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Method for expressing and identifying foreign genes in fungus

An exogenous gene and gene technology, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., to achieve the effect of simple technical operation and short cycle

Inactive Publication Date: 2010-12-15
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] In order to solve the problem that most fungi do not recognize the CAMV35S promoter used in plant expression vectors, the inventors have studied the ability of Fusarium oxysporum to recognize the CaMV35S promoter

Method used

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  • Method for expressing and identifying foreign genes in fungus
  • Method for expressing and identifying foreign genes in fungus
  • Method for expressing and identifying foreign genes in fungus

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1 Expression and functional identification of new glyphosate-resistant genes from prokaryotes

[0054] The plant expression vector pCAMBIA1300 used in the present invention comes from Cambia Research Institute of Australia, and the CAMV35S promoter carried on it is a strong promoter from cauliflower virus, which has been widely used in plant transgenic breeding.

[0055] The present invention obtains a novel highly glyphosate-resistant EPSP synthase (5-enol type pyruvyl shikimate-3-phosphate synthase) gene from prokaryotes, and its open reading frame nucleotide sequence has a full length of 1335 bases, encoding a total length of 445 amino acids. The expression and function of the gene in eukaryotes can be rapidly identified by using the method provided by the invention. The operation steps are as follows:

[0056] 1. Construction of plant expression vectors

[0057] 1.1 Using the plasmid containing the above-mentioned highly glyphosate-resistant EPSP synthase...

Embodiment 2

[0102] Example 2 Expression and functional identification of new salt-resistance-related genes from prokaryotes

[0103] The invention obtains a global regulatory gene IrrE from the extreme corrosion-resistant Deinococcus, which can improve the salt-tolerant ability of Escherichia coli. The expression and function of the gene in eukaryotes can be rapidly identified by using the method provided by the invention. The operation steps are as follows:

[0104] 1. Construction of plant expression vectors

[0105] 1.1 Use the genomic DNA of D.radiodurans R1 as a template to amplify the open reading frame of the IrrE gene by PCR. The 5' end primer used is complementary to the 3' end of the CAMV35S promoter by adding 25 bases, and the 3' end primer is introduced into HindIII restriction site;

[0106] 1.2 Design primers to amplify the CAMV35S promoter, the 5' end primer introduces the BamHI restriction site, and the 3' end primer adds 25 bases to complement the 5' end of the EPSP sy...

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Abstract

The invention relates to a plant transgenic breeding method. The foreign genes are expressed in fungus by using a fusarium oxysporum-identified CaMV 35S promoter so as to rapidly identify the gene function. The plant transgenic breeding method has the advantages of simpleness, convenience, rapidness, and capacities of shortening the plant transgenic breeding progress and finishing gene expression and preliminary functional identification within 24 days.

Description

Technical field: [0001] The invention relates to a plant transgenic breeding method, in particular to a method for expressing foreign genes in fungi by using CaMV 35S promoter and quickly identifying gene functions. Background technique: [0002] In the plant transgenic breeding methods of the prior art, the exogenous gene to be transferred usually needs to be expressed and verified in eukaryotes first, and then introduced into the target plant. The commonly used method at present is: by transforming model plants, such as tobacco or Arabidopsis, detecting its expression and preliminary function, and then transferring the gene into the target plant. This process of identification takes a long period, generally takes 6-8 months to complete, and is time-consuming and laborious. [0003] Fungi are the simplest eukaryotes. Compared with transformed plants, the genetic transformation of fungi eliminates the cumbersome operations such as sowing, harvesting, and tissue culture, an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/80C12N15/54C12Q1/68
Inventor 徐荣旗陆伟张维林敏
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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