CN104450895B - Fructus Capsici and Tomato Bacterial Scab bacterium PFGE molecular typing methods - Google Patents
Fructus Capsici and Tomato Bacterial Scab bacterium PFGE molecular typing methods Download PDFInfo
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Abstract
The invention discloses Bacterial Scab bacterium molecule classifying method on a kind of Fructus Capsici or Fructus Lycopersici esculenti, the method makes public for the first time employing particular restriction restriction endonucleaseSmlI (SwaI) in cutting blob of viscose, DNA combines pulsed field gel electrophoresis method (PFGE), and Fructus Capsici and Tomato Bacterial Scab bacterium carry out the operating technology of molecule parting.This pathogen suspension is made after blob of viscose, enzyme action through pulsed field gel electrophoresis after E.C. 3.4.21.64 processes, it is thus achieved that specific band, conversion stripes information carries out cluster analysis, the Bacterial Scab bacterium on Fructus Capsici and Fructus Lycopersici esculenti can effectively be divided into 4 kinds.The restriction endonuclease that the inventive method is used is for use first, and electrophoretic parameters, for optimize voluntarily, may be used for Fructus Capsici and Tomato Bacterial Scab bacterium taxonomic identification and genetic diversity Journal of Sex Research.
Description
Technical field
The present invention relates to phytopathogen molecule parting technology, be specifically related to by pulsed field gel electrophoresis (PFGE) method pair
Fructus Capsici and Tomato Bacterial Scab bacterium (Bacterial Spot caused by Xanthomonads) carry out the side of typing
Method.
Background technology
Fructus Capsici and Fructus Lycopersici esculenti shot hole are that a kind of bacterial disease on plant of Solanaceae generally occurs, and this pathogenic bacteria main harm is peppery
Green pepper and the blade of Fructus Lycopersici esculenti, stem are climing, fruit, especially universal to occur on blade.Blade is fallen ill, and the initial stage forms water stain shape, yellowish green
Fleck, becomes round after expansion or irregular shape, crineous, marginal swell and the scab of concavity, coarse in scab shape.
Yellow leaf time serious, dry up, rupture, early fallout.Stem and carpopodium morbidity, initial stage formation water soaked spots, gradually develops
Become brown billet speckle.Scab suberification is swelled, and lobe is ulcer shape scab speckle.Fruit is fallen ill, and is formed circular or oblong black
Color scab speckle.If this disease is prevented and treated not in time, the yield of Fructus Capsici and Fructus Lycopersici esculenti is not only greatly reduced, but also has a strong impact on Fructus Capsici
With the fruit quality of Fructus Lycopersici esculenti, cause heavy economic losses.
This pathogenic bacteria is at the U.S., Brazil, Australia, South Africa, Yugoslavia, Mexico, Costa Rica, A Gen at present
Many countries and regions, more than 60, the whole world such as the court of a feudal ruler occur, and are one of worldwide disease.Xinjiang of China, Gansu, the Inner Mongol, black dragon
Also there is generation in ten several areas such as river, Beijing, Shanxi, have caused people to pay close attention to widely.As far back as the sixties in 20th century, Dye etc.
(1964) just have been found that Fructus Capsici and Tomato Bacterial Scab pathogen exist quite abundant genetic diversity.2004
This pathogenic bacteria is divided into 4 kind: X. euvesicatoria, X. vesicatoria, X. perforans, X. by Jones etc.
gardneri 。
PFGE typing method is the restriction endonuclease bacterial digestion DNA utilizing restriction enzyme site few, produces big sheet
Section, carries out DNA fragmentation under the conditions of pulsed field gel electrophoresis and efficiently separates.PFGE is as a kind of very effective pathogen typing
Technology, has been widely used in the molecule parting research of many pathogen, and has been considered as " the gold of molecule parting by numerous researcheres
Standard " (Fakhr et al., 2005).At present, the Pulse field gel electrophoresis of Fructus Capsici and Tomato Bacterial Scab bacterium this
The research of aspect have not been reported.
Summary of the invention
It is an object of the invention to set up the Pulse field gel electrophoresis technology body of Fructus Capsici and Tomato Bacterial Scab bacterium
System, for Fructus Capsici and Tomato Bacterial Scab bacterium taxonomic identification and genetic diversity Journal of Sex Research, key issue to be solved is
The determination of the restricted enzyme that the method is used and the determination of electrophoresis parameters.
In order to solve above-mentioned technical problem, the technical solution adopted in the present invention is: bacillary on a kind of Fructus Capsici or Fructus Lycopersici esculenti
Streptomyces scabies molecular typing methods, the method screening low frequency restricted enzyme, use the electrophoretic parameters optimized, finally through pulse
Field gel electrophoresis, it is thus achieved that specific band, conversion stripes information carries out cluster analysis, is effectively distinguished by 4 kinds of pathogen.
Described low frequency restricted enzyme is SmlI (SwaI), and pulsed field gel electrophoresis parameters is:
Pattern is " TWO STATE ", voltage gradient: 6 V/cm, electric field angle: 140 °, the burst length: 20 ~ 80 s, electricity
The swimming time: 17 h, buffer: 0.5 × TBE, buffer temperature: 14 DEG C.
On described Fructus Capsici or Fructus Lycopersici esculenti, Bacterial Scab bacterium molecule classifying method, specifically comprises the following steps that
(1) blob of viscose is prepared: mixed with pulsed field level agarose colloidal sol by described pathogenic bacteria suspension, join and prepare blob of viscose
Mould solidifies；
(2) cell cracking: with SmlI, blob of viscose is carried out enzyme action and hatch, be carried out subsequently；
(3) by the DNA extraction in enzyme action blob of viscose out；
(4) DNA being proposed out is carried out pulsed field gel electrophoresis；
(5) acquisition of image: electrophoresis takes out blob of viscose after terminating, dye acquisition image of taking pictures；
(6) cluster analysis is carried out.
Further, described Fructus Capsici or Tomato Bacterial Scab bacterium molecule classifying method, specifically comprise the following steps that
(1) blob of viscose is prepared:
A. add the CSB liquid of pre-cooling at centrifuge tube, by the pathogen of exponential phase, be mixed into suspension, OD value to 3.6
~4.5；
The most separately taking centrifuge tube, by above-mentioned bacterial suspension in new pipe, water-bath adds Proteinase K Solution after hatching；
C. take 1% pulsed field level agarose colloidal sol and the bacterial suspension mixing in step B, add mixture to preparation
The mould of blob of viscose solidifies；
(2) cell cracking
Add the CLB liquid of pre-cooling the most on ice with centrifuge tube, add Proteinase K Solution, blob of viscose step (1) prepared
Move in centrifuge tube；
B. in water-bath, enzyme action is hatched；
(3) blob of viscose is cleaned
A. outwell CLB, add preheating pure water, 50 DEG C of water-bath 5-15min；
B., after pure water repeats to wash 2-3 time equally, preheating TE solution, 50 DEG C of water-bath 10-20min are added；
After C.TE solution repeats to wash 2-3 time, adding TE, stored refrigerated blob of viscose is standby；
(4) DNA in enzyme action blob of viscose is extracted
A. in the preparation every 150 μ l of buffer M:M buffer, pure water is 35 μ l, Buffer 15 μ l；
B. take out blob of viscose centrifuge tube TE liquid from refrigerator, be cut into 2mm width, put in M buffer, 30 DEG C of water-bath 15min；
C. preparation enzyme cutting buffering liquid H: each 1.5ml centrifuge tube needs H buffer: pure water 157 μ l+Buffer 20 μ l+
BSA20μl = 197μl；
D. with rifle head sucking-off each 1.5ml centrifuge tube buffer M, it is to avoid damage blob of viscose, adding buffer H is 197 l, then
Add 3 l restriction endonuclease Swa I (Smi I)；
E. 4h is hatched in 30 DEG C of water-baths；
(5) sample-adding and electrophoresis
A. take out the 1.5ml centrifuge tube pipettor careful sucking-off H buffer hatched, add 200 l 0.5 × tbe buffers
Liquid；
B. loading, it is ensured that all of blob of viscose is on a horizontal line, and blob of viscose contacts with the bottom surface of glue groove；
C. the 1% pulse level agarose colloidal sol of 60 DEG C that 100 ml have melted is poured into glue groove, set at room temperature；
D. open pulsed field gel electrophoresis instrument CHEF MAPPER XA system host and the switch of pump, open freezing machine, really
Protect preset temperature dynamo-electric swimming on 14 DEG C;
Electrophoretic parameters is set according to preliminary result as follows:
Pattern " TWO STATE "
Voltage gradient 6 V/cm
Electric field angle 140 °
Burst length 20 ~ 80 s
Electrophoresis time 17 h
Buffer 0.5 × TBE
Buffer temperature 14 DEG C
Pump speed 50
(6) acquisition of image: electrophoresis takes out blob of viscose after terminating, and uses EB(ethidium bromide) solution soaking gel, shaking table contaminates
Color 30 min takes pictures；
(7) cluster analysis: read tape electrophoresis pattern, according to the presence or absence of fingerprint band, is respectively converted into 2 numbers, i.e.
Have band to be designated as 1, without band for 0；The non-weighting group average method UPGMA using NTSYS2.1 computer software analyzes program, carries out
Molecular fingerprint cluster analysis, draws Dendrogram.
Bacterial Scab bacterium molecule classifying method on described Fructus Capsici or Fructus Lycopersici esculenti, cluster result at genetic similarity is
When 72.2%, all bacterial strains are divided into 4 subgroups, and the subgroup analysis of establishing criteria bacterial strain place understands, and group1 belongs to
Xanthomonas euvesicatoria, reference culture is ICMP98；Group2 group belongs to X. perforans, reference culture
Belonging to X. vesicatoria for ICMP16690, group 3 groups, reference culture is ICMP63；Group 4 groups belongs to X.
Gardneri, reference culture is ICMP16689, and the grouping result of this cluster is effective for 4 kinds of Fructus Capsici Tomato Bacterial Scab bacterium
Distinguish.
The present invention has a lower beneficial effect:
The present invention passes through software analysis low frequency restriction endonuclease to Fructus Capsici Tomato Bacterial Scab bacterium type strain Xcv85-10's
The restriction enzyme site of whole genome sequence and the size of endonuclease bamhi, have selected low frequency restricted enzyme SmlI (SwaI), and warp
Cross repetition test, it is determined that the Fructus Capsici Tomato Bacterial Scab every electrophoretic parameters of bacterium PFGE.The inventive method for Fructus Capsici and
Tomato Bacterial Scab bacterium taxonomic identification and genetic diversity Journal of Sex Research, solve this pathogenic bacteria because of classification and variation situation more complicated to
In real work, band serves problem, research and inquirement Fructus Capsici and Tomato Bacterial Scab bacterium population genetic variations and relationship is closed
They, for going to recognize Fructus Capsici and Tomato Bacterial Scab bacterium from the angle of genetic evolution, are classified by system from molecular level
With qualification, carry out this pathogenic bacteria virulence analysis and pathogenesis, developer molecule detection technique, improvement Breeding Strategies further
Deng offer scientific basis.
Accompanying drawing explanation
Fig. 1 is to carry out electrophoresis result figure, in figure with the restricted enzyme that present invention determine that and electrophoretic parameters: from left to right
Electrophoresis band be followed successively by: 1-Marker, 2-14 are pathogen.
Fig. 2: UPGMA dendrogram (right side bracket inner digital is bacterial strain number).
Detailed description of the invention
With the low frequency restricted enzyme Swa I of screening in the present invention and the pulsed field gel electrophoresis parameter that arranges to from
Domestic Fructus Capsici and the former bacterium of Tomato Bacterial Scab carry out electrophoresis, and result is shown in that Fig. 1, all bacterial strains present 15 specificitys altogether
Band, each bacterial strain presents the band about 3-8 bar, and size is between 180 ~ 2200 kb.By stripe information system in electrophoresis pattern
Become 0-1 form, have band to be designated as 1, be designated as 0 without band, use Ntsys 2.1 software to carry out cluster analysis according to UPGMA algorithm,
4 kinds of Fructus Capsicis and the former bacterium of Tomato Bacterial Scab effectively can be distinguished, restriction endonuclease and the electrophoretic parameters used in the present invention is described,
Fructus Capsici and Tomato Bacterial Scab bacterium can be carried out PFGE molecule parting.
Concrete operation method of the present invention is as follows:
1, prepared by blob of viscose
For colibacillary PFGE standard method (Ribot in this experimental technique Main Basis U.S. CDC-PulseNet
Et al., 2006), part steps to be changed, concrete operations are as follows:
(1) 1.5ml is centrifuged the CSB liquid adding 500 μ l pre-coolings, by the pathogen of exponential phase, is mixed into suspension,
OD value is to 3.6 ~ 4.5；
(2) separately taking 1.5ml centrifuge tube, by the 100 above-mentioned bacterial suspensions of l in new pipe, 5min is hatched in 37 DEG C of water-baths
After often pipe add 5 l Proteinase K Solution；
(3) take 100 μ l 1% pulsed field level agarose colloidal sols and the mixing of 100 l bacterial suspensions, add mixture to
Prepare in the mould of blob of viscose and solidify；
2, cell cracking
(1) add the CLB liquid of 5ml pre-cooling on ice with 50ml centrifuge tube, add 25 l E.C. 3.4.21.64s (20mg/ml) molten
Liquid, moves into the blob of viscose of above-mentioned preparation in centrifuge tube；
In (2) 54 DEG C of water-baths, enzyme action hatches 3h；
3, blob of viscose is cleaned
(1) outwelling CLB, often pipe adds preheating pure water 15ml, 50 DEG C of water-bath 10min；
(2), after pure water repeats to wash three times equally, add 15ml and preheat TE solution, 50 DEG C of water-bath 15min；
(3) after TE solution repeats to wash three times, adding 10ml TE, 4 DEG C of Refrigerator store blob of viscoses are standby；
4, the DNA in enzyme action blob of viscose
(1) preparation buffer M
Each 1.5ml centrifuge tube needs M buffer: pure water 135 μ l+Buffer 15 μ l=150 μ l；
(2) take out blob of viscose centrifuge tube TE liquid from refrigerator, be cut into 2mm width, put in M buffer, 30 DEG C of water-bath 15min；
(3) preparation enzyme cutting buffering liquid H
Each 1.5ml centrifuge tube needs H buffer: pure water 157 μ l+Buffer 20 μ l+ BSA20 μ l=197 μ l；
(4) with rifle head sucking-off each 1.5ml centrifuge tube buffer M, it is to avoid damage blob of viscose, adding buffer H is 197 l,
Add 3 l restriction endonuclease Swa I (Smi I)；
4h is hatched in (5) 30 DEG C of water-baths；
5, sample-adding and electrophoresis
(1) take out the 1.5ml centrifuge tube pipettor careful sucking-off H buffer hatched, add 200 l 0.5 × TBE and delay
Rush liquid；
(2) loading, it is ensured that all of blob of viscose is on a horizontal line, and blob of viscose contacts with the bottom surface of glue groove；
(3) the 1% pulse level agarose colloidal sol of 60 DEG C that 100 ml have melted is poured into glue groove, set at room temperature；
(4) open pulsed field gel electrophoresis instrument CHEF MAPPER XA system host and the switch of pump, open freezing machine, really
Protect preset temperature dynamo-electric swimming on 14 DEG C.
It is as follows that electrophoretic parameters is set voluntarily according to preliminary result:
Pattern " TWO STATE "
Voltage gradient 6 V/cm
Electric field angle 140 °
Burst length 20 ~ 80 s
Electrophoresis time 17 h
Buffer 0.5 × TBE
Buffer temperature 14 DEG C
Pump speed 50
6, the acquisition of image
Electrophoresis takes out blob of viscose after terminating, and uses EB(ethidium bromide) solution soaking gel, 30 min that shaking table dyes take pictures.
7, cluster analysis
Electrophoresis pattern is read tape, according to the presence or absence of fingerprint band, is respectively converted into 2 numbers, i.e. has band to be designated as 1, nothing
Band for 0；The non-weighting group average method UPGMA using NTSYS2.1 computer software analyzes program, carries out molecular fingerprint cluster
Analyze, draw Dendrogram such as Fig. 2.
All bacterial strain electrophoresis patterns are divided into 11 kinds of PFGE band Tupu types, are designated as A ~ K respectively.
Cluster result all bacterial strains when genetic similarity is 72.2% are divided into 4 subgroups, and establishing criteria bacterial strain place is sub-
Cluster analysis understands, and group1 belongs to Xanthomonas euvesicatoria, and reference culture is ICMP98；Group2 group belongs to
X. perforans, reference culture is that ICMP16690, group 3 groups belongs to X. vesicatoria, and reference culture is
ICMP63；Group 4 groups belongs to X. gardneri, and reference culture is ICMP16689.The grouping result energy handle of this cluster is described
4 kinds of Fructus Capsici Tomato Bacterial Scab bacterium efficiently differentiate out.
Claims (5)
1. Bacterial Scab bacterium molecule classifying method on a Fructus Capsici or Fructus Lycopersici esculenti, it is characterised in that: the method screening low frequency limit
Property restriction endonuclease processed, uses the electrophoretic parameters optimized, and finally through pulsed field gel electrophoresis, it is thus achieved that specific band, conversion stripes is taken a message
Breath carries out cluster analysis, is effectively distinguished by 4 kinds of pathogen；
Wherein, described low frequency restricted enzyme is SmlI (SwaI).
2. as claimed in claim 1 Bacterial Scab bacterium molecule classifying method on Fructus Capsici or Fructus Lycopersici esculenti, it is characterised in that: described arteries and veins
Rushing field gel electrophoresis parameters is:
Pattern is " TWO STATE ", voltage gradient: 6 V/cm, electric field angle: 140 °, and the burst length: 20 ~ 80 s, during electrophoresis
Between: 17 h, buffer: 0.5 × TBE, buffer temperature: 14 DEG C.
3. as claimed in claim 1 or 2 Bacterial Scab bacterium molecule classifying method on Fructus Capsici or Fructus Lycopersici esculenti, it is characterised in that:
(1) blob of viscose is prepared: mixed with pulsed field level agarose colloidal sol by described pathogenic bacteria suspension, join the mould preparing blob of viscose
Middle solidification；
(2) cell cracking: with SmlI, blob of viscose is carried out enzyme action and hatch, be carried out subsequently；
(3) by the DNA extraction in enzyme action blob of viscose out；
(4) DNA put forward is carried out pulsed field gel electrophoresis；
(5) acquisition of image: electrophoresis takes out blob of viscose after terminating, dye acquisition image of taking pictures；
(6) cluster analysis is carried out.
4. Fructus Capsici as claimed in claim 3 or Tomato Bacterial Scab bacterium molecule classifying method, it is characterised in that concrete steps
As follows:
(1) blob of viscose is prepared:
A. add the CSB liquid of pre-cooling at centrifuge tube, by the pathogen of exponential phase, be mixed into suspension, OD value to 3.6 ~
4.5；
The most separately taking centrifuge tube, by above-mentioned bacterial suspension in new pipe, water-bath adds Proteinase K Solution after hatching；
C. take 1% pulsed field level agarose colloidal sol and the bacterial suspension mixing in step B, add mixture to and prepare blob of viscose
Mould in solidify；
(2) cell cracking
Adding the CLB liquid of pre-cooling the most on ice with centrifuge tube, add Proteinase K Solution, blob of viscose step (1) prepared moves into
In centrifuge tube；
B. in water-bath, enzyme action is hatched；
(3) blob of viscose is cleaned
A. outwell CLB, add preheating pure water, 50 DEG C of water-bath 5-15min；
B., after pure water repeats to wash 2-3 time equally, preheating TE solution, 50 DEG C of water-bath 10-20min are added；
After C.TE solution repeats to wash 2-3 time, adding TE, stored refrigerated blob of viscose is standby；
(4) DNA in enzyme action blob of viscose is extracted
A. in the preparation every 150 μ l of buffer M:M buffer, pure water is 35 μ l, Buffer 15 μ l；
B. take out blob of viscose centrifuge tube TE liquid from refrigerator, be cut into 2mm width, put in M buffer, 30 DEG C of water-bath 15min；
C. preparation enzyme cutting buffering liquid H: each 1.5ml centrifuge tube needs H buffer: pure water 157 μ l+Buffer 20 μ l+
BSA20μl = 197μl；
D. with rifle head sucking-off each 1.5ml centrifuge tube buffer M, it is to avoid damage blob of viscose, adding buffer H is 197 l, adds
3 l restriction endonuclease Swa I (Smi I)；
E. 4h is hatched in 30 DEG C of water-baths；
(5) sample-adding and electrophoresis
A. take out the 1.5ml centrifuge tube pipettor careful sucking-off H buffer hatched, add 200 l 0.5 × tbe buffer liquid；
B. loading, it is ensured that all of blob of viscose is on a horizontal line, and blob of viscose contacts with the bottom surface of glue groove；
C. the 1% pulse level agarose colloidal sol of 60 DEG C that 100 ml have melted is poured into glue groove, set at room temperature；
D. open pulsed field gel electrophoresis instrument CHEF MAPPER XA system host and the switch of pump, open freezing machine, it is ensured that be pre-
If temperature is dynamo-electric swimming on 14 DEG C;
Electrophoretic parameters is set according to preliminary result as follows:
Pattern " TWO STATE "
Voltage gradient 6 V/cm
Electric field angle 140 °
Burst length 20 ~ 80 s
Electrophoresis time 17 h
Buffer 0.5 × TBE
Buffer temperature 14 DEG C
Pump speed 50
(6) acquisition of image: electrophoresis takes out blob of viscose after terminating, and uses EB(ethidium bromide) solution soaking gel, dyeing 30 on shaking table
Min takes pictures；
(7) cluster analysis: read tape electrophoresis pattern, according to the presence or absence of fingerprint band, is respectively converted into 2 numbers, i.e. has band
Be designated as 1, without band for 0；The non-weighting group average method UPGMA using NTSYS2.1 computer software analyzes program, carries out molecule
Fingerprint cluster is analyzed, and draws Dendrogram.
5. as claimed in claim 3 Bacterial Scab bacterium molecule classifying method on Fructus Capsici or Fructus Lycopersici esculenti, it is characterised in that: cluster knot
Fruit all bacterial strains when genetic similarity is 72.2% are divided into 4 subgroups, and the subgroup analysis of establishing criteria bacterial strain place understands,
Group1 belongs to Xanthomonas euvesicatoria, and reference culture is ICMP98；Group2 group belongs to X.
Perforans, reference culture is that ICMP16690, group 3 groups belongs to X. vesicatoria, and reference culture is ICMP63；
Group 4 groups belongs to X. gardneri, and reference culture is ICMP16689, and the grouping result of this cluster is thin for 4 kinds of Fructus Capsici Fructus Lycopersici esculenties
Bacterium property Streptomyces scabies efficiently differentiates out.
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Non-Patent Citations (3)
| Title |
|---|
| Development of a Diagnostic DNA Probe for Xanthomonads Causing Bacterial Spot of Peppers and Tomatoes;KUFLOM M. KUFLU AND DIANE A. CUPPELS;《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》;19971130;第63卷(第11期);4462-4470 * |
| Outbreaks of Bacterial Spot Caused by Xanthomonas gardneri on Processing Tomato in Central-West Brazil;Alice M. Quezado-Duval et al.;《Plant Disease》;20040229;第88卷(第2期);157-162 * |
| Systematic analysis of xanthomonads (Xanthomonas spp.) associated with pepper and tomato lesions;J. B. Jones et al.;《International Journal of Systematic and Evolutionary Microbiology》;20001231;第50卷;1211-1219 * |
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