Limonium bicolorum gene lbmyb75 and application thereof

Abstract

The present application relates to the field of plant genetic engineering, in particular to Limonium bicolorum gene LbMYB75 and its application. The present application discloses that transcription factor LbMYB75 is specifically highly expressed in the early stage of salt gland development and is significantly induced by salt. The present application proves that gene LbMYB75 positively regulates the development of salt glands and salt secretion of Limonium bicolorum. The density of salt glands in the overexpression strain of gene LbMYB75 of Limonium bicolorum is significantly increased, and the density of salt glands in the mutant strain of gene LbMYB75 is significantly reduced. The transcription factor LbMYB75 positively regulates the salt resistance of Limonium bicolorum, and compared with the wild type, the content of malondialdehyde (MDA), Na + and Na + / K + of the overexpression strain is significantly reduced, and the contents of K + and proline are increased in the high-salt environment; the present application explains the regulatory role of Limonium bicolorum LbMYB75 transcription factor in the development of salt glands, provides gene resources and theoretical basis for improving the salt tolerance of non-halophytic plants through transgenic means, and helps to promote the development and utilization of salinized soil.

Description

Technical Field

[0001] This invention relates to the field of plant genetic engineering, specifically to the LbMYB75 gene of *Limonium bicolor* and its applications. Background Technology

[0002] Soil salinization is a widespread problem globally. Soil salinization refers to the accumulation of salts in soil, and soil salinity is related to the amount of soluble salts and exchangeable sodium ions in the soil. By definition, a saturated solution at 25°C with a conductivity greater than 4 ds·m... -1 Soils with an exchangeable sodium ion ratio of less than 15% are called saline soils. Furthermore, rising sea levels caused by global warming exacerbate soil salinization. Soil salinization damages the natural soil ecosystem, leading to decreased soil fertility, reduced crop yields, and in some areas, even rendering the land uncultivable, resulting in land abandonment and significantly reducing agricultural resource utilization. The salinization of land is expanding year by year, while the cost of remediation is high, putting pressure on agricultural production and local economies. Saline land has become an important reserve of arable land for addressing global climate change and ensuring food security. Promoting the sustainable use of saline land and improving its ecological environment is of great significance for ensuring food and ecological security. Plants face salt stress, and the main harms they suffer can be divided into primary stress and secondary stress. Primary stress includes osmotic stress and ionic stress; secondary stress includes oxidative stress and nutrient stress. Specifically, salt stress initially triggers osmotic stress. This is because excessive salt in the soil increases the osmotic pressure of the soil solution, making it difficult for plant root cells to absorb water or even causing water loss, thus hindering normal plant growth and development. To alleviate the damage caused by salt stress, plants initiate a series of defense responses. To cope with osmotic stress under high salinity, plants synthesize or accumulate osmotic regulating substances to reduce their own osmotic potential and prevent excessive water loss from cells. To cope with ion stress, plants convert sodium... + Cl - and Ca 2+ Plants can alleviate ion homeostasis imbalances in the cell sap by efflux or vacuolar isolation. To combat oxidative damage, plants activate their antioxidant systems, primarily by increasing the activity of antioxidant enzymes and synthesizing antioxidants (such as glutathione) to scavenge reactive oxygen species (ROS). Furthermore, under salt stress, the levels of various hormones in plants change, acting as signaling molecules to regulate the plant's resistance to salt stress. For example, abscisic acid (ABA) levels increase rapidly under salt stress, promoting stomatal closure, reducing transpiration, and improving the plant's water retention capacity.

[0003] Halophytes can effectively absorb water and nutrients in soil with a NaCl concentration of not less than 200 mM, and grow and develop normally to complete their life cycle. Based on the different ion transport methods of halophytes, Breckle divides them into three categories: (1) Euhalophytes (such as Suaeda salsa), which transport NaCl to the soil through salt ion compartmentalization. + Transported and isolated into intracellular vacuoles or succulent leaves, while simultaneously absorbing large amounts of water and reducing Na+ + Concentration; (2) Salt-resistant halophytes (such as reeds), which will absorb Na + The parenchyma tissue of the xylem, which gathers at the junction of the root and stem, has a well-developed apoplastic barrier that prevents Na+ from entering the cell. + (3) Salt-secreting halophytes (such as Limonium bicolor and Limonium spp.) use salt glands or salt vesicles to expel excess salt ions from their bodies.

[0004] Salt glands are specialized functional epidermal structures evolved by halophytes during long-term adaptation to salt stress. They excrete excess salt absorbed by the plant, maintaining normal growth and resisting the adverse effects of salt stress. The salt gland of *Limonium bicolor* consists of 16 cells: secretory cells (SC) are located in the center, surrounded by adjacent cells (AC), which in turn are surrounded by inner cup cells (IC) and outer cup cells (OC). Yuan et al., by continuously sampling the first true leaf of *Limonium bicolor* and tracing its cell differentiation process, divided the developmental stages of the true leaf in detail and proposed for the first time that the salt gland develops earlier than the stomata. Based on the differentiation of the leaves of *Limonium bicolor*, its development process can be divided into five stages: the first stage is the undifferentiated stage (Stage A); the second stage is the salt gland differentiation stage (Stage B), which eventually forms a mature salt gland with 16 cells; the third stage is the stomatal differentiation stage (Stage C), where stomata begin to develop; the fourth stage is the epidermal cell differentiation stage (Stage D), where, after the stomata mature, the remaining stem cells develop into epidermal cells; and the fifth stage is the mature stage (Stage E), where the leaves enter the mature stage.

[0005] The molecular mechanisms of salt gland development in *Limonium bicolor* have been extensively studied and reported. Multiple molecules, including transcription factors, functional proteins, and small RNAs, are involved in salt gland development and salt secretion regulation, and a preliminary regulatory network for salt gland development has been revealed. Various transcription factors play a role in salt gland development in *Limonium bicolor*. However, current research on salt gland development in *Limonium bicolor* still has significant technical limitations. Therefore, in-depth analysis of the molecular regulatory mechanisms of salt gland development in *Limonium bicolor* is a core prerequisite for overcoming existing technical shortcomings and developing new technologies for the management and utilization of saline-alkali land, possessing significant theoretical research and practical application value. Summary of the Invention

[0006] The purpose of this invention is to provide the LbMYB75 gene of *Limonium bicolor* and its applications.

[0007] The R2R3 MYB family of genes is widely involved in plant growth, development, and stress responses, but their function and mechanism in the development of salt glands in *Limonium bicolor* have not been systematically elucidated. This invention obtained candidate R2R3 MYB sequences for *Limonium bicolor* from the *Limonium bicolor* genome annotation file using a Hidden Markov Model (HMM) based on R2R3 MYB, via local BLASTP, and validated the sequences using SMART. Subsequently, a systematic analysis of the *Limonium bicolor* R2R3 MYB family of genes revealed that only one R2R3 MYB gene was highly expressed in the early stages of salt gland development, induced by salt, and enriched in the epidermal development pathway by Gene Ontology analysis; this gene was named LbMYB75. In situ hybridization revealed that this gene is mainly located in the salt glands. Transgenic functional validation showed that the transcription factor LbMYB75 positively regulates salt gland development and salt tolerance. Heterologous overexpression of the LbMYB75 gene in the non-halophytic tomato revealed increased epidermal hair density and enhanced salt tolerance, providing a valuable gene for transforming crops and other non-halophytic plants, as well as for improving saline-alkali soils.

[0008] To achieve the above objectives, the present invention provides the following technical solution: This invention provides the LbMYB75 gene of *Limonium bicolor*, which is a gene encoding either protein (a) or (b): (a) The amino acid sequence shown in SEQ ID NO:1; or (b) Proteins derived from (a) with the sequence shown in SEQ ID NO: 1 substituted, deleted or added with one or more amino acids and having the same function.

[0009] The present invention cloned the gene LbMYB75 from Limonium bicolor. The coding region of the LbMYB75 gene, including the stop codon, is 1017 bp (SEQ ID NO:2) and encodes 338 amino acids (SEQ ID NO:1).

[0010] Secondly, the present invention provides biological materials containing the gene LbMYB75 or the promoter, wherein the biological materials include, but are not limited to, recombinant DNA, expression cassettes, transposons, plasmid vectors, viral vectors, engineered bacteria or non-renewable plant parts.

[0011] Thirdly, the present invention provides the application of the gene LbMYB75, or biological materials containing the gene LbMYB75, in the preparation of transgenic plants.

[0012] Fourthly, the present invention provides any of the following applications of the gene LbMYB75 or biological materials containing the gene LbMYB75: (1) Used to improve the salt tolerance of plants; (2) Used for plant variety improvement; (3) Used for the improvement of saline-alkali land.

[0013] In this invention, the plants include halophytes (such as *Limonium bicolor*) or Arabidopsis thaliana.

[0014] Fifthly, the present invention provides a method for improving the salt tolerance of plants, said method being selected from the following ① or ②: ① To enable plants to express the protein encoded by the gene LbMYB75; ② Overexpress the gene LbMYB75 in plants.

[0015] The overexpression mode is selected from the following 1) to 5), or any combination thereof: 1) By importing a plasmid containing the gene; 2) By increasing the copy number of the aforementioned genes on plant chromosomes; 3) By altering the promoter sequence of the aforementioned genes on plant chromosomes; 4) By operatively linking a strong promoter to the gene; 5) By introducing enhancers.

[0016] Sixthly, the present invention provides any of the following applications of the transgenic plants obtained according to the above method: i. Used in plant breeding; ii. Used for planting in saline-alkali land.

[0017] Besides *Limonium bicolor* or *Arabidopsis thaliana*, other plants that can include: (1) Cereal crops (wheat, rice, corn, sorghum, millet, foxtail millet, buckwheat); (2) Legumes (soybeans, broad beans, peas, mung beans, red beans, cowpeas, lentils, etc.); (3) Root and tuber crops (sweet potato, potato, cassava, yam, taro, etc.); (4) Fiber crops (cotton, ramie, jute, flax, sisal, etc.); (5) Oil crops (rapeseed, peanut, sunflower, sesame, camellia, oil palm, olive, etc.); (6) Medicinal crops (ginseng, wolfberry, honeysuckle, Panax notoginseng, mint, angelica); (7) Forage crops (alfalfa, ryegrass, clover, Sudan grass, etc.); (8) Green manure crops (purple clover, vetch, sesbania, sweet clover, etc.).

[0018] Furthermore, the breeding methods include, but are not limited to, transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

[0019] The beneficial effects of this invention are as follows: This invention screened a MYB transcription factor, LbMYB75, which is specifically highly expressed in the early stages of salt gland development and significantly induced by salt. This gene is highly expressed in the early stages of salt gland development, can be induced by salt, and its specific localization in salt glands was discovered through in situ hybridization. Transgenic functional verification showed that LbMYB75 positively regulates salt gland development and salt tolerance. Overexpression of the LbMYB75 gene in tomatoes enhances their salt tolerance. This invention lays the foundation for further exploration and elucidation of the developmental mechanism of salt glands, and also provides valuable genes for transforming crops and other non-halophytic plants and improving saline soils.

[0020] The above description is merely an overview of the technical solution of the present invention. In order to better understand the technical means of the present invention and to implement it in accordance with the contents of the specification, the preferred embodiments of the present invention are described in detail below with reference to the accompanying drawings. Attached Figure Description

[0021] Figure 1 To observe salt glands in leaves of wild-type (WT), LbMYB75 overexpression lines (LbMYB75 OE-1 and LbMYB75 OE-2), and knockout lines (lbmyb75-1 and lbmyb75-2) under ultraviolet light (358 nm) (scale bar = 125 μm). Figure 2 A diagram illustrating the positive regulation of salt gland development in *Limonium dichromatum* by the LbMYB75 gene; among which, Figure 2 In Figure A, the salt gland density in the leaves of wild-type, LbMYB75 gene overexpression lines, and knockout lines was quantitatively analyzed. Figure 2 Image B shows the secretory vacuoles on leaf discs of wild-type, LbMYB75 gene overexpression lines, and knockout lines (scale bar = 2.5 mm). Figure 2 C represents the volume of secretory vacuoles collected from wild-type, LbMYB75 gene overexpression lines and knockout lines for quantitative analysis; Figure 2D represents the calculation of sodium ions (Na) in a single salt gland. + Secretion rate; data are expressed as mean ± standard deviation (n=3), and statistical significance was tested using one-way ANOVA. Different letters indicate significant differences between groups. P <0.05); Figure 3 Phenotypes of wild-type, LbMYB75 gene overexpression lines and knockout lines (scale bar = 2cm). Figure 4 Phenotypic, total leaf area, 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining, and physiological and biochemical index detection images of wild-type, LbMYB75 gene overexpressing, and LbMYB75 gene knockout *Limonium bicolor* plants; wild-type, LbMYB75 gene overexpressing, and knockout lines were treated with 200 mM NaCl for two weeks (salt treatment), with plants under normal growth conditions serving as controls; among them... Figure 4 A is wild type. Total leaf area of ​​LbMYB75 gene overexpressing and knockout lines; Figure 4 Image B shows representative images of leaves collected from the same location on each plant that underwent DAB and NBT staining. Figure 4 C in the middle represents DAB staining. Figure 4 The optical density values ​​of NBT staining in D were quantitatively analyzed using ImageJ software. Figure 5 To determine the content of various components in wild-type, LbMYB75 gene overexpression lines and knockout lines; among them, Figure 5 A in the middle stands for malondialdehyde (MDA). Figure 5 In this context, B stands for hydrogen peroxide (H2O2). Figure 5 C represents proline, Figure 5 D represents sodium ions (Na+) + Data are expressed as mean ± standard deviation (n=3). A two-way ANOVA was used to test statistical significance. Different letters indicate significant differences between groups. P <0.05); Figure 6 To determine the K content in wild-type, LbMYB75 overexpressing, and knockout lines + Content and calculation of sodium ion / potassium ion (Na) + / K + ) ratio; where Figure 6 A represents potassium ions (K). + The content of ) Figure 6 B in the text is for calculating the sodium / potassium ion ratio (Na). + / K +The ratios were expressed as mean ± standard deviation (n=3). A two-way ANOVA was used to test statistical significance. Different letters indicate significant differences between groups. P <0.05); Figure 7 Trichomorphs of wild-type tomato and LbMYB75 gene overexpressing lines OE2 and OE3 (scale bar = 100 μm). Figure 8 Statistics on trichomes of wild-type tomato lines overexpressing the LbMYB75 gene; among them, Figure 8 In section A, the number of non-secretory trichomes is statistically analyzed. Figure 8 B represents the statistical count of secretory trichomes. Figure 8 C represents the statistical count of type IV trichomes. Figure 8 D represents the statistical count of type VI trichomes; Figure 9 Wild-type and LbMYB75 gene overexpressing tomato lines were treated with 120 mM NaCl for 12 days (salt treatment), with plants under normal growth conditions serving as controls. Images were taken (scale bar = 2 cm). Figure 9 A represents the control group. Figure 9 Group B represents the salt treatment group; Figure 10 For the determination of malondialdehyde (MDA) and proline content; among which Figure 10 In section A, malondialdehyde (MDA) content is determined. Figure 10 B represents the determination of proline content; data are expressed as mean ± standard deviation (n=3), and statistical significance was tested using one-way ANOVA. Different letters indicate significant differences between groups. P <0.05); Figure 11 The results are from multiple sequence alignment. The amino acid sequence similarity between the transcription factor LbMYB75 (Lb2G11025) of *Limonium bicolor* and the transcription factors AaMYB5 and CsMYB6 (known genes, derived from NCBI) is 45.03% and 38.42%, respectively. Detailed Implementation

[0022] This invention identified key genes involved in the development of salt glands in *Limonium bicolor*, revealing a MYB transcription factor, LbMYB75. LbMYB75 belongs to the 9th subgroup of R2R3 MYB transcription factors, which plays a crucial role in plant epidermal cell development. In non-halophytic plants like tomato and *Artemisia annua*, the 9th subgroup of transcription factors AaMYB17 and SlMX1 regulate the development of multicellular secretory trichomes. Transcriptome analysis of *Limonium bicolor* showed that the LbMYB75 gene is highly expressed in the early stages of salt gland development, and salt treatment significantly reduces its expression. Increased expression levels of the LbMYB75 gene suggest that LbMYB75 may play an important role in regulating the development of multicellular salt glands and salt tolerance in Limonium bicolor.

[0023] The screening process was as follows: The R2R3 MYB family genes of Limonium bicolor were systematically analyzed, and it was found that only one R2R3 MYB gene was highly expressed in the early stage of salt gland development and was induced by salt. Gene Ontology analysis showed that it was enriched in the epidermal development pathway, and it was named LbMYB75.

[0024] The present invention cloned the LbMYB75 gene from Limonium bicolor. The coding region of the LbMYB75 gene, including the stop codon, is 1017 bp (SEQ ID NO: 2) and encodes 338 amino acids (SEQ ID NO: 1).

[0025] The LbMYB75 gene of *Limonium bicolor* encodes either (a) or (b) the following protein: (a) The amino acid sequence shown in SEQ ID NO:1; or (b) A protein derived from (a) with the sequence shown in SEQ ID NO:1 substituted, deleted or added with one or more amino acids and having the same function.

[0026] The LbMYB75 gene coding region, including the stop codon, is a total of 1017 bp. The sequence is shown below in SEQ ID NO: 2. The amino acid sequence of the LbMYB75 transcription factor from *Limonium bicolor* is shown in SEQ ID NO:1. MGRSPCCEKKGLKRGPWTPEEDETLINYISKHGHGSWRSLPQLAGLRRCGKSCRLRWTNYLRPDIKRGPFTLEEEKLVIQLHAILGNRWAAIASQLPGRTDNEIKNLWNTHLKKRLLCMGIDPQTHEPAASSNPRLKPPASPSTRHMAQWESARLEAEARLSRESLLHM NSNHQCGHFSNNSSSSSGDQEGPDYFLKIWNSEVGESFRNFNNKTAEEEKDHHPTSCQMISPEASPSASSCTAKYSGAMADVKPPLFPSCSTGGNQTEDDAIMEECKVEEEHGNDHEGLNLNLGSDSSCTDELEDSSESALQLLLDFPGNNDMSFLEEDYNGYSFLSTR Related studies, through tracking the first true leaf of *Limonium bicolor*, divided the developmental stages of the true leaf and salt glands into: undifferentiated epidermal structure stage (Stage A), salt gland differentiation stage (Stage B), stomatal differentiation stage (Stage C), epidermal cell differentiation stage (Stage D), and maturity stage (Stage E). It was found that the salt gland is the earliest structure to develop on the leaf; therefore, investigating genes highly expressed in Stages A and B helps to identify key genes in the salt gland development process. This invention analyzes a large amount of transcriptome data from *Limonium bicolor* and identified the gene LbMYB75, which is highly expressed in the early stage of salt gland development (Stage A) and whose expression level increases after salt treatment. 。

[0027] This invention successfully screened and identified the key gene LbMYB75, which is specifically expressed in the early stage of salt gland development and positively regulates salt gland development and salt tolerance, from *Limonium bicolor* through a strategy combining whole-genome family analysis, in situ hybridization tissue localization, and transgenic function verification. This provides an important target gene and theoretical basis for elucidating the molecular regulatory mechanism of salt gland development.

[0028] 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.

[0029] The primers used in this invention are shown in the table below.

[0030] Table 1 Primers used in this invention .

[0031] Example 1: Cloning and Bioinformatics Analysis of Gene LbMYB75 1. Materials and Methods 1.1 Plant materials and growing conditions 1.1.1 Two-colored Blood-Nourishing Herb Wild *Limonium bicolor* seeds were collected from the saline-alkali land of the Yellow River Delta. Several dried, impurity-removed *Limonium bicolor* seeds were placed in 10mL centrifuge tubes containing 70% ethanol solution and shaken for 6 minutes for preliminary sterilization. The ethanol solution was discarded, and 6% sodium hypochlorite solution was added to the centrifuge tubes. The mixture was shaken for 10 minutes and repeated once for deep sterilization. In a clean bench, the sodium hypochlorite solution was discarded, and the seeds were rinsed 4-6 times with sterile water. After the solution became colorless and clear, the seeds were blotted dry with filter paper and transferred to MS agar plates. The plates were sealed and transferred to a sterile tissue culture room for cultivation. Nutrient soil culture of *Limonium bicolor*: For cultivation requiring observation of the *Limonium bicolor* phenotype, the dried seeds were evenly scattered in nutrient soil, sprayed with water, sealed with a film, and placed in a constant temperature climate chamber for cultivation. After about one week of germination, the seedlings were transplanted into appropriately sized flowerpots.

[0032] 1.1.2 Tomato Micro-Tomato 1.2 Genetic transformation of *Limonium bicolor* An LbMYB75 overexpression vector, a pCAMBIA1300-LbMYB75 gene expression vector, and an LbMYB75 gene knockout vector were constructed and transformed with Agrobacterium tumefaciens to carry out tissue infection and culture of Limonium bicolor.

[0033] 1.3 Observation and statistics of salt gland density Cut square sections from newly grown leaves of *Limonium bicolor* and observe the underside of the leaves. Observe the size and shape of the salt glands; normal salt glands show four luminescent spots under ultraviolet light. Calculate the salt gland density by counting the number of salt glands in multiple unit fields of view, converting it to the number of salt glands per square millimeter.

[0034] 1.4 Observation of secretory vesicles and statistics of secretion rate Perforations were made in the leaves of *Limonium bicolor* using a 1cm diameter punch to obtain leaf discs of different strains. Mineral oil was applied to the back of each leaf disc to coat it completely. Secretory vacuoles were observed and photographed after 24 hours. Depending on the salt gland density and secretion rate, the secretory vacuoles would exhibit different sizes and shapes.

[0035] Collect the secretory vesicles and mineral oil from the back of the leaf disc using a pipette. After centrifugation, remove the mineral oil and measure the volume of the secreted fluid using a pipette. Measure the Na+ content of the secreted fluid using a flame spectrophotometer.+ Concentration. Based on the volume of secreted fluid and Na+ + Concentration calculation of Na in secretions + Total amount. Calculate the Na content per salt gland based on the number of salt glands on the leaf disc. + Secretion rate.

[0036] 1.5 Measurement of physiological indicators 1.5.1 Determination of malondialdehyde (MDA) content Different strains of Limonium bicolor were treated with 200 mM NaCl for one week and were untreated. The MDA content in the tissues was calculated based on the OD value, supernatant volume, and tissue weight.

[0037] 1.5.2 Determination of H2O2 content Multiple strains of *Limonium bicolor*, treated with NaCl for one week and untreated, were sampled at the same location on the leaves, with 0.1g samples taken from each strain. Hydrogen peroxide content was determined using a hydrogen peroxide content assay kit.

[0038] 1.5.3 Determination of proline content Different Limonium bicolor strains were treated with 200mM NaCl for one week and untreated, and their OD values ​​were measured. The proline content was obtained according to the standard curve.

[0039] 1.5.4 Na + and K + Determination of content Different strains treated with 200 mM NaCl and those without treatment were sampled, boiled in water, and filtered. The resulting solutions were then analyzed by flame spectrophotometry for NaCl content. + and K + Content determination.

[0040] 1.6 Data Statistics and Analysis Each group was repeated in triplicate. The standard deviation (SD) was calculated, and the final data were expressed as mean ± standard deviation. Significance tests were performed using one-way or two-way ANOVA. The significance of differences between groups was assessed. P <0.05) are marked with different letters, while the same letter indicates no difference.

[0041] 2. Results 2.1 Functional Study of LbMYB75 Gene in Salt Gland Development and Salt Tolerance in Limonium bicolor 2.1.1 The LbMYB75 gene promotes the development of salt glands. LbMYB75 transgenic lines were obtained through Agrobacterium-mediated transformation, and LbMYB75 gene overexpression lines (LbMYB75 OE-1 and LbMYB75 OE-2) and two LbMYB75 gene knockout lines (lbmyb75-1 and lbmyb75-2) were constructed. Salt gland differences were observed in LbMYB75 gene overexpression, knockout, and wild-type *Limonium bicolor* plants of uniform growth size. The results showed that compared to the wild-type, the salt gland density of the overexpression lines increased by approximately 1.5 times, while the salt gland density of the knockout lines decreased by approximately 50%. Figure 1 and Figure 2 (A). However, no significant changes were observed in salt gland cell composition among wild-type, LbMYB75 overexpressing lines, and knockout lines. Figure 1 A leaf disc salt secretion experiment was conducted. The results showed that, compared to the wild type, the volume of secretory vacuoles produced by the salt glands in the LbMYB75 OE-1 and LbMYB75 OE-2 lines was significantly increased, while that in the lbmyb75-1 and lbmyb75-2 lines was significantly decreased. Figure 2 China B and Figure 2 (C). However, sodium ions (Na) in individual salt glands differed between wild-type, LbMYB75 overexpression lines, and knockout lines. + There was no significant difference in secretion rate. Figure 2 (D). These results indicate that the changes in leaf salt secretion in the LbMYB75 transgenic line are mainly due to changes in salt gland density, rather than the cell structure of the salt glands, the development of adjacent epidermal cells, or the secretion rate of individual salt glands.

[0042] 2.1.2 Salt tolerance of Limonium bicolor enhanced by LbMYB75 gene Wild-type, LbMYB75 overexpressing, and knockout lines were treated with 200 mM NaCl (salt treatment) for two weeks. Under normal growth conditions (control), no significant differences in phenotype and total leaf area were observed among the lines. Figure 3 and Figure 4 (A). However, under salt stress, compared with the wild type, the LbMYB75 gene overexpression lines showed better growth and larger total leaf area, while the LbMYB75 gene knockout lines showed poorer growth and smaller total leaf area. Figure 3 and Figure 4(Figure 4A). The degree of NaCl-induced oxidative stress was assessed using DAB and NBT staining: Under control conditions, the staining intensity was similar in all plants; after salt treatment, the DAB and NBT staining of the LbMYB75 gene overexpression lines was lighter than that of the wild type, while the staining of the LbMYB75 gene knockout lines was significantly darker (Figure 4B and Figure 4D). Related physiological index measurements showed that under control conditions, the levels of malondialdehyde (MDA), hydrogen peroxide (H2O2), proline, and sodium ions (Na+) in all plants were significantly higher. + ), potassium ions (K) + ) content and Na + / K + The ratios showed no significant difference. Figure 5-6 Under salt treatment, compared with the wild type, the LbMYB75 gene overexpression lines had lower MDA and H2O2 levels, higher proline content, and accumulated Na+. + Less, K + More, Na + / K + The ratio was lower, while the LbMYB75 gene knockout lines showed the opposite trend. Figure 5-6 These results indicate that the LbMYB75 gene positively regulates the salt tolerance of *Limonium bicolor*.

[0043] 2.2 Salt tolerance of tomatoes enhanced by the LbMYB75 gene 2.2.1 Overexpression of the LbMYB75 gene in tomato alters tomato trichome development. To overexpress the LbMYB75 gene in tomato plants, the LbMYB75 gene overexpression vector was transformed into Agrobacterium tumefaciens EHA105. This was used to genetically transform the Micro-Tom tomato variety, and two lines (LbMYB75 OE 2 and LbMYB75 OE 3) with relatively high LbMYB75 gene expression levels were selected for further analysis. During plant growth, overexpression of the LbMYB75 gene altered trichome development in tomatoes: compared to the wild type (WT), the number of trichomes in the LbMYB75 gene-expressing lines was significantly increased, with the most significant increase in type IV and VI glandular trichomes. Figure 7-8 ).

[0044] Related to the sodium bile acid family gene of tomatoes SlBASS4 Compared to the bileacid sodium symporter SlBASS4, which enhances tomato salt tolerance, overexpression of the LbMYB75 gene in tomatoes significantly improved tomato growth and salt tolerance under salt stress. Compared to their respective controls, tomatoes overexpressing the LbMYB75 gene were significantly taller. SlBASS4 Overexpression of the LbMYB75 gene in tomatoes had no significant effect. This indicates that overexpression of the LbMYB75 gene in tomatoes results in stronger salt tolerance.

[0045] 2.2.2 Overexpression of the LbMYB75 gene in tomatoes enhances their salt tolerance. Under normal growth conditions, there were no significant differences between wild-type and transgenic lines in terms of growth status, malondialdehyde (MDA) content, and proline content. Figure 9-10 However, after salt treatment, the genetically modified tomatoes grew better than the wild type, and had lower MDA content and higher proline content. Figure 9-10 These results indicate that overexpression of the LbMYB75 gene can enhance the salt tolerance of tomatoes.

[0046] Compared with other homologous genes, protein sequence alignment results showed that Lb2G11025 shared 45.03% and 38.42% sequence identity with AaMYB5 and CsMYB6 (known genes, derived from NCBI), respectively. Figure 11 ).

Claims

1. The LbMYB75 gene of *Limonium bicolor* is characterized by, The gene sequence containing the stop codon of the *Limonium bicolor* gene LbMYB75 is shown in SEQ ID NO: 2, and the amino acid sequence encoded by the *Limonium bicolor* gene LbMYB75 is shown in SEQ ID NO:

1.

2. A biomaterial containing the LbMYB75 gene of *Limonium bicolor* as described in claim 1, characterized in that, The biological material is recombinant DNA, expression cassette, transposon, plasmid vector, viral vector, or engineered bacteria.

3. The application of the LbMYB75 gene of *Limonium bicolor* as described in claim 1 or the biomaterial as described in claim 2 in the preparation of the salt-tolerant transgenic plant *Limonium bicolor*.

4. The application of transgenic plants overexpressing the LbMYB75 gene of *Limonium bicolor* as described in claim 1, characterized in that: i. Used for breeding salt-tolerant plants; ii. Used for planting in saline-alkali land; The plant in question is *Limonium bicolor*.

5. The application of the transgenic plant overexpressing the *Limonium dichromatum* gene LbMYB75 according to claim 4, characterized in that, Breeding methods include transgenic, hybridization, backcrossing, self-pollination, or asexual reproduction.

Images