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Method for improving storage resistance of tomatoes through gene editing

A gene editing and genome editing technology, which is applied in the field of double-site editing of the SlACS2 gene in tomato, can solve the problems of difficult to control the ripening process of tomato, rot and deterioration, and overripe and softening of the fruit.

Pending Publication Date: 2020-10-16
XINJIANG PRODION & CONSTR CORPS NO 6 DIV AGRI SCI INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

System II ethylene has an autocatalytic mechanism, which makes it difficult to control the tomato ripening process, resulting in overripe fruit softening, rot and deterioration

Method used

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  • Method for improving storage resistance of tomatoes through gene editing
  • Method for improving storage resistance of tomatoes through gene editing
  • Method for improving storage resistance of tomatoes through gene editing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Design and synthesis of sgRNA for specific targeting of SlACS2 gene in tomato SlACS2 gene knockout by CRISPR / Cas9

[0042] 1. Design of sgRNA targeting tomato SlACS2 gene

[0043] Use online tools to design SlACS2-sgRNA, the specific steps are:

[0044] ① Log on to the GenBank website, search for the SlACS2 gene sequence and download it, its nucleotide sequence is shown in SEQ ID NO:4.

[0045] ② Open the target site prediction website (https: / / crispr.dbcls.jp / ), use the target site online design tool "CRISPRdirect", enter the gene sequence in the text box, select the tomato species, and you can get all the gene sequences. Finally, a set of target sites was selected for targeting after comprehensive consideration of the scoring results, GC content, specificity of the target site, and the distance between the two target sites.

[0046] ③ The 19nt oligonucleotide sgRNA core sequence was designed according to the GG(N)19NGG sequence.

[0047] The obtained sgRNA ...

Embodiment 2

[0055] The construction of embodiment 2 plant expression vectors

[0056] The CP185 and CP178 vectors were provided by the Vegetable and Flower Research Institute of the Chinese Academy of Agricultural Sciences, which are public vectors. Using the CP185 vector as a template, PCR amplification was performed with the sense strand primer (SEQ ID NO:5) and the antisense strand primer (SEQ ID NO:6) to obtain a DNA fragment containing double sgRNA and ligate it to pEasy-blunt, and the sequence was correct Afterwards, it was digested with AarI and connected to the CP178 vector to form the plant expression vector pSlACS2-DsgRNA (for the structure see figure 1 , the nucleotide sequence is SEQ ID NO: 3). The Cas9 gene on the plant expression vector is driven by 35S, and the two sgRNA genes are driven by the tomato U6 promoter. The constructed plant expression vector was transformed into Escherichia coli DH5α strain by heat shock method and amplified, and the sequence of the vector was...

Embodiment 3

[0057] Example 3 Genetic transformation of tomato and acquisition of T0 generation SlACS2 gene edited plants

[0058] 1. Obtaining sterile vaccines

[0059] Treat the prepared tomato seeds with 75% alcohol for 30 seconds, rinse them twice with sterile water, add 10% bleach, place them on a shaker for 1 hour, take out the seeds and rinse them five times with double distilled water, and place them in Refrigerate at 4°C for 12 hours, inoculate into 1 / 2 MS medium (without sucrose) and cultivate for 6 days to obtain sterile vaccines.

[0060] 2. Preparation of bacterial solution

[0061] The pSlACS2-DsgRNA glycerol bacteria contained 50mg / L rifampicin and 100mg / L kanamycin YEB solid medium (yeast extract 5g / L, peptone 5g / L, beef extract 5g / L, magnesium sulfate heptahydrate 0.5g / L, sucrose 1g / L), and cultured at 28°C for two days, pick a single clone and inoculate it into 5ml liquid YEB medium containing 50mg / L rifampicin and 100mg / L kanamycin, at 28°C , 220rpm constant temperatu...

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Abstract

The invention provides a method for improving storage resistance of tomatoes through gene editing. A CRISPR / Cas9 system is used for editing a catalytic synthesis ethylene precursor regulatory gene SlACS2 in a tomato material genome, so that the performance of the catalytic synthesis ethylene precursor regulatory gene SlACS2 is lost, and the storage resistance of the tomatoes is improved. The system comprises two sgRNA target sites, namely sgRNA1 and sgRNA2; and target sequences recognized by the sgRNA1 and sgRNA2 are DNA fragments for encoding SlACS2 protein in the tomato material genome. By means of the editing sites, the SlACS2 gene of the tomatoes can be edited under mediation of endonuclease Cas9, and site-directed mutagenesis of the SlACS2 gene is formed. The method has a very important effect on promoting application of an endogenous gene knockout or exogenous gene site-directed integration technology in tomato gene breeding.

Description

technical field [0001] The invention belongs to the technical field of plant gene editing, in particular to a method for improving the storage resistance of tomato through gene editing, and in particular to performing double-site (197-215 and 275-257) modification of the S1ACS2 gene on tomato using a CRISPR-Cas9 system. Editing method. Background technique [0002] Tomato fruit tends to become overripe and soft, resulting in poor pressure resistance, shortened storage period, and reduced ability to resist pathogenic bacteria, which seriously affects production, storage, transportation, and processing quality. [0003] Hybrid breeding is a common way to improve tomato quality, but it takes a long time and is easily limited by undesired gene linkage and interspecific reproductive isolation. The introduction of exogenous genes into tomato to improve storability has made some progress, which shortens the breeding cycle and broadens the source of genes compared with conventional...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/60A01H5/00A01H6/82A01H1/02C12Q1/6895
CPCC12N15/8218C12N15/8261C12N9/88A01H1/02C12Q1/6895C12Y404/01014C12Q2600/13C12Q2600/156
Inventor 刘江娜张爱萍张西英李荣霞白云凤闫建俊薛丽萍刘伟
Owner XINJIANG PRODION & CONSTR CORPS NO 6 DIV AGRI SCI INST
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