Photoregulated proton pump expression module, method for constructing same and use thereof in enhancing plant salt tolerance

Abstract

The application provides a light-controlled proton pump expression module, a construction method thereof and application in enhancing plant salt tolerance, and belongs to the technical field of plant synthesis; the light-controlled proton pump expression module is constructed to realize specific expression of two light-controlled proton pumps from microorganisms in three root cell groups of Arabidopsis thaliana, and beta-carotene 15,15'-dioxygenase MbDio is also expressed in the light-controlled proton pump expression module; the light-controlled proton pump expression module can significantly improve the membrane localization efficiency of the light-controlled proton pump in root cells; the light-controlled proton pump expression module can significantly increase the acidification degree of the culture medium near the roots, and can specifically enhance the salt tolerance of plants; the application not only provides a theoretical basis for in-depth exploration of the salt tolerance molecular mechanism of different cell types of the root system, but also provides a new technical direction and idea for development and application of the salt tolerance characteristics of other aquatic planktonic plants, and has good practicability.

Description

Technical Field

[0001] This invention belongs to the field of plant synthesis technology, specifically relating to a light-controlled proton pump expression module and its construction method, and its application in enhancing plant salt tolerance. Background Technology

[0002] Optogenetics is a research technique that combines genetics and optics. It introduces exogenous photosensitive proteins into target cells, enabling their physiological activities to exhibit photoresponsive characteristics and thus regulating specific cellular physiology at different spatiotemporal scales. The core photosensitive protein in this technique is primarily derived from microbial rhodopsin, a type of opsin with a seven-transmembrane helical structure, which covalently binds all-trans retinal as a photosensitive chromophore. In recent years, optogenetics has seen initial applications in plants, such as regulating stomatal movement and pollen tube growth. However, whether this technique can enhance plant stress resistance, especially salt tolerance, has not yet been published in research.

[0003] Currently, research on the molecular regulatory mechanisms and genetic improvement of plant salt tolerance largely focuses on conventional genetic manipulations such as knocking out or overexpressing endogenous salt tolerance-related genes. While these traditional strategies have improved the salt tolerance phenotype of plants to some extent, they generally rely on endogenous regulatory pathways and gene elements, making it difficult to achieve precise and controllable spatiotemporally specific expression and functional regulation. Limitations include unadjustable expression intensity, restricted action sites, and inflexible responses to environmental signals. Furthermore, existing research primarily focuses on endogenous salt tolerance gene systems, with limited exploration of introducing novel regulatory elements such as exogenous light-controlled functional proteins and constructing precise salt tolerance regulatory systems that are not dependent on endogenous sources. This makes it difficult to meet the needs of crops for efficient, targeted, and dynamic adaptation under complex salt stress environments.

[0004] While optogenetic elements such as light-controlled ion channels or proton pumps have been gradually applied to plant cell signal regulation research, their universality and adaptability across different cell types remain significantly limited, often making it difficult to achieve precise activation and manipulation of ion signals specific to cell type. With the deepening of plant stress biology research, the specialization and synergistic effects of different tissues and cell types in salt stress responses are receiving increasing attention, and the spatiotemporal characteristics and cell specificity of ion signals have become key to elucidating salt tolerance mechanisms.

[0005] Therefore, using cell type-specific promoters to achieve the targeted expression of light-controlled channel proteins in target cells and precisely manipulate ion transport and signal output of specific cell populations is of great scientific significance and application value for systematically elucidating the functional roles of ion signals in plant salt stress responses of different cell types and constructing new cell-specific salt tolerance regulation strategies. Summary of the Invention

[0006] To address some shortcomings in existing technologies, this invention provides a light-controlled proton pump expression module, its construction method, and its application in enhancing plant salt tolerance. This invention achieves the specific expression of light-controlled proton pumps from two microorganisms in Arabidopsis root cells by constructing a light-controlled proton pump expression module. The module also expresses β-carotene 15,15'-dioxygenase MbDio. The light-controlled proton pump expression module significantly improves the membrane localization efficiency of the light-controlled proton pump in root cells. It also significantly increases the acidification of the culture medium near the roots and specifically enhances the salt tolerance of plants. This invention not only provides a theoretical basis for in-depth research into the molecular mechanisms of salt tolerance in different root cell types but also offers new technical directions and ideas for the development and application of salt tolerance characteristics in the roots of other aquatic phytoplankton, demonstrating significant practicality.

[0007] To achieve the above-mentioned technical objectives, the present invention employs the following technical means:

[0008] This invention first provides a light-controlled proton pump expression module, which includes: a light-controlled proton pump gene and a promoter;

[0009] The light-controlled proton pump gene includes ApOps2 from fungi or CvRH from algae;

[0010] The promoters include pRPL11C, pSCR, or pCO2.

[0011] Preferably, the nucleotide sequence of the fungal ApOps2 is as shown in SEQ ID No:1;

[0012] The nucleotide sequence of the algal-derived CvRH is shown in SEQ ID No:2;

[0013] The nucleotide sequence of pRPL11C is shown in SEQ ID No:4;

[0014] The nucleotide sequence of the pSCR is shown in SEQ ID No:5;

[0015] The nucleotide sequence of the pCO2 is shown in SEQ ID No:6;

[0016] The nucleotide sequence of the red fluorescent protein mScarlet is shown in SEQ ID No:7.

[0017] Preferably, the light-controlled proton pump expression plasmid also expresses β-carotene 15,15'-dioxygenase MbDio.

[0018] Preferably, β-carotene 15,15'-dioxygenase MbDio is expressed by linking the gene sequences of the chloroplast or plastid localization signal peptide RC2, the P2A self-cleaving polypeptide, and the β-carotene 15,15'-dioxygenase MbDio.

[0019] The ligated nucleotide sequence is shown in SEQ ID No:3.

[0020] The present invention also provides a light-controlled proton pump expression plasmid, wherein the light-controlled proton pump expression plasmid includes the above-mentioned light-controlled proton pump expression module.

[0021] Preferably, the expression vector of the light-controlled proton pump expression plasmid includes pFASTR-AG;

[0022] The present invention also provides a recombinant engineered bacterium, wherein the recombinant engineered bacterium comprises at least one of the above-described light-controlled proton pump expression modules or at least one of the above-described light-controlled proton pump expression plasmids.

[0023] This invention also provides applications of the above-mentioned light-controlled proton pump expression module or the above-mentioned recombinant engineered bacteria, wherein the applications include any of the following:

[0024] (1) To explore the molecular mechanisms of salt tolerance in different cell types of plant roots;

[0025] (2) To improve the salt tolerance of plants;

[0026] (3) Cultivate salt-tolerant plants.

[0027] Preferably, in (1) to (3), the plants include: Arabidopsis thaliana, maize, rice, wheat, and other closely related aquatic plant groups such as duckweed with roots.

[0028] Preferably, the application includes enhancing the salt tolerance of plant roots.

[0029] The present invention also provides a method for improving the salt tolerance of plants, the method comprising:

[0030] Using Agrobacterium-mediated transformation and infection, at least one light-controlled proton pump expression plasmid was introduced into plants and cultured continuously to obtain plants with improved salt tolerance.

[0031] Preferably, the plants include: Arabidopsis thaliana, maize, rice, wheat, and rooted aquatic plants such as Lemnae.

[0032] Preferably, during continuous culture, a β-carotene precursor is added to the liquid culture medium.

[0033] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0034] This invention is the first to express two proton pumps in three root cell groups of Arabidopsis thaliana, and enhance membrane localization through methods such as liquid culture. Using the pH-sensitive dye bromocresol purple (BCP), it was determined that light-induced proton efflux from root cells led to culture medium acidification. This transgenic line exhibited significant resistance to salt stress, indicating that the molecular mechanism regulating salt tolerance in the three cell groups is universal. The protein expression module of the light-controlled proton pump described in this invention can be directly used to enhance salt tolerance in other plants, especially to improve the light-controlled salt tolerance of rooted floating plants in water. Under light conditions, the heterologously expressed light-controlled proton pump can directionally pump H⁺ from the cytoplasm into the apoplast space, establishing and maintaining a high transmembrane proton kinetic potential. The SOS1 transporter on the cell membrane uses this proton gradient to carry out reverse Na⁺ / H⁺ exchange, using the H⁺ influx potential energy to drive the expulsion of excess Na⁺ from the cell to the extracellular space, reducing intracellular Na⁺ accumulation and ion toxicity, and ultimately enhancing the plant's salt tolerance.

[0035] This invention, through experimental verification, shows that compared to the control group (without NaCl treatment), the root length of homozygous plants expressing light-controlled proton pumps was significantly longer than that of wild-type plants under NaCl concentrations of 50 mM and 100 mM. This indicates that the expression of the two light-controlled proton pumps in all three root cell groups exhibits phenotypic characteristics of salt stress resistance. This technology mainly enables light-controlled regulation of plant salt tolerance, overcoming the limitations of existing technologies that rely on gene knockout or overexpression.

[0036] This invention not only provides a theoretical basis for in-depth research into the molecular mechanisms of salt tolerance in different cell types of plant roots (applicable to, but not limited to, the analysis of the molecular mechanisms of salt tolerance in crops such as corn, rice, and wheat), but also provides new technical directions and ideas for the development and application of salt tolerance characteristics of other aquatic phytoplankton roots (applicable to, but not limited to, the salt tolerance cultivation of closely related aquatic plant groups such as Lemnae), and has great practicality. Attached Figure Description

[0037] Figure 1 This is a schematic diagram of the construction mode of the light-controlled proton pump expression module.

[0038] Figure 2 The stable expression of the light-controlled proton pump expression module was shown in three root cell groups. Figure a shows the expression of proton pump ApOps2 in root meristem cells; Figure b shows the expression of proton pump ApOps2 in root endodermal cells; Figure c shows the expression of proton pump ApOps2 in root cortex cells; Figure d shows the expression of proton pump CvRH in root meristem cells; Figure e shows the expression of proton pump CvRH in root endodermal cells; Figure f shows the expression of proton pump CvRH in root cortex cells.

[0039] Figure 3Optimization of the expression of the light-controlled proton pump expression module containing ApOps2 to enhance membrane localization in three root cell groups. Figure a shows the expression of proton pump ApOps2 in root meristematic cells; Figure b shows the expression of proton pump ApOps2 in root endothelial cells; Figure c shows the expression of proton pump ApOps2 in root cortical cells.

[0040] Figure 4 Optimization of CvRH-containing light-controlled proton pump expression module for enhanced membrane localization in three root cell groups. Figure a shows the expression of CvRH proton pump in root meristem cells; Figure b shows the expression of CvRH proton pump in root endothelial cells; Figure c shows the expression of CvRH proton pump in root cortex cells.

[0041] Figure 5 To illustrate how two light-controlled proton pump expression modules, including ApOps2 and CvRH, mediate the acidification of culture media near different root cells in Arabidopsis thaliana. Figure a shows the acidification of culture media near root cells after specific expression of the proton pump ApOps2 in the wild-type Col-0 control group, endodermal cells, and cortical cells, respectively; Figure b shows the acidification of culture media near root cells after specific expression of the proton pump CvRH in the wild-type Col-0 control group, endodermal cells, and cortical cells, respectively.

[0042] Figure 6 This study analyzed the root length phenotype of mutants expressing the light-controlled proton pump ApOps2 and CvRH expression modules in root meristem cells, endothelial cells, and cortical cells (control group: no NaCl treatment). Figure a shows the root length phenotype of wild-type Col-0 mutants expressing the proton pump ApOps2 in the three root cell groups; Figure b shows the statistical results of the root length phenotype in Figure a, with no significant difference (n>10); Figure c shows the root length phenotype of wild-type Col-0 mutants expressing the proton pump CvRH in the three root cell groups; Figure d shows the statistical results of the root length phenotype in Figure c, with no significant difference (n>10).

[0043] Figure 7The salt stress response of the light-controlled proton pump ApOps2 and CvRH expression modules after broad-spectrum expression in root meristem cells (experimental groups: 50 mM, 100 mM NaCl treatment). Figure a shows the salt stress phenotype of wild-type Col-0; Figure b shows the salt stress phenotype of the proton pump ApOps2 expression mutant in root meristem cells; Figure c shows the salt stress phenotype of the proton pump CvRH expression mutant in root meristem cells; Figure d shows the root length measurements (n>10) of wild-type Col-0, root meristem cells expressing proton pump ApOps2, and root meristem cells expressing proton pump CvRH after 50 mM NaCl treatment; Figure e shows the root length measurements (n>10) of wild-type Col-0, root meristem cells expressing proton pump ApOps2, and root meristem cells expressing proton pump CvRH after 100 mM NaCl treatment.

[0044] Figure 8 The table shows the salt stress response of the light-controlled proton pump expression module after expression in root endothelial cells (treated with 50 mM and 100 mM NaCl). Figure a shows the salt stress phenotype of wild-type Col-0; Figure b shows the salt stress phenotype of the proton pump ApOps2 mutant expressed in root endothelial cells; Figure c shows the salt stress phenotype of the proton pump CvRH mutant expressed in root endothelial cells; Figure d shows the root length measurements (n>10) of wild-type Col-0, endothelial cells expressing proton pump ApOps2, and endothelial cells expressing proton pump CvRH mutants after 50 mM NaCl treatment; Figure e shows the root length measurements (n>10) of wild-type Col-0, endothelial cells expressing proton pump ApOps2, and endothelial cells expressing proton pump CvRH mutants after 100 mM NaCl treatment.

[0045] Figure 9 The table shows the salt stress response of the light-controlled proton pump expression module after expression in root cortex cells (treated with 50 mM and 100 mM NaCl). Figure a shows the salt stress phenotype of wild-type Col-0; Figure b shows the salt stress phenotype of the proton pump ApOps2 mutant expressed in root cortex cells; Figure c shows the salt stress phenotype of the proton pump CvRH mutant expressed in root cortex cells; Figure d shows the root length measurements (n>10) of wild-type Col-0, proton pump ApOps2 expressed in cortex cells, and proton pump CvRH expressed in cortex cells after 50 mM NaCl treatment; Figure e shows the root length measurements (n>10) of wild-type Col-0, proton pump ApOps2 expressed in cortex cells, and proton pump CvRH expressed in cortex cells after 100 mM NaCl treatment. Detailed Implementation

[0046] The present invention will be further described below with reference to the accompanying drawings and specific embodiments, but the scope of protection of the present invention is not limited thereto. In the following embodiments, various processes and methods not described in detail are conventional methods known in the art. The source, trade name, and components of the reagents used, if necessary, are indicated upon their first appearance. Unless otherwise specified, subsequent use of the same reagents will be based on the same information as initially indicated.

[0047] The vectors used in the following examples are derived from the cloning vector toolkit in addgene (#1000000036), including six intermediate vectors: pGGA, pGGB, pGGC, pGGD, pGGE, and pGGF; the plant expression vector pFASTR-AG was constructed in Decaestecker & Buono et al. Plant Cell (2019). All of the above vectors are commercially available.

[0048] Example 1: Construction of a light-controlled proton pump expression plasmid

[0049] In this embodiment, a cell-specific promoter and a light-controlled proton pump gene were fused into the pFASTR-AG expression vector using the Golden Gate cloning system to construct a light-controlled proton pump expression plasmid. To confirm the expression of the light-controlled proton pump gene in different root cell populations, this embodiment also fused the C-terminal red fluorescent protein mScarlet (SEQ ID No:7) into the constructed light-controlled proton pump expression plasmid. The specific steps are as follows:

[0050] S1. Based on the sequence information of the specific promoters pRPL11C (SEQ ID No:4), pSCR (SEQ ID No:5), and pCO2 (SEQ ID No:6), specific primers with BsaI restriction sites were designed. The fragments of pRPL11C, pSCR, and pCO2 were amplified by primer PCR, respectively. Then, the amplified pRPL11C, pSCR, and pCO2 fragments were constructed into the pGGA intermediate vector, respectively, to obtain three intermediate vectors with promoters: pGGA-pRPL11C, pGGA-pSCR, and pGGA-pCO2.

[0051] The sequences of the specific primers are shown below:

[0052] pRPL11C fw: aacaGGTCTCaacctGGAGTTGTTCGAGGCAGC (SEQ ID No: 8);

[0053] pRPL11C rev:aacaGGTCTCttgttCTCAAGATTAGGGGTTCGA (SEQ ID No:9);

[0054] pSCR fw: aacaGGTCTCaacctGACAAGCTCAATGTAAGC (SEQ ID No: 10);

[0055] pSCR rev: aacaGGTCTCttgttGGAGATTGAAGGGTTGTTG (SEQ ID No: 11);

[0056] pCO2 fw: aacaGGTCTCaacctGCAAACATATATTCTTAAAAT (SEQ ID No: 12);

[0057] pCO2 rev:aacaGGTCTCttgttTATCGTTATTAACTAGGGTT (SEQ ID No: 13).

[0058] S2. Based on the light-controlled proton pump genes ApOps2 (SEQ ID No:1) and CvRH (SEQ ID No:2), specific primers with BsaI restriction sites were designed. The ApOps2 and CvRH fragments were amplified by primer PCR, respectively. The amplified ApOps2 and CvRH fragments were then constructed into the pGGC intermediate vector to obtain two intermediate vectors with light-controlled proton pumps, pGGC-ApOps2 and pGGC-CvRH.

[0059] The sequences of the specific primers are shown below:

[0060] ApOps2 fw: aacaGGTCTCaggctATGGATTTCTTACAAAAGAG (SEQ ID No: 14);

[0061] ApOps2 rev: aacaGGTCTCtctgaGACCGTCTCTGCAGTCTG (SEQ ID No: 15);

[0062] CvRH fw: aacaGGTCTCaggctATGGCTGTGCACCAGATT (SEQ ID No: 16);

[0063] CvRH rev: aacaGGTCTCtctgaGCTGTTGATCAGCAGGCT (SEQ ID No: 17).

[0064] S3. The chloroplast or plastid localization signal peptide RC2 was ligated with the gene sequence of β-carotene 15,15'-dioxygenase MbDio using the gene sequence of a P2A self-cleaving polypeptide. The ligated sequence is shown in SEQ ID No:3. The ligated SEQ ID No:3 was then fused to the 5' end of the proton pump gene to obtain the intermediate expression vectors pGGC-RC2-MbDio-P2A-ApOps2 and pGGC-RC2-MbDio-P2A-CvRH.

[0065] S4. The red fluorescent protein mScarlet (SEQ ID No:7) was ligated into the pGGD intermediate vector to obtain the intermediate vector pGGD-mScarlet.

[0066] S5. The vectors constructed in steps S1, S3, and S4 are digested with BsaI. Other pGGB, pGGE, and pGGF intermediate vectors in Golden Gate containing N-tags, terminators, and selection marker genes are also digested with BsaI. Then, the resulting vectors are ligated to pFASTR-AG using T4 DNA ligase to obtain the light-controlled proton pump expression plasmid. Depending on the vectors selected in steps S1 and S3, different light-controlled proton pump expression plasmids are constructed, as follows:

[0067] (a) pFASTR-pRPL11C::RC2-MbDio-P2A-ApOps2-mScarlet;

[0068] (b) pFASTR-pRPL11C::RC2-MbDio-P2A-CvRH-mScarlet;

[0069] (c) pFASTR-pSCR::RC2-MbDio-P2A-ApOps2-mScarlet;

[0070] (d) pFASTR-pSCR::RC2-MbDio-P2A-CvRH-mScarlet;

[0071] (e) pFASTR-pCO2::RC2-MbDio-P2A-ApOps2-mScarlet;

[0072] (f) pFASTR-pCO2::RC2-MbDio-P2A-CvRH-mScarlet.

[0073] Taking pFASTR-pRPL11C::RC2-MbDio-P2A-ApOps2-mScarlet as an example, the construction of the light-controlled proton pump expression plasmid is described. The construction methods for other light-controlled proton pump expression plasmids are basically the same, with the only difference being the intermediate vectors used in steps S1 and S3. The specific steps are as follows:

[0074] Take 100 ng each of the six intermediate vectors obtained in step S1 (pGGA-pRPL11C), step S3 (pGGC-RC2-MbDio-P2A-ApOps2), step S4 (pGGD-mScarlet), pGGB, pGGE, and pGGF), and add them to a PCR tube. Then add 100 ng of the target vector pFASTR-AG, add ultrapure water, and then add buffer, BsaI enzyme, and T4 DNA ligase in sequence.

[0075] The enzyme digestion and ligation system is as follows: 6 μL of pGGA-pRPL11C, pGGC-RC2-MbDio-P2A-ApOps2 obtained in step S3, pGGD-mScarlet, pGGB, pGGE and pGGF obtained in step S4 (100 ng each of the 6 intermediate vectors), 1 μL of the target vector pFASTR-AG (approximately 100 ng), 1 μL of BsaI restriction endonuclease, 1 μL of T4 DNA ligase, 2 μL of 10x T4 DNA ligase buffer, and ddH2O to a final volume of 20 μL.

[0076] The assembly reaction procedure is as follows: enzyme digestion at 37℃ for 5 min, ligation at 16℃ for 5 min, and repeat the enzyme digestion and ligation steps 30 times; end trimming at 50℃ for 5 min, enzyme inactivation at 80℃ for 5 min, and then terminate the reaction.

[0077] After the reaction, the product can be directly used to transform *E. coli* DH5α competent cells, verifying the yield of pFASTR-pRPL11C::RC2-MbDio-P2A-ApOps2-mScarlet. This light-controlled proton pump expression plasmid comprises the FASTR target vector and the fusion of six expression modules, such as... Figure 1 As shown.

[0078] Similarly, five other light-controlled proton pump expression plasmids can be obtained.

[0079] Example 2: Genetic transformation and homozygous plant selection of light-controlled proton pump expression plasmid in Arabidopsis plants

[0080] S1. Agrobacterium-mediated transformation and Arabidopsis inflorescence infection transformation:

[0081] The six photosensitive proton pump expression plasmids correctly sequenced in Example 1 were transformed into Agrobacterium GV3101 competent cells using a liquid nitrogen freeze-thaw method. The transformed plasmids were then evenly spread onto LB agar plates containing 50 μg / mL spectinomycin and 25 μg / mL rifampin. The plates were incubated at 28 °C in the dark until single colonies appeared, yielding recombinant engineered bacteria 1-6, each carrying one of the six photosensitive proton pump expression plasmids.

[0082] Transformation using Arabidopsis thaliana inflorescence infection: Wild-type Arabidopsis thaliana Col-0 plants (laboratory-saved seed) in full bloom were selected, and mature siliques were removed. The constructed recombinant engineered bacteria 1-6 were cultured in LB medium containing 50 μg / mL spectinomycin and 25 μg / mL rifampin to the logarithmic growth phase. The bacterial cells were collected by centrifugation at 5000 rpm for 10 minutes and resuspended to an OD600 of 0.8-1.0. The inflorescences of the plants were completely immersed in the infection solution (100 mL aqueous solution containing 7 g sucrose and 30 μL surfactant Silwet-77) for 30 seconds, gently agitating during this time to ensure complete infection. The infected plants were laid flat and kept in the dark overnight, then cultured under normal light the next day until seed maturity.

[0083] S2. Once the infected Arabidopsis plants are fully mature and the pods have turned yellow, stop watering and collect the T0 generation seeds by dividing the plantlets. Place the seeds in 1.5 mL centrifuge tubes and add a desiccant to mature them for about a week, ensuring that the seeds are completely dried to guarantee a normal germination rate.

[0084] The Ruby reporter gene in the pFASTR vector system enables the expression of red betalains in the seed coat for non-destructive screening of homozygous plants. Dried T0 generation seeds were evenly dispersed on non-fluorescent white backing paper and observed using a stereomicroscope (ZEISS, Axio Zoom.V16). Seeds with bright and uniformly distributed red fluorescence signals were selected as positive materials under mRFP excitation light (excitation wavelength range approximately 530-570 nm). These positive seeds were directly sown in soil. Subsequent observation of fluorescence segregation in progeny seeds allowed for rapid identification and acquisition of homozygous T3 generation lines without phenotypic segregation. This method enables highly efficient initial screening of transgenic lines before sowing.

[0085] S3. This step uses a Zeiss laser scanning confocal microscope (ZEISS, LSM980) to verify the expression of the light-controlled proton pump in different cell groups (cortex, endodermis, and epidermis) of plant roots. The specific steps are as follows:

[0086] Five-day-old transgenic Arabidopsis seedlings (T3 generation homozygous lines without phenotypic segregation) were placed on a glass slide with deionized water. For lines with mScarlet fluorescent tags, the excitation wavelength was set to 561 nm, and the emission wavelength collection range was 580–630 nm. During imaging, the fluorescence signal of the light-controlled proton pump expression pump in three cell groups (cortex, endodermis, and epidermis) in the root was observed using a 20x or 40x objective lens. A bright-field channel was activated to calibrate cell contours, confirming whether the protein was stably expressed in the corresponding cell groups. The results are as follows: Figure 2 As shown. From Figure 2 It can be seen that the light-controlled proton pump labeled with red fluorescent protein is stably expressed in three root cell types. This result indicates that the light-controlled proton pump can be stably genetically expressed in plant cells, suggesting that it can also be stably expressed in root cells of other plants (including but not limited to crops such as maize, rice, and wheat, as well as closely related groups such as aquatic duckweed).

[0087] Example 3: Enhancing the membrane localization efficiency of light-controlled proton pumps in liquid culture

[0088] To enhance the membrane localization efficiency of the photocontrolled proton pump, this embodiment uses sterile liquid culture medium combined with 12-well plates to culture Arabidopsis seeds (including wild type and six transgenic homozygous mutants with expression modules; the specific culture method is as follows:)

[0089] First, the Arabidopsis seeds were sterilized by soaking them in a 75% (v / v) ethanol solution for 5 minutes, followed by disinfection with a 1% (v / v) sodium hypochlorite solution for 10 minutes. After disinfection, the seeds were rinsed repeatedly with sterile water 5 times to ensure that the seed surface was sterile.

[0090] Sterilized Arabidopsis thaliana seeds were sown in 12-well plates containing 1 / 2 MS liquid medium. Before sowing, the pH of the 1 / 2 MS liquid medium was adjusted to 5.8 with KOH solution. During the physiological experiment, after the liquid medium was autoclaved and cooled to room temperature, β-carotene precursor at a final concentration of 100 μM was added to each well. An appropriate amount of seeds (n>10) were sown in each well. Subsequent seedling culture and fluorescence imaging were performed. The results are as follows: Figure 3 and Figure 4 As shown.

[0091] As can be seen from the figure, confocal microscopy analysis of ApOps2 membrane localization efficiency ( Figure 3 ) and CvRH membrane positioning efficiency ( Figure 4 It can be seen that, compared to Figure 2In solid culture without β-carotene, the distribution of proton pump membrane localization fluorescence signals in root cells showed a significant increase in the fluorescence signals of the proton pumps ApOps2 and CvRH, especially... Figure 4 The photocontrolled proton pump CvRH derived from chlorophyll has the best membrane localization effect.

[0092] Example 4: Detection of acidification in the culture medium near roots expressing light-controlled proton pumps under light-induced conditions.

[0093] The color evolution characteristics of bromocresol purple (BCP) under different pH conditions were used to qualitatively evaluate the function of the photo-controlled proton pump in effluxing hydrogen ions. The specific steps included:

[0094] (1) To monitor rhizosphere acidification using an indicator colorimetric reaction, a rhizosphere pH monitoring medium was first prepared. The preparation steps were as follows: 0.006% (w / v) of BCP was added to 1 / 2 MS solid medium after sterilization. To improve the sensitivity of the monitoring, the initial pH of the medium was precisely adjusted to 6.6 using KOH / NaOH. At this point, the medium turned a deep purple color and was designated as the BCP colorimetric medium.

[0095] (2) Wild-type Col-0 was set up as a parallel control to eliminate the influence of other factors on the pH of the medium. Arabidopsis seeds were sown in 1 / 2 MS solid medium (without BCP) at pH 5.8 and cultured for 8 days under red light to allow the roots to develop normally and keep the proton pump in a low-activity state. Subsequently, without washing the roots, the seedlings were transferred as a whole to BCP chromogenic medium with an initial pH of 6.6. After induction with red and green light for 2 days, the change of the rhizosphere medium from purple (alkaline) to yellow (acidic) was recorded using a DSLR camera.

[0096] The results are as follows Figure 5 As shown, the ApOps2 proton pump, when used in conjunction with the CvRH proton pump, can acidify the root culture medium. The most acidic effect is observed in the proton pump driven by the widely expressed promoter pRPL11C in the meristematic zone, where the yellow color of the root culture medium is most pronounced. This result confirms that activated proton pumps can acidify the culture medium by effluxing intracellular hydrogen ions.

[0097] Example 5: Enhanced Salt Stress Tolerance Response of Photocontrolled Proton Pumps under Light-Induced Irradiation

[0098] This step evaluates the effect of the photocontrolled proton pump module on improving salt tolerance in Arabidopsis thaliana through gradient salt stress treatment. Using Arabidopsis thaliana as a model plant, it infers the role of the photocontrolled proton pump module in other plants. For example, in exploring the molecular mechanisms of salt tolerance in different cell types of plant roots, the application of photocontrolled proton pump salt tolerance stress can be extended to the analysis of the molecular mechanisms of salt tolerance in plants such as Arabidopsis thaliana, maize, rice, and wheat. In terms of salt tolerance cultivation of plants, the application of photocontrolled proton pump salt tolerance stress can be extended to the salt tolerance cultivation of closely related aquatic plant groups such as Lemnae with roots.

[0099] The experiment was conducted in a liquid shake culture mode using 12-well plates. 2 mL of liquid culture medium containing NaCl concentration gradients (0, 50 mM, 100 mM) was added to each well, and 10 Arabidopsis thaliana seeds (including wild-type and six mutants) were inoculated. Three biological replicates were performed. After inoculation, the 12-well plates were placed on a shaker at 100 rpm.

[0100] Illumination-induced mode settings: Two culture environments were set up: continuous red light and red-green light. Specific spectral physical parameters were: red LED spectrum 660 nm ± 85 nm, green LED spectrum 524 nm ± 65 nm, and light intensity set at 1.59 mW / cm². The ambient temperature was maintained at 22℃, and the light cycle was set to 16 h light / 8 h darkness.

[0101] Root length and germination rate were measured for each line on the 7th day after sowing. Plate images were acquired using a DSLR camera, and the taproot length of each line was measured using ImageJ software. Each experiment was repeated with a sample size of at least 30 seedlings. Relative germination rate and the degree of root length inhibition were calculated based on the measurement results. GraphPad Prism software was used for significant difference analysis (Student's t-test) to assess the contribution of proton pump expression to enhancing plant salt tolerance. The results showed that, compared with the wild type, both proton pumps (ApOps2, CvRH) exhibited stronger salt-tolerant phenotypes in all three root cell groups, including cells with broad-spectrum expression in the meristematic zone (such as...). Figure 7 As shown), endothelial cells (such as...) Figure 8 (as shown) and cortical cells (such as Figure 9 As shown). Seven days after the control group was not treated with NaCl, there was no significant difference in root growth between transgenic mutants expressing the two light-controlled proton pumps in the three root cell groups. Figure 6 After 7 days of treatment with 50 mM NaCl salt stress, the root growth of both light-controlled proton pump transgenic mutants was significantly better than that of the wild type, with root lengths 1.5–2 times that of the wild type. Figures 7-9When the NaCl concentration was increased to 100 mM, the CvRH light-controlled proton pump overexpression lines showed stronger salt tolerance, with root lengths reaching twice that of the wild type, and significant differences in salt tolerance phenotype. Figures 7-9 ).

[0102] In summary, this invention has achieved the specific expression of light-controlled proton pumps (PLPs) from two microorganisms in Arabidopsis root cells by constructing a PLP expression module. The PLP expression module also expresses β-carotene 15,15'-dioxygenase MbDio. The PLP expression module significantly improves the membrane localization efficiency of PLPs in root cells. It also significantly increases the acidification of the culture medium near the roots and specifically enhances the salt tolerance of the plant. Using Arabidopsis as a model plant, this invention not only provides a theoretical basis for in-depth research into the molecular mechanisms of salt tolerance in different root cell types but also offers new technical directions and ideas for the development and application of salt tolerance characteristics in the roots of other aquatic phytoplankton, demonstrating significant practicality.

[0103] The embodiments described above are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments. Any obvious improvements, substitutions or modifications that can be made by those skilled in the art without departing from the essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. A light-controlled proton pump expression module, characterized in that, The light-controlled proton pump expression module includes: a light-controlled proton pump gene and a promoter; The light-controlled proton pump gene includes ApOps2 from fungi or CvRH from algae; The promoters include pRPL11C, pSCR, or pCO2.

2. The photocontrolled proton pump expression module according to claim 1, characterized in that, The nucleotide sequence of the fungal ApOps2 is shown in SEQ ID No:1; The nucleotide sequence of the algal-derived CvRH is shown in SEQ ID No:2; The nucleotide sequence of pRPL11C is shown in SEQ ID No:4; The nucleotide sequence of the pSCR is shown in SEQ ID No:5; The nucleotide sequence of the pCO2 is shown in SEQ ID No:

6.

3. The photocontrolled proton pump expression module according to claim 1, characterized in that, The light-controlled proton pump expression plasmid also expresses β-carotene 15,15'-dioxygenase MbDio.

4. The photocontrolled proton pump expression module according to claim 3, characterized in that, β-carotene 15,15'-dioxygenase MbDio was expressed by linking the gene sequences of the chloroplast or plastid localization signal peptide RC2, the P2A self-cleaving polypeptide, and the β-carotene 15,15'-dioxygenase MbDio. The ligated nucleotide sequence is shown in SEQ ID No:

3.

5. A light-controlled proton pump expression plasmid, characterized in that, The light-controlled proton pump expression plasmid comprises the light-controlled proton pump expression module as described in any one of claims 1 to 4.

6. The light-controlled proton pump expression plasmid according to claim 5, characterized in that, The expression vector for the light-controlled proton pump expression plasmid includes pFASTR-AG.

7. Recombinant engineered bacteria, characterized in that, The recombinant engineered bacteria comprises at least one light-controlled proton pump expression module as described in any one of claims 1 to 4, or at least one light-controlled proton pump expression plasmid as described in any one of claims 5 or 6.

8. The application of the light-controlled proton pump expression module according to any one of claims 1 to 4, or the recombinant engineered bacteria according to claim 7, wherein the application includes any one of the following: (1) To explore the molecular mechanisms of salt tolerance in different cell types of plant roots; (2) To improve the salt tolerance of plants; (3) Cultivate salt-tolerant plants.

9. The application according to claim 8, characterized in that, (1) to (3) The plants mentioned include: Arabidopsis thaliana, maize, rice, wheat, and rooted duckweed family and other closely related aquatic plant groups; The applications include enhancing the salt tolerance of plant roots.

10. A method for improving the salt tolerance of plants, characterized in that, The method includes: Using Agrobacterium-mediated transformation and infection, at least one of the light-controlled proton pump expression plasmids described in any one of claims 1 to 4 is introduced into plants and continuously cultured to obtain plants with improved salt tolerance.

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