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Glyphosate-resistant EPSPS mutant gene, plant genetic transformation screening vector containing gene, and applications of vector

A glyphosate-resistant, genetic transformation technology, applied in the field of agricultural biology

Active Publication Date: 2020-07-10
HAINAN BOLIAN RICE GENE TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Another object of the present invention is to solve the problem of potential safety risks caused by the use of bacterial source screening markers in the current plant genetic transformation, and to provide a plant genetic transformation screening vector using plant source genes as screening markers

Method used

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  • Glyphosate-resistant EPSPS mutant gene, plant genetic transformation screening vector containing gene, and applications of vector
  • Glyphosate-resistant EPSPS mutant gene, plant genetic transformation screening vector containing gene, and applications of vector
  • Glyphosate-resistant EPSPS mutant gene, plant genetic transformation screening vector containing gene, and applications of vector

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Experimental program
Comparison scheme
Effect test

Embodiment 1E

[0088] Embodiment 1EiEPSPSm sequence analysis and optimization

[0089] The nucleotide sequence of the EPSPS mutant gene (EiEPSPSm) derived from goosegrass is shown in SEQ ID NO.6, and the amino acid sequence of the encoded protein is shown in SEQ ID NO.7. The present invention performs codon optimization and screening of expression elements on the basis of the sequence shown in SEQ ID NO.6, and screens the combination of the optimized EiEPSPSm sequence and expression elements suitable for crop expression and as a screening marker. During the optimization and screening process, the present invention found that the high-efficiency expression of the EPSPS mutant gene derived from goosegrass in crops could not be well achieved by adopting conventional codon optimization principles and optimization systems, and some codon-optimized sequences although It can be well expressed in crops, but the screening efficiency as a screening marker is not high, which limits its application as a...

Embodiment 2

[0090] Embodiment 2 Construction of Plant Genetic Transformation Screening Vector

[0091] 1. Preparation of plant transgenic screening expression cassettes

[0092] The construction method of the plant transgenic screening expression cassette ZmUbiP-oEiEPSPSm-OsUbiT (sequence shown in SEQ ID NO.2) of the present invention is as follows:

[0093] Design primer 0310-UEU-F / 0310-UEU-Rv1 to amplify the promoter ZmUbiP fragment from the maize genome; use primer 0310-UEU-F2 / 0310-UEU-Rv2 to synthesize the sequence-optimized goosegrass from Example 1 The target gene oEiEPSPSm fragment was amplified from the EPSPS mutant gene fragment; the terminator OsUbiT fragment was amplified from the rice genome using primers 0310-UEU-F3 / 0310-UEU-Rv. Among them, the 5' ends of primers 0310-UEU-F and 0310-UEU-Rv have about 15 nucleotide sequences repeated with the corresponding connection positions of the vector; the 5' ends of the upstream and downstream primers of adjacent fragments also have 15...

Embodiment 3

[0118] Embodiment 3 Agrobacterium transformation and identification

[0119] Take Agrobacterium EHA105 competent cells stored at -80°C, add 1 μl of the sequenced correct pCEiEPSPS plasmid obtained in Example 1, and transform by electroporation at 1.8KV. Spread on a YEP culture plate containing kanamycin, rifampicin and streptomycin, culture at 28°C for about 48 hours, pick a single colony and shake it overnight, and use specific primers (UEU-F1 and UEU-R1) to inoculate Liquid PCR verification (such as Figure 4 ), can amplify to obtain about 900bp target fragment, select the positive clone (engineering Agrobacterium), shake the bacteria for 36-48h, and save the bacterial liquid for infection.

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Abstract

The invention relates to the field of agricultural biotechnology, and in particular relates to a glyphosate-resistant EPSPS mutant gene, a plant genetic transformation screening vector containing thegene, and an application of the vector. The eleusine indica 5-enolpyruvyl shikimate-3-phosphate synthase mutant gene provided by the invention can endow plants with high resistance to glyphosate. According to the plant genetic transformation screening vector using an expression cassette as a selection marker and a screening method provided by the invention, callus can use the glyphosate as a screening agent in the tissue culture stage, and the obtained transgenic plants have high resistance of a glyphosate herbicide; the vector can be used as a transgenic screening vector to be used with additional functional elements, plant-derived genes can be used to replace exogenous screening marker genes such as bacterial sources in the transgenic process, so that the method for plant transgenic screening is enriched, potential safety risks of the transgenic plants caused by the exogenous genes can be effectively reduced, the commercial application of the transgenic plants is facilitated, and themarket value and social benefits are good.

Description

technical field [0001] The invention relates to the field of agricultural biotechnology, in particular to a glyphosate-resistant EPSPS mutant gene, a plant transgene screening expression cassette containing the gene, a plant genetic transformation screening carrier and applications thereof. Background technique [0002] With the rapid development of genetic engineering and molecular biology technology, the application of transgenic technology is becoming more and more extensive. Transgenic technology has many advantages, such as broadening available genetic resources and creating new germplasm resources; directional and fixed-point variation and selection of plant traits; providing new ways to breed high-yielding, high-quality, and high-resistant varieties. Genetically modified crops were approved for commercial planting in 1996. As of 2017, a total of 23 types of genetically modified crops have been approved for commercial production in the world, involving more than 16 ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N15/82A01H5/00A01H6/46A01H4/00
CPCC12N9/1092C12N15/821C12N15/8275A01H4/001A01H4/008C12Y205/01019
Inventor 安保光欧阳超陈思兰陈欣妍赵惠敏李新鹏龙湍曾翔吴永忠黄培劲
Owner HAINAN BOLIAN RICE GENE TECH CO LTD
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