Tomato salt stress gene SlGLUB, primer and application of tomato salt stress gene SlGLUB

Abstract

The invention relates to the field of gene technology application, in particular to a tomato salt stress gene SlGLUB, a primer and application of the tomato salt stress gene SlGLUB. In the invention, the SlGLUB gene can promote the root development of the tomato under normal conditions, and after salt stress treatment, the SlGLUB gene can improve the salt tolerance of the tomato by adjusting the root development of the tomato plant and increasing the total root length and the root surface area, and from the result of paraffin sections of roots, stems and leaves, the salt tolerance of the tomato can be improved on the cellular level. The SlGLUB gene can reduce the damage of salt stress to tomato plants, the SlGLUB gene can improve the capability of removing active oxygen by improving the activity of antioxidant enzyme, and the membrane lipid peroxidation degree can be reduced, so that the salt resistance of the tomato plants is improved.

Description

Technical Field

[0001] This invention relates to the field of gene technology applications, and in particular to a tomato salt stress gene SlGLUB, its primers, and their applications. Background Technology

[0002] Tomato is one of the main vegetable crops grown in greenhouses. It is one of the crops with the widest planting area, the highest yield, and the highest consumption in China. Moreover, tomatoes are an important model plant in plant research. Salt stress is one of the main factors affecting tomato cultivation, which seriously affects the yield and quality of tomatoes. Therefore, isolating and identifying salt-tolerant genes is of great significance for breeding new salt-tolerant varieties, increasing the yield of tomatoes in saline soils, and promoting sustainable development.

[0003] Currently, in the field of tomato production technology, research on salt tolerance focuses on β-1,3-glucanase, a class of enzymes that can specifically hydrolyze the β-1,3-glycosidic bonds in β-1,3-glucan. The products generated are a series of oligosaccharides or monosaccharides of different sizes. β-1,3-glucanase is widely found in bacteria, fungi, plants, and insects. Due to differences in origin and sequence evolution, its structure and catalytic function also exhibit diversity (see Wei Xiasen et al., 2023). The discovered β-1,3-glucanases mainly belong to the 12 lycoside hydrolase (GH) families, including GH16, GH17, GH55, GH64, GH81, GH128, and GH132. Based on their catalytic mechanism, β-1,3-glucanases can be divided into exo- and endo-types. Exo- and endo-types cleave the β-1,3-glycosidic bonds in the glucan chain sequentially, starting from the ends of the sugar residues at both ends and the inner side, respectively. The hydrolysis products are generally glucose or mono-oligosaccharides, playing a crucial role in the decomposition and reconstruction of β-1,3-glucan (see Wei et al., 2023; Wang, 2018). The function of the gene SlGLUB in salt tolerance has not yet been reported. Given the association of salt stress with tomato growth and development, this study proposes research on the regulation of tomato salt tolerance by the gene SlGLUB. Summary of the Invention

[0004] To overcome the above shortcomings, the purpose of this invention is to provide a tomato salt stress gene SlGLUB, primers, and their applications. The SlGLUB gene can reduce the damage caused by salt stress to tomato plants. The SlGLUB gene can improve the ability to scavenge reactive oxygen species by increasing the activity of antioxidant enzymes and reduce the degree of membrane lipid peroxidation, thereby improving the salt resistance of tomato plants.

[0005] The technical solution of this invention to solve its technical problem is: A tomato salt stress gene SlGLUB, wherein the nucleotide sequence of the gene SlGLUB is SEQ ID NO:1.

[0006] As an improvement of the present invention, SlGLUB-GFP-F and SlGLUB-GFP-R are included; The nucleotide sequence of SlGLUB-GFP-F is SEQ ID NO:2; The nucleotide sequence of SlGLUB-GFP-R is SEQ ID NO:3.

[0007] The silencing vector for the tomato salt stress gene SlGLUB is pTRV2-SlGLUB.

[0008] Primers for silencing the tomato salt stress gene SlGLUB include TRVSlGLUB-F and TRVSlGLUB-R. The nucleotide sequence of TRVSlGLUB-F is SEQ ID NO:4; The nucleotide sequence of TRVSlGLUB-R is SEQ ID NO:5.

[0009] Application of the tomato salt stress gene SlGLUB in improving root development of tomato plants.

[0010] Application of the tomato salt stress gene SlGLUB in improving the salt tolerance of tomatoes.

[0011] In this invention, the SlGLUB gene not only promotes root development in tomatoes under normal conditions, but also improves salt tolerance in tomatoes by regulating root development, increasing total root length and root surface area after salt stress treatment. Paraffin section results of roots, stems and leaves show that at the cellular level, the SlGLUB gene can reduce the damage caused by salt stress to tomato plants. The SlGLUB gene can also improve the ability to scavenge reactive oxygen species by increasing the activity of antioxidant enzymes and reducing the degree of membrane lipid peroxidation, thereby improving the salt tolerance of tomato plants. Attached Figure Description

[0012] For ease of explanation, the present invention will be described in detail below with reference to the preferred embodiments and accompanying drawings.

[0013] Figure 1 This is a schematic diagram of the construction of the tomato salt stress gene SlGLUB overexpression vector of the present invention; Figure 2 This is a diagram of the primer sequences for amplifying the tomato salt stress gene SlGLUB in this invention; Figure 3 This is a schematic diagram of the construction of the tomato salt stress gene SlGLUB silencing vector of the present invention; Figure 4 This is a diagram of the primers used to amplify the SlGLUB silencing vector for the tomato salt stress gene in this invention. Figure 5 This is an electrophoresis diagram of the silencing fragment of the tomato salt stress gene SlGLUB of the present invention, wherein M is a DNA molecular weight of DL2000; 1-3 are templates of salt-tolerant IL8-3 cDNA; and 4-6 are templates of salt-sensitive M82 cDNA. Figure 6 This is a colony PCR detection diagram of pTRV2-SlGLUB of the present invention, wherein M represents DNA molecular weight DL2000; 1-4 use salt-tolerant IL8-3 bacterial suspension as template; and 5-8 use salt-sensitive M82 bacterial suspension as template. Figure 7 This is a diagram of the pTRV2-SlGLUB sequencing results of the present invention; Figure 8 The image shows the electrophoretic detection diagram of the PCR amplification of the SlGLUB fragment of the present invention, wherein M is the DNA molecular weight DL2000; 1-3 are templates using salt-tolerant IL8-3 cDNA; and 4-6 are templates using salt-sensitive M82 cDNA. Figure 9 This is a diagram of the SlGLUB-GFP sequencing results of the present invention; Figure 10 This is a phenotypic diagram of tomato plants transiently silenced and overexpressed with SlGLUB under salt stress according to the present invention. In the diagram, A represents tomato plants transiently silenced with SlGLUB, and B represents tomato plants transiently overexpressing with SlGLUB. Scale bar: 1 cm. Figure 11 This diagram illustrates the plant height and root length of tomato plants transiently silenced and overexpressed with SlGLUB under salt stress according to the present invention. In this diagram, A and B represent tomato plants transiently silenced with SlGLUB, while C and D represent tomato plants transiently overexpressing with SlGLUB. Figure 12 This is a schematic diagram of the microstructure of tomato plants with transient silencing and overexpression of SlGLUB under salt stress according to the present invention, wherein A and B are the microstructures of leaves; C and D are the microstructures of stems; and E and F are the microstructures of roots. Figure 13 This is a root scan of tomato plants with transient silencing and overexpression of SlGLUB under salt stress according to the present invention (scale bar: 1 cm). Figure 14 This is a schematic diagram of the root surface area and total root length of tomato plants transiently silenced and overexpressed with SlGLUB under salt stress according to the present invention. In this diagram, A and B represent tomato plants transiently silenced with SlGLUB, and C and D represent tomato plants transiently overexpressing with SlGLUB. Figure 15This diagram illustrates the O2- production rate of tomato plants transiently silenced and overexpressed with SlGLUB under salt stress according to the present invention; wherein, A and B are tomato plants transiently silenced with SlGLUB; and C and D are tomato plants transiently overexpressing with SlGLUB. Figure 16 This diagram illustrates the H2O2 content in tomato plants transiently silenced and overexpressed with SlGLUB under salt stress according to the present invention; wherein, A and B represent tomato plants transiently silenced with SlGLUB; and C and D represent tomato plants transiently overexpressing with SlGLUB. Figure 17 This diagram illustrates the SOD activity of tomato plants transiently silenced and overexpressed with SlGLUB under salt stress according to the present invention; wherein, A and B represent tomato plants transiently silenced with SlGLUB; and C and D represent tomato plants transiently overexpressing with SlGLUB. Figure 18 This is a schematic diagram of CAT activity in tomato plants transiently silenced and overexpressed with SlGLUB under salt stress according to the present invention, wherein A and B represent tomato plants transiently silenced with SlGLUB; and C and D represent tomato plants transiently overexpressing with SlGLUB. Figure 19 This diagram illustrates the MDA content in tomato plants transiently silenced and overexpressed with SlGLUB under salt stress according to the present invention. A and B represent tomato plants transiently silenced with SlGLUB, while C and D represent tomato plants transiently overexpressing with SlGLUB. Detailed Implementation

[0015] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0016] This invention aims to explore the possible mechanisms of salt stress tolerance in tomatoes. Two tomato varieties with different salt tolerance were cultured in a light-controlled chamber (light intensity 300-320 μmol m⁻² s⁻¹, 24°C day / 22°C night, 16-hour photocycle) with a nutrient solution containing (2 mmol•L⁻¹ Ca(NO₃)₂, 1.25 mmol•L⁻¹ KCl, 1 mmol•L⁻¹ MgSO₄, 0.5 mmol•L⁻¹ NH₄H₂PO₄, iron salts (0.1 mmol•L⁻¹ FeSO₄ and 0.1 mmol•L⁻¹ EDTA-Na₂), and trace elements (115 µmol•L⁻¹ H₃BO₃, 22.5 µmol•L⁻¹ H₃BO₃, 0.16 µmol•L⁻¹ CuSO₄, 0.75 µmol•L⁻¹ ZnSO₄, 0.182 µmol•L⁻¹ MgSO₄, 0.182 µmol•L⁻¹ NH₄H₂PO₄, 0.15 µmol•L⁻¹ MgSO₄, 0.182 ... (NH4)6Mo7O24) was cultured for 30 days, and then the seedlings were transferred to a nutrient solution containing salt stress (200 mM NaCl) for 3 days. The root cell walls of the two genotype tomato seedlings were isolated and extracted, and proteins were isolated from the purified cell walls for proteomics analysis.

[0017] Within this invention, it was found that 82 and 81 cell wall proteins, respectively, in salt-tolerant tomato IL8-3 and salt-sensitive tomato M82 showed significant changes. Specifically, under salt stress, proteins related to signal transduction and changes in cell wall polysaccharides in the cell walls of IL8-3 and M82 increased. Among the differentially expressed proteins involved in cell wall metabolism, a glucan endo-1,3-beta-glucosidase B precursor (GLUB), belonging to the GH17 protein family, was found. It was upregulated 3.13-fold in salt-sensitive tomato (M82) and upregulated 8.05-fold in salt-tolerant tomato (IL8-3).

[0018] Based on the analysis of the above proteomics data, it is speculated that this GH protein plays an important role in the tomato's response to salt stress, and this gene will be used as the target gene for subsequent experimental research.

[0019] like Figures 1 to 19 As shown, this invention constructs transiently overexpressed and silenced SlGLUB gene plants to explore the effect of SlGLUB protein on the growth and development mechanism of tomato seedlings under salt stress, and further studies the role of SlGLUB protein in responding to salt stress. This has important practical significance for revealing the salt tolerance mechanism of tomatoes, improving the ecological environment for production, and increasing crop yield.

[0020] Specifically, to further explore the salt tolerance mechanism of the SlGlUB gene, using wild-type tomato AC (Alisa Craig) as a background, transiently silenced and transiently overexpressed SlGLUB plants were constructed respectively. The role of the SlGlUB gene in the response of tomato plants to salt stress was further explored at the physiological, cellular and molecular levels, providing a theoretical basis for molecular breeding and screening of salt-tolerant tomato varieties.

[0021] The transient overexpression vector pRI101-GFP and the virus-induced silencing expression vectors pTRV1 and pTRV2; Escherichia coli competent cells DH5α and Agrobacterium tumefaciens competent cells EHA105 were purchased from Shanghai Angyu Biotechnology Co., Ltd.

[0022] The nucleotide sequence of the tomato salt stress gene SlGLUB of the present invention is SEQ ID NO:1.

[0023] I. Construction of the transient overexpression vector for the SlGLUB gene: (1) Schematic diagram of the construction of the SlGLUB gene overexpression vector, as shown in the figure. Figure 1 As shown, the primer sequences are as follows: Figure 2 As shown, the SlGLUB amplification primer sequences include SlGLUB-GFP-F and SlGLUB-GFP-R; the nucleotide sequence of SlGLUB-GFP-F is SEQ ID NO:2; and the nucleotide sequence of SlGLUB-GFP-R is SEQ ID NO:3.

[0024] (2) Cloning of target fragments and gel recovery Using cDNA from salt-tolerant tomato IL8-3 and salt-sensitive tomato M82 as templates, respectively, amplification was performed using 2×Taq PCR MasterMix (catalog number: PC0903) from Beijing Adley Biotechnology Co., Ltd. SlGLUB The cDNA sequence was obtained, and the gel was recovered using the Tiangen Gel Recovery Kit (catalog number: DP219).

[0025] (3) Vector double enzyme digestion The pRI101-GFP vector was double-digested using Sal I and BamHI.

[0026] Enzyme digestion system (20 μL): Sal I 1 μL BamH I 1 μL 10× T Buffer 1.5 μL 3 μL of pRI101-GFP plasmid ddH2O 13.5 μL Enzyme digestion at 37℃ for 3 hours, followed by gel recovery using the Tiangen Gel Recovery Kit, and storage at -20℃ for later use.

[0027] (4) Ligation and transformation of the target gene with the vector The connection was performed using the Mona Biotechnology Hi-Fusion Cloning Mix V2 kit (catalog number: MC40101).

[0028] Connection system (13 μL): Hi-Fusion Cloning Mix V2 5 μL Linear carrier fragment 2 μL Fragment of interest 6 μL The mixed system was incubated in a 50°C metal bath for 15 minutes to obtain the recombinant plasmid, which was then transformed into E. coli. Escherichia coli DH5α competent cells.

[0029] (5) Plasmid extraction and sequencing verification Plasmid extraction was performed using the plasmid miniprep kit from Tiangen Biotech Co., Ltd. (catalog number: [catalog number missing]), and sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd.

[0030] II. Construction of the SlGLUB gene transient silencing expression vector: (1) Schematic diagram of SlGLUB gene silencing vector construction, as shown Figure 3 As shown, the silencing vector for the tomato salt stress gene SlGLUB is pTRV2-SlGLUB, and the primer sequences are as follows: Figure 4 As shown, the SlGLUB silencing vector amplification primers include TRVSlGLUB-F and TRVSlGLUB-R; The nucleotide sequence of TRVSlGLUB-F is SEQ ID NO:4; The nucleotide sequence of TRVSlGLUB-R is SEQ ID NO:5.

[0031] (2) Cloning of target fragments and gel recovery Using cDNA from salt-tolerant tomato IL8-3 and salt-sensitive tomato M82 as templates, respectively, amplification was performed using 2×Taq PCR MasterMix (catalog number: PC0903) from Beijing Adley Biotechnology Co., Ltd. SlGLUB The cDNA sequence was obtained. Gel recovery was performed using the Tiangen Gel Recovery Kit (catalog number: DP219).

[0032] (3) Vector double enzyme digestion use Eco R Ⅰ and Bam The pTRV2 vector was double-digested with HI.

[0033] Enzyme digestion system (20 μL): R Ⅰ 1 μL HI 1 μL 10× K Buffer 1.5 μL pTRV2 plasmid 3 μL ddH2O 13.5 μL Enzyme digestion at 37℃ for 3 h, followed by gel recovery using the Tiangen Gel Recovery Kit, and storage at -20℃ for later use.

[0034] (4) Ligation and transformation of the target gene with the vector Ligation was performed using the Mona Biotechnology Hi-Fusion Cloning Mix V2 kit (catalog number: MC40101). Ligation volume (13 μL): Hi-Fusion Cloning Mix V2 5 μL Linear carrier fragment 2 μL Fragment of interest 6 μL The mixed system was incubated in a 50°C metal bath for 15 minutes to obtain the recombinant plasmid, which was then transformed into E. coli. Escherichia coli DH5α competent cells.

[0035] (5) Plasmid extraction and sequencing verification Plasmid extraction was performed using the plasmid miniprep kit from Tiangen Biotech Co., Ltd. (catalog number: [catalog number missing]), and sequencing was performed by Sangon Biotech (Shanghai) Co., Ltd.

[0036] III. Agrobacterium-mediated transformation using recombinant plasmids: (1) Recombinant plasmid transformation of Agrobacterium The correctly sequenced recombinant plasmid (8 μL) was added to 100 μL of Agrobacterium tumefaciens competent cells EHA105. After mixing, the cells were incubated on ice for 30 minutes, then flash-frozen in liquid nitrogen for 5 minutes, and then incubated in a water bath at 37°C for 5 minutes. 800 μL of liquid YEP medium containing 50 mg·L⁻¹ Rif was added, and the cells were incubated at 28°C with shaking at 200 rpm for 6 hours. 100 μL of the shaken bacterial solution was evenly spread on YEP medium plates containing Rif and Kan, and incubated upside down at 28°C for 48 hours. Single colonies were picked and cultured for PCR identification. The bacterial solution containing the target vector was mixed with an equal volume of 50% sterile glycerol, aliquoted, and stored at -80°C.

[0037] (2) Construction of transiently silenced SlGLUB plants in tomatoes: Agrobacterium containing the pTRV2-SlGLUB recombinant plasmid and pTRV1 were each adjusted to 0.6 and then mixed 1:1. pTRV2 and pTRV1 without the target gene were each adjusted to 0.6 and then mixed 1:1 as controls. The mixed bacterial solution was placed in a dark environment at 28℃ for 3 hours. Germinated AC tomato seeds (root length about 0.2-0.6 cm) were immersed in this resuspension and placed in a vacuum environment for about 4 minutes. Then the seeds were sown in nutrient soil and placed in a light culture room for culture. The expression of the GLUB gene was detected by quantitative PCR in the later stage of phenotypic testing.

[0038] (3) Tomato SlGLUB Construction of transient overexpression plants: 100 μL of EHA105 bacterial culture containing the SlGLUB-GFP recombinant plasmid was added to 30 mL of YEP liquid medium containing 50 μg·mL⁻¹ Rif and 50 mg·mL⁻¹ Kan, and cultured at 200 rpm in a 28℃ incubator until the OD value reached 1.0–1.2. An empty vector without the target gene was used as a control. 20 mL of each bacterial culture was centrifuged at 4000 rpm for 15 minutes, and the precipitate was collected in 4 mL of infection solution (containing 10 mmol·L⁻¹). -1 MES and 10 mmol·L -1 Resuspend the sample in MgCl2·6H2O, centrifuge the resuspended liquid at 5000 rpm for 10 minutes, and precipitate with a staining buffer (containing 10 mmol·L⁻¹). -1 MES, 10 mmol·L -1 MgCl2·6H2O and 200 mmol·L -1 Acetyl eugenol was resuspended to achieve an OD value of 0.5. The resuspension was left to stand in the dark at 28°C for 3 hours. Germinated AC tomato seeds (root length about 0.2-0.6 cm) were then immersed in this resuspension and placed in a vacuum environment for about 4 minutes. The seeds were then sown in nutrient soil and cultured in a light-cured culture room. Quantitative PCR was used to detect GLUB gene expression in the later stages of phenotypic testing.

[0039] IV. Detection of physiological and biochemical indicators: (1) Antioxidant analysis: POD, SOD, CAT enzyme activity, MDA content, H2O2 and O2 -All assays were performed using kits from Suzhou CominBio Technology Co., Ltd., with catalog numbers POD-2-Y, SOD-2-Y, CAT-2-Y, MDA-2-Y, H2O2-2-Y, and SA-2-G, respectively. For detailed procedures, please refer to the instruction manual (CominBiotechnology, Suzhou, China, https: / / www.cominbio.com / index.html). (2) Paraffin section observation: The roots, stems and leaves of the plant were fixed with FAA fixative (5 mL formaldehyde, 5 mL acetic acid and 90 mL 50% alcohol) for one week. They were then dehydrated, cleared, embedded, sectioned and stained according to the method of Kang Yunyan et al. (2022).

[0040] This invention constructs a tomato system for transient silencing and overexpression of the SlGLUB gene, and applies salt stress of 180 mmol NaCl for 3 days. The following results were obtained by measuring growth morphology and physiological and biochemical indicators, and by preparing paraffin sections: (1) After NaCl treatment, the SlGLUB transiently silenced tomato plants were shorter, had yellow leaves, and were severely wilted compared to the control group. The plant height and root length were significantly lower than those of the control group. In contrast, the SlGLUB transiently overexpressed tomato plants had yellow leaves and were less wilted compared to the control group. The plant height and root length were significantly higher than those of the control group. This indicates that the SlGLUB gene plays an important role in salt tolerance of tomatoes.

[0041] (2) After NaCl treatment, the results of paraffin sections were observed and it was found that salt stress caused varying degrees of damage to the roots, stems and leaves of tomatoes. Compared with the control group, it can be seen that the SlGLUB gene improved the salt resistance of tomato plants at the cellular level.

[0042] (3) After NaCl treatment, the root system was photographed and analyzed by a root scanner. It was found that the number of roots of the tomato plants transiently silenced by SlGLUB was significantly lower than that of the control group, and the total root length and root surface area were also significantly lower than those of the control group. In contrast, the number of roots, total root length and root surface area of ​​the tomato plants transiently overexpressed by SlGLUB were significantly higher than those of the control group. This indicates that the SlGLUB gene improves the salt tolerance of tomato plants by affecting the growth and development of the root system.

[0043] (4) After NaCl treatment, the activities of antioxidant enzymes SOD, POD and CAT, and the contents of O2- and H2O2 in SlGLUB transiently silenced tomato plants were significantly lower than those in the control group, while the contents of MDA were higher. In contrast, the opposite was true for SlGLUB transiently overexpressed tomato plants. This indicates that the SlGLUB gene can reduce the damage caused by oxidative stress in tomatoes under salt stress by regulating the activity of antioxidant enzymes, thereby improving the salt resistance of tomato plants.

[0044] In this invention, the SlGLUB gene not only promotes root development in tomatoes under normal conditions, but also improves salt tolerance in tomatoes by regulating root development, increasing total root length and root surface area after salt stress treatment. Paraffin section results of roots, stems and leaves show that at the cellular level, the SlGLUB gene can reduce the damage caused by salt stress to tomato plants. The SlGLUB gene can also improve the ability to scavenge reactive oxygen species by increasing the activity of antioxidant enzymes and reducing the degree of membrane lipid peroxidation, thereby improving the salt tolerance of tomato plants.

[0045] This application contains a sequence list conforming to the WIPO ST.26 standard, which is submitted with this application as an XML file, the contents of which are incorporated herein by reference.

[0046] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A tomato salt stress gene SlGLUB, characterized in that, The nucleotide sequence of the gene SlGLUB is SEQ ID NO:

1.

2. Primers for amplifying the tomato salt stress gene SlGLUB, characterized in that, Including SlGLUB-GFP-F and SlGLUB-GFP-R; The nucleotide sequence of SlGLUB-GFP-F is SEQ ID NO:2; The nucleotide sequence of SlGLUB-GFP-R is SEQ ID NO:

3.

3. A silencing vector for the tomato salt stress gene SlGLUB, characterized in that, For pTRV2-SlGLUB.

4. Primers for amplifying the silencing vector of the tomato salt stress gene SlGLUB, characterized in that, Including TRVSlGLUB-F and TRVSlGLUB-R; The nucleotide sequence of TRVSlGLUB-F is SEQ ID NO:4; The nucleotide sequence of TRVSlGLUB-R is SEQ ID NO:

5.

5. Application of the tomato salt stress gene SlGLUB in improving root development of tomato plants.

6. Application of the tomato salt stress gene SlGLUB in improving the salt tolerance of tomatoes.

Images