CN106520648B - Recombination hillock monad and its construction method and application to chain sclerotinia sclerotiorum with inhibitory activity - Google Patents

Recombination hillock monad and its construction method and application to chain sclerotinia sclerotiorum with inhibitory activity Download PDF

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CN106520648B
CN106520648B CN201610960313.0A CN201610960313A CN106520648B CN 106520648 B CN106520648 B CN 106520648B CN 201610960313 A CN201610960313 A CN 201610960313A CN 106520648 B CN106520648 B CN 106520648B
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monad
hillock
recombination
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hemoglobin
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吴慧玲
刘伟成
刘朝蓬
刘霆
石玲玲
白雪莲
张涛涛
刘德文
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

The invention discloses the recombination hillock monad to chain sclerotinia sclerotiorum with inhibitory activity and its construction method and applications.The recombination hillock monad is prepared by assigning receptor hillock monad hemoglobin activity, and the receptor hillock monad is without the active hillock monad of hemoglobin；The receptor hillock monad and the recombination hillock monad have inhibitory activity to chain sclerotinia sclerotiorum, and in the activity for inhibiting chain sclerotinia sclerotiorum, the recombination hillock monad is higher than the receptor hillock monad.The recombination hillock monad has antibacterial activity more higher than receptor hillock monad, and recombination hillock monad is 2.3 times of receptor hillock monad to the bacteriostatic activity of Monilinia fructicola (Monilinia fructicola).Recombination hillock monad bacteriostasis of the invention significantly improves, and can be applied in the exploitation of peach brown rot biological prevention and control agent.

Description

To chain sclerotinia sclerotiorum have inhibitory activity recombination hillock monad and its construction method with Using
Technical field
The present invention relates to the recombination hillock monads and its structure in bioengineering field to chain sclerotinia sclerotiorum with inhibitory activity Construction method and application.
Background technique
The brown rot as caused by chain sclerotinia sclerotiorum (Monilinia spp.) is one of most important disease in peach production, and The most important postharvest disease of kernel approaches in world wide.This disease before adopting, after adopting and storage the transport phase can cause a large amount of underproduction, It is particularly acute after adopting, is up to 59-90% in America and Europe's loss.China's peach yield at 10,000,000 tons or more, for be only second to apple Two big fruit.Investigation shows that this disease of Beijing area can result in storage fruit year loss nearly 1/4, and decayed fruit rate is up to after outbound 84.6-100%.So far, chemical prevention is the main means for controlling peach brown rot, and the production of peach brown rot in this year is upper common Peach brown rot chemical insecticide produces drug resistance, so that the prevention and treatment of this disease receives great threat；Further, since chemical agent Caused environmental pollution, food safety and human health problems, green prevention and control have been increasingly becoming hot spot.Microorganism formulation tool Have the advantages that target specificity it is strong, to safety of human and livestock, environmental compatible, be ideal chemical agent substitute.
Grassy marshland hummock (ridge) monad (Collimonas pratensis) JZB120004 is from Fromlingzhi, tibet Mount Sejila Isolated Natural strains (the CN to Monilinia fructicola with special bacteriostasis in height above sea level 4530m mesophorbium, coryphile soil 104004680B), in order to which the biological and ecological methods to prevent plant disease, pests, and erosion for further increasing the bacterial strain is active, the better peach brown rot biological prevention and control agent of preventive effect is developed, is needed Research and develop the higher new strains of antibacterial activity.
Summary of the invention
A technical problem to be solved by this invention is how to construct to have more high bacteriostatic activity to plant brown rot Bacterial strain.
In order to solve the above technical problems, the present invention provides recombination hillock (hilllock) monads.
Recombination hillock (hilllock) monad provided by the present invention, it is living by assigning receptor hillock (hilllock) monad hemoglobin It is prepared by property；Receptor hillock (hilllock) monad and recombination hillock (hilllock) monad, which have chain sclerotinia sclerotiorum, to be inhibited to live Property, in the activity for inhibiting chain sclerotinia sclerotiorum, recombination hillock (hilllock) monad is higher than receptor hillock (hilllock) monad.
In above-mentioned recombination hillock monad, the receptor hillock monad can be for without the active hillock of hemoglobin (hilllock) monad.
In above-mentioned recombination hillock monad, the imparting receptor hillock monad hemoglobin activity is by by hemoglobin Encoding gene import in the receptor hillock monad and realize.
In above-mentioned recombination hillock monad, the hemoglobin can be following A1) or protein A2):
A1) amino acid sequence is the protein of SEQ ID No.1 in sequence table；
A2) by the substitution of one or several amino acid residues in sequence table in SEQ ID No.1 and/or missing and/or Addition and have hemoglobin it is active as A1) derived from protein.
Wherein, SEQ ID No.1 is made of 146 amino acid residues.
In above-mentioned recombination hillock monad, the receptor hillock monad can be grassy marshland hummock (ridge) monad (Collimonas pratensis), bacterial strain number is JZB120004, general in China Committee for Culture Collection of Microorganisms The number of registering on the books at logical microorganism center is CGMCC No.9193.
In above-mentioned recombination hillock monad, the encoding gene of the hemoglobin can be following A 11)-A13) in any Kind DNA molecular:
A11) coded sequence is the cDNA molecule or genomic DNA of SEQ ID No.2 in sequence table；
A12) under strict conditions with A11) limit DNA molecular hybridize and encode the hemoglobin cDNA molecule or Genomic DNA；
A13) and A11) or A12) DNA molecular that limits identity and the coding hemoglobin with 75% or more CDNA molecule or genomic DNA.
Wherein, SEQ ID No.2 is made of 441 nucleotide.
In above-mentioned recombination hillock monad, the chain sclerotinia sclerotiorum can be plant brown rot germ.The fruit of the plant is core Fruit, such as peach.Plant brown rot germ concretely U.S. Australia type drupe chain sclerotinia sclerotiorum (Monilinia fructicola).
In above-mentioned recombination hillock monad, recombination hillock (hilllock) monad can be grassy marshland hillock monad (Collimonas pratensis), bacterial strain number is ZV261, commonly micro- in China Committee for Culture Collection of Microorganisms The number of registering on the books of Bio-Centers is CGMCC No.13015.
In order to solve the above technical problems, the present invention provides contain recombination hillock (hilllock) monad or/and described heavy The microbial inoculum of the metabolin of group hillock (hilllock) monad, the microbial inoculum have inhibitory activity to chain sclerotinia sclerotiorum.
The active constituent of above-mentioned microbial inoculum can be recombination hillock (hilllock) monad or/and recombination hillock (hilllock) unit cell The metabolin of bacterium, the active constituent of above-mentioned microbial inoculum can also contain other biological ingredient or abiotic component, above-mentioned microbial inoculum other Active constituent those skilled in the art can determine according to the inhibitory effect to chain sclerotinia sclerotiorum.
In the microbial inoculum, in addition to the active constituent, also contain carrier.The carrier can it is common for pesticide field and It is biologically inert carrier.The carrier can be solid carrier or liquid-carrier；The solid carrier can for mineral material, Vegetable material or high-molecular compound；The mineral material can be clay, talcum, kaolin, montmorillonite, white carbon, zeolite, silica At least one of with diatomite；The vegetable material can be at least one of corn flour, bean powder and starch；The macromolecule Compound can be polyvinyl alcohol or/and polyglycols；The liquid-carrier can be organic solvent, vegetable oil, mineral oil or water；It is described Organic solvent can be decane or/and dodecane.
The dosage form of the microbial inoculum can be a variety of dosage forms, such as liquor, emulsion, suspending agent, pulvis, granule, wettable powder Or water dispersible granules.
As needed, surfactant (such as polysorbas20, Tween 80), adhesive, stabilization can be also added in the microbial inoculum Agent (such as antioxidant), pH adjusting agent.
Above, the metabolin of described recombination hillock (hilllock) monad can be from the fermentation of described recombination hillock (hilllock) monad It is obtained in liquid.The metabolin of described recombination hillock (hilllock) monad can be specifically prepared as follows: in liquid medium Recombination hillock (hilllock) monad is cultivated, recombination hillock (hilllock) monad removed in liquid culture (fermentation liquid) is Obtain the metabolin of described recombination hillock (hilllock) monad.
The present invention also provides the methods for constructing above-mentioned recombination hillock monad.
The method of building recombination hillock monad provided by the present invention, including by the encoding gene of hemoglobin import by In body hillock monad, the activity for the chain sclerotinia sclerotiorum that is inhibited is higher than the recombination hillock monad of the receptor hillock monad.
In the above method, the hemoglobin can be following A1) or protein A2):
A1) amino acid sequence is the protein of SEQ ID No.1 in sequence table；
A2) by the substitution of one or several amino acid residues in sequence table in SEQ ID No.1 and/or missing and/or Addition and have hemoglobin it is active as A1) derived from protein.
In the above method, the receptor hillock monad can be grassy marshland hummock (ridge) monad (Collimonas Pratensis), bacterial strain number is JZB120004, in China Committee for Culture Collection of Microorganisms's common micro-organisms center Number of registering on the books be CGMCC No.9193.
In the above method, the encoding gene of the hemoglobin is following A 11)-A13) in any DNA molecular:
A11) coded sequence is the cDNA molecule or genomic DNA of SEQ ID No.2 in sequence table；
A12) under strict conditions with A11) limit DNA molecular hybridize and encode the hemoglobin cDNA molecule or Genomic DNA；
A13) and A11) or A12) DNA molecular that limits identity and the coding hemoglobin with 75% or more CDNA molecule or genomic DNA.
In the above method, the chain sclerotinia sclerotiorum can be plant brown rot germ.The fruit of the plant is drupe, such as peach.It plants Object brown rot germ concretely U.S. Australia type drupe chain sclerotinia sclerotiorum (Monilinia fructicola).
In the above method, recombination hillock (hilllock) monad can be grassy marshland hillock monad (Collimonas Pratensis), bacterial strain number is ZV261, in stepping on for China Committee for Culture Collection of Microorganisms's common micro-organisms center Charging to volume number is CGMCC No.13015.Hereinafter abbreviation grassy marshland hillock monad (Collimonas pratensis) ZV261。
Following any applications of above-mentioned recombination hillock (hilllock) monad or above-mentioned microbial inoculum also belong to protection scope of the present invention:
A) the application of above-mentioned recombination hillock (hilllock) monad or above-mentioned microbial inoculum in preparation plant brown rot germ inhibitor；
B) above-mentioned recombination hillock (hilllock) monad or above-mentioned microbial inoculum are inhibiting the application in plant brown rot germ；
C) the application of above-mentioned recombination hillock (hilllock) monad or above-mentioned microbial inoculum in preparation plant brown rot inhibitor；
D) above-mentioned recombination hillock (hilllock) monad or above-mentioned microbial inoculum are inhibiting the application in plant brown rot.
In above-mentioned application, the fruit of the plant is drupe, such as peach.The plant brown rot germ concretely U.S. Australia type core Fruit chain sclerotinia sclerotiorum (Monilinia fructicola).
It is demonstrated experimentally that recombination hillock (hilllock) monad (grassy marshland hillock monad (Collimonas pratensis) ZV261) have more than wild type hillock monad (grassy marshland hillock monad (Collimonas pratensis) JZB120004) High antibacterial activity, recombination hillock (hilllock) monad are to the bacteriostatic activity of Monilinia fructicola (Monilinia fructicola) 2.3 times of wild type hillock monad.Recombination hillock monad bacteriostasis of the invention significantly improves, and it is brown to can be applied to peach In the exploitation of maize ear rot biological prevention and control agent.
Biomaterial preservation explanation
Classification naming: grassy marshland hummock monad (Collimonas pratensis)
Strain number: ZV261
Preservation mechanism: China Committee for Culture Collection of Microorganisms's common micro-organisms center
Preservation mechanism abbreviation: CGMCC
Address: Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Institute of Microorganism, Academia Sinica, postcode: 100101
Preservation date: on September 19th, 2016
Collection is registered on the books number: CGMCC No.13015
Detailed description of the invention
Fig. 1 is HindIII the and BamHI digestion result of hemoglobin gene expression vector pRK415-vhb.
Wherein, swimming lane M is DNA molecular amount standard, and 1 is pRK415-vhb.
Fig. 2 is that CO difference spectra method analyzes hillock monad (Collimonas pratensis) ZV261, hillock monad (Collimonas pratensis) JZB120004CGMCC No.9193 hemoglobin activity.
Wherein, underlying curve is grassy marshland hummock (ridge) monad (Collimonas pratensis) JZB120004CGMCC No.9193, the curve being located above are grassy marshland hillock monad (Collimonas pratensis) ZV261。
Fig. 3 is grassy marshland hillock monad (Collimonas pratensis) ZV261 and grassy marshland hillock monad The bacteriostatic activity of (Collimonas pratensis) JZB120004 compares.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Wide host expression vector pRK415 (NTS Keen, SD Tamaki, D Kobayashi, D in following embodiments Trollinger.Improved broad-host-range vectors for DNA cloning in Gram-negative Bacteria. " Gene ", 1988,70 (1): 191-7.) public can obtain from Beijing City Agriculture and Forestry Institute, and it is real to repeat the application It tests.
Escherichia coli S17-1 (E.coli S17-1) (Simon R, Priefer U, Puhler A in following embodiments (1983)A broad host range mobilization system for in vivo genetic engineering: Transposon mutagenesis in gram negative bacteria.Bio-Technology 1:784-791.) it is public Crowd can obtain from Beijing City Agriculture and Forestry Institute, to repeat the application experiment.
Pathogen Monilinia fructicola (Monilinia fructicola) (Liu Ting, Tao Wanqiang, king in following embodiments It closes, Hua Yupeng, Liu Weicheng.Active north gardening of the Antagonistic Fungi A02 metabolite to several fruit diseases, 2011,17:13- 16) public can obtain from Beijing City Agriculture and Forestry Institute, to repeat the application experiment.
Grassy marshland hummock (ridge) monad (Collimonas pratensis) JZB120004 in following embodiments in On May 23rd, 2014 is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, number of registering on the books It is the biomaterial authorized in CN 104004680B for CGMCC No.9193.
(Wen Ying, Li Jilun Vitreoscilla hemoglobin gene are in Avid kyowamycin by pRK404-vhb in following embodiments In .40 volumes of expression microorganism journal 1 phase .2000 2 months) public can obtain from Beijing City Agriculture and Forestry Institute, to repeat this Shen It please test.
The recombination hillock monad of embodiment 1, the anti-chain sclerotinia sclerotiorum of building
1, the building of hemoglobin gene expression vector pRK415-vhb
The entitled pRK415-vhb of hemoglobin gene expression vector of the present embodiment building, blood red egg is contained in the carrier The sequence of white gene and own promoter about 1.4kb.
The specific construction method of pRK415-vhb is as follows:
With HindIII and BamHI digestion pRK404-vhb, the 1.4kb piece of gene containing vhb and its own promoter is obtained Section, connects it on the pRK415 through same digestion, obtains containing hemoglobin gene (SEQ ID No.2) and itself starting The recombinant vector of son, is named as pRK415-vhb for the recombinant vector.PRK415-vhb expresses blood red shown in SEQ IDNo.1 Albumen (entitled vhb).
PRK415-vhb converts Escherichia coli S17-1, screens to obtain carrying hemoglobin gene with tetracyclin resistance Transformant, the transformant is with p1 (5'-ATGTTAGACCAGCAAACCA-3) and p2 (5'-TTATTCAACCGCTTGAGCGT- PCR amplification 3') is carried out for primer and obtains the hemoglobin ORF segment of 441bp, with HindIII and BamHI digestion pRK415-vhb The pRK415 plasmid fragments of 10.5kb and the hemoglobin gene of 1.4kb and own promoter segment (Fig. 1) are obtained, so that it is determined that Hemoglobin gene segment is connected correctly in pRK415 carrier.
2, hemoglobin gene is imported into grassy marshland hillock monad (Collimonas using pRK415-vhb Pratensis) JZB120004 is obtained with the active recombination hillock monad of hemoglobin
By pRK415-vhb carrier Transformed E .coli S17-1, the method engaged by amphiphilic converts hillock monad JZB120004 screens to obtain positive transformant by tetracyclin resistance, and PCR verifies to obtain correct transformant.Specific conversion side Method is as follows:
1) pRK415-vhb carrier is converted into demethylation E.coli S17-1
It is sieved by heat shock method by pRK415-vhb vector introduction E.coliS17-1 and by 12.5 μ g/ml tetracyclin resistances Choosing obtains the recombinant bacterium E.coliS17-1/pRK415-vhb for being transferred to pRK415-vhb carrier.
2) the recombination hillock monad of the anti-peach brown rot of amphiphilic engagement build
Bibliography (Bierman M et al.1992) carries out amphiphilic engagement, and the specific method is as follows:
(1) recombinant bacterium E.coliS17-1/pRK415-vhb is inoculated into the LB liquid medium containing tetracyclin resistance, 37 DEG C, 200rpm, overnight incubation, the next morning is connected in fresh LB by 1% inoculum concentration and is cultivated to OD600=0.4- 0.6, bacterium solution is collected, 4 DEG C, 4000rpm, 2min is centrifuged, abandons supernatant, is resuspended and is precipitated with the ice-cold pure LB culture solution of 20mL, 4 DEG C, 4000rpm is centrifuged 2min, abandons supernatant, it is therefore an objective to wash away antibiotic.Precipitating is resuspended with the ice-cold pure LB culture solution of 2mL. (2) grassy marshland hillock monad (Collimonas pratensis) JZB120004 that the inclined-plane PDA grows 3 days is inoculated into improvement King ' B (improvement KB culture medium) (Atlas R.M.1995.Handbook of media for environmental Microbiology.Boca Raton FL:CRC) on culture medium, 25 DEG C, 200rpm, overnight incubation, the next morning is by 1% Inoculum concentration is connected in fresh improvement KB culture medium and cultivates to OD600=0.8-1.0 collects bacterium solution, 4 DEG C, 4000rpm, is centrifuged 2min abandons supernatant, cleans thallus repeatedly with 20mL sterile water, it is therefore an objective to wash away bacterium surface mucus.(3) by step (1) and (2) Thallus be uniformly mixed, point to solid improvement KB culture medium on 4-6 hours, dilution spread changes in the solid containing tetracyclin resistance On good KB culture medium.Picking single bacterium is fallen on the improvement KB plate of the new above-mentioned antibiotic solution of addition after 1 day.Picking changes at this Bacterium colony on good KB plate carries out PCR with above-mentioned p1 and p2, by the recombination of the wherein amplified production of one plant of available 0.44kb Bacterium is named as grassy marshland hillock (hilllock) monad (Collimonas pratensis) ZV261.Grassy marshland hillock (hilllock) monad (Collimonas pratensis) ZV261 was preserved in Chinese microorganism strain preservation conservator on September 19th, 2016 Can common micro-organisms center, collection registers on the books number: CGMCC No.13015, hereinafter referred grassy marshland hillock monad (Collimonas pratensis)ZV261。
3) the hemoglobin activity of grassy marshland hillock monad (Collimonas pratensis) ZV261
By wild-type strain-grassy marshland hillock monad (Collimonas pratensis) JZB120004 and recombinant bacterial strain- Grassy marshland hillock monad (Collimonas pratensis) ZV261 transfers after being inoculated into improvement respectively KB solid medium 2 days It is cultivated 3 days in improvement KB fluid nutrient medium, carries out CO difference spectra method analysing haemoglobin activity.
The specific method is as follows:
The cell culture fluid of culture 3 days is centrifuged 10min with 4 000-6 000r/min, collects cell.It will be thin after centrifugation Born of the same parents are resuspended in phosphate buffer (100mmol/L, pH 7.5), after ultrasonic disruption 3min, are separately added into Na2S2O4Or NADH carries out reduction treatment, and then CO is bubbled into sample end certain time, scans 400~470nm's on spectrophotometer Difference spectra.The experimental results showed that the VHb albumen given expression in bacterial strain ZV261 has obvious absorption peaks, grassy marshland hillock at 420nm Monad (Collimonas pratensis) ZV261 is active with hemoglobin, and wild-type strain-grassy marshland hillock monad There is not characteristic absorption peak, wild-type strain-in the protein crude extract administration of (Collimonas pratensis) JZB120004 It is active (Fig. 2) that grassy marshland hillock monad (Collimonas pratensis) JZB120004 does not have hemoglobin.
Grassy marshland hillock monad (Collimonas pratensis) ZV261 of the present embodiment building is living with hemoglobin Property, for the recombination hillock monad of anti-chain sclerotinia sclerotiorum.
The suppression of embodiment 2, grassy marshland hillock monad (Collimonas pratensis) ZV261 (recombination hillock monad) Bacterium activity is higher than grassy marshland hillock monad (Collimonas pratensis) JZB120004 (wild type hillock monad)
1, the preparation of plant brown rot inhibitor
The preparation of 1.1 grassy marshland hillock monad (Collimonas pratensis) ZV261 fermentation liquids
Ring grassy marshland hillock monad (Collimonas pratensis) ZV261 is taken, is inoculated into and improves KB liquid equipped with 5mL In the test tube of body culture medium, in 26 DEG C of cultures to OD600nmFor 0.6 (not connect the improvement KB fluid nutrient medium of bacterium as blank pair According to), as seed liquor, the 500mL triangle of KB fluid nutrient medium is improved equipped with 200mL according to the access of 2% (volume ratio) inoculum concentration In bottle, is cultivated 3 days in improvement KB fluid nutrient medium at 26 DEG C, obtain grassy marshland hillock monad (Collimonas Pratensis) ZV261 fermentation liquid.
The preparation of 1.2 grassy marshland hillock monad (Collimonas pratensis) JZB120004 fermentation liquids
Difference with 1.1 is only that grassy marshland hillock monad (Collimonas pratensis) ZV261 replacing with grass Pasture hillock monad (Collimonas pratensis) JZB120004, the specific method is as follows:
Ring grassy marshland hillock monad (Collimonas pratensis) JZB120004 is taken, is inoculated into and is improved equipped with 5mL In the test tube of KB fluid nutrient medium, in 26 DEG C of cultures to OD600nmFor 0.6 (not connect the improvement KB fluid nutrient medium of bacterium as blank Control), the 500mL tri- as seed liquor, according to the access of 2% (volume ratio) inoculum concentration equipped with 200mL improvement KB fluid nutrient medium In the bottle of angle, is cultivated 3 days in improvement KB fluid nutrient medium at 26 DEG C, obtain grassy marshland hillock monad (Collimonas Pratensis) JZB120004 fermentation liquid.
The preparation of 1.3 plant brown rot inhibitor
By 1.1 grassy marshland hillock monad (Collimonas pratensis) ZV261 fermentation liquid and 1.2 grassy marshland hillock Monad (Collimonas pratensis) JZB120004 fermentation liquid distinguishes 5000rpm/min and is centrifuged 10-15min, on 0.45 μm of sterile filtering with microporous membrane of clear liquid, collects filtrate (without thallus in the filtrate), respectively obtains grassy marshland hillock unit cell Bacterium (Collimonas pratensis) ZV261 bacteria-free filtrate and grassy marshland hillock monad (Collimonas pratensis) JZB120004 bacteria-free filtrate, both bacteria-free filtrates are plant brown rot inhibitor.
2, bacteriostatic activity is tested
It is Monilinia fructicola (Monilinia fructicola) for examination target pathogens.
The conidium that the Monilinia fructicola (M.fructicola) of generation is cultivated on scraping PDA plate, with sterile water system It at bacteria suspension, is equably applied on freshly prepd PDA plate, sets drying in superclean bench；With the aseptic card punch of diameter 7mm It is punched in plate surrounding symmetric position, 1.3 grassy marshland hillock monad (Collimonas is then injected in every hole Pratensis) the grassy marshland hillock monad (Collimonas pratensis) of ZV261 bacteria-free filtrate 100 μ l or 1.3 JZB120004 bacteria-free filtrate 100 μ l is cultivated 3 days at 28 DEG C, and crossing method measures antibacterial circle diameter.Ten repetitions of every processing.
The experimental results showed that grassy marshland hillock monad (Collimonas pratensis) ZV261 bacteria-free filtrate and grassy marshland mountain Ridge monad (Collimonas pratensis) JZB120004 bacteria-free filtrate is on PDA plate to Monilinia fructicola (Monilinia fructicola) generates apparent inhibition zone, grassy marshland hillock monad (Collimonas pratensis) ZV261 bacteria-free filtrate is 1.8cm ± 0.08cm, grassy marshland hillock unit cell to Monilinia fructicola (M.fructicola) antibacterial circle diameter Bacterium (Collimonas pratensis) JZB120004 bacteria-free filtrate presses down Monilinia fructicola (Monilinia fructicola) Bacterium loop diameter is 0.8cm ± 0.06cm (Fig. 3).Illustrate grassy marshland hillock monad (Collimonas pratensis) ZV261 without Bacterium filtrate is grassy marshland hillock monad to the antibacterial circle diameter of Monilinia fructicola (Monilinia fructicola) Suppression of (Collimonas pratensis) the JZB120004 bacteria-free filtrate to Monilinia fructicola (Monilinia fructicola) 2.3 times of bacterium loop diameter；It recombinates hillock (hilllock) monad (grassy marshland hillock monad (Collimonas pratensis) ZV261) With than wild type hillock monad, (grassy marshland hillock monad (Collimonas pratensis) JZB120004 is higher anti- Bacterium activity, recombination hillock (hilllock) monad is wild type to the bacteriostatic activity of Monilinia fructicola (Monilinia fructicola) 2.3 times of hillock monad.
<110>Beijing City Agriculture and Forestry Institute
<120>the recombination hillock monad to chain sclerotinia sclerotiorum with inhibitory activity and its construction method and application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 146
<212> PRT
<213>artificial sequence
<220>
<223>
<400> 1
Met Leu Asp Gln Gln Thr Ile Asn Ile Ile Lys Ala Thr Val Pro Val
1 5 10 15
Leu Lys Glu His Gly Val Thr Ile Thr Thr Thr Phe Tyr Lys Asn Leu
20 25 30
Phe Ala Lys His Pro Glu Val Arg Pro Leu Phe Asp Met Gly Arg Gln
35 40 45
Glu Ser Leu Glu Gln Pro Lys Ala Leu Ala Met Thr Val Leu Ala Ala
50 55 60
Ala Gln Asn Ile Glu Asn Leu Pro Ala Ile Leu Pro Ala Val Lys Lys
65 70 75 80
Ile Ala Val Lys His Cys Gln Ala Gly Val Ala Ala Ala His Tyr Pro
85 90 95
Ile Val Gly Gln Glu Leu Leu Gly Ala Ile Lys Glu Val Leu Gly Asp
100 105 110
Ala Ala Thr Asp Asp Ile Leu Asp Ala Trp Gly Lys Ala Tyr Gly Val
115 120 125
Ile Ala Asp Val Phe Ile Gln Val Glu Ala Asp Leu Tyr Ala Gln Ala
130 135 140
Val Glu
145
<210> 2
<211> 441
<212> DNA
<213>artificial sequence
<220>
<223>
<400> 2
atgttagacc agcaaaccat taacatcatc aaagccactg ttcctgtatt gaaggagcat 60
ggcgttacca ttaccacgac tttttataaa aacttgtttg ccaaacaccc tgaagtacgt 120
cctttgtttg atatgggtcg ccaagaatct ttggagcagc ctaaggcttt ggcgatgacg 180
gtattggcgg cagcgcaaaa cattgaaaat ttgccagcta ttttgcctgc ggtcaaaaaa 240
attgcagtca aacattgtca agcaggcgtg gcagcagcgc attatccgat tgtcggtcaa 300
gaattgttgg gtgcgattaa agaagtattg ggcgatgccg caaccgatga cattttggac 360
gcgtggggca aggcttatgg cgtgattgca gatgtgttta ttcaagtgga agcagatttg 420
tacgctcaag cggttgaata a 441

Claims (4)

1. recombinating hillock monad, the recombination hillock monad is made by assigning receptor hillock monad hemoglobin activity It is standby；The receptor hillock monad and the recombination hillock monad have inhibitory activity to chain sclerotinia sclerotiorum, are inhibiting chain nuclear disk In the activity of bacterium, the recombination hillock monad is higher than the receptor hillock monad；The imparting receptor hillock monad blood Lactoferrin activity is realized by importing the encoding gene of hemoglobin in the receptor hillock monad；The receptor hillock list Born of the same parents bacterium be grassy marshland hummock monad (Collimonas pratensis), bacterial strain number is JZB120004, in China Microbiological The number of registering on the books of culture presevation administration committee common micro-organisms center is CGMCC No.9193；The chain sclerotinia sclerotiorum is Monilinia fructicola；The recombination hillock monad be grassy marshland hummock monad (Collimonas pratensis), bacterial strain number For ZV261；The hemoglobin is the protein that amino acid sequence is SEQ ID No.1 in sequence table.
2. recombination hillock monad according to claim 1, it is characterised in that: the encoding gene of the hemoglobin is to compile The cDNA molecule or genomic DNA that code sequence is SEQ ID No.2 in sequence table.
3. the microbial inoculum of the metabolin containing recombination hillock monad described in claim 1 or/and the recombination hillock monad, The microbial inoculum has inhibitory activity to chain sclerotinia sclerotiorum.
4. following any applications of recombination hillock monad of any of claims 1 or 2 or microbial inoculum as claimed in claim 3:
A) recombination hillock monad of any of claims 1 or 2 or microbial inoculum as claimed in claim 3 are in preparation plant brown rot germ Application in inhibitor；
B) recombination hillock monad of any of claims 1 or 2 or microbial inoculum as claimed in claim 3 are inhibiting plant brown rot germ In application；
C) recombination hillock monad of any of claims 1 or 2 or microbial inoculum as claimed in claim 3 are in preparation plant brown rot suppression Application in preparation；
D) recombination hillock monad of any of claims 1 or 2 or microbial inoculum as claimed in claim 3 are in inhibiting plant brown rot Application.
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