PCR (polymerase chain reaction) detection method and kit of ALS (acetolactate synthetase) inhibitor herbicide-resistant descurainia sophia

A technology of Artemisia indica and herbicides, which is applied in the field of PCR detection methods and kits for anti-ALS inhibitor herbicide Artemisia indica, can solve the problems of inability to detect in season, long time, heavy workload, etc., and meet the requirements of Rapid diagnosis, simple and fast operation, and strong practicability

Active Publication Date: 2014-07-16
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

This method can objectively evaluate the resistance of a certain weed to the corresponding herbicide, but it also has its limitations, mainly in the following two aspects: one is that it cannot be tested in the same season and at that time, and the other is that it takes up a lot of land and time long, heavy workload

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  • PCR (polymerase chain reaction) detection method and kit of ALS (acetolactate synthetase) inhibitor herbicide-resistant descurainia sophia
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  • PCR (polymerase chain reaction) detection method and kit of ALS (acetolactate synthetase) inhibitor herbicide-resistant descurainia sophia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Synthesis of PCR Primers for Detection of Anti-ALS Inhibitor Herbicide Artemisia annua

[0038]In the GenBank database, two ALS genes of Artemisia indica, namely B-ALS-1 (K C417457.1) and B-ALS-2 (FJ715633.1) gene sequences were queried, and primers were designed according to the sequence information found. Biological software, design PCR primers for specific amplification of B-ALS-1 and B-ALS-2 genes respectively, the primer sequences are as follows:

[0039] 1) Primer pair for specific amplification of the B-ALS-1 gene of Artemisia indica:

[0040] B-ALS-1F: 5'-TCTATCTCTCGCTCCTCTCC-3' (Seq ID No.1)

[0041] B-ALS-1R: 5'-CAAACAAACAGCAGTAGCG-3' (Seq ID No.2)

[0042] 2) Primer pair for specific amplification of the B-ALS-2 gene of Artemisia indica:

[0043] B-ALS-2F: 5'-CTTCTTCTCCTCCAACGA-3' (Seq ID No.3)

[0044] B-ALS-2R: 5'-GCCATCTCCCTTCCGTTATGA-3' (Seq ID No.4)

[0045] Primer synthesis was completed by Beijing Liuhe Huada Gene Technology Co., Ltd.

Embodiment 2

[0046] Example 2 PCR detection method of anti-ALS inhibitor herbicide Artemisia indica

[0047] Experimental materials: Artemisia sophia (Descuminia sophia) collected from different regions.

[0048] experimental method:

[0049] 1. Preparation of Artemisia indica DNA

[0050] Take 200 mg of fresh leaves of Artemisia indica, grind with liquid nitrogen, and extract the DNA of each Artemisia indica leaves by conventional CTAB method.

[0051] 2. Specific primers for detecting the mutation sites of Artemisia indica B-ALS-1 and B-ALS-2, see Example 1 for the primer sequences.

[0052] 3. PCR reaction system for detecting the ALS mutation site of Artemisia indica

[0053] 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 sterilized double-distilled water for the rest, with a final volume of 20 μL.

[0054] 4. PCR amplification program for detectin...

Embodiment 3

[0062] Example 3PCR method for detection of Artemisia indica against ALS inhibitors in the field

[0063] The detection method is as follows:

[0064] 1. Sample collection and DNA preparation

[0065] In order to verify the feasibility of the PCR detection method, samples were collected from the farmland where drug-resistant Artemisia indica was suspected to occur, and tested in the laboratory. Plants of Artemisia indica were collected from farmland where resistance was suspected, and after proper moisturizing treatment, they were mailed to the laboratory. Take 200 mg of fresh leaves of Artemisia indica, grind with liquid nitrogen, and extract the DNA of each Artemisia indica leaves by conventional CTAB method.

[0066] 2. Specific primers for detecting the mutation sites of Artemisia indica B-ALS-1 and B-ALS-2, see Example 1 for the primer sequences.

[0067] 3. PCR reaction system for detecting the ALS mutation site of Artemisia indica

[0068] With embodiment 2.

[006...

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Abstract

The invention provides a specific PCR (polymerase chain reaction) primer pair for detecting ALS (acetolactate synthetase) inhibitor herbicide-resistant descurainia sophia. The primer pair comprises a primer pair (Seq ID No.1-2) for specific amplification of a B-ALS-1 gene of the descurainia sophia and a primer pair (Seq ID No.3-4) for specific amplification of a B-ALS-2 gene of the descurainia sophia. A PCR detection method adopting the primer pair is excellent in specificity and sensitivity, and a specificity test proves that the two ALS genes of the descurainia sophia can be amplified (figures 1 and 2). As the PCR method is simple, convenient and quick to operate, the sampling detection can be carried out in season based on nucleotide detection, and only 2-4 days are required for a process from sampling to results. The sensitivity and specificity of the method can realize quick identification of suspended ALS inhibitor herbicide-resistant descurainia sophia in the field and confirmation of mutation sites, thus the scientific control of resistant weeds in production practice is guided.

Description

technical field [0001] The invention relates to a detection technology for weeds resistant to herbicides, in particular to a PCR detection method and a kit for resisting ALS inhibitor herbicides Artemisia indica. 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-methyl, thifensulfuron-methyl, iodosulfuron-methyl, flu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/686C12Q2531/113
Inventor 崔海兰李香菊王藏月
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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