Application of tomato SlARP6 gene as a negative regulatory factor in improving the low temperature tolerance of tomato

Abstract

This invention discloses tomatoes SlARP6 The application of genes as negative regulators in improving the cold tolerance of tomatoes, the aforementioned SlARP6 The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO: 1, and the application method is through knockout. SlARP6 The gene significantly enhances the low-temperature resistance of tomatoes. This invention provides key gene resources for the breeding of new low-temperature resistant tomato varieties, clarifying... SlARP6 This study explores the application value of genes as negative regulators of low-temperature resistance; it also lays an important theoretical foundation for elucidating the molecular pathways of tomato's response to stress signals and the molecular mechanisms of tolerance to adverse environments, and provides new ideas for molecular breeding practices to improve plant stress resistance.

Description

Technical Field

[0001] This application relates to fields such as genetic engineering, molecular biology and physiology, and specifically to the application of tomato SlARP6 as a negative regulator in improving the plant's cold tolerance. Background Technology

[0002] tomato( Solanum lycopersicum Originally from tropical South America, tomatoes are one of the most widely cultivated vegetable crops globally and a major warm-season crop in my country's greenhouse vegetable cultivation, boasting the largest cultivation area among greenhouse vegetables. Tomato growth and development are extremely sensitive to environmental changes, particularly temperature fluctuations. In recent years, with the increasing frequency of extreme weather events globally, coupled with the generally low level of equipment and poor controllability of temperature and light environments in my country's greenhouse tomato production, low-temperature stress has become a major factor restricting tomato growth and development, leading to reduced yield and quality, especially prominent in winter and spring. Therefore, effectively exploring the mechanisms and key genes involved in plant low-temperature tolerance is of significant theoretical importance, providing crucial theoretical support and germplasm resources for revealing the tomato temperature response regulatory network, improving the controllability of greenhouse cultivation environments, enhancing tomato low-temperature resistance, thereby increasing yield and quality, and reducing economic losses caused by low-temperature stress.

[0003] Chromatin remodeling factors, as core effector proteins in epigenetic regulation, can alter the interaction between DNA and histones, thereby changing the spatial conformation of chromatin. Their related complexes can utilize energy released from ATP hydrolysis to mediate nucleosome sliding, removal, and changes in nucleosome components, remodeling local or overall chromatin structure and thus regulating DNA accessibility and gene transcription status. They play an indispensable role in the regulation of plant growth, development, and stress adaptation. Chromatin remodeling factors can be divided into four subfamilies based on their Snf2 protein domains: ISWI (Imitation switch), CHD (chromodomain helicase DNA-binding), SWI / SNF (switch / sucrose nonfermentable), and INO80 (INOSITOL AUXOTROPHY 80). The INO80 family includes two highly conserved eukaryotic remodeling proteins, INO80 and SWR1, both of which can serve as integration scaffolds and form corresponding protein complexes with multiple other proteins. ARP6 is an essential core subunit of the SWR1 chromatin remodeling complex, participating in various biological processes such as plant growth and development, cell cycle progression, and stress response. In rice, OsARP6 interacts with OsPIE1, a core component of SWR1, to regulate cell cycle gene expression. osarp6 The mutant exhibited traits such as semi-dwarfism, reduced tillering, short leaves, malformed spikelets, and seed abortion. [Aziz Ul Ikram et al., “OsARP6 is involved in internode elongation by regulating cell-cycle-related genes.” Biomolecules, 2021, 11(8):1100.]; In Arabidopsis thaliana, the active form of the red light receptor phyB (Pfr) directly interacts with ARP6 to promote H2A.Z in auxin synthesis genes. YUC9 Site deposition inhibits its expression and auxin synthesis, thereby suppressing hypocotyl elongation. [Xuxu We et al., “Phytochrome B interacts with SWC6 and ARP6 to regulate H2A.Z deposition and photomorphogensis in Arabidopsis”. NewPhytologist, 2021, 63 (6): 1133-1146].

[0004] Although epigenetic factors play a crucial role in plant stress responses, research on their role in tomato's low-temperature resistance pathways remains limited, particularly regarding the function and regulatory mechanisms of the chromatin remodeling factor SlARP6 under tomato low-temperature stress. In recent years, the rapid development of CRISPR / Cas9 gene editing technology has provided new tools for plant genetic improvement. Compared to traditional breeding methods, this technology enables precise genome modification, efficiently improving crop traits and significantly shortening the breeding cycle by specifically knocking out target genes. Based on this, this study aims to utilize CRISPR / Cas9 technology to knock out SlARP6 in tomato, revealing its function under low-temperature stress and exploring potential stress-related gene resources, providing theoretical support and technical reserves for tomato low-temperature resistance breeding. Summary of the Invention

[0005] Therefore, embodiments of the present invention provide a tomato SlARP6 Application of genes as negative regulators in improving the low-temperature tolerance of tomatoes.

[0006] To achieve the objectives of this invention, the technical solution adopted is as follows: Firstly, tomatoes SlARP6 The application of genes as negative regulators in improving the low-temperature tolerance of tomatoes, wherein the application is through knockout SlARP6 The gene enhances the low-temperature resistance of tomatoes. SlARP6The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO: 1.

[0007] Furthermore, the knockout SlARP6 The gene knockout technology is as follows: In tomatoes SlARP6 The protein-coding region of the gene was selected from the target fragment containing the PAM motif adjacent to the pre-interstitial region sequence. Based on the first 20 bases of the target fragment, corresponding primers were designed to construct a CRISPR / Cas9 vector. The CRISPR / Cas9 vector was introduced into the host cell and then used to infect the target plant. Positive transgenic plants were screened to obtain low-temperature resistant transgenic plants.

[0008] Furthermore, the host cell is preferably a GV3101 Agrobacterium cell.

[0009] Furthermore, the aforementioned SlARP6 The nucleotide sequence of the first 20 bases of the gene containing the preinterspace sequence adjacent to the motif PAM structure is shown in SEQ ID NO: 3.

[0010] Furthermore, the CRISPR / Cas9 vector is pCAMBIA1301-U6-26-sgRNA-SlHTA9-35S-cas9 or pCAMBIA1301-U6-26-sgRNA-SlHTA11-35S-cas9.

[0011] Secondly, tomatoes SlARP6 The application of protein as a negative regulator in improving the low-temperature tolerance of tomatoes, wherein the application is through knockout SlARP6 The gene enhances the low-temperature resistance of tomatoes. SlARP6 The amino acid sequence of the protein is shown in SEQ ID NO: 2, and the low temperature is 4°C.

[0012] The beneficial effects of this invention are as follows: This invention provides the first analysis of chromatin remodeling factors. SlARP6 The role of tomatoes in low-temperature stress was investigated using gene knockout technology to construct tomatoes... slarp6 The mutant was studied for its low-temperature resistance function, and it was found that at low temperatures... slarp6 Knockout plants exhibited a cold-tolerant phenotype. This invention is the first to discover chromatin remodeling factors. SlARP6 It participates in plant low-temperature resistance, provides new gene resources for breeding new low-temperature tolerant tomato varieties, has good potential application value, and lays a theoretical foundation for studying the mechanism of tomato plants responding to stress signals and the molecular mechanism of tolerance to adverse environments.

[0013] 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

[0014] 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.

[0015] Figure 1 This is illustrated according to an exemplary embodiment. slarp6 Diagram of CRISPR / Cas9 transgenic material construction.

[0016] Figure 2 This is illustrated according to an exemplary embodiment. SlARP6 A graph showing gene expression levels at low temperatures.

[0017] Figure 3 This is illustrated according to an exemplary embodiment. slarp6 Leaf phenotype at 4℃.

[0018] Figure 4 This is illustrated according to an exemplary embodiment. slarp6 Chlorophyll fluorescence in leaves at a low temperature of 4℃.

[0019] Figure 5 This is illustrated according to an exemplary embodiment. slarp6 The maximum photochemical efficiency of PSII in leaves at a low temperature of 4℃.

[0020] Figure 6 This is illustrated according to an exemplary embodiment. slarp6 Relative electrical conductivity of the blade at a low temperature of 4℃. Detailed Implementation

[0021] 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.

[0022] 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.

[0023] Example 1: Tomato SlARP6 Construction of CRISPR / Cas9 gene knockout vector Website design using CRISPR-P SlARP6The gene target sequence is shown in SEQ ID No: 3. Forward and reverse primers for sgRNA were synthesized, and the sgRNA was annealed using a PCR instrument to form a double-stranded sgRNA with sticky-end adapters. 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 by heat-shock transformation of DH5α competent E. coli cells. Positive single colonies were selected 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 performed, followed by sequencing confirmation.

[0024] Example 2: Tomato slarp6 Construction and detection of genetically modified materials 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. SlARP6 Specific primers were designed by selecting 500 bp segments near the sgRNA sequence location of the gene to detect changes in the target gene sequence. slarp6 Six bases missing ( Figure 1 ).

[0025] Example 3: Tomato slarp6 Low-temperature resistance testing of genetically modified materials The tomato varieties selected for the experiment were wild-type AC (WT) and those obtained in Example 1. slarp6 The mutant was sown in seed trays containing a 3:1 mixture of peat moss and vermiculite, and watered with Hogrange nutrient solution to keep the substrate moist. When the tomato plants reached the five-leaf stage, they were subjected to a low-temperature treatment at 4°C, compared to a room-temperature control at 25°C, with a photoperiod of 12 h.

[0026] This experiment included four treatment groups: wild-type (WT) at room temperature, WT at low temperature, and... slarp6 mutant room temperature group slarp6 The mutant low-temperature group was treated at 4℃ for 7 days. Tissue samples from the fourth leaf of tomato plants were collected at 0 h, 3 h, 6 h, 9 h, and 24 h after the start of the low-temperature treatment for total RNA extraction and real-time quantitative PCR (RT-qPCR) analysis of cold tolerance-related genes. After the low-temperature treatment, plant phenotypic imaging, relative conductivity measurement, and maximum photochemical efficiency (Fv / Fm) of photosystem II (PSII) were simultaneously performed. PSII maximum photochemical efficiency (Fv / Fm) determination: After tomato plants were dark adapted for 30 min, functional leaves of uniform size were selected and placed on the detection tray. The maximum quantum yield (Fv / Fm) of PSII in tomato leaves after 7 days of low temperature treatment was determined using a chlorophyll fluorescence imaging spectrometer (IMAG-PAM). Relative conductivity determination: Leaves from the same functional part of the tomato plant were selected, and leaf strips of appropriate length were cut, avoiding the veins. Three parallel fresh samples (0.1 g each) were quickly weighed and placed in centrifuge tubes containing 20 mL of deionized water. After extraction in a constant temperature shaker at 28℃ and 200 rpm for 2 h, the conductivity of the extract was measured using a conductivity meter (R1). Subsequently, the samples were heated in a 95℃ hot water bath for 15 min, cooled to room temperature, and the conductivity was measured again (R2). The formula for calculating relative conductivity is: Relative conductivity (%) = (R1 / R2) × 100%.

[0027] RT-qPCR analysis: Detection was performed using a Roche Light Cycler® 480Ⅱ real-time quantitative PCR system (Roche, Switzerland) and a SYBR Green RT-PCR kit (Takara, catalog number RR420A). The total reaction volume was 20 μL, with the following components: 10 μL SYBR Green PCR MasterMix, 1 μL cDNA template, 0.4 μL forward primer, 0.4 μL reverse primer, and 8.2 μL ddH2O. The PCR program was set as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 10 s, 58℃ annealing for 30 s, and 72℃ extension for 30 s, for a total of 40 cycles; and a final extension at 72℃ for 5 min. Relative gene expression levels were analyzed using the delta-delta Ct method.

[0028] The results showed that SlARP6 Gene expression levels showed a gradual decreasing trend at low temperature treatment times of 0 h, 3 h, 6 h, and 9 h. Figure 2 This suggests that the gene may be involved in the regulation of low-temperature resistance in tomatoes. Furthermore, phenotypic and physiological indicators measured 7 days after low-temperature treatment showed that, compared with the wild-type (WT) control group, slarp6 The mutant exhibited a significantly enhanced cold resistance phenotype, specifically a significantly reduced degree of plant wilting. Figure 3 The maximum photochemical efficiency (Fv / Fm value) of photosystem II was significantly higher than that of the control group. Figures 4-5 ), and its relative conductivity was significantly lower than that of the control group ( Figure 6 The above results indicate that chromatin remodeling factors... SlARP6 Negative regulation of low-temperature resistance in tomatoes.

[0029] 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. Tomato SlARP6 The application of genes as negative regulators in improving the cold tolerance of tomatoes is characterized by, The application is through knockout SlARP6 The gene enhances the low-temperature resistance of tomatoes. SlARP6 The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO: 1, and the low temperature is 4°C.

2. The application according to claim 1, characterized in that, The knockout SlARP6 The gene knockout technology is as follows: In tomatoes SlARP6 The protein-coding region of the gene was selected from the target fragment containing the PAM motif adjacent to the pre-interstitial region sequence. Based on the first 20 bases of the target fragment, corresponding primers were designed to construct a CRISPR / Cas9 vector. The CRISPR / Cas9 vector was introduced into the host cell and then used to infect the target plant. Positive transgenic plants were screened to obtain low-temperature resistant transgenic plants.

3. The application according to claim 2, characterized in that, The host cell is preferably GV3101 Agrobacterium cells.

4. The application according to claim 2, characterized in that, The SlARP6 The nucleotide sequence of the first 20 bases of the gene containing the preinterspace sequence adjacent to the motif PAM structure is shown in SEQ ID NO:

3.

5. The application according to claim 2, characterized in that, The CRISPR / Cas9 vectors are pCAMBIA1301-U6-26-sgRNA-SlHTA9-35S-cas9 and pCAMBIA1301-U6-26-sgRNA-SlHTA11-35S-cas9.

6. The application of tomato SlARP6 protein as a negative regulator in improving the low-temperature tolerance of tomatoes, characterized in that, The application is through knockout SlARP6 The gene enhances the low-temperature resistance of tomatoes. The amino acid sequence of the SlARP6 protein is shown in SEQ ID NO:

2. The low temperature is 4°C.

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