Use of siabcg45 gene in improving orobanche and phelipanche resistance and yield of tomatoes

Abstract

The present invention relates to the technical field of biology, and specifically relates to the use of a SIABCG45 gene in improving the Orobanche and Phelipanche resistance and yield of tomatoes. Provided is a method for improving the Orobanche and Phelipanche resistance and tomato yield of a tomato plant in an Orobanche and Phelipanche parasitic environment. The method comprises: reducing the content of a SIABCG45 protein in a tomato plant or inhibiting the expression of a coding gene of the SIABCG45 protein in the tomato plant, wherein the amino acid sequence of the SIABCG45 protein is as shown in SEQ ID NO: 3. By using the method, the balance between the Orobanche and Phelipanche parasitic resistance and the growth and development of the tomato plant can be achieved, thereby significantly improving the yield of tomatoes in the Orobanche and Phelipanche parasitic environment.

Description

Application of the SlABCG45 gene in improving broomrape resistance and yield in tomatoes

[0001] Citation of relevant applications

[0002] This application claims priority to Chinese Patent Application No. 2025100436362, filed on January 10, 2025, entitled "Application of SlABCG45 Gene in Improving Orobanche Resistance and Yield in Tomato", and Chinese Patent Application No. 2025101209915, filed on January 24, 2025, entitled "Application of SlABCG45 Gene in Improving Orobanche Resistance and Yield in Tomato", the entire contents of which are incorporated herein by reference. Technical Field

[0003] This invention belongs to the field of biotechnology, specifically relating to the application of the SlABCG45 gene in improving broomrape resistance and yield in tomatoes. Background Technology

[0004] Orobanche (and Phellinanche spp.) are annual, holoparasitic weeds that parasitize the roots of plants. They lack root organs and chlorophyll, but possess haustoria. Their leaves are small and sessile, unable to perform photosynthesis. Orobanche obtains all the nutrients and water required for its growth from its host plant, thus causing significant damage to the host's growth. The inflorescence of orobanche is spike-like, with bell-shaped calyxes. Each flower contains a capsule, and each plant produces approximately 10,000 to 100,000 seeds. Using ITS (Internal Transscribed Spacer) sequencing analysis, different types of orobanche can be classified into two categories: Phellinanche and Orobanche. Phellinanche branches from the base or middle of the stem, while Orobanche is unbranched. Phelipanche, mainly including Egyptian broomcorn (P. aegyptiaca) and branching broomcorn (P. ramose), causes serious damage to economic crops such as tomatoes, melons, potatoes, tobacco, and rapeseed. Orobanche, mainly including sunflower broomcorn (O. cumana), Eurasian broomcorn (O. cernua), small broomcorn (O. minor), and serrated broomcorn (O. crenata), can parasitize sunflowers, broad beans, alfalfa, chickpeas, etc. Broomcorn species are numerous and widely distributed, with approximately 150-200 species found in Eurasia, Africa, and the Americas. In my country, there are 23 species, mainly distributed in the arid and semi-arid regions of Northwest China.

[0005] Globally, broomrape causes severe damage to the yields of important crops, threatening global food security. In the Mediterranean, North Africa, and Asia, approximately 2.6 million hectares of Solanaceae crops are infested with broomrape. In Israel, tomato yield reductions and quality degradation caused by broomrape infestation result in annual economic losses of approximately US$5 million, while branching broomrape inflicts devastating damage on the processing tomato industry in southern Italy. In Xinjiang, my country, Egyptian broomrape and branching broomrape are the most severely affected, infecting approximately 7,000 hectares of processing tomato fields annually, leading to yield reductions of 30% to 80%, and have become one of the major factors restricting the sustainable development of my country's processing tomato industry. Summary of the Invention

[0006] The inventors, through quantitative assessment and genome-wide association analysis of the parasitic phenotypes of natural tomato populations in fields contaminated with broomrape seeds, discovered that the SlABCG45 gene is associated with tomato's resistance to broomrape. The SlABCG45 gene encodes an ABCG family transporter protein (SlABCG45 protein), which was found to be involved in the exfoliation of strigolactones by tomatoes. Strigolactones are a class of plant hormones mainly synthesized in plant roots and secreted into the soil, inducing the germination of broomrape seeds in the soil. Further research demonstrated that knocking out the SlABCG45 gene significantly improved tomato plant resistance to broomrape and tomato yield in broomrape-infested environments.

[0007] Based on the above research results, the present invention provides a method for improving the resistance of tomato plants to orobanche and tomato yield in an orobanche parasitic environment, comprising: reducing the content of SlABCG45 protein in tomato plants or inhibiting the expression of the gene encoding SlABCG45 protein in tomato plants; the amino acid sequence of the SlABCG45 protein is shown in SEQ ID NO:3.

[0008] Preferably, the nucleotide sequence of the gene encoding the SlABCG45 protein is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0009] Preferably, the method is implemented by editing the gene encoding the SlABCG45 protein using a CRISPR gene editing system.

[0010] Preferably, the CRISPR gene editing system is a CRISPR-Cas9 system; the target sequence of the gene encoding the SlABCG45 protein used in the CRISPR-Cas9 system is target sequence 1 or target sequence 2; the nucleotide sequence of target sequence 1 is shown in SEQ ID NO:10; the nucleotide sequence of target sequence 2 is shown in SEQ ID NO:11.

[0011] Preferably, the method involves introducing the coding gene of sgRNA targeting target sequence 1 or target sequence 2 and the coding gene of Cas9 into tomato plants to obtain transgenic plants with increased resistance to orobanche and tomato yield in an orobanche parasitic environment.

[0012] The present invention also provides a product for improving the resistance of tomato plants to orobanche and tomato yield in an orobanche parasitic environment. The product is used to reduce the content of SlABCG45 protein in tomato plants or inhibit the expression of the gene encoding SlABCG45 protein in tomato plants. The amino acid sequence of the SlABCG45 protein is shown in SEQ ID NO:3.

[0013] Preferably, the nucleotide sequence of the gene encoding the SlABCG45 protein is shown in SEQ ID NO:1 or SEQ ID NO:2.

[0014] Preferably, the product is a reagent required for editing the gene encoding the SlABCG45 protein using a CRISPR gene editing system.

[0015] Preferably, the CRISPR gene editing system is a CRISPR-Cas9 system; the reagent is reagent 1 or reagent 2;

[0016] The reagent 1 is a composition of R1 or R2 and Cas9:

[0017] R1) targets the sgRNA of target sequence 1;

[0018] R2) targets the sgRNA of target sequence 2;

[0019] The reagent 2 is a recombinant vector containing the coding gene of R1 or R2 and the coding gene of Cas9.

[0020] The application of the SlABCG45 protein or the gene encoding the SlABCG45 protein in improving the resistance of tomato plants to broomrape parasitism and tomato yield is also within the scope of this invention.

[0021] Experiments have shown that, compared with wild-type tomato plants, tomato plants with the SlABCG45 gene knocked out not only exhibited enhanced broomrape resistance in broomrape parasitism environments, but also increased tomato yield by approximately 33%–36% (Example 3). Therefore, the method of the present invention can achieve a balance between broomrape parasitism resistance and growth and development, thereby significantly increasing tomato yield in broomrape parasitism environments. Attached Figure Description

[0022] Figure 1 shows the field survey process of broomrape parasitism on different tomato materials in Example 1. A is a schematic diagram of tomato planting in the field, and B is a flowchart of the broomrape parasitism phenotypic survey and correction.

[0023] Figure 2 shows the key genes affecting broomrape parasitism in tomato identified using genome-wide association analysis (GWAS) and transcriptome data in Example 1. A is the Manhattan plot drawn using a mixed linear model, and B is the significant SNP locus in the region (56.482-56.780 Mb) on chromosome 8 of the Manhattan plot, with linkage disequilibrium analysis performed on SNPs within a 200 Kb interval. In A and B, the observed value - Log 10 (P) = 8.638. C is the quantile plot (QQ plot), used to measure the data quality of GWAS. D shows the response of candidate genes in the region to phosphorus deficiency treatment. Genes whose expression level was induced by phosphorus deficiency by more than 2-fold are represented in red and blue, respectively.

[0024] Figure 3 shows that SlABCG45 and SlABCG44 proteins are localized on the cell membrane and possess strigolactone transport activity. In A, the bar values ​​represent the mean ± standard error (n = 3); *** indicates Student's t-test P < 0.001, * indicates Student's t-test P < 0.05. In B, FM4-64 is a cell membrane dye, and the scale bar is 20 μm.

[0025] Figure 4 shows the genome editing information of tomato SlABCG45 gene-edited materials (Slabcg45-1, Slabcg45-3, and Slabcg45-4), SlABCG44 gene-edited materials (Slabcg44-1 and Slabcg44-3), and SlCCD8 gene-edited material (Slccd8-1); PAM sequences are shown in blue, and inserted or deleted bases are shown in red; Slabcg45-1 and Slabcg44-1 were created in the wild currant tomato S. pimpinellifolium PI365967 background; Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1 were created in the cultivated tomato S. lycopersicum cv. Moneymaker background.

[0026] Figure 5 shows the strigolactone content in root extracts from wild-type (WT), SlABCG45 gene-edited material (Slabcg45-1), and SlABCG44 gene-edited material (Slabcg44-1) of tomato. Values ​​represent mean ± standard error (n=3), and *** indicates Student's t-test P<0.001.

[0027] Figure 6 shows the parasitic phenotypes of wild-type tomato (WT) and mutants Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1 in fields contaminated with broomrape seeds. A shows field photographs of wild-type tomato (WT) and mutants Slabcg45-3, Slabcg44-3, and Slccd8-1, with yellow arrows pointing to broomrape parasitizing the tomato plants. The upper left corner shows a magnified view of broomrape. Due to obstruction by field materials, some obscured broomrape could not be indicated by arrows, but these were counted and plotted in B. B represents the total number of broomrape parasitized in each plot, where values ​​represent the mean ± standard error (n = 4), and *** indicates a Student's t-test p < 0.001.

[0028] Figure 7 shows the fruit and yield phenotypes of wild-type (WT) tomato and mutants Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1. A: Fruit phenotype of wild-type (WT) tomato, mutants Slabcg45-3 and Slccd8-1; B: Average fruit size; C: Number of red fruits per plant; D: Number of green fruits per plant; E: Total number of fruits per plant; F: Weight of red fruits per plant; G: Weight of green fruits per plant; H: Total fruit weight per plant; The scale bar in A is 2.5 cm; The values ​​in B represent the mean ± standard error (n = 10); The values ​​in CH represent the mean ± standard error (n = 24). *** indicates Student's t-test P < 0.001, * indicates Student's t-test P < 0.05, and ns indicates no significant difference in the two-tailed Student's t-test.

[0029] Sequence Description

[0030] The genomic sequence of the SlABCG45 gene (SEQ ID NO:1);

[0031] The coding sequence (CDS) of the SEQ ID NO:2 SlABCG45 gene;

[0032] The amino acid sequence of the SEQ ID NO:3 SlABCG45 protein;

[0033] The genomic sequence of the SEQ ID NO:4 SlABCG44 gene;

[0034] SEQ ID NO:5 The coding sequence (CDS) of the SlABCG44 gene;

[0035] The amino acid sequence of the SlABCG44 protein; SEQ ID NO:6

[0036] The genomic sequence of the SlCCD8 gene (SEQ ID NO:7);

[0037] SEQ ID NO:8 Coding sequence (CDS) of the SlCCD8 gene;

[0038] SEQ ID NO:9 Amino acid sequence of SlCCD8 protein;

[0039] SEQ ID NO:10 Target sequence of the SlABCG45 gene in CRISPR gene editing 1;

[0040] SEQ ID NO:11 Target sequence 2 of the SlABCG45 gene in CRISPR gene editing;

[0041] SEQ ID NO:12 Target sequence of the SlABCG44 gene in CRISPR gene editing 1;

[0042] SEQ ID NO:13 Target sequence 2 of the SlABCG44 gene in CRISPR gene editing;

[0043] SEQ ID NO:14 Target sequence of the SlCCD8 gene in CRISPR gene editing 1;

[0044] SEQ ID NO:15 Target sequence 2 of the SlCCD8 gene in CRISPR gene editing;

[0045] SEQ ID NO:16 Target sequence of the SlCCD8 gene in CRISPR gene editing 3;

[0046] The nucleotide sequences of the primers used in the examples are shown in SEQ ID NO:17 to SEQ ID NO:50. Detailed Implementation

[0047] The present invention will be further described below with reference to embodiments. It should be understood that the following embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0048] The cultivated tomato *S. lycopersicum* cv. Moneymaker and the wild currant tomato *S. pimpinellifolium* PI365967 used in the following examples are described in the literature “Zhang, S., Yu, H., Wang, K., et al. (2018). Detection of major loci associated with the variation of important agronomic traits between *S. pimpinellifolium* and cultivated tomatoes. Plant J. 95:312-323.”, which are available to the public from the Institute of Genetics and Developmental Biology, Chinese Academy of Sciences.

[0049] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0050] In the quantitative experiments in the following examples, three replicate experiments were set up, and the average value of the results was taken.

[0051] The following examples used GraphPad Prism 8 statistical software to process the data. The experimental results are expressed as mean ± standard deviation. A two-tailed Student's t-test was used, and P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and ns (not significant) were recorded.

[0052] Example 1. Identification of key genes for resistance to orobanche in tomatoes

[0053] 1. Field parasitism survey

[0054] The population used in this study comprised 152 tomato accessions with good genome sequencing quality. One accession belonged to the wild tomato family, while the remaining 151 accessions belonged to the red-fruited clade. These accessions exhibited diverse geographical origins and rich genetic diversity. The genomic information of the population can be found in the reference "Zhu, G., Wang, S., Huang, Z., et al. (2018). Rewiring of the Fruit Metabolome in Tomato Breeding. Cell 172:249-261.e12". The number of broomrape parasitized on each tomato plant (broomrape parasitism) was used to reflect the resistance level of the tomato. The broomrape parasitism of this population was investigated in fields in Xinjiang contaminated with broomrape seeds. To eliminate errors caused by uneven distribution of broomrape seeds across different plots, five plots were designed, with each plot randomly planted with 16 individual plants of each material. The parasitism phenotype of each material was represented by dividing the total number of broomrape parasitized on each tomato material in each plot by the number of tomatoes planted in that plot. Meanwhile, to further improve the accuracy of the data, the parasitic phenotypes of some materials were investigated again in the field in Xinjiang in 2021 and 2022, and the parasitic phenotypes were statistically analyzed in a pot experiment under controlled greenhouse conditions in 2023. The phenotypic data from the above three years were used to calibrate the field survey data from 2015, and finally the calibrated 152 data points were used for genome-wide association analysis (GWAS) (Figure 1).

[0055] 2. Genome-wide association analysis

[0056] Figures 2A-2C show the results of genome-wide association analysis (GWAS) of broomrape parasitism in 152 tomato accessions. The Manhattan plot (Figure 2A) shows a signal on chromosome 8 that is clearly associated with the phenotype, with the SNP at the peak of this signal having a significance level of P = 4.66 × 10⁻⁶. -14 The SNP was significantly associated with orobanche parasitism. Linkage disequilibrium (LD) analysis of the region containing this signal revealed that the most associated SNP fell within a 200 kb interval, containing 21 candidate genes. A total of 23 SNPs significantly associated with orobanche parasitism exceeded the threshold (P < 2.301387 × 10⁻⁶). -9One SNP falls within an intron of the gene Solyc08g067560, 16 SNPs fall within introns or coding regions of the gene Solyc08g067620, and the remaining 6 SNPs fall in intergenic regions. Solyc08g067560 encodes a protein of unknown function. Solyc08g067620 encodes the ABCG family protein SlABCG45, which contains 13 transmembrane domains. Solyc08g067620 is abbreviated as the SlABCG45 gene, and its genomic sequence is shown in SEQ ID NO:1, while its coding sequence (CDS) is shown in SEQ ID NO:2. The amino acid sequence of the SlABCG45 protein encoded by the SlABCG45 gene is shown in SEQ ID NO:3.

[0057] 3. Identify candidate genes using transcriptome analysis (RNA-seq).

[0058] Phosphorus deficiency in the soil environment exacerbates the damage caused by orobanche. Given the close relationship between phosphorus deficiency and parasitism, candidate genes were analyzed using transcriptome data of phosphorus deficiency response in tomato roots. The phosphorus deficiency treatment experiment procedure is as follows: (1) Sterilization treatment of tomato seeds: Soak tomato seeds in 70% alcohol for 1 minute in a clean bench, discard the alcohol and wash them 3 times with sterile water. Then soak tomato seeds in 30% Kao bleach for 20 minutes, discard the alcohol and wash them 5 times with sterile water. Transfer the tomato seeds to a petri dish, keep a little water to keep them moist, and place them in a refrigerator at 4℃ for two days. (2) Prepare 1 / 2 MS solid medium in a sterile environment, sow tomato seeds on the medium, and culture for 10 days at 25℃ with a photoperiod of 16 / 8 hours. (3) Prepare 1 / 2 Hoagland liquid medium with normal phosphorus and phosphorus deficiency, transplant tomato seedlings from the solid medium to glass tubes, and culture for 10 days at 25℃ with a photoperiod of 16 / 8 hours. (4) Collect the root tissue of the tomato, absorb the moisture with toilet paper, wrap it in aluminum foil, and store it in liquid nitrogen.

[0059] 1 / 2MS solid medium (1L): Dissolve 2.2g MS salt and 10g sucrose in 990mL distilled water, adjust the pH to 5.8-6.0 with 1M KOH, bring the volume to 1L, add 8g imported agar powder, and autoclave for 15 minutes.

[0060] 1 / 2 Hoagland liquid medium (500mL): Potassium nitrate (500×) 101g, calcium nitrate tetrahydrate (500×) 236g, sodium iron ethylenediaminetetraacetate (1000×) 4.15g, magnesium sulfate heptahydrate (500×) 153g. Micronutrients (10000×): Boric acid 0.05g, manganese chloride tetrahydrate 0.035g, zinc sulfate heptahydrate 0.13g, copper sulfate pentahydrate 0.09g, molybdic acid 0.016g. Potassium dihydrogen phosphate (1000×) 89g. Adjust pH to 5.8-6.2. If preparing a phosphorus-deficient medium, do not add potassium dihydrogen phosphate.

[0061] The RNA extraction procedure from tomato roots is as follows. RNase has been removed from the reagents and consumables used in the RNA extraction process. (1) Grind the tomato root tissue sample into a uniform powder in a liquid nitrogen environment. (2) Weigh approximately 100 mg of the powder and transfer it to a 2 mL centrifuge tube. Add 1 mL of Trizol to each centrifuge tube, vortex until homogeneous, and let stand at room temperature for 5 minutes. (3) Add 200 μL of chloroform to each centrifuge tube, shake vigorously for 15 seconds, and let stand at room temperature for 2-3 minutes. (4) Centrifuge at 4℃ and 12000 rpm for 10 minutes. Take 600 μL of the supernatant and transfer it to a new 1.5 mL centrifuge tube. Add 600 μL of isopropanol to each centrifuge tube. Mix thoroughly and let stand at room temperature for 10 minutes. (5) Centrifuge at 4℃ and 12000 rpm for 10 minutes. Remove the supernatant; a white precipitate can be observed. Wash the precipitate once with 70% (v / v) ethanol and let stand at room temperature for 10 minutes to allow the precipitate to dry. (6) The precipitate was reconstituted with an appropriate amount of RNase-free water, and then the nucleic acid concentration was determined using Nanodrop. The RNA samples were sent to the DNBSEQ-T7 platform of BGI Genomics for transcriptome sequencing, with 2 μg of each sample used for library construction.

[0062] Transcriptome data showed that among the candidate genes, Solyc08g067620 (SlABCG45) responded most significantly to phosphorus deficiency treatment, with its expression upregulation exceeding 40-fold after phosphorus deficiency (Figure 2D). Therefore, SlABCG45 is considered a candidate gene influencing broomrape parasitism.

[0063] 4. SlABCG45 possesses strigolactone transport activity.

[0064] Protein sequence alignment revealed a protein in tomato, Solyc08g067610, which is highly homologous to the SlABCG45 protein, with an amino acid sequence similarity of 91.54%. The Solyc08g067610 gene is abbreviated as the SlABCG44 gene, and its genomic sequence is shown in SEQ ID NO:4, while its coding sequence (CDS) is shown in SEQ ID NO:5. The amino acid sequence of the SlABCG44 protein encoded by the SlABCG44 gene is shown in SEQ ID NO:6.

[0065] The transport activity of SlABCG45 and SlABCG44 proteins was detected using Xenopus oocytes. Xenopus oocyte vector construction: The CDS sequences of the SlABCG45 gene (SEQ ID NO:2) and SlABCG44 gene (SEQ ID NO:5) were synthesized by Hongxun Biotechnology Co., Ltd. The CDS sequences of the SlABCG45 and SlABCG44 genes were amplified using primers SlABCG45-XO-F / R and SlABCG44-XO-F / R, respectively, and then homologously recombined into the Xenopus oocyte expression vector pGHME2 (pG2). The restriction enzyme sites were BamHI / HindIII, resulting in the recombinant vectors SlABCG45-pG2 and SlABCG44-pG2. Vector pGHME2 is described in the doctoral dissertation "Qin Li. Study on the structure and activation mechanism of slow anion channels SLAHs [D]. Beijing: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 2021."

[0066] SlABCG45-XO-F:5'-GATCAATTCCCCGGGGATCCATGGAGGGTGGTGGAGAT-3' (SEQ ID NO: 17);

[0067] SlABCG45-XO-R:5'-GGTAACCAGATCAAGCTTCTATCTTTTCTGGAAGTTAAATGCTT-3' (SEQ ID NO: 18);

[0068] SlABCG44-XO-F:5'-CAATTCCCCGGGGATCCATGGAGGGTGGTGAAAATCTTGTG-3' (SEQ ID NO: 19);

[0069] SlABCG44-XO-R: 5'-GGTAACCAGATCAAGCTTCTATCTTTTCTGGAAGTTGAATGC-3' (SEQ ID NO: 20).

[0070] The preparation of cRNA mainly includes plasmid linearization, purification of linearized DNA, transcription reaction, and cRNA purification. The specific steps are as follows: (1) Use MluI enzyme to digest the pG2 vector (SlABCG45-pG2 and SlABCG44-pG2) containing the target gene. The 50μL digestion system contains 5μg of vector plasmid, 5μL of digestion buffer and 1.5μL of MluI-HF enzyme, and the remainder is made up with ddH2O. Incubate in a 37℃ water bath for 3 hours. (2) Add 450μL of DNase / RNase-free water to the digestion product, and then add 500μL of phenol / chloroform / isoamyl alcohol (25:24:1, pH 7.8), vortex to mix, and centrifuge at 14000rpm for 10 minutes. (3) After centrifugation, transfer the supernatant to a sterile 1.5 mL centrifuge tube (DNase / RNase deactivated), add 50 μL sodium acetate (pH 5.2), and mix well. (4) Add 1 mL of pre-cooled anhydrous ethanol, invert the tube to mix, and let stand at 4°C for 10 minutes. Then centrifuge at 14000 rpm for 20 minutes at 4°C. (5) After centrifugation, discard the supernatant, add 300 μL of 70% ethanol to the centrifuge tube, gently invert the tube 6-8 times, and centrifuge at 14000 rpm for 10 minutes at 4°C. After centrifugation, discard the supernatant and allow the remaining ethanol to evaporate at room temperature. (6) Prepare a 50 μL transcription reaction system: 8 μL cap structure mimic, 5 μL transcription buffer, 5 μL DTT, 2 μL rNTP mixture, 2 μL RNase inhibitor, 2 μL T7 RNA polymerase, and make up the remainder with DNase / RNase deactivated water. Add the above reaction solution to the centrifuge tube in (5) and incubate at 37°C for 3 hours. (7) After the reaction is complete, purify the cRNA using the same steps as (2)-(5), except that the pH of phenol / chloroform / isoamyl alcohol in step (2) is changed to 5.2. (8) Add an appropriate amount of DNA dehydrogenase / RNA-degrading water to dissolve the cRNA, aliquot it, and store it at -80°C.

[0071] cRNA injection and substrate injection: Under a microscope, select healthy oocytes and arrange them in a gridded petri dish, ready for microinjection. Inject 36 nL of SlABCG45 or SlABCG44 cRNA (36 ng) into each frog egg, and simultaneously inject an equal volume of sterile DNase / RNA-depleted water as a control. After adjusting the syringe scale, fill the injection needle with vegetable oil, minimizing air bubbles. Inject sequentially from left to right under the microscope. After injection, place three cells per well into a 24-well culture plate and incubate at 18°C ​​for 40 hours. Then, inject 23 nL (1.7 ng) of orobanchol (a natural form of strigolactone) into the frog eggs in the same manner. After 2.5 hours, collect the efflux fluid from the frog eggs for substrate content detection.

[0072] Purification and enrichment of strigolactones in frog egg efflux: (1) Add frog egg efflux to a pre-equilibrated Oasis HLB column (Waters). After all the efflux has eluted, wash the column 2-3 times with ddH2O. (2) After the column has dried slightly, elute the strigolactones into a 5mL Eppendorf tube with 2mL acetone and dry with nitrogen. (3) Redissolve the strigolactones in 2mL 50% acetonitrile, and then use it for mass spectrometry detection.

[0073] As shown in Figure 3A, in the frog egg cell system, both SlABCG45 and SlABCG44 proteins have the activity of transporting the natural form of strigolactone orobanchol.

[0074] Subcellular localization vector construction: Using SlABCG45-pG2 as a template, the fragment was amplified with primers SlABCG45-YFP-F / R and homologously recombined into vector YFP-HA to obtain the recombinant vector SlABCG45-YFP-HA; using SlABCG44-pG2 as a template, the fragment was amplified with primers SlABCG44-YFP-F / R and homologously recombined into vector YFP-HA to obtain the recombinant vector SlABCG44-YFP-HA. Vector YFP-HA is described in the literature "Wang, X., Liu, Z., Sun, S., et al. (2021). SISTER OF TM3 activates FRUITFULL1 to regulate inflorescence branching in tomato. Hortic. Res. 8:1-15."

[0075] SlABCG45-YFP-F:5'-GGACTCTTGAGGATCCATGGAGGGTGGTGGAGAT-3' (SEQ ID NO: 21);

[0076] SlABCG45-YFP-R:5'-TCGACAGATCCCCGGGTACCTCTTTTCTGGAAGTTAAATGCT-3' (SEQ ID NO: 22);

[0077] SlABCG44-YFP-F:5'-GGACTCTTGAGGATCCATGGAGGGTGGTGAAAATCTTGTG-3' (SEQ ID NO: 23);

[0078] S1ABCG44-YFP-R: 5'-TCGACAGATCCCCGGGTACCTCTTTTCTGGAAGTTGAATGCTTTG-3' (SEQ ID NO: 24).

[0079] Recombinant vectors SlABCG45-YFP-HA and SlABCG44-YFP-HA were introduced into Agrobacterium AGL1 via electroporation to obtain recombinant Agrobacterium AGL1-SlABCG45-YFP and AGL1-SlABCG44-YFP. Tomato callus tissue (wild currant tomato S. pimpinellifolium PI365967) was infected with recombinant Agrobacterium AGL1-SlABCG45-YFP and AGL1-SlABCG44-YFP, respectively, and tomato transgenic materials 35S:SlABCG45-YFP and 35S:SlABCG44-YFP were obtained through tissue culture.

[0080] To indicate cell membrane location, roots of transgenic materials 35S:SlABCG45-YFP and 35S:SlABCG44-YFP were immersed in 8 μM FM4-64 for 1 minute, then rinsed with water and observed as slides. Root imaging was performed using a laser scanning confocal microscope (Zeiss LSM 980 inverted microscope) with argon laser wavelengths of 514 nm (YFP) and 510 nm (FM4-64). Emission filters of 508–570 nm were used for YFP, and a 750 nm emission filter was used for FM4-64. Image analysis was performed using ZEN lite software.

[0081] As shown in Figure 3B, both SlABCG45-YFP and SlABCG44-YFP co-localize with the membrane-localizing dye FM4-64, indicating that SlABCG45 and SlABCG44 proteins are localized on the cell membrane. In conclusion, SlABCG45 and SlABCG44 proteins are strigolactone transporters located on the cell membrane.

[0082] Example 2. Creation of CRISPR gene editing materials

[0083] 1. Construction of CRISPR knockout vectors

[0084] The target sequences of genes SlABCG45, SlABCG44, and SlCCD8 were searched using the CRISPR-P v2.0 tool (http: / / cbi.hzau.edu.cn / CRISPR2 / ), and primers were designed. SlCCD8 (Solyc08g066650) is a reported strigolactone synthesis gene, whose genomic sequence is shown in SEQ ID NO:7, and its coding sequence (CDS) is shown in SEQ ID NO:8. The amino acid sequence of the SlCCD8 protein is shown in SEQ ID NO:9. The target sequences are as follows:

[0085] SlABCG45 target sequence 1:5'-AGCTCGATTTGGTGGAAAGG-3' (SEQ ID NO:10)

[0086] SlABCG45 target sequence 2:5'-GTCAGCGTGGAATGACGGG-3' (SEQ ID NO:11)

[0087] SlABCG44 target sequence 1:5'-GGCATATGCTCTGCCTACT-3' (SEQ ID NO:12)

[0088] SlABCG44 target sequence 2:5'-GATTCGAGGCAGACGTTGGG-3' (SEQ ID NO:13)

[0089] SlCCD8 target sequence 1:5'-CCGGGTCGAGCCACAAAGAA-3' (SEQ ID NO:14)

[0090] SlCCD8 target sequence 2: 5'-TATTTCTTGCCTAAATAAGC-3' (SEQ ID NO: 15)

[0091] SlCCD8 target sequence 3:5'-GCGGAGCTTGTCAAGGATGG-3' (SEQ ID NO:16)

[0092] To construct a dual-target knockout vector for SlABCG45 and SlABCG44, pCBC_DT1T2_SlU6p was used as a template. PCR was performed using Slabcg45 / 44-cri-F1 / R1, Slabcg45 / 44-cri-F2 / R2, and Slabcg45 / 44-cri-F3 / R3 to amplify the target fragment (PCR product 1, PCR product 2, and PCR product 3). The purified target fragment was digested with the restriction endonuclease BsaI, and then ligated into the BsaI-digested pTX041 vector using T4 ligase to construct the recombinant plasmid SlABCG45 / 44-Cas9.

[0093] To ensure the acquisition of the Slabcg44 single mutant, pCBC_DT1T2_SlU6p was used as a template, and PCR was performed using Slabcg44-cri-F1 / R1, Slabcg44-cri-F2 / R2, and Slabcg44-cri-F3 / R3 to amplify the target fragment (PCR product 4, PCR product 5, and PCR product 6). The purified target fragment was digested with the restriction endonuclease BsaI, and the target fragment was ligated into the pTX041 vector, which had also been digested with BsaI, using T4 ligase to construct the recombinant plasmid SlABCG44-Cas9.

[0094] To construct the SlCCD8 knockout vector, pCBC_DT1T2_SlU6p was used as a template. PCR was performed using Slccd8-cri-F1 / R1, Slccd8-cri-F2 / R2, and Slccd8-cri-F3 / R3 to amplify the target fragment (PCR product 7, PCR product 8, and PCR product 9). The purified target fragment was digested with the restriction endonuclease BsaI, and then ligated into the pTX041 vector, which had also been digested with BsaI, using T4 ligase to construct the recombinant plasmid SlCCD8-Cas9.

[0095] The plasmids pTX041 and pCBC_DT1T2_SlU6p mentioned above are described in the literature “Wang,X.,Liu,Z.,Sun,S.,etal.(2021).SISTER OF TM3activates FRUITFULL1to regulate inflorescence branching in tomato.Hortic.Res.8:1-15.”.

[0096] The nucleotide sequences of the primers are as follows:

[0097] Slabcg45 / 44-cri-F1:5'-ATATATGGTCTCGTTTGATTCGAGGCAGACGTTGGGGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:25);

[0098] Slabcg45 / 44-cri-R1:5'-ATTATTGGTCTCGTTCACAAACTACACTGTTAGATTC-3'(SEQ ID NO:26);

[0099] Slabcg45 / 44-cri-F2:5'-ATATATGGTCTCGTGAAGAGATTGTGGTCGAGGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:27);

[0100] Slabcg45 / 44-cri-R2:5'-ATTATTGGTCTCGGAGCCAAACTACACTGTTAGATTC-3'(SEQ ID NO:28);

[0101] Slabcg45 / 44-cri-F3:5'-ATATATGGTCTCGGCTCGATTTGGTGGAAAGGGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:29);

[0102] Slabcg45 / 44-cri-R3:5'-ATTATTGGTCTCGAAACGCCCGTCATTCCACGCTGACAAACTACACTGTTAGATTC-3'(SEQ ID NO:30);

[0103] Slabcg44-cri-F1:5'-ATATATGGTCTCGTTTGTGCTGCTCTAACCACCAAGGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:31);

[0104] Slabcg44-cri-R1:5'-ATTATTGGTCTCGTGCCCAAACTACACTGTTAGATTC-3'(SEQ ID NO:32);

[0105] Slabcg44-cri-F2:5'-ATATATGGTCTCGGGCATATGCTCTGCCTACTGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:33);

[0106] Slabcg44-cri-R2:5'-ATTATTGGTCTCGCCAACAAACTACACTGTTAGATTC-3'(SEQ ID NO:34);

[0107] slabcg44-cri-F3:5'-ATATATGGTCTCGTTGGATTCGAGGCAGACGTGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:35);

[0108] Slabcg44-cri-R3:5'-ATTATTGGTCTCGAAACCGCGATTTCAAGAATGATACAAACTACACTGTTAGATTC-3'(SEQ ID NO:36);

[0109] Slccd8-cri-F1:5'-ATATATGTCTCGTTTGCCCCTATGGGCTACATGGTTGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:37);

[0110] Slccd8-cri-R1:5'-ATTATTGGTCTCGCCGGCAAACTACACTGTTAGATTC-3'(SEQ ID NO:38);

[0111] Slccd8-cri-F2:5'-ATATATGGTCTCGCCGGGTCGAGCCACAAAGAAGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:39);

[0112] Slccd8-cri-R2:5'-ATTATTGGTCTCGAATACAAACTACACTGTTAGATTC-3'(SEQ ID NO:40);

[0113] Slccd8-cri-F3:5'-ATATATGGTCTCGTATTTCTTGCCTAAATAAGCGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:41);

[0114] Slccd8-cri-R3:5'-ATTATTGGTCTCGAAACCCATCCTTGACAAGCTCCGCCAAACTACACTGTTAGATTC-3'(SEQ ID NO:42)。

[0115] Enzyme digestion and ligation system 1 consisted of: 10X T4 DNA ligase buffer, 1 μL; 0.1% BSA (Takara), 1 μL; pTX041, 100 ng; PCR product 1, 30 ng; PCR product 2, 30 ng; PCR product 3, 30 ng; Bsa I (NEB), 0.5 μL; T4 DNA ligase (Promega), 0.5 μL; and ddH2O to a final volume of 10 μL.

[0116] Enzyme digestion and ligation system 2 consisted of: 10X T4 DNA ligase buffer, 1 μL; 0.1% BSA (Takara), 1 μL; pTX041, 100 ng; PCR product 4, 30 ng; PCR product 5, 30 ng; PCR product 6, 30 ng; Bsa I (NEB), 0.5 μL; T4 DNA ligase (Promega), 0.5 μL; and ddH2O was added to a final volume of 10 μL.

[0117] Enzyme digestion and ligation system 3 consisted of: 10X T4 DNA ligase buffer, 1 μL; 0.1% BSA (Takara), 1 μL; pTX041, 100 ng; PCR product 7, 30 ng; PCR product 8, 30 ng; PCR product 9, 30 ng; Bsa I (NEB), 0.5 μL; T4 DNA ligase (Promega), 0.5 μL; and ddH2O to a final volume of 10 μL.

[0118] The reaction program was as follows: (37℃, 5min → 16℃, 10min) 16 cycles; 50℃, 5min; 80℃, 5min.

[0119] 2. Transformation of Agrobacterium with plant expression vectors

[0120] Take 1 μL each of recombinant plasmids SlABCG45 / 44-Cas9, SlABCG44-Cas9, and SlCCD8-Cas9, and transform them into 100 μL of Agrobacterium tumefaciens competent cells AGL1 via high-voltage electroporation. Spread the transformed cells onto LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampin, and incubate at 28°C with inverted incubation for 2-3 days. Pick single colonies and place them in 3 mL of LB liquid medium containing the same antibiotics, and incubate overnight at 28°C with shaking at 220 rpm. The next day, transfer 200 μL of the bacterial culture to 50 mL of LB liquid medium with the same antibiotics, and incubate at 28°C with 220 rpm for 16 hours. When the bacterial culture OD... 600When the value is 0.7-0.8, bacterial cells are collected to obtain Agrobacterium AGL1-SlABCG45 / 44-Cas9, AGL1-SlABCG44-Cas9 and AGL1-SlCCD8-Cas9.

[0121] 3. Stable conversion of tomatoes

[0122] (1) Using cultivated tomato *S. lycopersicum* cv. Moneymaker and wild currant tomato *S. pimpinellifolium* PI365967 as recipients, plump, large seeds were selected. The seeds were soaked in 75% alcohol for 2 minutes, then the alcohol was discarded. The seeds were then soaked in 30% Kao bleach for 20 minutes, then the bleach was discarded. The seeds were washed four times with single-distilled sterile water. Finally, the seeds were spread on 1 / 2 MS solid medium and cultured at 25°C under 16 / 8 hour light. (2) After one week of growth on 1 / 2 MS solid medium, the cotyledons of the tomato seedlings expanded. Under aseptic conditions, the leaves were cut off with scissors and soaked in MSO for 1 hour. Excess MSO solution was blotted off the leaves with sterile filter paper, and then the leaves were placed on A1 solid medium for pre-culture in the dark at room temperature for 1 day. (3) Centrifuge the activated Agrobacterium AGL1-SlABCG45 / 44-Cas9, AGL1-SlABCG44-Cas9, and AGL1-SlCCD8-Cas9 at 200 rpm for 10 min, collect the cells, and discard the supernatant. (4) Resuspend the Agrobacterium AGL1-SlABCG45 / 44-Cas9, AGL1-SlABCG44-Cas9, and AGL1-SlCCD8-Cas9 cells in antibiotic-free LB liquid medium. Then centrifuge at 3800 rpm for 10 min to collect the cells and discard the supernatant. (5) Preparation of infection solution: Resuspend the Agrobacterium AGL1-SlABCG45 / 44-Cas9, AGL1-SlABCG44-Cas9, and AGL1-SlCCD8-Cas9 in liquid MSO medium and dilute to OD. 600 =0.3-0.4 and then add 50 μL of 0.074M acetylsyl syringone to obtain the infection solution. (6) Soak the tomato cotyledons that have been pre-cultured on A1 solid medium for 1 day in the infection solution for 15 min, then pour off the infection solution, blot the liquid on the cotyledons with sterile filter paper, and put them back on the original A1 solid medium to infect the leaves for 2-3 days. (7) Transfer the cotyledons to A2 solid medium. Culture them at 25℃ for two weeks under long-day conditions. (8) Observe the callus formation and transfer the callus tissue to A3 solid medium for inducing buds. (9) After inducing buds into strong seedlings, cut off the stem of the seedling and insert it into A4 solid medium for inducing rooting until it grows into a complete tomato transgenic material.

[0123] Culture medium and antibiotic preparation:

[0124] MSO (Agrobacterium tumefaciens suspension) (1L): MS Powder (M524) 4.33g, Sucrose 30g, bring the volume to 1L, adjust the pH to 5.9 with 1M NaOH / KOH, and autoclave for 15min.

[0125] MS medium (1L): MS Powder (M519) 4.43g, Sucrose 30g, bring to a final volume of 1L, adjust pH to 5.9 with 1M NaOH / KOH, and add 8g agar. Autoclave for 15min.

[0126] A1 solid medium (100 mL): 100 mL MS medium, 100 μL IAA (1 g / L), 175 μL ZT (1 g / L). Autoclave for 15 min.

[0127] A2 solid medium (100 mL): 100 mL MS medium, 100 μL IAA (1 g / L), 175 μL ZT (1 g / L), 225 μL Kan (50 mg / mL), 100 μL Tim (1 g / L). Autoclave for 15 min.

[0128] A3 solid medium (100 mL): 100 mL MS medium, 100 μL IAA (1 g / L), 175 μL ZT (1 g / L), 100 μL Kan (50 mg / mL), 100 μL Tim (1 g / L). Autoclave for 15 min.

[0129] A4 solid medium (100 mL): 100 mL MS medium, 100 μL Kan (50 mg / mL), 100 μL Tim (1 g / L). Autoclave for 15 min.

[0130] 1000× Rifampicin: Prepare a stock solution of 50 mg / mL with DMSO and filter to sterilize.

[0131] 1000× Kanamycin sulfate: Prepare a stock solution of 50 mg / mL with water and filter to sterilize.

[0132] In the above formula, IAA represents indoleacetic acid, ZT represents zeatin, Kan represents kanamycin, and Tim represents timentin.

[0133] 4. Identification of homozygous mutant materials

[0134] DNA was extracted from the leaves of tomato transformation materials. Single-plant samples were collected, and the leaves were placed in 2 mL Eppendof centrifuge tubes. 600 μL of CTAB extraction buffer was added, and the leaves were ground into powder using a sampler after adding a steel ball. The powder was then incubated at 65°C for 30 minutes. 200 μL of chloroform solution was added, and the mixture was shaken well and centrifuged at 9800 rpm for 10 minutes at 4°C. 400 μL of the supernatant was collected, and an equal volume of isopropanol was added. The mixture was inverted and mixed thoroughly, then centrifuged at 12000 rpm for 10 minutes at 4°C to precipitate the DNA. The DNA precipitate was washed with 70% ethanol, air-dried, and then dissolved in 100 μL of ddH2O. Using the obtained DNA as a template, the presence of bands was first detected using Cas9-F / R primers.

[0135] Cas9-F:5'-CACTATCCTTCGCAAGACCC-3'(SEQ ID NO:43);

[0136] Cas9-R: 5'-GAGATTCCCGAACAAGCCG-3' (SEQ ID NO: 44).

[0137] For materials that tested positive with Cas9 primers, the editing type was further identified using primers SlABCG45-seq-F / R, SlABCG44-seq-F / R, and SlCCD8-seq-F / R. The nucleotide sequences of the primers are as follows:

[0138] SlABCG45-seq-F:5'-TGGAGGGTGGTGGAGATATATTG-3' (SEQ ID NO: 45);

[0139] SlABCG45-seq-R:5'-TTAGCCTCTTTCTCTGCCCC-3' (SEQ ID NO: 46);

[0140] SlABCG44-seq-F:5'-CACGTCGTGATGAACCTTATAGG-3' (SEQ ID NO:47);

[0141] SlABCG44-seq-R:5'-TAGTAGGCGGATTCTTACGG-3' (SEQ ID NO:48);

[0142] SlCCD8-seq-F:5'-GAGACTATCTATTCAAAAGGTGGCA-3' (SEQ ID NO:49);

[0143] SlCCD8-seq-R: 5'-ACACATTCTATTACTGAGAGCAA-3' (SEQ ID NO: 50).

[0144] PCR reaction system (25μL): 1μL DNA template, 1.5μL 10μM forward primer, 1.5μL 10μM reverse primer and 21μL Gold Mix (Qingke Biotechnology).

[0145] PCR reaction program: 98℃ pre-denaturation for 3 min; (98℃ denaturation for 10 s, 55℃ annealing for 15 s, 72℃ extension for 30 s) 35 cycles; 72℃ extension for 5 min.

[0146] The PCR products were sent to Beijing Ruiboxingke Biotechnology Co., Ltd. for Sanger sequencing. After obtaining the sequencing results, the sequences were compared. As shown in Figure 4, 10 homozygous mutant materials with a 1bp insertion at position 1 of the SlABCG45 target sequence were identified under the PI365967 background, named Slabcg45-1; and 5 homozygous mutant materials with an 86bp deletion between positions 1 and 2 of the SlABCG44 target sequence were identified, named Slabcg44-1. Six homozygous mutants with a 1bp deletion at position 2 of the SlABCG45 target sequence were identified using the Moneymaker assay, named Slabcg45-3; five homozygous mutants with a 1bp insertion at position 2 of the SlABCG45 target sequence were identified, named Slabcg45-4; four homozygous mutants with a 7bp deletion at position 2 of the SlABCG44 target sequence were identified, named Slabcg44-3; and ten homozygous mutants with a 459bp deletion between positions 1 and 3 of the SlCCD8 target sequence were identified, named Slccd8-1. The transgenic materials were propagated in a greenhouse using a bagging method. The received seeds were planted, and DNA was extracted from the leaves. Materials that tested negative with Cas9 primers were transplanted for further propagation. Subsequent field trials were conducted using the received seeds.

[0147] 5. Determination of strigolactone content in Slabcg45-1 and Slabcg44-1 mutant materials

[0148] The material cultivation process for the strigolactone content determination experiment is as follows: (1) Sterilize wild-type (WT), Slabcg45-1 and Slabcg44-1 tomato seeds: Soak the seeds in 70% alcohol for 1 minute in a clean bench, discard the alcohol and wash them 3 times with sterile water. Then soak the seeds in 30% Kao bleach for 20 minutes, discard the alcohol and wash them 5 times with sterile water. Transfer the seeds to a petri dish, keep a little water to keep them moist, and place them in a refrigerator at 4℃ for two days. (2) Prepare 1 / 2 MS solid medium in a sterile environment, sow the tomato seeds on the medium, and culture them at 25℃ and 16 / 8 hour photoperiod for 10 days. (3) Transplant the tomato seedlings from the 1 / 2 MS solid medium to a glass tube containing 1 / 2 phosphorus-deficient Hoagland liquid medium and hydroponically culture them at 25℃ and 16 / 8 hour photoperiod for 12 days.

[0149] Collect the hydroponic solution after 5 hours and add GR24. 4DO As an internal standard, the hydroponic solution was transferred to a pre-equilibrated Oasis HLB column (Waters). The fraction containing strigolactones orobanchol and solanacol was eluted with acetone and dried under nitrogen. The eluent was reconstituted with 50% acetonitrile before LC-MS / MS analysis. Simultaneously, the fresh weight of the roots for each material was weighed to calculate the strigolactone content per unit mass of root exudate.

[0150] As shown in Figure 5, compared with the wild type (WT), the content of strigolactone in the root exudate of mutants Slabcg45-1 and Slabcg44-1 was significantly reduced, indicating that SlABCG45 and SlABCG44 proteins are crucial for the efflux of strigolactone, consistent with their function as strigolactone transporters.

[0151] Example 3. Determination of parasitism and yield of wild-type, Slabcg45-3, Slabcg45-4, Slabcg44-3 and Slccd8-1 mutants in fields contaminated with broomrape seeds.

[0152] 1. Parasitism statistics in fields contaminated with broomrape seeds

[0153] All transgenic materials planted in the field were homozygous mutants isolated from Cas9, i.e., Cas9-free materials. Since wild currant tomatoes have small fruits and no edible value, parasitism was only statistically analyzed in fields in Xinjiang contaminated with broomrape seeds, focusing on wild-type mutants (Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1) under the Moneymaker background. The specific method was as follows: Tomato seeds were transplanted to the field one month after seedling cultivation in plug trays. The land was prepared in advance, with raised beds and mulch. Drip irrigation was started immediately after transplanting to keep the soil moist. Six tomato plants were planted in two rows per plot, with 80cm wide beds, 40cm plant spacing, and 100cm distance between plots. Wild-type mutants (Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1) were planted in four plots each, spaced apart. Once the tomatoes reach about 0.5m in height, begin constructing a trellis, tying the main stem to bamboo poles, and regularly pruning and managing the vines. Later, perform routine management practices such as watering, fertilizing, and spraying herbicides.

[0154] The total number of broomrape parasites in each plot was counted during the tomato fruit ripening period. The fresh weight of broomrape in each plot was also recorded. As shown in Figure 6, compared with the wild type, the Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1 knockout mutants all exhibited enhanced broomrape resistance.

[0155] 2. Fruit and yield determination in fields contaminated with broomrape seeds.

[0156] Fruit traits and yield were measured for wild-type (WT), Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1. As shown in Figure 7, compared with wild-type (WT), the average size of individual fruits in Slabcg45-3 and Slabcg45-4 did not differ significantly, but the average size of individual fruits in Slabcg44-3 and Slccd8-1 was significantly smaller (Figure 7B). This indicates that the mutation of the SlABCG45 gene did not have a significant negative impact on fruit size, while the mutations of the SlABCG44 and SlCCD8 genes affected fruit development. Simultaneously, individual tomato plants were harvested from the above-mentioned field-grown tomatoes, and the number and weight of red fruits (red fruits), green fruits (green fruits), and all fruits (total fruits) on each plant were statistically analyzed. Statistical results showed that the number of fruits per plant and the fruit weight of Slabcg45-3 and Slabcg45-4 were significantly higher than those of the wild type (Figures 7C-7H); the total number of fruits and the total fruit weight of Slabcg44-3 did not change significantly compared with the wild type (Figures 7E and 7H); while Slccd8-1 showed a significant phenotype of fewer fruits and reduced fruit weight (Figures 7C-7H). Therefore, only knocking out the SlABCG45 gene can achieve a balance between broomrape resistance and tomato growth, that is, improving tomato resistance without causing a significant negative phenotype in tomato growth, showing potential for increased yield in the field. Finally, the total tomato yield of each plot was statistically analyzed, and compared with the wild type, the tomato materials with the SlABCG45 gene knocked out increased the yield by approximately 33% to 36% (Table 1). The above results indicate that the SlABCG45 gene has important application value in the cultivation of parasitic tomatoes, and that knocking out the SlABCG45 gene through gene editing technology can significantly increase the yield of tomatoes in parasitic environments.

[0157] Table 1

[0158] Note: The values ​​in the table are the average of the total tomato yield (g) of the four plots.

[0159] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including modifications made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A method for improving the resistance of tomato plants to broomrape parasitism and tomato yield in broomrape parasitism environments, comprising: The content of SlABCG45 protein in tomato plants or the expression of the gene encoding SlABCG45 protein in tomato plants are reduced; the amino acid sequence of the SlABCG45 protein is shown in SEQ ID NO:

3.

2. The method according to claim 1, characterized in that, The nucleotide sequence of the gene encoding the SlABCG45 protein is shown in SEQ ID NO:1 or SEQ ID NO:

2.

3. The method according to claim 1, characterized in that, The method is implemented by editing the gene encoding the SlABCG45 protein using the CRISPR gene editing system.

4. The method according to claim 3, characterized in that, The CRISPR gene editing system is a CRISPR-Cas9 system; the target sequence of the gene encoding the SlABCG45 protein used in the CRISPR-Cas9 system is target sequence 1 or target sequence 2; the nucleotide sequence of target sequence 1 is shown in SEQ ID NO:10; the nucleotide sequence of target sequence 2 is shown in SEQ ID NO:

11.

5. The method according to claim 4, characterized in that, The method involves introducing the coding gene for sgRNA targeting target sequence 1 or target sequence 2 and the coding gene for Cas9 into tomato plants to obtain transgenic plants with increased resistance to orobanche and tomato yield in an orobanche parasitic environment.

6. A product that improves the resistance of tomato plants to broomrape parasitism and tomato yield, wherein the product is used to reduce the content of SlABCG45 protein in tomato plants or inhibit the expression of the gene encoding SlABCG45 protein in tomato plants; the amino acid sequence of the SlABCG45 protein is shown in SEQ ID NO:

3.

7. The product according to claim 6, characterized in that, The nucleotide sequence of the gene encoding the SlABCG45 protein is shown in SEQ ID NO:1 or SEQ ID NO:

2.

8. The product according to claim 7, characterized in that, The product is a reagent required for editing the gene encoding the SlABCG45 protein using the CRISPR gene editing system.

9. The product according to claim 8, characterized in that, The CRISPR gene editing system is the CRISPR-Cas9 system; the reagent is reagent 1 or reagent 2; The reagent 1 is a composition of R1 or R2 and Cas9: R1) Targets the sgRNA of target sequence 1 as described in claim 4; R2) Targets the sgRNA of target sequence 2 as described in claim 4; The reagent 2 is a recombinant vector containing the coding gene of R1 or R2 and the coding gene of Cas9.

10. The application of the gene encoding the SlABCG45 protein of claim 1 or the SlABCG45 protein of claim 2 in improving the resistance of tomato plants to broomrape parasitism and tomato yield in tomato plants.

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