Application of slmyb113 related biological products in salt-tolerant stress of tomato

Abstract

This invention belongs to the field of plant genetic engineering technology, specifically relating to a... SlMYB113 The application of related biological products in the salt-alkali stress tolerance of tomatoes, the SlMYB113 The nucleotide sequence is shown in SEQ ID NO.1. This invention constructs... SlMYB113 Overexpression vectors were used and transformed into tomatoes to obtain overexpressing plants. After 6 days of treatment with 300 mM compound saline-alkali, the growth of overexpressing plants was significantly better than that of wild-type AC plants, with significantly reduced leaf yellowing, abscission, and wilting; SlMYB113 Knocking out the plants resulted in a more severe salt-alkali damage phenotype, proving that... SlMYB113 It has a positive regulatory effect on the salt and alkali resistance of tomatoes and can be used to improve the salt and alkali resistance of tomatoes, which is of great significance for creating salt and alkali tolerant tomato germplasm.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a... SlMYB113 Application of related biological products in the salt and alkali stress tolerance of tomatoes. Background Technology

[0002] tomato( Solanum lycopersicum As an important horticultural crop, tomatoes are not only a common vegetable in our daily diet, but also a model plant for research in genetics, stress resistance physiology and biochemistry, and biotechnology improvement. However, tomatoes are often affected by various environmental stresses during their growth and development, among which salt-alkali stress is one of the key factors limiting their quality and yield. Although saline-alkali land improvement technology can help expand arable land, its high cost severely restricts its large-scale application. Therefore, breeding salt-alkali tolerant crop varieties has become an economical and effective way to solve this problem.

[0003] To cope with salt and alkali stress, tomatoes have evolved complex adaptive mechanisms, encompassing morphological adaptation, physiological adaptation, and molecular regulation. Physiological adaptation mechanisms mainly involve osmotic regulation, ion and pH balance, antioxidant defense, and endogenous hormone responses. Molecular regulatory mechanisms are achieved through signal transduction, transcription factor regulation, expression of salt and alkali resistance genes, and epigenetic modifications. Transcriptome analysis shows that the expression levels of various transcription factors change significantly under salt and alkali stress, such as the CBF / DREB1, AP2 / ERF, NAC, bZIP, WRKY, and bHLH families, most of which have been confirmed to participate in the salt and alkali stress response. Under the regulation of these transcription factors, the expression of resistance genes related to osmotic regulation, ion homeostasis, redox balance, and hormone signal transduction is upregulated, thereby enhancing the salt and alkali tolerance of tomatoes.

[0004] However, although a large number of transcription factors have been identified as key regulators of tomato's response to salt and alkali stress, their effectiveness in responding to salt and alkali stress is poor. Therefore, there is an urgent need to provide a new strategy for tomatoes to cope with salt and alkali stress. Summary of the Invention

[0005] The purpose of this invention is to provide a SlMYB113 The application of related biological products in the salt and alkali stress tolerance of tomatoes has solved the problems existing in the prior art.

[0006] The technical solution adopted in this invention is: This invention provides SlMYB113 The application of related biological products in tomato salt-alkali stress tolerance provides a novel gene resource with significant regulatory potential for improving tomato salt-alkali resistance. SlMYB113 The nucleotide sequence is shown in SEQ ID NO.1.

[0007] Preferably, the biological product comprises at least one of the following: A. An overexpression vector containing the nucleotide sequence; B. Recombinant bacteria containing the overexpression vector described in A.

[0008] Preferably, the overexpression vector is constructed as follows: Using tomato leaves as a template, PCR amplification was performed using the primers shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the target fragment; The target fragment is ligated into a linearized expression vector to obtain an overexpression vector.

[0009] Preferably, the expression vector is pHellsgate8.

[0010] Preferably, the conditions for ligating the target fragment to the linearized expression vector are 37°C for 1 hour.

[0011] Preferably, the process of linearizing the expression vector is as follows: using EcoR I. The expression vector was digested with a single enzyme.

[0012] Preferably, the PCR amplification procedure is as follows: Denaturation at 98℃ for 10 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 1 minute, 35 cycles; Finally, extend the heat to 72℃ for 5 minutes.

[0013] Preferably, the PCR amplification system is as follows: 25 μL of 2×PrimeSTAR Max Premix, 2 μL of 10 μmol / L upstream primer, 2 μL of 10 μmol / L downstream primer, 1 μL of template DNA, and ddH2O to bring the total volume to 50 μL.

[0014] Compared with the prior art, the beneficial effects of the present invention are: This invention provides SlMYB113 The application of related biological products in the salt-alkali stress tolerance of tomatoes, the SlMYB113 The nucleotide sequence is shown in SEQ ID NO.1. This invention constructs... SlMYB113 Overexpression vectors were used and transformed into tomatoes to obtain overexpressing plants. After 6 days of treatment with 300 mM compound saline-alkali, the growth of overexpressing plants was significantly better than that of wild-type AC plants, with significantly reduced leaf yellowing, abscission, and wilting; SlMYB113 Knocking out the plants resulted in a more severe salt-alkali damage phenotype, proving that... SlMYB113 Overexpression can significantly enhance the tolerance of tomatoes to salt and alkali stress.

[0015] Meanwhile, under saline-alkali stress, SlMYB113 The levels of hydrogen peroxide and malondialdehyde in the leaves of overexpressing plants were significantly lower than those in AC plants, while the levels of these indicators were significantly higher in knockout plants, indicating that... SlMYB113 Overexpression can effectively alleviate oxidative damage and membrane lipid peroxidation caused by salt-alkali stress. Furthermore, SlMYB113 Under salt-alkali stress, the activities of superoxide dismutase, peroxidase, and catalase in overexpression plants were significantly higher than those in AC plants; the activities of these enzymes were significantly reduced in knockout plants. This indicates that... SlMYB113 By enhancing the activity of antioxidant enzyme systems, the plant's ability to scavenge reactive oxygen species is improved, thereby increasing its salt and alkali tolerance.

[0016] In summary, this invention improves... SlMYB113 The expression level of this gene significantly enhanced the tolerability of tomatoes to salt and alkali stress, specifically by reducing oxidative damage and membrane lipid peroxidation, and increasing the activity of antioxidant enzymes, thereby effectively alleviating the damage caused by salt and alkali stress. These results demonstrate... SlMYB113 It has a positive regulatory effect on the salt and alkali resistance of tomatoes and can be used to improve the salt and alkali resistance of tomatoes, which is of great significance for creating salt and alkali tolerant tomato germplasm. Attached Figure Description

[0017] Figure 1 for SlMYB113 Expression levels in overexpressing plants.

[0018] Figure 2 for SlMYB113 Phenotypic characteristics of overexpressing plants, knockout plants, and AC plants under salt stress.

[0019] Figure 3 for SlMYB113 H2O2 content in leaves of overexpressing plants, knockout plants and AC plants.

[0020] Figure 4 for SlMYB113 MDA content in leaves of overexpressing plants, knockout plants, and AC plants.

[0021] Figure 5 for SlMYB113 SOD enzyme activity in leaves of overexpressing plants, knockout plants and AC plants.

[0022] Figure 6 for SlMYB113 POD enzyme activity in leaves of overexpressing plants, knockout plants and AC plants.

[0023] Figure 7 for SlMYB113 CAT enzyme activity in leaves of overexpressing plants, knockout plants and AC plants. Detailed Implementation

[0024] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0025] The inventive concept of this invention is as follows: The MYB family is one of the largest transcription factor families, widely distributed in plants. Many members have been extensively studied, participating in the regulation of physiological and biochemical processes such as plant cell development, signal transduction, primary and secondary metabolism, and responses to various stresses. MYB transcription factors typically consist of three main domains: the DNA-binding domain (DBD), the transcriptional activation domain, and the negative regulatory domain. The DBD is the most conserved, generally containing 1–4 incomplete repetitive sequences, denoted as R1, R2, R3, and R1 / R2. Based on the number of adjacent incomplete repetitive sequences in the DBD, MYB TFs can be classified into four categories: 1R-MYB / MYB-like, R2R3-MYB, R1R2R3-MYB, and 4R-MYB. MYB113, as a member of the R2R3-MYB subfamily, primarily promotes anthocyanin accumulation in plants such as tomato, eggplant, and Arabidopsis thaliana. However, its role in the stress response of tomato remains unstudied. SlMYB113 The expression of [a specific substance] is significantly induced by salt-alkali stress, therefore the study... SlMYB113 This study aims to elucidate the role and function of genes in salt-alkali stress response, which is of great significance for breeding new salt-alkali tolerant tomato varieties.

[0026] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0027] Example 1 SlMYB113 The application of related biological products in tomato salt-alkali stress is as follows: 1. SlMYB113 Construction of overexpression vectors.

[0028] Found on the SNG tomato genome database website SlMYB113 The cDNA sequence of the gene is shown in SEQ ID NO.1; the design was performed using Primer Premier 5 software. SlMYB113 The amplification primers for the gene were prepared, and a pHellsgate8 vector-specific homologous recombination arm was added to the 5' end of the primers. The amplification primers are shown in SEQ ID NO.2 and SEQ ID NO.3.

[0029] SNG website: http: / / solgenomics.net / .

[0030] SEQ ID NO.1: ATGAACAGTACATCTATGTCTTCTTTGGGAGTAAGAAAAGGTTCATGGACTGAACAAGAAGATCTCCTCTTGAGGAAATGTATCAACAAGTATGGTGAAGGAAAGTGGCATCTTGTTCCCATAAGAGCTGGTCTGAATAGATGTCGGAAAAGTTGTAGACTAAGGTGGCTAAATTATCTAAGGCCACATATAAAGAGAGGTGACTTCGCTTCGGATGAAATAGATCTCATTTTGAGGCTTCATAAACTTTTAGGCAACAGATGGTCACTTATTGCTGGTAGACTTCCAGGAAGGACAGCAAACGATGTGAAAAACTATTGGAACACAAACCTTCTAAGGAAGGTAAATATTACTAAAATTGTTCCACGTGAAAAGTTTAAGAGTAAACGAGGAGAAATTAGTACTAAGATTGAAATAATAAAACCTCAAGCTAGGAAGTTCATATCGAACACAGAGAAGAATATTACAAACAATATTGTAATTGTAGACAAAGAGGAAGAATGTAAGGAAATAATAAGTGAGAAGCAAACTAGAGATGCATCGATAGACAACGGAGATGAATGGTGGGCAAATTTACTGGAAAATTGCAACGACGACGTTGTTGAAGAAGAAGAAGGAGGAGGAGGAGGTGTAACTGATTATGGAAAAACAATAACAAGTTTGTTACATGAGGAAATAACACCACCATTAAATGGTGGAGGTAACATCATGCAACAAGAACAAAGTGATGGTTGGGATGATTTTTTTGTTGATATTGATATATGGGATTTACTTAATTAA.

[0031] Upstream primer SlMYB113-OE-FW, SEQ ID NO.2: GATGACGATGACAAGGAATTCATGAACAGTACATCTATGTCTTC.

[0032] Downstream primer SlMYB113-OE-RV, SEQ ID NO.3: GTCCTTGTAATCCATGAATTCTTAATTAAGTAAATCCCATATATC.

[0033] SlMYB113 The gene amplification system is shown in Table 1, and the amplification procedure is shown in Table 2.

[0034] Table 1 PCR amplification system

[0035] Table 2 PCR amplification program

[0036] Note: In Table 2, "-" indicates that this item is not available.

[0037] use EcoR The pHellsgate8 vector was digested with a single enzyme, and the linearized vector was recovered by gel extraction and its concentration was determined. Homologous recombination was used to reconstitute the target fragment and the linearized vector. In a 10 μL system, the target fragment and the linearized vector were added, followed by 2×Basic Assembly Mix, with the remainder made up using ddH2O. After adding all reaction solutions, the mixture was gently aspirated and placed in a PCR instrument at 37°C for 1 h to perform homologous recombination and obtain the ligation product.

[0038] The ligation product was transformed into competent E. coli cells, and the obtained single clones were detected by colony PCR. The primers used for colony PCR were 35S and Gate8-RV. The detection system for colony PCR is shown in Table 3, and the detection procedure is shown in Table 4.

[0039] 35S, SEQ ID NO. 4: GATTTGGAGAGGACACGCTCGCA.

[0040] Gate8-RV, SEQ ID NO.5: CATAAAAATACGATAGTAACGGGTG.

[0041] Table 3 Bacterial Coagulation PCR Detection System

[0042] Table 4. Procedure for bacterial culture PCR detection

[0043] Single clones of *E. coli* with correct results from PCR detection and sequencing were amplified, and plasmids were extracted using a plasmid mini-extraction kit to obtain... SlMYB113The overexpression vector SlMYB113-pHellsgate8.

[0044] 2. SlMYB113 Construction of the knockout vector.

[0045] This invention selects two target sites for knockout, and the selected target site sequences are shown in SEQ ID NO.6 and SEQ ID NO.7.

[0046] Target 1, SEQ ID NO.6: TGTATCAACAAGTATGGTGA.

[0047] Target 2, SEQ ID NO.7: AAAGAGAGGTGACTTCGCTT.

[0048] The dual-target sites were amplified using the pCBC-DT1T2 vector plasmid as a template, and then the target bands were purified using a gel extraction kit. Based on the pBSE402 vector, a knockout vector was constructed using the Golden Gate method. The reaction system for constructing the knockout vector is shown in Table 5.

[0049] Table 5 Knockout Carrier Construction System

[0050] The PCR program was 37℃ for 5 hours and 80℃ for 10 minutes. After the reaction, the cells were transferred to *E. coli*, and single clones were selected for colony PCR detection and sequencing. The correct fusion expression vector was then used for stable genetic transformation of tomato via Agrobacterium-mediated transformation.

[0051] The primer sequences used for bacterial culture PCR detection are shown in SEQ ID NO.8 and SEQ ID NO.9.

[0052] U626-FW, SEQ ID NO. 8: aaaggcccctgggaatctga.

[0053] EDIT3-RV, SEQ ID NO.9: GGGAATCTGAAAGAAGAGAAGCAG.

[0054] 3. SlMYB113 Obtaining overexpressed and knockout plants of genes.

[0055] (1) The constructed SlMYB113 The overexpression vector and knockout vector were transformed into Agrobacterium competent cells GV3101 for later use.

[0056] (2) The above-mentioned Agrobacterium bacterial solution was used to infect the cotyledons of tomato AC.

[0057] (3) The infected explants were cultured in the dark.

[0058] (4) Explants were screened and cultured on a screening medium containing antibiotics, and resistant shoots with growth points were transferred to a rooting medium for rooting.

[0059] (5) After rooting, the resistant seedlings were transferred to nutrient soil and cultured for another month. Overexpression plants were tested for positive using primers shown in SEQ ID NO.4 and SEQ ID NO.5, and knockout plants were tested for positive using primers shown in SEQ ID NO.8 and SEQ ID NO.9.

[0060] (6) Continue to cultivate the positive seedlings until they bear fruit, and harvest the seeds of the T0 generation transgenic plants.

[0061] 4. SlMYB113 Identification of overexpression plants and knockout lines.

[0062] (1) Identification of overexpressing plants.

[0063] Take the above SlMYB113 RNA was extracted from leaves of the overexpression line using an RNA extraction kit, and cDNA was obtained by reverse transcription. Expression levels were then detected using quantitative real-time PCR. SlMYB113 The expression level was detected using primers as shown in SEQ ID NO.10 and SEQ ID NO.11. The real-time quantitative PCR system is shown in Table 6, and the real-time quantitative PCR program is shown in Table 7.

[0064] Expression level detection primers: Q_FW, SEQ ID NO. 10: GAGGTGACTTCGCTTCGGATG.

[0065] Q_RV, SEQ ID NO. 11: TAGTTTTTCACATCGTTTGCTGTCC.

[0066] Table 6 Real-time Quantitative PCR System

[0067] Table 7 Real-time Quantitative PCR Procedure

[0068] Through the above process, this invention obtained two overexpression plants, which are denoted as […]. SlMYB113 OE#1 and SlMYB113 OE#7. Expression level measurement results are as follows: Figure 1 The results showed that in the above-mentioned overexpressing plants SlMYB113 Gene expression levels were significantly increased.

[0069] (2) Identification of knocked-out plants.

[0070] Using the primers shown in SEQ ID NO.12 and SEQ ID NO.13, the target editing status of the knockout plants was detected. Two knockout plants were obtained by sequencing and comparing them with the reference gene sequence. These two knockout plants are denoted as […]. SlMYB113 KO#1 and SlMYB113 KO#9, Discover SlMYB113 KO-1 and SlMYB113 KO-9 cells all have missing segments to varying degrees, and the peaks of the missing segments are all single peaks.

[0071] SlMYB113 -KO-FW, SEQ ID NO. 12: CAATTGTGGACGGAGAGGGT.

[0072] SlMYB113 -KO-RV, SEQ ID NO. 13: TCGTTTGCTGTCCTTCCTGG.

[0073] 5. SlMYB113 Evaluation of salt and alkali stress resistance in overexpressing and knockout plants.

[0074] The cells that have grown normally in the light incubator for 45 days SlMYB113 Overexpressing plants, knockout plants, and AC plants were simultaneously treated with a 300 mM compound salt-alkali solution. The molar ratio of NaCl:Na2SO4:NaHCO3:Na2CO3 in the compound salt-alkali treatment solution was 1:9:9:1, and the pH was 8.90.

[0075] Six days after treatment, the leaves of the AC plants curled, turned yellow, and fell off. SlMYB113 The overexpressing plants showed significantly better growth than the AC plants, with leaves maintaining a better unfolded state, indicating that... SlMYB113 Overexpression of [specific gene] can effectively alleviate the inhibitory effect of salt-alkali stress on tomato growth. Conversely, SlMYB113 Knockout plants exhibited a more severe stress response, with significantly increased leaf yellowing and abscission, and the overall plant showed signs of wilting. This phenotypic difference indicates that... SlMYB113 It plays a positive regulatory role in the salt-alkali stress resistance of tomatoes, and its deficiency leads to a significant increase in the plant's sensitivity to stress. Results are shown below. Figure 2 .

[0076] To further reveal the degree of oxidative damage in different plants under stress, this invention further detected the contents of hydrogen peroxide (H2O2) and malondialdehyde (MDA) in leaves. H2O2, as an important component of reactive oxygen species (ROS), directly reflects the degree of oxidative stress; while MDA is a product of membrane peroxidation and is often used to assess the degree of cell membrane damage. The results showed that under salt-alkali stress, overexpression... SlMYB113 Significantly reduced the H2O2 and MDA content in the plant leaves; compared to AC, both plants SlMYB113 The H2O2 content in the overexpression plants was significantly reduced by 50.91% and 58.47%; the MDA content was significantly reduced by 28.24% and 29.77%. This result indicates that... SlMYB113 Overexpression of [specific ingredient] effectively inhibited stress-induced ROS burst, alleviated membrane peroxidation damage, and thus protected cell membrane integrity. Conversely, SlMYB113 The knockout plants showed significantly higher levels of H2O2 and MDA than the AC plants, indicating a large accumulation of reactive oxygen species and a significantly elevated level of oxidative stress. This is highly consistent with their severe wilting and leaf yellowing and abscission phenotypes. (See above results...) Figure 3 and Figure 4 .

[0077] Furthermore, this invention further tested the activities of three key antioxidant enzymes: SOD, POD, and CAT. SOD is the first line of defense in scavenging superoxide anions, while POD and CAT are responsible for further scavenging H2O2. The results showed that after salt-alkali treatment, SlMYB113 The activities of SOD, POD, and CAT in the overexpressing plants were significantly higher than those in the AC plants, indicating that... SlMYB113 Overexpression of this enzyme can systematically enhance the antioxidant enzyme system of tomato plants, thereby more efficiently scavenging stress-induced reactive oxygen species and maintaining intracellular redox balance. Meanwhile... SlMYB113 In the knockout plants, the activities of all three enzymes were significantly lower than those of AC, indicating a weakened ROS scavenging ability. This further explains why... SlMYB113 Knockout plants showed significantly increased levels of H2O2 and MDA, and exhibited more severe stress damage. See the results above. Figures 5-7 .

[0078] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0079] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. SlMYB113 The application of related biological products in the salt-alkali stress tolerance of tomatoes is characterized by, The SlMYB113 The nucleotide sequence is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The biological product includes at least one of the following: A. An overexpression vector containing the nucleotide sequence; B. Recombinant bacteria containing the overexpression vector described in A.

3. The application as described in claim 2, characterized in that, The overexpression vector is constructed as follows: Using tomato leaves as a template, PCR amplification was performed using the primers shown in SEQ ID NO.2 and SEQ ID NO.3 to obtain the target fragment; The target fragment is ligated into a linearized expression vector to obtain an overexpression vector.

4. The application as described in claim 3, characterized in that, The expression vector is pHellsgate8.

5. The application as described in claim 3, characterized in that, The target fragment was ligated to the linearized expression vector at 37°C for 1 hour.

6. The application as described in claim 3, characterized in that, The process of linearizing the expression vector is as follows: using EcoR I. The expression vector was digested with a single enzyme.

7. The application as described in claim 3, characterized in that, The procedure for PCR amplification is as follows: Denaturation at 98℃ for 10 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 1 minute, 35 cycles; Finally, extend the heat to 72℃ for 5 minutes.

8. The application as described in claim 3, characterized in that, The PCR amplification system is as follows: 25 μL of 2×PrimeSTAR Max Premix, 2 μL of 10 μmol / L upstream primer, 2 μL of 10 μmol / L downstream primer, 1 μL of template DNA, and ddH2O to bring the total volume to 50 μL.

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