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PCR detection method of photosystem II inhibitor herbicide resistant digitaria sanguinalis

A technology of inhibitors and herbicides, which is applied in the field of PCR detection methods and kits for the photosystem II inhibitor herbicide crabgrass, which can solve the problem of affecting the herbicide's herbicide effect, the decrease of the binding ability of D1 protein and herbicide, etc. problems, to achieve rapid detection, good specificity, and high efficiency

Inactive Publication Date: 2016-11-16
INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Referring to the sequence of the protein encoded by the psbA gene in Arabidopsis, mutations at amino acid positions 219, 228, 264, and 268 will lead to a decrease in the binding ability of the D1 protein to herbicides, thereby affecting the herbicide’s herbicidal effect and resulting in inhibition of PSⅡ. Generation of herbicide-resistant weeds

Method used

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  • PCR detection method of photosystem II inhibitor herbicide resistant digitaria sanguinalis
  • PCR detection method of photosystem II inhibitor herbicide resistant digitaria sanguinalis

Examples

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Effect test

Embodiment 1

[0037] Example 1 Synthesis of PCR primers for detecting resistance to photosystem II inhibitor herbicide crabgrass.

[0038] Inquire about the C. psB Gene (GenBank: KM453691.1) gene sequence, according to the queried sequence information, use the primer design software (Primer 5.0) to design a specific primer pair for PCR amplification of the psbA gene of crabgrass. The primer sequences are as follows:

[0039] MT-psbA-F:5'-GGTGTAGCTTGTTATATGGGTCGT-3'(Seq ID No.1)

[0040] MT-psbA-R:5'-GCCCAAGTATTAATAACACGTCC-3'(Seq ID No.2)

[0041] Primer synthesis was completed by Shanghai Bioengineering Co., Ltd.

Embodiment 2

[0042] The preparation of embodiment 2 PCR kit

[0043] According to the specific primer pair obtained in the embodiment, the necessary reagents for PCR reaction: dNTPS, TaqDNA polymerase and PCR reaction buffer containing Mg2+ were added to the kit, and combined with the synthesized primer pair, a PCR detection kit was made.

Embodiment 3

[0044] Example 3 PCR detection method for resistance to photosystem Ⅱ inhibitor herbicide crabgrass.

[0045] Planting or collection of crabgrass plants: the detected plants of the present invention can be field crabgrass plants in the current season, or plants planted with crabgrass seeds can be used as testing materials.

[0046] Preparation of the DNA of crabgrass crabgrass: 100-300 mg of fresh leaves of crabgrass crabgrass were taken, and the DNA of crabgrass crabgrass leaves was extracted by CTAB method.

[0047] PCR reaction system: use 25 μL of PCR reaction system, including 2.5 μL of 10×PCR reaction buffer containing Mg2+, 1 μL of 2.5 mM dNTPs, 1 μL of 10 μM primers, 2 U of TaqDNA polymerase, 1 μL of DNA template, sterilized Make up to a final volume of 25 μL with double distilled water.

[0048]PCR amplification program: pre-denaturation at 94°C for 4 min; denaturation at 94°C for 0.5 min, annealing at 53°C for 0.5 min, extension at 72°C for 1 min, a total of 30 cycl...

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Abstract

Photosystem II inhibitor herbicides belong to a main category in chemical herbicides, and psbA genes are target acting sites. The invention provides a specificity PCR (Polymerase Chain Reaction) primer pair (Seq ID No.1 and Seq ID No.2) for detecting the photosystem II inhibitor herbicide resistant digitaria sanguinalis and a PCR detection kit comprising the primer pair. The primer pair provided by the invention has the advantages of high specificity and high sensitivity; PCR experiments prove that (Figure 1) the psbA gene sequence of the digitaria sanguinalis can be amplified; and the amplified sequence contains reported all sites relevant to the resistance. The method has the advantages that the sensitivity and the specificity can realize the fast identification on the field suspected photosystem II inhibitor herbicide resistant digitaria sanguinalis, so that the guidance on the scientific management of resistance weeds in production practice can be realized.

Description

technical field [0001] The invention relates to the field of detection of herbicide-resistant weeds, in particular to a PCR detection method and kit for resistance to photosystem II inhibitor herbicide crabgrass. Background technique [0002] Crabgrass ( Digitaria sanguinalis ) is one of the main malignant weeds in my country's Huanghuaihai corn planting areas. Of the 7 main weed groups in the cornfields in the Huang-Huai-Hai area, 6 of the groups had crabgrass as the dominant species. At present, herbicides such as atrazine are mainly used for chemical control of crabgrass. However, due to the single molecular target of this type of herbicide, long-term use can easily lead to weed resistance. In recent years, the problem of resistant crabgrass has become very prominent in some corn fields in Huanghuaihai and other places in my country. For example, in some areas, spraying atrazine with conventional doses has been unable to effectively control crabgrass. [0003] If weed...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895
Inventor 李健李美高兴祥房锋
Owner INST OF PLANT PROTECTION SHANDONG ACAD OF AGRI SCI
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