A method for delaying leaf senescence by knocking out MUL8 gene using gene editing technology

Abstract

The application discloses a method for delaying leaf senescence by knocking out MUL8 genes by using a gene editing technology, and belongs to the technical field of genetic engineering. The method for delaying leaf senescence by knocking out MUL8 genes by using a gene editing technology is provided, and the leaf senescence is delayed by knocking out the MUL8 genes. The application provides a new way for delaying leaf senescence by using a gene editing technology and improving agricultural traits of crops.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and more specifically to a method for delaying leaf senescence by knocking out the MUL8 gene using gene editing technology. Background Technology

[0002] Leaf senescence is a genetically regulated process of programmed cell death, involving orderly changes such as differential gene expression, active degradation of cell structures, and recycling of nutrients. It plays a crucial role in plant reproductive growth, crop yield, and quality. The most prominent phenotype during leaf senescence is the change in leaf color from green to yellow until it withers and falls off. This is accompanied by chloroplast degradation, decreased photosynthesis, and a large accumulation of reactive oxygen species. Biomolecules such as proteins, nucleic acids, and polysaccharides are degraded into smaller molecules and transferred to other young or vigorously growing tissues and organs for reuse. The senescence rate and physiological molecular characteristics of leaves are closely influenced by both internal and environmental factors. Internal factors include age, reproductive development, and the levels of various plant hormones. Generally, ethylene (ETH), abscisic acid (ABA), jasmonic acid (JA), salicylic acid (SA), and brassinolide (BR) induce leaf senescence, while cytokinins (CTK), auxins (IAA), and gibberellins (GA) can delay it. Environmental factors influencing leaf senescence include high or low temperatures, drought, ozone, nutrient deficiency, pathogen infection, and shading. The combined effects of internal factors and the external environment form a complex network regulatory process.

[0003] With the maturation of genetic and omics analysis techniques, an increasing number of studies have revealed different molecular patterns controlling senescence at multiple regulatory levels. At the onset of senescence, genes related to photosynthesis are downregulated, while a subset of genes known as senescence-associated genes (SAGs) are upregulated transcriptionally. In recent years, tens of thousands of SAGs have been identified using transcriptomics techniques. Over the past two decades, significant progress has been made in understanding the molecular mechanisms of leaf senescence, and a preliminary molecular regulatory network for leaf senescence, centered on the NAC and WRKY transcription factor families, has been established. In addition to the transcriptional level, chromatin remodeling, post-transcriptional and post-translational modifications have been found to participate in the regulation of plant leaf senescence. In-depth exploration of new genes regulating leaf senescence has significant theoretical and practical value, providing a theoretical basis for breeding new senescence-resistant crop varieties.

[0004] Unknown functional domain proteins (DUFs) are a large group of proteins whose functions have not been characterized, and whose protein structures contain at least one highly conserved DUF domain. Research on the function of DUF genes contributes to a comprehensive and thorough understanding of the complex life mechanisms of plants and is one of the cutting-edge hot topics and challenges in the field of plant science. Currently, most research on the biological functions of plant DUF genes focuses on model plants Arabidopsis thaliana and rice. Through studies analyzing gene expression patterns, phenotypic differences and physiological changes in transgenic plants, and the expression patterns of downstream related genes, it has been demonstrated that they participate in the regulation of biological processes such as plant growth and development, plant defense responses to pests and diseases, and responses to abiotic stresses. MUL8 is a member of the DUF1997 family of proteins with unknown functions; however, the function of MUL8 protein in regulating leaf senescence has not yet been reported.

[0005] Therefore, providing a method for delaying leaf senescence by knocking out the MUL8 gene using gene editing technology is a problem that urgently needs to be solved by those skilled in the art. Summary of the Invention

[0006] In view of this, the present invention provides a method for delaying leaf senescence by knocking out the MUL8 gene using gene editing technology.

[0007] The MUL8 gene is a novel gene that influences leaf senescence in tomatoes. Overexpression of the MUL8 gene promotes leaf senescence, while knockout of the MUL8 gene delays leaf senescence. This invention, when applied to tomatoes, can delay leaf senescence, prolong photosynthetic time, and help increase tomato yield, providing a new approach to improving crop agricultural traits.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] Application of MUL8 gene knockout in delaying leaf senescence, the DNA sequence of the MUL8 gene is shown in SEQ ID NO.1.

[0010] Furthermore, a gene editing vector includes the MUL8 gene editing target; the MUL8 gene editing target is as follows:

[0011] sgRNA1: 5'-TTTGTGCAACGTTGCAGCA-3'; SEQ ID NO.6;

[0012] sgRNA2: 5'-GAGGGGCATGTCAGCTAAAA-3'; SEQ ID NO. 7.

[0013] Furthermore, a recombinant bacterium containing the aforementioned gene-editing vector.

[0014] Furthermore, a method for delaying leaf senescence by knocking out the MUL8 gene using gene editing technology includes the following steps:

[0015] (1) Construct the gene editing vector described above;

[0016] (2) The gene editing vector described in step (1) was transferred into Agrobacterium and then used to infect tomato explants;

[0017] (3) The tomato explants infected in step (2) were screened. After screening, callus tissue grew and further cultivation was carried out to obtain germplasm that delayed the senescence of tomato leaves.

[0018] Furthermore, the application of the aforementioned gene-editing vector or the aforementioned recombinant bacteria in delaying leaf senescence.

[0019] As can be seen from the above technical solution, compared with the prior art, this invention discloses a method for delaying leaf senescence by knocking out the MUL8 gene using gene editing technology. The tomato MUL8 protein affects the senescence process of tomato leaves; by knocking out the MUL8 gene, leaf senescence can be significantly delayed. This invention, on the one hand, clarifies the function of the unknown functional protein MUL8 in affecting leaf senescence; on the other hand, it provides new ideas for breeding new tomato varieties with delayed leaf senescence. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 The attached figure shows the editing pattern of the MUL8 gene-editing strain of this invention;

[0022] Figure 2 The attached figure shows the gene expression levels of the MUL8 overexpression line of this invention;

[0023] Figure 3 The attached figure shows the phenotypic diagram of the MUL8 gene-edited and overexpressed lines of this invention;

[0024] Among them, MUL8-OE: OE1; MUL8-KO: KO-8;

[0025] Figure 4 The attached figure shows the results of the physiological index measurement of the present invention;

[0026] Where A: chlorophyll content; B: MDA content;

[0027] Figure 5 The attached figure shows the phenotype of the blade treated with darkness according to the present invention;

[0028] Among them, MUL8-OE: OE1; MUL8-KO: KO-8. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] The DNA sequence of the MUL8 gene is shown in SEQ ID NO.1.

[0031] CAGAGTATCTGTCAATTACTATATAGGTTGAATAAGATGGAAATGTTTAGATGTAAAAAGAAGACGGGTAGGTCAATTACACAATCAACTAGTAAAGTAAATGGCGTCTAAACAACAAATACATGATGGCATAA CGCTTGCCCTTTCTCAAAATAGATGCAACTTCCACGCAAACAACTCCATTAGAGTATATATACATATTTTAAAACTCAAATTATCAACTAATCGATCATGAAAGTAGGTTTTGTGGCAGTGGCAGTTCCCTCTG TTTGTGCAACGTTGCAGCA GGGGAAAATCAGAAGCAGAGGGTTTCAATTATTCAAGAGAAATATACGAGTGTTGACTCCATTCCATGAATATGAAACAAAATACTATAATCCCATTAAGGCTCATCAACAGAGTTACGATTCACAAATCACCA TTTTAGCTGACATGCCCCTC

[0032] The MUL8 gene cDNA sequence is shown in SEQ ID NO.2.

[0033]

[0034] In SEQ ID NO.2, 1-231bp is the 5'UTR, 232-984bp is the ORF sequence, and 985-1284bp is the 3'UTR.

[0035] The amino acid sequence of the protein encoded by the MUL8 gene is shown in SEQ ID NO.3.

[0036] MKVGFVAVAVPSVCATLQQGKIRSRGFQLFKRNIRVLTPFHEYETKYYNPIKAHQQSYDSQITILADMPLFESPHASFDRYMEDKPRVLKAISPDNRGTQRINEEEWRIRMEPIGFLFLTAWPVVNM RLRCKTNGKEYPPGVPNHTSMVLEFKITKWDLEGVTEGKNKPSEFRLSMEGVLYPDRRISRIKGRLHMSISFAPPPMLALVPPHVHKDVTQAVMKNMAESMQHKVRNNLLADYAKFKKENPQP; SEQ ID NO.3.

[0037] Example 1

[0038] The method for constructing the MUL8 gene editing vector includes the following steps:

[0039] Using primers MUL8-PTX-Fw and MUL8-PTX-Rv, the target fragment containing the MUL8 gene editing target was amplified by PCR with pCBC-DT1T2 plasmid as a template.

[0040] The primer sequences for MUL8-PTX-Fw and MUL8-PTX-Rv are as follows:

[0041] MUL8-PTX-Fw:

[0042] 5'-GAATCTAACAGTGTAGTTTG TTTGTGCAACGTTGCAGCA GTTTTAGAGCTAGAAATAGC-3';SEQID NO.4;

[0043] MUL8-PTX-Rv:

[0044] 5'-GCTATTTCTAGCTCTAAAAC TTTTAGCTGACATGCCCCTC AAACTACACTGTTAGATTC-3'; SEQ ID NO. 5.

[0045] The target sequence of the MUL8 gene editing vector is as follows:

[0046] sgRNA1: 5'-TTTGTGCAACGTTGCAGCA-3'; SEQ ID NO.6;

[0047] sgRNA2: 5'-GAGGGGCATGTCAGCTAAAA-3'; SEQ ID NO. 7.

[0048] The PCR amplification system consisted of: 2×Phanata Max Buffer 25 μL, Phanata Max Super-Fidelity DNA Polymerase 1 μL, dNTP Mix (10 mmol / L) 1 μL, MUL8-PTX-Fw (10 μmol / L) 1 μL, MUL8-PTX-Rv (10 μmol / L) 1 μL, template DNA 1 μL, and ddH2O 20 μL.

[0049] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min.

[0050] After electrophoresis detection of the PCR products, the gel blocks were cut and recovered.

[0051] The PTX041 vector was digested with the restriction endonuclease BsaI from NEB. The reaction mixture consisted of 30 μL of PTX041 vector plasmid, 5 μL of 10×CutSmart Buffer, 1.5 μL of BsaI-HF, and 13.5 μL of ddH2O. Digestion was carried out at 37°C for 2 h. After electrophoresis, the linearized vector was recovered using a gel extraction kit from TAKARA.

[0052] The PCR-recovered product was incorporated into the PTX041 vector using the ClonExpress II One Step Cloning Kit (C112) ligase. The ligation mixture consisted of 3 μL of the PCR amplified fragment product, 4 μL of the digested PTX041 vector, 2 μL of 5×CE II buffer, and 1 μL of Exnase. The mixture was incubated at 37°C for 40 min and then placed on ice to complete the ligation.

[0053] The ligation product was introduced into competent E. coli DH5α cells and plated on LB agar plates containing 100 μg / mL kanamycin, then incubated overnight at 37°C. Single colonies were picked and PCR was performed using universal vector primers PTX-Fw and PTX-Rv. Positive clones were cultured, plasmids were extracted, and sent to Xi'an Qingke Biotechnology Co., Ltd. for sequencing verification, yielding the MUL8-PTX041 gene editing vector.

[0054] The primer sequences for PTX-Fw and PTX-Rv are as follows:

[0055] PTX-Fw: 5'-AGCGGATAACAATTTCACACAGGA-3'; SEQ ID NO.8;

[0056] PTX-Rv: 5'-GCAGGCATGCAAGCTTATTGG-3'; SEQ ID NO. 9.

[0057] Example 2

[0058] The method for constructing a MUL8 gene overexpression vector includes the following steps:

[0059] Forward and reverse primers for amplifying the MUL8 gene ORF sequence were designed using Primer3 (https: / / bioinfo.ut.ee / primer3), with the pHellsgate8 vector adapter sequence added before the primers, labeled MUL8-gate8-Fw and MUL8-gate8-Rv. The MUL8 gene containing the pHellsgate8 vector adapter sequence was amplified by PCR using cDNA reverse transcribed from total RNA of mature wild tomato cultivar Micro-Tom as a template, yielding the target fragment.

[0060] The primer sequences for MUL8-gate8-Fw and MUL8-gate8-Rv are as follows:

[0061] MUL8-gate8-Fw:

[0062] 5'-CATTTGGAGAGGACACGCTCGAG ATGAAAGTAGGTTTTGTGGCAGT -3';SEQ ID NO.10;

[0063] MUL8-gate8-Rv:

[0064] 5'-TCTCATTAAAGCAGGACTCTAGA TTATGGCTGGGGATTTTCCT -3';SEQ ID NO.11。

[0065] The PCR amplification system consisted of: 2×Phanata Max Buffer 25 μL, Phanata Max Super-FidelityDNA Polymerase 1 μL, dNTP Mix (10 mmol / L) 1 μL, MUL8-gate8-Fw (10 μmol / L) 1 μL, MUL8-gate8-Rv (10 μmol / L) 1 μL, template cDNA 1 μL, and ddH2O 20 μL.

[0066] The PCR amplification program was as follows: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s, 60℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; 72℃ final extension for 5 min.

[0067] After electrophoresis detection of the PCR products, the gel blocks were cut and recovered.

[0068] The pHellsgate8 vector was double-digested with XhoI and XbaI. The reaction mixture consisted of 30 μL of pHellsgate8 vector plasmid, 5 μL of 10×CutSmart Buffer, 1.0 μL of XhoI, 1.0 μL of XbaI, and 13.0 μL of ddH2O. Digestion was carried out at 37°C for 2 h. The linearized vector was recovered using a gel extraction kit from TAKARA after electrophoresis.

[0069] Homologous recombination was performed on the recovered target fragment and the linearized vector. The PCR product was ligated into the pHellsgate8 vector using the ClonExpress II One Step Cloning Kit (C112) ligase. The ligation mixture consisted of 3 μL of PCR amplified fragment product, 4 μL of digested pHellsgate8 vector, 2 μL of 5×CEⅡ buffer, and 1 μL of Exnase. The mixture was incubated at 37°C for 40 min and then placed on ice to complete the ligation.

[0070] The ligation product was introduced into competent E. coli cells DH5α and plated on LB agar plates containing 100 μg / mL spectinomycin, then incubated overnight at 37°C. Single colonies were picked and PCR was performed using universal vector primers pHellsgate8-Fw and pHellsgate8-Rv. Positive clones were cultured, plasmids were extracted, and sent to Xi'an Qingke Biotechnology Co., Ltd. for sequencing verification, yielding the MUL8 overexpression vector MUL8-pHellsgate8.

[0071] The primer sequences for pHellsgate8-Fw and pHellsgate8-Rv are as follows:

[0072] pHellsgate8-Fw: 5'-ACGCACAATCCCACTATCCTTC-3'; SEQ ID NO.12;

[0073] pHellsgate8-Rv: 5'-ATAGGCGTCTCGCATATCTCAT-3'; SEQ ID NO. 13.

[0074] Example 3

[0075] The genetic transformation method for MUL8 gene overexpression and gene editing vectors includes the following steps:

[0076] (1) Transformation of Agrobacterium: The tested MUL8 gene-editing plasmid and overexpression plasmid were introduced into competent Agrobacterium tumefaciens cells GV3101, respectively, and plated on LB agar plates containing Kan / Spe and Rif resistance (gene editing: 100 μg / mL Kan + 50 μg / mL Rif; overexpression: 100 μg / mL Spe + 50 μg / mL Rif), and incubated upside down at 28℃ for 2–3 days. Single colonies were picked for PCR identification of positive clones, and positive clones were selected and cultured in liquid medium containing LB agar containing Kan / Spe and Rif resistance, respectively.

[0077] (2) Inoculation: Soak the seeds of the tomato variety Micro-Tom in distilled water for 30-120 minutes, and wash the seeds in turn with 75% alcohol and sodium hypochlorite solution (84 disinfectant: purified water = 1:1); pour out the disinfectant in the clean bench, wash the seeds with sterile water 4-5 times, and filter out the seeds floating on the water surface; inoculate the washed seeds onto 1 / 2 MS medium, and culture in the dark at 25℃ for 3 days, and then culture in the light for 6 days until the two cotyledons are fully expanded.

[0078] (3) Explant preparation and infection: After removing the cotyledons of sterile seedlings, cut them into 4-6 explants and culture them in the dark on KCMS medium for 1 day; add Agrobacterium suspension and infect for 4 minutes, then discard the suspension and put the infected explants into a new KCMS medium with a layer of filter paper and culture them in the dark for 2 days.

[0079] (4) Screening: The explants that have been co-cultured are screened with the leaf face up. They are cultured for about 15 days under light and constant temperature of 25℃ until the callus tissue redifferentiates and grows adventitious buds.

[0080] (5) Subculture: Place the explants face up and subculture for about 15 days; for explants that grow well during subculture, cut off the leaf parts, leaving only the callus tissue and adventitious buds, and place them in a new subculture medium to continue culturing for about 15 days.

[0081] (6) Rooting culture: When the adventitious buds grow to 2-3cm, cut them off from the base of the root and transfer them to the rooting culture medium to induce rooting.

[0082] Example 4

[0083] The identification of MUL8 gene overexpression and MUL8 gene editing patterns includes the following steps:

[0084] (1) Identification of MUL8 gene editing forms: DNA was extracted from the leaves of the transformed gene-edited lines. The DNA from the leaves of the transformed plants was amplified using the detection primers (MUL8-KO-Fw / Rv). The product was then sent to Xi'an Qingke Biotechnology Co., Ltd. for sequencing. Three homozygous lines with different editing forms were finally obtained. The three homozygous gene-edited lines KO-8, KO-9, and KO-11 had three mutation types of -4, -1, and +1 at the target site (sgRNA2), respectively. Figure 1 ).

[0085] MUL8-KO-Fw: 5'-GCATAACGCTTGCCCTTTCTC-3'; SEQ ID NO.14;

[0086] MUL8-KO-Rv: 5'-CCACGCTGTGAGGAACAAGAA-3'; SEQ ID NO. 15.

[0087] (2) Identification of MUL8 overexpression levels in the transformed MUL8 overexpression lines: RNA was extracted from the leaves of the transformed MUL8 overexpression lines, reversed to cDNA, and the expression level of MUL8 was detected using the following primers (MUL8-Q-Fw / Rv). Finally, three overexpression lines (OE1, OE10, and OE19) were obtained. The MUL8 gene expression level in the MUL8 overexpression lines was about 2000-3000 times higher than that in the wild type. Figure 2 ).

[0088] MUL8-Q-Fw: 5'-AGAATTAGCAGGATCAAAGGTCG-3'; SEQ ID NO.16;

[0089] MUL8-Q-Rv: 5'-GGCTGGGGATTTTCCTTCTT-3'; SEQ ID NO. 17.

[0090] Example 5

[0091] The identification of MUL8 gene overexpression and gene-edited plant phenotypes includes the following aspects:

[0092] 1) Phenotypic observation

[0093] Wild-type plants with four leaves and one bud, MUL8 overexpression lines (OE1, OE10, OE19), and MUL8 gene-edited lines (KO-8, KO-9, KO-11) were transplanted into a glass greenhouse for phenotypic observation. At approximately 50 days of age, the MUL8 overexpression lines began to show signs of senescence, characterized by yellowing of the lower leaves and relatively smaller plants. At approximately 65 days of age, the MUL8 overexpression plants showed severe senescence, while the wild-type plants exhibited signs of senescence and yellowing leaves, whereas the MUL8 gene-edited plants remained green. Figure 3 The MUL8 overexpression lines (OE1, OE10, OE19) showed basically the same phenotype. Figure 3 Only OE1 was shown; the MUL8 gene-edited lines (KO-8, KO-9, KO-11) showed basically the same plant phenotype. Figure 3 Only KO-8 was shown in the video.

[0094] 2) Measurement of physiological indicators

[0095] Wild-type, MUL8 overexpression, and gene-edited plants were transplanted into a glass greenhouse and managed with normal water and fertilizer. Plant growth was observed, and physiological indicators, including chlorophyll and malondialdehyde (MDA), were measured when senescence symptoms (leaf yellowing) began to appear. Results showed that chlorophyll content in the leaves of MUL8 overexpression plants was significantly reduced at all three stages. At 65 days of seedling age, the chlorophyll content in the leaves of the MUL8 gene-edited line was significantly higher than that of the wild-type plant. Figure 4 A). As the plants grew, the malondialdehyde (MDA) content gradually increased, and the content in MUL8-overexpressing plants was higher than that in wild-type plants, while the content in MUL8-edited plants was lower than that in wild-type plants. Figure 4 B). The specific measurement method is as follows:

[0096] (1) Chlorophyll content determination

[0097] Weigh 0.2g of leaf material, cut it into small pieces, and place it in a mortar. Add 1-2mL of 95% ethanol and grind thoroughly until the tissue turns white. Let it stand for 3-5 minutes, then transfer it to a 2mL centrifuge tube. Centrifuge at 4000rpm for 5 minutes. Pipette the supernatant (extract) and measure the absorbance. Zero the centrifuge with 95% ethanol. Measure the absorbance of the extract at wavelengths of 665nm and 649nm. The chlorophyll content in the leaf is calculated using the following formula:

[0098] C 总 (mg / L)=18.08A 649 +6.63A 665

[0099] Chlorophyll content (mg / g) = [C 总[(mg / L)×Vt(mL)] / (FW×1000)

[0100] In the formula: C 总 : Chlorophyll concentration; Vt: Extraction volume; Fw: Fresh weight of sample.

[0101] The results of chlorophyll content determination were statistically analyzed using Microsoft Excel, and the mean, standard deviation, and t-test value were calculated. The results were then used to create a graph.

[0102] (2) Determination of malondialdehyde (MDA)

[0103] Pre-cool the mortar and mortar rod with liquid nitrogen. Take about 0.5g of leaves and put them into the mortar to grind into powder. Transfer the powder to a 50mL centrifuge tube, add 2mL of 10% TCA to rinse the mortar twice, and make up to 10mL with 10% TCA. Centrifuge at 4000g for 10min. The supernatant is the extract.

[0104] Pipette 2 mL of supernatant (add 2 mL of distilled water to the control) and 2 mL of 0.6% TBA solution into a 15 mL centrifuge tube. Mix well and incubate in a boiling water bath for 15–30 min. Immediately cool on ice and centrifuge at 13,000 rpm for 5 min. Take the supernatant and measure the extinction at wavelengths of 450 nm, 532 nm, and 600 nm. Each sample was tested three times. Results are expressed as mean ± standard error.

[0105] MDA (mmol / g) = [6.45 × (A) 532 -A 600 )-0.559×A 450 ]×Vt / (Vs×Fw)

[0106] Where: A: absorbance; Vt: volume of extract; Vs: volume of sample; Fw: fresh weight of sample.

[0107] The results of MDA content determination were statistically analyzed using Microsoft Excel, and the mean, standard deviation, and t-test value were calculated. The results were then used to create a graph.

[0108] 3) Dark processing

[0109] Mature leaves at the same node were harvested from wild-type tomato seedlings, the MUL8 overexpression line, and the edited line, which were two months old after transplanting. These leaves were placed in glass petri dishes containing three layers of sterile, moistened filter paper for induced senescence experiments. The plants were cultured in darkness at 25°C for 12 days, and leaf color changes were observed. The MUL8 overexpression line senescent earlier than the wild-type, while the gene-edited line remained largely green after 12 days. Figure 5 The leaf phenotypes of the MUL8 overexpression lines (OE1, OE10, OE19) were basically the same. Figure 5 Only OE1 was shown; the leaf phenotypes of the MUL8 gene-edited lines (KO-8, KO-9, KO-11) were basically the same. Figure 5 Only KO-8 was shown in the video.

[0110] In summary, MUL8, a member of the DUF1997 family of proteins with unknown function, influences the senescence process of plants. Overexpression of the MUL8 gene leads to premature senescence, while knocking out the MUL8 gene through gene editing can delay plant senescence. Therefore, by regulating the expression of the MUL8 gene in plants, the senescence process of plant leaves can be controlled. This invention provides a method for delaying leaf senescence by knocking out the MUL8 gene using gene editing technology.

[0111] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. Knock out the tomato MUL8 The application of genes in delaying leaf senescence is characterized by, The MUL8 The DNA sequence of the gene is shown in SEQ ID NO.

1.

2. A gene editing vector, characterized in that, Contains a DNA sequence encoding sgRNA; the DNA sequence encoding sgRNA is as follows: sgRNA1: 5'-TTTGTGCAACGTTGCAGCA-3'; SEQ ID NO.6; sgRNA2: 5'-GAGGGGCATGTCAGCTAAAA-3'; SEQ ID NO.

7.

3. A recombinant bacterium, characterized in that, Contains the gene editing vector as described in claim 2.

4. A method for knocking out [a gene] using gene editing technology MUL8 The method for delaying leaf senescence using genes is characterized by, The specific steps are as follows: (1) Construct the gene editing vector as described in claim 2; (2) The gene editing vector described in step (1) is transferred into Agrobacterium and then used to infect tomato explants; (3) The tomato explants infected in step (2) were screened. After screening, callus tissue grew and further cultivation was carried out to obtain germplasm that delayed the senescence of tomato leaves.

5. The application of the gene editing vector of claim 2 or the recombinant bacteria of claim 3 in delaying the senescence of tomato leaves.

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