JP7383136B2 - Use of genes to improve tomato Botrytis blight resistance - Google Patents

Description

The present invention relates to the use of genes to improve tomato Botrytis blight resistance and belongs to the field of crop disease resistance breeding.

Tomatoes are an important fruit crop in the world due to their high edible, medicinal and economic value. Botrytis is a common and serious fungal disease of tomatoes that can affect the stems, leaves, flowers, and fruits of the plant. Usually, the pathogen enters the plant through wounds or aging tissue and gradually spreads to other healthy parts of the plant, severely impacting tomato yield and quality. Using disease resistance genes to control crop diseases is the most economical and effective method. In recent years, more and more genes related to disease resistance in tomato have been discovered. For example, Yu et al. (2018) (Yu WQ, Zhao RR, Sheng JP, et al. SlERF2 is associated with Methyl Jasmonate-mediated defense response again t Botrytis cinerea in tomato fruit. J Agric Food Chem, 2018, 66(38): 9923 -9932.) found that the ethylene response factor SlERF2 is involved in regulating the defense of tomato against botrytis, and overexpression of SlERF2 improves the resistance to botrytis in tomato fruits; Sun et al. (2017) ) (Sun K, Tuinen A, van Kan JA, et al. Silencing of DND1 in potato and tomato impedes conidial germination, attachment and hyphal growth of Botrytis cinerea. BMC Plant Biol, 2017, 17(1): 235.) Silencing of the DND1 gene in tomato resulted in the silenced plants having a significantly reduced diameter of Botrytis blight lesions and a significant reduction in the number of Botrytis conidia on leaves, compared to control plants; It was discovered that hyphal growth was suppressed. This shows that the use of gene editing technology to discover more genes related to disease resistance in plants is an effective means for improving crop resistance and reducing economic losses. .

The problem to be solved by the present invention is how to effectively improve tomato Botrytis blight resistance.

In order to solve the above problems, the present invention provides a tomato gene Solyc05g004600 whose nucleotide sequence is represented by SEQ ID NO: 1.

The present invention also provides the use of the Solyc05g004600 gene in tomato to effectively improve botrytis leaf mold resistance by knocking out the gene, and the nucleotide sequence of the mu-1 Solyc05g004600 gene as shown in SEQ ID NO: 2 and the nucleotide sequence of the Solyc05g004600 gene of mu-2 is shown in SEQ ID NO:3.

Furthermore, the present invention provides a method for knocking out the gene Solyc05g004600 in tomato, comprising the following steps.
1), designing the target sequence sgRNA: 5'-GTTGTTCAACATGAGCAGAG-3' in CRISPR/Cas9 editing;
2), a step of synthesizing primers with the sequence obtained in step 1) and constructing a CRISPR/Cas9 vector;
3) Transform tomatoes (wild type tomato cultivar MicroTom) with the vector obtained in step 2) to generate transgenic tomato plants mu-1 and mu-2, which are tomato plants in which the corresponding Solyc05g004600 gene has been knocked out. (Identification of plants in which the Solyc05g004600 gene mutation has occurred from the transgenic tomato plants).

Although the sequence of the gene Solyc05g004600 has already been registered in the database, there are currently no studies or reports on the function or action of the gene, so it is unlikely that the gene is related to the resistance of tomato to Botrytis. This is the first discovery made by the present invention.

The technical solution of the present invention is as follows.
Using CRISPR/Cas9 gene editing technology, an sgRNA sequence that specifically targets the Solyc05g004600 gene was synthesized based on the nucleotide sequence of the Solyc05g004600 gene (SEQ ID NO: 1), a corresponding CRISPR/Cas9 vector was constructed, and a wild-type tomato variety MicroTom was genetically transformed, the Solyc05g004600 gene in the genome was directionally edited, transgenic plants were obtained, and the Solyc05g004600 gene in these transgenic plants was subjected to PCR amplification and sequencing, and as a result, Solyc05g004600 was identified. Mutant strains mu-1 and mu-2 (Fig. 1) with two different types of genes were obtained. The mutant sequence of the Solyc05g004600 gene in mu-1 plants is SEQ ID NO: 2, and the mutant sequence of the Solyc05g004600 gene in mu-2 plants is SEQ ID NO: 3. After inoculation with Botrytis tomato fungus, the wild-type control cultivar MicroTom developed disease, whereas the two strains with mutations in the Solyc05g004600 gene both had improved disease resistance (Fig. 2) and lesions on leaves. The area of MicroTom is significantly smaller than that of the control variety MicroTom (Figure 3). It has been revealed that mutation of the Solyc05g004600 gene in tomato can effectively improve Botrytis blight resistance in tomato.

Hereinafter, specific embodiments of the present invention will be described in further detail with reference to the drawings.
Sequence analysis of CRISPR/Cas9 target site of tomato Solyc05g004600 gene mutant. MicroTom: wild-type control variety; mu-1 and mu-2: two different types of mutant strains of the Solyc05g004600 gene. It is a comparison diagram of the phenotypes of leaves inoculated with Botrytis mold fungus between a tomato Solyc05g004600 gene mutant plant and a wild-type control MicroTom. Fig. 2 shows statistical results of lesions on leaves of tomato Solyc05g004600 gene mutant plants and wild type control MicroTom. In Figure 3, the values are mean ± standard deviation, and ** indicates a significant difference determined by t-test in the comparison between tomato Solyc05g004600 gene mutant plants and wild-type control MicroTom (P<0.01). Show that.

Hereinafter, the present invention will be further explained with reference to specific examples, but the protection scope of the present invention is not limited thereto.

Example 1:
Step 1, Construction of Solyc05g004600 gene knockout vector Based on the coding sequence of Solyc05g004600 gene (SEQ ID NO: 1), CRISPR/Cas9 target site analysis software (http://crispr.mit.edu/) is used to perform CRISPR/Cas9 on Solyc05g004600 gene. The sgRNA sequence for Cas9 editing: 5'-GTTGTTCAACATGAGCAGAG-3' was designed, and corresponding primers were synthesized based on the sequence.

upstream 5'-TGATTGTTGTTCAACATGAGCAGAG-3',
Downstream 5'-AAACCTCTGCTCATGTTGAACAACA-3'.

Then, the corresponding CRISPR/Cas9 vector was constructed with a CRISPR/Cas9 kit (Biogle, China). The construction method was performed according to the product instructions.

Step 2: Genetic transformation of Solyc05g004600 gene knockout vector into tomato Tomato cultivar MicroTom was genetically transformed with the CRISPR/Cas9 vector constructed in Step 1. Regarding the transgenic method, a corresponding series of unidentified transgenic tomato plants were obtained by the method of Kimura et al. (Kimura et al, CHS Protoc, 2008).

Step 3, Identification of Solyc05g004600 gene knockout plants Take 0.1g of tomato leaves, crush them with liquid nitrogen, and add the extracts (Tris-cl 15.76g, Nacl 29.22g, SDS powder 15.0g to ultrapure water. 600 μl of the solution was added and the volume was adjusted to 1 L, and the pH was adjusted to 8.0), and the mixture was incubated at 65° C. for 60 minutes. After adding 200 μl of 5M KAC and mixing uniformly, the mixture was cooled in an ice bath for 10 minutes. Furthermore, 500 μl of chloroform was added, mixed uniformly, and centrifuged at 10,000 rpm for 5 minutes. The supernatant was taken, 500 μl of isopropanol was added, mixed uniformly, centrifuged at 12,000 rpm for 3 minutes, and the supernatant was discarded. The precipitate was washed with 75% ethanol, centrifuged at 12,000 rpm for 3 minutes, and the supernatant was discarded. After the DNA was left upside down at room temperature and air-dried, 30 μl of pure water was added to dissolve the DNA.

Primers for PCR amplification of the Solyc05g004600 gene (upstream 5'-GCCTCTATTAATGACATC-3', downstream 5'-ATAAGCTAAATCGACTA-3') were synthesized, the genomic DNA of the transgenic tomato plant and its control variety MicroTom were used as templates, and GoTaq (registered trademark) was used. ) PCR amplification was performed using Master Mix (Promega, USA). The PCR amplification system was operated according to the product instructions.

In the PCR amplification program, an initial denaturation of 5 min at 94°C; 35 cycles of 30 s denaturation at 94°C, 30 s annealing at 55°C, and 30 s extension at 72°C; 10 s at 72°C; Extend by a minute.

The PCR amplification system consists of 10 μl of 2×Mix, 0.5 μl of upstream primer, 0.5 μl of downstream primer, 2 μl of template, and 7 μl of ddH ₂ O.

When sequence analysis was performed on the PCR product, two types of mutations, mu-1 and mu-2, in the Solyc05g004600 gene were successfully identified. In mu-1 plants, one base was inserted into the coding region of the Solyc05g004600 gene (Figure 1), and its nucleotide sequence is SEQ ID NO: 2. In mu-2 plants, three bases in the coding region of the Solyc05g004600 gene are deleted (Fig. 1), and the nucleotide sequence thereof is SEQ ID NO: 3.

Step 4. Identification of resistance of Solyc05g004600 gene knockout plants to gray mold fungus The Solyc05g004600 gene mutant strains mu-1, mu-2 and control variety MicroTom identified above were planted in a greenhouse, irradiated with light for 16 hours at 25°C, Cultured in the dark for hours. After about 45 days of growth, randomly select 3 plants from mu-1, mu-2 and the control variety, take 4 leaves per plant and soak them in 0.8% agar (more than 8g agar powder). (added to pure water to make a constant volume of 1 L) and fixed in a petri dish. Select a botrytis fungus with a volume of approximately 2 ml, put it in 5 ml of 1% SMB (10 g peptone for mold, 40 g maltose added to ultrapure water to make a constant volume of 1 L) solution, shake evenly, and filter. As a result, a spore stock solution was obtained as a filtrate. Calculate the concentration of the spore stock solution with a 25 × 16 standard hemocytometer, dilute it with 1% SMB solution so that there are approximately 16 spores in each medium block out of 25 medium blocks, and add an additional 4 spores at the time of inoculation. After dilution, a spore suspension was obtained. Using a 1% SMB solution without spores as a control, 5 μl of the spore suspension was dropped onto each leaf, covered with a lid, and placed in an environment suitable for tomato growth. Two days later, the leaves inoculated with the fungus were picked, and the area of the lesion was calculated using Image J software. The results showed that the Solyc05g004600 gene mutant strains mu-1 and mu-2 had a lower degree of disease onset on the leaves (Figure 2) and a significantly smaller area of lesions on the leaves (Figure 3) than the control variety. It was done.

As a result of the above, it was proved that the tomato plants mu-1 and mu-2 in which the Solyc05g004600 gene was knocked out had effectively improved leaf resistance against gray mold fungus.

It should be noted that what has been listed above are just some specific embodiments of the present invention. It is clear that the invention is not limited to the embodiments described above, but that many variations are possible. Any modification that a person skilled in the art directly derives or comes up with based on the disclosure of the present invention falls within the protection scope of the present invention.

Claims (4)

A method for improving the resistance of tomato leaves against botrytis fungus by knocking out the gene Solyc05g004600 whose nucleotide sequence is shown by SEQ ID NO: 1 in tomato. The method according to claim 1, wherein the nucleotide sequence of the Solyc05g004600 gene after the knockout is shown by SEQ ID NO: 2. Resistance to gray mold fungus on tomato leaves is obtained by modifying the gene Solyc05g004600 whose nucleotide sequence is shown in SEQ ID NO: 1 in tomato, and the nucleotide sequence of the modified Solyc05g004600 gene is shown as SEQ ID NO: 3. How to improve. Method according to any one of claims 1 to 3 , characterized in that it comprises the following steps.
1) Step of designing sgRNA:5'-GTTGTTCAACATGAGCAGAG-3' in CRISPR/Cas9 editing,
2) Synthesizing primers using the sgRNA sequence obtained in step 1) and constructing a CRISPR/Cas9 vector using the synthesized primers,
3) Transforming tomatoes with the vector obtained in step 2) to obtain a tomato mutant in which the Solyc05g004600 gene is knocked out or modified .

Images