Application of PaVAMP711-b protein in regulating and controlling potassium stress resistance of plants

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of a PaVAMP711-b protein in regulating and controlling potassium stress resistance of plants. The PaVAMP711-b protein disclosed by the invention is a vesicle transport related protein and belongs to a class of SNARE proteins, and the potassium stress resistance of plants can be improved by knocking out the PaVAMP711-b gene. Through a gene editing technology, the PaVAMP711-b gene in the poplar is knocked out, two obtained transgenic lines have high potassium stress resistance phenotypes, and the fresh weight and the plant height of the transgenic lines are remarkably increased compared with those of wild types. Theoretical basis and technical support are provided for reasonable fertilization, soil improvement and cultivation of high-potassium-stress-resistant forest tree varieties in production, so that the growth performance and yield of crops in an adverse soil environment are improved, and sustainable development of forestry is guaranteed.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to the application of PaVAMP711-b protein in regulating plant resistance to potassium stress. Background Technology

[0002] Potassium ions (K) + K is the most abundant cation in plants. + It participates in various enzymatic reactions, protein synthesis, and photosynthesis. K + It remains a key factor influencing cell turgor pressure, xylem and phloem transport, pH homeostasis, and membrane potential. Furthermore, maintaining adequate potassium levels in plant cell cytoplasm is also crucial. + Concentration is crucial for balancing the flux of cations and anions. However, when the concentration of potassium ions in the soil is too high, this nutrient element, which is normally beneficial to plants, may turn into a stress factor and have adverse effects on them.

[0003] Studies have shown that K + It may not trigger Na + Under the same physiological or metabolic stress, plants may require unique genetic pathways beyond typical salt response pathways (Pantha P, Oh DH, Longstreth D, Dassanayake M. Living with high potassium: Balance between nutrient acquisition and K-induced salt stress signaling. Plant Physiol. 2023 Feb 12;191(2):1102-1121. doi: 10.1093 / plphys / kiac564.) to survive high potassium-induced salt stress. Currently, for K... + There are few systematic studies on the molecular phenotypes and related mechanisms behind the toxicity, therefore, there is an urgent need to find more genes to cope with high potassium stress in plants. Summary of the Invention

[0004] The purpose of this invention is to apply the PaVAMP711-b protein to the regulation of potassium stress resistance in plants. The PaVAMP711-b protein described in this invention can negatively regulate the potassium stress resistance of plants and can be used to cultivate new potassium stress tolerant germplasm.

[0005] This invention provides the application of PaVAMP711-b protein in regulating plant resistance to potassium stress, and the amino acid sequence of PaVAMP711-b protein is shown in SEQ ID NO.1.

[0006] As a preferred embodiment, the regulation includes: reducing the expression level of PaVAMP711-b protein to improve the plant's resistance to potassium stress.

[0007] As a preferred option, the plant includes silver poplar.

[0008] As a preferred embodiment, the nucleotide sequence of the gene encoding the PaVAMP711-b protein is shown in SEQ ID NO.2.

[0009] This invention provides a targeted knockout PaVAMP711-b The target sequence of the gene, as shown in SEQ ID NO.3 and / or SEQ IN NO.4; The PaVAMP711-b The nucleotide sequence of the gene is shown in SEQ ID NO.2.

[0010] This invention provides a targeted knockout PaVAMP711-b A gene editing vector, wherein the editing vector contains the target sequence described in the above scheme.

[0011] This invention provides a targeted knockout PaVAMP711-b The engineered bacteria for gene generation, wherein the engineered bacteria include the editing vector described in the above scheme.

[0012] This invention provides the application of the target sequence, the editing vector, or the engineered bacteria described above in improving the potassium stress resistance of plants or cultivating potassium stress-resistant plants.

[0013] As a preferred embodiment, the improvement of plant resistance to potassium stress includes: increasing plant fresh weight and / or plant height in potassium stress environments.

[0014] This invention provides a method for improving the potassium stress resistance of plants by introducing the editing vector or the engineered bacteria described in the above scheme into the plant.

[0015] This invention provides the application of PaVAMP711-b protein in regulating plant potassium stress resistance. The amino acid sequence of the PaVAMP711-b protein is shown in SEQ ID NO.1. The PaVAMP711-b protein of this invention is a vesicle transport-related protein, belonging to a class of SNARE proteins. Knockout... PaVAMP711-b Genes can enhance a plant's resistance to potassium stress. This invention utilizes gene editing technology to modify genes in *Populus alba* to improve the plant's resistance to potassium stress. PaVAMP711-bGene knockout yielded two transgenic lines, both exhibiting high potassium stress tolerance. Compared to the wild type, the transgenic lines showed significantly increased fresh weight and plant height. This invention provides a theoretical basis and technical support for rational fertilization, soil improvement, and the cultivation of high potassium stress-tolerant tree varieties in production, thereby improving crop growth performance and yield under unfavorable soil conditions and ensuring the sustainable development of forestry. Attached Figure Description

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

[0017] Figure 1 Subcellular localization analysis of PaVAMP711-b protein from Populus tomentosa; from left to right: green fluorescence image of GFP-PaVAMP711-b, red fluorescence image of vacuolar membrane marker mCherry-INT1, bright field image, images synthesized from different channels, and Pearson coefficient image. Figure 2 For Realtime-PCR analysis PaVAMP711-b Expression patterns in various organs of Arabidopsis thaliana; Figure 3 Construct a map for the target gene knockout vector; Figure 4 For CRISPR-Cas9 technology PaVAMP711-b A schematic diagram of gene mutants, in which the target sequence is represented by blue text, the PAM sequence is in red, the pink dashed line is the base deletion and the asterisk is the stop codon; Figure 5 mutant PaVAMP711-b- 1. PaVAMP711-b- Phenotypic analysis of salt stress in 2; Figure 6 mutant PaVAMP711-b- 1. PaVAMP711-b- 2 of K + Ca 2+ Calcium ion content analysis. Detailed Implementation

[0018] This invention provides an application of PaVAMP711-b protein in regulating plant resistance to potassium stress. The amino acid sequence of the PaVAMP711-b protein is shown in SEQ ID NO.1: MAILYALVARGSVVLAEFTSTATNASAIARQILDKIPGNDDSNVSYSQDRYIFHVKRTDGLTVLCMADETAGRRIPFAFLEDIHQRFVRTYGRAVITAQAYAMNDEFSRVLSQQMEYYTNDPNADRINRLKGEMSQVRNVMIENIDKVLERGDRLELLVDKTANMQGNTFRFRKQARRFRSTVWWRNVKLTVALILLLLVIIYVVLAFVCHGLALPTCLK.

[0019] In one embodiment, the regulation includes: reducing the expression level of PaVAMP711-b protein to improve plant resistance to potassium stress. In another embodiment, the plant includes *Populus tomentosa*. PaVAMP711-b, as described in this invention, is a vesicle transport-related protein belonging to the SNARE protein class, located on the vacuolar membrane. Reducing the expression level of PaVAMP711-b protein can improve the plant's resistance to potassium stress. Through gene editing technology, the expression level of PaVAMP711-b in *Populus tomentosa* is reduced... PaVAMP711-b The gene was knocked out, and the two transgenic lines obtained both exhibited a high potassium stress tolerance phenotype, providing a theoretical basis and technical support for breeding high potassium stress tolerant tree varieties.

[0020] As one embodiment, the nucleotide sequence of the gene encoding the PaVAMP711-b protein is shown in SEQ ID NO.2: 5'--3'.

[0021] This invention also provides a targeted knockout PaVAMP711-b The target sequence of the gene, as shown in SEQ ID NO.3 and / or SEQ IN NO.4; wherein SEQ ID NO.3: 5'-GTGAGGACATATGGTCGCG-3', SEQ ID NO.4: 5'-GCTAGCGGAATTTACCTCGA-3'. The present invention... PaVAMP711-b The nucleotide sequence of the gene is shown in SEQ ID NO. 2. The target of this invention is... PaVAMP711-b The specific sequences in nucleic acid sequences enable gene editing to achieve high specificity and low off-target rate.

[0022] This invention also provides a targeted knockout PaVAMP711-b The gene editing vector contains the target sequence described in the above-mentioned scheme. In a specific embodiment of the present invention, the base vector of the editing vector is pYLCRISPR / Cas9-DH. The vector construction process is described in [Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, QiuR, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. MolPlant. 2015 Aug;8(8):1274-84. doi: 10.1016 / j.molp.2015.04.007. Epub 2015 Apr24.]. The present invention uses pYLCRISPR / Cas9-DH as the base vector to improve editing efficiency. As one implementation, the target sequence is inserted into the BsaⅠ site of the base vector.

[0023] This invention also provides a targeted knockout PaVAMP711-b The engineered bacteria for gene generation include the editing vector described in the above-described scheme. In a specific embodiment of the present invention, the base bacteria of the engineered bacteria include Agrobacterium.

[0024] This invention also provides the application of the target sequence, the editing vector, or the engineered bacteria described above in improving the potassium stress tolerance of plants or cultivating potassium stress-tolerant plants. As one embodiment, improving the potassium stress tolerance of plants includes increasing the fresh weight and / or plant height of plants under potassium stress. As one embodiment, the plant includes *Populus alba*.

[0025] This invention also provides a method for improving the potassium stress resistance of plants by introducing the editing vector or the engineered bacteria described in the above-described scheme into the plant. As one embodiment, the plant includes *Populus tomentosa*.

[0026] To further illustrate the present invention, the technical solutions provided by the present invention will be described in detail below with reference to the accompanying drawings and embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0027] Example 1 1. Subcellular localization of PaVAMP71-a protein in Populus tomentosa To investigate the subcellular localization of PaVAMP711-b, this embodiment performed subcellular co-localization analysis on GFP-PaVAMP711-b, the fusion protein, and the mCherry-INT1 (INT1 is located on the vacuolar membrane) fusion protein. The analysis method is described in [chneider S, Beyhl D, Hedrich R, Sauer N. Functional and physiological characterization of Arabidopsis INOSITOL TRANSPORTER1, a novel tonoplast-localized transporter for myo-inositol. Plant Cell. 2008 Apr;20(4):1073-87. doi: 10.1105 / tpc.107.055632. Epub 2008 Apr 25. PMID: 18441213; PMCID:PMC2390729.].

[0028] Construction of the GFP-PaVAMP711-b expression vector: The PaVAMP711-b CDS sequence was amplified using KOD high-fidelity enzyme and then ligated into the 1390-Proubq10:GFP vector via KpnⅠ / SalⅠ restriction enzyme digestion, resulting in the 1390-Proubq10:GFP-PaVAMP711-b vector. The 1390-Proubq10:GFP vector is based on pCAMBIA1390, with a UBQ promoter inserted between HindIII and PstI, followed by the GFP sequence after the PstI restriction site.

[0029] Construction of the mCherry-INT1 expression vector: The INT1 CDS sequence (NCBI publication number AT2G43330) was amplified using KOD high-fidelity enzyme and then ligated into the 1390-Proubq10:mCherry vector via KpnⅠ / SalⅠ restriction enzyme digestion, resulting in the 1390-Proubq10:mCherry-INT1 vector. The 1390-Proubq10:mCherry vector is based on pCAMBIA1390, with a UBQ promoter inserted between HindIII and PstI, followed by a GFP sequence after the PstI restriction site and an mCherry sequence after the PstI restriction site.

[0030] UBQ promoter sequence (SEQ ID NO.13): 5’-aagcttcgacgagtcagtaataaacggcgtcaaagtggttgcagccggcacacacgagtcgtgtttatcaactcaaagcacaaatacttttcctcaacctaaaaataaggcaattagccaaaaacaactttgcgtgtaaacaacgctcaatacacgtgtcattttattattagctattgcttcaccgccttagctttctcgtgacctagtcgtcctcgtcttttcttcttcttcttctataaaacaatacccaaagagctcttcttcttcacaattcagatttcaatttctcaaaatcttaaaaactttctctcaattctctctaccgtgatcaaggtaaatttctgtgttccttattctctcaaaatcttcgattttgttttcgttcgatcccaatttcgtatatgttctttggtttagattctgttaatcttagatcgaagacgattttctgggtttgatcgttagatatcatcttaattctcgattagggtttcatagatatcatccgatttgttcaaataatttgagttttgtcgaataattactcttcgatttgtgatttctatctagatctggtgttagtttctagtttgtgcgatcgaatttgtcgattaatctgagtttttctgattaacagttcgaa-3’; GFP nucleic acid sequence (SEQ ID NO.14): 5’-atggtgagcaagggcgaggagctgttcaccggggtggtgcccatcctggtcgagctggacggcgacgtaaacggccacaagttcagcgtgtccggcgagggcgagggcgatgccacctacggcaagctgaccctgaagttcatctgcaccaccggcaagctgcccgtgccctggcccaccctcgtgaccaccctgacctacggcgtgcagtgcttcagccgctaccccgaccacatgaagcagcacgacttcttcaagtccgccatgcccgaaggctacgtccaggagcgcaccatcttcttcaaggacgacggcaactacaagacccgcgccgaggtgaagttcgagggcgacaccctggtgaaccgcatcgagctgaagggcatcgacttcaaggaggacggcaacatcctggggcacaagctggagtacaactacaacagccacaacgtctatatcatggccgacaagcagaagaacggcatcaaggtgaacttcaagatccgccacaacatcgaggacggcagcgtgcagctcgccgaccactaccagcagaacacccccatcggcgacggccccgtgctgctgcccgacaaccactacctgagcacccagtccgccctgagcaaagaccccaacgagaagcgcgatcacatggtcctgctggagttcgtgaccgccgccgggatcactctcggcatggacgagctgtacaag-3’; mCherry nucleic acid sequence (SEQ ID NO.15): 5'--3'.

[0031] Tobacco leaves were co-transfected with *Agrobacterium* containing the GFP-PaVAMP711-b expression vector and *Agrobacterium* containing the mCherry-INT1 expression vector, according to the method described in [Fireflyase Complementation Assay for Protein Interaction, Zhao Yan, Zhou Jianmin, Acta Botanica Sinica, Vol. 1, 2020]. After 48–72 hours of growth, the fluorescence signals of GFP and mCherry were detected under a microscope, with mCherry-INT1 used as a marker for the vacuolar membrane. Scale bar: 50 μm. Experimental results are shown below. Figure 1 As shown. According to Figure 1 It can be seen that PaVAMP711-b is mainly located in the vacuolar membrane.

[0032] 3. Silver Poplar PaVAMP71-a Analysis of gene expression patterns In order to study the target gene PaVAMP711-b To investigate the tissue expression patterns in *Populus tomentosa*, mature leaves, young leaves, buds, stems, and roots of wild-type *Populus tomentosa* plants grown to the top of tissue culture flasks were collected. Total RNA was extracted and reverse transcribed into cDNA for further processing. PtACTIN As an internal reference, testing PaVAMP711-b The expression levels of the gene in different tissue sites, and the primer sequences used are shown below: RT-PaVAMP711-bF (SEQ ID NO.5): 5'-ATTTACCTCGACGGCGACAA-3'; RT-PaVAMP711-bR (SEQ ID NO.6): 5'-GTGAGGACATATGGTCGCGC-3'; RT-PaACTIN-F (SEQ ID NO.7): 5'-ATTTACCTCGACGGCGACAA-3'; RT-PaACTIN-R (SEQ ID NO. 8): 5'-GTGAGGACATATGGTCGCGC-3'.

[0033] The PCR reaction system is in 10 μL: TB Green Premix Ex Taq II 5 μL, forward primer (10 μM) 0.4 μL, reverse primer (10 μM) 0.4 μL, cDNA template 1 μL, ddH2O 3.6 μL.

[0034] The PCR reaction program was: 95℃ for 30s; 95℃ for 5s, 60℃ for 30s, 40 cycles; 95℃ for 5s.

[0035] Test results as follows Figure 2 As shown in Table 1, the results show PaVAMP711-b It is expressed in large quantities in mature leaves, but at lower levels in several other tissues.

[0036] Table 1 PaVAMP711-b Expression of various organs in Populus tomentosa

[0037] Example 2 Genetic transformation of Populus tomentosa 1. Constructing silver poplar PaVAMP711-b Gene editing materials (1) Based on the CRIPSPR / Cas9 gene editing principle, in Populus tomentosa PaVAMP711-b Two suitable targets were selected from the gene: target 1 (SEQ ID NO.3): 5'-GTGAGGACATATGGTCGCG-3', and target 2 (SEQ ID NO.4): 5'-GCTAGCGGAATTTACCTCGA-3'.

[0038] (2) Using the restriction enzyme BsaⅠ, target sites 1 and 2 were ligated into the pYLCRISPR / Cas9-DH expression vector containing the U3d promoter. The vector map is shown below. Figure 3 As shown.

[0039] The pYLCRISPR / Cas9-DH expression vector has a backbone of pCAMBIA-1300, which is safe and non-pathogenic in Escherichia coli, Agrobacterium, and plants. For the construction process of the pYLCRISPR / Cas9-DH vector, please refer to [Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, Lin Y, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant. 2015 Aug;8(8):1274-84. doi:10.1016 / j.molp.2015.04.007. Epub 2015 Apr 24.].

[0040] 2. Transformation of silver poplar trees The gene-editing vector constructed in step 1 was transformed into Agrobacterium EHA105, and then transformed into Populus alba using the Agrobacterium-mediated transformation method. The specific steps are as follows: (1) Agrobacterium containing gene editing vector was inoculated into YEB liquid medium (Kana, 100 mg / L; Rif, 50 mg / L) and cultured in a shaker at 28°C until OD600 = 0.8~1.0.

[0041] (2) Collect bacterial cells by centrifugation at 4000 rpm at 4℃. Resuspend the bacterial cells in a resuspension solution and incubate with shaking at 28℃ for at least 2 h. The OD600 of the bacterial solution is approximately 0.6. Place the infection solution on ice for later use. The resuspension solution is: 2.41 g / L WPM powder + 30.0 g / L sucrose + 100.0 μmol / L acetylsylgenone, pH 5.8~6.0.

[0042] (3) Select a seedling of Populus tomentosa that has grown for about one month, cut off tender leaves and stems, then divide the leaves into small pieces of 0.5×0.5cm, divide the young stems into stem segments of 0.5cm, and place the leaves and stem segments in the infection solution for 10~15min. (4) Remove the leaves and stem segments and place them on sterile filter paper to absorb the bacterial solution on the surface of the explants. Then spread these explants evenly on CM1 medium and incubate them in the dark at 25°C for 24 hours. The CM1 medium is composed of: 2.41 g / L WPM powder + 1.0 mg / L NAA + 2.0 mg / L ZT + 30.0 g / L sucrose + 100.0 μmol / L acetylsylgenone + 6.0 g / L agar powder, pH 5.8~6.0.

[0043] (5) Place the explants on CM2 medium and incubate in the dark at 25°C for 3-4 weeks, replacing the medium every 14 days. The CM2 medium is composed of 2.41 g / L WPM powder + 1.0 mg / L NAA + 2.0 mg / L ZT + 30.0 g / L sucrose + 400.0 mg / L cephalosporin + 10.0 mg / L hygromycin + 6.0 g / L agar powder, pH 5.8-6.0.

[0044] (6) Once callus has formed at the explant incision site, place the explant on CM3 medium and induce adventitious bud differentiation under light at 25°C. The CM3 medium consists of: 2.41 g / L WPM powder + 0.1 mg / L NAA + 2.0 mg / L ZT + 30.0 g / L sucrose + 400.0 mg / L cefotaxime + 10.0 mg / L hygromycin + 6.0 g / L agar powder, pH 5.8~6.0. (7) The callus from the above-mentioned adventitious buds was inoculated onto CM4 medium for adventitious bud development culture to obtain rootless seedlings. The CM4 medium consisted of 2.41 g / L WPM powder + 0.1 mg / L IBA + 15.0 g / L sucrose + 400.0 mg / L cephalosporin + 10.0 mg / L hygromycin + 6.0 g / L agar powder, pH 5.8~6.0.

[0045] (8) When the adventitious buds grow to 2-3 cm, cut them off and insert them into CM5 medium to induce the formation of adventitious roots. The CM5 medium is: 2.47 g / L 1 / 2 MS medium + 0.5 mg / L IBA + 15.0 g / L sucrose + 400.0 mg / L cephalosporin + 10.0 mg / L hygromycin + 6.0 g / L agar powder, pH 5.8~6.0.

[0046] (9) When the rooted seedlings grow to 8-10cm, they are transferred to soil and cultivated in a greenhouse.

[0047] Obtaining positive plants: RNA was extracted from transgenic poplar trees and reverse transcribed into cDNA. Primers were designed at highly specific sequences at both ends of target sites 1 and 2, and sequences containing the target sites were amplified. The primers used are shown below: PaVAMP711-b-T1-F (SEQ ID NO.9): 5'-GATTGTGAGGACATATGGTCGCG-3'; PaVAMP711-b-T1-R (SEQ ID NO. 10): 5'-AAACGTGAGGACATATGGTCGCG-3'; PaVAMP711-b-T2-F (SEQ ID NO. 11): 5'-GATTGCTAGCGGAATTTACCTCGA-3'; PaVAMP711-b-T2-R (SEQ ID NO. 12): 5'-AAACTCGAGGTAAATTCCGCTAGC-3'.

[0048] The PCR reaction system is in 100 μL: 50 μL of 2×KOD MIX buffer, 6 μL of forward primer (10 μM), 6 μL of reverse primer (10 μM), 2 μL of template cDNA / DNA, and ddH2O to 100 μL.

[0049] PCR reaction program: 98℃ for 5 min; 98℃ for 30 s, 56℃ for 30 s, 68℃ for 1 min, 35 cycles, 68℃ for 10 min; store at 20℃ for later use.

[0050] After amplification, the sample was sent to the company for sequencing. The sequencing results are as follows: Figure 4 As shown, two were ultimately obtained. PaVAMP711-b The transgenic lines that undergo gene editing are named PaVAMP711-b- 1 and PaVAMP711-b- 2.

[0051] Example 3: Silver Poplar PaVAMP711-b Phenotypic analysis of high potassium stress in loss-of-function mutants Take wild-type Populus tomentosa plants that have grown to the top of the tissue culture flask. PaVAMP711-b- 1. PaVAMP711-b- Two plants, planted in high K + The growth of different plants was observed in the stress culture medium, and the fresh weight and height of the plants were recorded. The results are as follows: Figure 5 As shown in Table 2. High K + The stress medium was: 2.47 g / L nitrogen-deficient 1 / 2 MS medium + 20 mM KNO3 + 20 mM KCl + 10 mM K2SO4 + 0.5 mg / L IBA + 15.0 g / L sucrose + 6.0 g / L agar powder, pH 5.8~6.0.

[0052] according to Figure 5 As shown in Table 2, at a high K of 50 mM + After growing in the culture medium for about 30 days, compared with wild-type plants, Pavamp711-b- 1. Pavamp711-b- 2. The plants grow well; for wild-type plants, Pavamp711-b- 1. Pavamp711-b- 2. Statistical analysis of plant height was performed. Pavamp711-b- 1. Pavamp711-b- The plant height of the two plants was significantly greater than that of wild-type Populus spp. ( Figure 5 (A, B); for wild-type plants, Pavamp711-b- 1. Pavamp711-b- 2. Fresh weight statistical analysis of the plants. Pavamp711-b- 1. Pavamp711-b- The fresh weight of the two plants was significantly higher than that of wild-type Populus tomentosa. Figure 5 (C). The above experimental results show that, Pavamp711-b- 1. Pavamp711-b- 2 mutants are resistant to high K + The phenotype of coercion.

[0053] Table 2 Mutants PaVAMP711-b- 1. PaVAMP711-b- Phenotypic analysis of salt stress in 2

[0054] For further testing PaVAMP711-b Genes on high K + The response to stress, in this embodiment, was detected. PaVAMP711-b- 1. PaVAMP711-b- 2 mutants in high K + The potassium ion content after stress treatment was as follows: Figure 6 As shown in A~C and Table 3. Experimental results indicate that wild-type Populus tomentosa and... PaVAMP711-b- 1. PaVAMP711-b- There was no significant difference in potassium ion content in the roots, stems, and leaves of the mutant 2.

[0055] Calcium ions, acting as second messengers, play a crucial role in plant responses to various abiotic stresses. Vacuoles are the primary reservoirs of calcium ions within plant cells, maintaining cytoplasmic calcium homeostasis. This study further investigated the effect of vacuole-localized PaVAMP711-b gene deletion on calcium ion content. The results are as follows: Figure 6 As shown in Tables D-F and 3, calcium ion content was detected in the leaves of Pavamp711-b-1 and Pavamp711-b-2 mutants, which were significantly lower than those in wild-type Populus tomentosa. However, there was no significant difference in calcium ion content in the stems and roots. Plant calcium signaling is widely involved in plant stress resistance regulation. This invention found that knocking out… PaVAMP711-b Genes can enhance a plant's resistance to potassium stress through calcium signaling regulation.

[0056] For methods of determining calcium and potassium ions, please refer to [Y Wang, YJ Jiao, Y Zhu, YF Wang, J Yang, Determination of sodium and potassium in Dan Hong injection by atomic absorption spectrometry, Tianjin Journal of Traditional Chinese Medicine, 2016-01]. Table 3 Mutants PaVAMP711-b- 1. PaVAMP711-b- 2 of K + Ca 2+ Calcium ion content analysis

[0057] In summary, the PaVAMP711-b negatively regulates the potassium stress resistance of plants, as described in this paper. Through gene editing technology, the PaVAMP711-b gene in *Populus tomentosa* can be used to enhance the potassium stress resistance of plants. PaVAMP711-b Gene knockout yielded two transgenic lines, both exhibiting high potassium stress tolerance. Compared to the wild type, the transgenic lines showed significantly increased fresh weight and plant height, providing a theoretical basis and technical support for rational fertilization, soil improvement, and the cultivation of high potassium stress-tolerant tree varieties in production.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Application of PaVAMP711-b protein in regulating plant resistance to potassium stress, wherein the amino acid sequence of PaVAMP711-b protein is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that, The regulation includes: reducing the expression level of PaVAMP711-b protein to improve the plant's resistance to potassium stress.

3. The application according to claim 1, characterized in that, The plants mentioned include silver poplar.

4. The application according to any one of claims 1 to 3, characterized in that, The nucleotide sequence of the gene encoding the PaVAMP711-b protein is shown in SEQ ID NO.

2.

5. A targeted knockout method PaVAMP711-b The target sequence of the gene, characterized in that, The target sequence is shown in SEQ ID NO.3 and / or SEQ IN NO.4; The PaVAMP711-b The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

6. A targeted knockout method PaVAMP711-b Gene editing vectors, characterized in that, The editing vector comprises the target sequence as described in claim 5.

7. A targeted knockout method PaVAMP711-b Genetically engineered bacteria, characterized by, The engineered bacteria include the editing vector described in claim 6.

8. The application of the target sequence of claim 5, the editing vector of claim 6, or the engineered bacteria of claim 7 in improving the potassium stress resistance of plants or cultivating potassium stress resistant plants.

9. The application according to claim 8, characterized in that, The improvement of plant resistance to potassium stress includes: increasing plant fresh weight and / or plant height in potassium stress environments.

10. A method for improving the resistance of plants to potassium stress, characterized in that, The editing vector of claim 6 or the engineered bacteria of claim 7 are introduced into plants.

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