CN112646819A - Use of gene to enhance resistance to tomato gray mold - Google Patents

Abstract

The invention relates to an application of a gene for increasing botrytis cinerea resistance of tomatoes, belonging to the field of crop disease-resistant breeding. The invention discloses application of a tomato gene Solyc05g004600, and the gene Solyc05g004600 is knocked out in a tomato, so that the resistance of tomato leaves to botrytis cinerea can be effectively improved. The invention also discloses a method for knocking out the mutant gene Solyc05g004600 in the tomato.

Description

Use of gene to enhance resistance to tomato gray mold

Technical Field

The invention relates to an application of a gene for increasing botrytis cinerea resistance of tomatoes, belonging to the field of crop disease-resistant breeding.

Background

Tomatoes are important vegetable and fruit crops in the world due to their high edible, medicinal and economic values. Botrytis is a common and serious fungal disease in tomatoes, and stems, leaves, flowers and fruits of plants can catch disease. Often the pathogen invades from the wound or aged tissue of the plant and gradually spreads to other healthy parts, causing a huge impact on the yield and quality of the tomato. The disease resistance gene is the most economic and effective method for preventing and treating crop diseases. In recent years, more and more tomato disease resistance related genes are excavated, for example, Yu et al (2018) find that an ethylene response factor SlERF2 participates in the defense regulation of tomatoes on gray mold, and overexpression SlERF2 can enhance the resistance of tomato fruits to gray mold; sun et al (2017) silenced DND1 gene in tomato, and found that the diameter of botrytis cinerea spot is significantly reduced, the number of conidia of botrytis cinerea on leaves is significantly reduced, and hypha growth is inhibited in the silenced plants compared with control plants. Therefore, by utilizing the gene editing technology, more plant disease-resistant related genes are excavated, and the method is an effective means for improving the crop resistance and reducing the economic loss.

Disclosure of Invention

The invention aims to solve the technical problem of how to effectively improve the resistance of tomatoes to gray mold.

In order to solve the technical problem, the invention provides a tomato gene Solyc05g004600, and the nucleotide sequence of the gene Solyc05g004600 is shown as SEQ ID NO. 1.

The invention also provides the application of the gene: the Solyc05g004600 gene is knocked out from the tomato, so that the resistance of tomato leaves to gray mold can be effectively increased.

Improvement as an application of the tomato gene Solyc05g004600 of the invention:

the nucleotide sequence of the Solyc05g004600 gene of mu-1 is shown in SEQ ID NO:2,

the nucleotide sequence of the Solyc05g004600 gene of mu-2 is shown in SEQ ID NO 3.

The invention also provides a method for knocking out mutant gene Solyc05g004600 in tomato, which comprises the following steps:

1) designing a target sequence sgRNA edited by CRISPR/Cas 9:

5'-GTTGTTCAACATGAGCAGAG-3’；

2) synthesizing a primer by using the sequence obtained in the step 1), and constructing the primer into a CRISPR/Cas9 vector;

3) transforming tomato (wild type tomato variety MicroTom) with the vector obtained in step 2), thereby obtaining corresponding transgenic tomato plants (plants with Solyc05g004600 gene mutation identified from the transgenic tomato plants): mu-1 and mu-2; the transgenic tomato plant is a tomato plant with a Solyc05g004600 gene knocked out.

Although the sequence of the gene Solyc05g004600 is registered in a database, no research report on related functions and effects of the gene exists at present, so that the gene is found to have the function of resisting botrytis cinerea for the first time.

The technical scheme of the invention is as follows:

a CRISPR/Cas9 gene editing technology is adopted, a sgRNA sequence of a specific target Solyc05g004600 gene is synthesized according to a nucleotide sequence (SEQ ID NO:1) of the Solyc05g004600 gene, a corresponding CRISPR/Cas9 vector is constructed and is genetically transformed into a wild tomato variety MicroTom, the Solyc05g004600 gene in a genome is directionally edited to obtain transgenic plants, PCR amplification and sequencing are carried out on the Solyc05g004600 gene in the transgenic plants, and 2 different types of mutant strains mu-1 and mu-2 of the Solyc05g004600 gene are identified and obtained (figure 1). The mutant sequence of the Solyc05g004600 gene in mu-1 plant is SEQ ID NO. 2; the mutant sequence of the Solyc05g004600 gene in mu-2 plants is SEQ ID NO 3. After the botrytis cinerea of the tomato is inoculated, a wild type control variety MicroTom is infected, disease resistance of two lines with mutation of the Solyc05g004600 gene is improved (figure 2), the scab area of the leaves of the two lines is lower than that of the control variety MicroTom (figure 3), and the fact that the resistance of the tomato to the botrytis cinerea can be effectively improved by the mutation of the Solyc05g004600 gene in the tomato is shown.

Drawings

The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.

FIG. 1 is CRISPR/Cas9 target site sequencing analysis of tomato Solyc05g004600 gene mutant;

MicroTom: wild type control variety; mu-1 and mu-2: 2 different types of mutant strains of the Solyc05g004600 gene.

FIG. 2 is a graph comparing the phenotype of tomato Solyc05g004600 gene mutant plants and wild type control MicroTom leaf ashbya.

FIG. 3 shows the statistics of tomato Solyc05g004600 gene mutation plants and wild type control MicroTom leaf spots;

the values in figure 3 are mean ± sd, indicating that there is a very significant (P <0.01) difference in t-test in tomato solany 05g004600 gene mutant plants compared to wild type control MicroTom.

Detailed Description

The invention will be further described with reference to specific examples, but the scope of the invention is not limited thereto:

example 1:

step 1, construction of Solyc05g004600 gene knockout vector

According to the coding sequence of the Solyc05g004600 gene (SEQ ID NO:1), a sgRNA sequence edited by CRISPR/Cas9 is designed in the Solyc05g004600 gene by using CRISPR/Cas9 target analysis software (http:// CRISPR. mit. edu /): 5'-GTTGTTCAACATGAGCAGAG-3', respectively; and synthesizing corresponding primers according to the sequence:

upstream of the flow path 5'-TGATTGTTGTTCAACATGAGCAGAG-3', it is preferred that,

downstream 5'-AAACCTCTGCTCATGTTGAACAACA-3'.

Then, a CRISPR/Cas9 kit (Biogle, China) is adopted to construct a corresponding CRISPR/Cas9 vector, and the construction method is operated according to the product instruction.

Step 2, tomato genetic transformation of Solyc05g004600 gene knockout vector

And (2) genetically transforming the CRISPR/Cas9 vector constructed in the step (1) into a tomato variety MicroTom, and obtaining a series of corresponding unidentified transgenic tomato plants by a transgenic method according to a Kimura et al method (Kimura S et al, CHS Protoc, 2008).

Step 3, identifying Solyc05g004600 gene knockout plants

Grinding 0.1g of tomato leaf with liquid nitrogen, adding 600 μ L of extractive solution (15.76g of Tris-cl, 29.22g of NaCl, 15.0g of SDS powder, adding ultrapure water to constant volume to 1L, adjusting pH to 8.0), and incubating at 65 deg.C for 60 min; adding 200 μ l 5M KAC, mixing, and ice-cooling for 10 min; adding 500 μ l chloroform, mixing, centrifuging at 10000rpm for 5 min; taking the supernatant, adding 500 mul of isopropanol, mixing uniformly, centrifuging at 12000rpm for 3min, and discarding the supernatant; washing the precipitate with 75% ethanol, centrifuging at 12000rpm for 3min, and discarding the supernatant; after the DNA was air-dried at room temperature by inversion, 30. mu.l of pure water was added to dissolve the DNA.

Synthesizing primers for PCR amplification of Solyc05g004600 gene: upstream 5'-GCCTCTATTAATGACATC-3' and downstream 5'-ATAAGCTAAAATCGACTA-3', using transgenic tomato plant and its control variety MicroTom genome DNA as template

Performing PCR amplification by a Master Mix (Promega, USA), wherein a PCR amplification system is operated according to the instruction of a product;

the PCR amplification procedure was: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 30sec, annealing at 55 ℃ for 30sec, extension at 72 ℃ for 30sec, 35 cycles; extension at 72 ℃ for 10 min.

The PCR amplification system is as follows: 2 XMix 10. mu.l, forward primer 0.5. mu.l, reverse primer 0.5. mu.l, template 2. mu.l, ddH₂O 7μl。

After sequencing analysis of the PCR products, 2 mutation types of the Solyc05g004600 gene are successfully identified: mu-1 and mu-2. In mu-1 plants, 1 base is inserted into the coding region of Solyc05g004600 gene (figure 1), and the nucleotide sequence is SEQ ID NO: 2; in mu-2 plants, the Solyc05g004600 gene coding region lacks 3 bases (FIG. 1), and the nucleotide sequence is SEQ ID NO. 3.

Step 4, identifying botrytis cinerea resistance of Solyc05g004600 gene knockout plants

The Solyc05g004600 gene mutant strains mu-1 and mu-2 identified above and a control variety MicroTom were planted in a greenhouse at 25 ℃ for 16h in light and 8h in dark. After the seedlings grow for about 45 days, 3 strains of mu-1, mu-2 and the reference variety are randomly selected, and 4 leaves of each strain are fixed on a 0.8% agar (8g of agar powder and ultrapure water are added to the agar powder to reach a constant volume of 1L) culture dish. Selecting Botrytis cinerea with a volume of about 2ml, adding into 5ml of 1% SMB (10g of mould peptone, 40g of maltose and ultrapure water to a constant volume of 1L) solution, shaking, mixing, and filtering to obtain filtrate (spore stock solution). The spore stock solution concentration was calculated using a 25 × 16 format blood cell counting plate, diluted with 1% SMB solution until 25 middle compartments, each containing approximately 16 spores, were inoculated, diluted 4-fold again, and the spore suspension was obtained after dilution. Using a 1% SMB solution without spores as a control, 5. mu.l of spore suspension was added dropwise to each leaf, and placed in an environment suitable for tomato growth with a lid. After 2 days, the inoculated leaf was harvested and the lesion area was calculated using Image J software. The result is that the pathogenic degree of the leaves of the Solyc05g004600 gene mutant strains mu-1 and mu-2 is lower than that of the control strain (figure 2), and the lesion area of the leaves is obviously smaller than that of the control strain (figure 3).

According to the above, it can be proved that: the leaves of tomato plants mu-1 and mu-2 with the Solyc05g004600 gene knocked out effectively increase the resistance to botrytis cinerea.

Finally, it is also noted that the above-mentioned lists merely illustrate a few specific embodiments of the invention. It is obvious that the invention is not limited to the above embodiments, but that many variations are possible. All modifications which can be derived or suggested by a person skilled in the art from the disclosure of the present invention are to be considered within the scope of the invention.

Sequence listing

<110> university of chessman in Zhejiang

Application of <120> gene in enhancing resistance to tomato gray mold

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 330

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgggtgatt cttcggctca atacatccac ttggtgcatc gcttgatcga agagtgttta 60

ttgttcaaca tgagcagaga ggaatgcatg gaagcacttt ccaaacatgc aaatattcag 120

ccagttatca catccacagt gtggaaagag ttggagaagg agaacaaaga gtttttcaag 180

tcatacaaga aaaaagacga ggatgaagaa tcaacatcaa tcaataataa caataataat 240

aataatatta tagaatccaa agtggagata acaagacaaa gaatccacaa tttgctattg 300

aattcccaac tctctaaata caaaccatag 330

<210> 2

<211> 331

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggtgatt cttcggctca atacatccac ttggtgcatc gcttgatcga agagtgttta 60

ttgttcaaca tgagcaagag aggaatgcat ggaagcactt tccaaacatg caaatattca 120

gccagttatc acatccacag tgtggaaaga gttggagaag gagaacaaag agtttttcaa 180

gtcatacaag aaaaaagacg aggatgaaga atcaacatca atcaataata acaataataa 240

taataatatt atagaatcca aagtggagat aacaagacaa agaatccaca atttgctatt 300

gaattcccaa ctctctaaat acaaaccata g 331

<210> 3

<211> 327

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

atgggtgatt cttcggctca atacatccac ttggtgcatc gcttgatcga agagtgttta 60

ttgttcaaca tgagagagga atgcatggaa gcactttcca aacatgcaaa tattcagcca 120

gttatcacat ccacagtgtg gaaagagttg gagaaggaga acaaagagtt tttcaagtca 180

tacaagaaaa aagacgagga tgaagaatca acatcaatca ataataacaa taataataat 240

aatattatag aatccaaagt ggagataaca agacaaagaa tccacaattt gctattgaat 300

tcccaactct ctaaatacaa accatag 327

Claims (4)

1. Tomato gene Solyc05g004600, characterized by: the nucleotide sequence of the gene is shown as SEQ ID NO:1 is shown.

2. The use of the tomato gene Solyc05g004600 according to claim 1, which is characterized in that: the gene Solyc05g004600 is knocked out in tomato, so that the resistance of tomato leaves to botrytis cinerea can be effectively increased.

3. The use of the tomato gene Solyc05g004600 according to claim 2, characterized in that:

the nucleotide sequence of the Solyc05g004600 gene of mu-1 is shown in SEQ ID NO:2,

the nucleotide sequence of the Solyc05g004600 gene of mu-2 is shown in SEQ ID NO 3.

4. The method for knocking out mutant gene Solyc05g004600 in tomato is characterized by comprising the following steps:

1) designing a target sequence sgRNA edited by CRISPR/Cas 9:

5'-GTTGTTCAACATGAGCAGAG-3’；

2) synthesizing a primer by using the sequence obtained in the step 1), and constructing the primer into a CRISPR/Cas9 vector;

3) transforming the tomato with the vector obtained in the step 2), thereby obtaining a corresponding transgenic tomato plant: mu-1 and mu-2; the transgenic tomato plant is a tomato plant with a Solyc05g004600 gene knocked out.

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