CN106834173A

CN106834173A - One plant of Ralstonia solanacearum of bioluminescence

Info

Publication number: CN106834173A
Application number: CN201710049934.8A
Authority: CN
Inventors: 徐秀兰; 杜和山; 陈斌; 耿三省; 张晓芬
Original assignee: Beijing Academy of Agriculture and Forestry Sciences
Current assignee: Beijing Academy of Agriculture and Forestry Sciences
Priority date: 2017-01-23
Filing date: 2017-01-23
Publication date: 2017-06-13

Abstract

The invention discloses one plant of Ralstonia solanacearum of bioluminescence.Ralstonia solanacearum provided by the present invention is Ralstonia solanacearum Rs BL#7, and it is CGMCC No.13637 in the deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center.The capsicum N1508 and susceptible material C alifonia Wonder of pepper ralstonia solanacearum is identified using Ralstonia solanacearum Rs BL#7 provided by the present invention, result shows, capsicum N1508 is resistance to bacterial wilt capsicum material, the susceptible material C alifonia Wonder of pepper ralstonia solanacearum are sense bacterial wilt capsicum material, completely the same with actual conditions.Therefore, the Ralstonia solanacearum Rs BL#7 that the present invention is provided can identify resistance of the capsicum material to bacterial wilt, with important application value.

Description

One plant of Ralstonia solanacearum of bioluminescence

Technical field

The present invention relates to biological technical field, and in particular to one plant of Ralstonia solanacearum of bioluminescence.

Background technology

Smith is described to the morphology and systematics property of plant Stalk Rot first within 1896, and will Stalk Rot is named as Bacterium solanacearum.Nineteen ninety-five Yabuucgi etc. is by further investigation, foundation table Type feature, cell lipid, aliphatic acid composition, rRNA-DNA homologys and 16S rRNA the sequencing results, by Causal Organism of The Bacterial Wilt It is Ralstonia solanacearum that bacterium renames.In the 3rd bacterial wilt international conference, for ease of description Ralstonia solanacearum Following difference is planted, Fegan and Prior proposes Evolution Type taxonomy model jointly, i.e., based on conservative egl, mutS, hrpBh Or ITS single-gene sequence polymorphisms, plant-bacterial-wilt pathogen is divided into four evolutions closely related with geographic origin Type-Evolution Type I, Evolution Type II, Evolution Type III and Evolution Type IV, wherein Evolution Type I include all biochemical changes from Asia Kind, Evolution Type II includes being isolated from the biochemical variant in America, and Evolution Type III includes being isolated from the biochemical variant in Africa, Evolution Type IV Biochemical variant including being isolated from Indonesia and Japan.

Pepper ralstonia solanacearum is to infect one kind soil for causing by Ralstonia solanacearum (Ralstonia solanacearum) to pass Bacterial disease, the disease is a kind of typical vascular bundle diseases, to endanger based on root and stem.Ralstonia solanacearum can be by rain Water, irrigation water, subterranean pest-insect, operation instrument etc. are propagated, and are invaded from host root or basal part of stem hole skin or wound more, and early stage belongs to Latency infects state, can rapidly be bred in the spiral duct of vascular bundle when condition is suitable, and expands to overground part along conduit Exhibition, makes whole conducting tissue destroyed and loses transmitting function, and stem, leaf are wilted because cannot get moisture supply, and then dead.By In the host of Ralstonia solanacearum is more, distribution is wide abundant with genetic diversity, cause pepper ralstonia solanacearum once occurring just to be difficult to prevent Control, so plantation disease-resistant variety is prevention pepper ralstonia solanacearum most efficient method.

The content of the invention

The technical problems to be solved by the invention are how to identify the resistance for treating measuring plants to bacterial wilt.

In order to solve the above technical problems, present invention firstly provides one plant of bacterium.

Bacterium provided by the present invention be Ralstonia solanacearum (Ralstonia solanacearum) Rs-BL#7, its The deposit number of China Committee for Culture Collection of Microorganisms's common micro-organisms center is CGMCC No.13637.

In order to solve the above technical problems, present invention also offers a kind of microbial inoculum.The microbial inoculum can contain the blue or green withered Lei Er Salmonella (Ralstonia solanacearum) Rs-BL#7CGMCC No.13637.

The preparation method of the microbial inoculum falls within protection scope of the present invention.The preparation method of the microbial inoculum may include as follows Step：The Ralstonia solanacearum (Ralstonia solanacearum) Rs-BL#7CGMCC No.13637 are seeded to carefully Bacterium culture medium is simultaneously cultivated, and the bacterium solution of acquisition is microbial inoculum.

In above-mentioned preparation method, the bacteria culture media can be NA fluid nutrient mediums.Every liter of NA fluid nutrient medium Composition is as follows：Beef extract 3.0g, glucose 10.0g, peptone 5.0g, dusty yeast 0.5g, balance of water.The NA liquid training Support the pH value concretely 7.0 of base.

Ralstonia solanacearum (Ralstonia solanacearum) the Rs-BL#7CGMCC No.13637 or described Application of the microbial inoculum in resistance to bacterial wilt plant is screened falls within protection scope of the present invention.

In the application, with the Ralstonia solanacearum (Ralstonia solanacearum) Rs-BL#7CGMCC Measuring plants are treated in No.13637 inoculations, then treat whether measuring plants are that resistance to bacterial wilt plant (examine by live body by the luminous judgement of observation Survey).The plant that bioluminescence signal can not be detected is or candidate is resistance to bacterial wilt plant；Bioluminescence signal can be detected Plant is or candidate is sense bacterial wilt plant.

Ralstonia solanacearum (Ralstonia solanacearum) the Rs-BL#7CGMCC No.13637 or described Microbial inoculum treats that the application in resistance of the measuring plants to bacterial wilt falls within protection scope of the present invention in identification.

In the application, with the Ralstonia solanacearum (Ralstonia solanacearum) Rs-BL#7CGMCC Measuring plants are treated in No.13637 inoculations, then by the luminous Resistance to bacterial wilt for judging to treat measuring plants of observation.Biology can not be detected Luminous signal, then plant resistance to bacterial wilt to be measured；Bioluminescence signal is detected, then treats the susceptible bacterial wilt of measuring plants.Bioluminescence Signal is stronger, then treat the more susceptible bacterial wilt of measuring plants.

The mode of the inoculation concretely soaks plant root.

Also need to place a plant into before " observation luminous " more than 20min at dark (purpose be make plant chlorophyll oneself Luminescence queenching).

Any of the above-described plant can be capsicum.

Any of the above-described capsicum can be Capsicum annuum N1508 or its offspring.

Any of the above-described capsicum can be the susceptible material C alifonia Wonder of pepper ralstonia solanacearum or its offspring.

Any of the above-described capsicum can be capsicum grafting.

Any of the above-described capsicum can be the seedling with Capsicum annuum N1508 as stock, pepper ralstonia solanacearum sense The capsicum grafting that the seedling of sick material C alifonia Wonder obtains for scion.

Any of the above-described capsicum can be the seedling with the susceptible material C alifonia Wonder of pepper ralstonia solanacearum as stock, The capsicum grafting that the seedling of Capsicum annuum N1508 obtains for scion.

Any of the above-described capsicum can be the capsicum grafting that Capsicum annuum N1508 are obtained from root graft.

Any of the above-described capsicum can be what the susceptible material C alifonia Wonder of pepper ralstonia solanacearum were obtained from root graft Capsicum grafting.

Any of the above-described capsicum can be disease-resistant material MC-4 or its offspring.

The Capsicum annuum N1508 are in China Committee for Culture Collection of Microorganisms's common micro-organisms center Deposit number be CGMCC No.13597.

Ralstonia solanacearum (Ralstonia solanacearum) the Rs-BL#7CGMCC No.13637 or described Application of the microbial inoculum in the chemical agent that screening prevents and treats bacterial wilt falls within protection scope of the present invention.

Using Ralstonia solanacearum provided by the present invention (Ralstonia solanacearum) Rs-BL#7CGMCC No.13637 identifies the Capsicum annuum N1508 and susceptible material C alifonia Wonder of pepper ralstonia solanacearum, as a result table Bright, Capsicum annuum N1508 are resistance to bacterial wilt capsicum material, the susceptible material C alifonia Wonder of pepper ralstonia solanacearum It is sense bacterial wilt capsicum material, it is completely the same with actual conditions.Therefore, the Ralstonia solanacearum (Ralstonia that the present invention is provided Solanacearum) Rs-BL#7CGMCC No.13637 can identify resistance of the capsicum material to bacterial wilt, should with important With value.

Brief description of the drawings

Fig. 1 is in the step 2 of embodiment 11 experimental result.

Fig. 2 is the experimental result of C in the step 22 of embodiment 1.

Fig. 3 is the experimental result of step one in embodiment 2.

Fig. 4 is in the step 2 of embodiment 21 experimental result.

Fig. 5 is in the step 2 of embodiment 22 experimental result.

Fig. 6 is the experimental result of step 3 in embodiment 2.

Fig. 7 is the experimental result of step 4 in embodiment 2.

Fig. 8 is the experimental result of step 2 in embodiment 3.

Fig. 9 is the experimental result of step 3 in embodiment 3.

Preservation explanation

Strain name：Ralstonia solanacearum

Latin name：Ralstonia solanacearum

Strain number：Rs-BL#7

Preservation mechanism：China Committee for Culture Collection of Microorganisms's common micro-organisms center

Preservation mechanism is referred to as：CGMCC

Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3

Preservation date：On January 17th, 2017

Collection is registered on the books numbering：CGMCC No.13637

Plant variety title：Capsicum annuum

Strain number：N1508

Preservation mechanism is referred to as：CGMCC

Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3

Preservation date：On January 17th, 2017

Collection is registered on the books numbering：CGMCC No.13597

Specific embodiment

The present invention is further described in detail with reference to specific embodiment, the embodiment for being given is only for explaining The bright present invention, rather than in order to limit the scope of the present invention.

Experimental technique in following embodiments, unless otherwise specified, is conventional method.

Material used, reagent etc. in following embodiments, unless otherwise specified, commercially obtain.

NA fluid nutrient mediums：Beef extract 3.0g, glucose 10.0g, peptone 5.0g and dusty yeast 0.5g are dissolved in steaming Distilled water, 1L, regulation pH value to 7.0 are settled to distilled water；Then 121 DEG C sterilizing 20min.

NA solid mediums：To adding agar in NA fluid nutrient mediums, the culture medium for making its concentration be obtained for 18g/L.

NA solid plates：About 50 DEG C of NA solid mediums are poured into sterile petri dish, NA solid plates are obtained after solidification.

TZC solid mediums：It is red the four of 1g/100mL to 5mL concentration is added in about 50 DEG C of 1L NA solid mediums The nitrogen azoles aqueous solution, the culture medium for obtaining.Concentration is that the preparation method of the red tetrazolium aqueous solution of 1g/100mL is：By red four nitrogen of 1g Azoles is dissolved in distilled water, and 100mL is settled to distilled water；Then 121 DEG C sterilizing 8min；Lucifuge, 4 DEG C of preservations.

TZC solid plates：About 50 DEG C of TZC solid mediums are poured into sterile petri dish, TZC solids is obtained after solidification and is put down Plate.

The step of detecting pepper seedling using biological living imaging system is as follows：First nutritive cube is placed in darkroom 30min (purpose is to make the spontaneous optical quenching of chlorophyll of pepper seedling), then takes pepper seedling, and gently shake removal root surface is attached The vermiculite, is then detected that (program is set to using biological living imaging system：4 × 4binning, time delay 3min, expose Light time 180s).After detection is finished, pepper seedling is replaced in the nutritive cube relaying equipped with vermiculite and Hoagland nutrient solutions Continuous culture.

Biological living imaging system is the Berthold NightSHADE LB985 plant living bodies of German Bai Tuo companies production Molecular imaging system.

The condition of the alternation of light and darkness culture (i.e. illumination cultivation and dark culturing replace) in following embodiments is：Illumination cultivation Shi Wendu is 27 DEG C, and temperature is 25 DEG C during dark culturing.The cycle of alternation of light and darkness culture is specially：14h illumination cultivations/10h is black Light culture.

BL-Rs1, BL-Rs2, BL-Rs3, BL-Rs4, BL-Rs5, BL-Rs6 or BL-Rs7 are with Ralstonia solanacearum Rs- SY1 is the bacterium that sets out, and imports what plasmid pXX3 was obtained.

The pepper ralstonia solanacearum evaluation that a situation arises is carried out using disease severity grade scale.The disease severity point Level standard is as follows：0 grade：Without wilting symptom；1 grade：Slight wilting symptom, i.e., 1 leaf withering；2 grades：Moderate wilting symptom, i.e., 2~ 3 leaf witherings；3 grades：Serious wilting symptom, other blades are wilted in addition to the spire of top；4 grades：Whole blades are wilted.

Disease index (DSI)=∑ (diseased plant numbers at different levels × disease severities at different levels)/investigation total strain number.

Bacterial genomes DNA extraction kit is the product of precious bioengineering (Dalian) Co., Ltd, and catalog number is 9763.Ultraviolet specrophotometer is the product of Thermo Fisher Scientific companies.Multi-function microplate reader is Germany The product of Berthold Technologeis, product type is TriStar2LB 942.

Aqueous sodium hypochlorite solution：By 6.14 parts by volume liquor natrii hypochloritises (effective chlorine is 8%-12%) and 13.86 volumes Part water is mixed.

The separation and identification of embodiment 1, Ralstonia solanacearum Rs-HN1

First, separate

1st, take pick up from the plantation of China Hainan province Sanya Malus spectabilis Wan Tengqiao towns Beijing City Agriculture and Forestry Institute Experimental Base be in The pepper plant of wilting shape, first fully rinses rhizome with sterilized water, then takes the basal part of stem tissue that is about 2cm and uses aseptic water logging Bubble 3h (has the dense spilling of bacterium of white) in immersion process.

2nd, the soak for completing step 1 is dipped, is rule on TZC solid plates, 28 DEG C of constant temperature are inverted culture 48h.Select Center is the flowing shape single bacterium colony that pink, outer rim are white, is purified 4-5 times repeatedly.The one plant of bacterium that will be screened is named as Bacterium Rs-HN1.

2nd, identify

1st, Morphological Identification

Bacterium Rs-HN1 is seeded to TZC solid plates, the form of single bacterium colony is observed in 28 DEG C of cultures after 48h.

Experimental result is shown in Fig. 1.Result shows that the single bacterium colony rat of bacterium Rs-HN1 is central for pink, outer rim are White, with mobility.

2nd, Molecular Identification

The extraction of A, the genomic DNA of bacterium Rs-HN1

(1) single bacterium colony of bacterium Rs-HN1 is inoculated in 5mL NA fluid nutrient mediums, 28 DEG C, 180rpm shaken cultivation 12h, Obtain cultivating bacterium solution.

(2) genomic DNA of bacterium solution is cultivated using bacterial genomes DNA extraction kit extraction step (1), bacterium is obtained The genomic DNA of Rs-HN1.

B, 16S rDNA sequence homology analysis

The genomic DNA of the bacterium Rs-HN1 extracted to step 1 carries out 16S rDNA sequence homology analysis.

The 16S rDNA of bacterium Rs-HN1 are as shown in the sequence 1 in sequence table.By comparing, bacterium Rs-HN1 and blue or green withered thunder You are Salmonella (Ralstonia solanacearum) EP1, Ralstonia solanacearum (Ralstonia solanacearum) FJAT- The 16S rDNA sequence homologies of 1458 grades are 100%.

C, Evolution Type detection

(1) genomic DNA of the bacterium Rs-HN1 extracted with step 1 is template, with primer 759：5’- GTCGCCGTCAACTCACTTTCC-3 ', primer 760：5’-GTCGCCGTCAGCAATGCGGAATCG-3’、Nmult21F：5’- CGTTGATGAGGCGCGCAATTT-3’、Nmult22F：5’-AAGTTATGGACGGTGGAAGTC-3’、Nmult23F：5’- ATTACSAGAGCAATCGAAAGATT-3 ' (S is C or G), Nmult24F：5 '-ATTGCCAAGACGAGAGAAGTA-3 ' and Nmult2R：5 '-TCGCTTGACCCTATAACGAGTA-3 ' enter performing PCR amplification for primer, obtain pcr amplification product.

According to above-mentioned steps, " genomic DNA of the bacterium Rs-HN1 that step 1 is extracted " is replaced with into water, it is right as feminine gender According to.

(2) pcr amplification product that step (1) is obtained is entered into row agarose gel electrophoresis, is then made the following judgment：If Contain two bands in pcr amplification product and size is respectively 280bp and 144bp, bacterium Rs-HN1 belongs to Ralstonia solanacearum to drill The class of change type I；If containing two bands in pcr amplification product and size being respectively 280bp and 372bp, bacterium Rs-HN1 belongs to blue or green The withered class of Lei Er Salmonellas Evolution Type II；If containing two bands in pcr amplification product and size being respectively 280bp and 91bp, bacterium Rs-HN1 belongs to the class of Ralstonia solanacearum Evolution Type III；If containing two bands in pcr amplification product and size being respectively 280bp And 213bp, bacterium Rs-HN1 belong to the class of Ralstonia solanacearum Evolution Type IV.

Experimental result is shown in Fig. 2 (1 is negative control, and 2 and 3 is the genomic DNA of bacterium Rs-HN1, and M is Marker).As a result Show, two bands are contained in pcr amplification product and size is respectively 280bp and 144bp, therefore bacterium Rs-HN1 belongs to blue or green withered thunder The class of that Salmonella Evolution Type I.

Summary each qualification result, bacterium Rs-HN1 is Ralstonia solanacearum (Ralstonia Solanacearum), Evolution Type I classes are belonged to.Hereinafter, bacterium Rs-HN1 is referred to as Ralstonia solanacearum Rs-HN1 or Rs- HN1。

Embodiment 2, the acquisition of bioluminescence bacterium and the identification of pathogenicity

First, the acquisition and identification of bioluminescence bacterium

1st, the preparation of Ralstonia solanacearum Rs-HN1 competence

(1) single bacterium colony of Ralstonia solanacearum Rs-HN1 is inoculated in 5mL NA fluid nutrient mediums, 28 DEG C, 200rpm vibrations Culture 12h, obtains cultivating bacterium solution 1.

(2) 1mL culture bacterium solutions 1 are taken, 50mL NA fluid nutrient mediums, 28 DEG C, 200rpm shaken cultivations to OD is inoculated in_600nm It is 0.6~0.8, obtains cultivating bacterium solution 2.

(3) the ice bath 30min of bacterium solution 2,4 DEG C, 5000rpm centrifugation 10min, collects thalline will be cultivated.

(4) thalline for collecting step (3) is washed three times, and the step of washing is as follows every time：The thalline of collection is taken, is used 100mL10% (v/v) glycerine water solution suspends (need to be operated on ice), 4 DEG C, 5000rpm centrifugation 15min, collects thalline.

(5) thalline of step (4) collection is taken, is suspended (need to be operated on ice) with 1mL 10% (v/v) glycerine water solution, so (100 μ L/EP pipes) is dispensed afterwards, that is, obtains Ralstonia solanacearum Rs-HN1 competence.

After by Ralstonia solanacearum Rs-HN1 competence liquid nitrogen flash freezers, -80 DEG C of preservations are placed in.

2nd, the acquisition and identification of bioluminescence bacterium

Bioluminescence (bioluminescence) is that chemical energy is converted into light by photobacteria using the chemical reaction of itself A kind of redox reaction of energy.Bioluminescent gene luxCDABE is by luxA genes, luxB genes, luxC genes, luxD bases Cause and luxE genomic constitutions.LuxA gene code luciferase α subunits, luxB gene code fluorescein β subunits, luxA and luxB It is collectively forming luciferase, luxC gene code fatty acid reduction enzymes, luxD gene code fatty acid transferases, luxE coding fat Fat acid enzyme, luxC, luxD, luxE are collectively forming fluorescent enzyme substrate synzyme, to maintain luminescence-producing reaction to provide continually Substrate.

Artificial synthesized plasmid pXX3 (Xu X, Miller SA, et al.Bioluminescence imaging of Clavibacter michiganensis subsp.michiganensis infection of tomato seeds and plants.Appl Environ Microbiol.2010Jun；76(12)：3978-88.), the nucleotides of plasmid pXX3 (annular) Sequence is as shown in the sequence 2 in sequence table.Sequence 2 in sequence table is chlorampenicol resistant for the 3299th to 4474 from 5 ' ends The encoding gene (abbreviation chloramphenicol resistance gene) of albumen, the 4795th to 6237 for fatty acid reduction enzyme encoding gene (i.e. LuxC genes), the 6249th to 7172 is the encoding gene (i.e. luxD genes) of fatty acid transferases, and the 7221st to 8303 is The encoding gene (i.e. luxA genes) of luciferase α subunits, the 8318th to 9301 for fluorescein β subunits encoding gene (i.e. LuxB genes), the 9480th to 10592 is the encoding gene (i.e. luxE genes) of fatty acid synthetase.Sequence 2 in sequence table In, also contain transposons Tn1409.Sequence 2 in sequence table is bioluminescent gene for the 4795th to 10592 from 5 ' ends The nucleotide sequence of luxCDABE.

The amino acid sequence of chlorampenicol resistant albumen is as shown in the sequence 3 in sequence table.The amino acid of fatty acid reduction enzyme Sequence is as shown in the sequence 4 in sequence table.The amino acid sequence of fatty acid transferases is as shown in the sequence 5 in sequence table.Fluorescence The amino acid sequence of plain enzyme α subunits is as shown in the sequence 6 in sequence table.In the amino acid sequence of fluorescein β subunits such as sequence table Sequence 7 shown in.The amino acid sequence of fatty acid synthetase is as shown in the sequence 8 in sequence table.

A, the acquisition of bioluminescence bacterium

Experiment is repeated 7 times, every time the luminous most strong single bacterium colony of screening one.The step of repeating every time is as follows：

(1) to 1 μ g plasmids pXX3 is added in 100 μ L Ralstonia solanacearum Rs-HN1 competence, electroporated (electric shock is joined Number：Voltage 1.8KV, electric capacity 25 μ F, the Ω of resistance 600) after, add 0.6mL NA fluid nutrient mediums, 28 DEG C, 140rpm shaken cultivations 3h, obtains cultivating bacterium solution.

(2) 100 μ L cultures bacterium solution is uniformly coated on the NA solid plates containing 40 μ g/mL chloramphenicol, 28 DEG C of cultures 48h.Then using the luminous most strong single bacterium colony of biological living imaging system screening.

7 single bacterium colonies screened are named as Rs-BL#1~Rs-BL#7 successively.Rs-BL#1~Rs-BL#7 is biology Photogen.

B, the identification of bioluminescence bacterium

(1) by bioluminescence bacterium (Rs-BL#1, Rs-BL#2, Rs-BL#3, Rs-BL#4, Rs-BL#5, Rs-BL#6 or Rs- BL#7 single bacterium colony) is inoculated in 5mL NA fluid nutrient mediums, and 28 DEG C, 180rpm shaken cultivation 12h obtain cultivating bacterium solution；Then The genomic DNA of culture bacterium solution is extracted using bacterial genomes DNA extraction kit, the genomic DNA of bioluminescence bacterium is obtained.

(2) genomic DNA with bioluminescence bacterium is as template, with 5 '-GTTGGCAGTGGTCGCATCTCA-3 ' and 5 '- ACGAATGTATGTCCTGCGTCTTG-3 ' enters performing PCR amplification for primer, obtains pcr amplification product；Pcr amplification product is carried out Agarose gel electrophoresis, then makes the following judgment：If containing a band in pcr amplification product and size being 894bp, give birth to Thing bioluminescence gene luxCDABE inserts bioluminescence bacterium；If not containing the bar that a size is 894bp in pcr amplification product Band, then bioluminescent gene luxCDABE be not inserted into bioluminescence bacterium.

Result shows, containing a band and size is 894bp in pcr amplification product, therefore Rs-BL#1~Rs-BL#7 Carry bioluminescent gene luxCDABE.

By Ralstonia solanacearum Rs-HN1, Rs-BL#1, Rs-BL#2, Rs-BL#3, Rs-BL#4, Rs-BL#5, Rs-BL#6 Or Rs-BL#7 is seeded on NA solid plates, the form of single bacterium colony is observed in 28 DEG C of cultures after 48h.It is imaged using biological living and is System detection Ralstonia solanacearum Rs-HN1, Rs-BL#1, Rs-BL#2, Rs-BL#3, Rs-BL#4, Rs-BL#5, Rs-BL#6 or Rs- The luminous situation of BL#7.

Experimental result is shown in Fig. 3 (Rs-HN1 is Ralstonia solanacearum Rs-HN1).Result shows that the bacterium colony of Rs-BL#1 is central For peony, edge white halo are less, without mobility；And the colonial morphology of Rs-BL#2~Rs-BL#7 and blue or green withered Lei Er Salmonella Rs-HN1's is basically identical；Rs-BL#1~Rs-BL#7 can detect bioluminescence signal；Ralstonia solanacearum Rs- HN1 can't detect bioluminescence signal.

2nd, the luminous intensity of bioluminescence bacterium compares

1st, one is compared

Bioluminescence bacterium be Rs-BL#1, Rs-BL#2, Rs-BL#3, Rs-BL#4, Rs-BL#5, Rs-BL#6, Rs-BL#7, BL-Rs1, BL-Rs2, BL-Rs3, BL-Rs4, BL-Rs5, BL-Rs6 or BL-Rs7.

(1) single bacterium colony of bioluminescence bacterium is inoculated in 5mL NA fluid nutrient mediums, 28 DEG C, 200rpm shaken cultivation 12h, Obtain cultivating bacterium solution.

(2) culture bacterium solution is inoculated in 200mL NA fluid nutrient mediums, 28 DEG C, 200rpm shaken cultivations 72h.Incubation In every 6h sampling, using ultraviolet specrophotometer determine in OD_600nmThe light absorption value at place, determines relative using multi-function microplate reader Number of photons (relative light units, RLU), measurement result in triplicate, is averaged.

According to the method described above, bioluminescence bacterium is replaced with into Ralstonia solanacearum Rs-HN1, as control.

Part of test results is shown in Fig. 4.Result shows, with the propagation of bioluminescence bacterium, increases therewith with respect to number of photons, can See that bioluminescent gene luxCDABE can be expressed in bioluminescence bacterium stabilization；Ralstonia solanacearum Rs-HN1 does not light.

2nd, two are compared

Due to the transposons Tn1409 in plasmid pXX3 in bacterial genomes be randomness insertion, insertion point upstream is opened The difference that mover starts ability can influence the expression of bioluminescent gene luxCDABE, so as to influence luminous intensity.In order to Luminous most strong bacterial strain is filtered out from Rs-BL#1~Rs-BL#7, is tested as follows：Carry out in step 1 during (2), When the bacteria concentration of bioluminescence bacterium about reaches 2 × 10⁸During CFU/mL, determined in OD using ultraviolet specrophotometer_600nmThe suction at place Light value, RLU is determined using multi-function microplate reader, and measurement result in triplicate, is averaged；Then luminous intensity is further calculated (luminous intensity=Log RLU/OD_600nm)。

Part of test results is shown in Fig. 5.Result shows that luminous intensity power is：Rs-BL#7>Rs-BL#2>Rs-BL#5>Rs- BL#6>Rs-BL#3>Rs-BL#4>Rs-BL#1>BL-Rs7>BL-Rs-2>BL-Rs5>BL-Rs6>BL-Rs3>BL-Rs4>BL- Rs1.After selection Rs-BL#7, Rs-BL#2, Rs-BL#5, Rs-BL#6, Rs-BL#3, BL-Rs7, BL-Rs-2 and BL-Rs5 are carried out Continuous pathogenicity identification experiment.

3rd, the pathogenicity identification of bioluminescence bacterium

The susceptible material C alifonia Wonder of pepper ralstonia solanacearum are recorded in following document：Y Mimura, M Yoshikawa, M Hirai.Pepper accession LS2341is highly resistant to Ralstonia Solanacearum strains from Japan.Hortscience, 2009,44：Hereinafter, capsicum is blue or green for 2038-2040. The susceptible material C alifonia Wonder abbreviations Califonia Wonder of rot.

1st, 30 seeds of Califonia Wonder are taken, is first sterilized 3-5min with aqueous sodium hypochlorite solution, then with aseptic Water is rinsed 3-4 times, is finally placed in the 5min that soaked seed in 55 DEG C of sterilized waters.

2nd, the seed after step 1 is taken into, culture dish is put into after the gauze wrapped with moistening, then culture dish is placed in culture Case, 25 DEG C of dark culturing to seeds show money or valuables one carries unintentionally (about 7~10d).

3rd, take into the seed after step 2, be seeded in equipped with Nutrition Soil (2 parts by volume turfs and 1 parts by volume vermiculite mixing and Into) 72 hole disks in, then alternation of light and darkness culture, obtain in the 4-6 leaf phases pepper seedling.

4th, the pepper seedling for obtaining step 3 is gently extracted from 72 hole disks, first rinses root with running water, then will It is 2 × 10 that root is soaked in concentration⁸30min in the bioluminescence bacterium aqueous solution of CFU/mL.Bioluminescence bacterium is Rs-BL#2, Rs- BL#3, Rs-BL#5, Rs-BL#6, Rs-BL#7, BL-Rs7, BL-Rs-2 or BL-Rs5.

5th, after completing step 4, pepper seedling is colonized in into the nutritive cube equipped with vermiculite and Hoagland nutrient solutions, and (specification is 8cm × 10cm), then alternation of light and darkness culture.5d is colonized, pepper seedling is detected using biological living imaging system.Field planting the 7d, investigates the disease index of pepper seedling.

According to the method described above, by " concentration is 2 × 10⁸The bioluminescence bacterium aqueous solution of CFU/mL " replace with " concentration be 2 × 10⁸The Ralstonia solanacearum Rs-HN1 aqueous solution of CFU/mL ", other steps are constant, used as control.

The partial results detected using biological living imaging system are shown in Fig. 6.Result shows, is inoculated with Rs-BL#2, Rs-BL#3 Or bioluminescence signal is can detect in the stalk of the pepper seedling of Rs-BL#7, and it is inoculated with the peppery of Rs-BL#5 or Rs-BL#6 Green pepper seedling can detect a small amount of bioluminescence signal only in root.

The survey showed that for disease index, and the disease index of the pepper seedling of inoculation Ralstonia solanacearum Rs-HN1 is 4.00, the disease index for being inoculated with the pepper seedling of Rs-BL#7 is 3.84, and is inoculated with the disease of the pepper seedling of other bioluminescence bacterium Feelings index is all higher than 2.00 and less than 3.00, and wilting symptom also there occurs delay or weaken.Therefore, Rs-BL#2, Rs-BL#3 In can invading the stalk of Califonia Wonder with Rs-BL#7, but the pathogenicity of Rs-BL#7 is better than Rs-BL#2 and Rs- BL#3。

Summary result, Rs-BL#7 luminous intensities are relatively strong and disease index with Ralstonia solanacearum Rs-HN1 is without aobvious Write difference (i.e. pathogenicity is without significant difference), therefore using Rs-BL#7 as this experiment screening ideal bioluminescence bacterium.

Rs-BL#7 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms on January 17th, 2017 (abbreviation CGMCC, address is at center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.13637. The full name of Rs-BL#7 is Ralstonia solanacearum (Ralstonia solanacearum) Rs-BL#7CGMCC No.13637, referred to as It is Rs-BL#7.

4th, the corresponding relation of pathogen quantity and luminous intensity in capsicum body is quantified

In order to quantify the corresponding relation of pathogen quantity and luminous intensity in capsicum body, the present inventor also have detected The clump count at different luminous intensity positions in the stalk of the pepper seedling of Rs-BL#7 is inoculated with, regression analysis is then carried out.Experiment knot Fruit sees Fig. 7 (cts is number of photons).Result shows, luminous intensity and clump count positive correlation；When in capsicum body clump count be 8 × 10⁷During more than CFU/g bioluminescence signal is clearly detected using biological living imaging system.

Embodiment 3, using bioluminescence dientification of bacteria capsicum material to the resistance of bacterial wilt

First, the acquisition and preservation of resistance to bacterial wilt capsicum material

Disease-resistant material MC-4 is recorded in following document：CA Lopes, SIC Carvalho, LS Boiteux, et al.Search for resistance to bacterial wilt in a Brazilian Capsicum germplasm Collection.Bacterial Wilt Disease＆the Species Complex, 2005.

The present inventor filters out resistance to bacterial wilt Capsicum annuum N1508 from a large amount of capsicum germplasm, tool Body screening step is as follows：

1st, 30 seeds of capsicum germplasm are taken, first with aqueous sodium hypochlorite solution sterilization 3-5min, then aseptic water washing 3-4 is used It is secondary, finally it is placed in the 5min that soaked seed in 55 DEG C of sterilized waters；

2nd, the seed of step 1 is taken into, culture dish is put into after the gauze wrapped with moistening, then culture dish is placed in culture Case, 25 DEG C of dark culturing to seeds show money or valuables one carries unintentionally (about 7~10d).

3rd, the seed of the capsicum germplasm of step 2 is taken into, is seeded in equipped with Nutrition Soil (2 parts by volume turfs and 1 parts by volume leech Stone is mixed) 72 hole disks in, then alternation of light and darkness culture, obtain in the 4-6 leaf phases pepper seedling.

4th, the pepper seedling for obtaining step 3 is gently extracted from 72 hole disks, first rinses root with running water, then will It is 2 × 10 that root is soaked in concentration⁸30min in the Ralstonia solanacearum Rs-HN1 aqueous solution of CFU/mL.

5th, after completing step 4, pepper seedling is colonized in into the nutritive cube equipped with vermiculite and Hoagland nutrient solutions, and (specification is 8cm × 10cm), then alternation of light and darkness culture.The disease index of each capsicum germplasm is investigated after field planting 40d and identify each capsicum Whether germplasm is resistance to bacterial wilt capsicum material.Standard of perfection is：If disease index is less than 2, the capsicum germplasm is anti-green grass or young crops Rot capsicum material；If disease index is more than 2, the capsicum germplasm is sense bacterial wilt capsicum material.

Result shows that after inoculation Ralstonia solanacearum Rs-BL#7, the disease index of Capsicum annuum N1508 is only It is 1.2, the disease index of disease-resistant material MC-4 is better than disease-resistant material for the Resistance to bacterial wilt of 1.8, Capsicum annuum N1508 Material MC-4.

Capsicum annuum N1508 are preserved in Chinese microorganism strain preservation management committee on January 17th, 2017 (abbreviation CGMCC, address is member's meeting common micro-organisms center：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3), deposit number is CGMCC No.13597.The full name of Capsicum annuum N1508 is Capsicum annuum N1508CGMCC No.13597, referred to as N1508 or capsicum N1508.

2nd, using bioluminescence dientification of bacteria capsicum material to the resistance of bacterial wilt

Capsicum material is Califonia Wonder or N1508.

Experiment in triplicate, each repetition the step of it is as follows：

1st, 50 seeds of capsicum material are taken, first with aqueous sodium hypochlorite solution sterilization 3-5min, then aseptic water washing 3-4 is used It is secondary, finally it is placed in 55 DEG C of seed soaking 5min in sterilized water.

3rd, the seed of step 2 is taken into, is seeded in equipped with Nutrition Soil (by 2 parts by volume turfs and the mixing of 1 parts by volume vermiculite Into) 72 hole disks in, then alternation of light and darkness culture obtains the pepper seedling in the capsicum 4-6 leaf phases.

4th, the pepper seedling for obtaining step 3 is gently extracted from 72 hole disks, first rinses root with running water, then will It is 2 × 10 that root is soaked in concentration⁸30min in the Rs-BL#7 aqueous solution of CFU/mL.

5th, after completing step 4, pepper seedling is placed in the nutritive cube equipped with vermiculite and Hoagland nutrient solutions, Ran Houguang Dark alternate culture 10d.During culture, daily using biological living imaging system detection pepper seedling.

6th, after completing step 4, pepper seedling is placed in the nutritive cube equipped with vermiculite and Hoagland nutrient solutions, Ran Houguang Dark alternate culture.Respectively in inoculation 1d, inoculation 3d, inoculation 5d, inoculation 7d, inoculation 9d, inoculation 11d and inoculation 13d, is counted using the method for plate culture count to the sick Microflora in pepper seedling different tissues, and specific steps are such as Under：Take 5 plants of pepper seedlings, first use aseptic water washing root, then with blade cutting tissue (2cm roots, the rhizome of 0.5cm, middle stem or Stem apex), it is placed in after weighing in the centrifuge tube equipped with 1mL sterilized waters (specification is 2mL), ground with glass bar, with aseptic after mixing Water carries out 10 times and is serially diluted, and dilution is applied on the NA solid plates containing 45 μ g/mL chloramphenicol, and each diluted concentration applies 3 Individual culture dish, cultivates 2-3d at 28 DEG C, counts clump count.The bacterium colony for calculating the tissue per g finally according to extension rate forms list Position, and change into lg logarithm values.

The experimental result of step 5 is shown in Fig. 8.2d is inoculated with, is detected in the root of Califonia Wonder and N1508 micro- Weak bioluminescence signal；3d is inoculated with, strong bioluminescence signal is detected in the stalk of Califonia Wonder, But it is not detected by optical signal at the rhizome and stalk of N1508；It is inoculated with 5d to start, Califonia Wonder start to occur withering Obvious bioluminescence signal is detected in listless symptom and stalk and stem apex, with the exacerbation of wilting symptom, bioluminescence signal Also it is remarkably reinforced；It is inoculated with 5d to start, wilting symptom does not occur in N1508, also not in plant vivo detection to optical signal.

The statistics of step 6 is shown in Table 1.1d is inoculated with, be can detect in the root of Califonia Wonder and N1508 Ralstonia solanacearum, and Microflora in two parts of materials without significant difference, but Microflora is less than the Monitoring lower-cut of system, so observation Less than bioluminescence signal.It is inoculated with 3d, the root of Califonia Wonder, rhizome and the quantity of Ralstonia solanacearum compares respectively in middle stem N1508 correspondence tissue it is high by 10,10²With 10⁷Times.Compared with Califonia Wonder, Rs-BL#7 is in the root and rhizome of N1508 Propagation slowly, and 9d reaches peak value after inoculation, and lg CFU/g are respectively 7.12 and 5.70, then in 11d and 13d When reduce；And Rs-BL#7 breeds in Califonia Wonder and spread very fast, 10 are risen to when 5d is inoculated with⁸CFU/ G, and before Califonia Wonder death, Rs-BL#7 reaches 10 in rhizome, middle stem and stem-tip tissue¹⁰CFU/g More than.

Above-mentioned qualification result shows that N1508 is resistance to bacterial wilt capsicum material, and Califonia Wonder are peppery for sense bacterial wilt Green pepper material, it is completely the same with actual conditions.Therefore, resistance of the capsicum material to bacterial wilt can be identified using Rs-BL#7.

Table 1

Note：ND is represented because plant death is not so detect colony count.

3rd, using bioluminescence dientification of bacteria capsicum grafting to the resistance of bacterial wilt

1st, the acquisition of the seedling of Califonia Wonder

(1) seed of Califonia Wonder is taken, first with aqueous sodium hypochlorite solution sterilization 3-5min, then is rushed with sterilized water Wash 3-4 times, be finally placed in 55 DEG C of seed soaking 5min in sterilized water.

(2) seed of step (1) is taken into, culture dish is put into after the gauze wrapped with moistening, then culture dish is placed in training Case is supported, 25 DEG C of dark culturing to seeds show money or valuables one carries unintentionally (about 7~10d).

(3) seed of step (2) is taken into, is seeded in and (is mixed by 2 parts by volume turfs and 1 parts by volume vermiculite equipped with Nutrition Soil Conjunction is formed) 72 hole disks in, then alternation of light and darkness culture obtains the Califonia Wonder in capsicum 4-6 leaf phases Seedling (the hereinafter referred to as seedling of Califonia Wonder).

2nd, the acquisition of the seedling of N1508

According to the method for above-mentioned steps 1, the seed of Califonia Wonder is replaced with the seed of N1508, other steps It is rapid constant, obtain the seedling (the hereinafter referred to as seedling of N1508) of the N1508 in the capsicum 4-6 leaf phases.

3rd, grafting

(1) four grafting combination uses cleft graft grafting.Each grafting is combined in triplicate, 10 plants every time.

Four grafting combinations are specific as follows：

Califonia Wonder/N1508：As stock, the seedling of Califonia Wonder is to connect to seedling with N1508 Fringe；

N1508/Califonia Wonder：As stock, the seedling of N1508 is to connect to seedling with Califonia Wonder Fringe；

N1508/N1508：The seedling of N1508 is from root graft；

Califonia Wonder/Califonia Wonder：The seedling of Califonia Wonder is from root graft.

(2) after completing step (1), grafting is placed in culturing room, temperature be 28 DEG C, humidity be 78% under conditions of train Support.After 30d, grafting is extracted, first rinse root with running water, it is 2 × 10 that root then is soaked in into concentration⁸CFU/mL's 30min in the Rs-BL#7 aqueous solution.

5d, 7d, 9d, 10d, 11d, the 15d or 30d of step (2) to be done, daily using biological living Body imaging system detects pepper seedling.

Experimental result is shown in that (A is N1508/Califonia for Califonia Wonder/Califonia Wonder, B to Fig. 9 Wonder, C are N1508/N1508 for Califonia Wonder/N1508, D).

Result shows that the resistance to Rs-BL#7 is：Califonia Wonder/Califonia Wonder<N1508/ Califonia Wonder<Califonia Wonder/N1508<N1508/N1508.It is inoculated with 5d to start, Califonia The bioluminescence signal detectable in vivo of Wonder/Califonia Wonder.It is inoculated with 9d to start, N1508/ The bioluminescence signal detectable in vivo of Califonia Wonder.Califonia Wonder/N1508 and N1508/ N1508 can complete normal solid in field, be not detected by bioluminescence signal in vivo before flowering.

The above results show that N1508 can effectively prevent and treat bacterial wilt as disease-resistant stock.Can be identified using Rs-BL#7 Resistance of the capsicum grafting to bacterial wilt.

<110>Beijing City Agriculture and Forestry Institute

<120>One plant of Ralstonia solanacearum of bioluminescence

<160> 8

<170> PatentIn version 3.5

<210> 1

<211> 1482

<212> DNA

<213>Ralstonia solanacearum（Ralstoniasolanacearum）

<400> 1

tacggctacc ttgttacgac ttcaccccag tcatgaaccc taccgtggta atcgccctcc 60

ttgcggttag gctaactact tctggtaaag cccactccca tggtgtgacg ggcggtgtgt 120

acaagacccg ggaacgtatt caccgcggca tgctgatccg cgattactag cgattccagc 180

ttcacgtagt cgagttgcag actacgatcc ggactacgat gcattttctg ggattagctc 240

cacctcgcgg cttggcaacc ctctgtatgc accattgtat gacgtgtgaa gccctaccca 300

taagggccat gaggacttga cgtcatcccc accttcctcc ggtttgtcac cggcagtctc 360

tctagagtgc cctttcgtag caactagaga caagggttgc gctcgttgcg ggacttaacc 420

caacatctca cgacacgagc tgacgacagc catgcagcac ctgtgtccac tttctctttc 480

gagcacctaa tgcatctctg cttcgttagt ggcatgtcaa gggtaggtaa ggtttttcgc 540

gttgcatcga attaatccac atcatccacc gcttgtgcgg gtccccgtca attcctttga 600

gttttaatct tgcgaccgta ctccccaggc ggtcaacttc acgcgttagc tacgttacta 660

aggaaatgaa tccccaacaa ctagttgaca tcgtttaggg cgtggactac cagggtatct 720

aatcctgttt gctccccacg ctttcgtgca tgagcgtcag tgttatccca ggaggctgcc 780

ttcgccatcg gtattcctcc acatctctac gcatttcact gctacacgtg gaattctacc 840

tccctctgac acactctagc cgtgcagtca ccaatgcaat tcccaggtta agcccgggga 900

tttcacatcg gtcttgcaca accgcctgcg cacgctttac gcccagtaat tccgattaac 960

gcttggaccc tacgtattac cgcggctgct ggcacgtagt tagccggtcc ttattcttcc 1020

ggtaccgtca tccacaccag gtattaacca gtgcgatttc tttccggaca aaagtgcttt 1080

acaacccgaa ggccttcttc acacacgcgg cattgctgga tcagggttgc ccccattgtc 1140

caaaattccc cactgctgcc tcccgtagga gtctgggccg tgtctcagtc ccagtgtggc 1200

tgatcgtcct ctcagaccag ctactgatcg tcgccttggt gggcctttac cccaccaact 1260

agctaatcag acatcggccg ctcctatagc atgaggcctt gcggtccccc actttcaccc 1320

tcaggtcgta tgcggtatta gctagtcttt cgactagtta tcccccacta cagggcacgt 1380

tccgatgtat tactcacccg ttcgccactc gccggcaggt agcaagctac ccccgctgcc 1440

gttcgacttg catgtgtaag gcatgccgcc agcgttcaat ct 1482

<210> 2

<211> 12344

<212> DNA

<213>Artificial sequence

<220>

<223>

<400> 2

ggggatcctc tagagtcgac ctgcagccca agcttggcgt aatcatggtc atagctgttt 60

cctgtgtgaa attgttatcc gctcacaatt ccacacaaca tacgagccgg aagcataaag 120

tgtaaagcct ggggtgccta atgagtgagc taactcacat taattgcgtt gcgctcactg 180

cccgctttcc agtcgggaaa cctgtcgtgc cagctgcatt aatgaatcgg ccaacgcgcg 240

gggagaggcg gtttgcgtat tgggcgctct tccgcttcct cgctcactga ctcgctgcgc 300

tcggtcgttc ggctgcggcg agcggtatca gctcactcaa aggcggtaat acggttatcc 360

acagaatcag gggataacgc aggaaagaac atgtgagcaa aaggccagca aaaggccagg 420

aaccgtaaaa aggccgcgtt gctggcgttt ttccataggc tccgcccccc tgacgagcat 480

cacaaaaatc gacgctcaag tcagaggtgg cgaaacccga caggactata aagataccag 540

gcgtttcccc ctggaagctc cctcgtgcgc tctcctgttc cgaccctgcc gcttaccgga 600

tacctgtccg cctttctccc ttcgggaagc gtggcgcttt ctcatagctc acgctgtagg 660

tatctcagtt cggtgtaggt cgttcgctcc aagctgggct gtgtgcacga accccccgtt 720

cagcccgacc gctgcgcctt atccggtaac tatcgtcttg agtccaaccc ggtaagacac 780

gacttatcgc cactggcagc agccactggt aacaggatta gcagagcgag gtatgtaggc 840

ggtgctacag agttcttgaa gtggtggcct aactacggct acactagaag gacagtattt 900

ggtatctgcg ctctgctgaa gccagttacc ttcggaaaaa gagttggtag ctcttgatcc 960

ggcaaacaaa ccaccgctgg tagcggtggt ttttttgttt gcaagcagca gattacgcgc 1020

agaaaaaaag gatctcaaga agatcctttg atcttttcta cggggtctga cgctcagtgg 1080

aacgaaaact cacgttaagg gattttggtc atgagattat caaaaaggat cttcacctag 1140

atccttttaa attaaaaatg aagttttaaa tcaatctaaa gtatatatga gtaaacttgg 1200

tctgacagtt accaatgctt aatcagtgag gcacctatct cagcgatctg tctatttcgt 1260

tcatccatag ttgcctgact ccccgtcgtg tagataacta cgatacggga gggcttacca 1320

tctggcccca gtgctgcaat gataccgcga gacccacgct caccggctcc agatttatca 1380

gcaataaacc agccagccgg aagggccgag cgcagaagtg gtcctgcaac tttatccgcc 1440

tccatccagt ctattaattg ttgccgggaa gctagagtaa gtagttcgcc agttaatagt 1500

ttgcgcaacg ttgttgccat tgctacaggc atcgtggtgt cacgctcgtc gtttggtatg 1560

gcttcattca gctccggttc ccaacgatca aggcgagtta catgatcccc catgttgtgc 1620

aaaaaagcgg ttagctcctt cggtcctccg atcgttgtca gaagtaagtt ggccgcagtg 1680

ttatcactca tggttatggc agcactgcat aattctctta ctgtcatgcc atccgtaaga 1740

tgcttttctg tgactggtga gtactcaacc aagtcattct gagaatagtg tatgcggcga 1800

ccgagttgct cttgcccggc gtcaatacgg gataataccg cgccacatag cagaacttta 1860

aaagtgctca tcattggaaa acgttcttcg gggcgaaaac tctcaaggat cttaccgctg 1920

ttgagatcca gttcgatgta acccactcgt gcacccaact gatcttcagc atcttttact 1980

ttcaccagcg tttctgggtg agcaaaaaca ggaaggcaaa atgccgcaaa aaagggaata 2040

agggcgacac ggaaatgttg aatactcata ctcttccttt ttcaatatta ttgaagcatt 2100

tatcagggtt attgtctcat gagcggatac atatttgaat gtatttagaa aaataaacaa 2160

ataggggttc cgcgcacatt tccccgaaaa gtgccacctg acgtctaaga aaccattatt 2220

atcatgacat taacctataa aaataggcgt atcacgaggc cctttcgtct cgcgcgtttc 2280

ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg agacggtcac agcttgtctg 2340

taagcggatg ccgggagcag acaagcccgt cagggcgcgt cagcgggtgt tggcgggtgt 2400

cggggctggc ttaactatgc ggcatcagag cagattgtac tgagagtgca ccatatgcgg 2460

tgtgaaatac cgcacagatg cgtaaggaga aaataccgca tcaggcgcca ttcgccattc 2520

aggctgcgca actgttggga agggcgatcg gtgcgggcct cttcgctatt acgccagctg 2580

gcgaaagggg gatgtgctgc aaggcgatta agttgggtaa cgccagggtt ttcccagtca 2640

cgacgttgta aaacgacggc cagtgaattc cggtaccatg gctcttcaga attgagggtg 2700

tagtgggcgc aactgcatgc agcgccgagg gctagagctt gggctgcagg tcgacctgca 2760

ggacatggtc aaagacctca acaccgagca gctaaagacc atcacatggg accaaggcgc 2820

agaaatggct gtcacagcac aagtccagat taaagacggc tgccaggtgt ttttctgtga 2880

accgcactca ccgtggcaga gaccaaccaa cgagaacacc aacggcgaga tcaggcgcag 2940

gttctacaaa aagggcaccg actttgccac cgtcacaccc gaacatgtcg cgtgggtgca 3000

aaatgagctc aacgaaacac cccgacaaat cctcggcggc gcaaccccac gtgaaatact 3060

taacgaacta ttcaagcgtg gcgcatccac cgcttgattc cgccttgaag gcatgttaga 3120

gtttggggca tgtcgagtcc cgaatcagac aggtcagggg ttgtcggtca caccgccagc 3180

cccttgaacc actagttacg acgcccacga tctgtgtggg cgtatgtctg gcgtacccgg 3240

ggcgctggcc gtggtgacaa gaagaaccat ttcttgatct cacacctcgg agtactcgat 3300

gccttttgcc ctctacatgc ttgccctggc ggtcttcgtc atgggcactt cagaattcat 3360

gctcgcggga ttgctccccg cgatcgcgac cgaacttgac gtctcggtcg gcactgcggg 3420

cctgctgacc tccgcattcg cagtcggtat ggtcgtcggc gcgccagtga tggcggcatt 3480

cgctcgccgt tggccaccgc ggctcacatt gatcgtttgc cttctcgtgt tcgcgggaag 3540

ccacgtcatc ggagcgatga caccagtgtt ctctctcctg ctcatcaccc gggtgctcag 3600

cgctctcgca aacgcaggat tcctcgccgt agcactgagc acggccacta ccctcgtgcc 3660

agcgaaccag aaggggcgtg cactgtcgat cctgctctcc ggcacgacga tcgcaaccgt 3720

cgtgggcgtc cccgccgggg cactgctcgg cacagcgctg ggctggcgaa cgacgttctg 3780

ggcgatcgcc atcctctgta ttcccgcggc cgttggagtc attcgtggcg tcacgaacaa 3840

tgttggtcgg agcgagacta gcgcgacctc accaaggctc cgtgtcgagc tcagccagtt 3900

ggcgacgccg cggctcatcc tggccatggc actcggagcg ctgatcaacg gagggacctt 3960

tgcggcattc accttcctgg cacccatcgt gaccgagacc gcgggcttgg ccgaagcgtg 4020

ggtgtccgtc gcgctggtga tgttcggcat cggatcgttc cttggcgtca cgatcgcagg 4080

acgactatca gatcaacgac ctggcctcgt gctcgcagtc ggcggaccgc tattgctgac 4140

aggctggatc gtgttggcag tggtcgcatc tcatcccgtt gcgcttatcg tcctcgtcct 4200

cgttcaggga ttcctgtcgt tcggcgtcgg cagtactctg atcacgcgtg tgctgtatgc 4260

agcatcgggt gcgccaacga tgggcggttc gtacgcaacc gcagcattga atatcggagc 4320

tgcagcgggg cccgtgcttg gtgcgctcgg gctcgcgacc gggctggggc tgctcgcgcc 4380

ggtttgggtc gcttcggtgc tgacagcgat cgctctcgtc atcatgcttc tcaccagacg 4440

cgcgcttacg aagaccgcgg cggaggccaa ttgatgaccc atccgaacgc tcttctcact 4500

cctcgtgccc gtctccggtt agctcggctg attgtcgaag acggctatcc ggccacgatc 4560

gccgcaaaga tgttcatggt ctccccgatc actgcccgga aatgggcagg ccgctaccgg 4620

gaagagggtg agtttgggat gcaggatcgc tccagcaagc cgcaccggat ccgtcgacct 4680

gcagcccaag ctctagcggc gtgatactcg agtcgaggtc gacggtatcg ataagcttga 4740

tatcgaattc ctgcagcccg ggggatccgg ggaattcagg cttggaggat acgtatgact 4800

aaaaaaattt cattcattat taacggccag gttgaaatct ttcccgaaag tgatgattta 4860

gtgcaatcca ttaattttgg tgataatagt gtttacctgc caatattgaa tgactctcat 4920

gtaaaaaaca ttattgattg taatggaaat aacgaattac ggttgcataa cattgtcaat 4980

tttctctata cggtagggca aagatggaaa aatgaagaat actcaagacg caggacatac 5040

attcgtgact taaaaaaata tatgggatat tcagaagaaa tggctaagct agaggccaat 5100

tggatatcta tgattttatg ttctaaaggc ggcctttatg atgttgtaga aaatgaactt 5160

ggttctcgcc atatcatgga tgaatggcta cctcaggatg aaagttatgt tcgggctttt 5220

ccgaaaggta aatctgtaca tctgttggca ggtaatgttc cattatctgg gatcatgtct 5280

atattacgcg caattttaac taagaatcag tgtattataa aaacatcgtc aaccgatcct 5340

tttaccgcta atgcattagc gttaagtttt attgatgtag accctaatca tccgataacg 5400

cgctctttat ctgttatata ttggccccac caaggtgata catcactcgc aaaagaaatt 5460

atgcgacatg cggatgttat tgtcgcttgg ggagggccag atgcgattaa ttgggcggta 5520

gagcatgcgc catcttatgc tgatgtgatt aaatttggtt ctaaaaagag tctttgcatt 5580

atcgataatc ctgttgattt gacgtccgca gcgacaggtg cggctcatga tgtttgtttt 5640

tacgatcagc gagcttgttt ttctgcccaa aacatatatt acatgggaaa tcattatgag 5700

gaatttaagt tagcgttgat agaaaaactt aatctatatg cgcatatatt accgaatgcc 5760

aaaaaagatt ttgatgaaaa ggcggcctat tctttagttc aaaaagaaag cttgtttgct 5820

ggattaaaag tagaggtgga tattcatcaa cgttggatga ttattgagtc aaatgcaggt 5880

gtggaattta atcaaccact tggcagatgt gtgtaccttc atcacgtcga taatattgag 5940

caaatattgc cttatgttca aaaaaataag acgcaaacca tatctatttt tccttgggag 6000

tcatcattta aatatcgaga tgcgttagca ttaaaaggtg cggaaaggat tgtagaagca 6060

ggaatgaata acatatttcg agttggtgga tctcatgacg gaatgagacc gttgcaacga 6120

ttagtgacat atatttctca tgaaaggcca tctaactata cggctaagga tgttgcggtt 6180

gaaatagaac agactcgatt cctggaagaa gataagttcc ttgtatttgt cccataatag 6240

gtaaaagtat ggaaaatgaa tcaaaatata aaaccatcga ccacgttatt tgtgttgaag 6300

gaaataaaaa aattcatgtt tgggaaacgc tgccagaaga aaacagccca aagagaaaga 6360

atgccattat tattgcgtct ggttttgccc gcaggatgga tcattttgct ggtctggcgg 6420

aatatttatc gcggaatgga tttcatgtga tccgctatga ttcgcttcac cacgttggat 6480

tgagttcagg gacaattgat gaatttacaa tgtctatagg aaagcagagc ttgttagcag 6540

tggttgattg gttaactaca cgaaaaataa ataacttcgg tatgttggct tcaagcttat 6600

ctgcgcggat agcttatgca agcctatctg aaatcaatgc ttcgttttta atcaccgcag 6660

tcggtgttgt taacttaaga tattctcttg aaagagcttt agggtttgat tatctcagtc 6720

tacccattaa tgaattgccg gataatctag attttgaagg ccataaattg ggtgctgaag 6780

tctttgcgag agattgtctt gattttggtt gggaagattt agcttctaca attaataaca 6840

tgatgtatct tgatataccg tttattgctt ttactgcaaa taacgataat tgggtcaagc 6900

aagatgaagt tatcacattg ttatcaaata ttcgtagtaa tcgatgcaag atatattctt 6960

tgttaggaag ttcgcatgac ttgagtgaaa atttagtggt cctgcgcaat ttttatcaat 7020

cggttacgaa agccgctatc gcgatggata atgatcatct ggatattgat gttgatatta 7080

ctgaaccgtc atttgaacat ttaactattg cgacagtcaa tgaacgccga atgagaattg 7140

agattgaaaa tcaagcaatt tctctgtctt aaaatctatt gagatattct atcactcaaa 7200

tagcaatata aggactctct atgaaatttg gaaacttttt gcttacatac caacctcccc 7260

aattttctca aacagaggta atgaaacgtt tggttaaatt aggtcgcatc tctgaggagt 7320

gtggttttga taccgtatgg ttactggagc atcatttcac ggagtttggt ttgcttggta 7380

acccttatgt cgctgctgca tatttacttg gcgcgactaa aaaattgaat gtaggaactg 7440

ccgctattgt tcttcccaca gcccatccag tacgccaact tgaagatgtg aatttattgg 7500

atcaaatgtc aaaaggacga tttcggtttg gtatttgccg agggctttac aacaaggact 7560

ttcgcgtatt cggcacagat atgaataaca gtcgcgcctt agcggaatgc tggtacgggc 7620

tgataaagaa tggcatgaca gagggatata tggaagctga taatgaacat atcaagttcc 7680

ataaggtaaa agtaaacccc gcggcgtata gcagaggtgg cgcaccggtt tatgtggtgg 7740

ctgaatcagc ttcgacgact gagtgggctg ctcaatttgg cctaccgatg atattaagtt 7800

ggattataaa tactaacgaa aagaaagcac aacttgagct ttataatgaa gtggctcaag 7860

aatatgggca cgatattcat aatatcgacc attgcttatc atatataaca tctgtagatc 7920

atgactcaat taaagcgaaa gagatttgcc ggaaatttct ggggcattgg tatgattctt 7980

atgtgaatgc tacgactatt tttgatgatt cagaccaaac aagaggttat gatttcaata 8040

aagggcagtg gcgtgacttt gtattaaaag gacataaaga tactaatcgc cgtattgatt 8100

acagttacga aatcaatccc gtgggaacgc cgcaggaatg tattgacata attcaaaaag 8160

acattgatgc tacaggaata tcaaatattt gttgtggatt tgaagctaat ggaacagtag 8220

acgaaattat tgcttccatg aagctcttcc agtctgatgt catgccattt cttaaagaaa 8280

aacaacgttc gctattatat tagctaagga gaaagaaatg aaatttggat tgttcttcct 8340

taacttcatc aattcaacaa ctgttcaaga acaaagtata gttcgcatgc aggaaataac 8400

ggagtatgtt gataagttga attttgaaca gattttagtg tatgaaaatc atttttcaga 8460

taatggtgtt gtcggcgctc ctctgactgt ttctggtttt ctgctcggtt taacagagaa 8520

aattaaaatt ggttcattaa atcacatcat tacaactcat catcctgtcg ccatagcgga 8580

ggaagcttgc ttattggatc agttaagtga agggagattt attttagggt ttagtgattg 8640

cgaaaaaaaa gatgaaatgc atttttttaa tcgcccggtt gaatatcaac agcaactatt 8700

tgaagagtgt tatgaaatca ttaacgatgc tttaacaaca ggctattgta atccagataa 8760

cgatttttat agcttcccta aaatatctgt aaatccccat gcttatacgc caggcggacc 8820

tcggaaatat gtaacagcaa ccagtcatca tattgttgag tgggcggcca aaaaaggtat 8880

tcctctcatc tttaagtggg atgattctaa tgatgttaga tatgaatatg ctgaaagata 8940

taaagccgtt gcggataaat atgacgttga cctatcagag atagaccatc agttaatgat 9000

attagttaac tataacgaag atagtaataa agctaaacaa gagacgcgtg catttattag 9060

tgattatgtt cttgaaatgc accctaatga aaatttcgaa aataaacttg aagaaataat 9120

tgcagaaaac gctgtcggaa attatacgga gtgtataact gcggctaagt tggcaattga 9180

aaagtgtggt gcgaaaagtg tattgctgtc ctttgaacca atgaatgatt tgatgagcca 9240

aaaaaatgta atcaatattg ttgatgataa tattaagaag taccacatgg aatataccta 9300

atagatttcg agttgcagcg aggcggcaag tgaacgaatc cccaggagca tagataacta 9360

tgtgactggg gtgagtgaaa gcagccaaca aagcagcagc ttgaaagatg aagggtataa 9420

aagagtatga cagcagtgct gccatacttt ctaatattat cttgaggagt aaaacaggta 9480

tgacttcata tgttgataaa caagaaatta cagcaagctc agaaattgat gatttgattt 9540

tttcgagcga tccattagtg tggtcttacg acgagcagga aaaaatcaga aagaaacttg 9600

tgcttgatgc atttcgtaat cattataaac attgtcgaga atatcgtcac tactgtcagg 9660

cacacaaagt agatgacaat attacggaaa ttgatgacat acctgtattc ccaacatcgg 9720

tttttaagtt tactcgctta ttaacttctc aggaaaacga gattgaaagt tggtttacca 9780

gtagcggcac gaatggttta aaaagtcagg tggcgcgtga cagattaagt attgagagac 9840

tcttaggctc tgtgagttat ggcatgaaat atgttggtag ttggtttgat catcaaatag 9900

aattagtcaa tttgggacca gatagattta atgctcataa tatttggttt aaatatgtta 9960

tgagtttggt ggaattgtta tatcctacga catttaccgt aacagaagaa cgaatagatt 10020

ttgttaaaac attgaatagt cttgaacgaa taaaaaatca agggaaagat ctttgtctta 10080

ttggttcgcc atactttatt tatttactct gccattatat gaaagataaa aaaatctcat 10140

tttctggaga taaaagcctt tatatcataa ccggaggcgg ctggaaaagt tacgaaaaag 10200

aatctctgaa acgtgatgat ttcaatcatc ttttatttga tactttcaat ctcagtgata 10260

ttagtcagat ccgagatata tttaatcaag ttgaactcaa cacttgtttc tttgaggatg 10320

aaatgcagcg taaacatgtt ccgccgtggg tatatgcgcg agcgcttgat cctgaaacgt 10380

tgaaacctgt acctgatgga acgccggggt tgatgagtta tatggatgcg tcagcaacca 10440

gttatccagc atttattgtt accgatgatg tcgggataat tagcagagaa tatggtaagt 10500

atcccggcgt gctcgttgaa attttacgtc gcgtcaatac gaggacgcag aaagggtgtg 10560

ctttaagctt aaccgaagcg tttgatagtt gaagttgatg acctgcaggc atgcaagctt 10620

gcgcgcggcc gccagacaag gtgagggcct cgggagagaa tcagattgtc tagatcttga 10680

attttctacc gcaaggccct catgtccaac gctactctcc agcgccctga cctgaccaca 10740

ttctgtcgcc tcgatgagct cggcctggaa gccactgggc agtgtctgta ccctgatcgt 10800

gctgtcatta cctgccgtgt ggtggatgct gatgagtggt gttccagatg tgggtgcgag 10860

ggcatcccgc gggatactgt gacccggcgg ctgggccatg aacccttcgg ttggcggccc 10920

acgatattgc tggtgaggct ccgacgttac cgctgcaccg ggtgcggtta tgtgtggcgg 10980

caggagacct cgaaggctgc tgaaccaaga gcgaagcttt cccgccgtgg actgcgctgg 11040

gcattggaag gcatcgtctg tcagcatctg acggtggccc gggttgccga gggcctgggc 11100

gtgtcgtgga ataccgcgaa caacgccgtt ctggccgagg gccggcgcgt attgatcgat 11160

gacccgcacc gcttcgatgg agtgaaggtc attggtgttg atgagcatgt ttggcggcac 11220

acccgccgcg gggacaagta cgtcaccgtc atcatcgatc tgacaccgat ccgtgacggc 11280

acgggccctt cccggctgct ggacatggtc gagggccgct caaagcaggt cttcgccacg 11340

tggctgaacg aacgccctga agcgtggcgg gaaggggtgg aagtcgtggc catggacgga 11400

ttctccggct tcaaaagcgc cgctgccgaa gagcttcccg aggctgttcc ggtcatggat 11460

cccttccacg tcatccgctt ggcaggcgat gcactcgatg tctgcaggcg acgcgttcag 11520

caggcaacgt gcgggcatcg tggacgggcc ggcgatcctc tctacaaagc gaggcggacg 11580

ctgcacacgg gtgctgacct gctcaccgac aaacaacgcc aacgccttcg ggacttgttc 11640

gccaccgatg cgcacgtcga ggtcgaagcc agctggggca tctatcagcg catgattgcc 11700

gcgtaccggg agcaggaccg catccaagga aagaagctca tgcaggcagt ggtcaattcc 11760

atcagcacag gcgtcccggc cgctctcacc gagctcaaga cgctggggcg gaccctcaaa 11820

cgacgggcag aagacgtgct ggcgtacttc gatcggcccg gaacatcgaa cggccccaca 11880

gaagcaatta acgggagact ggaacacctc cgcggctcag ccctcgggtt ccggaacctc 11940

accaactaca tagcccgctc gctcctggaa tccggcggat tcagaccgaa actacaccct 12000

caattctgaa gagcctcatt gcccaccctc ggcaagacta cctattgctg tttatgaggt 12060

tcgtagcgcg cgttcggcca agcaggtctc gaattacaaa tgacctggcc cgccgtcgtt 12120

cgccttgtct gacttcaaac tttctcttcg gctcacagcc gcctgcgacc tggtcgcttg 12180

ccggtccggg cgccgcggtc gtcacacgcc tgggtgaact tggccagatc cgtcaaggtc 12240

agctgcttga gtaagcgatc ggtctggatc aacagccgcg ccgagacctg tgaccatcgt 12300

gaaccaggcc cgttcggtga agccgagttc ggccgcagcg ttcg 12344

<210> 3

<211> 391

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 3

Met Pro Phe Ala Leu Tyr Met Leu Ala Leu Ala Val Phe Val Met Gly

1 5 10 15

Thr Ser Glu Phe Met Leu Ala Gly Leu Leu Pro Ala Ile Ala Thr Glu

20 25 30

Leu Asp Val Ser Val Gly Thr Ala Gly Leu Leu Thr Ser Ala Phe Ala

35 40 45

Val Gly Met Val Val Gly Ala Pro Val Met Ala Ala Phe Ala Arg Arg

50 55 60

Trp Pro Pro Arg Leu Thr Leu Ile Val Cys Leu Leu Val Phe Ala Gly

65 70 75 80

Ser His Val Ile Gly Ala Met Thr Pro Val Phe Ser Leu Leu Leu Ile

85 90 95

Thr Arg Val Leu Ser Ala Leu Ala Asn Ala Gly Phe Leu Ala Val Ala

100 105 110

Leu Ser Thr Ala Thr Thr Leu Val Pro Ala Asn Gln Lys Gly Arg Ala

115 120 125

Leu Ser Ile Leu Leu Ser Gly Thr Thr Ile Ala Thr Val Val Gly Val

130 135 140

Pro Ala Gly Ala Leu Leu Gly Thr Ala Leu Gly Trp Arg Thr Thr Phe

145 150 155 160

Trp Ala Ile Ala Ile Leu Cys Ile Pro Ala Ala Val Gly Val Ile Arg

165 170 175

Gly Val Thr Asn Asn Val Gly Arg Ser Glu Thr Ser Ala Thr Ser Pro

180 185 190

Arg Leu Arg Val Glu Leu Ser Gln Leu Ala Thr Pro Arg Leu Ile Leu

195 200 205

Ala Met Ala Leu Gly Ala Leu Ile Asn Gly Gly Thr Phe Ala Ala Phe

210 215 220

Thr Phe Leu Ala Pro Ile Val Thr Glu Thr Ala Gly Leu Ala Glu Ala

225 230 235 240

Trp Val Ser Val Ala Leu Val Met Phe Gly Ile Gly Ser Phe Leu Gly

245 250 255

Val Thr Ile Ala Gly Arg Leu Ser Asp Gln Arg Pro Gly Leu Val Leu

260 265 270

Ala Val Gly Gly Pro Leu Leu Leu Thr Gly Trp Ile Val Leu Ala Val

275 280 285

Val Ala Ser His Pro Val Ala Leu Ile Val Leu Val Leu Val Gln Gly

290 295 300

Phe Leu Ser Phe Gly Val Gly Ser Thr Leu Ile Thr Arg Val Leu Tyr

305 310 315 320

Ala Ala Ser Gly Ala Pro Thr Met Gly Gly Ser Tyr Ala Thr Ala Ala

325 330 335

Leu Asn Ile Gly Ala Ala Ala Gly Pro Val Leu Gly Ala Leu Gly Leu

340 345 350

Ala Thr Gly Leu Gly Leu Leu Ala Pro Val Trp Val Ala Ser Val Leu

355 360 365

Thr Ala Ile Ala Leu Val Ile Met Leu Leu Thr Arg Arg Ala Leu Thr

370 375 380

Lys Thr Ala Ala Glu Ala Asn

385 390

<210> 4

<211> 480

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 4

Met Thr Lys Lys Ile Ser Phe Ile Ile Asn Gly Gln Val Glu Ile Phe

1 5 10 15

Pro Glu Ser Asp Asp Leu Val Gln Ser Ile Asn Phe Gly Asp Asn Ser

20 25 30

Val Tyr Leu Pro Ile Leu Asn Asp Ser His Val Lys Asn Ile Ile Asp

35 40 45

Cys Asn Gly Asn Asn Glu Leu Arg Leu His Asn Ile Val Asn Phe Leu

50 55 60

Tyr Thr Val Gly Gln Arg Trp Lys Asn Glu Glu Tyr Ser Arg Arg Arg

65 70 75 80

Thr Tyr Ile Arg Asp Leu Lys Lys Tyr Met Gly Tyr Ser Glu Glu Met

85 90 95

Ala Lys Leu Glu Ala Asn Trp Ile Ser Met Ile Leu Cys Ser Lys Gly

100 105 110

Gly Leu Tyr Asp Val Val Glu Asn Glu Leu Gly Ser Arg His Ile Met

115 120 125

Asp Glu Trp Leu Pro Gln Asp Glu Ser Tyr Val Arg Ala Phe Pro Lys

130 135 140

Gly Lys Ser Val His Leu Leu Ala Gly Asn Val Pro Leu Ser Gly Ile

145 150 155 160

Met Ser Ile Leu Arg Ala Ile Leu Thr Lys Asn Gln Cys Ile Ile Lys

165 170 175

Thr Ser Ser Thr Asp Pro Phe Thr Ala Asn Ala Leu Ala Leu Ser Phe

180 185 190

Ile Asp Val Asp Pro Asn His Pro Ile Thr Arg Ser Leu Ser Val Ile

195 200 205

Tyr Trp Pro His Gln Gly Asp Thr Ser Leu Ala Lys Glu Ile Met Arg

210 215 220

His Ala Asp Val Ile Val Ala Trp Gly Gly Pro Asp Ala Ile Asn Trp

225 230 235 240

Ala Val Glu His Ala Pro Ser Tyr Ala Asp Val Ile Lys Phe Gly Ser

245 250 255

Lys Lys Ser Leu Cys Ile Ile Asp Asn Pro Val Asp Leu Thr Ser Ala

260 265 270

Ala Thr Gly Ala Ala His Asp Val Cys Phe Tyr Asp Gln Arg Ala Cys

275 280 285

Phe Ser Ala Gln Asn Ile Tyr Tyr Met Gly Asn His Tyr Glu Glu Phe

290 295 300

Lys Leu Ala Leu Ile Glu Lys Leu Asn Leu Tyr Ala His Ile Leu Pro

305 310 315 320

Asn Ala Lys Lys Asp Phe Asp Glu Lys Ala Ala Tyr Ser Leu Val Gln

325 330 335

Lys Glu Ser Leu Phe Ala Gly Leu Lys Val Glu Val Asp Ile His Gln

340 345 350

Arg Trp Met Ile Ile Glu Ser Asn Ala Gly Val Glu Phe Asn Gln Pro

355 360 365

Leu Gly Arg Cys Val Tyr Leu His His Val Asp Asn Ile Glu Gln Ile

370 375 380

Leu Pro Tyr Val Gln Lys Asn Lys Thr Gln Thr Ile Ser Ile Phe Pro

385 390 395 400

Trp Glu Ser Ser Phe Lys Tyr Arg Asp Ala Leu Ala Leu Lys Gly Ala

405 410 415

Glu Arg Ile Val Glu Ala Gly Met Asn Asn Ile Phe Arg Val Gly Gly

420 425 430

Ser His Asp Gly Met Arg Pro Leu Gln Arg Leu Val Thr Tyr Ile Ser

435 440 445

His Glu Arg Pro Ser Asn Tyr Thr Ala Lys Asp Val Ala Val Glu Ile

450 455 460

Glu Gln Thr Arg Phe Leu Glu Glu Asp Lys Phe Leu Val Phe Val Pro

465 470 475 480

<210> 5

<211> 307

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 5

Met Glu Asn Glu Ser Lys Tyr Lys Thr Ile Asp His Val Ile Cys Val

1 5 10 15

Glu Gly Asn Lys Lys Ile His Val Trp Glu Thr Leu Pro Glu Glu Asn

20 25 30

Ser Pro Lys Arg Lys Asn Ala Ile Ile Ile Ala Ser Gly Phe Ala Arg

35 40 45

Arg Met Asp His Phe Ala Gly Leu Ala Glu Tyr Leu Ser Arg Asn Gly

50 55 60

Phe His Val Ile Arg Tyr Asp Ser Leu His His Val Gly Leu Ser Ser

65 70 75 80

Gly Thr Ile Asp Glu Phe Thr Met Ser Ile Gly Lys Gln Ser Leu Leu

85 90 95

Ala Val Val Asp Trp Leu Thr Thr Arg Lys Ile Asn Asn Phe Gly Met

100 105 110

Leu Ala Ser Ser Leu Ser Ala Arg Ile Ala Tyr Ala Ser Leu Ser Glu

115 120 125

Ile Asn Ala Ser Phe Leu Ile Thr Ala Val Gly Val Val Asn Leu Arg

130 135 140

Tyr Ser Leu Glu Arg Ala Leu Gly Phe Asp Tyr Leu Ser Leu Pro Ile

145 150 155 160

Asn Glu Leu Pro Asp Asn Leu Asp Phe Glu Gly His Lys Leu Gly Ala

165 170 175

Glu Val Phe Ala Arg Asp Cys Leu Asp Phe Gly Trp Glu Asp Leu Ala

180 185 190

Ser Thr Ile Asn Asn Met Met Tyr Leu Asp Ile Pro Phe Ile Ala Phe

195 200 205

Thr Ala Asn Asn Asp Asn Trp Val Lys Gln Asp Glu Val Ile Thr Leu

210 215 220

Leu Ser Asn Ile Arg Ser Asn Arg Cys Lys Ile Tyr Ser Leu Leu Gly

225 230 235 240

Ser Ser His Asp Leu Ser Glu Asn Leu Val Val Leu Arg Asn Phe Tyr

245 250 255

Gln Ser Val Thr Lys Ala Ala Ile Ala Met Asp Asn Asp His Leu Asp

260 265 270

Ile Asp Val Asp Ile Thr Glu Pro Ser Phe Glu His Leu Thr Ile Ala

275 280 285

Thr Val Asn Glu Arg Arg Met Arg Ile Glu Ile Glu Asn Gln Ala Ile

290 295 300

Ser Leu Ser

305

<210> 6

<211> 360

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 6

Met Lys Phe Gly Asn Phe Leu Leu Thr Tyr Gln Pro Pro Gln Phe Ser

1 5 10 15

Gln Thr Glu Val Met Lys Arg Leu Val Lys Leu Gly Arg Ile Ser Glu

20 25 30

Glu Cys Gly Phe Asp Thr Val Trp Leu Leu Glu His His Phe Thr Glu

35 40 45

Phe Gly Leu Leu Gly Asn Pro Tyr Val Ala Ala Ala Tyr Leu Leu Gly

50 55 60

Ala Thr Lys Lys Leu Asn Val Gly Thr Ala Ala Ile Val Leu Pro Thr

65 70 75 80

Ala His Pro Val Arg Gln Leu Glu Asp Val Asn Leu Leu Asp Gln Met

85 90 95

Ser Lys Gly Arg Phe Arg Phe Gly Ile Cys Arg Gly Leu Tyr Asn Lys

100 105 110

Asp Phe Arg Val Phe Gly Thr Asp Met Asn Asn Ser Arg Ala Leu Ala

115 120 125

Glu Cys Trp Tyr Gly Leu Ile Lys Asn Gly Met Thr Glu Gly Tyr Met

130 135 140

Glu Ala Asp Asn Glu His Ile Lys Phe His Lys Val Lys Val Asn Pro

145 150 155 160

Ala Ala Tyr Ser Arg Gly Gly Ala Pro Val Tyr Val Val Ala Glu Ser

165 170 175

Ala Ser Thr Thr Glu Trp Ala Ala Gln Phe Gly Leu Pro Met Ile Leu

180 185 190

Ser Trp Ile Ile Asn Thr Asn Glu Lys Lys Ala Gln Leu Glu Leu Tyr

195 200 205

Asn Glu Val Ala Gln Glu Tyr Gly His Asp Ile His Asn Ile Asp His

210 215 220

Cys Leu Ser Tyr Ile Thr Ser Val Asp His Asp Ser Ile Lys Ala Lys

225 230 235 240

Glu Ile Cys Arg Lys Phe Leu Gly His Trp Tyr Asp Ser Tyr Val Asn

245 250 255

Ala Thr Thr Ile Phe Asp Asp Ser Asp Gln Thr Arg Gly Tyr Asp Phe

260 265 270

Asn Lys Gly Gln Trp Arg Asp Phe Val Leu Lys Gly His Lys Asp Thr

275 280 285

Asn Arg Arg Ile Asp Tyr Ser Tyr Glu Ile Asn Pro Val Gly Thr Pro

290 295 300

Gln Glu Cys Ile Asp Ile Ile Gln Lys Asp Ile Asp Ala Thr Gly Ile

305 310 315 320

Ser Asn Ile Cys Cys Gly Phe Glu Ala Asn Gly Thr Val Asp Glu Ile

325 330 335

Ile Ala Ser Met Lys Leu Phe Gln Ser Asp Val Met Pro Phe Leu Lys

340 345 350

Glu Lys Gln Arg Ser Leu Leu Tyr

355 360

<210> 7

<211> 327

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 7

Met Lys Phe Gly Leu Phe Phe Leu Asn Phe Ile Asn Ser Thr Thr Val

1 5 10 15

Gln Glu Gln Ser Ile Val Arg Met Gln Glu Ile Thr Glu Tyr Val Asp

20 25 30

Lys Leu Asn Phe Glu Gln Ile Leu Val Tyr Glu Asn His Phe Ser Asp

35 40 45

Asn Gly Val Val Gly Ala Pro Leu Thr Val Ser Gly Phe Leu Leu Gly

50 55 60

Leu Thr Glu Lys Ile Lys Ile Gly Ser Leu Asn His Ile Ile Thr Thr

65 70 75 80

His His Pro Val Ala Ile Ala Glu Glu Ala Cys Leu Leu Asp Gln Leu

85 90 95

Ser Glu Gly Arg Phe Ile Leu Gly Phe Ser Asp Cys Glu Lys Lys Asp

100 105 110

Glu Met His Phe Phe Asn Arg Pro Val Glu Tyr Gln Gln Gln Leu Phe

115 120 125

Glu Glu Cys Tyr Glu Ile Ile Asn Asp Ala Leu Thr Thr Gly Tyr Cys

130 135 140

Asn Pro Asp Asn Asp Phe Tyr Ser Phe Pro Lys Ile Ser Val Asn Pro

145 150 155 160

His Ala Tyr Thr Pro Gly Gly Pro Arg Lys Tyr Val Thr Ala Thr Ser

165 170 175

His His Ile Val Glu Trp Ala Ala Lys Lys Gly Ile Pro Leu Ile Phe

180 185 190

Lys Trp Asp Asp Ser Asn Asp Val Arg Tyr Glu Tyr Ala Glu Arg Tyr

195 200 205

Lys Ala Val Ala Asp Lys Tyr Asp Val Asp Leu Ser Glu Ile Asp His

210 215 220

Gln Leu Met Ile Leu Val Asn Tyr Asn Glu Asp Ser Asn Lys Ala Lys

225 230 235 240

Gln Glu Thr Arg Ala Phe Ile Ser Asp Tyr Val Leu Glu Met His Pro

245 250 255

Asn Glu Asn Phe Glu Asn Lys Leu Glu Glu Ile Ile Ala Glu Asn Ala

260 265 270

Val Gly Asn Tyr Thr Glu Cys Ile Thr Ala Ala Lys Leu Ala Ile Glu

275 280 285

Lys Cys Gly Ala Lys Ser Val Leu Leu Ser Phe Glu Pro Met Asn Asp

290 295 300

Leu Met Ser Gln Lys Asn Val Ile Asn Ile Val Asp Asp Asn Ile Lys

305 310 315 320

Lys Tyr His Met Glu Tyr Thr

325

<210> 8

<211> 370

<212> PRT

<213>Artificial sequence

<220>

<223>

<400> 8

Met Thr Ser Tyr Val Asp Lys Gln Glu Ile Thr Ala Ser Ser Glu Ile

1 5 10 15

Asp Asp Leu Ile Phe Ser Ser Asp Pro Leu Val Trp Ser Tyr Asp Glu

20 25 30

Gln Glu Lys Ile Arg Lys Lys Leu Val Leu Asp Ala Phe Arg Asn His

35 40 45

Tyr Lys His Cys Arg Glu Tyr Arg His Tyr Cys Gln Ala His Lys Val

50 55 60

Asp Asp Asn Ile Thr Glu Ile Asp Asp Ile Pro Val Phe Pro Thr Ser

65 70 75 80

Val Phe Lys Phe Thr Arg Leu Leu Thr Ser Gln Glu Asn Glu Ile Glu

85 90 95

Ser Trp Phe Thr Ser Ser Gly Thr Asn Gly Leu Lys Ser Gln Val Ala

100 105 110

Arg Asp Arg Leu Ser Ile Glu Arg Leu Leu Gly Ser Val Ser Tyr Gly

115 120 125

Met Lys Tyr Val Gly Ser Trp Phe Asp His Gln Ile Glu Leu Val Asn

130 135 140

Leu Gly Pro Asp Arg Phe Asn Ala His Asn Ile Trp Phe Lys Tyr Val

145 150 155 160

Met Ser Leu Val Glu Leu Leu Tyr Pro Thr Thr Phe Thr Val Thr Glu

165 170 175

Glu Arg Ile Asp Phe Val Lys Thr Leu Asn Ser Leu Glu Arg Ile Lys

180 185 190

Asn Gln Gly Lys Asp Leu Cys Leu Ile Gly Ser Pro Tyr Phe Ile Tyr

195 200 205

Leu Leu Cys His Tyr Met Lys Asp Lys Lys Ile Ser Phe Ser Gly Asp

210 215 220

Lys Ser Leu Tyr Ile Ile Thr Gly Gly Gly Trp Lys Ser Tyr Glu Lys

225 230 235 240

Glu Ser Leu Lys Arg Asp Asp Phe Asn His Leu Leu Phe Asp Thr Phe

245 250 255

Asn Leu Ser Asp Ile Ser Gln Ile Arg Asp Ile Phe Asn Gln Val Glu

260 265 270

Leu Asn Thr Cys Phe Phe Glu Asp Glu Met Gln Arg Lys His Val Pro

275 280 285

Pro Trp Val Tyr Ala Arg Ala Leu Asp Pro Glu Thr Leu Lys Pro Val

290 295 300

Pro Asp Gly Thr Pro Gly Leu Met Ser Tyr Met Asp Ala Ser Ala Thr

305 310 315 320

Ser Tyr Pro Ala Phe Ile Val Thr Asp Asp Val Gly Ile Ile Ser Arg

325 330 335

Glu Tyr Gly Lys Tyr Pro Gly Val Leu Val Glu Ile Leu Arg Arg Val

340 345 350

Asn Thr Arg Thr Gln Lys Gly Cys Ala Leu Ser Leu Thr Glu Ala Phe

355 360 365

Asp Ser

370

Claims

1. Ralstonia solanacearum (Ralstonia solanacearum) Rs-BL#7, it is managed in Chinese microorganism strain preservation The deposit number of committee's common micro-organisms center is CGMCC No.13637.

2. a kind of microbial inoculum, it is characterised in that：The microbial inoculum contains Ralstonia solanacearum (Ralstonia described in claim 1 solanacearum)Rs-BL#7 CGMCC No.13637。

3. microbial inoculum as claimed in claim 2, it is characterised in that：The preparation method of the microbial inoculum is will be blue or green described in claim 1 Withered Lei Er Salmonellas (Ralstonia solanacearum) Rs-BL#7 CGMCC No.13637 are seeded to bacteria culture media and go forward side by side Row culture, the bacterium solution of acquisition is microbial inoculum.

4. the preparation method of microbial inoculum described in Claims 2 or 3, comprises the following steps：By Ralstonia solanacearum described in claim 1 (Ralstonia solanacearum) Rs-BL#7 CGMCC No.13637 are seeded to bacteria culture media and are cultivated, and obtain Bacterium solution be microbial inoculum.

5. Ralstonia solanacearum described in claim 1 (Ralstonia solanacearum) Rs-BL#7 CGMCC Application of the microbial inoculum described in No.13637 or Claims 2 or 3 in resistance to bacterial wilt plant is screened.

6. Ralstonia solanacearum described in claim 1 (Ralstonia solanacearum) Rs-BL#7 CGMCC Application of the microbial inoculum described in No.13637 or Claims 2 or 3 in resistance of the measuring plants to bacterial wilt is treated in identification.

7. application as claimed in claims 6 or 7, it is characterised in that：It is described to treat that measuring plants are capsicum.

8. Ralstonia solanacearum described in claim 1 (Ralstonia solanacearum) Rs-BL#7 CGMCC Application of the microbial inoculum described in No.13637 or Claims 2 or 3 in the chemical agent that screening prevents and treats bacterial wilt.