SlHAT5 gene and its application in tomato's resistance to high temperature stress

Abstract

The present invention belongs to the field of genetic engineering technology, and specifically relates to a SlHAT5 gene and its application in tomato resistance to high temperature stress. The present invention studies the tomato HD-ZIP family transcription factor SlHAT5 strain, observes the phenotypic differences between transgenic plants and wild-type plants under high temperature stress, and screens the genes and their interacting proteins that induce their expression through the differences in physiological indicators and related gene expression levels of the treated samples, clarifies the mechanism of action of the gene in tomato high temperature stress feedback, and finds that the SlHAT5 gene negatively regulates the tolerance of tomatoes to high temperature stress. Overexpression of SlHAT5 can reduce the plant's resistance to high temperature and antioxidant levels, and at the same time participates in the ABA pathway, and under high temperature stress, by regulating the genes related to the above pathways, the sensitivity of tomato plants to high temperature stress is increased. This patent lays the foundation for the study of high temperature stress feedback of tomatoes and the cultivation of high temperature-resistant tomato germplasm resources.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a SlHAT5 gene and application thereof in tomato resistance to high temperature stress. Background Art

[0002] As the greenhouse effect intensifies, the frequency of extreme weather events around the world is increasing. High temperatures, a common natural disaster, are experiencing an increasing trend in both severity and frequency. Heat stress, a type of abiotic stress, is a significant challenge to plant survival. Severe heat stress can cause physiological and biochemical damage to plants, damaging their cells and disrupting their normal molecular regulatory mechanisms, resulting in irreversible harm. Therefore, studying plant adaptation and resistance mechanisms to heat stress, as a key component of plant stress tolerance research, has become a hot topic.

[0003] As one of the most widely grown vegetables in the world, tomato's yield and quality are the focus of people's attention. Existing studies have shown that high temperature stress has a very serious impact on the normal growth of tomatoes. Therefore, studying the high temperature resistance of tomatoes, one of the model plants for gene function research in higher plants, not only helps people discover and clarify the feedback mechanism of high temperature stress in their bodies, but also lays a certain molecular foundation for breeding new high temperature resistant tomato varieties. Studies have found that HD-ZIP transcription factors play an indispensable role in plant growth and development and various stress feedback. In tomatoes, a large number of transcription factors have been confirmed to be involved in the plant's response to high temperature stress, but the HD-ZIP family, as an important type of transcriptional regulatory factor, has rarely been reported to play an important role in high temperature stress.

[0004] Many studies have found that HD-ZIP family transcription factors are closely related to high temperature stress in plants. For example, in maize, overexpression of the sunflower Hah-4 gene effectively enhances its resistance to heat stress (Colombo et al., 2017). LpHOX21 is upregulated in heat-tolerant perennial ryegrass varieties, suggesting that it may be involved in enhancing ryegrass's resistance to heat stress (Wang et al., 2019). Screening of HD-ZIP family genes in cucumber revealed that two members of this subfamily, CsHDZ02 and CsHDZ33, were induced to express in response to heat stress (Sharif et al., 2020). Transgenic soybean plants overexpressing the HaHB4 gene exhibited improved heat stress tolerance by activating the transcriptional activity of the heat shock proteins AT-HSC70-1, AT-HSFB2A, and Hsp81.4. This reduced heat damage during pod setting and resulted in higher yields (Ribichich et al., 2020).

[0005] In the early stages of this patent's screening for transcription factors that respond to stress, the transcription factor SlHAT5 was identified. Further research revealed that SlHAT5, a member of the HD-ZIP family, plays a crucial role in tomato's response to high temperature stress. Based on this, this patent explores the mechanism by which the HD-ZIP transcription factor SlHAT5 contributes to tomato's heat resistance through a variety of methods, including bioinformatics analysis, transgenic technology, physiological and biochemical assays, and transcriptional analysis. Summary of the Invention

[0006] One of the objectives of the present invention is to provide an HD-ZIP transcription factor SlHAT5 gene that improves the high temperature resistance of tomatoes. The present invention found that the SlHAT5 gene overexpression strain can significantly increase the sensitivity of tomato plants to high temperature stress.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] The HD-ZIP transcription factor SlHAT5 gene improves the high temperature resistance of tomatoes. The amino acid sequence of the SlHAT5 gene is shown in SEQ ID NO.1.

[0009] Furthermore, the nucleotide sequence of the SlHAT5 gene is shown in SEQ ID NO.2.

[0010] Furthermore, the expression of the SlHAT5 gene is induced by hormones; the hormones include any one or more of ABA, Eth, JA, GA, and SA.

[0011] Furthermore, the expression level of the SlHAT5 gene was significantly downregulated after 12 hours of Eth treatment; significantly upregulated after 6 hours of ABA treatment; significantly upregulated after 6 hours of JA treatment, and significantly downregulated after 12 hours of JA treatment; when treated with GA for 1 hour and 24 hours, the expression level of the SlHAT5 gene was significantly higher than that of the wild type, and was temporarily downregulated at 12 hours; when treated with SA for 1 hour, the expression level of the SlHAT5 gene was significantly upregulated, and the upward trend gradually slowed down over time, and the expression level was downregulated at 24 hours.

[0012] Furthermore, the Eth inhibits the expression of S1HAT5.

[0013] Furthermore, the 3000bp promoter of the SlHAT5 gene contains many different types of cis-acting elements, including light-responsive cis-acting elements, cis-acting elements involved in defense and stress responses, and hormone-responsive elements such as salicylic acid, jasmonic acid, abscisic acid, and auxin.

[0014] Furthermore, the expression level of the SlHAT5 gene is highest in various parts of the floral organs (especially petals), while the expression level of the SlHAT5 gene is lower in roots, stems, leaves of different maturity and fruits at different stages, about 1 / 100 of the expression level in flowers.

[0015] A second object of the present invention is to provide a gene that regulates the response of the SlHAT5 gene to plant adverse stress.

[0016] To achieve the above object, the present invention adopts the following technical solutions:

[0017] The genes that regulate the SlHAT5 gene's response to plant stress are SlPLL (NCBI accession number: XM_004233304), SlPC (NCBI accession number: NM_001309263), SlDD2 (NCBI accession number: XM_004230247), SlRBCS4 (NCBI accession number: NM_001309210), SlGAPDH (NCBI accession number: XM_004236801) and DnaJ (NCBI accession number: XM_004239689).

[0018] The third object of the present invention is to provide a screening method for regulating the gene described in the second object.

[0019] To achieve the above object, the present invention adopts the following technical solutions:

[0020] A method for screening genes that regulate the response of the SlHAT5 gene to plant adverse stress comprises: using SlHAT5 as a bait protein, utilizing yeast one-hybrid technology, screening a homogenized yeast library, screening for genes that regulate the response of the SlHAT5 gene to plant adverse stress, and performing yeast point-to-point verification.

[0021] A fourth object of the present invention is to provide an interacting protein of the SlHAT5 gene.

[0022] To achieve the above object, the present invention adopts the following technical solutions:

[0023] The interacting proteins of the SlHAT5 gene are SlVDAC (NCBI accession number: XM_004233301), SlCP3L (NCBI accession number: XM_004243659), Dp-1 (NCBI accession number: XM_004249087), SlNBRL1 (NCBI accession number: XM_004241451), SlBELL6 (NCBI accession number: XM_026028738) and DnaK (NCBI accession number: XM_004230397).

[0024] A fifth object of the present invention is to provide a method for screening the interacting proteins.

[0025] To achieve the above object, the present invention adopts the following technical solutions:

[0026] The method for screening interacting proteins comprises the following steps: using SlHAT5 as bait protein, utilizing yeast two-hybrid technology, screening a homogenized yeast library, screening for interacting proteins of the SlHAT5 gene, and performing yeast point-to-point verification.

[0027] A sixth object of the present invention is to provide a method for preparing a tomato strain that overexpresses the SlHAT5 gene.

[0028] To achieve the above object, the present invention adopts the following technical solutions:

[0029] A method for preparing a tomato strain overexpressing the SlHAT5 gene comprises the following steps:

[0030] S1: Construction of overexpression vector: The SlHAT5 gene and the pVCT2024 vector were double-digested with restriction endonucleases BamHI and Sad, and the two digested fragments were ligated with T4 ligase to construct the PVCT2024-SlHAT5 overexpression vector;

[0031] S2: Genetic transformation of tomato: The SlHAT5 gene overexpression vector obtained in S1 was transferred into Agrobacterium LBA4404, and tomato cotyledons were infected by Agrobacterium-mediated method for tomato transformation;

[0032] S3: Tomato lines overexpressing the SlHAT5 gene were screened using qRT-PCR technology.

[0033] Furthermore, under high temperature stress, the chlorophyll content in the leaves of tomato plants overexpressing the SlHAT5 gene was significantly lower than that in the leaves of wild-type tomato plants; high temperature stress inhibited the accumulation of chlorophyll in the overexpression strain.

[0034] Furthermore, under high temperature stress, the overexpression strains showed severe wilting characteristics and the leaves withered.

[0035] Furthermore, the malondialdehyde content in tomato plants overexpressing the SlHAT5 gene was twice that of wild-type tomato plants; overexpression of the SlHAT5 gene increased the damage to plant cell membranes caused by high temperature stress.

[0036] Furthermore, overexpression of the SlHAT5 gene interferes with the clearance of ROS in plant cells.

[0037] The seventh object of the present invention is to provide an application of the tomato transcription factor SlHAT5 gene in improving the high temperature resistance and antioxidant level of tomatoes, and to improve the high temperature resistance and antioxidant level of tomatoes by constructing a tomato strain with low expression of the SlHAT5 gene.

[0038] An eighth object of the present invention is to provide an application of the tomato transcription factor SlHAT5 gene in breeding high-temperature-resistant tomatoes, and to obtain high-temperature-resistant tomato seeds by screening out tomato plants with low expression of the SlHAT5 gene.

[0039] The beneficial effects of the present invention are:

[0040] 1. This study discovered the SlHAT5 gene in tomatoes and found that it plays an important role in plant responses to stress. This study preliminarily clarifies the mechanism of action of the SlHAT5 gene in tomato heat stress response, laying a foundation for research on heat stress response in tomatoes and the cultivation of heat-tolerant tomato germplasm resources.

[0041] 2. This study found that overexpression of SlHAT5 reduced cell membrane stability and enhanced permeability under high temperature stress, significantly increasing the sensitivity of tomatoes to heat stress and increasing the degree of damage caused by heat stress. Furthermore, the study found that under high temperature stress, overexpression of SlHAT5 significantly reduced the activity of multiple enzymes in the plant's reactive oxygen species scavenging system, exacerbating cell wall damage and thereby increasing the sensitivity of SlHAT5-overexpressing tomato plants to heat stress. Furthermore, the study found that SlHAT5 plays a key role in high-temperature-induced hormone pathways in the body.

[0042] 3. The present study found that overexpression of SlHAT5 altered tomato's sensitivity to heat stress by affecting genes related to heat stress, antioxidant activity, and the ABA pathway. Heat stress affects genes in the plant, including SlPLL, SlPC, SlDD2, SlRBCS4, SlGAPDH, and DnaJ. These genes regulate the function of SlHAT5, which in turn inhibits the expression of genes in the ABA pathway that would otherwise be induced under heat stress. Furthermore, SlHAT5 interacts with SlVDAC, SlCP3L, Dp-1, SlNBRL1, SlBELL6, and DnaK, regulating the SUMOylation of HSFA1, thereby affecting the expression of HsfA2 and HsfB1. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 This is the result of SlHAT5 gene amplification;

[0044] Figure 2 is the SlHAT5 promoter amplification result;

[0045] Figure 3 is the phylogenetic tree of SlHAT5 and homologous proteins;

[0046] Figure 4 The expression pattern of SlHAT5 in various parts of tomato AC++ (R: root; S: stem; YL: young leaf; ML: mature leaf; SL: old leaf; Sp: sepal; St: stamen; Pe: petal; Pi: pistil; AZ: abscission layer; IMG: green fruit stage; MG: green ripe stage; B: color breaking stage; B+4: 4 days after color breaking; B+7: 7 days after color breaking);

[0047] Figure 5 is the relative expression pattern of SlHAT5 in tomato leaves under photoperiod and exogenous hormone treatments, Figure 5 -A: photoperiod; Figure 5 -B: 1 mM Eth treatment; Figure 5 -C: 100 μM ABA treatment; Figure 5 -D: 100 μM JA treatment; Figure 5 -E: 100 μM GA treatment; Figure 5 -F: 100 μM SA treatment, significant difference*: p < 0.05, **: p < 0.01;

[0048] Figure 6 The expression pattern of SlHAT5 in tomato induced by different stress treatments is shown in Figure 2. Figure 6 -A~ Figure 6 -C: high and low temperature stress treatment; Figure 6 -D: drought-rehydration treatment; Figure 6 -E: flooding treatment; Figure 6 -F: high salt stress treatment (leaves), significant difference*: p<0.05, **: p<0.01;

[0049] Figure 7 For pVCT2024-SlHAT5 colony PCR detection (M: DL2000+Marker; 1-9: pVCT2024-SlHAT5 colony samples; 10: CK+, positive control; 11: CK-, negative control);

[0050] Figure 8 For CRISPR / Cas9-SlHAT5 vector colony PCR detection (M: DL2000+Marker; 1-2: SlHAT5-A colony sample; 3: SlHAT5-B colony sample; 4: SlHAT5-C colony sample; 5: CK+, positive control; 6: CK-, negative control);

[0051] Figure 9 Genetic transformation of tomato (A: tomato cotyledons in differentiation culture; B: resistant callus; C: tomato plants in rooting culture);

[0052] Figure 10 For the positive screening of some transgenic plants (M: DL2000+Marker; 1: CK+, positive control; 2: CK-, negative control; 3-9: independent lines transfected with PVCT2024-SlHAT5; 10-13: CRISPR-Cas9 lines);

[0053] Figure 11 Detection of SlHAT5 gene expression in T0 generation overexpression plants (significant difference*: p<0.05, **: p<0.01);

[0054] Figure 12 The phenotypes of the SlHAT5 overexpressing strain under high temperature stress treatment and normal temperature control (Before: before treatment; After: after treatment; Heat Stress: high temperature treatment; Control: control);

[0055] Figure 13 Expression level detection and physiological index determination of SlHAT5 overexpressing strains under high temperature stress (A: SlHAT5 expression level detection in overexpressing strains and AC++ under high temperature treatment and normal temperature control; B: chlorophyll content; C: malondialdehyde content; D: superoxide dismutase activity; E: peroxidase activity; F: catalase activity; significant difference *: p < 0.05, **: p < 0.01);

[0056] Figure 14Screening of aurobacitin (AbA) concentrations (ng / mL) for inhibition of pAbAi-SlHAT5 yeast growth;

[0057] Figure 15 For monoclonal positive PCR detection (M: DL2000+Marker; 1-9: positive colony samples; 10: CK-, negative control)

[0058] Figure 16 Yeast one-hybrid verification of genes regulating SlHAT5;

[0059] Figure 17 Construction of the pGBKT7-SlHAT5 yeast expression vector (M: DL2000+Marker; 1-8: positive colony samples; 9: CK+, positive control; 10: CK-, negative control);

[0060] Figure 18 For partial monoclonal positive PCR detection (M: DL2000+Marker; 1-39: positive monoclonal samples; 40: negative control);

[0061] Figure 19 Yeast two-hybrid verification of SlHAT5 interacting proteins;

[0062] Figure 20 The expression profile of the corresponding gene of SlHAT5 interacting protein in tomato leaves under high temperature stress (CK: control group; HT: high temperature stress treatment group);

[0063] Figure 21 Detection of the expression levels of high temperature stress-related genes in SlHAT5 overexpressing strains under high temperature stress treatment (significant difference*: p<0.05, **: p<0.01);

[0064] Figure 22 Detection of the expression levels of antioxidant-related genes in SlHAT5 overexpressing strains under high temperature stress (significant difference*: p<0.05, **: p<0.01);

[0065] Figure 23 Expression detection of ABA-related genes in SlHAT5 overexpressing strains under high temperature stress (significant difference*: p<0.05, **: p<0.01);

[0066] Figure 24 Schematic diagram of the regulatory network of SlHAT5 gene in response to high temperature stress in tomato. DETAILED DESCRIPTION

[0067] The technical solutions of the present invention will be further described in detail below with reference to specific embodiments. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Therefore, all other embodiments derived by those skilled in the art based on the embodiments of the present invention without inventive effort are intended to fall within the scope of protection of the present invention.

[0068] Example 1. Cloning and bioinformatics analysis of the S1HAT5 gene sequence

[0069] 1. Cloning of the SlHAT5 gene and its promoter and gel electrophoresis detection of PCR products

[0070] (1) Cloning of the SlHAT5 gene and its promoter: Based on the obtained CDS and promoter sequences of the SlHAT5 gene, cDNA and gDNA of tomato AC++ leaves were used as templates, and gene-specific primers SlHAT5-F / SlHAT5-R and proSlHAT5-F / proSlHAT5-R were used as amplification primers. The two sequences were PCR amplified using high-fidelity enzymes. The amplification system was: 5μL SYBR+0.5μL Primer-F (5μM)+0.5μL Primer-R (5μM)+2μL cDNA+2μL RNase-free H2O; the reaction procedures were: ① 94℃ reaction for 20s; ② 94℃ reaction for 10s; ③ optimal annealing temperature reaction for 20s; ④ melting curve procedure, in which procedures ② and ③ were cycled 40 times.

[0071] Real-time fluorescence quantitative PCR: To ensure the accuracy of the results, the optimal annealing temperature of the designed fluorescence quantitative primers was explored, with a temperature gradient ranging from 55°C to 65°C, and three technical replicates were set.

[0072] Primer design: Primer Premier 5.0 primer design software was used to design SlHAT5 gene-specific amplification and related quantitative detection primers, see Table 1 for details.

[0073] After the PCR product (target gene fragment) was purified and recovered, the purified target fragment was ligated with the linearized blunt-end cloning vector pESAY-Blunt to further verify the amplified target fragment. The ligation system was: 20 ng SlHAT5 gene / promoter fragment + 1 μL pEASY-Blunt Cloning Vector + to 5 μL ddH2O, and the ligation was carried out at 25°C for 20 min.

[0074] Table 1. Specific amplification and detection primer sequences related to the SlHAT5 gene

[0075]

[0076]

[0077] (2) Gel electrophoresis of PCR products: Weigh 0.5 g of agarose powder into 50 mL of 1×TAE solution and heat until no oil droplets remain in the buffer. After cooling slightly, add 1 μL of 10×Gene Green nucleic acid dye, mix well, and pour into a gel casting tank. Allow to solidify and set aside. Pipette 5 μL of the above PCR product and add 1 μL of 6×Loading Buffer. Apply sample and perform electrophoresis at 150 V for 12 min. Observe the electrophoresis results.

[0078] Results: The SlHAT5 CDS sequence was amplified and detected by electrophoresis. The product band size was 867 bp. It was ligated into the pEASY-Blunt cloning vector and transformed into Escherichia coli and sequenced. The SlHAT5 gene amplification results were as follows: Figure 1 As shown, Figure 1 M stands for DL2000+Marker; 1 and 2 are amplified products. The sequencing results show 100% similarity to the SlHAT5 gene sequence obtained from the Solanaceae genome website. The SlHAT5 promoter sequence was amplified and detected by electrophoresis. The product band size was 1432 bp. It was ligated into the pEASY-Blunt cloning vector, transformed into E. coli, and sequenced. The SlHAT5 promoter amplification results are shown in Figure 2. Figure 2 As shown, Figure 2 M in the figure represents DL2000+Marker; 1 and 2 are amplified products, and the sequencing results have a 100% similarity with the SlHAT5 gene promoter sequence obtained from the Solanaceae genome website.

[0079] 2. Bioinformatics Analysis

[0080] To predict the conserved structure of the transcription factor SlHAT5 and infer its transcription factor family, we used NCBI to predict conserved domains. We also analyzed the amino acid sequence of SlHAT5 using ExPASy-Translatetool, SOPMA, and ExPASy-SWISS-MODEL, and predicted its physicochemical properties and structure. To clarify the phylogenetic position of SlHAT5 and its developmental relationship with other homologous proteins, we identified SlHAT5 homologs using NCBI-Blastp. A phylogenetic tree was constructed using the Newton-Joint Joint (NJ) method in MEGA 6.0 and enhanced using FigTree v1.4.3. Furthermore, to infer the specific cellular location of SlHAT5, we used Predict-Protein to predict its subcellular localization. To predict the potential functions of SlHAT5 in plants, we downloaded a 3000-bp sequence from the promoter region of SlHAT5 from NCBI and inserted it into PlantCARE to analyze its components and their specific functions.

[0081] result:

[0082] (1) Properties and structure prediction of SlHAT5 protein: The amino acid sequence of SlHAT5 translated by ExPASY was compared with the sequence downloaded from NCBI, and the similarity was 100%. Analysis by the software revealed that SlHAT5 encodes 288 amino acids, has a molecular weight of 33416.99 Da, and a theoretical isoelectric point of 4.94. It is a negatively charged, unstable hydrophobic protein containing 36 positively charged amino acid residues (Arg + Lys) and 52 negatively charged amino acid residues (Asp + Glu). Its amino acid composition includes Ala, Arg, Asn, Asp, Cys, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, and Val. 77.8% of the amino acid residues are exposed externally, and 22.22% are deeply buried internally. SlHAT5 contains 31.25% helices, 9.03% extended chains, 1.74% β-turns and 57.99% random coils, and is predicted to be located in the cell nucleus.

[0083] (2) Homologous alignment and phylogenetic analysis of SlHAT5 protein: NCBI shows that SlHAT5 protein has typical HD and LZ domains and can be classified as HD-ZIP family protein. Multalin was used to align the tomato SlHAT5 protein (XP_004238764.1) with homologous protein sequences in other species, such as pepper CaHOX16 (KAF3648869.1), tobacco NtHB54-like (XP_009594284.1), Medicago truncatula MtHAT5 (XP_003608986.1), Arabidopsis AtHAT5 (NP_186796.1), and maize ZmHAT5 (AQK74514.1). Phylogenetic analysis of these sequences revealed that SlHAT5 clustered with pepper CaHOX16, suggesting that their functions are similar. For details, see Figure 3 .

[0084] (3) Analysis of cis-acting elements in the SlHAT5 gene promoter: There are many different types of cis-acting elements in the 3000bp promoter of the SlHAT5 gene. In addition to a large number of light-responsive cis-acting elements, it also contains cis-acting elements involved in defense and stress responses, as well as hormone response elements such as salicylic acid, jasmonic acid, abscisic acid, and auxin. See Table 2 for details.

[0085] Table 2. Analysis of cis-acting elements in the 3000 bp promoter region of SlHAT5

[0086] Site name sequence number Function TC-rich repeats ATTCTCTAAC 1 Involved in defense and stress responses ARE AAACCA 3 Participate in anaerobic induction GC-motif CCCCCG 1 Participate in anaerobic induction GATA-motif AAGGATAAGG / GATAGGA 2 Participate in light response AT1-motif AATTATTTTTTATT 2 Participate in light response chs-CMA1a TTACTTAA 1 Participate in light response G-box TACGTG 5 Participate in light response TCT-motif TCTTAC 1 Participate in light response Box 4 ATTAAT 8 Participate in light response I-Box AGATAAGG / GATAAGGTC 2 Participate in light response TCA-element CCATCTTTTT 1 Participate in salicylic acid reaction TGACG-motif TGACG 2 Participate in jasmonic acid reaction CGTCA-motif CGTCA 2 Participate in jasmonic acid reaction ABRE ACGTG 3 Participate in abscisic acid reaction AuxRE TGTCTCAATAAG 1 Participates in auxin response

[0087] Example 2. Expression pattern of SlHAT5 gene in various parts of tomato and analysis of expression level under different induction factors

[0088] 1. Acquisition of zero-treatment samples and expression analysis of the SlHAT5 gene in various parts of tomato AC++

[0089] Tomatoes AC++ were planted in a net house, and fruit samples were collected at five different maturity stages: roots, stems, leaves of different maturity (young leaves, mature leaves, old leaves), various parts of flowers (stamens, petals, pistils, abscission layer, sepals), and fruit samples at five different maturity stages: green fruit stage, green ripe stage, color breaking stage, 4 days after color breaking (4 days after breaker, B+4), and 7 days after color breaking (7 days after breaker, B+7). The samples were quick-frozen in liquid nitrogen and stored in a -80°C refrigerator after collection.

[0090] This patent uses qRT-PCR to detect the expression pattern of SlHAT5 gene in various tissues of tomato to clarify the expression level of SlHAT5 gene in various parts of tomato. The results are as follows Figure 4As shown. The SlHAT5 gene is expressed at its highest levels in all parts of the floral organs (especially the petals). However, its expression levels in the roots, stems, leaves of varying maturity, and fruits at different stages are relatively low, at approximately 1 / 100th the level expressed in flowers. Furthermore, this patent observed and statistically analyzed indicators such as flowering period, flower morphology, fruit color, morphology, and maturity of transgenic SlHAT5 tomato plants. No significant differences were found compared to wild-type plants, so further research on flower-related aspects was not conducted.

[0091] 2. Acquisition of hormone-treated samples and expression analysis of the SlHAT5 gene under photoperiod and multiple hormone treatments

[0092] Tomato AC++ was sown in pots. When the plants had five leaves and one heart, a 100 μM solution of salicylic acid (SA), gibberellin (GA), jasmonic acid (JA), abscisic acid (ABA), and 1 mM ethylene (Eth) was prepared and sprayed onto the upper surface of the tomato leaves until the leaves were moist and not dripping. Root, stem, and leaf samples were collected at 0, 1, 6, 12, and 24 hours post-incubation (with a photoperiod of 16 hours light / 8 hours dark). Each sample was quickly frozen with liquid nitrogen and stored at -80°C.

[0093] In order to determine whether the expression of SlHAT5 gene is induced by hormones, after exogenous spraying of ABA, Eth, JA, GA and SA on tomato AC++, the expression levels of SlHAT5 gene in tomato leaves at different time periods under hormone induction were analyzed respectively with the light cycle samples as control. Figure 5 As shown in the figure, the expression level of the SlHAT5 gene was significantly downregulated after 12 hours of Eth treatment and significantly upregulated after 6 hours of ABA treatment. Under JA treatment, the expression level of the SlHAT5 gene was upregulated at 6 hours, but showed a significant downregulation trend after 12 hours. The expression level of the SlHAT5 gene was significantly higher than that of the wild type at 1 hour and 24 hours of GA treatment, but showed a transient downregulation at 12 hours. The expression level of the SlHAT5 gene showed a significant upregulation after 1 hour of SA treatment, and the upregulation trend slowed down over time, and the expression level showed a downregulation at 24 hours. Based on this, it is speculated that the expression of the SlHAT5 gene in tomato may be induced by hormones such as ABA, JA, GA, and SA, and that Eth can inhibit the expression of SlHAT5.

[0094] 3. Acquisition of stress-treated samples and expression analysis of the SlHAT5 gene under abiotic stress

[0095] Tomato AC++ was sown in nutrient pots. When the plants grew to five leaves and one heart, plants with consistent growth were selected and subjected to temperature, drought, flooding, and high salt stress treatments (with a photoperiod of 16 hours light / 8 hours dark). After a period of time, roots, stems, and leaves were collected and stored in a -80°C freezer. The specific treatments and sampling times are as follows:

[0096] ① Temperature treatment: Place the plants in a constant temperature incubator and adjust the temperature to 42℃ / 4℃ for 24h, with 24℃ as the control, and take samples at 0h, 4h, 8h, and 24h.

[0097] ② Drought treatment: When the plants have five leaves and one heart, stop watering until the plants show signs of drought-induced wilting. Rewater the plants for 24 hours. Samples are collected at 0 hours, at the end of drought treatment, and 2 hours, 4 hours, 8 hours, and 24 hours after rewatering.

[0098] ③ Flooding treatment: Place the plants in a tray filled with water (keep the drain port of the nutrient pot always below the water surface) for 8 days, and take samples at 0d, 2d, 4d, and 8d of treatment.

[0099] ④ Salt stress treatment: The plants were placed in salt solutions of different concentrations (100 mM, 200 mM, 300 mM, 400 mM, 500 mM), with the flooded samples as the control, and samples were taken 24 h after treatment.

[0100] This patent tests the expression level of SlHAT5 gene in tomato AC++ under different abiotic stresses to clarify the effects of various abiotic stresses on the expression level of SlHAT5 gene. Figure 6 As shown, under both 37°C and 4°C treatments, SlHAT5 gene expression showed a significant upregulation. However, when tomatoes were subjected to drought stress, expression levels in leaves and stems decreased significantly. After rehydration, expression only increased in roots. After 2 and 4 days of flooding, SlHAT5 expression levels were significantly upregulated in both roots and leaves, and after 8 hours, expression levels in stems and roots also showed a significant increase. When NaCl concentrations exceeded 300 mM, SlHAT5 expression levels in tomato plants increased significantly. Based on this, we hypothesize that high and low temperatures, flooding, and high salt stress promote SlHAT5 gene expression, while drought stress has the opposite effect. Among these, high temperature, low temperature, and salt stress all have a stronger effect on promoting SlHAT5 gene expression in tomato plants. Chongqing experiences high summer temperatures, so this patent selected high temperature stress as a research topic for the function of the SlHAT5 gene.

[0101] Example 3. Construction of plant expression vector and genetic transformation of tomato

[0102] 1. Overexpression and Construction of CRISPR / Cas9 Vectors

[0103] (1) Construction of pVCT2024-SlHAT5 overexpression vector

[0104] The SlHAT5 gene and the pVCT2024 vector connected to the cloning vector were double-digested with restriction endonucleases BamHI and Sad, and the digested fragments were recovered by gel recovery. The two fragments were ligated using T4 ligase to construct the PVCT2024-SlHAT5 overexpression vector.

[0105] ① Enzyme digestion and ligation

[0106] The enzyme digestion system is: 2μg plasmid / target gene + 2μL restriction endonuclease + 5μL restriction endonuclease Buffer + to 50μL ddH2O, overnight enzyme digestion at 37℃, purification and recovery of the enzyme digestion product, and ligation after concentration determination. The ligation system is: 0.01pmol of vector plasmid after enzyme digestion + 0.03~0.1pmol of target gene fragment after enzyme digestion + 0.7μL T4 DNA ligase + 1μL 10×T4 Buffer + to 10μL ddH2O, overnight ligation at 16℃.

[0107] ②Conversion of ligation products

[0108] 1) Add 10 μL 5× KCM and 35 μL ddH2O to 5 μL of ligation product and mix well;

[0109] 2) Take out the stored DH5α competent cells and place them on ice. When they are thawed to the point where ice and water coexist, add the mixture in (1);

[0110] 3) After standing on ice for 25 min, incubate at 37°C in a water bath for 5 min, add 700 μL of LB liquid, and incubate at 37°C with shaking at 220 rpm for 1 h;

[0111] 4) Centrifuge at 6000 rpm for 5 min, retain 100 μL of supernatant and resuspend the cells;

[0112] 5) Spread the resuspended liquid on LB+Kan (50 mg / L) solid culture medium and culture inverted at 37°C in the dark overnight.

[0113] ③Positive colony detection

[0114] The overnight cultured bacterial plate was taken out, and single colonies with regular growth and size were picked out and placed in 10 μL of sterile water. They were thoroughly pipetted and mixed, and 2 μL was aspirated for PCR detection. The remaining bacterial solution was stored in a refrigerator at 4°C. Single colony detection was performed using the universal primer M13F and the gene-specific amplification primers SlHAT5-R and proSlHAT5-R, respectively. The detection system was: 2.50 μL 10× Taq Buffer + 0.5 μL dNTPs + 1.00 μL Primer-F (5 μM) + 1.00 μL Primer-R (5 μM) + 0.25 μL Taq enzyme + 2.00 μL bacterial solution + 17.75 μL ddH2O. The positive colony detection procedure was as follows: 1) pre-denaturation, 95°C for 3 min; 2) denaturation, 95°C for 15 s; 3) annealing, 55°C for 15 s; 4) extension, 72°C at 1 kb / min; 5) post-extension, 72°C for 5 min. Procedures 2), 3), and 4) were cycled 35 times. PCR products were subjected to agarose gel electrophoresis to verify positive results and correct fragment size. Inoculate the bacterial suspension corresponding to the positive amplification product of the correct fragment size into 10 mL of LB + Kan (50 mg / L) liquid medium and expand for 10-16 hours. Aspirate 600 μL of the expanded bacterial suspension and add it to 300 μL of pre-prepared 60% glycerol for storage as glycerol culture. Centrifuge the remaining bacterial suspension and retain the dried cells for plasmid extraction. For detailed instructions, refer to the Novozymes Plasmid Extraction Kit instructions. Aspirate 10 μL of the extracted plasmid for sequencing verification, and store the remaining plasmid sample at -20°C for later use.

[0115] Results: The SlHAT5 gene fragment connected to pEASY-Blunt and the pVCT2024 vector were digested and connected to form a recombinant vector. After transformation into Escherichia coli, PCR detection was performed and the band size was 867 bp. Figure 7 As shown, the plasmid was extracted and sequenced for verification. The sequencing results were consistent with the CDS sequence of the SlHAT5 gene, indicating that the pVCT2024-SlHAT5 overexpression vector was successfully constructed.

[0116] (2) CRISPR / Cas9 vector construction

[0117] The CRISPR / Cas9 vector construction process is as follows:

[0118] ① Amplification of target fragment at mutation site

[0119] A qualified Cas9 recognition site was identified upstream and downstream of the SlHAT5 gDNA sequence to construct the CRISPR / Cas9 vectors SlHAT5-A, SlHAT5-B, and SlHAT5-C. The target mutation site fragments SlHAT5-a, b, and c were synthesized by PCR. The amplification system for the SlHAT5 gene and its promoter was: 2 μL PrimeSTAR Max Premix (2x) + 2 μL Primer-F (5 μM) + 2 μL Primer-R (5 μM) + 20 ng cDNA in 50 μL ddH2O. The amplification procedure for the SlHAT5 gene and its promoter was: 1) pre-denaturation, 98°C for 2 minutes; 2) denaturation, 98°C for 30 seconds; 3) annealing, 55°C for 30 seconds; 4) extension, 72°C at 1 kb / min; 5) post-extension, 72°C for 2 minutes. Procedures 2), 3), and 4) were cycled 35 times. Add 10 μL of each primer, mix well, and use the double-stranded DNA fragment synthesis program to complement each other to form double-stranded DNA fragments. The double-stranded DNA fragment synthesis program is as follows: 1) 95°C reaction for 30 seconds; 2) 50°C reaction for 30 seconds; 3) hold at 4°C.

[0120] ② Enzyme digestion: Use the enzyme digestion system to digest the vector pKSE401 at 37℃ for 3-4h, recover the large fragments after digestion by gel gel, and store at -20℃ for later use; the enzyme digestion system is: 2μg pKSE401+5μL BsaⅠBuffer+1μL BsaⅠ+to 50μLddH2O.

[0121] ③ Ligation: Ligate the double-stranded DNA fragment obtained in ① with the enzyme-digested pKSE401 obtained in ②. The ligation system is the same as ① in (1) Construction of pVCT2024-SlHAT5 overexpression vector.

[0122] ④ Transformation of ligation products and detection of positive colonies: The steps are the same as ② and ③ in (1) construction of pVCT2024-SlHAT5 overexpression vector.

[0123] Results: The fragments SlHAT5-A, SlHAT5-B, and SlHAT5-C were connected to the pKSE401 vector after enzyme digestion. After transformation, PCR detection was performed and the band size was 423 bp. Figure 8 As shown, the plasmids were extracted and sequenced for verification, and the results showed that the CRISPR / Cas 9 knockout vectors SlHAT5-A, SlHAT5-B and SlHAT5-C of the SlHAT5 gene were successfully constructed.

[0124] (3) Construction of pAbAi-SlHAT5 vector

[0125] The proSlHAT5 fragment ligated to pEASY-Blunt and the pAbAi empty vector were digested with SacI and SmaI, and the recovered products were recovered by gel cleavage. The concentrations of the recovered products were determined, and the two fragments were ligated with T4 ligase to construct the pAbAi-SlHAT5 yeast expression vector. The digestion, ligation system, and timing are detailed in ① in (1) Construction of the pVCT2024-SlHAT5 overexpression vector. After ligation, BstBI was used for digestion. The system and timing are detailed in ① in (1) Construction of the pVCT2024-SlHAT5 overexpression vector.

[0126] (4) Construction of pGBKT7-SlHAT5 vector

[0127] The SlHAT5 fragment ligated to pEASY-Blunt and the pGBKT7 empty vector were digested with NcoI and BamHI, and the recovered products were recovered by gel excision. The concentration of the recovered products was determined, and the two fragments were ligated with T4 ligase to construct the pGBKT7-SlHAT5 yeast expression vector. The details of the enzyme digestion, ligation system, and time are shown in ① in (1) Construction of the pVCT2024-SlHAT5 overexpression vector.

[0128] 2. Genetic Transformation of Tomatoes Cultivation and Positive Identification of SlHAT5 Transgenic Tomatoes

[0129] Genetic transformation is carried out by infecting tomato cotyledons using Agrobacterium-mediated infection. After resistant plants have established roots in the rooting medium, the film covering them is removed and hardened for 3-5 days. Once the plants have fully adapted to the external environment, the remaining medium is washed off and the plants are transplanted into nutrient pots for subsequent screening. After screening, they are transplanted into a screenhouse.

[0130] Specifically, the SlHAT5 gene overexpression vector and the CRISPR-Cas9 vectors with three different mutation sites were respectively transferred into Agrobacterium LBA4404. The sterile cotyledons of tomato explants were infected by Agrobacterium-mediated method. After 48 hours of co-culture, the calli were transferred to the screening medium for further differentiation culture to obtain independent resistant calli. Finally, rooting culture was carried out and the seedlings were planted in soil after hardening. For details, see Figure 9 .

[0131] Through genetic transformation of tomatoes, a total of 49 independent lines of the SlHAT5 gene were obtained, including 30 lines of the PVCT2024-SlHAT5 gene and 12 lines of the SlHAT5 gene CRISPR-Cas9 gene. Using the leaf DNA of the transgenic rooted plants as a template, the primers NPTⅡ-F / NPTⅡ-R designed in the Kanr region were used to detect the integration of the exogenous gene into the tomato genome. The test results showed that the 30 independent lines of the PVCT2024-SlHAT5 gene and the 12 independent lines of the CRISPR-Cas9 gene were all transgenic positive plants. The PCR test results of some transgenic positive plants are as follows: Figure 10 shown.

[0132] 3. Screening of SlHAT5 Overexpression and CRISPR / Cas9 Tomato Lines

[0133] (1) Screening of SlHAT5 gene-positive tomato plants

[0134] DNA from transgenic plants was obtained using the rapid DNA release method. 30 μL of TPS buffer was added to a 1.5 mL centrifuge tube. An appropriate amount of young tomato leaves were mashed until uniform and free of lumps. The extract was infused in a boiling water bath for 10 minutes, cooled on ice to room temperature, and then 70 μL of ddH₂O was added. The extract was centrifuged at high speed for 2 minutes to obtain a crude DNA extract. Taking advantage of the fact that ordinary tomatoes do not contain the Kanr region, PCR amplification of the Kanr region in transgenic plants was performed, and the amplification results were detected by electrophoresis. The PCR reaction system was: 10 μL 2×Taq Master Mix + 1 μL Primer-F (5 μM) + 1 μL Primer-R (5 μM) + 8 μL crude DNA extract; the detection procedure was: ① pre-denaturation, 94°C for 2 min; ② denaturation, 94°C for 30 s; ③ annealing, 55°C for 30 s; ④ extension, 72°C at 2 kb / min; ⑤ post-extension, 72°C for 2 min, and procedures ②, ③, and ④ were cycled 30 times.

[0135] (2) Screening of SlHAT5 gene expression in transgenic positive plants

[0136] Total RNA was extracted from the SlHAT5 gene-overexpressing plants and reverse-transcribed to obtain cDNA. Real-time fluorescence quantitative PCR was used to detect the expression level of the SlHAT5 gene in 30 independent lines of PVCT2024-SlHAT5 transgenic plants. The primers are detailed in Table 1. Young leaf tissues of these lines were collected for testing, and the expression levels of the SlHAT5 gene in these lines were compared with the expression levels of the gene in tomato AC++. The results are shown in Figure 1. Figure 11As shown in the figure, the seven PVCT2024-SlHAT5 gene-transfected lines, OE-2, OE-3, OE-5, OE-9, OE-11, OE-14, and OE-54, were overexpression lines, with expression multiples of 2.9×, 3.0×, 3.0×, 3.7×, 6.1×, 6.1×, and 5.0×, respectively. These three overexpression lines (3 times higher than the wild type) were mainly used for subsequent research.

[0137] (3) Identification of SlHAT5 knockout strains

[0138] For the identification of CRISPR knockout lines, gDNA from 12 transgenic CRISPR-Cas9-positive plants and AC++ was first extracted using the CTAB method. The knockout region was then amplified using a high-fidelity enzyme. The amplification system was: 2 μL PrimeSTAR Max Premix (2x) + 2 μL Primer-F (5 μM) + 2 μL Primer-R (5 μM) + 20 ng cDNA + to 50 μL ddH2O. The amplification procedure was: 1) pre-denaturation, 98°C for 2 min; 2) denaturation, 98°C for 30 s; 3) annealing, 55°C for 30 s; 4) extension, 72°C for 1 kb / min; 5) post-extension, 72°C for 2 min. Procedures 2), 3), and 4) were cycled 35 times. After electrophoresis detection, sequencing was performed and the sequencing results were compared with the same sequence of tomato AC++. It was found that one strain of SlHAT5-B, SlHAT5-B-10, had a mutation in the sequence, with a base T added at 102bp. Due to the preference of gene editing, the remaining 11 strains were not successfully edited.

[0139] This patent predicts the protein sequence corresponding to the SlHAT5-B-10 mutation to infer its effect on the function of the SlHAT5 gene. The results indicate that the addition of the base T causes a frameshift. Prediction suggests that only 26 amino acids remain in the SlHAT5-B-10 protein sequence, or the first 35 amino acids compared to the wild-type. Further investigation is needed to determine the potential differences in the reagents. However, phenotypic observations of the flowers and fruits of the T0 generation plants revealed no differences compared to the wild-type. This suggests that this gene may be redundant in flower and fruit development, and its function remains to be determined.

[0140] Example 4. Phenotypic Observation and Sample Collection of Transgenic SlHAT5 Tomato Plants under High Temperature Stress

[0141] 1. Preparation of plant materials:

[0142] Tomato AC++ seeds with full grains and tomato seeds of the three transgenic SlHAT5 strains with the highest expression levels were selected and germinated in a constant temperature incubator at 28°C; the white seeds were sown in nutrient pots (3 seeds per pot), and positive plants were screened when two true leaves grew. After screening, the seedlings were transplanted into 10 cm nutrient pots; overexpressing plants were screened when four true leaves grew; the plants were cultured for a total of about 45 days (16h light / 8h dark, 24°C) and subjected to high temperature stress treatment.

[0143] 2. High temperature stress treatment and sample collection:

[0144] Tomato plants that had been cultured for 45 days were subjected to high-temperature treatment. Prior to treatment, the seedlings and potting soil were adequately watered. The seedlings (all strains and wild-type) were divided equally into two groups: an experimental group and a control group. The treated groups were placed in a 42°C constant-temperature incubator (16 hours light / 8 hours dark, 42°C), while the control group was placed in a 24°C incubator (16 hours light / 8 hours dark, 24°C). Plants were kept adequately hydrated during the treatment period. After approximately 54 hours of high-temperature treatment, until significant phenotypic differences between the transgenic plants in the heat-stressed group and the wild-type plants appeared, leaf (3rd, 4th, and 5th) and root and stem samples were collected from all plants in both groups and stored in a -80°C freezer for subsequent experiments.

[0145] Results: The three overexpression lines with the highest SlHAT5 expression levels, OE-11, OE-14, and OE-54, were subjected to high temperature treatment. Using tomato plant AC++ as the control, it can be seen that the wild-type leaves lost water to a lesser extent and showed slight wilting characteristics; the overexpression line plants showed more severe wilting characteristics, and their leaves were basically withered. For details, see Figure 12 From the morphological characteristics of each strain after high temperature stress, it can be seen that overexpression of the SlHAT5 gene can increase the sensitivity of tomato to high temperature stress. At the same time, high temperature stress does not affect the relative expression trend of SlHAT5 in transgenic plants. Figure 12 .

[0146] In the preliminary experiment of high temperature stress, it was found that the SlHAT5 overexpression line had a more significant wilting phenotype under high temperature stress than tomato AC++, while the phenotype of the CRISPR-Cas9 line SlHAT5-B-10 under stress was not significantly different from that of AC++. Therefore, subsequent high temperature stress-related experiments were all carried out using the SlHAT5 overexpression line as the material.

[0147] Example 5. Physiological index determination of S1HAT5 transgenic tomato plants after high temperature stress

[0148] The different physiological indicators of the treated samples collected in Example 4 were measured, including chlorophyll content, malondialdehyde content, proline content, peroxidase activity, catalase activity, superoxide dismutase activity, etc. The results are as follows: Figure 16 As shown, Figure 13 -A is the expression level detection of SlHAT5 in overexpression strains and AC++ under high temperature treatment and normal temperature control.

[0149] 1. Determination of chlorophyll content:

[0150] Chlorophyll is an important photosynthetic pigment for plants to carry out photosynthesis and provide nutrients for normal plant growth. It is also an important indicator for judging whether plants are damaged by abiotic stress. In the control group, there was no significant difference in chlorophyll content between wild-type plants and overexpression plants, both of which were around 2.5 mg / g; under high temperature stress treatment, the chlorophyll content in overexpression plants was significantly lower than that in wild-type plants, the latter was about 3.6 mg / g, and the former was about 2.0 mg / g. This shows that: after being subjected to high temperature stress, the accumulation rate of chlorophyll content in plants slows down or is even inhibited, and normal photosynthesis cannot be carried out, thereby inhibiting the growth of tomato plants. In the experiment, overexpression of the SlHAT5 gene reduced the plant's tolerance to high temperature stress, and the chlorophyll accumulation rate and amount were lower than those of wild-type plants, and even yellowing and wilting occurred. It is speculated that high temperature stress inhibits the accumulation of chlorophyll in the overexpression strain. For details, see. Figure 13 -B.

[0151] 2. Determination of malondialdehyde content

[0152] Malondialdehyde (MDA) content is one of the important indicators for measuring the degree of cell membrane damage after plant stress. After testing leaf samples from the treatment and control groups, it was found that in the control group, the MDA content in the leaves of the overexpression strain and AC++ was relatively consistent and low, approximately 0.009 μmol / g; in the treatment group, while the overall MDA content increased, the MDA accumulation in the overexpression strain was approximately 0.03 μmol / g, which was twice that of the wild type. This result shows that overexpression of the SlHAT5 gene increased the damage to plant cell membranes caused by high temperature stress. For details, see Figure 13 -C.

[0153] 3. Detection of superoxide dismutase (SOD) activity

[0154] The present invention detected the activity of superoxide dismutase in the treated and control groups and found that under high temperature treatment, the superoxide dismutase activity in the cells of AC++ and transgenic plants showed a downward trend, decreasing from about 100u / g FW to less than 85u / g FW; at the same time, the SOD activity in the transgenic plants (about 40u / g FW) was significantly lower than that in AC++ (about 70u / g FW). This result also shows that the overexpression of the SlHAT5 gene affects the scavenging of ROS in the body by plant cells to a certain extent. Figure 13 -D.

[0155] 4. Detection of peroxidase (POD) and catalase (CAT) activity

[0156] The present invention detected the activities of peroxidase and catalase in the treatment and control groups. The results showed that in the control group, the activities of peroxidase and catalase were relatively low, with the former being approximately 16u / g FW and the latter being around 30u / g FW, and there was no significant difference between AC++ and transgenic plants. Under high temperature treatment, the activities of these two enzymes were greatly increased in both AC++ and transgenic plants, increasing to 1.5 times that at room temperature in AC++, and the enzyme activity in transgenic plants was significantly lower than that in AC++, which was only increased to 1.1 to 1.3 times that at room temperature. This result shows that the overexpression of the SlHAT5 gene interferes with the clearance of ROS in the body by plant cells to a certain extent, see for details. Figure 13 -E and Figure 13 -F.

[0157] Example 6. Screening of yeast one- and two-hybrid cDNA libraries of S1HAT5

[0158] 1. Obtain candidate genes and proteins from yeast cDNA library

[0159] The yeast cDNA library was screened using the mating method. After mating, the bacterial liquid corresponding to the positive plaques on SD / -Leu / Aba (single hybrid) and SD / -Trp / -Leu / -His / -Ade / X-α-Gal (double hybrid) solid culture media was sequenced. The sequencing results were compared with the gene sequences of various genes in NCBI, and genes were selected. Then, the functions of these genes were analyzed to screen out genes that may regulate the function of SlHAT5 and proteins that interact with SlHAT5 as alternatives for verification.

[0160] 2. Preliminary verification of candidate genes and proteins

[0161] Yeast plasmids were extracted from bacterial cultures corresponding to candidate genes and proteins, and then transformed into E. coli, where the plasmids were extracted. pAbAi-proSlHAT5 and plasmids corresponding to regulatory genes from single-hybrid screening were transformed into Y1H competent cells and plated onto SD / -Leu / Aba solid medium. Proteins from double-hybrid screening were transformed into Y187 competent cells and plated onto SD / -Leu solid medium. Simultaneously, pGADT7-T was transformed into Y187 competent cells, and pGBKT7-SlHAT5, pGBKT7-53, and pGBKT7-Lam plasmids were transformed into Y2H competent cells and plated onto SD / -Trp and SD / -Leu solid medium, respectively.

[0162] The following combinations were used for site-by-site interaction testing to preliminarily verify the interaction between the proteins corresponding to the candidate genes and SlHAT5: single-hybrid positive control pGADT7-53 + pAbAi-proSlHAT5, negative control pGADT7-T + pAbAi-proSlHAT5, and candidate gene verification pAbAi-proSlHAT5 + pGADT7-X (X represents the candidate gene); two-hybrid positive control pGBKT7-53 + pGADT7-T, negative control pGBKT7-Lam + pGADT7-T, and candidate gene verification pGBKT7-SlHAT5 + pGADT7-X (X represents the candidate protein). The bacterial broth from the single-hybrid site-by-site verification was plated on SD / -Leu / Aba solid medium, and its growth was observed. The bacterial solution verified by double hybrid dot-to-dot was spread on SD / -Trp / -Leu solid medium. After cultured until positive colonies appeared, it was spotted again on SD / -Trp / -Leu / -His / -Ade / X-α-Gal solid medium to observe its growth.

[0163] Result analysis:

[0164] (1) Screening of upstream genes regulating SlHAT5 expression

[0165] ① Construction of upstream gene screening vector regulating SlHAT5 expression: The amplified proSlHAT5 1432bp sequence was connected to the vector pAbAi by double enzyme digestion. After transformation into Escherichia coli, positive single colonies were screened using target fragment-specific primers. PCR detection was performed after shaking the bacteria to confirm the completion of the construction of the pAbAi-SlHAT5 recombinant vector.

[0166] ② Screening of gold basilin (AbA) concentrations that inhibit the growth of pAbAi-SlHAT5 yeast: The constructed pAbAi-SlHAT5 yeast expression vector was linearized and transferred into Y1HGold. The AbA concentration that could completely inhibit the normal growth of yeast was screened. It was found that when the AbA concentration in SD / -Ura solid medium was 1000 ng / mL, it could completely inhibit the normal growth of yeast. For details, see Figure 14 .

[0167] ③ Screening of upstream genes regulating SlHAT5 expression in yeast library: Through the toxicity and self-activation test of proSlHAT5 in yeast, it was found that it has no toxicity and self-activation. Therefore, proSlHAT5 was used as bait protein to screen the tomato cDNA library, and a total of 8 positive clones were screened. The PCR test results are as follows Figure 15 As shown. After these PCR products were sent for sequencing, the sequencing results were BLASTed on the Solanaceae genome website, and 8 genes were compared and analyzed. Subsequently, 6 genes were screened out for interaction verification through functional analysis. The functions of these six genes involve plant photosynthesis, respiration, redox and thermal response. Specifically, SlPLL promotes stomatal movement, SlPC and SlRBCS4 are involved in photosynthesis, SlDD2 is involved in photosynthesis and photorespiration, GAPDH is involved in redox metabolism, and DnaJ is involved in thermal response. Therefore, after extracting the yeast plasmid, multiple yeast expression vectors such as AD-SlPLL, AD-SlPC, AD-SlDD2, AD-SlRBCS4, AD-SlGAPDH, AD-DnaJ were constructed, and point-to-point interaction verification was performed with SlHAT5. After point-to-point verification, it was found that these proteins can interact with SlHAT5, see for details. Figure 16 , it is speculated that these genes are involved in regulating the response of SlHAT5 to tomato high temperature stress.

[0168] (2) Screening of SlHAT5-interacting proteins

[0169] ① Construction of SlHAT5 interacting protein screening vector: The amplified SlHAT5 867bp gene sequence was ligated to the vector pGBKT7 by double enzyme digestion. After transformation into E. coli, positive single colonies were screened using target fragment specific primers. After shaking the bacteria, PCR detection was performed. The results showed that the pGBKT7-SlHAT5 recombinant vector was constructed. For details, see Figure 17 .

[0170] ② Screening of SlHAT5 interacting proteins in yeast library: Through the toxicity and self-activation test of SlHAT5 in yeast, it was found that it has no toxicity and self-activation. Therefore, SlHAT5 was used as bait protein to screen the tomato cDNA library, and a total of 210 positive clones were screened. Some PCR test results are as follows Figure 18 These PCR products were sequenced, and then the sequencing results were BLAST-matched on the Solanaceae genome website, resulting in 41 genes.

[0171] ③Verification of SlHAT5 interacting proteins: Based on the description of the functions of the above 41 genes in the SGN gene database and NCBI, 6 gene fragments were selected for point-to-point verification. The corresponding genes include SlVDAC (gene number: Solyc02g067460), SlCP3L (gene number: Solyc07g041910), Dp-1 (gene number: Solyc10g078430), SlNBRL1 (gene number: Solyc06g071770), SlBELL6 (gene number: Solyc01g109980), and DnaK (gene number: Solyc01g103450). These genes are involved in plant photosynthesis, high temperature stress and cell apoptosis. Therefore, after extracting yeast plasmids, multiple yeast expression vectors including pGADT7-SlVDAC, pGADT7-SlCP3L, pGADT7-Dp-1, pGADT7-SlNBRL1, pGADT7-SlBELL6, and pGADT7-DnaK were constructed and tested for site-to-site interaction with SlHAT5. After site-to-site verification, it was found that these proteins could interact with SlHAT5. Figure 19 .

[0172] This patent also used the online transcriptome data website TomExpress to predict the expression levels of the above six genes in tomato leaves after high temperature treatment, and found that the expression levels of DnaK and SlNBRL1 decreased under high temperature treatment, while the remaining four genes showed upregulated expression levels, such as Figure 20 Given that these genes play important roles in photosynthesis, high temperature stress, and apoptosis in plants, it is speculated that these genes have important functions in the high temperature stress response network of tomatoes, but their specific location and main role still need further study.

[0173] (3) Analysis of expression levels of related genes in SlHAT5 overexpression lines under high temperature stress

[0174] When plants are subjected to high temperature stress, reactive oxygen species (ROS) increase significantly. To maintain homeostasis and protect the system, the activity of antioxidant enzymes in the ROS scavenging system increases. Simultaneously, ABA levels in the body change, affecting the ABA anabolic pathway. Furthermore, high temperatures induce the expression of heat shock transcription factors and heat shock proteins. Based on this, this patent uses qRT-PCR technology to analyze the expression levels of high temperature stress-related genes, antioxidant-related genes, and ABA-related genes in treated leaves.

[0175] ① Analysis of the expression of genes related to high temperature stress: Heat shock transcription factors HsfA2 and HsfB1 and the high temperature response gene SlSIZ1 have been reported to be closely related to the high temperature stress process. To further clarify the possible regulatory mechanism of SlHAT5 in regulating the response of tomato to high temperature stress, we analyzed the expression changes of these genes in the overexpression lines and wild type under high temperature stress treatment and control experiments. For details, see Figure 21 .

[0176] like Figure 21 As shown in the figure, after high temperature stress, the expression levels of HsfA2, HsfB1, and SlSIZ1 in each strain were increased compared with the control group. In both the treatment and control groups, the expression level of HsfA2 gene in the overexpression plants was significantly lower than that in the wild type. However, the difference in the expression level of this gene in the overexpression plants in the treatment group compared with the wild type increased from 1 / 30 in the control group to 1 / 20. For details, see Figure 21 -A; The expression level changes of HsfB1 and SlSZ1 genes under treatment were similar to those of HsfA2 gene. The expression fold of overexpression strains showed a significant increase compared with wild type after high temperature treatment, and the difference fold increased from 1 / 70 in the control group to 1 / 2 to 3 / 4. Figure 21 -B. Figure 21 -C). The results showed that when tomatoes are subjected to high temperature stress, the expression of the above genes is affected by regulating the expression of the SlHAT5 gene, thereby affecting their stress resistance.

[0177] ② Analysis of the expression of antioxidant-related genes: To explore the effects of high temperature stress on the active oxygen system of transgenic tomato plants, the present invention selected ascorbate peroxidase genes APX1, APX2 and superoxide dismutase gene SOD2 for detection. In the overexpression plants, the expression levels of APX1 and APX2 genes in the control group were higher than those in the wild type, while under high temperature treatment, their expression levels were significantly downregulated to no significant difference from AC++ or even slightly lower than AC++. For details, see Figure 22 -A. Figure 22 -B; while SOD2 levels showed a completely opposite trend. At room temperature, the expression level of SOD2 gene in the overexpression group was significantly lower than that of the wild type, while under high temperature treatment, its expression level was significantly increased to slightly higher than AC++. Figure 22 -C. The results showed that when tomatoes were subjected to high temperature stress, the SlHAT5 gene regulated the expression of the above genes, thereby affecting their stress resistance.

[0178] ③ Analysis of ABA-Related Gene Expression: Based on the expression profile of SlHAT5 under hormone-induced conditions in Example 2 (Analysis of SlHAT5 Expression under Photoperiod and Multiple Hormone Treatments), we speculated that this gene might be induced by ABA. Therefore, we screened for ABA signaling pathway-related genes SlPP2C2, SlNCED, SlAREB1, and SlTAS14, and performed qRT-PCR on plants treated with high temperature stress and the control to analyze changes in their expression.

[0179] The results are as follows Figure 23 As shown, in the control group, NCED expression in the overexpressing plants was significantly lower than that in the wild type. However, in the high temperature stress group, although the expression level of this gene in the overexpressing plants was still significantly lower than that in the wild type, the expression fold decreased compared to the wild type at the same time, increasing from 1 / 5 to about 1 / 4. In contrast, the expression levels of TAS14, PP2C2, and AREB1 in the overexpressing plants were on par with AC++ to about 3 times higher than AC++ in the control group, but were rapidly downregulated under high temperature treatment to a level significantly lower than AC++ (about 1 / 3) at the same time. This result indicates that the SlHAT5 gene in tomato is involved in the ABA pathway in response to high temperature stress.

[0180] In summary, the regulatory network of SlHAT5 under high temperature stress in tomato is as follows: Figure 24 As shown in the data, high temperature stress affects genes such as SlPLL, SlPC, SlDD2, SlRBCS4, SlGAPDH and DnaJ in plants. These genes regulate the function of SlHAT5, which inhibits the expression of genes in the ABA pathway that should be induced under high temperature stress. At the same time, SlHAT5 interacts with SlVDAC, SlCP3L, Dp-1, SlNBRL1, SlBELL6 and DnaK, and regulates the SUMOylation of HSFA1, thereby affecting the expression of HsfA2 and HsfB1. In addition, the expression of SlHAT5 also affects the expression of antioxidant enzyme-related genes in tomatoes under high temperature stress, ultimately reducing the sensitivity of tomatoes to high temperature stress. Figure 24 In the figure, the direction of the arrow indicates the direction of regulation; solid line: confirmed by literature; dotted line: conclusion of this paper; dotted lines 1 and 5: positive regulation; dotted lines 2, 3, 4, and 6: negative regulation.

Claims

1. Application of the tomato transcription factor SlHAT5 gene in improving the high temperature resistance and antioxidant level of tomatoes, characterized in that: The amino acid sequence of the S1HAT5 gene is shown in SEQ ID NO.1; By constructing a tomato line with low expression of the SlHAT5 gene, the high temperature resistance and antioxidant level of tomatoes can be improved.

2. The use according to claim 1, characterized in that The nucleotide sequence of the S1HAT5 gene is shown in SEQ ID NO.

2.

3. Application of the tomato transcription factor SlHAT5 gene in breeding of high-temperature-resistant tomatoes, characterized in that: By screening tomato plants with low expression of the SlHAT5 gene, high-temperature-resistant tomato seeds are obtained; the amino acid sequence of the SlHAT5 gene is shown in SEQ ID NO.1.

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