CN105367637B - The regulation and control Wuyiencin producing strains growth albumen of phenotype and encoding gene thereof and application - Google Patents

Abstract

The invention discloses a kind of regulate and control the Wuyiencin producing strains growth albumen of phenotype and encoding gene thereof and application.Protein provided by the present invention, is following (a) or (b): (a) aminoacid sequence is the protein of sequence 1 in sequence table；(b) by the aminoacid sequence shown in sequence 1 through the replacement of one or several amino acid residue and/or disappearance and/or interpolation and the protein that by sequence 1 derives relevant to Wuyiencin biosynthesis.Suppress described protein expression, the growth rate of Wuyiencin producing strains can be accelerated, do not interfere with the total output of Wuyiencin simultaneously, it is thus achieved that the time of identical Wuyiencin total output is greatly shortened.Ensureing on the premise of Wuyiencin yield, not only saving the time, also a saving the consuming cost of culture medium and instrument and equipment.The present invention has important theoretical and practical significance for cultivating Wuyiencin high yield new strains.The present invention has broad application prospects in field of biological pesticide.

Description

The regulation and control Wuyiencin producing strains growth albumen of phenotype and encoding gene thereof and application

Technical field

The invention belongs to biological technical field, relate to a kind of regulate and control the Wuyiencin producing strains growth albumen of phenotype and volume thereof Code gene and application.

Background technology

Biological pesticide is that the major technique of diseases and pests of agronomic crop green prevention and control and agricultural product security supports.Wherein, antibiotic Series bactericidal agent is one of product that biological pesticide industrialized development is the fastest, has decomposition efficient, easy good with environment intermiscibility etc. excellent Select it is considered to be reduce or replace the major technique of chemical pesticide, be also the focus of plant pest Biological control research.

Streptomycete is the gram-positive bacterium that a class derives from soil, and it can produce abundant secondary metabolite, Can be widely applied to the aspect such as people, animal and agricultural antibiotic element, antifungal, the mankind are had important using value.Due to agriculture With antibiotic, there is noresidue, the advantage such as degradable, play an increasingly important role in agricultural production.Application is exhausted at present Most antibiotics is all the secondary metabolites of streptomycete, such as avilamycin, jingganmycin, kasugarnycin etc..Streptomycete is secondary The synthesis of metabolite is that one huge and the regulated and control network of complexity, is regulated and controled by biological synthesis gene cluster, and in gene cluster Comprise a lot of controlling gene.Therefore, for the route of synthesis of its metabolite clear and definite, the research of controlling gene function becomes closely The focus studied over Nian.Avilamycin biosynthetic positive regulating factor AveR, is come by the transcriptional expression of adjustment structure gene Affect the generation of avilamycin.Pimaricin as the Typical Representative of polyene macrolide antifungal agent, that tower of its producing strains The controlling gene pimM of streptomycete can regulate and control the generation of pimaricin, and this result is the controlling gene research of LuxR family protein Provide theoretical foundation.The functional study of regulatory factor CSRs in streptomyces coelicolor, also at the regulation and control net of streptomyces coelicolor Network research plays an important role.In sum, by the research of regulatory factor in streptomycete, can be specified it anti- Effect in raw element route of synthesis.This is genetic engineering breeding from now on, and directed screening cultivates new strains, has established good work Basis.

Wuyiencin is by S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus Var.wuyiensis) a kind of high-efficiency broad spectrum low toxicity novel agricultural antibiotic produced, is by Chinese Academy of Agricultural Sciences's plant protection A kind of biological pesticide with independent intellectual property right of Research Institute, has efficient, the feature of wide spectrum, low toxicity, in the ecological agriculture It is widely used with in pollution-free food production.Within 2000, win to obtain national five ministries and commissions " national key new product " issued Certification.Since two thousand one, it is put into vegetable insect disease safe prevention technical specification (national standard), it is intended that be used for for years The preventing and treating multiple diseases such as solanaceous vegetables, melon, Green leaf vegetable powdery mildew, gray mold, leaf mold.Within 2010, Wuyiencin product obtains Obtain national organic products certification (COFCC-R-0903-0070).At present, Wuyiencin has become as China pollution-free vegetable and has Machine vegetable prevents and treats the high-performance bio pesticide of fungal disease in producing.

Early stage is mainly screened Wuyiencin superior strain by traditional approach (physically or chemically mutation etc.) and obtains one The method of fixed achievement, such as protoplast fusion improves titer, uses nitrosoguanidine mutagenesis and the side of mental retardation carbon ion implatation Method mutation Wuyiencin producing strains, uses 60Co gamma-ray and mutagenesis Wuyiencin producing strains L7, and these methods are all based on tradition The method screening superior strain of breeding, but the shortcoming that there is non-directional back mutation due to traditional breeding method, waste time and energy, in order to Improve Wuyiencin production further, by the method for molecular biology, Wuyiencin producing strains can be carried out biological engineering and change Make, to obtaining the bacterial strain of high and stable yields.

Summary of the invention

It is an object of the invention to provide a kind of regulate and control the Wuyiencin producing strains growth albumen of phenotype and encoding gene thereof with Application.

Albumen provided by the present invention, entitled WysR3, derive from the mutation of S. ahygroscopicus Wuyi (Streptomyces ahygroscopicus var.wuyiensis) CK-15, concretely following (a) or (b):

A () aminoacid sequence is the protein of sequence 1 in sequence table；

(b) by shown in sequence 1 aminoacid sequence through one or several amino acid residue replacement and/or disappearance and/ Or add and the protein that by sequence 1 derives relevant to Wuyiencin biosynthesis.

Protein in above-mentioned (b) can synthetic, it is possible to first synthesize its encoding gene, then carries out biological expression and obtain. The encoding gene of the protein in above-mentioned (b) can be by lacking one or several in the DNA sequence shown in sequence in sequence table 2 The codon of amino acid residue, and/or carry out the missense mutation of one or several base pair.

Wherein, in sequence table, sequence 1 is made up of 253 amino acid residues.

The nucleic acid molecules encoding described WysR3 albumen falls within protection scope of the present invention.

Described nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；Described nucleic acid molecules can also be RNA, such as mRNA, hnRNA or tRNA etc..

In one embodiment of the invention, described nucleic acid molecules is that to encode the gene of described WysR3 albumen (named WysR3 gene)；Described wysR3 gene following 1) to 4) in arbitrary described DNA molecular:

1) DNA molecular shown in 20-781 position of sequence 2 in sequence table；

2) DNA molecular shown in sequence 2 in sequence table；

3) under strict conditions with 1) or 2) DNA molecule hybridize that limits and the DNA molecular encoding described WysR3 albumen；

4) with 1) or 2) or 3) DNA molecular that limits has more than 90% homology and encode the DNA of described WysR3 albumen Molecule.

Above-mentioned stringent condition can be the solution with 6 × SSC, 0.5%SDS, hybridizes at 65 DEG C, then with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

Wherein, sequence 2 is made up of 782 nucleotide, and 20-781 position is ORF, in polynucleotide shown in sequence 1 WysR3 albumen.

Recombinant vector, expression cassette or recombinant bacterium containing described nucleic acid molecules fall within protection scope of the present invention.

Described recombinant vector can be recombinant expression carrier, it is possible to for recombinant cloning vector.

In an embodiment of the present invention, described recombinant expression carrier starts the promoter tool of described wysR3 genetic transcription Body is P_SF14Promoter.

More specifically, described recombinant expression carrier is the multiple clone site (such as BamH I and Spe I) at pSETC carrier Between insert described wysR3 gene after the recombiant plasmid that obtains.

Described expression cassette by starting the promoter of described wysR3 gene expression, described wysR3 gene, and transcribing Terminator sequence forms.

In one embodiment of the invention, described recombinant bacterium specially carries the restructuring strepto-of described wysR3 gene Bacterium；Described streptomycete is specially S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis)CK-15。

Described WysR3 albumen, or described nucleic acid molecules, or described recombinant expression carrier, expression cassette or recombinant bacterium are in regulation and control Application in the strain growth phenotype of Wuyiencin producing strains falls within protection scope of the present invention.

Wherein, described strain growth phenotype is mainly reflected in strain growth speed.

The strain growth phenotype of described regulation and control Wuyiencin producing strains is embodied as: the content of described WysR3 albumen is more Height, the growth rate of the most described Wuyiencin producing strains is the slowest；The content of described WysR3 albumen is the lowest, the most described Wuyiencin The growth rate of producing strains is the fastest.

It is a further object to provide a kind of method obtaining transgenic strain.

The method of acquisition transgenic strain provided by the present invention, for as follows (A) or (B):

(A) acquisition has the method for the transgenic strain of at least one in following character, comprises the steps: to recipient bacterium Importing aminoacid sequence in strain is the encoding gene of the protein shown in sequence 1 in sequence table, obtains transgenic strain；Described turn Gene bacterial strain has character shown in following (a1) and/or (a2) compared with described F-strain:

(a1) growth rate slows down；

(a2) on the premise of not affecting Wuyiencin total output, extend Wuyiencin and produce the cycle；

(B) acquisition has the method for the transgenic strain of at least one in following character, comprises the steps: recipient bacterium Strain is in sequence table, the encoding gene of the protein shown in sequence 1 carries out suppression expression to aminoacid sequence, obtains transgenic Bacterial strain；Described transgenic strain has character shown in following (b1) and/or (b2) compared with described F-strain:

(b1) growth rate is accelerated；

(b2) on the premise of not affecting Wuyiencin total output, shorten Wuyiencin and produce the cycle；

In the method for described (A) and described (B), described F-strain is Wuyiencin producing strains.

In described (A), described WysR3 albumen expression in described transgenic strain is higher than described F-strain； In described (B), described WysR3 albumen expression in described transgenic strain is less than described F-strain.

In described method (B), described transgenic strain has compared with described F-strain " is not affecting Wuyiencin On the premise of total output, shorten Wuyiencin produce the cycle " character, be embodied as: compared with described F-strain, described The Wuyiencin total output of transgenic strain remains basically stable (no difference of science of statistics), but due to the growth of described transgenic strain Speed accelerates compared with described F-strain, and for comparing described F-strain, described transgenic strain produces identical total product Time used by the Wuyiencin of amount (i.e. Wuyiencin produces the cycle) shortens.

In described method (A), described transgenic strain has compared with described F-strain " is not affecting Wuyiencin On the premise of total output, extend Wuyiencin produce the cycle " character, be embodied as: compared with described F-strain, described The Wuyiencin total output of transgenic strain remains basically stable (no difference of science of statistics), but due to the growth of described transgenic strain Speed slows down compared with described F-strain, and for comparing described F-strain, described transgenic strain produces identical total product Time used by the Wuyiencin of amount (i.e. Wuyiencin produces the cycle) extends.

In the process, described encoding gene (i.e. wysR3 gene) is following 1) to 4) in arbitrary described DNA divide Son:

1) DNA molecular shown in 20-781 position of sequence 2 in sequence table；

2) DNA molecular shown in sequence 2 in sequence table；

3) under strict conditions with 1) or 2) DNA molecule hybridize that limits and the DNA molecular encoding described WysR3 albumen；

4) with 1) or 2) or 3) DNA molecular that limits has more than 90% homology and encode the DNA of described WysR3 albumen Molecule.

Above-mentioned stringent condition can be the solution with 6 × SSC, 0.5%SDS, hybridizes at 65 DEG C, then with 2 × SSC, 0.1%SDS and 1 × SSC, 0.1%SDS respectively wash film once.

In described method (A), described wysR3 gene specifically can be subject to described in the importing of the above recombinant expression carrier In body bacterial strain, obtain described transgenic strain.

In described method (B), described " is the egg shown in sequence 1 in sequence table to aminoacid sequence in F-strain The encoding gene of white matter carries out suppression expresses ", can be any to reduce the expression of encoding gene described in described F-strain Method.

In the present invention, described " is the protein shown in sequence 1 in sequence table to aminoacid sequence in F-strain Encoding gene carries out suppression expresses ", particular by real in F-strain as described in the DNA fragmentation shown in following formula (I) is proceeded to Existing:

SEQ_{Upstream homology arm}-X–SEQ_{Downstream homology arm} (I)

Described SEQ_{Upstream homology arm}It is the 1-590 position nucleotide of sequence 3 in sequence table；

Described SEQ_{Downstream homology arm}It is the 1353-1777 position nucleotide of sequence 3 in sequence table；

Described X antibiotic coding gene sequence (the specifically gentamycin encoding gene as shown in sequence 4 in sequence table Sequence).

In the present invention, the sequence 5 that the nucleotides sequence of the DNA fragmentation shown in described formula (I) is classified as in sequence table.

Sequence 5 is made up of 1882 nucleotide.Wherein, 1-590 position is described SEQ_{Upstream homology arm}, 597-1451 position is celebrating Big mycin coding gene sequence (i.e. sequence 4), 1458-1882 position is described SEQ_{Downstream homology arm}。

Further, the DNA fragmentation shown in described formula (I) is that the form by recombinant vector proceeds in described F-strain 's.Concrete, described recombinant vector is insertion sequence between the restriction enzyme site (such as Hind III and BamH I) of pKC1139 carrier The recombiant plasmid obtained after DNA fragmentation shown in sequence 5 in table.

In above-mentioned application or method, described Wuyiencin producing strains is the mutation of S. ahygroscopicus Wuyi further (Streptomyces ahygroscopicus var.wuyiensis), specially S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis)CK-15。

The transgenic strain being beneficial to the acquisition of described method falls within protection scope of the present invention.

It is demonstrated experimentally that suppression WysR3 albumen and the expression of encoding gene thereof, the growth of Wuyiencin producing strains can be accelerated Speed, does not interferes with the total output of Wuyiencin simultaneously, it is thus achieved that the time of identical Wuyiencin total output is greatly shortened.Ensureing On the premise of Wuyiencin yield, not only save the time, also a saving the consuming cost of culture medium and instrument and equipment.This Bright have important theoretical and practical significance for cultivation Wuyiencin high yield new strains.The present invention has in field of biological pesticide Wide application prospect.

Accompanying drawing explanation

Fig. 1 is the PCR amplification of wysR3 gene.

Fig. 2 is BamH I and the Spe I double digestion qualification result of recombinant expression carrier pSTEC-wysR3.

Fig. 3 is wysR3 gene overexpression bacterial strain, △ wysR3 deletion mutation strain and the PCR qualification result of complemented strain.Its In, A is wysR3 gene overexpression bacterial strain (swimming lane 1)；B is △ wysR3 deletion mutation strain；C is complemented strain, and M is molecular weight mark Standard, 1 is complemented strain, and 2 is original strain CK-15.

Fig. 4 is original strain CK-15, wysR3 gene overexpression bacterial strain, wysR3 gene deletion mutants and complemented strain Phenotypic growth to when speed of growth measurement result.A and B is original strain CK-15 and the table of wysR3 gene overexpression bacterial strain Type growth contrast；A is growth 24h bacterial strain, and B is bacterial strain after cultivation 96h；Left side is original strain CK-15, and right side is wysR3 base Because of process LAN bacterial strain (experiment 1).C is the phenotypic growth contrast of original strain CK-15 and wysR3 gene deletion mutants, for training Support bacterial strain after 96h；Left side is wysR3 gene deletion mutants, and right side is original strain CK-15 (experiment 1).D is original strain Phenotypic growth contrast (the experiment 2, D of CK-15, wysR3 gene overexpression bacterial strain, wysR3 gene deletion mutants and complemented strain A in figure represents that original strain CK-15, b represent wysR3 gene deletion mutants, and c represents that complemented strain, d represent wysR3 base Because of process LAN bacterial strain).E is original strain CK-15, wysR3 gene overexpression bacterial strain and the growth of wysR3 gene deletion mutants Velocity determination result.

Fig. 5 is original strain CK-15, wysR3 process LAN bacterial strain, wysR3 gene deletion mutants and complemented strain fermentation The HPLC testing result of liquid.A and B is experiment 1, and A is original strain CK-15, and B is wysR3 process LAN bacterial strain.C, D, E and F are real Testing 2, C is original strain CK-15, and D is wysR3 gene deletion mutants, and E is wysR3 process LAN bacterial strain, and F is complemented strain.

Fig. 6 is wysR3 process LAN bacterial strain, wysR3 gene deletion mutants, complemented strain and original strain CK-15 fermentation The liquid inhibitory action to rhodothece rubra.A is that rhodothece rubra is pressed down by wysR3 process LAN bacterial strain with original strain CK-15 fermentation liquid Making use, left side is original strain CK-15 inhibition zone, and right side is wysR3 process LAN bacterial strain inhibition zone (experiment 1).B is wysR3 Gene deletion mutants and the original strain CK-15 fermentation liquid inhibitory action to rhodothece rubra, left side is that original strain CK-15 presses down Bacterium circle, right side is wysR3 gene deletion mutants inhibition zone (experiment 1).C is wysR3 process LAN bacterial strain, wysR3 gene delection Mutant, complemented strain and the original strain CK-15 fermentation liquid inhibitory action to rhodothece rubra, WT represents original strain CK-15, △ wysRIII represents wysR3 gene deletion mutants, and comRIII represents that complemented strain, ooRIII represent wysR3 process LAN bacterium Strain (experiment 2).

Fig. 7 is glucose standard curve.

Detailed description of the invention

Experimental technique used in following embodiment if no special instructions, is conventional method.

Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.

S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK-15: It is recorded in " Wang Wanqun, Wuyiencin producing strains CK-15 bacterial artificial chromosome (BAC) library construction, 2008.6.1 " literary composition, The public can obtain from Plant Protection institute, Chinese Academy of Agricultral Sciences.

E.coli ET12567/pUZ8002: be recorded in " Kieser TBM, Buttner MJ, Chater KF, HopwoodDA(2000)Practical Streptomyces Genetics.The John Innes Foundation, Norwich " in a literary composition, the public can obtain from Plant Protection institute, Chinese Academy of Agricultral Sciences.

PKC1139 carrier: be recorded in " Bierman M, Logan R, O ' Brien K, Seno ET, Rao RN, SchonerBE(1992)Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli toStreptomyces spp.Gene 116:43 49 " in a literary composition, the public can be from Chinese agriculture section Institute's Plant Protection Institute obtains.PKC1139 is temperature-sensitive plasmid, with a temperature from s.ghanaensis Responsive type replicon, when cultivation temperature is more than 34 DEG C, this free plasmid can not self replication in streptomycete.

PSETC carrier: be recorded in " Keqiang Fan, Guohui Pan, Xiaojing Peng, Jianting Zheng, Wubin Gao,Juan Wang,Weishan Wang,Yue Li,and Keqian Yang.(2012)Identification ofJadG as the B Ring Opening Oxygenase Catalyzing the Oxidative C-C Bond CleavageReaction in Jadomycin Biosynthesis.Chemistry&Biology 19,1381 1390 " one Wen Zhong, the public can obtain from Plant Protection institute, Chinese Academy of Agricultral Sciences.

PBBR1MCS-5 carrier: be recorded in " Liu Yuying. the clone of denitrogenation key gene, qualification and efficient denitrification bacterial strain Structure. the Chinese Academy of Agricultural Sciences, Master's thesis in 2010 " in a literary composition, the public can grind from Chinese Academy of Agricultural Sciences's plant protection Study carefully and obtained.

PEASY-T1 simple carrier: Quan Shijin biological reagent company.

Rhodothece rubra AS2.282 (Rhodotorula rubra): Wuyiencin estimation of biological potency, with indicator bacteria, is recorded In " once flood plum forests moral Xin stone justice duckweed, agriculture anti-Wuyiencin producing strains Protoplast Mutation breeding. Chinese biological is prevented and treated, 1996 01 phase, 48-49. " in a literary composition, the public can obtain from Plant Protection institute, Chinese Academy of Agricultral Sciences.

Embodiment 1, the acquisition of wysR3 gene

With S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK- 15 is starting strain, by the positive monoclonal in screening CK-15 genome fosmid library, and checks order, obtains one section The sequence of about 50kb, utilizes the comparison of bioinformatic analysis and open reading frame, obtains a G/C content the highest, meets chain The gene of mycete gene expression characteristics, is wysR3 by this unnamed gene.The 20-of sequence 2 in the ORF sequence such as sequence table of wysR3 gene Shown in 781.

Extract S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) The genomic DNA of CK-15, with it as template, uses primer 1 and primer 2 to carry out PCR amplification.

Primer 1:5'-GGATCCGTAGCGCAGGAGGAACAC-3'(underscore part is the identification of restriction enzyme site BamH I Sequence, sequence thereafter is the 1-18 position of sequence 2)；

Primer 2: 5'-ACTAGTGTCAGACCCGGATCACC-3'(underscore part is the identification sequence of restriction enzyme site Spe I Row, sequence thereafter is the reverse complementary sequence of the 766-782 position of sequence 2).

After reaction terminates, pcr amplification product carrying out 1% agarose gel electrophoresis detection (Fig. 1), result shows through PCR Amplification obtains the purpose fragment that length is about 760bp.Reclaim and purified pcr product, check order.Result shows, this fragment A length of 794bp, nucleotides sequence be classified as "GGATCC+ sequence 2+ACTAGT", the 20-781 position of sequence 2 is wysR3 gene Coding region sequence (CDS), the protein shown in sequence 1 in polynucleotide, by named for this albumen WysR3.

Embodiment 2, wysR3 gene overexpression strain construction and qualification

One, the structure of recombinant expression carrier pSTEC-wysR3

With restricted enzyme BamH I and Spe I, the purified product of the pcr amplification product of embodiment 1 carried out double enzyme Cut, reclaim purpose fragment, with through as double digestion pSETC carrier framework large fragment be connected, obtain recombiant plasmid.By BamH I and Spe I double digestion identifies correct recombiant plasmid (Fig. 2 obtains two bands that size is about 6636bp and 794bp) sample presentation Order-checking.To show between restriction enzyme site BamH I and the Spe I of pSETC carrier in insertion sequence table shown in sequence 2 through order-checking The named pSTEC-wysR3 of plasmid after DNA fragmentation.

In recombinant expression carrier pSTEC-wysR3, the promoter of the wysR3 genetic transcription shown in initiating sequence 2 is P_SF14Promoter.

Two, the structure of wysR3 gene overexpression bacterial strain

Recombinant expression carrier pSTEC-wysR3 step one built proceeds to E.coli ET12567/ by electric shocking method PUZ8002, the named ETpKC-wysR3 of recombinant bacterium obtained.The method shifted by joint again, is imported ETpKC-wysR3 In S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK-15, specifically grasp See " Yang Zhenjuan, Sun Lei, Wu Zhe etc. the exploration of S. ahygroscopicus Wuyi mutation CK-15 genetic conversion system and just building. Biotechnology is circulated a notice of, 2012, (10): 193-198 " literary composition, as follows: to be taken as the S. ahygroscopicus Wuyi mutation into starting strain The ripe spore of (Streptomyces ahygroscopicus var.wuyiensis) CK-15 makes spore suspension, will weight Group bacterium ETpKC-wysR3 makes competence, is coated with MS flat board, cultivates 16 in 30 DEG C after mixing with CK-15 spore suspension equal-volume After hour, (pSETC carrier carries apramycin resistance to cover the apramycin containing the nalidixic acid of 25 μ g/ml and 50 μ g/ml Gene) 1ml sterilized water in, continue cultivate to grow joint transfer son, obtain wysR3 transgenic strain.

The comparison proceeding to pSETC empty carrier in starting strain CK-15, the named CK-of gained recombinant bacterial strain are set simultaneously 15/pSETC。

Three, the qualification of wysR3 gene overexpression bacterial strain

Single bacterium colony of the wysR3 transgenic strain that picking step 2 builds, carries out liquid culture, extracts the genome of bacterial strain DNA, with it as template, uses following primer to carry out PCR amplification, by PCR primer size and sequencing result to wysR3 transgenic Carry out Molecular Identification.Simultaneously using the S. ahygroscopicus Wuyi mutation (Streptomyces as starting strain Ahygroscopicus var.wuyiensis) CK-15 is as comparison.

The 1-19 position of primer 3:5'-GTGCAATACGAATGGCGAA-3'(sequence 6)；

The reverse complementary sequence of the 731-750 position of primer 4:5'-GCATTCTTCGCATCCCGCCT-3'(sequence 6).

In theory, in wysR3 transgenic strain, owing to recombinant expression carrier pSTEC-wysR3 there being apramycin resist Property gene, with its genomic DNA as template, uses above primer to carrying out PCR amplification, can obtain and apramycin resistance gene The amplified fragments (750bp, the 1-750 position of sequence 6) that sequence size is coincide；And as comparison starting strain CK-15 due to Do not contain apramycin resistance gene, thus use above primer to carrying out PCR amplification, purpose band will not be obtained.

The PCR primer of wysR3 transgenic strain and starting strain CK-15 is carried out agarose gel electrophoresis, and result shows, The PCR primer size of wysR3 transgenic strain (swimming lane 1) is about 750bp (as shown in A in Fig. 3), and starting strain CK-15 without Amplified band, consistent with expected results.Further, the PCR primer sample presentation of wysR3 transgenic strain is checked order, its nucleotides sequence Row are being just the 1-750 position of sequence 6.Visible, the wysR3 transgenic strain obtained by step 2 is positive.

Embodiment 3, the structure of wysR3 gene deletion mutants and qualification

One, the structure of homologous recombination plasmid pKC-WG

By S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK- The upstream and downstream fragment of the wysR3 gene in the genomic DNA of 15 expands out respectively, obtains two homology arms, then at two It is connected into resistant maker gene gentamycin gene (Gm) between homology arm, then inserts it in pKC1139 carrier framework, obtain For building the homologous recombination plasmid pKC-WG of wysR3 gene deletion mutants.Concrete operations are as follows:

1, for expanding the design of the primer of two homology arms

According to the mutation of S. ahygroscopicus Wuyi (Streptomyces ahygroscopicus var.wuyiensis) WysR3 gene upstream and downstream sequence (sequence 3) design primer in CK-15 genomic DNA, for two homology arms of amplification.

As follows for expanding the primer of upstream homology arm (being called for short ArmA):

ArmA-F:5'-AAGCTTGCTTCGTTGCCCTGTT-3'(underscore part is the recognition sequence of Hind III, its It is the 1-16 position of sequence 3 afterwards)；

ArmA-R:5'-TCTAGAGGTGTTCCTCCTGCG-3'(underscore part is the recognition sequence of Xba I, thereafter The reverse complementary sequence of 576-590 position for sequence 3).

As follows for expanding the primer of downstream homology arm (being called for short ArmB):

ArmB-F:5'-TCTAGACCCCCTACGGGCGTG-3'(underscore part is the recognition sequence of Xba I, is followed by The 1353-1367 position of sequence 3)；

ArmB-R:5'-GGATCCCCCCGACACCGTGCG-3'(underscore part is the recognition sequence of BamH I, thereafter The reverse complementary sequence of 1763-1777 position for sequence 3).

2, PCR amplification obtains two homology arms

With S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK- 15 genome fosmid library 6E11 plasmids are template, use primer that ArmA-F/ArmA-R is carried out PCR amplification, amplified production Size is about 590bp, and after order-checking, its nucleotides sequence is classified as the 1-590 position of sequence 3 in sequence table.

With S. ahygroscopicus Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK- 15 genomic DNAs are template, use primer that ArmB-F/ArmB-R carries out PCR amplification, and amplified production size is about 425bp, surveys After sequence, its nucleotides sequence is classified as the 1353-1777 position of sequence 3 in sequence table.

3, the structure of recombiant plasmid pEASY-W

With Hind III and Xba I double digestion upstream homology arm, reclaim digestion products, with through as the pEASY-of double digestion T1simple carrier framework large fragment is connected, and obtains intermediate carrier；With Xba I and BamH I double digestion downstream homology arm, reclaim Digestion products, with through as double digestion intermediate carrier skeleton large fragment be connected, obtain recombiant plasmid.To show through order-checking Insert between the Hind III and BamH I of pEASY-T1simple carrier " the 1-590 position of sequence 3+TCTAGAThe of+sequence 3 1353-1777 position " the named pEASY-W of recombiant plasmid.

4, the structure of recombiant plasmid pEASY-WG

With pBBR1MCS-5 carrier as template, primer Gm-F and Gm-R is used to carry out PCR amplification, it is thus achieved that resistant gene celebrating is big Mould plain gene (Gm).Wherein, the sequence of primer GM-F and GM-R is as follows:

GmS:5'-TCTAGAGACGCACACCGTGGAAA-3'(underscore part is the recognition sequence of Xba I, is followed by The 1-17 position of sequence 4)；

GmA:5'-TCTAGAGCGGCGTTGTGACAATTT-3'(underscore part is the recognition sequence of Xba I, is followed by The reverse complementary sequence of the 838-855 position of sequence 4)；

Sequencing will be carried out through above-mentioned PCR amplification products therefrom resistant gene gentamycin gene (Gm), its Nucleotides sequence be classified as "TCTAGA+ sequence 4+TCTAGA”。

By gained PCR primer Xba I single endonuclease digestion, reclaim digestion products, with through as the pEASY-of enzyme action T1simple carrier framework large fragment is connected, and obtains recombiant plasmid, by its named pEASY-TGM.

With Xba I single endonuclease digestion recombiant plasmid pEASY-TGM, with through as the skeleton of recombiant plasmid pEASY-W of enzyme action Large fragment forward is connected, and obtains recombiant plasmid pEASY-WG.

The structure of recombiant plasmid pEASY-WG is described as: between the Hind III and BamHI of pEASY-T1simple carrier The recombiant plasmid obtained after insertion sequence 5.

5, the structure of homologous recombination plasmid pKC-WG

With Hind III and BamH I double digestion recombiant plasmid pEASY-WG, reclaim the purpose fragment that size is about 1870bp, By its with through as the skeleton large fragment of pKC1139 carrier of double digestion be connected, obtain recombiant plasmid.Enzyme action is identified correct Plasmid sample presentation order-checking.Insertion sequence between restriction enzyme site the Hind III and BamH I of pKC1139 carrier will be shown through order-checking The named pKC-WG of plasmid after DNA fragmentation shown in sequence 5 in table.

Two, the structure of wysR3 gene deletion mutants

Homologous recombination plasmid pKC-WG step one built proceeds to E.coli ET12567/pUZ8002 by electric shocking method In, the named ETpKC-WG of recombinant bacterium obtained.The method shifted by joint again, imports S. ahygroscopicus by ETpKC-WG In Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK-15, concrete operations see " Yang Zhen Beautiful, Sun Lei, Wu Zhe etc. the exploration of S. ahygroscopicus Wuyi mutation CK-15 genetic conversion system and just building. biotechnology is led to Report, 2012, (10): 193-198 " literary composition, as follows: to be taken as the S. ahygroscopicus Wuyi mutation into starting strain The ripe spore of (Streptomyces ahygroscopicus var.wuyiensis) CK-15 makes spore suspension, will weight Group bacterium ETpKC-WG makes competence, is coated with MS flat board, cultivates 16 hours in 30 DEG C after mixing with CK-15 spore suspension equal-volume After, cover the nalidixic acid (suppression Escherichia coli Growth) containing 25 μ g/ml and apramycin (the pKC1139 carrier of 50 μ g/ml Carry apramycin resistance gene) and 50 μ g/ml gentamycin 1ml sterilized water in, continue to put 37 DEG C of incubator constant temperature trainings Support to growing joint transfer.The bacterium colony grown may be incorporated on streptomycete CK-15 chromosome for recombiant plasmid pKC-WG Recombinant bacterial strain, then picking list bacterium colony is respectively at the flat lining out of the MS containing gentamycin and apramycin, is chosen at celebrating the most mould Grow in element resistant panel, and the bacterial strain not grown in apramycin resistant panel, obtain wysR3 gene deletion mutants, By its named △ wysR3 deletion mutation strain.

Three, the qualification of wysR3 gene deletion mutants

Single bacterium colony of the △ wysR3 deletion mutation strain that picking step 2 builds, carries out liquid culture, extracts the gene of bacterial strain Group DNA, with it as template, is used following primer to carrying out PCR amplification, is lacked by PCR primer size and sequence pair △ wysR3 Mutant carries out Molecular Identification.Simultaneously using the S. ahygroscopicus Wuyi mutation (Streptomyces as starting strain Ahygroscopicus var.wuyiensis) CK-15 is as comparison.

The 573-589 position of forward primer VF:5'-TAGCGCAGGAGGAACAC-3'(sequence 5, the 573-of sequence 3 589)；

The 1522-1538 position of downstream primer VR:5'-TGGACGAGGAGGGCTAC-3'(sequence 5, the of sequence 3 The reverse complementary sequence of 1417-1433 position).

In theory, in △ wysR3 deletion mutation strain, instead of original genes of interest owing to celebrating big gene Gm (855bp) WysR3 (762bp, i.e. the 591-1352 position of sequence 3), respectively adds one in the upstream and downstream celebrating big gene Gm (855bp) simultaneously , there is mutant 105bp longer than the starting strain sequence (855-of homologous recombination double crossing in the recognition sequence (6bp) of individual Xba I 762+6+6=105).The clip size using above primer that mutant carries out PCR amplification should be 966bp (the of sequence 5 573-1538 position)；And the PCR primer size as the starting strain CK-15 of comparison should be the 861bp (573-1433 of sequence 3 Position).

The PCR primer of △ wysR3 deletion mutation strain and starting strain CK-15 is carried out agarose gel electrophoresis, and result shows Showing, the PCR primer size of △ wysR3 deletion mutation strain is about 966bp, and the PCR primer size of starting strain CK-15 is about 861bp (as shown in B in Fig. 3), consistent with expected results.Further, the PCR primer sample presentation of △ wysR3 deletion mutation strain is surveyed Sequence, its nucleotide sequence is being just the 573-1538 position of sequence 5；The PCR primer sample presentation of starting strain CK-15 is checked order, its core Nucleotide sequence is being just the 573-1433 position of sequence 3.Visible, the △ wysR3 deletion mutation strain obtained by step 2 is positive.

Embodiment 4, the structure of complemented strain of wysR3 gene deletion mutants and qualification

One, the structure of the complemented strain of wysR3 gene deletion mutants

The recombinant expression carrier pSTEC-wysR3 that embodiment 2 step builds is proceeded to by electric shocking method E.coliET12567/pUZ8002, the named ETpKC-wysR3 of recombinant bacterium obtained.The method shifted by joint again, will ETpKC-wysR3 imports in the △ wysR3 deletion mutation strain that embodiment 3 builds, and concrete operations see " Yang Zhenjuan, Sun Lei, Wu Zhe Deng the exploration of. S. ahygroscopicus Wuyi mutation CK-15 genetic conversion system and just to build. biotechnology is circulated a notice of, and 2012, (10): 193-198 " literary composition, obtain the complemented strain of wysR3 gene deletion mutants.

Two, the qualification of complemented strain

Single bacterium colony of the complemented strain that picking step one builds, carries out liquid culture, extracts the genomic DNA of bacterial strain, with It is template, uses following primer to carry out PCR amplification, by PCR primer size and sequencing result, complemented strain is carried out molecule Identify.Simultaneously using S. ahygroscopicus Wuyi mutation (the Streptomyces ahygroscopicus as starting strain Var.wuyiensis) CK-15 is as comparison.

The 1-19 position of primer 3:5'-GTGCAATACGAATGGCGAA-3'(sequence 6)；

The reverse complementary sequence of the 731-750 position of primer 4:5'-GCATTCTTCGCATCCCGCCT-3'(sequence 6).

In theory, in complemented strain, owing to recombinant expression carrier pSTEC-wysR3 having apramycin resistance gene, With its genomic DNA as template, use above primer to carrying out PCR amplification, can obtain big with apramycin resistance gene sequence Little identical amplified fragments (750bp, the 1-750 position of sequence 6)；And as the starting strain CK-15 compareed owing to not containing Apramycin resistance gene, thus use above primer to carrying out PCR amplification, purpose band will not be obtained.

The PCR primer of complemented strain and starting strain CK-15 is carried out agarose gel electrophoresis, and result shows, wysR3 turns The PCR primer size of gene bacterial strain (swimming lane 1) is about 750bp (as shown in C in Fig. 3), and starting strain CK-15 is without amplification bar Band, consistent with expected results.Further, being checked order by the PCR primer sample presentation of complemented strain, its nucleotide sequence is being just sequence 6 1-750 position.Visible, the complemented strain obtained by step one is positive.

The speed of growth mirror of embodiment 5, wysR3 gene overexpression bacterial strain, △ wysR3 deletion mutation strain and complemented strain Fixed

One, phenotypic evaluation

(1) experiment 1

WysR3 gene overexpression bacterial strain and △ wysR3 disappearance that embodiment 2 and 3 obtains is cultivated respectively in MS culture medium Mutant, is observed Strain phenotypes, including colonial morphology, the speed of growth, colony colour etc..S. ahygroscopicus Wuyi is set simultaneously Mutation (Streptomyces ahygroscopicus var.wuyiensis) CK-15 is as comparison.

Result shows, wysR3 gene overexpression strain growth speed is considerably slower than original strain CK-15 (A in Fig. 4), produces Spore amount declines, and some bacterial strains even do not produce the raw spore of Lycoperdon polymorphum Vitt gas, the only substrate mycelium (B in Fig. 4) of white.And △ wysR3 Deletion mutation strain growth rate is significantly faster than that original strain CK-15 (C in Fig. 4).These results suggest that wysR3 effect gene Wuyi The phenotypic growth of rhzomorph producing strains S.wuyiensis CK-15.

(2) experiment 2

WysR3 gene overexpression bacterial strain, the △ of embodiment 3 acquisition that embodiment 2 obtains is cultivated respectively in MS culture medium The complemented strain that wysR3 deletion mutation strain, embodiment 4 obtain, observes Strain phenotypes, including colonial morphology, the speed of growth, bacterium colony Color etc..Mutation (Streptomyces ahygroscopicus in S. ahygroscopicus Wuyi is set simultaneously Var.wuyiensis) CK-15 is as comparison.

Result is as shown in D in Fig. 4, and △ wysR3 deletion mutation strain relatively wild-type strain CK-15 mycelial growth is very fast, produces spore Amount increases and produces spore time advance, and growth 3-4d can complete to produce spore.The phenotype of complemented strain is recovered to corresponding wild type, shows This character mutation is not that polar effect causes, but caused by the disappearance of wysR3 gene.WysR3 gene overexpression bacterium Strain growth rate is slack-off, produces spore and is obstructed, and the most no longer produces the substrate mycelium of the raw spore of gas of Lycoperdon polymorphum Vitt, only white and sparse Aerial hyphae.The phenotypic growth of wysR3 effect gene Wuyiencin producing strains S.wuyiensis CK-15 is described, thus it is speculated that it can The regulation process of CK-15 Morphological Differentiation can be participated in.

Two, dry weight method measures

With embodiment 2 obtain wysR3 process LAN bacterial strain, embodiment 3 obtain △ wysR3 deletion mutation strain, to described △ wysR3 deletion mutation strain imports the complemented strain of gained after wysR3 gene, and not absorbing water as starting strain again Streptomycete Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK-15 is experiment material.

The each bacterial strain slant pore taking fresh mature is inoculated in 50mL M3G neat liquid culture medium (formula: every liter of culture medium In containing (NH₄)₂SO₄10g, glucose 50g, yeast powder 5.0g, KH₂PO₄1.36g, K₂PO₄·3H₂O 0.8g, MgSO₄· 7H₂00.5g, ZnSO₄·7H₂O 0.04g, FeSO₄·7H₂O 0.03g；PH 7.0) in, 28 DEG C, 220r/min cultivate 48h conduct Seed liquor.Each bacterial strain seed liquor is inoculated in 100mL M3G neat liquid culture medium respectively with 2% ratio that (identical spore connects Plant amount), 28 DEG C, 220r/min shaken cultivation.100ml is sampled every 6h, with drying to the filter paper filtering of constant weight in advance, will filter Paper is dried again to constant weight in being placed in 90 DEG C of baking ovens.Twice weight is subtracted each other and is mycelial dry weight in sampling fermentation liquid.

Result is as shown in E in Fig. 4, and as seen from the figure, wild-type strain is lag phase at 0-18h, and 18-36h is logarithm Trophophase, growth quantity of mycelium is most, and complemented strain is roughly the same with wild-type strain growth tendency.△ wysR3 deletion mutation Strain growth rate is accelerated, and i.e. enters exponential phase, relatively wild-type strain thalline raised growth time advance about 6h at 12-36h, And wysR3 process LAN strain growth is slow, being lag phase at 0-24h, 24h-48h just enters exponential phase, relatively wild-type bacteria Strain thalline raised growth time retardation about 6h.

The titer mirror of embodiment 6, wysR3 process LAN bacterial strain, △ wysR3 deletion mutation strain and complemented strain tunning Fixed

One, experiment 1

(1) fermentation culture of each wysR3 related strain

The wysR3 process LAN bacterial strain that obtains with embodiment 2, proceed to the control strain CK-15/pSETC of pSETC empty carrier, And S. ahygroscopicus Wuyi mutation (the Streptomyces ahygroscopicus as starting strain Var.wuyiensis) CK-15 is experiment material.

The slant pore taking each bacterial strain fresh mature is inoculated in shaken cultivation in 50mL seed culture medium (28 DEG C, 220r/ min)20h.By seed liquor with 10% (volume ratio) be inoculated in 100mL fermentation medium (each bacterial strain keep identical spore connect Plant amount), shaken cultivation (28 DEG C, 220r/min) 64h, fermentation liquid is collected by filtration.Fermentation liquid is centrifuged 10min in 6000r/min, takes Supernatant is again through the microfilter of 0.22 μm, it is thus achieved that aseptic ferment filtrate, and 4 DEG C save backup.

(2) in fermentation liquid, the HPLC of Wuyiencin content analyzes

High performance liquid chromatography (high-performance liquid chromatography, HPLC) is utilized to analyze each bacterium The Wuyiencin fermentation liquid of strain, instrument model is Agilend 1100.Come by the peak area size of Wuyiencin chromatogram Represent each bacterial strain Wuyiencin change of production.Chromatographic column is ZORBAX SB-AQ (4.6mm × 250mm, 5 μm)；Flowing is mutually 1.4g/L trichloroacetic acid solution, flow velocity 1mL/min；Column temperature 25 DEG C；Sample size 20 μ L；Detection wavelength 254nm.

1, standard curve making

Take Wuyiencin standard substance, with the solution of pure water preparation series concentration.By the Wuyiencin standard substance of series concentration Solution carries out high performance liquid chromatography detection according to as above condition.With the concentration of Wuyiencin standard solution as abscissa, with peak Area is vertical coordinate, makes standard curve.

2, the Wuyiencin assay in fermentation liquid to be measured

Each bacterial strain fermentation liquor of step (one) gained is carried out high performance liquid chromatography detection according to as above condition the most respectively, will The peak area of the chromatographic peak consistent with Wuyiencin standard substance retention time substitutes into step 1 gained standard curve, thus calculates The content of the Wuyiencin in fermentation liquid to be measured.Test in triplicate, results averaged.

3, result

Result shows, the appearance time of Wuyiencin standard substance is 18min, other each bacterial strains produce Wuyiencin go out peak Time is also 18min, and in original strain CK-15 fermentation liquid, Wuyiencin content is that 18.16mg/L, wysR3 process LAN bacterial strain is sent out In ferment liquid, Wuyiencin content is 19.36mg/L.The HPCL detection knot of original strain CK-15, wysR3 process LAN bacterial strain fermentation liquor Fruit is as shown in A and B in Fig. 5.WysR3 process LAN bacterial strain, proceed to the control strain CK-15/pSETC of pSETC empty carrier all with former In beginning bacterial strain CK-15 fermentation liquid, Wuyiencin content is basically identical, no difference of science of statistics.These results suggest that wysR3 gene exists Wuyiencin biosynthesis pathway does not play regulating and controlling effect.

Two, experiment 2

(1) fermentation culture of each wysR3 related strain

With embodiment 2 obtain wysR3 process LAN bacterial strain, embodiment 3 obtain △ wysR3 deletion mutation strain, embodiment 4 The complemented strain obtained, the control strain CK-15/pSETC proceeding to pSETC empty carrier, and not absorbing water as starting strain Streptomycete Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK-15 is experiment material.

The slant pore taking each bacterial strain fresh mature is inoculated in shaken cultivation in 50mL seed culture medium (28 DEG C, 220r/ min)20h.By seed liquor with 10% (volume ratio) be inoculated in 100mL fermentation medium (each bacterial strain keep identical spore connect Plant amount), shaken cultivation (28 DEG C, 220r/min) 64h, fermentation liquid is collected by filtration.Fermentation liquid is centrifuged 10min in 6000r/min, takes Supernatant is again through the microfilter of 0.22 μm, it is thus achieved that aseptic ferment filtrate, and 4 DEG C save backup.

(2) in fermentation liquid, the HPLC of Wuyiencin content analyzes

High performance liquid chromatography (high-performance liquid chromatography, HPLC) is utilized to analyze each bacterium The Wuyiencin fermentation liquid of strain, instrument model is LC-1260.Represent each by the peak area size of Wuyiencin chromatogram Bacterial strain Wuyiencin change of production.Chromatographic column is ZORBAX SB-AQ (4.6mm × 250mm, 5 μm)；Flowing is 1.4g/L tri-mutually Monoxone aqueous solution, flow velocity 1mL/min；Column temperature 25 DEG C；Sample size 5 μ L；Detection wavelength 254nm.

1, standard curve making

Take Wuyiencin standard substance, with the solution of pure water preparation series concentration.By the Wuyiencin standard substance of series concentration Solution carries out high performance liquid chromatography detection according to as above condition.With the concentration of Wuyiencin standard solution as abscissa, with peak Area is vertical coordinate, makes standard curve.

2, the Wuyiencin assay in fermentation liquid to be measured

Each bacterial strain fermentation liquor of step (one) gained is carried out high performance liquid chromatography detection according to as above condition the most respectively, will The peak area of the chromatographic peak consistent with Wuyiencin standard substance retention time substitutes into step 1 gained standard curve, thus calculates The content of the Wuyiencin in fermentation liquid to be measured.Test in triplicate, results averaged.

3, result

Result is as shown in C-F in Fig. 5, and the appearance time of Wuyiencin standard substance is about 20min, and other each bacterial strains produce force The appearance time of smooth rhzomorph is also 20min.Wild-type strain, △ wysR3 deletion mutation strain, wysR3 gene overexpression bacterial strain and Wuyiencin content corresponding in complemented strain fermentation liquid is respectively 1.193g/L, 1.236g/L, 1.18g/L and 1.213g/L, The Wuyiencin content that the most each bacterial strain is produced does not has significant change.Illustrating under this condition, wysR3 gene the most directly participates in The biosynthetic regulation and control of Wuyiencin, have not significant impact the yield of Wuyiencin.

Embodiment 7, wysR3 process LAN bacterial strain and the Antibacterial Activity of △ wysR3 deletion mutation strain

One, experiment 1

(1) wysR3 process LAN bacterial strain and the fermentation culture of △ wysR3 deletion mutation strain

With embodiment 2 obtain wysR3 process LAN bacterial strain, embodiment 3 obtain △ wysR3 deletion mutation strain, proceed to The control strain CK-15/pSETC of pSETC empty carrier, and the S. ahygroscopicus Wuyi mutation as starting strain (Streptomyces ahygroscopicus var.wuyiensis) CK-15 is experiment material.

The slant pore taking each bacterial strain fresh mature is inoculated in shaken cultivation in 50mL seed culture medium (28 DEG C, 220r/ min)20h.Seed liquor is inoculated in 100mL fermentation medium with 10% (volume ratio), shaken cultivation (28 DEG C, 220r/min) 64h, is collected by filtration fermentation liquid.Fermentation liquid is centrifuged 10min in 6000r/min, takes supernatant again through the microporous filter of 0.22 μm Device, it is thus achieved that aseptic ferment filtrate, 4 DEG C save backup.

(2) cup-plate method detection fermentation liquid bacteriostatic activity

After being filtered by each bacterial strain fermentation liquor, survey fermentation liquid bacteriostatic activity by cup-plate method.With can be by Wuyiencin Developing restraint Rhodothece rubra AS2.282 (Rhodotorula rubra) as indicator bacteria, thus detect the antibacterial work of each bacterial strain fermentation liquor Property.Concrete operations are as follows:

(1) rhodothece rubra AS2.282 is inoculated in PDA slant medium, 28 DEG C of constant temperature culture 5 days, treat on inclined-plane uniformly Cover with after one layer of bacterium with lawn under the aseptic washing of 50mL, make bacteria suspension standby；

(2) gained bacteria suspension is inoculated in the PDA culture medium being cooled to about 40 DEG C with 2% (volume ratio) inoculum concentration, mixed After even, this PDA culture medium containing rhodothece rubra AS2.282 is sucked in the sterile petri dish of a diameter of 9cm by every ware 12.5mL, At symmetrically placed 4 the Oxford cups of media surface after culture medium solidifying；

(3) filtered each bacterial strain fermentation liquor 300 μ L to be measured is joined Oxford cup, observe also after 28 DEG C of cultivation 24h Antibacterial circle diameter is measured with slide gauge.

Experiment is repeated 6 times, results averaged.

Result shows, each bacterial strain fermentation liquor inhibition zone does not all have notable difference with the inhibition zone size of original strain CK-15 (A and B in Fig. 6), utilizes DPS Mathematical Statistics Analysis software to carry out the standard variance analysis of completely randomized experiment design, and enters Row Duncan ' s multiple comparisons analyzes the antibacterial circle diameter of each bacterial strain fermentation liquor inhibition zone and original strain does not has significant difference The antibacterial circle diameter comparative result of wysR3 process LAN bacterial strain and original strain (table 1 be), this is consistent with HPLC analysis result 's.Illustrate not affect in wysR3 gene pairs Wuyiencin total output.

Table 1 wysR3 process LAN bacterial strain and the original strain CK-15 fermentation liquid bacteriostasis to rhodothece rubra

Note: after data, letter representation 0.01 level difference is notable.

Two, experiment 2

(1) wysR3 process LAN bacterial strain, △ wysR3 deletion mutation strain and the fermentation culture of complemented strain

With embodiment 2 obtain wysR3 process LAN bacterial strain, embodiment 3 obtain △ wysR3 deletion mutation strain, embodiment 4 The complemented strain obtained, the control strain CK-15/pSETC proceeding to pSETC empty carrier, and not absorbing water as starting strain Streptomycete Wuyi mutation (Streptomyces ahygroscopicus var.wuyiensis) CK-15 is experiment material.

The slant pore taking each bacterial strain fresh mature is inoculated in shaken cultivation in 50mL seed culture medium (28 DEG C, 220r/ min)48h.Seed liquor is inoculated in 100mL fermentation medium with 2% (volume ratio), shaken cultivation (28 DEG C, 220r/min) 72h, is collected by filtration fermentation liquid.Fermentation liquid is centrifuged 10min in 6000r/min, takes supernatant again through the microporous filter of 0.22 μm Device, it is thus achieved that aseptic ferment filtrate, 4 DEG C save backup.

(2) cup-plate method detection fermentation liquid bacteriostatic activity

After being filtered by each bacterial strain fermentation liquor, survey fermentation liquid bacteriostatic activity by cup-plate method.With can be by Wuyiencin Developing restraint Rhodothece rubra AS2.282 (Rhodotorula rubra) as indicator bacteria, thus detect the antibacterial work of each bacterial strain fermentation liquor Property.Concrete operations see step one (experiment 1).

Result shows: each bacterial strain fermentation liquor inhibition zone does not all have notable difference with the inhibition zone size of original strain CK-15 (C in Fig. 6), utilizes DPS Mathematical Statistics Analysis software to carry out the standard variance analysis of completely randomized experiment design, and carries out Duncan ' s multiple comparisons analyzes the antibacterial circle diameter of each bacterial strain fermentation liquor inhibition zone and original strain does not has significant difference (table 2 be wysR3 process LAN bacterial strain, △ wysR3 deletion mutation strain, the antibacterial circle diameter of complemented strain and original strain compare knot Really), this is consistent with HPLC analysis result.Illustrate not affect in wysR3 gene pairs Wuyiencin total output.

Table 2 wysR3 process LAN bacterial strain, △ wysR3 deletion mutation strain, complemented strain and original strain CK-15 fermentation The liquid bacteriostasis to rhodothece rubra

Note: after data, letter representation 0.01 level difference is notable.

The soluble sugar of embodiment 8, wysR3 process LAN bacterial strain and △ wysR3 deletion mutation strain tunning measures

One, wysR3 process LAN bacterial strain and the fermentation culture of △ wysR3 deletion mutation strain

With embodiment 2 obtain wysR3 process LAN bacterial strain, embodiment 3 obtain △ wysR3 deletion mutation strain, proceed to The control strain CK-15/pSETC of pSETC empty carrier, and the S. ahygroscopicus Wuyi mutation as starting strain (Streptomyces ahygroscopicus var.wuyiensis) CK-15 is experiment material.

The slant pore taking each bacterial strain fresh mature is inoculated in shaken cultivation in 50mL seed culture medium (28 DEG C, 220r/ min)20h.Seed liquor is inoculated in 100mL fermentation medium with 10% (volume ratio), shaken cultivation (28 DEG C, 220r/min) 64h, is collected by filtration fermentation liquid.Fermentation liquid is centrifuged 10min in 6000r/min, takes supernatant again through the microporous filter of 0.22 μm Device, it is thus achieved that aseptic ferment filtrate, 4 DEG C save backup.

Two, the soluble sugar of tunning is identified

1, the formulation of glucose standard curve

Take 6 25mL volumetric flask numberings, take 1.0mg/mL Glucose standards solution, add different reagent (table 3).After shaking up Boiling water bath heats 10min colour developing, is settled to the reverse mixing of 25mL after cold water cooling, exists with UV-75B type spectrophotometer 520nm wavelength surveys absorbance, with No. 0 pipe for comparison zeroing, determines the light absorption value of 1～No. 5 pipe.It is vertical with the light absorption value of mensuration Coordinate, is that abscissa draws glucose standard curve with glucose content (mg).

The series of values of table 3 Glucose standards solution

2, the assay method of total sugar in fermentation liquid

List of references " research [J] of Han Dequan, Zhang Jiajia .DNS method sugar determination in pullulan fermentation liquid. food Industrial technology, 2008,29 (2): 285-290. ", specifically comprise the following steps that

(1) hydrolysis of total sugar in fermentation liquid

Take fermentation filtrate 0.5mL to be placed in 50mL volumetric flask, add the hydrochloric acid solution 2mL of 6mol/L, in boiling water bath Heating 15min hydrolysis, adds the sodium hydroxide solution 1.8mL of 6mol/L, and is settled to 50mL after cold water cooling, obtain total sugar hydrolysis Liquid.

(2) mensuration of total sugar in fermentation liquid

Taking total sugar hydrolyzed solution 2mL in 25mL volumetric flask, add and heat 5min in DNS reagent 1.5mL boiling water bath, cold water is cold It is settled to 25mL the most afterwards shake up, compares with blank and survey light absorption value at 520nm wavelength.It is right with glucose light absorption value standard curve According to, calculate total sugar content in fermentation liquid.

3, result

Obtaining glucose standard curve (Fig. 7), regression equation Y=0.631X-0.028 according to determination data, in equation, Y is Light absorption value, X is glucose content, coefficient R²=0.998, illustrate that with glucose as a standard product are in 0.2～1mg/mL scope Inside can be used for the mensuration of sugar content.

The measurement result of each bacterial strain fermentation liquor shows, the fermentation liquid of original strain CK-15 contains with sugar in each bacterial strain fermentation liquor Amount does not has significant change.Table 4 is the measurement result of sugar content in wysR3 process LAN bacterial strain and original strain CK-15 fermentation liquid.With Upper result explanation wysR3 gene is not directly dependent upon with sugar content in fermentation liquid, does not affect the change of sugar content.

Total sugar content in table 4 fermentation liquid

Claims (12)

1. protein, be aminoacid sequence be the protein of sequence 1 in sequence table.

2. the nucleic acid molecules of protein described in coding claim 1.

Nucleic acid molecules the most according to claim 2, it is characterised in that: described nucleic acid molecules is described in coding claim 1 The gene of protein；Described gene is following 1) or 2) described in DNA molecular:

1) DNA molecular shown in 20-781 position of sequence 2 in sequence table；

2) DNA molecular shown in sequence 2 in sequence table.

4. contain the recombinant vector of nucleic acid molecules described in Claims 2 or 3.

5. contain the expression cassette of nucleic acid molecules described in Claims 2 or 3.

6. contain the recombinant bacterium of nucleic acid molecules described in Claims 2 or 3.

Recombinant vector the most according to claim 4, it is characterised in that: described recombinant vector is recombinant expression carrier or restructuring Cloning vehicle.

8. the protein described in claim 1 or the nucleic acid molecules described in Claims 2 or 3 or the weight described in claim 4 or 7 Expression cassette described in group carrier or claim 5 or the recombinant bacterium described in claim 6 are the bacterium regulating and controlling Wuyiencin producing strains Application in strain growth phenotype；

Described strain growth phenotype is presented as strain growth speed.

Application the most according to claim 8, it is characterised in that: described Wuyiencin producing strains is S. ahygroscopicus Wuyi Mutation (Streptomyces ahygroscopicus var.wuyiensis).

10. the method obtaining transgenic strain, has the side of the transgenic strain of at least one in following character for obtaining Method, comprises the steps: to be the encoding gene of the protein shown in sequence 1 in sequence table to aminoacid sequence in F-strain Carry out suppression to express, obtain transgenic strain；Described transgenic strain have compared with described F-strain following (b1) and/or (b2) character shown in:

(b1) growth rate is accelerated；

(b2) on the premise of not affecting Wuyiencin total output, shorten Wuyiencin and produce the cycle；

Described F-strain is Wuyiencin producing strains.

11. methods according to claim 10, it is characterised in that: described Wuyiencin producing strains is that S. ahygroscopicus is military Smooth mutation (Streptomyces ahygroscopicus var.wuyiensis).

12. utilize the transgenic strain that method described in claim 10 or 11 obtains.