A method for breeding tomato plants with enhanced high temperature resistance

Abstract

The application discloses a breeding method for enhancing high-temperature resistance of tomatoes and belongs to the technical field of biotechnology. PARP3 The breeding method is to down-regulate expression or delete the function of a gene in a tomato plant by using a biological technical means so as to enhance the resistance of the tomato plant to high-temperature stress. PARP3 The application discloses the use of the gene in the negative regulation of the high-temperature stress resistance of tomatoes for the first time. PARP3 It is found that the resistance of a mutant plant with the function of the gene being deleted to high temperature is significantly enhanced by performing function verification on the gene by using a CRISPR / Cas9 gene editing technology. The application obtains the high-temperature resistant plant without introducing an exogenous gene by using the CRISPR technology to carry out tomato gene editing germplasm resource innovation, and has important theoretical and practical application values.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a method for enhancing the high-temperature resistance of tomatoes using gene editing technology. Background Technology

[0002] tomato( Solanum lycopersicum Tomato (Solanum lycopersicum) is an annual or perennial herbaceous plant belonging to the Solanaceae family and the Solanaceae genus. It thrives in sunny and warm conditions and is highly adaptable. However, hot and humid climates inhibit respiration and photosynthesis, affecting stem and leaf growth. In severe cases, this can cause sunburn, whitening, and necrosis of leaves. High temperatures also reduce tomato flower spikes, cause flower and fruit drop, shorten the growing season, and lead to premature fruit coloring before full development. This significantly reduces the time for nutrient accumulation in the fruit, severely impacting tomato yield and farmers' income (Lan Zihan et al. Long-term soilless cultivation technology and industrial significance of tomatoes in Hainan high-tech greenhouses. Anhui Agricultural Sciences, 2021, 49:41-44.). Especially in recent years, global warming has led to more frequent extreme heat waves, posing even greater challenges to tomato cultivation.

[0003] Therefore, further research into the response and regulation mechanisms of tomatoes to high temperatures, the cultivation of heat-resistant varieties, and the development of economical and effective cultivation techniques to improve the high-temperature resistance of tomatoes are of great significance for increasing tomato yield and quality and ensuring the year-round supply of tomatoes and other vegetables.

[0004] Poly-ADP ribosylation is an important protein modification pathway. Poly-ADP ribosylation is mainly catalyzed by poly-ADP ribose polymerases such as PARP, which catalyze the oxidative degradation of multiple nucleotides derived from nicotinamide adenine dinucleotide (NAD). + The transfer of ADP-ribose from Žaja to target proteins alters their physical and biochemical properties, thereby regulating physiological processes. This process also participates in various other processes, including transcriptional regulation, DNA damage and repair, apoptosis, cell cycle, and cell proliferation. et al. Poly(ADP-ribose)Recognition and Processing. Biomolecules, 2012,3:1-17.).

[0005] In plants, ADP-polyglycosylation modification has been shown to play a role in various biological stresses. For example, in Arabidopsis thaliana, knockout... AtPARP1 , AtPARP2 The constructed Arabidopsis double mutants exhibited significantly impaired immune gene activation and enhanced susceptibility to pathogens in microbial-associated molecular modeling, demonstrating that ADP-polyglycosylation plays a crucial role in plant immune gene expression and defense against pathogen attack (Kong, et al.Noncanonical mono(ADP-ribosyl)ation of zinc finger SZF proteins counteracts ubiquitination for protein homeostasis in plant immunity. Molecular cell, 2021, 22:4591-4604.); Dong et al. found that AtPARP2 in Arabidopsis can interact with the E2 ubiquitin-binding enzyme UBC13 and modify it with poly(ADP-ribosyl)ation, thereby enhancing the plant's resistance to pathogens (Yao, et al. Coordinated regulation of plant immunity by poly(ADP-ribosyl)ation and K63-linkedubiquitination. Molecular Plant, 2021,14:2088-2103.).

[0006] Currently, there is considerable research on the role of polyADP ribosylation in animal immunity and plant biotic stress, but there is still a lack of in-depth research on its role in plant abiotic stress. Summary of the Invention

[0007] The purpose of this invention is to discover genes in the tomato genome that regulate the plant's resistance to high-temperature stress, and to provide gene resources for the creation of new heat-resistant tomato germplasm.

[0008] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides PARP3 The application of genes in regulating the resistance of tomatoes to high temperature stress, the aforementioned PARP3 The CDS sequence of the gene is shown in SEQ ID NO.1, and the length of this protein-coding region is 3039 bp. The complete genome DNA sequence is shown in SEQ ID NO.3.

[0009] Should PARP3 The gene encodes a poly(ADP-ribose) polymerase, which consists of 1012 amino acids and its sequence is shown in SEQ ID NO.2.

[0010] This invention has found that by adding tomato genome... PARP3 After gene knockout, tomato plants showed significantly enhanced resistance to high-temperature stress. Specifically, under the same high-temperature conditions, compared to wild-type tomato plants, parp3The relative electrolyte leakage (REL) and malondialdehyde (MDA) content in the mutant plants were significantly reduced, and the damage to the mutant plants was significantly weakened. parp3 The maximum photochemical quantum yield of Photosystem II (Fv / Fm) in the mutant plants was significantly higher than that in the wild-type plants, and the mutant plants could still maintain high photosynthetic performance under high temperature stress. This result indicates that... PARP3 Genes are negative regulators of heat resistance in tomatoes.

[0011] High-temperature stress refers to an ambient temperature higher than the optimal temperature for plant growth and development, with the temperature range of heat stress being 35–45℃. For example, the optimal temperature for tomato plant growth is 15–35℃, and the temperature that causes heat stress is generally above 35℃. Temperatures exceeding 35℃ will have a negative impact on the growth and yield of tomatoes.

[0012] Furthermore, the application includes: using biological techniques to induce the formation of the aforementioned [product / process] in tomato plants. PARP3 Gene downregulation or loss of function can enhance the resistance of tomato plants to high-temperature stress.

[0013] In this invention, the biological techniques described may include, but are not limited to, CRISPR / Cas9 technology.

[0014] The indicators of resistance to high temperature stress include at least one of the following: plant phenotype, REL, MDA content, and Fv / Fm.

[0015] This invention also provides a method for enhancing the high-temperature resistance of tomatoes, comprising the following steps: (1) In tomatoes PARP3 The protein-coding region of the gene was selected to target a fragment containing a PAM structure. Primers were designed based on the first 20 bases of the PAM structure in the target fragment to construct a CRISPR / Cas9 vector; the tomato... PARP3 The nucleotide sequence of the gene is shown in SEQ ID NO.1; (2) Then, using Agrobacterium-mediated genetic transformation technology, the cells were transformed into tomato recipient tissues, and the resulting tissues were cultured and screened to obtain... PARP3 Homozygous mutant lines with gene loss of function.

[0016] The PAM structure is NGG, where N represents any base. The 20 bp sequence preceding NGG is defined as sgRNA, and a highly specific sgRNA sequence located in the gene protein coding region is selected. Furthermore, the nucleotide sequence of the first 20 bases of the target fragment PAM structure is shown in SEQ ID NO.4.

[0017] Furthermore, the nucleotide sequences of the primer pairs used to construct the CRISPR / Cas9 vector are shown in SEQ ID NO.5 and SEQ ID NO.6.

[0018] Furthermore, the Agrobacterium used is Agrobacterium GV3101.

[0019] Furthermore, the tomato receptor tissue was prepared using cotyledons.

[0020] Furthermore, the recipient tomato variety is Condine Red.

[0021] The PARP3 The homozygous mutant line with loss of gene function is caused by a base deletion at the sgRNA position. PARP3 A homozygous mutant line exhibiting premature termination of gene translation, lacking exogenous Cas9 protein, and stably inherited. The mutant line demonstrates significantly superior heat resistance compared to the wild-type plant.

[0022] The beneficial effects of this invention are as follows: This invention is reported for the first time. PARP3 The role of this gene in negatively regulating tomato resistance to high-temperature stress was verified using CRISPR / Cas9 gene editing technology. PARP3 Mutant plants with gene loss of function exhibit significantly enhanced resistance to high temperatures. This invention utilizes CRISPR technology to innovate tomato gene-edited germplasm resources, obtaining heat-resistant plants without introducing exogenous genes, which has significant theoretical and practical application value. Attached Figure Description

[0023] Figure 1 This is a schematic diagram of the gene editing sites in the T1 generation mutant plants obtained in Example 2. Compared to unedited normal tomatoes (hereinafter referred to as the control), the gene-edited mutants exhibit base deletions at the sgRNA position. Tomato parp3 The mutant lacks four bases compared to the control, resulting in the premature formation of a stop codon in the translation region, which leads to premature termination of translation.

[0024] Figure 2 Tomato in Example 3 parp3 Phenotypic images of mutant and wild-type plants after 18 h of high-temperature treatment at 45°C; the higher the degree of leaf wilting, the more severe the damage caused by high-temperature stress.

[0025] Figure 3 Tomato in Example 4 parp3 REL bar graphs of leaves from mutant and wild-type plants after 18 h of high-temperature treatment at 45°C; the more severe the high-temperature stress, the higher the REL value; the REL of control plant leaves was significantly higher than that of mutant plant; lowercase letters a, b, and c represent significant differences in REL values ​​among different plants at the p-value ≤ 0.001 level.

[0026] Figure 4 Tomato in Example 5 parp3 A bar graph showing the MDA content in leaves of mutant and wild-type plants after 18 h of high-temperature treatment at 45°C; the more severe the high-temperature stress, the higher the MDA; the MDA in the leaves of the control plants was significantly higher than that of the mutant plants; lowercase letters a, b, and c represent significant differences in MDA among different plants at the p-value ≤ 0.001 level.

[0027] Figure 5 Tomato in Example 6 parp3 The Fv / Fm ratios of mutant and wild-type plants after 18 h of high-temperature treatment at 45°C are shown in the figure on the right. The colors from red to blue-purple represent the numerical range from 0 to 1. The closer the color is to red-orange, the lower the Fv / Fm value, indicating more severe high-temperature stress.

[0028] Figure 6 This is a laser confocal microscopy image of the subcellular localization of the tomato PARP3 protein in Example 7; where green fluorescence indicates the location of the PARP3 protein with the GFP tag, and red fluorescence indicates the location of the nucleus of the tobacco cell with nuclear localization; when green fluorescence and red fluorescence overlap, yellow fluorescence appears. Detailed Implementation

[0029] The present invention will be further described below with reference to specific embodiments. The following examples are only specific embodiments of the present invention, but the scope of protection of the present invention is not limited thereto. Unless otherwise specified, the technical means used in the embodiments are well known to those skilled in the art, and the raw materials and reagent kits used are commercially available products.

[0030] The tomato variety used in the following examples is the conventional variety "Condine Red", and ordinary tomatoes that have not undergone gene editing are used as a control.

[0031] The nucleotide sequence runs from the 5' end to the 3' end from left to right.

[0032] Example 1: Construction of a CRISPR / Cas9 vector containing specific sgRNA It was found in the Slycopersicum_ITAG5_0 genome database on the Phytozome website: https: / / phytozome-next.jgi.doe.gov / info / Slycopersicum_ITAG5_0 PARP3 The DNA sequence of the (Solyc03G003077) gene is shown in SEQ ID NO.3. Using the website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR, a 20 bp base sequence ATTCCACTGCAGCCGGAGGA (SEQ ID NO.4) located before the PAM structure in the protein coding region was identified. This sequence has a high Onscore score, GC content >40%, Offtargetscore score <0.4.

[0033] Design CRISPR primers as follows: CRISPR preprime: gcGGTCTCTATTGaacaaagcaccagtggtctagtg (SEQ ID NO.5); CRISPR post primer: gcGGTCTCTAAACTCCTCCGGCTGCAGTGGAATtgcaccagccgggaatcg (SEQID NO.6); The above primers were used to perform PCR amplification with pBAtC-tRNA plasmid as template. After the product was purified by a conventional DNA purification kit, the amplified fragment was ligated to the pHEE401 plasmid with BsaI enzyme. The product was transformed into E. coli at 42°C and plated with kanamycin as the antibiotic.

[0034] Single colonies were selected and verified by PCR using the universal front primer M13-F: TGTAAAACGACGGCCAGT (SEQ ID NO.7) and the universal back primer M13-R: GGTATTGGTTTATCTCATCGGAACTGCA (SEQ ID NO.8) for the pHEE401 vector.

[0035] The bacterial culture with the correct band size was sent to a sequencing company for sequencing. The sequencing results showed that the vector contained one sgRNA sequence. After plasmid extraction, it was electroporated into Agrobacterium GV3101 competent cells. After two days of incubation at 28°C, the plasmids were picked for PCR verification, and Agrobacterium strains that can be used to construct CRISPR / Cas9 gene editing materials were obtained.

[0036] Example 2: parp3 Preparation and identification of mutant materials Sterilized tomato seeds were sown on a sowing medium, and the cotyledons were cut off after 7 days. The final plasmid prepared in Example 1 was transformed into the cotyledons using the Agrobacterium infection method, and the T0 generation gene-edited tomatoes were obtained by utilizing the totipotency of plant cells.

[0037] Detection of T0 generation gene-edited tomato seedlings: Genomic DNA was extracted from T0 generation plants using the CTAB method and used as a template. Primers were designed approximately 150-200 bp before and after the DNA sequence containing sgRNA, and PCR amplification and sequencing were performed for verification. Pre-seedling primer: GCTAACTGCATCTTGAGGAAAGC (SEQ ID NO.9); Primer for seedling verification: GCACATACTGTTCAGGTGCTTGC (SEQ ID NO.10); The obtained PCR products were sent to a sequencing company for sequencing. The sequencing results were compared with the original gene sequence using Snapgene software. Plants with base deletions in the sgRNA sequence and single-peak sequencing were selected for self-pollination to obtain seeds of generation T0.

[0038] The T0 generation seeds were planted in a growth chamber to obtain T1 generation plants. The sgRNA sequence editing status of the T1 generation plants was detected using the same method as described above. Simultaneously, PCR amplification of the DNA of the T1 generation plants was performed using Cas9 gene primers to detect the presence of the Cas9 sequence. T1 generation plants with mutated sgRNA and lacking the Cas9 protein were selected and identified as gene-edited lines, named […]. parp3 Its gene editing sites, such as Figure 1 As shown. parp3 Compared to the control plants, four bases were missing.

[0039] After sowing the seeds of the T1 generation of this line, stable T2 generation plants with no exogenous Cas9 gene and sgRNA mutations were obtained.

[0040] The following examples all used the above-mentioned homozygous T2 generation plants as materials for the experiment.

[0041] Example 3: Tomato parp3 High-temperature resistance study of mutants tomatoes parp3 Condine Red seeds were soaked in 55℃ warm water for 15 min, then placed in a shaker at 28℃ and 200 rpm for 2-3 days to germinate, with the water changed every 12 h. Once the seeds showed signs of germination, they were sown in 72-cell trays and cultured in an artificial climate chamber. The artificial climate chamber temperature was 25 / 20℃ (day / night), the photoperiod was 12 h light / 12 h dark, and the light intensity was 200 μmol / m². -2 s-1 When the tomato seedlings have grown a true leaf, transplant them into planting pots and water them with an appropriate amount of Hoagland nutrient solution every 3 days.

[0042] When tomato seedlings reached the stage of four leaves and one bud, healthy plants of similar size were selected and randomly divided into two groups. The control group was placed in an artificial climate chamber for continued cultivation, while the experimental group was moved into an artificial climate chamber for high-temperature treatment at 45°C. After 18 hours, the extent of high-temperature damage to the plants was observed and measured.

[0043] The results are as follows Figure 2 As shown, after treatment at 45℃ for 18 h, the WT wild-type plants showed obvious wilting, with all leaves drooping, most leaves severely curled, and the growing point bent; while parp3 The mutant wilted much less than the wild-type plant, with leaves drooping slightly and only the edges curling. It was significantly less affected by high-temperature stress than the wild-type plant.

[0044] Example 4: Tomato parp3 Determination of REL in mutant and wild-type leaves According to Example 3, in cultivation parp3 After treating mutant and control tomatoes at 45℃ for 18 h, leaves from the second and third nodes of the functional leaves were taken, cut into uniform strips, mixed thoroughly, and 0.2 g was weighed and placed in a 50 mL centrifuge tube containing 20 mL ddH2O, ensuring the leaves were completely submerged in ultrapure water. Three replicates were taken for each treatment. The samples were placed in a shaker at 28℃ and 200 rpm for 2-3 h, and the EC1 value was measured using a digital conductivity meter (DDS-307 digital conductivity meter, Shanghai Yueping). Then, the samples were placed in a water bath at 95℃ for 15 min, cooled to room temperature, and the total conductivity value EC2 was measured. The conductivity REL (%) was calculated using the following formula. REL(%) = EC1 / EC2 × 100%.

[0045] The results are as follows Figure 3 As shown, WT without high-temperature treatment and parp3 The relative efficiency (REL) of the mutant plants was similar, with no significant difference, both around 22%. However, after treatment at 45°C for 18 h, the conductivity content of both types of plants increased, and was significantly higher than that of the untreated plants. In particular, the REL of the WT plants increased to 70%, which was extremely significantly higher than that of the untreated plants. parp3 The mutant count was 40%, demonstrating that the WT plant cell membrane damage was more severe than that of other plants. parp3 The mutant is more severely affected by high temperature stress.

[0046] Example 5: Tomato parp3 Determination of MDA content in mutant and wild-type plants According to Example 3, in cultivation parp3After treating mutant and control tomatoes at 45℃ for 18 h, 0.3 g (w) of functional leaves from the second and third nodes of the tomato plants treated at 45℃ for 18 h were taken, and 6 mL of 10% TCA solution was added and ground in an ice bath at 12000 rpm. g Centrifuge for 20 min, and the resulting supernatant is the extract (V). Take 2 mL (V2) of the supernatant and add 2 mL of 0.6% TBA reaction solution. Boil in a water bath for 15 min, then immediately cool in an ice bath. g Centrifuge at 4℃ for 10 min, take the supernatant (V1) and measure its absorbance at 532 nm, 600 nm and 450 nm. Calculate the MDA content according to the following formula.

[0047] MDA content (nmol g) -1 FW)=[6.452×(A532-A600)-0.56×A450]×(V1×V) / (V2×W).

[0048] The results are as follows Figure 4 As shown, the MDA content of the two plants without high-temperature treatment was similar, both around 0.06 mmol / g, with no significant difference; after high-temperature treatment at 45°C for 18 h, the WT and parp3 The MDA levels in mutant plants were all elevated and significantly higher than in untreated plants. Specifically, the MDA level in WT plants was approximately 0.17 mmol / g, which was significantly higher than that in untreated plants. parp3 The mutant's 0.11 mmol / g indicates that the WT plant exhibits more severe membrane lipid peroxidation and greater damage than the standard mutant. parp3 The mutant is more severely affected by high temperature stress.

[0049] Example 6: Tomato parp3 Determination of Fv / Fm in mutant and wild-type plants Tomato plants before and after high-temperature treatment were dark-treated for 0.5 h. Then, the second and third functional leaves of the plants under different treatments were placed in a Dual-PAM-100 dual-channel chlorophyll fluorescence imaging system (Walz, Germany) for irradiation to detect the minimum fluorescence Fo and maximum fluorescence Fm of the leaves. Fv / Fm was obtained by the following formula.

[0050] Fv / Fm = (Fm-Fo) / Fm.

[0051] The results are as follows Figure 5 As shown, before high-temperature treatment, the Fv / Fm ratios of the two plants were similar, with no significant difference; after 18 hours of high-temperature treatment at 45°C, the WT and Fm ratios... parp3 Both the Fv / Fm ratios of the plants decreased significantly compared to the untreated plants. parp3 The mutant Fv / Fm ratio was significantly higher than that of the WT wild-type plant, indicating thatparp3 The mutant exhibits significantly higher photosystem II photoenergy conversion efficiency than the wild type under high-temperature stress, and can still maintain high photosynthetic performance under high-temperature stress conditions.

[0052] Example 7: Subcellular localization of PARP3 protein Constructing SlPARP3 pAC402 vector for gene CDS: Using tomato cDNA as a template, PCR amplification was performed using the following primers to obtain... SlPARP3 CDS sequence of the gene.

[0053] Preprimer GFP-PARP3-F: tctctctcgagctttcgcgagctcATGGCGAATCCTCCGAAGCCATGG (SEQ ID NO. 11); The back primer GFP-PARP3-R: ctcgcccttgctcaccatggatccCCGCTTGAAATTAAACCTCACTTTG (SEQ ID NO.12); The pAC402 vector (containing the reporter gene GFP) was double-digested with SacⅠ and BamHI restriction endonucleases. The digested vector was then homologously recombined with the PCR product and transformed into Escherichia coli DH5α competent cells.

[0054] The correctly sequenced plasmid was transformed into Agrobacterium competent cells GV3101 and plated. After two days of incubation at 28°C, single colonies were picked for PCR verification to obtain Agrobacterium strains that can be used for transient expression in tobacco.

[0055] After transferring the strain to LB liquid medium for propagation and activation, centrifugation was performed to obtain bacterial sediment. The culture was then resuspended in MES infection solution and the OD was adjusted. 600 After reaching 1.3-1.5 h, the culture was allowed to stand for 3 h. The resuspended bacterial solution was then injected into the nuclear-localized tobacco leaves using a syringe. This nuclear-localized tobacco had been genetically modified to incorporate the NLS nuclear localization marker gene carrying the red fluorescent protein RFP, enabling the cell nucleus to emit red fluorescence under 561 nm laser irradiation. After 36 h, tobacco leaf protoplasts were extracted using a tobacco protoplast kit (PPT201-10T, Beijing Coollab Technology Co., Ltd.) and observed and photographed under a laser confocal microscope (Nikon A1plus, Japan) using laser irradiation at wavelengths of 488 nm and 561 nm, respectively.

[0056] The results are as follows Figure 6As shown, when only GFP protein is expressed, green fluorescence appears in all parts of the cell, while yellow fluorescence overlapping with the red marker appears in the cell nucleus, indicating that GFP protein is expressed in all parts of the cell; when PARP3-GFP binding protein is expressed, only yellow fluorescence appears, that is, the green fluorescence of PARP3-GFP overlaps with the red fluorescence of the cell nucleus marker, indicating that PARP3-GFP is translated and expressed in the cell nucleus.

[0057] In summary, knockout PARP3 The tomato mutant of the gene exhibited significantly less wilting under high-temperature stress than the wild type, with significantly lower REL and MDA content in leaves, and maintained a higher Fv / Fm ratio, demonstrating stronger overall high-temperature resistance. Furthermore, gene knockout had no other impact on normal tomato growth and development. Subcellular localization results confirmed that the PARP3 protein can be translated and expressed within the plant cell nucleus. These results demonstrate... PARP3 Genes can negatively regulate tomato heat resistance, while knocking out tomatoes... PARP3 Genetics is a scientific and efficient technique for creating heat-resistant tomato germplasm resources.

Claims

1. Knock off the tomatoes PARP3 The application of genes in enhancing the resistance of tomatoes to heat stress is characterized by, The PARP3 The CDS sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The PARP3 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2.

3. A cultivation method for enhancing the high-temperature resistance of tomatoes, characterized in that, Includes the following steps: (1) In tomatoes PARP3 The protein-coding region of the gene was selected to target a fragment containing a PAM structure. Primers were designed based on the first 20 bases of the PAM structure in the target fragment to construct a CRISPR / Cas9 vector; the tomato... PARP3 The nucleotide sequence of the protein-coding region of the gene is shown in SEQ ID NO.1; (2) Then, using Agrobacterium-mediated genetic transformation technology, the cells were transformed into tomato recipient tissues, and the resulting tissues were cultured and screened to obtain... PARP3 Homozygous mutant lines with gene loss of function.

4. The cultivation method as described in claim 3, characterized in that, In step (1), the nucleotide sequence of the first 20 bases of the target fragment PAM structure is shown in SEQ ID NO.

4.

5. The cultivation method as described in claim 4, characterized in that, The nucleotide sequences of the primer pairs used to construct the CRISPR / Cas9 vector are shown in SEQ ID NO.5 and SEQ ID NO.

6.

6. The cultivation method as described in claim 3, characterized in that, In step (2), Agrobacterium GV3101 is used.

7. The cultivation method as described in claim 3, characterized in that, In step (2), the tomato recipient tissue is made from cotyledons.

8. The cultivation method as described in claim 3, characterized in that, In step (2), the recipient tomato variety is CondineRed.

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