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Process for using plant gene as tomato transformation safe marker gene and applications

A technology for marking genes and gene transformation, which is applied in the fields of plant genetic improvement, botany equipment and methods, biochemical equipment and methods, etc. It can solve the problems of not establishing a genetic transformation screening marker system, etc., to improve fruit quality or stress resistance , high screening efficiency and safe operation

Inactive Publication Date: 2011-06-29
CHONGQING UNIV
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  • Summary
  • Abstract
  • Description
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  • Application Information

AI Technical Summary

Problems solved by technology

Tomato is an important economic crop grown all over the world, and it is a model plant for the study of vegetable genetic engineering and fruit ripening mechanism. However, no genetic transformation screening marker system of plant origin and ecological environment has been established so far.

Method used

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  • Process for using plant gene as tomato transformation safe marker gene and applications
  • Process for using plant gene as tomato transformation safe marker gene and applications
  • Process for using plant gene as tomato transformation safe marker gene and applications

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example 1

[0034] 1 Construction of expression vector

[0035] (1) Primer design and PCR amplification

[0036] The sequence of the Arabidopsis gene AtTPS1 (Accession No.: Y08568) was obtained from Genbank, and primers were designed using the software primer 5. See the attached table for the primers, 1 and 2 are the primer sequences for PCR cloning of the AtTPS1 gene.

[0037] The RNA of Arabidopsis and rice was extracted with Trizol reagent, the first-strand cDNA was synthesized according to the conventional M-MLV reverse transcriptase system, 3ul was taken as a template and added to a 50ul PCR reaction system, and the full-length gene was amplified with high-fidelity enzymes. Conventional PCR reaction parameters are: pre-denaturation at 94°C for 4 min, deformation at 94°C for 30 s, annealing at 50°C for 1 min, extension at 72°C for 3 min, and extension at 72°C for 7 min after 30 cycles. PCR instrument is Bio-Rad My Clycler TM, perform PCR amplification in the Algorithmic Measurement...

example 2

[0049] 1 Construction of expression vector

[0050] (1) Primer design and PCR amplification

[0051] Login to Genbank to obtain the sequence of rice gene OsDREB2A (AF300971), and use primers 5 and 6 for PCR amplification. The rest of the operations are the same as above.

[0052] (2) Transformation vector construction

[0053] The pAI-35s-OsDREB2A vector ( Figure 4 ). Carrier primer 3 was paired with OsDREB2A gene reverse primer 8 for PCR detection, and other operations were the same as above.

[0054] 2 Determination of Sodium Chloride Concentration in Tomato Transformation Screening

[0055] Sow sterilized tomatoes on MS / 2 medium with different concentrations of sodium chloride. In the range of 100mM, it basically has no effect on the germination and rooting of tomato; when it exceeds 150mM, the germination rate of tomato decreases and the root growth is inhibited.

[0056] Select 0.5×0.4cm tomato leaves and place them in culture media containing 0, 50, 100, 150, 200...

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Abstract

The invention discloses a method and application for adopting a plant gene to select a marker gene for tomato genetic transformation. The method comprises the steps of: clone of a plant gene, construction of an expression vector, transformation of a tomato, identification of the transgenic tomato and so on. The method is simple, has safe operation, creates an approach utilizing a plant gene to safely select the marker gene for the tomato genetic transformation, can replace the disadvantages caused by the widely used heterogenous genes such as antibiotic genes, weedicide resistant genes, xyl A, pmi, GUS and so on, taken as marker genes, obtains a transgenic tomato with food safety, biological safety and ecological safety, relieves the public from anxiety of marker gene safety, and inevitably facilitates the commercialized application of the transgenic tomato.

Description

technical field [0001] The invention relates to a marker gene and its screening and application in transgenic plants, in particular to a method and application of a plant origin gene as a safe screening marker gene for tomato genetic transformation. Background technique [0002] Since the first genetically modified plant product in the United States—the genetically modified tomato variety "Flavr-Savr" (extended ripening and fresh-keeping transgenic tomato) was approved for marketing in 1994, genetically modified tomatoes have been commercially grown in many regions and countries around the world, such as the United States, the European Union, Australia, Asia, Latin America, Japan, Mexico, etc. The traits of the transgenic tomato planted include storage resistance, anti-virus, anti-fungal, insect resistance, herbicide resistance, frost resistance, salt tolerance, quality improvement and high yield. Tomato is one of the vegetables with high economic benefits in the world, acc...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/29C12N15/82A01H1/00A01H5/00
Inventor 李正国符勇耀邓伟杨迎伍
Owner CHONGQING UNIV
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