Stomata-specific promoter-driven gene expression vector and application of stomata-specific promoter-driven gene expression vector

Abstract

The invention discloses a stomata-specific promoter-driven gene expression vector and application thereof, and belongs to the technical field of plant genetic engineering. In order to improve the application value of ABA in forest tree breeding and improve the genetic improvement of poplar trees, the invention provides a stomatal specific promoter driver gene expression vector, and the expression vector comprises a stomatal specific promoter ProAtOST1 (SEQ ID NO.1) and a PagPYL4 gene (SEQ ID NO.2). According to the expression vector provided by the invention, the PagPYL4 gene can only respond to a drought signal in pore-related tissues, so that pore opening and closing are accurately regulated and controlled, and the water utilization efficiency is improved; the method has the advantages that adverse effects of constitutive overexpression on photosynthetic efficiency and biomass accumulation are avoided, synergistic regulation and control of drought resistance improvement, photosynthetic product accumulation and biomass increase are realized, and the method can be applied to plant genetic improvement, in particular to cultivation of drought-resistant poplar varieties.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular relates to a stomatal-specific promoter-driven gene expression vector and an application thereof. Background Art

[0002] At present, there is still a severe shortage of tree varieties that can be used for afforestation in difficult areas in my country. Cultivating new tree varieties that can grow in arid, semi-arid and other water-scarce and saline-alkali wastelands, improving and utilizing arid, semi-arid and saline-alkali wastelands, promoting the development and utilization of land resources, and improving environmental resources have become current research hotspots.

[0003] Poplar (Populus spp.) is one of the main tree species for plantation in my country and is widely used in forestry system. There are about 6.67 million hectares of poplar plantations in China. 2 , which is four times the total area of poplar plantations in other countries. It has enormous production potential and significant economic and ecological benefits. Among them, the 84K poplar, a clone derived from a hybrid of Populus alba and Populus glandularus (Populus alba × Populus glandulosa), has a well-developed root system, rapid growth, and strong adaptability, leading to its widespread application in soil and water conservation, windbreak and sand fixation, bioenergy, and vegetation restoration.

[0004] To improve the adaptability of trees to adverse conditions, expand their suitable habitats, and provide new, high-quality, drought-resistant tree varieties for difficult resource environments such as drought and semi-arid areas, modern technology primarily uses two approaches to obtain new strains: traditional breeding through natural genetic variation from wild relatives, and molecular breeding through bioengineering. However, the main strategy of traditional breeding is selection through genetic diversity analysis of adaptive traits, which has technical problems such as long breeding cycles, low efficiency, and limited shape improvement. Therefore, using genomic technology and gene mapping tools to improve poplars so that they can grow and develop normally in difficult resource environments such as drought and semi-arid areas is one of the effective ways to genetically improve poplars.

[0005] The accumulation of ABA (abscisic acid) activates downstream signaling pathways, leading to plant responses to drought stress and optimized water use. ABA regulation also alters the turgor pressure of stomatal guard and auxiliary cells, affecting stomatal aperture and the efficiency of gas-water exchange. While constitutive overexpression of genes involved in the ABA biosynthesis or signaling pathways can enhance plant drought resistance, the continued activation of stress responses disrupts normal plant growth and development, impacting plant biomass accumulation and thus limiting the application of ABA in forestry breeding. Summary of the Invention

[0006] In order to solve the above-mentioned technical problems, the present invention provides a stomatal-specific promoter-driven gene expression vector and its application.

[0007] One of the objects of the present invention is to provide a stomatal-specific promoter-driven gene expression vector, wherein the expression vector comprises a stomatal-specific promoter and a PagPYL4 gene, wherein the stomatal-specific promoter is any one of ProAtOST1, ProPagOST1.1 or ProPagCNGC6.1.

[0008] In a preferred embodiment of the present invention, the nucleotide sequence of the stomatal-specific promoter ProAtOST1 is shown in SEQ ID NO.1, the nucleotide sequence of the stomatal-specific promoter ProPagOST1.1 is shown in SEQ ID NO.22, and the nucleotide sequence of the stomatal-specific promoter ProPagCNGC6.1 is shown in SEQ ID NO.23.

[0009] In a preferred embodiment of the present invention, the nucleotide sequence of the PagPYL4 gene is shown as SEQ ID NO.2.

[0010] In a preferred embodiment of the present invention, the nucleotide sequence of the stomatal-specific promoter ProAtOST1 amplification primer set is shown as SEQ ID NO.3-4; the nucleotide sequence of the stomatal-specific promoter ProPagOST1.1 amplification primer set is shown as SEQ ID NO.9-10; the nucleotide sequence of the stomatal-specific promoter ProPagCNGC6.1 amplification primer set is shown as SEQ ID NO.11-12.

[0011] In a preferred embodiment of the present invention, the nucleotide sequence of the PagPYL4 gene amplification primer set is shown as SEQ ID NO.5-6.

[0012] In a preferred embodiment of the present invention, the starting vector of the expression vector is pCAMBIA1301.

[0013] A second object of the present invention is to provide the use of the above expression vector in plant genetic improvement.

[0014] In a preferred embodiment of the present invention, the plant genetic improvement refers to the cultivation of drought-resistant poplar varieties.

[0015] A third object of the present invention is to provide a method for creating transgenic plants, the method comprising the following steps: transferring the above-mentioned expression vector into the plant genome to obtain a transgenic plant that specifically expresses the PagPYL4 gene.

[0016] In a preferred embodiment of the present invention, the plant is 84K poplar.

[0017] Beneficial effects of the present invention: The present invention provides a stomatal-specific promoter-driven gene expression vector, which includes the PagPYL4 gene (nucleotide sequence shown in SEQ ID NO.2) and any one of the stomatal-specific promoters ProAtOST1 (nucleotide sequence shown in SEQ ID NO.1), ProPagOST1.1 (nucleotide sequence shown in SEQ ID NO.22) or ProPagCNGC6.1 (nucleotide sequence shown in SEQ ID NO.23).

[0018] The present invention uses the stomatal guard cell-specific promoter ProAtOST1 to drive the expression of the drought-resistant functional gene PagPYL4, so that the PagPYL4 gene responds to drought signals only in stomatal-related tissues, thereby accurately regulating stomatal opening and closing and improving water use efficiency; on this basis, it effectively avoids the adverse effects of constitutive overexpression on photosynthetic efficiency and biomass accumulation, and achieves the coordinated regulation of improved drought resistance and increased photosynthetic product accumulation and biomass.

[0019] Effect experiments show that compared with the transgenic strain in which the constitutive promoter CaMV35S drives the expression of the PagPYL4 gene, the transgenic strain in which the stomatal-specific promoter ProAtOST1 drives the expression of the PagPYL4 gene reduces the growth inhibitory effect on the plant; the transgenic strain containing the stomatal-specific promoter-driven gene expression vector provided by the present invention is more sensitive to water loss, can quickly respond to ABA signals, and reduce water loss; and can accurately regulate the expression of the PagPYL4 gene through the stomatal-specific promoter, achieve a rapid response of stomatal aperture under drought conditions, while maintaining a photosynthetic rate and biomass accumulation comparable to the wild type.

[0020] It can be seen that the stomatal-specific promoter-driven gene expression vector provided by the present invention can be applied to plant genetic improvement, specifically to the cultivation of drought-resistant poplar varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the double element promoter in Example 1;

[0022] Figure 2 This is the staining image of the GUS reporter gene driven by different promoters in Example 2; bar = 5 μm;

[0023] Figure 3Figures 1 and 2 show the phenotypic observation results of the 84K poplar transgenic strain and wild type in Example 3; A shows the phenotypic observation at 60 days; B shows the plant height measurement results; and C shows the ground diameter measurement results. The x-axis represents the growth of the 84K poplar at different times; the y-axis represents the length of different strains. Data are expressed as mean ± SD; a and b represent the significant differences between different strains at P < 0.05.

[0024] Figure 4 This is the water loss specific gravity diagram in Example 3;

[0025] Figure 5 This is a diagram showing phenotypic observation under short-term drought stress in Example 3;

[0026] Figure 6 This is a graph showing the results of measuring the net photosynthetic rate of different strains under short-term drought stress in Example 3;

[0027] Figure 7 This is a graph showing the stomatal conductance measurement results of different strains under short-term drought stress in Example 3. DETAILED DESCRIPTION

[0028] Those skilled in the art can refer to the content of this document and appropriately improve the process parameters. It is particularly important to note that all similar substitutions and modifications are obvious to those skilled in the art and are considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. It is obvious that relevant persons can modify or appropriately change and combine the methods and applications described herein without departing from the content and scope of the present invention to implement and apply the technology of the present invention.

[0029] In order to make the purpose, technical solutions and advantages of the present invention more clear, the present invention is further described in detail below in conjunction with specific embodiments. The experimental methods used in the following examples are all conventional methods unless otherwise specified, and the materials, reagents, methods and instruments used are all conventional materials, reagents, methods and instruments in the art unless otherwise specified, and can be obtained from commercial channels by those skilled in the art.

[0030] The experimental materials involved in the following examples include: 84K poplar (Populus alba × Populus glandulosa '84K') and Arabidopsis thaliana (Arabidopsis thaliana), all of which were cultured in an artificial climate chamber. The culture conditions were set as follows: temperature 22±1°C, relative humidity 35±5%, light intensity 10,000 Lux, and photoperiod 16 h light / 8 h dark.

[0031] The instruments involved in the following embodiments are:

[0032] Electric constant temperature drying oven (model: HWL-125, Tianjin Laboterui Instrument Equipment Co., Ltd.), vertical full-temperature oscillating incubator (model: ZQPL-200, Tianjin Laboterui Instrument Equipment Co., Ltd.), vertical automatic pressure steam sterilizer (model: GI54DWS, Xiamen Zhiwei Instrument Co., Ltd.), clean workbench (model: DL-CJ-2NDⅠ, Beijing Donglian Har Instrument Manufacturing Co., Ltd.), RDN type artificial climate chamber (model: RDN-1000D-N, Ningbo Southeast Instrument Co., Ltd.), stereo microscope (model: Discovery, Zeiss).

[0033] The culture medium involved in the following examples:

[0034] Poplar co-culture medium: 2.41 g / L WPM, 25 g / L Sucrose, 0.5 g / L MES, 6 g / L Agar, pH = 5.8, plus 100 μM AS after sterilization;

[0035] Poplar rooting medium: 2.41 g / L WPM, 25 g / L Sucrose, 0.5 g / L MES, 6 g / L Agar, pH = 5.8, add 200 mg L-1 Cef after sterilization;

[0036] Poplar callus induction medium: 2.41 g / L WPM, 25 g / L Sucrose, 1 mg / L 2,4-D, 0.1 mg / L Kinetin, 6 g / L Agar, pH = 5.8, add 200 mg L-1 Cef after sterilization;

[0037] Poplar callus induction medium (liquid): 2.41 g / L WPM, 2.5 g / L Sucrose, 1 mg / L 2,4-D, 0.1 mg / L Kinetin, pH = 5.8, add 200 mg L-1 Cef after sterilization;

[0038] Poplar bud induction medium: 2.41 g / L WPM, 25 g / L Sucrose, 1 mg / L 2,4-D, 0.1 mg / L Kinetin, 6 g / L Agar, pH = 5.8, add 200 mg L-1 Cef after sterilization;

[0039] YEB liquid culture medium: 5g / L Beef Extract, 1g / L Yeast Extract, 5g / L Peptone, 5g / L Sucrose, 0.4g / L MgSO4·7H2O, pH=7.4, 15g / L Agar.

[0040] The main reagents involved in the following examples are:

[0041] High-fidelity DNA polymerase 2×Phanta Max Master Mix-P515 (Norvozymes), reverse transcription kit HiScript II RT SuperMix for qPCR (Norvozymes), DNA polymerase 2×Taq Master Mix (Chemical Biotechnology), competent Escherichia coli DH5α (Sangon Biotechnology), competent Agrobacterium tumefaciens GV3101 (Sangon Biotechnology), plasmid miniprep kit (Welling Biotechnology), universal plant RNA extraction kit (Chemical Biotechnology), GUS staining premix (Coolaber), gel recovery kit (Omega, USA), T4 DNA ligase (NEB, USA), and restriction endonucleases (NEB, USA); pCAMBIA1300-NOS vector and pCAMBIA1301-GUS vector were all maintained by our laboratory.

[0042] Example 1: Stomatal-specific promoter screening and element analysis

[0043] In this example, the following genes related to stomatal specificity and stomatal high expression characteristics were screened from Arabidopsis: AtOST1 (Open Stamata 1), AtGC1 (Guard Cell 1), and CNGC6 (CYCLIC NUCLEOTIDE-GATED CHANNEL 6). Geneious 9.0 software was used to perform protein sequence alignment of the homologous genes with 84K poplar. The two genes with the highest relative scores, including gene sequence coverage, average alignment score, sequence group equivalence ratio, and E value, were analyzed for subsequent experiments. The results are shown in Table 1.

[0044] Furthermore, the target promoter was analyzed by NewPlace (https: / / www.dna.affrc.go.jp / PLACE / ?action=newplace), drawn using TB TOOLS2.5 software, and further adjusted using Powerpoint; Figure 1 As shown in Figure 3, PLANTCARE analysis results showed that the target genes all contained stomatal-specific promoter elements (5'-TAAAG-3') and ABA response element (ABRE) double elements.

[0045] Table 1

[0046]

[0047] Example 2: Cloning of stomatal-specific promoters and validation in poplar

[0048] In this example, the promoter sequences (about 2000 bp) of ProAtGC1 (nucleotide sequence shown in SEQ ID NO.21), ProAtOST1 (nucleotide sequence shown in SEQ ID NO.1), ProPagOST1.1 (nucleotide sequence shown in SEQ ID NO.22), and ProPagCNGC6.1 (nucleotide sequence shown in SEQ ID NO.23) were determined based on the known DNA library sequence. The promoter-specific primers with added restriction sites were designed using Geneious 9.0 using the genomic DNA of ProAtGC1, ProAtOST1, ProPagOST1.1, and ProPagCNGC6.1 as templates, respectively. The specific primers are shown in Table 2. Among them, the nucleotide sequence of the ProAtGC1 stomatal-specific promoter amplification primer set is shown in SEQ ID NO.7-8, the nucleotide sequence of the ProAtOST1 stomatal-specific promoter amplification primer set is shown in SEQ ID NO.3-4, and the nucleotide sequence of the ProPagOST1.1 stomatal-specific promoter amplification primer set is shown in SEQ ID NO. NO.9-10, the nucleotide sequence of the ProPagCNGC6.1 stomatal-specific promoter amplification primer set is shown in SEQ ID NO.11-12; using 2×Phanta Max Master Mix-P515 high-fidelity enzyme, a promoter with stomatal guard cell-specific high expression was cloned to obtain a PCR amplification product. The PCR amplification reaction system is shown in Table 3, the PCR amplification program is shown in Table 4, and it is fused with the reporter gene GUS (β-glucuronidase, β-glucuronidase).

[0049] Table 2

[0050]

[0051]

[0052] Table 3

[0053] Component Volume / Concentration 2×phanta Max Master MIX 12.5μL Forward Primer, 10 μM 1 μL Reverse Primer, 10 μM 1 μL DNA (70 ng / μL) 2μL <![CDATA[ddH2O]]> 8.5μL

[0054] Table 4

[0055]

[0056] The PCR products obtained above were subjected to 1.0% agarose gel electrophoresis to ensure that the target fragments were of the correct size. The gel was then excised and recovered using a Gel Extraction Kit (OMEGA, USA). The products were stored in a 4°C refrigerator. After the above amplification products were purified, they were ligated with the pCAMBIA1301-GUS linear vector after enzyme digestion and then recombined. The above recombinant plasmids were transformed into Escherichia coli competent DH5α (Sangon Biotechnology). When single colonies grew, PCR amplification was performed and agarose electrophoresis gel detection was performed. The primer sequences used for DNA identification are shown in Table 2, wherein the primer sequences for identifying the ProAtGC1 gene are shown in SEQ ID NO.13 and SEQ ID NO.19, the primer sequences for identifying the ProAtOST1 gene are shown in SEQ ID NO.14 and SEQ ID NO.19, the primer sequences for identifying the ProPagOST1.1 gene are shown in SEQ ID NO.16 and SEQ ID NO.19, and the primer sequences for identifying the ProPagCNGC6.1 gene are shown in SEQ ID NO.15 and SEQ ID NO.19.

[0057] The positive clone products obtained above were sent to Harbin Ruiboxingke (Northeast) for sequencing, and the sequencing results were consistent with the known sequence alignment results, proving that the ProAtGC1:GUS, ProAtOST1:GUS, ProPagOST1.1:GUS, and ProPagCNGC6.1:GUS vectors had been successfully constructed; the ProAtGC1:GUS, ProAtOST1:GUS, ProPagOST1.1:GUS, and ProPagCNGC6.1:GUS recombinant vectors obtained above were genetically transformed into 84K poplar, and the transgenic 84K poplar genomic DNA was extracted using the CTAB method for identification of positive seedlings; the lower epidermis of the leaves at the 5th-6th stem nodes of the transgenic positive seedlings cultured in soil for 14 days were taken, incubated in 1×GUS stain at 37°C for 4 hours, and the chlorophyll of the samples was removed with 70% ethanol. The samples were then immersed in ethanol for 1-3 hours until the negative control turned white. Observed under an optical microscope, the blue on the white background indicates the GUS expression site.

[0058] Effect data:

[0059] This example uses bioinformatics to analyze and screen genes that are specifically and highly expressed in stomatal guard cells. A promoter with high stomatal guard cell expression was cloned and fused to the reporter gene GUS. The constructed vector was used to construct transgenic poplar trees. The lower epidermis of 84K poplar leaves was attached to a coverslip using medical gel and GUS staining was performed.

[0060] The results are as follows Figure 2As shown, it was observed that ProAtGC1:GUS was not expressed or expressed at a low level. In this example, a total of three promoters that can be expressed in 84K poplar stomatal guard cells were identified, namely ProAtOST1:GUS, ProPagOST1.1:GUS and ProPagCNGC6.1:GUS, which can accurately trace stomata and have high expression levels; among them, ProPagOST1.1 and ProPagCNGC6.1 are endogenous promoters of 84K.

[0061] The above results show that among promoters from multiple different sources, this example successfully found a promoter that can be specifically and highly expressed in the stomata of 84K poplar leaves; among them, ProAtOST1:GUS has the best effect. Therefore, the present invention subsequently uses ProAtOST1 to construct transgenic plants.

[0062] Example 3: Construction of transgenic poplar

[0063] In this study, in order to construct a vector that drives the stress resistance gene PagPYL4 with the stomatal-specific promoter ProAtOST1 and compares it with the constitutive CaMV35S-driven PagPYL4 gene, the ABA receptor ATPYL4 (Pyrabactin Resistance-Like 4) gene and protein sequence were obtained through NCBI (www.ncbi.nlm.nih.). Geneious9.0 was used for homology comparative analysis, and the gene Pop_A16G090069.T1 with the highest homology to ATPYL4 was selected and named PagPYL4. The nucleotide sequence of the PagPYL4 gene is shown in SEQ ID NO.2.

[0064] In this example, PCR amplification was performed using 84K poplar cDNA as a template. Specific upstream and downstream primers were designed based on the published 84K poplar genome sequence information and the Arabidopsis gene sequence information. As shown in Table 2, the PagPYL4 gene was recombined with the linear vectors pCAMBIA1300-35S-NOS and pCAMBIA1300-ProAtOST1-NOS, respectively, to construct the 35S strong promoter vector pCAMBIA1300-35S-PagPYL4 and the stomatal-specific promoter-driven vector pCAMBIA1301-ProAtOST1-PagPYL4, respectively. Among them, the primers for constructing the pCAMBIA1300-ProAtOST1-NOS vector using pCAMBIA1300-35S-NOS as a template are shown in SEQ ID NO.17 and SEQ ID NO.18; the primers for constructing the pCAMBIA1301-ProAtOST1-PagPYL4-NOS vector are shown in SEQ ID NO.17-18 and SEQ ID NO.5-6.

[0065] The pCAMBIA1300-35S-PagPYL4 and pCAMBIA1301-ProAtOST1-PagPYL4 recombinant plasmids were transformed into Escherichia coli competent DH5α (Sanggong Biotechnology), and the Agrobacterium culture solution after overnight culture was added to 100 mL YEB liquid medium (containing 100 mg / L Kan and 33 mg / L rifampicin), cultured in a 28°C incubator for 3-5 h until the OD value of the culture solution was in the range of 0.3-0.5, centrifuged at 60,000 × g for 10 min, and the Agrobacterium precipitate was collected; the collected Agrobacterium precipitate was resuspended in liquid poplar callus induction medium and incubated for 2 h; 84K poplar callus tissue was placed in the liquid culture medium with Agrobacterium suspension, co-cultured for 20 minutes, and the water was controlled; the 84K poplar callus tissue treated above was placed in poplar co-culture medium (100 mM AS), placed in the dark for co-cultivation for 48 hours; the callus tissues after co-cultivation for 48 hours were placed in sterile tissue culture bottles, 200 mL of sterile RO water was added, and the cells were shaken at 900 × g for 30 minutes. During this period, the sterile RO water was replaced according to the turbidity of the water body; after shaking for 30 minutes, the callus tissues were shaken and poured onto a sterile colander, and the sterile RO water was replaced, and this was repeated twice; 200 mL of sterile RO water and 2 / 1000 Timentin with a final concentration of 2‰ were added again, and the cells were shaken at 900 × g for 40 minutes. After this, the callus tissues that had been dried were transferred to 84K shoot induction medium, and the medium was replaced every 14 days until young clustered shoots appeared. The clustered shoots were then peeled off into individual shoots and placed in 84K poplar resistance shoot rooting medium. After rooting, the transgenic seedlings were identified by DNA. The primer sequences used are shown in Table 2; wherein, the sequence of the primer for identifying the pCAMBIA1300-35S-PagPYL4 vector is SEQ ID NO.5 and SEQ ID NO.20, and the sequences of primers for identifying the pCAMBIA1301-ProAtOST1-PagPYL4 vector are shown in SEQ ID NO.17 and SEQ ID NO.20.

[0066] Example 4: Evaluation of Transgenic Poplar Growth and Drought Resistance Phenotype

[0067] In this example, the 84K transgenic lines of OE-PagPYL4 and ProAtOST1-PagPYL4 prepared in Example 3 were planted in a plant greenhouse with a photoperiod of 16 / 8 h, a temperature of 24°C, a light intensity of 175-100 μmol, and a humidity of 45-50%. A systematic physiological and ecological experiment was conducted on the transgenic plants cultured to 60 days of age to explore the effects of the growth and development and abiotic stress responses of the two transgenic lines.

[0068] (1) Changes in plant height and ground diameter:

[0069] The transgenic plants of OE-PagPYL4, ProAtOST1-PagPYL4 and wild type were tracked and measured at 60 days of growth, and the plant height and ground diameter on the 15th, 30th, 45th and 60th days were counted.

[0070] The results are as follows Figure 3 As shown in the figure, the plant height and ground diameter of the OE-PagPYL4 transgenic line were significantly lower than those of the wild type WT at the 30th, 45th and 60th days; while the plant height and ground diameter of the ProAtOST1-PagPYL4 transgenic line were lower than those of the wild type WT at the 30th and 45th days, but the significant differences were not obvious. However, the plant height of the ProAtOST1-PagPYL4 transgenic line was significantly higher than that of the OE-PagPYL4 transgenic line.

[0071] As can be seen, the plant height growth of the OE-PagPYL4 transgenic lines was significantly lower than that of the WT group, indicating that the PagPYL4 gene inhibits the growth and development of 84K poplar. However, there was no significant difference between ProAtOST1-driven gene expression and wild-type plants. Comparison of plant height and ground diameter showed that WT>ProAtOST1-PagPYL4>OE-PagPYL4, indicating that compared with expression of the PagPYL4 gene driven by the constitutive promoter CaMV35S, expression of the PagPYL4 gene driven by the stomatal-specific promoter ProAtOST1 reduced the growth inhibitory effect on the plant.

[0072] (2) Leaf water loss rate:

[0073] In this example, 60-day-old transgenic plants of OE-PagPYL4, ProAtOST1-PagPYL4, and wild-type were selected. Leaves at the 6th to 7th nodes were taken, and 10 biological replicates were taken from each line. After being taken, the leaves were immediately placed in Stomata-opening buffer and allowed to stand under a supplementary light for 30 minutes. After 30 minutes, the leaves were taken out and immediately dried, and placed at a temperature of 24°C and a light intensity of 75-100 μmol·m -2 ·s -1 , in a greenhouse with a humidity of 70%, weighing statistics are carried out every 1 hour.

[0074] The results are as follows Figure 4As shown in the figure, the OE-PagPYL4 transgenic line lost weight more slowly than the wild-type WT, with significant differences at each hour. The ProAtOST1-PagPYL4 transgenic line lost less weight than the wild-type WT at 1 and 5 hours, with significant differences. This indicates that both the OE-PagPYL4 and ProAtOST1-PagPYL4 transgenic lines are more sensitive to water loss than the wild-type WT, responding quickly to ABA signals and reducing water loss.

[0075] (3) Phenotypic observation after drought treatment:

[0076] To verify the effects of different gene expression patterns on plant drought resistance, three independent lines of 60-day-old transgenic plants (OE-PagPYL4, ProAtOST1-PagPYL4, and wild-type) were selected as experimental materials for natural drought treatment. After 10 days of watering, the plants were re-watered for 3 days to observe phenotypic changes.

[0077] The results are as follows Figure 5 As shown in the figure, after 10 days of drought treatment, both wild-type WT and transgenic 84K poplars with OE-PagPYL4 and ProAtOST1-PagPYL4 exhibited wilting, but all leaves in the wild-type WT plants exhibited severe wilting. While similar to the wild-type WT, the ProAtOST1-PagPYL4 transgenic line retained a few leaves at the top that had not lost water and remained morphologically intact. In contrast, in the OE-PagPYL4 transgenic line, although all leaves failed to stand upright, only the basal leaves wilted. After watering resumed, damaged leaves at nodes 1-8 in the OE-PagPYL4 transgenic line returned to green and remained stiff. In the ProAtOST1-PagPYL4 transgenic line, most leaves were damaged, with leaves at nodes 2-5 regaining their upright state. In contrast, damaged leaves in the wild-type WT plant fell completely and could not recover. Finally, a count of the surviving leaves from each strain was performed. The OE-PagPYL4 transgenic strain had the highest number of surviving leaves, and the ProAtOST1-PagPYL4 transgenic strain also had significantly higher numbers of surviving leaves than the wild-type WT. This indicates that while the wild-type 84K poplar has a certain foundation of drought resistance, its stomatal closure response to drought stress is slow, resulting in a higher transpiration rate. Both the OE-PagPYL4 and ProAtOST1-PagPYL4 transgenic strains provided by the present invention can improve the 84K poplar's tolerance to drought stress.

[0078] (4) Net photosynthetic rate and stomatal conductance:

[0079] During short-term natural drought treatment, photosynthetic parameters and stomatal conductance under the photosynthetic light response curve were measured using an infrared gas analysis system (Li-Cor-6400; USA) 6400 photosynthetic meter at the 6th and 7th stem nodes of the OE-PagPYL4 and ProAtOST1-PagPYL4 transgenic lines and the wild-type WT line under normal conditions (photoperiod: 16 h light, 8 h dark; day and night temperature: 25 / 20°C; relative humidity: 40-45%). Three plants were replicated in each group. The photosynthetic parameters and stomatal conductance were measured under the photosynthetic light response curve of 1200 μmol·m-2·sε1 (i.e., reaching the light saturation point) in a plant cultivation greenhouse under long-day conditions. The data were measured from 08:00 to 11:00 when photosynthesis was active. Two-way ANOVA was performed using GraphPad Prism.

[0080] The results are as follows Figure 6 As shown, the net photosynthetic rate of the OE-PagPYL4 transgenic line was generally lower than that of the wild-type WT from day 0 to day 4, similar to that of the wild-type WT on day 6, and higher than that of the wild-type WT from day 7 to day 9. The ProAtOST1-PagPYL4 transgenic line showed a similar pattern to that of the OE-PagPYL4 transgenic line, but remained on par with the wild-type WT at day 7. In subsequent time periods, the net photosynthetic rate was higher than that of the wild-type WT, but the difference was not significant. This suggests that the OE-PagPYL4 and ProAtOST1-PagPYL4 transgenic lines may have affected stomatal water loss, ensuring that they are more likely to maintain normal physiological activities during periods of extreme drought than the wild-type WT.

[0081] The results are as follows Figure 7 As shown, the overall stomatal conductance changes in the OE-PagPYL4 and ProAtOST1-PagPYL4 transgenic lines were similar to those in the wild-type WT. Specifically, stomatal conductance in the OE-PagPYL4 and ProAtOST1-PagPYL4 transgenic lines was lower than that in the wild-type WT during the first four days. After four days, the patterns of change were similar, reaching the same level as that of the wild-type WT on day 7. In the following period, stomatal conductance was higher than that of the wild-type WT, but the difference was not significant. This indicates that the wild-type WT was in an extreme drought state after eight days and could no longer maintain a normal biological rhythm. However, the expression of the PagPYL4 gene affected stomatal water loss, ensuring that the transgenic lines were more likely to maintain normal physiological activities during drought than the wild-type.

[0082] In summary, although the OE-PagPYL4 line significantly enhanced the stomatal closure speed through global expression of the PagPYL4 gene, it caused a decrease in photosynthetic rate and inhibition of biomass accumulation due to non-specific expression; in contrast, the ProAtOST1-PagPYL4 transgenic line precisely regulated the expression of the PagPYL4 gene through a stomatal-specific promoter, achieved a rapid response of stomatal aperture under drought conditions, and maintained a photosynthetic rate and biomass accumulation comparable to the wild type.

[0083] Any matters not described in detail in this specification are well known to those skilled in the art. Although the present invention has been disclosed above with reference to preferred embodiments, these are not intended to limit the present invention. Anyone skilled in the art may make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention shall be determined by the claims.

Claims

1. A stomatal-specific promoter-driven gene expression vector, characterized in that: The expression vector comprises a stomatal-specific promoter and a PagPYL4 gene, wherein the stomatal-specific promoter is any one of ProAtOST1, ProPagOST1.1 or ProPagCNGC6.

1.

2. The expression vector according to claim 1, characterized in that The nucleotide sequence of the stomatal-specific promoter ProAtOST1 is shown in SEQ ID NO.1, the nucleotide sequence of the stomatal-specific promoter ProPagOST1.1 is shown in SEQ ID NO.22, and the nucleotide sequence of the stomatal-specific promoter ProPagCNGC6.1 is shown in SEQ ID NO.

23.

3. The expression vector according to claim 1, characterized in that The nucleotide sequence of the PagPYL4 gene is shown in SEQ ID NO.

2.

4. The expression vector according to claim 1, characterized in that The nucleotide sequence of the stomatal-specific promoter ProAtOST1 amplification primer set is shown in SEQ ID NO.3-4; the nucleotide sequence of the stomatal-specific promoter ProPagOST1.1 amplification primer set is shown in SEQ ID NO.9-10; the nucleotide sequence of the stomatal-specific promoter ProPagCNGC6.1 amplification primer set is shown in SEQ ID NO.11-12.

5. The expression vector according to claim 1, characterized in that The nucleotide sequence of the PagPYL4 gene amplification primer set is shown in SEQ ID NO.5-6.

6. The expression vector according to claim 1, characterized in that The starting vector of the expression vector is pCAMBIA1301.

7. Use of the expression vector according to any one of claims 1 to 6 in plant genetic improvement.

8. The use according to claim 7, characterized in that The plant genetic improvement refers to the cultivation of drought-resistant poplar varieties.

9. A method for creating a transgenic plant, characterized in that: The method comprises the following steps: transferring the expression vector according to any one of claims 1 to 6 into a plant genome to obtain a transgenic plant that specifically expresses the PagPYL4 gene.

10. The method according to claim 9, characterized in that The plant is 84K poplar.

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