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Shepherd's purse drug resistance-related molecular marker and detection kit thereof

A technology of molecular markers and kits, which is applied in the determination/testing of microorganisms, DNA/RNA fragments, recombinant DNA technology, etc., to achieve the effects of simple and fast operation, fast diagnosis, and strong practicability

Active Publication Date: 2015-03-04
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the research report, only one ALS gene of shepherd's purse was cloned, but we found in the study that shepherd's purse has two ALS genes, named JC-ALS-1 and JC-ALS-2, both of which have functions, and any A gene mutation in a conserved region can cause drug resistance

Method used

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  • Shepherd's purse drug resistance-related molecular marker and detection kit thereof
  • Shepherd's purse drug resistance-related molecular marker and detection kit thereof
  • Shepherd's purse drug resistance-related molecular marker and detection kit thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0037] Example 1 Synthesis of PCR primers for detection of anti-ALS inhibitor herbicide shepherd's purse

[0038] Query the ALS gene sequence of shepherd's purse in the GenBank database (GenBank accession number: H Q880660.1), according to the sequence information found, use the primer design biology software to design for the specific amplification of JC-ALS-1 and JC-ALS -2 PCR primers for the conserved region sequence, and the two ends of the sequence were obtained by 3'RACE and chromosome walking methods, and finally the two ALS gene sequences of shepherd's purse were obtained.

[0039] The primer sequences are as follows:

[0040] Forward primer JC-ALS-F: 5'-TATTCGCTTACCCAGGTG-3';

[0041] Reverse primer JC-ALS-R: 5'-GGTTCTGAGTTTCATCTCTCA-3'.

[0042] Primer synthesis was completed by Beijing Liuhe Huada Gene Technology Co., Ltd.

Embodiment 2

[0043] Example 2 PCR detection method of anti-ALS inhibitor herbicide shepherd's purse

[0044] Experimental materials: Shepherd's purse (Capsella bursa-pastoris) collected from different regions.

[0045] experimental method:

[0046] 1. Preparation of shepherd's purse DNA

[0047] Take 200 mg of fresh leaves of shepherd's purse, grind with liquid nitrogen, and extract the DNA of each shepherd's purse leaf by conventional CTAB method.

[0048] 2. Specific primers for detecting mutation sites of shepherd's purse JC-ALS-1 and JC-ALS-2, see Example 1 for the primer sequences.

[0049] 3. PCR reaction system for detecting ALS mutation site of shepherd's purse

[0050] PCR reaction system, in which 10×PCR reaction buffer 2μL, 15mM MgCl 2 0.5 μL, 1 μL of 2.5 mM dNTPs, 0.5 μL of each 10 μM primer, 1 U of Taq DNA polymerase, 1 μL of DNA template, and the rest in sterile double distilled water, the final volume is 20 μL.

[0051] 4. PCR amplification program for detecting ALS mut...

Embodiment 3

[0060] Embodiment 3 PCR method detects the shepherd's purse of anti-ALS inhibitor in the field

[0061] The detection method is as follows:

[0062] 1. Sample collection and DNA preparation

[0063] In order to verify the feasibility of the PCR detection method, samples were collected from farmland where drug-resistant shepherd's purse was suspected to have occurred for two consecutive years in 2013 and 2014, and tested in the laboratory. Tribesulfuron-methyl was applied once to control shepherd's purse in these fields after the wheat turned green, but it was ineffective, so it was suspected that there might be resistance to tribenuron-methyl. Therefore, the shepherd's purse plants were collected from these suspected drug-resistant farmlands, and after proper moisturizing treatment, they were mailed to the laboratory for testing. Take 200 mg of fresh leaves of shepherd's purse, grind with liquid nitrogen, and extract the DNA of each shepherd's purse leaf by conventional CTAB...

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Abstract

The invention provides a shepherd's purse drug resistance related molecular marker and a specific PCR (polymerase chain reaction) primer pair for detecting anti-acetolactate synthase inhibitor weedicide shepherd's purse. The PCR primer pair comprises a primer pair for specifically amplifying shepherd's purse JC-ALS gene. The PCR detection method by using the primers has excellent specificity and sensitivity, can implement quick identification of suspected anti-ALS inhibitor shepherd's purse in the field and conformation of mutant sites, thereby instructing the scientific treatment on resistant weed in production practice.

Description

technical field [0001] The invention relates to a detection technology for herbicide-resistant weeds, in particular to a molecular marker related to drug resistance of shepherd's purse and a detection kit thereof. Background technique [0002] Acetolactate synthase (ALS for short), also known as acetohydroxyacid synthase (AHAS for short), is a biosynthetic process that induces valine, leucine, and isoleucine in plants and microorganisms. A key enzyme and an important herbicide target. The development and use of ALS inhibitor herbicides began in the early 1980s. Due to the obvious advantages of such herbicides, such as super high activity, low toxicity to humans and animals, and environmental friendliness, they have attracted widespread attention. At present, 50 Multiple varieties were developed and used. Since the 1980s, my country has successively promoted the use of tribenuron-methyl, metsulfuron-methyl, chlorsulfuron, florasulam, acesulfame, etc. in winter wheat fields ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q2600/13C12Q2600/156
Inventor 崔海兰李香菊张宏军
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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