Use of the gene scprt1 of the tooth rib erysimum in regulating the response of plants to abscisic acid

Abstract

This invention discloses the application of the *Rhizopus dentata* ScPRT1 gene in regulating plant responses to abscisic acid (ABA), the nucleotide sequence of which is shown in SEQ ID NO:1. This invention found that *Arabidopsis thaliana* lines overexpressing the *Rhizopus dentata* ScPRT1 gene, or *Rhizopus dentata* lines transiently expressing the *Rhizopus dentata* ScPRT1 gene, exhibited significantly reduced ABA sensitivity and significantly enhanced ABA response. Therefore, the *Rhizopus dentata* ScPRT1 gene negatively regulates plant responses to ABA signals. This invention provides resources for the discovery of stress-resistance genes, improves the regulatory network of *Rhizopus dentata* in response to ABA signals, and offers a new perspective for understanding the molecular mechanisms of stress adaptation in desert bryophytes.

Description

Technical Field

[0001] This invention belongs to the field of bio-agricultural technology, and more specifically, this invention relates to the application of the E3 ubiquitin ligase ScPRT1 gene of *Bryum esculentum* in regulating the plant response to abscisic acid. Background Technology

[0002] Syntrichia caninervis, belonging to the genus Syntrichia in the family Pottiaceae, is an early-established non-vascular plant, mainly distributed in arid desert regions such as the Gurbantünggüt Desert in northern Xinjiang, China, the Tengger Desert in Inner Mongolia, and the Mojave Desert in the southwestern United States. Its strong adaptability to extreme environments stems from the rich genetic code for stress resistance contained in its genome. Researchers have cloned and identified several key genes involved in stress resistance regulation based on the genome, gradually revealing the multi-level, multi-gene synergistic molecular regulatory network of Syntrichia caninervis.

[0003] The abscisic acid (ABA) signaling pathway is a core regulatory network in plant responses to abiotic stress. Current research on ABA signaling mainly focuses on higher plants such as Arabidopsis thaliana, while research on the ABA signaling regulation mechanism in bryophytes is relatively limited. The ABA signaling pathway of Bryophytum esculentum may have evolved a unique regulatory mechanism during its long-term adaptation to desert environments. Summary of the Invention

[0004] Based on this, the purpose of this invention is to provide a RING-type E3 ubiquitin ligase gene of *Rhodops dentata* that can regulate the plant's response to abscisic acid.

[0005] The specific technical solutions for achieving the above-mentioned objectives are as follows.

[0006] In a first aspect, the present invention provides the application of the *Rhizoctonia solani* ScPRT1 gene, the protein encoded therein, an overexpression vector of the *Rhizoctonia solani* ScPRT1 gene, a transient expression vector of the *Rhizoctonia solani* ScPRT1 gene, an engineered bacterium transformed with the overexpression vector of the *Rhizoctonia solani* ScPRT1 gene, and the engineered bacterium transformed with the transient expression vector of the *Rhizoctonia solani* ScPRT1 gene in regulating the plant response to abscisic acid, wherein the nucleotide sequence of the *Rhizoctonia solani* ScPRT1 gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein encoded therein is shown in SEQ ID NO:2.

[0007] In a second aspect, the present invention provides a method for improving the response of plants to abscisic acid, comprising the steps of: increasing the expression level of the ScPRT1 gene or its homolog in the plant, wherein the nucleotide sequence of the ScPRT1 gene is shown in SEQ ID NO:1.

[0008] The inventors of this invention discovered that Arabidopsis lines overexpressing the *ScPRT1* gene of *Rhizopus dentata*, or those transiently expressing the *ScPRT1* gene, exhibited significantly reduced sensitivity to abscisic acid (ABA) and significantly increased ABA tolerance. Therefore, the *ScPRT1* gene negatively regulates the plant's response to ABA signaling. This invention provides resources for the discovery of stress-resistance genes, refines the regulatory network of *Rhizopus dentata* in response to ABA signaling, and offers a new perspective for understanding the molecular mechanisms of stress adaptation in desert bryophytes. Attached Figure Description

[0009] Figure 1 The results of bioinformatics analysis of *Syntrichiacaninervis* ScPRT1 are shown below. A: Multiple sequence alignment of *Syntrichiacaninervis* ScPRT1 with other known species; conservation is indicated by different colors (dark blue > pink > light blue > gray). The overline represents the C3HC4 domain. B: Conserved domain analysis. C: Phylogenetic tree analysis. Sc: *Syntrichiacaninervis*; Pp: *Physcomitrella patens*; At: *Arabidopsis*; Os: *Oryza sativa*; Zm: *Zea mays*; Gm: *Glycine max*; Pd: *Phoenix dactylifera*; Pa: *Populus alba*.

[0010] Figure 2 The results show the seed germination and cotyledon greening of Arabidopsis thaliana overexpressing ScPRT1 under different concentrations of ABA treatment. A: Seed germination phenotype; B: Cotyledon greening phenotype; C: Seed germination rate statistics; D: Cotyledon greening rate statistics.

[0011] Figure 3 The results show the root length and fresh weight of Arabidopsis seedlings overexpressing ScPRT1 under different concentrations of ABA treatment. A: Seedling growth phenotype; B: Main root length statistics; C: Single plant fresh weight statistics.

[0012] Figure 4 Phenotypic images of Arabidopsis seedlings overexpressing ScPRT1 under different concentrations of ABA treatment.

[0013] Figure 5 The results of physiological parameters of Arabidopsis thaliana overexpressing ScPRT1 under different concentrations of ABA treatment are shown. A: H2O2 content; B: malondialdehyde (MDA) content; C: proline (Pro); D: soluble sugar (SS) content; E: catalase (CAT) activity; F: peroxidase (POD) activity; G: superoxide dismutase (SOD) activity.

[0014] Figure 6 The staining results of Arabidopsis thaliana overexpressing ScPRT1 under different concentrations of ABA treatment. A: NBT staining; B: DAB staining.

[0015] Figure 7 The results show the photosynthetic pigment content of Arabidopsis thaliana overexpressing ScPRT1 under different concentrations of ABA treatment. A: chlorophyll extract from leaves of WT and OE lines under different concentrations of ABA treatment; B: chlorophyll a content; C: chlorophyll b content; D: carotenoid content; E: total chlorophyll content.

[0016] Figure 8 The results show the chlorophyll fluorescence parameters of Arabidopsis thaliana overexpressing ScPRT1 under different concentrations of ABA treatment. A: Fv / Fm chlorophyll fluorescence imaging; B: Fv / Fm value statistics; C: actual photosynthetic efficiency Y(II); D: photoprotection capacity Y(NPQ); E: light damage index Y(NO).

[0017] Figure 9 The results show the stomatal movement and water loss rate of Arabidopsis leaves overexpressing ScPRT1 under different concentrations of ABA treatment. A: Stomatal phenotype of wild-type WT and overexpression lines OE-1 and OE-2 under different concentrations of ABA treatment; B: Stomatal opening and closing ratio statistics; C: Water loss phenotype; D: Water loss rate statistics.

[0018] Figure 10 The results show the senescence and ABA signaling pathway-related gene expression analysis in Arabidopsis thaliana overexpressing ScPRT1 under different ABA treatments. A: Relative expression level of AtNYE1; B: Relative expression level of AtNYC1; C: Relative expression level of AtSAG29; D: Relative expression level of AtABI5.

[0019] Figure 11 Molecular identification results of *Bryum dentata* lines transiently transformed with ScPRT1. A: DNA level identification of *Bryum dentata* lines transiently transformed with ScPRT1; B: Relative expression level of ScPRT1 in each line detected by RT-qPCR. M: DL2000 molecular weight standard; 1: EV; 2: ScPRT1-RNAi; 3: ScPRT1-OE. Different letters indicate significant differences at the P < 0.05 level.

[0020] Figure 12 Phenotypic images of the ScPRT1 transgenic *Brachys dentata* under different concentrations of ABA treatment. The first row shows the growth status of each line before ABA treatment; the second row shows the growth status of each line after ABA treatment.

[0021] Figure 13The results show the chlorophyll content and related gene expression of the ScPRT1 transgenic *Brachys dentata* under ABA treatment. Where A: chlorophyll a content; B: chlorophyll b content; C: total chlorophyll content; D: relative expression level of ScNYE1; E: relative expression level of ScNYC1.

[0022] Figure 14 The staining results of ScPRT1 transgenic *Rhizopus dentata* under ABA treatment. A: NBT staining; B: DAB staining.

[0023] Figure 15 The results of fluorescent staining of ScPRT1 transgenic *Brachys dentata* under ABA treatment are shown. A: DHE fluorescent probe staining to detect superoxide anions; B: H2DC-FDA fluorescent probe staining to detect overall reactive oxygen species levels.

[0024] Figure 16 The results show the physiological parameters of the ScPRT1 transgenic *Sclerotium dentatum* under ABA treatment. A: Hydrogen peroxide (H2O2) content; B: Malondialdehyde (MDA) content; C: Proline (Pro); D: Soluble sugar (SS) content.

[0025] Figure 17 Bioinformatics analysis results for *ScPYL4* and *ScPYL6* of *Rhizopus dentata*. A: Amino acid sequence alignment of *ScPYL4* with other species; B: Amino acid sequence alignment of *ScPYL6* with other species; C: Conserved domain analysis of *ScPYL4*; D: Conserved domain analysis of *ScPYL6*; E: Phylogenetic analysis of *ScPYL4*; F: Phylogenetic analysis of *ScPYL6*.

[0026] Figure 18 The results of subcellular localization analysis of ScPYL4 and ScPYL6.

[0027] Figure 19 To verify the interaction results between ScPYL4 and ScPYL6 in yeast two-hybrid assays.

[0028] Figure 20 Molecular identification results of transiently transformed *Brachys dentata* lines using ScPYL4 / ScPYL6. A: DNA level identification of transformed lines; B: Relative expression level of ScPYL4; C: Relative expression level of ScPYL6. M: DL2000 molecular weight standard; 1: EV; 2: OE-ScPYL4; 3: OE-ScPYL6; 4: RNAi-ScPYL4; 5: RNAi-ScPYL6. Different letters indicate significant differences at the P < 0.05 level.

[0029] Figure 21The results of reactive oxygen species (ROS) staining in ABA-treated ScPYL4 / ScPYL6 transgenic *Brachys dentata* are shown. A: DAB staining of each ScPYL4 line; B: NBT staining of each ScPYL4 line; C: DAB staining of each ScPYL6 line; D: NBT staining of each ScPYL6 line.

[0030] Figure 22 The results of fluorescent staining of ABA-treated ScPYL4 / ScPYL6 transgenic *Rhizopus dentata* are shown. A: DHE fluorescent probe staining to detect superoxide anions; B: H2DC-FDA fluorescent probe staining to detect overall reactive oxygen species levels.

[0031] Figure 23 The results of physiological index analysis of ScPYL4 / ScPYL6 transgenic *Brachys dentata* under ABA treatment are shown. A: MDA content of each ScPYL4 strain; B: H2O2 content of each ScPYL4 strain; C: Proline content of each ScPYL4 strain; D: Soluble sugar content of each ScPYL4 strain; E: MDA content of each ScPYL6 strain; F: H2O2 content of each ScPYL6 strain; G: Proline content of each ScPYL6 strain; H: Soluble sugar content of each ScPYL6 strain.

[0032] In the above figures, WT represents wild-type lines, and OE represents transgenic lines. Data are expressed as mean ± SE (n≥6). *, **, and *** indicate statistically significant differences at p<0.05, p<0.01, and p<0.001, respectively; ns indicates no significant difference. Detailed Implementation

[0033] To facilitate understanding of the present invention, a more complete description will be provided below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0034] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this specification is for the purpose of describing particular embodiments only and is not intended to limit the invention. The term "and / or" as used in this invention includes any and all combinations of one or more of the associated listed items.

[0035] Unless otherwise specified, all examples were performed under standard experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2013), or as recommended by the manufacturer.

[0036] In this invention, by comparing wild-type Arabidopsis thaliana lines and Arabidopsis thaliana lines overexpressing ScPRT1, it was found that the Arabidopsis thaliana lines overexpressing ScPRT1 showed significantly reduced sensitivity to ABA at multiple developmental stages, weakening ABA-inhibited seed germination, cotyledon chlorosis, taproot elongation, and biomass accumulation, as well as ABA-induced stomatal closure, leaf senescence, and oxidative damage. By comparing the transient transformation of *Rhizoctonia solani* lines overexpressing ScPRT1 and the transient transformation of *Rhizoctonia solani* lines with ScPRT1 interference, it was found that overexpression of ScPRT1 reduced the sensitivity of *Rhizoctonia solani* to ABA (exhibiting milder yellowing, higher chlorophyll content, and lower oxidative damage), while interference with ScPRT1 expression made *Rhizoctonia solani* highly sensitive to ABA (exhibiting severe yellowing and wilting, rapid chlorophyll degradation, reactive oxygen species bursts, and exacerbated membrane system damage), which is consistent with the results obtained in Arabidopsis thaliana. This indicates that when ScPRT1 is absent, ABA signaling is overactivated, leading to rapid chloroplast degradation and an oxidative burst. In natural environments, this overreaction may cause bryophytes to initiate an irreversible senescence process even under mild drought, thus losing their ability to recover and grow after rehydration. Furthermore, this invention found that the attenuating effect of ScPRT1 overexpression on ABA sensitivity appears to be more significant in *Bryophytum dentatum* than in *Arabidopsis thaliana*. This may reflect differences in the ABA signaling network structure between bryophytes and angiosperms—bryophyte ABA signaling pathways are relatively simple, and perturbations of negative regulators may produce more drastic phenotypic changes; or, *Bryophytum dentatum* genomes may contain fewer ScPRT1 homologs, resulting in lower functional redundancy and thus a more pronounced overexpression effect.

[0037] Therefore, in both the higher plant Arabidopsis thaliana and the bryophyte *Bryum argenteum*, ScPRT1 negatively regulates the response of *Bryum argenteum* to ABA signaling (weakening the response to ABA / reducing sensitivity to ABA / increasing tolerance to ABA), acting as a negative regulator of the ABA signaling pathway and finely modulating the intensity of ABA signaling. Precise regulation of ABA signaling intensity is crucial for the survival of bryophytes in extreme environments. As a negative regulator, ScPRT1 may control the ABA signaling intensity within an optimal tolerance window that activates sufficient defense responses without causing excessive senescence and oxidative damage, thereby optimizing the plant's "survival-growth" balance under stress. This functional conservation suggests that ScPRT1 may target certain core and conserved components of the ABA signaling pathway.

[0038] Based on this, the present invention identified the ABA receptors ScPYL4 / ScPYL6 as ubiquitination degradation substrates of ScPRT1. Furthermore, after transiently transforming *Rhizoctonia solani* with ScPYL4 and ScPYL6, the present invention found that lines overexpressing *Rhizoctonia solani* ScPYL4 and ScPYL6 were sensitive to ABA treatment, indicating that ScPYL4 and ScPYL6 can enhance the sensitivity of *Rhizoctonia solani* to ABA and increase oxidative stress caused by ABA overdose. ScPYL4 and ScPYL6, as positive regulators of the ABA signaling pathway in *Rhizoctonia solani*, function in the ABA response of *Rhizoctonia solani*, suggesting that ScPRT1 may negatively regulate the ABA signaling pathway by degrading the two positive regulators ScPYL4 and ScPYL6. ScPRT1 plays a crucial role in the stress adaptation and growth balance of *Rhizoctonia solani* by finely regulating the intensity and duration of ABA signaling through the stability of the ABA receptors ScPYL4 and ScPYL6 proteins.

[0039] In some embodiments of the present invention, the application of the ScPRT1 gene of *Rhizopus dentata* in regulating the plant response to abscisic acid is disclosed, the nucleotide sequence of which is shown in SEQ ID NO:1.

[0040] In one embodiment, the plant is a moss or Arabidopsis thaliana.

[0041] In one embodiment, the plant is *Brachys dentata*.

[0042] In other embodiments of the present invention, the application of a protein encoded by the ScPRT1 gene of *Bryum dentata* in regulating the plant response to abscisic acid is disclosed, the amino acid sequence of which is shown in SEQ ID NO:2.

[0043] In one embodiment, the plant is a moss or Arabidopsis thaliana.

[0044] In one embodiment, the plant is *Brachys dentata*.

[0045] In other embodiments of the present invention, the application of an overexpression vector or a transient expression vector of the ScPRT1 gene of *Rhizopus dentata* in improving the plant response to abscisic acid is disclosed, wherein the nucleotide sequence of the ScPRT1 gene of *Rhizopus dentata* is shown in SEQ ID NO:1.

[0046] In one embodiment, the plant is a moss or Arabidopsis thaliana.

[0047] In one embodiment, the plant is *Brachys dentata*.

[0048] In other embodiments of the present invention, the application of engineered bacteria transformed with an overexpression vector of the ScPRT1 gene of *Rhizopus dentata* or an intermediate expression vector of the ScPRT1 gene of *Rhizopus dentata* is disclosed in improving the plant response to abscisic acid, wherein the nucleotide sequence of the ScPRT1 gene of *Rhizopus dentata* is shown in SEQ ID NO:1.

[0049] In one embodiment, the plant is a moss or Arabidopsis thaliana.

[0050] In one embodiment, the plant is *Brachys dentata*.

[0051] In other embodiments of the present invention, a method for improving the plant response to abscisic acid is disclosed, comprising the following steps: increasing the expression level of the ScPRT1 gene of *Bryum dentata* in the plant, wherein the nucleotide sequence of the ScPRT1 gene is shown in SEQ ID NO:1.

[0052] In one embodiment, the plant is a moss or Arabidopsis thaliana.

[0053] In one embodiment, the plant is *Brachys dentata*.

[0054] In the following embodiments of the present invention, abscisic acid (ABA), DAB, and NBT staining agents were purchased from Beijing Cooler Biotech Co., Ltd.; the main physiological indicator detection kit was purchased from Nanjing Jiancheng Biotechnology Institute; the total RNA extraction kit and cDNA synthesis reagent were purchased from Beijing TransGen Biotech Co., Ltd.; the fluorescence quantitative reagent and PCR amplification enzyme premix were purchased from Nanjing Novizan Biotechnology Co., Ltd.; the fluorescent probe dyes etidium hydrogen fluorescein (DHE) and 2′,7′-dichlorodihydrofluorescein diacetate (H2DC-FDA) were purchased from Shanghai Haoyuan Biotechnology Co., Ltd.; acetylsyleugenone (AS) was purchased from Beijing Solarbio Biotechnology Co., Ltd.; other raw materials or reagents not specified were all commercially available.

[0055] In the following embodiments of the present invention, SPSS 18.0 and GraphPad Prism 10 software were used to process and analyze the data. For multivariate data, two-way ANOVA was used to analyze the main effects and interaction effects, followed by post-hoc multiple comparison tests to analyze the significance of differences between groups. For univariate data, one-way ANOVA was used to test the overall difference, followed by multiple comparison tests to perform pairwise comparisons between groups. Different letters indicate significant differences at each level. All experiments were performed in at least three independent biological replicates.

[0056] The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0057] Example 1: Bioinformatics Analysis and Promoter Regulatory Element Analysis of ScPRT1 in *Rhizopus dentata*

[0058] Homologs of ScPRT1 obtained using the BLAST program (http: / / www.ncbi.nlm.nih.gov / BLAST / ) include PpPRT1 (Scleroderma purpurea, XM_024525392.1), AtPRT1 (Arabidopsis thaliana, XM_080799.1), OsPRT1 (Rice, XM_015767241.2), ZmPRT1 (Maize, XM_020538608.2), GmPRT1 (Soybean, XM_006602468.4), PdPRT1 (Dateberry, XM_008786960.4), and PaPRT1 (Poplar, XM_035060044.1). PRT1 amino acid sequences were aligned using DNAMAN 8.0 software, and phylogenetic analysis was performed using the neighborhood linking method in MEGA software. Amino acid sequence alignment results indicate that ScPRT1 is a RING-type E3 ubiquitin ligase with a C3HC4 structure, and contains highly conserved cysteine ​​and histidine functional domains. Figure 1 (A and B). The ScPRT1 sequence of *Rhizopus dentata* showed the lowest homology with the OsPRT1 sequence of rice and the highest sequence similarity with the PpPRT1 sequence of *Bryum simonii*. Figure 1 (C). The amino acid sequence of the ScPRT1 protein of *Erythrina esculenta* is shown in SEQ ID NO:2, and the nucleotide sequence is shown in SEQ ID NO:1.

[0059] The promoter region (2kb upstream of the transcription start site) of the ScPRT1 gene was analyzed using the PlantCARE database. The results showed that the promoter region of ScPRT1 contains multiple ABA response elements (ABREs), suggesting that it may be involved in the ABA response. This provides important clues and evidence for further research on the function of ScPRT1 in the ABA signaling pathway.

[0060] SEQ ID NO:1

[0061] ATGAACACGGAGACGGTGTCGCACTGCCCGGTGTGCCAGAAGTCGTACGTGCACCTCCCCCGGATCTCGCCGCAGCTGCACCACCTCCTGGAGCACACGTACCCGCGGGAGTACGCGGCGCGCGAGCTGGAGGTGTCCACGGAGGAGCGCGACCAGAACAACTACTCCCCAGAGATTCCTGCGGGCGCGTCCTCGCTCGCGTGCACCGTGTGCAGGCAGGTCCTGCACAAGCCGGTGGCGATGAACTGCGGCCACCTGATGTGCCAGGGGTGCGCGGCGGGCGGCCCGGCCAACAGCTGCCGTGTCTGCGGGGTGCCGCACCCGGGCGCGTTCCCGCAGGTGTGCGTGGAGCTGCAGCAGTACCTGGAGGAGCGGCCGGGCTACAACAGCAGCAGGAAGGCAGCTCCTGACGTGCGCGCGGCCGCGCGCACGTGGAAGGCGCCGCCTGCGGATGGGGTGGGCGACGCGGTGGCCCTCGCGCAGAACCCGATTCACCCCAGCGTGGGCTGCGACGGCTGTGGCATGATGCCCATCCTTGGCAAGCGCTTCCACTGCCTGGACTGCCCCGAGAGCTGCGGGTACGACCTCTGCCAGAGCTGCCAGGACCGCGGCAGCAGCCTGCCCGGCCGCTTCAACCAGCGCCACGGCCCGCGCCACCGCATGGAGGAGCGCTCGCTGCGCAGCCCGTGGATCTTCGTCTGGGCCTGGTGA

[0062] SEQ ID NO:2

[0063] MNTETVSHCPVCQKSYVHLPRISPQLHHLLEHTYPREYAARELEVSTEERDQNNYSPEIPAGASSLACTVCRQVLHKPVAMNCGHLMCQGCAAGGPANSCRVCGVPHPGAFPQVCVEL QQYLEERRPGYNSSRKAAPDVRAAARTWKAPPADGVGDAVALAQNPIHPSVGCDGCGMMPILGKRFHCLDCPESCGYDLCQSCQDRGSSLPGRFNQRHGPRHRMEERSLRSPWIFVWAW*

[0064] Example 2: Functional analysis of ABA signal response in transgenic Arabidopsis thaliana overexpressing the ScPRT1 gene.

[0065] In this embodiment, the functional analysis of ScPRT1 in response to Arabidopsis thaliana ABA signal was performed using transgenic Arabidopsis T3 homozygous lines (OE-1 and OE-2, preserved in the applicant's laboratory) that overexpress the ScPRT1 gene.

[0066] 1. Arabidopsis seed germination rate and cotyledon chlorophyll rate

[0067] Transgenic Arabidopsis thaliana T3 homozygous seeds overexpressing the ScPRT1 gene and wild-type (WT) seeds were sterilized and sown on MS medium containing 0 μM ABA, 0.5 μM ABA, 0.8 μM ABA, and 1 μM ABA. Thirty seeds were allocated to each line, with at least three biological replicates per treatment. Seeds were vernalized at 4°C for 2 days and then transferred to a 22 ± 2°C, 16 h light / 8 h dark incubator for cultivation. Seed germination rate was assessed after 7 days, and cotyledon chlorophyll rate was assessed after 21 days.

[0068] The results are as follows Figure 2 As shown, under the control condition (0 μM ABA), there were no significant differences in seed germination rate and cotyledon greening rate between the WT and OE lines. The germination rate was close to 100% on day 7 and the cotyledon greening rate was close to 100% on day 21. With increasing ABA concentration, seed germination and cotyledon greening of both WT and OE lines were inhibited to varying degrees, but the inhibition level of the OE line was significantly lower than that of the WT line. Statistical results showed that under 0.5–1.0 μM ABA treatment, the germination rate and cotyledon greening rate of the OE line were significantly higher than those of the WT line. These results indicate that overexpression of ScPRT1 significantly weakened the inhibitory effect of ABA on seed germination and cotyledon greening, reducing the sensitivity of Arabidopsis thaliana to ABA in its early developmental stages.

[0069] 2. Arabidopsis thaliana seedlings have long roots and high fresh weight.

[0070] After sterilization, homozygous seeds of transgenic Arabidopsis thaliana T3 generation overexpressing the ScPRT1 gene and wild-type seeds were sown on MS medium. Once cotyledons emerged, seedlings with uniform growth and root length were selected and transplanted to MS vertical media containing 0 μM ABA, 0.5 μM ABA, 0.8 μM ABA, and 1 μM ABA. Three seedlings were placed in each line, with at least three biological replicates per treatment. The plants were cultured at 22 ± 2℃, 16 h light / 8 h dark for 7 days. Root length was observed and photographed, and root length and fresh weight were measured.

[0071] The results are as follows Figure 3 As shown, under the control condition (0 μM ABA), there were no significant differences in seedling growth, taproot length, and fresh weight between the WT and OE lines. After ABA treatment, the root length and fresh weight of all lines showed a decreasing trend, but the inhibition level of the OE line was significantly lower than that of the WT line. Under the treatment of 0.5–1.0 μM ABA, the taproot length of the OE line was significantly longer than that of the WT line, and the fresh weight was also significantly higher than that of the WT line. These results indicate that overexpression of ScPRT1 significantly weakened the inhibitory effect of ABA on taproot elongation and biomass accumulation in seedlings, reducing the sensitivity of Arabidopsis seedlings to ABA.

[0072] 3. Phenotype of Arabidopsis seedlings

[0073] Healthy wild-type and transgenic Arabidopsis thaliana plants, transplanted into the soil for about 3-4 weeks, were selected and photographed before treatment. Subsequently, the same amount of H2O, 50 μM ABA, 100 μM ABA, and 150 μM ABA solutions were sprayed each time. After 3 days of treatment, photographs were taken again.

[0074] The results are as follows Figure 4 As shown, under the control condition (H2O), both WT and OE lines grew well, with fully expanded leaves and normal color, showing no significant phenotypic differences. After ABA treatment, all lines exhibited varying degrees of yellowing and wilting, indicating senescence, and the degree of senescence gradually intensified with increasing ABA concentration. Consistent with the trend observed in the seedling stage, at all ABA treatment concentrations, the degree of leaf yellowing in the OE line was significantly less than that in the WT line. This result indicates that overexpression of ScPRT1 can reduce the sensitivity of Arabidopsis seedlings to ABA.

[0075] 4. Physiological indicators and photosynthetic capacity

[0076] (1) Physiological indicators

[0077] Leaves from ABA-treated wild-type and transgenic Arabidopsis seedlings were used to determine the contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), proline (Pro), soluble sugar (SS), and the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD). All physiological indicators were measured using commercially available kits (Nanjing Jiancheng Biotechnology Research Institute, China).

[0078] The results are as follows Figure 5 As shown, the H2O2 content of the OE strain was significantly lower than that of the WT strain after ABA treatment. Simultaneously, the content of malondialdehyde (MDA), a membrane lipid peroxidation product, was also significantly lower in the OE strain than in the WT strain. This indicates that overexpression of ScPRT1 can effectively alleviate ABA-induced reactive oxygen species accumulation and membrane lipid peroxidation damage. Under control conditions, there were no significant differences in proline (Pro) and soluble sugar (SS) contents between the WT and OE strains. After ABA treatment, the Pro and SS contents increased in all strains, but the Pro and SS contents of the OE strain were significantly higher than those of the WT strain, indicating that the OE strain has a stronger osmotic regulation capacity to cope with ABA-induced stress. Under control conditions, there were no significant differences in CAT, POD, and SOD activities between the WT and OE strains. After ABA treatment, the CAT, POD, and SOD activities of the OE strain were significantly higher than those of the WT strain. The above results indicate that ABA may induce premature leaf senescence and accumulation of reactive oxygen species, thereby leading to increased oxidative stress, membrane system damage, and decreased defense capabilities in Arabidopsis thaliana. Overexpression of ScPRT1 can attenuate ABA-induced premature leaf senescence and oxidative damage.

[0079] (2) Arabidopsis NBT and DAB staining

[0080] 0.1 g of NBT powder and 0.1 g of DAB powder were weighed into 100 mL of phosphate buffer (0.1 mM) to prepare 1 mg / mL NBT staining solution and 1 mg / mL DAB staining solution (pH=3.0). Transgenic and wild-type Arabidopsis leaves treated with H2O, 50 μM ABA, 100 μM ABA, and 150 μM MABA solutions were placed in the staining solutions and incubated at 37°C in the dark for 12 h. Chlorophyll was removed by boiling in 95% ethanol solution for 10 min. The destained leaves were then observed and photographed on a white luminescent plate. Five replicates were taken from each group and each strain.

[0081] NBT and DAB staining results are as follows Figure 6 As shown, the results indicate that under control conditions, the leaves of both WT and OE lines were lightly stained, with no significant difference. After ABA treatment, WT leaves exhibited deep blue (NBT) and dark brown (DAB) staining, while the staining depth of the OE line was significantly lighter than that of WT, indicating that the OE line accumulated less superoxide anions (O2). •-) and hydrogen peroxide (H2O2).

[0082] (3) Photosynthetic pigment content

[0083] 0.1 g of wild-type and transgenic Arabidopsis leaves were weighed and placed into 50 mL centrifuge tubes. 25 mL of 95% ethanol solution was added, the tubes were sealed, wrapped with aluminum foil, and extracted at room temperature in the dark for 24 h until the leaves turned pale green. The absorbance was measured at wavelengths of 665 nm and 649 nm, respectively. 95% ethanol solution was used as a blank. Six replicates were taken for each treatment, and the content of each pigment in the leaves was calculated.

[0084] The results are as follows Figure 7 As shown, under control conditions, there were no significant differences in chlorophyll a, chlorophyll b, total chlorophyll, and carotenoid contents between the WT and OE lines. After ABA treatment, the pigment contents of all lines showed a decreasing trend, but the decrease in the OE line was significantly smaller than that of the WT line. Under ABA treatment, the contents of chlorophyll a, chlorophyll b, carotenoids, and total chlorophyll in the OE line were significantly higher than those in the WT line. The color of the chlorophyll extract clearly shows that the WT extract became significantly lighter in color after ABA treatment, while the OE extract remained a darker green. These results indicate that overexpression of ScPRT1 weakens ABA-induced chlorophyll degradation.

[0085] (4) Photosynthetic capacity

[0086] The chlorophyll fluorescence of Arabidopsis thaliana treated with H2O, 50 μM ABA, 100 μM ABA, and 150 μM ABA solutions was measured using an IMAGING-PAM chlorophyll fluorescence meter (Heinz Walz GmbH, Germany). The fourth mature leaf of wild-type and overexpression lines was selected and pretreated in darkness for 30 min before measurement. The maximum photosynthetic efficiency (Fv / Fm) and the actual photosynthetic efficiency (ΦPSⅡ) were calculated.

[0087] Chlorophyll fluorescence imaging, such as Figure 8 As shown, the results indicated that under control conditions, the leaves of both WT and OE lines exhibited a uniform blue color. With increasing ABA treatment concentration, the leaves of WT changed from blue to green or even yellowish-green, indicating severe PSII damage. Under normal growth conditions, there were no significant differences in Fv / Fm, Y(II), Y(NPQ), and Y(NO) between wild-type and transgenic Arabidopsis, maintaining similar levels. After ABA treatment, Fv / Fm, Y(II), and Y(NPQ) were higher in transgenic lines than in wild-type, while Y(NO) was lower in transgenic lines than in wild-type. This suggests that ABA treatment increased ScPRT1 levels, thereby improving the maximum photochemical efficiency, actual photosynthetic efficiency, and photosynthetic capacity of Arabidopsis by increasing chlorophyll content, and mitigating light damage.

[0088] (5) Stomatal diameter and water loss rate of Arabidopsis thaliana leaves

[0089] Leaves from 1-month-old wild-type and transgenic Arabidopsis thaliana were collected and placed in stomatal opening buffer (5 mM MES, 10 mM KCl, 50 mM CaCl2) for 2 h. After treatment, the leaves were transferred to solutions of H2O, 50 μM ABA, 100 μM ABA, and 150 μM ABA. The lower epidermis of the Arabidopsis leaves was peeled off and photographed under a microscope. At least 6 leaves were collected for each treatment, and 10-15 stomata were randomly selected from each sample for measurement. The experimental data were analyzed using ImageJ software to determine the stomatal diameter and its width-to-length ratio. A ratio less than 0.2 was defined as a completely closed stoma, a ratio between 0.3 and 0.5 as a partially closed stoma, and a ratio greater than 0.5 as an open stoma.

[0090] Leaves of healthy wild-type and transgenic Arabidopsis thaliana with consistent growth were selected for the experiment. Immediately after removal, the leaves were placed on an analytical balance and their initial fresh weight (FW) was accurately measured. o After recording the data, the leaves were laid flat in a petri dish and allowed to dehydrate naturally in a well-ventilated environment without direct sunlight. At 0, 1, 2, 3, 4, and 5 hours of dehydration, the leaves were removed and their fresh weight (FW) was quickly measured at each time point. t Take photos and record the results. After weighing, immediately return the leaves to the original petri dish to continue losing water, avoiding further moisture loss. According to the formula: Water loss rate (%) = (FW) / (FW) o -FW t ) / FW o × 100%, calculate the water loss rate of detached leaves of wild-type and transgenic Arabidopsis thaliana at each time point, and plot the water loss curve with water loss time as the x-axis and water loss rate as the y-axis. Analyze the curves to compare the differences in water loss rate and water retention capacity between the two. The experiment was set up with 3 biological replicates, and 3-6 leaves were selected for each replicate.

[0091] The results are as follows Figure 9 As shown, under control conditions, the stomata of both the WT and OE lines were mostly open, with a high proportion of open stomata, and no significant differences among the lines. After ABA treatment, the proportion of closed stomata increased in all lines, but the response of the OE line was significantly weaker than that of the WT line. Under ABA treatment, the proportion of closed stomata in the WT line was significantly higher than that in the OE line, while the proportion of open stomata in the OE line was significantly higher than that in the WT line, indicating that overexpression of ScPRT1 weakened the ABA-induced stomatal closure response. The water loss rate of leaves in both the WT and OE lines gradually increased with time, but the water loss rate of the OE line was significantly higher than that of the WT line at all time points, indicating that its leaf water retention capacity decreased and the water loss rate accelerated. These results indicate that ScPRT1 overexpression weakens the sensitivity of stomata to ABA, leading to faster water loss from leaves under natural conditions.

[0092] 5. Real-time quantitative PCR of leaf senescence genes

[0093] ABA-treated wild-type and transgenic Arabidopsis leaves were selected, and total RNA was extracted after liquid nitrogen flash freezing and reverse-engineered into cDNA. RT-qPCR was performed using an Applied Biosystems 7500 real-time quantitative PCR system, with AtActin selected as an internal control gene. Based on changes in fluorescence signal during the reaction, 2... -ΔΔCt The relative expression levels of genes (chlorophyll degradation genes AtNYE1 and AtNYC1, aging response gene AtSAG29, and key gene AtABI5 in the ABA signaling pathway) were calculated using a method with 3 technical replicates and 3 biological replicates for each sample.

[0094] The results are as follows Figure 10 As shown, under control conditions, the expression levels of AtNYE1, AtNYC1, AtSAG29, and AtABI5 in both WT and OE lines were extremely low, with no significant differences among the lines. Under ABA treatment, the expression levels of AtNYE1, AtNYC1, AtSAG29, and AtABI5 in the OE line were consistently significantly lower than those in WT. These results indicate that ScPRT1 overexpression may attenuate ABA-induced leaf senescence by inhibiting the expression of chlorophyll degradation genes, senescence response genes, and key genes in the ABA signaling pathway.

[0095] 6. Differentially expressed gene analysis

[0096] Wild-type and transgenic Arabidopsis seedlings with homogeneous phenotypes, grown on MS solid medium for 7 days, were selected and transferred to MS vertical medium containing 0 μM (control) and 1.0 μM ABA, respectively, for 7 days of treatment before sampling. Three replicates were taken from each line, for a total of 12 samples. Transcriptome sequencing was performed by Beijing Biomarker Biotechnology Co., Ltd., using the Illumina HiSeq-PE150 high-throughput sequencing platform. Transcriptome analysis data were obtained from the bioinformatics analysis platform BMKCloud (www.biocloud.net). Gene expression levels were quantified using FPKM values. Genes with a corrected P-value < 0.01 and Fold Change ≥ 2, as analyzed by DESeq2, were designated as differentially expressed genes (DEGs). GO, KEGG functional annotation, and pathway enrichment were used to classify differentially expressed genes. Heatmaps were generated using software such as TBtools to screen differentially expressed genes. Quantitative analysis of differentially expressed genes was performed, with AtActin selected as an internal control gene. 2 -△△Ct The relative expression levels of genes were calculated using a method with three biological replicates and three technical replicates for each experiment.

[0097] Comparative analysis of differentially expressed genes (DEGs) under ABA treatment revealed 181 differentially expressed genes in the ScPRT1 overexpressing lines compared to the wild type, including 118 upregulated and 63 downregulated genes. GO enrichment bubble plots showed that differentially expressed genes were significantly enriched in multiple biological processes related to signal transduction, stress response, and hormone regulation, such as the "response to abscisic acid" and "hormone-mediated signaling pathway," demonstrating ScPRT1's involvement in the regulation of the ABA signaling pathway. KEGG bubble enrichment plots indicated that the differentially expressed genes were mainly enriched in pathways related to "plant-pathogen interaction," "plant MAPK signaling pathway," "RNA degradation," and "plant hormone signal transduction." This suggests that ScPRT1 overexpression not only affects the ABA signaling pathway itself but may also synergistically participate in the plant's response to ABA by regulating the MAPK cascade.

[0098] Further screening and comparison of differentially expressed genes between wild-type and overexpressing lines under ABA treatment and control conditions were conducted. The C_WT vs C_OE group had 154 specific differentially expressed genes (DEGs), and the A_WT vs A_OE group had 128 specific differentially expressed genes, with 53 overlapping differentially expressed genes between the two groups. These 53 overlapping genes reflect the transcriptional changes common to ScPRT1 overexpression under ABA treatment conditions, implying that ScPRT1, as an E3 ligase, may degrade some positive regulators of the ABA signaling pathway, leading to the specific upregulation of some negative regulators of the ABA signaling pathway. Heatmap visualization of these 53 overlapping DEGs showed that negative regulators of ABA signaling were significantly upregulated, including AtERF4, AtWRKY33, and AtWRKY40. Among the 128 specifically expressed DEGs in A_WT vs A_OE, AtERF8 and AtPUB19 were upregulated, while stress response genes such as AtKIN1 were downregulated. These results suggest that ScPRT1 may negatively regulate ABA signal transduction by upregulating inhibitory factors in the ABA signaling pathway and downregulating stress-response genes.

[0099] In summary, the results of this embodiment show that overexpression of ScPRT1 significantly reduced the sensitivity of Arabidopsis to ABA at multiple developmental stages, weakening ABA-inhibited seed germination and taproot elongation, as well as ABA-induced stomatal closure and leaf senescence. ScPRT1, as a negative regulator of the ABA signaling pathway, attenuates the plant's response to ABA by regulating the expression of key transcription factors.

[0100] Example 3: Functional analysis of ScPRT1 transgenic *Rhizopus dentata* in response to ABA signaling

[0101] In this embodiment, the transient transformation system of *Brachys dentata* was used to obtain transiently transformed plants with ScPRT1 overexpression (OE) and RNA interference (RNAi), respectively. The growth phenotype, physiological and biochemical indicators and related gene expression changes under ABA treatment were analyzed, and the function of ScPRT1 in the ABA signal response of *Brachys dentata* was analyzed.

[0102] The gametophytes of *Rhizopus dentata* used in this embodiment were collected from the Gurbantünggüt Desert in Xinjiang, China (44°32′30″N, 88°6′42″E). Before the experiment, the gametophytes were washed to remove sand and planted on a fully moist peat substrate. They were cultured in an artificial climate incubator for 3-4 weeks under the following conditions: 12 h light (18°C) and 12 h dark (15°C).

[0103] 1. Constructing ScPRT1 overexpression and ScPRT1-RNAi interference vectors

[0104] Interference vectors were constructed using homologous recombination. Two pairs of homologous recombination primers (SEQ ID NO:3: CAATTACATTTACAATTACCATGGGCGCAGAACCCGATTCAC and SEQ ID NO:4: TCATCGATTGGGCGCGCCCCATGGCCAGGCCCAGACGAAGAT, SEQ ID NO:5: TTGGATCCTAGGTGAGTCTAGACCAGGCCCAGACGAAGAT and SEQ ID NO:6: GGGTCTTAATTAACTCTCTAGAGCGCAGAACCCGATTCAC) were designed to amplify the forward (cis) fragment. The primers for amplifying the forward (cis) fragment had overlapping arms at both ends, each approximately 15 bp homologous to the NcoI restriction site on the pFGC5941 vector. The primers for amplifying the reverse (anti) fragment had overlapping arms at both ends, each approximately 15 bp homologous to the XbaI restriction site.

[0105] Using the pBI121-ScPRT1 recombinant plasmid as a template, PCR amplification was performed using the homologous recombination primers containing homologous arms, yielding the forward target fragment and the reverse complementary target fragment of ScPRT1 containing the vector overlapping sequence. The PCR reaction system is shown in Table 1.

[0106] Table 1

[0107] Components volume cDNA 1 mL Forward Primer (10 mM) 1 mL Reverse Primer (10 mM) 1 mL 2×FastPfu PCR SuperMix (-dye) 10 mL <![CDATA[ddH2O]]> 7 mL Total 20 mL

[0108] Meanwhile, the pFGC5941 empty vector was digested with the restriction endonuclease NcoI (reaction system shown in Table 2) to obtain a linearized vector fragment for the first round of ligation.

[0109] Table 2

[0110] Components volume plasmid DNA 1 μg FlyCut NcoⅠ 1 mL 10×FlyCut buffer 2 mL <![CDATA[ddH2O]]> To 20 μL

[0111] The purified ScPRT1 forward fragment was ligated into the NcoI-linearized pFGC5941 vector. The ligation product was transformed into *E. coli* DH5α competent cells and plated on LB agar containing kanamycin, then incubated overnight at 37°C. The next day, single colonies were picked for colony PCR identification. Positive clones with correct band size and high brightness were sent for sequencing verification. After expansion culture of the correctly sequenced strains, plasmids were extracted to obtain the intermediate vector pFGC5941-ScPRT1-cis.

[0112] The pFGC5941-ScPRT1-cis intermediate vector was digested with the restriction endonuclease XbaI (digestion system as shown in Table 2) to obtain a linearized vector. The purified ScPRT1 inverse complementary fragment was homologously ligated into the linearized vector, and the resulting ligation was repeated in DH5α competent cells. After plating, single colonies were picked for colony PCR identification, and positive clones were sequenced. Finally, the ScPRT1 interference expression vector pFGC5941-ScPRT1-RNAi, with both the forward and inverse complementary fragments correctly inserted, was obtained.

[0113] 2. Obtaining plants of *Rhizopus dentata* by transient transformation with ScPRT1

[0114] pBI121-ScPRT1-GFP (preserved in the applicant's laboratory and constructed using standard methods) and the successfully constructed pFGC5941-ScPRT1 vector were transformed into Agrobacterium GV3101, respectively. After colony PCR identification, Agrobacterium culture containing the recombinant vector was obtained. The above culture was incubated overnight at 28°C until OD... 600=1.0, centrifuged at 4000 rpm for 5 min, and collected the bacterial pellet. The bacterial pellet was resuspended in a resuspension containing 1 / 2 MS, sucrose, and acetylsyleugenone, and cultured in the dark at 80 rpm for 2 h at 24℃. Healthy *Rhizoctonia solani* growing on the substrate was taken, washed with water, and then soaked in a resuspension of *Agrobacterium* containing the recombinant vector, and co-cultured at 80 rpm for 1 h at 24℃. After culturing, residual *Agrobacterium* on the *Rhizoctonia solani* was washed away with distilled water containing termethin, and the transformed *Rhizoctonia solani* was transferred to clean culture dishes and cultured in the dark for 24-48 h to obtain transiently transformed plants overexpressing ScPRT1 (OE) and ScPRT1-RNAi interference (RNAi).

[0115] 3. Identification of ScPRT1 transiently transformed *Rhizopus dentata* strains

[0116] To confirm the successful transformation of the exogenous vector into *Rhizoctonia solani*, DNA level identification was performed on the transiently transformed plants. Approximately 0.05–0.1 g of gametophytes from normally growing empty vector control (EV), ScPRT1 overexpressing (OE), and ScPRT1-RNAi interference (RNAi) lines were rapidly frozen in liquid nitrogen and then thoroughly ground into powder. Genomic DNA was extracted from each line using the CTAB method. Using the extracted genomic DNA as templates, PCR amplification was performed using pBI121 universal primers (for detecting EV and OE lines, SEQ ID NO:7: TTTCATTTGGAGAGAACACG and SEQ ID NO:8: CGACCAGGATGGGCACCAC) and pFGC5941 universal primers (for detecting RNAi lines, SEQ ID NO:9: CACGCTCGAGTATAAGAGCTC and SEQ ID NO:10: CTACCTTCCCACAATTCGTCGG) (reaction system referenced in Table 1). After the amplification products were separated by 1.0% agarose gel electrophoresis, they were observed and photographed in a gel imaging system. The presence or absence of the target band determined the positive transiently transformed plants.

[0117] The transcriptional level of the ScPRT1 gene in DNA-identified transiently transformed plants was detected by real-time quantitative PCR (RT-qPCR). Total RNA was extracted from the EV, OE, and RNAi lines of *Rhizopus dentata* according to the instructions of the total RNA extraction kit from Beijing TransGen Biotech Co., Ltd. The concentration and purity of RNA were determined using a nucleic acid protein analyzer, and equal amounts of total RNA were used to synthesize first-strand cDNA according to the reverse transcription kit instructions. Using the diluted cDNA as a template, RT-qPCR was performed using ScPRT1 gene-specific primers (SEQ ID NO:11: AACAGCGTGCCGTGTCTG and SEQ ID NO:12: TTCCTGCTGCTGTTGTAGC), with the *Rhizopus dentata* Scβ-Tubulin gene used as an internal control (SEQ ID NO:13: CGGTCATTACACCGTGGGAA and SEQ ID NO:14: CCTTCCAGCAACAGCGAA). The reaction was performed in an Applied Biosystems 7500 real-time quantitative PCR system, with three technical replicates and three independent biological replicates for each sample. The relative expression level of genes is measured using 2 -ΔΔCt The method was used to calculate and analyze the relative expression level changes of ScPRT1 in the OE group and RNAi group, using the EV group as the calibration sample.

[0118] The results are as follows Figure 11 As shown, the results indicated that the target band of the expected size could be amplified in the empty vector control group (EV), overexpression group (OE), and interference group (RNAi), while no band was observed in the wild type (WT), indicating that each vector had been successfully transformed into *Rhizopus dentata* (a type of thorny plant). Figure 11 (A) Compared with the EV group, the expression level of ScPRT1 was significantly increased in the OE group, while the expression level of ScPRT1 was decreased in the RNAi group. Figure 11 (B in the example) demonstrates that the *Rhizophora dentata* material with transient overexpression and interference of ScPRT1 has been successfully obtained and can be used for subsequent functional analysis.

[0119] 4. Phenotypic analysis of ScPRT1 transgenic *Rhizopus dentata* under ABA treatment

[0120] OE, RNAi, and EV lines with uniform growth were selected and treated with H2O, 50 μM ABA, 100 μM ABA, and 150 μM ABA for 3 days. The growth phenotypes of each line before and after treatment were observed and recorded. The results are as follows: Figure 12As shown, the results indicated that before treatment, all strains exhibited a bright green color and vigorous growth, showing a healthy state, with no significant phenotypic differences among EV, RNAi, and OE strains under different ABA concentrations. After treatment with different concentrations of ABA, the sensitivity of each strain to ABA varied significantly. Under control conditions, all strains remained green. Under ABA treatment, the yellowing of the OE strain was much less than that of the EV and RNAi strains, while the yellowing of the RNAi strain was more pronounced than that of the EV and OE strains. This suggests that the expression level of ScPRT1 regulates the sensitivity of *Brachys dentata* to ABA.

[0121] 5. Chlorophyll content of ScPRT1 transgenic *Rhizopus dentata* under ABA treatment

[0122] Gametophytes of *Brachys dentata* strains treated with H2O, 50 μM ABA, 100 μM ABA, and 150 μM ABA for 3 days (empty control (EV), ScPRT1 overexpression (OE), and ScPRT1-RNAi interference (RNAi)) were placed into 2 mL centrifuge tubes, 2 mL of 95% ethanol solution was added, the tubes were sealed, wrapped in aluminum foil, and extracted at room temperature in the dark for 24 h until the gametophytes turned pale green. The absorbance of the extract was measured at wavelengths of 665 nm and 649 nm, and the contents of chlorophyll a, chlorophyll b, and total chlorophyll were calculated.

[0123] Total RNA was extracted from each line after ABA treatment and reverse transcribed into cDNA. The expression levels of chlorophyll degradation-related genes ScNYE1 (primers SEQ ID NO:15: GCGATTGGAGGAAATGGAGAA and SEQ ID NO:16: GTCTTGGGCAGTGGAATCA) and ScNYC1 (primers SEQ ID NO:17: GACAACTTTCGGCGTCTTTG and SEQ ID NO:18: CGAGGAGCAGATCAGTAAGAAC) were detected by RT-qPCR. Scβ-Tubulin was used as an internal reference gene, and the expression levels were calculated using a 2-1... -ΔΔCt Law.

[0124] The results are as follows Figure 13As shown, the results indicated that with increasing ABA concentration, the contents of chlorophyll a, chlorophyll b, and total chlorophyll in all lines decreased, but the magnitude of the decrease varied significantly among different lines. Under control conditions, there was no significant difference in chlorophyll content among the EV, RNAi, and OE lines. At 50 μM ABA treatment, chlorophyll content began to differentiate among the lines, with the OE line slightly higher than the EV and RNAi lines. When the ABA concentration increased to 100 μM and 150 μM, the contents of chlorophyll a, chlorophyll b, and total chlorophyll in the OE line were significantly higher than those in the EV line, while the contents of all chlorophyll contents in the RNAi line were significantly lower than those in the EV and OE lines. Under ABA treatment, the expression levels of ScNYE1 and ScNYC1 in the OE line were significantly lower than those in the EV line, while the expression levels of these two genes were significantly upregulated in the RNAi line. This suggests that ScPRT1 overexpression may slow down ABA-induced chlorophyll degradation by inhibiting the transcriptional upregulation of chlorophyll degradation genes.

[0125] 6. Reactive oxygen species staining of transgenic tooth-ribbed erythroplasia under ABA treatment

[0126] (1) NBT and DAB staining

[0127] Gametophytes of *Bryum dentata* strains treated with different concentrations of ABA were immersed in 1 mg / mL NBT staining solution and 1 mg / mL DAB staining solution, respectively. The samples were incubated at 37°C in the dark for 24 h, gently shaking several times during incubation to ensure uniform staining. After incubation, the staining solution was discarded, and 95% ethanol + 5% acetic acid was added. The samples were then decolorized in a boiling water bath for 10-15 min to remove chlorophyll until the material was completely decolorized. The decolorized samples were observed and photographed under a stereomicroscope.

[0128] NBT staining is used to detect superoxide anion (O2). •- The formation of H2O2 is observed, and DAB staining is used to detect the accumulation of H2O2. The staining results are as follows: Figure 14 As shown, the results indicated that under control conditions, the EV, RNAi, and OE lines all stained lightly, with no significant difference among them. With increasing ABA treatment concentration, the staining depth of all lines gradually increased, but there were significant differences in staining intensity among different lines. Under ABA treatment, the OE line showed the lightest NBT and DAB staining, the RNAi line showed the deepest staining, and the EV line was in the middle. These results suggest that overexpression of ScPRT1 can effectively alleviate ABA-induced superoxide anion and hydrogen peroxide accumulation.

[0129] (2) Staining with DHE and H2DC-FDA fluorescent probes

[0130] Gametophytes of each ABA-treated strain of *Bryum dentata* were placed in 10 μM DHE working solution (diluted with 10 mM phosphate buffer) and 50 μM H2DC-FDA working solution, respectively, and incubated at 37°C in the dark for 30 min. After incubation, the samples were washed three times with phosphate buffer for 5 min each time to remove background fluorescence. The treated samples were then subjected to fluorescence imaging on a Tianneng 5200 multifunctional imaging system, and the relative fluorescence intensity was calculated using ImageJ software.

[0131] The DHE fluorescent probe can specifically detect intracellular superoxide anion levels, while the H2DC-FDA probe is used to detect the overall levels of intracellular hydrogen peroxide and reactive oxygen species. Results are as follows: Figure 15 As shown, the results indicated that under control conditions, all lines exhibited weak background fluorescence. After ABA treatment, the fluorescence intensity of all lines increased, but the RNAi line showed the highest fluorescence intensity, while the OE line showed the lowest. Relative fluorescence intensity statistics showed that under 100 μM and 150 μM ABA treatments, the fluorescence intensities of DHE and H2DC-FDA in the RNAi line were significantly higher than those in the EV line, while the fluorescence intensity of the OE line was significantly lower than that in the EV line. These results suggest that ScPRT1 can reduce ABA-induced ROS accumulation by attenuating the plant's response to ABA.

[0132] 7. Detection of physiological indicators of transgenic *Erythrosporium dentatum* under ABA treatment

[0133] Approximately 0.1 g of gametophytes from *Brachys dentata* strains treated with H2O, 50 μM ABA, 100 μM ABA, and 150 μM ABA (empty vector control, EV, ScPRT1 overexpression, OE, and ScPRT1-RNAi interference, RNAi) were rapidly frozen in liquid nitrogen and thoroughly ground into powder. Tissue homogenates were prepared by adding the corresponding extraction solutions. The contents of hydrogen peroxide (H2O2), malondialdehyde (MDA), proline (Pro), and soluble sugars (SS) were determined according to the instructions of the corresponding commercially available kits provided by Nanjing Jiancheng Biotechnology Research Institute.

[0134] The results are as follows Figure 16As shown, the results indicated that under control conditions, there was no significant difference in H2O2 content among the different lines. With increasing ABA treatment concentration, the H2O2 content of all lines showed an increasing trend, but the RNAi line showed the largest increase, while the OE line showed the smallest increase. Under ABA treatment, the H2O2 content of the RNAi line was significantly higher than that of the EV line, while the H2O2 content of the OE line was significantly lower than that of the EV line. MDA content, as an indicator of membrane lipid peroxidation, showed a trend consistent with H2O2. Under high ABA treatment, the RNAi line had the highest MDA accumulation, while the OE line had the lowest, indicating that overexpression of ScPRT1 can effectively alleviate ABA-induced membrane lipid peroxidation damage, while interfering with its expression exacerbates membrane system damage. Proline and soluble sugars, as important osmotic regulators, play crucial roles in plant responses to abiotic stress. After ABA treatment, the Pro and SS contents of all lines increased, but the accumulation levels differed significantly among different lines. Under ABA treatment, the Pro and SS contents of the RNAi line were significantly lower than those of the EV and OE lines, while the Pro and SS contents of the OE line were higher than those of the EV line, indicating that overexpression of ScPRT1 can alleviate the damage caused by excessive ABA to plants. These results suggest that ScPRT1 can reduce the sensitivity of *Brachys dentata* to ABA and decrease oxidative stress caused by excessive ABA.

[0135] Based on the results of this embodiment, overexpression of ScPRT1 enhanced the tolerance of *Rhizoctonia solani* to ABA (ROS staining and physiological index measurements confirmed that ScPRT1 overexpression effectively alleviated ABA-induced oxidative stress and protected cell membrane integrity), while interference with ScPRT1 expression made *Rhizoctonia solani* highly sensitive to ABA (exhibiting severe yellowing and wilting, rapid chlorophyll degradation, reactive oxygen species bursts, and aggravated membrane system damage). Expression analysis of the chlorophyll degradation genes ScNYE1 and ScNYC1 further revealed that ScPRT1 delays ABA-induced chlorophyll degradation by inhibiting the transcriptional upregulation of these genes. ScPRT1 negatively regulates the response of *Rhizoctonia solani* to ABA signaling, acting as a negative regulator of the ABA signaling pathway and finely modulating the intensity of ABA signaling.

[0136] Example 4: Identification of ScPRT1 substrate proteins and analysis of degradation mechanisms

[0137] This embodiment utilizes yeast two-hybrid technology, using ScPRT1 as bait to screen a *Rhizopus dentata* cDNA library, obtaining 71 candidate interacting proteins. Based on this, the following experiments were conducted:

[0138] 1. Through point-to-point verification with yeast, it was found that ScPRT1 interacts with ScHDA5, ScPYL4, and ScPYL6 in yeast, while other potential proteins do not interact. However, after the addition of ABA, the interaction between ScPRT1 and ScPYL4 and ScPYL6 is stronger, indicating that ABA can promote the interaction between ScPRT1 and ScPYL4 and ScPYL6.

[0139] 2. Verification via bimolecular fluorescence complementarity (BiFC): Using homologous recombination technology, the full-length sequences of ScPRT1 (with stop codons removed) and ScPYL4 and ScPYL6 were constructed into pXY106-nYFP and pXY104-cYFP vectors, respectively, and transformed into Agrobacterium GV3101 (pSoup-p19). ScPRT1, ScPYL4, and ScPYL6 were transiently expressed in tobacco leaves using recombinant Agrobacterium GV3101 (pSoup-p19). Tobacco leaves injected with Agrobacterium scPYL4 / PYL6-pXY106 and ScPRT1-pXY104 emitted green fluorescence under 488 nm excitation light, and the positions of the green fluorescence and the blue fluorescence of the nuclear dye DAPI overlapped, confirming that ScPRT1 interacts with ScPYL4 and ScPYL6 in the tobacco cell nucleus.

[0140] 3. Validation using luciferase complementarity imaging (LCA): ScPRT1 was fused with cLUC, and ScPYL4 and ScPYL6 were fused with nLUC, respectively. These were co-injected into tobacco leaves using Agrobacterium. After 48 h, the leaves were treated with either buffer (-ABA) or buffer containing 100 μMABA (+ABA), and luminescence imaging was performed. Results showed that, without ABA, luminescence signals were detected only in the regions co-injected with nLUC-ScPYL4+cLUC-ScPRT1 and nLUC-ScPYL6+cLUC-ScPRT1, indicating a specific interaction between ScPRT1 and ScPYL4 / ScPYL6 in the plant. After ABA treatment, the luminescence signal in the experimental groups was significantly enhanced, indicating that ABA promotes the interaction between ScPRT1 and ScPYL4 / ScPYL6.

[0141] 4. Validation via GST Pull-down: Purified GST-ScPRT1 was co-incubated with MBP-ScPYL4 or MBP-ScPYL6, using GST and MBP as negative controls. After precipitating the complex using GST affinity resin, Western blot analysis was performed using Anti-GST and Anti-MBP antibodies. Results showed that all proteins in the Input group were expressed normally. In the Pull-down group, only the lanes co-incubated with GST-ScPRT1 and MBP-ScPYL4 showed a MBP-ScPYL4 band, while no corresponding bands were observed in the GST+MBP-ScPYL4 and GST-ScPRT1+MBP control lanes. The results for ScPRT1 and ScPYL6 were consistent, with a MBP-ScPYL6 band detected only in the lanes co-incubated with GST-ScPRT1 and MBP-ScPYL6. Anti-GST analysis showed that the bait proteins in each lane were effectively enriched. The results showed that ScPRT1 can interact specifically and directly with ScPYL4 and ScPYL6 in vitro.

[0142] 5. Validation by co-immunoprecipitation (Co-IP): ScPRT1-Myc was co-expressed with either ScPYL4-Flag or ScPYL6-Flag in leaves of *Nicotiana benthamiana*, with an empty vector (Flag) as a negative control. After total protein extraction, immunoprecipitation was performed using Flag magnetic beads, followed by Western blot analysis with Anti-Myc and Anti-Flag antibodies. Results showed that in the Input group, ScPRT1-Myc was expressed in both the co-expressed ScPYL4-Flag and empty vector control samples. ScPYL4-Flag was detected only in the co-expression group, while the empty vector control showed no band. In the IP products, Anti-Flag detection showed successful precipitation of ScPYL4-Flag with the Flag antibody, while Anti-Myc detection showed that the ScPRT1-Myc band was detected only in the co-expressed ScPYL4-Flag sample, and no band was found in the empty vector control group, indicating a specific interaction between ScPRT1 and ScPYL4 in the plant. The Co-IP results for ScPRT1 and ScPYL6 were consistent with this, with the ScPRT1-Myc band detected only in the IP product co-expressing ScPYL6-Flag. These results indicate that ScPRT1 interacts with both ScPYL4 and ScPYL6 in plants.

[0143] 6. Interaction site prediction of ScPRT1 and ScPYL4 / ScPYL6: The structure of the ScPRT1-ScPYL4 and ScPRT1-ScPYL6 complexes was predicted using AlphaFold3, and the visualization analysis was performed using PyMOL software. ScPRT1 and ScPYL4 bind at multiple positions, including 15 GLU(ScPRT1)-181 LYS(ScPYL4), 37 SER(ScPRT1)-65ARG(ScPYL4), 202SER(ScPRT1)-3GLU(ScPYL4), and 226 THR(ScPRT1)-4 LYS(ScPYL4); ScPRT1 and ScPYL6 bind at multiple positions, including 42 THR(ScPRT1)-174 LYS(ScPYL6), 202 SER(ScPRT1)-3 PHE(ScPYL6), 204GLY(ScPRT1)-1MET(ScPYL6), and 230 ASP(ScPRT1)-2ARG(ScPYL6). These predictions provide a theoretical basis for subsequent verification of key interaction sites and analysis of the molecular mechanism by which ScPRT1 regulates the ABA signaling pathway through site-directed mutagenesis.

[0144] 7. Analysis of ScPRT1 and Substrate Protein Gene Transcriptional Levels: Wild-type *Brachys dentata* was treated with 100 μM ABA, and samples were taken at 0, 0.5, 1.5, 3, 6, 12, 24, and 48 h. The relative expression levels of the three genes were detected by RT-qPCR. The results showed that the expression of all three genes initially increased and then decreased, without any sustained dramatic upregulation or complete silencing. This suggests that the regulation of ScPRT1, ScPYL4, and ScPYL6 expression levels may primarily occur at the post-translational level, rather than the transcriptional level.

[0145] 8. ScPRT1 Self-Ubiquitination and In Vitro Ubiquitination: Purified His-tagged ScPRT1 protein was incubated with E1, E2, and ubiquitin (Ub) in a reaction system. Ubiquitination modification was detected by anti-Ub antibody, and the amount of E3 protein loaded was detected by anti-His antibody. Results showed that in the intact reaction system, anti-Ub detected high-molecular-weight ladder-like and tailed bands. No obvious ubiquitination bands were detected when E1, E2, or Ub were missing. This indicates that ScPRT1 is a functional E3 ubiquitin ligase with complete enzymatic activity. Purified MBP-tagged ScPYL4 or ScPYL6 protein was co-incubated with E1, E2, Ub, and ScPRT1. Substrate ubiquitination modification was detected by anti-Ub antibody, the amount of substrate protein loaded was detected by anti-MBP antibody, and the addition of E3 protein was detected by anti-His antibody. The results showed that both ScPYL4 and ScPYL6 exhibited distinct high-molecular-weight ladder-like and tailed bands in the intact reaction system, indicating that ScPRT1 successfully catalyzed the ubiquitination modification of ScPYL4 and ScPYL6. These results demonstrate that ScPRT1 possesses E3 ubiquitin ligase activity, is capable of autoubiquitination, and can ubiquitinate ScPYL4 and ScPYL6 in vitro.

[0146] 9. Detection of ScPRT1 ubiquitination degradation in vivo: ScPYL4 and ScPYL6 were fused with LUC to construct ScPYL4-LUC and ScPYL6-LUC reporter vectors, respectively. These vectors were co-transformed into *Nicotiana benthamiana* leaves with GFP-ScPRT1 or an empty GFP vector. An MG132 treatment group was also included. The accumulation levels of ScPYL4 and ScPYL6 proteins were reflected by the luminescence intensity of LUC. Results showed that compared with the control group expressing LUC fusion proteins alone, LUC fluorescence decreased when ScPRT1-GFP was co-expressed with ScPYL4-LUC or ScPYL6-LUC, indicating that ScPRT1 promotes the degradation of ScPYL4 and ScPYL6 in vivo. After MG132 treatment, the LUC signal intensity was enhanced in samples co-expressing ScPRT1-GFP and ScPYL4-LUC / ScPYL6-LUC, indicating that this degradation process was inhibited by the proteasome inhibitor MG132. This indicates that ScPRT1 ubiquitinates ScPYL4 and ScPYL6 in vivo and targets and degrades ScPYL4 and ScPYL6 via the 26S proteasome pathway.

[0147] 10. ScPRT1-mediated stability assay of ScPYL4 / ScPYL6 proteins: ScPYL4-Flag or ScPYL6-Flag was co-expressed with ScPRT1-Myc or the empty vector (EV) in *Nicotiana benthamiana* leaves, and treated with MG132, CHX, or H2O, respectively. Substrate protein accumulation levels were detected by Western blot. Results showed that co-expression of ScPRT1-Myc significantly reduced ScPYL4-Flag protein accumulation, while MG132 treatment restored protein accumulation. Co-expression of ScPRT1-Myc with ScPYL6-Flag reduced ScPYL6-Flag protein accumulation, showing the same trend as ScPYL4-Flag. This further demonstrates that ScPRT1 can mediate the protein stability of ScPYL4 and ScPYL6 via the 26S proteasome pathway.

[0148] 11. Detection of ScPRT1-mediated degradation rate of ScPYL4 / ScPYL6 proteins: ScPYL4-Flag or ScPYL6-Flag was co-expressed with ScPRT1-Myc or the empty vector (EV) in *Nicotiana benthamiana* leaves. After CHX treatment to inhibit new protein synthesis, samples were collected at 0, 1.5, 3, 6, and 12 h, and the expression levels of the substrate proteins were detected by Western blot. The results showed that in the EV control group, the ScPYL4-Flag protein content was basically stable, indicating that ScPYL4 protein was highly stable and degraded slowly without ScPRT1. After co-expression of ScPRT1-Myc, the ScPYL4-Flag protein content decreased over time, indicating that ScPRT1 significantly accelerated the degradation of ScPYL4. ScPYL6-Flag showed the same trend. The results indicate that ScPRT1 significantly shortens the protein half-life of ScPYL4 and ScPYL6 by promoting their degradation.

[0149] Based on the results of this embodiment, the ABA receptors ScPYL4 / ScPYL6 were identified as ubiquitinated degradation substrates of ScPRT1, and the molecular mechanism by which ScPRT1 negatively regulates the ABA signaling pathway by targeting and degrading the ABA receptor was initially revealed.

[0150] Example 5: ABA signaling response function analysis of ScPRT1 substrate proteins ScPYL4 and ScPYL6

[0151] This embodiment performs bioinformatics analysis on ScPYL4 and ScPYL6 and transiently transforms them into *Bryophyta erythroptera* to elucidate the functions of ScPYL4 and ScPYL6 in the ABA signaling pathway, providing new insights into the evolution and function of PYL receptors in bryophytes.

[0152] 1. Bioinformatics analysis of ScPYL4 and ScPYL6 of *Rhizopus dentata*

[0153] Amino acid sequences of ScPYL4 and ScPYL6 were obtained from the *Rhizopus dentata* T2T genome database. PYL homologous protein sequences from *Arabidopsis thaliana*, rice (*Oryzasativa*), maize (*Zea mays*), soybean (*Glycine max*), and *Marchantia polymorpha* were downloaded from the NCBI database (https: / / www.ncbi.nlm.nih.gov / ). Multiple sequence alignment of *Rhizopus dentata* ScPYL4 and ScPYL6 sequences, as well as PYL protein sequences from other species, was performed using DNAMAN software, with alignment parameters set to default values. The ScPYL4 / ScPYL6 protein sequences were aligned online using the NCBI Conserved Domains Database (CDD, https: / / www.ncbi.nlm.nih.gov / Structure / cdd / cdd.shtml) to verify the integrity of the PYR_PYL_RCAR_like domain. The alignment results were imported into MEGA software, and a phylogenetic tree was constructed using the neighbor-joining method. The PYL4 and PYL6 protein sequences of all drought-tolerant plants were obtained from the website of drought-tolerant plants (http: / / desiccation.novogene.com / ), and domain analysis was performed in Tbtools software. Subsequently, a phylogenetic tree was constructed in MEGA software.

[0154] The results are as follows Figure 17 As shown, the results indicate that ScPYL4 is highly conserved in key regions compared to PYL4 proteins from other species, and ScPYL6 also exhibits highly homologous sequence regions. Conserved domain predictions show that ScPYL4 and ScPYL6, like other higher plants, contain complete PYR_PYL_RCAR_like domains. Phylogenetic trees show that PYL4 in angiosperms (At, Os, Zm, Gm) clusters into one branch, while PYL4 in bryophytes / mosses / liverworts (Mp, Pp, Sc) clusters into another independent branch. The phylogenetic tree for PYL6 shows a similar pattern. This reflects the early evolutionary differentiation between terrestrial and vascular plants, suggesting that the ABA receptor in bryophytes may have originated from an independent expansion event, forming a bryophyte-specific subfamily.

[0155] 2. Subcellular localization of ScPYL4 and ScPYL6 in *Erythrina variegata*

[0156] Overexpression vectors PHG-ScPYL4-EGFP and PHG-ScPYL6-EGFP for ScPYL4 and ScPYL6 respectively were constructed. PHG-EGFP served as an empty vector control and was mixed in equal volumes with labeling vectors CBL (plasma membrane localization) and HY5 (nuclear localization), and injected into tobacco leaves of appropriate age. Temporary slides were prepared from the lower epidermis of tobacco leaves 48 h after transformation, and observed and images were acquired using a laser scanning confocal microscope to analyze the colocalization relationship between the target protein and the subcellular label.

[0157] The results are as follows Figure 18 As shown, the results indicated that in the control group, free EGFP green fluorescence diffusely distributed throughout the cell without specific localization. The dotted fluorescence signal of EGFP-ScPYL4 completely overlapped with the HY5-RFP-labeled nucleus, while the diffuse signal was distributed in the cytoplasm, partially overlapping with CBL plasma membrane labeling. This suggests that ScPYL4 is localized in both the nucleus and cytoplasm. The results for EGFP-ScPYL6 were similar to those for EGFP-ScPYL4. These results demonstrate that both ScPYL4 and ScPYL6 are localized in both the nucleus and cytoplasm.

[0158] 3. Verification of two-hybrid yeast strains ScPYL4 and ScPYL6

[0159] ScPYL4 was constructed into the pGBKT7 vector, and ScPYL6 was constructed into the pGADT7 vector. These were co-transformed into the Y2HGold yeast strain and screened on SD / -Trp / -Leu, SD / -Trp / -Leu / -His, and SD / -Trp / -Leu / -His / -Ade / X-α-Gal media, respectively. pGBKT7-p53+pGADT7-T served as the positive control, pGBKT7-Lam+pGADT7-T as the negative control, and pGBKT7-ScPYL4+pGADT7 as the self-activation group.

[0160] The results are as follows Figure 19 As shown, the results indicated that all transformation combinations grew normally on double-deficient medium, indicating successful co-transformation. On triple-deficient and quadruple-deficient medium, the positive control grew normally and showed a blue color, while the negative control and the self-activated group showed no colony growth, proving the effectiveness of the experimental system and that ScPYL4 has no self-activation activity. The experimental group pGBKT7-ScPYL4+pGADT7-ScPYL6 showed no colony growth on triple-deficient and quadruple-deficient medium, and no blue color reaction, indicating that ScPYL4 and ScPYL6 do not directly interact in yeast and do not form a dimer structure.

[0161] 3. Obtaining and identifying the ScPYL4 and ScPYL6 transiently transformed *Rhizopus dentata* strains.

[0162] Overexpression vectors of ScPYL4 and ScPYL6 (PHG-ScPYL4-EGFP and PHG-ScPYL6-EGFP) and RNAi interference vectors (pFGC5941-ScPYL4 and pFGC5941-ScPYL6) were constructed and transiently transformed into gametophytes of *Brachys dentata* using Agrobacterium-mediated transformation to obtain empty vector control (EV), ScPYL4 overexpression (OE-ScPYL4), ScPYL6 overexpression (OE-ScPYL6), ScPYL4 interference (RNAi-ScPYL4), and ScPYL6 interference (RNAi-ScPYL6) lines. Genomic DNA was extracted from each line for PCR identification, and total RNA was extracted for RT-qPCR to detect the expression level of the target genes.

[0163] DNA level identification and RT-qPCR detection results are as follows: Figure 20 As shown, the results indicated that the EV, OE-ScPYL4, OE-ScPYL6, RNAi-ScPYL4, and RNAi-ScPYL6 lines all amplified specific bands of the expected size, indicating that each recombinant vector had been successfully transformed into *Rhizopus dentata*. Compared with the EV control, the expression levels of the target gene were increased in the OE-ScPYL4 and OE-ScPYL6 lines, while the expression levels of the target gene were decreased in the RNAi-ScPYL4 and RNAi-ScPYL6 lines.

[0164] 4. Reactive oxygen species staining of ScPYL4 and ScPYL6 transgenic *Erythrina variegata* under ABA treatment

[0165] Transiently transformed lines (EV, OE-ScPYL4, OE-ScPYL6, RNAi-ScPYL4, RNAi-ScPYL6) were treated with different concentrations of ABA, and ROS accumulation in each line was detected by histochemical staining and fluorescence staining. Results are as follows: Figure 21 and Figure 22 As shown, histochemical staining revealed that all lines stained more deeply with increasing ABA concentration. Under ABA treatment, the OE-ScPYL4 and OE-ScPYL6 lines showed the deepest DAB and NBT staining, indicating that overexpression of ScPYL4 and ScPYL6 significantly promoted ABA-induced ROS accumulation. DHE staining showed that after ABA treatment, the OE line exhibited the strongest red fluorescence, the RNAi line the weakest, and EV in the middle. H2DC-FDA staining showed that the OE line exhibited the strongest green fluorescence, the RNAi line the weakest, and EV in the middle. These results indicate that lines overexpressing ScPYL4 and ScPYL6 in *Brachys dentata* are sensitive to ABA treatment.

[0166] 5. Physiological parameters of ScPYL4 and ScPYL6 transgenic *Rhizopus dentata* under ABA treatment

[0167] Different concentrations of ABA were applied to each transiently transformed line, and the contents of malondialdehyde (MDA), hydrogen peroxide (H2O2), proline (Pro), and soluble sugars (SS) were measured. The results are as follows: Figure 23 As shown, the results indicated that there were no significant differences in any physiological indicators among the strains under the control conditions. After ABA treatment, the MDA and H2O2 contents of the OE-ScPYL4 strain were higher than those of the RNAi-ScPYL4 and EV strains; the Pro and SS contents of the RNAi-ScPYL4 strain were higher than those of the EV and OE-ScPYL4 strains. The results for the ScPYL6 strains after ABA treatment were similar to those for ScPYL4. These results suggest that ScPYL4 and ScPYL6 can enhance the sensitivity of *Rhizopus dentata* to ABA and increase the oxidative stress caused by ABA overdose.

[0168] The results of this embodiment indicate that ScPYL4 and ScPYL6, as positive regulators of the ABA signaling pathway in *Rhizoctonia solani*, function in the ABA response of *Rhizoctonia solani*. This suggests that ScPRT1 may negatively regulate the ABA signaling pathway by degrading the two positive regulators ScPYL4 and ScPYL6. ScPRT1 plays a crucial role in the stress adaptation and growth balance of *Rhizoctonia solani* by finely regulating the intensity and duration of ABA signaling through the stability of the ABA receptors ScPYL4 and ScPYL6 proteins.

[0169] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. Application of the ScPRT1 gene of *Bryum dentata* in regulating plant response to abscisic acid, wherein the nucleotide sequence of the ScPRT1 gene of *Bryum dentata* is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that, The plant is a moss or Arabidopsis thaliana; preferably, the plant is *Bryophytum gracilis*.

3. Application of the protein encoded by the ScPRT1 gene of *Bryum dentata* in regulating the plant response to abscisic acid, the amino acid sequence of which is shown in SEQ ID NO:

2.

4. The application according to claim 3, characterized in that, The plant is a moss or Arabidopsis thaliana; preferably, the plant is *Bryophytum gracilis*.

5. The application of an overexpression vector or a transient expression vector of the ScPRT1 gene of *Rhizopus dentata* in improving the plant response to abscisic acid, wherein the nucleotide sequence of the ScPRT1 gene of *Rhizopus dentata* is shown in SEQ ID NO:

1.

6. The application according to claim 5, characterized in that, The plant is a moss or Arabidopsis thaliana; preferably, the plant is *Bryophytum gracilis*.

7. The application of engineered bacteria transformed with an overexpression vector of the ScPRT1 gene of *Rhizopus dentata* or an engineered bacteria transformed with a transient expression vector of the ScPRT1 gene of *Rhizopus dentata* in improving the plant response to abscisic acid, wherein the nucleotide sequence of the ScPRT1 gene of *Rhizopus dentata* is shown in SEQ ID NO:

1.

8. The application according to claim 7, characterized in that, The plant is a moss or Arabidopsis thaliana; preferably, the plant is *Bryophytum gracilis*.

9. A method for improving the plant response to abscisic acid, characterized in that, Includes the following steps: To increase the expression level of the ScPRT1 gene in *Bryophytum comosum*, the nucleotide sequence of which is shown in SEQ ID NO:

1.

10. The method according to claim 9, characterized in that, The plant is a moss or Arabidopsis thaliana; preferably, the plant is *Bryophytum gracilis*.

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