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Molecular marker related with chemical resistance of shepherd's purse herbs and detection kit of molecular marker

A kit and drug resistance technology are applied in the field of molecular markers and detection kits to achieve the effects of rapid diagnosis, strong practicability, and simple and quick operation.

Active Publication Date: 2016-06-29
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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  • Molecular marker related with chemical resistance of shepherd's purse herbs and detection kit of molecular marker
  • Molecular marker related with chemical resistance of shepherd's purse herbs and detection kit of molecular marker
  • Molecular marker related with chemical resistance of shepherd's purse herbs and detection kit of molecular marker

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Effect test

Embodiment 1

[0037] Embodiment 1 is used for detecting the synthesis of the PCR primer of anti-ALS inhibitor class herbicide shepherd's purse

[0038] Query the shepherd's purse ALS gene sequence (GenBank accession number: HQ880660.1) in the GenBank database, according to the sequence information found, use primer design biology software, design for specific amplification JC-ALS-1 and JC-ALS- The PCR primers of the conserved region sequences of 2 were used, and the sequences at both ends were obtained by 3'RACE and chromosome walking methods, and finally the two ALS gene sequences of shepherd's purse were obtained respectively.

[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] The PCR detection method of embodiment 2 anti-ALS inhibitor class herbicide shepherd's purse

[0044] Experimental material: Shepherd's purse (Capsellabursa-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 TaqDNA polymerase, 1 μL of DNA template, and the rest in sterile double-distilled water, with a final volume of 20 μL.

[0051] 4. PCR amplification program for dete...

Embodiment 3

[0060] Embodiment 3PCR 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 is a divisional application of a patent application named molecular marker related with chemical resistance of shepherd's purse herbs and detection kit of molecular marker in the number of 201410643602.9 and provides a specific DNA (deoxyribonucleic acid) fragment related with chemical resistance of shepherd's purse herbs and a specific PCR (polymerase chain reaction) primer pair of the shepherd's purse herbs resisting ALS (acetolactate synthase) inhibitor herbicides, wherein the specific PCR primer pair comprises a primer pair used for specifically amplifying JC-ALS genes of the shepherd's purse herbs. A PCR detection method adopting the primers has great specificity and sensitivity, can quickly identify the shepherd's purse herbs probably resisting ALS inhibitors in fields and confirm mutation sites and accordingly provides an instruction for scientific treatment of resistant weeds in production practices.

Description

technical field [0001] The present invention is a divisional application of the patent application with the application number 201410643602.9 and the title "Molecular markers related to drug resistance of shepherd's purse and its detection kit". Molecular markers related to drug resistance and their detection kits. Background technique [0002] Acetolactate synthase (ALS for short), also known as acetohydroxyacid synthase (AHAS for short), is the key to inducing the biosynthesis of valine, leucine and isoleucine in plants and microorganisms. The enzyme is also 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 ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/54C12N9/10C12N15/11C12Q1/68
Inventor 崔海兰李香菊张宏军
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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