Sugarcane ScPP2C49 gene and application thereof

Abstract

The invention discloses a sugarcane ScPP2C49 gene and application thereof, and relates to the technical field of plant biology, the nucleotide sequence of the ScPP2C49 gene is as shown in SEQ ID No.1, and the amino acid sequence of protein coded by the ScPP2C49 gene is as shown in SEQ ID No.2. The gene is induced to express by drought and abscisic acid, and the encoded protein of the gene is positioned in a cell nucleus. Functional studies show that after overexpression of the ScPP2C49 gene in the plant, the drought resistance of the plant is negatively regulated through various mechanisms of inhibiting an abscisic acid signal channel, hindering stomatal closure under drought stress, weakening active oxygen scavenging ability, reducing photosynthetic efficiency, inhibiting root growth and the like. The invention also provides a recombinant overexpression vector containing the gene and a method for obtaining a transgenic plant with reduced drought resistance by using the vector through an agrobacterium-mediated method. According to the invention, a new gene resource is provided for deep analysis of a plant drought-resistant molecular mechanism, and an important target gene is provided for cultivation of high-stress-resistance crop varieties through a reverse genetics means (such as gene knockout).

Description

Technical Field

[0001] This invention belongs to the field of plant molecular biology, and particularly relates to a sugarcane ScPP2C49 Genes and their applications. Background Technology

[0002] In recent years, global warming has intensified the terrestrial water cycle, leading to a significant increase in the frequency and severity of drought events in many regions. Drought stress has become one of the major abiotic stress factors limiting crop growth and affecting global agricultural productivity. Sugarcane, as the most important sugar crop in my country and even the world, accounts for approximately 75% of global sugar production, playing a crucial role in the agricultural economy and food industry. In my country, sugarcane is mainly grown on hilly, dry slopes. These areas often lack adequate irrigation facilities, and coupled with seasonally uneven rainfall distribution, frequent soil droughts directly result in a significant decline in sugarcane yield and sugar content in sugarcane stalks, becoming a key bottleneck restricting the sustainable development of my country's sugar industry. Therefore, in-depth exploration of sugarcane's drought-resistant genetic resources, identification of key drought-resistant functional genes, and analysis of their molecular regulatory mechanisms are of great theoretical and practical significance for accelerating the breeding of high-yield, drought-resistant new sugarcane varieties using molecular breeding techniques.

[0003] Plant drought resistance is a complex physiological process regulated by multiple genes. Among these, the plant hormone abscisic acid (ABA) plays a central role in responding to drought stress. When plants sense water deficiency, ABA levels rapidly accumulate, enhancing drought resistance through a series of physiological and molecular responses, such as promoting stomatal closure to reduce water transpiration loss, regulating root architecture to enhance water absorption, and activating the expression network of specific stress-responsive genes. In model plants Arabidopsis thaliana and rice, the ABA signal transduction pathway has been relatively well elucidated, with a conserved ABA-PYR / PYL / RCAR-PP2C-SnRK2 signal transduction chain at its core. In this pathway, PYR / PYL / RCAR family proteins act as ABA receptors, while PP2C protein phosphatase and SnRK2 protein kinase constitute the core signal switches. Under normal growth conditions, the ABA level in plants is low. PP2C binds to SnRK2 and inhibits its kinase activity, thereby blocking downstream stress responses. When encountering adverse conditions such as drought, ABA accumulates in large quantities and binds to receptors, thereby inhibiting the activity of PP2C, releasing and activating SnRK2, and ultimately initiating the expression of a series of ABA response genes to help plants adapt to the stress environment.

[0004] Protein phosphatase 2C (PP2C), a key negative regulator of the ABA signaling pathway, has formed a large gene family in plants. For example, 80, 78, 181, 78, and 89 PP2C family members have been identified in Arabidopsis thaliana, rice, upland cotton, potato, and *Brachys edulis*, a close wild relative of sugarcane, respectively. Their expansion patterns differ among species; in potato, tandem replication is predominant, while in other species, whole-genome or fragment replication is more common. Numerous studies have shown that PP2C genes play multiple roles in plant drought resistance. Members of the Arabidopsis A subfamily, such as ABI1, ABI2, PP2CA, and HAB1, have been confirmed as negative regulators of ABA signaling, and their loss-of-function mutants exhibit enhanced tolerance to drought and salt stress. Conversely, some studies have shown that certain PP2C members have positive regulatory effects; for example, overexpression of *Brachys bipinnatus* BdPP2CA6 in Arabidopsis promotes stomatal closure and enhances salt tolerance. In rice, OsPP108 is induced by ABA and salt stress, and its overexpression enhances the drought and salt tolerance of Arabidopsis. Furthermore, while overexpression of wheat TaPP2C1 in tobacco reduces the plant's sensitivity to ABA, it confers salt tolerance by activating the antioxidant system. These studies suggest that the PP2C gene family has complex and diverse functions, and its specific roles may vary depending on the gene, species, and type of stress.

[0005] With the successive publication of the genomes of the wild sugarcane species *Gesoumi* and the cultivated variety *Zhe 1*, our understanding of the origin, evolution, and genetic basis of important agronomic traits in sugarcane hybrids has deepened, providing valuable genomic resources for molecular design breeding of sugarcane. Although the functions of the PP2C gene family in species such as *Arabidopsis thaliana* and rice have been extensively studied, confirming their important roles in plant growth, development, and abiotic stress responses, and their potential as candidate genes for improving crop resistance, systematic research and functional analysis of PP2C genes in sugarcane, an important sugar crop, remain quite limited. Currently, we know very little about the expression characteristics of sugarcane PP2C gene family members and their specific functions in the ABA signaling pathway and drought response, which greatly restricts our progress in using this gene family for genetic improvement of sugarcane drought resistance. Therefore, identifying and elucidating the functions of key PP2C genes in sugarcane is of urgent need and significant value for revealing the molecular mechanisms of sugarcane drought resistance and discovering new breeding targets. Summary of the Invention

[0006] To solve the above-mentioned technical problems, the present invention proposes a sugarcane ScPP2C49 The gene and its application: This gene can negatively regulate the drought resistance of plants by inhibiting stomatal closure, weakening the ability to scavenge reactive oxygen species, and affecting plant photosynthesis.

[0007] To achieve the above objectives, the present invention provides a sugarcane ScPP2C49 Genes, the sugarcane ScPP2C49 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0008] The present invention also provides a sugarcane derived from the above-mentioned sugarcane ScPP2C49 The ScPP2C49 protein is encoded by a gene, and the amino acid sequence of the ScPP2C49 protein is shown in SEQ ID No. 2.

[0009] The present invention also provides a sugarcane as described above. ScPP2C49 Application of the gene or the aforementioned ScPP2C49 protein in the negative regulation of plant drought resistance.

[0010] Furthermore, the negative regulation of plant drought resistance is achieved by inhibiting plant root elongation, inhibiting stomatal closure under drought stress, or inhibiting the abscisic acid signaling pathway.

[0011] Furthermore, the plants include Arabidopsis thaliana and / or sugarcane.

[0012] The present invention also provides a method for cultivating sugarcane with stronger drought resistance, including the step of reducing the expression level of ScPP2C49 protein in recipient sugarcane; wherein the ScPP2C49 protein is a protein with an amino acid sequence as shown in SEQ ID No. 2.

[0013] Further, the method includes the following steps: suppressing the expression of the gene encoding the ScPP2C49 protein in the recipient sugarcane to obtain transgenic sugarcane; the transgenic sugarcane exhibits stronger drought resistance compared to the recipient sugarcane.

[0014] The present invention also provides a method for cultivating sugarcane with weaker drought resistance, including the step of increasing the expression level of ScPP2C49 protein in recipient sugarcane; wherein the ScPP2C49 protein is a protein with an amino acid sequence as shown in SEQ ID No. 2.

[0015] Further, the method includes the following steps: overexpressing the gene encoding the ScPP2C49 protein in the recipient sugarcane to obtain transgenic sugarcane; the transgenic sugarcane is less drought resistant than the recipient sugarcane.

[0016] The present invention also provides the above-mentioned sugarcane ScPP2C49 The application of the gene or the aforementioned ScPP2C49 protein in sugarcane breeding, wherein the sugarcane ScPP2C49 Genes are used to regulate drought resistance traits in sugarcane; by using sugarcane ScPP2C49 Transforming sugarcane plants with gene overexpression vectors yields transgenic sugarcane with weakened drought resistance; or by transforming sugarcane... ScPP2C49Gene knockout vectors were used to transform sugarcane plants, resulting in transgenic sugarcane with enhanced drought resistance.

[0017] Compared with the prior art, the present invention has the following advantages and technical effects: This invention is the first to clone and functionally identify [a specific type of substance] from sugarcane. ScPP2C49 This gene, induced by drought and ABA, encodes a protein located in the cell nucleus. Heterologous overexpression of this gene in Arabidopsis thaliana inhibits the ABA signaling pathway, leading to a series of negative physiological effects, including impaired stomatal closure, accelerated water loss, reduced reactive oxygen species scavenging capacity, impaired photosynthetic function, and inhibited root growth, thus significantly reducing the plant's drought resistance. Therefore, ScPP2C49 This gene is a key regulator of drought resistance in plants. This discovery not only enriches our understanding of the molecular network of ABA signal transduction and drought response in sugarcane and even monocotyledonous plants, but also provides important candidate targets and theoretical basis for breeding new drought-resistant sugarcane varieties through gene editing strategies (such as CRISPR / Cas9 knockout or inhibition of this gene expression). Attached Figure Description

[0018] Figure 1 for ScPP2C49 Analysis of tissue expression patterns and expression patterns after PEG and ABA treatment; Figure A represents... ScPP2C49 Gene expression levels in roots, stems, and leaves, as shown in Figures B and C respectively. ScPP2C49 Gene expression levels induced by PEG and ABA at different time points; Figure 2 Subcellular localization of ScPP2C49 protein; Figure A shows that ScPP2C49 protein is localized in the cell nucleus, with RFP as the nuclear localization marker; Figure B shows the grayscale analysis of fluorescence intensity in Figure A using ImageJ software; Figure 3 for ScPP2C49 Seed germination of transgenic Arabidopsis thaliana under different concentrations of mannitol stress; Figure A shows the RT-qPCR experiment detecting mannitol in transgenic Arabidopsis thaliana. ScPP2C49 The expression levels, Figure B represents wild type and ScPP2C49 The germination phenology of transgenic Arabidopsis seeds after 6 days of growth in media containing 0, 200, and 300 mM mannitol. Figure C is a statistical representation of the germination rate in Figure B. Figure 4 for ScPP2C49 The root length of the main root in transgenic Arabidopsis thaliana under different concentrations of mannitol stress; where the AC diagram represents the wild type and... ScPP2C49 The phenotype of the main root of transgenic Arabidopsis thaliana on medium containing 0, 200, and 300 mM mannitol. Figure D shows the statistics of main root length. Figure 5 for ScPP2C49 Heterologous overexpression of genes regulates the phenotype of drought resistance in plants; Figure A represents the wild type and... ScPP2C49 Phenotypes of transgenic Arabidopsis thaliana before and after drought treatment. Figure B represents the wild type and... ScPP2C49 Root length phenotypes of transgenic Arabidopsis thaliana before and after drought treatment; Figure C shows wild type and... ScPP2C49 Fresh weight statistics of transgenic Arabidopsis before and after drought treatment; Figure D is the root length statistics in Figure B. Figure 6 for ScPP2C49 Physiological parameters and DAB determination in transgenic Arabidopsis thaliana; Figures A and B represent wild-type and... ScPP2C49 Relative conductivity and POD activity of transgenic Arabidopsis thaliana before and after drought treatment; Figure C shows the results under normal conditions and drought treatment for wild-type and transgenic Arabidopsis thaliana. ScPP2C49 DAB staining of transgenic Arabidopsis leaves; Figure 7 for ScPP2C49 Water loss rate and stomata in transgenic Arabidopsis thaliana; Figure A represents wild-type and... ScPP2C49 Statistics on aboveground water loss rate of transgenic Arabidopsis thaliana; Figures B and C represent the wild-type and [other species] under normal conditions and drought treatment, respectively. ScPP2C49 Measurement of stomatal phenotype and pore size ratio in transgenic Arabidopsis leaves; Figure 8 for ScPP2C49 Analysis of chlorophyll fluorescence parameters in transgenic Arabidopsis thaliana before and after drought; Figure A represents wild-type and... ScPP2C49 Chlorophyll imaging of transgenic Arabidopsis thaliana; BE diagrams represent... ScPP2C49 Fv / Fm, NPQ, qp, and Rfd in transgenic Arabidopsis thaliana under normal growth and drought stress; Figure 9 for ScPP2C49 Seed germination of transgenic Arabidopsis thaliana under ABA treatment; Figure A represents wild-type and... ScPP2C49 Germination phenology of transgenic Arabidopsis seeds at ABA concentrations of 0, 0.5, and 1 μM. Figure B is a statistical representation of the germination rate in Figure A. Figure 10 for ScPP2C49 Root length of transgenic Arabidopsis thaliana under ABA treatment; where the AC diagram represents... ScPP2C49 Root length phenotypes of transgenic Arabidopsis thaliana at ABA concentrations of 0, 0.5, and 1 μM. Figure D shows the statistical data on root length. Figure 11 Figure A shows the RT-qPCR results for genes in the ABA signaling pathway. AtABI2 Gene, Figure B is AtHAI1 Gene, Figure C is AtHAB2 Gene, D diagram is AtSnRK2.4 Gene. Detailed Implementation

[0019] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0020] All raw materials used in this invention are not particularly restricted in their source; they can be purchased from the market or prepared using conventional methods known to those skilled in the art.

[0021] There are no particular restrictions on the purity of any of the raw materials used in this invention. However, this invention preferably uses raw materials of analytical grade or purity commonly used in the field of chemical synthesis.

[0022] This invention provides a method derived from sugarcane ( Saccharum The gene of the drought-resistant cultivar 'Zhongzhe No. 1' (spp.) was named ScPP2C49 It encodes a protein of the protein phosphatase 2C (PP2C) family. The open reading frame (ORF) nucleotide sequence of this gene is shown in SEQ ID No. 1, and the amino acid sequence of the protein it encodes is shown in SEQ ID No. 2.

[0023] SEQ ID No. 1: SEQ ID No. 2: MGAGAEVMHQVVPLLEPPFHRCVVKSVDVMEEVVAVAPGQVQPATSPKAVVEVAVEVPDLEFKRASNAGSSVSAEQLQFVPSIRSGSFADIGPRRFMEDEHIRIDDLSGHLGSLLMVSAPSAFYGVFDGHGGSDAVAYMKTHAMRLFFEDADFPQASQEDEIFAESVEESVRKAFLRADLALADDSVINRSSGTTALV LGRQLLVANAGDCRAVLCRKGIAVEMSKDHRPTYDAERQRVIECGGYIEDGYLNGVLSVTRALGDWDMKLPQGLPSPLIAEPEIHWTTLMEDDEFLIIGCDGIWDVMSSQHAVSMVRKGLRRHDDPERCARELAMEAKRLKTFDNLTVIIVCFVPDLAGASAAAPSSEQAPAPAQAGRIRCCKSLSPEALCKLRRWLESDH This invention also provides the sugarcane gene described in the above technical solution. ScPP2C49 Alternatively, the application of the proteins described in the above-mentioned technical solutions in reducing plant drought resistance. In this invention, the plant preferably includes Arabidopsis thaliana and / or sugarcane. This invention preferably reduces plant drought resistance by inhibiting root elongation. This invention preferably reduces plant drought resistance by inhibiting stomatal closure. This invention preferably reduces plant drought resistance by inhibiting the abscisic acid signaling pathway.

[0024] This invention provides an overexpression vector, comprising the sugarcane gene described in the above technical solution. ScPP2C49 The sugarcane gene was obtained by insertion into the pGWB418 vector. This invention relates to the sugarcane gene... ScPP2C49 There are no special limitations on the method of insertion into the pGWB418 vector; those skilled in the art can follow conventional methods.

[0025] This invention also provides a method for obtaining drought-resistant plants, comprising the following steps: 1) The overexpression vector described in the above technical solution is transformed into Agrobacterium to obtain the transformed bacteria; 2) The transforming bacteria obtained in step 1) are transformed into plants by the flower dipping method to obtain drought-resistant plants.

[0026] This invention does not specifically limit the method for transforming the overexpression vector into Agrobacterium; those skilled in the art can use conventional methods. In this invention, Agrobacterium is preferably Agrobacterium GV3101.

[0027] This invention does not specifically limit the method of transforming the transforming bacteria into plants via the flower-dipping method; conventional methods can be used by those skilled in the art. Preferably, this invention uses conventional culture methods after transformation to obtain drought-resistant plants. In this invention, the plants preferably include Arabidopsis thaliana and / or sugarcane.

[0028] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0029] Unless otherwise specified, all experimental materials, reagents, and consumables used in the following examples are commercially available. The primer sequences used in the examples are shown in Table 1.

[0030] Example 1: Sugarcane ScPP2C49 Gene expression pattern analysis and subcellular localization To gain a deeper understanding ScPP2C49 The function of the gene was first analyzed, including its basal expression pattern in different sugarcane tissues and its induced expression pattern under simulated drought stress (PEG treatment) and abscisic acid (ABA) treatment. Simultaneously, the intracellular localization of the ScPP2C49 protein was investigated.

[0031] 1. ScPP2C49 Tissue-specific and stress-induced expression analysis Root, stem, and leaf tissues were collected from 'Zhongzhe No. 1' sugarcane seedlings. The seedlings were treated with either 20% PEG-6000 solution (simulating drought) or 100 μM ABA solution, and samples were taken at 0, 1, 3, 6, and 12 hours after treatment. Total RNA was extracted from each sample using TRIzol reagent. Genomic DNA was digested with DNase I, and then cDNA first strand was synthesized by reverse transcription.

[0032] according to ScPP2C49 Gene sequence, and specific quantitative primers designed using Primer Premier 6.0 software ( ScPP2C49 -qPCR-F / R, sequence shown in Table 1). Sugarcane housekeeping gene. ScGAPDH (Primer sequences are shown in Table 1) As internal controls, real-time quantitative PCR (RT-qPCR) analysis was performed using SYBR Green Master Mix on a quantitative PCR instrument. Three technical replicates were set up for each sample, and the relative expression level of the gene was calculated using the 2^(-ΔΔCt) method.

[0033] The results are as follows Figure 1 As shown. Figure 1 A shows, ScPP2C49The gene is expressed in the root, stem, and leaf tissues of sugarcane, with the highest expression level in the leaves, suggesting that the gene may mainly function in the leaf tissues. Figure 1 B shows that after PEG treatment... ScPP2C49 The expression level of the gene was rapidly upregulated, reaching a peak after 1 hour of treatment. Although it subsequently declined, it was still significantly higher than that of the untreated control, indicating that the gene was strongly and rapidly induced by drought stress. Figure 1 C shows that ABA treatment can also induce... ScPP2C49 Expression was upregulated, reaching its peak at 3 hours of treatment, and then decreased at 12 hours, indicating that this gene is also involved in the ABA-mediated signaling pathway response.

[0034] 2. Subcellular localization of ScPP2C49 protein To clarify the functional sites of the ScPP2C49 protein in cells, subcellular localization analysis was performed.

[0035] First, using 'Zhongzhe No. 1' cDNA as a template, specific primers with homologous recombination adapters were used. ScPP2C49 -pGDG-F / R amplification ScPP2C49 The complete ORF sequence of the gene (excluding the stop codon) was obtained. The PCR product was inserted into the plant transient expression vector pGDG using homologous recombination cloning technology. This vector carries the CaMV 35S strong promoter and a green fluorescent protein (GFP) tag, thus constructing the 35S-ScPP2C49-GFP fusion expression vector.

[0036] The recombinant plasmid was introduced into *Agrobacterium tumefaciens* strain GV3101 via electroporation. Single colonies were picked and expanded, and the cells were collected and resuspended in infection buffer (10 mM MES, 10 mM MgCl2, 150 μM acetylsylcholine, pH 5.6). Simultaneously, *Agrobacterium* culture containing only the empty vector 35S-GFP was prepared as a control. *Nicotiana benthamiana* (Tobacco Benzoinum) was used as a control. Nicotiana benthamiana Approximately 4 weeks after seedling planting, the above-mentioned Agrobacterium tumefaciens bacterial solution (OD200) was injected using a sterile syringe. 600 ≈0.8 g of the solution was injected under the epidermal cells on the underside of healthy tobacco leaves. To ensure accurate localization, Agrobacterium-mediated transformation (Agrobacterium) containing the nuclear localization marker protein AtHY5-RFP was injected along with the 35S-ScPP2C49-GFP bacterial solution. The injected tobacco plants were then cultured in a light incubator for 48-72 hours after injection.

[0037] A sample of the lower epidermis of the leaf from the injection site was placed on a confocal microscope slide for observation. The results of the laser confocal microscope observation are as follows: Figure 2 As shown. Figure 2As shown in Figure A, in control cells expressing only the empty GFP vector, the GFP fluorescence signal was distributed throughout the entire cell, including the nucleus and cytoplasm. However, in cells co-expressing ScPP2C49-GFP and AtHY5-RFP, the green fluorescence (ScPP2C49-GFP) and red fluorescence (AtHY5-RFP, nuclear labeling) signals highly overlapped, and the fluorescence signal was mainly concentrated in the nuclear region. Figure 2 B shows the colocalization grayscale analysis of the fluorescence signal in the selected region of Figure A using ImageJ software. The curves show a very high degree of agreement between the peak positions of GFP and RFP signals. These results collectively demonstrate that the ScPP2C49 protein is a nucleus-localized protein, consistent with its hypothetical function as a transcriptional regulation-related phosphatase or involvement in nuclear signal transduction.

[0038] Example 2: Overexpression ScPP2C49 Reduced tolerance of Arabidopsis thaliana to drought stress To verify ScPP2C49 The function of the gene in plant drought resistance was investigated. A plant overexpression vector of the gene was constructed and heterologously overexpressed in the model plant Arabidopsis thaliana. The phenotype of the transgenic plants under simulated drought conditions was systematically analyzed.

[0039] 1. ScPP2C49 Obtaining Arabidopsis thaliana overexpression lines Using primers ScPP2C49 -418-F / R, amplified from sugarcane cDNA by PCR ScPP2C49 The full-length CDS sequence was obtained. The purified PCR product was cloned into the entry vector pDONR207 via Gateway BP reaction, and then recombined into the plant binary overexpression vector pGWB418 via LR reaction. This vector carries the CaMV 35S promoter and a hygromycin resistance selection marker, and finally the recombinant plasmid 35S-ScPP2C49-pGWB418 was obtained.

[0040] The recombinant plasmid was transformed into Agrobacterium tumefaciens strain GV3101 using a freeze-thaw method. The plasmid was then transformed into Agrobacterium-mediated inflorescence infection. ScPP2C49 Genes were introduced into wild-type Arabidopsis thaliana of the Columbia ecotype (Col-0). Transformed seeds (T0 generation) were harvested and subjected to resistance selection on 1 / 2 MS solid medium containing 25 μg / mL hygromycin. Surviving seedlings were transplanted into soil, and T1 generation seeds were harvested. Homozygous T2 or T3 overexpression lines were obtained through hygromycin selection and molecular identification.

[0041] Total RNA was extracted from various transgenic lines and wild-type (WT) Arabidopsis seedlings, reverse transcribed into cDNA, and then analyzed using transgenic RNA extraction methods. ScPP2C49 Gene-specific primers ( ScPP2C49 -qPCR-F / R) and Arabidopsis thaliana internal reference gene AtACTIN2 Primers were used for RT-qPCR detection.

[0042] The results are as follows Figure 3 As shown in A, no detection was found in the wild-type (WT) and one transgenic line (OE-5). ScPP2C49 The gene was expressed, and high-level expression of this gene was detected in multiple independent transgenic lines (OE-1, OE-2, OE-3, OE-4, OE-6). The two lines with the highest expression levels, OE-1 and OE-2, were selected for all subsequent functional analysis experiments.

[0043] 2. Overexpression ScPP2C49 Inhibiting seed germination of Arabidopsis thaliana under mannitol stress Mannitol is a commonly used osmotic regulator that can simulate drought stress by increasing the osmotic pressure of the culture medium. To evaluate overexpression... ScPP2C49 To investigate the effect of mannitol stress on drought tolerance during the germination stage of Arabidopsis thaliana seeds, a germination experiment was conducted.

[0044] After surface sterilization, seeds of the WT, OE-1, and OE-2 lines were evenly sown on 1 / 2 MS solid medium plates containing 0 (control), 200 mM, and 300 mM mannitol. The plates were vernalized at 4°C for 2 days, then transferred to a light incubator (22°C, 16 h light / 8 h dark) for vertical cultivation. After 6 days of cultivation, the germination rate was calculated (based on the radicle breaking through the seed coat).

[0045] Experimental results are as follows Figure 3 B and Figure 3 As shown in Figure C, on the control medium without mannitol, the seed germination rates of WT and both transgenic lines were close to 100%, with no significant difference. However, on stress media containing 200 mM and 300 mM mannitol, the germination of all seeds was inhibited, and this inhibition was more pronounced in the transgenic lines. Specifically, under 300 mM mannitol stress, the germination rate of WT seeds was approximately 66%, while the germination rates of OE-1 and OE-2 decreased to approximately 57% and 46%, respectively, significantly lower than that of WT. This indicates that overexpression... ScPP2C49 It significantly enhanced the sensitivity of Arabidopsis seeds to germination under osmotic stress, i.e. reduced their drought resistance during the germination period.

[0046] 3. Overexpression ScPP2C49 Inhibiting root growth in Arabidopsis thaliana under mannitol stress The root system is the main organ for plant water absorption, and its growth status directly reflects the plant's ability to adapt to soil drought. Further analysis was conducted on the response of seedling roots to mannitol stress.

[0047] Seeds of WT, OE-1, and OE-2 were sown separately on normal 1 / 2 MS medium and grown vertically for 3 days. Seedlings with uniform growth and straight radicles were selected and transferred to 1 / 2 MS medium containing 0, 200 mM, and 300 mM mannitol, respectively, and continued vertical cultivation for 4 days. Subsequently, the growth phenotype of the seedling taproot was photographed and the taproot length was measured using ImageJ software.

[0048] The results are as follows Figure 4 As shown. Figure 4 AC shows the representative phenotypes of seedlings under different treatments. Figure 4 D represents the statistical analysis of root length. Under no stress conditions, there was no significant difference in taproot length between WT and transgenic lines. Under 200 mM and 300 mM mannitol stress, taproot growth was inhibited in all seedlings. However, the inhibition was more severe in transgenic lines. Under 300 mM mannitol stress, the taproot length of OE-1 and OE-2 decreased by an average of approximately 24% compared to WT, a highly significant difference. These results indicate that overexpression... ScPP2C49 It weakened the root elongation ability of Arabidopsis seedlings under osmotic stress.

[0049] 4. Overexpression ScPP2C49 Weakening the tolerance of Arabidopsis seedlings to soil drought To more closely approximate natural arid conditions, drought treatment experiments were conducted on Arabidopsis seedlings under soil cultivation conditions.

[0050] WT, OE-1, and OE-2 seeds were sown in nutrient soil and cultured for 3 weeks under normal moisture conditions to obtain seedlings with good and uniform growth. Subsequently, the experimental groups were subjected to drought treatment by stopping watering, while the control group continued to be irrigated normally. After 15 days of drought treatment, the overall phenotype and degree of wilting of the plants were observed and recorded, and samples were taken for measurement of relevant indicators.

[0051] The results are as follows Figure 5 As shown. Figure 5 Visually, after 15 days of drought treatment, although the WT plants showed signs of wilting, most of the leaves still retained a certain degree of flexibility; while the two... ScPP2C49 Plants overexpressing the lines (OE-1, OE-2) exhibited more severe wilting, yellowing of leaves, and even overall drying, indicating that they were more severely affected by drought. Figure 5 B and 5D showed that before drought treatment, there was no difference in root length among the different genotypes; after drought treatment, the roots of WT plants continued to grow or maintain a certain degree in order to seek water, while the root growth of transgenic lines completely stopped or even shrank, and their root length was significantly shorter than that of WT. Figure 5Fresh weight statistics for C showed that there was no difference in aboveground fresh weight among the lines before drought treatment; after drought treatment, the fresh weight of all plants decreased, but the fresh weight loss of the transgenic lines was significantly greater than that of the total weight (WT). This comprehensive phenotypic evidence strongly suggests that overexpression... ScPP2C49 It significantly reduced the overall drought resistance of Arabidopsis seedlings.

[0052] Example 3: Overexpression ScPP2C49 Effects on drought resistance-related physiological indicators in Arabidopsis thaliana In order to explain from a physiological perspective ScPP2C49 To investigate the mechanisms by which plants reduce drought resistance, a series of key physiological indicators were measured in transgenic and WT Arabidopsis thaliana plants before and after drought treatment.

[0053] 1. Determination of relative conductivity and peroxidase (POD) activity Relative conductivity reflects the integrity of the cell membrane; increased conductivity under stress indicates cell membrane damage and increased electrolyte leakage. Peroxidase (POD) is one of the key enzymes in the reactive oxygen species scavenging system, and changes in its activity can indicate the plant's oxidative stress level and scavenging capacity.

[0054] Rosette leaves of Arabidopsis thaliana were collected after normal irrigation and 15 days of drought treatment. They were washed with deionized water and the surface moisture was absorbed. For conductivity determination, leaf discs were taken using a perforator, immersed in a certain amount of deionized water, and the initial conductivity (C1) and the final conductivity after boiling (C2) were measured. Relative conductivity (%) = (C1 / C2) × 100%. For POD activity determination, leaves were quick-frozen in liquid nitrogen, ground into powder, and crude enzyme solution was extracted. Enzyme activity was determined using the guaiacol method at a spectrophotometer at a wavelength of 470 nm.

[0055] The results are as follows Figure 6 As shown in A and 6B. Under normal conditions, there were no significant differences in relative conductivity and POD activity between WT and transgenic lines. After drought treatment, the relative conductivity of all plants increased, and POD activity was enhanced, but the increase in relative conductivity of transgenic lines was significantly greater than that of WT. Figure 6 A), while the increase in POD activity was significantly lower than that in WT ( Figure 6 B). This indicates that under drought stress, overexpression ScPP2C49 The plant cell membranes of these plants are more severely damaged, and their antioxidant enzyme systems are less responsive.

[0056] 2. Detection of reactive oxygen species accumulation (DAB staining) Drought stress induces the production of reactive oxygen species (ROS) in plants, and excessive accumulation can lead to oxidative damage. The diaminobenzidine (DAB) staining method can visually demonstrate the accumulation of hydrogen peroxide (H2O2) in leaves.

[0057] Arabidopsis leaves, both under normal and drought treatment, were immersed in a 1 mg / mL DAB-HCl (pH 3.8) staining solution. After vacuum permeation, the leaves were stained under light for 8 hours. The staining was then decolorized with 95% ethanol until the background was clear, and the leaves were observed and photographed.

[0058] The results are as follows Figure 6 As shown in Figure C. Under normal conditions, both WT and transgenic lines have leaves with only a very light background color. After drought treatment, all leaves were stained brownish-red, indicating H2O2 accumulation, but the staining depth of the transgenic lines (OE-1, OE-2) leaves was significantly deeper than that of the WT leaves. This directly proves that under drought stress, overexpression... ScPP2C49 The plants accumulated more hydrogen peroxide and had insufficient reactive oxygen species scavenging capacity, which corroborates the result of reduced POD activity.

[0059] 3. Determination of water loss rate of detached leaves Transpiration is the main pathway for water loss in plants under drought conditions. The water loss rate is an important indicator for measuring the water retention capacity of plants.

[0060] Three-week-old WT and transgenic Arabidopsis plants with uniform growth were selected, and the above-ground parts (roselet leaves) were cut off and weighed immediately to obtain the initial fresh weight (W0). The plants were then laid flat on a dry experimental surface (room temperature, humidity ~50%) and weighed (Wt) after 0.5, 1, 2, 4, and 6 hours, respectively. The water loss rate was calculated as follows: Water loss rate (%) = [(W0 - Wt) / W0] × 100%.

[0061] The results are as follows Figure 7 As shown in Figure A, throughout the entire in vitro dehydration process, the water loss rate of both transgenic lines was consistently higher than that of WT. Six hours after in vitro incubation, the water loss rate of WT plants was approximately 34%, while the water loss rates of OE-1 and OE-2 were as high as approximately 62% and 57%, respectively, significantly higher than that of WT. This indicates that overexpression... ScPP2C49 The plants in this category have a poorer ability to retain water and are more prone to water loss in arid environments.

[0062] 4. Stomatal aperture analysis Stomata are the main channels for gas exchange and water transpiration between plant leaves and the outside world, and their opening and closing movements are crucial to drought response.

[0063] The lower epidermis of Arabidopsis rosette leaves was collected after normal irrigation and drought treatment. The epidermis was peeled off and prepared into sections, which were immediately observed and photographed under an optical microscope. The pore size (aperture) of at least 30 stomata was randomly measured using ImageJ software, and the average stomatal pore size ratio (aperture under stress / aperture under normal conditions) was calculated.

[0064] The results are as follows Figure 7As shown in B and 7C. Under normal water supply conditions, the stomata of both WT and transgenic lines were open, with no significant difference in stomatal aperture. Drought stress caused stomatal closure in all plants. However, the degree of stomatal closure in the transgenic lines was significantly less than that in WT, meaning that their stomata maintained a relatively large aperture even under stress. Figure 7 (Statistical results). This indicates... ScPP2C49 Overexpression of this substance can accelerate water loss by regulating stomatal opening, thereby weakening the drought resistance of plants.

[0065] 5. Chlorophyll fluorescence parameter analysis Photosynthesis is one of the physiological processes most significantly affected by drought stress. Chlorophyll fluorescence parameters are an effective indicator for rapid and non-destructive detection of the functional status of photosynthetic system II (PSII).

[0066] A portable modulated chlorophyll fluorescence imaging system was used to measure the fluorescence of leaves from WT and transgenic Arabidopsis plants under normal and drought treatments. The following key parameters were selected and analyzed: maximum photochemical efficiency (Fv / Fm), non-photochemical quenching coefficient (NPQ), photochemical quenching coefficient (qp), and fluorescence decay ratio (Rfd).

[0067] The results are as follows Figure 8 As shown. Under normal growth conditions, there were no significant differences in Fv / Fm, NPQ, qp, and Rfd values ​​among the different genotypes ( Figure 8 BE). Following drought stress, all these fluorescence parameters decreased, but the decrease was significantly greater in transgenic lines than in WT. The significant decrease in Fv / Fm ( Figure 8 B) indicates that the PSII reaction center in transgenic plants suffered more severe photoinhibition damage; the decrease in NPQ ( Figure 8 C) This indicates that its ability to dissipate excess light energy for self-protection is weakened; the decrease in qp and Rfd ( Figure 8 D, E) reflect a decrease in the open ratio of its PSII reaction centers and light energy utilization efficiency. These data combined indicate that overexpression... ScPP2C49 This makes the photosynthetic apparatus of Arabidopsis thaliana more susceptible to damage under drought conditions, resulting in a more severe decline in photosynthetic function.

[0068] Example 4: Overexpression ScPP2C49 Reduce the sensitivity of Arabidopsis thaliana to ABA Given ScPP2C49 The gene is ABA-induced and belongs to the PP2C family in the ABA signaling pathway. Further research will be conducted to determine whether this gene affects the plant's sensitivity to ABA.

[0069] 1. Seed germination experiment under ABA treatment After surface sterilization, WT, OE-1, and OE-2 seeds were sown on 1 / 2 MS solid medium containing 0 (control), 0.5 μM, and 1.0 μM ABA. After vernalization, the seeds were placed in a light incubator. Seed germination rate was recorded after 6 days of culture.

[0070] The results are as follows Figure 9 As shown. On media without ABA, the germination rate of all genotypes was close to 100%, with no difference. On media containing 0.5 μM and 1.0 μM ABA, ABA significantly inhibited seed germination. However, this inhibitory effect was significantly weaker in transgenic lines than in WT (Wheat Root). Figure 9 A). For example Figure 9 Statistical analysis (B-s) showed that under 1.0 μM ABA conditions, the germination rate of WT was strongly inhibited, while the germination rates of OE-1 and OE-2 were significantly higher than those of WT. This indicates that overexpression of ABA... ScPP2C49 It reduced the sensitivity of Arabidopsis seed germination to ABA.

[0071] 2. Root growth inhibition experiment under ABA treatment Three-day-old, uniformly growing WT and transgenic Arabidopsis seedlings were transferred to 1 / 2 MS medium containing 0, 0.5 μM, and 1.0 μM MABA and cultured vertically for another 7 days. The length of their taproots was then measured.

[0072] The results are as follows Figure 10 As shown. Figure 10 AC showed representative phenotypes. Figure 10 D represents root length statistics. ABA treatment significantly inhibited taproot elongation in all seedlings, with higher concentrations showing stronger inhibition. However, comparative analysis revealed that ABA's inhibition of taproot elongation in transgenic lines was significantly less severe than its inhibition on WT. Under 1.0 μM ABA conditions, the root length of transgenic lines was significantly longer than that of WT. This result is consistent with the germination experiment conclusions, further confirming the overexpression... ScPP2C49 It weakened the sensitivity of Arabidopsis seedling root growth to ABA.

[0073] Example 5: Overexpression ScPP2C49 Effects on the expression of key genes in the ABA signaling pathway In order to explore from the molecular mechanism ScPP2C49 To investigate how ABA sensitivity and drought resistance are affected, RT-qPCR was used to analyze the expression changes of key genes in the ABA signaling pathway in transgenic Arabidopsis thaliana.

[0074] Extracting WT and under normal growth conditions ScPP2C49Total RNA from overexpression line (OE-1) seedlings was reverse transcribed, and the expression levels of several key genes in the ABA signaling pathway were detected using the specific primers listed in Table 1. These genes include negative regulators. AtABI2 , AtHAI1 , AtHAB2 and positive regulatory factors AtSnRK2.4 .

[0075] The results are as follows Figure 11 As shown. Compared to WT, in overexpression ScPP2C49 In Arabidopsis plants, the PP2C gene negatively regulates ABA signaling. AtABI2 , AtHAI1 and AtHAB2 The expression levels of these kinases were significantly upregulated; while α, a key kinase in the positive ABA signaling pathway, was significantly upregulated. AtSnRK2.4 Gene expression levels were significantly downregulated. This expression pattern clearly indicates that heterologous overexpression in sugarcane... ScPP2C49 Genes may, by regulating the expression of components of the endogenous ABA signaling pathway, strengthen the negative regulatory arm of the pathway while weakening the positive regulatory arm, thereby inhibiting the overall transduction efficiency of the ABA signaling pathway. This explains the overexpression at the molecular level. ScPP2C49 It will reduce the plant's sensitivity to ABA and ultimately lead to a decrease in drought resistance.

[0076] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A type of sugarcane ScPP2C49 Genes, characterized by, The sugarcane ScPP2C49 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

2. A sugarcane according to claim 1 ScPP2C49 The gene-encoded ScPP2C49 protein is characterized by, The amino acid sequence of the ScPP2C49 protein is shown in SEQ ID No.

2.

3. A sugarcane as described in claim 1 ScPP2C49 The application of the gene or the ScPP2C49 protein as described in claim 2 in the negative regulation of plant drought resistance.

4. The application according to claim 3, characterized in that, The negative regulation of plant drought resistance is achieved by inhibiting root elongation, inhibiting stomatal closure under drought stress, or inhibiting the abscisic acid signaling pathway.

5. The application according to claim 3, characterized in that, The plants include Arabidopsis thaliana and / or sugarcane.

6. A method for cultivating sugarcane with stronger drought resistance, characterized in that, The method includes a step of reducing the expression level of the ScPP2C49 protein in the recipient sugarcane; the ScPP2C49 protein is a protein with the amino acid sequence shown in SEQ ID No.

2.

7. The method according to claim 6, characterized in that, The process includes the following steps: suppressing the expression of the gene encoding the ScPP2C49 protein in the recipient sugarcane to obtain transgenic sugarcane; the transgenic sugarcane exhibits stronger drought resistance compared to the recipient sugarcane.

8. A method for cultivating sugarcane with weaker drought resistance, characterized in that, The method includes the step of increasing the expression level of ScPP2C49 protein in the recipient sugarcane; the ScPP2C49 protein is a protein with the amino acid sequence shown in SEQ ID No.

2.

9. The method according to claim 8, characterized in that, The process includes the following steps: overexpressing the gene encoding the ScPP2C49 protein in the recipient sugarcane to obtain transgenic sugarcane; the transgenic sugarcane is less drought resistant than the recipient sugarcane.

10. The sugarcane according to claim 1 ScPP2C49 The application of the gene or the ScPP2C49 protein as described in claim 2 in sugarcane breeding is characterized by, The sugarcane ScPP2C49 Genes are used to regulate drought resistance traits in sugarcane; by using sugarcane ScPP2C49 Transforming sugarcane plants with gene overexpression vectors yields transgenic sugarcane with weakened drought resistance; or by transforming sugarcane... ScPP2C49 Gene knockout vectors were used to transform sugarcane plants, resulting in transgenic sugarcane with enhanced drought resistance.

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