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Application of slbbx20 Gene in Regulating Tomato Botrytis Botrytis Resistance

A technology for tomato gray mold and gray mold, which is applied in the field of genetic engineering, can solve the problems of affecting food quality, environmental hazards, and the effect is not very good, and achieves the improvement of gray mold resistance and reduction of gray mold resistance. Effect

Active Publication Date: 2022-05-27
HUAZHONG AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For example, the selection of seedlings without disease beds, high-temperature stuffy sheds, reasonable fertilization, strengthening of cultivation management, implementation of crop rotation and ecological control and other control methods have certain effects, but affected by many factors, the effect is not very good, and the production is mainly Focus on chemical control
But chemical control not only affects the quality of our food, but also harms the environment, so it is especially important to breed disease-resistant varieties

Method used

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  • Application of slbbx20 Gene in Regulating Tomato Botrytis Botrytis Resistance
  • Application of slbbx20 Gene in Regulating Tomato Botrytis Botrytis Resistance
  • Application of slbbx20 Gene in Regulating Tomato Botrytis Botrytis Resistance

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Inducible expression profile of SlBBX20 gene in Botrytis cinerea resistant materials

[0042] Botrytis resistant material TS179 (variety selected from TGRC, American Tomato Genetic Resources Center) and botrytis susceptible material TS100 (variety selected from TGRC, American Tomato Genetic Resources Center) were sown, and the seedlings grown for 4 weeks were inoculated with Botrytis cinerea experiment. Take pictures to observe the disease-resistant material TS179 (see Figure 5 ) and susceptible material TS100 (see Image 6 ) incidence of Botrytis cinerea 72h inoculation. In the resistant material TS179, the lesions were smaller, but the lesions spread greatly after the susceptible material TS100 was inoculated with Botrytis cinerea for 72 hours. Samples were taken at different time points (0h, 24h, 48h and 72h) of inoculation of Botrytis cinerea, RNA was extracted, reverse transcribed into cDNA, and real-time quantitative PCR was performed to detect the presence of...

Embodiment 2

[0050] Preparation of SlBBX20 gene knockout vector

[0051] The DNA sequence of the SlBBX20 gene was searched on the SGN (https: / / www.sgn.cornell.edu / search / locus) website, and the DNA sequence of the gene was entered into the CRISPR design website (http: / / crispr.hzau.edu .cn / cgi-bin / CRISPR2 / CRISPR), select two suitable targets according to the off-target rate, etc.: including the first target fragment of the SlBBX20 gene and the second target fragment of the SlBBX20 gene, the first target fragment of the SlBBX20 gene The sequence is shown in SEQ ID NO.3, and the sequence of the second target fragment of the SlBBX20 gene is shown in SEQ ID NO.4.

[0052] Gene synthesis contains primers of two targets, the primer sequence of the first target fragment of the SlBBX20 gene is as shown in SEQ ID NO.5, and the primer sequence of the second target fragment of the SlBBX20 gene is as shown in SEQ ID NO.6 .

[0053] Use synthetic primers containing the target sequence, with the pCBC_D...

Embodiment 3

[0061] Vector preparation with ORF gene fragment of SlBBX20

[0062] The total RNA in tomato was extracted with Trizol (Vazyme, China) reagent, and then the cDNA of tomato gene (sequence shown in SEQ ID NO. Primer, the front primer sequence is shown in SEQ ID NO.7, the rear primer sequence is shown in SEQ ID NO.8, and the ORF (open reading frame) of the SlBBX20 gene is obtained by PCR amplification, and the sequence is shown in SEQ ID NO.2 , detected by 1.0% agarose gel, and recovered the target fragment with a recovery kit (OMEGA, USA).

[0063] The reverse transcription system is as follows:

(1)

[0064] RNase free ddH 2 O to 16μL

[0065] 4*gDNAwiperMix 4μL

[0066] Template RNA 1μg

[0067] Gently mix with a pipette, 42 °C for 2 min.

(2)

[0068] Directly add 4 μL of 5*HiScript II Qrt SuperMix II to the reaction tube in the first step, mix by gently pipetting, 50°C for 15min, 85°C for 5s. cDNA can be obtained after this procedure, and the product can be directly...

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Abstract

The present invention relates to the application of SlBBX20 gene in regulating tomato gray mold resistance, reducing the gray mold resistance of tomato by increasing the expression of SlBBX20 in tomato, and improving the gray mold resistance of tomato by reducing or eliminating the expression of SlBBX20 in tomato. Mildew resistance. Compared with the prior art, the beneficial effects of the present invention are: (1) using gene editing technology to obtain plants with strong gray mold resistance, which is more economical and effective than traditional breeding methods, and is a good method for creating disease-resistant materials; method, the breeding period is greatly shortened; (2) compared with the traditional control method, the resistance of the SlBBX20 knockout line of the present invention to Botrytis cinerea is significantly enhanced, the usage of pesticides can be reduced, and the environment is protected; (3) Plants and methods for reducing gray mold resistance are provided, and materials are provided for subsequent research and control of gray mold.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and particularly relates to the application of SlBBX20 gene in regulating tomato gray mold resistance. Background technique [0002] Botrytis cinerea is a broad-spectrum pathogenic fungus with more than 200 hosts, and can invade plant organs such as flowers, stems, leaves and fruits, posing a huge threat to the world's fruit and vegetable production and quality every year. Botrytis cinerea is a saprophytic pathogen. After invading plants, it secretes some toxic substances and uses its own defense mechanism to destroy host cells, obtain nutrients, and reproduce itself. [0003] As the second largest fungal disease in the world, Botrytis cinerea has caused great losses to agricultural production. Most of the domestic control of Botrytis cinerea is still in the research on the biological characteristics of Botrytis cinerea, and the methods of agricultural control, biological control and chemical ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/29C12N15/82A01H6/82
CPCC07K14/415C12N15/8218C12N15/8282
Inventor 王涛涛罗丹熊程孙文慧张小燕叶志彪张俊红杨长宪李汉霞
Owner HUAZHONG AGRI UNIV
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