CN103409533B - Detect primer and the method for the F.graminearum schw carbendazim resistance reference point sudden change E198K frequency of occurrences - Google Patents

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

Primers and method for detecting occurrence frequency of point mutation E198K associated with Fusarium graminearum carbendazim resistance

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 the point mutation E198K associated with Fusarium graminearum carbendazim resistance.

Background technique

In recent years, due to changes in straw returning to the field and climatic factors in my country, the common occurrence area of wheat scab caused by Fusarium graminearium has expanded from the middle and lower reaches of the Yangtze River to the north to Shandong, Henan, Hebei and other major wheat producing areas in the Yellow and Huaihe Rivers. , In 2010, the diseased area in the whole country exceeded 100 million mu, and the toxin of the scab in the wheat harvested in some areas seriously exceeded the standard, resulting in heavy losses. Due to the lack of germplasm materials with high level of resistance, the chemical agent carbendazim is mainly used to control wheat head blight at present. However, due to the long-term and extensive use of such agents (benzimidazoles), the problem of carbendazim resistance has emerged in Zhejiang, Jiangsu and Anhui.

Point mutations at positions 6, 50, 167, 198, 200 or 240 of β-tubulin are the main reason for the resistance of most pathogenic bacteria to benzimidazole fungicides. According to the resistance index (Resistantfactor, RF), Fusarium graminearum showed low, medium and high levels of resistance to carbendazim, in which 1≤RF<10 was low resistance, 10≤RF<50 was medium resistance, 50≤RF<100 is high resistance. Studies have shown that different point mutations on β-tubulin correspond to different levels of resistance: E(GAG)198L(CTG) leads to high levels of resistance, F(TTT)167Y(TAT), F(TTC) 200Y(TAC) and E(GAG)198K(AAG) resulted in moderate levels of resistance, and E(GAG)198Q(CAG) resulted in low levels of resistance. Field studies of carbendazim-resistant Fusarium graminearum have reported point mutations F167Y, E198/Q/L and F200Y. In 2011, Qiu et al. obtained two carbendazim-resistant strains containing new point mutations (Y50C and E198K) through the laboratory of site-directed mutagenesis (Qiu et al., 2011, Pest Management Science, 67:191-198). When our research group tested carbendazim resistance of 4320 scab samples collected in the field in 2012, a resistant strain containing the E198K point mutation was found in the Jingjiang wheat area of Jiangsu, which indicated that the Fusarium graminearum population in the field had been Resistant strains containing this point mutation appeared.

For the point mutation E198K, the Chinese patent document with the publication number CN101988122A discloses a method for genotype detection of Fusarium graminearum resistance to carbendazim, including: extracting the nuclear genomic DNA of the strain to be tested; using three A pair of primers was set for PCR reaction; the genotype of the strain's resistance to carbendazim was identified according to the electrophoresis pattern of the PCR product. In this patent, it is disclosed that the Tetra-primer ARMS-PCR method can be used to detect the point mutation qualitatively, but it cannot detect the point mutation quantitatively.

Therefore, it is necessary to establish a detection technology that can quantitatively detect the point mutation and accurately detect the occurrence frequency and change dynamics of the population containing the point mutation in the field, which is of guiding significance for the effective control of diseases and the rational use of pesticides. Under screening pressure, the study of the dynamic changes of different types of resistant populations provides a basis.

Contents of the invention

The invention provides a primer for detecting the occurrence frequency of the E198K point mutation related to Fusarium graminearum carbendazim resistance, which has strong specificity.

A kind of primer, base sequence is:

Forward primer: 5'-AGCTCGTCGAGAACTCTGAAA-3';

Reverse primer: 5'-GCAGCGGCCATGATGTTCTT-3'.

The present invention also provides a method for detecting the occurrence frequency of point mutation E198K related to Fusarium graminearum carbendazim resistance, which is characterized in that it comprises:

(1) Sampling, mixing samples and extracting DNA;

(2) using the DNA as a template, using the primers as claimed in claim 1 to carry out real-time fluorescent quantitative PCR amplification to obtain the weight of the DNA containing the E198K point mutation in the mixed sample;

(3) Design primers according to the actin gene of Fusarium graminearum, and use the DNA as a template to perform real-time fluorescent quantitative PCR amplification to obtain the weight of the total DNA in the mixed sample;

(4) Calculate the occurrence frequency of the point mutation E198K related to carbendazim resistance according to the following formula:

FRE _E198K = Q _E198K /Q _Fg × 100%;

Among them, FRE _E198K is the occurrence frequency of point mutation E198K related to carbendazim resistance, Q _E198K is the weight of DNA containing point mutation E198K in the mixed sample, and Q _Fg is the weight of the total DNA of Fusarium graminearum in the mixed sample.

The frequency of E198K point mutations related to carbendazim resistance to Fusarium graminearum is the frequency of the E198K mutation population of Fusarium graminearum. In order to ensure the accuracy of the results, the sampling should be carried out according to the method of biostatistics.

Since this application uses the ratio of DNA weight to reflect the occurrence frequency of the E198K point mutation related to Fusarium graminearum resistance, those skilled in the art can understand that when samples are mixed before DNA extraction, the weight of different samples should be as much as possible. be consistent.

In step (1), the DNA extraction method includes:

(a) Collect mycelia or asci shells, mix them and add the extract to grind them;

(b) Let the ground mixture stand for a period of time, and centrifuge to get the supernatant;

(c) The supernatant is precipitated with alcohol, and the precipitate is washed to obtain the DNA;

When extracting DNA from mycelium, the extracting solution includes: 200mM Tris-HCl, 50mM EDTA, 20mMNaCl and 1%SDS;

When extracting DNA from ascothecia, the extracting solution includes: 1-2% polyvinylpyrrolidone, 200mM Tris-HCl, 50mM EDTA, 200mMNaCl and 1% SDS.

Described alcohol is ethanol or Virahol.

The above DNA extraction method is simple and fast, does not require traditional DNA extraction and purification steps, and does not affect the accuracy of subsequent detection.

In steps (2) and (3), the real-time fluorescent quantitative PCR amplification system is: 1 μL DNA template, 10 μmol/L forward and reverse primers 0.4uL each, PremixExTaq ^TM 10 μL, make up to 20 μL with water.

In steps (2) and (3), the conditions for the real-time fluorescent quantitative PCR amplification are: pre-denaturation at 95°C for 3 minutes; denaturation at 95°C for 15 seconds, annealing at 63°C for 15 seconds, extension at 72°C for 20 seconds, and 40 cycles.

Actin, "actin", is an important skeleton protein of cells, which is highly conserved among different species of organisms. According to the design of actin gene primers for amplification, the total weight of Fusarium graminearum can be quantified.

In step (3), the base sequence of the primer is:

Forward primer: 5'-ATCCACGTCACCACTTTTCAA-3'

Reverse primer: 5'-TGCTTGGAGATCCACATTTG-3.

Compared with prior art, the beneficial effect of the present invention is:

(1) The primers provided in this application are designed for the mutation site E(GAG)198K(AAG) of the β-tubulin gene (GenBank accession number: KC765119) of Fusarium graminearum resistant to carbendazim, wherein , the designed forward primer is located at the mutation site, and the 3' terminal base is specially designed to increase the carbendazim-sensitive strains, high antibacterial strains, and the mutation site is F (TTT) 167Y (TAT) or F (TTC) 200Y (TAC) medium antibacterial strain and the mismatch probability of low antibacterial strain, improve the specificity of the mutation site E (GAG) 198K (AAG) against carbendazim-resistant Fusarium graminearum; design The reverse primer is located at base 1203-1222 of the β-tubulin gene, which is specific to Fusarium graminearum; and the amplified fragment is 337bp in size, which is easy to amplify. Therefore, after selecting the appropriate target sequence and designing the base sequence of the primer reasonably, the primers of the present application are used for PCR amplification, and the gene that is neutrally resistant to carbendazim and whose mutation site is (GAG) 198K can be detected. (AAG) Fusarium graminearum strain DNA was amplified, and the specificity of the primers was high.

In addition, the primers of the present invention can not only amplify the DNA sequence of carbendazim-resistant and mutation site (GAG) 198K (AAG) from the ascothecia of rice stump (the main source of field infection) not only from the mycelium .

(2) The detection method of the present invention can quantitatively detect (GAG) 198K (AAG) point mutation and can accurately detect the occurrence frequency and change dynamics of the population containing the point mutation in the field, the detection is fast and accurate, and the effective control of diseases and pesticides The rational use of is instructive, and it also provides a basis for the study of the dynamic changes of different types of resistant populations under the pressure of drug screening.

Description of drawings

Fig. 1 is the position sequence of primer sequence of the present invention in β-tubulin gene;

Fig. 2 is the position map of the primer sequence of the present invention on the β-tubulin gene sequence;

Fig. 3 is the specific detection electrophoresis figure of primer of the present invention;

Figure 4 is the standard curve (A) of the primer Fg198K-FC/Fgtub2-R of the present invention for the quantitative detection of the E198K point mutation and the standard curve of the primer F-actin-F/F-actin-R for the quantitative detection of all Fusarium graminearum DNA (B).

detailed description

The present invention will be further explained below in combination with specific embodiments.

Example 1

1. Strain type

F. oxysporum, F. avenaceum, F. acuminatum, F. semimitectum, F. subglutinans ), 1 strain each of F. culmorum, and 10 strains of F. graminearum. The 10 Fusarium graminearum strains included 2 carbendazim-sensitive strains (PH-1, HN9), 2 resistant strains containing the E198Q point mutation (YS13, BM2), and 2 resistant strains containing the F167Y point mutation ( JM12, JL22), 2 resistant strains containing F200Y point mutation (JQ82, HA16), 1 resistant strain JL45 containing E198K point mutation, and 1 resistant strain JM2 containing E198L point mutation.

The above-mentioned strains are all common plant pathogenic fungi, which are preserved in the Institute of Biotechnology of Zhejiang University, and can also be obtained by conventional methods of separation and purification of fungi.

2. Primer synthesis

The E(GAG) 198K (AAG) point mutation on β-tubulin can lead to a moderate level of resistance to carbendazim. According to the β-tubulin gene (Genebank accession number is KC765119), the point mutation E(GAG) 198K For the base change of (AAG), the forward PCR primer Fg198K-FC was designed, and the base A at the 3' end of the primer was paired with the mutant base A in the antibacterial strain of E(GAG)198K(AAG). , cannot pair with base G in sensitive strains, high antibacterial strains, medium antibacterial strains with mutation sites F(TTT) 167Y (TAT) or F(TTC) 200Y (TAC), and low antibacterial strains.

In order to further improve the specificity of the PCR primers, the second last base at the 3' end of the primers is changed from G to A (Fg198K-FC), so that for sensitive strains and high antibacterial strains, the mutation site is F(TTT)167Y (TAT) or F(TTC) 200Y (TAC) medium antibacterial strains and low antibacterial strains have two base mismatches, which are more specific than primers with only one base mismatch under the same PCR reaction conditions. high. The sequence of the reverse primer Fgtub2-R is located at bases 1203-1222 of the β-tubulin gene, and this segment of bases is found to be a sequence specific to Fusarium graminearum through alignment in NCBI. Therefore, the use of Fg198K-FC/Fgtub2-R allele-specific PCR primers can only amplify bands from Fusarium graminearum strains containing the E198K point mutation, but not from carbendazim-resistant strains containing other point mutations. , carbendazim-sensitive strains and any bands amplified in other fungi (the sequences of the primers are shown in Figure 1).

The corresponding relationship between the specific sequences of the above primers and the sequences in the sequence listing is shown in Table 1:

Table 1

The physical positions of the above primers on the β-tubulin gene sequence are shown in FIG. 2 .

The primers for the above sequences were synthesized by Shanghai Biotechnology Co., Ltd.

3. DNA extraction

What the present invention adopted is a kind of simple and quick DNA extraction method, concrete operation is as follows:

(1) Scrape mycelium (100mg) from the PDA plate with an inoculation needle and place it in a 1.5mL Eppendorf tube;

(2) Add 500 μL of DNA extraction lysate (200 mM Tris-HCl, 50 mM EDTA, 20 mM NaCl, 1% SDS, pH 8.0) to the EP tube, grind thoroughly with an electric drill, shake and mix, and stand at room temperature for 10 min; 4°C, 13200r/min, Centrifuge for 5min;

(3) Take about 400 μL of the supernatant into a new 1.5 mL Eppendorf tube, add 750 μL of absolute ethanol, mix well, and centrifuge at 13200 r/min for 5 min at 4 °C;

(4) Discard the supernatant, wash the precipitate with 70% ethanol, dry at room temperature for 5-10 minutes, dissolve in 30 μLTE (pH 8.0), and store at -20°C for later use.

The DNA of the 16 kinds of fungal strains in this example and the tested bacteria can be extracted by the above method.

4. Primer specificity verification

Using the DNA of the 16 fungi extracted above as templates, PCR reactions were carried out with Fg198K-FC/Fgtub2-R primers, and each reaction contained a negative control (use sterile water instead of DNA templates).

The PCR amplification system is: 1 μL DNA template (about 0.4ng), 0.2 μmoll ^-1 primers, 0.2 μmoll ^-1 dNTP, 2 mmoll ^-1 MgCl ₂ , 1× buffer (produced by Beijing Dongsheng Company), 1.5 U polymerase unit, make up to 25 μL with double distilled water.

The PCR reaction conditions were: pre-denaturation at 95°C for 3 min, denaturation at 94°C for 30 s, annealing at 63°C for 30 s, extension at 72°C for 30 s, 35 cycles, and finally extension at 72°C for 5 min.

The PCR products were electrophoresed in 1.5% agarose in 1×TAE buffer, and photographed by EB color development.

From the electrophoresis photos (as shown in Figure 3), only the carbendazim-resistant strain JL45 containing the E198K point mutation can give a 337bp band, while the other 7 carbendazim-resistant strains containing the point mutation ( YS13 and BM2 contained E198Q, JM12 and JL22 contained F167Y, JQ82 and HA16 contained F200Y, JM2 contained E198L), two carbendazim-sensitive strains (PH-1, HN9) and the other six pathogenic fungi did not amplify any bands. bring. Therefore, it was verified that this pair of allele-specific PCR primers could specifically detect the E198K point mutation of Fusarium graminearum.

5. Establish a standard curve for the quantitative detection of the E198K point mutation and the total DNA of Fusarium graminearum related to carbendazim resistance to Fusarium graminearum

Extract the DNA of the Fusarium graminearum carbendazim-sensitive strain PH-1 and the carbendazim-resistant strain JL45 containing the E198K point mutation (according to the above fungal DNA extraction method), and dilute the extracted DNA to the following concentration: 424ng/μL , 42.4ng/μL, 4.24ng/μL, 424pg/μL, 42.4pg/μL. Using the primers Fg198K-FC/Fgtub2-R of the present invention to carry out real-time fluorescence quantitative PCR amplification on the DNA of JL45 in different gradients. Using the reported primers F-actin-F/F-actin-R (Yine et al., Phytopathology, 2009, 99:487-497), real-time fluorescent quantitative PCR amplification was performed on DNA with different gradients of PH-1. Each concentration gradient was replicated in duplicate, and the entire PCR amplification was repeated in triplicate. in,

F-actin-F: 5'-ATCCACGTCACCACTTTTCAA-3';

F-actin-R: 5'-TGCTTGGAGATCCACATTTG-3'.

PCR amplification system: 1μL DNA template, 0.4uL each primer (10μmoll ^-1 ), PremixExTaq ^TM (manufactured by TaKaRa Company) 10uL, made up to 20uL with double distilled water.

The PCR reaction conditions were: pre-denaturation at 95°C for 3min, denaturation at 95°C for 15s, annealing at 63°C for 15s, extension at 72°C for 20s, and 40 cycles. The reaction was carried out on the Eppendorf MastercyclerepRealplex2 PCR instrument, and after the reaction was completed, a standard curve was established.

The PCR amplification system and conditions using F-actin-F/F-actin-R as primers and Fg198K-FC/Fgtub2-R as primers are the same.

6. Detection of the occurrence frequency of the point mutation E198K associated with carbendazim resistance in the ascothecia of rice in different regions

In April 2012, ascocarps were collected from rice piles in Hai'an and Jingjiang, Jiangsu, Fanchang and Chizhou wheat fields in Anhui, with 30-90 ascocarps in each area. Specific operation: scrape the same volume of ascus shell from each cluster of rice piles, and divide the ascus shell into two parts, one for real-time fluorescent quantitative PCR measurement, and the other for conventional drug sensitivity and Common PCR assay.

Place the ascus collected on the rice stump into a 1.5 mL Eppendorf tube, add 100 μl of DNA extraction buffer (1-2% polyvinylpyrrolidone (PVP), 200 mM Tris-HCl, 50 mM EDTA, 200 mM NaCl, ₁ % SDS, ddH2O as solvent , pH8.0), use a pointed glass rod to install it on a commonly used electric hand torch and grind for 1min, then add 400μl of DNA extraction buffer to the centrifuge tube, vortex to mix, and let stand at room temperature for 10min; the above mixture Centrifuge at 15,000 r/min at 4°C for 5 min, then transfer the supernatant to another centrifuge tube, add 750 μl absolute ethanol, mix well, and centrifuge at 15,000 r/min at 4°C for 2 min, discard the supernatant; The precipitated DNA was washed with 70% ethanol, dried at room temperature and dissolved in 50 μl sterile water to obtain the extracted DNA; the above DNA solution was purified by column purification with UNIQ-10 column PCR product recovery kit (produced by Shanghai Sangong)

When extracting DNA, the ascothecia of the same region used for real-time fluorescent quantitative PCR assay were mixed together for DNA extraction.

Using the primers Fg198K-FC/Fgtub2-R of the present invention and the reported primers F-actin-F/F-actin-R to carry out real-time fluorescent quantitative PCR amplification of the DNA of 4 field samples, the system and conditions of fluorescent quantitative PCR amplification As described in 5 (Establishment of a standard curve for quantitative detection of F. graminearum carbendazim resistance-associated E198K point mutation and total DNA of F. graminearum). According to the above established standard curve, calculate the amount of DNA containing the E198K point mutation (Q _E198K ) and the total DNA amount (Q _Fg ) in the field samples. The frequency of occurrence of point mutation E198K related to carbendazim graminearum resistance was calculated by the formula (FRE _E198K ), FRE _E198K =Q _E198K /Q _Fg ×100%.

Table 2

As shown in Table 2, among the four regions, carbendazim-resistant Fusarium graminearum containing point mutation E198K was only detected in field samples collected in Jingjiang, Jiangsu, and the frequency of this point mutation was 1.2%.

At the same time, we cultured the ascothecia collected in these four regions on a PDA plate containing 100 μg/ml streptomycin. Determination of the sensitivity to carbendazim, if it can grow, it shows that the bacterial strain is a carbendazim-resistant strain, and if it cannot grow, it shows that the bacterial strain is a carbendazim-sensitive bacterial strain. According to the primers Fg198K-FC/Fgtub2-R of the present invention, a PCR reaction is performed on the carbendazim-resistant strain obtained through screening to determine whether it contains the E198K point mutation. Use the formula to calculate the occurrence frequency of the point mutation E198K related to the resistance of Fusarium graminearum carbendazim (FRE _E198K ), FRE _E198K =N _E198K /N _Fg ×100% (N _E198K is the number of Fusarium graminearum containing the point mutation E198K, N _Fg is the number of all Fusarium graminearum isolated).

The PCR amplification system, reaction conditions and product detection were as described in 4 (primer specificity verification).

Through routine drug sensitivity and common PCR testing, the results are shown in Table 3. Among the four regions, only carbendazim-resistant Fusarium graminearum containing the point mutation E198K was detected in the field samples collected in Jingjiang, Jiangsu. The frequency of point mutations was 1.14%.

table 3

Compared with conventional drug sensitivity and common PCR methods, it is found that the method for detecting the occurrence frequency of the point mutation E198K related to carbendazim resistance established by the present invention 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:

FRE_E198K＝Q_E198K/Q_Fg×100％；

wherein, FRE_E198KThe frequency of occurrence of the point mutation E198K related to carbendazim resistance, Q_E198KThe weight of DNA containing the point mutation E198K, Q, in the mixed sample_FgIs 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,PremixExTaq^TM10uL 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.