Application of tomato histone variant H2A.Z in improving low temperature resistance of tomato
By knocking out the tomato histone variant H2A.Z using CRISPR/Cas9 gene editing technology, the sl_h2a.z double mutant was constructed, which solved the problem of poor growth of tomatoes under low temperature conditions, improved the low temperature resistance and fruit yield of tomatoes, and laid the molecular mechanism basis for tomatoes to respond to adverse signals.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- ZHEJIANG UNIV
- Filing Date
- 2025-06-25
- Publication Date
- 2026-07-21
Smart Images
Figure CN120796341B_ABST
Abstract
Description
Technical Field
[0001] This application relates to fields such as genetic engineering, molecular biology and physiology, and specifically to the application of a tomato histone variant H2A.Z in improving the low-temperature resistance of tomatoes. Background Technology
[0002] tomato( Solanum lycopersicum Originating in tropical South America, tomato is one of the most widely cultivated vegetable crops in the world and an important warm-season crop in greenhouse vegetable cultivation in my country. However, with the frequent occurrence of extreme weather events globally, problems such as low-level equipment and poor controllability of temperature and light environments are common in greenhouse tomato production. Tomatoes often suffer from low-temperature injury during winter and spring, leading to poor growth and development, reduced fruit yield, and lower quality. Therefore, studying the key regulatory factors of tomato's low-temperature response not only helps to reveal its temperature response molecular network but also provides important theoretical support for improving tomato yield and quality and reducing economic losses caused by low temperatures.
[0003] Epigenetic regulation plays a crucial role in plant stress responses, with histone variant substitution being a key area of epigenetic research. Besides the four conventional histones (H2A / H2B / H3 / H4), plant genomes contain histone variants with different amino acid sequences and biological functions. Histone variant H2A.Z is one such variant of H2A, replacing H2A through ATP-dependent chromatin remodeling factors (such as the SWR1 complex), thereby altering nucleosome structure and chromatin accessibility. The enrichment of H2A.Z in different gene regions affects gene transcription, thus playing a vital regulatory role in various important physiological pathways (such as plant development, flowering, and environmental responses). For example, in Arabidopsis thaliana, high-temperature stress reduces the enrichment of H2A.Z at nucleosome sites, promoting growth. HSP70 and HSFA1 Gene expression, thereby responding to high-temperature stress [SV Kumar et al., "H2A.Z - Containing Nucleosomes Mediate the Thermosensory Response in Arabidopsis." Cell, 2010, 140(1):136-147]; Under salt stress, Arabidopsis activates H2A.Z by reducing its enrichment. AtMYB44The transcription of H2A.Z, which in turn regulates the salt stress response [NH Nguyen et al., "H2A.Z-containing nucleosomes are evicted to activate AtMYB44 transcriptionin response to salt stress." Biochemical and Biophysical Research Communications, 2018, 499 (4): 1039-1043]; In addition, under drought stress, the enrichment of H2A.Z in the gene coding region significantly inhibits the transcription level of drought stress response genes [W Sura et al., "Dual Role of the Histone Variant H2A.Z in Transcriptional Regulation of Stress-Response Genes." The Plant Cell, 2017, 29(4):791-807].
[0004] Although the important role of epigenetic factors in plant stress response has been extensively studied, research on their role in tomato low-temperature resistance pathways is still relatively limited, especially the role and regulatory mechanism of histone variant H2A.Z in tomato low-temperature stress. Summary of the Invention
[0005] Therefore, embodiments of the present invention provide a method for knocking out the tomato histone variant H2A.Z to improve the low-temperature resistance of tomatoes.
[0006] To achieve the objectives of this invention, the technical solution adopted is as follows:
[0007] Application of a tomato histone variant H2A.Z in improving the low-temperature resistance of tomatoes, wherein the histone variant H2A.Z is composed of SlHTA8, SlHTA9, SlHTA11 Three genes encode, among which, SlHTA9 The nucleotide sequence is shown in SEQ ID NO: 1. SlHTA11 The nucleotide sequence is shown in SEQ ID NO: 2. The application involves knocking out the gene encoding histone H2A.Z using gene knockout technology. SlHTA9 and SlHTA11 This improves the low-temperature resistance of tomatoes.
[0008] Furthermore, the aforementioned SlHTA9, SlHTA11 The amino acid sequence of the encoded H2A.Z protein is shown in SEQ ID NO: 3 and SEQ ID NO: 4.
[0009] Furthermore, the gene knockout technology is specifically as follows:
[0010] In tomatoes SlHTA9 , SlHTA11 We selected target fragments containing PAM motifs adjacent to the pre-intermediate region sequence from the protein coding region, designed corresponding primers, and constructed a CRISPR / Cas9 recombinant expression vector.
[0011] The CRISPR / Cas9 recombinant expression vector was introduced into Agrobacterium, which was then used to infect tomato cotyledons. Positive transgenic plants were screened, and single mutant purified lines were obtained through self-pollination. Double mutants were obtained through hybridization. sl_h2a.z The sl_h2a.z By enhancing the cold response genes in tomatoes SlCBFs The expression of this substance can enhance the low-temperature resistance of tomatoes.
[0012] Furthermore, the recombinant expression vector is pCAMBIA1301-U6-26-sgRNA-SlHTA9-35S-cas9 or pCAMBIA1301-U6-26-sgRNA-SlHTA11-35S-cas9.
[0013] Furthermore, the aforementioned SlHTA9, SlHTA11 The nucleotide sequences containing the first 20 bases of the PAM motif adjacent to the preinterspace sequence are shown in SEQ ID NO: 5 and SEQ ID NO: 6.
[0014] Furthermore, the aforementioned sl_h2a.z By enhancing plant cold response genes SlCBF1, SlCBF2, SlCBF3 This increases the transcriptional level, thereby improving the cold resistance of tomatoes.
[0015] The technical solutions provided by the embodiments of this application may include the following beneficial effects:
[0016] As demonstrated by the above embodiments, this application reveals for the first time the regulatory role of the histone variant H2A.Z in tomato under low-temperature stress. A tomato gene editing system was constructed using CRISPR / Cas9 gene editing technology. sl_h2a.z The double mutant was investigated, and its low-temperature resistance function was studied in detail. The results showed that under low-temperature conditions... sl_h2a.z Knockout plants exhibited a significant cold-resistant phenotype. This invention is the first to discover that the histone variant H2A.Z participates in regulating plant cold resistance, providing a new gene resource for breeding new cold-resistant tomato varieties and possessing significant potential application value. Simultaneously, this invention lays a theoretical foundation for elucidating the molecular mechanisms of tomato response to stress signals and the molecular mechanisms of tolerance to adverse environments.
[0017] It should be understood that the above general description and the following detailed description are exemplary and explanatory only, and do not limit this application. Attached Figure Description
[0018] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the description, serve to explain the principles of this application.
[0019] Figure 1 The tomato in Example 1 of this invention sl_h2a.z Sequencing results of the sgRNA sequence of the gene knockout line.
[0020] Figure 2 Wild-type tomatoes and in Example 2 of this invention sl_h2a.z Double mutant plants at room temperature (25°C) o C) and low temperature (4) o C) Phenotype after 7 days of treatment.
[0021] Figure 3 Wild-type tomatoes and in Example 2 of this invention sl_h2a.z Double mutant plants at room temperature (25°C) o C) and low temperature (4) o C) Relative electrolyte permeability after 7 days of treatment.
[0022] Figure 4 Wild-type tomatoes and in Example 2 of this invention sl_h2a.z Double mutant plants at room temperature (25°C) o C) and low temperature (4) o C) Maximum photochemical efficiency (Fv / Fm) of photosystem II after 7 days of treatment.
[0023] Figure 5 Wild type and in Example 2 of this application sl_h2a.z Crispr / Cas9 gene knockout plants at room temperature (25°C) o C) and low temperature (4) o C) Expression level of cold resistance genes after 6 h of treatment, where A represents tomato SlCBF1 Gene expression levels, B represents tomato. SlCBF2 Gene expression, C for tomato SlCBF3 Gene expression levels. Detailed Implementation
[0024] The present invention will be further described below with reference to specific embodiments. It should be understood that the following examples are merely specific embodiments of the present invention, and the scope of protection of the present invention is not limited thereto.
[0025] Unless otherwise stated, the present invention will be practiced using conventional botanical techniques, tissue culture, molecular biology, physiological and biochemical techniques well known to those skilled in the art. The relevant techniques are fully explained in the literature, and the experimental reagents and materials used are commercially available.
[0026] Example 1: Tomato sl_h2a.z Construction of CRISPR / Cas9 gene knockout vector and obtaining homozygous double mutant plants.
[0027] The gene encoding the histone variant H2A.Z was found on the SGN website (http: / / solgenomics.net / ). SlHTA9, SlHTA11 The full-length DNA sequence is shown in SEQ ID NO:1 and SEQ ID NO:2.
[0028] Website design using CRISPR-P SlHTA9, SlHTA11 The sgRNA sequence was obtained, and forward and reverse primers for sgRNA were synthesized. The sgRNA was annealed using a PCR instrument to form a double-stranded sgRNA with sticky end linkers. This sgRNA was then ligated to the vector AtU6-sgRNA-AtUBQ-Cas9, which had been digested with BbsI, using T4 ligase. The ligation was performed on heat-shocked DH5α competent E. coli cells. Positive single colonies were selected and sent for sequencing. After confirming the sequencing results, the newly obtained AtU6-sgRNA-AtUBQ-Cas9 fragment was ligated to the pCAMBIA1301 vector, which had been digested with Hind III / KpnI, using T4 ligase. This fragment was then transformed into DH5α competent E. coli cells. Single colonies were picked and cultured in liquid LB medium containing 50 mg / L kanamycin at 37°C with shaking at 200 rpm overnight. PCR verification and identification of positive clones were then performed, followed by sequencing. Figure 1 ).
[0029] The correctly sequenced gene-editing vector was electroporated into GV3101 Agrobacterium competent cells. Callus was induced by infecting tomato cotyledons using the "leaf disc method," and resistance-inducing differentiation and rooting were achieved using hygromycin, yielding preliminary candidate transgenic plants. SlHTA9, SlHTA11 Specific primers were designed within 500 bp of the sgRNA sequence location of the gene to detect changes in the target gene sequence, resulting in positive lines. Homozygous lines were then obtained through self-crossing. slhta9, slhta11 After obtaining a single mutant line, a double mutant was obtained by hybridization. sl_h2a.z.
[0030] Example 2: sl_h2a.z Low-temperature resistance testing of genetically modified materials
[0031] The tomato varieties used in the experiment were wild-type '1479' (WT) and those obtained in Example 1. sl_h2a.z The double mutant was developed by sowing seeds in seed trays containing a 3:1 mixture of peat moss and vermiculite, watering with Hogrange nutrient solution to keep the substrate moist, and then subjecting the tomatoes to low-temperature treatment when they reached the five-leaf stage. The low-temperature treatment was 4℃, while the control was 25℃, with a photoperiod of 12 h.
[0032] The experiment consisted of four groups: WT room temperature group, WT low temperature group, and... sl_h2a.z room temperature group sl_h2a.z Low-temperature group: The low-temperature treatment lasted for 7 days, and young leaf tissue from the fourth leaf position of tomato was collected at 0 h, 3 h, 6 h, 9 h, and 24 h after the start of the low-temperature treatment for RNA extraction and RT-qPCR analysis of relevant cold-resistance genes. After the low-temperature treatment, phenotypic imaging, relative conductivity measurement, and maximum photochemical efficiency of photosystem II were performed.
[0033] The method for determining the maximum photochemical efficiency (Fv / Fm) of tomato photosystem II was as follows: after the tomato plants were dark adapted for 30 min, leaves of uniform size were placed on the test tray, and the maximum quantum yield of tomato PSII after 7 days of low temperature treatment was determined using a chlorophyll fluorescence imager (IMAG-PAM).
[0034] The method for determining the relative conductivity of tomatoes is as follows: Tomato leaves from the same functional part are taken, cut into strips of appropriate length avoiding the veins, and three fresh samples (0.1 g each) are quickly weighed and placed in 20 mL of deionized water. The samples are then extracted for 2 hours in a shaker at 28℃ and 200 rpm. The conductivity value R1 of the extract is measured using a conductivity meter. The extract is then heated in a 95℃ hot water bath for 15 minutes, and after cooling to room temperature, the conductivity value R2 is measured. Relative conductivity = R1 / R2 * 100%.
[0035] The real-time quantitative PCR (RT-qPCR) method was performed using a Roche Light Cycler® 480Ⅱ Real-Time PCR detection system (Roche, Swiss) and the SYBR Green RT-PCR Kit (Takara, RR420A). The total reaction volume was 20 μL, containing 10 μL of SYBR Green PCR MasterMix, 1 μL of cDNA template, 0.4 μL of forward primer, 0.4 μL of reverse primer, and 8.2 μL of dd H2O. The PCR reaction conditions were: 95℃ pre-denaturation for 3 min, 95℃ denaturation for 10 s, 58℃ annealing for 30 s, 72℃ extension for 30 s, 40 cycles, followed by a final extension at 72℃ for 5 min. Relative gene expression levels were analyzed using the delta-delta Ct method.
[0036] The results showed that, under low temperature conditions, compared with the control group WT, sl_h2a.z It exhibits a significant cold-hardy phenotype, with low levels of plant wilting. Figure 2 ), with relatively lower conductivity ( Figure 3 ), Fv / Fm value is higher (Figure 4 ), and cold-induced SlCBF1, SlCBF2, SlCBF3 Higher transcriptional expression ( Figure 5 This indicates that the histone variant H2A.Z negatively regulates the low-temperature resistance of tomatoes.
[0037] Although the present invention has been described in detail above with general descriptions and specific embodiments, the present invention is not limited to the above embodiments, and many modifications or improvements are possible, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.
Claims
1. The application of a tomato histone variant H2A.Z in improving the low-temperature resistance of tomatoes, characterized in that, The histone variant H2A.Z is composed of SlHTA8, SlHTA9, SlHTA11 Three genes encode, among which, SlHTA9 The nucleotide sequence is shown in SEQ ID NO:
1. SlHTA11 The nucleotide sequence is shown in SEQ ID NO:
2. The application involves knocking out the gene encoding histone H2A.Z using gene knockout technology. SlHTA9 and SlHTA11 This improves the low-temperature resistance of tomatoes; the low temperature is 4℃.
2. The application as described in claim 1, characterized in that, The SlHTA9, SlHTA11 The amino acid sequence of the encoded H2A.Z protein is shown in SEQ ID NO: 3 and SEQ ID NO:
4.
3. The application according to claim 2, characterized in that, The gene knockout technology is as follows: In tomatoes SlHTA9 , SlHTA11 We selected target fragments containing PAM motifs adjacent to the pre-intermediate region sequence from the protein coding region, designed corresponding primers, and constructed a CRISPR / Cas9 recombinant expression vector. The CRISPR / Cas9 recombinant expression vector was introduced into Agrobacterium, which was then used to infect tomato cotyledons. Positive transgenic plants were screened, and single mutant purified lines were obtained through self-pollination. Double mutants were obtained through hybridization. sl_h2a.z The sl_h2a.z By enhancing the cold response genes in tomatoes SlCBFs The expression of this substance can enhance the low-temperature resistance of tomatoes.
4. The application as described in claim 3, characterized in that, The recombinant expression vectors are pCAMBIA1301-U6-26-sgRNA-SlHTA9-35S-cas9 and pCAMBIA1301-U6-26-sgRNA-SlHTA11-35S-cas9.
5. The application as described in claim 3, characterized in that, The SlHTA9, SlHTA11 The nucleotide sequences containing the first 20 bases of the PAM motif adjacent to the preinterspace sequence are shown in SEQ ID NO: 5 and SEQ ID NO:
6.
6. The application as described in claim 3, characterized in that, The sl_h2a.z By enhancing plant cold response genes SlCBF1, SlCBF2, SlCBF3 This increases the transcriptional level, thereby improving the cold resistance of tomatoes.