CN102925565A - Method for identifying nucleotide point mutation of phytophthora sojae beta-microtubulin gene and use thereof for diagnosing zoxamide resistance - Google Patents
Method for identifying nucleotide point mutation of phytophthora sojae beta-microtubulin gene and use thereof for diagnosing zoxamide resistance Download PDFInfo
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- CN102925565A CN102925565A CN2012103987270A CN201210398727A CN102925565A CN 102925565 A CN102925565 A CN 102925565A CN 2012103987270 A CN2012103987270 A CN 2012103987270A CN 201210398727 A CN201210398727 A CN 201210398727A CN 102925565 A CN102925565 A CN 102925565A
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Abstract
The invention relates to the cloning of a phytophthora sojae beta-microtubulin gene, the detection of the mutation site of the gene and the use of the gene in monitoring the zoxamide resistance, and particularly discloses a molecular detection method for zoxamide resistance caused by mutation of site-716 in the phytophthora sojae beta-microtubulin and special primers thereof. The primer pair consists of DNA molecules represented by SEQID NO:1 and SEQID NO:2, and the polymerase chain reaction (PCR) annealing temperature is 67DEG C. The invention provides a molecular diagnosis method for zoxamide resistance of phytophthora sojae, which has the characteristics of high sensitivity, simplicity, speed and stability, can be used for detecting the zoxamide resistance development trend of field phytophthora sojae with high pass, so that an early warning for medicine resistance can be given. The method has a great significance for control of medicine resistance in pathogenic bacteria.
Description
Technical field
The present invention relates to soybean phytophthora 'beta '-tubulin gene cloning and the application in sterilant zoxamide resistance monitoring thereof, specifically disclose the gene nucleotide point mutation of a kind of Rapid identification soybean phytophthora 'beta '-tubulin and to the drug-fast molecular detecting method of zoxamide and primer special.Belong to technical field of molecular biology.
Background technology
Soybean blight is by soybean phytophthora (Phytophthora sojae Kaufmann﹠amp; What Gerdemann) cause a kind ofly has danger, a destructive oomycetes disease.It all can be fallen ill soybean each breeding time, the wherein general underproduction 25%～50% of susceptible variety, and high sense kind can reach more than 90%, even total crop failure; And grain protein content obviously descends, and use value is affected.This disease from 1948 after the Indiana, USA northeast is found first, expand to rapidly more than 20 countries such as Brazil, Argentina.Then mainly occur in the provinces such as Heilungkiang, Anhui, Fujian in China, be important outer inspection and interior inspection disease.
Soybean phytophthora is homothallic oomycetes, under the natural condition syngenesis can occur, and has accelerated restructuring and the exchange of gene, so that the genetic diversity of field soybean Mtr mutant is more and more abundanter; And soybean phytophthora physiological strain Toxicity Variation is obvious, and new microspecies emerge in an endless stream, and many soybean resistant genes are overcome by new microspecies, and disease-resistant resource loses effectiveness.And chemical prevention fast effectively, make it to become the measure of outbalance in this disease control.But the sterilant kind that is specifically designed at present the oomycetes disease control both at home and abroad is less, and wherein the most representative is benzamides sterilant take metaxanin as representative, is the important systemic fungicide of a large class that the first-generation is specifically designed to control oomycetes disease.Yet because the action site of this series bactericidal agent is comparatively single, and big area is widely used, and cause pathogenic bacteria that the resistance of such medicament is come one after another, and development rapidly, and pathogenic bacteria is particularly serious to the resistance problem of metaxanin at present.Zoxamide (zoxamide) then is arise at the historic moment under this background a kind of to the specific benzamide series bactericidal agent of oomycetes tool, is unique a kind of ovicide that acts on tubulin in the market.This series bactericidal agent begins commercialization in calendar year 2001, and is widely used in the European and American areas, in the middle of China also has been in the registration popularization.
Although zoxamide is mainly used to prevent and treat the oomycetes disease, it also has certain activity to pathogenic fungies such as Botrytis cinerea, Venturia inaequalis, Monilinia fructicola, Mycosphaerella fijiensis and Cercospora beticola.Have and studies show that zoxamide and benzimidazole germicide have similar mechanism of action, all act on the 'beta '-tubulin of target bacterium.Benzimidazole germicide take derosal as representative is after use in the field, and the resistance problem produces very soon and be day by day serious, and FRAC (Fungicide Resistance Action Committee) classifies it sterilant of high resistance risk as.Have been reported and show, the resistance of benzimidazole germicide mainly is because the 198th of pathogenic bacteria 'beta '-tubulin and 200 amino acids site mutation have occured caused, and set up corresponding field method for quick, comprise allele-specific PCR (Allele-specific polymerase chain reaction, AS-PCR) etc.At present, soybean phytophthora is also not clear and definite to the molecular basis of insecticide resistance of zoxamide, and clear and definite its concrete resistant gene, set up the resistance molecular detecting method, can carry out early detection by the antagonism bacterial strain, understand the dynamic of its resistance development, formulate rational plant disease management scheme, to delay drug-fast generation.
Traditional biological method and molecular biology method are two kinds of common methods during the phytopathogen resistance detects or monitors.The traditional biological method namely refers to measure medicament to the EC of pathogenic bacteria
50To distinguish sensitivity and resistant strain, need a plurality of chemicals treatment, repeatedly repeat, sense cycle is long, workload is large, manpower and material that consume are more; And this method detection sensitivity is low, requires the mutation frequency of drug-fast strain more than 1%.And molecular biology method, from carrying DNA to specific PCR or restriction analysis, detect required time short, detect highly sensitive, detect frequency 10
-5～10
-4, more be applicable to detect low-frequency drug resistance gene, therefore also be used as the Perfected process of field drug-fastness early diagnosis.
Summary of the invention
The present invention aim to provide a kind of detect or the auxiliary detection soybean phytophthora in the 'beta '-tubulin gene whether have the method in mutational site and primer special thereof pair.
Whether the 'beta '-tubulin gene exists the method in mutational site in detection provided by the present invention or the auxiliary detection soybean phytophthora, comprises the steps:
Take the genomic dna of soybean phytophthora to be measured as template, carry out pcr amplification with primer pair shown in SEQ ID NO:1 and the SEQ ID NO:2, if pcr amplification product is the fragment of 288bp, then there are the mutational site in the existence of 'beta '-tubulin gene or candidate in the described soybean phytophthora to be measured;
Described mutational site refers to that genome sequence the 716th Nucleotide from 5 ' end of 'beta '-tubulin gene in the soybean phytophthora is C.
In the described soybean phytophthora genome sequence of 'beta '-tubulin gene from 5 ' terminal rise the 716th Nucleotide namely among the SEQ ID NO:3 from 5 ' end the 716th Nucleotide.
In the said process, in the described pcr amplification, annealing temperature is 67 ℃.
Whether the 'beta '-tubulin gene exists the primer pair in mutational site in detection provided by the present invention or the auxiliary detection soybean phytophthora, is comprised of dna molecular shown in SEQ ID NO:1 and the SEQ ID NO:2.
Another object of the present invention provides the mutational site of 'beta '-tubulin gene in the soybean phytophthora or albumen, the primer of 'beta '-tubulin gene complete sequence of wherein increasing is comprised of dna molecular shown in SEQ ID NO:5 and the SEQ ID NO:6, and annealing temperature is 63.5 ℃.
The 'beta '-tubulin gene mutational site in the soybean phytophthora provided by the present invention, for the genome sequence of 'beta '-tubulin in the soybean phytophthora from 5 ' terminal 716 Nucleotide be C.The genome sequence of described 'beta '-tubulin gene is from 5 ' terminal namely the 716th Nucleotide from 5 ' end among the SEQ ID NO:3 of the 716th Nucleotide that rises.
'beta '-tubulin mutational site in the soybean phytophthora provided by the present invention is for the 239th amino acids from the N end of 'beta '-tubulin in the soybean phytophthora is Serine.Described 'beta '-tubulin from the 239th amino acids the N end namely among the SEQ ID NO:4 from 5 ' the 239th amino acids terminal.
The application of above arbitrary described mutational site in identifying the soybean phytophthora resistance also belongs to protection scope of the present invention; Described resistance is anti-zoxamide.
In the above-mentioned application, exist the soybean phytophthora in described mutational site to have or the candidate has resistance to zoxamide.
Albumen also belongs to protection scope of the present invention shown in gene shown in the SEQ ID NO:3 or the SEQ ID NO:4.
The method of the point mutation that detection soybean phytophthora provided by the invention develops immunity to drugs to zoxamide, can be used for detecting fast, delicately the mould resistance genesis to zoxamide of field soybean epidemic disease dynamic, for drug-fast improvement provides technical support, be convenient in time adjust the disease control strategy, to delay and to control drug-fast further developing.
Description of drawings
Fig. 1, Fig. 2 are to zoxamide performance resistance and responsive soybean phytophthora bacterial strain 'beta '-tubulin gene complete sequence and protein sequence comparison result, find a mutational site relevant with resistance.Wherein, Ps9, Ps13, Ps52, Ps53, AH1302 and PsJMS2 are sensitive strain, and RZ2-2, RZ5-1, RZ6-2, RZ9-2, RZ10-1, RZ12-1, RZ14-1 and RZ15-1 are resistant strain.
Fig. 3 shows that for adopting different AS-PCR primers to carry out thermograde pcr amplification electrophorogram the specificity of each primer there are differences.S: sensitive strain; R: resistant strain; From top to bottom forward primer is followed successively by RZ-FA, RZ-FT, RZ-FC and RZ-FG, and reverse primer is SRZ-A.
Fig. 4 is for adopting Auele Specific Primer RZ-FC and SRZ-A amplification to the AS-PCR electrophorogram of the soybean phytophthora bacterial strain of zoxamide performance sensitivity and resistance.Marker:DNA Marker II; RZ2-2, RZ6-2, RZ9-2, RZ10-1 and RZ8-2 are resistant strain; PsJMS2, Ps6, Ps4, Ps13 and Ps52 are sensitive strain; The negative contrast of CK.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
" resistant strain " all refers to the soybean phytophthora bacterial strain to the zoxamide resistance in the literary composition; " sensitive strain " all refers to the soybean phytophthora bacterial strain to the zoxamide sensitivity.
The soybean phytophthora bacterial strain that uses among the following embodiment is: resistant strain RZ2-2, RZ5-1, RZ6-2, RZ9-2, RZ10-1, RZ12-1, RZ14-1 and RZ15-1; Sensitive strain Ps4, Ps6, Ps9, Ps13, Ps52, Ps53, AH1302 and PsJMS2.
Above-mentioned bacterial strains Ps4, Ps6, Ps9, Ps13, Ps52 and Ps53 all pick up from the Fujian China area, wherein Ps4, Ps6, Ps9 and Ps13 are by the Chen Furu researcher of Fujian Academy of Agricultural Sciences present, and Ps52 and Ps53 are presented by the Zhu Zhendong researcher of the Chinese Academy of Agricultural Sciences; AH1302 picks up from Chinese Anhui province, is presented by Academy of Agri-Science and Technology Anhui Province Qi Rende researcher; PsJMS2 picks up from Chinese Heilongjiangdistrict, is presented by the Zhu Zhendong researcher of the Chinese Academy of Agricultural Sciences.Resistant strain RZ2-2, RZ5-1, RZ6-2, RZ9-2, RZ10-1, RZ12-1, RZ14-1 and RZ15-1 all obtain by the method for fungicide tame, and parent strain is PsJMS2.Above bacterial strain all is accredited as the soybean phytophthora bacterial strain through existing morphology and molecular biology method.Above-mentioned each bacterial strain all carries out sensitivity testing and resistance checking through the colony growth rate method.
Embodiment 1, soybean phytophthora are to the sensitivity testing of zoxamide
8 strain sensitive strain Ps4, Ps6, Ps9, Ps13, Ps52, Ps53, AH1302 and PsJMS2; 8 strain resistant strain RZ2-2, RZ5-1, RZ6-2, RZ9-2, RZ10-1, RZ12-1, RZ14-1 and RZ15-1.
The Radix Dauci Sativae substratum: Radix Dauci Sativae 200g, squeeze the juice, 4 layers of filtered through gauze, distilled water is settled to 1L, 121 ℃ of moist heat sterilizations 20 minutes.
1, experimental procedure is as follows:
1) zoxamide is made into 10 with dimethyl sulfoxide (DMSO) (dimethyl sulfoxide DMSO)
5The mother liquor of μ g/mL.The zoxamide stepwise dilution is become the concentration gradient of 200 μ g/mL, 300 μ g/mL, 400 μ g/mL, 500 μ g/mL, 600 μ g/mL and 700 μ g/mL, 1 ‰ DMSO are as blank, to measure the susceptibility of 8 strain soybean phytophthora sensitive strains.For the sensitivity testing of 8 strain soybean phytophthora resistant strains, 1 ‰ DMSO measure it 10 as blank
5Inhibiting rate under the μ g/mL concentration.
2) by from low to high order, the liquid 480 μ L that draw each concentration gradient with liquid-transfering gun add sterilized being cooled in 45 ℃ the 480mL Radix Dauci Sativae substratum, making solvent is 1 ‰, mixing, to pour into the substratum of medicine in the culture dish that diameter is 9cm, only to add the blank that is treated to of 480 μ L DMSO.Each concentration repeats 3 times.
3) get the soybean phytophthora bacterial strain of 25 ℃ of lower dark culturing after 4 days, buy the bacterium cake that cut-off directly is 0.5cm along colony edge with punch tool, mycelia faces down and is inoculated in step 2) in the band medicine and the contrast flat board in, place 25 ℃ of incubator dark culturing, measure colony diameter behind the 4d.
4) right-angled intersection method is measured colony diameter, calculates under each concentration inhibiting rate to the mycelial growth of strains tested according to bacterium colony mean diameter value.Then inhibiting rate is changed into probit value (Y), drug concentration converts denary logarithm value (X) to, in Microsoft Excel, make regression straight line, obtain respectively each for the toxicity regression curvilinear equation Y=a+bX of examination soybean phytophthora bacterial strain to zoxamide, and concentration EC in correlation coefficient r and the establishment
50Value,
2. result
Table 1.8 strain soybean phytophthora sensitive strain and 8 strain soybean phytophthora resistant strains are to the susceptibility of zoxamide
Sensitivity testing is the result show, 8 strain sensitive strains are to the EC of zoxamide
50All less than 0.1 μ g/mL, and 8 strain resistant strains on the band medicine flat board of 100 μ g/mL also without any inhibition, its EC
50All greater than 100 μ g/ml, the utmost point is significantly higher than the EC of sensitive strain
50, the resistant multiple is also all more than 1000 times.
The discovery in 'beta '-tubulin gene and corresponding protein mutation site thereof in embodiment 2, the soybean phytophthora
1, bacterial strain: resistant strain RZ2-2, RZ5-1, RZ6-2, RZ9-2, RZ10-1, RZ12-1, RZ14-1 and RZ15-1, sensitive strain Ps9, Ps13, Ps52, Ps53, AH1302 and PsJMS2.
2, method:
1) strain culturing: use Radix Dauci Sativae substratum (Radix Dauci Sativae 200g squeezes the juice, 4 layers of filtered through gauze, distilled water is settled to 1L, 121 ℃ of moist heat sterilizations 20 minutes are for subsequent use behind the moist heat sterilization) to cultivate phytophthora sojae kaufmann﹠gerdemanns in 25 ℃.Pre-incubated soybean phytophthora sensitive strain is seeded on the PDA substratum that is covered with glassine paper, collects mycelia behind 25 ℃ of dark culturing 5d, in-80 ℃ of preservations, be used for extracting genomic dna after liquid nitrogen is freezing.
2) extract respectively the genomic dna of resistant strain and sensitive strain.
3) pcr amplification of 'beta '-tubulin gene complete sequence, the primer is comprised of dna molecular shown in SEQ ID NO:5 and the SEQ ID NO:6.
The PCR reaction system: the purpose fragment length adopts Beijing Quanshijin Biotechnology Co., Ltd's reagent less than the PCR reaction of 2kb, comprise 1 μ L template DNA (30-50ng) in the 50 μ L reaction systems, 4 μ L 10mM dNTP, 5 μ L10 * amplification buffers (contain 20mM Mg
2+), 1 μ L Taq archaeal dna polymerase (5U/ μ L), every primer (10 μ M) 1 μ L, 37 μ L ddH
2O, reaction system increases and decreases at double according to the follow-up test needs.
PCR reaction conditions: 94 ℃ of 4min of denaturation; 94 ℃ of 30s, 63.5 ℃ of 30s, 72 ℃ of 1min 30s, 35 circulations; Last 72 ℃ of downward-extension 10min.Amplified production is electrophoresis detection in 1% sepharose.
4) order-checking
The PCR product is checked order.The 'beta '-tubulin gene complete sequence of resistant strain is 1341bp altogether, sequence shown in SEQID NO:3, the intronless sequence, 446 amino acid of encoding altogether, sequence is shown in SEQ ID NO:4.
5) sequence alignment
Respectively the 'beta '-tubulin gene complete sequence of resistant strain and sensitive strain is compared.The result shows: compare with sensitive strain, genome 'beta '-tubulin gene 716 Nucleotide at dna sequence dna from 5 ' end sport C (Fig. 1) by G in the resistant strain.This site base mutation causes aminoacid sequence to be undergone mutation at 239, sports Serine (Ser, S) (Fig. 2) by halfcystine (Cys, C).
PCR product sequencing result shows, resistant strain is analyzed the crest line figure of order-checking 716 sudden changes that C has all occured to be become by G, can find that this site crest line figure is clear single clear and definite, shows the homozygous mutation that sports in this site.The sudden change of inferring this site produces resistance relevant (Fig. 1) with soybean phytophthora to zoxamide.
The method in mutational site in embodiment 3, the detection soybean phytophthora
One, design of primers
Bacterial strain is bacterial strain uses therefor PsJMS2 and RZ2-2 among the embodiment 1.
Primer such as table 2, sequences Design allele specific-PCR primer according to mutant 'beta '-tubulin gene, 3 ' of forward primer RZ-FT-last base of end with the sudden change after base consistent, in order to increase the specificity of primer, introduce base mismatch (primer RZ-FA, RZ-FC and RZ-FG) in forward primer 3 '-second base of end.Reverse primer is SRZ-A.
Table 2PCR detects the employed primer of soybean phytophthora bacterial strain to zoxamide performance resistance
Identical among concrete reaction system and the embodiment 2.Primer is carried out the thermograde PCR of different annealing temperature, determine the annealing temperature that specificity is high.Response procedures is: 94 ℃ of 4min of denaturation; 94 ℃ of 30s, 55-70 ℃ of 30s, 72 ℃ of 30s, 35 circulations; Last 72 ℃ of downward-extension 10min.Amplified production is electrophoresis detection in 2% sepharose.
The result as shown in Figure 3, primer is along with its specificity of raising of annealing temperature all increases.Wherein primer pair RZ-FT and SRZ-A when annealing temperature is 67 ℃, can only amplify the purpose band of 288bp from resistant strain; And after second base of 3 '-end introduced base mismatch C (primer pair RZ-FC and SRZ-A), also can only in resistant strain, amplify the purpose band of 288bp, and can not from sensitive strain, amplify corresponding fragment, and this specificity to primer is better than RZ-FT and SRZ-A, shows that this primer can be used for detecting the mutational site.Therefore select RZ-FC and SRZ-A this is used for the detection of G716C resistance allele to primer.
The amplified production of primer pair RZ-FC and SRZ-A antagonism bacterial strain is checked order, order-checking PCR product sequence be among the SEQ ID NO:3 from 5 ' terminal the 695th to 982 nucleotide sequence.
Two, detect the mutational site
Take the genomic dna of soybean phytophthora to be measured as template, adopt primer pair RZ-FC and SRZ-A to carry out pcr amplification, if obtain the fragment of 288bp, judge then in the soybean phytophthora genome that detects that the 'beta '-tubulin gene order terminally plays the 716th Nucleotide as C (be among the SEQ ID NO:3 from 5 ' end the 716th Nucleotide be C) from 5 ', judges that further described bacterial strain is resistant strain; If do not amplify the fragment of 288bp, judge then in the soybean phytophthora genome that detects that the 'beta '-tubulin gene order terminally plays the 716th Nucleotide as G (be among the SEQ ID NO:3 from 5 ' end the 716th Nucleotide be G) from 5 ', judges that further described bacterial strain is sensitive strain.
Consistent in pcr amplification system and reaction conditions and the experiment one, annealing temperature is 67 ℃.
Bacterial strain to be measured: resistant strain is RZ2-2, RZ6-2, RZ9-2, RZ10-1 and RZ8-2, and sensitive strain is PsJMS2, Ps6, Ps4, Ps13 and Ps52.
The result as shown in Figure 4, the result shows, primer pair RZ-FC and SRZ-A only can amplify the specificity target fragment from the soybean phytophthora bacterial strain (RZ2-2, RZ6-2, RZ9-2, RZ10-1 and RZ8-2) to zoxamide performance resistance, and can from sensitive strain (PsJMS2, Ps6, Ps4, Ps13 and Ps52), amplify any band, therefore, adopt primer pair RZ-FC and SRZ-A, when annealing temperature is 67 ℃, carry out pcr amplification, can be used for the specific detection phytophthora sojae kaufmann﹠gerdemann and whether zoxamide produced resistance.
Claims (10)
- One kind detect or the auxiliary detection soybean phytophthora in 'beta '-tubulin gene or the corresponding albumen method of presence bit point mutation whether, step is as follows:Take the genomic dna of soybean phytophthora to be measured as template, carry out pcr amplification with primer pair shown in SEQ ID NO:1 and the SEQ ID NO:2, if pcr amplification product is the fragment of 288bp, then there are the mutational site in the existence of 'beta '-tubulin gene or candidate in the described soybean phytophthora to be measured;Described mutational site refers to that genome sequence the 716th Nucleotide from 5 ' end of 'beta '-tubulin gene in the soybean phytophthora is C.
- 2. method according to claim 1, it is characterized in that: in the described pcr amplification, annealing temperature is 67 ℃.
- One kind detect or the auxiliary detection soybean phytophthora in the 'beta '-tubulin gene whether have the primer pair in mutational site, formed by dna molecular shown in SEQ ID NO:1 and the SEQ ID NO:2.
- 4. the 'beta '-tubulin gene mutational site in the soybean phytophthora, for the genome sequence of 'beta '-tubulin gene in the soybean phytophthora from 5 ' terminal the 716th Nucleotide be C.
- 5. the primer pair of soybean phytophthora 'beta '-tubulin gene complete sequence that increases is comprised of dna molecular shown in SEQ ID NO:5 and the SEQID NO:6, and in the pcr amplification, annealing temperature is 63.5 ℃.
- 6. 'beta '-tubulin mutational site in the soybean phytophthora is for the 239th amino acids from the N end of 'beta '-tubulin in the soybean phytophthora is Serine (Ser, S).
- 7. claim 4 or the 6 described mutational sites application in identifying the soybean phytophthora resistance; Described resistance refers to the resistance to zoxamide.
- 8. application according to claim 7 is characterized in that: exist the soybean phytophthora in described mutational site to have or the candidate has resistance to zoxamide.
- 9.SEQ gene shown in the ID NO:3.
- 10.SEQ albumen shown in the ID NO:4.
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| CN107746894A (en) * | 2017-10-26 | 2018-03-02 | 中国农业大学 | Rapid identification soybean phytophthora β tubulin gene nucleotides point mutation and its to the drug-fast method of Guardian |
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| CN106191205A (en) * | 2016-09-14 | 2016-12-07 | 江苏丘陵地区镇江农业科学研究所 | A kind of ash arrhizus bacteria method for quick drug-fast to various medicaments |
| CN107746894A (en) * | 2017-10-26 | 2018-03-02 | 中国农业大学 | Rapid identification soybean phytophthora β tubulin gene nucleotides point mutation and its to the drug-fast method of Guardian |
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Application publication date: 20130213 |