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Detection gene of Fasarium graminearum for resisting carbendazim and its detection method

A technology of Fusarium graminearum and carbendazim, which is applied in the fields of genetic engineering, plant genetic improvement, chemical instruments and methods, etc., can solve the problem of inability to use short-term prediction of drug resistance, limited number of test samples, difficult drug-resistant subgroups, etc. problem, to achieve the effect of effective detection, simple detection and monitoring, and simple method

Inactive Publication Date: 2005-08-24
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method of measuring sensitivity to drugs usually takes a few weeks, and the workload is heavy. The number of samples tested is limited, and it is difficult to detect the existence of drug-resistant subpopulations early, and it cannot be used for short-term prediction of drug resistance in the current year.

Method used

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  • Detection gene of Fasarium graminearum for resisting carbendazim and its detection method
  • Detection gene of Fasarium graminearum for resisting carbendazim and its detection method
  • Detection gene of Fasarium graminearum for resisting carbendazim and its detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 PCR amplification to obtain β related to anti-multibacterial spirituality of Fusarium graminearum 2 -Tubulin gene

[0034] 1. Acquisition of detection genes: primers were designed according to the sequence of the unnamed tubulin whole gene (gene number: FG06611.1) of the wheat head blight reference strain NRRL31084. The primers are shown in Table 2. The 50 μL amplification system contains d 2 h 2 O 37.5μL, dNTP 0.2μM, 10×buffer (10mM Tris-HCl, 50mM KCl, 0.1% Triton 100) 5μL, MgCl 2 2mM, primer X (upstream primer of the desired amplified fragment) 1 μM, primer Y (downstream primer of the desired amplified fragment) 1 μM, Taq (Shenergy Biotechnology Co., Ltd.) 2.5U, template DNA 100ng. The amplification conditions for the primer pair uktF1 and uktR1 were 94°C for 5min; 94°C for 1min, 55°C for 1min, 72°C for 1min, 36 cycles; 72°C for 15min (primer secondary structure removal, hot start). The primer pair uktF2 and uktR2 amplification conditions were 94°C for ...

Embodiment 2

[0045] Example 2 Rapid detection of resistance of Fusarium graminearum to carbendazim-resistant strains and sensitive strains on wheat in Yancheng City, Jiangsu Province in 2004

[0046] Randomly select 100 diseased ears collected in 2004, pick a pink diseased wheat kernel from each ear, put it directly into 0.1mL double-distilled water, vibrate vigorously for 1min, take out the diseased wheat kernels, boil the suspension for 15min, vortex It can be directly used for ASO-PCR detection within 2 minutes.

[0047] ready to use

[0048] Upstream primer MBCRF (5'-TCCCCGATCGCATGATGGCCACCTA-3')

[0049] Downstream primer MBCRR3 (5'-CTTTCGCAGATCCGAGTTGAGCTGT-3'). 5 μL boiled conidia suspension in 50 μL reaction system, 5 μL 10× buffer, 0.2 mmol / L each dNTP, MgCl 2 2mmol / L, 0.5μmol / L each primer, 2U TaqDNA polymerase (Shanghai Promaga Company). The reaction was carried out on PTC-100TM (MJ Research, Inc.), reaction conditions: 94°C 5min; 56°C 60s, 72°C 60s, 94°C 60s, 35 cycles; 72...

Embodiment 3

[0052] Example 3 Rapid Detection of Resistance of Fusarium graminearum to Carbendazim Moderately Resistant and Sensitive Strains on Wheat in Yancheng, Jiangsu Province in 2004

[0053] Randomly select 80 diseased ears collected in 2004, pick the diseased grains or diseased tissues on the diseased ears to the potato sucrose medium under aseptic operation, and culture them at 25 degrees for 3-5 days for genomic DNA extraction : Scrape the aerial mycelia grown on the potato sucrose medium, add 1.0mL extraction buffer and 0.1-0.5g quartz sand, fully grind, incubate at 60°C for 50min, and centrifuge at 10000rpm for 8min, then use the same volume of the supernatant as above The extract was extracted twice, the supernatant was added with an equal volume of isopropanol to precipitate and dried, then dissolved in 50 μL TE, bathed in water at 37°C for 10 minutes, and 2 μL was used to detect bright bands in 0.7g / 100L agarose electrophoresis, indicating that it had been extracted Genomic ...

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Abstract

A Fusarium graminearum beta 2-microtubulin gene for detecting the resistance of Fusarium graminearum to carbendazim and its ASo-RCR detection method are disclosed. The whole length of said gene contains 997 nucleotides, one intron and the drug-resistant mutation site. It has high detecting speed (4 hr) and correctness (99%).

Description

1. Technical field [0001] The present invention is a detection gene and detection method of Fusarium graminearum (Gibberella zeae) resistant to carbendazim, which belongs to the detection method of plant pathogenic bacteria drug-resistant subgroups, and is specially used for detection of resistance to carbendazim and other benzene and imidazole fungicides of Fusarium graminearum. 2. Technical Background [0002] Wheat scab is a worldwide disease caused by Fusarium graminearum, which seriously affects the yield and quality of wheat. For a long time, benzimidazole fungicides or compound agents based on such agents have been used for chemical control. Benzimidazole fungicides are used in production as a class of high-efficiency, broad-spectrum systemic fungicides, which solve the environmental toxicity problem of protective fungicides and improve the ability of humans to control the disease. Benzimidazole fungicides include carbendazim, benomyl, thiabendazole, and thiophanate...

Claims

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Application Information

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IPC IPC(8): C07K14/37C12N15/31C12Q1/68
Inventor 陈长军周明国王建新李红霞
Owner NANJING AGRICULTURAL UNIVERSITY
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