Application of knockout SlABCB13 gene in improvement of soluble sugar content of tomato fruits

Abstract

The invention relates to the technical field of gene engineering, in particular to application of a knockout SlABCB13 gene in increasing the soluble sugar content of tomato fruits. The function of the SlABCB13 gene is deleted by mediating gene editing, so that the content of soluble sugar (cane sugar, fructose and glucose) and the content of soluble solids in tomato fruits are remarkably increased, and the SlABCB13 participates in sugar transport of the tomato fruits. Meanwhile, the mutant does not influence related yield indexes such as plant height, stem diameter, fruit size, fruit setting number and single-plant fruit yield, and a new thought is provided for the problem that yield and quality are difficult to consider in existing tomato breeding.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to knockout. SlABCB13 Application of genes in increasing the soluble sugar content of tomato fruits. Background Technology

[0002] Tomato is an important global economic fruit and vegetable crop, and also a major model plant for studying fruit development. In recent years, the pursuit of high yield, disease resistance, and storage tolerance in tomato breeding has led to a significant decline in its flavor and quality. Sugar accumulation is one of the key factors determining the quality of horticultural crop fruits; therefore, developing high-quality, high-sugar tomato varieties has become one of the primary goals of tomato breeding. Summary of the Invention

[0003] To address the above problems, the present invention provides a knockout mechanism. SlABCB13 Application of genes in increasing the soluble sugar content of tomato fruits.

[0004] To achieve the above objectives, the present invention provides the following technical solution: This invention provides knockout SlABCB13 Application of genes in increasing the soluble sugar content of tomato fruits.

[0005] Preferably, the soluble sugar includes one or more of sucrose, fructose, and glucose.

[0006] Preferably, the SlABCB13 The nucleotide sequence of the gene is shown in SEQ ID No. 1.

[0007] Preferably, the tomato fruit is a red-ripe tomato fruit.

[0008] The present invention also provides a knockout SlABCB13 The sgRNA of the gene, including sgRNA1 and sgRNA2; The nucleotide sequence of sgRNA1 is shown in SEQ ID No. 2, and the nucleotide sequence of sgRNA2 is shown in SEQ ID No. 3.

[0009] This invention also provides a method for obtaining plants with increased soluble sugar content in tomato fruits, comprising the following steps: 1) The sgRNA described in the above technical solution is ligated into a CRISPR / Cas9 vector to obtain a recombinant editing vector; 2) The recombinant editing vector described in step 1) is transformed into Agrobacterium to obtain the transformed bacteria; 3) The transforming bacteria described in step 2) are used to infect the cotyledons and hypocotyls of tomatoes to obtain tomato plants.

[0010] Preferably, the connection system in step 1) is: 4 µl of sgRNA, 2 µl of CRISPR / Cas9 vector at a concentration of 100 ng / µl, 1 µl of recombinase, 2 µl of recombinant buffer, and 1 µl of ddH2O; The concentrations of sgRNA1 and sgRNA2 in the 4 µl sgRNA were both 100 ng / µl. The CRISPR / Cas9 vector is a linearized CRISPR / Cas9 vector; The conditions for the connection include: operating at 37°C for 30 minutes.

[0011] Preferably, the recombinant editing vector in step 2) is transformed into Agrobacterium by electroporation.

[0012] Preferably, the soluble sugar includes one or more of sucrose, fructose, and glucose.

[0013] This invention also provides knockout SlABCB13 Application of genes in increasing the fructose and glucose content of green-ripe tomato fruits.

[0014] The beneficial effects of this invention are: This invention is disclosed in SlABCB13 Specific targets on genes were identified, and corresponding sgRNA1 and sgRNA2 were designed for this purpose. These sgRNAs can bind to the CRISPR / Cas9 system, specifically recognizing and cleaving them. SlABCB13 Gene target sites, through mediating gene editing to SlABCB13 The loss of gene function significantly increased the content of soluble sugars (sucrose, fructose, glucose) and soluble solids in tomato fruit, indicating that... SlABCB13 This invention participates in sugar transport in tomato fruits. Simultaneously, the mutant does not affect related yield indicators such as plant height, stem diameter, fruit size, fruit set number, and yield per plant, providing a new approach to addressing the current challenge of balancing yield and quality in tomato breeding. This invention utilizes CRISPR / Cas9 technology to provide important gene resources for molecular breeding of high-sugar tomatoes, and has significant application prospects. Attached Figure Description

[0015] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0016] Figure 1 for SlABCB13 Gene editing targets 1 (T1) and 2 (T2); Figure 2 for SlABCB13Electrophoretic image of hygromycin resistance gene in T2 generation homozygous mutants after gene editing; Figure 3 for SlABCB13 Sequencing peak diagram of target sites in gene-edited T2 generation homozygous lines; Figure 4 for SlABCB13 Soluble sugar content in leaves and fruits of gene-edited strains; Figure 5 for SlABCB13 Agronomic traits and soluble solids content in fruits of gene-edited strains; Figure 6 for SlABCB13 Comparison of gene-edited tomato lines with wild-type tomato plants and fruit phenotypes. Detailed Implementation

[0017] This invention provides knockout SlABCB13 Application of genes in increasing the soluble sugar content of tomato fruit. In this invention, the soluble sugar preferably includes one or more of sucrose, fructose, and glucose. In this invention, the tomato fruit is a red-ripe tomato fruit. In this invention, the... SlABCB13 The nucleotide sequence of the gene is shown in SEQ ID No. 1, as follows: Solyc08g076720 (SlABCB13) gene sequence Bold underlined portion: Target 1 (sgRNA1); Bold double underlined portion: Target 2 (sgRNA2); Dashed underlined portion: Exon; Wavy underlined portion: Exon UTR; TTTAAAATCACAAGAAACACATGTGTTGTATATATACTAATTTAAGATAACAAAGTTTATTTTTTTCTAAAACTTTTTTTCTTAATACATTTACTTTGATCAACTTAATTACCACTACTTTCTTACCACCCCCTCATAACTTTCTTTTCTTTTATAAGAAGGAAAACTTATGTTTCTTTTATTGCTCATAAAGTCTAGT TGAAAACCAAAAAAAAATGA GCCAACAACAAAGTCATGCATTATCTGTTGACAGCAGCAAAATTTCAAAAATGAATCAGAAAAATAATGAAGAAGA AGAAGAAAGGAAAAAAAAGACTCATAAAAAAGTTTCATTATTGAAGCTTTTCTCTTTTGCTGATTCCTATGATTAT TTGTTGATGTTTTTGGGATCTATTGGTGCTTGTTTACATGGTGCTTCTGTACCTGTTTTCTTCATTTTCTTTGGCA AAATGATTAATATTGCTGGCCTTGCTTACCTTTTTCCTGCTCAAACTTCTCACAAAATTGCCAAGGTAACATATCCAAAAATTTCTCACATAGTCGTTCAACTAATAATCGTAAAACTTAACAGATGGTTAAGTGACATTATAAGAATTAACCTTGATTTCTCGTATGGTTACTCAACTAAAAATTATTATTATCTCAGAAACGTGCTAATTCTAGCAAATAGATTTTGTTAACTATGGACTATTTAATTACAAATTAAGTATGGATTTTCATAGTTAATTTTTGTATTTTTTTTTTGAGGATGCAAAAAATAAAGATCCAATTAATTATTGCATGTAACTAATTAATTGTTCATATGAAGTGGAGCATATAATAACCTTAAAATAAGTAGATTAAGGAAATTACTTGCTACATTATATTAAACTAAACTGAAAATATAATGGATTAATTAATGTTTTATTATATATTGTGTGATGAACTTATGTTAATTTTGGATTGTTTGTTTGCAG TATTCATTGGATTTTG TGTACCTAAGCGTGGTGATATTATTTGCATCATGGATAG GTTTGAGCTTATTGCCCAACAATTATATATATTTTATTTTTTATTTAATTTATATATTAAAATTCCTAATATTTTTTTAATTAAACAAAAAAAAAAG AGGTGGCATGTTGGAT GCATAGTGGAGAAAGACAAGCAGCAAAAATAAGAATGGCATACTTAAAGTCTATGTTAAATCAAGATATAAGTCTT TTTGACACTGAAGCATCTACTGGAGAAGTCATTGCTGCAATTACCAGTGATATTATTATTGTTCAAGATGCTATTT CTGAGAAGGCA GGGAATTTCTTGCATTATATCAGCCGTTTCTTAGCTGGTTTTACAATTGGGTTCATAAGAGTATGGCAGATCAGTT TGGTGACGCTTTCAATTGTGCCTTTGATCGCTCTTGCTGGTGGTATTTATGCCTATGTTACCATTGGTCTCATTGC TCGAGTCCGCAAATCCTATATTAAAGCTGGAGAGATTGCTGAAGAG GTATGCACTCCAACTGTTTGACAAAAGTCTTTTCTTATCTATGTTGGTCGGACTCTTTTAAAAATTATCAATCATCGGTGCGTGTTAGATTCGCATATTTTTGGAGAATCTGACTCGAGTGCGGCATCAAAGTGAAGAGTCTGCACAACTAAGTTGCTTAGTATCTCATTCTAATTTATGAACGGAAAAGGCTCAAACACTATCATAACTAGTTTAAATATATTCTCGTTATACTTTTGATCCCAAATATACCTTGTCCGTTATACTTTATTACCCTTAATTTAGATTGACATATTATATATTAGTGATAGTGGAGGAGTGTCATATAAATTAGGACAATAGAGTAGTTTATTCAAGAAAGGATCATATGTTAAATGAGTATTGATAACAATAGTACCCTGTTTAACTTTTTATTTTCATAATCCTTGCTCTTTGCTTAGTGATCTTTTGCAATACTCAG GTTGTGGCTAATATTAGAACAGTGCAA GCATTTACTGGAGAAGAGAATGCGGTAAAATCGTATAAAGGTGCCTTGTTGAACACTTATAAGTATGGGAGAAAAG CTGGATTCGCCAAGGGCTTGGGTTTGGGCACCTTGCATTGTATCCTTTTCCTTTCTTGGTCATTGCTTGTGTGGTT TACTAGCATTGTTGTGCACAAGAACATTGCAAATGGAGGAGATTCTTTCACCACCATGCTCAATGTTGTCATTGCT GGCCT GTAAGTTTTTGCTTTCATACTCAATTTCGAACTGCTTTTACAGCATAATCTTAAACAATTAAACTATTACGAGTATTAACCGAGATTAGTGCTCTCTGTTCTTGAGAATCCGGTGCTTCTCTGAATTTCATCACTACTCATAGGCATATATGAATAACCTCTTTCATGCCAGTGTAGTGATTATTGATGGAATTGAGCTGTTATGTTAAGCCAATGCTTATTCATACTGCTATTGTGTTAACAAACCAAGAAACACTAGTTGATCTTAGTCCAATTGCTTCAAACGTTTGGAGTTGTTGGATGTGTAGAACACCAAGTGTTGTTTTGACTAGCTGAAAAGGCAGGTTATCCTGTTAGGCTCAACCAGTTGCACTGAATTGAAACAACATAATCTTGCTGTCTTGTTATGCA GAAAATTTCAATGACTTGATTTGTGTTTAAAGAAGTTGCTTCTTTCAATGTATCTCTATTGGTTAAAGAATAGATATTACGTCCATTAATCCTTTCTAGATTTCAGATTTTAGAGCAATTTATAAAGTTACCCTTTATAGGCCAGCTCTAATGAAGTCAGGAAGAAACCAGATGTTTATGACATCGTGTCTCATCCTTATTGATCAAGTTACTTGTTACTTCTATACTTGGTCAAACTTTTTCCTTAGTTTATAGAGGTCATTTCGTGCCTCAATGTCACTTGGAAAGTTTTTCATTGCATACATCTTGAATTCAGACTTTGTATGGTCATAAAGTAGGTGCTCCACTAGCTGCCCAGCTTTTGTTCTGTTTATGCAATCTTCTCTTCAATTTAACTTTAACTGCTTCTCAG GTCACTGG GTCAGGCAGCACCAGATATTACAGCATTCCTCAGGGCTAAGTCAGCTGCCTATCCTATTTTTGAGATGATTGAACG AGATACTATCAGTAAAACCAGCTCCAAGAGTGGTCAGAAGCTGAGCAAAGTAGATGGACACATACAATTTAAGGAT GTATGTTTTAGCTATCCATCTCGTCCCGATGTGGTGATATTTGACAAGCTCTCTCTTGATATTCCATCAGGGAAGA TTGTAGCTCTTGTGGGAGGAAGTGGATCAGGGAAAAGTACAGTTATATCTTTGATTGAACGCTTCTATGAACCACT CTCGGGACAGATATTATTGGATGGATTTGATATTAGGCACCTGGACTTGAAGTGGCTGAGGCAGCAAATAGGGCTA GTAAATCAAGAACCCGCTCTTTTCGCTACAACTATTAGAGAGAACATTCTCTATGGAAAAAGTGATGCTAGCCTTG AAGATATTGCACGTGCGGCCAAACTTTCCGAGGCAATGACTTTCATCAACAACCTCCCCGATCGTTTTGAAACTCA GGTACATTTTGTACTTCATAAACTATTTGTACATGCACTAGGAGATGCCACCGCTATAGCAAAGAGAATTTGGTGGATGCTTGTATTTATTTATTACATGCTAAGTACTTCGATTTGACTATACCCTGGAGTCGTTTGGTTGTTGGTTAGAAGGTATTAGGAATACATGGATTAGTTATGAAGAAATCTATGTATCA TTCTATGCATGTATTAGTTACCGGAGGGTTTACTTATTCATGTATTACTTATTCCATCTTTTATCCTGTATAAAACAATACATAGATTCCCTATATCCTTTCATTATCCGTTACCAGCATCATTTGAGTAGAAGAATTCTTGTGTTGCTTTTCTTTCTGTTAGCTGTTCAGAATTAGGCGATTTTTTACTATTGTT GTGGTTTATGAGTATCTCATGATTTCAAAATAAAAACACTCAAGATGCTTCACTCTCTCGAGTTGAAATGTTTGCCTCTAAATCAAGAGAGTTATTGAAAATATTGTGTTGAATATTCTTAATTAATATGTCAGTGAAAAAATATTAAGAAAATTGTGTAGAGTATTCCTATGATTATGACATAATTAAAAATCCCTAGGATTAAGTAACTTAACTCAAAACTCCTATTGAAGGAGGTCTCTTGTATATTGTTCAAAGAACAAGAAAATTCAATTAGAGTTTTTGTCTTCTTCTCTTTTCACAAAAATATTCAAGAAAGGCCTCATAAGACTGAAAATTTTCTTTAATATAACATAATATCATAGATATCTGAGGCCACGTCATTTACAG GTTGGTGAAAGAGGGGTTCAGTTATCTGGGGGGCAGAAGCAAAGAATTGCAATTT CTCGTGCTATAGTGAAGAATCCATCAATTCTTCTATTAGATGAAGCTACAAGTGCACTTGATGCTGAGTCAGAAAA AAGTGTACAAGATGCTCTTGATCGTGTGATGGTGGGAAGAACAACTGTAATTGTTGCTCATCGTCTTTCTACCATT AGGAATGCTGATATAATAGCGGTCGTAAACAATGGCAAGATAGTAGAAACTGGAAGTCACGAGGAGCTTATTTCAA AACCTAATAGTGCCTATGCATCACTTGTTCAACTTCAGCAAGCAGCTTCTTCACATCTCCATCCTTCTCAAGAGCC AACCATGGGACGACCTCATAG GTACAATATTTACCTGCTGATGCGTTGTATTCCTTACTATATCAGAGTGTGATGTATATCCTCTTTATTGTTGCGTTGGAGTCAGCTCAGTACTGTCATGCATTTGACAGCTAATTCTAGTGATACTTTGACAATTTCTGCAACTGAGGGTATAAAGTTCAAAAAAATAATAAAAAATGTCATTTCCTGCTTATAAGCAATTAGACTGATTTTTCTATCATTATATGCATAATTTGCTTAAACTTTTTTCTACAG CATACGGTATTCCCGTGAATTATCCCGA ACGACAACGCGAAGCCGTGGAGCTAGCTTTCGTTCTGAAAAATCTGTTAGCGGTATTGGTGCTGGTGATGTAGAGG ATGTTAAGTCACCAAATGTTTCCGCAGGGAGATTGTATTCTATGATTCGTCCTGAATGGCATTATGGAGTTATTGG AACTATCTGTGCATTTATTGCTGGAGCTCAGATGCCACTCTTTGCCTTAGGGGTGTCACAGGCCCTTGTGTCTTAT TACATGGATTGGGACACAACACGCCATGAAGTGAAGAAGATTTGTTTCCTGTTTTGTGTTGGTGCAGTATTAACCG TCGTCGTTCACGCCATTGCACATACTTGCTTTGGAATCATAGGGGAGCGGCTTACTCTTCGCGTGAGAGAAATGAT GTTCTCTG GTAATCATACAAAAACCAATGTGTTTGCTTTACGAGAGATTGCTTATCGTGTAAATATGTAGAGCTTTAATTGCATAATTACTTTCTTTGCAAGACTTTGGTTCACTGCGTTGATACTATTCATCATATTTAATTTTAGAAGATGATTTTTGTACTCTTTTCTGAAACTTTCCGCTCATCTCTGTTTTCTTAGTATCAGTCGTTAAAACAGACAATTAGCTTAACTTGTATCAATAAATGATTTCTAAATGTAAGAAAGCTACGATTCGTATTTGTGTGGAATCATTAATCCAGAAATAATTTTTTTCATGATTCAGATAAATATGAATTATACCTTTCCTACATTTGTTGATATGTTGCATTGGTATCTGTTTTCTTAGTATGTGTCCTTTTTGTCCATCTTATTCAAATAAAAAGTTCCCTAACATTCTTCATGATGTGGTTGCAG CTATGCTAAGAAATGAAATAGGGTGGTTTGATGAGGTGAACAACTCAAGTTCTACACTTGCATCACGATTGGAAAG TGATGCTACTCTATTGCGAACTGTAGTGGTTGATCGCTCTACTATTCTCCTACAGAATGTGGGGTTGGTGGCCACT TCCTTCATCATCGCATTCATCTTGAATTGGAGGCTCACCCTTGTTGTCATGGCTATGTACCCACTTATCGTTAGTG GTCACATCAGCGAG GTTCAGTCTTTTCCCCTTAATCAGCATCTGCCATATATCTTCCATATCATTATTTTAATAGACATTGCTTTCTTTCAG AAACTCTTTATGTCAGGTTTTGGTGGTGACTTGAGCAAAGCCTATCTTAGAGCAAACATGT TTGCTGGTGAGGCTGTAAGTAACATCAGAACTGTTGCTGCATTCTGTGCTGAGGAGAAGGTAACTGATCTCTATGC TCGTGAACTTGTTGAACCTGCAAAGCATTCATTTCGCAGGGGACAGACTGCTGGGATCCTTTATGGAGTCTCTCAG TTTTTCATCTTCTCCTCTTATGCCCTTGCCTTGTG GTATGTTGCTCAAGTTTTAGTTATCTAAATTGTTCATCTTGTTGCTTTTTGGTTGAGATTCTATGAGTCAGTGAGGCAGCTTGCAGTTCAATGAAGTGCTTTGGCATACTTGTACTTCACATGCTATGACATGATGACAATTTTGAGACTAGATAAATTTACCACCTTCTTTCA CTATTTTACAAACTATCCACTGGGGAAAATGTTACAACAAAAGTTTAGCAAAGTAGATAGCCAATCACATACATTATGACAGTTGTTTATTTTACTAGATTACAGTCTTGTACTAATAAGGTTTTCACTTGAGCTCGCAATAGTTTAACATGTATTTTTTCCTAAACCTTGTATAG GTATGGTTCTGTTTTGATGGGGAAGGAGCTAACCAGCTTCAAAGCTGTTATGAAATCGTTTATGGTTTTG ATTGTAACTGCTTTGGCTATGGGAGAGACCCTTGCTATGGCACCAGACCTTATAAAAGGAAACCAGATGGTAGCAT CAGTATTTGAAGTGCTTGATCGGAAGACAGAAATTGTAACCGACAGTGGGGAGGAGCTAACTGTGGTTGAGGGAAC AATTGAGTTTAAGGATGTTGAGTTCTGCTATCCTGCAAGACCCGATGTTCACATTTTCAGAGACTTCAATATGAGA GTGCATGCAGGAAAGAGTATGGCAATAGTAGGGCAGAGTGGTTCTGGTAAAAGCTCAGTATTGGCTCTCATTTTAC GGTTCTATGATCCTATATCTGGGAAAGTGATCATTGATG GTAAGTTTCATTTGCACCTATACTATTTCGAACTAGAATCATTCCAACGTTCATCATGGATTAGTTTAATCATATCGATGATGTGAATTTATACAG GCAAAGACATCAGGAAA CTAAAGCTCAATTCTCTAAGAAAGCACATTGGCCTGGTCCAACAAGAACCAGCACTCTTTGCCACAACAATCTATG AAAACATCCTTTATGGAAAAGAGGGAGCATCTGAAGCAGAAGTGATTCAAGCAGCTAAGCTTGCTAATGCACATAG TTTCATTAGTGCTCTTCCTGATGGCTACTCGACCCAAGTTGGGGAACGAGGAGTTCAATTGTCCGGTGGACAGAAG CAAAGAGTAGCCATTGCCCGAGCTGTTCTAAAGAACCCTGAAATCTTACTATTAGATGAAGCTACAAGTGCTCTAG ACGTGGAATCTGAGCGTATAGTTCAACAAGCATTGGACAGGTTGATGCGAAACAGGACTACTGTCATCGTGGCACA TCGACTATCCACCATAAAAGATGCAGATCAGATATCTGTTTTACAAGATGGGAAAATCGTCGATCAAGGGACTCAC TCTGCATTGATAGAGAACAGAGATGGAGCATACTTTAAGCTAATTCACTTACAGCAACAGCAGCAACAGTAATAGC ACACCAACATACTTGGAGATGTTTTTCTCATACAAACACTTAAAATGTGTAAAATTCTTTTCCCCTATACTTAATA TAATTGCAATAATATACTTTGTATTTAGAGTTGTTGTATAGGCTATTTTTTACTGTTCTTTCTTCCCCTGGAATAT TTTTTCGGTTGTTGAAGGCTTCACTAGTTGGATGGGGTTAAAAATTTAAGGCTCCTAAATGTAAAATTAGTTAAAC ACTTCGCATTTG ACACCCCAAAAATAAAAGGGCAAGTTAACACATTTTGCAGGTCAGCTAAAAATATTACTTCATTTCAATGTATATCTCTCGTTTTAATTTGATACGGAGTTTAAGAAGATAAATAAAATTTTGAATCTTGTAATTTTAAACTAAAGATATGTGAAAGATACCAAAATGTCTTTTAATCTTATGGTATTAAACAAAGACG.

[0018] This invention provides a knockout SlABCB13 The sgRNA of the gene, including sgRNA1 and sgRNA2; The nucleotide sequence of sgRNA1 is shown in SEQ ID No. 2, and the nucleotide sequence of sgRNA2 is shown in SEQ ID No. 3.

[0019] SEQ ID No. 2: 5'-TTTGACACTGAAGCATCTACTGG-3'; SEQ ID No. 3: 5'-GACTCGAGCAATGAGACCAATGG-3'.

[0020] This invention also provides a method for obtaining plants with increased soluble sugar content in tomato fruits, comprising the following steps: 1) The sgRNA described in the above technical solution is ligated into a CRISPR / Cas9 vector to obtain a recombinant editing vector; 2) The recombinant editing vector described in step 1) is transformed into Agrobacterium to obtain the transformed bacteria; 3) The transforming bacteria described in step 2) are used to infect the cotyledons and hypocotyls of tomatoes to obtain tomato plants.

[0021] In this invention, the preferred ligation system in step 1) is: 4 µl of sgRNA, 2 µl of CRISPR / Cas9 vector at a concentration of 100 ng / µl, 1 µl of recombinase (Novizan ClonExpress II One Step Cloning Kit), 2 µl of recombinant buffer, and 1 µl of ddH2O; the concentrations of sgRNA1 and sgRNA2 in the 4 µl of sgRNA are preferably both 100 ng / µl; the CRISPR / Cas9 vector is a linearized CRISPR / Cas9 vector; the ligation conditions include running at 37°C for 30 min.

[0022] In this invention, the recombinant editing vector is preferably converted into Agrobacterium via electroporation. In this invention, the soluble sugar preferably includes one or more of sucrose, fructose, and glucose.

[0023] This invention also provides knockout SlABCB13 Application of genes in increasing the fructose and glucose content of green-ripe tomato fruits.

[0024] To further illustrate the present invention, the following detailed description is provided in conjunction with embodiments, but these should not be construed as limiting the scope of protection of the present invention.

[0025] Example 1 SlABCB13 Design of gene-targeting sgRNAs, vector construction and Agrobacterium-mediated genetic transformation of tomato 1. SlABCB13 Gene-targeted sgRNA design Obtained from the tomato genome database SlABCB13 Gene (Solyc08g076720) The genome sequence. For example... Figure 1 As shown, targeting the exon region of this gene, two specific sgRNA sequences were designed on the 3rd and 4th exons downstream of the start codon using the online website http: / / crispr.hzau.edu.cn / CRISPR2 / (this part was commissioned to Weimi Biotechnology (Jiangsu) Co., Ltd.).

[0026] T1 (SEQ ID No. 2): 5'-TTTGACACTGAAGCATCTACTGG-3'; T2 (SEQ ID No. 3): 5'-GACTCGAGCAATGAGACCAATGG-3'; The above sgRNA sequence was cloned into the CRISPR / Cas9 vector (pCBSG012-slu61-DSG) to construct the recombinant editing vector (this part was commissioned to Weimi Biotechnology (Jiangsu) Co., Ltd.).

[0027] (1) Design a target based on the sequence obtained from the target gene number, recombine the target sequence with the linearized vector backbone to construct a knockout vector, and then proceed to transformation after the vector is constructed.

[0028] (2) The target fragment was artificially synthesized based on the target sequence and the linearized vector backbone recombinant arm sequence. The recombinant vector was obtained according to the recombination ligation system (on-ice mixing).

[0029] The ligation system consisted of: 4 µl of sgRNA (the target fragment in Table 1), 2 µl of CRISPR / Cas9 vector (the linearized vector template in Table 1) at a concentration of 100 ng / µl, 1 µl of recombinase (Novizan ClonExpress II One Step Cloning Kit), 2 µl of recombinant buffer, and 1 µl of ddH2O; the 4 µl of sgRNA contained sgRNA1 and sgRNA2 at a concentration of 100 ng / µl.

[0030] Run the recombination program on the PCR instrument: 37℃ for 30 min, then store on ice or at 4℃.

[0031] Table 1 System (3) Transformation of DH5α 10 μl or more of the ligation product + 100 μl of competent E. coli cells. After removing the competent DH5α cells from the refrigerator, quickly place them on ice. After 5 minutes, wait for the bacterial block to dissolve, then add the ligation product. Let stand on ice for 25 min, heat shock at 42℃ for 45 s, and place on ice for 2 min (do not shake). Add 100 μl of antibiotic-free LB, shake at 37℃ and 200 rpm for 1 h, plate, add LB + bacterial antibiotic, and incubate at 37℃ for one day (this part was outsourced to Weimi Biotechnology (Jiangsu) Co., Ltd.).

[0032] (4) Colony PCR Twelve single clones were randomly selected and placed into 2 ml sterile EP tubes. 500 μl of LB was added beforehand, and the cells were shaken for 6 h. 5 μl of bacterial culture was taken from each sample for PCR. One correct bacterial culture was selected for sequencing (this part was outsourced to Weimi Biotechnology (Jiangsu) Co., Ltd.).

[0033] F (SEQ ID No. 10): CAAAGATCGTTATGTTTATCGGCACT; R (SEQ ID No. 11): TTGGCGACCTCGTATTGGGAA.

[0034] Table 2 System (5) Transform Agrobacterium into plasmids with correct sequencing results. ① Agrobacterium transformation: 20 μl Agrobacterium (EHA105-WM) + 1 μl plasmid, incubate on ice for 5 min, flash freeze in liquid nitrogen for 5 min, incubate in water at 37℃ for 5 min, and incubate on ice for 5 min. Add 100 μl of antibiotic-free LB, shake at 28℃ and 200 rpm for 2 h, and directly plate onto a plate containing the corresponding bacterial antibiotic + rifampin, and incubate at 28℃ for two days.

[0035] ② Pick a single clone and shake it: Two days later, pick a single clone and put it into a 5 ml sterile EP tube. Add 2 ml of the corresponding bacterial antibiotic + rifampin in advance, and shake it overnight.

[0036] ③ Preservation of glycerol bacteria: 400 μl of bacterial culture + 100 μl of 75% sterile glycerol, label properly, and store in a -70℃ refrigerator. Extract plasmid from the remaining bacterial culture and transform it into E. coli (1 μl of plasmid + 20 μl of competent E. coli cells, incubate on ice for 30 min, heat shock at 42℃ for 35 s, incubate on ice for 2 min, add 100 μl of antibiotic-free LB, shake at 37℃ and 200 rpm for 1 h, plate, add LB and corresponding bacterial antibiotic, and incubate at 37℃ for one day).

[0037] ④ Sequencing: Select a single clone, shake the culture in the morning, and send the bacterial culture directly for sequencing in the evening. If the sequencing feedback is correct, verifying that there is no problem with Agrobacterium, the prepared Agrobacterium can be used for genetic transformation experiments (this part was outsourced to Weimi Biotechnology (Jiangsu) Co., Ltd.).

[0038] 2. Genetic transformation The successfully constructed recombinant editing vector was transformed into cotyledonary explants of the tomato variety 'Micro-Tom' using Agrobacterium electroporation. After tissue culture, resistant regenerated plants were obtained, and genomic DNA was extracted. PCR amplification and sequencing were performed to identify the mutation type of the SlABCB13 gene in the T0 generation plants (this part was commissioned to Weimi Biotechnology (Jiangsu) Co., Ltd.).

[0039] (1) The vector was transferred into Agrobacterium EHA105 by electroporation and identified by PCR. After seed germination, cotyledons and hypocotyls were used as infection materials. After pre-culturing for 2 days, they were placed in a suspension containing Agrobacterium for 5 min. After the bacterial suspension was dried, they were placed in a co-culture medium and co-cultured at 23℃ for 3 days. The explants were then transferred to the corresponding resistance selection medium for selection. After 3 weeks, they were subcultured into differentiation medium. Subculture was repeated every 3 weeks. Resistant shoots were obtained successively after about 7 weeks. The main stems that grew to 2-3 cm were subcultured into rooting glass tubes.

[0040] (2) The plants were tested for plant resistance (hygromycin) genes and target sites. The primer information is as follows: HYG-F1 (SEQ ID No. 4): CAAAGATCGTTATGTTTATCGGCACT; HYG-R1 (SEQ ID No. 5): TTGGCGACCTCGTATTGGGAA; The amplified size is 519 bp.

[0041] Primers for detecting target sites: Solyc08g076720-T1-F1 (SEQ ID No. 6): GGTGGCATGTTGGATGCATAG; Solyc08g076720-T1-R1 (SEQ ID No.7): CGTGAACCCCCACTTACCA; Solyc08g076720-T2-F1 (SEQ ID No. 8): GTTGGTGTTGATGCAGGAGAG; Solyc08g076720-T2-R1 (SEQ ID No.9): TCTTCACTTTGATGCCGCAC; The reaction system of 20 μL is as follows: Table 3 System Table 4 Reaction Procedure Example 2 SlABCB13 Validation of genetic stability of T2 generation plants of gene-edited homozygous mutant 1. Screening and identification of T1 generation homozygous mutants. T1 generation gene-edited plants were obtained through self-pollination, and genomic DNA was extracted. Unedited wild-type tomato plants were used as a control. SlABCB13 Two target sites of the gene were identified by PCR amplification and sequencing. The gene was then identified in T1 generation plants. SlABCB13 Gene mutation types were identified and plants without gene editing vector sequences were screened. Three T1 generation homozygous mutant lines with different editing types were selected (named...). slabcb13-7-16 , slabcb13-20-4 , slabcb13-24-21 (Single-plant seed saving).

[0042] 2. Planting and Homozygosity Verification of the T2 Generation Population. T2 generation seeds were sown as single-plant lines, with at least 20 plants planted from each T1 generation single plant in the T2 generation population. Wild-type tomatoes were planted as a control. When the T2 generation plants reached 3-4 true leaves, DNA was extracted from 12 randomly selected plants for analysis. SlABCB13 Gene target sites are subjected to PCR sequencing. For example... Figure 2 , Figure 3 As shown, the target site sequences of the tested plants were completely identical to those of the parental T1 generation homozygous mutants, with no wild-type sequences or other mutation types segregating, thus obtaining genetically stable T2 generation homozygous mutant lines.

[0043] Example 3 SlABCB13 Determination of sugar content in fruits of gene-edited homozygous mutant plants 1. Sampling: Select fruit tissues from mutants and wild-type tomatoes at the green and red ripe stages, as well as mature functional leaves of the plants, with consistent fruit maturity. Weigh 0.3 g / sample and store at -80℃.

[0044] 2. Place the sample in a test tube, add 5 mL of 80% ethanol solution, shake to mix, and place in an 80℃ water bath for 1 hour, shaking every 20 minutes to promote sugar dissolution. Repeat the extraction three times and combine the supernatants from the three extractions. Place the combined supernatant in an evaporating dish, evaporate the ethanol solution to dryness, and then add 1 mL of distilled water to the evaporated dish to dissolve the sugar. Centrifuge at 12000 rpm for 5 minutes at 4℃, collect the supernatant, filter it through a 0.22 μm organic phase filter membrane into an HPLC vial, and prepare for analysis.

[0045] 3. Detection of soluble sugars (glucose, fructose, and sucrose) using liquid chromatography (Agilent 1260) Mobile phase preparation: Acetonitrile-water (3:1, volume ratio) was used as the mobile phase. The column was degassed by sonication for 60 min to prevent the formation of air bubbles in the column. Instrument warm-up: The computer workstation, infusion pump, detector, and column oven were turned on in sequence. The column temperature was set to 30℃ and the detector temperature to 33℃. The column was warmed up for 30 min. Column equilibration: The flow rate was increased from 0.1 ml / min, and then 0.1 ml was added every 1 min until it reached 1 ml / min. The column was flushed for 30 min to 60 min until the baseline stabilized before injection.

[0046] 4. Measure and compare SlABCB13 The content of soluble sugars (glucose, fructose, sucrose) in the fruits of gene-edited T2 generation homozygous mutants and wild-type control plants.

[0047] like Figure 4 As shown, compared with wild-type plants, the soluble sugar content in the leaves of SlABCB13 gene-edited plants did not change significantly; during the green ripening stage, the fructose and glucose content in the fruit increased, while sucrose showed no significant change, and the total sugar content increased significantly; during the red ripening stage, both soluble sugar and total sugar content increased significantly. The fructose content of the three edited lines increased by 55%, 48%, and 46%, respectively; the glucose content increased by 71%, 51%, and 51%, respectively; the sucrose content increased by 157%, 93%, and 245%, respectively; and the total sugar content increased by 67%, 51%, and 58%, respectively. In conclusion, knocking out… SlABCB13 The gene can promote the accumulation of glucose, fructose and sucrose in tomato fruit, thereby significantly increasing the total sugar content of the fruit, clarifying that the gene has an important regulatory function in tomato sugar accumulation.

[0048] Example 4 SlABCB13 Other indicators of gene-edited lines were measured. 1. Soluble solids content: Soluble solids content: The fruit juice was directly measured using a handheld refractometer (PAL-fu, ATAGO).

[0049] 2. Fruit firmness: Select fruits of uniform maturity and use a fruit firmness tester (PUYAN) to measure the resistance when the probe penetrates the flesh, reflecting the firmness in N. Calculate the average firmness of the mutant and wild-type fruits.

[0050] 3. Fruit size: Select fruits of mutant and wild-type tomatoes with the same maturity, avoiding deformed or diseased fruits. Use an electronic digital caliper (0.01 mm accuracy) to measure the longest distance from the base of the fruit stalk to the navel and the maximum diameter of the fruit at the equator.

[0051] 4. Plant height and stem diameter: Select 10 plants with uniform growth from each line. Use a ruler to measure the distance from the base of the plant (soil surface) vertically to the growing point at the top of the plant, in cm, and take the average value. Use an electronic digital caliper to measure the stem diameter between the 3rd and 4th nodes of the plant (avoiding the nodes), in mm, and take the average value.

[0052] 5. Number of fruits set: Count the number of fruits per plant and take the average value.

[0053] 6. Fruit weight per plant: The total weight of the fruit per plant is calculated and the average value is taken.

[0054] like Figure 5 , Figure 6 As shown, compared to wild-type tomato plants, SlABCB13 From plant height and stem diameter (vegetative growth indicators) to fruit size, number of fruits, yield per plant, and fruit firmness (fruit phenotypic indicators), no significant regular differences were observed in the gene-edited lines. However, the soluble solids content, an important indicator of fruit sugar content, was significantly higher in all three gene-edited lines than in the wild type. Figure 4 The contents of glucose, fructose, and sucrose in the fruits of the gene-edited strains showed a significant increasing trend, confirming... SlABCB13 Targeted gene editing significantly increases the soluble sugar content of tomato fruits without adversely affecting the plant's vegetative and reproductive growth traits, and does not cause any loss in yield or growth and development.

[0055] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. Knockout SlABCB13 Application of genes in increasing the soluble sugar content of tomato fruits.

2. The application according to claim 1, characterized in that, The soluble sugars include one or more of sucrose, fructose, and glucose.

3. The application according to claim 1, characterized in that, The SlABCB13 The nucleotide sequence of the gene is shown in SEQ ID No.

1.

4. The application according to claim 1, characterized in that, The tomato fruit in question is a red-ripe tomato fruit.

5. A knockout method SlABCB13 The sgRNA of a gene is characterized by, Including sgRNA1 and sgRNA2; The nucleotide sequence of sgRNA1 is shown in SEQ ID No. 2, and the nucleotide sequence of sgRNA2 is shown in SEQ ID No.

3.

6. A method for obtaining plants with increased soluble sugar content in tomato fruits, characterized in that, Includes the following steps: 1) The sgRNA described in claim 5 is ligated into a CRISPR / Cas9 vector to obtain a recombinant editing vector; 2) The recombinant editing vector described in step 1) is transformed into Agrobacterium to obtain the transformed bacteria; 3) The transforming bacteria described in step 2) are used to infect the cotyledons and hypocotyls of tomatoes to obtain tomato plants.

7. The method for obtaining according to claim 6, characterized in that, Step 1) The connection system is as follows: 4 µl of sgRNA, 2 µl of CRISPR / Cas9 vector at a concentration of 100 ng / µl, 1 µl of recombinase, 2 µl of recombinant buffer, and 1 µl of ddH2O; The concentrations of sgRNA1 and sgRNA2 in the 4 µl sgRNA were both 100 ng / µl. The CRISPR / Cas9 vector is a linearized CRISPR / Cas9 vector; The conditions for the connection include: operating at 37°C for 30 minutes.

8. The acquisition method according to claim 6, characterized in that, Step 2) The recombinant editing vector is transformed into Agrobacterium using an electroporation method.

9. The acquisition method according to claim 6, characterized in that, The soluble sugars include one or more of sucrose, fructose, and glucose.

10. Knockout SlABCB13 Application of genes in increasing the fructose and glucose content of green-ripe tomato fruits.

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