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Mutant site of glufosinate-ammonium resisting Eleusine indica species, primer, detection method and application

A mutation site and detection method technology, which is applied in biochemical equipment and methods, microbial measurement/inspection, DNA/RNA fragments, etc., can solve time-consuming and labor-intensive problems, and achieve high detection accuracy, high detection efficiency, and specificity strong effect

Active Publication Date: 2020-05-08
PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the identification of glufosinate-resistant weeds is mainly determined by indoor pot bioassay. The method includes field sampling, seed collection, indoor cultivation, stem and leaf spraying, bioassay and other steps. The cycle is about 2 months, which is time-consuming and labor-intensive.

Method used

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  • Mutant site of glufosinate-ammonium resisting Eleusine indica species, primer, detection method and application
  • Mutant site of glufosinate-ammonium resisting Eleusine indica species, primer, detection method and application
  • Mutant site of glufosinate-ammonium resisting Eleusine indica species, primer, detection method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0053] The glufosinate-ammonium-resistant and sensitive goosegrass glutamine synthetase gene EiGS1-R and EiGS1-S clones provided in this example and the steps for finding the A-175-G mutation characteristics of the EiGS1-R gene are as follows:

[0054] (1.1) RNA extraction of goosegrass, take 3 glufosinate-sensitive goosegrass populations (10 years ago, collected in the farmland where glufosinate has never been applied, multiplied and preserved in this laboratory, available to the public issued for the verification test.) and 1 glufosinate-resistant goosegrass population (collected and screened from the farmland where glufosinate has been applied for a long time, and stored in this laboratory, and can be distributed to the public for verification test) leaf material (3 plants were taken from each glufosinate-ammonium sensitive goosegrass material, a total of 9 plants, and glufosinate-ammonium-ammonium-resistant goosegrass population materials were taken from 6 plants), RNA was ...

Embodiment 2

[0066] The dCAPs primer design for detecting the A-175-G gene mutation site of the glufosinate-resistant goosegrass population is as follows:

[0067] (2.1) Get glufosinate-ammonium-ammonium-resistant type and sensitive type goosegrass blade, adopt CTAB plant genome extraction method, obtain glufosinate-ammonium-ammonium-ammonium-resistant type and sensitive type goosegrass genomic DNA (gDNA) respectively;

[0068] (2.2) Using upstream primer GS1-F (shown in SEQ ID NO: 5) and downstream primer GS1-R (shown in SEQ ID NO: 6), using goosegrass gDNA as a template, clone EiGS1-R and EiGS1-S The genome sequence of the gene (respectively shown as SEQ ID NO: 2 and SEQ ID NO: 1); the PCR reaction system is as follows: 2×PCR Buffer 12.5 μL, 10 μM GS1-F (10 μM) and GS1-R (10 μM) primers Each 1 μL, goosegrass gDNA 2 μL (about 100ng), add 8.5 μL sterile distilled water until the reaction system is 25 μL.

[0069] The PCR program is as follows: 40 cycles at 95°C for 4 minutes, 94°C for 30s...

Embodiment 3

[0076] The detection method of the glufosinate-resistant goosegrass population provided by the present embodiment comprises the following steps:

[0077] (3.1) extract the genomic DNA (gDNA) of the leaves of glufosinate-ammonium-sensitive and resistant goosegrass (10 samples each);

[0078] (3.2) Using the primers dCaps-GS1-F and dCaps-GS1-R designed in Example 2, carry out PCR amplification with the gDNA of (3.1); PCR reaction system and its conditions: 2×PCR Buffer 12.5μL, 10μM 1 μL each of dCaps-GS1-F (10 μM) and dCaps-GS1-R (10 μM) primers, 2 μL (about 100 ng) of goosegrass gDNA, and 8.5 μL of sterile distilled water were added to make the reaction system 25 μL.

[0079] The PCR program is as follows: 40 cycles at 95°C for 4 minutes, 94°C for 30s, 58°C for 30s, and 72°C for 30s, and finally extended at 72°C for 7 minutes.

[0080] (3.3) Digest the PCR product of (3.2) and cut it with KpnⅠ-HF. The reaction system is as follows: 10×NEB. CustmerBuffer 3 μL, KpnⅠ-HF 1 μL, step ...

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Abstract

The invention discloses a mutant site of a glufosinate-ammonium resisting Eleusine indica species. The mutant site is located at a 175th position of a nucleotide sequence shown in SEQ ID NO: 1 of a target enzyme, i.e., glutamine synthetase gene EiGS1-S cloned from glufosinate-ammonium sensitive Eleusine indica, and generated mutation is A-G. The invention further discloses a primer dCAPs for detecting a gene mutation site of the glufosinate-ammonium resisting Eleusine indica species, a detection method for the glufosinate-ammonium resisting Eleusine indica species and application of the primerin preparation of a kit or biological preparation for detecting the glufosinate-ammonium resisting Eleusine indica species.

Description

technical field [0001] The invention belongs to the technical field of glufosinate-resistant beef tendon detection, and in particular relates to a mutation site, a primer, a detection method and an application of a glufosinate-resistant goosegrass population. Background technique [0002] Glufosinate ammonium (phosphinothricin) is a broad-spectrum contact-killing herbicide successfully developed in the 1980s by the former German company Aigerfort (later owned by Bayer). Glufosinate-ammonium is a phosphonic acid herbicide that can inhibit glutamine synthetase in the plant nitrogen metabolism pathway, thereby interfering with plant metabolism and causing plant death. Glufosinate-ammonium, glyphosate, and paraquat are the three largest broad-spectrum herbicides in the world. Among them, the production and sales of glyphosate rank first. However, due to the continuous increase of resistant populations, the global sales growth rate of glyphosate is nearly 5%. Since 2016, paraquat...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6895C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6895C12Q2600/13C12Q2600/156C12Q2531/113C12Q2521/301C12Q2565/125
Inventor 张纯田兴山张泰劼郭文磊冯莉
Owner PLANT PROTECTION RES INST OF GUANGDONG ACADEMY OF AGRI SCI
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