Gene related to eclipta prostrate drug resistance and application thereof

A drug resistance and gene technology, applied in the field of molecular biology, can solve the problems of inability to detect in season, heavy workload, and large land occupation, and achieve the effect of rapid diagnosis, strong practicability, good specificity and sensitivity

Active Publication Date: 2017-12-08
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
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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

Method used

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  • Gene related to eclipta prostrate drug resistance and application thereof
  • Gene related to eclipta prostrate drug resistance and application thereof
  • Gene related to eclipta prostrate drug resistance and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0066] Example 1 Cloning of Snakehead ALS Gene

[0067] Search for Helianthus annuus, accession number AY541454.1, cocklebur (Xanthium sibiricum, accession number U16280.1), and Conyza Canadensis (Conyza Canadensis, accession number HM067014), which are closely related to the snakehead in the NCBI website. 1) The ALS of Anthemis cotula (Accession No. JF327754.1) was used as a reference sequence, and the ALS gene of Arabidopsis thaliana (Accession No. AY042819.1) was used for sequence comparison. According to the principle of primer design, use DNA MAN6.0 software to design the first set of primers, see Table 1.

[0068] Table 1 Primers for amplification of ALS gene in snakehead intestine (first group)

[0069] direction

[0070] The PCR amplification system (15μl) is as follows: Channa genomic DNA 0.5μl, 10×PCR Buffer 1.5μl, 2.5mMdNTP Mixture 1μl, 10μM forward primer 0.3μl, 10μM reverse primer 0.3μl, Taq DNA polymerase 0.3μl, ddH 2 O11.1μl.

[0071] The temperature gradient PCR re...

Example Embodiment

[0119] Example 2 Design of PCR primers for detecting the anti-ALS inhibitor herbicide Channa intestines

[0120] According to the full-length sequence of the snakehead ALS gene and the principle of primer design, two pairs of primers were designed using DNA MAN6.0 software to amplify the full length of the snakehead ALS gene, as shown in Table 9.

[0121] Table 9 Primers for amplifying the ALS gene of snakehead intestine

[0122]

[0123]

[0124] The PCR amplification system (15μl) and reaction conditions were the same as the first group of PCR. Take 3μl of PCR reaction solution and use 1% Agarose gel electrophoresis, the primer pair temperature gradient amplification results are as follows Picture 9 , 10 As shown, the amplification effect is good, and the sequencing result shows the ALS gene sequence of Channa intestines, and the fragments cover eight SNP sites related to drug resistance: A122, P197, A205, D376, R377, W574, S653 and G654.

Example Embodiment

[0125] Example 3 PCR detection method of anti-ALS inhibitor herbicide Channa intestines

[0126] Experimental materials: Channa intestines collected from different regions.

[0127] Experimental method: ①Preparation of snakehead intestine DNA Take 200 mg of fresh snakehead intestine leaves, ground with liquid nitrogen, and extract DNA from each snakehead intestine leaves by conventional CTAB method.

[0128] ②Specific primers used to detect the mutation sites of LC-ALS in snakehead intestines. See Table 9 in Example 2 for the primer sequence.

[0129] ③The PCR reaction system used to detect the mutation site of ALS in the snakehead is shown in the first set of PCR in Example 1.

[0130] ④ The PCR amplification program for detecting ALS mutation sites in snakehead intestine is shown in the first set of PCR in Example 1, primer pair I and primer pair II.

[0131] ⑤Identification of PCR products Take 3μL of PCR products, separate them with 1% (weight / volume) agarose gel electrophoresis, sta...

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Abstract

The invention relates to a molecular marker related to eclipta prostrate drug resistance and application thereof, and the molecular marker is CCC-TCC base mutation of codon, encoded by LC-ALS gene shown in amino acid sequence such as SEQ ID NO.1, of the 176th amino acid of the amino acid sequence shown in SEQ ID NO.2. The invention further provides specific primers and a method for detecting the molecular marker, sequence of the specific primer pair is as shown in SEQ ID NO. 3-4 and SEQ ID NO. 5-6, field suspected anti-ALS inhibitor eclipta prostrate can be rapid identificated and mutation sites can be confirmed by adopting the primer pair, so that scientific management for resistance eclipta prostrate can be guided in production practice, and the molecular marker related to eclipta prostrate drug resistance has good specificity and sensitivity; and the method is accurate and reliable, and operation is easy.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a gene LC-ALS related to the drug resistance of the snakehead intestine, a SNP molecular marker related to the drug resistance of the snakehead intestine, and a method for detecting the LC-ALS gene. 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 metsulfuron-me...

Claims

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Application Information

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IPC IPC(8): C12N15/60C12N9/88C12Q1/68C12N15/11
CPCC12N9/88C12Q1/6895C12Q2600/13C12Q2600/156C12Y401/03
Inventor 崔海兰李丹李香菊于惠林张宏军
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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