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 diffi

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
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Example Embodiment

[0033] Example 1 PCR amplification to obtain β related to the resistance to carbendazim of Fusarium graminearum 2 -Tubulin gene

[0034] 1. Obtaining the detection gene: Design primers based on the unnamed full tubulin gene (gene number: FG06611.1) sequence of the reference strain NRRL31084 of wheat head blight. The primers are shown in Table 2. 50μL 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) 1μM, primer Y (downstream primer of the desired amplified fragment) 1μM, Taq (Sheneng Biotech Co., Ltd.) 2.5U, template DNA 100ng. The primer pair uktF1 and uktR1 amplification conditions 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 5min; 94°C for 1min, 54°C for 1min, 72°C for 1min, 3...

Example Embodiment

[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 and put it directly into 0.1 mL of re-distilled water, vigorously shake for 1 min, take out diseased wheat kernels, boil the suspension for 15 minutes, and 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'). 5μL boiled conidia suspension in 50μL reaction system, 5μL 10×buffer, dNTP each 0.2mmol / L, MgCl 2 2mmol / L, each primer 0.5μmol / L, TaqDNA polymerase 2U (Shanghai Promaga Company). The reaction was carried out on PTC-100TM (MJ Research, Inc.) under the reaction conditions: 94°C for 5min; 56°C for 60s, 72°C for 60s, 94°C for ...

Example Embodiment

[0052] Example 3 Rapid detection of resistance of Fusarium graminearum to carbendazim moderately resistant strains and sensitive strains on wheat such as 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 for 3-5 days at 25°C for genomic DNA extraction : Scrape the aerial hyphae grown on potato sucrose medium, add 1.0mL extraction buffer and 0.1~0.5g quartz sand, grind thoroughly, incubate at 60°C for 50min, centrifuge at 10000rpm for 8min, then use the same volume of the supernatant to extract the above The extract was extracted twice, the supernatant was precipitated with an equal volume of isopropanol and dried, then dissolved in 50μL TE, 37℃ water bath for 10min, take 2μL and use 0.7g / 100L agarose electrophoresis to detect all bright bands, indicating that it has been extracted The genomic DNA of the pa...

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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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