CN105039383B - Inhibit construction method and the application of the recombination streptomyces lydicus of plant pathogenic fungi - Google Patents
Inhibit construction method and the application of the recombination streptomyces lydicus of plant pathogenic fungi Download PDFInfo
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Abstract
The invention discloses the construction method for the recombination streptomyces lydicus for inhibiting plant pathogenic fungi and applications.The method of structure recombination streptomyces lydicus provided by the present invention includes the step of importing the encoding gene of protein as recombination streptomyces lydicus in the streptomyces lydicus of host strain, is obtained；The protein is a) or b)：A) amino acid sequence is the protein of SEQ ID No.1 in sequence table；B) by the amino acid sequence of SEQ ID No.1 in sequence table by the substitution of one or several amino acid residues and/or the protein with the same function lacked and ored add.It is demonstrated experimentally that streptomyces lydicus (Streptomyces lydicus) the AB01CGMCC No.10707 and RFRP of the present invention can be used for inhibiting plant pathogenic fungi and prepare plant pathogenic fungi inhibitor.
Description
Technical field
The present invention relates in bioengineering field inhibit plant pathogenic fungi recombination streptomyces lydicus construction method with
Using.
Background technology
China is the large agricultural country based on planting industry, many great diseases, such as gray mold, anthracnose, tomato leaf mold
Disease, watermelon blight, wheat scab etc. cause heavy losses (Chen Jun etc., 2010) to agricultural production.Every year by using
Pesticide can reduce economic loss up to 30,000,000,000 yuan or so, but chemical prevention plays a major role, and chemical pesticide amount of application accounts for pesticide use
80% or more of amount causes serious environmental pollution, pesticide residue, resistance increase etc., seriously threatens health and the life of the mankind
State environment.Therefore, biological control is the inexorable trend of Agrochemicals, since microorganism and its metabolite have height single-minded
Property, insecticide efficiency is high, does not kill natural enemy, and is easy degradation, and noresidue is nontoxic to people and animals, can enhance the disease resistance of plant, stimulation
Plant growth, while the drug resistance that chemical pesticide is brought can be effectively relieved etc. (Huang Dinggao, 2010).Microbial pesticide in recent years
Popularization and application in production have produced huge economic benefit, social benefit and ecological benefits, at present by many states
Family is included in state key scientific research planning.
According to the difference of purposes and controlling object, microbe species for biological control also many samples, Streptomyces
It is widely used in Strategies of Agricultural Bio-control since secondary metabolite is abundant.But as Natural strains, the generation of single bacterial strain
It is limited to thank to product species, just will appear phenomena such as antimicrobial mechanism is single, and bacteriostasis is poor, and antimicrobial spectrum is narrow.
Invention content
The technical problem to be solved by the present invention is to how improve antibacterial activity of the streptomyces lydicus to plant pathogenic fungi.
In order to solve the above technical problems, present invention firstly provides the methods of structure recombination streptomyces lydicus.
The method of structure recombination streptomyces lydicus provided by the present invention, includes that the encoding gene of protein is imported conduct
In the streptomyces lydicus of host strain, the step of obtaining recombinating streptomyces lydicus；
By the RFRP that is named as of the protein, RFRP be it is following a) or b)：
A) amino acid sequence is the protein of SEQ ID No.1 in sequence table；
B) by the amino acid sequence of SEQ ID No.1 in sequence table by one or several amino acid residues substitution and/
Or the protein with the same function lacked and ored add.
In order to which the protein in making a) is convenient for purifying, can in sequence table protein shown in SEQ ID No.1 amino
End or the upper label as shown in Table 1 of carboxyl terminal connection.
The sequence of table 1, label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being usually 5) | RRRRR |
| Poly-His | 2-10 (being usually 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
It is above-mentioned b) in protein can be artificial synthesized, also can first synthesize its encoding gene, then carry out biological expression and obtain.
It is above-mentioned b) in the encoding gene of protein can be by by DNA sequence dna shown in the 14-1618 nucleotide of SEQ ID No.2
The codon of middle one or several amino acid residues of missing, and/or the missense mutation of one or several base-pairs of progress obtain.
In the above method, the encoding gene of the protein is following a1) or a2) or a3) or a4) shown in gene：
A1) nucleotide sequence is the cDNA molecule or DNA molecular of SEQ ID No.2 in sequence table；
A2) nucleotide sequence is the 14-1618 cDNA molecule or DNA molecular of SEQ ID No.2 in sequence table；
A3) and a1) or a2) nucleotide sequence that limits has 75% or 75% or more homogeneity, and coded amino acid sequence
It is classified as the cDNA molecules or genomic DNA molecule of the protein of SEQ ID No.1；
A4) under strict conditions with a1) or a2) limit nucleotide sequence hybridization, and encoding amino acid sequence be SEQ
The cDNA molecules or genomic DNA molecule of the protein of ID No.1.
Wherein, SEQ ID No.2 are made of 1626 nucleotide, and the 8-13 nucleotide of SEQ ID No.2 are Nde I
Recognition site, the protein of the 14-1618 nucleotide coding SEQ ID No.1 of SEQ ID No.2, SEQ ID No.2
1619-1624 nucleotide be EcoR I recognition site.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair
The nucleotide sequence of the protein of amino acid sequence composition shown in bright coding SEQ ID No.1 has 75% or higher, or
85% or higher 90% or higher 95% or higher homogeneity nucleotide sequence.Homogeneity can with the naked eye or calculate
Machine software is evaluated.Using computer software, the homogeneity between two or more sequences can use percentage (%) to indicate,
It can be used for evaluating the homogeneity between correlated series.
In the above method, the stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film
2 times, each 5min, and in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 15min.
Above-mentioned 75% or 75% or more homogeneity can be 80%, 85%, 90% or 95% or more homogeneity.
In the above method, the encoding gene of the protein is imported as being to include in the streptomyces lydicus of host strain
The expression vector of the encoding gene of the RFRP imports in the streptomyces lydicus as host strain.
In the above method, it includes following step that the encoding gene by protein, which is imported as the streptomyces lydicus of host strain,
Suddenly：The expression vector is imported in Escherichia coli and obtains recombination bacillus coli, then by the recombination bacillus coli and the work
Parents' engagement acquisition recombination streptomyces lydicus is carried out for the streptomyces lydicus of host strain.
In an embodiment of the present invention, the construction step of the expression vector includes：With the 14- of SEQ ID No.2
DNA molecular shown in 1618 nucleotide replaces what the DNA fragmentation between Nde I and EcoR the I identification sequences in pIB139 obtained
Recombinant vector pIB139-RFRP.In the pIB139-RFRP, the expression of RFRP genes is by red mould in the pIB139-RFRP
Plain promoter (ermE*) starts, protein shown in expression SEQ ID No.1.The pIB139-RFRP is comprising described
The expression vector of the encoding gene of RFRP.
In the above method, the streptomyces lydicus as host strain can be the profit that deposit number is CGMCC No.1653
Enlightening streptomycete (Streptomyces lydicus) A01.The Escherichia coli can be demethylation E.coliET12567
(pUZ8002)。
In order to solve the above technical problems, the present invention also provides recombination streptomyces lydicus.
Recombination streptomyces lydicus provided by the present invention, for the recombination streptomyces lydicus obtained by the method.
In above-mentioned recombination streptomyces lydicus, the recombination streptomyces lydicus can be the 14-1618 containing SEQ ID No.2
The recombination streptomyces lydicus of DNA fragmentation shown in the nucleotide of position.The recombination streptomyces lydicus concretely contains described
The recombination streptomyces lydicus of pIB139-RFRP.
In above-mentioned recombination streptomyces lydicus, the bacterial strain number of the recombination streptomyces lydicus is AB01, in China Microbiological
The number of registering on the books of culture presevation administration committee common micro-organisms center is CGMCC No.10707.
In order to solve the above technical problems, the present invention also provides the products of following H1 or H2：
H1、RFRP；
H2, with the relevant biomaterials of RFRP, be following A 1) to A20) any one of：
A1 the nucleic acid molecules of RFRP) are encoded；
A2) contain A1) expression cassettes of the nucleic acid molecules；
A3) contain A1) recombinant vectors of the nucleic acid molecules；
A4) contain A2) recombinant vector of the expression cassette；
A5) contain A1) recombinant microorganisms of the nucleic acid molecules；
A6) contain A2) recombinant microorganism of the expression cassette；
A7) contain A3) recombinant microorganism of the recombinant vector；
A8) contain A4) recombinant microorganism of the recombinant vector；
A9) contain A1) genetically modified plants of the nucleic acid molecules or animal cell line；
A10) contain A2) genetically modified plants of the expression cassette or animal cell line；
A11) contain A3) genetically modified plants of the recombinant vector or animal cell line；
A12) contain A4) genetically modified plants of the recombinant vector or animal cell line；
A13) contain A1) genetically modified plants of the nucleic acid molecules or animal tissue；
A14) contain A2) genetically modified plants of the expression cassette or animal tissue；
A15) contain A3) genetically modified plants of the recombinant vector or animal tissue；
A16) contain A4) genetically modified plants of the recombinant vector or animal tissue；
A17) contain A1) genetically modified plants of the nucleic acid molecules or animal organ；
A18) contain A2) genetically modified plants of the expression cassette or animal organ；
A19) contain A3) genetically modified plants of the recombinant vector or animal organ；
A20) contain A4) genetically modified plants of the recombinant vector or animal organ.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules also may be used
To be RNA, such as mRNA or hnRNA.
Those of ordinary skill in the art can easily adopt by known method, for example, orthogenesis and point mutation side
Method is mutated the nucleotide sequence of the coding RFRP of the present invention.Those have and are detached with the present invention by manually modified
The nucleotide sequence 75% of obtained RFRP or the nucleotide of higher homogeneity, as long as encoding RFRP and there is identical function,
It is the nucleotide sequence derived from the present invention and is equal to the sequence of the present invention.
Term " homogeneity " used herein refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this hair
The nucleotide sequence of the protein of amino acid sequence composition shown in bright coding SEQ ID No.1 has 75% or higher, or
85% or higher 90% or higher 95% or higher homogeneity nucleotide sequence.Homogeneity can with the naked eye or calculate
Machine software is evaluated.Using computer software, the homogeneity between two or more sequences can use percentage (%) to indicate,
It can be used for evaluating the homogeneity between correlated series.
In the said goods, the stringent condition is to hybridize at 68 DEG C in 2 × SSC, the solution of 0.1%SDS and wash film
2 times, each 5min, and in 0.5 × SSC, the solution of 0.1%SDS, hybridize at 68 DEG C and wash film 2 times, each 15min.
Above-mentioned 75% or 75% or more homogeneity can be 80%, 85%, 90% or 95% or more homogeneity.
In the said goods, the carrier can be plasmid, sticking grain, bacteriophage or viral vectors.
In the said goods, the microorganism can be yeast, bacterium, algae or fungi, such as Escherichia coli.The Escherichia coli can
For bacillus coli DH 5 alpha.
In the said goods, the transgenic plant cells system, Transgenic plant tissue, genetically modified plants organ, transgenosis
Animal cell line, transgenic animals tissue and transgenic animal organ do not include propagating materials.
In an embodiment of the invention, encoding gene (the i.e. 14-1618 cores of SEQ ID No.2 of RFRP
DNA molecular shown in thuja acid) it is imported in bacillus coli DH 5 alpha by the recombinant vector of the expression cassette of the encoding gene containing RFRP.
The recombinant vector replaces the Nde in pIB139 for DNA molecular shown in the 14-1618 nucleotide with SEQ ID No.2
The recombinant vector pIB139-RFRP that DNA fragmentation between I and EcoR I identification sequences obtains.In the pIB139-RFRP, RFRP
The expression of gene is started by the erythromycin promoter (ermE*) in the pIB139-RFRP, and expression SEQ ID No.1's is shown
Protein.
In the said goods, A1) nucleic acid molecules are the a1) or the a2) or the a3) or the a4) shown in
Gene.
In order to solve the above technical problems, the present invention also provides recombination streptomyces lydicus or following of its metabolin
One application：
1) application of the recombination streptomyces lydicus or its metabolin in inhibiting plant pathogenic fungi；
2) application of the recombination streptomyces lydicus or its metabolin in preparing plant pathogenic fungi inhibitor；
3) application of the recombination streptomyces lydicus or its metabolin in preparing fungal diseases of plants inhibitor；
4) application of the recombination streptomyces lydicus or its metabolin in inhibiting fungal diseases of plants；
5) application of the recombination streptomyces lydicus or its metabolin in lichenin of degrading.
In above application, the plant pathogenic fungi is botrytis cinerea (Botrytis cinerea)；
The fungal diseases of plants is the disease caused by botrytis cinerea (Botrytis cinerea).
In above application, the metabolin of the recombination streptomyces lydicus can be from the zymotic fluid of the recombination streptomyces lydicus
It obtains.The metabolin of the recombination streptomyces lydicus can be specifically prepared as follows, in liquid medium described in culture
Streptomyces lydicus is recombinated, the recombination streptomyces lydicus in liquid culture (zymotic fluid) is removed and obtains the sharp enlightening strepto- of the recombination
The metabolin of bacterium.
In order to solve the above technical problems, the present invention also provides following any applications：
M1) application of the product in inhibiting plant pathogenic fungi；
M2) application of the product in preparing plant pathogenic fungi inhibitor；
M3) application of the product in preparing fungal diseases of plants inhibitor；
M4) application of the product in inhibiting fungal diseases of plants；
M5) application of the product in lichenin of degrading.
In above application, the plant pathogenic fungi is botrytis cinerea (Botrytis cinerea)；
The fungal diseases of plants is the disease caused by botrytis cinerea (Botrytis cinerea).
It is demonstrated experimentally that streptomyces lydicus (Streptomyces lydicus) the AB01 CGMCC No.10707 of the application
There is the ability of degradation lichenin with RFRP：Growth has streptomyces lydicus (Streptomyces lydicus) AB01CGMCC
The lichenin screening flat board of No.10707 and streptomyces lydicus (Streptomyces lydicus) AG01 generate transparent circle,
And growing has the lichenin screening flat board of streptomyces lydicus (Streptomyces lydicus) A01 not have transparent circle appearance,
Growth has the lichenin screening flat board of streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707 to produce
Raw transparent circle is 1.3 times of streptomyces lydicus (Streptomyces lydicus) AG01；Growth has DH5 α-pIB139-
The lichenin screening flat board of RFRP generates transparent circle, and growing has the lichenin screening flat board of bacillus coli DH 5 alpha not saturating
It is bright to iris out now.
It is demonstrated experimentally that streptomyces lydicus (Streptomyces lydicus) the AB01 CGMCC No.10707 of the application
There is higher bacteriostatic activity with RFRP：Streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707
Antibacterial circle diameter to botrytis cinerea is 3.98cm ± 0.36cm, streptomyces lydicus (Streptomyces lydicus)
AG01 fungal diseases of plants inhibitor is 3.4cm ± 0.26cm, streptomyces lydicus to the antibacterial circle diameter of botrytis cinerea
(Streptomyces lydicus) A01 is 2.2cm ± 0.15cm to the antibacterial circle diameter of botrytis cinerea.Streptomyces lydicus
(Streptomyces lydicus) AB01 CGMCC No.10707 are sharp enlightening chain respectively to the bacteriostatic activity of botrytis cinerea
1.2 times of mould (Streptomyces lydicus) AG01 and streptomyces lydicus (Streptomyces lydicus) A01 and
1.8 again.
It is demonstrated experimentally that streptomyces lydicus (Streptomyces lydicus) the AB01 CGMCC No.10707 of the application
It can be used for inhibiting plant pathogenic fungi with RFRP and prepare plant pathogenic fungi inhibitor.
Biomaterial preservation explanation
The Classification And Nomenclature of biomaterial：Streptomyces lydicus (Streptomyces lydicus)
The strain number of biomaterial：AB01
Depositary institution's title of biomaterial：China Committee for Culture Collection of Microorganisms's common micro-organisms center
The depositary institution of biomaterial is referred to as：CGMCC
The depositary institution address of biomaterial：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3, Chinese Academy of Sciences microorganism
Research institute, postcode：100101
The preservation date of biomaterial：On April 10th, 2015
The collection of biomaterial is registered on the books number：CGMCC No.10707
Specific implementation mode
The present invention is further described in detail With reference to embodiment, 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.
In following embodiments streptomyces expression vector pIB139 (Christopher J.Wilkinson, et al.,
Increasing the Efficiency of Heterologous Promoters in Actinomycetes,
J.Mol.Microbiol.Biotechnol.(2002)4(4):417-426.) public can obtain from Beijing City Agriculture and Forestry Institute,
The biomaterial is only attached most importance to used in the related experiment of duplicate invention, not can be used as other purposes and is used.
In following embodiments demethylation E.coliET12567 (pUZ8002) (Paget MSB, Chamberlin L,
Atrih A,Foster SJ,Buttner MJ.Evidence that the extracytoplasmic function
sigma factor sigma(E)is required for normal cell wall structure in
Streptomyces coelicolor A3(2).J Bacteriol.(1999)181:204-211) public can be from Beijing's agriculture
The woods academy of sciences obtains, which only attaches most importance to used in the related experiment of duplicate invention, not can be used as other purposes and uses.
Botrytis cinerea (Botrytis cinerea) ((Sui Qin, Liu Weicheng, Qiu Jiyan, Liu in following embodiments
Thunderbolt, the stability plant protection of the antimicrobial spectrum and its bacteriostatic activity of the quick Streptomyces lydicus A02s of Pan Zhengyan, Liu Xue, 2007,33
(5) 67-71) public can obtain from Beijing City Agriculture and Forestry Institute, and which only attaches most importance to used in the related experiment of duplicate invention,
Other purposes are not can be used as to use.
The preparation method of Gause I slant medium in following embodiments is：Respectively by K2HPO4 0.5g、
NaCl0.5g、KNO3 1.0g、FeSO4·7H2O 0.01g、MgSO4·7H2O 0.5g, soluble starch 20g, agar 20g are dissolved in
800mL distilled water adjusts pH to 7.2-7.4, and distilled water is used in combination to be settled to 1000ml, 121 DEG C of sterilizings.
The preparation method of lichenin screening flat board in following embodiments is：By 5g lichenins, 2.5g Na2HPO4、
2.5g KH2PO4, 2.5g peptones and 0.5g yeast extracts be dissolved in distilled water, then plus water is settled to 1000ml, 121 DEG C of sterilizings.
The preparation method of seed culture medium in following embodiments is：(distilled water is added to boil 15g soya beans soya bean filtrate
0.5-1h, filtering, obtain soya bean filtrate), 5g peptones, 2.5g ammonium sulfate, 20g glucose, 10g starch, 0.25g magnesium sulfate,
0.2g potassium dihydrogen phosphates, 5g sodium chloride, are dissolved in distilled water respectively, are made into aqueous solution, after adjusting pH to 7-8, add 1g calcium carbonate,
Then plus water is settled to 1000ml, 121 DEG C of sterilizings.
The preparation method of fermentation medium in following embodiments is：(distilled water is added to boil 15g soya beans soya bean filtrate
0.5-1h, filtering, obtain soya bean filtrate), 5g peptones, 2.5g ammonium sulfate, 10g sucrose, 10g starch, 0.25g magnesium sulfate,
0.2g potassium dihydrogen phosphates and 5g sodium chloride are dissolved in distilled water respectively, are made into aqueous solution, after pH is adjusted to 7-8, add 1g carbonic acid
Calcium, then plus water is settled to 1000ml, 121 DEG C of sterilizings.
A01 in following embodiments is streptomyces lydicus (Streptomyces lydicus) A01 CGMCC
No.1653, the bacterial strain are public in the Chinese patent literature that Authorization Notice No. is CN 100590194C and in 2 months 2010 17
Day authorizes.
Paenibacillus polymyxa JZB120001CGMCC No.5563 in following embodiments are in application publication number
It is announced in the Chinese invention patent application of CN102851243A.
Streptomyces lydicus (Streptomyces lydicus) AG01 in following embodiments be document (Li et al.,
Expression of Paenibacillus polymyxa b-1,3-1,4-glucanase in Streptomyces
Lydicus A01improves its biocontrol effect against Botrytis cinerea, Biological
Control 90 (2015) 141-147) in S.lydicus AG01, S.lydicus AG01 be that will contain to come from more viscous classes
The recombinant vector of gene shown in 14-730 of the SEQ ID No.3 of bacillus JZB120001 CGMCC No.10708
PIB139-CONTROL imports the recombinant bacterium obtained in streptomyces lydicus (Streptomyces lydicus) A01.Recombinant vector
PIB139-CONTROL is with DNA fragmentation (the i.e. sequence between Nde I and EcoR the I recognition sites of SEQ ID No.3 in sequence table
DNA fragmentation shown in the 14-730 nucleotide of SEQ ID No.3 in list) replace pIB139 in Nde I and EcoR I
Other sequences of DNA fragmentation between recognition site, pIB139 are constant, obtained recombinant vector.
Embodiment 1, structure recombination streptomyces lydicus
Present embodiments provide from Paenibacillus polymyxa JZB120001 CGMCC No.5563 protein and its
Encoding gene, by the protein, it is named as RFRP.In the amino acid sequence of RFRP such as sequence table shown in SEQ ID No.1,
In the sequence of encoding gene such as sequence table shown in the 14-1618 nucleotide of SEQ ID No.2.
1, the structure of recombinant vector
With DNA fragmentation (the i.e. SEQ in sequence table between Nde I and EcoR the I recognition sites of SEQ ID No.2 in sequence table
DNA fragmentation shown in the 14-1618 nucleotide of ID No.2) replace pIB139 in Nde I and EcoR I recognition sites between
DNA fragmentation, other sequences of pIB139 are constant, obtain the recombinant vector containing RFRP genes, which is named as
pIB139-RFRP.In pIB139-RFRP, the expression of RFRP genes is opened by the erythromycin promoter (ermE*) in the recombinant vector
It is dynamic, the RFRP of expression SEQ ID No.1.
2, the structure of streptomyces lydicus is recombinated
The structure of 2.1 recombination bacillus colis
Recombinant vector pIB139-RFRP is converted into demethylation E.coliET12567 (pUZ8002) by heat shock method and is passed through
The screening of 100 μ g/ml apramycin resistances is crossed, the recombinant bacterium E.coliET12567 for importing recombinant vector pIB139-RFRP is obtained
(pUZ8002)/pIB139-RFRP。
3.2 amphiphilic engagement build streptomyces lydicus (Streptomyces lydicus) AB01
Bibliography (Bierman M et al.1992) carries out amphiphilic engagement, and the specific method is as follows：
(1) by recombinant bacterium E.coliET12567 (pUZ8002)/pIB139-RFRP is inoculated into containing chloramphenicol, to block that mould
In the LB liquid medium of element and apramycin resistance, 37 DEG C, 200rpm, shake culture is overnight, and the next morning connects by 1%
Kind amount, which is connected in fresh LB, cultivates to OD600=0.4-0.6 collects bacterium solution, under 4 DEG C, 4000rpm, centrifuges 2min,
Supernatant is abandoned, precipitation is resuspended with the ice-cold pure LB liquid mediums of 20mL, under 4 DEG C, 4000rpm, centrifuges 2min, abandons supernatant,
Precipitation is resuspended with the ice-cold pure LB culture solutions of 2mL, obtains recombinant bacterium E.coliET12567 (pUZ8002)/pIB139-RFRP
Bacterium solution.
(2) 2 weeks streptomyces lydicus (Streptomyces lydicus) A01 spores will be grown on the inclined-planes PDA with sterile
Distilled water elutes, and is used in combination sample injector piping and druming uniform, obtains spore suspension.500 μ L 2 are added into 250 μ L spore suspensions
× YT culture solutions, gently after mixing, the thermal shock 10min at 50 DEG C, the streptomyces lydicus A01 spore suspensions activated.
(3) by above-mentioned 500 μ L recombinant bacteriums E.coliET12567 (pUZ8002)/pIB139-vhb-RFRP bacterium solutions and 500 μ L
The streptomyces lydicus A01 spore suspensions of activation gently mixing, and under 4000rpm, room temperature centrifuges 3min, removes supernatant, will precipitate
MgCl containing 10mM/L is coated on after mixing2MS culture mediums on, at 28 DEG C be inverted culture 18h, apply antibiosis on the tablet later
0.5mg nalidixic acids and 60 μ g apramycins (are dissolved in 1mL ddH by plain solution2The liquid obtained in O).Picking after 2-3 days
Single bacterium falls on new be coated on the MS tablets of above-mentioned antibiotic solution.Bacterium colony of the picking on the MS tablets carries out PCR screenings, will
The obtained recombinant bacterium containing DNA fragmentation shown in the 14-1618 nucleotide of SEQ ID No.2 in ordered list is named as profit
Enlightening streptomycete (Streptomyces lydicus) AB01.Streptomyces lydicus (Streptomyces lydicus) AB01 in
Be preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on April 10th, 2015, collection register into
Volume number：CGMCC No.10707, the bacterium are hereinafter referred to as AB01.
The morphological feature of AB01 thalline is as follows：Gram-positive；It (is not added in the YEME agar mediums for being not added with sucrose
The YEME agar mediums of sucrose are made of water, malt extract, peptone, yeast powder, glucose and agar, and wherein malt is taken out
The content of extract is 0.3g/100mL, and the content of peptone is 0.5g/100mL, and the content of yeast powder is 0.3g/100mL, grape
The content of sugar is 4g/100mL, and the content of agar is 1.5g/100mL) on grow 7 days after, substrate mycelium physically well develops, no cross
Every not broken；Aerial hyphae well-grown, multi-branched；Fibrillae of spores is flexible or bending, spore are oval.It is screened in lichenin
Apparent haloing can be generated on tablet.
The degradation lichens of embodiment 2, streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707
The ability of polysaccharide
In triplicate, the result for repeating experiment every time is as follows for experiment：
By streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707 of embodiment 1, sharp enlightening chain
Mould (Streptomyces lydicus) AG01 and streptomyces lydicus (Streptomyces lydicus) A01 are inoculated into respectively
It in lichenin screening flat board, cultivates 2 days, carries out congo red staining experiment.The result shows that growth has streptomyces lydicus
(Streptomyces lydicus) AB01 CGMCC No.10707 and streptomyces lydicus (Streptomyces lydicus)
The lichenin screening flat board of AG01 generates transparent circle, and growing has streptomyces lydicus (Streptomyces lydicus)
The lichenin screening flat board of A01 does not have transparent circle appearance, growth to have streptomyces lydicus (Streptomyces lydicus)
The transparent circle that the lichenin screening flat board of AB01 CGMCC No.10707 generates is streptomyces lydicus (Streptomyces
Lydicus) 1.3 times of AG01 show streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707's
Metabolin has the ability of degradation lichenin.
The pIB139-RFRP of 1 step 1 of embodiment is imported into bacillus coli DH 5 alpha (Tiangeng biochemical technology (Beijing) limited public affairs
Department, CB101-01), the recombinant bacterium containing pIB139-RFRP is obtained, DH5 α-pIB139-RFRP are named as.By DH5 α-
PIB139-RFRP and bacillus coli DH 5 alpha are inoculated into respectively in lichenin screening flat board, are cultivated 2 days, and congo red staining is carried out
Experiment.The result shows that growth has the lichenin screening flat board of DH5 α-pIB139-RFRP to generate transparent circle, and growing has large intestine
The lichenin screening flat board of bacillus DH5 α does not have transparent circle appearance, shows that the metabolin of DH5 α-pIB139-RFRP has degradation
The ability of lichenin.
The bacteriostatic activity of embodiment 3, streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707
1, the preparation of fungal diseases of plants inhibitor
Streptomyces lydicus (Streptomyces lydicus) the AB01 CGMCC No.10707 of embodiment 1 are inoculated into
On Gause I slant medium, 28 DEG C are cultivated 7-10 days, wait for that it generates enough spores, its spore is scraped with sterile platinum loop
2-3 rings are inoculated in the 50ml seed culture mediums in 250ml triangular flasks, are set on controllable temperature shaking table, under the conditions of 28 DEG C,
200rpm/min (radius of turn 13mm) constant-temperature shaking culture -30h for 24 hours；Then it is aseptically tapped in 10
(being 100ml per bottled liquid measure), OD after every bottle of inoculation in fermentation medium in 500ml triangular flasks600Value is all 0.1；After inoculation
Shaking flask under the conditions of 31 DEG C, with the speed oscillation culture 96h of 240rpm/min (radius of turn 13mm), obtain AB01 fermentations
Liquid is generated containing streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707 in the AB01 zymotic fluids
High concentration bacteriostatic activity metabolite.By AB01 zymotic fluids 5000rpm/min centrifugation 10-15min sedimentation mycelium and admittedly
Shape object abandons precipitation, and by 0.45 μm of sterile filtering with microporous membrane of obtained supernatant, it is (sterile in the filtrate to collect filtrate
Body), which is AB01 fungal diseases of plants inhibitor, and it is spare to set 4 DEG C of storages.
According to the method described above, by the streptomyces lydicus of embodiment 1 (Streptomyces lydicus) AB01 CGMCC
No.10707 replaces with streptomyces lydicus (Streptomyces lydicus) A01, other steps are constant, obtain A01 plants
Fungal disease inhibitor.
According to the method described above, by the streptomyces lydicus of embodiment 1 (Streptomyces lydicus) AB01 CGMCC
No.10707 replaces with streptomyces lydicus (Streptomyces lydicus) AG01, other steps are constant, obtains AG01 plants
Object fungal disease inhibitor.
2, the bacteriostatic activity of streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707
It is botrytis cinerea (Botrytis cinerea) for examination target pathogens, experiment in triplicate, repeats every time
Experiment is as follows：
The conidium that the botrytis cinerea (Botrytis cinerea) of generation is cultivated on scraping PDA plate, with nothing
Bacteria suspension is made in bacterium water, is equably applied on freshly prepd PDA plate, sets drying in superclean bench and obtains botrytis cinerea
Tablet；Punched in tablet symmetric position with the aseptic card punch of diameter 7mm, then respectively in every hole injection step 1 AB01
Fungal diseases of plants inhibitor, AG01 fungal diseases of plants inhibitor and each 50 μ l of A01 fungal diseases of plants inhibitor, not connect
The botrytis cinerea tablet of kind streptomyces lydicus is control, is cultivated 3 days at 28 DEG C, and crossing method measures the straight of inhibition zone
Diameter.Each three repetitions of processing.
The experimental results showed that AB01 fungal diseases of plants inhibitor, AG01 fungal diseases of plants inhibitor and A01 plants are true
Fungus diseases inhibitor can generate botrytis cinerea (Botrytis cinerea) on PDA plate apparent antibacterial
Circle, AB01 fungal diseases of plants inhibitor, AG01 fungal diseases of plants inhibitor and A01 fungal diseases of plants inhibitor are to tomato
Ash arrhizus bacteria (Botrytis cinerea) antibacterial circle diameter is respectively 3.98cm ± 0.36cm, 3.4cm ± 0.26cm, 2.2cm
The bacteriostatic activity of ± 0.15cm, streptomyces lydicus (Streptomyces lydicus) AB01 CGMCC No.10707 is respectively
Streptomyces lydicus (Streptomyces lydicus) AG01's and streptomyces lydicus (Streptomyces lydicus) A01
1.2 times and 1.8 times.
Claims (5)
1. the method for structure recombination streptomyces lydicus includes the sharp enlightening strepto- imported the encoding gene of protein as host strain
In bacterium, the step of obtaining recombinating streptomyces lydicus；
The protein is the protein that amino acid sequence is SEQ ID No.1 in sequence table；
The streptomyces lydicus as host strain is the streptomyces lydicus that deposit number is CGMCC No.1653
(Streptomyces lydicus)A01。
2. according to the method described in claim 1, it is characterized in that：The encoding gene of the protein is following a1) or a2) institute
The gene shown：
A1) nucleotide sequence is the DNA molecular of SEQ ID No.2 in sequence table；
A2) nucleotide sequence is the 14-1618 DNA moleculars of SEQ ID No.2 in sequence table.
3. the recombination streptomyces lydicus obtained by claims 1 or 2 the method.
4. recombination streptomyces lydicus according to claim 3, it is characterised in that：The bacterial strain number of the recombination streptomyces lydicus
It is CGMCC in the number of registering on the books of China Committee for Culture Collection of Microorganisms's common micro-organisms center for AB01
No.10707。
5. the following any applications of recombination streptomyces lydicus described in claim 3 or 4：
1) the recombination streptomyces lydicus inhibit botrytis cinerea (Botrytis cinerea) in application；
2) the recombination streptomyces lydicus prepare plant botrytis cinerea (Botrytis cinerea) answering in inhibitor
With；
3) application of the recombination streptomyces lydicus in preparing fungal diseases of plants inhibitor, the fungal diseases of plants are served as reasons
Botrytis cinerea (Botrytis cinerea) caused by disease；
4) application of the recombination streptomyces lydicus in inhibiting fungal diseases of plants, the fungal diseases of plants are by tomato ash
Mildew bacterium (Botrytis cinerea) caused by disease.
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