A method for creating tomato male sterile mutants based on SlFAR3 gene editing

Abstract

The invention provides a method for creating a tomato male sterile mutant based on SlFAR3 gene editing, belonging to the technical field of genetic engineering. A single base C or A is inserted after the 88 bp of the first exon of a wild-type tomato SlFAR3 gene to obtain a tomato male sterile mutant. The nucleotide sequence of the SlFAR3 gene in the tomato male sterile mutant is shown in SEQ ID NO.11 in the sequence table. The invention performs editing of specific sites on the SlFAR3 gene, so that the gene editing rate reaches 87%, and a homozygous tomato male sterile mutant is obtained. Compared with the wild type, the vegetative growth and pistil appearance of the mutant are not significantly different, but the stigma is more prominent, the early stigma is exposed, the anther is thin and light yellow-green, and TTC staining of mature pollen shows that the mutant strain has no pollen grains in the anther during the mature period, and the mutant is a typical pollenless male sterile mutant.

Description

Technical Field

[0001] The invention relates to a method for creating a tomato male sterile mutant based on SlFAR3 gene editing, and belongs to the technical field of genetic engineering. Background Art

[0002] Male sterility is a phenomenon that is widely found in flowering plants in nature. It is the loss of male function caused by abnormal development of male reproductive organs or pollen. Although male sterility is not conducive to the transmission of individual genes, the application of male sterile lines in agricultural production provides great productivity for agricultural production, and therefore has received close attention from scientific researchers.

[0003] As a global vegetable crop, tomato is a typical self-pollinating plant with significant hybrid vigor. Hybrids currently dominate tomato production, but traditional hybrid seed production is limited by artificial emasculation and pollination, which is not only time-consuming, labor-intensive, and costly, but also affects the purity of hybrids. Based on this, creating a completely sterile tomato male sterile line for use in hybrid seed production has production practice value.

[0004] As early as 1945, some scholars had applied male sterile lines to tomato hybrid seed production, such as ms10 , ms15 , ps12 and long style types, but the results were not ideal due to the low pollen sterility rate, the long time of sterile gene introduction and the difficulty in preserving sterile line materials.

[0005] CRISPR-Cas9-based gene editing technology can accurately locate the target gene by designing gRNA and achieve gene editing through cutting by Cas9 enzyme, allowing precise modification of genes at the molecular level. It is simple to construct, low-cost and highly efficient.

[0006] Acyl-CoA reductase (abbreviated as SlFAR3) in tomato is highly enriched in anther tissue, indicating that it is involved in the metabolism and formation of tomato anther and pollen wall. The SlFAR3 gene is a key gene regulating the early anther development of tomato.

[0007] CN117887754A discloses a method and application of creating tomato male sterile material through CRISPR-Cas9 gene editing, which uses pEGCas9Pubi-B vector as a skeleton, adopts a single targeted editing site, and performs SLFAC gene editing on the model tomato Micro-Tom, and finally obtains pollen abortion positive strains, but pollen is still present in the obtained tomato male sterile material. Summary of the invention

[0008] In view of the shortcomings of the prior art, the present invention provides a method for creating a tomato male sterile mutant based on SlFAR3 gene editing, to achieve the following invention objectives: to improve the editing rate of the SlFAR3 gene and to create a typical pollenless male sterile mutant.

[0009] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0010] A method for creating a tomato male sterile mutant based on SlFAR3 gene editing, wherein a single base C or A is inserted after the 88th bp of the first exon of the wild-type tomato SlFAR3 gene to obtain a tomato male sterile mutant; the nucleotide sequence of the SlFAR3 gene in the tomato male sterile mutant is shown in SEQ ID NO.11 in the sequence table.

[0011] The targeting sites for SlFAR3 gene editing include a first targeting site and a second targeting site, the nucleotide sequence of the first targeting site is shown in SEQ ID NO.3 in the sequence list; the nucleotide sequence of the second targeting site is shown in SEQ ID NO.4 in the sequence list.

[0012] The target fragment containing the first targeting site and the second targeting site was amplified and inserted into the PTX041 plasmid to construct a recombinant plasmid. After transformation with Agrobacterium, Agrobacterium was used to infect tomatoes to screen and obtain tomato male sterile mutants.

[0013] Compared with the prior art, the present invention achieves the following beneficial effects:

[0014] The present invention edits the SlFAR3 gene at specific sites, with a gene editing rate of 87%, and obtains a homozygous tomato male sterile mutant. Compared with the wild type, there is no obvious difference in its vegetative growth and pistil appearance, but the stigma is more prominent. Its early stigma is exposed, the anther is thin and light yellow-green, and TTC staining of mature pollen shows that the mutant strain has no pollen grains in the anther during the mature period, which is a typical pollenless male sterile mutant. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 is the structural diagram of the recombinant plasmid;

[0016] Figure 2 Flowchart for obtaining gene-edited plants by Agrobacterium infection of tomato explants;

[0017] A is the state diagram of tomato cotyledons after 2 days of pre-culture; B is the state diagram of tomato explants after 2 days of culture after Agrobacterium infection; C is the state diagram after 2 weeks of resistance screening; D is the state diagram after 2 weeks of culture in 0.2ZR medium; E is the state diagram after 4 weeks of culture in 0.2ZR medium; F is the state diagram after 4 weeks of rooting culture;

[0018] Figure 3 The test result is positive seedlings;

[0019] Wherein P is a band amplified using the recombinant plasmid constructed in step (3) of Example 1 as a template; N is a band amplified using ultrapure water as a template; 1-16 is a band amplified using gDNA of young leaves of 16 tomato seedlings obtained in Example 1 as a template;

[0020] Figure 4 Comparison of the phenotypes and pollen staining results of the wild type and slfar3-1 mutant;

[0021] Among them, (a) is the floral organ of the wild-type AC tomato plant; (b) is the outer side of the anther of the wild-type AC tomato plant; (c) is the inner side of the anther of the wild-type AC tomato plant; (d) is the pollen staining result of the wild-type AC tomato plant; (e) is the floral organ of the slfar3-1 mutant AC tomato plant; (f) is the outer side of the anther of the slfar3-1 mutant AC tomato plant; (g) is the inner side of the anther of the slfar3-1 mutant AC tomato plant; (h) is the pollen staining result of the slfar3-1 mutant AC tomato plant. DETAILED DESCRIPTION

[0022] The version of the tomato genome of the present invention is SL4.0 version (http: / / solgenomics.net / tomato / );

[0023] The nucleotide sequence of the tomato SlFAR3 gene is shown in SEQ ID NO.1 in the sequence listing, and the amino acid sequence is shown in SEQ ID NO.2 in the sequence listing;

[0024] The pCBC_DT1T2_SlU6p plasmid, PTX041 plasmid, and AC (Ailsa Craig) tomato seeds of the present invention were donated by Yang Changxian's research group at the Modern Agricultural Research Institute of Peking University.

[0025] Example 1 A method for creating a tomato male sterile mutant based on SlFAR3 gene editing

[0026] (1) Gene Editing

[0027] First, based on the gene information of SlFAR3, Cas9 target analysis was performed on the cDNA of SlFAR3 on CRISPR RGEN Tools (http: / / www.rgenome.net / ), and two targeting sites were designed. The first targeting site was located at the 73rd to 94th bases from the start codon atg; the second targeting site was located at the 228th to 249th bases from the start codon ATG.

[0028] The nucleotide sequence of the first targeting site is shown in SEQ ID NO.3 in the sequence listing; the nucleotide sequence of the second targeting site is shown in SEQ ID NO.4 in the sequence listing.

[0029] (2) Amplification of target fragment

[0030] Using the nucleotide sequences shown in SEQ ID NO.5 and SEQ ID NO.6 in the sequence table as amplification primers, a target fragment containing double target sites was amplified from the pCBC_DT1T2_SlU6p plasmid by PCR. The target fragment included a first targeting site, a terminator, a tomato U6 promoter, and a second targeting site.

[0031] (3) Construction of recombinant plasmid

[0032] use Bsa I (NEB) digested the PTX041 plasmid (37°C, >3h), and the digested product was detected by 1.0% agarose gel. The PTX041 (18K) large fragment was recovered by gel cutting using a recovery kit. The PTX041 (18K) large fragment was ligated with the target fragment containing dual target sites obtained in step (2) by homologous recombination to obtain a ligation product. The ligation product was transformed into Escherichia coli by heat shock method. The transformed colonies were subjected to PCR detection. The forward primer used for detection was PTX-FW (the nucleotide sequence is shown in SEQ ID NO.7 in the sequence table), and the rear primer was PTX-RV (the nucleotide sequence is shown in SEQ ID NO.8 in the sequence table). The PCR product was detected by 1.0% agarose gel. The size of the empty fragment was 1820bp, while the size of the recombinant vector with the target fragment was 1169bp. The positive plaque was selected for sequencing of the target site and its surrounding sequences. After the sequencing was correct, a positive single colony was selected for culture, and the recombinant plasmid was extracted by small-volume method.

[0033] The structure of the recombinant plasmid is as follows Figure 1 shown.

[0034] (4) Transformation of Agrobacterium

[0035] The obtained recombinant plasmid was transformed into Agrobacterium GV3101 by chemical transformation, and then screened with LB solid plate culture medium containing rifampicin with a final concentration of 100 mg / L and kanamycin with 50 mg / L, and positive clones were selected. The clones were shaken and cultured overnight at 28°C and 200 r / min. 1 μL of the Agrobacterium culture solution was taken as a template, and PCR detection was performed with vector primers PTX-FW (the nucleotide sequence is shown in SEQID NO.7 in the sequence table) and PTX-RV (the nucleotide sequence is shown in SEQID NO.8 in the sequence table). The amplified fragment size of 1169 bp was in line with expectations, and the clone was a positive Agrobacterium.

[0036] (5) Agrobacterium infection of tomatoes

[0037] A. Pre-cultivation

[0038] After disinfecting AC tomato seeds with sodium hypochlorite (2% available chlorine) for 15 minutes, sow them on 1 / 2MS medium (pH = 5.8), culture them at 25 ± 2 ° C in the dark until the seeds germinate, and then culture them under a photoperiod of 16 h light / 8 h dark (with a light intensity of 1800 lx). Cut the cotyledons (area 0.5 cm) of sterile seedlings at 7-8 days old. 2 ), and cultured in the pre-culture medium MS0 for 2 days. After 2 days of pre-culture, the explants were Figure 2 As shown in A.

[0039] The pre-culture medium MS0 is MS culture medium supplemented with ZR (zeatin riboside) at a final concentration of 2 mg / L and IAA (indole-3-acetic acid) at a pH of 5.8.

[0040] B. Infection

[0041] Take 10uL of the positive Agrobacterium bacterial solution, add it to 20mL of LB liquid medium containing rifampicin at a final concentration of 100mg / L and kanamycin at 50mg / L, place it in a 50mL centrifuge tube, and culture it overnight at 28℃ and 200r / min. After centrifugation, resuspend the Agrobacterium in MS liquid medium to OD600=0.5, and then infect the explants pre-cultured for 2d for 4min. Use sterile filter paper to absorb the excess bacterial solution on the explants, and put them back on the pre-culture medium MS0. Infect and culture for 2d under dark conditions. The explants after infection and culture for 2d are as follows Figure 2 As shown in B.

[0042] C. Resistance screening

[0043] After 2 days of infection and culture, the explants were transferred to the screening medium 1.0ZR for resistance screening, and subcultured every two weeks. The status after 2 weeks of resistance screening culture was as follows: Figure 2 As shown in C.

[0044] The screening medium 1.0ZR is a basic medium containing MS supplemented with ZR at a final concentration of 1.0 mg / L, 400 mg / L Cef (cephalosporin) and 100 mg / L kanamycin, with a pH of 5.8.

[0045] After the resistant buds appeared, the callus with buds was transferred to 0.2ZR medium. After 2 weeks of culture, the state was as follows: Figure 2 As shown in D, the status after 4 weeks of culture is as follows Figure 2 As shown in E.

[0046] The 0.2ZR medium is MS medium, to which ZR with a final concentration of 0.2 mg / L, IAA of 0.5 mg / L, cephalosporin of 200 mg / L, and kanamycin of 100 mg / L are added, and the pH value is 5.8.

[0047] D. Rooting culture

[0048] After culturing in 0.2ZR medium for 4 weeks, the resistant buds were cut from the callus and inserted into rooting medium RM to induce rooting. Figure 2 As shown in F, 16 tomato seedlings were finally obtained.

[0049] The rooting medium RM is MS medium, to which IAA with a final concentration of 0.1 mg / L, cephalosporin with a final concentration of 200 mg / L, and kanamycin with a pH of 5.8 is added.

[0050] Example 2 Detection and identification of tomato male sterile mutants

[0051] (1) Positive test

[0052] The gDNA of young tomato leaves was extracted by CTAB method. The gDNA of young tomato leaves was used as template, PTX-FW (nucleotide sequence is shown in SEQ ID NO.7 in the sequence list) was used as the front primer, and PTX-RV (nucleotide sequence is shown in SEQ ID NO.8 in the sequence list) was used as the back primer for amplification. The PCR results are shown in Figure 3 The results showed that all 16 plants had a band of 1169 bp, indicating that they were all positive seedlings.

[0053] (2) Gene editing detection

[0054] Using gDNA of tomato positive seedlings as template, the detection primers are as follows:

[0055] SeqFAR3-F2, the nucleotide sequence is shown in SEQ ID NO.9 in the sequence listing;

[0056] SeqFAR3-R2, the nucleotide sequence is shown in SEQ ID NO.10 in the sequence listing;

[0057] The PCR products were detected by 1% agarose gel and sent for sequencing. Sequencing comparison revealed that among the 16 positive seedlings, 14 plants had SlFAR3 gene sequences edited, with an editing rate of 87%. Two of them were detected as homozygous mutants, named slfar3-1 and slfar3-2. Slfar3-1 had a single base insertion of C or A after the 88th bp of the first exon of the SlFAR3 gene; the nucleotide sequence of the SlFAR3 gene in slfar3-1 is shown in SEQ ID NO.11 in the sequence table.

[0058] The mutation site of slfar3-2 occurs at the 5'UTR end, and the phenotype of slfar3-2 has no obvious change compared with the wild type.

[0059] (3) Phenotypic identification of gene-edited plants

[0060] Homozygous edited plants were further compared with wild-type AC.

[0061] The results show:

[0062] The slfar3-2 mutant showed no significant phenotype change compared with the wild type;

[0063] The slfar3-1 mutant has no obvious phenotypic defects compared with the wild type during the vegetative growth period, and there is no obvious difference in the pistil appearance during the reproductive growth period ( Figure 4 (b), (f)); however, compared with the wild type, the slfar3-1 mutant has a more prominent stigma, and its early stigma is exposed ( Figure 4 (c), (g)); Further observation revealed that compared with the wild type, the anthers of the slfar3-1 mutant were thin and pale yellow-green ( Figure 4 (a), (e)).

[0064] (4) TTC staining experiment of pollen

[0065] The pollen fertility of the wild type and slfar3-1 mutant was analyzed by TTC staining experiment. The experimental results showed that the wild type pollen was regular round and dark red, and its pollen vitality was strong; while the slfar3-1 mutant had no pollen grains in its anthers, which was a typical pollenless male sterile mutant ( Figure 4 (d), (h)).

Claims

1. A method for creating a tomato male sterile mutant based on SlFAR3 gene editing, characterized in that: A single base C or A is inserted after the 88th bp of the first exon of the wild-type tomato SlFAR3 gene to obtain a tomato male sterile mutant; the nucleotide sequence of the SlFAR3 gene in the tomato male sterile mutant is shown in SEQ ID NO.11 in the sequence table.

2. The method for creating a tomato male sterile mutant based on S1FAR3 gene editing according to claim 1, characterized in that: The targeting sites for SlFAR3 gene editing include a first targeting site and a second targeting site, the nucleotide sequence of the first targeting site is shown in SEQ ID NO.3 in the sequence list; the nucleotide sequence of the second targeting site is shown in SEQ ID NO.4 in the sequence list.

3. The method for creating a tomato male sterile mutant based on S1FAR3 gene editing according to claim 2, characterized in that: The target fragment containing the first targeting site and the second targeting site was amplified and inserted into the PTX041 plasmid to construct a recombinant plasmid. After transformation with Agrobacterium, Agrobacterium was used to infect tomatoes to screen and obtain tomato male sterile mutants.

Images