CN112575013B - 基因gnatb作为筛选标记基因在抗性筛选中的应用 - Google Patents
基因gnatb作为筛选标记基因在抗性筛选中的应用 Download PDFInfo
- Publication number
- CN112575013B CN112575013B CN202011552186.3A CN202011552186A CN112575013B CN 112575013 B CN112575013 B CN 112575013B CN 202011552186 A CN202011552186 A CN 202011552186A CN 112575013 B CN112575013 B CN 112575013B
- Authority
- CN
- China
- Prior art keywords
- gnatb
- gene
- cycloheximide
- screening
- saccharomyces cerevisiae
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 69
- 238000012216 screening Methods 0.000 title claims abstract description 36
- 239000003550 marker Substances 0.000 title claims abstract description 26
- YPHMISFOHDHNIV-FSZOTQKASA-N cycloheximide Chemical compound C1[C@@H](C)C[C@H](C)C(=O)[C@@H]1[C@H](O)CC1CC(=O)NC(=O)C1 YPHMISFOHDHNIV-FSZOTQKASA-N 0.000 claims abstract description 109
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
- 230000003115 biocidal effect Effects 0.000 claims description 7
- 239000003242 anti bacterial agent Substances 0.000 claims description 3
- 238000010276 construction Methods 0.000 claims description 3
- 241000235070 Saccharomyces Species 0.000 claims 2
- 240000004808 Saccharomyces cerevisiae Species 0.000 abstract description 32
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 abstract description 32
- 239000003814 drug Substances 0.000 abstract description 10
- 230000009471 action Effects 0.000 abstract description 8
- 230000007246 mechanism Effects 0.000 abstract description 7
- 108020002494 acetyltransferase Proteins 0.000 abstract description 6
- 230000006696 biosynthetic metabolic pathway Effects 0.000 abstract description 4
- 238000012258 culturing Methods 0.000 abstract 1
- 210000003527 eukaryotic cell Anatomy 0.000 abstract 1
- 230000001737 promoting effect Effects 0.000 abstract 1
- 239000013598 vector Substances 0.000 description 15
- 239000000047 product Substances 0.000 description 13
- 241000233866 Fungi Species 0.000 description 9
- 210000004027 cell Anatomy 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 7
- 239000002299 complementary DNA Substances 0.000 description 7
- 229940079593 drug Drugs 0.000 description 6
- 239000012634 fragment Substances 0.000 description 6
- 241000971116 Geosmithia pallida Species 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 238000012163 sequencing technique Methods 0.000 description 5
- 230000014616 translation Effects 0.000 description 5
- 238000012795 verification Methods 0.000 description 5
- 238000012408 PCR amplification Methods 0.000 description 4
- ISAKRJDGNUQOIC-UHFFFAOYSA-N Uracil Chemical compound O=C1C=CNC(=O)N1 ISAKRJDGNUQOIC-UHFFFAOYSA-N 0.000 description 4
- 102000005421 acetyltransferase Human genes 0.000 description 4
- 230000003321 amplification Effects 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000003199 nucleic acid amplification method Methods 0.000 description 4
- 239000013612 plasmid Substances 0.000 description 4
- 238000003752 polymerase chain reaction Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- 241000588724 Escherichia coli Species 0.000 description 3
- 230000001580 bacterial effect Effects 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 230000002950 deficient Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- 239000013642 negative control Substances 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 2
- 101100010928 Saccharolobus solfataricus (strain ATCC 35092 / DSM 1617 / JCM 11322 / P2) tuf gene Proteins 0.000 description 2
- 101150001810 TEAD1 gene Proteins 0.000 description 2
- 101150074253 TEF1 gene Proteins 0.000 description 2
- 102100029898 Transcriptional enhancer factor TEF-1 Human genes 0.000 description 2
- 239000004480 active ingredient Substances 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229940088710 antibiotic agent Drugs 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001962 electrophoresis Methods 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- BRZYSWJRSDMWLG-CAXSIQPQSA-N geneticin Chemical compound O1C[C@@](O)(C)[C@H](NC)[C@@H](O)[C@H]1O[C@@H]1[C@@H](O)[C@H](O[C@@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](C(C)O)O2)N)[C@@H](N)C[C@H]1N BRZYSWJRSDMWLG-CAXSIQPQSA-N 0.000 description 2
- 239000008103 glucose Substances 0.000 description 2
- 230000006801 homologous recombination Effects 0.000 description 2
- 238000002744 homologous recombination Methods 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 230000000670 limiting effect Effects 0.000 description 2
- 239000007788 liquid Substances 0.000 description 2
- 238000000034 method Methods 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 239000013600 plasmid vector Substances 0.000 description 2
- 238000001243 protein synthesis Methods 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000011550 stock solution Substances 0.000 description 2
- 238000013519 translation Methods 0.000 description 2
- 230000005945 translocation Effects 0.000 description 2
- 229940035893 uracil Drugs 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- MNULEGDCPYONBU-WMBHJXFZSA-N (1r,4s,5e,5'r,6'r,7e,10s,11r,12s,14r,15s,16s,18r,19s,20r,21e,25s,26r,27s,29s)-4-ethyl-11,12,15,19-tetrahydroxy-6'-[(2s)-2-hydroxypropyl]-5',10,12,14,16,18,20,26,29-nonamethylspiro[24,28-dioxabicyclo[23.3.1]nonacosa-5,7,21-triene-27,2'-oxane]-13,17,23-trio Polymers O([C@@H]1CC[C@@H](/C=C/C=C/C[C@H](C)[C@@H](O)[C@](C)(O)C(=O)[C@H](C)[C@@H](O)[C@H](C)C(=O)[C@H](C)[C@@H](O)[C@H](C)/C=C/C(=O)O[C@H]([C@H]2C)[C@H]1C)CC)[C@]12CC[C@@H](C)[C@@H](C[C@H](C)O)O1 MNULEGDCPYONBU-WMBHJXFZSA-N 0.000 description 1
- MNULEGDCPYONBU-DJRUDOHVSA-N (1s,4r,5z,5'r,6'r,7e,10s,11r,12s,14r,15s,18r,19r,20s,21e,26r,27s)-4-ethyl-11,12,15,19-tetrahydroxy-6'-(2-hydroxypropyl)-5',10,12,14,16,18,20,26,29-nonamethylspiro[24,28-dioxabicyclo[23.3.1]nonacosa-5,7,21-triene-27,2'-oxane]-13,17,23-trione Polymers O([C@H]1CC[C@H](\C=C/C=C/C[C@H](C)[C@@H](O)[C@](C)(O)C(=O)[C@H](C)[C@@H](O)C(C)C(=O)[C@H](C)[C@H](O)[C@@H](C)/C=C/C(=O)OC([C@H]2C)C1C)CC)[C@]12CC[C@@H](C)[C@@H](CC(C)O)O1 MNULEGDCPYONBU-DJRUDOHVSA-N 0.000 description 1
- MNULEGDCPYONBU-YNZHUHFTSA-N (4Z,18Z,20Z)-22-ethyl-7,11,14,15-tetrahydroxy-6'-(2-hydroxypropyl)-5',6,8,10,12,14,16,28,29-nonamethylspiro[2,26-dioxabicyclo[23.3.1]nonacosa-4,18,20-triene-27,2'-oxane]-3,9,13-trione Polymers CC1C(C2C)OC(=O)\C=C/C(C)C(O)C(C)C(=O)C(C)C(O)C(C)C(=O)C(C)(O)C(O)C(C)C\C=C/C=C\C(CC)CCC2OC21CCC(C)C(CC(C)O)O2 MNULEGDCPYONBU-YNZHUHFTSA-N 0.000 description 1
- MNULEGDCPYONBU-VVXVDZGXSA-N (5e,5'r,7e,10s,11r,12s,14s,15r,16r,18r,19s,20r,21e,26r,29s)-4-ethyl-11,12,15,19-tetrahydroxy-6'-[(2s)-2-hydroxypropyl]-5',10,12,14,16,18,20,26,29-nonamethylspiro[24,28-dioxabicyclo[23.3.1]nonacosa-5,7,21-triene-27,2'-oxane]-13,17,23-trione Polymers C([C@H](C)[C@@H](O)[C@](C)(O)C(=O)[C@@H](C)[C@H](O)[C@@H](C)C(=O)[C@H](C)[C@@H](O)[C@H](C)/C=C/C(=O)OC([C@H]1C)[C@H]2C)\C=C\C=C\C(CC)CCC2OC21CC[C@@H](C)C(C[C@H](C)O)O2 MNULEGDCPYONBU-VVXVDZGXSA-N 0.000 description 1
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- MNULEGDCPYONBU-UHFFFAOYSA-N 4-ethyl-11,12,15,19-tetrahydroxy-6'-(2-hydroxypropyl)-5',10,12,14,16,18,20,26,29-nonamethylspiro[24,28-dioxabicyclo[23.3.1]nonacosa-5,7,21-triene-27,2'-oxane]-13,17,23-trione Polymers CC1C(C2C)OC(=O)C=CC(C)C(O)C(C)C(=O)C(C)C(O)C(C)C(=O)C(C)(O)C(O)C(C)CC=CC=CC(CC)CCC2OC21CCC(C)C(CC(C)O)O2 MNULEGDCPYONBU-UHFFFAOYSA-N 0.000 description 1
- 102000057234 Acyl transferases Human genes 0.000 description 1
- 108700016155 Acyl transferases Proteins 0.000 description 1
- 108010006654 Bleomycin Proteins 0.000 description 1
- 101100064707 Caenorhabditis elegans eef-2 gene Proteins 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- ROHFNLRQFUQHCH-YFKPBYRVSA-N L-leucine Chemical compound CC(C)C[C@H](N)C(O)=O ROHFNLRQFUQHCH-YFKPBYRVSA-N 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- ROHFNLRQFUQHCH-UHFFFAOYSA-N Leucine Natural products CC(C)CC(N)C(O)=O ROHFNLRQFUQHCH-UHFFFAOYSA-N 0.000 description 1
- 101100062121 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) cyc-1 gene Proteins 0.000 description 1
- 108091005461 Nucleic proteins Proteins 0.000 description 1
- 239000001888 Peptone Substances 0.000 description 1
- 108010080698 Peptones Proteins 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 241000187392 Streptomyces griseus Species 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- RIOXQFHNBCKOKP-UHFFFAOYSA-N benomyl Chemical compound C1=CC=C2N(C(=O)NCCCC)C(NC(=O)OC)=NC2=C1 RIOXQFHNBCKOKP-UHFFFAOYSA-N 0.000 description 1
- MITFXPHMIHQXPI-UHFFFAOYSA-N benzoxaprofen Natural products N=1C2=CC(C(C(O)=O)C)=CC=C2OC=1C1=CC=C(Cl)C=C1 MITFXPHMIHQXPI-UHFFFAOYSA-N 0.000 description 1
- 229960001561 bleomycin Drugs 0.000 description 1
- OYVAGSVQBOHSSS-UAPAGMARSA-O bleomycin A2 Chemical compound N([C@H](C(=O)N[C@H](C)[C@@H](O)[C@H](C)C(=O)N[C@@H]([C@H](O)C)C(=O)NCCC=1SC=C(N=1)C=1SC=C(N=1)C(=O)NCCC[S+](C)C)[C@@H](O[C@H]1[C@H]([C@@H](O)[C@H](O)[C@H](CO)O1)O[C@@H]1[C@H]([C@@H](OC(N)=O)[C@H](O)[C@@H](CO)O1)O)C=1N=CNC=1)C(=O)C1=NC([C@H](CC(N)=O)NC[C@H](N)C(N)=O)=NC(N)=C1C OYVAGSVQBOHSSS-UAPAGMARSA-O 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 208000035475 disorder Diseases 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000009472 formulation Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 238000012268 genome sequencing Methods 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 230000002363 herbicidal effect Effects 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 230000005764 inhibitory process Effects 0.000 description 1
- 230000002452 interceptive effect Effects 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000005649 metathesis reaction Methods 0.000 description 1
- 238000012543 microbiological analysis Methods 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 238000010369 molecular cloning Methods 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 230000017095 negative regulation of cell growth Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 108020004707 nucleic acids Proteins 0.000 description 1
- 102000039446 nucleic acids Human genes 0.000 description 1
- 150000007523 nucleic acids Chemical class 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 229930191479 oligomycin Natural products 0.000 description 1
- MNULEGDCPYONBU-AWJDAWNUSA-N oligomycin A Polymers O([C@H]1CC[C@H](/C=C/C=C/C[C@@H](C)[C@H](O)[C@@](C)(O)C(=O)[C@@H](C)[C@H](O)[C@@H](C)C(=O)[C@@H](C)[C@H](O)[C@@H](C)/C=C/C(=O)O[C@@H]([C@@H]2C)[C@@H]1C)CC)[C@@]12CC[C@H](C)[C@H](C[C@@H](C)O)O1 MNULEGDCPYONBU-AWJDAWNUSA-N 0.000 description 1
- 235000019319 peptone Nutrition 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 108090000765 processed proteins & peptides Proteins 0.000 description 1
- 210000003705 ribosome Anatomy 0.000 description 1
- 210000004708 ribosome subunit Anatomy 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 239000013049 sediment Substances 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- 238000010200 validation analysis Methods 0.000 description 1
- 239000007222 ypd medium Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/10—Transferases (2.)
- C12N9/1025—Acyltransferases (2.3)
- C12N9/1029—Acyltransferases (2.3) transferring groups other than amino-acyl groups (2.3.1)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1096—Processes for the isolation, preparation or purification of DNA or RNA cDNA Synthesis; Subtracted cDNA library construction, e.g. RT, RT-PCR
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Mycology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Crystallography & Structural Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了基因GNATB作为筛选标记基因在抗性筛选中的应用及其应用。基因GNATB的核苷酸序列如SEQ ID NO.1所示。本发明的新型筛选标记基因GNATB能够高效协助酿酒酵母抵抗放线菌酮，从而为后期在酿酒酵母重构放线菌酮的生物合成通路和解析筛选标记基因GNATB与放线菌酮的作用机制奠定基础，提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础。此外，乙酰转移酶基因GNATB可开发成一种高效表达且高抗的筛选标记基因，用来筛选和维持培养成功转染放线菌酮抗性基因的真核细胞，应用于分子生物学、微生物学和医药等领域。
Description
技术领域
本发明属于基因工程领域，具体涉及基因GNATB作为筛选标记基因在抗性筛选中的应用。
背景技术
筛选标记基因可使转化子获得自身所不具备的新的遗传特性；是遗传转化载体所必备的基本元件；是利用特定的选择培养基筛选转化子的一类特殊标记基因。抗性筛选是将抗性基因转入受体菌中使其在一定药物浓度下生长而表现抗药性的一类筛选方式。常用的抗性筛选标记基因分为抗生素抗性基因和除草剂抗性基因两类。抗生素类药物对细胞生长的抑制作用主要在于影响细胞壁的形成和细胞膜的功能、抑制核酸或蛋白质的生物合成。常用的抗生素包括潮霉素、遗传霉素、寡霉素、博来霉素及苯菌灵。
放线菌酮(Cycloheximide)是灰色链霉菌(Streptomyces griseus)的一种产物，一种在真核生物中对蛋白质生物合成过程有抑制效应的化合物，它通过干扰蛋白质合成过程中的易位步骤而阻碍翻译过程。放线菌酮抗性由许多不同的基因产生，高水平的抗性常由编码L29核糖体亚基的cyh2基因座发生突变产生，在生物科研领域用于mRNA的合成，阻止肽链的进一步延伸。此外由于放线菌酮的生物合成研宄却是一片空白，这就极大的阻碍了通过组合生物合成对高活性放线菌酮类似物进行进一步改造和利用。据报道放线菌酮的有效成分环己酰亚胺(CHX)能结合核糖体并抑制eEF2介导的易位。令人惊讶的是，环己酰亚胺允许在停止任何进一步延伸之前进行一个完整的易位循环。推测环己酰亚胺需要E位点结合的脱酰基tRNA用于活性。目前尚未有放线菌酮抗性基因的相关报道，也尚未有深海真菌次级代谢产物生物合成基因作为筛选标记的相关研究。
发明内容
本发明的第一个目的是提供基因GNATB作为筛选标记基因在抗性筛选中的应用，所述的基因GNATB，其核苷酸序列如SEQ ID NO.1所示。
本发明根据深海真菌G.pallid FS140基因组测序结果显示，GNATB基因编码乙酰转移酶，通过成功导入酿酒酵母进行验证，发现乙酰转移酶GNATB能够高效协助酿酒酵母抵抗放线菌酮，从而为后期在酿酒酵母重构放线菌酮的生物合成通路和解析GNATB基因与放线菌酮的作用机制奠定基础；提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础。推测作用机制为GNATB基因能乙酰化放线菌酮的有效成分环己酰亚胺(CHX)抑制蛋白质合成，阻断真核翻译的延伸期，具体的作用机制有待进一步深入研究。
本发明抗放线菌酮的新型筛选标记基因GNATB通过以下方法获得的：通过转录组测序结果预测编码乙酰转移酶基因GNATB的序列，在其上下游设计特异性引物，其引物序列为GNATB-F:5'-ATGTCCTACCAGTCTACAATCTACACCT-3'；GNATB-R:5'-CTACACTATATCCTTATCGCAACCCACC-3'，以由深海真菌FS140转录组反转录而得的cDNA文库为模板，通过PCR扩增获得产物并纯化回收片段，获得目的基因GNATB，其核苷酸序列如SEQ IDNO.1所示。
本发明利用同源重组法将GNATB基因插入到酵母载体YEp352-TEF1-CYC1的表达盒内部。首先设计含有同源臂的GNATB基因的上下游引物，其引物序列为YEp352-GNATB-F:5'-TAGCAATCTAATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GNATB-R:5'-TACATGAT GCGGCCCGTCGACCTACACTATATCCTTATCGCAACCCA-3(下划线序列为同源臂片段)，通过PCR扩增获得产物并纯化回收片段。对已构建的YEp352-TEF1-CYC1载体采用内切酶Sal I和Xba I双酶切，然后使用ClonExpress II One Step Cloning Kit C112(Vazyme)将片段和酶切载体重组连接并转化至大肠杆菌感受态细胞，涂布于氨苄青霉素抗性平板筛选出阳性克隆。经过此轮分子克隆，目的基因GNATB(其核苷酸序列如SEQ ID NO.1所示)插入到启动子TEF1和终止子CYC1之间，构建得到YEp352-TEF1-GNATB载体，将其电转入酿酒酵母BJ5464细胞中，利用尿嘧啶缺陷型的SD培养基平板进行筛选和验证。与转入YEp352-TEF1-CYC1质粒(阴性对照)的酿酒酵母BJ5464相比，含有重组载体YEp352-TEF1-GNATB的酿酒酵母生长速度明显加快，相同培养时间内菌落密度更高，证明功能基因GNATB能有效协助酿酒酵母抵抗放线菌酮，从而为后期在酿酒酵母重构放线菌酮的生物合成通路和解析筛选标记基因GNATB与放线菌酮的作用机制奠定基础，提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础。
优选，所述的基因GNATB作为筛选标记基因在放线菌酮抗性筛选中的应用。
本发明的第二个目的是提供基因GNATB在协助宿主细胞在抵抗抗生素中的应用。
所述的抗生素为放线菌酮。
所述的宿主细胞优选为深海真菌Geosmithia pallida FS140或酿酒酵母Saccharomyces cerevisiae BJ5464。
与现有技术相比，本发明具有以下有益效果：
本发明所涉及的深海真菌G.pallida FS140分离自南海沉积物，本课题组前期对该菌株进行了转录组测序并对基因进行了注释。鉴于目前尚未有关于深海真菌基因作为分子筛选标记的相关报道。因此本发明从深海真菌FS140的cDNA文库中获得了筛选标记GNATB基因序列，并成功导入到酿酒酵母S.cerevisiae BJ5464中通过放线菌酮药物筛选进行功能验证，为后期在酿酒酵母重构放线菌酮的生物合成通路和解析筛选标记基因GNATB与放线菌酮的作用机制奠定基础，提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础，同时可开发成一种高效表达且高抗的筛选标记基因应用于分子生物学、微生物学和医药等领域。
本发明的深海真菌G.pallida FS140，其公开于文献：Zhang-Hua Sun,JiangyongGu,Wei Ye,Liang-Xi Wen,Qi-Bin Lin,Sai-Ni Li,Yu-Chan Chen,Hao-Hua Li,Wei-MinZhang.Geospallins A–C:New Thiodiketopiperazines with Inhibitory Activityagainst Angiotensin-Converting Enzyme from a Deep-Sea-Derived FungusGeosmithia pallida FS140.Marine Drugs,2018,16(12),464.https://doi.org/10.3390/md16120464。该菌种本申请人也持有，保证自发明的申请日起20年内向公众提供。
附图说明
图1是筛选标记GNATB基因序列的获得：以FS140 cDNA文库为模板，基因GNATB扩增产物的电泳图；
图2为实验所使用放线菌酮结构式及酿酒酵母BJ5464和酿酒酵母YEp352-TEF1-GNATB(GNATB)在YPD平板和含不同浓度的放线菌酮的YPD平板中培养30h的效果图。
图3为重组载体YEp352-TEF1-GNATB的构建；其中A为YEp352-TEF1-CYC1载体图谱；B为YEp352-TEF1-GNATB载体图谱；C为基因GNATB的菌落PCR扩增产物的电泳图；
图4是GNATB编码的蛋白质跨膜螺旋进行预测。
具体实施方式
以下实施例是对本发明的进一步说明，而不是对本发明的限制。
本实施例中所用的SD固体培养基的配方为：每升含有葡萄糖20g、Do supplement0.62g(-Leu/-Trp/-Ura，Clontech)、无氨基酵母氮源YNB 6.7g(普博欣)、亮氨酸0.06g、色氨酸0.04g和琼脂粉20g，余量为蒸馏水，其配制方法是将各成分混合均匀，灭菌制得。
本实施例中所用的YPD固体培养基的配方为：每升含有酵母粉10g、蛋白胨20g、葡萄糖20g和琼脂粉20g，余量为蒸馏水，其配制方法是将各成分混合均匀，灭菌制得。
本实施例中所用抗生素:放线菌酮购于Dr.Ehrenstorfer。
实施例1抗放线菌酮的新型筛选标记GNATB基因序列的获得
基因GNATB的扩增：将深海真菌G.pallida FS140接种于YPD培养基平板，于37℃培养72h，挑取新鲜的菌丝体，利用真菌RNA提取试剂盒提取RNA，再用All-in-one RT MasterKit逆转录获得cDNA。根据转录组测序结果预测编码乙酰转移酶GNATB基因序列，设计上下游引物YEp352-GNATB-F和YEp352-GNATB-R，其引物序列为YEp352-GNATB-F:5'-TAGCAATCT AATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GNATB-R:5'-TACATGATGCGGCCCGT CGACCTACACTATATCCTTATCGCAACCCA-3(下划线序列为同源臂片段)，以cDNA文库为模板扩增，获得PCR产物(图1)。回收产物并用pEASY-T1试剂盒进行TA克隆，转化至大肠杆菌感受态细胞，涂布于氨苄青霉素抗性平板筛选出阳性克隆，以通用引物M13-F(5′-GTAAAACGACGGCCAGT-3′)和M13-R(5′-CAGGAAACAGCTATGAC-3′)进行菌液PCR验证阳性克隆并测序，获得目的基因GNATB序列(其核苷酸序列如SEQ ID NO.1所示，atgtcctaccagtctacaatctacaccttcatcttcggctctcgcaccatacaatacaactacaaagtcagcaaacccaaaccaacagacagaaaaaaaaaaacataccttgcatgcatcccaaccatccacacactcaatctccctcagcaaccgaaaccccatcttctcatagatcaccagattgggcatccccttggaactctccaggtaacacggcaccccctccgcatccgcacgactggtcacaacctcaaccagtctcctcccgacccccatcccgcgcatctccgcactcaccgcaaccacattgcagaagtagtaaccccggtcatccgtccagagatcctgatgggtgcgcgcctgcagatccttccagatccagtaccggcggacattaagcccgccgcggccccagaaccggaggttgtacaggagctggcggacggagagtagccagtcctgggaccaggacgtccaagaaggggactgggaaggtggatgcggggcgaaccaccagcagacgccgacgatgggggatgagggtcgggggtgtttgtcagtggcagtggggcggtatttggcgatgtagatgggggcgttgcagctgaggccgtggaggaaatgggcggttatggaggctgcgttgcgttgggggttgtactacttgcattaacgggttgttttttttttttaaggagaagaggacgtactttggatgggtcatcaaagacccagaggaagtacgggtcgtcggcgaaggtcttctggatgcaggctacggcatcggggatgtcttctttggtcaatgggaggatatcgatagccatttttgagggagttcaatctctactggctctgcgggtgggttgcgataaggatatagtgtag)。通过生物学在线软件工具TMHMM Server,v.2.0对基因GNATB编码的蛋白质跨膜螺旋进行预测(图4)，结果表明GNATB基因有1个跨膜组分，其功能可推测乙酰转移酶GNATB乙酰化环己酰亚胺为低活性化合物从而保护宿主免受药物作用方面发挥着重要作用。
实施例2抗放线菌酮的新型筛选标记GNATB的功能验证
利用同源重组法将GNATB基因插入到酵母载体YEp352-TEF1-CYC1中(YEp352-TEF1-CYC1为早期构建质粒，携带有组成型启动子TEF1和终止子CYC1，载体图谱见图3A，为现有技术中的已知产品：Xiaodan Ouyang,Yaping Cha,Wen Li,Chaoyi Zhu,Muzi Zhu,Shuang Li,Min Zhuo,Shaobin Huang and Jianjun Li.Stepwise engineering ofSaccharomycescerevisiae to produce(+)-valencene and its relatedsesquiterpenes,RSC Adv.,2019,9,30171,DOI:10.1039/c9ra05558d)。首先设计针对基因GNATB(SEQ ID NO.1)扩增的上下游引物YEp352-GNATB-F和YEp352-GNATB-R，其引物序列为YEp352-GNATB-F:5'-TAGCAATCTAATCTAAGTCTAGAATGTCCTACCAGTCTACAATCTA-3'；YEp352-GNATB-R:5'-TACATGATGCGGCCCGTCGACCTACACTATATCCTTATCGCAACCCA-3'(下划线序列为同源臂片段)，以cDNA文库为模板扩增，通过PCR扩增获得产物。对载体YEp352-TEF1-CYC1采用Sal I和Xba I双酶切并回收产物，然后使用ClonExpress II One Step Cloning Kit C112(Vazyme)将两个产物重组连接并转化至DH5α中筛选阳性克隆。采用引物YEp352-GNATB-F和YEp352-GNATB-R进行菌落PCR验证，结果表明基因GNATB成功插入YEp352-TEF1-CYC1载体中(图3C)，并通过测序予以确认，得到YEp352-TEF1-GNATB载体(载体图谱见图3B)。
将YEp352-TEF1-GNATB质粒载体以及YEp352-TEF1-CYC1质粒载体(阴性对照)分别电转入酿酒酵母BJ5464细胞中(1500V，5ms)，均匀涂布于尿嘧啶缺陷型的SD平板中，在30℃培养2d，利用菌落PCR筛选阳性克隆，获得分别含有YEp352-TEF1-GNATB质粒以及YEp352-TEF1-CYC1质粒的酿酒酵母BJ5464细胞。
分别将酿酒酵母BJ5464(YEp352-TEF1-CYC1)、酿酒酵母BJ5464(YEp352-TEF1-GNATB)接种于相应缺陷型的SD培养基中，在30℃培养2d。用分光光度计测量各菌液OD600，将各菌液用无菌水稀释到OD600≈1.0作为原液，再以100μL的原液加900μL的无菌水的方式稀释成10-1，以同样的方式稀释成10-2、10-3、10-4。各取100μL不同菌株的10-4的稀释液分别在YPD无抗平板、各种浓度(100μg/mL、250μg/mL和500μg/mL)放线菌酮平板涂板，在30℃培养并实时观察。培养24h的平板结果显示(图2)，BJ5464为酿酒酵母BJ5464(YEp352-TEF1-CYC1)、GNATB为酿酒酵母BJ5464(YEp352-TEF1-GNATB)，酿酒酵母BJ5464(YEp352-TEF1-CYC1)和酿酒酵母BJ5464(YEp352-TEF1-GNATB)在不添加抗生素的YPD平板上生长状况近乎一致，但在含有放线菌酮的YPD平板上，阴性对照BJ5464(YEp352-TEF1-CYC1)生长均明显受阻，没有菌落生长。而导入酰基转移酶GNATB基因的酿酒酵母则生长良好，菌体密度与正常酿酒酵母相当，说明来源于深海真菌Geosmithia pallida FS140的GNATB基因部分或全部恢复了酿酒酵母BJ5464对添加放线菌酮的耐受性(耐受浓度＞500μg/mL)，有效帮助酿酒酵母在含有放线菌酮的环境下正常生长。
本发明从深海真菌FS140的cDNA文库中获得了筛选标记GNATB基因序列，并成功导入到酿酒酵母S.cerevisiae BJ5464中通过放线菌酮药物筛选进行功能验证，为后期在酿酒酵母重构放线菌酮的生物合成通路和解析筛选标记基因GNATB与放线菌酮的作用机制奠定基础，提升放线菌酮的异源表达水平并获得新型放线菌酮奠定分子生物学基础，同时可开发成一种高效表达且高抗的筛选标记基因应用于分子生物学、微生物学和医药等领域。
以上仅是本发明的优选实施方式，应当指出的是，上述优选实施方式不应视为对本发明的限制，本发明的保护范围应当以权利要求所限定的范围为准。对于本技术领域的普通技术人员来说，在不脱离本发明的精神和范围内，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。
序列表
<110> 广东省微生物研究所（广东省微生物分析检测中心）
<120> 基因GNATB作为筛选标记基因在抗性筛选中的应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 891
<212> DNA
<213> 深海真菌FS140(Geosmithia pallida)
<400> 1
atgtcctacc agtctacaat ctacaccttc atcttcggct ctcgcaccat acaatacaac 60
tacaaagtca gcaaacccaa accaacagac agaaaaaaaa aaacatacct tgcatgcatc 120
ccaaccatcc acacactcaa tctccctcag caaccgaaac cccatcttct catagatcac 180
cagattgggc atccccttgg aactctccag gtaacacggc accccctccg catccgcacg 240
actggtcaca acctcaacca gtctcctccc gacccccatc ccgcgcatct ccgcactcac 300
cgcaaccaca ttgcagaagt agtaaccccg gtcatccgtc cagagatcct gatgggtgcg 360
cgcctgcaga tccttccaga tccagtaccg gcggacatta agcccgccgc ggccccagaa 420
ccggaggttg tacaggagct ggcggacgga gagtagccag tcctgggacc aggacgtcca 480
agaaggggac tgggaaggtg gatgcggggc gaaccaccag cagacgccga cgatggggga 540
tgagggtcgg gggtgtttgt cagtggcagt ggggcggtat ttggcgatgt agatgggggc 600
gttgcagctg aggccgtgga ggaaatgggc ggttatggag gctgcgttgc gttgggggtt 660
gtactacttg cattaacggg ttgttttttt tttttaagga gaagaggacg tactttggat 720
gggtcatcaa agacccagag gaagtacggg tcgtcggcga aggtcttctg gatgcaggct 780
acggcatcgg ggatgtcttc tttggtcaat gggaggatat cgatagccat ttttgaggga 840
gttcaatctc tactggctct gcgggtgggt tgcgataagg atatagtgta g 891
Claims (2)
1.基因GNATB作为筛选标记基因在转入宿主细胞中筛选放线菌酮抗性中的应用，所述的基因GNATB，其核苷酸序列如SEQ ID NO.1所示，所述的宿主细胞为深海真菌Geosmithia pallida FS140或酿酒酵母Saccharomyces cerevisiae BJ5464。
2.基因GNATB在构建抵抗抗生素的宿主细胞中的应用，所述的基因GNATB，其核苷酸序列如SEQ ID NO.1所示，所述的抗生素为放线菌酮；所述的宿主细胞为深海真菌Geosmithia pallida FS140或酿酒酵母Saccharomyces cerevisiae BJ5464。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011552186.3A CN112575013B (zh) | 2020-12-24 | 2020-12-24 | 基因gnatb作为筛选标记基因在抗性筛选中的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011552186.3A CN112575013B (zh) | 2020-12-24 | 2020-12-24 | 基因gnatb作为筛选标记基因在抗性筛选中的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112575013A CN112575013A (zh) | 2021-03-30 |
| CN112575013B true CN112575013B (zh) | 2022-05-17 |
Family
ID=75139602
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011552186.3A Active CN112575013B (zh) | 2020-12-24 | 2020-12-24 | 基因gnatb作为筛选标记基因在抗性筛选中的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112575013B (zh) |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4857460A (en) * | 1985-08-27 | 1989-08-15 | Nikka Whisky Distilling Co., Ltd. | Cycloheximide resistant gene |
| US5849524A (en) * | 1994-05-25 | 1998-12-15 | Kirin Beer Kabushiki Kaisha | Transformation systems for the yeast candida utilis and the expression of heterologous genes therewith |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2002254203A1 (en) * | 2001-03-12 | 2002-09-24 | The General Hospital Corporation | Master activators of pathogen responsive genes |
| US8557746B2 (en) * | 2009-04-01 | 2013-10-15 | The General Hospital Corporation | High throughput screens for small-molecule inhibitors of the nuclear receptor-like pathway regulating multidrug resistance in fungi |
| JP7099967B2 (ja) * | 2016-07-01 | 2022-07-12 | リサーチ ディベロップメント ファウンデーション | 幹細胞由来移植片からの増殖性細胞の排除 |
-
2020
- 2020-12-24 CN CN202011552186.3A patent/CN112575013B/zh active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4857460A (en) * | 1985-08-27 | 1989-08-15 | Nikka Whisky Distilling Co., Ltd. | Cycloheximide resistant gene |
| US5849524A (en) * | 1994-05-25 | 1998-12-15 | Kirin Beer Kabushiki Kaisha | Transformation systems for the yeast candida utilis and the expression of heterologous genes therewith |
Non-Patent Citations (2)
| Title |
|---|
| Geospallins A–C: New Thiodiketopiperazines with Inhibitory Activity against Angiotensin-Converting Enzyme from a Deep-Sea-Derived Fungus Geosmithia pallida FS140;Zhang-Hua Sun et al.;《Marine Drugs》;20181231;第16卷(第12期);文献号：464 * |
| Puromycin resistance gene as an effective selection marker for ciliate Tetrahymena;Masaaki Iwamoto et al.;《Gene》;20140125;第534卷(第2期);第249-255页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112575013A (zh) | 2021-03-30 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Cacho et al. | Identification and characterization of the echinocandin B biosynthetic gene cluster from Emericella rugulosa NRRL 11440 | |
| CN110904125B (zh) | 一种露湿漆斑菌A553抗单端孢霉烯自我保护基因mfs1及其应用 | |
| CN107142272A (zh) | 一种控制大肠杆菌中质粒复制的方法 | |
| Amiri | Fibrillarin-like proteins occur in the domain Archaea | |
| Mankin et al. | Chloramphenicol resistance mutations in the single 23S rRNA gene of the archaeon Halobacterium halobium | |
| Wetzel et al. | The mating-related loci sexM and sexP of the zygomycetous fungus Mucor mucedo and their transcriptional regulation by trisporoid pheromones | |
| CN111019945B (zh) | 一种露湿漆斑菌A553单端孢霉烯合酶基因Tri12启动子及其应用 | |
| CN106916837B (zh) | 高渗透压甘油蛋白激酶基因RkHog1及其重组表达载体 | |
| CN114277046B (zh) | 一种合成四氢嘧啶的三基因串联表达载体及应用 | |
| CN112608931B (zh) | 一种深海真菌FS140抗胶霉毒素自我保护基因GliM及其应用 | |
| CN110343709B (zh) | 一种北极拟诺卡氏菌套索肽基因簇及其克隆与表达方法 | |
| CN112575013B (zh) | 基因gnatb作为筛选标记基因在抗性筛选中的应用 | |
| CN111117942B (zh) | 一种产林可霉素的基因工程菌及其构建方法和应用 | |
| CN112680469B (zh) | 抗胶霉毒素自我保护基因GliK在协助宿主细胞抵抗胶霉毒素中的应用 | |
| JP7252536B2 (ja) | ポリリジン生産菌及びそれを用いるポリリジン製造方法 | |
| WO2006046694A1 (ja) | 誘導可能に発現するエルゴステロール合成酵素を有する酵母を利用した遺伝子のスクリーニング法 | |
| US8927254B2 (en) | Pyrococcus furiosus strains and methods of using same | |
| CN112592954B (zh) | 基因GliT作为筛选标记基因在抗性筛选中的应用 | |
| WO2015092575A1 (en) | Gene cluster for biosynthesis of pamamycin | |
| Yoko-o et al. | Mating type switching, formation of diploids, and sporulation in the methylotrophic yeast Ogataea minuta | |
| CN112695044B (zh) | 一种深海真菌FS140抗胶霉毒素自我保护基因mfs-get及其应用 | |
| CN112626046A (zh) | 一种能抗b型和d型单端孢霉烯族毒素的酰基转移酶gantb及其应用 | |
| CN112725337B (zh) | 一种深海真菌FS140氧化还原酶基因GliT启动子及其应用 | |
| EP3802783A1 (en) | Microorganisms and the production of fine chemicals | |
| KR101550217B1 (ko) | 숙주세포의 생체 회로 간섭 없이 외래 유전자를 발현하기 위한 재조합 벡터 및 이의 용도 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CP01 | Change in the name or title of a patent holder |
Address after: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee after: Institute of Microbiology, Guangdong Academy of Sciences Address before: 510070 No.56 courtyard, No.100 Xianlie Middle Road, Yuexiu District, Guangzhou City, Guangdong Province Patentee before: GUANGDONG INSTITUTE OF MICROBIOLOGY (GUANGDONG DETECTION CENTER OF MICROBIOLOGY) |
|
| CP01 | Change in the name or title of a patent holder |