Application of SlKNOX7 gene in regulating flowering time of tomato

Abstract

This invention relates to the field of biotechnology, specifically to... SlKNOX7 The application of genes in regulating tomato flowering time, the aforementioned SlKNOX7 The sequence 5000 bp upstream of the start codon of the gene is shown in SEQ ID NO:1. SlKNOX7 The gene's stop codon downstream of 1000 bp is shown in SEQ ID NO:2. SlKNOX7 The gene sequence is shown in SEQ ID NO:3. SlKNOX7 The gene coding sequence is shown in SEQ ID NO:4. Using CRISPR-Cas9-mediated gene editing technology, this gene was mutated to lose its function. The mutant plants generally flowered about 8 days earlier than the control plants. Editing this gene can control the flowering time of tomatoes, shorten the plant growth cycle, and enable the breeding of varieties with different flowering periods.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, specifically involving SlKNOX7 Application of genes in regulating tomato flowering time. Background Technology

[0002] As a major vegetable crop, tomatoes rank among the top vegetable crops globally in terms of annual output, profoundly influencing the structure and development of the vegetable industry. The flowering process of tomatoes is a prerequisite for fruit and seed formation, and the timing of flowering, as a key factor regulating core agronomic traits such as plant quality, yield, and growth period, has always attracted widespread attention in the botanical community. Research on the regulation of flowering time in economic crops is not only a hot topic in plant biology but also an important direction for breeders striving to achieve early flowering and high yield through technological means. Therefore, in-depth exploration of the relevant mechanisms and regulation strategies of tomato flowering time has profound scientific significance and enormous application prospects. Through this type of research, we can expect to shorten the tomato's growth cycle, enabling it to adapt more quickly to changes in market demand; at the same time, optimizing flowering time can also promote the effective use of resources, increase tomato yield per unit area, and further promote the sustainable development of the vegetable industry. Therefore, conducting research on tomato flowering time is not only a manifestation of scientific and technological progress but also an important contribution to improving agricultural production efficiency and meeting the needs of socio-economic development.

[0003] In recent years, with the rapid development of traditional gene cloning and high-throughput sequencing technologies, and the successful acquisition of numerous flower development-related mutants, scientists have successfully cloned and identified several genes closely related to tomato flowering time. These genes include: SFT ( SINGLE FLOWER TRUSS ), J ( JOINTLESS ), UF ( UNIF LORA) S ( COMPOUND INFLORESCENCE ), SP5G ( SELF-PRUNING 5G ), TMF ( TERMINATING FLOWER )as well as FA ( FALSIFLORA )wait.

[0004] Currently, compared with other model plants such as Arabidopsis thaliana, research on tomato flowering genes is still very limited. New flowering genes are yet to be discovered, and the discovery of new flowering genes is beneficial for the creation of new early-flowering tomato germplasm, providing new gene resources and guidance for improving tomato flowering time, and has important application prospects. Summary of the Invention

[0005] Therefore, this invention utilizes CRISPR-Cas9-mediated gene editing technology to edit tomatoes. SlKNOX7 After the gene was edited and knocked out, resulting in loss of its function, the tomato plants exhibited an earlier flowering phenotype, thus confirming that the gene is related to the flowering time of tomatoes.

[0006] One of the objectives of this invention is to provide SlKNOX7 The application of genes in regulating tomato flowering time, the aforementioned SlKNOX7 The sequence 5000 bp upstream of the start codon of the gene is shown in SEQ ID NO:1. SlKNOX7 The gene's stop codon downstream of 1000 bp is shown in SEQ ID NO:2. SlKNOX7 The gene sequence is shown in SEQ ID NO:3. SlKNOX7 The coding sequence of the gene is shown in SEQ ID NO:4.

[0007] The second objective of this invention is to provide SlKNOX7 The application of gene-encoded proteins in regulating tomato flowering time, the aforementioned SlKNOX7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:5.

[0008] The third objective of this invention is to provide a method for regulating the flowering time of tomatoes, specifically by using gene editing technology. SlKNOX7 Genes are knocked out.

[0009] Furthermore, the gene editing technology is performed using the CRISPR / Cas9 system, which includes... SlKNOX7 A vector for the gRNA of a gene, the sequence of which is shown in SEQ ID NO:8.

[0010] The fourth objective of this invention is to provide the application of recombinant vectors, expression cassettes, or engineered bacteria containing the above-mentioned sequences in regulating the flowering time of tomatoes.

[0011] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0012] This invention uses IP-MS to screen interacting proteins, identify candidate genes, and perform gene function identification and verification, ultimately determining the genes related to tomato flowering time. SlKNOX7 Genes; through design SlKNOX7The target gene was identified, and a U6pro-Target-gRNA expression cassette was constructed and ligated into a pTX binary vector using homologous recombination. This cassette was then transformed into Agrobacterium, infecting tomato cotyledon explants, which differentiated into shoots and induced roots, resulting in transgenic plants. Statistical analysis of the target editing and the phenotypic characteristics of the mutant plants revealed that, compared to the control plants, the mutant plants flowered approximately 8 days earlier. This indicates that the gene plays a crucial role in regulating the tomato flowering pathway, and to some extent, can shorten the plant's growth and development cycle. This discovery created a new early-flowering tomato germplasm and provided genetic resources for improving tomato flowering time. Attached Figure Description

[0013] Figure 1 The figures show the results of the in vivo interaction experiment between KNOX7 and FAF1 / 2c; where A is the result of the luciferase complementation assay and B is the result of the Co-IP experiment.

[0014] Figure 2 The figure shows the results of the in vitro interaction experiment between KNOX7 and FAF1 / 2c.

[0015] Figure 3 for KNOX7 Schematic diagram of genomic DNA.

[0016] Figure 4 for KNOX7 Diagram showing target editing process.

[0017] Figure 5 AC for eight-week-old seedlings and KNOX7 -Real photo of CR plant.

[0018] Figure 6 For AC and KNOX7 Flowering time of -CR strains (-8, -10, -12) Detailed Implementation

[0019] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can more clearly understand the present invention.

[0020] Example 1

[0021] This embodiment provides SlKNOX7 The methods for gene screening and identification specifically include the following steps:

[0022] 1. Screening of interacting proteins using IP-MS:

[0023] Constructing CaMV-35S-driven FAF1 / 2c (early flowering gene discovered by our research group, already published in an article, EARLYFLOWERING is a dominant gain-of-function allele of) FANTASTIC FOUR 1 / 2c (This promotes early flowering in tomato, Plant Biotechnology Journal, doi: 10.1111 / pbi.14217) A transgenic tomato plant was obtained by fusing an mCherry fluorescent tag protein vector with an Agrobacterium-mediated tomato genetic transformation system. Apical meristems and young leaves were collected from the transgenic plants, and total protein was extracted. The protein was then enriched and purified using mCherry magnetic beads to obtain the protein from the transgenic plants. FAF1 / 2c -mCherry protein and its interacting proteins. The enriched samples were analyzed and identified by LC-MS, ultimately screening out several candidate proteins. Table 1 lists the genes involved in this patent. SlKNOX7 .

[0024] Table 1. One of the candidate proteins that interact with FAF1 / 2c, screened by LC-MS.

[0025]

[0026] 2. Interoperability verification between KNOX7 and FAF1 / 2c:

[0027] Interoperability verification between KNOX7 and FAF1 / 2c was performed using three methods: pull-down, co-IP, and LUC. The verification results are shown below. Figure 1 , 2 ,in:

[0028] Figure 1 (A) is the luciferase complementation assay (LUC). It can be seen that only the combination of FAF1 / 2c-nLUC+KNOX7-cLUC produces fluorescence, i.e., protein interaction. Figure 1 (B) shows the Co-IP experiment, where FAF1 / 2c fused with an mCherry tag and KNOX7 fused with an HA tag. Different combinations of Agrobacterium tumefaciens infected tobacco leaves. Proteins were immunoprecipitated (IP) using mCherry magnetic beads and detected with mCherry and HA antibodies. Flag-mCherry and GFP-HA served as controls. Figure 1 In (B), the red arrows from top to bottom indicate the positions of the expected protein bands of FAF1 / 2c-mCherry, Flag-mCherry, KNOX7-HA and GFP-HA, respectively, indicating that KNOX7 interacts with FAF1 / 2c protein.

[0029] Figure 2 For the pull-down experiment, FAF1 / 2c and KNOX7 were fused with MBP and GST tags, respectively. The recombinant proteins were expressed, purified, and immunoprecipitated in *E. coli* cells. After incubation, the protein mixtures were passed through glutathione agar beads and detected using GST and MBP antibodies, respectively. The red arrows from top to bottom represent the expected protein bands of KNOX7-GST, GST, and FAF1 / 2c-MBP, respectively. The experimental results indicate that KNOX7 and FAF1 / 2c interact both in vivo and in vitro in plants.

[0030] 3. Identify candidate genes:

[0031] KNOX The gene has been reported to participate in plant cell differentiation, cell morphogenesis, apical meristem and carpel development, and can also promote the synthesis of cytokinins. FAF1 / 2c It may influence cell differentiation and apical meristem development through interaction with KNOX7, thereby accelerating the flowering process in plants. Therefore, KNOX7 (Solyc08g041820) was selected from the above candidate proteins for further gene function identification and verification.

[0032] 4. SlKNOX7 Gene-related sequences:

[0033] SlKNOX7 Located on chromosome 8, with an amino acid sequence length of 349aa, it contains a conserved homebox domain, and its relevant sequence is as follows:

[0034] (1) SlKNOX7 The sequence 5000bp upstream of the gene start codon is as follows;

[0035]

[0036] (2) SlKNOX7 The sequence 1000 bp downstream of the gene stop codon is as follows;

[0037]

[0038] (3) SlKNOX7 The genome sequence of the gene, specifically;

[0039]

[0040] (4) SlKNOX7 The coding sequence of the gene is as follows;

[0041]

[0042] (5) SlKNOX7 The protein sequence encoded by the gene, specifically;

[0043] SEQ ID NO:5:MDMPDQNSPILMTSLPEQDVKPLEHQQPPPTSLNREILLPQTQYTGESNSNQWLSRSILQRNIQASNDSDLTKDEFGESEAVNWQNAGYKSEILAHPLFEQLLSAHVACLRIATPVDQLPRIDAQLAQSQQIVGKYSGLGHGNLSDDKELDQFLTHYVLLLCSFKEQLQQHVR VHAMEAVMACWEIEQSLQSLTGVSPGEGTGATMSDDEDEQVDSDANLFEGSLDGHDSMGFGPLIPTESERSLMERVRQELKHELKQGYKEKLVDIREEILRKRRAGKLPGDTTSVLKAWWQSHSKWPYPTEEDKAKLVEETGLQLKQINNWFINQRKRNWHSNPSSSTALKSKRKR*.

[0044] Example 2

[0045] This embodiment is based on the selection from Embodiment 1. SlKNOX7 To verify gene function, Ailsa Craig (AC) was selected as the background material in the tomato genetic transformation study. Its germplasm was obtained from the Tomato Genetic Resource Center (TGRC). The specific research process covered the following key steps:

[0046] 1. Build SlKNOX7 Gene editing vectors:

[0047] Through the CRISPR direct website SlKNOX7 The gene was designed with two targets. Two 20 bp targets were designed on the first exon, ensuring that the interval between the two targets did not exceed 700 bp. Two targets were selected from the generated target list. Primers were then designed for these targets, with a sequence for homologous recombination added to the 5' end of the primers and a universal primer sequence for ligation to the intermediate vector added to the 3' end. The specific sequences are as follows:

[0048] SlKNOX7 -g1-F:GAATCTAACAGTGTAGTTTGGATGACTAGTTTACCGGAACGTTTTAGAGCTAGAAATAG (SEQ ID NO.6);

[0049] SlKNOX7 -g2-R: GCTATTTCTAGCTCTAAAACGTTGAGCGACGTCGGTGGCGCAAACTACACTGTTAGATT (SEQ ID NO. 7).

[0050] To amplify the inclusion SlKNOX7 We used the intermediate vector pTX043 plasmid as a template to obtain the gRNA sequence targeting both genes (Deng L, Wang H, Sun C et al., 2018). Using specific primers SlKNOX7-g1-F and SlKNOX7-g2-R, we amplified the gRNA sequence containing... SlKNOX7 gRNA sequences targeting two gene sites.

[0051] The specific amplification steps are as follows: The PCR amplification system was set to 50 μL, containing 1 μL of 100-200 ng / μL DNA template, 25 μL of 2×PhantaMax buffer, 1 μL of 10 mM dNTP Mix, 2 μL each of 10 μM primers, 1 μL of PhantaMax Super-Fidelity DNA Polymerase, and 18 μL of ddH2O. The amplification program included 95℃ pre-denaturation for 3 minutes, followed by 35 cycles of 95℃ denaturation for 15 seconds, 56℃ annealing for 15 seconds, and 72℃ extension for 30 seconds, ending with a final extension at 72℃ for 5 minutes and an incubation at 12℃ for 5 minutes. The amplified sequence is shown in SEQ ID NO:8.

[0052] SEQ ID NO:8:

[0053] Amplification SlKNOX After sequencing the gRNA targeting the dual gene sites, the target fragment was recovered via gel extraction. The target fragment was then linearized by single-restriction digestion of the binary expression vector pTX041 (Deng L, Wang H, Sun C et al., 2018) with BsaI1 restriction endonuclease. Homologous recombination was then performed to recombine the recovered fragment with the linearized pTX041 vector under appropriate conditions. Following homologous recombination, heat shock was performed, and the recombinant vector was transformed into *E. coli* Trans-T1 (purchased from Beijing TransGen Biotech Co., Ltd.). Single clones containing the correct recombinant plasmid were detected and selected for expansion culture, and the plasmid was extracted. For subsequent tomato genetic transformation, this recombinant plasmid was efficiently transformed into *Agrobacterium* GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) using either electroporation or liquid nitrogen methods. After successful transformation with *Agrobacterium*, these *Agrobacterium* strains carrying the recombinant plasmid were used in subsequent tomato genetic transformation experiments to achieve…​ Precise editing of genes.

[0054] 2. Genetic transformation in tomatoes:

[0055] After selection and sterilization, seeds were inoculated onto 1 / 2 MS medium. Once the cotyledons unfolded, the seeds were cut into small explant segments and cultured in the dark on KCMS medium. Subsequently, the explants were infected with activated Agrobacterium and cultured in the dark again. Afterward, the explants were transferred to a selection medium and cultured under specific light and temperature conditions to select transformants and induce new shoot formation. When the new shoots reached an appropriate size, they were transferred to a rooting medium to induce rooting, and finally, transgenic seedlings were obtained and transplanted into pots containing a mixture of potting soil and vermiculite.

[0056] 3. ​ Detection of gene-edited plants:

[0057] Approximately 50-100 mg of young tissue from transgenic tomato plants was collected, and DNA was efficiently extracted using the CTAB method. First, the tissue was ground and broken up in a centrifuge tube containing steel balls, then heated in a 65°C water bath to promote DNA release. A mixed solvent of chloroform and isoamyl alcohol was added, and impurities were removed by vigorous shaking and centrifugation to obtain a pure DNA supernatant. Next, the DNA was precipitated with isopropanol, washed, and air-dried, then dissolved in ddH₂O to obtain a high-purity DNA solution, which was stored at -20°C for subsequent experiments.

[0058] Using the extracted DNA sample as a template, PCR amplification was performed using specific primers pTX-F and pTX-R (SEQ ID NO: 9~10) to detect whether the vector insertion was successful. After confirming vector insertion, [further steps were taken]. ​ A pair of target editing detection primers were designed and synthesized before and after the dual target regions of the gene. ​ -CR-F and ​ -CR-R (SEQ ID NO:11~12). Using this primer pair, PCR amplification is performed again for accurate detection. ​ The editing status of the gene at these target sites, including possible mutations, insertions, or deletions, is used to assess the effectiveness of gene editing. The specific primer sequences are as follows:

[0059] pTX-F: AGCGGATAACAATTTCACACAGGA (SEQ ID NO:9);

[0060] pTX-R: GCAGGCATGCAAGCTTATTGG (SEQ ID NO: 10);

[0061] ​-CR-F: ACCTTTGGGGACCACTGATGTATA (SEQ ID NO: 11);

[0062] ​ -CR-R: GGATTCCATAAAAGTTCAAACCTAA (SEQ ID NO: 12).

[0063] 4. Observation and Statistics ​ Phenotype of gene-edited plants.

[0064] against ​ Detailed phenotypic observations and statistical analyses were conducted on the mutant plants, with AC as the control material. After germination and sowing under the same conditions, when the plants grew to 2-3 true leaves, they were transplanted and subjected to uniform cultivation management. The changes in the number of true leaves were recorded in detail to assess growth rate and flowering time, with particular attention paid to the timing of the first flower bud differentiation.

[0065] The specific results of the above verification experiments can be found in [link to relevant documentation]. ​ ,in:

[0066] ​ for ​ A schematic diagram of genomic DNA, showing the start codon (ATG), two guide RNAs (target1 and target2), the direction of gene transcription, and the sequences of the two guide RNAs.

[0067] ​ for ​ A diagram illustrating target editing is shown in the image. ​ In WT(AC), ​ -CR-8, 10, and 12 contain genomic DNA sequences with two target sequences. Compared to the control material WT, ​ -CR-8 has a 1-base insertion at the first target site. ​ -CR-10 has a 1-base insertion at the first target site and a 2-base deletion at the second target site. ​ -CR-12 has a 7-base deletion at the first target site and a 1-base deletion at the second target site.

[0068] ​ For AC and ​ The mutant plant phenotypes are shown in the image above, which is the control AC and... ​ -CR-8, 10, and 12 lineages, scale bar is 10cm; the image below shows the control AC and... ​ - Enlarged view of the apical meristem of the CR line, where the red arrow indicates the first inflorescence and the white arrow indicates the second inflorescence. Compared with the control AC, ​ The mutant plants started flower bud differentiation earlier.

[0069] ​ For comparison with AC and ​ A chart showing the flowering time of mutant plants. The arrows indicate the time when the first inflorescence appears. The red arrow indicates that AC's flowering time was 48 days, and the blue arrows indicate... ​ -CR-12 flowers in 38 days, indicated by the yellow arrow. ​ -CR-10 has a flowering time of 40 days, indicated by the gray arrow. ​ -CR-8 has a flowering time of 42 days.

[0070] In summary, compared with the control plant AC, the three mutant strains... ​ -CR-8、 ​ -CR-10 ​ -CR-12 bloomed about 8 days earlier.

[0071] Unless otherwise specified, all raw materials used in this invention are existing substances that can be purchased directly from the market.

[0072] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. SlKNOX7 The application of genes in regulating tomato flowering time is characterized by, The SlKNOX7 The sequence 5000 bp upstream of the start codon of the gene is shown in SEQ ID NO:

1. SlKNOX7 The gene's stop codon downstream of 1000 bp is shown in SEQ ID NO:

2. SlKNOX7 The gene sequence is shown in SEQ ID NO:3; The application involves using gene editing technology to... SlKNOX7 Genes are knocked out, thus enabling tomatoes to flower earlier.

2. SlKNOX7 The application of gene-encoded proteins in regulating tomato flowering time is characterized by, The SlKNOX7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

5. The application is the use of gene editing technology to... SlKNOX7 Genes are knocked out, thus enabling tomatoes to flower earlier.

Images