CN101760554B - Primer sequence for detecting resistance frequency of botrytis cinerea population to strobilurin fungicide and method thereof - Google Patents

Abstract

The invention discloses two groups of primers and further discloses a method for utilizing the two group of the primers for detecting resistance frequency of a botrytis cinerea population to a strobilurin fungicide, and the method comprises the following steps: extracting DNA of the botrytis cinerea population to be detected; taking the DNA as a template, utilizing the first group of the primers and the second group of the primers for respectively carrying out real-time quantitative PCR amplification and recording the cycle number of achieving a fluorescence threshold; referring to a standard curve of DNA total content of the botrytis cinerea population to be detected of real-time quantitative PCR determination and the standard curve of the DNA content of botrytis cinerea resisting the strobilurin fungicide of the real-time quantitative PCR determination, and obtaining the DNA total content Q of the botrytis cinerea population to be detected and the DNA content QR of the botrytis cinerea resisting the strobilurin fungicide; and calculating the resistance frequency of the botrytis cinerea population to be detected to the strobilurin fungicide. The primers have high specificity and can accurately, rapidly and conveniently detect the resistance frequency of the field botrytis cinerea population.

Description

Detect primer sequence and the method for ash arrhizus bacteria colony to the methoxy acrylic bactericide fastness frequency

Technical field

The present invention relates to biology field, relate in particular to a kind of PCR method and detect primer sequence and the method for ash arrhizus bacteria colony the methoxy acrylic bactericide fastness frequency.

Background technology

Gray mold is infected and is caused by Deuteromycotina, hyphomycetes, hyphomycetales, Staphlosporonites pathogenic bacteria (Botrytis).This pathogenic bacteria is a kind of host facultative parasitism fungi very widely; It can parasitize in multiple fruit, vegetables and the flowers; Main harm inflorescence, young fruit and will mellow fruit; Also can infect carpopodium, young sprout and young leaflet tablet, this germ has become the main disease that storage, transportation, selling period cause fruit rot.

Methoxy acrylic bactericide (as: ICIA 5504, BAS 490F etc.) can compare the effectively preventing gray mold; This compounds is to be guide's synthetic new type bactericide with natural product Strobilurin, thereby they mainly are the synthetic sterilization effects that reaches of energy that destroys germ through the respiration that suppresses germ.The Amici reaches and has wider fungicidal spectrum, can prevent and treat the multiple kinds of crops disease, and can improve crop yield and quality, and its toxicity is merely 50% of salt, has been widely used in the disease control of high value vegetables or fruit abroad.

Abroad, because long-term a large amount of use of this medicament had more than ten kind of germ that this medicament has been produced tangible resistance problem.Recently, the ash arrhizus bacteria of the anti-ICIA 5504 of many strains (commodity are by name: the Amici reaches) also obtains in China's field separation, studies this germ the ICIA 5504 resistance mechanism is found, the 143rd amino acids sudden change of Cytb gene has caused ash arrhizus bacteria that ICIA 5504 is produced resistance.Owing to have cross resistance between the methoxy acrylic bactericide, after germ produces resistance to a kind of medicament, also can produce resistance to other kind of such medicament.

Along with the continuous development of Protocols in Molecular Biology (particularly real-time quantitative PCR), on the basis of clear and definite germ resistance molecular mechanism, utilize molecular detection technology can from a large amount of germ colonies, detect the anti-medicine individuality of frequency very low (0.1%) in recent years.

Summary of the invention

The invention provides a kind of primer sequence of ash arrhizus bacteria to the methoxy acrylic bactericide fastness frequency that be used to detect, this primer specificity is high, and ability rapid detection ash arrhizus bacteria is to the methoxy acrylic fastness frequency.

A kind ofly be used to detect the primer sequence of ash arrhizus bacteria colony to the methoxy acrylic bactericide fastness frequency; Comprise two groups of primers: the forward primer of first group of primer has the described base sequence of SEQ IDNO:1 in the sequence table, and the reverse primer of first group of primer has the described base sequence of SEQ ID NO:2 in the sequence table; The forward primer of second group of primer has the described base sequence of SEQ ID NO:3 in the sequence table, and the reverse primer of second group of primer has the described base sequence of SEQ ID NO:4 in the sequence table.

The present invention also provides a kind of and has utilized above-mentioned primer sequence to detect the method for ash arrhizus bacteria colony to the methoxy acrylic bactericide fastness frequency, may further comprise the steps:

(1) DNA of extraction ash arrhizus bacteria to be measured colony;

(2) DNA that obtains with step (1) is a template, utilizes first group of primer and second group of primer to carry out the real-time quantitative PCR amplification respectively and write down the cycle number that it reaches fluorescence threshold;

(3) cycle number that obtains according to step (2); Measure the typical curve of DNA amount of typical curve and the ash arrhizus bacteria that real-time quantitative PCR is measured anti-methoxy acrylic bactericide of the DNA total amount of ash arrhizus bacteria to be measured colony with reference to real-time quantitative PCR, obtain the DNA amount Q of the ash arrhizus bacteria of anti-methoxy acrylic bactericide in DNA total amount Q and the ash arrhizus bacteria to be measured colony of ash arrhizus bacteria to be measured colony _R

(4) calculate the fastness frequency FR=Q of ash arrhizus bacteria to be measured colony to methoxy acrylic bactericide _R/ Q.

The present invention is according to the drug-fast molecular mechanism of ash arrhizus bacteria; The primer that has designed two groups of high specifics is used for the high throughput testing fastness frequency; At the disease early period of origination; Just can detect the fastness frequency of field ash arrhizus bacteria colony accurately, fast, easily, technical support is provided the resistance management that instructs gray mold.

Description of drawings

Fig. 1 is the position sequence of second group of primer of the present invention on Cytb;

Fig. 2 is the position collection of illustrative plates of primer sequence of the present invention on the sequence of Cytb;

Fig. 3 is the gel electrophoresis figure of the specific detection of two groups of primers of the present invention;

Fig. 4 is the DNA real-time quantitative PCR amplification typical curve of ash arrhizus bacteria to be measured colony;

Fig. 5 is the DNA real-time quantitative PCR amplification typical curve of resistance ash arrhizus bacteria.

Embodiment

Primer is synthetic

Search ash arrhizus bacteria Cytb gene complete sequence through NBCI, analyze this sequence through GenScan again and draw, ash arrhizus bacteria Cytb sequence contains 3 introns.Because the Cytb coding region is more conservative, and this gene intron sequence has bigger variability in the Different Kinds of Pathogens fungi, therefore, according to the special PCR primer of intron sequences design ash arrhizus bacteria.

First group of primer Bc-F1 and Bc-R2 among the present invention just are positioned in the 2nd intron of ash arrhizus bacteria Cytb sequence; Utilize this to species-specific primer, just can detect the DNA total amount (comprising responsive and the resistance germ) of ash arrhizus bacteria in the DNA sample through real-time quantitative PCR.

Cytb gene the 143rd bit codon GGT is mutated into GCT and causes ash arrhizus bacteria that methoxy acrylate is produced resistance; Designed forward PCR primer Bc143-F according to this point mutation; This primer 3 ' terminal bases C can match with ash arrhizus bacteria bacterial strain (hereinafter to be referred as the resistant strain) DNA that methoxy acrylate is had resistance, can't match with the DNA to the responsive ash arrhizus bacteria bacterial strain (hereinafter to be referred as sensitive strain) of methoxy acrylate; Reverse primer Bc143-R sequence is the sequence special to ash arrhizus bacteria, is arranged in the 3rd intron.

Therefore, utilize this group equipotential specially to change the PCR primer, can from the DNA of resistant strain, amplify band, can not from the DNA of sensitive strain or other bacterial strain, amplify any band (primer is as shown in Figure 1 in the sequence at place).

The sequence corresponding relation is as shown in the table in the concrete sequence of above-mentioned primer and the sequence table:

The physical location of above-mentioned primer on the Cytb sequence is as shown in Figure 2, comprises that the primer of above-mentioned sequence is synthetic by the Shanghai lottery industry.

DNA extraction

Select bacterial strain for use: fusarium graminearum (Fusarium graminearum), rhizoctonia cerealis (Rhizoctonia cerealis), brown rot germ (Monilinia fructicola), sclerotium germ (Sclerotiniasclerotiorum), Pyricularia oryzae (Magnaporthe grisea), Botrytis cinerea bacterium (Botrytiscinerea), to the ash arrhizus bacteria (Bc143-1 of ICIA 5504 resistance; Bc143-2; Bc143-3), the ash arrhizus bacteria (Bc-S-1 responsive to ICIA 5504; Bc-S-2, Bc-S-3).

Above-mentioned bacterial strains is an experiment material, obtains through separation and purification at random, and the characteristic of bacterial strain is to not influence of experimental result, all cultivates on the PDA flat board before extracting DNA, and the DNA extraction process is following:

Scrape from the PDA flat board and to get the about 100mg of mycelia, place 1.5-mL Eppendorf pipe, (20mM NaCl 1%SDS), fully grinds with electric drill for 200mM Tris-HCl, 50mM EDTA, vibration mixing, the static 10min of room temperature to add 500 μ L DNA extraction lysates; 4 ℃ of 13200r/min, centrifugal 5min; Get the about 400 μ L of supernatant in new 1.5-mL Eppendorf pipe, add 750 μ L absolute ethyl alcohols, mixed mixing, 4 ℃ of 13200r/min, centrifugal 5min abandons supernatant; Deposition is used 70% washing with alcohol, and room temperature is placed dry 5-10min, is dissolved in 30 μ L TE (pH 8.0), and-20 ℃ of preservations are subsequent use.

The primer specificity checking

DNA with above-mentioned bacterial strains is a template, respectively species-specific primer Bc-F1+Bc-R2 and equipotential is specially changed PCR primer Bc143-F+Bc143-R and carries out specific assay, and each reaction all has a negative control (with sterilized water as template).

The system of PCR reaction is: 1 μ L dna profiling (about 0.4ng), each 0.2 μ mol l of primer ^-1, dNTP 0.2 μ mol l ^-1, MgCl22mmol l ^-1, 1 * damping fluid (east, Beijing victory company produces), a polysaccharase 1.5U unit, distilled water complement to 25 μ L.

Reaction conditions is: 95 ℃ of preparatory sex change 3min, and 94 ℃ of sex change 20s, 60 ℃ of annealing of 56 ℃/second group primer of first group of primer 30s, 72 ℃ are extended 30s, carry out 35 circulations, extend 5min at last.The PCR product behind the electrophoresis, is taken pictures with the EB colour developing in 1 * TAE damping fluid with 1.5% agarose.

See (as shown in Figure 3) from the electrophoresis photo, in PCR reaction, with first group of primer Bc-F1 and Bc-R2 from resistant strain (Bc143-1; Bc143-2; Bc143-3) and sensitive strain (Bc-S-1, Bc-S-2 Bc-S-3) all can arrive the band of 294-bp; Any band and other pathogenic fungi does not increase explains that this species-specific primer specially changes (Fig. 3 A) to ash arrhizus bacteria.In second group of PCR primer Bc143-F and the Bc143-R amplification PCR products; Can only be from the ash arrhizus bacteria bacterial strain (Bc143-1 of anti-ICIA 5504; Bc143-2, Bc143-3) the increase band of 260-bp is from sensitive strain and other pathogenic fungi any band (Fig. 3 B) that all do not increase.Show that this equipotential is specially changed the ash arrhizus bacteria that PCR primer ability specific detection goes out anti-methoxy acrylic bactericide.

The detection of fastness frequency

The ash arrhizus bacteria DNA of concentration known (is comprised sensitive strain and resistant strain; Ratio is any) become 6 concentration gradients (5fg～0.5ng) with 1: 10 dilution proportion; With these 6 concentration DNA as template; Carry out real-time quantitative PCR amplification with first group of primer, reach the difference of the required cycle number of fluorescence threshold values, draw and obtain the typical curve (as shown in Figure 4) that real-time quantitative PCR is measured ash arrhizus bacteria DNA amount according to the template DNA amplification of 6 concentration known

The ash arrhizus bacteria DNA of the anti-ICIA 5504 of concentration known is become 6 concentration gradients (5fg～0.5ng) with 1: 10 dilution proportion; With the template of these 6 concentration DNA as the known standard sample; Carry out the real-time quantitative PCR amplification with second group of primer; Amplification reaches the difference of the required cycle number of fluorescence threshold values according to the template DNA of 6 concentration known, draws to obtain the typical curve (as shown in Figure 5) that real-time quantitative PCR is measured anti-ICIA 5504 ash arrhizus bacteria DNA amount.

Ordinate zou is represented the concentration logarithmic value of real-time quantitative PCR reaction front template DNA among the figure, and X-coordinate is represented to reach the required cycle number of fluorescence threshold values after the DNA cloning, i.e. C (T) value.

Real-time quantitative PCR reaction system: 10 μ L SYBR Premix Ex Taq ^TM(production of TaKaRa company), template DNA 1 μ L, forward and reverse primer (0.2 μ mol l ^-1) each 1 μ L, distilled water complements to 20 μ L.

Reaction conditions: 95 ℃ of preparatory sex change 2min, 95 ℃ of sex change 20s, 60 ℃ (first group of primer) or 62 ℃ (second group of primer) annealing 25s, 72 ℃ are extended 35s, carry out 40 circulations, extend 7min at last.Real-time quantitative PCR instrument model: DNA Engine Opticons 4 (MJ Research.MA.USA)

Because the grey mold bacterial strain ratio of the anti-ICIA 5504 in field is very low at present; So; We press 1: 9,1: 1,9: 1 dilution proportion mixed extraction DNA with the spore of resistant strain and sensitive strain; Carry out real-time quantitative PCR reaction as simulation three field samples to be detected, calculate its fastness frequency then, whether the ratio of actual resistant strain is consistent in checking real-time quantitative PCR and the sample.Three samples utilize above-mentioned two groups of primers to carry out the real-time quantitative PCR reaction respectively, have obtained corresponding C (T) value respectively, according to its corresponding standard curve, obtain the DNA total amount Q of ash arrhizus bacteria in the testing sample and the DNA amount Q of resistance ash arrhizus bacteria _RAsh arrhizus bacteria is to the fastness frequency FR=Q of ICIA 5504 in detected three samples of real-time quantitative PCR _R/ Q, the result sees the following form:

This result shows that the real-time quantitative PCR result can more accurately detect the resistant strain ratio.This method can be used for the resistance situation of rapid detection ash arrhizus bacteria colony to methoxy acrylic bactericide.

SEQUENCE?LISTING

< 110>Zhejiang University

< 120>detect primer sequence and the method for ash arrhizus bacteria colony to the methoxy acrylic bactericide fastness frequency

<130>

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<170>PatentIn?version?3.3

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< 213>Botrytis cinerea bacterium (Botrytis)

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gcagctaagg?ccatagactg?t 21

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<211>20

<212>DNA

< 213>Botrytis cinerea bacterium (Botrytis)

<400>2

ccgtcgggtt?ataccacttt 20

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<211>19

<212>DNA

< 213>Botrytis cinerea bacterium (Botrytis)

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ggcaaatgtc?actgtgagc 19

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<212>DNA

< 213>Botrytis cinerea bacterium (Botrytis)

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accatctcca?tccaccatac?ct 22

Claims (3)

1. one kind is used to detect the primer sequence of ash arrhizus bacteria colony to the methoxy acrylic bactericide fastness frequency; It is characterized in that; Comprise two groups of primers: the base sequence of the forward primer of first group of primer is shown in SEQ ID NO:1, and the base sequence of the reverse primer of first group of primer is shown in SEQ IDNO:2; The base sequence of the forward primer of second group of primer is shown in SEQ ID NO:3, and the base sequence of the reverse primer of second group of primer is shown in SEQ ID NO:4.

2. one kind is utilized the described primer sequence of claim 1 to detect the method for ash arrhizus bacteria colony to the methoxy acrylic bactericide fastness frequency, may further comprise the steps:

(1) DNA of extraction ash arrhizus bacteria to be measured colony;

(2) DNA that obtains with step (1) is a template, utilizes first group of primer and second group of primer to carry out the real-time quantitative PCR amplification respectively and write down the cycle number that it reaches fluorescence threshold;

(3) cycle number that obtains according to step (2); Measure the typical curve of DNA amount of typical curve and the ash arrhizus bacteria that real-time quantitative PCR is measured anti-methoxy acrylic bactericide of the DNA total amount of ash arrhizus bacteria to be measured colony with reference to real-time quantitative PCR, obtain the DNA amount Q of the ash arrhizus bacteria of anti-methoxy acrylic bactericide in DNA total amount Q and the ash arrhizus bacteria to be measured colony of ash arrhizus bacteria to be measured colony _R

(4) calculate the fastness frequency F of ash arrhizus bacteria to be measured colony to methoxy acrylic bactericide _R=Q _R/ Q.

3. method according to claim 2 is characterized in that: described methoxy acrylic bactericide is an ICIA 5504.

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