Application of SlABCG45 gene in improvement of Orobanche coerulescens resistance and yield of tomato

Abstract

The invention belongs to the technical field of biology, and particularly relates to application of an SlABCG45 gene to improvement of Orobanche coerulescens resistance and yield of tomatoes. The invention provides a method for improving orobanche coerulescens resistance and tomato yield of a tomato plant in an orobanche coerulescens parasitic environment. The method comprises the following steps: reducing the content of SlABCG45 protein in the tomato plant or inhibiting the expression of a coding gene of the SlABCG45 protein in the tomato plant; the amino acid sequence of the SlABCG45 protein is as shown in SEQ ID NO: 3. By means of the method, balance of Orobanche coerulescens parasitic resistance and growth development of tomato plants can be achieved, and then the yield of tomatoes in an Orobanche coerulescens parasitic environment is remarkably increased.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to application of S1ABCG45 gene in improving resistance and yield of tomato broomrape. Background Art

[0002] Orobanche (Orobanche and Phelipanche spp.) is a type of annual fully parasitic weed that parasitizes the root system of plants. It has no root organs and chlorophyll, but has haustoria. The leaves are small and sessile, and cannot carry out photosynthesis. All the nutrients and water required for the growth of Orobanche come from its host plant, so the parasitism of Orobanche will cause great harm to the growth of the host. The inflorescence of Orobanche is spike-shaped, with a bell-shaped calyx. Each flower contains a capsule, and each Orobanche produces about 10,000 to 100,000 seeds. Using ITS (Internal transcribed spacer) sequence determination and analysis technology to classify and identify different Orobanche, it can be divided into two categories: Phelipanche and Orobanche. Among them, Phelipanche branches from the base or middle of the stem, while Orobanche has no branches. Phelipanche mainly includes Egyptian Orobanche (P.aegyptiaca) and Branched Orobanche (P.ramose), which cause serious damage to economic crops such as tomatoes, melons, potatoes, tobacco and rapeseed. Orobanche mainly includes Orobanche cumana, Orobanche cernua, Orobanche minor, Orobanche crenata, etc., which can parasitize sunflower, broad bean, alfalfa, chickpea, etc. There are many types of Orobanche and they are widely distributed.

[0003] Worldwide, Orobancha has caused serious damage to the yield of important crops and has become one of the important factors restricting the sustainable development of my country's processing tomato industry. Summary of the invention

[0004] The inventors conducted a quantitative assessment and whole-genome association analysis of the parasitic phenotypes of natural tomato populations in fields contaminated by Broomrape seeds, and found that the SlABCG45 gene was associated with the resistance of tomatoes to Broomrape and tomato yield. The SlABCG45 gene encodes an ABCG family transporter (SlABCG45 protein), and experiments have found that the SlABCG45 protein is involved in the process of tomato secreting strigolactones into the roots. Strigolactones are a class of plant hormones that are mainly synthesized in plant roots and secreted into the soil to induce the germination of Broomrape seeds in the soil. Further studies have shown that knocking out the SlABCG45 gene can significantly improve the resistance of tomato plants to Broomrape and tomato yield in a parasitic environment of Broomrape.

[0005] 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 environment parasitic on Orobanche, comprising: reducing the content of SlABCG45 protein in tomato plants or inhibiting the expression of the gene encoding the SlABCG45 protein in tomato plants; the amino acid sequence of the SlABCG45 protein is shown in SEQ ID NO:3.

[0006] Preferably, the nucleotide sequence of the gene encoding the S1ABCG45 protein is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0007] Preferably, the method is implemented by editing the gene encoding the SlABCG45 protein through the CRISPR gene editing system.

[0008] 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 the target sequence 1 is shown in SEQ ID NO: 10; the nucleotide sequence of the target sequence 2 is shown in SEQ ID NO: 11.

[0009] Preferably, the method obtains transgenic plants with improved resistance to Orobancha and tomato yield in an Orobancha parasitic environment by introducing the coding gene of the sgRNA targeting the target sequence 1 or the target sequence 2 and the coding gene of Cas9 into tomato plants.

[0010] The present invention also provides a product for improving the resistance of tomato plants to Orobanche and the tomato yield in an environment where Orobanche is parasitic. 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.

[0011] Preferably, the nucleotide sequence of the gene encoding the S1ABCG45 protein is shown in SEQ ID NO: 1 or SEQ ID NO: 2.

[0012] Preferably, the product is a reagent required for editing the gene encoding the SlABCG45 protein using the CRISPR gene editing system.

[0013] Preferably, the CRISPR gene editing system is a CRISPR-Cas9 system; the reagent is reagent 1 or reagent 2;

[0014] The reagent 1 is a composition consisting of R1) or R2) and Cas9:

[0015] R1) sgRNA targeting the target sequence 1;

[0016] R2) sgRNA targeting the target sequence 2;

[0017] The reagent 2 is a recombinant vector containing the coding gene of R1) or R2) and the coding gene of Cas9.

[0018] The use of the S1ABCG45 protein or the gene encoding the S1ABCG45 protein in improving the resistance of tomato plants to Orobancha parasitism and the tomato yield also falls within the scope of the present invention.

[0019] Experiments have shown that compared with wild-type tomato plants, tomato plants with the SlABCG45 gene knocked out not only showed enhanced resistance to Orobanche in a parasitic environment, but also increased tomato yield by about 33% to 36% (Example 3). Therefore, the method of the present invention can achieve a balance between resistance to Orobanche parasitism and growth and development, thereby significantly increasing the yield of tomatoes in a parasitic environment of Orobanche. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The process of investigating the parasitism of different tomato materials in the field is shown in Example 1. A is a schematic diagram of field tomato planting, and B is a flow chart of the phenotypic investigation and correction of the parasitism of Orobanche.

[0021] Figure 2 The key genes affecting the parasitism degree of broomrape in tomato were identified using genome-wide association analysis (GWAS) and transcriptome data in Example 1. A is a Manhattan plot drawn using a mixed linear model, and B is a significant SNP site in the region (56.482-56.780Mb) where the most significant peak is located on chromosome 8 in the Manhattan plot, and linkage disequilibrium analysis is performed on SNPs within the 200Kb interval. In A and B, the observed value -Log 10 (P) = 8.638. C is the quantile plot (QQ plot), which is used to measure the data quality of GWAS. D is the response of candidate genes in the region to phosphorus deficiency. Genes whose expression levels are induced by phosphorus deficiency by more than 2 times are represented in red and blue, respectively.

[0022] Figure 3 The results show that SlABCG45 and SlABCG44 proteins are localized on the cell membrane and have sclerotin transport activity. The values ​​of the bar graph in A represent the mean ± standard deviation (n = 3); *** indicates Student's t-test P < 0.001, and * indicates Student's t-test P < 0.05. FM4-64 in B is a cell membrane dye, and the scale is 20 μm.

[0023] Figure 4Genome 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 materials (Slccd8-1); the PAM sequence is represented by blue, and the inserted or deleted bases are represented by red; Slabcg45-1 and Slabcg44-1 were created in the background of wild currant tomato S.pimpinellifolium PI365967; Slabcg45-3, Slabcg45-4, Slabcg44-3 and Slccd8-1 were created in the background of cultivated tomato S.lycopersicum cv.Moneymaker.

[0024] Figure 5 The contents of strigolactone secreted by tomato wild type (WT), SlABCG45 gene-edited material (Slabcg45-1) and SlABCG44 gene-edited material (Slabcg44-1) are shown. The values ​​represent mean ± standard deviation (n=3), and *** indicates Student's t-test P<0.001.

[0025] Figure 6 The parasitic phenotypes of wild-type tomato (WT) and mutants Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1 in the field contaminated by Orobancha seeds are shown. A is a field photo of wild-type tomato (WT) and mutants Slabcg45-3, Slabcg44-3, and Slccd8-1. The yellow arrows point to the Orobancha parasitized on the tomato plants, and the upper left corner is an enlarged image of Orobancha parasitized on the tomato plants. Due to the obstruction of field materials, there are still some Orobancha that cannot be indicated by arrows, but they have been counted and plotted in B. B is the total number of Orobancha parasitized in each plot, where the values ​​represent the mean ± standard deviation (n = 4), and *** indicates Student's t-test P < 0.001.

[0026] Figure 7The fruit phenotype and yield phenotype of tomato wild type (WT) and mutants Slabcg45-3, Slabcg45-4, Slabcg44-3, Slccd8-1. A: Fruit phenotype of tomato wild type (WT), mutants Slabcg45-3 and Slccd8-1, B: Average fruit size per plant, C: Number of red fruits (red fruits) per plant, D: Number of green fruits (green fruits) per plant, E: Number of all fruits (total fruits) per plant, F: Weight of red fruits per plant, G: Weight of green fruits per plant, H: Weight of all fruits (total fruits) per plant; The scale bar in A is 2.5 cm; The values ​​in B represent the mean ± standard deviation (n = 10); The values ​​in CH represent the mean ± standard deviation (n = 24). *** indicates Student's t-test P < 0.001, * indicates Student's t-test P < 0.05, and ns indicates Student's t two-tailed test P value greater than 0.05 (no significant difference).

[0027] Sequence Description

[0028] SEQ ID NO:1 The genomic sequence of S1ABCG45 gene;

[0029] SEQ ID NO: 2S1ABCG45 gene coding sequence (CDS);

[0030] SEQ ID NO:3 Amino acid sequence of S1ABCG45 protein;

[0031] SEQ ID NO:4 The genomic sequence of S1ABCG44 gene;

[0032] SEQ ID NO:5 Coding sequence (CDS) of S1ABCG44 gene;

[0033] SEQ ID NO:6 Amino acid sequence of S1ABCG44 protein;

[0034] SEQ ID NO:7 Genomic sequence of S1CCD8 gene;

[0035] SEQ ID NO: 8S1CCD8 gene coding sequence (CDS);

[0036] SEQ ID NO:9 amino acid sequence of S1CCD8 protein;

[0037] SEQ ID NO: 10 Target sequence 1 of S1ABCG45 gene in CRISPR gene editing;

[0038] SEQ ID NO: 11 Target sequence 2 of S1ABCG45 gene in CRISPR gene editing;

[0039] SEQ ID NO: 12 Target sequence 1 of S1ABCG44 gene in CRISPR gene editing;

[0040] SEQ ID NO: 13 Target sequence 2 of S1ABCG44 gene in CRISPR gene editing;

[0041] SEQ ID NO: 14 Target sequence 1 of SlCCD8 gene in CRISPR gene editing;

[0042] SEQ ID NO: 15 Target sequence 2 of SlCCD8 gene in CRISPR gene editing;

[0043] SEQ ID NO: 16 Target sequence 3 of SlCCD8 gene in CRISPR gene editing;

[0044] SEQ ID NO: 17 to SEQ ID NO: 50 are nucleotide sequences of primers used in the Examples. DETAILED DESCRIPTION

[0045] The present invention is further described below in conjunction with examples. It should be understood that the following examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention.

[0046] The cultivated tomato S. lycopersicum cv. Moneymaker and the wild currant tomato S. pimpinellifolium PI365967 used in the following examples are recorded in the document “Zhang, S., Yu, H., Wang, K., et al. (2018). Detection of major loci associated with the variation of important agronomic traits between Solanum 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.

[0047] The experimental methods in the following examples, unless otherwise specified, are all conventional methods, and are performed according to the techniques or conditions described in the literature in the field or according to the product instructions. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial channels.

[0048] The quantitative tests in the following examples were all repeated three times, and the results were averaged.

[0049] The following examples used GraphPad Prism 8 statistical software to process the data, and the experimental results were expressed as mean ± standard deviation, and two-tailed Student's t-test was used, P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), ns (not significant).

[0050] Example 1. Identification of key genes for resistance to Orobancha parasitism in tomatoes

[0051] 1. Field parasitism survey

[0052] The population used in this study contains 152 tomato materials with good genome sequencing quality, one of which belongs to wild tomato and the other 151 materials belong to the red fruit branch. These materials have different geographical origins and contain rich genetic diversity. The genomic information of the population can be found in the literature "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 parasitoids on each tomato plant (broomrape parasitism degree) reflects the resistance level of tomato. The broomrape parasitism degree of this population was investigated in fields contaminated by broomrape seeds in Xinjiang. In order to eliminate the error caused by uneven broomrape seeds in different plots, we designed 5 plots, each of which randomly planted the population, and planted 16 individual plants for each material. The total number of broomrape parasitoids on each tomato material in each plot divided by the number of tomatoes planted in the plot represents the parasitism phenotype of the material. At the same time, in order to further improve the accuracy of the data, the parasitic phenotypes of some materials will be investigated again in the Xinjiang field in 2021 and 2022, and the parasitic phenotypes will be statistically analyzed using pot experiments under controlled conditions in the greenhouse in 2023. The phenotypic data of the above three years were used to calibrate the 2015 field survey data, and finally the calibrated 152 data were used for genome-wide association analysis (GWAS) ( Figure 1 ).

[0053] 2. Genome-wide association analysis

[0054] Figure 2 A-2C shows the results of a genome-wide association study of the parasitism of 152 tomato accessions. Figure 2 A) shows that a signal significantly associated with the phenotype appeared on chromosome 8, and the SNP with the highest point of the signal was significant at P = 4.66 × 10 -14, which was significantly associated with the parasitism of Orobancha. The linkage disequilibrium (LD) analysis of the signal interval found that the most associated SNP fell within the 200Kb interval, which contained 21 candidate genes. A total of 23 SNPs significantly associated with the parasitism of Orobancha exceeded the threshold (P<2.301387×10 -9 ). One SNP is located in the intron of gene Solyc08g067560, 16 SNPs are located in the intron or coding region of gene Solyc08g067620, and the remaining 6 SNPs are located in the intergenic region. Solyc08g067560 encodes a protein of unknown function.

[0055] Solyc08g067620 encodes the ABCG family protein S1ABCG45, which contains 13 transmembrane domains. Solyc08g067620 is referred to as the S1ABCG45 gene, and its genomic sequence is shown in SEQ ID NO: 1, and its coding sequence (CDS) is shown in SEQ ID NO: 2. The amino acid sequence of the S1ABCG45 protein encoded by the S1ABCG45 gene is shown in SEQ ID NO: 3.

[0056] 3. Identification of candidate genes using transcriptome analysis (RNA-seq)

[0057] Phosphorus deficiency in the soil environment can aggravate the damage caused by parasitism. Given the close relationship between phosphorus deficiency and parasitism, candidate genes were analyzed using transcriptome data of phosphorus deficiency responses in tomato roots. The phosphorus deficiency treatment process is as follows: (1) Sterilization of tomato seeds: Soak tomato seeds in 70% alcohol for 1 minute in a clean bench, pour out the alcohol and wash them three times with sterile water. Then soak the tomato seeds in 30% Kao bleach for 20 minutes, pour out and wash them five times with sterile water. Transfer the tomato seeds to a culture dish, keep a little water to keep them moist, and place them in a 4℃ refrigerator for two days. (2) Prepare 1 / 2MS solid culture medium in a sterile environment, sow tomato seeds on the culture medium, and culture them at 25℃, 16 / 8 h photoperiod for 10 days. (3) Prepare 1 / 2Hoagland liquid culture medium with normal phosphorus and phosphorus deficiency, transplant the tomato seedlings from the solid culture medium to a glass tube, and culture them at 25℃, 16 / 8 h photoperiod for 10 days. (4) Collect the root tissue of the tomato, absorb the moisture with toilet paper, wrap it in tin foil, and store it in liquid nitrogen.

[0058] 1 / 2MS solid culture 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, add 8g imported agar powder after the volume is fixed to 1L, and sterilize with high pressure steam for 15 minutes.

[0059] 1 / 2 Hoagland liquid medium (500mL): potassium nitrate (500×) 101g, calcium nitrate tetrahydrate (500×) 236g, sodium ferric ethylenediaminetetraacetate (1000×) 4.15g, magnesium sulfate heptahydrate (500×) 153g. Trace elements (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.

[0060] The RNA extraction process of tomato roots is as follows. The reagents and experimental consumables used in the RNA extraction process have been free of RNase. (1) Grind the tomato root tissue sample into a uniform powder in a liquid nitrogen environment. (2) Weigh approximately 100 mg of powder and transfer it to a 2 mL centrifuge tube. Add 1 mL of Trizol to each centrifuge tube, vortex evenly, and let it stand at room temperature for 5 minutes. (3) Add 200 μL of chloroform to each centrifuge tube, shake vigorously for 15 seconds, and let it stand at room temperature for 2-3 minutes. (4) Centrifuge at 4°C, 12000 rpm for 10 minutes. Pipette 600 μL of supernatant and transfer it to a new 1.5 mL centrifuge tube. Add 600 μL of isopropanol to each centrifuge tube. Mix evenly and let it stand at room temperature for 10 minutes. (5) Centrifuge at 4°C, 12000 rpm for 10 minutes. Remove the supernatant and a white precipitate can be observed. Wash the precipitate once with 70% (v / v) ethanol and let it stand at room temperature for 10 minutes to wait for the precipitate to dry. (6) The precipitate was re-dissolved with an appropriate amount of RNase-free water, and then the nucleic acid concentration was measured using Nanodrop. The RNA sample was sent to the DNBSEQ-T7 platform of BGI for transcriptome sequencing, and the amount of each sample used for library construction was 2 μg.

[0061] According to the transcriptome data, Solyc08g067620 (SlABCG45) was the most responsive to phosphorus deficiency among the candidate genes, with the gene being upregulated more than 40-fold after phosphorus deficiency ( Figure 2 D). In conclusion, SlABCG45 is a candidate gene that affects the parasitism degree of Orobanche deserticola.

[0062] 4. SlABCG45 has strigolactone transport activity

[0063] Through protein sequence comparison, it was found that there is a protein Solyc08g067610 in tomato that is highly homologous to the SlABCG45 protein, and its amino acid sequence similarity is 91.54%. The Solyc08g067610 gene is referred to as the SlABCG44 gene, and its genomic sequence is shown in SEQ ID NO:4, and 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.

[0064] The transport activity of SlABCG45 and SlABCG44 proteins was detected using Xenopus oocytes. Construction of Xenopus oocyte vector: The CDS sequence of the SlABCG45 gene (SEQ ID NO: 2) and the CDS sequence of the 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 homologously recombined into the African Xenopus oocyte expression vector pG2, with restriction sites of BamHI / HindIII, to obtain recombinant vectors SlABCG45-pG2 and SlABCG44-pG2. The vector pG2 is described in the doctoral thesis "Qin Li. Study on the structure and activation mechanism of the slow anion channel SLAH s [D]. Beijing: Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, 2021."

[0065] SlABCG45-XO-F:5'-GATCAATTCCCCGGGGATCCATGGAGGGTGGTGGAGAT-3'

[0066] (SEQ ID NO: 17);

[0067] SlABCG45-XO-R:

[0068] 5'-GGTAACCAGATCAAGCTTCTATCTTTTCTGGAAGTTAAATGCTT-3' (SEQ ID NO: 18);

[0069] SlABCG44-XO-F:5'-CAATTCCCCGGGGATCCATGGAGGGTGGTGAAAATCTTGTG-3' (SEQ IDNO: 19);

[0070] SlABCG44-XO-R:

[0071] 5'-GGTAACCAGATCAAGCTTCTATCTTTTCTGGAAGTTGAATGC-3' (SEQ ID NO: 20).

[0072] The preparation of cRNA mainly includes plasmid linearization, purification of linearized DNA, transcription reaction and purification of cRNA. 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 vector plasmid, 5μL digestion buffer and 1.5μL MluI-HF enzyme, and the rest is filled with ddH2O. Incubate in a 37℃ water bath for 3 hours. (2) Add 450μL DNase / RNase-free water to the digestion product, then add 500μL phenol / chloroform / isoamyl alcohol (25:24:1, pH 7.8), vortex to mix, and centrifuge at 14000rpm for 10 minutes. (3) After centrifugation, take the supernatant to a sterilized 1.5 mL centrifuge tube free of DNase / RNase, add 50 μL of sodium acetate (pH 5.2), and mix well. (4) Add 1 mL of pre-cooled anhydrous ethanol, mix well by inverting, and let stand at 4°C for 10 minutes. Then centrifuge at 14,000 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 centrifuge tube 6-8 times, and centrifuge at 14,000 rpm for 10 minutes at 4°C. After centrifugation, pour out the supernatant and wait for the remaining ethanol to evaporate at room temperature. (6) Prepare a 50 μL transcription reaction system: 8 μL of cap structure mimic, 5 μL of transcription buffer, 5 μL of DTT, 2 μL of rNTP mixture, 2 μL of RNase inhibitor, 2 μL of T7 RNA polymerase, and make up the rest with DNase / RNase-free water. Add the above reaction solution to the centrifuge tube in (5) and place it in a 37°C constant temperature incubator for reaction for 3 hours. (7) After the reaction is completed, purify the cRNA, the steps are the same as (2)-(5), and the pH of phenol / chloroform / isoamyl alcohol in step (2) is changed to 5.2. (8) Add an appropriate amount of DNase / RNA-free water to dissolve the cRNA, and store it at -80°C after aliquoting.

[0073] cRNA injection and substrate injection: Select oocytes in good condition under a microscope, arrange them in a grid-lined dish, and prepare for microinjection. Each frog egg is injected with 36nL SlABCG45 or SlABCG44 cRNA (36ng), and an equal volume of sterilized DNase / RNA-free water is injected as a control. After adjusting the scale of the injection instrument, fill the injection needle with vegetable oil, and try not to have bubbles. Under the microscope, inject from left to right. After the injection, place one well of every 3 cells in a 24-well culture plate and culture at 18℃ for 40 hours. Then, inject the substrate 23nL (1.7ng) orobanchol (orobanchol, a natural form of monocotyledon) into the frog eggs in the same way. After 2.5 hours, collect the frog egg exudate for substrate content detection.

[0074] Purification and enrichment of strigolactones in frog egg exudate: (1) Add the frog egg exudate to the pre-equilibrated Oasis HLB column (Waters), and after all the exudate has flowed through, rinse the column 2-3 times with ddH2O. (2) After the column is slightly dry, elute the strigolactones with 2 mL of acetone into a 5 mL Eppendorf tube and blow dry with nitrogen. (3) Redissolve with 2 mL of 50% acetonitrile and then use for mass spectrometry detection.

[0075] like Figure 3 As shown in A, in the frog egg cell system, both SlABCG45 and SlABCG44 proteins have the activity of transporting the natural form of orobanchol.

[0076] Subcellular localization vector construction: Using SlABCG45-pG2 as a template, the fragment was amplified with primers SlABCG45-YFP-F / R and homologously recombined into the 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 the vector YFP-HA to obtain the recombinant vector SlABCG44-YFP-HA. The vector YFP-HA is recorded in the document "Wang, X., Liu, Z., Sun, S., et al. (2021). SISTER OF TM3activates FRUITFULL1 to regulate inflorescence branching into mato. Hortic. Res. 8: 1-15."

[0077] SlABCG45-YFP-F:5'-GGACTCTTGAGGATCCATGGAGGGTGGTGGAGAT-3' (SEQ ID NO: 21);

[0078] SlABCG45-YFP-R:

[0079] 5'-TCGACAGATCCCCGGGTACCTCTTTTCTGGAAGTTAAATGCT-3' (SEQ ID NO: 22);

[0080] SlABCG44-YFP-F:5'-GGACTCTTGAGGATCCATGGAGGGTGGTGAAAATCTTGTG-3' (SEQ IDNO: 23);

[0081] SlABCG44-YFP-R:

[0082] 5'-TCGACAGATCCCCGGGTACCTCTTTTCTGGAAGTTGAATGCTTTG-3' (SEQ ID NO: 24).

[0083] The recombinant vectors S1ABCG45-YFP-HA and S1ABCG44-YFP-HA were introduced into Agrobacterium AGL1 by electroporation to obtain recombinant Agrobacterium AGL1-S1ABCG45-YFP and AGL1-S1ABCG44-YFP. The recombinant Agrobacterium AGL1-S1ABCG45-YFP and AGL1-S1ABCG44-YFP were used to infect tomato callus (wild currant tomato S.pimpinellifolium PI365967) to obtain tomato transgenic materials 35S:S1ABCG45-YFP and 35S:S1ABCG44-YFP by tissue culture.

[0084] To indicate the location of the cell membrane, the roots of the transgenic materials 35S:SlABCG45-YFP and 35S:SlABCG44-YFP were soaked in 8μM FM4-64 for 1 minute, then washed with water and pressed for observation. The roots were imaged using a laser scanning confocal microscope (Zeiss LSM 980 inverted microscope) with an argon laser wavelength of 514nm (YFP) and 510nm (FM4-64). An emission filter of 508-570nm was used for YFP and an emission filter of 750nm was used for FM4-64. Image analysis was performed using ZEN lite software.

[0085] like Figure 3As shown in B, both SlABCG45-YFP and SlABCG44-YFP co-localize with the membrane localization dye FM4-64, indicating that SlABCG45 and SlABCG44 proteins are localized on the cell membrane. In conclusion, SlABCG45 and SlABCG44 proteins are strigolactone transporters localized on the cell membrane.

[0086] Example 2. Creation of CRISPR gene editing materials

[0087] 1. Construction of CRISPR knockout vector

[0088] The website CRISPR-P v2.0 tool (http: / / cbi.hzau.edu.cn / CRISPR2 / ) was used to search for target sequences of genes SlABCG45, SlABCG44 and SlCCD8 and to design primers. SlCCD8 (Solyc08g066650) is a known strigolactone biosynthesis gene, and its 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 SlCCD8 protein is shown in SEQ ID NO:9. The target sequence is as follows:

[0089] S1ABCG45 target sequence 1: 5'-AGCTCGATTTGGTGGAAAGG-3' (SEQ ID NO: 10)

[0090] S1ABCG45 target sequence 2: 5'-GTCAGCGTGGAATGACGGG-3' (SEQ ID NO: 11)

[0091] S1ABCG44 target sequence 1: 5'-GGCATATGCTCTGCCTACT-3' (SEQ ID NO: 12)

[0092] S1ABCG44 target sequence 2: 5'-GATTCGAGGCAGACGTTGGG-3' (SEQ ID NO: 13)

[0093] S1CCD8 target sequence 1: 5'-CCGGGTCGAGCCACAAAGAA-3' (SEQ ID NO: 14)

[0094] S1CCD8 target sequence 2: 5'-TATTTCTTGCCTAAATAAGC-3' (SEQ ID NO: 15)

[0095] S1CCD8 target sequence 3: 5'-GCGGAGCTTGTCAAGGATGG-3' (SEQ ID NO: 16)

[0096] To construct a dual-target knockout vector for SlABCG45 and SlABCG44, pCBC_DT1T2_SlU6p was used as a template, and PCR was performed using Slabcg45 / 44-cri-F1 / R1, Slabcg45 / 44-cri-F2 / R2, and Slabcg45 / 44-cri-F3 / R3, respectively, to amplify the target fragments (PCR product 1, PCR product 2, and PCR product 3). The purified target fragments were digested with restriction endonuclease BsaI, and the target fragments were ligated to the pTX041 vector that was also digested with BsaI using T4 ligase to construct the recombinant plasmid SlABCG45 / 44-Cas9.

[0097] In order to ensure that the Slabcg44 single mutant can be obtained, 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, respectively, to amplify the target fragments (PCR product 4, PCR product 5, and PCR product 6). The purified target fragments were digested with restriction endonuclease BsaI, and the target fragments were ligated to the pTX041 vector that was also digested with BsaI using T4 ligase to construct the recombinant plasmid SlABCG44-Cas9.

[0098] To construct the knockout vector of SlCCD8, pCBC_DT1T2_SlU6p was used as a template, and PCR was performed using Slccd8-cri-F1 / R1, Slccd8-cri-F2 / R2, and Slccd8-cri-F3 / R3 to amplify the target fragments (PCR product 7, PCR product 8, and PCR product 9). The purified target fragments were digested with restriction endonuclease BsaI, and the target fragments were ligated to the pTX041 vector that was also digested with BsaI using T4 ligase to construct the recombinant plasmid SlCCD8-Cas9.

[0099] The above-mentioned plasmids pTX041 and pCBC_DT1T2_SlU6p are both recorded in the document “Wang, X., Liu, Z., Sun, S., et al. (2021). SISTER OF TM3 activates FRUITFULL1 to regulate inflorescencebranching in tomato. Hortic. Res. 8: 1-15.”.

[0100] The nucleotide sequences of the above primers are as follows:

[0101] Slabcg45 / 44-cri-F1:

[0102] 5’-ATATATGGTCTCGTTTGATTCGAGGCAGACGTTGGGGTTTTAGAGCTAGAAATAGC-3’(SEQ IDNO:25);

[0103] Slabcg45 / 44-cri-R1:5’-ATTATTGGTCTCGTTCACAAACTACACTGTTAGATTC-3’(SEQIDNO:26);

[0104] Slabcg45 / 44-cri-F2:

[0105] 5’-ATATATGGTCTCGTGAAGAGATTGTGGTCGAGGTTTTAGAGCTAGAAATAGC-3’

[0106] (SEQ ID NO:27);

[0107] Slabcg45 / 44-cri-R2:5’-ATTATTGGTCTCGGAGCCAAACTACACTGTTAGATTC-3’(SEQ IDNO:28);

[0108] Slabcg45 / 44-cri-F3:

[0109] 5’-ATATATGGTCTCGGCTCGATTTGGTGGAAAGGGTTTTAGAGCTAGAAATAGC-3’

[0110] (SEQ ID NO:29);

[0111] Slabcg45 / 44-cri-R3:

[0112] 5’-ATTATTGGTCTCGAAACGCCCGTCATTCCACGCTGACAAACTACACTGTTAGATTC-3’(SEQ IDNO:30);

[0113] Slabcg44-cri-F1:

[0114] 5’-ATATATGGTCTCGTTTGTGCTGCTCTAACCACCAAGGTTTTAGAGCTAGAAATAGC-3’(SEQ IDNO:31);

[0115] Slabcg44-cri-R1:5'-ATTATTGGTCTCGTGCCCAAACTACACTGTTAGATTC-3'(SEQ IDNO:32);

[0116] Slabcg44-cri-F2:

[0117] 5'-ATATATGGTCTCGGGCATATGCTCTGCCTACTGTTTTAGAGCTAGAAATAGC-3'(SEQ ID NO:33);

[0118] Slabcg44-cri-R2:5'-ATTATTGGTCTCGCCCAACAAACTACACTGTTAGATTC-3'(SEQ IDNO:34);

[0119] Slabcg44-cri-F3:

[0120] 5'-ATATATGGTCTCGTTGGATTCGAGGCAGACGTGTTTTAGAGCTAGAAATAGC-3'

[0121] (SEQ ID NO:35);

[0122] Slabcg44-cri-R3:

[0123] 5'-ATTATTGGTCTCGAAACCGCGATTTCAAGAATGATACAAACTACACTGTTAGATTC-3'(SEQ IDNO:36);

[0124] Slccd8-cri-F1:

[0125] 5'-ATATATGGTCTCGTTTGCCCCTATGGGCTACATGGTTGTTTTAGAGCTAGAAATAGC-3'(SEQID NO:37);

[0126] Slccd8-cri-R1:5'-ATTATTGGTCTCGCCGGCAAACTACACTGTTAGATTC-3'(SEQ ID NO:38);

[0127] Slccd8-cri-F2:

[0128] 5'-ATATATGGTCTCGCCGGGTCGAGCCACAAAGAAGTTTTAGAGCTAGAAATAGC-3'

[0129] (SEQ ID NO:39);

[0130] Slccd8-cri-R2:5'-ATTATTGGTCTCGAATACAAACTACACTGTTAGATTC-3' (SEQ ID NO:40);

[0131] Slccd8-cri-F3:

[0132] 5'-ATATATGGTCTCGTATTTCTTGCCTAAATAAGCGTTTTAGAGCTAGAAATAGC-3'

[0133] (SEQ ID NO:41);

[0134] Slccd8-cri-R3:

[0135] 5'-ATTATTGGTCTCGAAACCCATCCTTGACAAGTCCCGCCAAACTACACTGTTAGATTC-3' (SEQ ID NO: 42).

[0136] The enzyme digestion and ligation system 1 is: 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; make up to 10 μL with ddH2O.

[0137] Enzyme digestion and ligation system 2 is: 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; make up to 10 μL with ddH2O.

[0138] The enzyme digestion and ligation system 3 is: 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; make up to 10 μL with ddH2O.

[0139] The reaction procedure was: (37°C, 5 min→16°C, 10 min) 16 cycles; 50°C, 5 min; 80°C, 5 min.

[0140] 2. Plant expression vector transformation into Agrobacterium

[0141] Take 1 μL of recombinant plasmids SlABCG45 / 44-Cas9, SlABCG44-Cas9 and SlCCD8-Cas9, respectively, and transform them into 100 μL Agrobacterium competent cells AGL1 by high voltage electroporation, and spread them on LB solid medium containing 50 mg / L kanamycin and 25 mg / L rifampicin, and invert and culture at 28°C for 2-3 days. Pick a single clone and place it in 3mL LB liquid medium containing the same antibiotics, and shake and culture it at 28°C, 220rpm overnight. The next day, transfer 200 μL of bacterial solution to 50mL LB liquid medium with the same resistance, and culture it at 28°C, 220rpm for 16 hours. When the bacterial solution OD 600 When the value was 0.7-0.8, the bacteria were collected to obtain Agrobacterium AGL1-SlABCG45 / 44-Cas9, AGL1-SlABCG44-Cas9 and AGL1-SlCCD8-Cas9.

[0142] 3. Stable transformation of tomato

[0143] (1) Use cultivated tomato S.lycopersicum cv.Moneymaker and wild currant tomato S.pimpinellifolium PI365967 as receptors, select plump and large seeds, soak the seeds in 75% alcohol for 2 minutes, and pour out the alcohol. Soak the seeds in 30% Kao bleach for 20 minutes, and pour out the bleach. Wash the seeds with single-distilled sterilized water 4 times. Finally, spread the seeds on 1 / 2MS solid culture medium and culture at 25℃ and 16 / 8 hours of light. (2) After growing on 1 / 2MS solid culture medium for one week, the cotyledons of tomato seedlings flattened. Cut the leaves with scissors under sterile conditions and soak them in MSO for 1 hour. Use sterilized filter paper to absorb the excess MSO liquid on the leaves, and then place the leaves on A1 solid culture 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 LB liquid culture medium without antibiotics. Then centrifuge at 3800 rpm for 10 min to collect the cells and discard the supernatant. (5) Prepare the infection solution: Resuspend the Agrobacterium AGL1-SlABCG45 / 44-Cas9, AGL1-SlABCG44-Cas9 and AGL1-SlCCD8-Cas9 in liquid MSO culture medium and dilute to OD 600 =0.3-0.4, then add 50 μL of 0.074M acetosyringone to obtain the infection solution. (6) Soak the tomato cotyledons that have been pre-cultured on the A1 solid culture medium for 1 day in the infection solution for 15 minutes, then pour out the infection solution, use sterilized filter paper to absorb the liquid on the cotyledons, and put them back on the original A1 solid culture medium to infect the leaves for 2-3 days. (7) Transfer the cotyledons to the A2 solid culture medium. Culture at 25°C for two weeks with a long daylight cycle. (8) Observe the callus formation and transfer the callus tissue to the A3 solid culture medium for bud induction. (9) After inducing budding into strong seedlings, cut off the stems of the seedlings and insert them into the A4 solid culture medium for root induction until they grow into complete tomato transgenic materials.

[0144] Culture medium and antibiotics configuration:

[0145] MSO (Agrobacterium suspension) (1 L): MS Powder (M524) 4.33 g, Sucrose 30 g, dilute to 1 L, adjust pH to 5.9 with 1 M NaOH / KOH, and sterilize with high pressure steam for 15 min.

[0146] MS medium (1L): 4.43g MS Powder (M519), 30g Sucrose, adjust the volume to 1L with 1M NaOH / KOH to adjust the pH to 5.9, add 8g agar, and sterilize with high pressure steam for 15min.

[0147] A1 solid medium (100 mL): MS medium 100 mL, IAA (1 g / L) 100 μL, ZT (1 g / L) 175 μL. High pressure steam sterilization for 15 min.

[0148] A2 solid medium (100 mL): MS medium 100 mL, IAA (1 g / L) 100 μL, ZT (1 g / L) 175 μL, Kan (50 mg / mL) 225 μL, Tim (1 g / L) 100 μL. High pressure steam sterilization for 15 min.

[0149] A3 solid medium (100 mL): MS medium 100 mL, IAA (1 g / L) 100 μL, ZT (1 g / L) 175 μL, Kan (50 mg / mL) 100 μL, Tim (1 g / L) 100 μL. High pressure steam sterilization for 15 min.

[0150] A4 solid culture medium (100 mL): MS medium 100 mL, Kan (50 mg / mL) 100 μL, Tim (1 g / L) 100 μL. High pressure steam sterilization for 15 min.

[0151] 1000× Rifampicin: Prepare into 50 mg / mL stock solution with DMSO and filter sterilize.

[0152] 1000× Kanamycin sulfate: Prepare into 50 mg / mL stock solution with water and filter sterilize.

[0153] In the above formula, IAA represents indoleacetic acid, ZT represents zeatin, Kan represents kanamycin, and Tim represents Timentin.

[0154] 4. Identification of homozygous mutant material

[0155] Extract leaf DNA from tomato transformation materials. Take samples from individual plants, place the leaves in a 2mL Eppendof centrifuge tube, add 600μL CTAB extraction buffer, add steel beads and use a sampler to grind the leaves into powder, and place in a 65℃ water bath for 30 minutes. Add 200μL chloroform solution, shake well and centrifuge at 4℃9800rpm for 10 minutes. Pipette 400μL supernatant, add an equal volume of isopropanol, mix upside down, and centrifuge at 4℃12000rpm for 10 minutes to precipitate DNA. Wash the DNA precipitate with 70% ethanol, open the lid and dry, then dissolve the DNA precipitate with 100μL ddH2O. Using the obtained DNA as a template, first use Cas9-F / R primers to detect whether there are bands.

[0156] Cas9-F:5'-CACTATCCTTCGCAAGACCC-3'(SEQ ID NO:43);

[0157] Cas9-R: 5'-GAGATTCCCGAACAAGCCG-3' (SEQ ID NO: 44).

[0158] For materials that tested positive for Cas9 primers, primers SlABCG45-seq-F / R, SlABCG44-seq-F / R, and SlCCD8-seq-F / R were used to identify the editing type of the material. The nucleotide sequences of the primers are as follows:

[0159] SlABCG45-seq-F:5'-TGGAGGGTGGTGGAGATATATTG-3' (SEQ ID NO: 45);

[0160] SlABCG45-seq-R:5'-TTAGCCTCTTTCTCTGCCCC-3' (SEQ ID NO: 46);

[0161] SlABCG44-seq-F:5'-CACGTCGTGATGAACCTTATAGG-3' (SEQ ID NO:47);

[0162] SlABCG44-seq-R:5'-TAGTAGGCGGATTCTTACGG-3' (SEQ ID NO:48);

[0163] SlCCD8-seq-F:5'-GAGACTATCTATTCAAAAGGTGGCA-3' (SEQ ID NO:49);

[0164] SlCCD8-seq-R: 5'-ACACATTCTATTACTGAGAGCAA-3' (SEQ ID NO: 50).

[0165] PCR reaction system (25 μL): 1 μL DNA template, 1.5 μL 10 μM forward primer, 1.5 μL 10 μM reverse primer and 21 μL Gold Mix (Qingke Biotechnology).

[0166] PCR reaction program: pre-denaturation at 98°C for 3 min; 35 cycles of (denaturation at 98°C for 10 s, annealing at 55°C for 15 s, and extension at 72°C for 30 s); extension at 72°C for 5 min.

[0167] The PCR product was sent to Beijing Ruibo Xingke Biotechnology Co., Ltd. for Sanger sequencing. After the sequencing results were obtained, the sequences were compared. Figure 4 As shown, 10 homozygous mutant materials with 1 bp base insertion at SlABCG45 target sequence 1 were identified in the PI365967 background, named Slabcg45-1; 5 homozygous mutant materials with 86 bp base deletion between SlABCG44 target sequence 1 and 2 were identified, named Slabcg44-1. In the Moneymaker background, 6 homozygous mutant materials with 1 bp base deletion at SlABCG45 target sequence 2 were identified, named Slabcg45-3; 5 homozygous mutant materials with 1 bp base insertion at SlABCG45 target sequence 2 were identified, named Slabcg45-4; 4 homozygous mutant materials with 7 bp base deletion at SlABCG44 target sequence 2 were identified, named Slabcg44-3; 10 homozygous mutant materials with 459 bp base deletion between SlCCD8 target sequence 1 and 3 were identified, named Slccd8-1. The transgenic materials were planted in a greenhouse for bagging and propagation. The received seeds were planted, leaves were taken for DNA extraction, and materials that tested negative for Cas9 primers were transplanted and propagated. The received seeds were used for subsequent field trials.

[0168] 5. Determination of the content of strigolactone in Slabcg45-1 and Slabcg44-1 mutant materials

[0169] The material culture process for the test of determination of the content of strigolactones is as follows: (1) Sterilize the tomato seeds of wild type (WT), Slabcg45-1 and Slabcg44-1: Soak the seeds in 70% alcohol for 1 minute in a clean bench, pour out the alcohol and wash them 3 times with sterile water. Then soak the seeds in 30% Kao bleach for 20 minutes, pour out and wash them 5 times with sterile water. Transfer the seeds to a culture dish, keep a little water to keep them moist, and place them in a refrigerator at 4°C for two days. (2) Prepare 1 / 2MS solid culture medium in a sterile environment, sow tomato seeds on the culture medium, and culture them at 28°C and 16 / 8 hours photoperiod for 10 days. (3) Transplant the tomato seedlings from the 1 / 2MS solid culture medium into a glass tube containing 1 / 2 phosphorus-deficient Hoagland liquid culture medium, and culture them in water at 28°C and 16 / 8 hours photoperiod for 12 days.

[0170] Collect the hydroponic solution for 5 hours and add GR24 4DO As an internal standard. The hydroponic solution was transferred to an equilibrated Oasis HLB column (Waters). The components containing orobanchol and solanacol were eluted with acetone, and the eluate was blown dry with nitrogen. After reconstitution with 50% acetonitrile, LC-MS / MS analysis was performed. At the same time, the fresh weight of the roots of each material was weighed to convert the content of orobanchol secreted by the root system per unit mass.

[0171] like Figure 5 As shown, compared with the wild type (WT), the content of strigolactone in the root exudates of mutants Slabcg45-1 and Slabcg44-1 was significantly reduced, indicating that SlABCG45 and SlABCG44 proteins are essential for the export of strigolactone, which is consistent with their function as strigolactone transporters.

[0172] Example 3. Parasitism and yield determination of wild type, Slabcg45-3, Slabcg45-4, Slabcg44-3 and Slccd8-1 mutants in fields contaminated with Orobancha seeds

[0173] 1. Statistics of parasitism in fields contaminated by Orobancha seeds

[0174] All transgenic materials planted in the field were homozygous mutants of Cas9, i.e., Cas9-free materials. Since the fruits of wild currant tomatoes are small and have no edible value, the parasitism of wild-type, Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1 mutants in the Moneymaker background of cultivated tomatoes were only counted in fields contaminated by Orobanche seeds in Xinjiang. The specific method is as follows: tomato seeds were transplanted to the field one month after seedling cultivation on plug trays. The land was prepared, ridges were made, and mulched in advance. Drip irrigation was started immediately after the tomato materials were transplanted to keep the soil moist. Every 6 tomato materials were planted in two rows in one plot, with a ridge width of 80 cm, a plant spacing of 40 cm, and a distance of 100 cm between plots. Wild-type, Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1 were planted in 4 plots each, with intervals. When the tomatoes grow to about 0.5m, start to build the racks, tie the main stems of the tomatoes to the bamboo poles, and regularly manage them. In the later stage, carry out routine management such as applying water, fertilizer and spraying herbicides.

[0175] The total number of parasitic Orobancha in each plot was counted during the tomato fruit ripening period. The fresh weight of Orobancha in each plot was also counted. Figure 6 As shown, the Slabcg45-3, Slabcg45-4, Slabcg44-3, and Slccd8-1 knockout mutants all exhibited enhanced resistance to Orobancha cava compared with the wild type.

[0176] 2. Fruit and yield determination in fields contaminated with Orobancha seeds

[0177] The fruit traits and yield of wild type (WT), Slabcg45-3, Slabcg45-4, Slabcg44-3 and Slccd8-1 were determined. Figure 7 As shown, compared with the wild type (WT), the average size of individual fruits of Slabcg45-3 and Slabcg45-4 was not significantly different, but the average size of individual fruits of Slabcg44-3 and Slccd8-1 was significantly smaller ( Figure 7B), indicating that the mutation of SlABCG45 gene has no obvious negative effect on fruit size, while the mutation of SlABCG44 gene and SlCCD8 gene will affect fruit development. At the same time, the tomatoes planted in the above fields were harvested individually, and the number and weight of red fruits (red fruits), green fruits (green fruits) and all fruits (total fruits) on each tomato plant were counted. The statistical results showed that the number of red fruits per plant, the total number of fruits and the total fruit weight of Slabcg45-3 and Slabcg45-4 were significantly higher than those of the wild type; the total number of fruits and the total fruit weight of Slabcg44-3 did not change significantly compared with the wild type; and Slccd8-1 showed a significant phenotype of fewer fruits and reduced fruit weight ( Figure 7 C, 7E and 7H). Therefore, only by knocking out the SlABCG45 gene can the balance between broomrape resistance and tomato growth be achieved, that is, while improving tomato resistance, it will not cause obvious negative phenotypes on tomato growth, showing the potential for increasing field yield. Finally, the total tomato yield of each plot was counted. Compared with the wild type, the tomato material with the SlABCG45 gene knocked out can increase the yield by about 33% to 36% (Table 1). The above results show that the SlABCG45 gene has important application value in the cultivation of parasitic-resistant tomatoes, and knocking out the SlABCG45 gene by gene editing technology can significantly increase the yield of tomatoes in a parasitic environment.

[0178] Table 1

[0179]

[0180] Note: The values ​​in the table are the average values ​​of the total tomato yield (g) of the four plots.

[0181] The present invention has been described in detail above. For those skilled in the art, without departing from the purpose and scope of the present invention, the present invention can be implemented in a wide range under equivalent parameters, concentrations and conditions. Although the present invention provides specific embodiments, it should be understood that the present invention can be further improved. In a word, according to the principle of the present invention, the application is intended to include any changes, uses or improvements of the present invention, including departing from the disclosed scope in the application and the changes made with conventional techniques known in the art.

Claims

1. A method for improving the resistance of tomato plants to Orobancha parasites and the yield of tomatoes, comprising: Reducing the content of S1ABCG45 protein in tomato plants or inhibiting the expression of the gene encoding S1ABCG45 protein in tomato plants; the amino acid sequence of the S1ABCG45 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 S1ABCG45 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 achieved by editing the gene encoding the SlABCG45 protein through 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 the target sequence 1 is shown in SEQ ID NO: 10; the nucleotide sequence of the target sequence 2 is shown in SEQ ID NO:

11.

5. The method according to claim 4, characterized in that The method introduces the coding gene of the sgRNA targeting the target sequence 1 or the target sequence 2 and the coding gene of Cas9 into the tomato plant, thereby obtaining a transgenic plant with improved resistance to Orobancha and tomato yield in an Orobancha parasitic environment.

6. A product for improving the resistance of tomato plants to Orobanche and tomato yield in an environment parasitic on Orobanche, the product being 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 S1ABCG45 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 a CRISPR-Cas9 system; the reagent is reagent 1 or reagent 2; The reagent 1 is a composition consisting of R1) or R2) and Cas9: R1) an sgRNA targeting the target sequence 1 of claim 4; R2) an sgRNA targeting the target sequence 2 of claim 4; The reagent 2 is a recombinant vector containing the coding gene of R1) or R2) and the coding gene of Cas9.

10. Use of the S1ABCG45 protein of claim 1 or the gene encoding the S1ABCG45 protein of claim 2 in improving the resistance of tomato plants to Orobancha parasitism and improving the yield of tomato.

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