CN105087601B - 一种珠子参转录因子基因PjWRKY1的应用 - Google Patents
一种珠子参转录因子基因PjWRKY1的应用 Download PDFInfo
- Publication number
- CN105087601B CN105087601B CN201510561989.8A CN201510561989A CN105087601B CN 105087601 B CN105087601 B CN 105087601B CN 201510561989 A CN201510561989 A CN 201510561989A CN 105087601 B CN105087601 B CN 105087601B
- Authority
- CN
- China
- Prior art keywords
- panax japonicus
- japonicus majoris
- pjwrky1
- saponin
- gene
- 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.)
- Expired - Fee Related
Links
- 241000168720 Panax japonicus Species 0.000 title claims abstract description 83
- 235000003174 Panax japonicus Nutrition 0.000 title claims abstract description 83
- 108091023040 Transcription factor Proteins 0.000 title claims abstract description 31
- 229930182490 saponin Natural products 0.000 claims abstract description 49
- 239000001397 quillaja saponaria molina bark Substances 0.000 claims abstract description 48
- 150000007949 saponins Chemical class 0.000 claims abstract description 47
- 102000040945 Transcription factor Human genes 0.000 claims abstract description 26
- 230000015572 biosynthetic process Effects 0.000 claims abstract description 26
- 241000196324 Embryophyta Species 0.000 claims abstract description 17
- 206010020649 Hyperkeratosis Diseases 0.000 claims abstract description 16
- 230000014509 gene expression Effects 0.000 claims abstract description 11
- 108090000790 Enzymes Proteins 0.000 claims abstract description 9
- 239000013604 expression vector Substances 0.000 claims abstract description 9
- 238000013518 transcription Methods 0.000 claims abstract description 7
- 230000037361 pathway Effects 0.000 claims abstract description 5
- 101150044508 key gene Proteins 0.000 claims abstract description 4
- 230000002018 overexpression Effects 0.000 claims abstract description 4
- 230000009261 transgenic effect Effects 0.000 claims description 10
- 102000004190 Enzymes Human genes 0.000 claims description 6
- 238000011529 RT qPCR Methods 0.000 claims description 6
- 238000012216 screening Methods 0.000 claims description 6
- 230000037353 metabolic pathway Effects 0.000 claims description 5
- 230000035897 transcription Effects 0.000 claims description 5
- 230000001404 mediated effect Effects 0.000 claims description 4
- 238000001514 detection method Methods 0.000 claims description 3
- 239000013598 vector Substances 0.000 claims description 3
- 241000193830 Bacillus <bacterium> Species 0.000 claims description 2
- 239000002773 nucleotide Substances 0.000 claims description 2
- 108090000623 proteins and genes Proteins 0.000 abstract description 24
- 238000005516 engineering process Methods 0.000 abstract description 3
- 238000012269 metabolic engineering Methods 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 abstract description 2
- 235000017709 saponins Nutrition 0.000 description 36
- 238000000034 method Methods 0.000 description 22
- 238000006243 chemical reaction Methods 0.000 description 18
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 108020004414 DNA Proteins 0.000 description 14
- 241000894006 Bacteria Species 0.000 description 12
- 239000002299 complementary DNA Substances 0.000 description 11
- 238000002156 mixing Methods 0.000 description 11
- 239000013612 plasmid Substances 0.000 description 11
- 239000000243 solution Substances 0.000 description 11
- 239000000047 product Substances 0.000 description 10
- 241000589158 Agrobacterium Species 0.000 description 9
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 9
- 238000003786 synthesis reaction Methods 0.000 description 9
- 230000008569 process Effects 0.000 description 8
- 102100035111 Farnesyl pyrophosphate synthase Human genes 0.000 description 6
- 230000033228 biological regulation Effects 0.000 description 6
- 239000003292 glue Substances 0.000 description 6
- 230000024053 secondary metabolic process Effects 0.000 description 6
- 210000002966 serum Anatomy 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 5
- 240000005373 Panax quinquefolius Species 0.000 description 5
- 239000000872 buffer Substances 0.000 description 5
- 239000012634 fragment Substances 0.000 description 5
- 239000007787 solid Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- 102000012410 DNA Ligases Human genes 0.000 description 4
- 108010061982 DNA Ligases Proteins 0.000 description 4
- 241000180649 Panax notoginseng Species 0.000 description 4
- 235000003143 Panax notoginseng Nutrition 0.000 description 4
- 235000003140 Panax quinquefolius Nutrition 0.000 description 4
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 4
- 230000029087 digestion Effects 0.000 description 4
- 239000003814 drug Substances 0.000 description 4
- 230000000694 effects Effects 0.000 description 4
- 239000007788 liquid Substances 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000000203 mixture Substances 0.000 description 4
- 238000011084 recovery Methods 0.000 description 4
- NFZYDZXHKFHPGA-QQHDHSITSA-N Chikusetsusaponin-V Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@H](O[C@H]1O[C@H]1CC[C@]2(C)[C@H]3CC=C4[C@@]([C@@]3(CC[C@H]2C1(C)C)C)(C)CC[C@]1(CCC(C[C@H]14)(C)C)C(=O)O[C@H]1[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O1)O)C(O)=O)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O NFZYDZXHKFHPGA-QQHDHSITSA-N 0.000 description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 235000002791 Panax Nutrition 0.000 description 3
- 241000208343 Panax Species 0.000 description 3
- 238000013459 approach Methods 0.000 description 3
- 238000005119 centrifugation Methods 0.000 description 3
- -1 dammarane saponins Chemical class 0.000 description 3
- 238000001962 electrophoresis Methods 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000002068 genetic effect Effects 0.000 description 3
- 235000008434 ginseng Nutrition 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 238000011160 research Methods 0.000 description 3
- 238000003757 reverse transcription PCR Methods 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- NFZYDZXHKFHPGA-UHFFFAOYSA-N 17alpha-hydroxygofruside Natural products C12CC(C)(C)CCC2(C(=O)OC2C(C(O)C(O)C(CO)O2)O)CCC(C2(CCC3C4(C)C)C)(C)C1=CCC2C3(C)CCC4OC1OC(C(O)=O)C(O)C(O)C1OC1OC(CO)C(O)C(O)C1O NFZYDZXHKFHPGA-UHFFFAOYSA-N 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- QXQFFGOMXYKNBA-UHFFFAOYSA-N Chikusetsusaponin V Natural products CC1(C)CCC2(CCC3C(=CCC4C3(C)CCC5C(C)(C)C(CCC45C)OC6OC(C(O)C(O)C6OC7OC(CO)C(O)C(O)C7O)C(=O)O)C2C1)C(=O)OC8OC(CO)C(O)C(O)C8O QXQFFGOMXYKNBA-UHFFFAOYSA-N 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 102100031780 Endonuclease Human genes 0.000 description 2
- 108010022535 Farnesyl-Diphosphate Farnesyltransferase Proteins 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 2
- 235000005035 Panax pseudoginseng ssp. pseudoginseng Nutrition 0.000 description 2
- UIGMAMGZOJVTDN-WHFBIAKZSA-N Ser-Gly-Ser Chemical compound OC[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O UIGMAMGZOJVTDN-WHFBIAKZSA-N 0.000 description 2
- 240000003768 Solanum lycopersicum Species 0.000 description 2
- 102100037997 Squalene synthase Human genes 0.000 description 2
- 229960000583 acetic acid Drugs 0.000 description 2
- 238000000246 agarose gel electrophoresis Methods 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical class 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
- 108010062796 arginyllysine Proteins 0.000 description 2
- 230000001580 bacterial effect Effects 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000007796 conventional method Methods 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000012154 double-distilled water Substances 0.000 description 2
- 229940079593 drug Drugs 0.000 description 2
- 239000000284 extract Substances 0.000 description 2
- 238000013467 fragmentation Methods 0.000 description 2
- 238000006062 fragmentation reaction Methods 0.000 description 2
- 238000010353 genetic engineering Methods 0.000 description 2
- RBRANZURTULKJD-UHFFFAOYSA-N ginsenoside Ro Natural products CC1(C)CCC2(CCC3(C)C(=CCC4C5(C)CCC(OC6OC(C)(C(O)C(O)C6OC7OC(CO)C(O)C(O)C7O)C(=O)O)C(C)(C)C5CCC34C)C2C1)C(=O)OC8OC(CO)C(O)C(O)C8O RBRANZURTULKJD-UHFFFAOYSA-N 0.000 description 2
- 239000012362 glacial acetic acid Substances 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 239000012535 impurity Substances 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical class O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 2
- 210000004072 lung Anatomy 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- VLTRZXGMWDSKGL-UHFFFAOYSA-N perchloric acid Chemical compound OCl(=O)(=O)=O VLTRZXGMWDSKGL-UHFFFAOYSA-N 0.000 description 2
- 238000005498 polishing Methods 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 108010087432 terpene synthase Proteins 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 108030004879 (+)-delta-cadinene synthases Proteins 0.000 description 1
- FBFMBWCLBGQEBU-RXMALORBSA-N (2s,3r,4s,5s,6r)-2-[(2r,3r,4s,5s,6r)-2-[[(3s,5r,6s,8r,9r,10r,12r,13r,14r,17s)-3,12-dihydroxy-4,4,8,10,14-pentamethyl-17-[(2s)-6-methyl-2-[(2s,3r,4s,5s,6r)-3,4,5-trihydroxy-6-(hydroxymethyl)oxan-2-yl]oxyhept-5-en-2-yl]-2,3,5,6,7,9,11,12,13,15,16,17-dodecah Chemical compound O([C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(C[C@@H]([C@H]4C(C)(C)[C@@H](O)CC[C@]4(C)[C@H]3C[C@H]2O)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O[C@H]2[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O2)O)C)(C)CC1)[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O FBFMBWCLBGQEBU-RXMALORBSA-N 0.000 description 1
- VCNKUCWWHVTTBY-UHFFFAOYSA-N 18alpha-Oleanane Natural products C1CCC(C)(C)C2CCC3(C)C4(C)CCC5(C)CCC(C)(C)CC5C4CCC3C21C VCNKUCWWHVTTBY-UHFFFAOYSA-N 0.000 description 1
- VWFJDQUYCIWHTN-YFVJMOTDSA-N 2-trans,6-trans-farnesyl diphosphate Chemical compound CC(C)=CCC\C(C)=C\CC\C(C)=C\CO[P@](O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-YFVJMOTDSA-N 0.000 description 1
- FBFMBWCLBGQEBU-GYMUUCMZSA-N 20-gluco-ginsenoside-Rf Natural products O([C@](CC/C=C(\C)/C)(C)[C@@H]1[C@H]2[C@H](O)C[C@H]3[C@](C)([C@]2(C)CC1)C[C@H](O[C@@H]1[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O2)[C@@H](O)[C@H](O)[C@@H](CO)O1)[C@H]1C(C)(C)[C@@H](O)CC[C@]31C)[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 FBFMBWCLBGQEBU-GYMUUCMZSA-N 0.000 description 1
- 108020005345 3' Untranslated Regions Proteins 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- YLTKNGYYPIWKHZ-ACZMJKKPSA-N Ala-Ala-Glu Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O YLTKNGYYPIWKHZ-ACZMJKKPSA-N 0.000 description 1
- LWUWMHIOBPTZBA-DCAQKATOSA-N Ala-Arg-Lys Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@@H](CCCCN)C(O)=O LWUWMHIOBPTZBA-DCAQKATOSA-N 0.000 description 1
- IPZQNYYAYVRKKK-FXQIFTODSA-N Ala-Pro-Ala Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IPZQNYYAYVRKKK-FXQIFTODSA-N 0.000 description 1
- IYKVSFNGSWTTNZ-GUBZILKMSA-N Ala-Val-Arg Chemical compound C[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N IYKVSFNGSWTTNZ-GUBZILKMSA-N 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 241000208340 Araliaceae Species 0.000 description 1
- HJVGMOYJDDXLMI-AVGNSLFASA-N Arg-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CCCNC(N)=N HJVGMOYJDDXLMI-AVGNSLFASA-N 0.000 description 1
- SNBHMYQRNCJSOJ-CIUDSAMLSA-N Arg-Gln-Asn Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O SNBHMYQRNCJSOJ-CIUDSAMLSA-N 0.000 description 1
- QEHMMRSQJMOYNO-DCAQKATOSA-N Arg-His-Asn Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N QEHMMRSQJMOYNO-DCAQKATOSA-N 0.000 description 1
- OOIMKQRCPJBGPD-XUXIUFHCSA-N Arg-Ile-Leu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O OOIMKQRCPJBGPD-XUXIUFHCSA-N 0.000 description 1
- ATABBWFGOHKROJ-GUBZILKMSA-N Arg-Pro-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O ATABBWFGOHKROJ-GUBZILKMSA-N 0.000 description 1
- POZKLUIXMHIULG-FDARSICLSA-N Arg-Trp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)NC(=O)[C@H](CCCN=C(N)N)N POZKLUIXMHIULG-FDARSICLSA-N 0.000 description 1
- 208000006820 Arthralgia Diseases 0.000 description 1
- GOVUDFOGXOONFT-VEVYYDQMSA-N Asn-Arg-Thr Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GOVUDFOGXOONFT-VEVYYDQMSA-N 0.000 description 1
- RRKCPMGSRIDLNC-AVGNSLFASA-N Asp-Glu-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RRKCPMGSRIDLNC-AVGNSLFASA-N 0.000 description 1
- DWOGMPWRQQWPPF-GUBZILKMSA-N Asp-Leu-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O DWOGMPWRQQWPPF-GUBZILKMSA-N 0.000 description 1
- FAUPLTGRUBTXNU-FXQIFTODSA-N Asp-Pro-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O FAUPLTGRUBTXNU-FXQIFTODSA-N 0.000 description 1
- RVMXMLSYBTXCAV-VEVYYDQMSA-N Asp-Pro-Thr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMXMLSYBTXCAV-VEVYYDQMSA-N 0.000 description 1
- VNXQRBXEQXLERQ-CIUDSAMLSA-N Asp-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)O)N VNXQRBXEQXLERQ-CIUDSAMLSA-N 0.000 description 1
- 108091006146 Channels Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- LWTTURISBKEVAC-CIUDSAMLSA-N Cys-Cys-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CS)N LWTTURISBKEVAC-CIUDSAMLSA-N 0.000 description 1
- 102000004163 DNA-directed RNA polymerases Human genes 0.000 description 1
- 108090000626 DNA-directed RNA polymerases Proteins 0.000 description 1
- 206010013954 Dysphoria Diseases 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 241000305071 Enterobacterales Species 0.000 description 1
- 241000588722 Escherichia Species 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- VWFJDQUYCIWHTN-UHFFFAOYSA-N Farnesyl pyrophosphate Natural products CC(C)=CCCC(C)=CCCC(C)=CCOP(O)(=O)OP(O)(O)=O VWFJDQUYCIWHTN-UHFFFAOYSA-N 0.000 description 1
- 241000282326 Felis catus Species 0.000 description 1
- 108010026318 Geranyltranstransferase Proteins 0.000 description 1
- UFNDONGOJKNAES-UHFFFAOYSA-N Ginsenoside Rb1 Natural products CC(=CCCC(C)(OC1OC(COC2OC(CO)C(O)C(O)C2O)C(O)C(O)C1O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CC(O)C45C)C UFNDONGOJKNAES-UHFFFAOYSA-N 0.000 description 1
- HYPFYJBWSTXDAS-UHFFFAOYSA-N Ginsenoside Rd Natural products CC(=CCCC(C)(OC1OC(CO)C(O)C(O)C1O)C2CCC3(C)C4CCC5C(C)(C)C(CCC5(C)C4CC(O)C23C)OC6OC(CO)C(O)C(O)C6OC7OC(CO)C(O)C(O)C7O)C HYPFYJBWSTXDAS-UHFFFAOYSA-N 0.000 description 1
- AJDMYLOISOCHHC-YVNDNENWSA-N Gln-Gln-Ile Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O AJDMYLOISOCHHC-YVNDNENWSA-N 0.000 description 1
- QBLMTCRYYTVUQY-GUBZILKMSA-N Gln-Leu-Asp Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O QBLMTCRYYTVUQY-GUBZILKMSA-N 0.000 description 1
- KVQOVQVGVKDZNW-GUBZILKMSA-N Gln-Ser-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N KVQOVQVGVKDZNW-GUBZILKMSA-N 0.000 description 1
- ISXJHXGYMJKXOI-GUBZILKMSA-N Glu-Cys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CS)NC(=O)[C@@H](N)CCC(O)=O ISXJHXGYMJKXOI-GUBZILKMSA-N 0.000 description 1
- HPJLZFTUUJKWAJ-JHEQGTHGSA-N Glu-Gly-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HPJLZFTUUJKWAJ-JHEQGTHGSA-N 0.000 description 1
- QOXDAWODGSIDDI-GUBZILKMSA-N Glu-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)O)N QOXDAWODGSIDDI-GUBZILKMSA-N 0.000 description 1
- YYQGVXNKAXUTJU-YUMQZZPRSA-N Gly-Cys-His Chemical compound NCC(=O)N[C@@H](CS)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O YYQGVXNKAXUTJU-YUMQZZPRSA-N 0.000 description 1
- UEGIPZAXNBYCCP-NKWVEPMBSA-N Gly-Cys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CS)NC(=O)CN)C(=O)O UEGIPZAXNBYCCP-NKWVEPMBSA-N 0.000 description 1
- JUBDONGMHASUCN-IUCAKERBSA-N Gly-Glu-His Chemical compound NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O JUBDONGMHASUCN-IUCAKERBSA-N 0.000 description 1
- HAXARWKYFIIHKD-ZKWXMUAHSA-N Gly-Ile-Ser Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CO)C(O)=O HAXARWKYFIIHKD-ZKWXMUAHSA-N 0.000 description 1
- NGBGZCUWFVVJKC-IRXDYDNUSA-N Gly-Tyr-Tyr Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=C(O)C=C1 NGBGZCUWFVVJKC-IRXDYDNUSA-N 0.000 description 1
- 235000009438 Gossypium Nutrition 0.000 description 1
- 241000219146 Gossypium Species 0.000 description 1
- 240000001814 Gossypium arboreum Species 0.000 description 1
- 235000014751 Gossypium arboreum Nutrition 0.000 description 1
- 208000000616 Hemoptysis Diseases 0.000 description 1
- AWHJQEYGWRKPHE-LSJOCFKGSA-N His-Ala-Arg Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O AWHJQEYGWRKPHE-LSJOCFKGSA-N 0.000 description 1
- BXOLYFJYQQRQDJ-MXAVVETBSA-N His-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CN=CN1)N BXOLYFJYQQRQDJ-MXAVVETBSA-N 0.000 description 1
- CGAMSLMBYJHMDY-ONGXEEELSA-N His-Val-Gly Chemical compound CC(C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CC1=CN=CN1)N CGAMSLMBYJHMDY-ONGXEEELSA-N 0.000 description 1
- QDYPTQWAAOGCJD-UHFFFAOYSA-N Huzhangoside A Natural products OC1C(C)OC(OC2C(OCC(O)C2O)OC2C(C3C(C4C(C5(CCC6(CCC(C)(C)CC6C5=CC4)C(O)=O)C)(C)CC3)(C)CC2)(C)C)C(O)C1OC1OCC(O)C(O)C1O QDYPTQWAAOGCJD-UHFFFAOYSA-N 0.000 description 1
- HLYBGMZJVDHJEO-CYDGBPFRSA-N Ile-Arg-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N HLYBGMZJVDHJEO-CYDGBPFRSA-N 0.000 description 1
- KUHFPGIVBOCRMV-MNXVOIDGSA-N Ile-Gln-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N KUHFPGIVBOCRMV-MNXVOIDGSA-N 0.000 description 1
- LGMUPVWZEYYUMU-YVNDNENWSA-N Ile-Glu-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N LGMUPVWZEYYUMU-YVNDNENWSA-N 0.000 description 1
- YKLOMBNBQUTJDT-HVTMNAMFSA-N Ile-His-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YKLOMBNBQUTJDT-HVTMNAMFSA-N 0.000 description 1
- DSDPLOODKXISDT-XUXIUFHCSA-N Ile-Leu-Val Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O DSDPLOODKXISDT-XUXIUFHCSA-N 0.000 description 1
- IITVUURPOYGCTD-NAKRPEOUSA-N Ile-Pro-Ala Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O IITVUURPOYGCTD-NAKRPEOUSA-N 0.000 description 1
- ZNOBVZFCHNHKHA-KBIXCLLPSA-N Ile-Ser-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N ZNOBVZFCHNHKHA-KBIXCLLPSA-N 0.000 description 1
- IAJFFZORSWOZPQ-SRVKXCTJSA-N Leu-Leu-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O IAJFFZORSWOZPQ-SRVKXCTJSA-N 0.000 description 1
- UHNQRAFSEBGZFZ-YESZJQIVSA-N Leu-Phe-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N UHNQRAFSEBGZFZ-YESZJQIVSA-N 0.000 description 1
- VULJUQZPSOASBZ-SRVKXCTJSA-N Leu-Pro-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O VULJUQZPSOASBZ-SRVKXCTJSA-N 0.000 description 1
- CHJKEDSZNSONPS-DCAQKATOSA-N Leu-Pro-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O CHJKEDSZNSONPS-DCAQKATOSA-N 0.000 description 1
- 235000007688 Lycopersicon esculentum Nutrition 0.000 description 1
- IRNSXVOWSXSULE-DCAQKATOSA-N Lys-Ala-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN IRNSXVOWSXSULE-DCAQKATOSA-N 0.000 description 1
- ZTPWXNOOKAXPPE-DCAQKATOSA-N Lys-Arg-Cys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CS)C(=O)O)N ZTPWXNOOKAXPPE-DCAQKATOSA-N 0.000 description 1
- GAOJCVKPIGHTGO-UWVGGRQHSA-N Lys-Arg-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O GAOJCVKPIGHTGO-UWVGGRQHSA-N 0.000 description 1
- XTONYTDATVADQH-CIUDSAMLSA-N Lys-Cys-Asn Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(N)=O)C(O)=O XTONYTDATVADQH-CIUDSAMLSA-N 0.000 description 1
- ISHNZELVUVPCHY-ZETCQYMHSA-N Lys-Gly-Gly Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)NCC(O)=O ISHNZELVUVPCHY-ZETCQYMHSA-N 0.000 description 1
- WVJNGSFKBKOKRV-AJNGGQMLSA-N Lys-Leu-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O WVJNGSFKBKOKRV-AJNGGQMLSA-N 0.000 description 1
- QBHGXFQJFPWJIH-XUXIUFHCSA-N Lys-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCCCN QBHGXFQJFPWJIH-XUXIUFHCSA-N 0.000 description 1
- UQJOKDAYFULYIX-AVGNSLFASA-N Lys-Pro-Pro Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 UQJOKDAYFULYIX-AVGNSLFASA-N 0.000 description 1
- AETNZPKUUYYYEK-CIUDSAMLSA-N Met-Glu-Asn Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O AETNZPKUUYYYEK-CIUDSAMLSA-N 0.000 description 1
- LCPUWQLULVXROY-RHYQMDGZSA-N Met-Lys-Thr Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LCPUWQLULVXROY-RHYQMDGZSA-N 0.000 description 1
- 229930189092 Notoginsenoside Natural products 0.000 description 1
- 208000012868 Overgrowth Diseases 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- 240000004371 Panax ginseng Species 0.000 description 1
- 235000002789 Panax ginseng Nutrition 0.000 description 1
- 101710093888 Pentalenene synthase Proteins 0.000 description 1
- LZDIENNKWVXJMX-JYJNAYRXSA-N Phe-Arg-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC1=CC=CC=C1 LZDIENNKWVXJMX-JYJNAYRXSA-N 0.000 description 1
- IPFXYNKCXYGSSV-KKUMJFAQSA-N Phe-Ser-Lys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O)N IPFXYNKCXYGSSV-KKUMJFAQSA-N 0.000 description 1
- SHUFSZDAIPLZLF-BEAPCOKYSA-N Phe-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)O SHUFSZDAIPLZLF-BEAPCOKYSA-N 0.000 description 1
- ORPZXBQTEHINPB-SRVKXCTJSA-N Pro-Arg-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H]1CCCN1)C(O)=O ORPZXBQTEHINPB-SRVKXCTJSA-N 0.000 description 1
- AFXCXDQNRXTSBD-FJXKBIBVSA-N Pro-Gly-Thr Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O AFXCXDQNRXTSBD-FJXKBIBVSA-N 0.000 description 1
- AQGUSRZKDZYGGV-GMOBBJLQSA-N Pro-Ile-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O AQGUSRZKDZYGGV-GMOBBJLQSA-N 0.000 description 1
- ULWBBFKQBDNGOY-RWMBFGLXSA-N Pro-Lys-Pro Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N2CCC[C@@H]2C(=O)O ULWBBFKQBDNGOY-RWMBFGLXSA-N 0.000 description 1
- 108010076504 Protein Sorting Signals Proteins 0.000 description 1
- 108010092799 RNA-directed DNA polymerase Proteins 0.000 description 1
- 101710192189 Ribonuclease 1 Proteins 0.000 description 1
- 101710141795 Ribonuclease inhibitor Proteins 0.000 description 1
- 229940122208 Ribonuclease inhibitor Drugs 0.000 description 1
- 102100037968 Ribonuclease inhibitor Human genes 0.000 description 1
- 102100039832 Ribonuclease pancreatic Human genes 0.000 description 1
- 229920002684 Sepharose Polymers 0.000 description 1
- 238000012300 Sequence Analysis Methods 0.000 description 1
- ZUGXSSFMTXKHJS-ZLUOBGJFSA-N Ser-Ala-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O ZUGXSSFMTXKHJS-ZLUOBGJFSA-N 0.000 description 1
- ULVMNZOKDBHKKI-ACZMJKKPSA-N Ser-Gln-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ULVMNZOKDBHKKI-ACZMJKKPSA-N 0.000 description 1
- UFKPDBLKLOBMRH-XHNCKOQMSA-N Ser-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CO)N)C(=O)O UFKPDBLKLOBMRH-XHNCKOQMSA-N 0.000 description 1
- QBUWQRKEHJXTOP-DCAQKATOSA-N Ser-His-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O QBUWQRKEHJXTOP-DCAQKATOSA-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
- KQNDIKOYWZTZIX-FXQIFTODSA-N Ser-Ser-Arg Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQNDIKOYWZTZIX-FXQIFTODSA-N 0.000 description 1
- JCLAFVNDBJMLBC-JBDRJPRFSA-N Ser-Ser-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JCLAFVNDBJMLBC-JBDRJPRFSA-N 0.000 description 1
- XQJCEKXQUJQNNK-ZLUOBGJFSA-N Ser-Ser-Ser Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O XQJCEKXQUJQNNK-ZLUOBGJFSA-N 0.000 description 1
- 101710115850 Sesquiterpene synthase Proteins 0.000 description 1
- 235000002560 Solanum lycopersicum Nutrition 0.000 description 1
- DWYAUVCQDTZIJI-VZFHVOOUSA-N Thr-Ala-Ser Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O DWYAUVCQDTZIJI-VZFHVOOUSA-N 0.000 description 1
- AQAMPXBRJJWPNI-JHEQGTHGSA-N Thr-Gly-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O AQAMPXBRJJWPNI-JHEQGTHGSA-N 0.000 description 1
- BDGBHYCAZJPLHX-HJGDQZAQSA-N Thr-Lys-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O BDGBHYCAZJPLHX-HJGDQZAQSA-N 0.000 description 1
- VTMGKRABARCZAX-OSUNSFLBSA-N Thr-Pro-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O VTMGKRABARCZAX-OSUNSFLBSA-N 0.000 description 1
- WPSKTVVMQCXPRO-BWBBJGPYSA-N Thr-Ser-Ser Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WPSKTVVMQCXPRO-BWBBJGPYSA-N 0.000 description 1
- COYHRQWNJDJCNA-NUJDXYNKSA-N Thr-Thr-Thr Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O COYHRQWNJDJCNA-NUJDXYNKSA-N 0.000 description 1
- KPMIQCXJDVKWKO-IFFSRLJSSA-N Thr-Val-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O KPMIQCXJDVKWKO-IFFSRLJSSA-N 0.000 description 1
- PWONLXBUSVIZPH-RHYQMDGZSA-N Thr-Val-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N)O PWONLXBUSVIZPH-RHYQMDGZSA-N 0.000 description 1
- 206010053476 Traumatic haemorrhage Diseases 0.000 description 1
- MVHHTXAUJCIOMZ-WDSOQIARSA-N Trp-Arg-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCCN)C(=O)O)N MVHHTXAUJCIOMZ-WDSOQIARSA-N 0.000 description 1
- IXTQGBGHWQEEDE-AVGNSLFASA-N Tyr-Asp-Gln Chemical compound NC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 IXTQGBGHWQEEDE-AVGNSLFASA-N 0.000 description 1
- GVJUTBOZZBTBIG-AVGNSLFASA-N Val-Lys-Arg Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N GVJUTBOZZBTBIG-AVGNSLFASA-N 0.000 description 1
- KSFXWENSJABBFI-ZKWXMUAHSA-N Val-Ser-Asn Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O KSFXWENSJABBFI-ZKWXMUAHSA-N 0.000 description 1
- AEFJNECXZCODJM-UWVGGRQHSA-N Val-Val-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)NCC([O-])=O AEFJNECXZCODJM-UWVGGRQHSA-N 0.000 description 1
- JVGDAEKKZKKZFO-RCWTZXSCSA-N Val-Val-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](C(C)C)N)O JVGDAEKKZKKZFO-RCWTZXSCSA-N 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 238000002835 absorbance Methods 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 108010087924 alanylproline Proteins 0.000 description 1
- 150000001335 aliphatic alkanes Chemical class 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 238000009412 basement excavation Methods 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 230000008901 benefit Effects 0.000 description 1
- 238000007622 bioinformatic analysis Methods 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 238000010367 cloning Methods 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 235000009508 confectionery Nutrition 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- OORMXZNMRWBSTK-LGFJJATJSA-N dammarane Chemical compound C1CCC(C)(C)[C@@H]2CC[C@@]3(C)[C@]4(C)CC[C@H]([C@H](C)CCCC(C)C)[C@H]4CC[C@@H]3[C@]21C OORMXZNMRWBSTK-LGFJJATJSA-N 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000004821 distillation Methods 0.000 description 1
- 230000003828 downregulation Effects 0.000 description 1
- 238000004043 dyeing Methods 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000012252 genetic analysis Methods 0.000 description 1
- 230000005017 genetic modification Effects 0.000 description 1
- 235000013617 genetically modified food Nutrition 0.000 description 1
- GZYPWOGIYAIIPV-JBDTYSNRSA-N ginsenoside Rb1 Chemical compound C([C@H]1O[C@H]([C@@H]([C@@H](O)[C@@H]1O)O)O[C@@](C)(CCC=C(C)C)[C@@H]1[C@@H]2[C@@]([C@@]3(CC[C@H]4C(C)(C)[C@@H](O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O[C@H]5[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O5)O)CC[C@]4(C)[C@H]3C[C@H]2O)C)(C)CC1)O[C@@H]1O[C@H](CO)[C@@H](O)[C@H](O)[C@H]1O GZYPWOGIYAIIPV-JBDTYSNRSA-N 0.000 description 1
- TXEWRVNOAJOINC-UHFFFAOYSA-N ginsenoside Rb2 Natural products CC(=CCCC(OC1OC(COC2OCC(O)C(O)C2O)C(O)C(O)C1O)C3CCC4(C)C3C(O)CC5C6(C)CCC(OC7OC(CO)C(O)C(O)C7OC8OC(CO)C(O)C(O)C8O)C(C)(C)C6CCC45C)C TXEWRVNOAJOINC-UHFFFAOYSA-N 0.000 description 1
- PWAOOJDMFUQOKB-WCZZMFLVSA-N ginsenoside Re Chemical compound O[C@@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@H]1O[C@H]1[C@H](O[C@@H]2[C@H]3C(C)(C)[C@@H](O)CC[C@]3(C)[C@@H]3[C@@]([C@@]4(CC[C@@H]([C@H]4[C@H](O)C3)[C@](C)(CCC=C(C)C)O[C@H]3[C@@H]([C@@H](O)[C@H](O)[C@@H](CO)O3)O)C)(C)C2)O[C@H](CO)[C@@H](O)[C@@H]1O PWAOOJDMFUQOKB-WCZZMFLVSA-N 0.000 description 1
- AOGZLQUEBLOQCI-UHFFFAOYSA-N ginsenoside-Re Natural products CC1OC(OCC2OC(OC3CC4(C)C(CC(O)C5C(CCC45C)C(C)(CCC=C(C)C)OC6OC(CO)C(O)C(O)C6O)C7(C)CCC(O)C(C)(C)C37)C(O)C(O)C2O)C(O)C(O)C1O AOGZLQUEBLOQCI-UHFFFAOYSA-N 0.000 description 1
- 229930182470 glycoside Natural products 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- YQOKLYTXVFAUCW-UHFFFAOYSA-N guanidine;isothiocyanic acid Chemical compound N=C=S.NC(N)=N YQOKLYTXVFAUCW-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000008236 heating water Substances 0.000 description 1
- 230000023597 hemostasis Effects 0.000 description 1
- 241000411851 herbal medicine Species 0.000 description 1
- SIOMFBXUIJKTMF-UHFFFAOYSA-N hypoglauterpenic acid Natural products C1CC(O)C(C)(C)C2=CCC3(C)C4(C)CCC5(C(O)=O)CCC(C)(C)CC5C4=CCC3C21C SIOMFBXUIJKTMF-UHFFFAOYSA-N 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 208000014674 injury Diseases 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 210000000936 intestine Anatomy 0.000 description 1
- 239000010977 jade Substances 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 231100000518 lethal Toxicity 0.000 description 1
- 230000001665 lethal effect Effects 0.000 description 1
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 229910001629 magnesium chloride Inorganic materials 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- BPAWXSVOAOLSRP-UHFFFAOYSA-N oleanane Natural products CCCCCCCCCCCCCCCC(=O)OC1CCC2(C)C(CCC3(C)C2CC=C4C5CC(C)(C)CCC5(C)C(O)CC34C)C1(C)C BPAWXSVOAOLSRP-UHFFFAOYSA-N 0.000 description 1
- 229930182495 oleanane-type triterpene Natural products 0.000 description 1
- 238000011017 operating method Methods 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 230000003119 painkilling effect Effects 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108010082795 phenylalanyl-arginyl-arginine Proteins 0.000 description 1
- 108010089198 phenylalanyl-prolyl-arginine Proteins 0.000 description 1
- 230000029279 positive regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 108010015796 prolylisoleucine Proteins 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- TXUSTSJRABHHQP-UFGIQYKASA-N pulsatilla saponin D Natural products C[C@@H]1O[C@@H](O[C@@H]2[C@@H](O)[C@H](CO[C@H]2O[C@H]3CC[C@@]4(C)[C@@H](CC[C@]5(C)[C@@H]4CC=C6[C@@H]7CC(C)(C)CC[C@@]7(CC[C@@]56C)C(=O)O)[C@]3(C)O)O[C@@H]8O[C@H](CO)[C@@H](O)[C@H](O)[C@H]8O)[C@H](O)[C@H](O)[C@H]1O TXUSTSJRABHHQP-UFGIQYKASA-N 0.000 description 1
- 238000003762 quantitative reverse transcription PCR Methods 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 239000011535 reaction buffer Substances 0.000 description 1
- 238000003753 real-time PCR Methods 0.000 description 1
- 230000008844 regulatory mechanism Effects 0.000 description 1
- 239000011347 resin Substances 0.000 description 1
- 229920005989 resin Polymers 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 239000003161 ribonuclease inhibitor Substances 0.000 description 1
- 229930000044 secondary metabolite Natural products 0.000 description 1
- 238000012772 sequence design Methods 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- 230000003068 static effect Effects 0.000 description 1
- 239000008223 sterile water Substances 0.000 description 1
- 210000002784 stomach Anatomy 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000011426 transformation method Methods 0.000 description 1
- 230000008736 traumatic injury Effects 0.000 description 1
- 150000003648 triterpenes Chemical class 0.000 description 1
- 201000008827 tuberculosis Diseases 0.000 description 1
- 108010051110 tyrosyl-lysine Proteins 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- UOJAEODBOCLNBU-UHFFFAOYSA-N vinaginsenoside R4 Natural products C1CC(C2(CC(O)C3C(C)(C)C(OC4C(C(O)C(O)C(CO)O4)OC4C(C(O)C(O)C(CO)O4)O)CCC3(C)C2CC2O)C)(C)C2C1C(C)(CCC=C(C)C)OC1OC(CO)C(O)C(O)C1O UOJAEODBOCLNBU-UHFFFAOYSA-N 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明公开了一种珠子参转录因子基因PjWRKY1的用途，即在提高珠子参皂苷生物合成关键酶基因表达量和增加珠子参愈伤组织中皂苷含量的应用，PjWRKY1基因核苷酸序列如SEQ ID NO︰1 所示，编码WRKY类转录因子；本发明采用功能基因组学和代谢工程相关技术证明珠子参PjWRKY1转录因子具有正调控珠子参皂苷生物合成的功能。将本发明所述的珠子参PjWRKY1转录因子基因构建到植物表达载体上并转入珠子参愈伤组织中使其过量表达，增强了珠子参皂苷合成途径关键酶基因的表达量，提高了珠子参皂苷的产量。
Description
技术领域
本发明涉及分子生物学以及基因工程领域，尤其是一种珠子参皂苷生物合成转录因子基因PjWRKY1的应用。
背景技术
珠子参Panax japonicus C. A. Mey.var. major (Burk.) C. Y. Wu et K. M.Feng为五加科Araliaceae人参属Panax植物，因根茎节间纤细，节膨大成球形念珠状而得名，其根茎入药。珠子参作为药材，始载于《滇南本草》，为历版《中国药典》收载品种。药用珠子参主产于云南，属名贵常用中药材，是彝族、纳西族、白族、藏族、傈僳族等少数民族的传统用药。目前，野生珠子参主要分布于滇西北、滇东北地区，常见于海拔达2500-4000 m的亚高山针叶林及阔叶林下。珠子参性味苦、甘、微寒，归肝、肺、胃经，具有补肺养阴、祛瘀止痛，止血之功效，临床上应用于气阴两虚，烦热口渴，虚痨咳嗽，跌打损伤，关节疼痛，咳血，吐血，外伤出血等。
珠子参皂苷(Panax japonicus saponins, PJS)为珠子参的主要活性成分，包括达玛烷型、齐墩果烷型三萜皂苷。目前，已从珠子参根茎及叶中分离出了30多种皂苷成分，主要包括竹节参皂苷IVa、竹节参皂苷IV、竹节参皂苷V、人参皂苷Ro、人参皂苷Re、人参皂苷Rd、人参皂苷Rb1等。人参属的代表物种“人参”(Panax ginseng)主要含达玛烷型皂苷，齐墩果烷型皂苷仅发现含量极微的人参皂苷Ro；另一种人参属著名药材“三七”(Panax notoginseng)，只含达玛烷型皂苷，不含齐墩果烷型皂苷。珠子参所含皂苷组分与同属“人参”、“三七”相比，在成分种类以及各组分含量上均存在明显差异，因其含有大量齐墩果烷型皂苷，导致临床上的特殊用途。
珠子参为多年生草本植物，需生长6年以上方能入药。目前，珠子参药材多为野生品，无序采挖导致珠子参资源日渐枯竭，生境受到破坏。近些年，以珠子参为组分的药品市场迅速扩大，导致珠子参药材需求增长，供需矛盾突出。鉴于人工栽培时间长，化学合成机理和路线不够清晰等弊端，利用生物工程技术和基因调控的方法来生产珠子参皂苷逐渐成为研究热点。
转录因子对基因的转录激活是植物次生代谢过程重要的调控环节。利用转录因子作为改造植物代谢途径的工具，以其独有的“多点调控”优势，弥补了代谢工程操作中单个关键酶基因作用不足和多个关键酶基因可能产生组成性致死表达的情况，成为一种新的策略。次生代谢途径中多个功能相关的酶基因常被同一转录因子正调控或负调控，对转录因子进行基因修饰显然比多基因操作更容易改良目标次生代谢途径。
WRKY是植物特有的锌指型转录调控因子，是植物中比较大的转录因子家族，越来越多的研究表明该类转录因子对真核生物的次生代谢过程具有重要调控作用。例如木本棉(Gossypium arboreum)GaWRKY1转录因子可调节倍半萜烯合酶(+)-δ-杜松烯合酶A的活性；番茄(Solanum lycopersicum) SLWRKY73转录因子能激活其萜类合成酶基因的启动子，促进萜类合成酶基因的表达；西洋参(Panax quinquefolium)PqWRKY1转录因子可正调控三萜皂苷合成途径关键酶基因的表达。
随着对植物次生代谢网络的深入解析和调控机制的阐明，特别是调节特定次生代谢物合成的转录因子的分离和鉴定，基于转录因子的基因工程将为开发利用植物次生代谢物提供更加有效的手段。本发明以体外培养的珠子参愈伤组织为研究对象，克隆PjWRKY1转录因子基因，并对该转录因子进行分析和功能鉴定，明确PjWRKY1转录因子在珠子参皂苷生物合成过程中的地位和作用，为获得高效、稳定的珠子参皂苷合成调控技术和同源或异源高效表达系统的建立提供理论参考和依据。
发明内容
本发明的目的是提供一种珠子参转录因子基因PjWRKY1的用途，即珠子参转录因子基因PjWRKY1在提高珠子参皂苷合成代谢途径中关键酶基因表达量和增加珠子参愈伤组织中皂苷含量的应用；本发明从珠子参中克隆获得可调控珠子参皂苷生物合成的转录因子基因PjWRKY1以及明确该转录因子的应用。
本发明基于同源克隆的原理，从珠子参中克隆得到WRKY类转录因子基因的cDNA并对其编码蛋白进行功能鉴定。发明人将这个基因命名为PjWRKY1，基因登录号为KP890786，其中所述cDNA如SEQ ID NO︰1所示。对该基因进行序列分析，表明PjWRKY1 cDNA大小为820bp，具有807 bp的开放阅读框(Open reading frame，ORF)，编码含有268个氨基酸的蛋白质，氨基酸序列如SEQ ID NO︰2所示。利用植物表达载体，通过农杆菌介导法将本发明的PjWRKY1转录因子基因导入珠子参愈伤组织中，可提高珠子参皂苷合成途径关键酶基因的表达量，使珠子参皂苷的产量增加。
上述转录因子基因可应用于正调控珠子参皂苷的生物合成，具体操作如下：
（1）基因的获得：提取珠子参总RNA，反转录合成cDNA第一链。通过RT-PCR扩增出PjWRKY1的全长编码区，然后将其连接到pGEM-T easy载体上，经测序验证获得具有目的基因的克隆。
（2）植物表达载体构建与遗传转化：用限制性内切酶BamH I和XbaI酶切pGEM-T-PjWRKY1质粒，通过胶回收获得目的基因片段。用同样的内切酶消化植物表达载体pCAMBIA2300S，胶回收获得载体大片段。将目的基因片段与pCAMBIA2300S载体片段连接，构建植物超表达载体pCAMBIA2300S-PjWRKY1。通过液氮冻融法将pCAMBIA2300S-PjWRKY1质粒导入农杆菌菌株EHA105中。利用农杆菌介导的遗传转化法，将PjWRKY1导入珠子参愈伤组织中使其过量表达。通过抗生素筛选和qRT-PCR筛选阳性转基因细胞系。
（3）转基因细胞系皂苷含量检测：提取珠子参转基因和非转基因细胞系中的皂苷，分析转基因和非转基因细胞系间皂苷含量的差异。
本发明为提高珠子参中皂苷的含量提供了一种新方法，利用生物工程技术和基因调控的方法可更高效率合成珠子参皂苷，克服了人工栽培周期长、化学合成机理和路线不够清晰等缺点。将转录因子PjWRKY1基因导入珠子参细胞中表达，使珠子参皂苷生物合成途径关键酶基因的表达量提高，增加了珠子参皂苷的产量，为大规模产业化生产珠子参皂苷提供了理论参考和科学依据。
附图说明
图1为本发明中珠子参总RNA电泳图谱；
图2为本发明中PjWRKY1 RT-PCR检测结果。其中M为DL2000 DNA Marker，1为PjWRKY1的RT-PCR产物；
图3为本发明中PjWRKY1的三维结构预测；
图4为本发明中qRT-PCR结果分析图，表示珠子参皂苷合成途径中受PjWRKY1调控的FPS和SS基因在野生型和转基因细胞系中的表达水平，其中C为野生型细胞系，1-3为转基因细胞系；
图5为本发明中珠子参中皂苷含量测定结果，其中C为野生型细胞系，1-3为转基因细胞系。
具体实施方式
下面通过附图和实施例对本发明进一步说明，但本发明保护范围不局限于所述内容，实施例中方法如无特殊说明均为常规方法，使用的试剂如无特殊说明均为常规市售试剂或按常规方法配制的试剂。
实施例1：PjWRKY1基因的克隆以及生物信息学分析
因珠子参中含有较多的次生代谢物质，需采用两步法进行总RNA的提取。先采用改良的异硫氰酸胍法取进行粗提，接着使用DNA酶进行消化并利用氯仿抽提得到较纯的珠子参总RNA（图1）。采用逆转录酶M-MLV (promega)以珠子参总RNA为模板合成cDNA 第一链，反应体系和操作过程为：取5 μg Total RNA，依次加入50 ng oligo(dT) 15、2 μL dNTP (2.5mM each)、DEPC水至反应体积为13.5 μL；混匀后，70 ℃加热变性5 min后迅速在冰上冷却5min，然后依次加入4 μL 5×First-stand buffer、0.5 μL RNasin (200 U)、1 μL M-MLV(200 U)，混匀并瞬时离心，42 ℃温浴1.5h，取出后70 ℃加热10 min，终止反应。cDNA第一链合成后置于-20 ℃保存备用。
以合成的第一链cDNA为模板，根据三七中与调控三七皂苷生物合成相关的WRKY类转录因子基因cDNA序列设计特异性引物，扩增目的基因PjWRKY1，所用引物序列分别为5’-GGATCCCTGAAGATGGAAAATCATGTTGGGA-3’以及5’-TCTAGAGATTTCATCATTTC GACTCTACTAG-3’。采用AdvantageTM 2 PCR Enzyme (Clontech)扩增出目的基因。PCR反应条件：94 ℃ 5 min；94 ℃ 30 s，54 ℃ 30 s，72 ℃ 50 s，32 cycles；72 ℃ 10 min。反应体系(50 μL)为4 μL第一链cDNA、5 μL 10×Buffer、1 μL 50×dNTP Mix、0.5 μL 正向引物(10 μM)、0.5 μL 反向引物(10 μM)、1 μL AdvantageTM 2 PCR Enzyme、38 μL PCR-Grade Water。PCR结束后，取5 μL用于琼脂糖凝胶电泳，检测扩增产物的特异性以及大小（图2），其余PCR产物进行胶回收。将目的片段胶回收产物进行TA克隆，反应体系和操作过程为：取3.6 μL 胶回收产物，依次加入0.7 μL pGEM-T easy vector (50 ng/μL) 、5 μL 2×Rapid Ligation Buffer，和0.7 μL T4 DNA Ligase混匀置于8 ℃过夜反应。采用热激转化法将连接产物转入大肠杆菌Trans1-T1中。用含有氨苄青霉素(ampicillin，Amp)的LB固体培养基筛选阳性克隆。挑选若干个白色菌落，摇菌后用扩增PjWRKY1的特异引物鉴定出多克隆位点插入PjWRKY1的克隆，将鉴定的克隆进行测序。
最终获得的PjWRKY1 cDNA大小为820 bp，通过NCBI ORF finder (http://www.ncbi.nlm.nih.gov/gorf/gorf.html)分析发现其包含一个807 bp的ORF(见序列表)。PjWRKY1编码蛋白的分子量约为30.0 KD，等电点为9.95，不稳定系数为65.65，预测PjWRKY1编码的蛋白质不稳定。生物信息学预测PjWRKY1不包含跨膜区，不含信号肽，具有一个WRKY转录因子特征保守结构域。通过在线工具iPSORT预测PjWRKY1可能定位于细胞核。借助SWISS-MODEL工具，PjWRKY1以拟南芥AtWRKY4为模板进行三维结构建模（图3），结果显示PjWRKY1与AtWRKY4具有54.39%的序列相似性的空间结构。
实施例2：植物表达载体构建
采用SanPrep柱式质粒DNA小量抽提试剂盒(上海生工)提取插入PjWRKY1的大肠杆菌质粒pGEM-T- PjWRKY1以及植物表达载体pCAMBIA2300S的质粒，取1 μL用于琼脂糖凝胶电泳以检测所提取质粒的完整性和浓度高低。用BamHI (TaKaRa)和XbaI (TaKaRa)分别对质粒pGEM-T-PjWRKY1和pCAMBIA2300S进行双酶切(100 μL体系)，反应体系和操作过程为：取20 μL pGEM-T-PjWRKY1或pCAMBIA2300S质粒，依次加入10 μL 10×K buffer、5 μL BamH I、5 μL XbaI、60 μL ddH2O，混匀后短时离心，置于37 ℃过夜反应。将所有酶切产物点于琼脂糖凝胶中进行电泳，然后对PjWRKY1片段和pCAMBIA2300S大片段分别进行胶回收，整个过程使用SanPrep柱式DNA胶回收试剂盒(上海生工)。取1 μL回收产物通过琼脂糖凝胶电泳检测回收片段的大小以及浓度，置于-20 ℃保存备用。
利用T4 DNA Ligase (TaKaRa)，将回收的PjWRKY1DNA片段和pCAMBIA2300S载体片段连接起来，反应体系(20 μL)和操作过程为：取10 μL PjWRKY1DNA片段依次加入2 μLpCAMBIA2300S载体DNA、2 μL 10×T4 DNA Ligase Buffer、1 μL T4 DNA Ligase、5 μLddH2O，混匀后短时离心，置于16 ℃水浴过夜反应。接着采用热激转化法将连接产物转入大肠杆菌Trans1-T1中，用含有50 mg/L卡那霉素(kanamycin，Km)的固体培养基筛选阳性克隆。挑选单菌落摇菌，以菌液为模板用扩增PjWRKY1的特异引物进行PCR，挑选出PjWRKY1与pCAMBIA2300S成功连接的克隆，所检测的菌株若为阳性，加入20%甘油混匀后置于-80 ℃保存备用。
用试剂盒提取并纯化上述大肠杆菌中的pCAMBIA2300S-PjWRKY1质粒。制备农杆菌EHA105菌株的感受态细胞并分装于1.5 mL离心管中，每管150 μL，液氮速冻后置于-80 ℃保存备用。采用液氮冻融法将上述构建的植物表达载体pCAMBIA2300S-PjWRKY1转入所制备的农杆菌EHA105感受态细胞中。操作步骤为：取3 μg pCAMBIA2300S-PjWRKY1质粒加入含有150 μL感受态细胞的离心管中，轻轻混匀后冰浴30 min，接着转入液氮中速冻5 min，然后迅速置于37 ℃水浴5 min，之后立即冰浴2 min，加入600 μL LB液体培养基，28 ℃振荡培养4 h。将活化后的农杆菌涂于含有50 mg/L Km的LB固体培养基上，28 ℃静置培养。挑选单菌落摇菌，用扩增PjWRKY1的特异引物进行PCR，检测pCAMBIA2300S-PjWRKY1是否转入农杆菌中。对于阳性克隆，加入甘油后置于-80 ℃保存备用。
实施例3：农杆菌介导的珠子参遗传转化
从-80 ℃冰箱中取出保存的含有pCAMBIA2300S-PjWRKY1质粒的农杆菌EHA105菌种，接种于5 mL含有50 mg/L Km和25 mg/L利福平的LB液体培养基中，28 ℃培养至浑浊。吸取1 mL浑浊的菌液至含有50 mg/L Km的LB固体培养基上，28 ℃培养48 h。将LB固体培养基上的农杆菌刮下适量接种于MGL液体培养基中，附加40 mg/L的乙酰丁香酮，28 ℃振荡培养至OD600为0.6时停止摇菌，所得菌液用于侵染。
将生长状态较好的珠子参愈伤组织接到MS预培养基（含35 mg/L乙酰丁香酮）上进行预培养3天。预培养完成后，将愈伤组织完全浸泡于上述农杆菌菌液中进行振荡培养。浸染完毕，除去菌液，用无菌的滤纸吸取愈伤组织表面的菌液，最后将愈伤组织接种到共培养基中，于25 ℃进行暗培养，时间为三天。浸染后的愈伤组织接种于MS共培养基（含35 mg/L乙酰丁香酮）中，暗培养3天，然后用无菌水对愈伤组织进行洗涤，再转接于含400 mg/L头孢霉素的MS培养基上进行除菌培养，于25 ℃暗培养15天，防止农杆菌过度生长。最后将愈伤组织转接到筛选培养基中，每45天继代一次。经过5次筛选，最终分离出具有Km抗性的增值速度快的纯的细胞系，用于后续检测。
实施例4：PjWRKY1基因超表达对珠子参皂苷合成途径关键酶基因FPS和SS表达量的影
响
选取25天左右、生长状态良好的珠子参转基因细胞系和野生型细胞系，分别提取RNA，然后按照GoTaq® 2-Step RT-qPCR System试剂盒说明书合成cDNA，反应体系和操作过程为：在离心管中加入4 µg 总RNA和1 µL Oligo(dT)15，用Nuclease-free Water补齐至10µL，将反应体系放于70 ℃变性5 min，然后置于冰上5 min。随后将离心管在离心机中短暂离心，使反应液收集于管底，再向其中加入4 µL GoScriptTM 5×Reaction Buffer、2 µLMgCl2 (25 mM)、1 µL PCR Nucleotide Mix(10 mM)、0.5 µL Recombinant RNasinRibonuclease Inhibitor和1 µL GoScriptTM Reverse Transcriptase，将整个反应体系漩涡混匀，离心收集到管底，将反应物置于42 ℃恒温水浴锅中反应1 h，再于70 ℃水浴中维持15 min以终止反应，最后将合成的cDNA置于-20 ℃冰箱中保存备用。
以该cDNA为模板，根据珠子参18S rRNA基因（登录号：AB088018.1）、法尼基焦磷酸合成酶（Farnesyl diphosphate synthase, FPS）基因（登录号：KP684141）以及鲨烯合酶（Squalene synthase, SS）基因（登录号：KP890782）设计引物，依据GoTaq® 2-Step RT-qPCR System试剂盒说明书进行荧光半定量PCR扩增珠子参内参基因和FPS基因。所用引物序列为18S F：5’-GTTGTTGCAGTTAAAAAGCTCGTAG-3’，18S R：5’-ACCTCTGACTATGAAATACGAAT GC-3’；FPS F：5’-AGA ATGAGCGATCTGAAGACGAG-3’；FPS B：5’-ACAGACAACAACTTC CCCTCC AT-3’；SS F：5’-TAGAGAGAAAATGGGAAGTTTGGGG-3’；SS B：5’- TCACTGTTT GTTCGGTAGTAGGTTT-3’；具体反应体系和操作过程为：在PCR管中加入20 ng cDNA、25 µL GoTaq® qPCR Master Mix (2×)和0.2 µL qPCR Primers(18S F /18S R, FPS B / FPS F, SS B / SS F, 10 mM)，用Nuclease-Free Water补齐至50 µL。将反应体系漩涡混匀后，离心将其收集到管底，随后将其置于荧光定量PCR仪中进行反应，采用两步法进行荧光定量PCR，反应参数如下：热启动 95 ℃ 2min；变性 95 ℃ 15 s，退火/延伸 60 ℃ 1 min，共45个循环。每个样品对应的每个基因重复检测2次。
qRT-PCR结果显示，转PjWRKY1基因珠子参细胞中FPS和SS基因的表达量都比野生型的高（图4），说明PjWRKY1作为转录因子，能够促进珠子参皂苷合成代谢途径中关键酶基因FPS和SS的表达。图中，C表示对照组野生型细胞系，1、2和3分别表示不同的转基因细胞系实验组。
实施例5：PjWRKY1基因超表达对珠子参皂苷合成量的影响
选取生长35天左右的转基因细胞系和野生型细胞系，分别置于洗净的100 mL三角瓶中，加入20 mL的甲醇溶液浸泡过夜后，常温超声波处理1 h。过滤，收集滤液，将滤液挥干后再用甲醇溶解，定容至25 mL，得粗提液。将残渣在50 ℃下烘至恒重，称重。精密吸取粗提液5 mL，置50 mL烧杯中水浴蒸干。蒸干后用4倍体积的蒸馏水分次溶解，充分溶解后过滤，滤液全部转移至已处理好的Hsp100大孔树脂柱内，先用2个柱体积的蒸馏水慢慢洗去糖类等杂质。Molish反应检测糖类杂质是否去除干净，如果结果呈阳性，继续用蒸馏水洗至阴性，然后用75%乙醇溶液洗脱2个柱体积，收集醇液，水浴蒸干，残渣用甲醇溶液溶解，定容至25 mL。
精确吸取此样品150 μL至带塞的10 mL试管中（设3个重复），挥干溶剂，加入新配制的5%香草醛-冰醋酸溶液0.2 mL，高氯酸0.8 mL，混匀后60 ℃水浴加热15 min，立即用冰水冷却，加入5 mL冰醋酸，混匀静止10 min后554 nm测吸光度，参照标准曲线计算PJS含量。结果显示，转PjWRKY1基因珠子参细胞中皂苷含量明显高于野生型细胞中皂苷含量（图5），说明PjWRKY1转录因子参与了珠子参皂苷的合成代谢调控，有助于皂苷产量的提高。C表示对照组野生型细胞系，1、2和3分别表示不同的转基因细胞系实验组。
序列表
<110> 昆明理工大学
<120> 一种珠子参转录因子基因PjWRKY1的应用
<160> 10
<170> PatentIn version 3.5
<210> 1
<211> 820
<212> DNA
<213> Panax japonicus
<220>
<221> mRNA
<222> (1)..(820)
<220>
<221> 5'UTR
<222> (1)..(6)
<220>
<221> CDS
<222> (7)..(813)
<220>
<221> 3'UTR
<222> (814)..(820)
<400> 1
ctgaagatgg aaaatcatgt tgggattcat gaatcagcag cagcagcagc tgagatgaag 60
actattgaac aacaccttat tcgcatatta caatctcacc atcaacaaat tcaactcgat 120
ttctccaaaa aagccgtgaa tcgaaccggc cacgcccggt tccgccgtcg accatcagat 180
ccgtctactt cttctcagtc tgaaccattt acaccgattc agctcaaacc aatccctaaa 240
ccgtgcgact caaaaatatc tgaagaatgt aaaaccaaaa atactccgat atcgtccggg 300
agctcgtcga tcaccggaga ggaagggacc gtttccaatt gtaagcgagg attattaaac 360
accgcatcag caccggcacc gcgggtttat tcgtccagaa agccccctct tccgtcatct 420
cacaggaaaa gatgccgtga ccttgagccc atcgacggaa tttctgtcaa acgttcaatt 480
tcacgcggct gccactgttg caagagaagg aaaacagtgg agattagaag agtaacaaca 540
acaaaaggag gttcatcatc cattcctgcg gatgagtatt catggagaaa gtacgatcaa 600
aagttaatcc cgggcactct cttcccaaga ggatattaca aatgcaatac cgtaaaggga 660
tgcccggcga ggaagcacgc ggtgagatcc caagatgatc caacggtgct agtcgtgaca 720
tacgaaggag agcaccgtca taaccgttgg attctaccag agaggcaaaa taggagtggt 780
agtgttgttg gtatactagt agagtcgaaa tgatgaaatc 820
<210> 2
<211> 268
<212> PRT
<213> Panax japonicus
<400> 2
Met Glu Asn His Val Gly Ile His Glu Ser Ala Ala Ala Ala Ala Glu
1 5 10 15
Met Lys Thr Ile Glu Gln His Leu Ile Arg Ile Leu Gln Ser His His
20 25 30
Gln Gln Ile Gln Leu Asp Phe Ser Lys Lys Ala Val Asn Arg Thr Gly
35 40 45
His Ala Arg Phe Arg Arg Arg Pro Ser Asp Pro Ser Thr Ser Ser Gln
50 55 60
Ser Glu Pro Phe Thr Pro Ile Gln Leu Lys Pro Ile Pro Lys Pro Cys
65 70 75 80
Asp Ser Lys Ile Ser Glu Glu Cys Lys Thr Lys Asn Thr Pro Ile Ser
85 90 95
Ser Gly Ser Ser Ser Ile Thr Gly Glu Glu Gly Thr Val Ser Asn Cys
100 105 110
Lys Arg Gly Leu Leu Asn Thr Ala Ser Ala Pro Ala Pro Arg Val Tyr
115 120 125
Ser Ser Arg Lys Pro Pro Leu Pro Ser Ser His Arg Lys Arg Cys Arg
130 135 140
Asp Leu Glu Pro Ile Asp Gly Ile Ser Val Lys Arg Ser Ile Ser Arg
145 150 155 160
Gly Cys His Cys Cys Lys Arg Arg Lys Thr Val Glu Ile Arg Arg Val
165 170 175
Thr Thr Thr Lys Gly Gly Ser Ser Ser Ile Pro Ala Asp Glu Tyr Ser
180 185 190
Trp Arg Lys Tyr Asp Gln Lys Leu Ile Pro Gly Thr Leu Phe Pro Arg
195 200 205
Gly Tyr Tyr Lys Cys Asn Thr Val Lys Gly Cys Pro Ala Arg Lys His
210 215 220
Ala Val Arg Ser Gln Asp Asp Pro Thr Val Leu Val Val Thr Tyr Glu
225 230 235 240
Gly Glu His Arg His Asn Arg Trp Ile Leu Pro Glu Arg Gln Asn Arg
245 250 255
Ser Gly Ser Val Val Gly Ile Leu Val Glu Ser Lys
260 265
<210> 3
<211> 31
<212> DNA
<213> 人工序列
<400> 3
ggatccctga agatggaaaa tcatgttggg a 31
<210> 4
<211> 31
<212> DNA
<213> 人工序列
<400> 4
tctagagatt tcatcatttc gactctacta g 31
<210> 5
<211> 25
<212> DNA
<213> 人工序列
<400> 5
gttgttgcag ttaaaaagct cgtag 25
<210> 6
<211> 25
<212> DNA
<213> 人工序列
<400> 6
acctctgact atgaaatacg aatgc 25
<210> 7
<211> 23
<212> DNA
<213> 人工序列
<400> 7
agaatgagcg atctgaagac gag 23
<210> 8
<211> 23
<212> DNA
<213> 人工序列
<400> 8
acagacaaca acttcccctc cat 23
<210> 9
<211> 25
<212> DNA
<213> 人工序列
<400> 9
tagagagaaa atgggaagtt tgggg 25
<210> 10
<211> 25
<212> DNA
<213> 人工序列
<400> 10
tcactgtttg ttcggtagta ggttt 25
Claims (2)
1.一种珠子参转录因子基因PjWRKY1在提高珠子参皂苷合成代谢途径中关键酶基因FPS和SS表达量和增加珠子参愈伤组织中皂苷含量的应用，其特征在于：所述珠子参转录因子基因PjWRKY1的核苷酸序列如SEQ ID NO︰1 所示。
2.根据权利要求1所述珠子参转录因子基因PjWRKY1在提高珠子参皂苷合成代谢途径中关键酶基因FPS和SS表达量和增加珠子参愈伤组织中皂苷含量的应用，其特征在于具体操作如下：
（1）将珠子参转录因子基因PjWRKY1与植物超表达载体pCAMBIA2300S连接，构建植物超表达载体；
（2）将上述构建的表达载体通过农杆菌介导转入珠子参愈伤组织中；
（3）通过抗生素筛选珠子参转基因细胞系，qRT-PCR检测转基因细胞系中珠子参皂苷合成途径关键酶基因表达量；
（4）提取珠子参转基因和非转基因细胞系中的皂苷，分析转基因和非转基因细胞系间皂苷含量的差异，最后筛选出皂苷含量得到提高的阳性转基因细胞系。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510561989.8A CN105087601B (zh) | 2015-09-07 | 2015-09-07 | 一种珠子参转录因子基因PjWRKY1的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510561989.8A CN105087601B (zh) | 2015-09-07 | 2015-09-07 | 一种珠子参转录因子基因PjWRKY1的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105087601A CN105087601A (zh) | 2015-11-25 |
| CN105087601B true CN105087601B (zh) | 2018-06-15 |
Family
ID=54568933
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510561989.8A Expired - Fee Related CN105087601B (zh) | 2015-09-07 | 2015-09-07 | 一种珠子参转录因子基因PjWRKY1的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105087601B (zh) |
Families Citing this family (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441462B (zh) * | 2016-01-06 | 2018-10-23 | 昆明理工大学 | 一种三七转录因子基因PnERF1及其应用 |
| CN105441461B (zh) * | 2016-01-06 | 2019-02-05 | 昆明理工大学 | 一种三七转录因子基因PnWRKY1的应用 |
| CN105441460B (zh) * | 2016-01-06 | 2018-08-10 | 昆明理工大学 | 一种岷江百合WRKY转录因子基因LrWRKY1及应用 |
| CN105441463B (zh) * | 2016-01-06 | 2018-10-23 | 昆明理工大学 | 一种三七转录因子基因PnbHLH1及其应用 |
| CN109295069B (zh) * | 2018-09-19 | 2021-08-20 | 昆明理工大学 | 珠子参转录因子基因PjMYB1的应用 |
| CN109295080B (zh) * | 2018-09-19 | 2021-08-20 | 昆明理工大学 | 珠子参β-香树脂醇合成酶基因Pjβ-AS的用途 |
| CN110343678B (zh) * | 2019-06-12 | 2022-09-27 | 云南农业大学 | 一种珠子参糖基转移酶UGTPjm1基因及在制备人参皂苷Ro上的应用 |
| CN110747202B (zh) * | 2019-11-13 | 2021-09-14 | 昆明理工大学 | 一种岷江百合WRKY转录因子基因LrWRKY11及应用 |
| CN111217896B (zh) * | 2020-02-28 | 2022-04-29 | 天津大学 | 人参PgWRKY4X转录因子在调控人参中人参皂苷类化合物含量的应用 |
| CN112301038B (zh) * | 2020-10-26 | 2023-06-13 | 吉林农业大学 | 人参wrky64-04基因及其应用 |
| CN112831504B (zh) * | 2021-03-16 | 2023-03-24 | 昆明理工大学 | 三七WRKY转录因子基因PnWRKY9及其应用 |
| CN114058628A (zh) * | 2021-10-11 | 2022-02-18 | 浙江理工大学 | 基因PnWRKY1及其在调控三七皂苷合成中的应用 |
| CN115725620B (zh) * | 2022-09-12 | 2023-09-15 | 昆明理工大学 | 一种在三七细胞中合成竹节参皂苷的方法 |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463363A (zh) * | 2008-12-02 | 2009-06-24 | 吉林大学 | 一种提高人参种质皂苷含量的方法 |
| CN102888424A (zh) * | 2012-09-14 | 2013-01-23 | 吉林农业大学 | 一种植物双元表达载体pMHZ112及应用 |
| CN102899378A (zh) * | 2012-10-18 | 2013-01-30 | 昆明理工大学 | 红曲霉对三七药材进行生物转化的方法 |
| CN103088027A (zh) * | 2013-02-05 | 2013-05-08 | 中南大学 | 一种调控人参皂苷积累的pdr转运蛋白基因启动子及其应用 |
| CN103103194A (zh) * | 2013-02-05 | 2013-05-15 | 中南大学 | 茉莉酸甲酯应答的人参PgPDR3基因启动子及其应用 |
| CN103614411A (zh) * | 2013-11-02 | 2014-03-05 | 吉林农业大学 | 西洋参发状根诱导及植株再生研究方法 |
| WO2014195944A1 (en) * | 2013-06-05 | 2014-12-11 | Yeda Research And Development Co. Ltd. | Plant with altered content of steroidal glycoalkaloids |
| WO2015124620A1 (en) * | 2014-02-18 | 2015-08-27 | Vib Vzw | Means and methods for regulating secondary metabolite production in plants |
-
2015
- 2015-09-07 CN CN201510561989.8A patent/CN105087601B/zh not_active Expired - Fee Related
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101463363A (zh) * | 2008-12-02 | 2009-06-24 | 吉林大学 | 一种提高人参种质皂苷含量的方法 |
| CN102888424A (zh) * | 2012-09-14 | 2013-01-23 | 吉林农业大学 | 一种植物双元表达载体pMHZ112及应用 |
| CN102899378A (zh) * | 2012-10-18 | 2013-01-30 | 昆明理工大学 | 红曲霉对三七药材进行生物转化的方法 |
| CN103088027A (zh) * | 2013-02-05 | 2013-05-08 | 中南大学 | 一种调控人参皂苷积累的pdr转运蛋白基因启动子及其应用 |
| CN103103194A (zh) * | 2013-02-05 | 2013-05-15 | 中南大学 | 茉莉酸甲酯应答的人参PgPDR3基因启动子及其应用 |
| WO2014195944A1 (en) * | 2013-06-05 | 2014-12-11 | Yeda Research And Development Co. Ltd. | Plant with altered content of steroidal glycoalkaloids |
| CN103614411A (zh) * | 2013-11-02 | 2014-03-05 | 吉林农业大学 | 西洋参发状根诱导及植株再生研究方法 |
| WO2015124620A1 (en) * | 2014-02-18 | 2015-08-27 | Vib Vzw | Means and methods for regulating secondary metabolite production in plants |
Non-Patent Citations (5)
| Title |
|---|
| Analysis of the transcriptome of Panax notoginseng root uncovers putative triterpene saponin-biosynthetic genes and genetic markers;Hongmei Luo等;《BMC Genomics》;20111231;第12卷(第S5期);摘要，第7页左栏第1段 * |
| Production of ginseng saponins: elicitation strategy and signal transductions;Shadi Rahimi等;《Appl Microbiol Biotechnol》;20150721;第99卷(第15期);第1-13页 * |
| Transcript expression profiling for adventitious roots of Panax ginseng Meyer;Sathiyamoorthy Subramaniyam等;《Gene》;20141231;第1-8页 * |
| 植物萜类物质生物合成的相关转录因子及其应用前景;赵恒伟等;《中草药》;20121231;第43卷(第12期);摘要 * |
| 药用植物转录组研究专题;王尧龙等;《中国中药杂志》;20150630;第40卷(第11期);第2055-2061页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105087601A (zh) | 2015-11-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105087601B (zh) | 一种珠子参转录因子基因PjWRKY1的应用 | |
| CN105087599B (zh) | 一种珠子参转录因子基因PjERF1的应用 | |
| CN105441461B (zh) | 一种三七转录因子基因PnWRKY1的应用 | |
| CN106497939B (zh) | 一种三七转录因子基因PnMYB1及其应用 | |
| CA2390600A1 (en) | Seed trait genes | |
| CN105441463B (zh) | 一种三七转录因子基因PnbHLH1及其应用 | |
| CN105087600B (zh) | 一种珠子参转录因子基因PjbHLH1的应用 | |
| CN105441462B (zh) | 一种三七转录因子基因PnERF1及其应用 | |
| TW201040275A (en) | Manipulating fructan biosynthesis and enhancing plant biomass | |
| CN109295080A (zh) | 珠子参β-香树脂醇合成酶基因Pjβ-AS的用途 | |
| CN113265408B (zh) | 一种三七DOF转录因子基因PnDof1及其应用 | |
| CN114891803B (zh) | 一种受茉莉酸甲酯诱导的人参PgWRKY40基因及其应用 | |
| CN117683776B (zh) | 一种低温和干旱诱导的ProPgCOMT2启动子及其在人参皂苷生物合成中的应用 | |
| CN112430261A (zh) | Wrky32调控yft1表达影响番茄果色及其在番茄品质改良中应用 | |
| CN109487005A (zh) | 用于扩增山羊地方性鼻内肿瘤病毒全基因组序列的引物 | |
| CN105154420B (zh) | 赤芝萜类合酶GL22395编码基因cDNA序列及其应用 | |
| WO2010063162A1 (zh) | 一种干旱诱导的水稻花特异性启动子及其应用 | |
| CN114395566B (zh) | 甘薯ERF转录因子IbERF4在促进植物绿原酸类物质合成中的用途 | |
| CN106967161A (zh) | 调控蓝莓果实中花青素含量的蛋白及其编码基因与应用 | |
| CN109295069B (zh) | 珠子参转录因子基因PjMYB1的应用 | |
| CN114058632A (zh) | 基因PnCOX11及其在调控三七皂苷合成中的应用 | |
| CN114058628A (zh) | 基因PnWRKY1及其在调控三七皂苷合成中的应用 | |
| CN114058627A (zh) | 基因PnMYB2及其在调控三七皂苷合成中的应用 | |
| CN104928307B (zh) | 一种茎瘤芥硫苷酶基因myr1、茎瘤芥硫苷酶及其基因工程表达方法 | |
| WO2008148298A1 (en) | Improvement of cold tolerance in plants by protein kinase gene oscipk03 from rice |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20180615 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |