Application of LecRK26 gene in regulation and control of saline-alkaline tolerance of tomato
By overexpressing the LecRK26 gene in tomatoes, the problem of tomatoes' sensitivity to salt and alkali stress was solved, and their salt and alkali tolerance and drought resistance were significantly improved, providing an efficient genetic engineering improvement approach.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- CHONGQING UNIV OF ARTS & SCI
- Filing Date
- 2026-03-24
- Publication Date
- 2026-05-08
AI Technical Summary
Tomatoes are sensitive to salt and alkali stress, and traditional breeding and cultivation methods are inefficient and costly, lacking effective genetic engineering improvement pathways.
By overexpressing the LecRK26 gene, the LecRK26 gene was transferred into tomato plants using the Agrobacterium tumefaciens infection method to enhance their salt and alkali tolerance. A recombinant expression vector was constructed and transgenic tomato varieties with enhanced salt and alkali tolerance were bred.
It significantly improves the seed germination rate and survival rate of tomatoes under drought stress, reduces the number of leaf stomata and water loss rate, enhances the adaptability to saline-alkali environments, shortens the breeding cycle and reduces costs.
Smart Images
Figure CN121992028A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the field of plant genetic engineering technology, specifically to the application of the LecRK26 gene in regulating the salt and alkali tolerance of tomatoes. Background Technology
[0002] Tomato (Solanum lycopersicum L.) is an important economic crop widely cultivated globally, but salt stress is one of the major abiotic stresses limiting its yield and quality. Approximately 20% of arable land and 50% of irrigated land worldwide are affected by salinization, and this area continues to expand due to climate change and improper irrigation. Tomatoes are particularly sensitive to salt stress; saline-alkali environments can hinder root water absorption, cause ion imbalance, and exacerbate oxidative damage, ultimately leading to stunted plant growth, reduced yield, and even death.
[0003] Currently, improving the salt and alkali tolerance of tomatoes mainly relies on traditional breeding and cultivation methods. However, traditional breeding is time-consuming and inefficient, while cultivation methods (such as salt leaching and application of amendments) are costly and cannot fundamentally improve the salt and alkali tolerance of tomatoes themselves. Genetic engineering technology provides an efficient approach to improving crop salt and alkali tolerance, and identifying key salt and alkali tolerance genes is a core prerequisite. Lectin receptor-like protein kinases (LecRKs) are plant-specific transmembrane protein kinases that have been shown to participate in plant stress response processes, but the function and application of the tomato LecRK26 gene in salt and alkali tolerance regulation have not yet been reported.
[0004] Therefore, clarifying the role of the LecRK26 gene in salt-alkali tolerance of tomatoes and developing its application in molecular breeding is of great theoretical and practical value for breeding salt-alkali tolerant tomato varieties and ensuring tomato production in saline-alkali areas. Summary of the Invention
[0005] To address the aforementioned shortcomings of existing technologies, this invention provides the application of the LecRK26 gene in regulating the salt and alkali tolerance of tomatoes, clarifying that this gene can significantly improve the salt and alkali tolerance of tomatoes, and providing new gene resources and technical solutions for the molecular breeding of drought-resistant tomato varieties.
[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows: The LecRK26 gene is derived from tomato, and its nucleotide sequence is shown in SEQ ID NO.1, as well as its amino acid sequence is shown in SEQ ID NO.2. The regulation of tomato salt and alkali tolerance specifically refers to improving the salt and alkali tolerance of tomatoes, that is, by enhancing the expression of the LecRK26 gene, the survival ability and stress resistance of tomatoes under drought stress can be significantly improved.
[0007] Further, the application involves transferring the LecRK26 gene into tomato plants to induce overexpression of the LecRK26 gene. The method for transferring the LecRK26 gene into tomato plants is one of the following: Agrobacterium tumefaciens infection method, gene gun method, or polyethylene glycol method.
[0008] Furthermore, it can be applied to the breeding of transgenic tomato varieties with enhanced salt and alkali tolerance.
[0009] The present invention also provides a recombinant expression vector for regulating the salt and alkali tolerance of tomatoes, the nucleotide sequence of which is shown in SEQ ID NO. 3.
[0010] The present invention also provides a transgenic cell line or engineered bacteria containing the above-mentioned recombinant expression vector.
[0011] The present invention also provides a method for improving the salt and alkali tolerance of tomatoes, comprising the following steps: 1) constructing a recombinant expression vector containing the above-mentioned LecRK26 gene; 2) transferring the recombinant expression vector into tomato recipient cells to obtain transgenic tomato cells; 3) cultivating the transgenic tomato cells into complete plants and screening out transgenic tomato plants that overexpress the LecRK26 gene and have enhanced salt and alkali tolerance.
[0012] Furthermore, the enhanced salt and alkali tolerance in step 3) is manifested in the following ways: compared with wild-type tomatoes, transgenic tomatoes have a 25% to 40% higher seed germination rate and a 20% to 35% higher relative germination rate under drought stress, a 15% to 28% lower number of leaf stomata, and a 20% to 35% lower leaf water loss rate.
[0013] The beneficial effects of this invention are as follows: This invention is the first to discover and confirm the important role of the LecRK26 gene in regulating the salt and alkali tolerance of tomatoes, filling the gap in the research on the drought resistance function of the tomato LecRK26 gene, enriching the research content on the molecular mechanism of drought resistance in tomatoes, and providing a new perspective for the study of the functional diversity of the plant LecRK gene family.
[0014] The LecRK26 gene provided by this invention can significantly improve the salt and alkali tolerance of tomatoes. By overexpressing this gene, the seed germination rate and survival rate of tomatoes under drought stress can be significantly improved, while the number of leaf stomata, water loss rate and malondialdehyde content can be significantly reduced, thus enhancing the adaptability of tomatoes to saline-alkali environments and fundamentally improving the salt and alkali tolerance of tomatoes, solving the problem of poor salt and alkali tolerance of traditional tomato varieties.
[0015] The technical solution provided by this invention is simple and efficient to operate. It uses genetic engineering technology to transfer the LecRK26 gene into tomatoes to cultivate drought-resistant transgenic tomato varieties. The breeding cycle is short and highly targeted, and new tomato materials with stable salt and alkali tolerance can be obtained quickly. Compared with traditional breeding methods, it greatly improves the efficiency of improving the salt and alkali tolerance of tomatoes and reduces breeding costs.
[0016] The LecRK26 gene, recombinant expression vector, and transgenic method provided by this invention can not only be used for the breeding of drought-resistant tomato varieties, but also provide reference and gene resources for improving the salt and alkali tolerance of other solanaceous crops (such as peppers and eggplants). It has a wide range of applications and huge economic and social benefits, and is of great significance for ensuring safe crop production and responding to global drought and climate change. Attached Figure Description
[0017] Figure 1 This is a comparison of the relative expression levels of the LecRLK26 gene in different strains in Example 6; Figure 2 This is a comparison of the macroscopic characterization results of seed germination of different strains under NaCl stress in Example 7; Figure 3 This is a comparison of seed germination rates of different strains under NaCl stress in Example 7; Figure 4 This is a comparison of the macroscopic characterization results of leaves of different strains under NaCl stress in Example 8; Figure 5 This is a comparison chart of leaf water content and chlorophyll content of different strains under NaCl stress in Example 8. Detailed Implementation
[0018] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.
[0019] The tomato variety used in this embodiment of the invention is "Solanum lycopersicum L.cv.MicroTom", which was obtained from laboratory seed stock. The 8GWN intermediate vector and K303 vector were modified in the laboratory. *E. coli* DH5α and *Agrobacterium tumefaciens* GV3101 were preserved in our laboratory. The RNA extraction kit (Reagent) was provided by Solarbio; Taq DNA polymerase was provided by Chongqing Baoguang Biotechnology Co., Ltd.; the cDNA synthesis kit (PrimeScript™ RT reagent Kit with gDNA Eraser), restriction endonucleases, and PrimeSTAR high-fidelity DNA polymerase were all purchased from TaKaRa; the homologous recombinase (II One Step Cloning Kit-C112) was provided by Nanjing Novizan Biotechnology Co., Ltd.; GatewayLR Clanase was purchased from Thermo Fisher Scientific; the DNA extraction and purification kit (centrifuge column type) was a product of OMEGA; and all other reagents were commercially available analytical grade reagents.
[0020] Example 1: Cloning of the full-length LecRLK26 gene (1) Primer design: Using the cDNA sequence of tomato (Solanum lycopersicum L.) lectin receptor protein kinase LecRLK26 (Solyc10g006710.3.1) as a template, primers were designed to clone the full-length CDS sequence of the tomato LecRLK26 gene. At the same time, homologous recombination fragments were introduced at both ends of the primers according to the intermediate vector 8GWN. The primers were RK15-F and RK15-R, and the sequences are as follows: RK15-F: 5'-atacttccaactagtgcggccgctattaccacgacaaacacc-3' (SEQ ID No. 4); RK15-R: 5'-atggtcatcccgggacctgcaggtcgagcgtccaacaaagtga-3' (SEQ IDNo. 5); (2) PCR reaction: Total RNA was extracted from tomato leaves using an RNA extraction kit (TRIzol@Reagent), and cDNA was synthesized according to the instructions of a cDNA synthesis kit (PrimeScript RT reagent Kit with gDNA Eraser). Using the cDNA as a template and SEQ ID No. 4 and SEQ ID No. 5 as primers, the full-length LecRLK26 gene containing homologous fragments was amplified. The PCR reaction system is shown in Table 1.
[0021] Table 1
[0022] The PCR reaction program was as follows: pre-denaturation at 98℃ for 3 minutes; then denaturation at 98℃ for 15 seconds, annealing at 55℃ for 15 seconds, extension at 72℃ for 2 minutes, for a total of 35 cycles; and finally extension at 72℃ for 5 minutes.
[0023] (3) Preparation of recombinant plasmid 8GWN::35S-LecRLK26-GFP: The PCR amplification product was identified by agarose gel electrophoresis. The results showed that a target band of approximately 2500 bp was obtained. The amplification product was then purified using a DNA purification kit. The purified full-length fragment of the LecRLK26 gene was ligated to the 8GWN vector using homologous recombinase (ClonExpress@IIOneStep Cloning Kit-C112) to obtain the recombinant plasmid 8GWN::35S-LecRLK26-GFP. The plasmid was then transformed into DH5α Escherichia coli, and positive clones were identified. Sequencing was performed by Sangon Biotech Co., Ltd. The results showed that the full-length LecRLK26 gene is 2554 bp, and the nucleotide sequence is shown in SEQ ID No. 1.
[0024] Example 2: Construction of an overexpression vector for the LecRLK26 gene The 8GWN::35S-LecRLK26-GFP plasmid and the empty K303 plasmid were cloned using Gateway recombination cloning technology. The reaction system is as follows: In a 20 μL reaction system, 150 ng of 8GWN::35S-LecRLK26-GFP plasmid, 150 ng of K303 empty plasmid, 4 μL of 5×LR Clonase Reaction Buffer, 4 μL of LR Clonase enzyme mix, and TE buffer were added to a final volume of 20 μL. After mixing, the mixture was incubated at 25°C for 60 min. Then, 2 μL of Proteinase K solution was added and the reaction was terminated at 37°C for 10 min. 10 μL of the reaction product was then transformed into DH5α *E. coli*, and positive clones were identified. Single colonies were then selected for expansion culture, and plasmids were extracted and sequenced by Sangon Biotech Co., Ltd. If the sequence was correct, the K303::35S-LecRLK26-GFP vector was successfully constructed.
[0025] Example 3: Recombinant plasmid K303::35S-LecRLK26-GFP was transformed into Agrobacterium. (1) Construction of recombinant expression strain: 5 μL of plasmid was transformed into Agrobacterium tumefaciens GV3101 competent cells and plated on YEB solid medium (pH 7.2) containing 1.5% agar, 50 mg / L rifampin and 50 mg / L kanamycin. The cells were incubated upside down at 28±1℃ in the dark for 2-3 days until single colonies grew. 3-5 single colonies were picked for colony PCR. The K303::35S-LecRLK 26-GFP vector was detected using K303F and SC15R primers, and the product length was approximately 1600 bp.
[0026] The detection primer sequences for the K303::35S-LecRLK26-GFP plasmid are as follows: K303F: 5'-cgtcttgcgcactgatttga-3' (SEQ ID No. 6) SC15R: 5'-gctccactccatctaagaccattcc-3' (SEQ ID No. 7) The reaction system is shown in Table 2.
[0027] Table 2
[0028] The reaction conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 15 s, 55℃ annealing for 30 s, 72℃ extension for 30 s, 25 cycles, and 72℃ final extension for 5 min.
[0029] Positive colonies were selected and cultured in YEB liquid medium (pH 7.2) containing 50 mg / L rifampicin and 50 mg / L kanamycin at 28±1℃ in the dark and at 200 rpm for 1.5 days until the bacterial suspension was homogeneous and the OD600 was 0.8~1.0. Then, the bacterial suspension was centrifuged at 28±1℃ and the supernatant was discarded. The bacterial cells were washed with fresh YEB liquid medium and then resuspended in 100 mL of MS medium containing 3% sucrose at pH 5.8 to obtain the Agrobacterium-engineered bacterial suspension.
[0030] (2) Transformation of tomato cotyledons: Soak tomato seeds in 75% alcohol for 1 min and rinse with sterile water 6 times; then soak seeds in 1% (effective chlorine concentration) NaClO aqueous solution for 10 min and rinse with sterile water 7 times; soak seeds in sterile water for 4 h, sow on MS solid medium, and culture in a light incubator at 26℃ (16h light) / 18℃ (8h darkness) until the cotyledons are flattened. Cut off the flattened cotyledons to obtain tomato explants.
[0031] The cotyledons of tomato explants were cut and placed in MS solid medium containing 3 wt% sucrose, 0.8 wt% agar, 1 mg / L indoleacetic acid, 1 mg / L zeatin, and pH 5.8. After pre-culturing at 25°C (16 h light) / 20°C (8 h dark) for 1 day, they were immersed in Agrobacterium-mediated bacterial suspension for 15 min, then returned to the aforementioned MS solid medium containing 3 wt% sucrose, 1 wt% agar, 1 mg / L indoleacetic acid, 1 mg / L zeatin, and pH 5.8. They were co-cultured at 28±1°C in the dark for 48 hours, and then transferred to MS solid medium containing 3 wt% sucrose, 1 wt% agar, 1.0 mg / L indoleacetic acid, 1 mg / L zeatin, and 200 mg / L ticarcillin sodium. Transgenic tomato plants were cultured in MS solid medium containing potassium clavulanate, 120 mg / L amoxicillin sodium clavulanate, 100 mg / L kanamycin, and pH 5.8 at 25 ± 1 °C, with a photoperiod of 16 h / d and a light intensity of 3000–5000 lx until callus and resistant shoots appeared. The resistant shoots, which were 3–4 cm long, were cut off and transferred to MS solid medium containing 3 wt% sucrose, 1 wt% agar, 200 mg / L ticarcillin sodium clavulanate, 120 mg / L amoxicillin sodium clavulanate, and 50 mg / L kanamycin at pH 5.8. The plants were cultured in MS solid medium at 25 ± 1 °C, with a photoperiod of 16 h / d and a light intensity of 3000–5000 lx until roots appeared, thus obtaining transgenic tomato plants.
[0032] Example 4: Screening of positive transgenic tomato plants PCR detection to determine whether the LecRLK26 gene has been transferred into tomato plants Based on the gene sequence in the vector K303::35S-LecRLK26-GFP, the following specific detection primers were designed: 35SF: 5'-atgacgcacaatcccactatccttc-3' (SEQ ID No. 8); SC15R: 5'-gctccactccatctaagaccattcc-3' (SEQ ID No. 7).
[0033] Genomic DNA was extracted from both transgenic and non-transgenic tomato plants. Using the extracted genomic DNA as a template and the sequences shown in SEQ ID No. 7 and SEQ ID No. 8 as primers, PCR amplification was performed to detect whether the LecRLK26 gene had been transferred into the tomato plants. Agarose gel electrophoresis analysis of the PCR products showed a target DNA band at the 858 bp position in the PCR products of positive transgenic tomato plants. Sequencing confirmed that the band matched the LecRLK26 gene fragment sequence, confirming these plants as positive transgenic tomato plants. After two generations of selection, homozygous overexpressing transgenic tomato plants from the F1 generation were obtained.
[0034] Example 5: Preparation of gene knockout plants The accurate genome sequence of the tomato LecRLK26 gene (Solyc10g006710.3.1) was obtained from the public database Sol Genomics Network, with a focus on its exon regions, particularly the first or second exon near the 5′ end, as mutations in these regions are more likely to lead to loss of gene function. After obtaining the sequence, we will use the online tool CRISPR-P 2.0 from Huazhong Agricultural University for target design. The target gene sequence was input into the system, and tomato was selected as the reference genome. Priority was given to the target site with a high overall score, i.e., high cleavage efficiency and low off-target risk, namely 5′-ATGGAGTGGAGCACCAAACA-3′. After adding adapters with restriction enzyme sites (the same as those on the Cas9VL vector) to both ends, the corresponding oligonucleotide single strands were synthesized and annealed to form double-stranded DNA with sticky ends.
[0035] The Cas9VL vector was double-digested with enzymes, and the recovered sgRNA fragment was inserted into the vector via a ligation reaction. The ligation product was transformed into competent *E. coli* cells. Positive clones were initially screened by colony PCR, and the plasmid was extracted and subjected to Sanger sequencing to precisely verify that the sgRNA sequence was correctly inserted and free of any mutations. The correctly sequenced recombinant plasmid was named Cas9VL-LecRLK26-sgRNA. Subsequently, the plasmid was introduced into competent *Agrobacterium* GV1301 cells using a freeze-thaw method. The transformed bacterial culture was plated on LB agar containing rifampicin and kanamycin and incubated at 28°C for 2–3 days. After confirming the positive *Agrobacterium* clones again by colony PCR, they were selected for subsequent tomato genetic transformation experiments.
[0036] The genetic transformation experiment used the Micro-Tom tomato variety as the recipient material. Surface-sterilized Micro-Tom seeds were sown on 1 / 2 MS medium. Sterile seedlings with fully expanded cotyledons (7-10 days old) were used as explants, with cotyledon segments cut from them. Simultaneously, Agrobacterium GV1301 carrying the target vector was activated and cultured in LB medium containing acetylsyleugenol until the OD600 reached 0.6-0.8. The bacterial cells were collected by centrifugation and resuspended in the infection solution. The cotyledon explants were completely immersed in the infection solution for infection, then the surface bacterial solution was blotted dry with sterile filter paper, and the explants were inoculated onto co-culture medium and cultured in the dark at 22°C for 2-3 days. After co-culture, the explants were transferred to callus induction and differentiation medium containing hygromycin and termethin. After approximately 4-8 weeks of subculture and selection, resistant adventitious shoots differentiated from the explant cuts. When the adventitious buds grow to 1-2 cm in height, cut them off and insert them into a rooting medium containing hygromycin to induce rooting. After about 2 weeks, wash the medium off the regenerated plants with well-developed root systems, transplant them into nutrient soil, and cultivate them in a greenhouse. These plants are the T0 generation.
[0037] After obtaining T0 generation regenerated plants, genomic DNA was extracted from the leaves of the T0 generation plants. First, PCR detection was performed using primers with a vector-specific marker (Cas9 gene). Plants that amplified the expected band were identified as transgenic positive seedlings. Further, specific primers were designed targeting the sgRNA site for PCR amplification and sequencing. Analysis of the sequencing peaks near the target site was used to preliminarily determine whether editing had occurred in the plant. Seeds from individual T0 generation plants identified as transgenic positive and showing editing were harvested to obtain the T1 generation. The T1 generation was sown according to lines. All individual plants were first screened for hygromycin or tested for the Cas9 gene PCR to identify plants where the exogenous gene had been lost through genetic segregation (i.e., PCR negative). These candidate non-transgenic plants underwent target PCR and sequencing again to identify their mutation type (homozygous / heterozygous / wild-type). Cas9-negative plants with the target mutation in the T1 generation were selected for T2 generation seed harvesting. After planting the T2 generation, all individual plants were sequenced again for target sites. At this point, homozygous mutants should be able to be screened out in a Cas9-negative background. Finally, T3 generation seeds were harvested from the homozygous mutant plants selected from the T2 generation. After planting, individual plants were randomly selected for sequencing verification. If the mutation type of all tested individual plants was consistent with the T2 generation parents, it indicated that stable homozygous non-transgenic mutants rk26-1 and rk26-2 had been obtained. Sequencing results showed that they lost 1 and 2 bases, respectively, in the first exon region.
[0038] Example 6: qPCR detection of LecRLK26 gene expression in transgenic tomato plants Based on the LecRLK26 gene sequence and the tomato internal reference gene SlActin sequence, the following primers were designed: qRKC15-F: 5'-gacctcaggcagctatcggaatca-3' (SEQ ID No. 9); qRKC15-R: 5'-cg aagaatcaggcagactccaacc-3' (SEQ ID No. 10); qActin-F: 5'-tgggtgtgcctttctgaatg-3' (SEQ ID No. 11); qActin-R: 5'-gctaagaacgatggacctaatg-3' (SEQ ID No. 12).
[0039] Young leaves of the overexpressing transgenic tomato lines (OE-204, OE-214) selected in Example 4 and the gene knockout plants (rk26-1, rk26-2) selected in Example 5 were collected, and total RNA was extracted. The total RNA was reverse transcribed into cDNA. Then, using the obtained cDNA as a template, qPCR was performed with qRKC15-F and qRKC15-R, qActin-F and qActin-R as primers to detect the expression level of the LecRLK26 gene in the positive transgenic tomato plants. The results are as follows. Figure 1 As shown.
[0040] Depend on Figure 1 It can be seen that the expression level of the LecRLK26 gene in the overexpression transgenic tomato lines was significantly increased compared with the wild-type WT, that is, the overexpression of the LecRLK26 gene was successfully achieved; the expression level of the LecRLK26 gene in the gene knockout plants was effectively reduced compared with the wild-type WT, that is, the knockout of the LecRLK26 gene was successfully achieved.
[0041] Example 7: Identification of Salt and Alkali Tolerance During Seed Germination Mature seeds of wild-type tomato (WT), LecRK26 overexpression transgenic lines (OE#204, OE#214), and LecRK26 knockout lines (rk26-1, rk26-2) were selected, disinfected with 75% ethanol for 30 seconds, disinfected with 0.1% mercuric chloride for 10 minutes, and then rinsed 5 times with sterile water for later use.
[0042] After sterilization, the seeds were sown on MS solid medium containing 0 mM NaCl (normal control), 50 mM NaCl, and 100 mM NaCl, with 50 seeds sown per plate and three biological replicates. The plates were incubated in an artificial climate chamber at 25°C with 16 h of light and 8 h of darkness. After 14 days, the seed germination rate was calculated (based on the radicle breaking through the seed coat).
[0043] Seed germination results as follows Figure 2 As shown, by Figure 2 It can be seen that, under both 50 mM NaCl and 100 mM NaCl stress, the germination of the overexpressing transgenic lines (OE#204, OE#214) was better than that of WT, while the germination of the mutants was worse than that of WT.
[0044] Seed germination rate results after salt stress are as follows: Figure 3 As shown, where Figure 3 In the figure, 'a' represents a comparison of the germination rates of different strains under a 0 mM NaCl environment. Figure 3 b in the figure is a comparison of the germination rates of different strains under 50mM NaCl conditions; Figure 3 Figure c shows a comparison of the germination rates of different lines under 100 mM NaCl conditions. The results indicate that under normal (0 mM NaCl) conditions, there was no significant difference in seed germination rates among wild-type tomato (WT), LecRK26 overexpressing transgenic lines (OE#204, OE#214), and LecRK26 knockout lines (rk26-1, rk26-2). However, under both 50 mM and 100 mM NaCl stress, the germination rates of the overexpressing transgenic lines (OE#204, OE#214) were significantly higher than those of WT, while the germination rate of the mutants was significantly lower than that of WT. This suggests that overexpression of LecRK26 can significantly improve the tolerance of tomatoes to salt stress.
[0045] Example 8: Identification of salt and alkali tolerance in mature plants Micro-TOM tomato wild-type (WT), overexpression lines (OE#204, OE#214) and mutant lines ( rk26-1、 rk26-2 All of them were grown to maturity under the condition that the soil moisture content was maintained at 65%-75% of the field maximum water holding capacity.
[0046] Then stop watering. Water each culture pot daily with 10 mL of 50 mM NaCl solution, and weigh the soil to maintain a moisture content of 65%-75% (add water if necessary). Continue this stress for 7 days, then observe the growth of the tomatoes. Figure 4 .
[0047] Depend on Figure 4 It is evident that after 7 consecutive days of salt stress, the overexpression lines (OE#204, OE#214) showed good growth, with no drooping or wilting leaves and a lower degree of leaf yellowing; while the mutant lines ( rk26-1, rk26-2 The leaves of the WT and WT strains drooped and wilted significantly, with a deeper degree of yellowing, especially in the mutant strains.
[0048] Further measurements of leaf water content and chlorophyll content yielded the following results: Figure 5 As shown, where, Figure 5 In the figure, 'a' represents a comparison of leaf water content among different strains. Figure 5 In the figure, b is a comparison of chlorophyll a content in leaves of different strains. Figure 5 In the figure, 'c' represents a comparison of chlorophyll b content in leaves of different strains. Figure 5 The figure 'd' represents a comparison of the total chlorophyll content in leaves of different strains. Figure 5 As can be seen from a, after 7 days of continuous stress with 50 mM NaCl solution, the water content of the overexpression lines (OE#204, OE#214) was significantly higher than that of the WT; while the water content of the mutant lines ( rk26-1, rk26-2 The water content of the overexpression lines (OE#204, OE#214) was significantly lower than that of the WT line. After stress, the contents of chlorophyll a, chlorophyll b, and total chlorophyll were all significantly higher in the overexpression lines than in the WT line; while the mutant lines (…)… rk26-1, rk26- 2 All were significantly lower than WT ( Figure 5 (b, 5c, and 5d). This demonstrates that overexpression of LecRK26 significantly improves the tolerance of tomatoes to salt stress.
Claims
1. The application of the LecRK26 gene in regulating the salt and alkali tolerance of tomatoes, characterized by, The LecRK26 gene is derived from tomato, and its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.
2. The regulation of tomato salt and alkali tolerance specifically refers to improving the salt and alkali tolerance of tomatoes, that is, by enhancing the expression of the LecRK26 gene, the survival ability and stress resistance of tomatoes under drought stress can be significantly improved.
2. The application of the LecRK26 gene according to claim 1 in regulating the salt and alkali tolerance of tomatoes, characterized in that, The application involves transferring the LecRK26 gene into tomato plants to overexpress the LecRK26 gene. The method for transferring the LecRK26 gene into tomato plants is one of the following: Agrobacterium tumefaciens infection method, gene gun method, or polyethylene glycol method.
3. The application of the LecRK26 gene according to claim 1 in regulating the salt and alkali tolerance of tomatoes, characterized in that, The application is to cultivate transgenic tomato varieties with enhanced salt and alkali tolerance.
4. A recombinant expression vector for regulating the salt and alkali tolerance of tomatoes, characterized in that, The nucleotide sequence of the recombinant expression vector is shown in SEQ ID NO.
3.
5. A transgenic cell line or engineered bacterium containing the recombinant expression vector of claim 4.
6. A method for improving the salt and alkali tolerance of tomatoes, characterized in that, Includes the following steps: 1) Construct a recombinant expression vector containing the LecRK26 gene as described in claim 1; 2) Transform the recombinant expression vector into tomato recipient cells to obtain transgenic tomato cells; 3) Cultivate the transgenic tomato cells into complete plants and screen for transgenic tomato plants that overexpress the LecRK26 gene and have enhanced salt and alkali tolerance.
7. The method according to claim 6, characterized in that, The enhanced salt and alkali tolerance described in step 3) is manifested in the following ways: compared with wild tomatoes, transgenic tomatoes have a 25% to 40% higher seed germination rate and a 20% to 35% higher relative germination rate under drought stress, a 15% to 28% lower number of leaf stomata, and a 20% to 35% lower leaf water loss rate.