CN103409533B - Detect primer and the method for the F.graminearum schw carbendazim resistance reference point sudden change E198K frequency of occurrences - Google Patents
Detect primer and the method for the F.graminearum schw carbendazim resistance reference point sudden change E198K frequency of occurrences Download PDFInfo
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Abstract
The invention discloses a kind of primer and the method that detect the F.graminearum schw carbendazim resistance reference point sudden change E198K frequency of occurrences, does is the base sequence of described primer: forward primer: 5 '-AGC TCG TCG AGA ACT CTG AAA-3 '; Reverse primer: 5 '-GCA GCG GCC ATG ATG TTC TT-3 '.The invention also discloses the detection method of a kind of F.graminearum schw carbendazim resistance reference point sudden change E198K frequency of occurrences.Primer specificity of the present invention is strong, and detection method of the present invention can grasp the frequency of occurrences of this mutation type in field and dynamic change rapidly and accurately, for Disease management and resistance management provide technical support.
Description
Technical Field
The invention relates to the field of molecular biology, in particular to a primer and a method for detecting the occurrence frequency of a fusarium graminearum carbendazim resistance-related point mutation E198K.
Background
In recent years, due to the change of straw returning and climate factors, the common growing area of wheat scab caused by fusarium graminearum (fusarium graminearum) in China is expanded from the middle and lower reaches of Yangtze river to the north direction to the main growing areas of Huang-Huai wheat such as Shandong, Henan and Hebei, the disease area in China in 2010 exceeds 1 hundred million mu, and the gibberellic disease toxin in wheat harvested in part of areas seriously exceeds standard and is disastrous. Because of the lack of germplasm materials with high-level resistance, the chemical carbendazim is mainly used for preventing and treating wheat scab in the current production. However, due to the long-term use of a large amount of the medicines (benzimidazoles), the carbendazim resistance problem occurs in regions such as Zhejiang, Jiangsu and Anhui.
The 6 th, 50 th, 167 th, 198 th, 200 th or 240 th point mutation of beta-tubulin is the main reason causing the majority of pathogenic bacteria to generate the drug resistance of benzimidazole germicide. Fusarium graminearum exhibits low, medium and high levels of three types of resistance to carbendazim in terms of resistance index (RF), where 1 ≦ RF <10 is low resistance, 10 ≦ RF <50 is medium resistance, and 50 ≦ RF <100 is high resistance. It has been shown that different point mutations in β -tubulin correspond to different levels of resistance: e (GAG) 198L (CTG) results in high levels of resistance, F (TTT) 167Y (TAT), F (TTC) 200Y (TAC) and E (GAG) 198K (AAG) results in medium levels of resistance, E (GAG) 198Q (CAG) results in low levels of resistance. Point mutations F167Y, E198/Q/L and F200Y have been reported in field carbendazim-resistant Fusarium graminearum studies. In 2011 Qiau et al obtained two carbendazim-resistant strains containing new point mutations (Y50C and E198K) by site-directed mutagenesis method laboratories (Qiaetal., 2011, PestManagementscience,67: 191-198). When 2012 performed the carbendazim resistance test on 4320 gibberellic disease samples collected in the field, the subject group found a resistant strain containing the E198K point mutation in Jingjiang wheat area of Jiangsu, which indicates that the resistant strain containing the point mutation has appeared in the field fusarium graminearum population.
Aiming at the point mutation E198K, Chinese patent document with publication number CN101988122A discloses a typing detection method of fusarium graminearum to carbendazim drug resistance genotype, which comprises the following steps: extracting nuclear genome DNA of a strain to be detected; carrying out PCR reaction by using three groups of primer pairs; and (3) identifying the genotype of the bacterial strain for the carbendazim resistance according to the PCR product electrophoresis pattern. In this patent, it is disclosed that the point mutation can be qualitatively detected by using the Tetra-primaryarms-PCR method, but the point mutation cannot be quantitatively detected.
Therefore, a detection technology is needed to be established, the point mutation can be quantitatively detected, the occurrence frequency and the change dynamic state of the population containing the point mutation in the field can be accurately detected, the method has guiding significance for effective prevention and treatment of diseases and reasonable use of pesticides, and meanwhile, a foundation is provided for research on dynamic changes of different types of resistant populations under medicament screening pressure.
Disclosure of Invention
The invention provides a primer for detecting the occurrence frequency of E198K point mutation related to fusarium graminearum carbendazim resistance, and the specificity is strong.
A primer, the base sequence is:
a forward primer: 5'-AGCTCGTCGAGAACTCTGAAA-3', respectively;
reverse primer: 5'-GCAGCGGCCATGATGTTCTT-3' are provided.
The invention also provides a method for detecting the frequency of the point mutation E198K related to the resistance of fusarium graminearum carbendazim, which is characterized by comprising the following steps:
(1) sampling, namely mixing the samples and extracting DNA;
(2) performing real-time fluorescent quantitative PCR amplification by using the DNA as a template and the primer according to claim 1 to obtain the weight of the DNA containing the E198K point mutation in the mixed sample;
(3) designing a primer according to actin gene of fusarium graminearum, and carrying out real-time fluorescent quantitative PCR amplification by using the DNA as a template to obtain the weight of the total DNA in the mixed sample;
(4) the frequency of occurrence of the carbendazim resistance-associated point mutation E198K was calculated according to the following formula:
FREE198K=QE198K/QFg×100%；
wherein, FREE198KThe frequency of occurrence of the point mutation E198K related to carbendazim resistance, QE198KThe weight of DNA containing the point mutation E198K, Q, in the mixed sampleFgIs the weight of total DNA of Fusarium graminearum in the mixed sample.
The frequency of occurrence of E198K point mutation related to the resistance of the fusarium graminearum carbendazim is the frequency of occurrence of a fusarium graminearum E198K mutant population, and in order to ensure accurate results, sampling is carried out according to a biometrical method.
Since the present application reflects the frequency of E198K point mutations associated with Fusarium graminearum resistance as a ratio of DNA weights, one skilled in the art will appreciate that the weights of the different samples should be kept as consistent as possible when mixing the samples prior to DNA extraction.
In the step (1), the method for extracting DNA comprises the following steps:
(a) collecting mycelium or ascocarp shell, mixing, adding extractive solution, and grinding;
(b) standing the ground mixed solution for a period of time, centrifuging and taking supernatant;
(c) precipitating the supernatant with alcohol, and washing the precipitate to obtain the DNA;
when extracting DNA from hyphae, the extract comprises: 200mM Tris-HCl, 50mM EDTA, 20mM NaCl and 1% SDS;
when extracting DNA from an ascocarp, the extract comprises: 1-2% polyvinylpyrrolidone, 200mM Tris-HCl, 50mM EDTA, 200mM NaCl and 1% SDS.
The alcohol is ethanol or isopropanol.
The DNA extraction method is simple and quick, does not need the traditional DNA extraction and purification steps, and does not influence the accuracy of subsequent detection.
In the steps (2) and (3), the real-time fluorescent quantitative PCR amplification system comprises: 1 mu LDNA template, 0.4uL of forward primer and reverse primer of 10 mu mol/L respectively,PremixExTaqTM10 μ L of water was added to 20 μ L.
In the steps (2) and (3), the real-time fluorescent quantitative PCR amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 63 ℃ for 15s, and extension at 72 ℃ for 20s, for 40 cycles.
Actin, an important framework protein of cells, is highly conserved among different organisms, is amplified by designing primers according to Actin genes, and can quantify the total weight of fusarium graminearum.
In the step (3), the base sequence of the primer is as follows:
a forward primer: 5'-ATCCACGTCACCACTTTCAA-3'
Reverse primer: 5' -TGCTTGGAGATCCACATTTG-3.
Compared with the prior art, the invention has the beneficial effects that:
(1) the primer provided by the application is designed aiming at beta-tubulin gene (GenBank accession number is KC 765119) of the anti-carbendazim Fusarium graminearum and mutation site E (GAG) 198K (AAG), wherein the designed forward primer is positioned at the mutation site, and the base at the 3' end is specially designed, so that the mismatching probability of the anti-bacterial strain and the low-resistant strain of the anti-carbendazim sensitive strain, the high-resistant strain and the mutation site F (TTT) 167Y (TAT) or F (TTC) 200Y (TAC) is increased, and the specificity of the anti-carbendazim Fusarium graminearum of the anti-carbendazim Fusarium with the mutation site E (GAG) 198K 198 AAG) is improved; the designed reverse primer is positioned at the 1203-1222 th basic group of the beta-tubulin gene and is specific to fusarium graminearum; and the amplified fragment is 337bp in size and is easy to amplify. Therefore, after selecting a proper target sequence and reasonably designing the base sequence of the primer, the primer is used for PCR amplification, namely the DNA of the fusarium graminearum strain which is resistant to carbendazim and has a mutation site (GAG) 198K (AAG) can be amplified, and the specificity of the primer is high.
In addition, the primer of the invention can amplify DNA sequences with the mutation site (GAG) 198K (AAG) in carbendazim from hypha and rice pileus shell (main field infection source).
(2) The detection method can quantitatively detect (GAG) 198K (AAG) point mutation, can accurately detect the occurrence frequency and change dynamic of the population containing the point mutation in the field, has quick and accurate detection, has guiding significance for effective prevention and treatment of diseases and reasonable use of pesticides, and simultaneously provides a foundation for research on dynamic change of different types of resistant populations under medicament screening pressure.
Drawings
FIG. 1 shows the sequence of the primer of the present invention at the position of the beta-tubulin gene;
FIG. 2 is a map of the position of the primer sequences of the present invention on the beta-tubulin gene sequence;
FIG. 3 is an electrophoretogram for specific detection of primers of the present invention;
FIG. 4 shows a standard curve (A) for quantitative detection of E198K point mutation by the primer Fg198K-FC/Fgtub2-R and a standard curve (B) for quantitative detection of all Fusarium graminearum DNA by the primer F-actin-F/F-actin-R.
Detailed Description
The present invention will be further explained with reference to specific embodiments.
Example 1
1. Type of Strain
Fusarium oxysporum (f.oxysporum), fusarium avenaceum (f.avenacrum), fusarium acuminatum (f.acuminatum), fusarium semitectum (f.semitectum), fusarium moniliforme (f.subglutinans), fusarium flavum (f.culmorum), and 10 fusarium graminearum (f.graminearum) strains. The 10 Fusarium graminearum strains included 2 susceptible strains (PH-1, HN 9), 2 resistant strains with a mutation at E198Q (YS 13, BM 2), 2 resistant strains with a mutation at F167Y (JM 12, JL 22), 2 resistant strains with a mutation at F200Y (JQ 82, HA 16), 1 resistant strain with a mutation at E198K (JL 45), and 1 resistant strain with a mutation at E198L (JM 2).
The strains are all common plant pathogenic fungi, are stored in institute of biotechnology of Zhejiang university, and can also be obtained by conventional fungus separation and purification methods.
2. Primer synthesis
E (GAG) 198K (AAG) point mutation on beta-tubulin can cause middle-level resistance to carbendazim, and based on base change of beta-tubulin gene (Genebank accession No. KC 765119), point mutation E (GAG) 198K (AAG), a forward PCR primer Fg198K-FC is designed, the 3' end base A of the primer pairs with the mutant base A in the anti-strain with the mutation site E (GAG) 198K (AAG), but does not pair with the mutant base A in the sensitive strain, the high-resistant strain, the anti-strain with the mutation site F (TTT) 167Y (TAT) or F (TTC) 200Y (TAC), and the base G in the low-resistant strain.
To further improve the specificity of the PCR primers, the second base from the reciprocal of the 3' end of the primer is changed from G to A (Fg 198K-FC), so that there are two base mismatches for sensitive strains, high-resistance strains, resistant strains with mutation sites of F (TTT) 167Y (TAT) or F (TTC) 200Y (TAC) and low-resistance strains, and the specificity is higher than that of primers with only one base mismatch under the same PCR reaction conditions. The reverse primer Fgtub2-R sequence is located at 1203-1222 th bases of beta-tubulin gene, and the bases are found to be specific to Fusarium graminearum by alignment at NCBI. Thus, using the Fg198K-FC/Fgtub2-R allele-specific PCR primers, bands were amplified only from the F.graminearum strain containing the E198K point mutation, and not from any bands from the carbendazim-resistant, carbendazim-sensitive and other fungi strains containing other point mutations (the sequences in which the primers are located are shown in FIG. 1).
The specific sequence of the primer and the sequence in the sequence table are shown in the following table 1:
TABLE 1
The physical positions of the primers on the beta-tubulin gene sequence are shown in FIG. 2.
The primer of the sequence is synthesized by Shanghai Bocai.
3. Extraction of DNA
The invention adopts a simple and rapid DNA extraction method, which comprises the following specific operations:
(1) scraping hyphae (100 mg) from a PDA plate by using an inoculating needle, and placing the hyphae in a 1.5mLEppendorf tube;
(2) adding 500 μ LDNA into EP tube to extract lysate (200 mM Tris-HCl, 50mM EDTA, 20mM NaCl, 1% SDS, pH8.0), grinding with electric drill, shaking, mixing, standing at room temperature for 10 min; centrifuging at 13200r/min at 4 ℃ for 5 min;
(3) taking about 400 μ L of the supernatant, putting into a new 1.5mL LEPPendorf tube, adding 750 μ L of absolute ethanol, mixing, and centrifuging at 4 deg.C and 13200r/min for 5 min;
(4) removing supernatant, washing precipitate with 70% ethanol, drying at room temperature for 5-10min, dissolving in 30 μ LTE (pH 8.0), and storing at-20 deg.C.
The DNA of the 16 fungal strains and the DNA of the test bacteria can be extracted by the method.
4. Primer specificity verification
PCR reactions were carried out using 16 fungal DNAs extracted as described above as templates and primers Fg198K-FC/Fgtub2-R, each reaction containing a negative control (sterile water was used in place of the DNA template).
The PCR amplification system is as follows: 1 μ LDNA template (ca. 0.4 ng), 0.2 μmoll of each primer－1，dNTP0.2μmoll－1，MgCl22mmoll－11 × buffer solution (manufactured by Touchen, Beijing) and 1.5U polymeraseThen, the solution is made up to 25. mu.L with double distilled water.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 94 ℃ for 30s, annealing at 63 ℃ for 30s, extension at 72 ℃ for 30s, 35 cycles, and final extension at 72 ℃ for 5 min.
The PCR product was electrophoresed in 1 XTAE buffer with 1.5% agarose, and photographed by EB color development.
From the electrophotograph (as shown in FIG. 3), only the carbendazim-resistant strain JL45 containing the E198K point mutation gave a band of 337bp, while the other 7 carbendazim-resistant strains containing the point mutation (YS 13 and BM2 contain E198Q, JM12 and JL22 contain F167Y, JQ82 and HA JM 16 contain F200Y, and E198L in JM 2), 2 multi-strain susceptible strains (PH-1, HN 9) and other 6 pathogenic fungi did not amplify any band. Therefore, the pair of allele-specific PCR primers were verified to be capable of specifically detecting the E198K point mutation of Fusarium graminearum.
5. Establishing a standard curve for quantitatively detecting E198K point mutation related to the resistance of fusarium graminearum carbendazim and total DNA of fusarium graminearum
DNA of a Fusarium graminearum sensitive strain PH-1 and a carbendazim resistant strain JL45 containing E198K point mutations (as described above for fungal DNA extraction) was extracted and the extracted DNA was diluted to the following concentrations, respectively: 424 ng/. mu.L, 42.4 ng/. mu.L, 4.24 ng/. mu.L, 424 pg/. mu.L, 42.4 pg/. mu.L. The primer Fg198K-FC/Fgtub2-R of the invention is used for carrying out real-time fluorescent quantitative PCR amplification on DNA of different gradients JL 45. Real-time fluorescent quantitative PCR amplification was performed on DNA at different gradients of pH-1 using the reported primers F-actin-F/F-actin-R (Yin et al, Phytopathology,2009,99: 487-. Two replicates per concentration gradient, and three replicates of the entire PCR amplification. Wherein,
F-actin-F：5’-ATCCACGTCACCACTTTCAA-3’；
F-actin-R：5’-TGCTTGGAGATCCACATTTG-3’。
PCR amplification System: 1 μ LDNA template, 0.4uL each of primers (10 μmoll)－1），PremixExTaqTM(manufactured by TaKaRa Co., Ltd.) 10. mu.L of double distilled water was made up to 20. mu.L.
The PCR reaction conditions are as follows: pre-denaturation at 95 ℃ for 3min, denaturation at 95 ℃ for 15s, annealing at 63 ℃ for 15s, and extension at 72 ℃ for 20s, for 40 cycles. The reaction was carried out on an Eppendorf MastercycleRealplex 2PCR instrument and after the reaction was complete, a standard curve was established.
The PCR amplification system using F-actin-F/F-actin-R as primer and Fg198K-FC/Fgtub2-R as primer has the same conditions.
6. Detecting the occurrence frequency of carbendazim resistance related point mutation E198K in rice stump cyst shells in different regions
In 4 months of 2012, ascocarp is collected from rice stakes in Jiangsu Haian and Jingjiang, Anhui Mesona Chang and Chizhou wheat fields, and 30-90 ascocarp per region. The method comprises the following specific operations: scraping the same volume of the ascocarp from each cluster of rice stumps, and dividing the ascocarp into two parts, wherein one part is used for real-time fluorescent quantitative PCR determination, and the other part is used for conventional medicament sensitivity and common PCR determination.
The ascospores collected on the rice pegs were placed in 1.5ml LEPPendorf tubes, 100. mu.l of DNA extraction buffer (1-2% polyvinylpyrrolidone (PVP), 200mM Tris-HCl, 50mM EDTA, 200mM NaCl, 1% SDS) was added as ddH2O is solvent, pH8.0), grinding for 1min with a pointed glass rod installed on a common electrician flashlight, adding 400 μ l of DNA extraction buffer solution into the centrifuge tube, mixing, and standing at room temperature for 10 min; centrifuging the mixed solution at 4 deg.C and 15000r/min for 5min, transferring the supernatant into another centrifuge tube, adding 750 μ l anhydrous ethanol, mixing, centrifuging at 4 deg.C and 15000r/min for 2min, and removing the supernatant; washing the precipitated DNA with 70% ethanol, drying at room temperature, and dissolving in 50 μ l sterile water to obtain extracted DNA; the DNA solution was purified by column chromatography using UNIQ-10 column PCR product recovery kit (manufactured by Shanghai)
When extracting DNA, the ascocapsids used for real-time fluorescent quantitative PCR determination in the same region are mixed together for extracting DNA.
The primers Fg198K-FC/Fgtub2-R and the reported primers F-actin-F/F-actin-R are used for carrying out real-time fluorescent quantitative PCR amplification on 4 field sample DNAs respectively, and the system and conditions of the fluorescent quantitative PCR amplification are as described in 5 (establishing a standard curve for quantitatively detecting E198K point mutation related to the resistance of fusarium graminearum and the total DNA of fusarium graminearum). The amount of DNA (Q) containing the E198K point mutation in the field samples was calculated based on the standard curve established aboveE198K) And the amount of total DNA (Q)Fg). Calculating the frequency of occurrence (FRE) of the point mutation E198K related to the resistance of fusarium graminearum carbendazim by using a formulaE198K），FREE198K=QE198K/QFg×100%。
TABLE 2
As shown in table 2, in four regions, the carbendazim-resistant fusarium graminearum containing the point mutation E198K was detected only in field samples collected in jing jiang su, and the frequency of occurrence of the point mutation was 1.2%.
Meanwhile, the ascomycetes shells collected in the four areas are cultured on a PDA plate containing 100 mu g/ml streptomycin, after the fusarium graminearum colonies grow out, the sensitivity to carbendazim is further determined on the PDA plate containing 5 mu g/ml carbendazim, if the fusarium graminearum colonies can grow, the strain is a carbendazim resistant strain and cannot grow, and the strain is a carbendazim sensitive strain. The PCR reaction of the carbendazim resistant strain obtained by screening is carried out according to the primer Fg198K-FC/Fgtub2-R of the invention, and whether the strain contains E198K point mutation or not is judged. Calculating the frequency of occurrence (FRE) of the point mutation E198K related to the resistance of fusarium graminearum carbendazim by using a formulaE198K），FREE198K=NE198K/NFg×100%（NE198KNumber of Fusarium graminearum containing point mutation E198K, NFgObtaining the number of all Fusarium graminearum for isolation)。
The PCR amplification system, reaction conditions and product detection were as described in 4 (primer specificity verification).
As shown in table 3, in four regions, the occurrence frequency of the point mutation, which was detected only in field samples collected from jiangsu jing, to be resistant to carbendazim-resistant fusarium graminearum containing the point mutation E198K, was 1.14%.
TABLE 3
Compared with the conventional medicament sensitivity and the common PCR method, the method for detecting the frequency of the carbendazim resistance-related point mutation E198K is reliable and convenient.
Claims (5)
1. A method for detecting the frequency of a point mutation E198K associated with resistance to Fusarium graminearum, comprising:
(1) sampling, namely mixing the samples and extracting DNA;
(2) performing real-time fluorescent quantitative PCR amplification by using the DNA as a template and using a primer to obtain the weight of the DNA containing E198K point mutation in the mixed sample;
(3) designing a primer according to actin gene of fusarium graminearum, and carrying out real-time fluorescent quantitative PCR amplification by using the DNA as a template to obtain the weight of the total DNA in the mixed sample;
(4) the frequency of occurrence of the carbendazim resistance-associated point mutation E198K was calculated according to the following formula:
FREE198K＝QE198K/QFg×100％；
wherein, FREE198KThe frequency of occurrence of the point mutation E198K related to carbendazim resistance, QE198KThe weight of DNA containing the point mutation E198K, Q, in the mixed sampleFgIs the weight of total DNA of Fusarium graminearum in the mixed sample;
the base sequence of the primer is as follows:
a forward primer: 5'-AGCTCGTCGAGAACTCTGAAA-3', respectively;
reverse primer: 5'-GCAGCGGCCATGATGTTCTT-3' are provided.
2. The method of claim 1, wherein in step (1), the method for extracting DNA comprises:
(a) collecting mycelium or ascocarp shell, mixing, adding extractive solution, and grinding;
(b) standing the ground mixed solution for a period of time, centrifuging and taking supernatant;
(c) precipitating the supernatant with alcohol, and washing the precipitate to obtain the DNA;
when extracting DNA from hyphae, the extract comprises: 200mM Tris-HCl, 50mM EDTA, 20mM NaCl and 1% SDS;
when extracting DNA from an ascocarp, the extract comprises: 1-2% polyvinylpyrrolidone, 200mM Tris-HCl, 50mM EDTA, 200mM NaCl and 1% SDS.
3. The method of claim 1, wherein in the steps (2) and (3), the real-time fluorescent quantitative PCR amplification system is as follows: 1 mu LDNA template, 0.4uL of forward primer and reverse primer of 10 mu mol/L respectively,PremixExTaqTM10uL of water was supplemented to 20. mu.L.
4. The method of claim 1, wherein in steps (2) and (3), the conditions for real-time fluorescent quantitative PCR amplification are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 15s, annealing at 63 ℃ for 15s, and extension at 72 ℃ for 20s, for 40 cycles.
5. The method according to claim 1, wherein in the step (3), the base sequence of the primer is:
a forward primer: 5'-ATCCACGTCACCACTTTCAA-3', respectively;
reverse primer: 5' -TGCTTGGAGATCCACATTTG-3.
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| Characterization of Sterol Demethylation Inhibitor-Resistant Isolates of Fusarium asiaticum and F. graminearum Collected from Wheat in China;Y.Yin等;《PHYTOPATHOLOGY》;20090531;第99卷(第5期);487-497 * |
| β-Tubulins in Gibberella zeae_ their characterization and contribution to carbendazim resistance;JianBo Qiu;《Pest Manag Sci》;20120420;第68卷;1191-1198 * |
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