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Method for making solanum lycopersicum materials having high fruit lycopene content

A high lycopene, fruit technology, applied in the field of plant genetic engineering and genetic modification, can solve the time-consuming and labor-intensive problems of tomatoes, achieve good application prospects, and increase the effect of content

Pending Publication Date: 2020-02-11
HORTICULTURE INST OF XINJIANG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a method for creating tomato materials with high lycopene content through gene editing, so as to overcome the time-consuming and labor-consuming shortage of creating high lycopene tomatoes in the prior art

Method used

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  • Method for making solanum lycopersicum materials having high fruit lycopene content
  • Method for making solanum lycopersicum materials having high fruit lycopene content
  • Method for making solanum lycopersicum materials having high fruit lycopene content

Examples

Experimental program
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Effect test

Embodiment 1

[0010] Example 1: (1) The gene SISGR1, which controls the lycopene content of tomato fruit, is subjected to sequence analysis, and the CRISPR-Plant online design tool is used to design two target sites according to the principle of CRISPR / Cas9 technology design target sites. On the first exon and the second exon, the distance between the two target sequences is 260bp; the sequence of the first gRNA target site is: 5'-TCTAAGCTCAACAATGAACA-3'; (located in SEQ ID NO. 222-241 from the 5' end in 1); the sequence of the second gRNA target site is: 5'-AGAACATATACACTGACTCA-3'; (located at positions 502-521 from the 5' end in SEQ ID NO.1)

Embodiment 2

[0011] Example 2. The method for creating tomato material with high lycopene content in fruit: (1) by enzyme cutting and ligation, two gRNAs are integrated into the constructed plant expression vector pCGR1301-CRISPR / Cas9 to obtain pCGR1301-CRISPR / Cas9-gRNA vector; (2) import the pCGR1301-CRISPR / Cas9-gRNA expression vector into Agrobacterium to obtain pCGR1301-CRISPR / Cas9-gRNA Agrobacterium; (3) tomato with low lycopene content (containing SISGR1 gene ) as material, infect the hypocotyl tissue with pCGR1301-CRISPR / Cas9-gRNA Agrobacterium; (4) Induce callus, and screen for kanamycin resistance to obtain transgenic positive plants; (5) Obtain positive transgenic After the T0 generation of the strain, extract the genomic DNA, design primers on both sides of the target site, perform PCR amplification on the target fragment, purify the PCR amplification product and perform sequencing, screen homozygous mutants according to the sequencing results, and detect the T0 generation The l...

Embodiment 3

[0012] Example 3. The application of creating mutants with high lycopene content based on CRISPR / Cas9 technology in tomato breeding. Specifically, the tomato mutant with high lycopene content, which is homozygous for mutation of SISGR1 and has no foreign aid gene insertion, is selected as material, and tomato breeding with high lycopene content is carried out through breeding techniques such as hybridization; (1) SISGR1 gene sequence is as follows: Shown in SEQ ID NO.1 (2629bp); wherein, the coding sequence (CDS) of the SISGR1 gene consists of the 189th to 305th base, the 406th to 579th base, the 406th to 579th base, The 1698th to 1865th base and the 2033rd to 2392th base are composed; the CRISPR / Cas9 backbone vector described in (2) is pCGR1301; the Agrobacterium described in the step (3) is Rhizobacillus GV3101.

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Abstract

The invention belongs to the field of a solanum lycopersicum biotechnology and a transgenic technology, and discloses a method for making solanum lycopersicum materials having high fruit lycopene content through gene editing. According to the method disclosed by the invention, through designing an editing target site of an SISGR1 gene, a pCGR1301-CRISPR / Cas9-gRNA expression vector is constructed,through an agrobacterium tumefaciens mediating method, the pCGR1301-CRISPR / Cas9-gRNA expression vector is transformed into a solanum lycopersicum material having low fruit lycopene content, and through antibiotic screening, obtaining positive T0 generation transgenic solanum lycopersicum plants; and performing self cross on the T0 generations to obtain T1 generation plants, screening plants whichdo not contain exogenous transgenic component, but of which SISGR1 genes are subjected to homozygosis mutation and of which fruit lycopene content is high from the T1 generations so that the high lycopene materials are obtained. Through the adoption of the method, the lycopene content in solanum lycopersicum fruits can be markedly increased, and the method has favorable application prospects in solanum lycopersicum high-quality molecular breeding.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering and genetic modification, and specifically relates to a method for editing a gene that inhibits the increase of lycopene through a gene editing system and creating tomato mutant materials with high lycopene content in fruits. Background technique [0002] Tomato (Solanum lycopersicum) is an important vegetable and economic crop, which is popular all over the world. my country is the largest producer of fresh tomatoes and the third largest producer of processed tomatoes, playing a pivotal role in the world tomato market. In recent years, due to economic reasons and quality problems, the export volume of tomato paste in my country has been greatly reduced, and the cultivated area of ​​processed tomatoes has also shrunk seriously. The content of lycopene is an important index to measure the quality of tomato fruit, and the red color of tomato comes from it. As a functional natural pigment, ...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113A01H5/00A01H6/82
CPCC12N15/113C12N15/8218C12N15/825C12N2310/20
Inventor 王娟王柏柯李宁余庆辉黄少勇
Owner HORTICULTURE INST OF XINJIANG ACAD OF AGRI SCI
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