CN102115790B - Primer sequence and method for detecting anti-imazalil-Penicillium-digitatum system by PCR (Polymerase Chain Reaction) process - Google Patents

Abstract

The invention discloses a primer sequence and method for detecting an anti-imazalil-Penicillium-digitatum system by a PCR (Polymerase Chain Reaction) process. The primer sequence comprises two pairs of primers, wherein the base sequence of the forward primer of the first pair of primers is disclosed as SEQ ID NO.1, and the base sequence of the reverse primer is disclosed as SEQ ID NO.2; and the base sequence of the forward primer of the second pair of primers is disclosed as SEQ ID NO.3, and the base sequence of the reverse primer is disclosed as SEQ ID NO.4. The method disclosed by the invention is simple to operate, and has the advantages of time saving, labor saving and reliable result; and the detection result can be used as a scientific instruction for fruit growers to select chemicals.

Description

The PCR method detects anti-primer sequence and the method that presses down mould azoles oranges and tangerines green mold bacterium fungus strain

Technical field

The present invention relates to biology field, relate in particular to a kind of PCR method and detect anti-primer sequence and the method that presses down mould azoles oranges and tangerines green mold bacterium fungus strain.

Background technology

China is first big country that world's oranges and tangerines are produced, and the cultivated area (2,700 ten thousand mu) and the output (2,500 ten thousand tons) of China's oranges and tangerines all rank first in the world at present.The citrus common green mold that is caused by citrus common green mold bacterium (Penicilliumdigitatum) is the main disease of oranges and tangerines postharvest storage and selling period, if do not use chemicals treatment, general this disease of time can cause the back oranges and tangerines of adopting of 20-30% to rot.

Nineteen ninety, China's widespread use sterilant presses down mould azoles (imazalil) processing and adopts the back oranges and tangerines with the control green mould generation of storage period.But because life-time service, the anti-citrus common green mold fungus strain that presses down mould azoles finds in each oranges and tangerines producing region successively, at the storage that resistant strain exists, routine dose press down the control poor of mould azoles to green mould even complete failure.Therefore, the Fast Detection Technique of setting up resistant strain guarantees that to instructing the medication of orchard worker's science the control effect utmost point is necessary.

Existing discovering, there are 2 kinds of molecular mechanisms in the citrus common green mold bacterium to the resistance that presses down mould azoles.A 126-bp transcriptional enhancer of (1) 14 α-demethylase gene CYP51 promoter region has simply been connected repetition 5 times; Duoed (Hamamoto H 4 times than sensitive strain; Hasegawa K; NakauneR; Et al.Tandem repeat of a transcriptional enhancer upstream of the stetol14 α-demethylase gene (CYP51) in Penicillium digitatum [J] .Applied andEnvironmental Microbiology, 2000,66 (8): 3421-3426; Li H Y; Xie Q Y; SongA H.Sequence comparison of CYP51 genes between imazalil-sensitive andimazalil-resistant strains of Penicillium digitatum (in Chinese) [J] .Mycosystema (fungus system); 2003,22 (1): 153-256.), we are referred to as to press down mould azoles resistance I type; (2) inserted one section 199-bp nucleotide sequence (Ghosoph J M in the 126-bp transcriptional enhancer of CYP51 promoter region; Schmidt L S; Margosan D A, et al.Imazalil resistancelinked to a unique insertion sequence in the PdCYP51 promoter region ofPenicillium digitatum [J] .Postharvest Biology and Technology, 2007; 44:9-18), we are referred to as to press down mould azoles resistance II type.These extra insertions cause pressing down mould azoles target (CYP51) expression of gene rises, thereby the germ resistance is occurred.Based on above resistance molecular mechanism; Set up Molecular Detection system (HamamotoH fast with domestic (this laboratory) in the world; Hasegawa K, Nakaune R, et al.PCR-based detection of steroldemethylation inhibitor-resistant strains of Penicillium digitatum [J] .PestManagement Science; 2001,57:839-843.; Chen G J; Zhang Z F; Jiang L Y; Etal.Real-time PCR assay for detection of the frequency of i mazalil-resistanceof Penicillium digitatum (in Chinese) [J] .ACTA PHYTOPATHOLOGICASINICA (Plant Pathology), 2008,38 (6): 561-569).

Yet we find recently, though above-mentioned resistance molecular mechanism also exists in China, most there is not any extra insertion in the isolating CYP51 gene promoter area that presses down mould azoles resistance fungus strain from Zhejiang, and resistance neither belongs to resistance I type or II type.Find that through analyzing oranges and tangerines green mold bacterium EST storehouse there are two homologous genes in citrus common green mold bacterium CYP51 gene, is referred to as CYP51B and CYP51C.Relatively the sequence of CYP51B full-length gene and upper reaches 1Kb thereof between sensitive strain and the resistant strain find that the upper reaches-all there is the insertion of a 199bp nucleotide sequence at the 174bp place to resistant strain CYP51B promoter region, and this sequence does not exist all in sensitive strain.Real-time fluorescence quantitative PCR (qPCR) analysis revealed, the CYP51B expression amount of resistant strain than 6 times of sensitive organism plant heights or more than.To comprise the CYP51B gene importing sensitive strain that this 199bp Nucleotide inserts, sensitive strain is strengthened the resistance that presses down mould azoles.It is to cause this type fungus strain to pressing down the molecular mechanism of mould azoles resistance that above statement of facts, the 199bp sequence of CYP51B promoter region insert, and is referred to as to press down mould azoles resistance III type.

Summary of the invention

The invention provides one group of primer sequence, utilize this group primer sequence that bacterium DNA to be measured is increased, can identify the anti-type that presses down mould azoles of oranges and tangerines green mold bacterium.

A kind of PCR method detects the anti-primer sequence that presses down mould azoles oranges and tangerines green mold bacterium fungus strain; Comprise two pairs of primers; Wherein the base sequence of the upstream primer of first pair of primer is shown in SEQ ID NO.1, and the base sequence of the downstream primer of first pair of primer is shown in SEQ ID NO.2; The base sequence of the upstream primer of second pair of primer is shown in SEQ ID NO.3, and the base sequence of the downstream primer of second pair of primer is shown in SEQ ID NO.4.

The present invention also provides and has utilized above-mentioned primer sequence to detect the anti-method that presses down mould azoles oranges and tangerines green mold bacterium fungus strain, comprising:

(1) DNA of extraction bacterium to be measured;

(2) be template with the DNA that extracts, utilize above-mentioned primer sequence to carry out pcr amplification;

(3) pcr amplification product carries out gel electrophoresis separation, dyeing;

(4) according to band number on the gel and the anti-type that presses down mould azoles of location determination bacterium to be measured thereof.

Resistance primer specificity of the present invention is high, and whether utilize primer of the present invention can in a PCR reaction system, can detect bacterial strain to be measured is the molecule type that presses down mould azoles resistant strain and resistance, and easy to operate, consuming time few.

Based on above-mentioned this detected result, can grasp the development trend of resistance fungus strain population in the storage effectively, instruct the medication of orchard worker's science, avoid blindly medication, reduce cost, reduce pesticide residue, increase economic benefit and social benefit.

Description of drawings

Fig. 1 is a primer sequence design diagram of the present invention;

Fig. 2 detects gel electrophoresis spectrum synoptic diagram, M:DNA Marker for resistance of the present invention; S: sensitive strain; I: resistance I type bacterial strain; II: resistance II type bacterial strain; III: resistance III type bacterial strain;

Fig. 3 detects gel electrophoresis figure, M:DNA Marker for primer specificity of the present invention; 1:PdKH8 (S); 2:Pd19D (II); 3:Pdw03 (III); 4:Pd01 (I);

Fig. 4 detects the true collection of illustrative plates of gel electrophoresis, A for resistance of the present invention: use CYP51A1 and CYP51A2 primer amplification; B: use B1 and B2 primer amplification.

Embodiment

(1) reference culture

Press down mould azoles responsive (Pd23 or PdKH8), resistance I type (Pd01); The oranges and tangerines green mold bacterium reference culture of resistance II type (Pd19D) and resistance III type (Pdw03); The resistance level of these bacterial strains confirms through the laboratory evaluation of resistance that all the resistance molecular mechanism confirms through gene clone and order-checking.And above-mentioned bacterial strains is experiment material, no specificity requirement.Be preserved in the Li Hongye of biotechnology research institute of Zhejiang University professor laboratory, bacterial classification opens to the public.

(2) testing sample and DNA extraction

From the oranges and tangerines garden, the morbidity fruit gathered of storage, intermediate links and merchandising location or separates the pure growth that obtains from the morbidity fruit, scrape the mould layer of getting on morbidity fruit or the pure growth (conidium of green mold bacterium), extraction DNA, concrete grammar is following:

A. scrape the conidium of getting (and mycelia) (about 50-200mg, having a small amount of substratum does not influence DNA extraction) and place 2mL Eppendorf to manage above-mentioned, add 800 μ L SLS lysate (200mmol/L Tris-HCl; 50mmol/L EDTA, 200mmol/L NaCl, 2g/100ml N-sodium lauroyl sareosine; PH 8.0), with the abundant dispersed with stirring mycelia of aseptic toothpick, 55 ℃ of water-bath 10min; Vibrate therebetween mixing 2-3 time, the centrifugal 10min of 13200r/min;

B. get supernatant 750 μ L in 1.5-mL Eppendorf pipe, add the mixed-solvent extraction DNA of equal-volume chloroform and primary isoamyl alcohol (volume ratio 24: 1), the centrifugal 10min of 13200r/min;

C. get supernatant in new 1.5-mL Eppendorf pipe, add 2 times of volume absolute ethyl alcohol mixing mixings ,-20 ℃ leave standstill 10min after, in 4 ℃, the centrifugal 4min of 13200r/min, deposit D NA;

D. with 70% absolute ethanol washing deposition, be deposited in room temperature and place seasoning 5-10min, be dissolved in 40 μ L TE solution (pH 8.0) ,-20 ℃ of preservations are subsequent use.

(3) primer is synthetic

Design primer referring to Fig. 1, obtain following 4 Auele Specific Primer B1, B2, CYP51A1 and CYP51A2, specifically see the following form:

The primer title	Sequence	Sequence number in the sequence table
			B1	5’-TATAGCGACATTAGTTTGGC-3’	SEQ?ID?NO.1
B2	5’-AGGAAAGTTGCAGAGAGACCCAT-3’	SEQ?ID?NO.2
			CYP51A1	5’-TAGCTCCAAAACAAATCGTCTGCC-3’	SEQ?ID?NO.3
CYP51A2	5’-GGTGAAGATATTGCCGTACTAGAC-3’	SEQ?ID?NO.4

Primer entrusts Shanghai Sangon Biological Engineering Technology And Service Co., Ltd (or other commercial companies) synthetic according to the sequence in the sequence table, and also available ordinary method is synthetic.

(4) primer specificity detects

Synthetic B1, B2, CYP51A1 and two pairs of primers of CYP51A2 are used reference culture PdKH8 respectively, and Pd01, Pd19d and Pdw03 carry out PCR reaction (20 μ L systems; Each 1 μ L of 10mmol/L primer; 10 * PCR buffer, 2 μ L, 2mmol/L dNTP 2 μ L, 25mmol/L MgCl ₂1.6 μ L, 5U/ μ L Taq enzyme 0.2 μ L, distilled water 12.2 μ L), the PCR product is through the specificity of 1% agarose gel electrophoresis detection primer, and the primer specificity detected result is referring to Fig. 3.

PCR product band is single and clear and legible, shows that these two pairs of primers can be used in the check and analysis of subsequent P CR method resistance.

(5) PCR reaction

DNA to extract is a template, utilizes above-mentioned two pairs of primers that carried out specific detection, uses the method for PCR to increase.

PCR reaction system: with each 2 μ L of primer B1, B2, CYP51A1 and CYP51A2 (primer concentration 5mmol/L), 10 * PCR buffer, 2 μ L, dNTP (2mmol/L) 2 μ L, MgCl ₂(25mmol/L) 1.6 μ L, Taq enzyme (5U/ μ L) 0.2 μ L, distilled water 6.2 μ L, mixing is put conventional pcr amplification appearance,

The PCR reaction conditions: 94 ℃ of preparatory sex change 5min, 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, carry out 32 circulations, and last 72 ℃ are extended 10min.

(100V 30min), after EB dyeing, observes product band (Fig. 4) to the agarose gel electrophoresis of PCR product use 1%.

In sensitive strain, expand the stripe size that and be about 500bp and 400bp (1), resistance I type is about 1000bp and 400bp (4), and resistance II type is about 700bp and 400bp (2), resistance III type be about 500bp and 600bp (3).The pcr amplification band is clear, and specificity is good, and detected result is consistent with the gene sequencing result, shows that this primer can be good at as the means that detect novel resistance.

Claims (2)

1. a PCR method detects the anti-primer sets that presses down mould azoles oranges and tangerines green mold bacterium fungus strain; It is characterized in that; Comprise two pairs of primers: the base sequence of the upstream primer of first pair of primer is shown in SEQ ID NO.1, and the base sequence of the downstream primer of first pair of primer is shown in SEQ ID NO.2; And the base sequence of the upstream primer of second pair of primer is shown in SEQ ID NO.3, and the base sequence of the downstream primer of second pair of primer is shown in SEQ ID NO.4.

2. one kind is utilized the described primer sets of claim 1 to detect the anti-method that presses down mould azoles oranges and tangerines green mold bacterium fungus strain, comprising:

(1) DNA of extraction bacterium to be measured;

(2) be template with the DNA that extracts, utilize the described primer sets of claim 1 to carry out pcr amplification;

(3) pcr amplification product carries out gel electrophoresis separation, dyeing;

(4) according to band number on the gel and the anti-type that presses down mould azoles of location determination bacterium to be measured thereof.

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