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Method for preparing tomato strains containing transgene sites

A transgenic and tomato technology, applied in botany equipment and methods, biochemical equipment and methods, plant gene improvement, etc., can solve problems such as inability to integrate, achieve the effects of improving safety, avoiding gene silencing, and reducing interference

Inactive Publication Date: 2011-10-12
WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition to the above three recombination systems, there are two irreversible recombination systems, one is the β-six and γδ-res recombination systems, which have the same recombinase recognition site, but can only catalyze the recombination of DNA fragments. deletion, but does not catalyze DNA integration

Method used

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  • Method for preparing tomato strains containing transgene sites
  • Method for preparing tomato strains containing transgene sites
  • Method for preparing tomato strains containing transgene sites

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] A method for preparing a tomato target gene strain containing a specific transgenic site, the steps of which are:

[0058] (1) Sterilize tomato seeds in 75% (v / v) alcohol for 28-34s, then pour out the alcohol.

[0059] (2) Transfer the seeds to a sterilized Erlenmeyer flask with tweezers, and sterilize with NaClO for 10-14min.

[0060] (3) Pour out the NaClO and wash it with sterilized distilled water for 3 to 4 times. Inoculate in 1 / 2MS medium and culture for 8-9 days.

[0061] (4) The cotyledons are fully stretched, and the tomato tender seedlings whose true leaves have not yet grown are spread on the filter paper of a large petri dish (the filter paper is moistened with sterilized water in advance). Cut off the hypocotyl and root with a razor blade, then cut off the cotyledon with a small petiole, and cut off the tip, leaving a wound for bacterial infection. Then use tweezers to transfer the cut cotyledons to the 0.2Z pre-medium (MS medium added with 2 μg / ml zeati...

Embodiment 2

[0073] The extraction of tomato genome DNA, its step is:

[0074] (1) Collect 50-100 mg tomato young leaves.

[0075] (2) Prepare freshly prepared DNA extraction solution and preheat it in a 65°C water bath.

[0076] (3) The leaves were ground in liquid nitrogen, then transferred to a 2 ml centrifuge tube, and 800 μl of DNA extraction solution was added. Water bath at 65°C for 30-120 minutes, shaking occasionally during the period.

[0077] (4) Add 600 μl of chloroform:isoamyl alcohol (24:1), mix well, shake up and down 50-100 times, and centrifuge at 10,000 rpm for 5 minutes.

[0078] (5) Pipette the aqueous phase into a new centrifuge tube and repeat step (4) once.

[0079] (6) Pipette the water phase into a new centrifuge tube, add 2 / 3-1 times the volume of pre-cooled isopropanol, and turn it upside down until the DNA precipitates.

[0080] (7) Immediately centrifuge at 10,000 rpm for 5 minutes, pour out the isopropanol, and add 70% (v / v) alcohol to wash.

[0081] (8) ...

Embodiment 3

[0088] PCR amplification of specific genes and loci in the tomato genome:

[0089] Genomic DNA is used as a template for PCR amplification, and the DNA stock solution is diluted 100 times, and T 0 Transformed seedlings and F 1 The primers selected for individual screening are the upstream primers designed in the 35S promoter, and the downstream primers designed in the nptII gene (35Sd-F and nptII-R primer pair) (Tm=57°C):

[0090] 35Sd-F: 5′-CCCAAGCTTCCCAGATTAGCCTTTTCAATTTC-3′

[0091] nptII-R: 5′-TCGATCCGAACCCCAGAGTC-3′

[0092] The PCR program is: 94°C, 5min; 94°C, 30s, 57°C, 30s; 72°C, 1min; 72°C, 8min. 16°C hold.

[0093] f 1 Integrity detection of T-DNA insertion in generation individuals, the primer pair on the left border is P1 and P2 (Tm=55°C); the primer pair on the right border is P3 and P4 (Tm=54°C). Figure 6 Shown are the detection results for the right boundary.

[0094] The primer pair sequences for PCR detection are as follows:

[0095] P1: 5′-TAAACGCTC...

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Abstract

The invention discloses a method for preparing tomato strains containing transgene sites, and the method comprises the following steps: A. preparing a transformed tomato seedling containing transgene sites of T0 generation; B. screening stable expression individuals from the transformed tomato seedling of T0 generation: a) carrying out PCR (polymerase chain reaction) screening on the transformed tomato seedling of T0 generation; b) carrying out hybridization detection on the copy number of foreign genes in the transformed tomato seedling of T0 generation; and c) carrying out GUS (glucuronidase) staining on the transformed seedling of T0-11; C. carrying out hybridization on T0-11 strains and tomato without transformation to obtain F1 generation: collecting and smearing pollen of tomatoes without transformation on the pistil of the T0-11 strains, thus obtaining seeds of F1 generation; and D. identifying the genetic stability of individuals of F1 generation: a) separating out individuals without transgene sites through primary PCR screening; b) identifying the T-DNA (triplex-deocyribose nucleic acid) integrity of individuals containing transgene sites of F1 generation; and c) identifying the genetic stability of the T-DNA of individuals of F1 generation.

Description

technical field [0001] The invention relates to the technical field of plant transformation in genetic engineering, in particular to a method for preparing a tomato strain containing a specific transgenic site. Background technique [0002] Site-directed recombination technology is a new technology developed based on site-directed recombination system in transgenic research to delete sequences from the genome or integrate foreign genes into specific sites in the genome. The basic principle of site-directed recombination is described in two aspects: gene deletion and gene integration. What they have in common is that they all include a recombinase and a DNA sequence that can be recognized by the enzyme. For gene deletion, there are two direct repeat sequences in the same DNA molecule, which can be recognized by specific recombinases, so that fusion and exchange of DNA occurs at the recognition sites of these two recombinases. The gene between the two sequences is deleted. ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12Q1/68A01H1/02A01H5/00
Inventor 王瑛周银
Owner WUHAN BOTANICAL GARDEN CHINESE ACAD OF SCI
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