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Glyphosate-resistant selective marker gene and application thereof in maize transgenic technology

A technology for screening marker genes and glyphosate resistance, which can be used in applications, genetic engineering, plant genetic improvement, etc., and can solve the problem of high false positive rate

Inactive Publication Date: 2016-08-24
BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the use of bar,CP4-EPSPS gene as a screening marker gene for transgenic maize positive plants has been relatively mature and the screening efficiency has been improved, but the false positive rate is still high in actual transgenic research. Expressed and highly glyphosate-resistant genes are of great significance for further improving the screening efficiency of transgenic positive plants

Method used

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  • Glyphosate-resistant selective marker gene and application thereof in maize transgenic technology
  • Glyphosate-resistant selective marker gene and application thereof in maize transgenic technology
  • Glyphosate-resistant selective marker gene and application thereof in maize transgenic technology

Examples

Experimental program
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Effect test

Embodiment 1

[0026] Example 1 Obtaining of glyphosate-resistant screening marker gene G23V-EPSPS

[0027]The fusion sequence of the chloroplast leader peptide sequence derived from the maize rbcS gene (ie, the 491-631 base sequence in Genbank: Y00322.1, a total of 141bp) and the EPSPS gene (1245bp) of GenBank: GM718572.1 is highly expressed according to the maize genome The codon of the gene was optimized, and a BamHI site was added at the 5' end of the gene coding sequence, and KpnI and SacI sites were added at the 3' end of the coding sequence. The G23V-EPSPS gene with added restriction sites was synthesized by Shanghai Jierui Bioengineering Co., Ltd. The synthesized EPSPS gene is named G23V-EPSPS, and its base sequence is shown in SEQ ID NO.1, wherein the 1st-6th base sequence from the 5' end is the BamHI site, and the 13th-15th base sequence The sequence ATG is the translation initiation codon, the 154th-156th base sequence ATG is the EPSP translation initiation codon, the 1393-1395th...

Embodiment 2

[0029] The glyphosate resistance of the G23V-EPSPS gene synthesized in embodiment 2 in escherichia coli (E.Coli)

[0030] The plasmid pGH-G23V containing the target gene G23V-EPSPS and the plasmid pGH-CP4 containing the CP4 type EPSPS gene (prepared by Shanghai Jierui Bioengineering Co., Ltd.) were respectively transferred into different Escherichia coli DH5α, and the G23V-EPSPS gene was combined with The CP4-type EPSPS gene was compared to detect the glyphosate resistance of the G23V-EPSPS gene and the CP4-type EPSPS gene in Escherichia coli. The specific experimental steps are as follows:

[0031] 1) The plasmids pGH-G23V and pGH-CP4 were transformed into different Escherichia coli DH5α by heat shock method, and cultured overnight at 37° C. on solid LB medium containing 50 mg / L ampicillin.

[0032] 2) Pick a single colony and culture it overnight at 37°C in liquid LB medium containing 50 mg / L ampicillin, then extract the plasmid for enzyme digestion verification, and then p...

Embodiment 3

[0039] Example 3 Construction of recombinant expression vector pBAC9200

[0040] The pGH-G23V plasmid synthesized by Shanghai Jierui Bioengineering Co., Ltd. was digested with BamHI / KpnI to recover the 1.4kbp target gene, and then inserted the target gene into the pBAC823 vector (see the construction method of the invention patent ZL201110294326.6). Between the BamHI / KpnI sites, the expression vector pBAC9200 (base number 5895bp) driven by the CaMV 35S promoter and maize Adh1intron1 to drive the target gene G23V-EPSP is obtained. For the map of its T-DNA region, see figure 1 .

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Abstract

The invention discloses a glyphosate-resistant selective marker gene and an application thereof in a maize transgenic technology. The glyphosate-resistant selective marker gene G23V-EPSPS has a base sequence represented as SEQ ID NO.1 in a sequence table and can be used for selecting positive plants of transgenic maize. The gene G23V-EPSPS has stronger glyphosate resistance, a maize variety with the high glyphosate-resistant characteristic is acquired when the maize is genetically modified with the gene G23V-EPSPS, and the positive rate is up to 35.7%.

Description

technical field [0001] The invention relates to a selection marker gene, in particular to a glyphosate resistance selection marker gene and its application in plant transgenic technology. Background technique [0002] At present, the types of genetically modified crops mainly include soybean, corn, cotton, and rapeseed, and their traits are mainly herbicide resistance, insect resistance, and disease resistance. Among them, corn (Zea mays L.) is one of the three major food crops in the world, which occupies an important position in agriculture, animal husbandry and industry, and the global demand for corn is constantly rising. In recent years, maize, as the main research object of transgenic technology, has been widely concerned by scientists from all over the world. A number of available agronomic traits have been obtained by applying transgenic technology, such as resistance to corn borer, herbicides, root-eating pests, and improvement of nutritional quality of corn. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/62C12N15/84C12N15/65C12N1/21A01H5/00
CPCC07K2319/02C12N9/1092C12N15/65C12N15/8275C12Y205/01019
Inventor 张晓东江颖张立全徐摇光姜志军
Owner BEIJING ACADEMY OF AGRICULTURE & FORESTRY SCIENCES
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