Application of potassium ion channel KAT1 gene BrKAT1.1 in plant stress resistance regulation

Abstract

The application discloses a kind of potassium ion channel KAT1 gene BrK AT1.1 In plant stress resistance regulation application.The application first discovers and verifies potassium ion channel KAT1 gene BrK AT1.1 Can enhance the organic acid metabolism response of plant under aluminum stress, promote the secretion and secretory chelation of organic acid, significantly improve the aluminum tolerance of plant, and then significantly improve the biomass of plant under aluminum stress, significantly reduce the aluminum accumulation in plant, can efficiently create high aluminum-tolerant crop new germplasm, and has important role in excellent crop new variety cultivation and agricultural production popularization.

Description

Technical Field

[0001] This invention relates to plant genetic engineering, and more particularly to the potassium ion channel KAT1 gene. BrKAT1.1 Application in the regulation of plant stress resistance. Background Technology

[0002] Acidic soils are a widespread type of stress soil in agricultural production. Soil acidification processes cause a large amount of active aluminum ions to be released into the soil solution. When the soil pH drops below 5.5, aluminum-containing minerals dissolve, releasing trivalent aluminum ions (Al2+). 3+ Active aluminum becomes the main form of aluminum in the soil solution. Active aluminum has strong phytotoxicity and can rapidly inhibit the elongation and division of root cells, leading to thicker and shorter roots, blunt root tips, and poor root hair development. This hinders the plant's absorption and utilization of water and mineral nutrients such as nitrogen, phosphorus, and potassium, ultimately resulting in slow growth of the above-ground parts, wilting and yellowing of leaves, and a significant decrease in biomass. In severe cases, it can even cause the plant to wither and die. It is a major factor limiting crop production in acidic soils.

[0003] Currently, conventional methods for alleviating aluminum toxicity stress in plants during agricultural production mainly rely on chemical amendments, including the application of lime, organic fertilizers, and soil conditioners. Lime application is the most widespread, as it can quickly raise the pH of topsoil to above 5.5. However, this approach has significant limitations: while it lowers acidity quickly, it also causes rapid acidification, making the effect difficult to sustain; its effects are mostly limited to the topsoil, offering little benefit to subsoil acidification; and it requires repeated application year after year, resulting in high costs. Furthermore, long-term, large-scale application of lime can lead to soil compaction and secondary environmental pollution.

[0004] Compared to traditional chemical improvement, discovering key aluminum-tolerant genes in plants and breeding new aluminum-tolerant crop varieties through genetic engineering is the fundamental way to effectively solve aluminum toxicity stress in the long term, and it is also a current research hotspot in stress-resistant crop breeding. Summary of the Invention

[0005] Purpose of the invention: The purpose of this invention is to provide a potassium ion channel KAT1 gene. BrKAT1.1 Application in the regulation of plant stress resistance.

[0006] Technical solution: The potassium ion channel KAT1 gene described in this invention BrKAT1.1 Application in the regulation of plant stress resistance.

[0007] Preferably, the potassium ion channel KAT1 gene BrKAT1.1 The encoded proteins include: a. A protein having the amino acid sequence shown in SEQ ID NO: 1; b. Homologous proteins that have at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO: 1.

[0008] Preferably, the plant is a cruciferous plant.

[0009] Preferably, the stress resistance is aluminum stress tolerance; more preferably, the application is overexpression of the potassium ion channel KAT1 gene. BrKAT1.1 Improve plant tolerance to aluminum stress.

[0010] Preferably, the application steps include: (1) Cloning isolation BrKAT1.1 Gene nucleic acid fragments; (2) Based on the genes isolated in step 1, a recombinant overexpression vector was constructed; (3) Introduce the recombinant overexpression vector obtained in step 2 into the target plant; (4) Screening to obtain stable overexpression BrKAT1.1 Genetically modified plants.

[0011] Preferably, the clone obtained in step 1 is... BrKAT1.1 The sequence of the gene nucleic acid fragment is shown in SEQ ID NO: 2.

[0012] Preferably, the expression vector in step 2 is a vector containing any one of the CaMV 35S promoter, U6 promoter, Actin promoter, UBQ10 promoter, or other constitutive high-expression promoters; more preferably, the expression vector is PHB.

[0013] Preferably, the method for introducing the target plant in step 3 is any one of Agrobacterium-mediated transformation, protoplast transformation, viral vector delivery, pollen tube pathway, or gene gun method; more preferably, the method for introducing the target plant is Agrobacterium-mediated inflorescence infection.

[0014] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages: The present invention is the first to discover and verify the potassium ion channel KAT1 gene. BrKAT1.1 It can enhance the organic acid metabolic response of plants under aluminum stress, promote the secretion of organic acids and secretory chelation, significantly improve the aluminum tolerance of plants, and thus significantly increase the biomass of plants under aluminum stress and significantly reduce the amount of aluminum accumulated in plants. It can efficiently create new germplasm of highly aluminum-tolerant crops and plays an important role in the breeding of superior new crop varieties and the promotion of agricultural production. Attached Figure Description

[0015] Figure 1 for BrKAT1.1 Overexpressing plants and wild-type plants BrKAT1.1 Graph of relative expression level measurement results; Figure 2 For plate test BrKAT1.1Representative images of the growth of overexpressing plants and wild-type plants; Figure 3 For plate test BrKAT1.1 Figure showing the biomass statistics of overexpressing plants and wild-type plants; Figure 4 For plate test BrKAT1.1 Statistical results of total root length of overexpressing plants and wild-type plants; Figure 5 For soil cultivation experiment BrKAT1.1 Representative images of the growth of overexpressing plants and wild-type plants (left) and biomass statistics (right); Figure 6 For soil cultivation experiment BrKAT1.1 Statistical results of aluminum content in overexpressing plants and wild-type plants; Figure 7 For soil cultivation experiment BrKAT1.1 The statistical results of organic acid secretion in overexpressing plants and wild-type plants are shown in the figure. The left figure shows the statistical results of malic acid secretion, and the right figure shows the statistical results of citric acid secretion. Detailed Implementation

[0016] The technical solution of the present invention will be further described below.

[0017] Example 1: KAT1 gene, a potassium ion channel BrKAT1.1 Cloning and isolation and construction of overexpression vectors 1. Potassium ion channel KAT1 gene BrKAT1.1 Cloning of nucleic acid fragments Design and synthesis BrKAT1.1 Amplification primers, sequences as follows: BrKAT1.1-F, the sequence is: 5'-accagtctctctctcaagcttATGTCCATCTCTTGCACCAGAAACTTC-3'; BrKAT1.1-R, the sequence is: 5'-ctagaggatcaattcgagctcTCAGCTTGATGAGAAAAAAAATGATC-3'.

[0018] Take cabbage ( Brassica rapa var. chinensis Total RNA was extracted from *L.* Kitamura* leaves using the Novizum SuperFastPure Cell RNA Isolation Kit (catalog number RC102), followed by reverse transcription to obtain cDNA using the Novizum Full-Length cDNA One-Strand Synthesis Kit (catalog number R211). The obtained cDNA was then used as a template for... BrKAT1.1The amplification primers were used for PCR amplification using the Novizan 2× Phanta Max Master Mix kit (catalog number P515). The reaction program was as follows: 95℃ pre-denaturation for 5 min; 95℃, 15 s, 65℃, 15 s, 72℃, 2 min, 30 cycles; and 72℃ final extension for 5 min.

[0019] After the PCR reaction, the amplification products were detected by electrophoresis on a 0.8% agarose gel. The band at approximately 2000 bp was recovered using the Tiangen Agarose Gel DNA Recovery Kit (catalog number DP209-03). BrKAT1.1 The nucleic acid fragment, the CDS sequence of which is shown in SEQ ID NO: 2, and the amino acid sequence it encodes is shown in SEQ ID NO: 1.

[0020] 2. Potassium ion channel KAT1 gene BrKAT1.1 Construction of overexpression vectors PHB (sequence shown in SEQ ID NO: 3) was digested with HindIII and SacI and then subjected to agarose gel electrophoresis. The band at 12000 bp was recovered using the Tiangen Agarose Gel DNA Recovery Kit to obtain the linearized vector.

[0021] The aforementioned cloning kit was ligated using the Novizan ClonExpress II one-step cloning kit (catalog number C112). BrKAT1.1 Nucleic acid fragments and linearized vectors were then used, and the ligation products were subsequently transformed into... E. coli DH5α competent cells were plated on LB plates containing 100 μg / mL kanamycin and incubated at 37°C for 12 h.

[0022] After the culture was completed, a single colony was picked and cultured in LB liquid containing 100 μg / mL kanamycin. The plasmid was extracted and sent to Nanjing Qingke Biotechnology Co., Ltd. for sequencing to verify its correctness. The recombinant overexpression vector PHB-BrKAT1.1 was obtained for subsequent experiments.

[0023] Example 2: Agrobacterium-mediated potassium channel KAT1 gene BrKAT1.1 Genetic transformation 1. Preparation of Agrobacterium infection solution Add 5 μL of PHB-BrKAT1.1 to every 100 μL of EHA105 Agrobacterium competent cells, mix well, and then sequentially incubate on ice for 30 min, flash freeze in liquid nitrogen for 5 min, and heat shock at 37°C for 5 min. Add 800 μL of antibiotic-free YEB liquid medium and incubate at 28°C with shaking at 200 rpm for 3 h. Spread the recovered bacterial culture onto YEB solid culture plates containing 50 μg / mL rifampicin and 50 μg / mL kanamycin, and incubate at 28°C in the dark for 2 days. Pick single colonies with good growth and inoculate them into YEB liquid medium containing 50 μg / mL rifampicin and 50 μg / mL kanamycin, and incubate overnight at 28°C with shaking at 200 rpm to obtain EHA105 / PHB-BrKAT1.1 bacterial culture. Centrifuge at 5000 rpm for 20 min, collect the bacterial pellet, and resuspend it in infection buffer to obtain OD. 600 =0.8 Agrobacterium infection solution. The infection buffer contains 500 μL / L Silwet L-77, 5% (m / v) sucrose, and the remainder is deionized water.

[0024] 2. Genetic transformation in Arabidopsis thaliana The Arabidopsis Col-0 inflorescences were immersed in the aforementioned Agrobacterium infection solution for 1 min. After removal, they were covered with polyethylene film to maintain humidity and placed in the dark in a 22°C culture room for 24 h. Then the covering was removed, and the inflorescences were transferred to a normal photoperiod (12 h light / 12 h dark) for culture. The seeds were harvested after the siliques matured.

[0025] The harvested seeds were placed in 5 mL centrifuge tubes, and 50% (v / v) sodium hypochlorite disinfectant containing 0.1% Triton X-100 was added. The mixture was shaken for 15 min for disinfection. The seeds were then rinsed five times with deionized water in a sterile operating table, and then evenly spread on sterile filter paper and air-dried in a laminar flow hood for 1 h. The air-dried seeds were then evenly dispersed and sown on the surface of a selection medium and cultured under light at 22℃, 70% RH, and 8000 lx light intensity for 2 weeks. Healthy, positive seedlings were selected and transplanted into a cultivation substrate consisting of nutrient soil and vermiculite in a 2:1 volume ratio. The seedlings were covered with polyethylene film to maintain moisture for 3 days, and then cultured in a light incubator at 22℃, 70% RH, 8000 lx light intensity, and normal photoperiod until the siliques matured. The T1 generation seeds were then harvested. The above resistance screening was repeated with the T1 generation seeds to obtain the T2 generation seeds. Hygromycin resistance was tested on each T2 generation line using a selection medium. If all seeds of a line survived (100% survival rate), the line was determined to be a homozygous T2 generation line, and its seeds were considered homozygous seeds. The selection medium was 1 / 2 MS solid medium (containing 15 g / L sucrose and 8 g / L agar) with a final concentration of 50 μg / mL hygromycin added.

[0026] Example 3: Homozygous BrKAT1.1 Evaluation of stress resistance of Arabidopsis thaliana overexpression The test materials were wild-type Arabidopsis thaliana seeds (control, denoted as WT) and BrKAT1.1 Transgenic T2 generation homozygous seeds, wherein the T2 generation homozygous seeds were derived from the three independent [genomes / plants] in Example 2 above. BrKAT1.1 The homozygous overexpression lines were numbered OE#1, OE#2, and OE#3, respectively.

[0027] Each test seed was sown separately on the surface of 1 / 4 MS solid medium (containing 7.5 g / L sucrose and 8 g / L agar) at pH 5.8 and cultured for 14 days at 22℃, 70% RH, 8000 lx light intensity, and normal photoperiod. Total RNA was extracted using the Novizan Super FastPure Cell RNA Isolation Kit, and then reverse transcribed into cDNA using the Novizan full-length cDNA one-strand synthesis kit. Using the obtained cDNA as a template, primers were used... BrKAT1.1 Upstream primer: 5'-acttggttcgccttcgatgt-3'; BrKAT1.1 Downstream primer: 5'-ccgtctgagcctccataacc-3'; AtKAT1 Upstream primer: 5'-tcatgctgacgatggacgag-3'; AtKAT1 Downstream primer: 5'-gtgtcggaagtcggattcgt-3', AtACTIN2 Upstream primer: 5'-tgctgttgactacgagcagg-3'; Downstream primer: 5'-cgagggctggaacaagactt-3'. qPCR detection was performed using Novizan ChamQ Blue Universal SYBR qPCR Master Mix reagent (catalog number Q312). Reaction conditions were: 95℃ pre-denaturation for 30 s; 95℃ denaturation for 5 s, 60℃ annealing and extension for 30 s, for 40 cycles. Fluorescence values ​​were collected during the extension phase of each cycle. AtACTIN2 As an intrinsic parameter, the relative Ct value is calculated via 2 -ΔΔCt Method calculation BrKAT1.1 Compared to endogenous genes AtKAT1 The relative expression level.

[0028] The results are as follows Figure 1 As shown, each strain BrKAT1.1 Expression levels in order AtKAT1 The dosage was increased by 17.7, 13.8, and 35.9 times, while the three strains showed differences. BrKAT1.1There was no significant difference in overexpression levels, ruling out the influence of differences in overexpression levels on subsequent experiments. Therefore, these three lines were used for subsequent phenotypic assays.

[0029] 1. Plate culture experiment Each tested seed was sown separately on the surface of 1 / 4 MS solid medium (containing 7.5 g / L sucrose and 8 g / L agar) at pH 5.8. After 14 days of cultivation at 22℃, 70% RH, 8000 lx light intensity, and normal photoperiod, the seedlings were transferred in the order WT, OE#1, OE#2, and OE#3 to either 1 / 4 MS solid medium plates at pH 4.2 (Control group, containing 7.5 g / L sucrose and 8 g / L agar) or 1 / 4 MS solid medium plates supplemented with a final concentration of 500 μM AlCl3 (Al...). 3+ The group (containing 7.5 g / L sucrose and 8 g / L agar) was cultured for another 14 days, and the phenotype was recorded by photographing. Various physiological indicators were also measured.

[0030] Phenotypes after plate culture, such as Figure 2 As shown, under control conditions without aluminum stress, BrKAT1.1 No significant differences in growth phenotype were observed between the overexpression lines and wild-type plants; however, under aluminum stress, the growth vigor of the transgenic lines was significantly better than that of the wild-type control. Biomass (by fresh weight) after plate culture is shown in the following figures. Figure 3 As shown, under aluminum-free control conditions, there was no significant difference in growth phenotype between the transgenic lines and wild-type plants; however, under aluminum stress conditions, the biomass of the transgenic lines was significantly higher than that of the wild-type control, with the fresh weight of the OE#1, OE#2, and OE#3 lines increasing by 28.3%, 29.8%, and 39.1% respectively compared to the wild-type. These results confirm... BrKAT1.1 Gene overexpression can significantly enhance the aluminum tolerance of transgenic Arabidopsis thaliana.

[0031] The statistical results of total root length after plate culture are as follows: Figure 4 As shown, under aluminum-free control conditions, there was no statistically significant difference in total root length between transgenic lines and wild-type plants; however, under aluminum stress conditions, the total root length of all overexpression lines was significantly better than that of the wild-type control, with OE#1, OE#2, and OE#3 increasing by 35.9%, 29.8%, and 50.6%, respectively. These results confirm... BrKAT1.1 Gene overexpression can effectively enhance the root growth ability of Arabidopsis thaliana under aluminum stress.

[0032] 2. Soil cultivation experiment The tested seeds were sown in nutrient soil and cultured for 14 days at 22℃, 70% RH, 8000 lx light intensity, and normal photoperiod. Seedlings with uniform growth were selected and transplanted into seedling trays containing nutrient soil in the order WT, OE#1, and OE#2. After 3 days of acclimatization, the seedlings were watered every 7 days with either 1 / 4 Hoagland nutrient solution (pH=4.2, Control group) or 1 / 4 Hoagland nutrient solution with a final concentration of 2 mM AlCl3 (Al...). 3+ (Group), 1.5 L per tray each time. After 14 days of treatment, plant phenotypes were recorded by photograph, samples were harvested, and various physiological and biochemical indicators were measured.

[0033] For the determination of aluminum content, the following steps were taken: The aboveground tissues of Arabidopsis thaliana from each group were harvested, dried to constant weight, and then ground into powder. 0.05 g of powder was weighed into a polytetrafluoroethylene digestion vessel, and 5 mL of nitric acid was added for overnight soaking. The inner lid was closed and the stainless steel outer casing was tightened. The vessel was placed in a constant temperature drying oven and maintained at 80℃ for 2 h, 120℃ for 2 h, and 160℃ for 4 h. After naturally cooling to room temperature, the product was heated to volatilize to near dryness. The digestion product was washed into a 10 mL volumetric flask with 1% nitric acid solution for later use. Subsequently, the aluminum content was determined by inductively coupled plasma mass spectrometry (ICP-MS) according to the method described in GB5009.268-2025, "National Food Safety Standard - Determination of Multiple Elements in Food".

[0034] For the determination of organic acids, the following steps were taken: Arabidopsis thaliana plants from each group were taken, and after cleaning the roots, they were placed in 40 mL of sterile water with the aboveground parts exposed above the water surface. They were cultured at 22℃, RH 70%, and light intensity 8000 lx for 7 h to collect root exudates. The collected liquid was filtered through a 0.22 μm filter membrane and then subjected to high performance liquid chromatography (HPLC) according to the method described by Wang, P., Zhou, R., Cheng, J., & Bi, S. (2007). LC determination of trace short-chain organic acids in wheat root exudates under aluminum stress. Chromatographia, 66(11), 867-872.

[0035] The statistical results of phenotypic and biomass (based on fresh weight) of each tested line after soil culture are as follows: Figure 5As shown, under aluminum-free control conditions, there was no significant difference in growth phenotype between the overexpression lines and wild-type plants; however, under aluminum stress conditions, the biomass of the overexpression lines was significantly higher than that of the wild-type control, with the fresh weight of lines OE#1 and OE#2 increasing by 78.2% and 52.5% respectively compared to the wild type. This result further confirms... BrKAT1.1 Gene overexpression can significantly enhance the aluminum tolerance of Arabidopsis thaliana.

[0036] The results of aluminum content determination for each tested strain after soil cultivation are as follows: Figure 6 As shown, under aluminum stress conditions, BrKAT1.1 The aluminum content in the overexpression lines OE#1 and OE#2 decreased by 80% and 73% respectively compared with the wild-type control, and the difference was statistically significant; this result confirms... BrKAT1.1 Gene overexpression can significantly reduce aluminum accumulation levels in Arabidopsis thaliana under aluminum stress.

[0037] The results of the organic acid secretion content determination of each test line after soil cultivation are as follows: Figure 7 As shown, under aluminum stress conditions, BrKAT1.1 The overexpression lines OE#1 and OE#2 showed a 2.2-fold increase in malic acid secretion and a 6-fold and 7-fold increase in citric acid content, respectively, compared to the wild-type control, with statistically significant differences. These results confirm... BrKAT1.1 Gene overexpression can significantly enhance the organic acid metabolism response of Arabidopsis thaliana under aluminum stress.

[0038] In summary, BrKAT1.1 Gene overexpression can significantly enhance the aluminum tolerance of plants by promoting the secretion of organic acids and secretory chelation, thereby increasing biomass and reducing aluminum accumulation under aluminum stress conditions.

Claims

1. A potassium ion channel KAT1 gene BrKAT1.1 Application in the regulation of plant stress resistance.

2. The application according to claim 1, characterized in that, The potassium ion channel KAT1 gene BrKAT1.1 The encoded proteins include: a. A protein having the amino acid sequence shown in SEQ ID NO: 1; b. Homologous proteins that have at least 90% sequence identity with the amino acid sequence shown in SEQ ID NO:

1.

3. The application according to claim 1, characterized in that, The plant in question is a member of the Brassicaceae family.

4. The application according to claim 1, characterized in that, The stress resistance mentioned refers to aluminum stress tolerance.

5. The application according to claim 4, characterized in that, The application involves overexpressing the potassium ion channel KAT1 gene. BrKAT1.1 Improve plant tolerance to aluminum stress.

6. The application according to claim 5, characterized in that, The steps of the application include: (1) Cloning isolation BrKAT1.1 Gene nucleic acid fragments; (2) Based on the genes isolated in step 1, a recombinant overexpression vector was constructed; (3) Introduce the recombinant overexpression vector obtained in step 2 into the target plant; (4) Screening to obtain stable overexpression BrKAT1.1 Genetically modified plants.

7. The application according to claim 6, characterized in that, The clone obtained in step 1 BrKAT1.1 The CDS sequence of the gene nucleic acid fragment is shown in SEQ ID NO:

2.

8. The application according to claim 6, characterized in that, The expression vector described in step 2 is a vector containing any one of the CaMV 35S promoter, U6 promoter, Actin promoter, UBQ10 promoter, or other constitutive high-expression promoters.

9. The application according to claim 8, characterized in that, The starting vector for the recombinant overexpression vector is PHB.

10. The application according to claim 6, characterized in that, The method for introducing the target plant in step 3 is any one of Agrobacterium-mediated transformation, protoplast transformation, viral vector delivery, pollen tube pathway, or gene gun method.

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