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Method for creating tomato ginkgo material by gene editing technology

A genome editing and tomato technology, applied in the biological field, can solve the problems of long cycle time and achieve the effect of shortening the transfer time and high editing efficiency

Inactive Publication Date: 2019-06-25
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The cycle of this method is long, generally takes 5-10 years, and is affected by factors such as chain burden

Method used

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  • Method for creating tomato ginkgo material by gene editing technology
  • Method for creating tomato ginkgo material by gene editing technology
  • Method for creating tomato ginkgo material by gene editing technology

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1, Construction of CRIPSR / Cas9 gene editing vector L2-PTX041

[0063] 1. Obtaining the target sequence of Lutescent2 gene sgRNA

[0064] According to the Lutescent2 (L2 for short) gene sequence registered in the SGN database (http: / / solgenomics.net / ) (the accession number is Solyc10g081470.1.1, and the nucleotide sequence is sequence 1 in the sequence list), the structure of the L2 gene was analyzed . Analysis results such as figure 1 As shown, the L2 gene consists of 10 exons (marked as E1-E10 respectively) and 9 introns. Since the natural mutation occurred in exon 10, the sequence of exon 10 was submitted to the CRISPRdirect online target analysis database (http: / / crispr.dbcls.jp / ) for CRIPSR / Cas9 target design, and the PAM sequence design Set as NGG, species data set as Tomato (Solanum lycopersicum) str.Heinz 1706genome SL2.50. The final selected sgRNA targets such as figure 1 As shown, the L2 gene sgRNA target sequence is as follows: 5'-GAGGAAAGCAGCTCTT...

Embodiment 2

[0074] Embodiment 2. A method of converting tomato powder fruit material into ginkgo fruit material by gene editing

[0075] 1. Transform the gene editing vector L2-PTX041 into Agrobacterium

[0076] Take 1 μg of the gene editing vector L2-PTX041 prepared in Example 1 and place it in 100 μL of LBA4404 competent cells (Beijing Huayueyang Biology, NRR01270), freeze in liquid nitrogen for 3 minutes, bathe in water at 37°C for 5 minutes, and then add 1 mL of YEB medium (YEB medium is made up of solute and solvent, and solvent is water, and the concentration of solute and in medium is: yeast extract 5g / L, peptone 5g / L, beef extract 5g / L, magnesium sulfate heptahydrate 0.5g / L L, sucrose 1g / L), cultured at 28°C for 2-4 hours; centrifuged at 10,000×g for 30 seconds, discarded the supernatant, added 0.1ml YEB medium to resuspend the cells, and spread on a medium containing 50μg / mL kanamycin, 500μg / mL streptomycin and 50μg / mL rifampicin on the YEB plate, cultured in the dark at 28°C for...

Embodiment 3

[0088] Example 3, the acquisition and identification of non-transgenic tomato ginkgo material

[0089] 1. Detection of exogenous DNA fragments

[0090] The TB0748-2 L2 gene homozygous target plant was selfed to obtain the F1 generation seed; the F1 generation seed was sown; after the true leaves grew, the offspring of the TB0748-2 L2 gene homozygous target plant (F1 generation ). Using the genomic DNA of the progeny of the TB0748-2 L2 gene homozygous target plant as a template, and using the nucleotide sequences shown in Sequence 7 and Sequence 8 as primers, the exogenous DNA fragment (cas9) was subjected to PCR amplification and electrophoresis. The progeny without PCR amplification products were selected, that is, the progeny of TB0748-2 L2 gene homozygous target plants that did not carry exogenous DNA fragments.

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Abstract

The invention discloses a method for creating a tomato ginkgo material by a gene editing technology. The method is characterized in that a sgRNA target sequence for editing the CRIPSR / Cas9 gene for the L2 gene is designed, and then a gene editing vector containing two of the above target sequences is constructed. The experiment proves that the L2 gene can be precisely edited by transforming the gene editing vector into a tomato powder fruit material, the tomato gingko material is obtained, and the non-transgenic tomato ginkgo material with L2 gene homozygous mutation can be screened from the self-bred progeny. The method of the invention is applicable to different varieties of tomato powder fruit or a red fruit material, and has high editing efficiency. Compared with the traditional l2-site backcross transformation, the method can greatly shorten the transformation time and achieve rapid and accurate conversion of parental fruit color traits within 1 year, is not limited by factors such as linkage drag and transgenic safety, and has great breeding application prospects and economic value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a method for creating tomato ginkgo material through gene editing technology. Background technique [0002] Tomatoes are both vegetable and fruit crops that are widely grown and loved around the world. Tomatoes are very rich in fruit color, including red, pink, yellow, orange, coffee, green and white. But the tomatoes sold in the market are mainly red and pink. Ginkgo tomatoes are very rare and expensive in the market. Lutescent2 (L2) is an important gene controlling ginkgo traits, which encodes a chloroplast-localized zinc metalloprotease. The accumulation of chloroplast in the fruit of the l2 mutant decreased significantly, and the fruit was white. The breeding of ginkgo tomato mainly depends on the high-generation backcross of the l2 gene locus. This method has a long cycle, usually 5-10 years, and is affected by factors such as chain burden. [0003] The gene ed...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/113A01H1/02
Inventor 杨天霞邓磊李常保李传友
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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