CN109022299B - 一种erg1基因缺陷酵母工程菌、其构建方法及其运用 - Google Patents
一种erg1基因缺陷酵母工程菌、其构建方法及其运用 Download PDFInfo
- Publication number
- CN109022299B CN109022299B CN201810896549.1A CN201810896549A CN109022299B CN 109022299 B CN109022299 B CN 109022299B CN 201810896549 A CN201810896549 A CN 201810896549A CN 109022299 B CN109022299 B CN 109022299B
- Authority
- CN
- China
- Prior art keywords
- erg1
- gene
- yeast
- leu
- squalene epoxidase
- 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
- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 103
- 241000894006 Bacteria Species 0.000 title claims abstract description 77
- 101150093680 ERG1 gene Proteins 0.000 title claims abstract description 53
- 230000007547 defect Effects 0.000 title claims abstract description 31
- 238000010276 construction Methods 0.000 title abstract description 7
- 108020003891 Squalene monooxygenase Proteins 0.000 claims abstract description 36
- 108091033409 CRISPR Proteins 0.000 claims abstract description 21
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 94
- 108020004414 DNA Proteins 0.000 claims description 43
- 210000004027 cell Anatomy 0.000 claims description 36
- 108090000623 proteins and genes Proteins 0.000 claims description 35
- 101001047090 Homo sapiens Potassium voltage-gated channel subfamily H member 2 Proteins 0.000 claims description 31
- 235000015398 thunder god vine Nutrition 0.000 claims description 25
- 108020005004 Guide RNA Proteins 0.000 claims description 24
- 238000000034 method Methods 0.000 claims description 24
- 230000006870 function Effects 0.000 claims description 22
- 241000830536 Tripterygium wilfordii Species 0.000 claims description 20
- 102000005782 Squalene Monooxygenase Human genes 0.000 claims description 13
- OILXMJHPFNGGTO-UHFFFAOYSA-N (22E)-(24xi)-24-methylcholesta-5,22-dien-3beta-ol Natural products C1C=C2CC(O)CCC2(C)C2C1C1CCC(C(C)C=CC(C)C(C)C)C1(C)CC2 OILXMJHPFNGGTO-UHFFFAOYSA-N 0.000 claims description 12
- RQOCXCFLRBRBCS-UHFFFAOYSA-N (22E)-cholesta-5,7,22-trien-3beta-ol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CCC(C)C)CCC33)C)C3=CC=C21 RQOCXCFLRBRBCS-UHFFFAOYSA-N 0.000 claims description 12
- OQMZNAMGEHIHNN-UHFFFAOYSA-N 7-Dehydrostigmasterol Natural products C1C(O)CCC2(C)C(CCC3(C(C(C)C=CC(CC)C(C)C)CCC33)C)C3=CC=C21 OQMZNAMGEHIHNN-UHFFFAOYSA-N 0.000 claims description 12
- DNVPQKQSNYMLRS-NXVQYWJNSA-N Ergosterol Natural products CC(C)[C@@H](C)C=C[C@H](C)[C@H]1CC[C@H]2C3=CC=C4C[C@@H](O)CC[C@]4(C)[C@@H]3CC[C@]12C DNVPQKQSNYMLRS-NXVQYWJNSA-N 0.000 claims description 12
- 102100025560 Squalene monooxygenase Human genes 0.000 claims description 12
- DNVPQKQSNYMLRS-SOWFXMKYSA-N ergosterol Chemical compound C1[C@@H](O)CC[C@]2(C)[C@H](CC[C@]3([C@H]([C@H](C)/C=C/[C@@H](C)C(C)C)CC[C@H]33)C)C3=CC=C21 DNVPQKQSNYMLRS-SOWFXMKYSA-N 0.000 claims description 12
- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 claims description 10
- 239000001301 oxygen Substances 0.000 claims description 10
- 229910052760 oxygen Inorganic materials 0.000 claims description 10
- 241000235342 Saccharomycetes Species 0.000 claims description 9
- 239000013604 expression vector Substances 0.000 claims description 9
- 230000014509 gene expression Effects 0.000 claims description 8
- 238000011084 recovery Methods 0.000 claims description 8
- 239000007787 solid Substances 0.000 claims description 8
- 102000053602 DNA Human genes 0.000 claims description 6
- 238000000137 annealing Methods 0.000 claims description 6
- 238000010367 cloning Methods 0.000 claims description 6
- 230000009466 transformation Effects 0.000 claims description 5
- 108090000790 Enzymes Proteins 0.000 claims description 4
- 230000034431 double-strand break repair via homologous recombination Effects 0.000 claims description 4
- 102000003960 Ligases Human genes 0.000 claims description 3
- 108090000364 Ligases Proteins 0.000 claims description 3
- 230000008827 biological function Effects 0.000 claims description 3
- 230000002950 deficient Effects 0.000 claims description 3
- 238000013461 design Methods 0.000 claims description 3
- 238000012795 verification Methods 0.000 claims description 3
- 210000005253 yeast cell Anatomy 0.000 claims 2
- 238000010354 CRISPR gene editing Methods 0.000 abstract description 5
- 238000005516 engineering process Methods 0.000 abstract description 4
- 238000010362 genome editing Methods 0.000 abstract description 4
- 238000012217 deletion Methods 0.000 abstract description 2
- 230000037430 deletion Effects 0.000 abstract description 2
- 230000035772 mutation Effects 0.000 abstract description 2
- 230000008439 repair process Effects 0.000 abstract description 2
- 101100445499 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) erg-1 gene Proteins 0.000 abstract 1
- 239000000243 solution Substances 0.000 description 37
- 239000000499 gel Substances 0.000 description 19
- 102100022807 Potassium voltage-gated channel subfamily H member 2 Human genes 0.000 description 18
- 239000002609 medium Substances 0.000 description 18
- 239000013612 plasmid Substances 0.000 description 18
- 239000001963 growth medium Substances 0.000 description 17
- 239000000047 product Substances 0.000 description 17
- 239000007788 liquid Substances 0.000 description 15
- 238000006243 chemical reaction Methods 0.000 description 14
- 238000001179 sorption measurement Methods 0.000 description 14
- 241000196324 Embryophyta Species 0.000 description 13
- 238000012216 screening Methods 0.000 description 12
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 11
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 10
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 10
- 238000000746 purification Methods 0.000 description 10
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 9
- 101100008069 Rattus norvegicus Cuzd1 gene Proteins 0.000 description 9
- 101100517253 Rattus norvegicus Nsf gene Proteins 0.000 description 9
- 101150076597 SQLE gene Proteins 0.000 description 9
- 229930182558 Sterol Natural products 0.000 description 9
- 150000003432 sterols Chemical class 0.000 description 9
- 235000003702 sterols Nutrition 0.000 description 9
- 239000013598 vector Substances 0.000 description 9
- 238000012360 testing method Methods 0.000 description 8
- GEWDNTWNSAZUDX-WQMVXFAESA-N (-)-methyl jasmonate Chemical compound CC\C=C/C[C@@H]1[C@@H](CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-WQMVXFAESA-N 0.000 description 7
- 230000001580 bacterial effect Effects 0.000 description 7
- 238000005119 centrifugation Methods 0.000 description 7
- 238000012258 culturing Methods 0.000 description 7
- 238000000605 extraction Methods 0.000 description 7
- 239000012530 fluid Substances 0.000 description 7
- 239000012634 fragment Substances 0.000 description 7
- GEWDNTWNSAZUDX-UHFFFAOYSA-N methyl 7-epi-jasmonate Natural products CCC=CCC1C(CC(=O)OC)CCC1=O GEWDNTWNSAZUDX-UHFFFAOYSA-N 0.000 description 7
- 235000015097 nutrients Nutrition 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- 239000006228 supernatant Substances 0.000 description 7
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 229920000297 Rayon Polymers 0.000 description 6
- 230000015572 biosynthetic process Effects 0.000 description 6
- 235000019441 ethanol Nutrition 0.000 description 6
- 108700026244 Open Reading Frames Proteins 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000000284 extract Substances 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 230000008569 process Effects 0.000 description 5
- 108091008146 restriction endonucleases Proteins 0.000 description 5
- PUERAZFQIXLYDR-CQNOQWDGSA-N 3-[(4e,8e,12e,16e)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenylidene]oxetane Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CC\C=C(/C)CC\C=C(/C)CCC=C1COC1 PUERAZFQIXLYDR-CQNOQWDGSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 239000012148 binding buffer Substances 0.000 description 4
- 239000003153 chemical reaction reagent Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- 238000011161 development Methods 0.000 description 4
- 239000003292 glue Substances 0.000 description 4
- 230000006801 homologous recombination Effects 0.000 description 4
- 238000002744 homologous recombination Methods 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 238000002360 preparation method Methods 0.000 description 4
- 150000003648 triterpenes Chemical class 0.000 description 4
- 239000011534 wash buffer Substances 0.000 description 4
- 238000005406 washing Methods 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- YYGNTYWPHWGJRM-UHFFFAOYSA-N (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene Chemical compound CC(C)=CCCC(C)=CCCC(C)=CCCC=C(C)CCC=C(C)CCC=C(C)C YYGNTYWPHWGJRM-UHFFFAOYSA-N 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 241000588724 Escherichia coli Species 0.000 description 3
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 238000012181 QIAquick gel extraction kit Methods 0.000 description 3
- BHEOSNUKNHRBNM-UHFFFAOYSA-N Tetramethylsqualene Natural products CC(=C)C(C)CCC(=C)C(C)CCC(C)=CCCC=C(C)CCC(C)C(=C)CCC(C)C(C)=C BHEOSNUKNHRBNM-UHFFFAOYSA-N 0.000 description 3
- 230000002759 chromosomal effect Effects 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000005520 cutting process Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- PRAKJMSDJKAYCZ-UHFFFAOYSA-N dodecahydrosqualene Natural products CC(C)CCCC(C)CCCC(C)CCCCC(C)CCCC(C)CCCC(C)C PRAKJMSDJKAYCZ-UHFFFAOYSA-N 0.000 description 3
- 230000002255 enzymatic effect Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000013467 fragmentation Methods 0.000 description 3
- 238000006062 fragmentation reaction Methods 0.000 description 3
- 238000001502 gel electrophoresis Methods 0.000 description 3
- BTIJJDXEELBZFS-QDUVMHSLSA-K hemin Chemical compound CC1=C(CCC(O)=O)C(C=C2C(CCC(O)=O)=C(C)\C(N2[Fe](Cl)N23)=C\4)=N\C1=C/C2=C(C)C(C=C)=C3\C=C/1C(C)=C(C=C)C/4=N\1 BTIJJDXEELBZFS-QDUVMHSLSA-K 0.000 description 3
- 229940025294 hemin Drugs 0.000 description 3
- 239000012160 loading buffer Substances 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 229930014626 natural product Natural products 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 230000004044 response Effects 0.000 description 3
- TUHBEKDERLKLEC-UHFFFAOYSA-N squalene Natural products CC(=CCCC(=CCCC(=CCCC=C(/C)CCC=C(/C)CC=C(C)C)C)C)C TUHBEKDERLKLEC-UHFFFAOYSA-N 0.000 description 3
- 229940031439 squalene Drugs 0.000 description 3
- YYGNTYWPHWGJRM-AAJYLUCBSA-N squalene group Chemical group CC(C)=CCC\C(\C)=C\CC\C(\C)=C\CC\C=C(/C)\CC\C=C(/C)\CCC=C(C)C YYGNTYWPHWGJRM-AAJYLUCBSA-N 0.000 description 3
- 239000000725 suspension Substances 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 238000012549 training Methods 0.000 description 3
- 238000011144 upstream manufacturing Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 2
- HOVPGJUNRLMIOZ-CIUDSAMLSA-N Ala-Ser-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@H](C)N HOVPGJUNRLMIOZ-CIUDSAMLSA-N 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 2
- 108010045510 NADPH-Ferrihemoprotein Reductase Proteins 0.000 description 2
- 101150063416 add gene Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 230000007812 deficiency Effects 0.000 description 2
- 229960000935 dehydrated alcohol Drugs 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 2
- 239000006210 lotion Substances 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 238000002156 mixing Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 238000012856 packing Methods 0.000 description 2
- 239000002244 precipitate Substances 0.000 description 2
- 108010053725 prolylvaline Proteins 0.000 description 2
- 239000011435 rock Substances 0.000 description 2
- 239000013049 sediment Substances 0.000 description 2
- 230000035939 shock Effects 0.000 description 2
- 238000010583 slow cooling Methods 0.000 description 2
- 239000011800 void material Substances 0.000 description 2
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 1
- HGRBNYQIMKTUNT-XVYDVKMFSA-N Ala-Asn-His Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N HGRBNYQIMKTUNT-XVYDVKMFSA-N 0.000 description 1
- IYCZBJXFSZSHPN-DLOVCJGASA-N Ala-Cys-Phe Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O IYCZBJXFSZSHPN-DLOVCJGASA-N 0.000 description 1
- PAIHPOGPJVUFJY-WDSKDSINSA-N Ala-Glu-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O PAIHPOGPJVUFJY-WDSKDSINSA-N 0.000 description 1
- MQIGTEQXYCRLGK-BQBZGAKWSA-N Ala-Gly-Pro Chemical compound C[C@H](N)C(=O)NCC(=O)N1CCC[C@H]1C(O)=O MQIGTEQXYCRLGK-BQBZGAKWSA-N 0.000 description 1
- NZGRHTKZFSVPAN-BIIVOSGPSA-N Ala-Ser-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N NZGRHTKZFSVPAN-BIIVOSGPSA-N 0.000 description 1
- WQKAQKZRDIZYNV-VZFHVOOUSA-N Ala-Ser-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O WQKAQKZRDIZYNV-VZFHVOOUSA-N 0.000 description 1
- IETUUAHKCHOQHP-KZVJFYERSA-N Ala-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)N)[C@@H](C)O)C(O)=O IETUUAHKCHOQHP-KZVJFYERSA-N 0.000 description 1
- UISQLSIBJKEJSS-GUBZILKMSA-N Arg-Arg-Ser Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(O)=O UISQLSIBJKEJSS-GUBZILKMSA-N 0.000 description 1
- OTCJMMRQBVDQRK-DCAQKATOSA-N Arg-Asp-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O OTCJMMRQBVDQRK-DCAQKATOSA-N 0.000 description 1
- NKNILFJYKKHBKE-WPRPVWTQSA-N Arg-Gly-Val Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O NKNILFJYKKHBKE-WPRPVWTQSA-N 0.000 description 1
- CVXXSWQORBZAAA-SRVKXCTJSA-N Arg-Lys-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N CVXXSWQORBZAAA-SRVKXCTJSA-N 0.000 description 1
- GRRXPUAICOGISM-RWMBFGLXSA-N Arg-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O GRRXPUAICOGISM-RWMBFGLXSA-N 0.000 description 1
- OMKZPCPZEFMBIT-SRVKXCTJSA-N Arg-Met-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O OMKZPCPZEFMBIT-SRVKXCTJSA-N 0.000 description 1
- JQHASVQBAKRJKD-GUBZILKMSA-N Arg-Ser-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCCN=C(N)N)N JQHASVQBAKRJKD-GUBZILKMSA-N 0.000 description 1
- JPAWCMXVNZPJLO-IHRRRGAJSA-N Arg-Ser-Phe Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O JPAWCMXVNZPJLO-IHRRRGAJSA-N 0.000 description 1
- WCZXPVPHUMYLMS-VEVYYDQMSA-N Arg-Thr-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(O)=O WCZXPVPHUMYLMS-VEVYYDQMSA-N 0.000 description 1
- ISVACHFCVRKIDG-SRVKXCTJSA-N Arg-Val-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O ISVACHFCVRKIDG-SRVKXCTJSA-N 0.000 description 1
- HLTLEIXYIJDFOY-ZLUOBGJFSA-N Asn-Cys-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(O)=O HLTLEIXYIJDFOY-ZLUOBGJFSA-N 0.000 description 1
- DDPXDCKYWDGZAL-BQBZGAKWSA-N Asn-Gly-Arg Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N DDPXDCKYWDGZAL-BQBZGAKWSA-N 0.000 description 1
- OPEPUCYIGFEGSW-WDSKDSINSA-N Asn-Gly-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O OPEPUCYIGFEGSW-WDSKDSINSA-N 0.000 description 1
- QXOPPIDJKPEKCW-GUBZILKMSA-N Asn-Pro-Arg Chemical compound C1C[C@H](N(C1)C(=O)[C@H](CC(=O)N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O QXOPPIDJKPEKCW-GUBZILKMSA-N 0.000 description 1
- XLDMSQYOYXINSZ-QXEWZRGKSA-N Asn-Val-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N XLDMSQYOYXINSZ-QXEWZRGKSA-N 0.000 description 1
- VTYQAQFKMQTKQD-ACZMJKKPSA-N Asp-Ala-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCC(N)=O)C(O)=O VTYQAQFKMQTKQD-ACZMJKKPSA-N 0.000 description 1
- PJERDVUTUDZPGX-ZKWXMUAHSA-N Asp-Cys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CC(O)=O PJERDVUTUDZPGX-ZKWXMUAHSA-N 0.000 description 1
- NYQHSUGFEWDWPD-ACZMJKKPSA-N Asp-Gln-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N NYQHSUGFEWDWPD-ACZMJKKPSA-N 0.000 description 1
- KHBLRHKVXICFMY-GUBZILKMSA-N Asp-Glu-Lys Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O KHBLRHKVXICFMY-GUBZILKMSA-N 0.000 description 1
- QPDUWAUSSWGJSB-NGZCFLSTSA-N Asp-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N QPDUWAUSSWGJSB-NGZCFLSTSA-N 0.000 description 1
- 102000014914 Carrier Proteins Human genes 0.000 description 1
- 241000251730 Chondrichthyes Species 0.000 description 1
- VZKXOWRNJDEGLZ-WHFBIAKZSA-N Cys-Asp-Gly Chemical compound SC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O VZKXOWRNJDEGLZ-WHFBIAKZSA-N 0.000 description 1
- CHRCKSPMGYDLIA-SRVKXCTJSA-N Cys-Phe-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O CHRCKSPMGYDLIA-SRVKXCTJSA-N 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 241000206602 Eukaryota Species 0.000 description 1
- ZEEPYMXTJWIMSN-GUBZILKMSA-N Gln-Lys-Ser Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@@H](N)CCC(N)=O ZEEPYMXTJWIMSN-GUBZILKMSA-N 0.000 description 1
- QKWBEMCLYTYBNI-GVXVVHGQSA-N Gln-Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(N)=O QKWBEMCLYTYBNI-GVXVVHGQSA-N 0.000 description 1
- VCUNGPMMPNJSGS-JYJNAYRXSA-N Gln-Tyr-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O VCUNGPMMPNJSGS-JYJNAYRXSA-N 0.000 description 1
- FLQAKQOBSPFGKG-CIUDSAMLSA-N Glu-Cys-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CCCN=C(N)N FLQAKQOBSPFGKG-CIUDSAMLSA-N 0.000 description 1
- AUTNXSQEVVHSJK-YVNDNENWSA-N Glu-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O AUTNXSQEVVHSJK-YVNDNENWSA-N 0.000 description 1
- MFNUFCFRAZPJFW-JYJNAYRXSA-N Glu-Lys-Phe Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 MFNUFCFRAZPJFW-JYJNAYRXSA-N 0.000 description 1
- BXSZPACYCMNKLS-AVGNSLFASA-N Glu-Ser-Phe Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O BXSZPACYCMNKLS-AVGNSLFASA-N 0.000 description 1
- FIQQRCFQXGLOSZ-WDSKDSINSA-N Gly-Glu-Asp Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FIQQRCFQXGLOSZ-WDSKDSINSA-N 0.000 description 1
- SOEATRRYCIPEHA-BQBZGAKWSA-N Gly-Glu-Glu Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O SOEATRRYCIPEHA-BQBZGAKWSA-N 0.000 description 1
- AAHSHTLISQUZJL-QSFUFRPTSA-N Gly-Ile-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AAHSHTLISQUZJL-QSFUFRPTSA-N 0.000 description 1
- ULZCYBYDTUMHNF-IUCAKERBSA-N Gly-Leu-Glu Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O ULZCYBYDTUMHNF-IUCAKERBSA-N 0.000 description 1
- BXICSAQLIHFDDL-YUMQZZPRSA-N Gly-Lys-Asn Chemical compound [H]NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O BXICSAQLIHFDDL-YUMQZZPRSA-N 0.000 description 1
- UWQDKRIZSROAKS-FJXKBIBVSA-N Gly-Met-Thr Chemical compound [H]NCC(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O UWQDKRIZSROAKS-FJXKBIBVSA-N 0.000 description 1
- CUVBTVWFVIIDOC-YEPSODPASA-N Gly-Thr-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)O)NC(=O)CN CUVBTVWFVIIDOC-YEPSODPASA-N 0.000 description 1
- 101150074296 HEM1 gene Proteins 0.000 description 1
- 101150009006 HIS3 gene Proteins 0.000 description 1
- SGLXGEDPYJPGIQ-ACRUOGEOSA-N His-Phe-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CC=CC=C2)C(=O)O)NC(=O)[C@H](CC3=CN=CN3)N SGLXGEDPYJPGIQ-ACRUOGEOSA-N 0.000 description 1
- VCBWXASUBZIFLQ-IHRRRGAJSA-N His-Pro-Leu Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O VCBWXASUBZIFLQ-IHRRRGAJSA-N 0.000 description 1
- STGQSBKUYSPPIG-CIUDSAMLSA-N His-Ser-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 STGQSBKUYSPPIG-CIUDSAMLSA-N 0.000 description 1
- QLBXWYXMLHAREM-PYJNHQTQSA-N His-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC1=CN=CN1)N QLBXWYXMLHAREM-PYJNHQTQSA-N 0.000 description 1
- NCSIQAFSIPHVAN-IUKAMOBKSA-N Ile-Asn-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H]([C@@H](C)O)C(=O)O)N NCSIQAFSIPHVAN-IUKAMOBKSA-N 0.000 description 1
- LPXHYGGZJOCAFR-MNXVOIDGSA-N Ile-Glu-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N LPXHYGGZJOCAFR-MNXVOIDGSA-N 0.000 description 1
- CSQNHSGHAPRGPQ-YTFOTSKYSA-N Ile-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCCN)C(=O)O)N CSQNHSGHAPRGPQ-YTFOTSKYSA-N 0.000 description 1
- NUKXXNFEUZGPRO-BJDJZHNGSA-N Ile-Leu-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N NUKXXNFEUZGPRO-BJDJZHNGSA-N 0.000 description 1
- HUORUFRRJHELPD-MNXVOIDGSA-N Ile-Leu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HUORUFRRJHELPD-MNXVOIDGSA-N 0.000 description 1
- FHPZJWJWTWZKNA-LLLHUVSDSA-N Ile-Phe-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N FHPZJWJWTWZKNA-LLLHUVSDSA-N 0.000 description 1
- 108010065920 Insulin Lispro Proteins 0.000 description 1
- 101150068332 KIT gene Proteins 0.000 description 1
- PMGDADKJMCOXHX-UHFFFAOYSA-N L-Arginyl-L-glutamin-acetat Natural products NC(=N)NCCCC(N)C(=O)NC(CCC(N)=O)C(O)=O PMGDADKJMCOXHX-UHFFFAOYSA-N 0.000 description 1
- ZRLUISBDKUWAIZ-CIUDSAMLSA-N Leu-Ala-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O ZRLUISBDKUWAIZ-CIUDSAMLSA-N 0.000 description 1
- WNGVUZWBXZKQES-YUMQZZPRSA-N Leu-Ala-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O WNGVUZWBXZKQES-YUMQZZPRSA-N 0.000 description 1
- STAVRDQLZOTNKJ-RHYQMDGZSA-N Leu-Arg-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O STAVRDQLZOTNKJ-RHYQMDGZSA-N 0.000 description 1
- WGNOPSQMIQERPK-UHFFFAOYSA-N Leu-Asn-Pro Natural products CC(C)CC(N)C(=O)NC(CC(=O)N)C(=O)N1CCCC1C(=O)O WGNOPSQMIQERPK-UHFFFAOYSA-N 0.000 description 1
- NHHKSOGJYNQENP-SRVKXCTJSA-N Leu-Cys-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N NHHKSOGJYNQENP-SRVKXCTJSA-N 0.000 description 1
- DZQMXBALGUHGJT-GUBZILKMSA-N Leu-Glu-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O DZQMXBALGUHGJT-GUBZILKMSA-N 0.000 description 1
- KVMULWOHPPMHHE-DCAQKATOSA-N Leu-Glu-Gln Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O KVMULWOHPPMHHE-DCAQKATOSA-N 0.000 description 1
- LAPSXOAUPNOINL-YUMQZZPRSA-N Leu-Gly-Asp Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O LAPSXOAUPNOINL-YUMQZZPRSA-N 0.000 description 1
- VWHGTYCRDRBSFI-ZETCQYMHSA-N Leu-Gly-Gly Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)NCC(O)=O VWHGTYCRDRBSFI-ZETCQYMHSA-N 0.000 description 1
- MUCIDQMDOYQYBR-IHRRRGAJSA-N Leu-Pro-His Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N MUCIDQMDOYQYBR-IHRRRGAJSA-N 0.000 description 1
- BTEMNFBEAAOGBR-BZSNNMDCSA-N Leu-Tyr-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCCN)C(=O)O)N BTEMNFBEAAOGBR-BZSNNMDCSA-N 0.000 description 1
- RDFIVFHPOSOXMW-ACRUOGEOSA-N Leu-Tyr-Phe Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O RDFIVFHPOSOXMW-ACRUOGEOSA-N 0.000 description 1
- CGHXMODRYJISSK-NHCYSSNCSA-N Leu-Val-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(O)=O CGHXMODRYJISSK-NHCYSSNCSA-N 0.000 description 1
- TUIOUEWKFFVNLH-DCAQKATOSA-N Leu-Val-Cys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CS)C(O)=O TUIOUEWKFFVNLH-DCAQKATOSA-N 0.000 description 1
- VHXMZJGOKIMETG-CQDKDKBSSA-N Lys-Ala-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CCCCN)N VHXMZJGOKIMETG-CQDKDKBSSA-N 0.000 description 1
- VHNOAIFVYUQOOY-XUXIUFHCSA-N Lys-Arg-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VHNOAIFVYUQOOY-XUXIUFHCSA-N 0.000 description 1
- AAORVPFVUIHEAB-YUMQZZPRSA-N Lys-Asp-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O AAORVPFVUIHEAB-YUMQZZPRSA-N 0.000 description 1
- QZONCCHVHCOBSK-YUMQZZPRSA-N Lys-Gly-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O QZONCCHVHCOBSK-YUMQZZPRSA-N 0.000 description 1
- MIMXMVDLMDMOJD-BZSNNMDCSA-N Lys-Tyr-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(C)C)C(O)=O MIMXMVDLMDMOJD-BZSNNMDCSA-N 0.000 description 1
- QAHFGYLFLVGBNW-DCAQKATOSA-N Met-Ala-Lys Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN QAHFGYLFLVGBNW-DCAQKATOSA-N 0.000 description 1
- CTVJSFRHUOSCQQ-DCAQKATOSA-N Met-Arg-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(O)=O)C(O)=O CTVJSFRHUOSCQQ-DCAQKATOSA-N 0.000 description 1
- WNJXJJSGUXAIQU-UFYCRDLUSA-N Met-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)CCSC)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 WNJXJJSGUXAIQU-UFYCRDLUSA-N 0.000 description 1
- MPCKIRSXNKACRF-GUBZILKMSA-N Met-Pro-Asn Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(O)=O MPCKIRSXNKACRF-GUBZILKMSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 108010047562 NGR peptide Proteins 0.000 description 1
- 101100123655 Neurospora crassa (strain ATCC 24698 / 74-OR23-1A / CBS 708.71 / DSM 1257 / FGSC 987) alv-1 gene Proteins 0.000 description 1
- 108020002230 Pancreatic Ribonuclease Proteins 0.000 description 1
- 102000005891 Pancreatic ribonuclease Human genes 0.000 description 1
- MIICYIIBVYQNKE-QEWYBTABSA-N Phe-Ile-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N MIICYIIBVYQNKE-QEWYBTABSA-N 0.000 description 1
- OQTDZEJJWWAGJT-KKUMJFAQSA-N Phe-Lys-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O OQTDZEJJWWAGJT-KKUMJFAQSA-N 0.000 description 1
- MGLBSROLWAWCKN-FCLVOEFKSA-N Phe-Phe-Thr Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MGLBSROLWAWCKN-FCLVOEFKSA-N 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- XQLBWXHVZVBNJM-FXQIFTODSA-N Pro-Ala-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 XQLBWXHVZVBNJM-FXQIFTODSA-N 0.000 description 1
- LCRSGSIRKLXZMZ-BPNCWPANSA-N Pro-Ala-Tyr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O LCRSGSIRKLXZMZ-BPNCWPANSA-N 0.000 description 1
- HJSCRFZVGXAGNG-SRVKXCTJSA-N Pro-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H]1CCCN1 HJSCRFZVGXAGNG-SRVKXCTJSA-N 0.000 description 1
- FKYKZHOKDOPHSA-DCAQKATOSA-N Pro-Leu-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FKYKZHOKDOPHSA-DCAQKATOSA-N 0.000 description 1
- WOIFYRZPIORBRY-AVGNSLFASA-N Pro-Lys-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O WOIFYRZPIORBRY-AVGNSLFASA-N 0.000 description 1
- RFWXYTJSVDUBBZ-DCAQKATOSA-N Pro-Pro-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 RFWXYTJSVDUBBZ-DCAQKATOSA-N 0.000 description 1
- RCYUBVHMVUHEBM-RCWTZXSCSA-N Pro-Pro-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RCYUBVHMVUHEBM-RCWTZXSCSA-N 0.000 description 1
- SNGZLPOXVRTNMB-LPEHRKFASA-N Pro-Ser-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N2CCC[C@@H]2C(=O)O SNGZLPOXVRTNMB-LPEHRKFASA-N 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 101100394989 Rhodopseudomonas palustris (strain ATCC BAA-98 / CGA009) hisI gene Proteins 0.000 description 1
- 241000793189 Saccharomyces cerevisiae BY4741 Species 0.000 description 1
- YQHZVYJAGWMHES-ZLUOBGJFSA-N Ser-Ala-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YQHZVYJAGWMHES-ZLUOBGJFSA-N 0.000 description 1
- XSYJDGIDKRNWFX-SRVKXCTJSA-N Ser-Cys-Phe Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XSYJDGIDKRNWFX-SRVKXCTJSA-N 0.000 description 1
- GZFAWAQTEYDKII-YUMQZZPRSA-N Ser-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CO GZFAWAQTEYDKII-YUMQZZPRSA-N 0.000 description 1
- UIPXCLNLUUAMJU-JBDRJPRFSA-N Ser-Ile-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O UIPXCLNLUUAMJU-JBDRJPRFSA-N 0.000 description 1
- QMCDMHWAKMUGJE-IHRRRGAJSA-N Ser-Phe-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(O)=O QMCDMHWAKMUGJE-IHRRRGAJSA-N 0.000 description 1
- PPCZVWHJWJFTFN-ZLUOBGJFSA-N Ser-Ser-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O PPCZVWHJWJFTFN-ZLUOBGJFSA-N 0.000 description 1
- KKKVOZNCLALMPV-XKBZYTNZSA-N Ser-Thr-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O KKKVOZNCLALMPV-XKBZYTNZSA-N 0.000 description 1
- VFEHSAJCWWHDBH-RHYQMDGZSA-N Thr-Arg-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O VFEHSAJCWWHDBH-RHYQMDGZSA-N 0.000 description 1
- TZKPNGDGUVREEB-FOHZUACHSA-N Thr-Asn-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O TZKPNGDGUVREEB-FOHZUACHSA-N 0.000 description 1
- KBLYJPQSNGTDIU-LOKLDPHHSA-N Thr-Glu-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N)O KBLYJPQSNGTDIU-LOKLDPHHSA-N 0.000 description 1
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 1
- XYFISNXATOERFZ-OSUNSFLBSA-N Thr-Ile-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H]([C@@H](C)O)N XYFISNXATOERFZ-OSUNSFLBSA-N 0.000 description 1
- ODXKUIGEPAGKKV-KATARQTJSA-N Thr-Leu-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CS)C(=O)O)N)O ODXKUIGEPAGKKV-KATARQTJSA-N 0.000 description 1
- XSEPSRUDSPHMPX-KATARQTJSA-N Thr-Lys-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O XSEPSRUDSPHMPX-KATARQTJSA-N 0.000 description 1
- GUHLYMZJVXUIPO-RCWTZXSCSA-N Thr-Met-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(O)=O GUHLYMZJVXUIPO-RCWTZXSCSA-N 0.000 description 1
- AHERARIZBPOMNU-KATARQTJSA-N Thr-Ser-Leu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O AHERARIZBPOMNU-KATARQTJSA-N 0.000 description 1
- KIMOCKLJBXHFIN-YLVFBTJISA-N Trp-Ile-Gly Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O)=CNC2=C1 KIMOCKLJBXHFIN-YLVFBTJISA-N 0.000 description 1
- TVOGEPLDNYTAHD-CQDKDKBSSA-N Tyr-Ala-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 TVOGEPLDNYTAHD-CQDKDKBSSA-N 0.000 description 1
- HSVPZJLMPLMPOX-BPNCWPANSA-N Tyr-Arg-Ala Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O HSVPZJLMPLMPOX-BPNCWPANSA-N 0.000 description 1
- PRONOHBTMLNXCZ-BZSNNMDCSA-N Tyr-Leu-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 PRONOHBTMLNXCZ-BZSNNMDCSA-N 0.000 description 1
- NSGZILIDHCIZAM-KKUMJFAQSA-N Tyr-Leu-Ser Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N NSGZILIDHCIZAM-KKUMJFAQSA-N 0.000 description 1
- SOEGLGLDSUHWTI-STECZYCISA-N Tyr-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=C(O)C=C1 SOEGLGLDSUHWTI-STECZYCISA-N 0.000 description 1
- BIWVVOHTKDLRMP-ULQDDVLXSA-N Tyr-Pro-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(O)=O BIWVVOHTKDLRMP-ULQDDVLXSA-N 0.000 description 1
- PWKMJDQXKCENMF-MEYUZBJRSA-N Tyr-Thr-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O PWKMJDQXKCENMF-MEYUZBJRSA-N 0.000 description 1
- JOQSQZFKFYJKKJ-GUBZILKMSA-N Val-Arg-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N JOQSQZFKFYJKKJ-GUBZILKMSA-N 0.000 description 1
- JIODCDXKCJRMEH-NHCYSSNCSA-N Val-Arg-Gln Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N JIODCDXKCJRMEH-NHCYSSNCSA-N 0.000 description 1
- SCBITHMBEJNRHC-LSJOCFKGSA-N Val-Asp-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](C(C)C)C(=O)O)N SCBITHMBEJNRHC-LSJOCFKGSA-N 0.000 description 1
- URIRWLJVWHYLET-ONGXEEELSA-N Val-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)C(C)C URIRWLJVWHYLET-ONGXEEELSA-N 0.000 description 1
- WJVLTYSHNXRCLT-NHCYSSNCSA-N Val-His-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N WJVLTYSHNXRCLT-NHCYSSNCSA-N 0.000 description 1
- VXDSPJJQUQDCKH-UKJIMTQDSA-N Val-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N VXDSPJJQUQDCKH-UKJIMTQDSA-N 0.000 description 1
- APQIVBCUIUDSMB-OSUNSFLBSA-N Val-Ile-Thr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)O)NC(=O)[C@H](C(C)C)N APQIVBCUIUDSMB-OSUNSFLBSA-N 0.000 description 1
- AEMPCGRFEZTWIF-IHRRRGAJSA-N Val-Leu-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(O)=O AEMPCGRFEZTWIF-IHRRRGAJSA-N 0.000 description 1
- LJSZPMSUYKKKCP-UBHSHLNASA-N Val-Phe-Ala Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](C)C(O)=O)CC1=CC=CC=C1 LJSZPMSUYKKKCP-UBHSHLNASA-N 0.000 description 1
- YQMILNREHKTFBS-IHRRRGAJSA-N Val-Phe-Cys Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)O)N YQMILNREHKTFBS-IHRRRGAJSA-N 0.000 description 1
- CKTMJBPRVQWPHU-JSGCOSHPSA-N Val-Phe-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(=O)O)N CKTMJBPRVQWPHU-JSGCOSHPSA-N 0.000 description 1
- SJRUJQFQVLMZFW-WPRPVWTQSA-N Val-Pro-Gly Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O SJRUJQFQVLMZFW-WPRPVWTQSA-N 0.000 description 1
- UGFMVXRXULGLNO-XPUUQOCRSA-N Val-Ser-Gly Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O UGFMVXRXULGLNO-XPUUQOCRSA-N 0.000 description 1
- VHIZXDZMTDVFGX-DCAQKATOSA-N Val-Ser-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N VHIZXDZMTDVFGX-DCAQKATOSA-N 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 108010086434 alanyl-seryl-glycine Proteins 0.000 description 1
- 108010045350 alanyl-tyrosyl-alanine Proteins 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 150000001336 alkenes Chemical class 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 108010008355 arginyl-glutamine Proteins 0.000 description 1
- 108010060035 arginylproline Proteins 0.000 description 1
- 108010036533 arginylvaline Proteins 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 108091008324 binding proteins Proteins 0.000 description 1
- 230000000975 bioactive effect Effects 0.000 description 1
- 230000003115 biocidal effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 239000006143 cell culture medium Substances 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 238000005859 coupling reaction Methods 0.000 description 1
- 108010069495 cysteinyltyrosine Proteins 0.000 description 1
- 230000006378 damage Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- 238000010790 dilution Methods 0.000 description 1
- 239000012895 dilution Substances 0.000 description 1
- FSXRLASFHBWESK-UHFFFAOYSA-N dipeptide phenylalanyl-tyrosine Natural products C=1C=C(O)C=CC=1CC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FSXRLASFHBWESK-UHFFFAOYSA-N 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 239000005712 elicitor Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- XKUKSGPZAADMRA-UHFFFAOYSA-N glycyl-glycyl-glycine Natural products NCC(=O)NCC(=O)NCC(O)=O XKUKSGPZAADMRA-UHFFFAOYSA-N 0.000 description 1
- 108010048994 glycyl-tyrosyl-alanine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 108010040030 histidinoalanine Proteins 0.000 description 1
- 108010036413 histidylglycine Proteins 0.000 description 1
- 108010092114 histidylphenylalanine Proteins 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 235000003642 hunger Nutrition 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 108010057821 leucylproline Proteins 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000012982 microporous membrane Substances 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 239000002547 new drug Substances 0.000 description 1
- 238000001921 nucleic acid quantification Methods 0.000 description 1
- 244000039328 opportunistic pathogen Species 0.000 description 1
- 108010064486 phenylalanyl-leucyl-valine Proteins 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000009465 prokaryotic expression Effects 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010015796 prolylisoleucine Proteins 0.000 description 1
- 238000012827 research and development Methods 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 108010026333 seryl-proline Proteins 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 238000009987 spinning Methods 0.000 description 1
- 108010005652 splenotritin Proteins 0.000 description 1
- 108010088324 squalene cyclase Proteins 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 230000001954 sterilising effect Effects 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000005720 sucrose Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 241001148471 unidentified anaerobic bacterium Species 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0071—Oxidoreductases (1.) acting on paired donors with incorporation of molecular oxygen (1.14)
- C12N9/0083—Miscellaneous (1.14.99)
-
- 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
- C12N15/87—Introduction of foreign genetic material using processes not otherwise provided for, e.g. co-transformation
- C12N15/90—Stable introduction of foreign DNA into chromosome
- C12N15/902—Stable introduction of foreign DNA into chromosome using homologous recombination
- C12N15/905—Stable introduction of foreign DNA into chromosome using homologous recombination in yeast
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y114/00—Oxidoreductases acting on paired donors, with incorporation or reduction of molecular oxygen (1.14)
- C12Y114/99—Miscellaneous (1.14.99)
- C12Y114/99007—Squalene monooxygenase (1.14.99.7)
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Microbiology (AREA)
- Mycology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明涉及一种ERG1基因缺陷酵母工程菌及其构建方法,所述ERG1基因缺陷型酵母工程菌通过CRISPR/Cas9基因编辑技术对酵母进行改造,实现了Erg1基因序列的完全删除,避免了酵母自身修复产生的回复突变现象,得到一种可以适合验证其它来源鲨烯环氧化酶基因的工程菌。
Description
技术领域
本发明涉及一种ERG1基因缺陷型酵母工程菌,以及一种敲除酵母菌中ERG1基因的方法,通过CRISPR/Cas9基因编辑技术对酿酒酵母进行改造,将酵母染色体中的鲨烯环氧化酶(Squalene epoxidase,ERG1)基因进行敲除,使之成为一个适合验证其它来源的鲨烯环氧化酶基因功能的缺陷菌,并通过该酵母缺陷菌成功验证了从雷公藤中克隆得到的鲨烯环氧化酶基因(Twse),属于基因工程领域。
背景技术
中药或其它来源的活性天然产物的获取主要依赖于对原物种的提取,但这种方法所获取的天然产物远远无法满足需求,也容易对环境和资源造成破坏。通过在微生物中构建重要次生代谢物的生物合成途径,实现天然产物低成本、高产量的生产,这将促进天然产物的应用和新药的研发以及资源的可持续发展。这种方式能够实行的前提是基因原件的获取以及合成途径的解析。
ERG1是鲨烯环氧化酶,因为是位于内质网Endoplasmic Reticulum上的基因,所以用Erg命名。鲨烯环氧化酶是三萜和甾醇生物合成途径上的关键酶基因,它的活性和功能影响着三萜和甾醇的生物合成。鲨烯在鲨烯环氧化酶和其辅酶细胞色素P450还原酶(Cytochrome P450 Reductase,CPR)的作用下生成环氧鲨烯(图1),环氧鲨烯再进一步转化为三萜或甾醇类物质。在植物中,鲨烯环氧化酶通过影响植物的甾醇和三萜类活性成分的合成进一步影响植物的生长和发育以及应对生物和非生物的压力。研究表明在拟南芥中发现的环氧鲨烯酶SE1基因影响着拟南芥的根和种子的发育而SE3基因影响胚芽的发育和大小。
由于鲨烯环氧化酶属于内质网膜结合蛋白,在大肠杆菌中很难表达出能够发挥功能的鲨烯环氧化酶,因而很难通过原核表达,体外酶促的方法来验证植物或其它来源的鲨烯环氧化酶的功能。酵母属于真核生物,能够很好的表达植物或其它来源的鲨烯环氧化酶,但是酵母本身也有鲨烯环氧化酶基因,因而对植物和其它来源的鲨烯环氧化酶基因功能的鉴定造成干扰。早期在研究酵母中ERG1基因在酵母染色体上的位置时,通过同源重组的方法对酵母中的ERG1基因进行了敲除(菌命名为KLN1:MATa,ERG1::URA3,Ieu2,ura3,trp1)。酵母虽然是兼性厌氧菌,但是它需要氧气用于合成两类必须成分:一是甾醇,另一类是不饱和脂肪酸。前期人们在研究酵母的时候发现,有氧条件下,酵母不会从培养基里摄取麦角固醇,而在无氧的条件下酵母会从培养基里摄取麦角甾醇。后期在验证植物的鲨烯环氧化酶基因一般都是通过KLN1这个菌来做的,这个菌是通过将筛选标记***ERG1基因的编码区然后通过同源重组的方法整合到酵母染色体当中构建的,存在的缺点是ERG1基因没有完全敲除,在验证外源se基因的功能时可能存在假阳性。也有个别使用另一种改造的菌RXY6(MATaerg7::HIS3 erg1::KanMX4 hem1::TRP1 ura3-52 trp1-△63 leu2–3,112 his3-△200ade2 Gal+),这种菌的主要优势是敲除了ERG1的前提下也敲除了Hem1,敲除Hem1可以使酵母在有氧的条件下从培养基里吸收麦角固醇,但是同时敲除ERG1和Hem1难度较大,而且Hem1对血晶素合成十分重要,血晶素参与体内多种代谢反应,因此敲除了Hem1基因的酵母需要在培养基里补充血晶素。
本研究采用的基因编辑方法实现了ERG1基因序列的完全删除,避免了酵母自身修复产生的回复突变现象,使之成为适合验证其它来源鲨烯环氧化酶基因的工程菌。另外,该方法敲除过程中使用的质粒可以通过高温处理去除,保留了原菌株的筛选标记,适用范围更广,且实验操作比以往的同源重组方法更加简单快捷。
发明内容
本发明一方面提供了一种敲除酵母中自身的ERG1基因的方法,所述方法为通过CRISPR/Cas9基因编辑技术对酵母进行改造,敲除酵母中自身的鲨烯环氧化酶基因,得到一种可以适合验证其它来源鲨烯环氧化酶基因的工程菌。可以通过如下方式构建并进行基因功能的验证:
利用酵母CRISPR/Cas9gRNA设计网站http://yeastriction.tnw.tudelft.nl/#!/对ERG1基因进行gRNA位点筛选,将筛选得到的gRNA位点构建到gRNA载体中,制备酵母BY4741感受态细胞,将构建好的gRNA载体和Cas9载体以及同源片段一同转入BY4741酵母感受态细胞,在固体筛选培养基中30℃无氧培养。对长出的单菌落进行PCR验证和测序验证。成功构建的erg1酵母缺陷菌在液体筛选培养基中30℃无氧培养,制备erg1酵母感受态细胞。
在对植物细胞中的鲨烯环氧化酶基因功能进行验证时,比如验证雷公藤中的鲨烯环氧化酶基因功能,可以从雷公藤悬浮细胞中克隆得到雷公藤鲨烯环氧化酶基因,将Twse基因构建到pESC-his真核表达载体中,转化入erg1酵母缺陷菌,通过功能互补的方式验证Twse基因功能。
具体地,所述敲除酵母中自身的ERG1基因的方法,包括:
(1)筛选ERG1基因的gRNA位点;
(2)构建敲除ERG1基因的表达载体;
(3)改造Cas9载体,将p414-TEF1p-Cas9-CYC1t的筛选标记TRP替换为LEU;
(4)获取ERG1基因的双链DNA(dsOligo)
(5)制备酵母化学感受态细胞;
(6)将含有酵母ERG1基因特异性识别位点的gRNA载体、改造好的Cas9载体以及合成的ERG1双链DNA转化至骤(5)中的感受态细胞中,在筛选培养基SC-Leu-Ura上无氧培养,获得突变成功的ERG1基因缺陷的酵母菌。
一个更为具体的方案是:
(1)筛选ERG1基因的gRNA位点,所述gRNA位点的基因序列为:
TATCTTTTCACGTTTCAGAA;
(2)以EGR1-F和EGR1-R为引物,以p426-SNR52p-gRNA真核表达载体为模板克隆得到线性载体,并以T4连接酶进行平末端连接,构建得到敲除ERG1基因的gRNA表达载体,
所述引物序列为:
ERG1-F:TATCTTTTCACGTTTCAGAAGTTTTAGAGCTAGAA,
ERG1-R:GATCATTTATCTTTCACTGCGGAGAAG;
(3)改造Cas9载体,将p414-TEF1p-Cas9-CYC1t的筛选标记TRP替换为LEU;
(4)设计敲除ERG1基因的dsOligo,通过同源重组修复对目的基因进行敲除,所ERG1-Oligo-F及ERG1-Oligo-R的序列为:
ERG1-Oligo-F:
CAGGTTATTTCGAACAATTGAAAAAAAAAAATCACAGAAAAACATATCGAGAAAAGGGTCCTACAGCTTATAAGGGAGAGAGGATAGGAACCGTCAAACATTAAGCTGCACCTTTTTTTT,
ERG1-Oligo-R:
AAAAAAAAGGTGCAGCTTAATGTTTGACGGTTCCTATCCTCTCTCCCTTATAAGCTGTAGGACCCTTTTCTCGATATGTTTTTCTGTGATTTTTTTTTTTCAATTGTTCGAAATAACCTG,
所述ERG1基因的dsOligo获得通过将ERG1-Oligo-F及ROX1-Oligo-R退火形成DNA双链,并回收纯化既得;
(5)根据Frozen-EZ Yeast Transformation IITM试剂盒说明书制备酵母化学感受态细胞;
(6)将含有酵母ERG1基因特异性识别位点的gRNA载体、改造好的Cas9载体以及合成的ERG1双链DNA转化至骤(5)中的感受态细胞中,在筛选培养基SC-Leu-Ura上无氧培养,获得突变成功的ERG1基因缺陷的酵母菌。
对ERG1基因缺陷的酵母菌进行PCR验证和测序验证,以确认得到的是正确的基因缺陷型酵母。成功构建的erg1酵母缺陷菌在液体筛选培养基中30℃无氧培养,制备erg1酵母感受态细胞。
克隆欲验证的植物中的鲨烯环氧化酶基因,而后将基因构建到真核表达载体中,再转化入erg1酵母缺陷菌,通过功能互补的方式验证鲨烯环氧化酶基因功能。
本发明中,所述的酵母工程菌,可以选自GEN.PK系酿酒酵母菌或BY系酿酒酵母菌,优选为BY系酿酒酵母,如BY4741酿酒酵母。
本发明的再一目的在于提供一种ERG1基因缺陷型酵母工程菌,所述的酵母可以选自GEN.PK系酿酒酵母菌或BY系酿酒酵母菌,优选为BY系酿酒酵母,如BY4741酿酒酵母。
一个更具体的实施方案中,本发明提供了一种ERG1基因缺陷型的BY4741酵母工程菌,该基因缺陷型酵母工程菌可以用于验证植物细胞中的鲨烯环氧化酶基因功能,如用于验证雷公藤鲨烯环氧化酶基因(Twse)功能。
本发明所述的ERG1基因缺陷型酵母工程菌,如ERG1缺陷型BY4741酵母工程菌,可以通过上述方法构建得到。
本发明的再一目的在于提供本发明所述ERG1基因缺陷型酵母工程菌,如ERG1基因缺陷型BY4741酵母菌,在验证鲨烯环氧化酶基因功能中的运用,如验证植物细胞中鲨烯环氧化酶基因功能,更具体地一个实施方式中验证了植物雷公藤中鲨烯环氧化酶基因功能。
本发明的又一目的在于提供一种验证植物细胞(如雷公藤)中鲨烯环氧化酶基因功能的方法,所述方法包括:分别将含有植物鲨烯环氧化酶基因的表达载体(如pESC-his-Twse载体)及相应不含植物鲨烯环氧化酶基因的空载体(pESC-his空载体)转化到ERG1基因缺陷型酵母工程菌(如ERG1基因缺陷型BY4741酵母菌)感受态细胞中,然后均在筛选培养基上,分别于30℃有氧和无氧条件下培养,若转入含鲨烯环氧化酶基因载体的erg1酵母缺陷菌在有氧和无氧条件下均可生长,确认具有生物学功能,所述筛选培养基为SC-his加麦角固醇培养基。
附图说明
图1鲨烯环化酶催化反应图。
图2Erg1敲除示意图和凝胶电泳图。
图3Erg1酵母缺陷菌验证图,其中1为有氧条件下erg1缺陷酵母生长情况,2为无氧条件下Erg1缺陷酵母生长情况。
图4雷公藤总RNA提取结果图。
图5雷公藤Twse基因凝胶电泳图。
图6雷公藤Twse基因功能互补图,其中1和2分别为无氧条件及有氧条件下不同类型erg1酵母菌生长情况,1(a)及2(a)为未转入任何载体的erg1酵母缺陷菌,1(b)及2(b)为转入空载体pESC-his的erg1酵母缺陷菌,1(c)及2(c)为转入pESC-his-Twse的erg1酵母缺陷菌。
具体实施方式
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
高保真酶High-Fidelity DNA Polymerase、限制性内切酶和T4连接酶均来自(NEB公司),Trans-T1大肠杆菌感受态细胞、EasyTaq DNA Polymerase、pEASY-BluntZero Cloning Kit、无缝拼接试剂盒UniSeamLess Cloning and Assembly Kit(北京全式金生物技术有限公司),AraI内切酶和琼脂糖凝胶DNA回收试剂盒Gene JET GelExtraction Kit(美国Thermo Scientific公司),快速质粒小提试剂盒(北京天根生化科技有限公司),Annealing Buffer(美国Origene公司),T4DNA Ligase(HC)和Super总RNA提取试剂盒(Promega(北京)生物技术有限公司),SC-His酵母培养基和SC-Ura-Leu酵母培养基(北京泛基诺科技有限公司)、酵母培养基酿酒酵母BY4741实验室保存,p426-SNR52p-gRNA真核表达载体和p414-TEF1p-Cas9-CYC1t真核表达载体购自Addgene,引物合成和测序由北京睿博公司完成。
实施实例1、Erg1酵母缺陷菌的构建
一、利用酵母CRISPR/Cas9gRNA设计网站http://yeastriction.tnw.tudelft.nl/#!/对ERG1基因进行gRNA位点筛选,ERG1基因gRNA序列如下表。
表1 ERG1基因gRNA序列
二、gRNA载体构建
通过引物设计的方法将gRNA序列加到gRNA骨架前端,采用RF克隆的方法以p426-SNR52p-gRNA真核表达载体(Addgene官网购买)为模板克隆得到线性载体序列,并通过T4连接酶进行平末端连接,即可得到含酵母ERG1基因特异性识别的位点的gRNA载体。引物信息如下:
表2 gRNA载体构建引物
1、RF克隆
体系:
反应条件:
*Tm值为引物的退火温度,延伸时间根据引物的长度设定
2、线性载体纯化
将上述PCR产物取出,各取5uL加2μL的6×Loading Buffer,以8k Marker做对照,跑1.0%的琼脂糖凝胶(180V,10min)。看到目的扩增条带后,在剩余产物中加入1uL的DMT酶,混匀,37℃下消化一小时(目的是去除模板质粒DNA,以减少假阳性)。将反应完的产物中加入10uL的6×Loading Buffer,以8k Marker做对照,跑1.5%的琼脂糖凝胶(120V,30min)。在紫外切胶仪下,将目的条带切下,用琼脂糖凝胶DNA回收试剂盒回收片段,步骤如下:
(1)向胶块中加入等倍体积溶液Binding Buffer(如果凝胶重为0.1g,其体积可视为100μL,则加入100μL Binding Buffer溶液),55℃水浴放置,其间不断温和地上下翻转离心管,以确保胶块充分溶解。如果还有未溶的胶块,可继续放置几分钟或再补加一些溶胶液,直至胶块完全溶解(若胶块的体积过大,可事先将胶块切成碎块)。
(2)将完全溶解的胶液加入吸附柱中(吸附柱放入收集管中),12000rpm离心60s,倒掉收集管中的废液,将吸附柱放入收集管中。
(4)向吸附柱中加入700μl漂洗液Wash Buffer(使用前先检查是否已加入无水乙醇),12000rpm离心60s,倒掉收集管中的废液,将吸附柱放入收集管中。
(5)12000rpm空柱离心1min,尽量除尽漂洗液。将吸附柱置于新的1.5mL离心管中,挥醇。
(6)向吸附柱滤膜中加入20μl预热的无菌水,室温放置1min。12000rpm离心1min收集DNA溶液。
3、载体连接
使用NanoDrop测量回收得到的载体片段浓度,按照如下比例配制连接反应体系:
条件:25℃2h
4、连接产物转化
(1)将上述连接产物全部加入到100μL的DMT感受态细胞中,冰中放置30min;
(2)42℃热激1min后,再放置冰中2min;
(3)加入1mL不含抗生素的LB培养基,30℃,180rpm振荡培养1h;
(4)吸取200μL菌液涂在LB+Amp固定培养基上,30℃*培养14h,观察菌落生长情况。
*该质粒为温敏质粒,因此采用低温30℃对其宿主菌进行培养。
5、菌液PCR验证阳性菌落
在超净台中,用灭过菌的10μL白枪头挑取平板中的单一菌落,置于LB+Amp培养基中,30℃,250rpm恒温振荡培养约1-2h后,取1μL菌液进行菌液PCR,检测目的片段是否与载体有效连接上。
PCR反应体系:
PCR反应条件:
PCR产物经过1%琼脂糖凝胶电泳检测后,凝胶成像仪上观察。将出现700bp左右目的片段条带的菌液送测序公司测序;选取测序结果阳性的菌株,加入20%的甘油保存于-80℃冰箱中,并划LB+Amp平板待用。
三、Cas9载体改造
由于p414-TEF1p-Cas9-CYC1t载体的筛选标记TRP不适用于BY4741酵母,因此本实验中采用无缝拼接的方式将TRP筛选标记替换为LEU,LEU序列模板为实验室所存pESC-LEU质粒。
表3 Cas9载体改造引物
(1)分别以pESC-LEU质粒为模板扩增LEU序列、以p414-TEF1p-Cas9-CYC1t质粒为模板扩增TRP上游序列、下游序列,引物分别是LEU-F/R、U-F/R、D-F/R。
(2)p414-TEF1p-Cas9-CYC1t质粒双酶切
选取TRP1 ORF上游148bp的SnaBI限制性内切酶位点和下游323bp的DraIII限制性内切酶位点。
条件:37℃2h
(3)切胶回收各片段,操作同上。
(4)In-Fusion反应
注:n(载体):n(片段)=1:2
条件:50℃孵育15min;冰上放置。
(5)将10μL拼接产物转化到50μL Trans1-T1感受态细胞中,30℃复苏,涂LB+Amp固体培养基,30℃培养过夜。
(6)挑取单菌落,LB+Amp液体培养基30℃,250rpm摇培4~6h,菌液PCR验证,引物如下,若PCR产物经琼脂糖凝胶电泳检测出现1740bp附近的条带,则将对应菌液送公司测序。
表8 Cas9载体改造菌液PCR引物
(7)将测序正确的菌液摇培,提取质粒,并将菌液中添加20%的甘油,-80℃冻存。
四、dsOligo的获得
为了获得特定基因型的突变菌株,需要双链DNA(dsOligo)介导同源重组修复。在酿酒酵母中,同源臂长度为60bp时即可获得80%以上得的同源重组效率,本实验中采取删除整条基因的方式达到基因敲除的目的,即在基因ORF上下游各设计60bp的同源臂(共120bp)通过同源重组修复对目的基因进行敲除。ERG1基因的dsOligo采用合成的120nt长链Oligo退火形成DNA双链,回收纯化获得。
Oligo退火形成双链:
(1)将4OD oligo合成一管,用ddH2O稀释到10pmol/μL,oligo序列如下表。
表3-9 dsOligo序列
条件:95℃ 5min
95~25℃ -1℃/min 71cycles
10℃ hold
(2)dsOligo纯化
将退火产物按照Gene JET Gel Extraction Kit胶回收试剂盒回收纯化,步骤如下:
①按照体积比退火体系:异丙醇:Binding Buffer=1:1:1的量混合;
②将混合液转移到Gene JET基因回收纯化柱中,12000rpm离心1min,弃去流出液,然后将柱放回相同的收集管;
③加入700μLWash Buffer(已用乙醇稀释)到Gene JET纯化柱。12000rpm离心1min,弃去流出液,然后将柱放回相同的收集管;
④离心空GeneJET纯化柱12000rpm离心1min,彻底去除残留的Wash Buffer;
⑤将Gene JET纯化柱转移到一个干净的1.5mL离心管,加20μL ddH2O(可60℃预热)于纯化柱膜,12000rpm离心1min,弃纯化柱,离心所得液体即为纯化的目的dsOligo。
(3)测浓度,用于后续的酵母转化实验。
五、BY4741化学感受态细胞制备
根据Frozen-EZ Yeast Transformation IITM试剂盒说明书进行酵母化学感受态细胞的制备,步骤如下:
(1)将BY4741菌液划YPD固体培养平板,48h后挑取单菌落于10mL YPD液体培养基中30℃,220rpm过夜培养,至OD600=0.8-1.0。
(2)500×g离心4min沉淀菌体,弃上清;
(3)10mL EZ1重悬、洗涤菌体,500×g离心4min,弃上清;
(4)1mL EZ2重悬菌体,每50μL分装一管,备用。
注:制备完的感受态细胞可以直接转化,或缓慢降温冻存至-80℃(4℃,1h;-20℃,1h;-80℃)。
六、Cas9载体、gRNA载体及同源片段的转化
(1)取0.2-1μg(≤5μL)Cas9质粒pZJW01,gRNA约500ng,Erg1dsOligo 1μg,根据不同敲除目的配成混合体系加入到100μL BY4741感受态细胞中;
(2)加入500μL EZ3溶液,彻底混匀;
(3)30℃孵育45min,过程中摇晃2-3次;
(4)取菌液200μL涂布在筛选培养基SC-Leu-Ura加麦角甾醇(20ug/mL)固体培养基上,30℃无氧培养48~72h。
(5)挑取长出的单菌落,于YPD加麦角甾醇(20ug/mL)液体培养基中培养,至培养基浑浊,以未改造的菌株作为对照,用以下引物对菌液进行PCR,检测目的基因是否发生突变。
表11 突变检测引物
琼脂糖凝胶电泳检测,将阳性PCR产物送公司测序,测序正确的菌株即为突变成功菌株。凝胶电泳和测序结果显示,成功将酵母BY4741中的ERG1基因敲除(见图2)。
(6)将测序成功的erg1缺陷酵母菌按千分之一扩大5mL培养,在30℃无氧培养条件下(YPD加麦角固醇(20ug/mL)液体培养基)培养至浑浊。取菌液在YPD加麦角固醇固体培养基上划板子,分别于有氧或者无氧条件30℃培养,进一步验证ERG1基因是否完全敲除。
由于ERG1基因对酵母中甾醇的生物合成起着重要的作用,因此敲除ERG1基因的酵母在缺乏甾醇的情况下无法存活。另外,由于酵母在有氧条件下,对外源甾醇具有排他性,因此,在有氧条件下,即使培养基中添加麦角固醇也无法吸收利用和生长。本实验结果显示,敲除ERG1基因的酵母缺陷菌在有氧的条件下,即使培养基中加入麦角固醇,也无法生长(见图3)。实验结果证明,erg1酵母缺陷菌构建成功。
实施实例2、雷公藤环氧鲨烯环化酶基因全长克隆
一、雷公藤悬浮细胞总RNA提取
1、雷公藤悬浮细胞的培养及诱导
(1)MS培养(含激素)配制:4.43g·L-1MS,0.5mg.L-1 2,4-D,0.1mg·L-1KT,0.5mg.L-1IBA,30g·L-1蔗糖,调节pH=5.8。然后121℃,高压蒸汽锅灭菌20min.
(2)诱导子茉莉酸甲酯(MeJA)溶液配制:将115μL 95%MeJA溶于885μL DMSO中,配制成0.5M MeJA母液。配制中所用的MeJA和DMSO均用0.22μm微孔滤膜过滤,并于无菌环境中混合。
(3)取MS液体培养保存的雷公藤悬浮细胞,在无菌条件下将约2g悬浮细胞接种于装有25mL MS液体培养基的100mL三角瓶中进行继代培养,培养至10d后作为试验材料。培养条件为25±1℃,120rpm,黑暗条件下悬浮培养。
(4)MeJA处理:向培养10d的雷公藤悬浮细胞培养基中分别加入MeJA终浓度为50μmol·L-1进行诱导处理,对照组加入相同量的溶剂DMSO。在处理后的12、24和48h分别抽滤收获悬浮细胞,每个时间点3个生物学重复,用滤纸吸干后于液氮速冻后-80℃保存(提取总RNA)。
2、Super总RNA提取试剂盒提取雷公藤悬浮细胞:
(1)取约50mg悬浮细胞于2mL EP中,液氮环境下粉碎。
(2)将粉碎好的样品加入500μL RNA裂解液,颠倒离心管3~4次,混匀。
(3)加入500μL稀释液,移液枪混匀,14000rpm离心5min。
(4)离心后的上清中加入0.5倍体积的无水乙醇,混匀。
(5)将混合液转入离心柱中,12000~14000rpm离心1min,弃滤液。
(6)向离心柱中加入600μL洗液,12000~14000rpm离心45s,弃滤液。
(7)加入50μL DNA酶I,孵育15min。
μL RNase-free水,静置两分钟,12000~14000rpm离心1min,收集RNA,-70℃保存。(RNA提Total RNA
(8)向离心柱中加入600μL洗液,12000~14000rpm离心45s,弃滤液。重复一次。
(9)空柱离心2min。
(10)向离心柱中加入50~200μL RNase-free水,静置两分钟,12000~14000rpm离心1min,收集RNA,-70℃保存。(RNA提取结果见图4)
二、反转获得第一链cDNA
(1)去除基因组DNA
42℃,3min。
(2)反转第一链cDNA
42℃,15min;95℃,3min。
三、Twse基因全长克隆
根据转录组数据,设计雷公藤鲨烯环氧化酶基因特异引物:
表3 Twse全长克隆引物
全长克隆PCR反应体系:
*模板DNA为上述实施实例2中反转的雷公藤cDNA
全长PCR反应程序:
*Tm值为引物的退火温度,延伸时间根据引物的长度设定
PCR产物取5μL加2μL的6×Loading Buffer(天根),5μL的2k plus Marker做对照,跑1%的琼脂糖凝胶(180V,10min),检测条带大小(图5)。
确认条带大小后,剩余产物各加9μL的6xLoading Buffer,10μL的2k plus Marker做对照,跑1.5%的琼脂糖凝胶(120V,30min)。
在紫外切胶仪下,将目的条带切下,用琼脂糖凝胶DNA回收试剂盒回收片段:
(1)用解剖刀或剃刀片切割含有DNA片段的凝胶,尽可能的靠近DNA片段切割,将胶片放在事先称重的1.5mL离心管并称重。记录胶片的重量。
(2)加1:1量的Binding Buffer到胶片中(量以重量计,如每100mg琼脂糖凝胶加100μL的Binding Buffer);
(3)在50-60℃的条件下温育凝胶混合物10min,期间颠倒混匀2-3次,促进胶融化,保证胶全部溶解;
(4)转移最多800μL凝胶溶解液到基因回收纯化柱,12000rpm离心1min,弃去流出液,然后将柱放回相同的收集管;
(5)加入700μL Wash Buffer(已用乙醇稀释)到Gene JET纯化柱。12000rpm离心1min,弃去流出液,然后将柱放回相同的收集管;
(6)离心空GeneJET纯化柱12000rpm离心1min,彻底去除残留的Wash Buffer;
(7)将Gene JET纯化柱转移到一个干净的1.5mL离心管,加30-50μL ddH2O(可60℃预热)于纯化柱膜,12000rpm离心1min;
(8)丢掉Gene JET纯化柱并储存纯化的DNA在-20℃。
四、T载体连接、感受态转化及阳性克隆PCR鉴定
使用pEASY-T3Cloning Vector或pEASY-Blunt Vector(北京全式金生物公司),实验前合成通用引物(M13Forward Primer:5’-GTAAAACGACGGCCAGT-3’;M13Reverse Primer:5’-CAGGAAACAGCTATGAC-3’);或使用片段特异引物进行阳性克隆PCR鉴定。
(1)参考pEASY-T3Cloning Vector或pEASY Blunt Vector使用说明书,进行T载连接;
(2)将全部T载连接产物(5μL)加入到50μL的DH5α感受态细胞中,冰中放置30min;
(3)42℃加热60-90s,再放置冰中2min;
(4)加入500μL的LB培养基,置于摇床中培养45-60min,培养条件为37℃,180rpm;
(5)取出后,在超净台中,将菌液用涂布器均匀涂布于LB+AMP平板上(AMP终浓度为100mg·L-1,后续所用LB+AMP均为此浓度),待菌液吸收完全后,在37℃恒温箱中倒置培养12-16h;
(6)待菌落长出后,在超净台中挑取单克隆菌落,转到含有1mL LB+AMP液体培养基的EP管中;
(7)在摇床上培养3-6h,培养条件为37℃,250rpm;
(8)取1μL菌液作为PCR模板,检测目的片段是否连接上载体;
PCR反应体系:
PCR反应条件:
(9)琼脂糖凝胶电泳检测PCR产物,将出现目的片段条带的菌液,送测序公司测序。
五、大肠杆菌质粒提取
(1)取阳性克隆并测序正确的菌液,按千分之一的比例转接到20mL LB+AMP液体培养基中,在摇床上培养12-16h,培养条件为37℃,250rpm;
(2)利用Plasmid Mini Kit试剂盒(OMEGA)提取菌液中的质粒,详悉步骤参考说明书;
(3)用核酸定量仪测定质粒浓度,存放于-20℃。
五、真核表达载体构建
(1)Twse基因开放阅读框(ORF)扩增:
表3 Twse ORF克隆引物
PCR体系,反应条件及DNA片段回收步骤同上。
(2)pESC-his载体双酶切
双酶切反应体系:
*DNA为pESC-his载体,内切酶为NotI和PacI各1μL。
条件:37℃,2h。
酶切产物加9μL的6xloading Buffer,10μL的2k plus Marker做对照,跑1.5%的琼脂糖凝胶(120V,30min)。在紫外切胶仪下,将目的条带切下,用琼脂糖凝胶DNA回收试剂盒回收片段步骤同上。
(3)载体与片段无缝拼接
连接体系:
*10μL反应体系中,载体与各***片段加入量在0.01-0.25pmols,载体与各个片段的最佳摩尔比为1:2。pmols=质量ng/(片段长度bp×0.65kDa)
条件:50℃,15min。
五、转化,验证,提质粒
(1)取上述连接体系加入到50μL的Trans1-T1感受态细胞中,冰中放置30min;
(2)42℃热激1min后,再放置冰中静置2min;
(3)加入500μL 42℃预热的不含Amp的LB培养基,37℃,180rpm振荡培养60min;
(4)吸取约200μL菌液涂布于LB+Amp培养平板上,37℃黑暗条件下倒置培养12-16h;
(5)挑取单菌落过程在紫外线灭菌后的超净台中操作:挑取平板中的单菌落,置于LB+Amp液体培养基中,37℃,250rpm恒温振荡培养约1-2h后,取1μL菌液进行PCR,检测目的片段是否与载体有效连接。
PCR反应体系:
PCR反应条件:
(备注:*表示PCR产物的片段>3kb,且每加1kb则延伸时间延长1min)
产物经过1%琼脂糖凝胶电泳检测后,凝胶成像仪上观察。将出现目的片段条带的菌液,送公司测序;
(6)根据测序结果,选取测序结果正确的菌液按千分之一的比例扩大培养,250rpm,37℃培养10-12h。
(7)取1-4mL过夜培养的菌液,加入离心管中,12,000rpm(~13,400×g)离心1min,尽量吸除上清(菌液较多时可以通过多次离心将菌体沉淀收集到一个离心管中)。
(8)向留有菌体沉淀的离心管中加入150μL溶液P1(检查是否已加入RNase A和TIANRed),使用移液器或涡旋振荡器彻底悬浮细菌沉淀。
(9)向离心管中加入150μL溶液P2,温和地上下翻转6-8次使菌体充***解。
(10)向离心管中加入350μL溶液P5,立即快速地上下颠倒混匀12-20次,充分混匀,此时将出现絮状沉淀。12,000rpm(~13,400×g)离心2min。
(11)将上一步收集的上清液用移液器转移到吸附柱CP3中(吸附柱放入收集管中),注意尽量不要吸出沉淀。12,000rpm(~13,400×g)离心30sec,倒掉收集管中的废液,将吸附柱CP3放入收集管中。
(12)向吸附柱CP3中加入300μL漂洗液PWT(已加入无水乙醇),12,000rpm(~13,400×g)离心30sec,倒掉收集管中的废液,将吸附柱CP3放入收集管中。
(13)将吸附柱CP3放入收集管中,12,000rpm(~13,400×g)离心1min。
(14)将吸附柱CP3置于一个干净的离心管中,向吸附膜的中间部位滴加30μL无菌水,12,000rpm(~13,400×g)离心1min将质粒溶液收集到离心管中。三管合一管,-20℃保存。
实施实例3、雷公藤鲨烯环氧化酶基因功能验证
一、Erg1缺陷酵母感受态制备
根据Frozen-EZ Yeast Transformation IITM试剂盒说明书进行酵母化学感受态细胞的制备,步骤如下:
(1)挑取构建成功的erg1单菌落,于20mL YPD+麦角固醇(20ug/mL)30℃无氧条件下培养至OD600=0.8-1.0。
(2)500×g离心4min沉淀菌体,弃上清;
(3)10mL EZ1重悬、洗涤菌体,500×g离心4min,弃上清;
(4)1mL EZ2重悬菌体,每50μL分装一管,备用。
注:制备完的感受态细胞可以直接转化,或缓慢降温冻存至-80℃(4℃,1h;-20℃,1h;-80℃)。
二、功能互补实验
(1)取0.2-1μg(≤5μL)pESC-his-Twse质粒或pESC-his质粒(作为对照)分别加入到50μL erg1感受态细胞中;
(2)加入500μL EZ3溶液,彻底混匀;
(3)30℃孵育45min,过程中摇晃2-3次;
(4)取菌液200μL涂布在筛选培养基SC-his+麦角固醇(20ug/mL)固体培养基上,分别于30℃有氧和无氧条件下培养48~72h。
实验结果显示:无氧条件下(见图6左1),erg1酵母缺陷菌无法在SC-his加麦角固醇培养基中生长(a),转入pESC-his空载体的erg1酵母缺陷菌和转入pESC-his-Twse载体的erg1酵母缺陷菌都可以在SC-his加麦角固醇培养基中生长。在有氧的条件下(图6右2),只有转入pESC-his-Twse载体的erg1酵母缺陷菌可以在中生长,转入空载和不转入载体的erg1缺陷酵母菌都无法生长。实验结果证明:雷公藤Twse基因具有生物学功能。
序列表
<110> 首都医科大学
<120> 一种ERG1基因缺陷酵母工程菌、其构建方法及其运用
<141> 2018-08-08
<160> 21
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1771
<212> DNA
<213> 人工序列()
<400> 1
ttcggtagag acaggggagc gagagagact gttattgtgt tccttgcttc tatctggatt 60
tgcatttcga aatggtggat cactgtttgc tcggctggat cctggcctcg gtgctcgggt 120
tcttcacttt gtattttttg gtgttgaagc ggacggatga cgagaagaga gcggttttgg 180
aggcgagaag agagggcatg gagagtctca acacgacaaa cggagaatgt agatctagtg 240
acggcgaaga cgtcgacgtc atcattgtag gagctggcgt cgccggagcc gctcttgctc 300
acactctcgg caaggatgga cgtagagtac gtgtaattga aagagacttg acagagcctg 360
accgcatagt tggtgaattg ctacaaccag ggggctacct caaattgatt gagttgggac 420
ttgaagattg tgttgaagaa attgatgctc aacgagtgtt tggctatgcc ttgtttaagg 480
atgggaaaaa tactcgactt tcttatccct tggaaaagtt tcattcagac gtctcaggga 540
gaagttttca caatggacgt ttcatacaga ggatgcgaca gaaatctgca tcgcttccaa 600
atgtacgatt ggagcaagga actgttactt ctctgctaga agcagatggg ataattaggg 660
gtgtgcagta caaaactaaa agtggggaag aacttaaagc atatgcttct ctgaccattg 720
tgtgtgatgg ctgtttctca aacttgcgac gctccctctg caaaccaaag gttgatgtac 780
cttcttgttt tgttggtttg attctcgaga attgcaacct tccacatgca aatcatggac 840
atgttatcct tggagatcct tcccctattt tgtgttatcc tatcagtagt actgaggttc 900
gctgtctagt tgatgtacct ggtcagaagg ttccctcaat ctcaaacggc gaaatggcaa 960
agtatttgaa gaccatggtg gctccacagc ttcctcctga agtccacgat tcctttgtgg 1020
ctgctgttga taagggaaac cttagaacaa tgccaaaccg aagcatgcca gcttctccct 1080
atcatactcc tggagctttg ttgatggggg atgcattcaa catgcgccac cctctaacag 1140
ggggaggaat gactgtggca ctcgctgata ttgtcgtgct gcggaatctt ctgagacctt 1200
tgcatgactt gaatgacgca cctacccttt gcaaatatct tgaatctttc tacacactgc 1260
gtaagcccgt ggcatccaca atcaacacac tggcaggtgc tttatacaag gtgttttgtg 1320
cctcacctga tcaagcaaga aaggaaatgc gtgaggcttg ctttgattat ttgagtcttg 1380
gaggggtttt cgctgcagga cctgtctctt tgctgtcggg tttaaaccca cgccccttaa 1440
gtttagtttg ccacttcttt gctgttgctg tattcggtgt tggacgttta ttattgccat 1500
tcccttcacc taaacgaatc tggatcggag ctagagtgat cacgagtgca tcaggaatca 1560
tcttccccat cattaaggct gaaggagtaa ggcaaatgtt ctttccggca acggttccag 1620
catactatcg agctcctccc accaagtgag atccaagtat taaggcaact gttaatccta 1680
gtccctagtt attatcatct acgttcccac ccttaaagtg aagggcaagg ttgaatgttt 1740
catcaaaaag ggtgtccctt accgcatcta a 1771
<210> 2
<211> 525
<212> PRT
<213> 人工序列()
<400> 2
Met Val Asp His Cys Leu Leu Gly Trp Ile Leu Ala Ser Val Leu Gly
1 5 10 15
Phe Phe Thr Leu Tyr Phe Leu Val Leu Lys Arg Thr Asp Asp Glu Lys
20 25 30
Arg Ala Val Leu Glu Ala Arg Arg Glu Gly Met Glu Ser Leu Asn Thr
35 40 45
Thr Asn Gly Glu Cys Arg Ser Ser Asp Gly Glu Asp Val Asp Val Ile
50 55 60
Ile Val Gly Ala Gly Val Ala Gly Ala Ala Leu Ala His Thr Leu Gly
65 70 75 80
Lys Asp Gly Arg Arg Val Arg Val Ile Glu Arg Asp Leu Thr Glu Pro
85 90 95
Asp Arg Ile Val Gly Glu Leu Leu Gln Pro Gly Gly Tyr Leu Lys Leu
100 105 110
Ile Glu Leu Gly Leu Glu Asp Cys Val Glu Glu Ile Asp Ala Gln Arg
115 120 125
Val Phe Gly Tyr Ala Leu Phe Lys Asp Gly Lys Asn Thr Arg Leu Ser
130 135 140
Tyr Pro Leu Glu Lys Phe His Ser Asp Val Ser Gly Arg Ser Phe His
145 150 155 160
Asn Gly Arg Phe Ile Gln Arg Met Arg Gln Lys Ser Ala Ser Leu Pro
165 170 175
Asn Val Arg Leu Glu Gln Gly Thr Val Thr Ser Leu Leu Glu Ala Asp
180 185 190
Gly Ile Ile Arg Gly Val Gln Tyr Lys Thr Lys Ser Gly Glu Glu Leu
195 200 205
Lys Ala Tyr Ala Ser Leu Thr Ile Val Cys Asp Gly Cys Phe Ser Asn
210 215 220
Leu Arg Arg Ser Leu Cys Lys Pro Lys Val Asp Val Pro Ser Cys Phe
225 230 235 240
Val Gly Leu Ile Leu Glu Asn Cys Asn Leu Pro His Ala Asn His Gly
245 250 255
His Val Ile Leu Gly Asp Pro Ser Pro Ile Leu Cys Tyr Pro Ile Ser
260 265 270
Ser Thr Glu Val Arg Cys Leu Val Asp Val Pro Gly Gln Lys Val Pro
275 280 285
Ser Ile Ser Asn Gly Glu Met Ala Lys Tyr Leu Lys Thr Met Val Ala
290 295 300
Pro Gln Leu Pro Pro Glu Val His Asp Ser Phe Val Ala Ala Val Asp
305 310 315 320
Lys Gly Asn Leu Arg Thr Met Pro Asn Arg Ser Met Pro Ala Ser Pro
325 330 335
Tyr His Thr Pro Gly Ala Leu Leu Met Gly Asp Ala Phe Asn Met Arg
340 345 350
His Pro Leu Thr Gly Gly Gly Met Thr Val Ala Leu Ala Asp Ile Val
355 360 365
Val Leu Arg Asn Leu Leu Arg Pro Leu His Asp Leu Asn Asp Ala Pro
370 375 380
Thr Leu Cys Lys Tyr Leu Glu Ser Phe Tyr Thr Leu Arg Lys Pro Val
385 390 395 400
Ala Ser Thr Ile Asn Thr Leu Ala Gly Ala Leu Tyr Lys Val Phe Cys
405 410 415
Ala Ser Pro Asp Gln Ala Arg Lys Glu Met Arg Glu Ala Cys Phe Asp
420 425 430
Tyr Leu Ser Leu Gly Gly Val Phe Ala Ala Gly Pro Val Ser Leu Leu
435 440 445
Ser Gly Leu Asn Pro Arg Pro Leu Ser Leu Val Cys His Phe Phe Ala
450 455 460
Val Ala Val Phe Gly Val Gly Arg Leu Leu Leu Pro Phe Pro Ser Pro
465 470 475 480
Lys Arg Ile Trp Ile Gly Ala Arg Val Ile Thr Ser Ala Ser Gly Ile
485 490 495
Ile Phe Pro Ile Ile Lys Ala Glu Gly Val Arg Gln Met Phe Phe Pro
500 505 510
Ala Thr Val Pro Ala Tyr Tyr Arg Ala Pro Pro Thr Lys
515 520 525
<210> 3
<211> 20
<212> DNA
<213> 人工序列()
<400> 3
tatcttttca cgtttcagaa 20
<210> 4
<211> 35
<212> DNA
<213> 人工序列()
<400> 4
tatcttttca cgtttcagaa gttttagagc tagaa 35
<210> 5
<211> 27
<212> DNA
<213> 人工序列()
<400> 5
gatcatttat ctttcactgc ggagaag 27
<210> 6
<211> 38
<212> DNA
<213> 人工序列()
<400> 6
atagcttgtc accttacgta caatcttgat ccggagct 38
<210> 7
<211> 35
<212> DNA
<213> 人工序列()
<400> 7
cttaggggca gacatactcc aagctgcctt tgtgt 35
<210> 8
<211> 35
<212> DNA
<213> 人工序列()
<400> 8
aaggcagctt ggagtatgtc tgcccctaag aagat 35
<210> 9
<211> 41
<212> DNA
<213> 人工序列()
<400> 9
tactactcag taataactta agcaaggatt ttcttaactt c 41
<210> 10
<211> 39
<212> DNA
<213> 人工序列()
<400> 10
agaaaatcct tgcttaagtt attactgagt agtatttat 39
<210> 11
<211> 35
<212> DNA
<213> 人工序列()
<400> 11
agggtgatgg ttcacgtagt gggccatcgc cctga 35
<210> 12
<211> 23
<212> DNA
<213> 人工序列()
<400> 12
gcaccataaa cgacattact ata 23
<210> 13
<211> 21
<212> DNA
<213> 人工序列()
<400> 13
accccaaaaa acttgattag g 21
<210> 14
<211> 120
<212> DNA
<213> 人工序列()
<400> 14
caggttattt cgaacaattg aaaaaaaaaa atcacagaaa aacatatcga gaaaagggtc 60
ctacagctta taagggagag aggataggaa ccgtcaaaca ttaagctgca cctttttttt 120
<210> 15
<211> 120
<212> DNA
<213> 人工序列()
<400> 15
aaaaaaaagg tgcagcttaa tgtttgacgg ttcctatcct ctctccctta taagctgtag 60
gacccttttc tcgatatgtt tttctgtgat tttttttttt caattgttcg aaataacctg 120
<210> 16
<211> 21
<212> DNA
<213> 人工序列()
<400> 16
cattacgcct atctaatctc c 21
<210> 17
<211> 19
<212> DNA
<213> 人工序列()
<400> 17
ttgacggttc ctatcctct 19
<210> 18
<211> 19
<212> DNA
<213> 人工序列()
<400> 18
ttcggtagag acaggggag 19
<210> 19
<211> 19
<212> DNA
<213> 人工序列()
<400> 19
ttagatgcgg taagggaca 19
<210> 20
<211> 37
<212> DNA
<213> 人工序列()
<400> 20
aaccctcact aaagggcatg gtggatcact gtttgct 37
<210> 21
<211> 37
<212> DNA
<213> 人工序列()
<400> 21
ctggcgaaga attgttaatt cacttggtgg gaggagc 37
Claims (1)
1.一种雷公藤鲨烯环氧化酶基因功能验证方法,包括:分别将含有雷公藤鲨烯环氧化酶基因的表达载体pESC-his-Twse及相应不含雷公藤鲨烯环氧化酶基因的空载体pESC-his转入到ERG1基因缺陷型酵母工程菌感受态细胞中,然后将所得感受态细胞涂布在“SC-his+麦角固醇”固体培养基上,分别于有氧和无氧条件下培养,若有氧和无氧下,转入含雷公藤鲨烯环氧化酶基因表达载体的ERG1酵母缺陷菌均可生长,确认雷公藤中鲨烯环氧化酶基因具有生物学功能,其中所述ERG1基因缺陷型酵母工程菌感受态细胞是完全敲除了ERG1基因的酵母工程菌感受态细胞,所述雷公藤鲨烯环氧化酶基因序列如SEQ ID NO:1所示,所述酵母为BY4741酿酒酵母,并且所述ERG1缺陷型酵母工程菌的基因的敲除是采用CRISP/Cas9方法敲除,包括:
(1)筛选酵母ERG1基因的gRNA位点,所述gRNA位点的基因序列为:
TATCTTTTCACGTTTCAGAA;
(2)构建敲除酵母ERG1基因的表达载体:
以EGR1-F和EGR1-R为引物,以p426-SNR52p-gRNA真核表达载体为模板克隆得到线性载体,并以T4连接酶进行平末端连接,构建得到敲除酵母ERG1基因的gRNA表达载体,
所述引物序列为:
ERG1-F:TATCTTTTCACGTTTCAGAAGTTTTAGAGCTAGAA,
ERG1-R:GATCATTTATCTTTCACTGCGGAGAAG;
(3)改造Cas9载体,将p414-TEF1p-Cas9-CYC1t的筛选标记TRP替换为LEU;
(4)获取ERG1基因的双链DNA:
设计敲除酵母ERG1基因的dsOligo,通过同源重组修复对目的基因进行敲除,ERG1-Oligo-F及ERG1-Oligo-R的序列为:
ERG1-Oligo-F:
CAGGTTATTTCGAACAATTGAAAAAAAAAAATCACAGAAAAACATATCGAGAAAAGGGTCCTACAGCTTATAAGGGAGAGAGGATAGGAACCGTCAAACATTAAGCTGCACCTTTTTTTT,
ERG1-Oligo-R:
AAAAAAAAGGTGCAGCTTAATGTTTGACGGTTCCTATCCTCTCTCCCTTATAAGCTGTAGGACCCTTTTCTCGATATGTTTTTCTGTGATTTTTTTTTTTCAATTGTTCGAAATAACCTG,
所述酵母ERG1基因的dsOligo通过将ERG1-Oligo-F及ERG1-Oligo-R退火形成DNA双链,并回收纯化获得;
(5)根据Frozen-EZ Yeast Transformation IITM试剂盒说明书制备酵母感受态细胞;
(6)将步骤(2)中敲除酵母ERG1基因的gRNA表达载体、步骤(3)中改造好的Cas9载体以及步骤(4)中合成的酵母ERG1双链DNA转化至骤(5)中的酵母感受态细胞中,获得突变成功的ERG1基因缺陷的酵母菌。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810896549.1A CN109022299B (zh) | 2018-08-08 | 2018-08-08 | 一种erg1基因缺陷酵母工程菌、其构建方法及其运用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201810896549.1A CN109022299B (zh) | 2018-08-08 | 2018-08-08 | 一种erg1基因缺陷酵母工程菌、其构建方法及其运用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109022299A CN109022299A (zh) | 2018-12-18 |
CN109022299B true CN109022299B (zh) | 2019-09-10 |
Family
ID=64649601
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201810896549.1A Active CN109022299B (zh) | 2018-08-08 | 2018-08-08 | 一种erg1基因缺陷酵母工程菌、其构建方法及其运用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109022299B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN114774503B (zh) * | 2022-06-20 | 2022-10-14 | 中国中医科学院中药研究所 | 角鲨烯环氧化酶及其编码基因与应用 |
Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108359615A (zh) * | 2018-02-09 | 2018-08-03 | 首都医科大学 | 一种高产柳杉二醇的酵母工程菌 |
-
2018
- 2018-08-08 CN CN201810896549.1A patent/CN109022299B/zh active Active
Patent Citations (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108359615A (zh) * | 2018-02-09 | 2018-08-03 | 首都医科大学 | 一种高产柳杉二醇的酵母工程菌 |
Non-Patent Citations (3)
Title |
---|
Building Terpene Production Platforms in Yeast;Xun Zhuang等;《Biotechnology and Bioengineering》;20150930;第112卷(第9期);1854-1864 |
ERGl, Encoding Squalene Epoxidase, is Located on the Right Arm of Chromosome VII of Saccharomyces cerevisiae;KARINA M. LANDL等;《YEAST》;19961231;第12卷;609-613 |
MultiplexmetabolicpathwayengineeringusingCRISPR/Cas9 in Saccharomyces cerevisiae;TadasJakočiūnas等;《Metabolic Engineering》;20150128;第28卷;213-222 |
Also Published As
Publication number | Publication date |
---|---|
CN109022299A (zh) | 2018-12-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104560742B (zh) | 农杆菌介导茭白黑粉菌转化子菌株及其制备方法和应用 | |
CN108866093A (zh) | 一种利用CRISPR/Cas9***对紫花苜蓿基因定点突变的方法 | |
CN106459985B (zh) | 一种分枝杆菌基因工程菌在制备甾体化合物中的应用 | |
CN107287272A (zh) | 一种牛磺熊去氧胆酸的制备方法 | |
CN111057654B (zh) | 一种适用于巴戟天内生真菌A761的Cas9基因敲除载体及其构建方法和应用 | |
CN109609537A (zh) | 一种基因编辑方法在东方拟无枝酸菌中的应用 | |
CN109022299B (zh) | 一种erg1基因缺陷酵母工程菌、其构建方法及其运用 | |
CN109293757B (zh) | 具有控制叶片卷曲功能的毛竹PeTCP10蛋白及其应用 | |
CN112553238B (zh) | 一种适用于拟盾壳霉FS482的CRISPR/Cas9载体及其构建方法和应用 | |
Balabanova et al. | Development of host strains and vector system for an efficient genetic transformation of filamentous fungi | |
CN103173368A (zh) | 一种达玛烯二醇的生物合成方法及其生产菌株 | |
CN103146722B (zh) | 一种来源于矮沙冬青的i型液泡膜焦磷酸酶的基因序列及其克隆方法 | |
CN104212787A (zh) | 一种竹节参β-香树素合酶基因及其应用 | |
CN116218799A (zh) | 催化β-香树脂醇16α位羟基化的CYP450酶蛋白及编码基因与应用 | |
CN105886415A (zh) | 一种生产白桦脂酸的酿酒酵母工程菌及其构建方法 | |
CN109694892A (zh) | 制备红景天苷的方法和试剂盒 | |
CN106520766A (zh) | 一种海藻内源组成型启动子及其应用 | |
CN113956990A (zh) | 产二氢尼洛替星的重组酿酒酵母及其制备方法和应用 | |
CN107903227B (zh) | 琥珀酸酐类化合物、与其相关的基因和蛋白及其制备方法 | |
CN114958637B (zh) | 一种产β-桉叶醇工程菌及其构建方法、应用 | |
CN114438109B (zh) | 桂花基因OfTPS13.2及其应用 | |
CN113430194B (zh) | 一种基于CRISPR/Cas9的白桦基因编辑方法 | |
CN112481290B (zh) | 一种基于形态基因共干扰提高柠檬酸发酵生产水平的方法 | |
CN107858362B (zh) | 产甘油假丝酵母热休克蛋白基因CgYDJ1及其应用 | |
CN107858361B (zh) | 产甘油假丝酵母热休克蛋白基因CgHsp10及其应用 |
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 |