Lbckx3 and application thereof

Abstract

This invention belongs to the field of plant genetic engineering and provides the LbCKX3 gene related to cytokinin degradation in *Limonium bicolor* and its applications. This invention utilizes virus-induced gene silencing technology to verify the function of key genes in the cytokinin signal transduction pathway. It was found that silencing the cytokinin degradation gene LbCKX3 significantly increased the number of salt glands and the amount of salt secreted by leaves, but reduced Na... + The secretion rate was not affected. These results indicate that inhibiting or silencing the cytokinin degradation-related gene LbCKX3 can promote the development of salt glands in *Limonium bicolor*, thus providing an important theoretical basis and candidate gene for improving crop salt tolerance, and opening up a new path for cultivating salt-tolerant crops.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering, and specifically relates to the LbCKX3 gene related to the degradation of cytokinin in Limonium bicolor and its application. Background Technology

[0002] Soil salinization severely restricts crop production, and improving plant salt tolerance is key to the development and utilization of saline land. High salt concentrations trigger osmotic stress, ion imbalance, and reactive oxygen species accumulation, disrupting normal plant physiological activities. Plants respond to salt stress through various strategies, among which plant hormones play a crucial role in the plant salt stress response by regulating plant growth and development (Lv B, Yan Z, Tian H, et al. Local auxin biosynthesis mediates plant growth and development [J]. Trends Plant Sci, 2019, 24(1): 6-9).

[0003] Plants under salt stress typically exhibit decreased cytokinin levels (Nishiyama R, Watanabe Y, Fujita Y, et al. Analysis of cytokinin mutants and regulation of cytokininmetabolic genes reveals important regulatory roles of cytokinins in drought, salt and abscisic acid responses, and abscisic acid biosynthesis [J]. PlantCell, 2011, 23(6): 2169-2183). *Limonium bicolor* (Bunge) Kuntze is a typical halophyte with multicellular salt glands on its leaf epidermis. However, the molecular mechanisms by which cytokinin-related genes participate in and regulate salt gland development remain unclear, and the core molecular modules regulating salt gland development need further clarification. Summary of the Invention

[0004] The purpose of this invention is to provide the LbCKX3 gene, which is related to the degradation of cytokinin in *Limonium bicolor*, and its applications.

[0005] This invention investigated the newly discovered LbCKX3 gene related to the degradation of cytokinin in *Limonium bicolor*, and performed virus-induced gene silencing (VIGS) on it to obtain LbCKX3-silenced lines. It was found that the LbCKX3-silenced lines exhibited phenotypes with increased salt gland density and salt secretion capacity, thus leading to this invention.

[0006] Therefore, on the one hand, the present invention provides the LbCKX3 gene related to the degradation of cytokinin in *Limonium bicolor*, which is a gene encoding the following protein (a) or (b):

[0007] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or

[0008] (b) A protein derived from (a) with one or more amino acids substituted, deleted or added in the amino acid sequence shown in SEQ ID NO:2 and having an equivalent function.

[0009] In an embodiment of the present invention, the nucleotide sequence of the LbCKX3 gene, which is related to the degradation of cytokinin in *Limonium bicolor*, is shown in SEQ ID NO:1.

[0010] Secondly, the present invention provides a cytokinin-decomposing factor LbCKX3 from *Limonium bicolor*. In an embodiment of the present invention, the amino acid sequence of the cytokinin-decomposing factor LbCKX3 from *Limonium bicolor* is shown in SEQ ID NO:2.

[0011] Thirdly, the present invention provides biomaterials containing the LbCKX3 gene, which is related to the degradation of cytokinin in *Limonium bicolor*.

[0012] In embodiments of the present invention, the biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, or engineered bacteria.

[0013] Fourthly, the present invention provides the application of the LbCKX3 gene related to the degradation of cytokinin in *Limonium bicolor* or the biological material described in the third aspect in the preparation of transgenic plants.

[0014] Fifthly, the present invention provides any of the following applications of the LbCKX3 gene, which is related to the degradation of cytokinin in *Limonium bicolor*, or biological materials containing this gene:

[0015] (1) Used to improve the growth and development of salt glands in halophytes;

[0016] (2) Used to enhance the salt-secreting capacity of halophytes;

[0017] (3) Used to improve the stress resistance of halophytes that secrete salts;

[0018] (4) Used for plant variety improvement.

[0019] In embodiments of the present invention, improving the growth and development of salt glands in halophytes can be achieved, for example, by increasing the number of salt gland cells and promoting the differentiation of meristematic cells into the initial structure of multicellular salt glands.

[0020] In an embodiment of the present invention, the preferred plant is *Limonium bicolor*.

[0021] In a sixth aspect, the present invention provides a method for promoting the development of salt glands in *Limonium bicolor* (including increasing the number of salt gland cells and promoting the differentiation of meristematic cells into the initial structure of multicellular salt glands), including using genetic engineering to weaken the *Limonium bicolor* cytokinin degradation-related gene LbCKX3 to obtain gene-weakened plants; weakening includes knocking out the gene LbCKX3 or reducing the expression of the gene.

[0022] In the embodiments of the present invention, the genetic engineering method is selected from any one of mutagenesis, site-directed mutagenesis, and homologous recombination.

[0023] In a specific implementation plan, the method includes using VIGS technology to silence the LbCKX3 gene, which is related to the degradation of cytokinin in *Limonium sibiricum*.

[0024] In a further specific implementation plan, the method includes constructing a recombinant virus containing a fragment of the LbCKX3 gene related to the degradation of cytokinin in Limonium bicolor, infecting Limonium bicolor, and screening for LbCKX3 gene-silenced lines.

[0025] In this invention, the LbCKX3 gene related to the degradation of cytokinin in *Limonium bicolor* can also be referred to as the *Limonium bicolor* gene LbCKX3 or gene LbCKX3.

[0026] This invention marks the first time that the cytokinin degradation-related gene LbCKX3 has been cloned from *Limonium bicolor*, and its biological function was investigated. Experiments showed that LbCKX3-silenced lines exhibited a phenotype with increased salt gland density and salt secretion capacity. This invention provides a preliminary understanding of the regulatory network of cytokinin on salt gland development in halophytes, offering important theoretical basis and candidate genes for improving crop salt tolerance through genetic engineering, and also opening new pathways for cultivating salt-tolerant crops (including species lacking salt glands). Attached Figure Description

[0027] Figure 1 This is a molecular verification electrophoresis image of the VIGS vector construction of gene LbCKX3 in a preferred embodiment of the present invention.

[0028] Figure 2This figure shows the expression level determination of the LbCKX3 gene-silenced lines in a preferred embodiment of the present invention. The expression levels were measured in the *Limonium bicolor* control line (TRV::0) and the gene-silenced lines (TRV::LbCKX3-1~7). The three dots in the figure indicate the three groups of lines with lower expression levels, with three biological replicates for each line. The values ​​in the figure are mean ± standard deviation (n = 3), and different lowercase letters indicate significant differences at the Duncan multiple test level (P < 0.05).

[0029] Figure 3 Analysis of salt gland phenotype and salt secretion function in gene-silenced *Limonium bicolor* lines. A: Salt gland images of leaves of the control line (TRV::0) and each gene-silenced line (TRV::LbCKX3-1 / -2 / -4) under UV excitation, scale bar = 50 μm. B: Total number of salt glands in leaf discs, Na... + Secretion rate and 24-hour salt secretion volume statistics. The values ​​in the figure are mean ± standard deviation (n = 3), and different lowercase letters indicate significant differences at the Duncan multiple test level (P < 0.05). Detailed Implementation

[0030] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the examples are conducted under conventional experimental conditions, such as those described in Sambrook et al., Molecular Cloning: a Laboratory Manual (Sambrook J & Russell DW, 2012), or as recommended by the manufacturer's instructions.

[0031] Example

[0032] 1. Experimental Materials and Methods

[0033] 1.1. Experimental Materials

[0034] The seeds of *Limonium bicolor* were collected in October 2023 from the saline-alkali land in the Yellow River Delta in Dongying City, Shandong Province, China. After rubbing the seeds out, large, plump, and uniformly sized seeds were selected and stored in a refrigerator at 4°C for 6 months before use to minimize individual differences between seeds.

[0035] 1.2. Experimental Methods

[0036] 1.2.1 Cultivation of Limonium bicolor

[0037] After drying the preserved seeds, select uniform and plump seeds and vernalize them at 4℃ for 3 days. After vernalization, evenly scatter the seeds on nutrient soil (Shouguang Jiaheng Seedling Substrate Processing Plant, Shandong Province), cover with a film, and place under the following cultivation conditions: 26 / 22℃ (day / night), photoperiod 16 / 8 h (day / night), light intensity 600 μmol / m². 2 They were cultivated in a greenhouse ( / s) until the sixth true leaf grew.

[0038] 1.2.2 Gene Cloning

[0039] RNA was extracted from the leaves of *Limonium bicolor* using the FastPure Plant Total RNA Isolation Kit RC401-01 (Nanjing Novizan Biotechnology Co., Ltd.), and cDNA templates were obtained by reverse transcription using the HiScript III 1stStrand cDNA Synthesis Kit (+gDNA wiper) (Nanjing Novizan Biotechnology Co., Ltd.).

[0040] Based on the nucleotide sequence of the LbCKX3 gene (SEQ ID NO:1), amplification primers were designed (Table 1) for PCR.

[0041] Table 1 Primers used for cloning 5' DNA flanking sequences of genes

[0042]

[0043] The reaction was performed using high-fidelity enzyme 2 × Phanta Max Master Mix (Dye Plus) (Nanjing Novizan Biotechnology Co., Ltd.), with leaf cDNA as a template. The reaction steps are as follows:

[0044] (1) PCR reaction: PCR reaction was performed using LbCKX3-5 as the upstream primer and LbCKX3-3 as the downstream primer.

[0045] ① Prepare the PCR reaction solution according to the following components:

[0046]

[0047] ② The PCR reaction conditions are as follows:

[0048]

[0049] (2) Take 50 μL of each PCR reaction solution and perform electrophoresis on a 1% agarose gel.

[0050] (3) After DNA electrophoresis, use a blade to quickly cut off clear electrophoretic bands under UV light for gel recovery. The gel recovery method is based on FastPure Gel DNA Extraction Mini Kit DC301 (Vazyme). Use LbCKX3-5 and LbCKX3-3 as primers to sequence the DNA of the PCR products. Proceed to the next step of the experiment for DNA samples with correct sequencing.

[0051] 1.2.3 Construction and infection of VIGS vectors

[0052] (1) Based on the pTRV2 vector (Hunan Fenghui Biotechnology Co., Ltd.), a non-conserved sequence of about 300 bp of the LbCKX3 gene was selected as the target (SEQ ID NO: 3) and ligation primers were designed.

[0053] Table 2 Primers used to construct the VIGS vector

[0054]

[0055] (2) PCR amplification of the insert fragment, in the following system (50 μL):

[0056]

[0057] PCR reaction procedure:

[0058]

[0059] (3) The pTRV2 vector was digested using New England Biolabs (NEB) restriction enzymes and the corresponding buffer. The reaction mixture was 50 μL as follows:

[0060]

[0061] Reaction conditions: 37°C for 2-3 hours; after completion, the enzyme digestion product was purified and recovered.

[0062] (4) The PCR product was purified and recovered, and the pTRV2 vector was ligated using the ClonExpress Ultra One Step Cloning Kit V3 (Nanjing Novizan Biotechnology Co., Ltd.). The reaction system was as follows (10 μL):

[0063]

[0064] Reaction conditions: 50°C for 15 min.

[0065] (5) Take 5 μL of the ligation product and transform it into Escherichia coli DH5α (Shanghai Beyotime Biotechnology Co., Ltd.). Spread it on LB solid medium containing 50 μg / mL kanamycin (Beijing Cooler Technology Co., Ltd.). After incubating at 37℃ for one day, pick single colonies. After identifying positive single clones using primers LbCKX3-5 and LbCKX3-3, expand the positive colonies and extract plasmids using FastPure Plasmid Mini Kit DC201 (Nanjing Novizan Biotechnology Co., Ltd.). Transform the correctly sequenced recombinant plasmid into K599 Agrobacterium rhizogenes (Beijing Cooler Technology Co., Ltd.).

[0066] (6) Agrobacterium-mediated genetic transformation of *Limonium bicolor*: Single clones of *Agrobacterium* positive for the target gene were selected and cultured by shaking. Simultaneously, pTRV1 and pTRV2 (Hunan Fenghui Biotechnology Co., Ltd.) bacterial suspensions were cultured. After culturing the above strains at 29°C for 16-18 h, 1 mL of each bacterial suspension was added to 50 mL of YEB liquid medium (Beijing Coollab Technology Co., Ltd.) for further propagation. This medium was pre-contained with kanamycin and rifampin at a final concentration of 50 μg / mL, and acetylsyl syringone at a final concentration of 100 μM. After the second culture, the bacterial suspension was enriched by centrifugation in 50 mL centrifuge tubes at 3000 rpm for 10 min. The supernatant was discarded after centrifugation. A resuspension solution (MgCl2 2.033 mg / mL, acetylsyl syringone 500 μM, MES 2.132 mg / mL) was prepared, and the bacterial suspension was fully resuspended to OD. 600 The value was 1.0, and subsequent infection experiments were conducted after 3 hours of darkness and stillness.

[0067] (7) Infection of *Limonium bicolor*: Resuspension of pTRV1 and pTRV2 containing the target gene fragment (experimental group), and resuspension of pTRV1 and pTRV2 (control group) were mixed at a 1:1 ratio. *Limonium bicolor* seedlings with consistent growth were selected, and the mixed bacterial solution was injected onto the underside of their leaves until the solution penetrated the entire leaf. After injection, the plants were kept in darkness for 48 h. Leaves from mature *Limonium bicolor* seedlings that had been cultured for two weeks were collected to determine the silencing efficiency (RT-qPCR experiment). Total RNA was extracted using the FastPure Plant Total RNA Isolation Kit RC401-01 (Nanjing Novizan Biotechnology Co., Ltd.) according to the kit instructions. To eliminate genomic DNA contamination, RNA was reverse transcribed into cDNA template using HiScriptIII RT SuperMix for qPCR (+gDNA wiper) (Nanjing Novizan Biotechnology Co., Ltd.). The reagent used was ChamQ Universal SYBR qPCR Master Mix Q711 (Nanjing Novizan Biotechnology Co., Ltd.).

[0068] Table 3 Primers and internal controls used for LbCKX3 silencing efficiency assay

[0069]

[0070] 1.2.4 Observation of salt glands in leaves of *Limonium bicolor* under ultraviolet excitation light

[0071] The leaves of *Limonium bicolor* were washed with deionized water to remove salt from the leaf surface. The leaves were then fixed using a fixative (ethanol:acetic acid = 3:1, reagent purchased from Sinopharm Chemical Reagent Co., Ltd.). Transparent leaves were placed on glass slides and observed using a fluorescence microscope under ultraviolet light (330-380 nm) with differential interference microscopy (DIC, ECLIPSE 80i, Nikon, Japan). Three leaves from each strain were selected to calculate the number of salt glands. Five different fields of view were observed on each leaf, and the average value of these three leaf observations was taken as the number of salt glands on each leaf.

[0072] 1.2.5 Real-time quantitative PCR (RT-qPCR)

[0073] Gene quantification primers and internal control primers were designed using Beacon Designer software. Using the primers shown in Table 3 above, control lines and gene-silenced lines were amplified using the ChanQ Universal SYBR qPCR Master Mix Q711 (Vazyme) fluorescent quantitative enzyme system in a 96 ppm fluorescence quantitative PCR instrument, following the formula Relative expression level = 2. −ΔΔC(T) Calculate the expression level.

[0074] 1.2.6 Determination of Salt Secretion Capacity (Leaf Disc Method)

[0075] Leaves of *Limonium bicolor* with similar developmental stages were selected and rinsed with distilled water to remove surface salts. Discs with a diameter of 10 mm were isolated from the same position on each leaf using a perforator. After drying the leaves on filter paper, they were placed in petri dishes and floated in a 200 mM NaCl solution with the underside facing upwards. A thin layer of mineral oil was then applied to the leaf discs. After 18–24 h of incubation, salt droplets were collected from the leaf surface and centrifuged. The volume of secreted fluid above the surface of each leaf disc was measured using a micropipette. The collected salt droplets were diluted appropriately, and the Na+ content in the secreted fluid was determined using a flame photometer. + concentration.

[0076] 1.2.7 Statistical Analysis

[0077] To ensure the reliability and reproducibility of the experimental results, at least three biological replicates were performed. Data were statistically analyzed using SPSS Statistics 17.0 software and Excel. The Shapiro-Wilk test was used to assess the normality of the residuals for each group (p > 0.05 was considered to be normally distributed). One-way ANOVA was used if there was only one independent variable; two-way ANOVA was used if two independent variables and their interaction were involved. For the experimental data, the mean ± standard deviation was calculated to represent the average level and range of fluctuation of the data. Duncan's multiple test was used, and p < 0.05 indicated a significant difference. Data graphs were created using tools such as Excel.

[0078] 2. Experimental Results

[0079] To verify the function of the LbCKX3 gene, this invention employs viral-induced gene silencing (VIGS) technology, extracting the first 300 bp of LbCKX3 as a pTRV2 vector ligation fragment (SEQ ID NO: 3). Figure 1 ), through statistical measurement of strains with high silencing efficiency ( Figure 2 The study of salt gland number and salt secretion revealed that silencing the cytokinin degradation gene significantly increased both the number of salt glands and the amount of salt secreted from leaves, but Na...+ The secretion rate was not affected. Figure 3 These results suggest that the cytokinin pathway may play a crucial role in the development and functional maintenance of salt glands. Specifically, the silencing of LbCKX3, a key gene for cytokinin degradation, may promote the proliferation and differentiation of salt glands by reducing cytokinin degradation.

[0080] This invention preliminarily reveals the negative regulatory function of LbCKX3 in salt gland development and provides new molecular evidence for understanding the evolution and regulation of specialized epidermal structures in halophytes. More importantly, as a cytokinin degradation factor, LbCKX3 shows promise as a potential target for improving crop salt tolerance through gene editing.

[0081] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, such as obtaining overexpression or knockout lines, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. The LbCKX3 gene related to the degradation of cytokinin in *Limonium bicolor*, characterized in that... It is a gene encoding either protein (a) or (b) as follows: (a) A protein consisting of the amino acid sequence shown in SEQ ID NO:2; or (b) A protein derived from (a) with one or more amino acids substituted, deleted or added in the amino acid sequence shown in SEQ ID NO:2 and having an equivalent function.

2. The LbCKX3 gene related to the degradation of cytokinin in *Limonium bicolor* is characterized by, The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. The cytokinin decomposition factor LbCKX3 of *Limonium sibiricum*, characterized in that... The amino acid sequence of the factor is shown in SEQ ID NO:

2.

4. A biological material containing the LbCKX3 gene of claim 1, wherein the biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

5. The use of the gene LbCKX3 of claim 1 or the biomaterial of claim 4 in the preparation of transgenic plants.

6. Any of the following applications of the gene LbCKX3 of claim 1 or biological materials containing the gene: (1) Used to improve the growth and development of salt glands in halophytes; (2) Used to enhance the salt-secreting capacity of halophytes; (3) Used to improve the stress resistance of halophytes that secrete salts; (4) Used for plant variety improvement.

7. The use according to claim 6, characterized in that, The plant in question is *Limonium bicolor*.

8. A method for increasing the number of salt gland cells in *Limonium bicolor* and promoting salt gland development, characterized in that, Using genetic engineering techniques, the LbCKX3 gene, which is related to the degradation of cytokinin in *Limonium bicolor*, was weakened to obtain gene-weakened plants; the weakening included knocking out the LbCKX3 gene or reducing the expression of the LbCKX3 gene; the LbCKX3 gene is as described in claim 1.

9. The method according to claim 8, characterized in that, The genetic engineering method is selected from any one of mutagenesis, site-directed mutagenesis, and homologous recombination.

10. Any one of the following applications of the transgenic plant obtained by the method of claim 8: i. Used in plant breeding; ii. Used for planting in saline-alkali land.

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