO.2.

[0006] SPX4 is an important negative regulatory component in the ABA signaling system, participating in negative regulation of stress response processes such as drought resistance and salt tolerance.

[0007] SPX4 in this invention can function as a gene or protein, exerting a negative regulatory capacity during drought response.

[0008] Genetic improvement of germplasm or new varieties can be achieved by utilizing the SPX4 protein and targeting it.

[0009] Secondly, the application of the SPX4 gene in negatively regulating plant drought tolerance is provided, the application including obtaining plants overexpressing SPX4 to achieve negative regulation of plant drought tolerance.

[0010] The application of the SPX4 gene in the negative regulation of drought tolerance in rice plants was presented.

[0011] Furthermore, the SPX4 overexpressing plants were obtained through the following steps: The SPX4 gene, as shown in SEQ ID NO.1, was cloned, and the SPX4 gene was introduced into an expression vector to construct a recombinant expression vector. The recombinant expression vector was introduced into the rice recipient variety Zhonghua 11, and transgenic rice plants overexpressing SPX4 were obtained after screening and cultivation.

[0012] Furthermore, the expression vector is pCAMBIA1300-221-FLAG.

[0013] Furthermore, the negative regulation of plant drought resistance was achieved using subcellular localization analysis of rice protoplasts. Under ABA induction, SPX4 caused transcription factors to remain in the cytoplasm.

[0014] SPX4 is an important negative regulatory component in the stress response process, which can regulate the nuclear entry of downstream core transcription factors.

[0015] Transient expression of SPX4 protein in rice protoplasts followed by ABA treatment prevented the transcription factor from entering the cell nucleus.

[0016] Furthermore, the negative regulation of plant drought tolerance is achieved by obtaining SPX4 overexpressing plants, which, under ABA treatment, suppress the expression of stress response genes OsNCED4, OsPSY3, OsPM1, DREB1C, OsbHLH120, OsNAC52, OsHsfC2a and OsNAC6.

[0017] Under ABA treatment, compared to the wild type, SPX4 overexpression inhibited the expression of multiple stress response genes, including OsNCED4, OsPSY3 and OsPM1 (ABA synthesis and transport-related genes), DREB1C and OsbHLH120 (development-related genes), OsNAC52 and OsHsfC2a (stress-related genes) and OsNAC6 (glucosinolate metabolism-related gene), suggesting that it plays a negative regulatory role in the stress response process.

[0018] Furthermore, the negative regulation of plant drought tolerance is achieved by obtaining SPX4-overexpressing plants, whose root growth is sluggish under ABA treatment.

[0019] Plants overexpressing SPX4 showed a dulling effect compared to ABA treatment in root growth experiments.

[0020] Furthermore, the negative regulation of plant drought tolerance is achieved by obtaining SPX4-overexpressing plants, whose survival rate decreases after PEG-simulated drought treatment.

[0021] After PEG 6000 simulated drought treatment, SPX4-overexpressing plants showed drought sensitivity, further demonstrating the negative regulatory capacity of SPX4 in the drought response process.

[0022] Furthermore, the negative regulation of plant drought tolerance is achieved by obtaining SPX4 overexpressing plants, which exhibit reduced yield under low-nitrogen or high-nitrogen drought field conditions.

[0023] In the field, under drought conditions with low or high nitrogen, SPX4-OE plants showed a significant decrease in yield compared to the wild type, especially under low nitrogen conditions, where the yield loss was particularly significant, demonstrating that it participates in the plant stress tolerance process as a negative regulatory component.

[0024] Furthermore, the CDS sequence of SPX4 is shown in SEQ ID NO.1, and the sequence of the protein encoded by SPX4 is shown in SEQ ID NO.2.

[0025] The beneficial effects of this invention are: This invention provides an SPX4 gene that negatively regulates plant drought tolerance and its applications. Experimental results show that SPX4 is an important negative regulatory component in the stress response process, regulating the nuclear entry of downstream core transcription factors. Specifically, SPX4 can cause transcription factors to remain in the cytoplasm and prevent them from entering the nucleus, thus blocking the transmission of drought resistance commands from transcription factors. Furthermore, by obtaining SPX4-overexpressing plants, it was found that under ABA treatment, compared with the wild type, SPX4 overexpression inhibited the expression of multiple stress response genes, including ABA synthesis and transport-related genes OsNCED4, OsPSY3, and OsPM1, development-related genes DREB1C and OsbHLH120, stress-related genes OsNAC52 and OsHsfC2a, and glucosinolate metabolism-related gene OsNAC6, suggesting that it plays a negative regulatory role in the stress response process. It was found that SPX4-overexpressing plants showed ABA insensitivity in root growth experiments, and SPX4 made the plants lose sensitivity to drought signals. Under a simulated drought treatment of 6000, plants overexpressing SPX4 exhibited drought sensitivity, wilting faster and more severely, and with lower survival rates, further demonstrating the negative regulatory capacity of SPX4 in the drought response process; that is, SPX4 overexpression significantly reduces plant drought tolerance. In the field, under low-nitrogen / high-nitrogen drought conditions, compared to the wild type, SPX4-OE plants showed a significant decrease in yield, especially under low-nitrogen conditions, where yield loss was particularly significant, proving that it participates in the plant stress tolerance process as a negative regulatory component. This invention provides a new target and effective strategy for crop stress resistance breeding.

[0026] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description

[0027] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with the invention and, together with the description, serve to explain the principles of the invention.

[0028] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, for those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0029] Figure 1 This is a subcellular localization analysis of rice protoplasts in an embodiment of the present invention; Figure 2 In this embodiment of the invention, overexpression of SPX4 significantly inhibited the ABA response in plants; Figure 3In this embodiment of the invention, plants overexpressing SPX4 showed insensitivity to ABA treatment. Figure 4 The phenotype and survival rate of SPX4-overexpressing plants before and after PEG-simulated drought treatment in this embodiment of the invention; Figure 5 This invention relates to the phenotype and yield per plant of SPX4-overexpressing plants under low-nitrogen or high-nitrogen arid field conditions. Detailed Implementation

[0030] Preferred embodiments of the invention will now be described in more detail. While preferred embodiments of the invention have been shown, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided so that the invention will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.

[0031] Example 1: Subcellular localization analysis of rice protoplasts Through experimental methods in this field, we obtained Figure 1 The results shown, where for Figure 1 The experiments involved are only briefly described. Those skilled in the art can operate the corresponding experiments based on the experiment names, so only a brief description is given here.

[0032] After rice seedlings were grown in basal nutrient solution for 10 days, rice protoplasts were isolated and transfected according to the method described in the reference. To achieve efficient transfection, protoplasts were gently resuspended in MMg solution (containing 0.4 M mannitol, 15 mM MgCl2, 4 mM MES, pH 5.7), and counted under an optical microscope using a hemocytometer. The suspension volume was adjusted to achieve a protoplast concentration of 2–5 × 10⁻⁶. 6 per milliliter.

[0033] To investigate the nuclear localization of NLP4 protein in rice protoplasts under ABA induction, wild-type ZH11 protoplasts were transfected with the NLP4-eGFP plasmid and cultured at 28℃ in W5 solution (containing 154 mM NaCl, 125 mM CaCl2, 5 mM KCl, 2 mM MMES, pH 5.7) for 12 hours. The transfected protoplasts were centrifuged at 200 g for 3 minutes and treated with W5 solution containing either 100 nM ABA or ethanol (as a control) for 30 minutes. Fluorescence signals were then acquired using a confocal laser scanning microscope (Leica STELLARIS5, Germany).

[0034] To investigate the effect of ABA (abscisic acid)-induced SPX4 on the localization pattern of NLP4, wild-type ZH11 protoplasts were transfected with SPX4-RFP and NLP4-eGFP plasmids and incubated in W5 solution at 28°C for 12 hours. The protoplasts were then centrifuged at 200×g for 3 minutes and treated with W5 solution containing 100 nM ABA (or ethanol control) for 30 minutes, and observations were performed as described above.

[0035] Figure 1 In this process, SPX4 can cause transcription factors to remain in the cytoplasm. A. Transcription factors can enter the cell nucleus under the induction of ABA.

[0036] B. When SPX4 protein is transiently expressed in rice protoplasts and then treated with ABA, the transcription factor cannot enter the cell nucleus.

[0037] In summary, subcellular localization experiments on rice protoplasts confirmed that, under ABA treatment, core transcription factors can be translocated from the cytoplasm to the nucleus. Figure 1 A). However, when SPX4 protein is transiently expressed in rice protoplasts followed by ABA treatment, the transcription factor is arrested in the cytoplasm and cannot enter the nucleus to function. Figure 1 B). This indicates that SPX4 is an important negative regulatory component in the stress response process, which can regulate the nuclear translocation of downstream core transcription factors. Specifically, SPX4 negatively regulates the plant stress response by inhibiting the activation of core transcription factors under stress signals.

[0038] Protoplast subcellular localization analysis: Protoplasts are plant cells with their cell walls removed, enabling efficient transfection of exogenous macromolecules (DNA, RNA, proteins), providing a universal single-cell functional analysis platform for rapidly elucidating the functions of these macromolecules. Through a mature reporter gene and marker gene detection system, it was confirmed that high-quality protoplasts isolated from rice leaf sheath tissue exhibited highly consistent response characteristics to various abiotic and biotic stress signals, as well as hormones and nutrients, with intact plants. This transient expression system has been widely proven to be a key and reliable technique for elucidating transcription factor activity, protein kinase function, protein subcellular localization and transport, protein-protein interactions, and protein stability in signal transduction pathways. The key to successfully utilizing this technology lies in obtaining viable plant tissue protoplasts and achieving efficient transfection.

[0039] References: Trinidad JL, Longkumer T, Kohli A. Rice protoplast isolation and transfection for transient gene expression analysis. Methods Mol. Biol. 2021,2238: 313-324. Example 2: Overexpression of SPX4 significantly inhibited the ABA response in plants. Obtaining genetically modified rice involves the following steps: (1) RNA extraction from plant samples In a liquid nitrogen-pre-cooled mortar, the rice material was rapidly frozen in liquid nitrogen and ground into a fine powder. 50–100 mg of the powder was placed in a centrifuge tube free of RNase. 1 mL of Trizol was added, vortexed, and allowed to stand for 5 min. 200 µL of chloroform was added, vortexed, and allowed to stand for 3 min. The mixture was then centrifuged at 12000 rpm, 4°C, for 15 min. Approximately 400 µL of the supernatant was transferred to a new RNase-free centrifuge tube. An equal volume (400 µL) of isopropanol was added, inverted, and allowed to stand for 20–30 min. The mixture was then centrifuged at 12000 rpm, 4°C, for 10 min. The supernatant was discarded, and 1 mL of 75% ethanol (prepared with RNase-free water) was added to wash the precipitate. The precipitate was centrifuged at 7500 rpm, 4°C, for 5 min. Depending on the amount of precipitate, this washing process could be repeated once. The supernatant was discarded, and the mixture was allowed to stand at room temperature for 5–10 min to air dry. Note that the precipitate should not be allowed to dry completely, as this would affect RNA dissolution. 100 µL of RNase-free water was added. For better dissolution of RNA, water can be used, and the solution can be heated in a metal bath at 55°C for 10 minutes to fully dissolve it.

[0040] (2) cDNA synthesis First-strand cDNA was synthesized in vitro using the Toyobo Reverse Transcription Kit ReverTra Ace qPCR RT with gDNA Remover Kit (TOYOBO, FSQ-301). The specific steps were as follows: 1 μg RNA was added to a certain amount of RNase-free water to bring the volume to 6 µL, and the mixture was briefly centrifuged; the RNA was denatured at 65°C for 5 min, and then immediately placed on ice; 2 µL of 4 × DN Master Mix (gDNA Remover was added at a 1:50 ratio and mixed before use) was added, and the mixture was gently mixed and briefly centrifuged; the mixture was reacted at 37°C for 5 min to remove residual genomic DNA, and then placed on ice; 2 µL of 5 × RT Master Mix II was added, and the mixture was gently mixed and briefly centrifuged; the mixture was reacted at 37°C for 15 min; at 50°C for 5 min; at 98°C for 5 min; and then incubated at 4°C.

[0041] (3) Construction of SPX4 overexpression vector Total RNA was extracted from the japonica rice variety ZH11 and reverse transcribed into cDNA. Using the cDNA as a template, the CDS region of OsSPX4 was amplified using the SPX4-OE-F / SPX4-OE-R primer combination. The primer sequences are as follows: SPX4-OE-F: CACGGGGGACTCTAGAATGAAATTCGGGAAGGATTTC SPX4-OE-R: TGTAGTCCATGTCGACGCCGCCGCTGCCGCCGCCTTCATCACGTGGCTGGCC The underlined sequences in the above SPX4-OE-F / SPX4-OE-R primer sequences are the restriction enzyme sites on the vector, namely XbaI (TCTAGA) and SalI (GTCGAC) restriction sites, respectively.

[0042] The amplified sequence was ligated between the XbaI and SalI restriction sites of the vector pCAMBIA1300-221-FLAG to obtain the SPX4 overexpression vector.

[0043] (4) Genetic transformation of rice The recombinant plant expression vector pCAMBIA1300-221-OsSPX4-FLAG was transformed into Agrobacterium AGL1 and then infected the callus tissue of the japonica rice variety ZH11. After transformation and screening, SPX4 overexpressing plants, namely SPX4-OE3 and SPX4-OE6, were obtained.

[0044] For specific transformation and screening methods, please refer to the literature "Yi Zili, Cao Shouyun, Wang Li, He Sijie, Chu Chengcai, Tang Zuoshun, Zhou Puhua, Tian Wenzhong. Study on increasing the frequency of Agrobacterium-mediated transformation of rice. Acta Genetica Sinica, 2001, 28(4):352-358". (5) Real-time quantitative PCR analysis The Toyobo THUNDERBIRD SYBR qPCR Mix (QPS-201) was used. A 20 μl reaction mixture was prepared as follows: 6.5 μl ddH2O, 10 μl 2×SYBR qPCR Mix, 3 μl Primers (5 μM), and 0.5 μl cDNA. PCR was performed using a Bio-Rad CFX96. The reaction procedure followed the manufacturer's instructions, employing a two-step PCR method. Melting curve analysis was performed according to the CFX96 standard settings. Data processing was performed using the Comparative CT method (Schmittgen and Livak, 2008; Bala et al., 2020). OsUBQ1 was used as the primer for quantitative PCR internal control, and each gene was amplified in triplicate. The primers used for quantitative PCR are shown in Table 1.

[0045] Table 1 Primer List Primer name Forward sequence (5'-3') Reverse sequence (5'-3') OsUBQ1 AACCAGCTGAGGCCCAAGA ACGATTGATTTAACCAGTCCATGA NRT1.1B GGCAGGCTCGACTACTTCTA AGGCGCTTCTCCTTGTAGAC SPX4 ATCGAGAGGGAGGAGTGGTA AGGGAGCTATAGGTTTGCAG OsNCED4 AATGTCGTGTCCAAGCCGTA GACGGCATAGTTCTCGGTGA OsPSY3 CAATGGACGAGCTCGAGATG GTCGGTACAGAAGTAGAGAA OsPM1 TCTACGTCGCCATGCTCCAC GGTGTGTCGGGGTCAAATTC OsDREB1C CATGATGATGCAGTACCAGGA GATCATCAGTAGCTCCAGAGTG OsbHLH120 CCTTACCCATGCAGCACCAC CTCATCACCATCTATCCATG OsNAC52 GTACTGGGGCAAGCCGTTC CATTCCATCTCCCATCTGAC OsHsfC2a GGTGGACAGCGGCTACTAAA AAACACTTTGGGGAGGGCAA OsNAC6 TAGCCCAGTGGTTGAAGTGT CACGCTTTTTCCTCGCATGG In summary, by obtaining SPX4-overexpressing plants, RT-qPCR analysis revealed that, as Figure 2 As shown, under ABA treatment, compared with the wild type, SPX4 overexpression inhibits the expression of multiple stress response genes, including ABA synthesis and transport-related genes OsNCED4, OsPSY3, and OsPM1, development-related genes DREB1C and OsbHLH120, stress-related genes OsNAC52 and OsHsfC2a, and glucosinolate metabolism-related gene OsNAC6, suggesting that it plays a negative regulatory role in the stress response process.

[0046] References: Bala, M., Sinha, R., Mallick, MA, Sharma, TR, and Singh, AK (2020). Methods of gene expression profiling to understand abiotic stressperception and response in legume crops. In Legume Genomics: Methods andProtocols, M. Jain, and R. Garg, eds. (New York, NY: Springer US), pp. 99-126. Schmittgen, TD, and Livak, KJ (2008). Analyzing real-time PCRdata by the comparative C(T) method. Nat. Protoc. 3, 1101-1108 Example 3: Plants overexpressing SPX4 showed insensitivity to ABA treatment Through experimental methods in this field, we obtained Figure 3 The results shown, where for Figure 3 The experiments involved are only briefly described. Those skilled in the art can operate the corresponding experiments based on the experiment names, so they will not be described in detail here.

[0047] By obtaining SPX4-overexpressing plants (the method used in Example 2 is not repeated here), the phenotype and root length of the SPX4-overexpressing plants were statistically analyzed under different concentrations of ABA treatment. The results are as follows: Figure 3 As shown, A. Phenotypes of SPX4-OE plants under different concentrations of ABA treatment.

[0048] B. Statistical results of root length of SPX4-OE plants under different concentrations of ABA treatment.

[0049] In summary, in root growth experiments with different concentrations of ABA, the root growth of SPX4-OE plants was significantly inhibited by ABA to a lesser extent than that of wild-type plants, exhibiting an "insensitive" phenotype to ABA. This suggests that SPX4 negatively regulates the ABA response in plants.

[0050] Example 4: Phenotype and survival rate of SPX4-overexpressing plants before and after PEG-simulated drought treatment Through experimental methods in this field, we obtained Figure 4 The results shown, where for Figure 4 The experiments involved are only briefly described. Those skilled in the art can operate the corresponding experiments based on the experiment names, so only a brief description is given here.

[0051] First, wild-type Zhonghua 11 and SPX4 overexpression rice seeds were surface-sterilized and sown on 96-well perforated PCR plates. Seedlings were allowed to germinate and grow for 10–14 days until they reached the three-leaf stage, i.e., the third leaf was still developing. At the three-leaf stage, the PCR plates containing seedlings were transferred to nitrogen-free Kimura B nutrient solution containing 25% PEG 6000 (w / v) for simulated drought treatment.

[0052] Then, 72 hours after the stress treatment, the PCR plate with seedlings was placed in a tray filled with plenty of double-distilled water, and the rice seedling roots were gently rinsed to remove the PEG adhering to the roots. The PCR plate with seedlings was then transferred to a standard Kimura B nutrient solution, and the seedlings were allowed to recover for 5-7 days. The survival rate after PEG-induced drought was assessed, and the results were photographed and recorded.

[0053] SPX4-overexpressing plants were obtained using the method described in Example 2, which will not be repeated here. PEG6000 was used to simulate drought stress during treatment. Figure 4 The results showed that A. Phenotypic changes of SPX4-OE plants before and after PEG-simulated drought treatment.

[0054] During recovery after PEG-simulated drought treatment, SPX4-OE plants showed more severe wilting, yellowing, and drying compared to wild-type Zhonghua 11.

[0055] B, Statistical results of plant survival rate during recovery after PEG treatment.

[0056] Under PEG-simulated drought treatment, the survival rate of SPX4-OE plants was significantly lower than that of wild-type Zhonghua 11.

[0057] In summary, under PEG 6000 simulated drought treatment, SPX4-overexpressing plants showed drought sensitivity, further demonstrating the negative regulatory capacity of SPX4 in the drought response process.

[0058] Example 5: Phenotypic characteristics and yield per plant of SPX4-overexpressing plants under low-nitrogen or high-nitrogen drought field conditions. Through experimental methods in this field, we obtained Figure 5 The results shown, where for Figure 5 The experiments involved are only briefly described. Those skilled in the art can operate the corresponding experiments based on the experiment names, so they will not be described in detail here.

[0059] SPX4 overexpressing plants were obtained using the method described in Example 2, which will not be repeated here. Under field conditions, different nitrogen levels were set and drought treatment was applied. Figure 5 The results showed that, under low or high nitrogen conditions, compared with the control group wild-type Zhonghua 11, SPX4-OE plants in arid land exhibited dwarf phenotypes with fewer grains per spike. Under low or high nitrogen conditions, the yield per plant of SPX4-OE plants in arid land was significantly lower than that of the control group wild-type.

[0060] In summary, in the field, under drought conditions with low or high nitrogen, SPX4-OE plants showed a significant decrease in yield compared to the wild type, especially under low nitrogen conditions, where the yield loss was particularly significant, demonstrating that it participates in the plant stress tolerance process as a negative regulatory component.

[0061] In practical applications, adjustments can be made according to the actual situation. By knocking out the SPX4 gene, a large number of positive regulatory factors are released into the cell, activating the stress response and improving the plant's tolerance to stress. Alternatively, gene expression can be moderately knocked down to coordinate the balance between plant stress resistance and growth.

[0062] The CDS sequence of SPX4 mentioned above is shown in SEQ ID NO.1, which is as follows: ATGAAATTCGGGAAGGATTTCAGGAGCCACCTGGAGGAGACGCTGCCGGCGTGGAGGGACAAGTATCTGGCGTACAAGTCCCTCAAGAAGCTAATCAAGAACCTGCCCCCCGATGGAGATCCCCCTCCCGTCGCCGCGGCGGCGGAGGTGCCTGCGGGAGATGGGGATGGGGATGGGGGCATCGCCCTGGGGAACTGGTTCGCCAGGGTCCTCGACATGGAGCTCCAGAAGCTCAACGACTTCTACATCGAGAGGGAGGAGTGGTACGTCATCCGCCTCCAGGTGCTCAAAGAAAGAATTGAACGTGTCAAAGCTAAGAAGAATGGGGCTTTTACATCTAAGAGCGAATTCACTGAAGAGATGTTAGAGATACGCAAAGCTTTTGTCATCATCCATGGGGAGATGATTCTTCTGCAAACCTATAGCTCCCTAAATTTTGCCGGACTTGTGAAGATACTGAAAAAATATGACAAGAGAACAGGTGGTTTGCTCAGCCTACCTTTCACTCAACGTGCTCGACACCAACCATTTTTCACAACGGAACCTTTAACAAGGCTTGTTCGAGAATGTGAGGCTAATCTTGAGCTCCTGTTTCCTATCGAAGCAGAAGTACTTGAGTCTGCTAGCTCCTCTGCTAAGTTGCAACCTCAGAATGATGATGCGGCTAGCCATGACCCAGCGTCATCTGTCGATGTTGAAACCTCGGACGTGTACCGAAGCACACTCGCGGCAATGAAGGCGATTCAGGGCCTCCGCAAAGCCAGCTCTACTTACAATCCCTTGTCGCTCGCCAGGTTCTTCCATGGAGAGGACGGTGAAGCCTGCTCTGGAGCCATCACCTCTGAAAGTGATTCCTATTCTGATTCCCAGATTGAAGATGCTGAAGATGATGATAAAGAGGTGCAATCAAGGGAGCAGAATACGGCTCAAAATGCTGCAGAAGGCCAGCCACGTGATGAATGA The sequence of the protein encoded by SPX4 mentioned above is shown in SEQ ID NO.2, which is as follows: MKFGKDFRSHLEETLPAWRDKYLAYKSLKKLIKNLPPDGDPPPVAAAAEVPAGDGDGDGGIALGNWFARVLDMELQKLNDFYIEREEWYVIRLQVLKERIERVKAKKNGAFTSKSEFTEEMLEIRKAFVIIHGEMILLQTYSSLNFAGLVKILKKYDKRT GGLLSLPFTQRARHQPFFTTEPLTRLVRECEANLELLFPIEAEVLESASSSAKLQPQNDDAASHDPASSVDVETSDVYRSTLAAMKAIQGLRKASSTYNPLSLARFFHGEDGEACSGAITSESDSYSDSQIEDAEDDDKEVQSREQNTAQNAAEGQPRDE* The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. An SPX4 gene, characterized in that, The CDS sequence of SPX4 is shown in SEQ ID NO.1, and the sequence of the protein encoded by SPX4 is shown in SEQ ID NO.

2.

2. The application of the SPX4 gene in the negative regulation of plant drought tolerance, characterized by: The applications include achieving negative regulation of plant drought tolerance by obtaining SPX4-overexpressing plants.

3. The application of the SPX4 gene in the negative regulation of plant drought tolerance according to claim 2, characterized in that, The SPX4 overexpressing plants were obtained through the following steps: The SPX4 gene, as shown in SEQ ID NO.1, was cloned, and the SPX4 gene was introduced into an expression vector to construct a recombinant expression vector. The recombinant expression vector was introduced into the rice recipient variety Zhonghua 11, and transgenic rice plants overexpressing SPX4 were obtained after screening and cultivation.

4. The application of the SPX4 gene in the negative regulation of plant drought tolerance according to claim 3, characterized in that, The expression vector is pCAMBIA1300-221-FLAG.

5. The application of the SPX4 gene in the negative regulation of plant drought tolerance according to claim 2, characterized in that, The negative regulation of plant drought resistance was achieved using subcellular localization analysis of rice protoplasts. Under ABA induction, SPX4 caused transcription factors to remain in the cytoplasm.

6. The application of the SPX4 gene in the negative regulation of plant drought tolerance according to claim 2, characterized in that, The negative regulation of plant drought tolerance was achieved by obtaining SPX4 overexpressing plants, which, under ABA treatment, suppressed the expression of stress response genes OsNCED4, OsPSY3, OsPM1, DREB1C, OsbHLH120, OsNAC52, OsHsfC2a and OsNAC6.

7. The application of the SPX4 gene in the negative regulation of plant drought tolerance according to claim 2, characterized in that, The negative regulation of plant drought tolerance was achieved by obtaining SPX4-overexpressing plants, whose root growth was sluggish under ABA treatment.

8. The application of the SPX4 gene in the negative regulation of plant drought tolerance according to claim 2, characterized in that, The negative regulation of plant drought tolerance was achieved by obtaining SPX4-overexpressing plants, whose survival rate decreased after PEG-simulated drought treatment.

9. The application of the SPX4 gene in the negative regulation of plant drought tolerance according to claim 2, characterized in that, The negative regulation of plant drought tolerance is achieved by obtaining SPX4 overexpressing plants, which exhibit reduced yield under low-nitrogen or high-nitrogen drought field conditions.

10. The application of the SPX4 gene according to any one of claims 2-9 in the negative regulation of plant drought tolerance, characterized in that, The CDS sequence of SPX4 is shown in SEQ ID NO.1, and the sequence of the protein encoded by SPX4 is shown in SEQ ID NO.2.

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