CN104988081A - Saccharopolyspora spinosa recombinant strain with double bldD genes - Google Patents
Saccharopolyspora spinosa recombinant strain with double bldD genes Download PDFInfo
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Abstract
The invention discloses a saccharopolyspora spinosa recombinant strain with double bldD genes. The provided saccharopolyspora spinosa recombinant strain with double bldD genes is prepared by integrating bldD gene into saccharopolyspora spinosa genome. The provided saccharopolyspora spinosa recombinant strain with double bldD genes is named as S.spinosa-BldD, and is preserved in the China Center for Type Culture Collection (CCTCC) in May 31st, 2015, and the preservation number is CCTCC No. M2015347. The PCR detection results show that bldD gene has been successfully integrated into a target bacterial strain chromosome. In the plate culture, people find that in BHI and TSB plates, the spores of recombinant strains are prominently inhibited. The results of shaking flask fermentation show that the spinosad yield of recombinant strain is increased by 1.35 times compared with that of a referential strain.
Description
Technical field
The present invention relates to gene recombination, being specifically related to by increasing in thorn saccharopolyspora strain
bldDgene copy number, obtains pleocidin superior strain
s. spinosa-BldD.
Background technology
Ecological environmental protection and food-safety problem are the focuses that society is paid close attention to, and have the use of chemical residue agricultural chemicals to cause pollution of agricultural products, environmental destruction, the negative impact brought for the mankind can not be ignored.The exploitation of environmental type biotic pesticide is inexorable trends of agricultural chemicals industry development.Thorn saccharopolyspora strain secondary metabolite pleocidin insecticidal spectrum is wide, insecticidal mechanism is unique, have splendid killing effect to multiple kinds of crops insects such as Coleoptera, lepidopteran, Homoptera, Thysanopteras, its safety, fast, characteristic of low residue makes it to become one of novel biopesticide most with prospects.But the ability of wild-type thorn saccharopolyspora strain synthesis pleocidin is relatively weak and fermentation period is long, how to shorten fermentation period, improves the hot issue that pleocidin output becomes scientific research.Pleocidin output can be improved preferably by traditional method improvement bacterial classifications such as medium optimization and physics and chemistry mutagenesis, protoplast fusions.And utilize genetic engineering means to carry out to thorn saccharopolyspora strain the advantage that genetic modification has directional transformation on a molecular scale, also can yet be regarded as and improve the desirable approach of pleocidin output.
bldgene is the typical regulatory factor of overall importance of the one in streptomycete, and in type strain streptomyces coelicolor, have material impact to the Morphological Differentiation of cell and the synthesis of secondary metabolite, its gene family member comprises
bldA,
bldD,
bldH,
bldN,
bldKdeng.In streptomyces coelicolor,
bldDgenes encoding contains the small-molecular-weight DBP of 167 amino-acid residues, and molecular weight is about 18 kDa.Generally, BldD, as transcription inhibitory factor, can not only be combined with the promoter region of self and check oneself expression, Hai Nengyu
bldN,
sigH,
whiGdeng promotor combine, affect the expression of the growth period important sigma factor, thus regulates and controls streptomycete sporulation and antibiotic generation.The research such as Chng shows,
bldDgene is positioned at
erythe upstream of gene cluster, the conduct of BldD albumen
erya common transcription regulaton factor of gene cluster, can with whole
erythe promoter region of gene cluster combines, thus promotes
erythe expression of gene cluster.
bldDthe red saccharopolyspora mutant strain of genetically deficient, its yield of erythrocin significantly declines and is only 1/7 of original bacteria, and the Morphological Differentiation of bacterial strain is also subject to obvious suppression.Overexpression baldness type gene in red saccharopolyspora
bldD, the output of erythromycin can be significantly improved.
At present, the method that genetic modification mainly adopts protoplast transformation and conjugal transfer is carried out to thorn saccharopolyspora strain, but the two efficiency is all very low, exogenous origin gene integrator is existed certain difficulty to thorn saccharopolyspora strain genome.
Summary of the invention
The technical problem to be solved in the present invention is, overcomes the deficiencies in the prior art, provides a kind of
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, is intended to applying gene recombinant technology, will
bldDgene integration, to thorn saccharopolyspora strain genome, increases in thorn saccharopolyspora strain to passing through
bldDthe copy number of gene, realizes
bldDthe overexpression of gene, promotes the biosynthesizing of pleocidin.
The technical scheme that the present invention solves the employing of its technical problem is, a strain
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, described in
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, by baldness type gene
bldDbe incorporated into thorn saccharopolyspora strain genome to produce.
The present invention it
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, namely stings saccharopolyspora strain
s. spinosa-BldD(Saccharopolyspora spinosa
s. spinosa-BldD) on May 31st, 2015 in China typical culture collection center (be called for short CCTCC, address: Wuhan, China Wuhan University) preservation, culture presevation number is CCTCC NO:M 2015347.
(1) described baldness type gene
bldD,clone is from stinging saccharopolyspora strain, and its sequence is as follows:
atgggcgact acgccaaggc gctgggcagc aagctccgcg ctatccgcca gcagcagggt 60
ctgtcgctgca cggcgtcgag cagaagtctg gcgggcggtg gaaggccgtg gtcgtcgggt 120
cctatgagcga ggcgaccgtg cggtgaccgt gcagaagctg gccgaactgg ccgacttcta 180
cggggttccg gtcgcggaac tgcttcccga gggtcgggtg ccgtccggcg ccgagcccgc 240
caccaaagtt gtgatcaacct ggagcggct gcagcagcta cctgcggaga aggtgggccc 300
gctggcccgc tacgcggccc catccagagc cagcggggtg actacaacgg caaggtgctg 360
tccatccgca ccgaggacct gcgatccctg gccatcatct acgacatgac gccaggtgag 420
ctcaccgagc agctcatcga ctggggcgtg cttcccccgg aggcccgccc agcccgggag 480
gagtga 486
(2) construction process of described recombinant bacterial strain:
(1) overlapping pcr is adopted, will
bldDgene is placed in erythromycin strong promoter
p ermE control under, subclone, to pUC-spn carrier (preservation of this laboratory), obtains recombinant vectors pUC-spn-P
ermE-bldD;
(2) by step (1) gained recombinant vectors pUC-spn-P
ermE-bldD is electroporated
e. colis17, obtains transformant
e. colis17 (pUC-spn-P
ermE-bldD);
(3) by transformant (donor bacterium)
e. colis17 (pUC-spn-P
ermE-bldD) sting saccharopolyspora strain SP06081 Conjugal transfer with recipient bacterium, obtain recombinant bacterial strain, called after
s. spinosa-BldD.
(3) recombinant bacterial strain
s. spinosa-BldDpCR qualification.
(4)
bldDthe impact that process LAN grows on thorn saccharopolyspora strain.
(5)
bldDprocess LAN is on the impact of thorn saccharopolyspora strain pleocidin output.
(6) the genetic stability analysis of recombinant bacterial strain.
The present invention is by increasing regulatory gene of overall importance in thorn saccharopolyspora strain
bldDcopy number, build
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication
s. spinosa-BldD.
The present invention for template, designs pair of primers cm-reg-1/cm-ermE-reg-2 with plasmid pIJ86-cm:
cm-reg-1: gacgtcccgaaggcgtggcgcggcttccccgtgccggagcaatcgccctgATCTAAAGGAAGCGGATGT GAC；
cm-ermE-reg-2: GGATCCTACCAACCGGCACGAT
Wherein there is phiC31 attP site upstream homology arm in the 5' end band of cm-reg-1, amplification chloramphenicol resistance gene and erythromycin strong promoter gene fragment cm-P
ermE, to sting saccharopolyspora strain SP06081 genome for template, design pair of primers cm-ermE-BldD-3/ cm-BldD-4, wherein has erythromycin strong promoter P in the 5' end band of cm-ermE-BldD-3
ermEhomology arm, the 5' end band of cm-BldD-4 has phiC31 integrase gene
intdownstream homology arm, amplification
bldDgene.Reclaim product for template with above-mentioned 2 kinds of PCR, cm-reg-1/ cm-BldD-4 is primer, adopts over-lap PCR (Splicing by overlap extension PCR, SOE-PCR) method, by cm-P
ermEgene fragment and
bldDgene fragment " stitching ", obtains the cm-P with homology arm
ermE-bldD gene fragment.Utilize Red/ET homologous recombination technique by cm-P
ermE-bldD subclone, on intestinal bacteria-streptomycete shuttle vectors pUC-spn, obtains recombinant plasmid pUC-spn-P
ermE-bldD, and carry out order-checking confirmation.By conjugal transfer mode by pUC-spn-P
ermE-bldD imports in thorn saccharopolyspora strain SP06081, utilizes 28 kb part pleocidin synthetic genes bunch on carrier to be incited somebody to action by homologous recombination
bldDbe incorporated on thorn saccharopolyspora strain karyomit(e), obtain the recombinant bacterial strain that heritability is stable
s. spinosa-BldD.To be integrated with the thorn saccharopolyspora strain of initial carrier pUC-spn
s. spinosa-1 is control strain.To the PCR detection display of recombinant bacterial strain
bldDsuccessful integration is on karyomit(e); Slat chain conveyor observes growth and the colonial morphology difference of control strain and recombinant bacterial strain, and slat chain conveyor is observed and found, soaks meat soup at the BHI(brain heart) and TSB(pancreas peptone soybean broth) on flat board, the sporulation of recombinant bacterial strain is subject to obvious suppression; Shake flask fermentation result shows, and recombinant bacterial strain pleocidin output brings up to 1.35 times compared with control strain.This explanation
bldDthe overexpression of gene can suppress the sporulation of stinging saccharopolyspora strain to a certain extent, and effectively can promote the biosynthesizing of pleocidin in thorn saccharopolyspora strain.
The recombinant bacterial strain that the present invention obtains possesses following advantage:
The first,
bldDadopt erythromycin strong promoter
p ermE ,
p ermE can highly drive transcribing of downstream gene, can effectively improve
bldDexpression.
The second, increase in thorn saccharopolyspora strain
bldDcopy number, realize
bldDoverexpression, significantly can promote the biosynthesizing of pleocidin, and recombinant bacterial strain
s. spinosa-BldDthere is good genetic stability.
The explanation of microbial preservation situation
Sting saccharopolyspora strain described in specification sheets and deliver China typical culture collection center (being called for short CCTCC, address: Wuhan, China Wuhan University) preservation in September, 2006, deposit number is: CCTCC M208034.
The present invention it
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, namely stings saccharopolyspora strain
s. spinosa-BldD(Saccharopolyspora spinosa
s. spinosa-BldD), on May 31st, 2015 in China typical culture collection center (being called for short CCTCC, address: Wuhan, China Wuhan University) preservation, culture presevation number is CCTCC NO:M 2015347.
Accompanying drawing explanation
Fig. 1 is recombinant vectors pUC-spn-P
ermEthe structure schema of-bldD;
Fig. 2 is restructuring strain construction schema;
Fig. 3 is recombinant vectors and thorn saccharopolyspora strain genome recombination schematic diagram;
Fig. 4 is
bldDthe effect diagram that process LAN grows to thorn saccharopolyspora strain;
Fig. 5 and Fig. 6 is
bldDto the effect diagram of thorn saccharopolyspora strain pleocidin synthesis, (Fig. 5 is the thorn saccharopolyspora strain being integrated with initial carrier pUC-spn to process LAN
s. spinosa-1, be control strain; Fig. 6 is integrated with recombinant vectors pUC-spn-P
ermEthe recombinant bacterial strain of-bldD
s. spinosa-BldD).
Embodiment
Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail.
The present embodiment it
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, described in
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, by baldness type gene
bldDbe incorporated into thorn saccharopolyspora strain genome to produce.
The present embodiment it
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, namely stings saccharopolyspora strain
s. spinosa-BldD(Saccharopolyspora spinosa
s. spinosa-BldD) on May 31st, 2015 in China typical culture collection center (be called for short CCTCC, address: Wuhan, China Wuhan University) preservation, culture presevation number is CCTCC NO:M 2015347.
(1) described baldness type gene
bldD,clone is from stinging saccharopolyspora strain, and its sequence is as follows:
atgggcgact acgccaaggc gctgggcagc aagctccgcg ctatccgcca gcagcagggt 60
ctgtcgctgca cggcgtcgag cagaagtctg gcgggcggtg gaaggccgtg gtcgtcgggt 120
cctatgagcga ggcgaccgtg cggtgaccgt gcagaagctg gccgaactgg ccgacttcta 180
cggggttccg gtcgcggaac tgcttcccga gggtcgggtg ccgtccggcg ccgagcccgc 240
caccaaagtt gtgatcaacct ggagcggct gcagcagcta cctgcggaga aggtgggccc 300
gctggcccgc tacgcggccc catccagagc cagcggggtg actacaacgg caaggtgctg 360
tccatccgca ccgaggacct gcgatccctg gccatcatct acgacatgac gccaggtgag 420
ctcaccgagc agctcatcga ctggggcgtg cttcccccgg aggcccgccc agcccgggag 480
gagtga 486
(2) recombinant vectors pUC-spn-P
ermEthe structure (see Fig. 1) of-bldD
With pIJ86-cm plasmid for template, design pair of primers cm-reg-1/cm-ermE-reg-2,
cm-reg-1: gacgtcccgaaggcgtggcgcggcttccccgtgccggagcaatcgccctgATCTAAAGGAAGCGGATGT GAC
cm-ermE-reg-2: GGATCCTACCAACCGGCACGAT
Wherein there is phiC31 attP site upstream homology arm (lower-case portion is homology arm) in the 5' end band of cm-reg-1, amplification cm-P
ermEgene fragment, PCR reacts total system 50 microlitre (pure water 17.5 microlitre, 2 × GC buffer 25 microlitre, 10 mM dNTP mixture 4 microlitres, each 1 microlitre of primer cm-reg-1 and cm-ermE-reg-2, DNA profiling 1 microlitre, PrimeSTAR
?hS DNA Polymerase 0.5 microlitre), loop parameter is as follows: start following circulation after 94 DEG C of denaturation 5 min: 94 DEG C of 45 s, 60 DEG C of 45 s, 72 DEG C of 90 s, 30 circulations, and 72 DEG C extend 10 min.
To sting saccharopolyspora strain SP06081 genome plate for template, design pair of primers cm-ermE-BldD-3/ cm-BldD-4,
cm-ermE-BldD-3: atcgtgccggttggtaggatccATGGGCGACTACGCCAAGGCGCTG
cm-BldD-4:ctacgccgctacgtcttccgtgccgtcctgggcgtcgtcttcgtcgtcgtGGTGCCCCTCGTACGTAACTG
Wherein there is erythromycin strong promoter in the 5' end band of cm-ermE-BldD-3
p ermE homology arm, the 5' end band of cm-BldD-4 has phiC31 integrase gene
intdownstream homology arm, amplification
bldDgene.PCR reaction system and cm-P
ermEgene fragment is consistent.Loop parameter is as follows: start following circulation after 94 DEG C of denaturation 5 min: 94 DEG C of 45 s, 64 DEG C of 45 s, 72 DEG C of 60 s, 30 circulations, and 72 DEG C extend 10 min.
Reclaim product for template with above-mentioned 2 kinds of PCR, cm-reg-1/ cm-BldD-4 is primer, adopts overlapping PCR method, by cm-P
ermEgene fragment and
bldDgene fragment " stitching ", obtains the cm-P with homology arm
ermE-bldD gene fragment.By cm-P
ermE-bldD fragment electricity proceeds to containing original plasmid pUC-spn's
e.coliin GBRed, under the effect of Red α/Red β, cm-P
ermEphiC31 attP site on-bldD gene fragment replacement initial carrier pUC-spn and phiC31 integrase gene, obtain recombinant plasmid pUC-spn-P
ermE-bldD, and carry out order-checking confirmation.
(2) recombinant bacterial strain
s. spinosa-BldDstructure (see Fig. 2)
First by recombinant plasmid pUC-spn-P
ermE-bldD is electroporated
e. colis17, through Apr(apramycin), Cm(paraxin), Str(Streptomycin sulphate) three anti-screening positive transformants, extract plasmid enzyme restriction qualification obtain transformant (donor bacterium)
e. colis17 (pUC-spn-P
ermE-bldD) and carry out culture presevation.
Intestinal bacteria
e. coliengage between S17 and thorn saccharopolyspora strain SP06081 kind referral document [Matsushima P, Broughton M C, Turner J R,
et al. Conjugal transfer of cosmid DNA from
escherichia colito
saccharopolyspora spinosa: effects of chromosomal insertions on macrolide A83543 production. Gene, 1994,146:39-45], slightly make an amendment: by recipient bacterium
s. spinosasP06081(culture presevation number: CCTCC NO:M208034) at CSM (g/L:Tryptic Soy Broth (pancreas peptone soybean broth) 30, Yeast Powder (yeast powder) 3, Glucose(glucose) 5, Maltose (maltose) 4) activate 48 h in substratum; Ratio switching TSB(pancreas peptone soybean broth with 10%) liquid nutrient medium (g/L:Tryptic Soy Broth(pancreas peptone soybean broth) 30), 30 DEG C, 250 rpm shaking culture 20 h; Transfer bacterium liquid to TSB(pancreas peptone soybean broth again with the inoculum size secondary of 25%) substratum, continues cultivation 4 h.Glass homogenizer grinding bacterium liquid and packing 1.8 mL bacterium liquid in EP pipe, collected by centrifugation mycelium is also resuspended in 450 μ L(microlitres) TSB(pancreas peptone soybean broth) in.Meanwhile, 900 μ L(microlitres are got) through the donor bacterium transformant of incubated overnight
e. colis17 (pUC-spn-P
ermE-bldD) bacterium liquid divides and is filled in EP pipe, collected by centrifugation mycelium, uses TSB(pancreas peptone soybean broth) substratum cleaning mycelium twice to remove microbiotic, then is resuspended in 1.8 mL TSB(pancreas peptone soybean broths) in.Donor bacterium and recipient bacterium are with 3:1(i.e. 300 μ L:100 μ L) ratio mix, be spread evenly across the R6(g/L:Sucrose(sucrose of antibiotic-free) 200, BHI(brain heart infusion broth) 26, Dextrin(dextrin) 10, Casamino acids(casamino acids) 1, (potassium sulfate) 0.1, FeSO47H2O(ferric sulfate) 0.1, MgSO47H2O(magnesium sulfate) 0.05, MnCl
24H
2o (Manganous chloride tetrahydrate) 0.001, ZnSO
47H
2o(zinc sulfate) 0.001, Agar(agar powder) 12, add distilled water and be settled to 877 mL, 115 DEG C of sterilizing 30 min.Treat that substratum is cooled to 70 DEG C, then add following three kinds with the degerming material of 0.22 μm of membrane filtration, 1 M L-glutamic acid (L-glutamic acid) 65 mL, 1 M CaCl
22H
2o (calcium chloride) 48 mL and 1 M MOPS (propanesulfonic acid) 5 mL.) on flat board, be inverted cultivation 20 h for 30 DEG C; Apr (apramycin) and NA (nalidixic acid) is added into 1 mL TSB(pancreas peptone soybean broth) in liquid nutrient medium, cover flat board after mixing, make final concentration reach 50 micrograms/mL and 25 micrograms (microgram)/mL respectively.Dry up in Bechtop, be inverted in 30 DEG C of constant incubators and cultivate 10-15 days.Treat R6 flat board grows mono-clonal, picking zygote is forwarded to dull and stereotyped (the g/L:BHI(brain heart leach liquor meat soup) 37 of the BHI (brain heart leach liquor meat soup) being added with Apr (apramycin) and NA (nalidixic acid), Agar(agar powder) 12), with transfering loop by even for thalline coating, cultivate 3-5 days for 30 DEG C; Until BHI(brain heart infusion soup) after bacterium colony on flat board fully grows, picking part thalline is forwarded to and is added with Apr(apramycin) and NA(nalidixic acid) TSB(pancreas peptone soybean broth) in liquid nutrient medium, cultivate 3-4 days for 30 DEG C.
Collect zygote (i.e. recombinant bacterial strain) and wild fungus liquid respectively, extract genome as template, carry out pcr amplification A Baila mycin resistant gene fragment with primer pair Apra-F/Apra-R.With wild strain genome for negative control, with recombinant vectors pUC-spn-P
ermE-bldD is positive control.PCR identifies correct zygote (recombinant bacterial strain), called after
s. spinosa-BldD.-80 DEG C of preservation of bacteria strains, in order to tests such as follow-up further detection, Analysis offermehtations.Result shows, recombinant bacterial strain
s. spinosa-BldDbe positive with above-mentioned primer PCR amplification, and with original strain
s. spinosagenome is that the negative control of template occurs without object band, explanation
bldDgene Successful integration on thorn saccharopolyspora strain karyomit(e).
(3)
bldDthe impact that process LAN grows on thorn saccharopolyspora strain
Get 40 μ L(microlitres) control strain
s. spinosa-1(is integrated with initial carrier pUC-spn) and recombinant bacterial strain
s. spinosa-BldD,-80 DEG C of culture presevation liquid are spread evenly across on BHI (brain heart infusion soup) and TSB (pancreas peptone soybean broth) solid medium respectively, and 30 DEG C of constant temperature culture observe its colonial morphology and sporulation situation on different culture media.Shown in experimental result Fig. 4, recombinant bacterial strain and the colonial morphology of control strain on BHI (brain heart infusion soup) and TSB (pancreas peptone soybean broth) substratum do not have notable difference, and cultivate on BHI (brain heart infusion soup) and TSB (pancreas peptone soybean broth) substratum after 3 days, control strain has produced more white spore, and recombinant bacterial strain only produces the spore of seldom amount.Compared with control strain, the sporulation of recombinant bacterial strain is subject to obvious suppression.
(4)
bldDprocess LAN is on the impact of thorn saccharopolyspora strain pleocidin synthesis
Get 1 mL-80 DEG C to sting saccharopolyspora strain culture presevation liquid to be seeded to liquid amount be 50 mL seed culture mediums (g/L:Trypticase Soy Broth (pancreas peptone soybean broth) 45, Glucose glucose 10, Yeast Extract(yeast powder) 9, MgSO
47H
2o (magnesium sulfate) 2.2) 300 mL triangular flasks in, 30 DEG C of 250 r/min cultivates 2 days; Ratio switching seed liquor to liquid amount with 10% is 30 mL synthesis fermention medium (g/L:Glucose(glucose) 20, Tryptone(peptones) 4, Yeast Powder (yeast powder) 4, KNO
3(saltpetre) 1, K
2hPO
43H
2o(dipotassium hydrogen phosphate) 0.5, MgSO
47H
2o(magnesium sulfate) 0.5, FeSO
4(ferric sulfate) 0.01) 300 mL triangular flasks in, 30 DEG C of 250 r/min ferments 10 days.
Get 700 μ L(microlitres) fermented liquid add isopyknic acetone mixing, 4 DEG C of lixiviate 24 h.Centrifuging and taking supernatant, through 0.22 μm of (micron) filtering with microporous membrane, uses Agilent 1290 Ultra Performance Liquid Chromatography systems measurement pleocidin content.Chromatographic column specification is ZORBAX SB-C18(5 μm, 4.6 × 150 mm); Column temperature: room temperature; Moving phase: methyl alcohol/acetonitrile/2% ammonium acetate solution (volume ratio 45/45/10); Flow velocity: 1.5 mL/min; Loading volume: 10 μ L(microlitres); Determined wavelength: 250 nm.Carry by detection system the response peak area that analysis software calculates A83543A, D, just can draw the content of pleocidin in sample with standard substance comparison.Result shows (see Fig. 5, Fig. 6), recombinant bacterial strain in synthesis fermention medium
s. spinosa-BldDretention time is that the chromatographic peak of 5.5 min and 6.7min significantly improves.Control strain
s. spinosa-1 A83543A+D peak area is 145.9 mAU*s, recombinant bacterial strain
s. spinosa-BldDa83543A+D peak area is 196.5 mAU*s, calculating is learnt, in synthesis fermention medium, the output of the A83543A+D of control strain S. spinosa-1 is 99.2 mg/L, the output of recombinant bacterial strain S. spinosa-BldD A83543A+D is 133.6 mg/L, compared to control strain pleocidin output increased to 1.35 times.
(5) recombinant bacterial strain
s. spinosa-BldDgenetic stability detects
By recombinant bacterial strain
s. spinosa-BldDbe seeded in CSM substratum and activate 2 days, the bacterium liquid got after appropriate dilution is spread evenly across TSB(pancreas peptone soybean broth) on dull and stereotyped (antibiotic-free), cultivate 5-7 d for 30 DEG C; Picking 70 mono-clonals be forwarded to be added with A Baila mycin (50 μ g/mL) TSB (pancreas peptone soybean broth) flat board on, cultivate 3-4 days for 30 DEG C, calculate resistance clone number; And therefrom random picking 20 mono-clonals, carry out colony PCR amplification A Baila mycin resistant gene fragment, research recombinant bacterial strain
s. spinosa-BldDgenetic stability.Result shows: recombinant bacterial strain
s. spinosa-BldDunder non-resistant selective pressure, continuous passage cultivates 8 times, and single bacterium colony of more than 98% still has A Baila chloramphenicol resistance, and colony PCR amplification A Baila mycin resistant gene result is positive, and shows in the process of continuous passage, integration
bldDgene is not lost,
s. spinosa-BldDgenetic stability good.
Claims (4)
1. a strain
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, is characterized in that, by baldness type gene
bldDbe incorporated into thorn saccharopolyspora strain genome to produce.
2. according to claim 1
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, is characterized in that, described in
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, namely stings saccharopolyspora strain
s. spinosa-BldD, Saccharopolyspora spinosa
s. spinosa-BldD, on May 31st, 2015 in China typical culture collection center preservation, culture presevation number is CCTCC NO:M 2015347.
3. according to claim 1 and 2
bldDthe thorn saccharopolyspora strain recombinant bacterial strain of gene duplication, is characterized in that, described baldness type gene
bldD,clone is from stinging saccharopolyspora strain, and its sequence is as follows:
atgggcgact acgccaaggc gctgggcagc aagctccgcg ctatccgcca gcagcagggt 60
ctgtcgctgca cggcgtcgag cagaagtctg gcgggcggtg gaaggccgtg gtcgtcgggt 120
cctatgagcga ggcgaccgtg cggtgaccgt gcagaagctg gccgaactgg ccgacttcta 180
cggggttccg gtcgcggaac tgcttcccga gggtcgggtg ccgtccggcg ccgagcccgc 240
caccaaagtt gtgatcaacct ggagcggct gcagcagcta cctgcggaga aggtgggccc 300
gctggcccgc tacgcggccc catccagagc cagcggggtg actacaacgg caaggtgctg 360
tccatccgca ccgaggacct gcgatccctg gccatcatct acgacatgac gccaggtgag 420
ctcaccgagc agctcatcga ctggggcgtg cttcccccgg aggcccgccc agcccgggag 480
gagtga 486
4. as described in one of claim 1-3
bldDthe application of thorn saccharopolyspora strain recombinant bacterial strain in biosynthesizing pleocidin of gene duplication.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108085290A (en) * | 2018-01-04 | 2018-05-29 | 哈尔滨瀚邦医疗科技有限公司 | A kind of detection method of Hepatitis E virus genetic stability and application |
| CN110029069A (en) * | 2019-04-29 | 2019-07-19 | 湖南师范大学 | The palpus saccharopolyspora strain engineered strain and its application that one plant of flaviolin gene cluster knocks out |
| CN110713964A (en) * | 2019-04-30 | 2020-01-21 | 湖南师范大学 | Saccharopolyspora whiskers engineering strain with cspA gene doubled and application thereof |
| CN111454975A (en) * | 2020-04-17 | 2020-07-28 | 长沙微智生物科技有限公司 | Application of ECH gene related to spinosad |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108085290A (en) * | 2018-01-04 | 2018-05-29 | 哈尔滨瀚邦医疗科技有限公司 | A kind of detection method of Hepatitis E virus genetic stability and application |
| CN110029069A (en) * | 2019-04-29 | 2019-07-19 | 湖南师范大学 | The palpus saccharopolyspora strain engineered strain and its application that one plant of flaviolin gene cluster knocks out |
| CN110029069B (en) * | 2019-04-29 | 2022-11-29 | 湖南师范大学 | Saccharopolyspora sinensis engineering strain with light flavomycin gene cluster knocked out and application thereof |
| CN110713964A (en) * | 2019-04-30 | 2020-01-21 | 湖南师范大学 | Saccharopolyspora whiskers engineering strain with cspA gene doubled and application thereof |
| CN110713964B (en) * | 2019-04-30 | 2022-08-30 | 湖南师范大学 | Saccharopolyspora whiskers engineering strain with cspA gene doubled and application thereof |
| CN111454975A (en) * | 2020-04-17 | 2020-07-28 | 长沙微智生物科技有限公司 | Application of ECH gene related to spinosad |
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