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Method and application for creating purple fruit tomato mutant using CRISPR/Cas9 gene editing system

A gene editing and mutant technology, applied in the field of tomato biotechnology and genetic modification, can solve the problems of scarcity of purple fruit tomato resources, long technical cycle, low efficiency, etc., achieve good application prospects and increase the effect of content

Active Publication Date: 2018-12-28
SOUTH CHINA AGRI UNIV
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Problems solved by technology

[0005] In order to overcome the problems of long period and low efficiency of traditional purple tomato breeding technology and the shortage of purple tomato resources, the object of the present invention is to provide a method for creating purple tomato mutants using the CRISPR / Cas9 gene editing system

Method used

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  • Method and application for creating purple fruit tomato mutant using CRISPR/Cas9 gene editing system

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Embodiment 1

[0043] 1. Selection of SlMYBATV gene target site sequence and design of upstream and downstream primers for gRNA fragments

[0044] According to literature reports, there are three transcripts of SlMYBATV (SlMYBATV-X1, SlMYBATV-X2 and SlMYBATV-X3), and there is no difference in the first two exons of the three transcripts. Therefore, using the CRISPR-Plant online design tool, according to the principle of CRISPR / cas9 technology design target sites, the two target sites are designed on the second exon of the SlMYBATV gene (see SEQ ID NO: 1), And the distance between the two target site sequences does not exceed 100bp. The sequence of the first gRNA target site of SlMYBATV is: 5'-gagtggttgcattagagac-3'; the sequence of the second gRNA target site is: 5'-acgaagaaacctctaaact-3'.

[0045] 2. According to the sequence of gRNA target site, design gRNA PCR primers:

[0046] Upstream primer gRNA-F: 5'-ATATATGGTCTCGTTTGgtctctaatgcaaccactcGTTTTGAGCTAGAAATAG-3';

[0047] Downstream pri...

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Abstract

The invention discloses a method and application for creating a purple fruit tomato mutant using a CRISPR / Cas9 gene editing system, and relates to the field of tomato biotechnology and transgenic technology. The method includes constructing a CRISPR / Cas9-gRNA (pHSE401-gRNA) expression vector by designing a mutant target site of SlMYBATV, introducing a cultivated tomato LA1996 containing an Aft site by an agrobacterium-mediated method and conducting kanamycin resistance marker screening to obtain a positive transgenic plant. A strain with homozygous mutation determined by gene sequencing and phenotypic observation and containing no foreign aid gene insertion is a tomato mutant that completely loses the SlMYBATV function and has purple fruit. According to the method and application, the content of anthocyanins in tomato peels can be significantly improved. The method and application have good application prospects in high quality molecular breeding of tomatoes.

Description

technical field [0001] The present invention relates to the field of tomato biotechnology and transgenic technology, in particular to a method and application of using the CRISPR / Cas9 gene editing system to mutate the tomato SlMYBATV gene and create purple fruit tomato mutants. Background technique [0002] Anthocyanins are a class of natural antioxidant substances widely present in plants. Long-term consumption of foods rich in anthocyanins will help people improve their eyesight and reduce the incidence of cardiovascular diseases, obesity, diabetes and cancer. Anthocyanins can make plant fruits present rich colors (red, blue and purple), which can meet the needs of different consumers. At the same time, anthocyanins can also improve the resistance of plants to environmental stress, such as low temperature, drought and UV damage Wait. [0003] Tomato is one of the most important vegetables in China and even in the world, and it is also a model crop in the study of plant mo...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/08A01H6/82
CPCC07K14/415C12N15/825C12N2310/10C12N2310/20
Inventor 邱正坤曹必好侯军晓黄泽军崔霞
Owner SOUTH CHINA AGRI UNIV
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