Application of the SlNAC66 gene and its encoded protein in regulating salt tolerance in tomatoes

Abstract

This invention is applicable to the field of molecular biology technology and provides... SlNAC66 Application of genes and their encoded proteins in regulating salt tolerance in tomatoes. SlNAC66 The nucleotide sequence of the gene is shown in SED ID NO:1, and the amino acid sequence of the SlNAC66 transcription factor it encodes is shown in SED ID NO:2. This invention obtains the gene through genetic transformation. SlNAC66 Tomato plants overexpressing this gene showed significantly higher salt tolerance than wild-type tomato plants. Under salt stress, SlNAC66 Overexpression resulted in tomato plants exhibiting higher SOD and POD activities and proline content, stronger reactive oxygen species scavenging ability, less leaf damage, better growth status, and lower inhibition rates of fresh and dry weight under salt stress. This invention lays a theoretical foundation for improving tomato stress resistance through genetic engineering, provides a new application method for regulating plant salt tolerance, and contributes to further research on tomato salt tolerance signaling pathways.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, and particularly relates to... SlNAC66 Application of genes and their encoded proteins in regulating salt tolerance in tomatoes. Background Technology

[0002] Salt stress, a key abiotic stress factor restricting plant growth and development, refers to the phenomenon of high osmotic potential experienced by plants growing in high-salinity habitats. When plants are under salt stress, the increase in soil salinity leads to an increase in the osmotic potential of plant cells, causing cell dehydration, physiological drought, and consequently osmotic stress, making it difficult for plants to absorb water. Simultaneously, high soil salinity causes plants to absorb and accumulate excessive salt ions, damaging biofilm integrity and disrupting the homeostasis of reactive oxygen species within the plant, resulting in oxidative stress. Currently, approximately 6% of the world's land and 30% of irrigated fields suffer from soil salinization, severely limiting plant growth and distribution, reducing crop yield and quality, and posing a significant challenge to sustainable agricultural development. The same applies to tomato cultivation and industry development. With the increasing market demand for vegetables, the expansion of production scale, and the popularization of greenhouse cultivation techniques, the area of ​​greenhouse tomato cultivation has expanded rapidly, leading to increasingly severe secondary soil salinization in greenhouses, becoming a critical challenge that urgently needs to be addressed in tomato cultivation. Therefore, exploring the mechanism of tomato's response to salt stress is of great significance for the discovery and breeding of new tomato varieties with strong resistance.

[0003] NAC transcription factors are a plant-specific family of transcription factors and one of the largest families of transcription factors in plants. NAC transcription factors typically possess a highly homologous and conserved NAC domain at their N-terminus, consisting of approximately 150 amino acids and five conserved subdomains: A, B, C, D, and E. This NAC domain can bind to DNA. The C-terminus of NAC transcription factors is a highly variable transcriptional activation region. Related studies have shown that NAC transcription factors are widely involved in various physiological processes in plants, including growth and development, hormone signaling, plant senescence, fruit ripening, and biotic and abiotic stresses. Their functional diversity is closely related to the specificity of their protein structure.

[0004] However, the NAC transcription factor family contains numerous genes, and their functions vary. Although various biological functions of this family in plants have been reported, their specific functions and molecular mechanisms in regulating salt tolerance in tomatoes remain unclear. In particular, the specific function of the NAC transcription factor SlNAC66, which also belongs to the NAC family, has not yet been reported. Given the severe impact of salt stress on tomato cultivation and the gaps in research on the role of NAC transcription factors in regulating salt tolerance in tomatoes, especially... SlNAC66The lack of understanding of gene function necessitates in-depth exploration of the tomato's response mechanism to salt stress. Therefore, this invention proposes... SlNAC66 Application of genes and their encoded proteins in regulating salt tolerance in tomatoes. Summary of the Invention

[0005] The purpose of this invention is to provide SlNAC66 The application of genes and their encoded proteins in regulating salt tolerance in tomatoes aims to address the problems raised in the background section.

[0006] The objective of this invention is achieved through the following technical solution:

[0007] SlNAC66 The application of genes in regulating salt tolerance in tomatoes, the SlNAC66 The nucleotide sequence of the gene is shown in SEQ ID NO:1, or it encodes the SlNAC66 transcription factor having the amino acid sequence shown in SEQ ID NO:2.

[0008] SlNAC66 The application of genes in the breeding of salt-tolerant transgenic plants includes: [the following is a list of genes containing...] SlNAC66 Gene overexpression vectors were transferred into tomato plants via Agrobacterium-mediated transformation to obtain transgenic plants, which showed enhanced salt tolerance compared to wild-type tomato plants.

[0009] Furthermore, the enhanced salt tolerance is specifically manifested in the following ways: under 250 mmol / L NaCl treatment, compared with wild-type tomato plants, transgenic plants also showed less leaf wilting, less yellowing, less flower drop, and lower fresh weight inhibition rate, dry weight inhibition rate, as well as higher SOD activity, POD activity and proline content.

[0010] Furthermore, the method for constructing the overexpression vector includes: using... Bam HI and Xba The pCAMBIA1300-YFP vector was digested with enzyme I, and then ligated via seamless cloning. SlNAC66 Gene fragments and enzyme-digested vectors.

[0011] Compared with the prior art, the beneficial effects of the present invention are:

[0012] This invention uses genetic transformation to obtain tomato plants overexpressing the SlNAC66 transcription factor gene, and then investigates its function in tomato salt tolerance. The study will... SlNAC66 The overexpression vector was transformed into tomato plants via Agrobacterium-mediated transformation. The resulting positive overexpression plants showed significantly higher salt tolerance than wild-type tomato plants. After treatment with 250 mM NaCl for 12 days, SlNAC66The SOD and POD activities and proline content in overexpressed tomato plants were significantly higher than those in wild-type tomato plants, indicating a stronger ability to scavenge reactive oxygen species. One month after treatment, wild-type tomato plants exhibited yellowing leaves, severe wilting, and flower drop. SlNAC66 Overexpression resulted in less leaf damage and better growth in tomato plants, with significantly lower inhibition rates of fresh and dry weight under salt stress compared to wild-type tomato plants. This invention lays a theoretical foundation for improving tomato stress resistance through genetic engineering and provides a new application method for regulating plant salt tolerance, contributing to further research on tomato salt tolerance signaling pathways. Attached Figure Description

[0013] Figure 1 for SlNAC66 Gene amplification products and detection results after transformation of E. coli with pCAMBIA1300-SlNAC66-YFP vector; Figure a shows the full-length CDS product amplified by PCR, where M: DL2000 Marker, and 1 and 2 are PCR amplification products; Figure b shows the PCR results of bacterial culture after transformation of E. coli with pCAMBIA1300-YFP vector, where M: DL2000 Marker, and 1-6 are bacterial culture PCR products.

[0014] Figure 2 These are the results of positive seedling detection, where M: Marker, P: Plasmid positive control, N: Wild-type tomato plant (MT), 1-6: Different SlNAC66 Overexpression in tomato plants.

[0015] Figure 3 for SlNAC66 Overexpression of tomato plant phenotypes (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6) and wild-type tomato plant (MT) phenotypes (1-month seedling age).

[0016] Figure 4 for SlNAC66 Flowering phenotypes of overexpressing tomato plants (OE-1, OE-2) and wild-type tomato plants (MT) (45-day seedling age).

[0017] Figure 5 for SlNAC66 Phenotypes of overexpressing tomato plants (OE-1, OE-2) and wild-type tomato plants (MT) after salt stress; Figure a shows the phenotypes of OE-1, OE-2 and MT lines after 30 days of salt stress treatment; b shows the fresh weight inhibition rate of OE-1, OE-2 and MT lines after salt stress treatment; c shows the dry weight inhibition rate of OE-1, OE-2 and MT lines after salt stress treatment.

[0018] Figure 6 Under salt stress SlNAC66Changes in reactive oxygen species (ROS) accumulation and antioxidant enzyme activity in overexpressing tomato plants (OE-1, OE-2) and wild-type tomato plants (MT); Figure a shows DAB staining results; b shows NBT staining results; c shows changes in SOD activity before and after salt stress treatment in OE-1, OE-2, and MT lines; d shows changes in POD activity before and after salt stress treatment in OE-1, OE-2, and MT lines.

[0019] Figure 7 Under salt stress SlNAC66 Changes in proline content in overexpressing tomato plants (OE-1, OE-2) and wild-type tomato plants (MT). Detailed Implementation

[0020] In order to provide a clearer understanding of the technical features, objectives and beneficial effects of the present invention, the technical solution of the present invention will now be described in detail below, but it should not be construed as limiting the scope of implementation of the present invention.

[0021] SlNAC66 The nucleotide sequence of the gene is as follows:

[0022]

[0023] SlNAC66 The amino acid sequence of the SlNAC66 transcription factor encoded by the gene is as follows:

[0024] MNLSVNGQSQVPPGFRFHPTEEELLHYYLRKKIANEKIDLDVIREVDLNKLEPWDIQEKCKIGSTPQNDWYLFSHKDKKYPSGSRTNRATAAGFWKATGRDKVIYGNCKRIGMRKTLVFYKGRAPGLKLDWIMHEYRLDDISTPQHQPSLNFCASESAALEEGWVVCRVFKKKSTL QSSSAVSKTLIHENTNTHHDGVLDQILMYMGRSSKQQQQHEIKSNNIVQQVNNNDNENIQFDNTEDGFLQLPELVNHHQDCNFNDEMIIMSTGNDDQHYSCMINNYQENNEAADHKKNGPTCDWLAMVASQLNGHHLEPPTNNNTTRSNDDDVEFWSYAQSSTFDPLSQFTV (such as SED ID NO:2).

[0025] The specific implementation of the present invention will be described in detail below with reference to specific embodiments.

[0026] Example 1: Obtaining transgenic plants;

[0027] 1. Construct overexpression vectors;

[0028] (1) Material and template preparation: The wild-type tomato plant used in this embodiment is Micro-Tom (MT). The leaves of 4-week-old tomato seedlings were used as materials to extract total RNA and reverse transcribe it to obtain cDNA.

[0029] (2) Primer design:

[0030] Upstream primer SlNAC66-F:

[0031] TACTCGACCCCGGGGGATCCATGAATCTCTCTGTAAATGGTCAATC (as shown in SED ID NO:3);

[0032] Downstream primer SlNAC66-R:

[0033] CTCACCATACTAGTTCTAGATACAGTGAACTGACTTAGTGGA (as shown in SED ID NO:4).

[0034] (3) PCR amplification and purification: PCR amplification was performed using cDNA as a template, and the target fragment was then recovered via gel extraction. The results are as follows: Figure 1 As shown in Figure a, lanes 1 and 2 contain PCR amplification products, which are about 1 kb in size, consistent with the size of the target product.

[0035] (4) Carrier construction: using Bam HI and Xba The pCAMBIA1300-YFP vector was digested with enzyme I, and the target gene fragment was ligated to the digested vector through seamless cloning to obtain the complete overexpression vector (pCAMBIA1300-SlNAC66-YFP).

[0036] 2. Plasmid transformation of E. coli;

[0037] (1) Take 50 μL of Trans1-T1 competent cells and thaw them on ice. Add 2 μL of recombinant product to the competent cells on a clean bench and gently mix with a 200 μL pipette.

[0038] (2) After standing on ice for 30 min, immediately heat shock at 42℃ for 45 s, then cool on ice for 2 min;

[0039] (3) Add 450 ml of antibiotic-free LB liquid medium to the centrifuge tube and incubate at 37°C and 200 r / min for 1 h;

[0040] (4) Take 100 μL of bacterial culture and spread it on kanamycin-resistant LB agar plates, and incubate overnight at 37°C;

[0041] (6) After selecting positive colonies for verification, plasmids were extracted and sequenced.

[0042] The results are as follows Figure 1 As shown in Figure b, lanes 1-6 all have a single band, which is the same size as the target fragment, indicating that bacterial solutions 1-6 were successfully ligated. Bacterial solutions 3 and 6 were selected for plasmid extraction and sequencing identification.

[0043] 3. Agrobacterium-mediated genetic transformation was used to obtain overexpressing tomato plants;

[0044] (1) Take 50 μL of GV3101 Agrobacterium competent cells stored in a -80℃ refrigerator, thaw them at room temperature, insert them on ice, add 1 μL of plasmid in a clean bench, and mix well by whisking.

[0045] (2) The treatment was carried out in sequence: 5 min on ice, 5 min in liquid nitrogen, 5 min in a 37℃ water bath, and 5 min in an ice bath;

[0046] (3) Add 500 μL of antibiotic-free LB liquid culture medium to the centrifuge tube and shake in a shaker at 28℃ and 200 r / min for 3h;

[0047] (4) Take about 100 μL of bacterial culture and spread it on an LB agar plate containing rifampicin and kanamycin. Invert the plate and incubate it in a 28°C incubator for 2-3 days.

[0048] (5) After picking a single colony to verify the positive result, add glycerol and store it in a -80℃ refrigerator for later use.

[0049] 4. Obtained by leaf disc method SlNAC66 Overexpression tomato plants;

[0050] (1) Sterile sowing of tomatoes to obtain sterile tomato leaf explants;

[0051] (2) Perform Agrobacterium infection, subculture, and rooting culture in sequence;

[0052] (3) Test the cultured plants for positive seedlings.

[0053] The results are as follows Figure 2 As shown, strains 1-6 all amplified specific bands of the same size as the positive control, indicating that six strains were successfully obtained. SlNAC66 Overexpression in tomato plants.

[0054] Example 2: Salt tolerance assessment;

[0055] 1. Cultivation of plant materials;

[0056] Prepare a petri dish lined with filter paper, moisten it with water, spread tomato seeds of different strains evenly, treat them in the dark for about 4-5 days, and then sow them in the soil. When the seedlings have 2-3 true leaves, transplant them. After testing positive, transfer them to the substrate and place them in an incubator for cultivation. The cultivation conditions are: cultivation temperature 25 / 20℃ (day / night), photoperiod 16 / 8 h light-dark cycle, and watering with 1 / 2 Hogland nutrient solution.

[0057] 2. Identification of transgenic positive seedlings: DNA was extracted from different plants and PCR was used to detect positive plants;

[0058] (1) Primer design:

[0059] Upstream primer 35SF:

[0060] GACGCACAATCCCACTATCC (as shown in SED ID NO:5);

[0061] Downstream primer SlNAC66-RTR1:

[0062] TATCACGACCCGTAGCCTTC (as shown in SED ID NO:6).

[0063] (2) Perform PCR amplification. The PCR reaction system (10 μL) is as follows:

[0064] Table 1 PCR reaction system

[0065] Reagents Usage 2×MS HiPer plus Taq HiFi PCR Mix 5 μL 35SF 0.5 μL SlNAC66-RTR1 0.5 μL DNA 1 μL <![CDATA[ddH2O]]> Up to 10 μL

[0066] The reaction conditions are as follows:

[0067] Table 2 PCR reaction conditions

[0068] step temperature time Pre-variation 94℃ 3 min transsexual 94℃ 5 s annealing 55℃ 15 s extend 72℃ 5 s Final extension 72℃ 5 min

[0069] (3) Prepare a gel by mixing agarose and TAE buffer at a ratio of 1 g: 100 mL, add 2 μL of EB substitute dye, and perform electrophoresis detection. Use plasmid as a positive control and DNA from wild-type tomato plants (MT) as a negative control. Plants with corresponding bands to the positive control are considered positive plants.

[0070] 3. SlNAC66 Observation of overexpression phenotype in tomato plants;

[0071] Using T1 generation SlNAC66 Phenotypic observations were conducted on tomato plants overexpressing the gene, and data such as plant height and leaf size were collected comparing them with wild-type tomato plants (MT). The flowering rate on day 45 was also recorded to investigate... SlNAC66 The effect of gene overexpression on the flowering and fruiting process of tomatoes.

[0072] Results: Observation of wild-type tomato plants (MT) at 1 month of age and SlNAC66 Overexpressing tomato plants (OE-1, OE-2, OE-3, OE-4, OE-5, OE-6) (T1 generation) showed significantly reduced height and smaller leaf size compared to the MT lines. Figure 3 (a and b). At the same time, it was observed that the OE-1 and OE-2 lines exhibited a phenotype of later flowering ( Figure 4 ).

[0073] 4. SlNAC66 Analysis of salt tolerance in tomato plants overexpressing the gene;

[0074] SlNAC66 Overexpressing tomato plants and wild-type tomato plants were cultured in a culture room. After about one month of growth, plants with uniform growth were selected. SlNAC66Overexpression was used in tomato plants (OE-1, OE-2) and wild-type tomato plants (MT). 250 mmol / L NaCl solution was used as the salt stress treatment reagent, with water as the control. Each experimental group had three replicates. The control group (Control) was watered every 3 days, with tomato plants placed in trays and 1000 mL of water poured into the trays. The experimental group (Salt) was watered with 1000 mL of 250 mmol / L NaCl solution in the same manner. After 12 days of treatment, leaf samples were collected from leaves of similar age at the same time point. The collected samples were immediately frozen in liquid nitrogen, then ground in a pre-cooled mortar in an ice bath and aliquoted into 0.1 g centrifuge tubes, stored at -80°C, for the determination of physiological indicators such as peroxidase (POD) activity, superoxide dismutase (SOD) activity, and proline content. Phenotypic observation and dry / fresh weight determination were also performed on plant materials one month after salt stress treatment.

[0075] Results: One month after salt stress treatment, all three strains (OE-1, OE-2, and MT) showed some degree of wilting. However, the MT strain exhibited more pronounced yellowing of leaves, severe wilting, and flower drop, while the OE-1 and OE-2 strains showed relatively less leaf damage and better growth than the MT strain. Figure 5 (a) Because transgenic plants exhibit dwarfing characteristics under normal conditions, the fresh weight, dry weight, and inhibition rate of the OE-1, OE-2, and MT lines before and after salt stress were further measured. The results showed that salt stress treatment inhibited the fresh weight of the MT line by 50.65%, and the inhibition rates of the OE-1 and OE-2 lines were 11.53% and 14.43%, respectively, significantly lower than that of the MT line. Figure 5 (b); Similarly, salt stress treatment inhibited the dry weight of the MT line by 45.65%, and the inhibition rates of the OE-1 and OE-2 lines were 13.58% and 12.35%, respectively, which were also significantly lower than those of the MT line. Figure 5 (c) The above results show that SlNAC66 Overexpression reduced the impact of salt stress on tomato plants, decreased their sensitivity to salt stress, and improved the salt tolerance of tomatoes.

[0076] To investigate the differences in reactive oxygen species (ROS) metabolism under salt stress, the accumulation of hydrogen peroxide and superoxide anions in tomato leaves was analyzed using DAB and NBT staining methods. The results showed that under control conditions, there were no significant differences in DAB and NBT staining between the OE-1, OE-2, and MT lines; however, after salt stress treatment, the leaves of the OE-1 and OE-2 lines showed lighter colors after DAB and NBT staining, indicating... SlNAC66 The accumulation of hydrogen peroxide and superoxide anions in overexpressed tomato plants was significantly lower than that in wild-type tomato plants, demonstrating superior reactive oxygen species scavenging ability. Figure 6 (a and b). Antioxidant enzyme activity was also measured after 12 days of salt stress treatment. SlNAC66 The SOD and POD activities of overexpressing tomato plants were significantly higher than those of wild-type tomato plants. The SOD activities of the OE-1 and OE-2 lines were increased by 37.45% and 38.78%, respectively, and the POD activities were increased by 46.88% and 54.50%, respectively, compared with the MT line. Figure 6 (c and d in the middle). Explanation. SlNAC66 Overexpression in tomato plants can enhance the activity of antioxidant enzymes, reduce oxidative stress by efficiently scavenging reactive oxygen species, and improve the plant's tolerance to salt stress.

[0077] The proline content in leaves of OE-1, OE-2, and MT lines was determined after 12 days of salt stress treatment. The results showed that under control conditions, there was no significant difference in proline content in leaves between the OE-1, OE-2, and MT lines; however, after salt stress treatment, the proline content in leaves of the OE-1 and OE-2 lines was significantly higher than that of the MT line, increasing by 50.72% and 36.92%, respectively. Figure 7 This indicates that under salt stress, SlNAC66 Overexpression can promote the accumulation of proline in tomato leaves, which helps improve the plant's salt tolerance through osmotic regulation.

[0078] The above are merely preferred embodiments of the present invention. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention, and these should also be considered within the scope of protection of the present invention. These modifications and improvements will not affect the effectiveness of the implementation of the present invention or the practicality of the patent.

Claims

1. Overexpression SlNAC66 The application of genes in improving the salt tolerance of tomatoes is characterized by, The SlNAC66 The nucleotide sequence of the gene is shown in SEQ ID NO:1, or the SlNAC66 transcription factor encoding the amino acid sequence shown in SEQ ID NO:

2.

2. SlNAC66 The application of genes in the breeding of salt-tolerant transgenic plants is characterized by, include: Will contain SlNAC66 The gene overexpression vector was transferred into tomato plants via Agrobacterium-mediated transformation to obtain transgenic plants, which showed enhanced salt tolerance compared to wild-type tomato plants. The SlNAC66 The nucleotide sequence of the gene is shown in SEQ ID NO:

1.

3. The application according to claim 2, characterized in that, The enhanced salt tolerance is specifically manifested in the following ways: under 250 mmol / L NaCl treatment, compared with wild-type tomato plants, transgenic plants also showed less leaf wilting, less yellowing, less flower drop, and lower fresh weight inhibition rate, dry weight inhibition rate, as well as higher SOD activity, POD activity and proline content.

4. The application according to claim 2, characterized in that, The method for constructing the overexpression vector includes: using... Bam HI and Xba The pCAMBIA1300-YFP vector was digested with enzyme I, and then ligated via seamless cloning. SlNAC66 Gene fragments and enzyme-digested vectors.

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