Application of Tomato SlbZIP10 Gene in Improving High Temperature Tolerance of Tomato

Abstract

The present invention belongs to the technical field of plant molecular biology, and specifically relates to the application of tomato SlbZIP10 gene in improving high temperature tolerance of tomatoes. In the present invention, the SlbZIP10 gene was screened, and overexpression plants and knockout plants of the SlbZIP10 gene were successfully created. It was found that after high temperature treatment, the plants overexpressing the SlbZIP10 gene wilted less. 2 ·‑ and H 2 O 2 The accumulation of O 2 ·‑ and H 2 O 2 The accumulation of SlbZIP10 gene is less, indicating that the SlbZIP10 gene positively regulates the heat resistance of tomatoes. The present invention provides gene resources for breeding new high temperature resistant tomato varieties, has good potential application value, and lays a theoretical foundation for studying the mechanism of tomato plants responding to adverse signals and the molecular mechanism of tolerating high temperature environments.

Description

Technical Field

[0001] The invention belongs to the technical field of plant molecular biology, and particularly relates to application of a tomato SlbZIP10 gene in improving high temperature tolerance of tomatoes. Background Art

[0002] Tomato (Solanum lycopersicum L.) is a herbaceous plant of the Solanaceae family, native to South America. It likes light and warmth and can be cultivated in both open fields and facilities. Tomato fruits are rich in nutrients, vitamins and trace mineral nutrients. They can be eaten fresh or used as cooking ingredients. They can also be processed into light industrial products such as tomato sauce, tomato sauce, and tomato juice, and are deeply loved by the people.

[0003] When tomatoes are exposed to temperatures above 35°C during their vegetative growth and flowering and fruiting stages, their growth, development, yield and quality will be greatly affected, mainly manifested in growth stagnation, falling flowers and fruits or deformed fruits, blocked lycopene synthesis, and even death of the entire plant. Currently, the growth temperature of tomatoes in summer facility cultivation can reach above 40°C, making them vulnerable to high temperature stress.

[0004] The bZIP family is a class of transcription factors containing a basic leucine zipper domain, which plays an important role in plant growth and development and abiotic stress response. In recent years, the biological functions of plant bZIP genes have been gradually revealed. For example, silencing SlbZIP1 in tomato reduced the drought tolerance of transgenic tomato plants under drought stress, and the expression of abscisic acid biosynthesis and signal transduction genes in the silenced plants was downregulated. Overexpression of IbbZIP1 in sweet potato enhanced the salt tolerance of transgenic Arabidopsis plants, and the expression of genes related to proline synthesis and reactive oxygen scavenging was upregulated in the overexpressed plants. The maize bzip60 mutant reduced the heat shock response under high temperature stress and inhibited the up-regulated expression of a series of heat shock proteins. However, the role of tomato bZIP genes in high temperature stress is still poorly understood. Summary of the invention

[0005] The present invention provides a new use of the SlbZIP10 gene, and specifically relates to the use of the tomato SlbZIP10 gene in improving the high temperature tolerance of tomatoes.

[0006] In order to solve the above technical problems, the present invention adopts the following technical solutions.

[0007] The present invention provides the use of SlbZIP10 gene or its related biological materials in improving high temperature tolerance of tomatoes:

[0008] a. The protein encoded by the SlbZIP10 gene, the amino acid sequence of which is shown in SEQ ID NO.4;

[0009] b. a recombinant plasmid comprising the SlbZIP10 gene;

[0010] c. a recombinant microbial strain comprising the SlbZIP10 gene;

[0011] The nucleotide sequence of the SlbZIP10 gene is shown in SEQ ID NO.3.

[0012] In some technical schemes of the present invention, the SlbZIP10 gene is overexpressed to increase superoxide dismutase activity, peroxide dismutase activity and conductivity in the plant, and reduce the accumulation of hydrogen peroxide, superoxide anion and malondialdehyde content in the plant.

[0013] In some technical solutions of the present invention, the original vector of the recombinant plasmid includes: pHellsgate2, pCAMBIA1300, pCAMBIA2300, and pCAMBIA3300.

[0014] In some technical solutions of the present invention, the starting strains of the recombinant microorganism include Escherichia coli and Agrobacterium.

[0015] The present invention also provides the application of the SlbZIP10 gene or related biological materials in cultivating high temperature resistant tomatoes.

[0016] In some technical solutions of the present invention, the biomaterial comprises:

[0017] a. The protein encoded by the SlbZIP10 gene, the amino acid sequence of which is shown in SEQ ID NO.4;

[0018] b. a recombinant plasmid comprising the SlbZIP10 gene;

[0019] c. a recombinant microbial strain comprising the SlbZIP10 gene;

[0020] The nucleotide sequence of the SlbZIP10 gene is shown in SEQ ID NO.3.

[0021] The present invention also provides a method for improving the high temperature tolerance of plants, which increases the expression level of the SlbZIP10 gene in plants to obtain high temperature resistant plants.

[0022] Some technical schemes of the present invention include the steps of transferring the SlbZIP10 gene into plants to construct plants that highly express the SlbZIP10 gene.

[0023] In some technical solutions of the present invention, the method of transfer includes Agrobacterium-mediated method.

[0024] In some technical solutions of the present invention, the plant is tomato.

[0025] In some technical schemes of the present invention, tomato genomic DNA is used as a template, and the SlbZIP10 gene is amplified using specific primers with sequences such as those shown in SEQ ID NO.1-2, and the amplified product is recovered, and the amplified SlbZIP10 gene fragment is connected to the plant expression vector pHellsgate2 vector to obtain an overexpression vector of the SlbZIP10 gene; the overexpression vector is used to transform Agrobacterium, and then the transformed Agrobacterium bacterial liquid is used to infect tomato cotyledons, and the infected tomato cotyledons are cultured to obtain tomato plants overexpressing the SlbZIP10 gene.

[0026] In some technical solutions of the present invention, the OD value of the transformed Agrobacterium bacterial solution is 0.5-0.7.

[0027] In some technical solutions of the present invention, the OD value of the transformed Agrobacterium bacterial solution is 0.6.

[0028] In some technical solutions of the present invention, 25-35 mL of the transformed Agrobacterium liquid is used to infect tomato cotyledons.

[0029] In some technical solutions of the present invention, 30 mL of the transformed Agrobacterium liquid is used to infect tomato cotyledons.

[0030] In some technical solutions of the present invention, the plant expression vector is pHellsgate2.

[0031] In some technical solutions of the present invention, the Agrobacterium is Agrobacterium GV3101.

[0032] In some technical schemes of the present invention, the infected tomato cotyledons are subjected to callus induction using a tomato screening medium, adventitious bud differentiation using a subculture medium, and rooting culture using a rooting medium, to finally obtain rooted tissue culture seedlings.

[0033] In some technical schemes of the present invention, the tomato screening culture medium comprises raw materials of the following concentrations: 4-5 g / L MS powder, 28-32 g / L sucrose, 7-8 g / L agar, 0.08-0.12 mg / L IAA, 1.8-1.2 mg / L zeatin, 98-102 mg / L kana, and 358-362 mg / L TMT.

[0034] In some technical schemes of the present invention, the tomato screening culture medium comprises raw materials of the following concentrations: 4.43 g / L MS powder, 30 g / L sucrose, 7.5 g / L agar, 0.1 mg / L IAA, 2 mg / L zeatin, 100 mg / L kana, and 360 mg / L TMT.

[0035] In some technical schemes of the present invention, the tomato subculture culture medium comprises raw materials of the following concentrations: 4-5 g / L MS powder, 28-32 g / L sucrose, 7-8 g / L agar, 0.18-0.22 mg / L zeatin, 98-102 mg / L kana, and 358-362 mg / L TMT.

[0036] In some technical schemes of the present invention, the tomato subculture culture medium includes raw materials with the following concentrations: 4.43 g / L MS powder, 30 g / L sucrose, 7.5 g / L agar, 0.2 mg / L zeatin, 100 mg / L kana, and 360 mg / L TMT.

[0037] In some technical schemes of the present invention, the rooting medium includes raw materials with the following concentrations: 4-5g / LMS powder, 28-32g / L sucrose, 1.8-2.2g / L agar, 48-52mg / L kana, and 358-362mg / L TMT.

[0038] In some technical schemes of the present invention, the rooting medium includes raw materials with the following concentrations: 4.43 g / L MS powder, 30 g / L sucrose, 2.0 g / L agar, 50 mg / L kana, and 360 mg / L TMT.

[0039] Compared with the prior art, the present invention has the following beneficial effects: The present invention mines differentially expressed genes from the transcriptome of high temperature treatment and finds that the SlbZIP10 gene is strongly upregulated by high temperature treatment, and it is speculated that it plays an important role in high temperature response. The present invention successfully creates overexpression plants and knockout plants of the SlbZIP10 gene, and finds that after high temperature treatment, the plants overexpressing the SlbZIP10 gene wilt less, and O2 ·- and H2O2 accumulation was low, and the overexpression plants had less O2 ·- The accumulation of H2O2 is low, indicating that the SlbZIP10 gene positively regulates the heat resistance of tomatoes. The present invention provides gene resources for breeding new high-temperature resistant tomato varieties, has good potential application value, and lays a theoretical foundation for studying the mechanism of tomato plants responding to adverse signals and the molecular mechanism of tolerance to adverse environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0040] Figure 1 This is the result of detecting the expression level of SlbZIP10 in tomato leaves of wild-type tomato variety M82 treated at 42°C for different periods of time.

[0041] Figure 2 This is the expression level detection diagram of SlbZIP10 gene in wild-type plants and T0 generation overexpression plants.

[0042] Figure 3This is a diagram showing the sequencing results of the T0 generation knockout plants obtained in Example 4; wherein, Figure 3 A represents the gene editing site map of the T0 generation knockout plants; Figure 3 B represents the genetic difference map between the wild-type plant, the knockout line KO-2 and the knockout line KO-5; Figure 3 C represents the amino acid difference map between the wild-type plant, the knockout line KO-2 and the knockout line KO-5.

[0043] Figure 4 The high temperature stress phenotype diagram of the overexpression strain, knockout strain and wild-type plant AC under normal temperature and high temperature treatment in Example 5; among them, OE-3 and OE-9 are overexpression strains, and KO-2 and KO-5 are knockout strains.

[0044] Figure 5 The results of DAB and NBT staining of leaves of overexpression strains, knockout strains and wild-type plants AC under normal temperature and high temperature treatment in Example 5 are shown; among them, OE-3 and OE-9 are overexpression strains, and KO-2 and KO-5 are knockout strains.

[0045] Figure 6 The results of the high temperature tolerance test of the overexpression strain, knockout strain and wild-type plant AC under normal temperature and high temperature treatment in Example 5 are shown in FIG. Figure 6 A represents the conductivity measurement result diagram;

[0046] Figure 6 B represents the result of MDA content determination; Figure 6 C represents the result of SOD enzyme activity determination;

[0047] Figure 6 D represents the result of POD enzyme activity assay; among them, OE-3 and OE-9 are overexpression strains, and KO-2 and KO-5 are knockout strains. DETAILED DESCRIPTION

[0048] The present invention is described in detail below in conjunction with specific examples, but it should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0049] When tomatoes are exposed to high temperatures above 35°C during their vegetative growth and flowering and fruiting periods, their growth, development, yield and quality will be greatly affected, mainly manifested as growth stagnation, falling flowers and fruits or producing deformed fruits, blocked lycopene synthesis, and even death of the entire plant. Currently, the growth temperature of tomatoes in summer facility cultivation can reach above 40°C, making them vulnerable to high temperature stress. The bZIP family is a class of transcription factors containing a basic leucine zipper domain, which plays an important role in plant growth and development and abiotic stress response.

[0050] The present invention provides an application of the SlbZIP10 gene in improving high temperature tolerance of tomatoes. In the present invention, overexpression plants and knockout plants of the SlbZIP10 gene were successfully constructed, and it was found that after high temperature treatment, the plants overexpressing the SlbZIP10 gene wilted less, and the temperature of O2 ·- and H2O2 accumulation was low, and the overexpression plants had less O2 ·- and H2O2 accumulation was less, indicating that the SlbZIP10 gene positively regulates tomato heat resistance.

[0051] Example 1: Sequence and cloning of the SlbZIP10 gene

[0052] Screening of SlbZIP10 gene:

[0053] Differentially expressed genes were mined from the transcriptome under high temperature treatment, and SlbZIP10 was strongly upregulated by high temperature treatment, which is speculated to play an important role in high temperature response.

[0054] Methods: Wild-type tomato variety M82 that grew normally to 1 month old was used for high temperature treatment, 42℃, 16h light / 8h dark. Tomato leaves were sampled at 0h, 2h, 4h, 12h, and 24h for transcriptome sequencing analysis.

[0055] Results: Figure 1 As shown, analysis of the transcriptome results showed that the FPKM value of SlbZIP10 expression at high temperature treatment for 2 h was 3.25 times that at 0 h, 2.86 times at 4 h, 10.61 times at 12 h, and 14.08 times at 24 h.

[0056] The SlbZIP10 gene does not have introns and can be cloned from tomato genomic DNA. According to the SlbZIP10 gene sequence, specific primers OE-SlbZIP10-F and OE-SlbZIP10-R were designed, and the sequences are shown in SEQ ID NO.1-2. The SlbZIP10 gene fragment was amplified using a high-fidelity DNA polymerase (Nanjing Novozyme Super-Fidelity DNA Polymerase, catalog number: P501-d1), and the nucleotide sequence of the SlbZIP10 gene is shown in SEQ ID NO.3, and the amino acid sequence of the SlbZIP10 protein is shown in SEQ ID NO.4. The specific primers, SlbZIP10 gene and SlbZIP10 protein sequences are shown in Table 1.

[0057] PCR amplification conditions: high-fidelity DNA polymerase 1 μL, buffer 12.5 μL, dNTP (10 mM) 0.5 μL, forward primer (10 μM) 1 μL, reverse primer (10 μM) 1 μL, template (tomato genomic DNA) 1 μL, sterile water to 25 μL.

[0058] PCR amplification system: pre-denaturation at 98°C for 3 min; 98°C for 10 s; 57°C for 30 s; 72°C for 40 s, 35 cycles; 72°C for 5 min.

[0059] Table 1 Specific primers, SlbZIP10 gene and SlbZIP10 protein sequences

[0060]

[0061]

[0062] Example 2: Construction of SlbZIP10 gene overexpression vector

[0063] The SlbZIP10 gene does not have introns, and the SlbZIP10 gene can be cloned from tomato genomic DNA. As shown in Table 1, specific primers OE-SlbZIP10-F and OE-SlbZIP10-R were designed according to the SlbZIP10 gene sequence, and the sequence is shown in SEQ ID NO.1-2. The SlbZIP10 gene fragment was amplified using a high-fidelity DNA polymerase (Nanjing Novagene Super-Fidelity DNA Polymerase, Catalog No.: P501-d1), and then the pHellsgate2 vector was double-digested with Xba I and Xho I to linearize it, and the SlbZIP10 gene fragment was connected to the linearized vector using a homologous recombinase (Nanjing Novagene ClonExpress Ultra One Step Cloning Kit, Catalog No.: C115-01) to obtain a 35S promoter-driven overexpression vector. The recombinant plasmid was sent to Xi'an Qingke Company for sequencing, and the detection primer sequences were shown in SEQ ID NO.5-6. The results showed that they were completely consistent with the sequence published in the tomato genome, the nucleotide sequence of SlbZIP10 was shown in SEQ ID NO.3, and the encoded amino acid sequence was shown in SEQ ID NO.4. This indicated that the construction of the SlbZIP10 gene overexpression vector was successful.

[0064] Table 1 Overexpression vector construction primers and detection primers

[0065] name sequence serial number Detection Primer-F 5'-GACGCACAATCCCACTATCC-3' SEQ ID NO.5 Detection Primer-R 5'-TCGACTAATATTGTCATCACGTCAG-3' SEQ ID NO.6

[0066] Example 3: Construction of CRISPR / Cas9 vector containing SlbZIP10 gene-specific sgRNA

[0067] The SlbZIP10 gene-specific knockout target was designed through the CRISPR-P website (http: / / crispr.hzau.edu.cn / CRISPR / ), and the sequence was shown in SEQ ID NO.7 as TTCAGCAATTAATGGATCAG. After the target sequence was annealed, it was connected with the AtU6-sgRNA-AtUBQ-Cas9 vector linearized by BbsI restriction enzyme digestion, and the plasmid was extracted after transformation of Escherichia coli. The constructed plasmid and pCAMBIA2301 vector were double-digested with EcoRI and HindIII, and the plasmid was extracted after transformation of Escherichia coli after ligation. The final vector was sent to Xi'an Qingke Company for sequencing.

[0068] Table 2 Primers for constructing SlbZIP10 gene knockout vector and detection primers

[0069]

[0070]

[0071] Example 4: Obtaining tomato SlbZIP10 gene overexpression plants and knockout plants

[0072] The correctly sequenced overexpression vector and knockout vector were transformed into Agrobacterium GV3101. The tomato cotyledons were infected with 30 mL of Agrobacterium GV3101 culture solution transformed with the overexpression vector and knockout vector at OD 0.6, and then callus induction was performed using tomato screening medium, adventitious bud differentiation was performed using subculture medium, and rooting culture was performed using rooting medium, and finally rooted tissue culture seedlings were obtained.

[0073] Tomato screening medium: Weigh 4.43g MS powder (Phytotech, M519), dissolve in 800mL ultrapure water, add 30g sucrose, and dilute to 1000mL. Adjust pH to 5.8, add 7.5g agar, autoclave at 121℃ for 21min, and add 0.1mg / L IAA + 2mg / L zeatin (ZR) + 100mg / L kana + 360mg / L TMT after cooling.

[0074] Callus induction culture conditions: 16h light / 8h dark, 25 degrees, culture for 14 days.

[0075] Tomato subculture medium: Weigh 4.43g MS powder (Phytotech, M519), dissolve in 800mL ultrapure water, add 30g sucrose, and dilute to 1000mL. Adjust pH to 5.8, add 7.5g agar, autoclave at 121℃ for 21min, and add antibiotics 0.2mg / L zeatin (ZR) + 100mg / L kana + 360mg / L TMT after cooling.

[0076] Adventitious bud differentiation culture conditions: 16h light / 8h dark, 25 degrees, culture for 14 days.

[0077] Rooting medium: Weigh 4.43 g MS powder (Phytotech, M519), dissolve in 800 mL ultrapure water, add 30 g sucrose, 2 mg / L IBA, and dilute to 1000 mL. Adjust pH to 5.8, add 7.5 g agar, autoclave at 121°C for 21 min, and add 360 mg / L TMT+50 mg / L kana antibiotics after cooling.

[0078] Rooting culture conditions: 16h light / 8h dark, 25 degrees, culture for 21 days.

[0079] The overexpressed plants were tested using primers with sequences as shown in SEQ ID NO.5-6. The target site sequences of the candidate knockout plants were amplified by PCR using Taq enzyme, and the primer sequences were as shown in SEQ ID NO.12-13, and sent to Xi'an Qingke Company for sequencing to detect whether editing occurred.

[0080] Real-time fluorescence quantitative PCR was used to detect the expression level of the overexpressing plants, and the primer sequences were shown in SEQ ID NO.14-15.

[0081] Table 3 Fluorescence quantitative PCR primers and knockout plant editing amplification detection primers

[0082]

[0083] The results are as follows Figure 2 As shown, the results of real-time fluorescence quantitative PCR analysis showed that the expression levels of the SlbZIP10 gene in the OE-3 and OE-9 lines were approximately 13 times and 8 times that of the wild-type AC plants, respectively, and two independent overexpression plants were successfully obtained for subsequent research.

[0084] like Figure 3 As shown in Figure 5A-C, the wild-type plant SlbZIP10 gene structure was found to have only one exon and no introns. Sequencing results showed that KO-2 had one base inserted compared to the wild-type plant, causing an amino acid frameshift and premature translation termination, and KO-5 had one base missing compared to the wild-type plant, also causing an amino acid frameshift and premature translation termination, indicating that two independent knockout plants, KO-2 and KO-5, were successfully obtained.

[0085] Example 5: Detection of high temperature tolerance of tomato SlbZIP10 gene overexpression strains and knockout strains

[0086] Wild-type tomato AC, SlbZIP10 gene overexpression plants and SlbZIP10 gene knockout plants grown to five leaves and one heart were treated in a light incubator at 42°C for 0h, 36h and 48h, and the high temperature stress phenotypes of each strain were observed. Hydrogen peroxide and superoxide anions in leaves were detected by DAB and NBT staining, and conductivity, malondialdehyde (MDA), superoxide dismutase activity (SOD) and peroxidase activity (POD) were measured.

[0087] The phenotypic observation results of each strain are as follows Figure 4 As shown in Figure 2, before high temperature treatment, the wild type, SlbZIP10 overexpression plants and SlbZIP10 knockout plants all grew well. However, after 36 h of high temperature treatment, the SlbZIP10 overexpression plants wilted slightly, the wild type plants wilted more severely, and the SlbZIP10 knockout plants wilted the most severely, with dry leaves. Figure 5As shown, O2 ·- and H2O2 are two major reactive oxygen species (ROS) molecules that usually accumulate under abiotic stress. ·- ) and DAB staining (to detect H2O2) were used to detect the O2 in AC, overexpression plants and knockout plants under control and high temperature conditions. ·- and H2O2 accumulation. Under control conditions, O2 ·- There was no difference in H2O2 accumulation. Under high temperature treatment, knockout plants had no significant difference in H2O2 accumulation. ·- and H2O2 accumulation was higher, and the overexpression plants had ·- The accumulation of SOD and POD in wild-type plants was lower than that in wild-type plants. Figure 6 As shown in AD, the overexpression plants showed increased activities of antioxidant enzymes SOD and POD and reduced membrane damage (manifested by reduced electrolyte leakage and reduced malondialdehyde content) under high temperature stress. The above results indicate that the SlbZIP10 gene positively regulates tomato heat resistance.

[0088] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0089] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. Application of SlbZIP10 gene or its related biological materials in improving high temperature tolerance of tomatoes, characterized in that: The biological material comprises: a. The protein encoded by the SlbZIP10 gene, the amino acid sequence of which is shown in SEQ ID NO.4; b. a recombinant plasmid comprising the SlbZIP10 gene; c. a recombinant microbial strain comprising the SlbZIP10 gene; The nucleotide sequence of the SlbZIP10 gene is shown in SEQ ID NO.

3.

2. The use according to claim 1, characterized in that Overexpression of the SlbZIP10 gene increased the superoxide dismutase activity and peroxide dismutase activity in the plants and reduced the accumulation of hydrogen peroxide, electrical conductivity, superoxide anion, and malondialdehyde content in the plants.

3. The use according to claim 1, characterized in that The original vectors of the recombinant plasmid include: pHellsgate2, pCAMBIA1300, pCAMBIA2300, and pCAMBIA3300.

4. The use according to claim 1, characterized in that The starting strains of the recombinant microorganism include Escherichia coli and Agrobacterium.

5. Use of the SlbZIP10 gene or its related biological materials as claimed in claim 1 in cultivating high temperature resistant tomatoes.

6. A method for improving high temperature tolerance of plants, characterized in that: The expression level of the SlbZIP10 gene in claim 1 is increased in tomatoes to obtain high temperature resistant tomato plants.

7. The method according to claim 6, characterized in that The method comprises the steps of transferring the SlbZIP10 gene into plants to construct plants with high expression of the SlbZIP10 gene.

8. The method according to claim 7, characterized in that The methods of transfer include Agrobacterium-mediated transfer.

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