Gene editing method using CRISPR/Cas9 system to create pink fruit tomato

Abstract

The invention relates to construction of a tomato transgenic material, and aims to provide a gene editing method using a CRISPR / Cas9 system to mutate a tomato SlMYB12 gene in order to transform red fruit tomato into pink fruit tomato. The method includes the following steps: designing an oligo primer corresponding to the gRNA target site of the SlMYB12 gene, and recombining and connecting an oligo dimer with a Cas9 / gRNA vector; transforming Escherichia Coli competent cells, transforming a plasmid with a correct sequencing result into Agrobacterium tumefaciens, and mediating and transforming tomato calluses to obtain a transgenic plant in order to obtain a transgenic positive strain; and carrying out gene sequencing and phenotype observation to determine a pure mutant strain which is an SlMYB12 function completely lost tomato mutant for pink fruits. The method can effectively knock out the transcriptional translation of the SlMYB12 gene, and is an ideal gene editing system for successfully transforming the tomato red fruits into the pink fruits.

Description

technical field [0001] The invention relates to the construction of tomato transgenic materials, in particular to a gene editing method for converting red-fruit tomato materials into pink-fruit tomato materials by using the CRISPR / Cas9 gene editing system. Background technique [0002] Tomato is one of the most important vegetable crops in the world, and it is also a model plant for fruit research. At present, in terms of tomato production, the consumption habits of European and American regions are dominated by bright red fruit tomatoes, while some Asian countries, especially China and Japan, have certain consumption habits of pink fruit tomatoes. Due to the early development of red-fruit tomato breeding and abundant materials, the related characteristics of red-fruit tomato materials such as high-yield, disease resistance and commerciality are generally better than pink-fruit tomato. Therefore, there are far more red fruit germplasm resources than powder fruit germplasm r...

AI Technical Summary

Problems solved by technology

[0005] The problem to be solved by the present invention is to overcome the shortcomings of the existing traditional tomato breeding technology, such as the long cycle and low efficiency and the inability of the existing transgenic technology to knock out genes, and provide a method for knocking out the red fruit tomato material SlMYB12 gene by using the CRISPR / Cas9 system A gene editing method to obtain an ideal pink-fruited tomato mutant that completely loses the function of SlMYB12, is genetically stable, and has no foreign gene insertion

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