Application of SlKNOX7 gene in regulation and control of tomato flowering time

Abstract

The invention relates to the technical field of biology, in particular to application of an S1KNOX7 gene in regulation and control of tomato flowering time, the sequence of upstream 5000bp of an initiation codon of the S1KNOX7 gene is shown as SEQ ID NO: 1, the sequence of downstream 1000bp of a termination codon of the S1KNOX7 gene is shown as SEQ ID NO: 2, the sequence of the S1KNOX7 gene is shown as SEQ ID NO: 3, and the coding sequence of the S1KNOX7 gene is shown as SEQ ID NO: 4. The gene is mutated through a CRISPR-Cas9 mediated gene editing technology, so that the function of the gene is lost, the flowering time of a mutant plant is generally advanced by about 8 days compared with that of a control plant, the flowering time of tomatoes can be controlled by editing the gene, the growth cycle of the plant can be shortened, and variety cultivation work in different flowering periods can be realized.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and specifically relates to SlKNOX7 Application of genes in regulating flowering time in tomato. Background Art

[0002] As an important bulk vegetable crop, tomato ranks among the top vegetable crops in the world in terms of annual output, which has a profound impact on the pattern and development of the vegetable industry. The flowering process of tomatoes is a prerequisite for the formation of fruits and seeds, and flowering time, as a key factor in regulating core agronomic traits such as plant quality, yield and growth period, has always attracted widespread attention in the botanical community. The study of flowering time regulation of economic crops is not only one of the hot topics in the field of plant biology, but also an important direction for breeders to achieve the goals of early flowering and high yield through technical means. Therefore, in-depth exploration of the relevant mechanisms and regulatory strategies of tomato flowering time has profound scientific significance and great application prospects. Through this type of research, we hope to shorten the growth cycle of tomatoes and enable them to adapt to changes in market demand more quickly; 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 reflection of scientific and technological progress, but also an important contribution to improving agricultural production efficiency and meeting the needs of social and economic development.

[0003] In recent years, with the rapid development of traditional gene cloning technology and high-throughput sequencing technology, as well as the successful acquisition of a large number of 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] At present, compared with other model plants such as Arabidopsis, research on tomato flowering genes is still very limited, and new flowering genes are still to be discovered. In addition, the discovery of new flowering genes is conducive to the creation of new early-flowering tomato germplasm, providing new genetic resources and guidance for the improvement of tomato flowering period, and has important application prospects. Summary of the invention

[0005] In view of this, the present invention uses CRISPR-Cas9 mediated gene editing technology to SlKNOX7 After gene editing and knocking out the gene to cause loss of its function, tomato plants showed an early flowering phenotype, thus confirming that the gene is related to the flowering time of tomatoes.

[0006] One of the objects of the present invention is to provide SlKNOX7 Application of genes in regulating tomato flowering time, the SlKNOX7 The sequence of 5000 bp upstream of the start codon of the gene is shown in SEQ ID NO: 1. SlKNOX7 The 1000 bp downstream of the stop codon of the gene 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 object of the present invention is to provide SlKNOX7 Application of a gene-encoded protein in regulating tomato flowering time, the SlKNOX7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:5.

[0008] The third object of the present invention is to provide a method for regulating the flowering time of tomatoes, specifically using gene editing technology to SlKNOX7 Gene knockout.

[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 the gRNA being shown in SEQ ID NO:8.

[0010] A fourth object of the present invention is to provide a recombinant vector, expression cassette or engineered bacteria containing the above sequence for use in regulating the flowering time of tomatoes.

[0011] Compared with the prior art, the present invention has the following beneficial effects: The present invention screened the interacting proteins through IP-MS, identified the candidate genes, and performed gene function identification and verification, and finally determined the genes related to the flowering time of tomato. SlKNOX7 Genes; by design SlKNOX7The target of the gene was constructed, and the U6pro-Target-gRNA expression cassette was connected to the pTX binary vector by homologous recombination; it was further transformed into Agrobacterium, infected tomato cotyledon explants, further differentiated buds, induced roots, and obtained transgenic plants. Through the statistics of target editing and mutant plant phenotypes, it was found that compared with the control plants, the mutant plants flowered about 8 days earlier, indicating that the gene plays an important role in regulating the flowering pathway of tomatoes, and can shorten the growth and development cycle of plants to a certain extent, creating a new early-flowering tomato germplasm and providing genetic resources for the improvement of tomato flowering period. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 The figures are the results of the in vivo interaction experiment between KNOX7 and FAF1 / 2c; A is the result of the split-luciferase complementation assay, and B is the result of the Co-IP experiment.

[0013] Figure 2 This is a diagram showing the results of an in vitro interaction experiment between KNOX7 and FAF1 / 2c.

[0014] Figure 3 for KNOX7 Schematic diagram of genomic DNA.

[0015] Figure 4 for KNOX7 Schematic diagram of target editing.

[0016] Figure 5 For eight-week-old AC and KNOX7 -Real photos of CR plants.

[0017] Figure 6 For AC and KNOX7 Flowering time of -CR strains (-8, -10, -12) DETAILED DESCRIPTION The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand the present invention more clearly.

[0018] Example 1 This embodiment provides SlKNOX7 The method for gene screening and determination specifically comprises the following steps: 1. IP-MS screening of interacting proteins: Construct CaMV-35S driven FAF1 / 2c (early flowering gene discovered by the research group, published article, EARLYFLOWERING is a dominant gain-of-function allele of FANTASTIC FOUR 1 / 2cThe mCherry fluorescent tag protein vector was fused to the tomato plant, and the Agrobacterium-mediated tomato genetic transformation system was used to obtain positive transgenic tomato plants. The apical meristem and young leaves were taken to extract total protein. The mCherry magnetic beads were used for enrichment and purification to obtain the protein in the transgenic plants. FAF1 / 2c -mCherry protein and its interacting proteins. The samples obtained after enrichment were subjected to mass spectrometry detection and identification by LC-MS method, and finally several candidate proteins were screened out. Table 1 shows the genes involved in this patent. SlKNOX7 .

[0019] Table 1 One of the candidate proteins interacting with FAF1 / 2c screened by LC-MS

[0020] 2. Verification of the interaction between KNOX7 and FAF1 / 2c: The interaction between KNOX7 and FAF1 / 2c was verified by Pull-down, Co-IP, and LUC. The verification results are shown in Figure 1 , 2 ,in: Figure 1 (A) Split luciferase complementation assay (LUC), showing that only the combination of FAF1 / 2c-nLUC+KNOX7-cLUC produces fluorescence, indicating protein interaction; Figure 1 (B) Co-IP experiment, FAF1 / 2c was fused with mCherry tag, KNOX7 was fused with HA tag, and different combinations of Agrobacterium were used to infect tobacco leaves. Proteins were immunoprecipitated (IP) with mCherry magnetic beads and detected with mCherry antibody and HA antibody, and Flag-mCherry and GFP-HA were used as controls. Figure 1 In (B), the red arrows indicate the positions of the expected protein bands of FAF1 / 2c-mCherry, Flag-mCherry, KNOX7-HA and GFP-HA from top to bottom. The results show that KNOX7 interacts with FAF1 / 2c protein.

[0021] Figure 2For the pull-down experiment, FAF1 / 2c and KNOX7 were fused with MBP tags and GST tags, respectively. The recombinant proteins were expressed in E. coli cells, purified, and immunoprecipitated. After incubation with the protein mixture, it was passed through glutathione agarose beads and detected with GST and MBP antibodies, respectively. The red arrows indicate the expected protein bands of KNOX7-GST, GST, and FAF1 / 2c-MBP from top to bottom, respectively. The above experimental results show that KNOX7 interacts with FAF1 / 2c both in plants and in vitro.

[0022] 3. Identify candidate genes: KNOX The gene has been reported to be involved in plant cell differentiation, cell morphology, apical meristem and carpel development, and can also promote the synthesis of cytokinins. FAF1 / 2c It may affect cell differentiation and apical meristem development by interacting with KNOX7, thereby accelerating the flowering process of plants. Therefore, KNOX7 (Solyc08g041820) was selected from the above candidate proteins for subsequent gene function identification and verification.

[0023] 4. SlKNOX7 Gene related sequences: SlKNOX7 Located on chromosome 8, the amino acid sequence is 349aa long and contains the Homebox conserved domain. The relevant sequences are as follows: (1) SlKNOX7 The sequence 5000 bp upstream of the gene start codon is specifically:

[0024] (2) SlKNOX7 The sequence 1000 bp downstream of the gene stop codon is specifically;

[0025] (3) SlKNOX7 The genomic sequence of the gene, specifically;

[0026] (4) SlKNOX7 The coding sequence of the gene, specifically;

[0027] (5) SlKNOX7 The protein sequence encoded by the gene, specifically; SEQ ID NO:5:MDMPDQNSPILMTSLPEQDVKPLEHQQPPPTSLNREILLPQTQYTGESNSNQWLSRSILQRNIQASNDSDLTKDEFGESEAVNWQNAGYKSEILAHPLFEQLLSAHVACLRIATPVDQLPRIDAQLAQSQQIVGKYSGLGHGNLSDDKELDQFLTHYVLLLCSFKEQLQQHVR VHAMEAVMACWEIEQSLQSLTGVSPGEGTGATMSDDEDEQVDSDANLFEGSLDGHDSMGFGPLIPTESERSLMERVRQELKHELKQGYKEKLVDIREEILRKRRAGKLPGDTTSVLKAWWQSHSKWPYPTEEDKAKLVEETGLQLKQINNWFINQRKRNWHSNPSSSTALKSKRKR*.

[0028] Example 2 This embodiment is based on the screened SlKNOX7 To verify the function of the gene, Ailsa Craig (AC) was selected as the background material in the tomato genetic transformation research. Its germplasm came from the International Tomato Genetic Resource Center (TGRC). The specific research process covers the following key steps: 1. Build SlKNOX7 Gene Editing Vectors: CRISPR direct SlKNOX7 Design dual target sites of the gene. Design two targets with a length of 20 bp on the first exon, and ensure that the interval between the two targets does not exceed 700 bp. Select two targets from the generated target list, and then design primers for these targets, add sequences for homologous recombination at the 5' end of the primers, and add universal primer sequences connected to the intermediate vector at the 3' end. The specific sequences are as follows: SlKNOX7 -g1-F: GAATCTAACAGTGTAGTTTGGATGACTAGTTTACCGGAACGTTTTAGAGCTAGAAATAG (SEQ ID NO.6); SlKNOX7-g2-R: GCTATTTCTAGCTCTAAAACGTTGAGCGACGTCGGTGGCGCAAACTACACTGTTAGATT (SEQ ID NO. 7).

[0029] To expand the inclusion SlKNOX7 For the gRNA sequences of dual gene targets, we used the intermediate vector pTX043 plasmid as a template (Deng L, Wang H, Sun C et al, 2018). Using specific primers SlKNOX7-g1-F and SlKNOX7-g2-R, we amplified the SlKNOX7 gRNA sequences for dual gene targets.

[0030] The specific amplification steps are as follows: the PCR amplification system is set to 50 μL, including 1 μL of 100-200 ng / μL DNA template, 25 μL of 2×Phanta Max buffer, 1 μL of 10 mM dNTP Mix, 2 μL of 10 μM primers, 1 μL of PhantaMax Super-Fidelity DNA Polymerase, and 18 μL of ddH2O. The amplification program includes 95°C pre-denaturation for 3 minutes, followed by 35 cycles of 95°C denaturation for 15 seconds, 56°C annealing for 15 seconds, and 72°C extension for 30 seconds, and finally 72°C extension for 5 minutes and 12°C incubation for 5 minutes. The amplified sequence is shown in SEQ ID NO:8.

[0031] SEQ ID NO:8:.

[0032] Amplification SlKNOX7 After the gRNA sequences of the dual gene targets were obtained, gel excision was performed to obtain the purified target fragments, and then the binary expression vector pTX041 (Deng L, Wang H, Sun C et al., 2018) was treated with a single enzyme digestion using the BsaI1 restriction endonuclease to ensure that it was linearized. Using homologous recombination technology, the recovered fragments were recombined with the linearized pTX041 vector under appropriate conditions. After homologous recombination, heat shock was performed, and the recombinant vector was transferred into Escherichia coli Trans-T1 (purchased from Beijing Quanshijin Biotechnology Co., Ltd.), and single clones containing the correct recombinant plasmid were detected and selected for expansion culture, and the plasmid was extracted. In order to carry out subsequent tomato genetic transformation, this recombinant plasmid was efficiently transformed into Agrobacterium GV3101 (purchased from Shanghai Weidi Biotechnology Co., Ltd.) by electroporation or liquid nitrogen method. After confirming that the Agrobacterium transformation was successful, these Agrobacteria carrying the recombinant plasmid will be used in subsequent tomato genetic transformation experiments to achieveSlKNOX7 Precise editing of genes.

[0033] 2. Tomato genetic transformation: After the seeds are selected and disinfected, they are inoculated in 1 / 2MS medium. After the cotyledons are unfolded, they are cut into small explants and placed in KCMS medium for dark culture. Subsequently, the explants are infected with activated Agrobacterium and cultured again in the dark. After that, the explants are transferred to the screening medium and cultured under specific light and temperature conditions to screen transformants and induce the formation of new shoots. When the new shoots grow to an appropriate size, they are transferred to the rooting medium to induce rooting, and finally the transgenic seedlings are obtained and transplanted into a pot mixed with nutrient soil and vermiculite for planting.

[0034] 3. SlKNOX7 Detection of gene-edited plants: About 50-100 mg of young tissue from transgenic tomato plants was taken and DNA was efficiently extracted using the CTAB method. First, the tissue was ground and crushed in a centrifuge tube filled with steel balls, and 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, and after washing and air drying, it was dissolved in ddH2O to finally obtain a high-purity DNA solution, which was stored in a -20°C refrigerator for subsequent experiments.

[0035] Using the extracted DNA sample as a template, specific primers pTX-F and pTX-R (SEQ ID NO: 9-10) were used for PCR amplification to detect whether the vector was successfully inserted. SlKNOX7 A pair of target editing detection primers were designed and synthesized before and after the double target region of the gene SlKNOX7 -CR-F and SlKNOX7 -CR-R (SEQ ID NO: 11~12). Using this pair of primers, PCR amplification was performed again to accurately detect SlKNOX7 The editing of genes at these target sites, including possible mutations, insertions or deletions, can be used to evaluate the effect of gene editing. The specific primer sequences are as follows: pTX-F: AGCGGATAACAATTTCACACAGGA (SEQ ID NO:9); pTX-R: GCAGGCATGCAAGCTTATTGG (SEQ ID NO: 10); SlKNOX7 -CR-F: ACCTTTGGGGACCACTGATGTATA (SEQ ID NO: 11); SlKNOX7-CR-R: GGATTCCATAAAAGTTCAAACCTAA (SEQ ID NO: 12).

[0036] 4. Observation Statistics SlKNOX7 Phenotypes of gene-edited plants.

[0037] against SlKNOX7 The mutant plants were observed and counted in detail, and the control material was AC. After germination and sowing under the same conditions, when the plants grew to 2-3 true leaves, they were divided and uniformly cultivated and managed. The changes in the number of true leaves were recorded to evaluate the growth rate and flowering time, and special attention was paid to the time of the first flower bud differentiation.

[0038] The specific results of the above verification experiments can be found in Figures 3-6 ,in: Figure 3 for KNOX7 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.

[0039] Figure 4 for KNOX7 Schematic diagram of target editing. KNOX7 In WT (AC), KNOX7 -CR-8, 10, 12 contain two target sequences of the genomic DNA sequence. Among them, compared with the control material WT, KNOX7 -CR-8 has 1 base insertion at the first target site, KNOX7 -CR-10 has a 1-base insertion at the first target site and a 2-base deletion at the second target site, KNOX7 -CR-12 has a 7-base deletion at the first target site and a 1-base deletion at the second target site.

[0040] Figure 5 For AC and SlKNOX7 Phenotypes of mutant plants. The upper picture shows the control AC and KNOX7 -CR-8, 10, 12 strains, the scale is 10cm; the figure below is the control AC and KNOX7 -CR strain apical meristem enlargement, where the red arrow indicates the first inflorescence and the white arrow indicates the second inflorescence. Compared with the control AC, SlKNOX7 The onset of flower bud differentiation in mutant plants is advanced.

[0041] Figure 6 AC and SlKNOX7 Flowering time statistics of mutant plants. Arrows indicate the time when the first inflorescence appears. The red arrow indicates that AC flowers at 48 days, and the blue arrow indicatesKNOX7 - CR-12 flowering time is 38d, as indicated by the yellow arrow KNOX7 - CR-10 flowering time is 40d, as indicated by the grey arrow KNOX7 - The flowering time of CR-8 is 42 days.

[0042] In summary, compared with the control plants AC, the three mutants KNOX7 -CR-8, KNOX7 -CR-10, KNOX7 - The flowering time of CR-12 was advanced by about 8 days.

[0043] The specific raw materials not described in the present invention are all existing materials and can be directly purchased from the market.

[0044] The above are only preferred embodiments of the present invention and are not intended to limit the protection scope of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. SlKNOX7 The application of the gene in regulating the flowering time of tomatoes is characterized in that: Said SlKNOX7 The sequence of 5000 bp upstream of the start codon of the gene is shown in SEQ ID NO:

1. SlKNOX7 The 1000 bp downstream of the stop codon of the gene 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.

2. SlKNOX7 The use of a gene-encoded protein in regulating the flowering time of tomatoes is characterized in that: Said SlKNOX7 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO:

5.

3. A method for regulating the flowering time of tomatoes, characterized in that: The method is to knock out the gene described in claim 1 using gene editing technology.

4. The method according to claim 3, characterized in that The gene editing technology is performed using the CRISPR / Cas9 system, which includes SlKNOX7 A vector of a gRNA for a gene, the sequence of which is shown in SEQ ID NO:

8.

5. Use of a recombinant vector, expression cassette or engineered bacteria containing the sequence of claim 1 in regulating the flowering time of tomatoes.

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