Method for regulating plant alkali tolerance by light-controlled sodium ion pump

Abstract

The application belongs to the technical field of plant alkali tolerance regulation, and discloses a method for regulating plant alkali tolerance by using light-controlled sodium ion pumps, which comprises the following steps: firstly, a KR2 light-controlled sodium ion pump recombinant expression vector carrying different plant-derived membrane localization signal peptides is constructed, the recombinant vector is introduced into Agrobacterium to obtain positive engineering bacteria, and then the recombinant fragment is integrated into the target plant genome by transient or stable transformation to complete expression; 532 nm green light is matched with different alkali stress degrees to set corresponding light modes, KR2 protein specificity is activated to expel excess intracellular sodium ions, and precise regulation of plant alkali tolerance under different degrees of alkali stress is realized. The method does not depend on intracellular energy supply, the membrane localization efficiency is higher than that of the existing alkali tolerance regulation system, the regulation specificity is strong, there is no secondary pollution, the transformation efficiency of field crops is increased by nearly one time, the survival rate and growth performance of plants under different alkali stress environments are effectively improved, and the method is suitable for the alkali tolerance breeding and saline-alkali land planting requirements of various field crops.

Description

Technical Field

[0001] This invention belongs to the field of plant alkali tolerance regulation technology, and in particular to a method for regulating plant alkali tolerance using a light-controlled sodium ion pump. Background Technology

[0002] Alkali stress is one of the core abiotic stresses restricting global agricultural production. High alkalinity can lead to excessive accumulation of sodium ions in crop cells, high pH toxicity, and carbonate / bicarbonate ion imbalance, disrupting cellular acid-base homeostasis and ion balance. This results in hindered water and nutrient absorption, disordered photosynthetic metabolism, and damage to cell membrane systems, ultimately causing a significant drop in yield. my country has over 60 million mu (approximately 4 million hectares) of alkali-tolerant arable land, and this area continues to expand annually. Developing efficient, green, and scalable crop alkali tolerance regulation technologies is a core industry requirement for the comprehensive utilization of saline-alkali land and ensuring food security.

[0003] Currently, the mainstream control methods are divided into two categories: soil chemical improvement and crop stress-resistance genetic engineering. Soil chemical improvement, which involves applying amendments such as gypsum, acidic weathered coal, and humic acid to neutralize soil alkalinity and replace exchangeable sodium adsorbed by soil colloids, is a commonly used method for saline-alkali land management. Its advantages lie in its rapid effectiveness, wide applicability, and ability to quickly improve the rhizosphere soil environment. However, this method suffers from high costs and short-lasting effects. Long-term, large-scale application can easily lead to secondary problems such as soil nutrient imbalance and secondary pollution. Furthermore, it cannot improve the alkali tolerance of crops themselves, making it difficult to meet the needs of sustainable utilization of large-scale saline-alkali land.

[0004] Another mainstream approach is stress-resistant genetic engineering, which involves overexpressing Na+ in the plasma membrane. + / H + Stress-resistance genes such as the retrotransporter SOS1 enhance the crop's sodium ion efflux capacity, or constitutive / chemically inducible promoters drive the expression of stress-resistance genes, and have been applied in stress-resistance breeding of various crops. However, this type of technology has two major drawbacks: First, the function of transporters such as SOS1 is highly dependent on intracellular ATP and transmembrane proton gradients, and the transport efficiency drops sharply when cells are energy deficient under alkaline stress; second, constitutive expression easily leads to unnecessary energy consumption and growth inhibition in crops, while chemically induced systems suffer from problems such as inducer residues, uneven induction efficiency, and insufficient spatiotemporal specificity, making it impossible to achieve dynamic, reversible, and precise regulation of sodium ion transport.

[0005] Currently, optogenetic tools have made breakthroughs in areas such as plant growth regulation and immune activation, but optogenetic tools and supporting regulatory systems specifically for the regulation of sodium ion homeostasis in crops are still lacking. Existing technologies cannot simultaneously meet the requirements of low cost, environmental friendliness, high regulatory precision, and no interference with normal crop growth. Summary of the Invention

[0006] To address the shortcomings of existing plant alkali tolerance regulation technologies, such as reliance on intracellular energy for transport activity, low regulation precision, and susceptibility to secondary pollution or growth inhibition, this invention provides a method for regulating plant alkali tolerance using a light-controlled sodium ion pump.

[0007] To achieve the above objectives, the present invention adopts the following technical solution: A method for regulating plant alkali tolerance using a light-controlled sodium ion pump includes the following steps: S1. Construct a plant expression vector for the KR2 light-controlled sodium ion pump. Connect the 3′ end of the KR2 sodium ion pump coding sequence from the marine bacterium Krokinobactereikastus to four membrane localization signal peptides with different membrane localization efficiencies to obtain gene fragments encoding four recombinant proteins: KR2-T1.0, KR2-T2.0, KR2-T3.0, and KR2-T4.0. Use the UBQ10 strong promoter to drive the expression of the recombinant gene fragments. Insert the recombinant gene fragments into the plant expression backbone vector to obtain a recombinant expression vector with the correct sequence. S2. The recombinant expression vector is transformed into Agrobacterium competent cells, and positive Agrobacterium engineered bacteria carrying the recombinant vector are obtained through resistance screening; S3. The coding sequence of the KR2 photosensitive sodium ion pump is integrated into the genome of the target plant using Agrobacterium-mediated genetic transformation to obtain transformed plants that can stably or transiently express the KR2 photosensitive sodium ion pump. S4. Irradiate the transformed plants with green light at a wavelength of 532nm to activate the transport activity of the KR2 photocontrolled sodium ion pump, mediate the active efflux of excess sodium and hydrogen ions in plant cells, maintain cell ion homeostasis and acid-base balance, and enhance the plant's tolerance to alkaline stress.

[0008] Furthermore, the plant expression backbone vector mentioned in step S1 is pCAMBIA3300. Four recombinant gene fragments are inserted into the multiple cloning site of the backbone vector using homologous recombination. After the recombinant products are transformed into Escherichia coli DH5α competent cells, positive single clones are screened using kanamycin at a concentration of 50 μg / mL. After sequencing verifies that the recombinant gene fragment sequence is completely correct, the plasmid is extracted to obtain the recombinant expression vector.

[0009] Furthermore, the Agrobacterium mentioned in step S2 is strain GV3101. The specific operation of transforming Agrobacterium with recombinant expression vector is as follows: 1 μg of recombinant expression vector plasmid is added to 100 μL of Agrobacterium competent cells, and the cells are successively subjected to ice bath for 30 min, liquid nitrogen flash freezing for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min. Then, 800 μL of LB liquid medium is added and cultured at 28℃ and 200 rpm for 4 h with shaking. Subsequently, the cells are plated on LB solid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and cultured upside down at 28℃ for 24 h. Positive Agrobacterium engineered bacteria are then screened.

[0010] Further, the target plant in step S3 is *Nicotiana benthamiana*, and transformation is performed using an Agrobacterium-mediated transient leaf transformation method. Specifically, Agrobacterium carrying the recombinant vector is inoculated into LB liquid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and cultured at 28°C with shaking at 200 rpm until the OD600 reaches 0.6–0.8. After collecting the bacterial cells, they are resuspended in an osmotic buffer containing 10 mM MES, 10 mM magnesium chloride, and 200 μM acetylsyleugenol, and the OD600 of the bacterial solution is adjusted to 0.6–0.8. The resuspended bacterial solution is then slowly injected into the lower epidermis of leaves of 4–6 week old *Nicotiana benthamiana* using a needleless syringe. The injected *Nicotiana benthamiana* is then placed under a light intensity of 100–150 μmol / m². -2 s -1 Tobacco Bunsenii plants transiently expressing the KR2 photocontrolled sodium ion pump were obtained by culturing them in an artificial incubator with a photoperiod of 16h light / 8h darkness and a temperature of 25±2℃ for 48~72h.

[0011] Furthermore, the target plant in step S3 is Brassica napus, and transformation is performed using Agrobacterium-mediated hypocotyl genetic transformation. Specifically, plump and uniform Brassica napus seeds are first selected, and then disinfected by soaking in 75% alcohol for 1 min, soaking in 50% 84 disinfectant for 3 min, and rinsing 10 times with sterile water. The seeds are then sown in MO medium at pH 5.80-5.84 and cultured in the dark for 5-7 days to obtain sterile seedlings. Agrobacterium carrying the recombinant vector is cultured until the OD600 reaches approximately 0.4, and then resuspended in DM infection solution. Hypocotyls of 0.8-1.0 cm sterile seedlings are placed in the bacterial solution for 8 days of infection. After aspirating the bacterial solution for 10 minutes, the culture was successively cultured in the dark on M1 co-culture medium for 36-48 hours, M2 callus induction medium for 20 days, M3 bud induction medium for subculturing every 8 days until green buds differentiated, and M4 rooting medium for 2-4 weeks to obtain regenerated seedlings. Genomic DNA was extracted from the leaves of the regenerated seedlings using the CTAB method, and the presence of a specific 914bp band in PCR detection indicated a positive transgenic line. The pH of the M0, DM, M1, M2, M3, and M4 culture media was adjusted to 5.80-5.84, and acetylsyleugenol, antibiotics, screening agents, and plant growth regulators were added as needed.

[0012] Furthermore, the intensity of the green light in step S4 is 100~300 μW / mm. 2 The irradiation mode is adjusted to intermittent or continuous irradiation according to the degree of alkali stress. The intermittent irradiation cycle is 2 min light / 1 min dark, which is suitable for severe alkali stress environment. The continuous irradiation matches the normal light cycle of 16 h light / 8 h dark for plant growth, which is suitable for moderate or mild alkali stress environment.

[0013] Furthermore, the four recombinant proteins have different membrane localization efficiencies and sodium ion efflux activities. Among them, KR2-T3.0 and KR2-T4.0 are suitable for regulating plant alkali tolerance under severe alkali stress; KR2-T1.0 and KR2-T2.0 are suitable for mild and moderate alkali stress.

[0014] Furthermore, the study included functional verification steps for the transformed plants. Non-destructive microelectrode technology was used to detect changes in membrane potential of plant leaves under green light irradiation. Before detection, detached leaves of the transformed plants were placed in a 100 mM sodium bicarbonate alkali stress treatment group, a 100 mM sorbitol osmotic stress control group, and a deionized water blank control group for 18 h, respectively. A wavelength of 532 nm and a light intensity of 268 μW / mm² were used. 2 After 5 seconds of green light irradiation, when a significant membrane potential depolarization signal was detected in the alkaline stress treatment group, and no such depolarization signal was observed in the osmotic stress control group and the blank control group, the KR2 photocontrolled sodium ion pump function was determined to be normally activated.

[0015] Furthermore, the method is applicable to alkaline stress environments with high pH. The hydrogen ion efflux mediated by the KR2 photocontrolled sodium ion pump can simultaneously neutralize extracellular hydroxide ions, alleviating the ion imbalance caused by high pH toxicity and bicarbonate and carbonate ions. The activation of the KR2 photocontrolled sodium ion pump does not depend on the ATP energy supply in plant cells, but directly uses light energy as the transport power. Under non-alkaline stress conditions, the sodium ion efflux activity can be stopped by turning off green light irradiation.

[0016] The present invention has the following beneficial effects: This invention constructs a membrane-localized optimized KR2 photocontrolled sodium ion pump expression system, which directly uses light energy as the transport driving force. It mediates the active efflux of excess intracellular sodium and hydrogen ions without relying on intracellular ATP supply or transmembrane proton gradients. Even under alkaline stress leading to cellular energy depletion, it maintains stable transport activity, effectively solving the problem of existing sodium ion pumps. + / H + The defect of reverse transport proteins whose function declines sharply under high stress conditions is overcome by significantly improving sodium ion efflux efficiency.

[0017] This invention triggers the transport activity of KR2 with green light of a specific wavelength. The intensity of sodium ion efflux can be dynamically and reversibly controlled by adjusting the light mode and light intensity parameters. The function can be activated by turning on the light only when alkaline stress occurs. Turning off the light during non-stress periods will not interfere with the normal ion balance and growth metabolism of plants. No chemical inducers are required, and there is no risk of inducer residues. This invention effectively solves the defects of existing constitutive / chemically induced expression systems, such as insufficient spatiotemporal specificity and easy generation of non-specific growth inhibition and secondary pollution.

[0018] The KR2 recombination pump system developed in this invention has gradient membrane positioning efficiency and sodium ion efflux activity. It can select the appropriate recombination system for different degrees of alkaline stress environment and is compatible with the genetic transformation system of various crops. It can be used for laboratory research on the physiological mechanism of plant stress resistance and for the breeding of alkaline-tolerant crop varieties in the field. It can improve the alkaline resistance of crops without the need for additional soil conditioners, avoiding the problems of high transformation cost and soil structure damage caused by traditional chemical improvement. It meets the needs of large-scale application in saline-alkali land agricultural production and has broad prospects for promotion. Attached Figure Description

[0019] Figure 1 This is a schematic diagram illustrating the structural optimization of the KR2-T1.0~KR2-T4.0 recombinant expression vectors proposed in this invention; Figure 2 This is a bar chart comparing the average fluorescence intensity of different KR2 recombinant proteins proposed in this invention on plant cell membranes. Figure 3 The graphs show the dynamic changes in membrane potential after green light irradiation in the alkaline stress treatment group and the control group proposed in this invention. Figure 4 The images show the leaf growth phenotypes of the embodiments proposed in this invention, the empty control group, and the blank control group after 0 days of alkali stress. Figure 5 The images show the leaf growth phenotypes of the embodiments proposed in this invention, the empty control group, and the blank control group after 6 days of alkali stress. Figure 6 These are electrophoresis images of the positive transgenic lines of Brassica napus described in Examples 1-4 of this invention for PCR identification. Detailed Implementation

[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0021] (a) General culture medium formulation: The culture medium formulations used for genetic transformation of Brassica napus in the following examples are uniformly as follows, and the pH of all culture media is adjusted to 5.80~5.84: M0 medium: 2.2 g / L 1 / 2 MS medium, 30.0 g / L sucrose, 7.0 g / L agar powder; DM infection solution: 4.4 g / L MS medium, 30 g / L sucrose, 100 μM acetylsalicylic acid; M1 co-culture medium: 4.4 g / L MS medium, 30 g / L sucrose, 18 g / L mannitol, 1 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 mg / L KT kinetin, 100 μM acetylsalicylic acid, 8 g / L agar powder; M2 callus induction medium: 4.4 g / L MS medium, 30 g / L sucrose, 18 g / L mannitol, 1 mg / L 2,4-dichlorophenoxyacetic acid, 0.3 mg / L KT kinetin, 8 g / L agar powder, STS components including 15 μM silver nitrate and 15 μM sodium thiosulfate, 300 mg / L termethin, 6 mg / L glufosinate; M3 bud induction medium: 4.4 g / L MS medium, 10 g / L glucose, 2 g / L sucrose, 0.25 g / L xylitol, 0.6 g / L 2-morpholine ethanesulfonic acid, 8 g / L agar powder, 2.25 mg / L trans-zeatin, 0.09375 mg / L indoleacetic acid, 300 mg / L termethin, 6 mg / L glufosinate, 6 mg / L silver nitrate; M4 rooting medium: 4.4 g / L MS medium, 10 g / L sucrose, 0.8 mg / L indolebutyric acid, 8 g / L agar, 300 mg / L termethin.

[0022] (II) Examples and Comparative Examples Example 1 Instantaneous conversion of Tobacco Benzoinus in the KR2-T1.0 recombinant system The specific steps in this embodiment are as follows: S1. Construction of the KR2-T1.0 recombinant expression vector: The KR2 sodium ion pump coding sequence with GenBank accession number AB738960 was obtained, and the T1.0 membrane localization signal peptide was ligated to its 3′ end to obtain the KR2-T1.0 coding fragment. The expression of this fragment was driven by the UBQ10 strong promoter. The fragment was inserted into the multiple cloning site of the pCAMBIA3300 backbone vector by homologous recombination. After the recombinant product was transformed into E. coli DH5α competent cells, positive single clones were selected by kanamycin at a concentration of 50 μg / mL. After sequencing to verify that the recombinant fragment sequence was completely correct, the plasmid was extracted to obtain the KR2-T1.0 recombinant expression vector with the correct sequence.

[0023] S2. Preparation of positive Agrobacterium engineered bacteria: Take 1 μg of the above recombinant expression vector plasmid and add it to 100 μL of GV3101 Agrobacterium competent cells. After successively incubating on ice for 30 min, quick-freezing in liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min, add 800 μL of LB liquid medium and culture at 28℃ and 200 rpm for 4 h. Then, spread it on LB solid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and culture upside down at 28℃ for 24 h. After screening, positive Agrobacterium engineered bacteria are obtained.

[0024] S3. Agrobacterium-mediated transient transformation of Nicotiana benthamiana: Positive Agrobacterium tumefaciens bacteria were inoculated into LB liquid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and cultured at 28°C with shaking at 200 rpm until the OD600 reached 0.6. After collecting the bacterial cells, they were resuspended in osmotic buffer containing 10 mM MES, 10 mM magnesium chloride, and 200 μM acetylsyleugenol, and the OD600 was adjusted to 0.6. The resuspended bacterial solution was then slowly injected into the lower epidermis of 4-week-old Nicotiana benthamiana leaves using a needleless syringe. The injected Nicotiana benthamiana was then placed under a light intensity of 100 μmol / m². -2 s -1 Tobacco Bunsenii plants transiently expressing KR2-T1.0 were obtained by culturing in an artificial incubator with a photoperiod of 16h light / 8h darkness and a temperature of 25℃ for 48h.

[0025] S4. Green light activation regulates alkali resistance: A wavelength of 532nm and a light intensity of 100μW / mm² are used. 2 Transformed plants were irradiated with green light, with the irradiation pattern matching the plant's normal 16h light / 8h dark light cycle, adapting to a mild alkali stress environment. Functional validation was conducted simultaneously: detached leaves from the transformed plants were placed in a 50mM sodium bicarbonate alkali stress treatment group, a 100mM sorbitol osmotic stress control group, and a deionized water blank control group for 18h, respectively, using a wavelength of 532nm and a light intensity of 268μW / mm². 2 After 5 seconds of green light irradiation, a membrane potential depolarization signal of 2.3 mV was detected only in the alkaline stress treatment group. No such signal was observed in the osmotic stress control group and the blank control group, indicating that the KR2-T1.0 function was normally activated.

[0026] Example 2: Instantaneous conversion of Tobacco Benzoinus in the KR2-T2.0 recombination system The specific steps in this embodiment are as follows: S1. Construction of the KR2-T2.0 recombinant expression vector: The KR2 sodium ion pump coding sequence with GenBank accession number AB738960 was obtained, and the T2.0 membrane localization signal peptide was ligated to its 3′ end to obtain the KR2-T2.0 coding fragment. The expression of this fragment was driven by the UBQ10 strong promoter. The fragment was inserted into the multiple cloning site of the pCAMBIA3300 backbone vector by homologous recombination. After the recombinant product was transformed into E. coli DH5α competent cells, positive single clones were selected by kanamycin at a concentration of 50 μg / mL. After sequencing verification that the recombinant fragment sequence was completely correct, the plasmid was extracted to obtain the KR2-T2.0 recombinant expression vector with the correct sequence.

[0027] S2. Preparation of positive Agrobacterium engineered bacteria: Take 1 μg of the above recombinant expression vector plasmid and add it to 100 μL of GV3101 Agrobacterium competent cells. After successively incubating on ice for 30 min, quick-freezing in liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min, add 800 μL of LB liquid medium and culture at 28℃ and 200 rpm for 4 h. Then, spread it on LB solid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and culture upside down at 28℃ for 24 h. After screening, positive Agrobacterium engineered bacteria are obtained.

[0028] S3. Agrobacterium-mediated transient transformation of Nicotiana benthamiana: Positive Agrobacterium tumefaciens were inoculated into LB liquid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and cultured at 28°C with shaking at 200 rpm until the OD600 reached 0.8. After collecting the bacterial cells, they were resuspended in osmotic buffer containing 10 mM MES, 10 mM magnesium chloride, and 200 μM acetylsyleugenol, and the OD600 of the bacterial suspension was adjusted to 0.8. The resuspended bacterial suspension was then slowly injected into the lower epidermis of 6-week-old Nicotiana benthamiana leaves using a needleless syringe. The injected Nicotiana benthamiana was then placed under a light intensity of 150 μmol / L. -2 s -1 Nicotiana benthamiana plants transiently expressing KR2-T2.0 were obtained by culturing in an artificial incubator with a photoperiod of 16h light / 8h darkness and a temperature of 27℃ for 72h. Quantitative fluorescence detection showed that the average fluorescence intensity of the KR2-T2.0 cell membrane was 81.83, which was 3.11 times higher than that of the wild type, verifying the optimized membrane localization effect of the T2.0 signal peptide.

[0029] S4. Green light activation regulates alkali resistance: A wavelength of 532nm and a light intensity of 200μW / mm² are used. 2Transformed plants were irradiated with green light, with the irradiation pattern matching the plant's normal 16h light / 8h dark light cycle, adapting to a moderate alkali stress environment. Functional validation was conducted simultaneously: detached leaves from the transformed plants were placed in a 100mM sodium bicarbonate alkali stress treatment group, a 100mM sorbitol osmotic stress control group, and a deionized water blank control group for 18h, respectively, using a wavelength of 532nm and a light intensity of 268μW / mm². 2 After 5 seconds of green light irradiation, a membrane potential depolarization signal of 2.5 mV was detected only in the alkaline stress treatment group. No such signal was observed in the osmotic stress control group and the blank control group, indicating that the KR2-T2.0 function was normally activated.

[0030] Example 3: Stable transformation of Brassica napus into rapeseed using the KR2-T3.0 recombination system The specific steps in this embodiment are as follows: S1. Construction of the KR2-T3.0 recombinant expression vector: The KR2 sodium ion pump coding sequence with GenBank accession number AB738960 was obtained, and the T3.0 membrane localization signal peptide was ligated to its 3′ end to obtain the KR2-T3.0 coding fragment. The expression of this fragment was driven by the UBQ10 strong promoter. The fragment was inserted into the multiple cloning site of the pCAMBIA3300 backbone vector by homologous recombination. After the recombinant product was transformed into E. coli DH5α competent cells, positive single clones were selected by kanamycin at a concentration of 50 μg / mL. After sequencing verification that the recombinant fragment sequence was completely correct, the plasmid was extracted to obtain the KR2-T3.0 recombinant expression vector with the correct sequence.

[0031] S2. Preparation of positive Agrobacterium engineered bacteria: Take 1 μg of the above recombinant expression vector plasmid and add it to 100 μL of GV3101 Agrobacterium competent cells. After successively incubating on ice for 30 min, quick-freezing in liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min, add 800 μL of LB liquid medium and culture at 28℃ and 200 rpm for 4 h. Then, spread it on LB solid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and culture upside down at 28℃ for 24 h. After screening, positive Agrobacterium engineered bacteria are obtained.

[0032] S3. Agrobacterium-mediated stable transformation of Brassica napus: Plump and uniform Brassica napus ZS11 seeds were selected, disinfected by soaking in 75% alcohol for 1 min, soaking in 50% 84 disinfectant for 3 min, and rinsing with sterile water 10 times. The seeds were then sown on the aforementioned M0 medium and cultured in the dark for 7 days to obtain sterile seedlings. Agrobacterium carrying the recombinant vector was cultured until the OD600 was 0.4 and then resuspended in the aforementioned DM infection solution. The hypocotyl of the sterile seedlings, cut off at 1.0 cm, was placed in the bacterial solution for 10 min. After the bacterial solution was dried, the seedlings were cultured in the aforementioned M1 co-culture medium in the dark for 48 h, the aforementioned M2 callus induction medium for 20 days, the aforementioned M3 bud induction medium for subculture every 8 days until green buds differentiated, and the aforementioned M4 rooting medium for 4 weeks to obtain regenerated seedlings. Genomic DNA was extracted from the leaves of the regenerated seedlings using the CTAB method. The presence of a specific band of 914 bp in PCR detection indicated a positive transgenic line.

[0033] S4. Green light activation regulates alkali resistance: A wavelength of 532nm and a light intensity of 300μW / mm² are used. 2 Transformed plants were irradiated with green light in an intermittent cycle of 2 minutes of light and 1 minute of darkness to adapt to a severe alkaline stress environment. Functional validation was conducted simultaneously: detached leaves from the transformed plants were placed in a 100 mM sodium bicarbonate alkaline stress treatment group, a 100 mM sorbitol osmotic stress control group, and a deionized water blank control group for 18 hours, respectively, using a wavelength of 532 nm and a light intensity of 268 μW / mm². 2 After 5 seconds of green light irradiation, a membrane potential depolarization signal of 7.4 mV was detected only in the alkaline stress treatment group. No such signal was observed in the osmotic stress control group and the blank control group, indicating that the KR2-T3.0 function was normally activated.

[0034] Example 4: Stable transformation of Brassica napus into rapeseed using the KR2-T4.0 recombination system The specific steps in this embodiment are as follows: S1. Construction of the KR2-T4.0 recombinant expression vector: The KR2 sodium ion pump coding sequence with GenBank accession number AB738960 was obtained, and the T4.0 membrane localization signal peptide was ligated to its 3′ end to obtain the KR2-T4.0 coding fragment. The expression of this fragment was driven by the UBQ10 strong promoter. The fragment was inserted into the multiple cloning site of the pCAMBIA3300 backbone vector by homologous recombination. After the recombinant product was transformed into E. coli DH5α competent cells, positive single clones were selected by kanamycin at a concentration of 50 μg / mL. After sequencing verification that the recombinant fragment sequence was completely correct, the plasmid was extracted to obtain the KR2-T4.0 recombinant expression vector with the correct sequence.

[0035] S2. Preparation of positive Agrobacterium engineered bacteria: Take 1 μg of the above recombinant expression vector plasmid and add it to 100 μL of GV3101 Agrobacterium competent cells. After successively incubating on ice for 30 min, quick-freezing in liquid nitrogen for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min, add 800 μL of LB liquid medium and culture at 28℃ and 200 rpm for 4 h. Then, spread it on LB solid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and culture upside down at 28℃ for 24 h. After screening, positive Agrobacterium engineered bacteria are obtained.

[0036] S3. Agrobacterium-mediated stable transformation of Brassica napus: Plump and uniform Brassica napus ZS11 seeds were selected, disinfected by soaking in 75% alcohol for 1 min, soaking in 50% 84 disinfectant for 3 min, and rinsing with sterile water 10 times. The seeds were then sown on the aforementioned M0 medium and cultured in the dark for 5 days to obtain sterile seedlings. Agrobacterium carrying the recombinant vector was cultured until the OD600 was 0.4 and then resuspended in the aforementioned DM infection solution. The hypocotyl of the sterile seedlings, cut at 0.8 cm, was placed in the bacterial solution for 8 min. After the bacterial solution was dried, the seedlings were cultured in the aforementioned M1 co-culture medium in the dark for 36 h, the aforementioned M2 callus induction medium for 20 days, the aforementioned M3 bud induction medium for subculture every 8 days until green buds differentiated, and the aforementioned M4 rooting medium for 2 weeks to obtain regenerated seedlings. Genomic DNA was extracted from the leaves of the regenerated seedlings using the CTAB method. The presence of a specific band of 914 bp in PCR detection indicated a positive transgenic line.

[0037] S4. Green light activation regulates alkali resistance: A wavelength of 532nm and a light intensity of 300μW / mm² are used. 2 Transformed plants were irradiated with green light in an intermittent cycle of 2 minutes of light and 1 minute of darkness to adapt to a severe alkaline stress environment. Functional validation was conducted simultaneously: detached leaves from the transformed plants were placed in a 100 mM sodium bicarbonate alkaline stress treatment group, a 100 mM sorbitol osmotic stress control group, and a deionized water blank control group for 18 hours, respectively, using a wavelength of 532 nm and a light intensity of 268 μW / mm². 2 After 5 seconds of green light irradiation, a membrane potential depolarization signal of 7.9 mV was detected only in the alkaline stress treatment group. No such signal was observed in the osmotic stress control group and the blank control group, indicating that the KR2-T4.0 function was normally activated.

[0038] Constitutive overexpression of SOS1 comparative example This comparative example uses a CaMV35S compositional promoter to drive the SOS1 Na. + / H +The ZS11 line of Brassica napus expressing the reverse transporter protein was cultured for 6 days under the same 100mM NaHCO3 severe alkaline stress environment as in Examples 3 and 4, without any special light treatment. Functional verification results showed that after 18 hours of alkaline stress, the sodium ion efflux activity of this line was only 22% of that of the KR2-T3.0 line, and no specific membrane potential depolarization signal was generated. After 6 days of culture, the leaves showed extensive yellowing and wilting, with a survival rate of only 42%.

[0039] Blank control group The blank control group consisted of wild-type Brassica napus ZS11 plants that had not undergone any genetic transformation. These plants were cultured for 6 days under severe alkaline stress (100 mM NaHCO3) as described in Examples 3, 4, and the comparative examples, without any special light treatment. (eYFP empty control group) This control group used Brassica napus ZS11 plants transformed with the empty pCAMBIA3300-eYFP vector (without the KR2 coding sequence). Functional verification and alkali stress treatment were performed under the same conditions as in Examples 3 and 4 and the comparative example. No depolarization signal was observed in membrane potential detection. After 6 days of alkali stress, leaf survival rate, chlorophyll retention rate, and fresh weight retention rate were not significantly different from the wild-type blank control, indicating that fluorescent protein expression and Agrobacterium transformation itself did not produce alkali tolerance. (III) Experimental Data Tables and Explanations Table 1. Core construction parameters and functional activity parameters for each embodiment and comparative example.

[0040] As shown in the table, the membrane localization efficiency of the four KR2 recombinant proteins constructed in this invention is higher than that of the wild type and the existing SOS1 overexpression lines. Among them, KR2-T3.0 has the highest membrane localization efficiency. Different recombinant proteins have different membrane localization efficiencies and sodium ion efflux activities, which can adapt to the regulatory needs of different degrees of alkaline stress. The aforementioned optimized culture medium formula has increased the positive transformation rate of Brassica napus by nearly 1 times compared with the existing technology, effectively solving the problems of low KR2 heterologous expression membrane uptake rate and insufficient transformation efficiency in field crops.

[0041] Table 2. Growth performance parameters of each embodiment and comparative example under alkali stress for 6 days.

[0042] As shown in the table, the growth performance of each embodiment under alkali stress was better than that of the comparative example and the wild-type blank control. The KR2 recombinant system directly uses light energy as the transport power, without relying on intracellular ATP supply. It can achieve dynamic regulation through green light alone, without the non-specific growth inhibition effect of constitutive expression. It effectively solves the defects of existing alkali tolerance regulation technology, such as transport activity relying on intracellular energy, insufficient regulation precision, and easy to induce growth inhibition.

[0043] (iv) Verification of experimental results All test results in this section are derived from parallel replicate experiments in the aforementioned embodiments, and the detection methods conform to standard operating procedures in the field of plant molecular biology. Membrane positioning efficiency verification The results of mean fluorescence intensity measurements of the cell membrane showed that KR2-T3.0 had the highest membrane localization efficiency, which was 3.44 times that of the wild type. Figure 2 As shown, KR2-T2.0 was the second most efficient, being 3.11 times that of the wild type; KR2-T1.0 and KR2-T4.0 were 2.39 times and 2.17 times that of the wild type, respectively, both significantly higher than the 1.07 times efficiency of existing constitutive overexpression SOS1 lines. The gradient membrane localization efficiency can be adapted to regulatory scenarios with different levels of alkaline stress.

[0044] Specificity verification of light control function Under 532nm green light irradiation, specific membrane potential depolarization signals were detected only in the sodium bicarbonate alkali stress treatment group of detached leaves from the transformed plants in each embodiment. Specifically, the depolarization peak value was 2.3mV in Example 1, 2.5mV in Example 2, 7.4mV in Example 3, and 7.9mV in Example 4. This signal was not detected in the osmotic stress control group and the blank control group. Typical curves of membrane potential dynamic changes are shown below. Figure 3 As shown, the KR2 pump function is strictly regulated by green light, does not respond to non-sodium ion-related osmotic stress, and has no off-target effect.

[0045] Verification of growth protection effect under alkali stress like Figure 4 and Figure 5 As shown, after 0 days of light treatment, the leaf growth phenotypes of the plants in each example were basically the same. After continuous activation of the corresponding light mode for 6 days, the leaf survival rate, chlorophyll retention rate, and fresh weight retention rate of the plants in each example were all better than those of the comparative example and the blank control, and the growth phenotypes were as follows. Figure 5 As shown, under severe alkali stress, the leaf survival rates of Examples 3 and 4 reached 94% and 92%, respectively, while the existing SOS1 overexpression lines only achieved 42%, and the wild type only 18%. The leaves of the comparative examples and wild type showed large-scale yellowing and wilting, demonstrating the significant alkali resistance protection effect of this scheme.

[0046] Validation of conversion efficiency of Brassica napus The optimized genetic transformation system of this invention increases the success rate of obtaining positive transgenic lines of Brassica napus to 30%–32%, nearly double that of the existing SOS1 overexpression system (17%). PCR identification results of some positive lines are shown below. Figure 6 As shown, a target-specific band of 914 bp is clearly visible, confirming that the recombinant vector was successfully integrated into the rapeseed genome.

[0047] The above results demonstrate that the plant-derived signal peptide-guided KR2 photocontrolled sodium ion pump recombination system constructed in this invention successfully overcomes the shortcomings of existing technologies, such as low membrane localization efficiency, transport activity dependent on intracellular ATP, insufficient regulatory precision, and difficulty in field crop transformation. It achieves precise, efficient, and low-energy regulation of alkali tolerance under different degrees of alkali stress.

[0048] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A method for regulating plant alkali tolerance using a light-controlled sodium ion pump, characterized in that, Includes the following steps: S1. Construct a plant expression vector for the KR2 light-controlled sodium ion pump. Connect the 3′ end of the KR2 sodium ion pump coding sequence from the marine bacterium Krokinobacter eikastus to four membrane localization signal peptides with different membrane localization efficiencies to obtain gene fragments encoding four recombinant proteins: KR2-T1.0, KR2-T2.0, KR2-T3.0, and KR2-T4.

0. Use the UBQ10 strong promoter to drive the expression of the recombinant gene fragments. Insert the recombinant gene fragments into the plant expression backbone vector to obtain a recombinant expression vector with the correct sequence. S2. The recombinant expression vector is transformed into Agrobacterium competent cells, and positive Agrobacterium engineered bacteria carrying the recombinant vector are obtained through resistance screening; S3. The coding sequence of the KR2 photosensitive sodium ion pump is integrated into the genome of the target plant using Agrobacterium-mediated genetic transformation to obtain transformed plants that stably or transiently express the KR2 photosensitive sodium ion pump. S4. Irradiate the transformed plant with green light at a wavelength of 532nm to activate the transport activity of the KR2 photocontrolled sodium ion pump.

2. The method for regulating plant alkali tolerance using a light-controlled sodium ion pump according to claim 1, characterized in that, The plant expression backbone vector described in step S1 is pCAMBIA3300. Four recombinant gene fragments were inserted into the multiple cloning site of the backbone vector using homologous recombination. After the recombinant products were transformed into Escherichia coli DH5α competent cells, positive single clones were screened using kanamycin at a concentration of 50 μg / mL. After sequencing verification that the recombinant gene fragment sequences were completely correct, the plasmid was extracted to obtain the recombinant expression vector.

3. The method for regulating plant alkali tolerance using a light-controlled sodium ion pump according to claim 1, characterized in that, The Agrobacterium mentioned in step S2 is strain GV3101. The specific operation of transforming Agrobacterium with recombinant expression vector is as follows: 1 μg of recombinant expression vector plasmid is added to 100 μL of Agrobacterium competent cells, and the cells are successively subjected to ice bath for 30 min, liquid nitrogen flash freezing for 5 min, water bath at 37℃ for 5 min, and ice bath for 5 min. Then, 800 μL of LB liquid medium is added and cultured at 28℃ and 200 rpm for 4 h with shaking. Subsequently, the cells are plated on LB solid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and cultured upside down at 28℃ for 24 h. Positive Agrobacterium engineered bacteria are then screened.

4. The method for regulating plant alkali tolerance using a light-controlled sodium ion pump according to claim 1, characterized in that, The target plant in step S3 is *Nicotiana benthamiana*, and transformation is performed using Agrobacterium-mediated transient leaf transformation. Specifically, Agrobacterium carrying the recombinant vector is inoculated into LB liquid medium containing 50 μg / mL kanamycin and 10 μg / mL rifampin, and cultured at 28°C with shaking at 200 rpm until the OD600 reaches 0.6–0.

8. After collecting the bacterial cells, they are resuspended in osmotic buffer containing 10 mM MES, 10 mM magnesium chloride, and 200 μM acetylsyleugenol, and the OD600 of the bacterial solution is adjusted to 0.6–0.

8. The resuspended bacterial solution is then slowly injected into the lower epidermis of leaves of 4–6 week old *Nicotiana benthamiana* using a needleless syringe. The injected *Nicotiana benthamiana* is then placed under light intensity of 100–150 μmol / m². -2 s -1 Tobacco Bunsenii plants transiently expressing the KR2 photocontrolled sodium ion pump were obtained by culturing them in an artificial incubator with a photoperiod of 16h light / 8h darkness and a temperature of 25±2℃ for 48~72h.

5. The method for regulating plant alkali tolerance using a light-controlled sodium ion pump according to claim 1, characterized in that, The target plant in step S3 is Brassica napus, which is transformed using Agrobacterium-mediated hypocotyl genetic transformation. Specifically, plump and uniform Brassica napus seeds are first selected, and after being disinfected by soaking in 75% alcohol for 1 min, soaking in 50% 84 disinfectant for 3 min, and rinsing with sterile water 10 times, they are sown in Mo medium at pH 5.80~5.84 and cultured in the dark for 5~7 days to obtain sterile seedlings. Agrobacterium carrying the recombinant vector was cultured to an OD600 of approximately 0.4 and then resuspended in DM infection medium. Hypocotyls of sterile seedlings (0.8–1.0 cm in length) were placed in the bacterial solution for 8–10 minutes. After drying the bacterial solution, the seedlings were successively cultured in M1 co-culture medium in the dark for 36–48 hours, M2 callus induction medium for 20 days, M3 bud induction medium for subculture every 8 days until green buds differentiated, and M4 rooting medium for 2–4 weeks to obtain regenerated seedlings. Genomic DNA was extracted from the leaves of the regenerated seedlings using the CTAB method, and the presence of a 914 bp specific band in PCR detection indicated a positive transgenic line. The pH of all M0, DM, M1, M2, M3, and M4 culture media was adjusted to 5.80–5.84, and acetylsyleugenol, antibiotics, screening agents, and plant growth regulators were added as needed.

6. The method for regulating plant alkali tolerance using a light-controlled sodium ion pump according to claim 1, characterized in that, The intensity of the green light in step S4 is 100~300 μW / mm. 2 The irradiation mode is adjusted to intermittent or continuous irradiation according to the degree of alkali stress. The intermittent irradiation cycle is 2 min light / 1 min dark, which is suitable for severe alkali stress environment. The continuous irradiation matches the normal light cycle of 16 h light / 8 h dark for plant growth, which is suitable for moderate or mild alkali stress environment.

7. The method for regulating plant alkali tolerance using a light-controlled sodium ion pump according to claim 1, characterized in that, The four recombinant proteins have different membrane localization efficiencies and sodium ion efflux activities. Among them, KR2-T3.0 and KR2-T4.0 are suitable for regulating plant alkali tolerance under severe alkali stress; KR2-T1.0 and KR2-T2.0 are suitable for mild and moderate alkali stress.

8. The method for regulating plant alkali tolerance using a light-controlled sodium ion pump according to claim 1, characterized in that, The study also included functional verification steps for the transformed plants. Non-invasive microelectrode technology was used to detect changes in membrane potential of plant leaves under green light irradiation. Before the test, detached leaves of the transformed plants were placed in a 100 mM sodium bicarbonate alkali stress treatment group, a 100 mM sorbitol osmotic stress control group, and a deionized water blank control group for 18 h, respectively. A wavelength of 532 nm and a light intensity of 268 μW / mm² were used. 2 After 5 seconds of green light irradiation, when a significant membrane potential depolarization signal was detected in the alkaline stress treatment group, and no such depolarization signal was observed in the osmotic stress control group and the blank control group, the KR2 photocontrolled sodium ion pump function was determined to be normally activated.

9. The method for regulating plant alkali tolerance using a light-controlled sodium ion pump according to claim 1, characterized in that, The method is applicable to alkaline stress environments with high pH; the activation of the KR2 photocontrolled sodium ion pump does not depend on the ATP energy supply in plant cells, but uses light energy as the transport power, and the sodium ion efflux activity is stopped when green light irradiation is turned off under non-alkaline stress conditions.

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