CN103509090B - Participate in albumen glpks4 and the encoding gene thereof of the synthesis of lung sac Kangding - Google Patents
Participate in albumen glpks4 and the encoding gene thereof of the synthesis of lung sac Kangding Download PDFInfo
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Abstract
The invention discloses a kind of the albumen glpks4 and the encoding gene thereof that participate in the synthesis of lung sac Kangding.Protein provided by the invention is following (a) or (b): the protein that (a) is made up of the aminoacid sequence shown in sequence in sequence table 1; B aminoacid sequence shown in sequence in sequence table 1 is participated in the protein derived by sequence 1 of lung sac Kangding synthesis through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by ().Do you suppress Glarea? the expression of the encoding gene of albumen described in lozoyensis, can not produce lung sac Kangding B
0with lung sac Kangding A
0.The present invention provides theoretical foundation for further investigation lung sac Kangding biosynthetic pathway and/or pathways metabolism, can be used for obtaining different lung sac Kangding derivatives by biosynthetic pathway, therefrom screens more efficient echinocandin antifungal agent thing.
Description
Technical field
The present invention relates to a kind of the albumen glpks4 and the encoding gene thereof that participate in the synthesis of lung sac Kangding.
Background technology
Caspofungin (Caspofungin) is first echinocandin be approved listing by U.S. FDA (Echinocandin) class antifungal drug, can specific Antifungi cell walls β-(1,3) synthesis of-D-dextran, fungi is dissolved, there is good anti-mycotic activity, and the cytotoxicity of the medicines such as similar amphotericin B can not be produced patient, be therefore also described as the penicillin in antifungal drug.
Caspofungin is used for the treatment of clinically to fail to respond to any medical treatment to other or the monilial peritoneal abscess of not tolerant Aspergillosis and candidiasis microbemia and secondary and peritonitis patient.Within 2009, its global marketing volume is 6.17 hundred million dollars, and in the whole antifungal drug hospital market of China, portion is 12.45%, occupies the 4th.At present, there is no domestic Caspofungin product, domestic use all needs import, and single price is up to thousand yuan.But Merck & Co., Inc. will expire in 2013 for the patent right of Caspofungin, Caspofungin production domesticization will when the time comes be imperative, and this will bring huge economic worth.
The precursor of Caspofungin is lung sac Kangding (pneumocandin) B produced by Glarealozoyensis
0, therefore lung sac Kangding B in Glarealozoyensis
0synthesize most important.Glarealozoyensis belongs to Ascomycota (Ascomycota) Leotia guiding principle (Leotiomycetes) Helotiales (Helotiales), is anamorph filamentous fungus.1991, Adefarati etc. are lung sac Kangding B by technical Analysis such as NMR
0structure, find that this compound is by Threonine, trans-4-oxyproline, 3,4-dihydroxyl height tyrosine, 3-hydroxyglutamine, trans-3-oxyproline and 4, the ring six peptide main chain of 5-dihydroxyl ornithine composition and the polyketone side chain of 10, a 12-dimethyl-myristate are formed.
Lung sac Kangding comprises and lung sac Kangding A
0with lung sac Kangding B
0, structural formula is as follows:
Work as R=CH
3time, compound shown in formula I is lung sac Kangding A
0; As R=H, compound shown in formula I is lung sac Kangding B
0.
Molecular modification and metabolic regulation are the methods increasing substantially meta-bolites, find and lung sac Kangding B
0the gene that synthesis is relevant, is operated by molecular genetics and effectively improves lung sac Kangding B
0output be key issue anxious to be resolved.
Summary of the invention
The object of this invention is to provide a kind of the albumen glpks4 and the encoding gene thereof that participate in the synthesis of lung sac Kangding.
Protein provided by the invention (glpks4 albumen), from Glarealozoyensis, is following (a) or (b):
A protein that () is made up of the aminoacid sequence shown in sequence in sequence table 1;
B aminoacid sequence shown in sequence in sequence table 1 is participated in the protein derived by sequence 1 of lung sac Kangding synthesis through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation by ().
The replacement of one or several amino-acid residue described and/or disappearance and/or interpolation refer to the replacement and/or disappearance and/or interpolation that are no more than 10 amino-acid residues.
In order to make the albumen in (a) be convenient to purifying, the N-terminal of the protein that the aminoacid sequence shown in sequence 1 forms or C-terminal label as shown in table 1 can be connected in by sequence table.
The sequence of table 1 label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Replacement in above-mentioned (b) and/or disappearance and/or interpolation, can be caused by natural variation or induced mutations.
Albumen in above-mentioned (a) or (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of the albumen in above-mentioned (b) is by the codon by lacking one or several amino-acid residue in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence connecting the label shown in table 1 is held to obtain at its 5 ' end and/or 3 '.
The gene (glpks4 gene) of encoding said proteins also belongs to protection scope of the present invention.
Described gene can be (1) or (2) or (3) or the DNA molecular described in (4) as follows:
(1) DNA molecular shown in sequence 2 in sequence table;
(2) DNA molecular shown in sequence 3 in sequence table;
(3) DNA sequence dna limited with (1) or (2) is under strict conditions hybridized and is encoded and participates in the DNA molecular of the albumen that lung sac Kangding is synthesized;
(4) DNA sequence dna limited with (1) or (2) at least has more than 90% homology and encodes and participates in the DNA molecular of the albumen that lung sac Kangding is synthesized.
Above-mentioned stringent condition can be at 0.1 × SSPE(or 0.1 × SSC), in the solution of 0.1%SDS, hybridize under 65 DEG C of conditions and wash film.
Recombinant vectors containing described gene, expression cassette, transgenic cell line or recombinant bacterium all belong to protection scope of the present invention.
Available existing expression vector establishment contains the recombinant expression vector of described gene.Described expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of micropellet bombardment.Described expression vector also can comprise 3 ' end untranslated region of foreign gene, namely comprises the DNA fragmentation of polyadenylation signals and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylation signals joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any one enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can be used alone or are combined with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also enhanser can be used, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to ensure the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthesis.Translation initiation region can from transcription initiation region or structure gene.For the ease of qualification and screening, can processing expression carrier used thereof, enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the chemical resistance reagent marker gene etc. of colour-change can be produced as added coding.Also any selected marker can not be added, directly according to phenotypic screen.
Described protein can participate in the synthesis of lung sac Kangding.
The product of described genetic expression is suppressed to can be used for blocking the synthesis of lung sac Kangding.
The product of the described genetic expression of described suppression specifically can be and suppresses the specific DNA fragment of described genetic expression or the recombinant plasmid containing described specific DNA fragment by homologous recombination.
Recombinant plasmid containing described specific DNA fragment can be following recombinant plasmid: skeleton carrier is carrier pAg1-H3, in the sequence 2 of the different multiple clone site insertion sequence tables respectively of described skeleton carrier from the sequence 2 of the DNA fragmentation first shown in 5 ' end the 104 to 2802 Nucleotide and sequence table from the DNA fragmentation second shown in 5 ' end the 3518 to 6261 Nucleotide.
Recombinant plasmid containing described specific DNA fragment specifically can be following recombinant plasmid: skeleton carrier is carrier pAg1-H3, the sequence 2 inserting sequence table between PvuII and ApaI restriction enzyme site, from the DNA fragmentation first shown in 5 ' end the 104 to 2802 Nucleotide, inserts the sequence 2 of sequence table from the DNA fragmentation second shown in 5 ' end the 3518 to 6261 Nucleotide between AscI and SbfI restriction enzyme site.
The present invention also protects the application in the expression of above arbitrary described recombinant plasmid containing described specific DNA fragment encoding gene of arbitrary described glpks4 albumen more than suppressing.
The encoding gene of described suppression glpks4 albumen expresses the expression referring to the encoding gene suppressing glpks4 albumen described in Glarealozoyensis.
Described lung sac Kangding can be lung sac Kangding B
0and/or lung sac Kangding A
0
The present invention has found participation lung sac Kangding B by analyzing Glarealozoyensis genome
0with lung sac Kangding A
0the albumen of synthesis and encoding gene thereof.Suppress the expression of this gene in Glarealozoyensis, lung sac Kangding B can not be produced
0with lung sac Kangding A
0.If by this gene clone on integrative vector, be inserted into genome by integrative vector, lung sac Kangding B may be improved
0with lung sac Kangding A
0output.The present invention provides theoretical foundation for further investigation lung sac Kangding biosynthetic pathway and/or pathways metabolism, can be used for obtaining different lung sac Kangding derivatives by biosynthetic pathway, therefrom screens more efficient echinocandin antifungal agent thing.
Accompanying drawing explanation
Fig. 1 is the partial results of PCR qualification.
Fig. 2 is HPLC collection of illustrative plates.
Fig. 3 is the result of bacteriostatic test; 1: lung sac Kangding B
0standard substance; 2: wild mushroom; 4: recombinant bacterium; 5:DMSO.
Embodiment
Following embodiment is convenient to understand the present invention better, but does not limit the present invention.Experimental technique in following embodiment, if no special instructions, is ordinary method.Test materials used in following embodiment, if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples, all arranges and repeats experiment for three times, results averaged.
Glarealozoyensis(wild mushroom): (ATCC, network address is American Type Culture collection warehousing
www.atcc.org/), ATCC is numbered 20868.
Carrier pAg1-H3: the public can obtain from Institute of Microorganism, Academia Sinica; Reference: A.Zhangetal., Efficientdisruptionofapolyketidesynthasegene (pks1) requiredformelaninsynthesisthroughAgrobacterium-mediated transformationofGlarealozoyensis.MolecularGeneticsandGen omics268,645 (2003)..
Agrobacterium AGL-1(AgrobacteriumtumefaciensAGL-1): the public can obtain from Institute of Microorganism, Academia Sinica; Reference: X.Chen, M.Stone, C.Schlagnhaufer, C.P.Romaine, AFruitingBodyTissueMethodforEfficientAgrobacterium-Media tedTransformationofAgaricusbisporus.ApplEnvironMicrob66,4510 (2000)..
Candida albicans SC5314(CandidaalbicansSC5314): the public can obtain from Institute of Microorganism, Academia Sinica; Reference: J.G ó mez-Raja, E.Andaluz, B.Magee, R.Calderone, G.Larriba, AsingleSNP, G929T (Gly310Val), determinesthepresenceofafunctionalandanon-functionalalle leofHIS4inCandidaalbicansSC5314:detectionofthenon-functi onalalleleinlaboratorystrains.FungalGenetBiol45,527 (2008)..
IMAS liquid nutrient medium (200mL): 80mL2.5 × MM, 0.36g glucose, 1mL glycerine, add water to 192mL, add 8mL1MMES and 4mL10mMAS after sterilizing.
IMAS solid medium (200mL): 80mL2.5 × MM, 0.18g glucose, 1mL glycerine, 3g agar, add water to 188mL, add 8mL1MMES and 4mL10mMAS after sterilizing.
1MMES (100mL): 21.325gMES and 100mL water mixes, adjusts pH to 5.3 with potassium hydroxide, filtration sterilization.
10mMAS (100mL): 0.1962g Syringylethanone and the mixing of 100mL water, adjusts pH to 8.0 with potassium hydroxide, filtration sterilization.
2.5 × MM (1L): 3.625g dipotassium hydrogen phosphate, 5.125g potassium primary phosphate, 1.25g magnesium sulfate, 0.375g sodium-chlor, 0.165g calcium chloride, 0.0062g ferrous sulfate and 1.25g ammonium sulfate, water-soluble and be settled to 1L.
M-100 substratum (1L): 62.5mLM-100 salts solution, 10g glucose, 3g saltpetre and 15g agar, water-soluble and be settled to 1L.
M-100 salts solution (1L): 16g potassium primary phosphate, 4g sodium sulfate, 8g Repone K, 2g magnesium sulfate, 1g calcium chloride and 8mLM-100 trace element solution, water-soluble and be settled to 1L.
M-100 trace element solution (500mL): 30mg boric acid, 70mg Manganous chloride tetrahydrate, 200mg zinc chloride, 20mg Sodium orthomolybdate, 50mg iron(ic) chloride and 200mg copper sulfate, water-soluble and be settled to 500mL.
LYCP-5 produces spore substratum (pH6.0): by 2.5g glucose, 0.9g potassium primary phosphate, 0.5g yeast extract, 1g cottonseed meal, 0.2mL85g/100mL lactic acid aqueous solution, 1mL trace element mixture, water-soluble and be settled to 100mL.
FGY substratum (pH5.3): by 4g fructose, 0.8g Sodium Glutamate, 0.8g yeast extract, 1.5g proline(Pro), 0.15g potassium primary phosphate, 0.04g magnesium sulfate, 1mL trace element mixture, water-soluble and be settled to 100mL.
Trace element mixture: 0.1g ferrous sulfate, 0.1g manganous sulfate, 0.02g zinc sulfate, 0.01g calcium chloride, 0.0056g boric acid, 0.0025g copper sulfate, 0.0019g ammonium molybdate, 12M aqueous hydrochloric acid 5ml, water-soluble and be settled to 100mL.
The discovery of embodiment 1, glpks4 albumen and encoding gene thereof
Genome sequencing is carried out to Glarealozoyensis, sequencing result through assembling, predictive genes and annotation found one may be relevant to the biosynthesizing of lung sac Kangding gene cluster, therefrom found a participation lung sac Kangding A
0with lung sac Kangding B
0the new albumen of route of synthesis, this albumen of bioinformatic analysis belongs to polyketide synthase, is glpks4 albumen by this protein designations.
Glpks4 albumen is as shown in the sequence 1 of sequence table.Be glpks4 gene by the unnamed gene of coding glpks4 albumen, its genomic dna is as shown in the sequence 2 of sequence table, and its open reading frame is as shown in the sequence 3 of sequence table.
The functional verification of embodiment 2, glpks4 albumen and encoding gene thereof
One, the structure of glpks4 knockout carrier
1, according to the sequence of glpks4 gene, two pairs of primers are designed as follows:
Pks4-S1 and pks4-R1 forms primer pair first, and target sequence is that the sequence 2 of sequence table is from 5 ' end the 51 to 2903 Nucleotide.
pks4-S1：5’-TCTCGCTATCGTGGGTATG-3’；
pks4-R1：5’-ATCTTGAGTATGCTTTCGCC-3’。
Pks4-S2 and pks4-R2 forms primer pair B, and target sequence is that the sequence 2 of sequence table is from 5 ' end the 3518 to 6261 Nucleotide.
Pks4-S2:5 '-C
gGCGCGCCtGAAGAAAGTCGGTCTC-3 ' (underscore mark AscI recognition sequence);
Pks4-R2:5 '-C
cCTGCAGGcCGAATGTGTCCATCAGG-3 ' (underscore mark SbfI recognition sequence).
2, with the genomic dna of Glarealozoyensis for template, carry out pcr amplification by primer pair first, obtain pcr amplification product.
3, restriction enzyme PvuII(CAG^CTG is used) and the ApaI(G^GGCCC) pcr amplification product that obtains of double digestion step 2, obtain digestion products.Sequence between digestion products PvuII recognition sequence and ApaI recognition sequence is that the sequence 2 of sequence table is from 5 ' end the 104 to 2802 Nucleotide.
4, with restriction enzyme PvuII and ApaI double digestion carrier pAg1-H3, carrier framework (about 6.5kb) is reclaimed.
5, the digestion products of step 3 is connected with the carrier framework of step 4, obtains recombinant plasmid pAg1-H3-pks4-1.
6, with the genomic dna of Glarealozoyensis for template, carry out pcr amplification by primer pair B, obtain pcr amplification product.
7, with the pcr amplification product that restriction enzyme A scI and SbfI double digestion step 6 obtain, digestion products is obtained.Sequence between digestion products AscI recognition sequence and SbfI recognition sequence is that the sequence 2 of sequence table is from 5 ' end the 3518 to 6261 Nucleotide.
8, with restriction enzyme A scI and SbfI double digestion recombinant plasmid pAg1-H3-pks4-1, carrier framework (about 9.3kb) is reclaimed.
9, the digestion products of step 7 is connected with the carrier framework of step 8, obtains recombinant plasmid pAg1-H3-pks4.According to sequencing result, structrual description carries out to recombinant plasmid pAg1-H3-pks4 as follows: skeleton carrier is carrier pAg1-H3, the sequence 2 of sequence table is inserted from the DNA fragmentation first shown in 5 ' end the 104 to 2802 Nucleotide between PvuII and ApaI restriction enzyme site, the sequence 2 inserting sequence table between AscI and SbfI restriction enzyme site, from the DNA fragmentation second shown in 5 ' end the 3518 to 6261 Nucleotide, has hygromycin gene between DNA fragmentation first and DNA fragmentation second; DNA fragmentation first and DNA fragmentation second can with the genomic dna generation homologous recombination of Glarealozoyensis, replace the glpks4 gene fragment of genomic dna by hygromycin gene, thus make glpks4 gene inactivation.
Two, the acquisition of recombinant bacterium
1, recombinant plasmid pAg1-H3-pks4 is imported Agrobacterium AGL-1, obtain recombinational agrobacterium.
2, recombinational agrobacterium is resuspended in IMAS liquid nutrient medium makes OD
660value is 0.15,28 DEG C, 200rpm shaking culture 4-6 hour, obtains OD
660the recombinational agrobacterium bacterium liquid of value between 0.6-0.8.
3, by resuspended for the spore distilled water of Glarealozoyensis, obtaining spore concentration is 10
6the spore suspension of individual spore/mL.
4, the recombinational agrobacterium bacterium liquid that the spore suspension 100 μ L steps 3 obtained and 100 μ L steps 2 obtain mixes, coat on IMAS solid medium, cultivate 2 days for 28 DEG C, then cover with the M-100 substratum containing 300 μ g/mL Reflins and 200 μ g/mL Totomycin, cultivate 2-3 week for 25 DEG C.
5, the bacterium colony of growth in picking step 4, is seeded on the PDA solid medium containing 300 μ g/mL Reflins and 200 μ g/mL Totomycin, cultivates 2-3 week, obtains the bacterial strain of pure culture for 25 DEG C.
6, the genomic dna of the bacterial strain of the pure culture of extraction step 5 acquisition, carry out PCR qualification by primer pair third and primer pair fourth, if be the positive with the PCR qualification result of primer pair third and primer pair fourth, then this bacterial strain is recombinant bacterium.
Primer pair third is made up of (distinguishing the sequence in corresponding DNA fragment first and DNA fragmentation second) 4G and 4H, if having the pcr amplification product of about 3.5kb, PCR qualification result is positive.
4G：5’-AGTGTAACGCTTTCTGGCG-3’；
4H：5’-CCTCGGATGCTCTTTCAAC-3’。
Primer pair fourth is made up of (the corresponding carrier framework of 4I, the corresponding glpks4 gene of 4J) 4I and 4J, if having the pcr amplification product of about 4.3kb, PCR qualification result is positive.
4I：5’-CGAGGGCAAAGGAATAGAGTAG-3’；
4J：5’-GTTCTGTTCTGGGATTGTGAC-3’。
Partial results is shown in Fig. 1.In Fig. 1, swimming lane a is DNAmarker, and swimming lane b to increase recombinant bacterium for adopting primer pair third for adopting increase Glarealozoyensis, swimming lane c of primer pair third, and swimming lane d is for adopting primer pair fourth amplification recombinant bacterium.
Three, the acquisition of bacterium is contrasted
Replace recombinant plasmid pAg1-H3-pks4 to carry out the operation of step 2 with carrier pAg1-H3, obtain contrasting bacterium.
Four, the functional verification of glpks4 albumen and encoding gene thereof
Respectively recombinant bacterium, contrast bacterium and wild mushroom are carried out following steps (parallel processing):
1, the colony inoculation of 0.5cm × 0.5cm is produced spore substratum to 25mLLYCP-5, then 25 DEG C, 200rpm shaking culture 4 days, the spore liquid obtained.
2, by the spore liquid of step 1 with 5%(volume ratio) inoculum size be forwarded to FGY substratum, 25 DEG C, 200rpm shaking culture 14 days.
3, get the culture system of completing steps 2, with the lixiviate of equal-volume methyl alcohol (25 DEG C, 200rpm shaking culture 2 hours), then use 0.22 μm of membrane filtration, collect filtrate.
4, LC-MS is adopted to detect in filtrate whether have lung sac Kangding A
0with lung sac Kangding B
0.
Chromatographic analyzer of liquid phase device is Agilent company 1200 high performance liquid chromatography, and chromatographic column is AgilentZorbaxExtend-C
181.8 μm, the C18 post of 2.1 × 50mm; Moving phase overall flow rate is 0.3mL/min; Moving phase is the mixture of mobile phase A, Mobile phase B or mobile phase A and Mobile phase B, and mobile phase A is 0.1%(volume ratio) aqueous formic acid, Mobile phase B be acetonitrile; Total elution time is 25 minutes; Elution process is: 0 ~ 0.5min, the volume ratio that Mobile phase B accounts for moving phase is 30%, the volume ratio that 0.5 ~ 4min Mobile phase B accounts for moving phase linearly rises to 70% by 30%, the volume ratio that 4 ~ 12min Mobile phase B accounts for moving phase linearly rises to 100% by 70%, the volume ratio that 12 ~ 17min Mobile phase B accounts for moving phase is 100%, the volume ratio that 17 ~ 17.5min Mobile phase B accounts for moving phase linearly drops to 30% by 100%, and the volume ratio that 17.5 ~ 25min Mobile phase B accounts for moving phase is 30%; Column temperature 40 DEG C, the sample size of sample is 10 μ L, and after post, effluent liquid directly enters mass spectrometric detection without shunting.
Mass spectrometer is Agilent6520 quadrupole flight time mass spectrum (Agilent, USA), adopts electric spray ion source positive ion mode to detect; Cracked voltage and capillary voltage are respectively 130V and 3500V; Desolventizing gas is high pure nitrogen, and temperature is 300 DEG C, and flow is 10L/min; Nebulizer pressure is 25psi; Mass scan range is 80 ~ 1200m/z; Within every 0.97 second, gather a secondary data.
HPLC collection of illustrative plates is shown in that (peak 1 is lung sac Kangding B to Fig. 2
0, peak 2 is lung sac Kangding A
0).
Wild mushroom all demonstrates peak two desired location; The Mass Spectrometric Identification result that first peak is corresponding is: m/z value is 1065.57, is lung sac Kangding B
0; The Mass Spectrometric Identification result that second peak is corresponding is: m/z value is 1079.21, is lung sac Kangding A
0.
The result of contrast bacterium is consistent with wild mushroom.
Recombinant bacterium does not demonstrate peak two desired location.
Above result shows, after suppressing glpks4 genetic expression, recombinant bacterium can not synthesize lung sac Kangding A
0with lung sac Kangding B
0, namely glpks4 albumen participates in lung sac Kangding A
0with lung sac Kangding B
0route of synthesis.
5, bacteriostatic test
(1) culture system of completing steps 2 is got, vacuum freeze drier freeze-drying, sample adds equal-volume methyl alcohol by freeze-drying front volume, lixiviate (25 DEG C, 200rpm shaking culture 1 hour), then centrifugal (5000rpm, 5min) gets supernatant 10mL in glass beaker, and adding 2mLDMSO, to be placed on dried overnight to volume in stink cupboard be 2ml.
(2) picking Candida albicans SC5314 is placed in husky Bao Shi glucose broth (SDB substratum), 30 DEG C of incubated overnight are 0.4 to OD660, then with 3%(volume ratio) inoculum size be seeded to shake up in husky Bao Shi glucose agar medium (SDA substratum) after pour in 9cm plastic board.
(3) get the solution that 10 μ L steps (1) obtain, join on 0.5cm filter paper, be then positioned on flat board that step (2) obtains, cultivate after 20 hours for 30 DEG C and observe inhibition zone.Adopt the lung sac Kangding B of 5mg/mL
0the positive control of the solution that standard solution obtains as step (1), the negative control of the solution adopting DMSO to obtain as step (1).
The results are shown in Figure 3.
Lung sac Kangding B
0the extract of standard substance and wild mushroom culture all can observe the generation of inhibition zone, and the extract of recombinant bacterium culture and DMSO all can not observe the generation of inhibition zone, and this can not synthesize lung sac Kangding B just because of recombinant bacterium
0cause.
Embodiment 2 carries out three revision tests, and result is consistent.
Claims (3)
1. a recombinant plasmid, its skeleton carrier is carrier pAg1-H3, at the recombinant plasmid that the sequence 2 of the different multiple clone site insertion sequence tables respectively of described skeleton carrier obtains from the DNA fragmentation second shown in 5 ' end the 3518 to 6261 Nucleotide from the sequence 2 of the DNA fragmentation first shown in 5 ' end the 104 to 2802 Nucleotide and sequence table.
2. the application of recombinant plasmid described in claim 1 in suppressing the encoding gene of glpks4 albumen to be expressed; Described glpks4 albumen is made up of the aminoacid sequence shown in sequence in sequence table 1.
3. apply as claimed in claim 2, it is characterized in that: the encoding gene of described glnrps4 albumen is following (1) or the DNA molecular described in (2):
(1) DNA molecular shown in sequence 2 in sequence table;
(2) DNA molecular shown in sequence 3 in sequence table.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210202161.XA CN103509090B (en) | 2012-06-15 | 2012-06-15 | Participate in albumen glpks4 and the encoding gene thereof of the synthesis of lung sac Kangding |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201210202161.XA CN103509090B (en) | 2012-06-15 | 2012-06-15 | Participate in albumen glpks4 and the encoding gene thereof of the synthesis of lung sac Kangding |
Publications (2)
| Publication Number | Publication Date |
|---|---|
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| CN103509090B true CN103509090B (en) | 2016-01-20 |
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| ENA Sequence:AGUE01000179.1;无;《EMBL-EBI》;20011215;全文 * |
| Genome sequence of the fungus Glarea lozoyensis:the first genome sequence of a species from the Helotiaceae family;Loubna Youssar et al.;《Eukaryot cell》;20120229;第11卷(第2期);第250页第1栏第3段 * |
| 抗生素结构修饰的进展;张致平;《中国抗生素杂志》;20060228;第31卷(第2期);第90-99页 * |
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