Application of Pucipk11 gene in regulating drought resistance of plants

Abstract

The application relates to the technical field of genetic engineering, and particularly relates to PuCIPK11 Application of a gene in regulating drought resistance of plants. PuCIPK11 The gene plays a key role in enhancing drought resistance by regulating stomatal closure, and overexpression PuCIPK11 The gene can promote the depolymerization of the microfilament of the stomatal pore from a radial structure and reconfigure into a longitudinal structure, thereby accelerating the stomatal closure and enhancing the drought resistance. PuCIPK11 The gene is disclosed as a key regulating factor of the drought resistance of poplar, and provides a potential target for genetic improvement of the poplar.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to... PuCIPK11 Application of genes in regulating plant drought resistance. Background Technology

[0002] Abiotic stresses such as drought, salinity, and low temperature severely limit plant growth and development. Nearly 50% of the annual yield loss of major crops worldwide is related to abiotic stress. Understanding the physiological and biochemical responses of plants under abiotic stress is an important indicator for evaluating the effectiveness of crop resistance to abiotic stress. Poplar is one of the main tree species for plantation in my country, possessing significant economic and ecological value. However, compared to other tree species, poplar is a high water-consuming species. Large-scale planting of poplar plantations in water-scarce arid and semi-arid regions not only results in low tree survival rates but may also exacerbate regional water shortages. Therefore, cultivating new poplar varieties that combine strong drought resistance with high water use efficiency is an urgent problem to be solved to ensure the success of plantation construction and ecological security in my country.

[0003] The study of stomatal movement mechanisms has always been a focus of academic attention. In recent years, key signaling nodes in the stomatal regulatory network have been continuously discovered and identified. These elements, through multiple levels such as subcellular localization and migration, post-translational protein modification, intracellular redox homeostasis changes, ion channel opening and closing, and gene transcription regulation, achieve precise regulation of plant stomata under specific habitat and continuously fluctuating environmental factors. At the same time, studies have also shown that temporal and spatial control of certain signaling elements through genetic engineering can improve crop yield and stress tolerance.

[0004] The core driving force behind stomatal movement comes from the dynamic remodeling of the microfilament and microtubule cytoskeleton within guard cells. Microfilaments (F-actin), as an important component of the plant cytoskeleton, are polymerized from actin and widely participate in physiological processes such as cell morphology maintenance, substance transport, and signal transduction. In Arabidopsis thaliana… Arabidopsis thaliana In plants such as [list of plants], the mechanism by which microfilaments regulate stomatal opening and closing is relatively well understood: when stomata are fully open, the microfilaments within guard cells are highly bundled and distributed radially from the inner wall to the outer wall; while when stomata are closed, the microfilaments are mostly arranged along the long axis of the cell or are randomly distributed. This indicates that the aggregation dynamics and spatial conformation changes of the guard cell microfilament cytoskeleton are key links in regulating stomatal movement, and changes in these two dimensions may involve the functional synergy of a series of upstream and downstream signaling elements. Although there is substantial evidence that the microfilament cytoskeleton is an important node in the stomatal regulatory network, its molecular regulatory mechanisms still require further in-depth research.

[0005] Various abiotic stress signals (ABA, drought, high salinity, etc.) induce calcium oscillations in the cytoplasm of guard cells. Under these stimuli, calcium ions participate in multiple signal transduction processes in the guard cell response, acting as a crucial second messenger. Changes in calcium ion concentration can regulate the activity of various ion channels. For example, high intracellular calcium ion concentrations stimulate the activation of calcium-dependent protein kinases (CPKs), thereby regulating the activity of other potassium and anion channels and triggering stomatal closure. Changes in calcium ion concentration in guard cells mainly depend on the action of calcium channel proteins and calcium transporters on the cell membrane and vacuolar membrane. Cyclic nucleotide-gated calcium channels (CNGCs) on the guard cell membrane serve as the link between cyclic nucleotides and calcium signals, participating in multiple calcium-related physiological processes. Studies have shown that multiple CNGC family members (CNGC5, CNGC6, CNGC9, and CNGC12) exist in Arabidopsis stomatal guard cells, forming calcium ion channels in a functionally redundant manner. These channels respond to ABA signals induced by various stress environments, dynamically regulating stomatal opening and closing movements by modulating intracellular calcium ion signals, thus participating in plant responses to biotic and abiotic stresses. Vacuoles are the largest calcium reservoir in guard cells, and their cell membranes contain abundant calcium ion channels and transport proteins. For example, TPC1 (Two Pore Calcium Channel 1) is a non-selective cation channel protein in the guard cell vacuolar membrane. Increased calcium ion concentration can activate TPC1, triggering calcium ion efflux and vacuolar membrane depolarization, further promoting TPC1 opening in a positive feedback manner.

[0006] Members of the calcium signal decoding group play a deeper regulatory role in the calcium signal transduction pathway. Plant calcium... 2+ Sensors are mainly classified into three categories: calmodulin (CaM) and calmodulin-like (CML) families, calcium-dependent protein kinases (CDPKs), and calmodulin B-like proteins (CBL). CBL interacts with CBL-like protein kinases (CIPKs) to form a functional complex. When CBL binds to CIPK, the self-inhibiting NAF domain (named for its characteristic amino acids N, A, and F) dissociates from the kinase domain, thus forming the enzyme's active conformation. This CBL / CIPK network characteristic is thought to facilitate efficient signal channeling in calcium signal transduction. Studies have found that the CBL / CIPK complex plays a role in various physiological processes, including ion transport regulation, osmotic regulation, and stress responses such as drought. For example, salt stress-induced calcium... 2+ Transiently increased phosphorylation activity of the Arabidopsis thaliana AtCBL4-AtCIPK24 complex, activating plasma membrane (PM)-localized Na++ / H + The antitransferor AtSOS1 leads to Na + It is excreted from the root into the environment, and Na+ is released therefrom. + Loaded into the xylem for long-distance transport. Similarly, the AtCBL1 / AtCBL9-AtCIPK23 composite is subjected to different external K... + At concentrations, via phosphorylation of K + Transporters HAK5 or AKT1 positively regulate potassium in Arabidopsis roots. + The uptake of iron is also involved. Furthermore, this complex participates in the iron starvation response, affecting iron absorption by regulating the phosphorylation and degradation of the IRT1 protein. In drought resistance mechanisms, CIPK proteins directly participate in stomatal movement by regulating cytoskeleton dynamics. Recent studies have revealed that in Arabidopsis thaliana... AtCIPK20 Under drought stress, stomatal closure is mediated by regulating microtubule stability. This study found that AtCIPK20 interacts with microtubule-binding proteins and stabilizes microtubule structure through phosphorylation, thereby promoting stomatal closure, reducing water loss, and enhancing plant drought resistance. This mechanism directly links calcium signaling with cytoskeleton remodeling, elucidating a novel pathway of action of the CBL / CIPK module in drought response. Besides stress response, CBL / CIPK also participates in plant development processes, such as CBL2 / 3-CIPK12 regulating pollen tube growth. CIPK6 and CIPK25 can promote root growth. The CBL2-CIPK6-TST2 (tonoplast-localization Sugar Transporter2) signaling network formed in vacuoles can significantly promote glucose accumulation. However, how the CBL-CIPK module regulates microfilament cytoskeleton remodeling during stomatal movement is poorly understood and requires further investigation.

[0007] Against the backdrop of frequent droughts caused by global warming, how trees sense moisture and precisely regulate their stomata to adapt to adversity is a key scientific question in forest biology and ecological restoration. In-depth analysis. PuCIPK11 Understanding the molecular mechanisms of drought resistance and stomatal regulation in forest trees is of great significance for breeding highly resistant tree species and improving the survival rate of afforestation. Summary of the Invention

[0008] To address the above problems, the present invention provides PuCIPK11 Application of genes in regulating plant drought resistance, overexpression PuCIPK11 Genes can promote the depolymerization of stomatal microfilaments from a radial structure and their remodeling into a longitudinal structure, thereby accelerating stomatal closure and enhancing drought resistance. This study reveals the mechanism regulating stomatal cytoskeleton dynamics and identifies... PuCIPK11 Genes, as key regulators of drought resistance in poplar trees, provide potential targets for the genetic improvement of poplar trees.

[0009] To achieve the above objectives, the present invention provides the following technical solution: This invention provides PuCIPK11 Application of genes in regulating plant drought resistance.

[0010] Preferred, overexpression PuCIPK11 Genes positively regulate plant drought resistance.

[0011] Preferred, overexpression PuCIPK11 Genes promote stomatal closure, enhancing plant drought resistance.

[0012] Preferred, overexpression PuCIPK11 Genes promote stomatal closure by arranging themselves longitudinally after depolymerization of stomatal microfilaments.

[0013] Preferably, the plant includes Populus tomentosa.

[0014] Preferably, the PuCIPK11 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0015] The beneficial effects of this invention are: This invention identifies and characterizes *Populus alba* (a type of poplar). Populus ussuriensis A CBL-interacting protein kinase gene in Kom. PuCIPK11 This gene plays a crucial role in enhancing drought resistance by regulating stomatal closure. Results indicate... PuCIPK11 The gene is primarily expressed in guard cells and can be induced by drought. Overexpression PuCIPK11 Genes can promote the depolymerization of stomatal microfilaments from a radial structure and their remodeling into a longitudinal structure, thereby accelerating stomatal closure and enhancing drought resistance. This study reveals the mechanism regulating stomatal cytoskeleton dynamics and identifies... PuCIPK11 Genes, as key regulators of drought resistance in poplar trees, provide potential targets for the genetic improvement of poplar trees. Attached Figure Description

[0016] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0017] Figure 1 For the evolutionary analysis of CIPK proteins in Arabidopsis thaliana, Populus pilosa, and Populus tomentosa, a neighbor-joining (NJ) phylogenetic tree was constructed using 25 CIPK proteins from Arabidopsis thaliana, 27 CIPK proteins from Populus pilosa, and 48 CIPK proteins from Populus tomentosa, and 1000 bootstrapping analyses were performed. Green asterisks, purple squares, and orange circles represent Arabidopsis thaliana, Populus pilosa, and Populus tomentosa, respectively.

[0018] Figure 2 drought stress Lower PuCIPK11 The spatiotemporal expression mode; (A) PuCIPK11 promoter regulationGUS Gene expression and staining results; plant scale bar = 2 cm; stomatal scale bar = 50 μm; (B) PuCIPK11 Tissue-specific expression in *Populus alba*; values ​​are the mean ± standard deviation of three biological replicates (n=3); asterisks indicate significant differences (one-way ANOVA). (P<0.01); (C) Leaves of *Populus alba* under 0–48 hours of drought stress (5% PEG6000 nutrient solution) PuCIPK11 Expression changes; data are the mean ± standard deviation of three biological replicates (n=3); asterisks indicate significant differences (one-way ANOVA, P<0.0001).

[0019] Figure 3 for PuCIPK11 Obtaining transgenic lines; (A) PuCIPK11 - RT-qPCR of OE strains; values ​​are the mean ± standard deviation of three biological replicates (n=3). Asterisks indicate significant differences (one-way ANOVA, (P<0.0001); (B) CRISPR / Cas9 editing to obtain deletion mutants.

[0020] Figure 4 for PuCIPK11 Conferring drought resistance to Populus tomentosa; (A) Three-month-old wild type (WT), PuCIPK11 -OE and pucipk11 Growth status of mutant lines before drought stress, after drought stress, and one week after rehydration, scale bar = 5 cm; (B) Three-month-old wild type, PuCIPK11 -OE and pucipk11 Optical microscopic observation of leaf stomata in mutant plants before and after drought stress. Optical microscopic image scale bar = 50 μm.

[0021] Figure 5 For WT and PuCIPK11 Stomatal parameters and physiological parameters of each strain were measured; (A) WT, PuCIPK11 -OE and pucipk11 Statistical analysis of leaf stomatal aperture of the strains before and after two weeks of drought stress; 50 stomata were analyzed in each group (n=50); asterisks indicate significant differences (two-way ANOVA, "ns" indicates no significant difference). P<0.05, (P<0.0001); (B) WT and different values ​​during drought stress PuCIPK11 Monitoring of relative soil moisture content of transgenic lines; (C) WT and different values ​​before and after drought stress PuCIPK11Determination of malondialdehyde (MDA) content in leaves of transgenic strains; data are the mean ± standard deviation of three biological replicates (n=3); statistical significance was determined by two-way ANOVA and indicated by different lowercase letters; (D) Determination of WT and different PuCIPK11 Proline (PRO) content in leaves of transgenic lines; data are the mean ± standard deviation of three biological replicates (n=3); statistical significance was determined by two-way ANOVA and marked with different lowercase letters.

[0022] Figure 6 For WT and PuCIPK11 Observation of stomatal microfilament distribution in various strains; three-month-old wild type (WT) PuCIPK11 -OE and pucipk11 Distribution of microfilaments in stomata of mutant lines before and after drought stress. Fluorescence microscopy image scale bar = 10 μm. Detailed Implementation

[0023] This invention provides PuCIPK11 Application of genes in regulating plant drought resistance. In this invention, overexpression... PuCIPK11 Gene selection positively regulates plant drought resistance. In this invention, overexpression... PuCIPK11 Gene selection promotes stomatal closure and enhances plant drought resistance. In this invention, overexpression... PuCIPK11 The gene preferably promotes stomatal closure by depolymerizing stomatal microfilaments. In this invention, the plant preferably includes Populus tomentosa.

[0024] In this invention, the PuCIPK11 The nucleotide sequence of the gene is shown in SEQ ID No. 1, as follows:

[0025] To make the objectives, technical solutions, and advantages of this invention clearer, the invention is further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art. Unless otherwise specified, the materials, reagents, enzymes, competent cells, and plasmids used are all commercially available.

[0026] This application uses pBI121- GFP The pYLCRISPR-Cas9-DN series vectors were preserved by the National Key Laboratory of Forest Genetics and Breeding, Northeast Forestry University.

[0027] 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.

[0028] Example 1 1. Materials and Methods 1.1 Plant materials, growth conditions and treatment methods Selected Populus tomentosa ( Populus ussuriensis Poplar (Kom.) seedlings were used as genetic transformation materials. Under in vitro culture conditions, Poplar shoot cultures were placed on half-concentration MS medium and cultured at 25°C under long-day conditions (16 hours light / 8 hours dark), with subculturing every three weeks. For drought treatment, rooted Poplar seedlings were transferred to pots containing a soil / vermiculite (2:1) mixture and cultured in a greenhouse with a 16-hour light / 8-hour dark cycle at 25°C. Excess water was drained after each pot had fully absorbed the soil. Subsequently, the seedlings were cultured under water-deficient conditions (soil moisture content <10%) for 2–3 weeks. During this period, the pots were changed regularly to avoid position effects. When differential drought phenotypes appeared, phenotypic analysis, photography, and physiological testing were performed, followed by rehydration for one week and imaging again.

[0029] 1.2 Populus tomentosa PuCIPK11 Gene family identification and phylogenetic tree analysis By searching reference sequences from three sources, preliminary candidates for CIPK homologous genes were identified: (1) Arabidopsis thaliana from the TAIR database (https: / / www.Arabidopsis.org / index.jsp) Arabidopsis thaliana(1) AtCIPK protein sequence; (2) PtrCIPK homolog (Populus trichocarpa v4.1) obtained by Phytozome v13.0 (annotated by P. trichocarpa v4.1); and (3) PuCIPK genome data from NCBI BioProject SUB13708257 (https: / / www.ncbi.nlm.nih.gov). The genomes of *Populus trichocarpa* were then cross-validated for orthogonal homologs of CIPK proteins between *Arabidopsis thaliana* and *Populus trichocarpa* using a batch BLAST search (https: / / cottongen.org / tools / batch_blast). In phylogenetic analysis, protein sequences of all identified CIPK proteins in *Arabidopsis thaliana*, and sequences of *Populus trichocarpa* and *Populus trichocarpa* were aligned using ClustalX (v1.83) software with default parameters. Evolutionary relationships were inferred using the neighbor-joining method in MEGA7.0 software and validated by 1000 guided iterations. Pairwise distances were calculated using the Poisson correction method to account for multiple substitution effects.

[0030] 1.3 RNA extraction and RT-qPCR analysis To explore PuCIPK11 To investigate the expression pattern under stress conditions, three-week-old Populus tomentosa tissue cultures were cultured in half-concentration MS liquid medium containing 5% PEG6000. Total RNA was extracted from leaves at different time points (0, 3, 6, 12, 24, and 48 hours) using the cetyltrimethylammonium bromide (CTAB) method. Simultaneously, the above method was used to extract RNA from transgenic cells. PubCIPK11 -OE, Wild Type and pucipk11 Total RNA was extracted from the mutant lines. The extracted total RNA was reverse transcribed using a reverse transcription kit (Vazyme, Nanjing, China). Real-time quantitative PCR (RT-qPCR) was performed on a Bio-Rad CFX96 real-time PCR system. The reaction mixture contained: 10 μl Green One-Step qRT-PCR SuperMix (TransGene, Beijing, China), 8 μl double-distilled water, 1 μl complementary DNA (cDNA) template, and 0.5 μl each of forward and reverse primers (Table 1). Two... -ΔΔCt Quantitative analysis of relative gene expression levels and selection PuActin As an internal reference gene, three biological replicates were set up for the experiment.

[0031] 1.4 GUS histochemical detection Amplification using specific primers (Table 1) PuCIPK11The target sequence, 1.6 kb upstream of the start codon, was inserted into the pENTR / D-TOPO vector and recombinated into the target vector pBGW3 via Gateway linear recombination (LR) reaction. This resulted in the creation of [a specific product / technology] using Agrobacterium-mediated transformation. Pro CIPK11 : GUS Transgenic lines. Three-week-old whole transgenic plants were immersed in pre-cooled 90% acetone solution and vacuumed at room temperature for 10 min. The samples were then washed three times with GUS staining buffer to remove excess acetone. The fixed material was then completely immersed in GUS staining solution and stained overnight at 37°C in the dark. The stained tissue was then transferred to 70% ethanol solution for destaining 2-3 times, 30 min each time.

[0032] Table 1 Primer Information 1.5 Construction of expression vectors and gene transformation PuCIPK11 The gene was obtained by amplification from cDNA using PCR technology, employing KOD DNA polymerase (TOYOBO, Osaka, Japan). PuCIPK11 The full-length coding sequence was constructed into pBI121- GFP Overexpression was achieved in the vector under the control of the CaMV 35S promoter. Screening was performed using online bioinformatics tools (http: / / www.genome.arizona.edu / crispr / CRISPRsearch.html). PuCIPK11 Target site. Cas9- PuCIPK11 The recombinant fragments were amplified from the pYLCRISPR / Cas9 vector by PCR. The recombinant plasmids were transformed into Agrobacterium 'GV3101' competent cells to obtain engineered bacteria for later use. Using *Populus alba* leaves as recipient material, the leaves were cut into approximately 1 cm lengths and infected with *Agrobacterium* containing the recombinant plasmids for 20 min. After infection, the liquid on the leaf surface was blotted dry with paper towels, and then the leaves were spread evenly on differentiation medium (WPM + 0.1 g / L 6-BA + 0.01 g / L NAA + 0.2 g / L AS) and incubated in the dark for 48 hours. After dark incubation, a sterilization step was performed. The leaves spread on the differentiation medium were collected and washed three times with sterile water for 3 min each time. Then, they were washed six times with 1 / 2 MS liquid medium containing cephalosporin (Cef, 0.2 mg / L) for 5 min each time. After sterilization, the leaves were laid flat on the selection medium (WPM + 0.1 g / L 6-BA + 0.01 g / L NAA + 0.05 g / L Kana + 0.2 mg / L Cef). Resistant shoots obtained from differentiation screening were cut and transferred to 1 / 2 MS rooting medium. After rooting, the resistant shoots yielded… PubCIPK11 -OE andpucipk11 Transgenic mutant resistant seedlings. Aseptic techniques were used throughout the process. All transgenic lines were validated by PCR. See Table 1 for detailed primer information.

[0033] 1.6 Stomatal aperture measurement and observation of guard cell microfilaments Two-month-old wild-type (WT) animals were observed before and after drought stress. PuCIPK11 -OE and pucipk11 Stomatal opening degree of transgenic lines. Leaves of each line were immersed in stomatal opening solutions (OS) containing 0.01 M KCl, 0.1 M CaCl2, and 0.01 mM MES-KOH, respectively, for 0.5 hours in the dark and 2 hours in the light. The epidermal cells on the underside of the leaves were immediately peeled off by hand. Stomatal images were acquired using a Hitachi S-3400N scanning electron microscope. Stomatal opening was calculated by dividing stomatal width by stomatal length. More than 50 guard cells were measured in each sample. Simultaneously, leaves of 2-month-old Populus tomentosa treated with various lines were stained with 5 μM rhodamine-phalloidin for 10 minutes, and the microfilament structure in the stomata was observed using a fluorescence microscope (LSM800, Zeiss, Germany) equipped with a 60× oil immersion objective.

[0034] 1.7 Measurement of physiological indicators The concentration of proline (PRO) in each strain was quantitatively determined using the sulfosalicylic acid method. Tissue homogenate was mixed with 3% sulfosalicylic acid and centrifuged to remove the precipitate. The supernatant was heated with acidic ninhydrin reagent at 100°C for 30 minutes. After cooling, the chromophore was extracted with toluene. The absorbance of the toluene phase was measured at 520 nm. For the determination of malondialdehyde (MDA) content, the homogenate sample was first mixed with 10% (w / v) thiobarbituric acid (TBA) and incubated at 4°C for 12 hours, then centrifuged (12000×g, 10 minutes). The supernatant was mixed with an equal volume of 0.6% (w / v) TBA and heated at 95°C for 15 minutes. After cooling and centrifugation, the absorbance was measured at 530 nm using water as a blank control. The proline and MDA contents were calculated using the following formula: 1.8 Statistical Analysis All experiments were performed at least three times independently. Data were analyzed using one-way or two-way ANOVA. Statistical significance was considered to be achieved at the P-level (<0.05).

[0035] 2 Results 2.1 Drought-responsive genes PuCIPK11 spatiotemporal expression analysis Based on multiple sequence alignment analysis, a total of 48 PuCIPK genes were identified. (This is from Populus alba var. spp.) Populus ussuriensis Identified in the genome of Kom. PuCIPK11 Genes. For example... Figure 1 As shown, this gene is classified into group III. To determine... PuCIPK11 The expression mode adopts Pro CIPK11 : GUS The recombinant vector was used to transform poplar trees using Agrobacterium-mediated transformation to obtain transgenic lines. Histochemical GUS staining of the transgenic lines showed... PuCIPK11 Mainly expressed in guard cells ( Figure 2 (A). RT-qPCR results showed... PuCIPK11 It is mainly expressed in leaves, consistent with the results of GUS staining. Figure 2 (B) Three-week-old poplar seedlings were hydroponically treated with a 5% PEG6000 solution to simulate osmotic stress. RT-qPCR results showed that with prolonged stress, the concentration of PEG6000 in the leaves decreased. PuCIPK11 The expression level showed an overall upward trend, reaching a peak at 48 hours. Figure 2 (C). These results indicate that in Populus tomentosa... PuCIPK11 It exhibits stomatal cell-specific localization and undergoes drought-induced upregulation. Therefore, we will next explore the molecular mechanism of its drought response using a transgenic approach.

[0036] 2.2 PuCIPK11 Enhance the drought resistance of poplar trees by regulating stomatal closure. Two high-expression [proteins] were successfully constructed using RT-qPCR technology. PuCIPK11 Overexpression lines (OE-#3 and OE-#5; see Figure 3 (A), and pucipk11 Mutant lines (#1-1 and #1-3). Figure 3 Figure B demonstrates the validation results of the base deletion in the knockout mutant, confirming the success of the targeted genomic region editing operation. Subsequently, the phenotypic and physiological responses of each line after two weeks of natural drought treatment (seedling age of three months) were analyzed. Phenotypic results showed no significant difference in the growth of potted seedlings among all transgenic lines before drought treatment. Figure 4 (Middle A, left). After two weeks of drought stress, compared with wild-type (WT) plants, PuCIPK11 -OE strains showed leaf wilting but no obvious dehydration or leaf drop. Figure 4 (A). After a week of rehydration, its terminal buds recovered turgor pressure and regrowed, demonstrating significant drought resistance. Conversely, pucipk11The mutant lines exhibited near-complete leaf wilting and dehydration. Seven days after rehydration, almost all leaves fell off and growth ceased, indicating their sensitivity to drought. Figure 4 (Right side of A in the middle) Optical microscopy showed that under non-stress conditions, stomata were open in all genotypes, with no significant difference in stomatal diameter. Figure 4 B; Figure 5 (A) After drought stress, transgenic lines exhibited varying degrees of stomatal closure. Figure 4 (B) Quantitative analysis showed that compared with the wild type, PuCIPK11 -OE strains showed significantly enhanced stomatal closure, while pucipk11 The degree of closure in mutant lines is reduced ( Figure 5 (A). Soil relative water content (RWC) was monitored daily for all potted seedlings of all genotypes following drought treatment. Measurements were discontinued after seven days due to unreliable subsequent assessments caused by severe soil compaction and structural degradation. During the monitoring period, soil relative water content showed a gradual decreasing trend for all lines, but... PuCIPK11 -OE strains consistently maintained higher levels than wild types and pucipk11 mutant strains ( Figure 5 (B) In addition, the levels of the physiological markers malondialdehyde (MDA) and proline (PRO) were measured. Under adequate irrigation conditions, there were no significant differences in MDA and PRO levels among the different genotypes. However, after drought stress, PuCIPK11 Overexpression lines compared to wild type and pucipk11 The mutant lines significantly reduced the accumulation levels of MDA and PRO. Figure 5 The presence of C and D in these samples indicates reduced oxidative damage and enhanced osmotic regulation. These findings collectively demonstrate that: PuCIPK11 By promoting stomatal closure to reduce water loss due to transpiration, the drought resistance of Populus tomentosa is enhanced.

[0037] 2.3 PuCIPK11 Promoting pore closure through microfilament depolymerization Wild-type (WT) and PuCIPK11 Leaves of transgenic lines before and after drought stress were used for microfilament visualization analysis. The dorsal epidermis of these leaves was stained with Phalloidin-iFluor 488, a fluorescent probe that specifically labels filamentous actin (F-actin). Figure 6 As shown, under non-stress conditions, the microfilament bundles in guard cells are arranged in an open, transverse, radial pattern. After drought stress, PuCIPK11 The -OE strain exhibits complete stomata closure, accompanied by the disaggregation of transverse microfilament bundles, which then reorganize into a longitudinal arrangement aligned with the long axis of the guard cells. In contrast, the wild-type and pucipk11The mutant's stomata failed to close completely, and its microfilament bundles exhibited a disordered arrangement. These observations suggest that under drought stress, *Populus tomentosa*... PuCIPK11 Genes play a crucial role in regulating the dynamic changes of microfilament bundles during stomatal movement.

[0038] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. PuCIPK11 Application of genes in regulating plant drought resistance.

2. The application according to claim 1, characterized in that, overexpression PuCIPK11 Genes positively regulate plant drought resistance.

3. The application according to claim 2, characterized in that, overexpression PuCIPK11 Genes promote stomatal closure, enhancing plant drought resistance.

4. The application according to claim 3, characterized in that, overexpression PuCIPK11 Genes promote stomatal closure by depolymerizing stomatal microfilaments.

5. The application according to any one of claims 1 to 4, characterized in that, The plant mentioned includes Populus tomentosa.

6. The application according to claim 1, characterized in that, The PuCIPK11 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

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