Preparation method of pink tomato material

A tomato and gene technology, applied in the field of plant genetic engineering and genetic modification, can solve time-consuming and labor-intensive problems, and achieve the effect of simple and easy method

Active Publication Date: 2015-10-21
INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] It takes at least 3-4 years to transform an excellent red-fruited tomato inbred line into a pink-fruited tomato inbred line through the traditional backcrossing method, including one crossing, at least five backcrossings and one selfing, which is time-consuming and labor-intensive

Method used

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  • Preparation method of pink tomato material
  • Preparation method of pink tomato material
  • Preparation method of pink tomato material

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Example 1 Construction of CRISPR / Cas9 vector containing specific sgRNA

[0025] Carry out sequence analysis to the transcription factor SlMYB12 that controls tomato pericarp, look for the sequence that contains PAM sequence, i.e. 5'-NGG-3' (N represents any nucleotide of A, T, C, G), the 19bp before NGG Sequences are defined as sgRNAs. Using the sgRNA as the query sequence, BLAST was carried out in the tomato reference genome to obtain a specific matching sgRNA sequence, its DNA sequence is shown in SEQ ID NO.2, which is located in the third exon of the gene (such as SEQ ID NO. 1) above.

[0026] The sgRNA sequence was synthesized in vitro, connected to the CRISPR / Cas9 vector by Infusion enzyme, and the recombinant vector was constructed, and the recombinant vector was transformed into the Agrobacterium GV3101 strain.

Embodiment 2

[0027] Example 2 Preparation and identification of powdery tomato material

[0028] The vector prepared in Example 1 was transformed into the cotyledons of the red tomato Moneymaker by the Agrobacterium infection method, and T0 generation transgenic tomato plants were obtained through regeneration.

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Abstract

The invention provides a preparation method of a pink tomato material, and belongs to the field of plant gene engineering. The method comprises the following steps: 1, carrying out sequence analysis on the transcription factor S1MYB12 for controlling tomato skins to determine a specific sgRNA sequence; 2, constructing an expression vector containing sgRNA and a Cas9 protein; 3, transforming the obtained recombinant vector to red tomato through an Agrobacterium infection technology to obtain T0 generation transgenic tomato plants; and 4, planting the T0 generation plants with S1MYB12 gene sequence mutation in a greenhouse, selfing to obtain T1 generation plants, and screening plants free from exogenous transgenic components, with S1MYB12 gene sequence homozygous mutation and with pink fruits from the T1 generation plants in order to obtain the pink tomato material. The method has the advantages of low cost, preparation cycle shortening, and obtaining of the pink tomato material within one year; and the pink tomato does not contain exogenous gene, is safe and is free from risk.

Description

technical field [0001] The invention belongs to the fields of plant genetic engineering and genetic modification, and in particular relates to a method for preparing powdery tomato materials using a CRISPR / Cas9 system. Background technique [0002] Tomato is one of the most important vegetable crops widely grown worldwide. Fruit color is an important economic trait of tomato, and consumers in different regions have different preferences for tomato fruit color. In Europe and North America, consumers prefer red tomatoes, while consumers in Asia prefer pink tomatoes. Tomato commercial breeding in the past few decades has mainly focused on the improvement of red-fruited tomatoes, and there are relatively few excellent pink-fruited tomatoes. At present, Chinese breeders mostly focus on cultivating pink fruit resources with red fruit background to meet the needs of consumers. [0003] The difference between red and pink tomatoes is mainly determined by the flavonoids in the pee...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/113C12N15/82C12N15/84C12Q1/68A01H5/00
Inventor 黄三文张春芝宋晓燕伦尧尧
Owner INST OF VEGETABLE & FLOWERS CHINESE ACAD OF AGRI SCI
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