CN109232726B - 蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用 - Google Patents
蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用 Download PDFInfo
- Publication number
- CN109232726B CN109232726B CN201811318436.XA CN201811318436A CN109232726B CN 109232726 B CN109232726 B CN 109232726B CN 201811318436 A CN201811318436 A CN 201811318436A CN 109232726 B CN109232726 B CN 109232726B
- Authority
- CN
- China
- Prior art keywords
- rice
- osvpe2
- inorganic phosphorus
- protein
- vacuole
- 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
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 title claims abstract description 109
- 239000011574 phosphorus Substances 0.000 title claims abstract description 109
- 229910052698 phosphorus Inorganic materials 0.000 title claims abstract description 109
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 85
- 210000003934 vacuole Anatomy 0.000 title claims abstract description 65
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 56
- 230000033228 biological regulation Effects 0.000 title claims abstract description 11
- 241000209094 Oryza Species 0.000 claims abstract description 110
- 235000007164 Oryza sativa Nutrition 0.000 claims abstract description 102
- 235000009566 rice Nutrition 0.000 claims abstract description 99
- 230000001965 increasing effect Effects 0.000 claims abstract description 25
- 230000009261 transgenic effect Effects 0.000 claims abstract description 23
- 230000014509 gene expression Effects 0.000 claims abstract description 19
- 230000002829 reductive effect Effects 0.000 claims abstract description 19
- 230000000694 effects Effects 0.000 claims abstract description 17
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 5
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 238000000034 method Methods 0.000 claims description 29
- 108020004414 DNA Proteins 0.000 claims description 22
- 230000001105 regulatory effect Effects 0.000 claims description 15
- 150000007523 nucleic acids Chemical class 0.000 claims description 13
- 102000039446 nucleic acids Human genes 0.000 claims description 12
- 108020004707 nucleic acids Proteins 0.000 claims description 12
- 230000001276 controlling effect Effects 0.000 claims description 11
- 102000053602 DNA Human genes 0.000 claims description 10
- 239000004575 stone Substances 0.000 claims description 7
- 230000003247 decreasing effect Effects 0.000 claims description 4
- 238000009395 breeding Methods 0.000 claims 4
- 230000001488 breeding effect Effects 0.000 claims 2
- 241001060517 Dicranolaius bellulus Species 0.000 claims 1
- 241000196324 Embryophyta Species 0.000 abstract description 102
- 238000003209 gene knockout Methods 0.000 abstract description 10
- 235000018102 proteins Nutrition 0.000 description 40
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 30
- 206010020649 Hyperkeratosis Diseases 0.000 description 26
- 239000013598 vector Substances 0.000 description 19
- 239000002773 nucleotide Substances 0.000 description 17
- 125000003729 nucleotide group Chemical group 0.000 description 17
- 238000005286 illumination Methods 0.000 description 13
- 239000001963 growth medium Substances 0.000 description 12
- 239000013612 plasmid Substances 0.000 description 12
- 238000012408 PCR amplification Methods 0.000 description 11
- 238000012258 culturing Methods 0.000 description 9
- 108091033409 CRISPR Proteins 0.000 description 8
- OJOBTAOGJIWAGB-UHFFFAOYSA-N acetosyringone Chemical compound COC1=CC(C(C)=O)=CC(OC)=C1O OJOBTAOGJIWAGB-UHFFFAOYSA-N 0.000 description 8
- 239000000499 gel Substances 0.000 description 8
- 150000002505 iron Chemical class 0.000 description 7
- 238000005481 NMR spectroscopy Methods 0.000 description 6
- 239000012153 distilled water Substances 0.000 description 6
- 208000015181 infectious disease Diseases 0.000 description 6
- 108091008146 restriction endonucleases Proteins 0.000 description 6
- 238000002791 soaking Methods 0.000 description 6
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 5
- 241000589158 Agrobacterium Species 0.000 description 5
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 5
- 229930006000 Sucrose Natural products 0.000 description 5
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 5
- 125000000539 amino acid group Chemical group 0.000 description 5
- 239000005018 casein Substances 0.000 description 5
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 5
- 235000021240 caseins Nutrition 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 235000015097 nutrients Nutrition 0.000 description 5
- 239000005720 sucrose Substances 0.000 description 5
- 238000010354 CRISPR gene editing Methods 0.000 description 4
- ONIBWKKTOPOVIA-BYPYZUCNSA-N L-Proline Chemical compound OC(=O)[C@@H]1CCCN1 ONIBWKKTOPOVIA-BYPYZUCNSA-N 0.000 description 4
- ZDXPYRJPNDTMRX-VKHMYHEASA-N L-glutamine Chemical compound OC(=O)[C@@H](N)CCC(N)=O ZDXPYRJPNDTMRX-VKHMYHEASA-N 0.000 description 4
- 229930182816 L-glutamine Natural products 0.000 description 4
- 229930182821 L-proline Natural products 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 4
- 108010092528 Phosphate Transport Proteins Proteins 0.000 description 4
- 102000016462 Phosphate Transport Proteins Human genes 0.000 description 4
- 150000001413 amino acids Chemical group 0.000 description 4
- 239000002299 complementary DNA Substances 0.000 description 4
- 239000012634 fragment Substances 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 229960002429 proline Drugs 0.000 description 4
- 230000009466 transformation Effects 0.000 description 4
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 3
- 108091026890 Coding region Proteins 0.000 description 3
- 240000007594 Oryza sativa Species 0.000 description 3
- 108091027544 Subgenomic mRNA Proteins 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 239000001110 calcium chloride Substances 0.000 description 3
- 229910001628 calcium chloride Inorganic materials 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 239000012881 co-culture medium Substances 0.000 description 3
- 210000000805 cytoplasm Anatomy 0.000 description 3
- 230000004069 differentiation Effects 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 238000010362 genome editing Methods 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 239000002609 medium Substances 0.000 description 3
- NLKNQRATVPKPDG-UHFFFAOYSA-M potassium iodide Chemical compound [K+].[I-] NLKNQRATVPKPDG-UHFFFAOYSA-M 0.000 description 3
- 238000011084 recovery Methods 0.000 description 3
- 239000012883 rooting culture medium Substances 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 229930186147 Cephalosporin Natural products 0.000 description 2
- ZGTMUACCHSMWAC-UHFFFAOYSA-L EDTA disodium salt (anhydrous) Chemical compound [Na+].[Na+].OC(=O)CN(CC([O-])=O)CCN(CC(O)=O)CC([O-])=O ZGTMUACCHSMWAC-UHFFFAOYSA-L 0.000 description 2
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 2
- PXKACEXYLPBMAD-JBDRJPRFSA-N Ile-Ser-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PXKACEXYLPBMAD-JBDRJPRFSA-N 0.000 description 2
- HGCNKOLVKRAVHD-UHFFFAOYSA-N L-Met-L-Phe Natural products CSCCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 HGCNKOLVKRAVHD-UHFFFAOYSA-N 0.000 description 2
- 241000880493 Leptailurus serval Species 0.000 description 2
- INCJJHQRZGQLFC-KBPBESRZSA-N Leu-Phe-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)NCC(O)=O INCJJHQRZGQLFC-KBPBESRZSA-N 0.000 description 2
- XXXXOVFBXRERQL-ULQDDVLXSA-N Leu-Pro-Phe Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 XXXXOVFBXRERQL-ULQDDVLXSA-N 0.000 description 2
- 241000209510 Liliopsida Species 0.000 description 2
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 2
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 description 2
- 108091028043 Nucleic acid sequence Proteins 0.000 description 2
- 239000004677 Nylon Substances 0.000 description 2
- 229910019142 PO4 Inorganic materials 0.000 description 2
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 description 2
- XYSXOCIWCPFOCG-IHRRRGAJSA-N Pro-Leu-Leu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O XYSXOCIWCPFOCG-IHRRRGAJSA-N 0.000 description 2
- 239000005708 Sodium hypochlorite Substances 0.000 description 2
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 2
- 210000004027 cell Anatomy 0.000 description 2
- 229940124587 cephalosporin Drugs 0.000 description 2
- 150000001780 cephalosporins Chemical class 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000012217 deletion Methods 0.000 description 2
- 230000037430 deletion Effects 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- IDGUHHHQCWSQLU-UHFFFAOYSA-N ethanol;hydrate Chemical compound O.CCO IDGUHHHQCWSQLU-UHFFFAOYSA-N 0.000 description 2
- 241001233957 eudicotyledons Species 0.000 description 2
- 230000005284 excitation Effects 0.000 description 2
- 108010084389 glycyltryptophan Proteins 0.000 description 2
- 210000002288 golgi apparatus Anatomy 0.000 description 2
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 238000003780 insertion Methods 0.000 description 2
- 230000037431 insertion Effects 0.000 description 2
- 229930027917 kanamycin Natural products 0.000 description 2
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound 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
- 229960000318 kanamycin Drugs 0.000 description 2
- 229930182823 kanamycin A Natural products 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 235000019341 magnesium sulphate Nutrition 0.000 description 2
- 210000001161 mammalian embryo Anatomy 0.000 description 2
- 238000005259 measurement Methods 0.000 description 2
- 108010068488 methionylphenylalanine Proteins 0.000 description 2
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 2
- 235000019796 monopotassium phosphate Nutrition 0.000 description 2
- 229920001778 nylon Polymers 0.000 description 2
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 2
- 239000010452 phosphate Substances 0.000 description 2
- 239000002686 phosphate fertilizer Substances 0.000 description 2
- 238000003976 plant breeding Methods 0.000 description 2
- 230000037039 plant physiology Effects 0.000 description 2
- GNSKLFRGEWLPPA-UHFFFAOYSA-M potassium dihydrogen phosphate Chemical compound [K+].OP(O)([O-])=O GNSKLFRGEWLPPA-UHFFFAOYSA-M 0.000 description 2
- FGIUAXJPYTZDNR-UHFFFAOYSA-N potassium nitrate Chemical compound [K+].[O-][N+]([O-])=O FGIUAXJPYTZDNR-UHFFFAOYSA-N 0.000 description 2
- LWIHDJKSTIGBAC-UHFFFAOYSA-K potassium phosphate Substances [K+].[K+].[K+].[O-]P([O-])([O-])=O LWIHDJKSTIGBAC-UHFFFAOYSA-K 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 2
- 229960001225 rifampicin Drugs 0.000 description 2
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 2
- 239000002689 soil Substances 0.000 description 2
- 238000009331 sowing Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000006467 substitution reaction Methods 0.000 description 2
- 238000011426 transformation method Methods 0.000 description 2
- NWONKYPBYAMBJT-UHFFFAOYSA-L zinc sulfate Chemical compound [Zn+2].[O-]S([O-])(=O)=O NWONKYPBYAMBJT-UHFFFAOYSA-L 0.000 description 2
- 229910000368 zinc sulfate Inorganic materials 0.000 description 2
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 101150028074 2 gene Proteins 0.000 description 1
- PIPTUBPKYFRLCP-NHCYSSNCSA-N Ala-Ala-Phe Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 PIPTUBPKYFRLCP-NHCYSSNCSA-N 0.000 description 1
- IFTVANMRTIHKML-WDSKDSINSA-N Ala-Gln-Gly Chemical compound C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)NCC(O)=O IFTVANMRTIHKML-WDSKDSINSA-N 0.000 description 1
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 1
- NIZKGBJVCMRDKO-KWQFWETISA-N Ala-Gly-Tyr Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 NIZKGBJVCMRDKO-KWQFWETISA-N 0.000 description 1
- DVJSJDDYCYSMFR-ZKWXMUAHSA-N Ala-Ile-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O DVJSJDDYCYSMFR-ZKWXMUAHSA-N 0.000 description 1
- GMGWOTQMUKYZIE-UBHSHLNASA-N Ala-Pro-Phe Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 GMGWOTQMUKYZIE-UBHSHLNASA-N 0.000 description 1
- MMLHRUJLOUSRJX-CIUDSAMLSA-N Ala-Ser-Lys Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCCCN MMLHRUJLOUSRJX-CIUDSAMLSA-N 0.000 description 1
- JPOQZCHGOTWRTM-FQPOAREZSA-N Ala-Tyr-Thr Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JPOQZCHGOTWRTM-FQPOAREZSA-N 0.000 description 1
- 102100036475 Alanine aminotransferase 1 Human genes 0.000 description 1
- 101100136613 Arabidopsis thaliana PHT1-3 gene Proteins 0.000 description 1
- 101100136616 Arabidopsis thaliana PHT1-4 gene Proteins 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
- AFNHFVVOJZBIJD-GUBZILKMSA-N Arg-Met-Asp Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O AFNHFVVOJZBIJD-GUBZILKMSA-N 0.000 description 1
- PFOYSEIHFVKHNF-FXQIFTODSA-N Asn-Ala-Arg Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PFOYSEIHFVKHNF-FXQIFTODSA-N 0.000 description 1
- FVKHEKVYFTZWDX-GHCJXIJMSA-N Asn-Ile-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC(=O)N)N FVKHEKVYFTZWDX-GHCJXIJMSA-N 0.000 description 1
- NVWJMQNYLYWVNQ-BYULHYEWSA-N Asn-Ile-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)NCC(O)=O NVWJMQNYLYWVNQ-BYULHYEWSA-N 0.000 description 1
- HCZQKHSRYHCPSD-IUKAMOBKSA-N Asn-Thr-Ile Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HCZQKHSRYHCPSD-IUKAMOBKSA-N 0.000 description 1
- JBDLMLZNDRLDIX-HJGDQZAQSA-N Asn-Thr-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O JBDLMLZNDRLDIX-HJGDQZAQSA-N 0.000 description 1
- PBVLJOIPOGUQQP-CIUDSAMLSA-N Asp-Ala-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O PBVLJOIPOGUQQP-CIUDSAMLSA-N 0.000 description 1
- IXIWEFWRKIUMQX-DCAQKATOSA-N Asp-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(O)=O IXIWEFWRKIUMQX-DCAQKATOSA-N 0.000 description 1
- YDJVIBMKAMQPPP-LAEOZQHASA-N Asp-Glu-Val Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O YDJVIBMKAMQPPP-LAEOZQHASA-N 0.000 description 1
- VIRHEUMYXXLCBF-WDSKDSINSA-N Asp-Gly-Glu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O VIRHEUMYXXLCBF-WDSKDSINSA-N 0.000 description 1
- SNDBKTFJWVEVPO-WHFBIAKZSA-N Asp-Gly-Ser Chemical compound [H]N[C@@H](CC(O)=O)C(=O)NCC(=O)N[C@@H](CO)C(O)=O SNDBKTFJWVEVPO-WHFBIAKZSA-N 0.000 description 1
- TZOZNVLBTAFJRW-UGYAYLCHSA-N Asp-Ile-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC(=O)O)N TZOZNVLBTAFJRW-UGYAYLCHSA-N 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- 239000003109 Disodium ethylene diamine tetraacetate Substances 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- XJKAKYXMFHUIHT-AUTRQRHGSA-N Gln-Glu-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N XJKAKYXMFHUIHT-AUTRQRHGSA-N 0.000 description 1
- OUBUHIODTNUUTC-WDCWCFNPSA-N Gln-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)O OUBUHIODTNUUTC-WDCWCFNPSA-N 0.000 description 1
- LGYCLOCORAEQSZ-PEFMBERDSA-N Glu-Ile-Asp Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O LGYCLOCORAEQSZ-PEFMBERDSA-N 0.000 description 1
- CQAHWYDHKUWYIX-YUMQZZPRSA-N Glu-Pro-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O CQAHWYDHKUWYIX-YUMQZZPRSA-N 0.000 description 1
- UUTGYDAKPISJAO-JYJNAYRXSA-N Glu-Tyr-Leu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(C)C)C(O)=O)CC1=CC=C(O)C=C1 UUTGYDAKPISJAO-JYJNAYRXSA-N 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- OVSKVOOUFAKODB-UWVGGRQHSA-N Gly-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OVSKVOOUFAKODB-UWVGGRQHSA-N 0.000 description 1
- BGVYNAQWHSTTSP-BYULHYEWSA-N Gly-Asn-Ile Chemical compound [H]NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BGVYNAQWHSTTSP-BYULHYEWSA-N 0.000 description 1
- SCWYHUQOOFRVHP-MBLNEYKQSA-N Gly-Ile-Thr Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SCWYHUQOOFRVHP-MBLNEYKQSA-N 0.000 description 1
- LRQXRHGQEVWGPV-NHCYSSNCSA-N Gly-Leu-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN LRQXRHGQEVWGPV-NHCYSSNCSA-N 0.000 description 1
- UHPAZODVFFYEEL-QWRGUYRKSA-N Gly-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)CN UHPAZODVFFYEEL-QWRGUYRKSA-N 0.000 description 1
- JNGHLWWFPGIJER-STQMWFEESA-N Gly-Pro-Tyr Chemical compound NCC(=O)N1CCC[C@H]1C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JNGHLWWFPGIJER-STQMWFEESA-N 0.000 description 1
- FFJQHWKSGAWSTJ-BFHQHQDPSA-N Gly-Thr-Ala Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(O)=O FFJQHWKSGAWSTJ-BFHQHQDPSA-N 0.000 description 1
- JQFILXICXLDTRR-FBCQKBJTSA-N Gly-Thr-Gly Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)NCC(O)=O JQFILXICXLDTRR-FBCQKBJTSA-N 0.000 description 1
- SQUHHTBVTRBESD-UHFFFAOYSA-N Hexa-Ac-myo-Inositol Natural products CC(=O)OC1C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C(OC(C)=O)C1OC(C)=O SQUHHTBVTRBESD-UHFFFAOYSA-N 0.000 description 1
- ORERHHPZDDEMSC-VGDYDELISA-N His-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N ORERHHPZDDEMSC-VGDYDELISA-N 0.000 description 1
- VIJMRAIWYWRXSR-CIUDSAMLSA-N His-Ser-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CC1=CN=CN1 VIJMRAIWYWRXSR-CIUDSAMLSA-N 0.000 description 1
- JUCZDDVZBMPKRT-IXOXFDKPSA-N His-Thr-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC1=CN=CN1)N)O JUCZDDVZBMPKRT-IXOXFDKPSA-N 0.000 description 1
- UPJODPVSKKWGDQ-KLHWPWHYSA-N His-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N)O UPJODPVSKKWGDQ-KLHWPWHYSA-N 0.000 description 1
- 101000928460 Homo sapiens Alanine aminotransferase 1 Proteins 0.000 description 1
- AQCUAZTZSPQJFF-ZKWXMUAHSA-N Ile-Ala-Gly Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C)C(=O)NCC(O)=O AQCUAZTZSPQJFF-ZKWXMUAHSA-N 0.000 description 1
- WNQKUUQIVDDAFA-ZPFDUUQYSA-N Ile-Gln-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCSC)C(=O)O)N WNQKUUQIVDDAFA-ZPFDUUQYSA-N 0.000 description 1
- CDGLBYSAZFIIJO-RCOVLWMOSA-N Ile-Gly-Gly Chemical compound CC[C@H](C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O CDGLBYSAZFIIJO-RCOVLWMOSA-N 0.000 description 1
- LWWILHPVAKKLQS-QXEWZRGKSA-N Ile-Gly-Met Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CCSC)C(=O)O)N LWWILHPVAKKLQS-QXEWZRGKSA-N 0.000 description 1
- RWYCOSAAAJBJQL-KCTSRDHCSA-N Ile-Gly-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N RWYCOSAAAJBJQL-KCTSRDHCSA-N 0.000 description 1
- ZXIGYKICRDFISM-DJFWLOJKSA-N Ile-His-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ZXIGYKICRDFISM-DJFWLOJKSA-N 0.000 description 1
- PWDSHAAAFXISLE-SXTJYALSSA-N Ile-Ile-Asp Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O PWDSHAAAFXISLE-SXTJYALSSA-N 0.000 description 1
- VLCMCYDZJCWPQT-VKOGCVSHSA-N Ile-Met-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N VLCMCYDZJCWPQT-VKOGCVSHSA-N 0.000 description 1
- IIWQTXMUALXGOV-PCBIJLKTSA-N Ile-Phe-Asp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(=O)O)C(=O)O)N IIWQTXMUALXGOV-PCBIJLKTSA-N 0.000 description 1
- XLXPYSDGMXTTNQ-UHFFFAOYSA-N Ile-Phe-Leu Natural products CCC(C)C(N)C(=O)NC(C(=O)NC(CC(C)C)C(O)=O)CC1=CC=CC=C1 XLXPYSDGMXTTNQ-UHFFFAOYSA-N 0.000 description 1
- CAHCWMVNBZJVAW-NAKRPEOUSA-N Ile-Pro-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)O)N CAHCWMVNBZJVAW-NAKRPEOUSA-N 0.000 description 1
- KFKWRHQBZQICHA-STQMWFEESA-N L-leucyl-L-phenylalanine Natural products CC(C)C[C@H](N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 KFKWRHQBZQICHA-STQMWFEESA-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
- XBBKIIGCUMBKCO-JXUBOQSCSA-N Leu-Ala-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O XBBKIIGCUMBKCO-JXUBOQSCSA-N 0.000 description 1
- UILIPCLTHRPCRB-XUXIUFHCSA-N Leu-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(C)C)N UILIPCLTHRPCRB-XUXIUFHCSA-N 0.000 description 1
- OIARJGNVARWKFP-YUMQZZPRSA-N Leu-Asn-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O OIARJGNVARWKFP-YUMQZZPRSA-N 0.000 description 1
- MMEDVBWCMGRKKC-GARJFASQSA-N Leu-Asp-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N MMEDVBWCMGRKKC-GARJFASQSA-N 0.000 description 1
- PPBKJAQJAUHZKX-SRVKXCTJSA-N Leu-Cys-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CS)C(=O)N[C@H](C(O)=O)CC(C)C PPBKJAQJAUHZKX-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
- 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
- VGPCJSXPPOQPBK-YUMQZZPRSA-N Leu-Gly-Ser Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O VGPCJSXPPOQPBK-YUMQZZPRSA-N 0.000 description 1
- HYMLKESRWLZDBR-WEDXCCLWSA-N Leu-Gly-Thr Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O HYMLKESRWLZDBR-WEDXCCLWSA-N 0.000 description 1
- ZALAVHVPPOHAOL-XUXIUFHCSA-N Leu-Ile-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@H](CC(C)C)N ZALAVHVPPOHAOL-XUXIUFHCSA-N 0.000 description 1
- LIINDKYIGYTDLG-PPCPHDFISA-N Leu-Ile-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LIINDKYIGYTDLG-PPCPHDFISA-N 0.000 description 1
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 1
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 1
- LCNASHSOFMRYFO-WDCWCFNPSA-N Leu-Thr-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CCC(N)=O LCNASHSOFMRYFO-WDCWCFNPSA-N 0.000 description 1
- UCRJTSIIAYHOHE-ULQDDVLXSA-N Leu-Tyr-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N UCRJTSIIAYHOHE-ULQDDVLXSA-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
- NKKFVJRLCCUJNA-QWRGUYRKSA-N Lys-Gly-Lys Chemical compound NCCCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCCN NKKFVJRLCCUJNA-QWRGUYRKSA-N 0.000 description 1
- SQJSXOQXJYAVRV-SRVKXCTJSA-N Lys-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](CCCCN)N SQJSXOQXJYAVRV-SRVKXCTJSA-N 0.000 description 1
- LJADEBULDNKJNK-IHRRRGAJSA-N Lys-Leu-Val Chemical compound CC(C)C[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O LJADEBULDNKJNK-IHRRRGAJSA-N 0.000 description 1
- MSSJJDVQTFTLIF-KBPBESRZSA-N Lys-Phe-Gly Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](Cc1ccccc1)C(=O)NCC(O)=O MSSJJDVQTFTLIF-KBPBESRZSA-N 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- GLFNIEUTAYBVOC-UHFFFAOYSA-L Manganese chloride Chemical compound Cl[Mn]Cl GLFNIEUTAYBVOC-UHFFFAOYSA-L 0.000 description 1
- MUYQDMBLDFEVRJ-LSJOCFKGSA-N Met-Ala-His Chemical compound CSCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC1=CNC=N1 MUYQDMBLDFEVRJ-LSJOCFKGSA-N 0.000 description 1
- CHQWUYSNAOABIP-ZPFDUUQYSA-N Met-Glu-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCSC)N CHQWUYSNAOABIP-ZPFDUUQYSA-N 0.000 description 1
- SLQDSYZHHOKQSR-QXEWZRGKSA-N Met-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCSC SLQDSYZHHOKQSR-QXEWZRGKSA-N 0.000 description 1
- RSOMVHWMIAZNLE-HJWJTTGWSA-N Met-Phe-Ile Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RSOMVHWMIAZNLE-HJWJTTGWSA-N 0.000 description 1
- GWADARYJIJDYRC-XGEHTFHBSA-N Met-Thr-Ser Chemical compound CSCC[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O GWADARYJIJDYRC-XGEHTFHBSA-N 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- WUGMRIBZSVSJNP-UHFFFAOYSA-N N-L-alanyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C)C(O)=O)=CNC2=C1 WUGMRIBZSVSJNP-UHFFFAOYSA-N 0.000 description 1
- XZFYRXDAULDNFX-UHFFFAOYSA-N N-L-cysteinyl-L-phenylalanine Natural products SCC(N)C(=O)NC(C(O)=O)CC1=CC=CC=C1 XZFYRXDAULDNFX-UHFFFAOYSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 108010002311 N-glycylglutamic acid Proteins 0.000 description 1
- 229910002651 NO3 Inorganic materials 0.000 description 1
- 101150023810 PHO1 gene Proteins 0.000 description 1
- 101150010967 PHT3 gene Proteins 0.000 description 1
- VLZGUAUYZGQKPM-DRZSPHRISA-N Phe-Gln-Ala Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VLZGUAUYZGQKPM-DRZSPHRISA-N 0.000 description 1
- HBGFEEQFVBWYJQ-KBPBESRZSA-N Phe-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=CC=C1 HBGFEEQFVBWYJQ-KBPBESRZSA-N 0.000 description 1
- PBXYXOAEQQUVMM-ULQDDVLXSA-N Phe-His-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CC=CC=C2)N PBXYXOAEQQUVMM-ULQDDVLXSA-N 0.000 description 1
- CMHTUJQZQXFNTQ-OEAJRASXSA-N Phe-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC1=CC=CC=C1)N)O CMHTUJQZQXFNTQ-OEAJRASXSA-N 0.000 description 1
- KNYPNEYICHHLQL-ACRUOGEOSA-N Phe-Leu-Tyr Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(O)=O)C1=CC=CC=C1 KNYPNEYICHHLQL-ACRUOGEOSA-N 0.000 description 1
- INHMISZWLJZQGH-ULQDDVLXSA-N Phe-Leu-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 INHMISZWLJZQGH-ULQDDVLXSA-N 0.000 description 1
- GPLWGAYGROGDEN-BZSNNMDCSA-N Phe-Phe-Ser Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CO)C(O)=O GPLWGAYGROGDEN-BZSNNMDCSA-N 0.000 description 1
- KIQUCMUULDXTAZ-HJOGWXRNSA-N Phe-Tyr-Tyr Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(=O)N[C@@H](Cc1ccc(O)cc1)C(O)=O KIQUCMUULDXTAZ-HJOGWXRNSA-N 0.000 description 1
- CDHURCQGUDNBMA-UBHSHLNASA-N Phe-Val-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 CDHURCQGUDNBMA-UBHSHLNASA-N 0.000 description 1
- GOUWCZRDTWTODO-YDHLFZDLSA-N Phe-Val-Asn Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O GOUWCZRDTWTODO-YDHLFZDLSA-N 0.000 description 1
- FEPSEIDIPBMIOS-QXEWZRGKSA-N Pro-Gly-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H]1CCCN1 FEPSEIDIPBMIOS-QXEWZRGKSA-N 0.000 description 1
- HAEGAELAYWSUNC-WPRPVWTQSA-N Pro-Gly-Val Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O HAEGAELAYWSUNC-WPRPVWTQSA-N 0.000 description 1
- ITUDDXVFGFEKPD-NAKRPEOUSA-N Pro-Ser-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ITUDDXVFGFEKPD-NAKRPEOUSA-N 0.000 description 1
- PRKWBYCXBBSLSK-GUBZILKMSA-N Pro-Ser-Val Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O PRKWBYCXBBSLSK-GUBZILKMSA-N 0.000 description 1
- 101100243924 Pseudomonas putida pht4 gene Proteins 0.000 description 1
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 108020004511 Recombinant DNA Proteins 0.000 description 1
- 108091006595 SLC15A3 Proteins 0.000 description 1
- 108091006597 SLC15A4 Proteins 0.000 description 1
- 101100271429 Saccharomyces cerevisiae (strain ATCC 204508 / S288c) ATP6 gene Proteins 0.000 description 1
- LVVBAKCGXXUHFO-ZLUOBGJFSA-N Ser-Ala-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(O)=O LVVBAKCGXXUHFO-ZLUOBGJFSA-N 0.000 description 1
- GXXTUIUYTWGPMV-FXQIFTODSA-N Ser-Arg-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C)C(O)=O GXXTUIUYTWGPMV-FXQIFTODSA-N 0.000 description 1
- LALNXSXEYFUUDD-GUBZILKMSA-N Ser-Glu-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LALNXSXEYFUUDD-GUBZILKMSA-N 0.000 description 1
- 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 1
- HMRAQFJFTOLDKW-GUBZILKMSA-N Ser-His-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O HMRAQFJFTOLDKW-GUBZILKMSA-N 0.000 description 1
- NNFMANHDYSVNIO-DCAQKATOSA-N Ser-Lys-Arg Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O NNFMANHDYSVNIO-DCAQKATOSA-N 0.000 description 1
- VIIJCAQMJBHSJH-FXQIFTODSA-N Ser-Met-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CO)C(O)=O VIIJCAQMJBHSJH-FXQIFTODSA-N 0.000 description 1
- NQZFFLBPNDLTPO-DLOVCJGASA-N Ser-Phe-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CO)N NQZFFLBPNDLTPO-DLOVCJGASA-N 0.000 description 1
- BVLGVLWFIZFEAH-BPUTZDHNSA-N Ser-Pro-Trp Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O BVLGVLWFIZFEAH-BPUTZDHNSA-N 0.000 description 1
- 102100021485 Solute carrier family 15 member 3 Human genes 0.000 description 1
- 102100021484 Solute carrier family 15 member 4 Human genes 0.000 description 1
- XSLXHSYIVPGEER-KZVJFYERSA-N Thr-Ala-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H](C(C)C)C(O)=O XSLXHSYIVPGEER-KZVJFYERSA-N 0.000 description 1
- NAXBBCLCEOTAIG-RHYQMDGZSA-N Thr-Arg-Lys Chemical compound NC(N)=NCCC[C@H](NC(=O)[C@@H](N)[C@H](O)C)C(=O)N[C@@H](CCCCN)C(O)=O NAXBBCLCEOTAIG-RHYQMDGZSA-N 0.000 description 1
- UBDDORVPVLEECX-FJXKBIBVSA-N Thr-Gly-Met Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O UBDDORVPVLEECX-FJXKBIBVSA-N 0.000 description 1
- JQAWYCUUFIMTHE-WLTAIBSBSA-N Thr-Gly-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JQAWYCUUFIMTHE-WLTAIBSBSA-N 0.000 description 1
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 1
- SCSVNSNWUTYSFO-WDCWCFNPSA-N Thr-Lys-Glu Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O SCSVNSNWUTYSFO-WDCWCFNPSA-N 0.000 description 1
- STUAPCLEDMKXKL-LKXGYXEUSA-N Thr-Ser-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O STUAPCLEDMKXKL-LKXGYXEUSA-N 0.000 description 1
- IEZVHOULSUULHD-XGEHTFHBSA-N Thr-Ser-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O IEZVHOULSUULHD-XGEHTFHBSA-N 0.000 description 1
- KAJRRNHOVMZYBL-IRIUXVKKSA-N Thr-Tyr-Gln Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O KAJRRNHOVMZYBL-IRIUXVKKSA-N 0.000 description 1
- PELIQFPESHBTMA-WLTAIBSBSA-N Thr-Tyr-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=C(O)C=C1 PELIQFPESHBTMA-WLTAIBSBSA-N 0.000 description 1
- CYCGARJWIQWPQM-YJRXYDGGSA-N Thr-Tyr-Ser Chemical compound C[C@@H](O)[C@H]([NH3+])C(=O)N[C@H](C(=O)N[C@@H](CO)C([O-])=O)CC1=CC=C(O)C=C1 CYCGARJWIQWPQM-YJRXYDGGSA-N 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- RNFZZCMCRDFNAE-WFBYXXMGSA-N Trp-Asn-Ala Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C)C(O)=O RNFZZCMCRDFNAE-WFBYXXMGSA-N 0.000 description 1
- YRXXUYPYPHRJPB-RXVVDRJESA-N Trp-Gly-Trp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)NCC(=O)N[C@@H](CC3=CNC4=CC=CC=C43)C(=O)O)N YRXXUYPYPHRJPB-RXVVDRJESA-N 0.000 description 1
- XGFGVFMXDXALEV-XIRDDKMYSA-N Trp-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N XGFGVFMXDXALEV-XIRDDKMYSA-N 0.000 description 1
- OSYOKZZRVGUDMO-HSCHXYMDSA-N Trp-Lys-Ile Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O OSYOKZZRVGUDMO-HSCHXYMDSA-N 0.000 description 1
- 101100115751 Trypanosoma brucei brucei dnaaf11 gene Proteins 0.000 description 1
- QARCDOCCDOLJSF-HJPIBITLSA-N Tyr-Ile-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N QARCDOCCDOLJSF-HJPIBITLSA-N 0.000 description 1
- QFHRUCJIRVILCK-YJRXYDGGSA-N Tyr-Thr-Cys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)N)O QFHRUCJIRVILCK-YJRXYDGGSA-N 0.000 description 1
- FZSPNKUFROZBSG-ZKWXMUAHSA-N Val-Ala-Asp Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CC(O)=O FZSPNKUFROZBSG-ZKWXMUAHSA-N 0.000 description 1
- LTFLDDDGWOVIHY-NAKRPEOUSA-N Val-Ala-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C)NC(=O)[C@H](C(C)C)N LTFLDDDGWOVIHY-NAKRPEOUSA-N 0.000 description 1
- ZLFHAAGHGQBQQN-GUBZILKMSA-N Val-Ala-Pro Natural products CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N1CCC[C@H]1C(O)=O ZLFHAAGHGQBQQN-GUBZILKMSA-N 0.000 description 1
- CELJCNRXKZPTCX-XPUUQOCRSA-N Val-Gly-Ala Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](C)C(O)=O CELJCNRXKZPTCX-XPUUQOCRSA-N 0.000 description 1
- NXRAUQGGHPCJIB-RCOVLWMOSA-N Val-Gly-Asn Chemical compound CC(C)[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O NXRAUQGGHPCJIB-RCOVLWMOSA-N 0.000 description 1
- PIFJAFRUVWZRKR-QMMMGPOBSA-N Val-Gly-Gly Chemical compound CC(C)[C@H]([NH3+])C(=O)NCC(=O)NCC([O-])=O PIFJAFRUVWZRKR-QMMMGPOBSA-N 0.000 description 1
- MDYSKHBSPXUOPV-JSGCOSHPSA-N Val-Gly-Phe Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N MDYSKHBSPXUOPV-JSGCOSHPSA-N 0.000 description 1
- OTJMMKPMLUNTQT-AVGNSLFASA-N Val-Leu-Arg Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](C(C)C)N OTJMMKPMLUNTQT-AVGNSLFASA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- KTEZUXISLQTDDQ-NHCYSSNCSA-N Val-Lys-Asp Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(=O)O)C(=O)O)N KTEZUXISLQTDDQ-NHCYSSNCSA-N 0.000 description 1
- JAKHAONCJJZVHT-DCAQKATOSA-N Val-Lys-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)O)N JAKHAONCJJZVHT-DCAQKATOSA-N 0.000 description 1
- YKNOJPJWNVHORX-UNQGMJICSA-N Val-Phe-Thr Chemical compound CC(C)[C@H](N)C(=O)N[C@H](C(=O)N[C@@H]([C@@H](C)O)C(O)=O)CC1=CC=CC=C1 YKNOJPJWNVHORX-UNQGMJICSA-N 0.000 description 1
- CEKSLIVSNNGOKH-KZVJFYERSA-N Val-Thr-Ala Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](C)C(=O)O)NC(=O)[C@H](C(C)C)N)O CEKSLIVSNNGOKH-KZVJFYERSA-N 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010070944 alanylhistidine Proteins 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 108010040443 aspartyl-aspartic acid Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 230000003851 biochemical process Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 239000004327 boric acid Substances 0.000 description 1
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 1
- 210000000170 cell membrane Anatomy 0.000 description 1
- 229910052927 chalcanthite Inorganic materials 0.000 description 1
- 210000003763 chloroplast Anatomy 0.000 description 1
- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 230000002950 deficient Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 239000003085 diluting agent 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
- 235000019301 disodium ethylene diamine tetraacetate Nutrition 0.000 description 1
- 210000002472 endoplasmic reticulum Anatomy 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003746 feather Anatomy 0.000 description 1
- 239000011790 ferrous sulphate Substances 0.000 description 1
- 235000003891 ferrous sulphate Nutrition 0.000 description 1
- 108020001507 fusion proteins Proteins 0.000 description 1
- 102000037865 fusion proteins Human genes 0.000 description 1
- 230000009368 gene silencing by RNA Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 108010079547 glutamylmethionine Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010078326 glycyl-glycyl-valine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010077515 glycylproline Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 229960000367 inositol Drugs 0.000 description 1
- CDAISMWEOUEBRE-GPIVLXJGSA-N inositol Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@H](O)[C@@H]1O CDAISMWEOUEBRE-GPIVLXJGSA-N 0.000 description 1
- BAUYGSIQEAFULO-UHFFFAOYSA-L iron(2+) sulfate (anhydrous) Chemical compound [Fe+2].[O-]S([O-])(=O)=O BAUYGSIQEAFULO-UHFFFAOYSA-L 0.000 description 1
- 229910000359 iron(II) sulfate Inorganic materials 0.000 description 1
- 238000012933 kinetic analysis Methods 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 108010044056 leucyl-phenylalanine Proteins 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 230000004807 localization Effects 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 229940099596 manganese sulfate Drugs 0.000 description 1
- 239000011702 manganese sulphate Substances 0.000 description 1
- 235000007079 manganese sulphate Nutrition 0.000 description 1
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 108020004999 messenger RNA Proteins 0.000 description 1
- 239000002207 metabolite Substances 0.000 description 1
- 210000003470 mitochondria Anatomy 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 235000001968 nicotinic acid Nutrition 0.000 description 1
- 229960003512 nicotinic acid Drugs 0.000 description 1
- 239000011664 nicotinic acid Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 108010070409 phenylalanyl-glycyl-glycine Proteins 0.000 description 1
- 230000008288 physiological mechanism Effects 0.000 description 1
- 230000008121 plant development Effects 0.000 description 1
- 230000008635 plant growth Effects 0.000 description 1
- 239000001103 potassium chloride Substances 0.000 description 1
- 235000011164 potassium chloride Nutrition 0.000 description 1
- 239000004323 potassium nitrate Substances 0.000 description 1
- 235000010333 potassium nitrate Nutrition 0.000 description 1
- OTYBMLCTZGSZBG-UHFFFAOYSA-L potassium sulfate Chemical compound [K+].[K+].[O-]S([O-])(=O)=O OTYBMLCTZGSZBG-UHFFFAOYSA-L 0.000 description 1
- 229910052939 potassium sulfate Inorganic materials 0.000 description 1
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 description 1
- 108010087846 prolyl-prolyl-glycine Proteins 0.000 description 1
- 108010015796 prolylisoleucine Proteins 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- ZUFQODAHGAHPFQ-UHFFFAOYSA-N pyridoxine hydrochloride Chemical compound Cl.CC1=NC=C(CO)C(CO)=C1O ZUFQODAHGAHPFQ-UHFFFAOYSA-N 0.000 description 1
- 229960004172 pyridoxine hydrochloride Drugs 0.000 description 1
- 235000019171 pyridoxine hydrochloride Nutrition 0.000 description 1
- 239000011764 pyridoxine hydrochloride Substances 0.000 description 1
- 238000012113 quantitative test Methods 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- CDAISMWEOUEBRE-UHFFFAOYSA-N scyllo-inosotol Natural products OC1C(O)C(O)C(O)C(O)C1O CDAISMWEOUEBRE-UHFFFAOYSA-N 0.000 description 1
- 108010048818 seryl-histidine Proteins 0.000 description 1
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012360 testing method Methods 0.000 description 1
- DPJRMOMPQZCRJU-UHFFFAOYSA-M thiamine hydrochloride Chemical compound Cl.[Cl-].CC1=C(CCO)SC=[N+]1CC1=CN=C(C)N=C1N DPJRMOMPQZCRJU-UHFFFAOYSA-M 0.000 description 1
- 229960000344 thiamine hydrochloride Drugs 0.000 description 1
- 235000019190 thiamine hydrochloride Nutrition 0.000 description 1
- 239000011747 thiamine hydrochloride Substances 0.000 description 1
- 108010031491 threonyl-lysyl-glutamic acid Proteins 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 108010038745 tryptophylglycine Proteins 0.000 description 1
- 108010077037 tyrosyl-tyrosyl-phenylalanine Proteins 0.000 description 1
- 108010073969 valyllysine Proteins 0.000 description 1
- 229960001763 zinc sulfate Drugs 0.000 description 1
- 239000011686 zinc sulphate Substances 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biochemistry (AREA)
- Zoology (AREA)
- Biophysics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Cell Biology (AREA)
- Plant Pathology (AREA)
- Nutrition Science (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
本发明公开了蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用。蛋白质OsVPE2的氨基酸序列如序列表中序列3所示。提高出发植物中蛋白质OsVPE2的表达量和/或活性,得到转OsVPE2基因植物;与水稻品种石狩白毛相比,转OsVPE2基因植物的液泡无机磷输出能力提高,液泡里的无机磷浓度降低。抑制出发植物中蛋白质OsVPE2的表达量和/或活性,得到OsVPE2基因敲除水稻植株;与水稻品种石狩白毛相比,OsVPE2基因敲除水稻植株的液泡无机磷输出能力降低,液泡里的无机磷浓度增加。由此可见,蛋白质OsVPE2可以调控植物液泡无机磷输出能力,进而调节液泡里的无机磷浓度。本发明具有重要的应用价值。
Description
技术领域
本发明属于生物技术领域,具体涉及蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用。
背景技术
磷素是植物生长发育所必需的大量元素,是许多代谢物和生物大分子的重要组分,并且在能量转化和信号转导等生化过程中起重要作用。当土壤缺磷时,植物一般会出现生长矮小、分蘖减少和产量降低等现象。因此,提高作物磷肥利用率,减少磷肥施用量,对水稻优质高产、保护环境都有重要的作用。而充分了解植物磷素吸收和利用的分子生理机制,是提高植物磷素吸收和利用效率的前提。
植物通过磷酸盐转运体从外界吸收磷酸盐并分配到不同的器官。对植物进行磷吸收动力学分析表明,高等植物中同时存在两种磷酸盐吸收转运***:一类是在磷供应充足时负责磷吸收和转运的低亲和磷酸盐转运体;另一类是在磷供应不足时诱导表达,增加有效磷的吸收和转运的高亲和磷酸盐转运体,高亲和磷酸盐转运体是植物从根部吸收磷的主要***。目前已知的磷酸转运体有:质膜定位的PHT1家族,主要负责从根部吸收土壤中的无机磷;PHT3家族负责将细胞质中的无机磷向线粒体内转运;PHT2家族,PHT4家族,XPT,TPT,PPT,GPT1主要负责叶绿体和高尔基体的无机磷转运;PHO1定位于内质网和高尔基体,主要在根的木质部表达,负责无机磷从根到地上部的转移。
植物中的大部分无机磷储存于液泡中,当无机磷缺乏时,液泡中的无机磷被释放到细胞质来维持细胞质中的无机磷水平。所以,液泡中磷素的储存和输出对于维持植物中的磷素平衡非常重要。提高作物中液泡磷的输出,有利于提高磷素的利用效率。
发明内容
本发明的目的是提高植物液泡磷的输出能力,从而提高磷素的利用效率。
本保护首先保护蛋白质OsVPE2的应用可为X1)或X2)或X3)或X4):
X1)调控植物液泡无机磷输出能力;
X2)调控植物液泡里的无机磷浓度;
X3)调控植物无机磷输出能力;
X4)调控植物的无机磷浓度。
上述应用中,所述蛋白质OsVPE2可为a1)或a2)或a3):
a1)氨基酸序列是序列表中序列3所示的蛋白质;
a2)在序列表中序列3所示的蛋白质的N端或/和C端连接标签得到的融合蛋白质;
a3)将序列表中序列3所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加得到的与植物液泡无机磷输出能力和/或植物液泡里的无机磷浓度和/或植物无机磷输出能力和/或植物的无机磷浓度相关的蛋白质。
其中,序列表中序列3由499个氨基酸残基组成。
为了使a1)中的蛋白质便于纯化,可在序列表中序列3所示的蛋白质的氨基末端或羧基末端连接上如表1所示的标签。
表1.标签的序列
标签 | 残基 | 序列 |
Poly-Arg | 5-6(通常为5个) | RRRRR |
FLAG | 8 | DYKDDDDK |
Strep-tag II | 8 | WSHPQFEK |
c-myc | 10 | EQKLISEEDL |
上述a3)中的蛋白质,所述一个或几个氨基酸残基的取代和/或缺失和/或添加为不超过10个氨基酸残基的取代和/或缺失和/或添加。
上述a3)中的蛋白质可人工合成,也可先合成其编码基因,再进行生物表达得到。
上述a3)中的蛋白质的编码基因可通过将序列表中序列2所示的DNA序列中缺失一个或几个氨基酸残基的密码子,和/或进行一个或几个碱基对的错义突变,和/或在其5′端和/或3′端连上表1所示的标签的编码序列得到。
本发明还保护编码上述任一所述蛋白质OsVPE2的核酸分子的应用,可为X1)或X2)或X3)或X4):
X1)调控植物液泡无机磷输出能力;
X2)调控植物液泡里的无机磷浓度;
X3)调控植物无机磷输出能力;
X4)调控植物的无机磷浓度。
上述应用中,所述编码蛋白质OsVPE2的核酸分子可为如下b1)或b2)或b3)或b4)或b5)所示的DNA分子:
b1)编码区如序列表中序列2所示的DNA分子;
b2)核苷酸序列是序列表中序列2所示的DNA分子;
b3)核苷酸序列是序列表中序列1所示的DNA分子;
b4)与b1)或b2)或b3)限定的核苷酸序列具有75%或75%以上同一性,且编码所述蛋白质OsVPE2的DNA分子;
b5)在严格条件下与b1)或b2)或b3)限定的核苷酸序列杂交,且编码所述蛋白质OsVPE2的DNA分子。
其中,所述核酸分子可以是DNA,如cDNA、基因组DNA或重组DNA;所述核酸分子也可以是RNA,如mRNA或hnRNA等。
其中,序列表中序列1由1585个核苷酸组成,序列表中序列2由1500个核苷酸组成,序列表中序列2的核苷酸编码序列表中序列3所示的氨基酸序列。
本领域普通技术人员可以很容易地采用已知的方法,例如定向进化和点突变的方法,对本发明的编码所述蛋白质OsVPE2的核苷酸序列进行突变。那些经过人工修饰的,具有与本发明分离得到的所述蛋白质OsVPE2的核苷酸序列75%或者更高同一性的核苷酸,只要编码所述蛋白质OsVPE2,均是衍生于本发明的核苷酸序列并且等同于本发明的序列。
这里使用的术语“同一性”指与天然核酸序列的序列相似性。“同一性”包括与本发明的编码序列表的序列3所示的氨基酸序列组成的蛋白质OsVPE2的核苷酸序列具有75%或更高,或80%或更高,或85%或更高,或90%或更高,或95%或更高同一性的核苷酸序列。同一性可以用肉眼或计算机软件进行评价。使用计算机软件,两个或多个序列之间的同一性可以用百分比(%)表示,其可以用来评价相关序列之间的同一性。
上述任一所述的应用中,所述调控植物液泡无机磷输出能力可为提高植物液泡无机磷输出能力或降低植物液泡无机磷输出能力。所述调控植物液泡里的无机磷浓度可为增加植物液泡里的无机磷浓度或降低植物液泡里的无机磷浓度。所述调控植物无机磷输出能力可为提高植物无机磷输出能力或降低植物无机磷输出能力。所述调控植物的无机磷浓度可为增加植物的无机磷浓度或降低植物的无机磷浓度。
本发明还保护一种培育转基因植物甲的方法,可包括如下步骤:提高出发植物中上述任一所述蛋白质OsVPE2的表达量和/或活性,得到转基因植物甲;与出发植物相比,转基因植物甲的液泡无机磷输出能力提高和/或液泡里的无机磷浓度降低和/或无机磷输出能力提高和/或无机磷浓度降低。
上述方法中,所述“提高出发植物中所述蛋白质OsVPE2的表达量和/或活性”可通过转基因、多拷贝、改变启动子、调控因子等本领域熟知的方法,达到增加植物中所述蛋白质OsVPE2表达量和/或活性的效果。
上述方法中,所述“提高出发植物中所述蛋白质OsVPE2的表达量和/或活性”具体可通过向出发植物中导入编码所述蛋白质OsVPE2的核酸分子实现。
上述方法中,所述“向出发植物中导入编码所述蛋白质OsVPE2的核酸分子”可通过向出发植物中导入重组载体实现;所述重组载体可为向表达载体***编码所述蛋白质OsVPE2的核酸分子,得到的重组质粒。所述重组载体可为实施例提及的重组质粒pCAMBIA1300-35S-OsVPE2-rbcs。所述重组质粒pCAMBIA1300-35S-OsVPE2-rbcs具体是将序列表中的序列2所示的DNA分子***pCAMBIA1300-35S-rbcs载体的限制性内切酶KpnI和SalI识别位点之间,得到的重组质粒。
所述转基因植物甲具体可为实施例提及的OsVPE2-OE-1至OsVPE2-OE-28。
本发明还保护一种培育转基因植物乙的方法,可包括如下步骤:抑制出发植物中上述任一所述蛋白质OsVPE2的表达量和/或活性,得到转基因植物乙;与出发植物相比,转基因植物乙的液泡无机磷输出能力降低和/或液泡里的无机磷浓度增加和/或无机磷输出能力降低和/或无机磷浓度增加。
上述方法中,所述“抑制出发植物中所述蛋白质OsVPE2的表达量和/或活性”可通过基因定点编辑、RNA干扰、同源重组、基因敲除等本领域熟知的方法,达到抑制所述蛋白质OsVPE2的表达量和/或活性的目的。
上述方法中,所述“抑制出发植物中所述蛋白质OsVPE2的表达量和/或活性”具体可为使出发植物含有CRISPR-Cas9***;所述CRISPR-Cas9***中含有sgRNA;所述sgRNA识别的靶标可为出发植物的OsVPE2基因,对出发植物中的OsVPE2基因进行基因编辑。
上述方法中,所述“使出发植物含有CRISPR-Cas9***”具体可通过向出发植物中导入含有CRISPR-Cas9***的载体实现。
上述方法中,所述sgRNA识别的靶位点可为5’-CAAAGTTGGGGTCCTCTCCA-3’。
上述方法中,所述“含有CRISPR-Cas9***的载体”可为实施例提及的载体pYLCRISPR/Cas9-OsVPE2。所述载体pYLCRISPR/Cas9-OsVPE2可为根据植物CRISPR/Cas9载体***靶位点的要求,选择OsVPE2基因的靶序列为5’-CAAAGTTGGGGTCCTCTCCA-3’,然后按照文献(Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y,Xie Y,Shen R,Chen S,Wang Z,Chen Y,Guo J,Chen L,Zhao X,Dong Z,Liu YG(2015)A RobustCRISPR/Cas9 System for Convenient,High-Efficiency Multiplex Genome Editing inMonocot and Dicot Plants.Mol Plant 8:1274-1284)中的方法构建而成。
所述转基因植物乙具体可为实施例提及的水稻纯合突变体Osvpe2。与水稻品种石狩白毛相比,水稻纯合突变体Osvpe2两条同源染色体上OsVPE2基因的相同位置(具体为序列表中序列1自5’末端起第195位)均***一个碱基C。由于碱基C的***,导致OsVPE2基因的阅读框移码,进而蛋白质OsVPE2的功能失活,蛋白质OsVPE2的表达量和/或活性受到抑制。
本发明还保护一种植物育种方法,可包括如下步骤:增加植物中上述任一所述蛋白质OsVPE2的含量和/或活性,从而提高液泡无机磷输出能力和/或降低液泡里的无机磷浓度和/或提高无机磷输出能力和/或降低无机磷浓度。
本发明还保护一种植物育种方法,可包括如下步骤:降低植物中上述任一所述蛋白质OsVPE2的含量和/或活性,从而降低液泡无机磷输出能力和/或增加液泡里的无机磷浓度和/或降低无机磷输出能力和/或增加无机磷浓度。
上述任一所述植物可为如下c1)至c5)中的任一种:c1)双子叶植物;c2)单子叶植物;c3)禾本科植物;c4)水稻;c5)水稻品种石狩白毛。
实验证明,向水稻品种石狩白毛中导入重组质粒pCAMBIA1300-35S-OsVPE2-rbcs,得到转OsVPE2基因植物;与水稻品种石狩白毛相比,转OsVPE2基因植物的液泡无机磷输出能力提高,液泡里的无机磷浓度降低。向水稻品种石狩白毛中导入载体pYLCRISPR/Cas9-OsVPE2,得到OsVPE2基因敲除水稻植株;与水稻品种石狩白毛相比,OsVPE2基因敲除水稻植株的液泡无机磷输出能力降低,液泡里的无机磷浓度增加。由此可见,蛋白质OsVPE2可以调控植物液泡无机磷输出能力,进而调节液泡里的无机磷浓度。本发明具有重要的应用价值。
附图说明
图1为pCAMBIA1300-35S-rbcs载体的图谱。
图2为水稻品种石狩白毛植株和OsVPE2-OE-23超表达植株根中液泡内的无机磷浓度测定结果。
图3为水稻品种石狩白毛植株和纯合突变体Osvpe2植株根中液泡内的无机磷浓度测定结果。
具体实施方式
以下的实施例便于更好地理解本发明,但并不限定本发明。
下述实施例中的实验方法,如无特殊说明,均为常规方法。
下述实施例中所用的试验材料,如无特殊说明,均为自常规生化试剂商店购买得到的。
以下实施例中的定量试验,均设置三次重复实验,结果取平均值。
光照交替培养即光培养和暗培养交替,条件为:30℃;12小时光照培养/22℃;12小时黑暗培养;光照培养时的光照强度为3000Lux。
诱导培养基:取N6大量50mL、B5微量10mL、NB有机10mL、铁盐10mL、2.0mg 2,4-D、0.5g L-谷氨酰胺、2.8g L-脯氨酸、0.3g水解酪蛋白和30g蔗糖,用蒸馏水定容至1L,然后调节pH值至5.8,加入4g植物凝胶。121℃高压灭菌15min。
共培养基:取N6大量50mL、B5微量10mL、NB有机10mL、铁盐10mL、2.0mg 2,4-D、0.5gL-谷氨酰胺、2.8g L-脯氨酸、0.6g水解酪蛋白、10g葡萄糖和30g蔗糖,用蒸馏水定容至1L,然后调节pH值至5.2,加入4g植物凝胶。121℃高压灭菌15min。恢复至常温后,加入乙酰丁香酮并使其在体系中的浓度为200μmol/L。
选择培养基:取N6大量50mL、B5微量10mL、NB有机10mL、铁盐10mL、2.0mg 2,4-D、0.5g L-谷氨酰胺、2.8g L-脯氨酸、0.6g水解酪蛋白和30g蔗糖,用蒸馏水定容至1L,然后调节pH值至5.8,加入4g植物凝胶。121℃高压灭菌15min。恢复至常温后,加入潮霉素和头孢,并使潮霉素在体系中的浓度为50mg/L,头孢在体系中的浓度为500mg/L。
分化培养基:取N6大量50mL、B5微量10mL、NB有机10mL、铁盐10mL、0.5g L-谷氨酰胺、0.5g L-脯氨酸、1.0g水解酪蛋白、3.0mg 6-BA、0.5mg NAA、30g蔗糖和20g山梨醇,用蒸馏水定容至1L,然后调节pH值至5.8,加入4g植物凝胶。121℃高压灭菌15min。
生根培养基:取N6大量50mL、B5微量10mL、NB有机10mL、铁盐10mL和20g蔗糖,用蒸馏水定容至1L,然后调节pH值至5.8,加入3.5g植物凝胶。121℃高压灭菌15min。
AAM转化液:取AA大量100mL、B5微量10mL、NB有机10mL、铁盐10mL、0.3g水解酪蛋白和30g麦芽糖,用蒸馏水定容至1L,然后调节pH值至5.5。121℃高压灭菌15min。恢复至常温后,加入乙酰丁香酮并使其在体系中的浓度为200μmol/L。
N6大量:将硝酸钾56.6g、氯化钙3.32g、硫酸镁2.70g、磷酸二氢钾8.0g和硫酸铵9.26g溶于水,然后用水定容至1L。
B5微量:将碘化钾0.0750g、硼酸0.30g、硫酸锰1.0g、硫酸锌0.2g和硫酸铜0.0025g溶于水,然后用水定容至1L。
NB有机:将烟酸1g、盐酸吡哆醇1g、盐酸硫胺素10g和肌醇10g溶于水,然后用水定容至1L。
铁盐:将硫酸亚铁2.78g和乙二铵四乙酸二钠3.73g溶于水,然后用水定容至1L。
AA大量:将氯化钾2.95g、氯化钙0.15g、硫酸镁0.25g和磷酸二氢钾0.15g溶于水,然后用水定容至1L。
水稻标准营养液的配方见表2。
表2
试剂 | g/10L |
NH<sub>4</sub>NO<sub>3</sub> | 0.914 |
CaCl<sub>2</sub> | 0.886 |
MgSO<sub>4</sub>·7H<sub>2</sub>O | 3.240 |
K<sub>2</sub>SO<sub>4</sub> | 0.714 |
NaH<sub>2</sub>PO<sub>4</sub>·2H<sub>2</sub>O | 0.403 |
Na<sub>2</sub>EDTA·2H<sub>2</sub>O | 0.09315 |
FeSO<sub>4</sub>·7H<sub>2</sub>O | 0.06965 |
MnCl<sub>2</sub>·4H<sub>2</sub>O | 0.015 |
H<sub>3</sub>BO<sub>3</sub> | 0.00934 |
(NH<sub>4</sub>)<sub>6</sub>·Mo<sub>7</sub>O<sub>24</sub>·4H<sub>2</sub>O | 0.000074 |
ZnSO<sub>4</sub>·7H<sub>2</sub>O | 0.000035 |
CuSO<sub>4</sub>·5H<sub>2</sub>O | 0.000031 |
水稻基因组中,OsVPE2基因的核苷酸序列如序列表中序列1所示,其编码区的核苷酸序列如序列表中序列2所示。序列表中的序列2所示的DNA分子编码序列表中的序列3所示的OsVPE2蛋白。
实施例1、转OsVPE2基因水稻的液泡输出无机磷的能力提高
一、重组质粒pCAMBIA1300-35S-OsVPE2-rbcs的构建
1、取水稻品种日本晴的叶片,先用液氮充分研磨,然后用QIAGEN公司的RNeasyPlant Mini KitRNA提取日本晴的总RNA(详细的提取步骤见试剂盒的说明书),用NanoDropND-1000核酸检测仪测量RNA样品的浓度及质量。
2、在离心管(规格为0.2mL)中加入日本晴的总RNA2μg、1μLdNTP(浓度为10mM)和1μLOligo(dT)20,加水补足体积到10μL,65℃温育5min后,立即放置冰上冷却。然后依次添加2μL 10×RT buffer、4μL MgCl2(浓度为25mM)、2μL DTT水溶液(浓度为0.1M)、1μL RNaseOUT和1μIII RT,50℃温育50min,85℃加热5min终止反应,得到反转录产物。向反转录产物中加入40μL ddH2O进行稀释,得到稀释液(稀释液中含日本晴的cDNA)。-20℃保存样品。
3、以步骤2得到的稀释液为模板,以引物F:5’-GACGAGCTGAGCTCGGTACCATGGCTCATTCTCATGAAA(下划线为限制性内切酶KpnI的识别位点)和引物R:5’-GAACCTGCAGGTCGACTTACACCTCATCTGAGTAGG-3’(下划线为限制性内切酶SalI的识别位点)进行PCR扩增,得到PCR扩增产物。
4、完成步骤3后,将PCR扩增产物进行琼脂糖凝胶电泳,然后用凝胶回收试剂盒(QIAGEN公司的产品)回收约1500bp的DNA片段。
5、取步骤4回收的DNA片段,用限制性内切酶KpnI和SalI双酶切,用凝胶回收试剂盒回收酶切产物。
6、取pCAMBIA1300-35S-rbcs载体(pCAMBIA1300-35S-rbcs载体的核苷酸序列如序列表中序列4所示,图谱见图1),用限制性内切酶KpnI和SalI双酶切,用凝胶回收试剂盒回收约10107bp的载体骨架。
7、将步骤6得到的酶切产物和步骤7得到的载体骨架进行连接,得到重组质粒pCAMBIA1300-35S-OsVPE2-rbcs。
将重组质粒pCAMBIA1300-35S-OsVPE2-rbcs进行测序。根据测序结果,对重组质粒pCAMBIA1300-35S-OsVPE2-rbcs进行结构描述如下:将序列表中的序列2所示的DNA分子(OsVPE2基因)***pCAMBIA1300-35S-rbcs载体的限制性内切酶KpnI和SalI识别位点之间,得到的重组质粒。重组质粒pCAMBIA1300-35S-OsVPE2-rbcs表达序列表中序列3所示的OsVPE2蛋白。
二、T0代拟转OsVPE2基因水稻的获得
1、采用电激转化法,将重组质粒pCAMBIA1300-35S-OsVPE2-rbcs导入根癌农杆菌EHA105,得到重组农杆菌,命名为EHA105/35S-OsVPE2。
2、将EHA105/35S-OsVPE2单克隆接种于20mL含卡那霉素50mg/L和利福平50mg/L的YEB液体培养基,28℃、220rpm震荡培养12~16h后,然后按2%(v/v)的比例接种于AAM转化液,28℃、220rpm振荡培养至OD600值达到0.5左右,得到农杆菌侵染液。
3、水稻品种石狩白毛的种子去壳脱粒,置于100mL三角瓶中,加入70%(v/v)乙醇水溶液浸泡30sec,再置于25%(v/v)次氯酸钠水溶液中,120rpm震荡灭菌30min,无菌水冲洗3次,用滤纸吸干水分,然后将种子胚朝下置于诱导培养基上诱导愈伤,30℃全光照诱导7天,长出的愈伤掐芽后用于水稻转化。
4、完成步骤3后,取生长状态较好的胚性愈伤组织,浸泡于步骤2得到的农杆菌侵染液,28℃、80rpm摇床轻摇愈伤,侵染30min,然后,放在铺有一层灭菌滤纸的共培养基上,25℃暗培养4天。
5、取步骤4得到的愈伤组织,置于选择培养基,光照交替培养2周后,更换新的选择培养基,愈伤组织继续光照交替培养2周,将原愈伤组织上长出的抗性愈伤组织转移至选择培养基上,光照交替培养2周。
6、完成步骤5后,取生长旺盛的抗性愈伤组织,置于分化培养基,光照交替培养3周,抗性愈伤组织上分化出抗性小芽。
7、完成步骤6后,取抗性小芽,置于生根培养基,光照交替培养,得到抗性植株。待抗性植株长至6~10cm时,进行开放式水培养,长出新根后移栽温室,得到T0代拟转OsVPE2基因水稻植株。
三、T0代拟转OsVPE2基因水稻的鉴定
1、分别提取T0代拟转OsVPE2基因水稻叶片的基因组DNA并以其作为模板,采用引物F:5’-GGCTTGGAAGATACCTGGAGT-3’和引物R:5’-ACAGTTTTCCCAATGCCATAATACT-3’组成的引物对进行PCR扩增,得到PCR扩增产物。如果PCR扩增产物中含有约750bp的DNA片段,则相应的T0代拟转OsVPE2基因水稻鉴定为T0代转OsVPE2基因水稻。
经鉴定,获得28株T0代转OsVPE2基因水稻,依次命名为OsVPE2-OE-1至OsVPE2-OE-28。
2、分别提取OsVPE2-OE-1至OsVPE2-OE-28、水稻品种石狩白毛的总RNA,将总RNA反转录出第一链cDNA。以cDNA为模板,RT-PCR检测OsVPE2基因的表达量。鉴定OsVPE2基因的引物为:5’-GGCTTGGAAGATACCTGGAGT-3’和5’-AGGTACTCACCGCCAATAGGT-3。
结果表明,与水稻品种石狩白毛相比,24株T0代转OsVPE2基因水稻的OsVPE2基因的表达量增强10倍以上。选取OsVPE2基因的表达量明显增强的OsVPE2-OE-23进行后续实验。
四、表型分析
分别取水稻品种石狩白毛种子或OsVPE2-OE-23的T2代纯合体种子,剥壳后,用清水浸泡,37℃浸种至露白(2-3天),期间早晚换水。将露白的水稻种子播种于装有水稻标准营养液的尼龙网纱上面(pH值约5.5),在水稻培养室(白天30℃,晚上22℃,光强3000Lux,光照时间12个h)中培养7天,然后挑取长势一致的水稻苗转到水稻培养槽(规格为32L)里继续在水稻标准营养液中培养一周,得到水稻植株。
利用核磁共振技术测定水稻植株根中液泡内的无机磷浓度。核磁共振实验条件和方法参照文献:刘于.(2015).基于核磁共振的水稻根细胞内pH值的测定方法.植物生理学报51(5):792-796。
结果如图2所示((WT为水稻品种石狩白毛植株,OsVPE2-OE-23为OsVPE2-OE-23植株)。结果表明,与水稻品种石狩白毛植株相比,OsVPE2-OE-23植株液泡输出无机磷的能力显著提高,导致液泡里的无机磷浓度显著下降。
实施例2、OsVPE2基因敲除水稻液泡输出无机磷的能力降低
一、OsVPE2基因敲除载体的构建
根据植物CRISPR/Cas9载体***靶位点的要求,选择OsVPE2基因的靶序列为5’-CAAAGTTGGGGTCCTCTCCA-3’,然后按照文献(Ma X,Zhang Q,Zhu Q,Liu W,Chen Y,Qiu R,Wang B,Yang Z,Li H,Lin Y,Xie Y,Shen R,Chen S,Wang Z,Chen Y,Guo J,Chen L,ZhaoX,Dong Z,Liu YG(2015)A Robust CRISPR/Cas9 System for Convenient,High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants.Mol Plant 8:1274-1284)中的方法,构建载体pYLCRISPR/Cas9-OsVPE2。
二、T0代OsVPE2基因敲除水稻的获得
1、采用电激转化法,将pYLCRISPR/Cas9-OsVPE2导入根癌农杆菌EHA105,得到重组农杆菌,命名为EHA105/Cas9-OsVPE2。
2、将EHA105/Cas9-OsVPE2单克隆接种于20mL含卡那霉素50mg/L和利福平50mg/L的YEB液体培养基,28℃、220rpm震荡培养12~16h后,然后按2%(v/v)的比例接种于AAM转化液,28℃、220rpm振荡培养至OD600值达到0.5左右,得到农杆菌侵染液。
3、水稻品种石狩白毛的种子去壳脱粒,置于100mL三角瓶中,加入70%(v/v)乙醇水溶液浸泡30sec,再置于25%(v/v)次氯酸钠水溶液中,120rpm震荡灭菌30min,无菌水冲洗3次,用滤纸吸干水分,然后将种子胚朝下置于诱导培养基上诱导愈伤,30℃全光照诱导7天,长出的愈伤掐芽后用于水稻转化。
4、完成步骤3后,取生长状态较好的胚性愈伤组织,浸泡于步骤2得到的农杆菌侵染液,28℃、80rpm摇床轻摇愈伤,侵染30min,然后,放在铺有一层灭菌滤纸的共培养基上,25℃暗培养4天。
5、取步骤4得到的愈伤组织,置于选择培养基,光照交替培养2周后,更换新的选择培养基,愈伤组织继续光照交替培养2周,将原愈伤组织上长出的抗性愈伤组织转移至选择培养基上,光照交替培养2周。
6、完成步骤5后,取生长旺盛的抗性愈伤组织,置于分化培养基,光照交替培养3周,抗性愈伤组织上分化出抗性小芽。
7、完成步骤6后,取抗性小芽,置于生根培养基,光照交替培养,得到抗性植株。待抗性植株长至6~10cm时,进行开放式水培养,长出新根后移栽温室,得到T0代OsVPE2基因敲除水稻植株。
三、T0代OsVPE2基因敲除水稻的鉴定
1、分别提取T0代OsVPE2基因敲除水稻叶片的基因组DNA并以其作为模板,以5’-GCTGATCTTCGTCTCATCTACC-3’和5’-GCTCCACCGTCAATGTAACCGG-3’为引物进行PCR扩增,得到PCR扩增产物。如果PCR扩增产物中含有655bp的DNA片段,则相应的T0代OsVPE2基因敲除水稻鉴定为T0代OsVPE2基因敲除水稻阳性苗。
2、分别以T0代OsVPE2基因敲除水稻阳性苗叶片的基因组DNA为模板,以5’-TTCATATGACTAGGAAGATACC-3’和5’-GACAGAAGTGTGTGCATTCCAA-3’为引物进行PCR扩增,得到长度为461bp的PCR扩增产物。
3、将步骤2得到的PCR扩增产物进行测序,测序结果与序列表中序列1所示的核苷酸序列进行比对,然后判断T0代OsVPE2基因敲除水稻阳性苗中OsVPE2基因的突变位置与形式。
经检测,获得了一个纯合突变体Osvpe2。纯合突变体Osvpe2两条同源染色体上OsVPE2基因的相同位置(序列表中序列1自5’末端起第195位)***一个碱基C。由于碱基C的***,导致OsVPE2基因的阅读框移码,进而OsVPE2蛋白的功能失活。
四、表型分析
分别取水稻品种石狩白毛种子或纯合突变体Osvpe2种子,剥壳后,用清水浸泡,37℃浸种至露白(2-3天),期间早晚换水。将露白的水稻种子播种于装有水稻全营养液的尼龙网纱上面(pH值约5.5),在水稻培养室(白天30℃,晚上22℃,光强3000Lux,光照时间12个h)中培养7天,然后挑取长势一致的水稻苗转到水稻培养槽(规格为32L)里继续在水稻标准营养液中培养一周,得到水稻植株。
利用核磁共振技术测定水稻植株根中液泡内的无机磷浓度。核磁共振实验条件和方法参照文献:刘于.(2015).基于核磁共振的水稻根细胞内pH值的测定方法.植物生理学报51(5):792-796。
结果如图3所示(WT为水稻品种石狩白毛植株,Osvpe2为纯合突变体Osvpe2植株)。结果表明,与水稻品种石狩白毛植株相比,纯合突变体Osvpe2植株液泡输出无机磷的能力显著降低,导致液泡里的无机磷浓度显著升高。
<110> 蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用
<120> 中国农业科学院农业资源与农业区划研究所
<160> 4
<170> PatentIn version 3.5
<210> 1
<211> 1585
<212> DNA
<213> 水稻(Oryza sativa L.)
<400> 1
atggctcatt ctcatgaaat gacgagtagg aaacctcctg gtattcgatt attcggaggc 60
attacggtgc tacggacgta ccagacactt gttttggtgc ttacatttgt cgcgtatact 120
tgctttcata tgactaggaa gataccgagt attgttaaga gcgtgcttga tccacagaca 180
aagttggggt cctctccatg gggacgactg cacacaaaaa acactcttaa catcggttgg 240
ttgccgttta acaccattga tggctcagca ttgcttggtg agatagatgt ggcgtttctt 300
gcggtttatt ctgttgggat gttctttgct ggacatcttg gtgaccgcat ggatttaagg 360
atcttcctga caattggcat gtttggaact gctgtgttca ctgccctttt tggtgctgga 420
tattggctaa atatccacaa tttctactat ttcctggtca ttcagatgat tgccggttta 480
ttccaagcaa ttggatggcc ttcagttgtc gcgattgttg gaaactggtt tggaaaaagc 540
aagaggggat tgattatggg catttggaat gcacacactt ctgtcggcaa catatctggt 600
tcactgctgg ctgcatttct gctgaagttt gggtggggct ggtcatttgc catccccagc 660
ttaatcatgg ttgctgtcgg gttgttggtg tttgtcttct taccggttag cccagaggtg 720
atggagatag acattgatga tggggagata agctctgtta aggatactac caaggagccc 780
ctcttggaac caggacaaga agtgaaacac aatgcagtag gtttcttaga ggcttggaag 840
atacctggag ttgcgccctt tgctctgtgc ctcttcttct ccaaattggt tgcttacacc 900
ttcttgtatt ggctaccgtt ctacatcagt catacacgta agcgctcttt ttccaacttc 960
cttcaacttg tgtgcaaaac tgtttttagt gtgcttttct aacagaactt cacttgtttc 1020
agctattggc ggtgagtacc tctcagatgc tttggctggc agcctatcaa caatctttga 1080
tgttggaggc gtgttgggtg gagtccttgc tggtcacatc tcggatcgct taaatgcacg 1140
agcggttaca gctgccagct tcatgtactg cgcgatacct gccctcttcc tataccgcac 1200
atacggcagc atgtcgataa tgtggaacat ttgcctcatg ttcatcaccg ggatgttcgt 1260
caatggtcct tatgccctaa tcacaactgc agtgtcagct gaccttggca ctcacagctc 1320
attgaatgga aattcccggg cattggctac tgtgacagcc atcattgacg ggaccgggtc 1380
tgttggcgcc gccattgggc cattgctgac aggctacatc tcgtcgagta gttggagcgc 1440
tgtgttcacg atgcttatgg cagcagctct ccttgctggg ctcctcttga cacaacttgt 1500
gtgcagtgaa ctaaaaggaa aggcgacctc caatgcgagc aaggatgtcg ccgatgctca 1560
aggtacctac tcagatgagg tgtaa 1585
<210> 2
<211> 1500
<212> DNA
<213> 水稻(Oryza sativa L.)
<400> 2
atggctcatt ctcatgaaat gacgagtagg aaacctcctg gtattcgatt attcggaggc 60
attacggtgc tacggacgta ccagacactt gttttggtgc ttacatttgt cgcgtatact 120
tgctttcata tgactaggaa gataccgagt attgttaaga gcgtgcttga tccacagaca 180
aagttggggt cctctccatg gggacgactg cacacaaaaa acactcttaa catcggttgg 240
ttgccgttta acaccattga tggctcagca ttgcttggtg agatagatgt ggcgtttctt 300
gcggtttatt ctgttgggat gttctttgct ggacatcttg gtgaccgcat ggatttaagg 360
atcttcctga caattggcat gtttggaact gctgtgttca ctgccctttt tggtgctgga 420
tattggctaa atatccacaa tttctactat ttcctggtca ttcagatgat tgccggttta 480
ttccaagcaa ttggatggcc ttcagttgtc gcgattgttg gaaactggtt tggaaaaagc 540
aagaggggat tgattatggg catttggaat gcacacactt ctgtcggcaa catatctggt 600
tcactgctgg ctgcatttct gctgaagttt gggtggggct ggtcatttgc catccccagc 660
ttaatcatgg ttgctgtcgg gttgttggtg tttgtcttct taccggttag cccagaggtg 720
atggagatag acattgatga tggggagata agctctgtta aggatactac caaggagccc 780
ctcttggaac caggacaaga agtgaaacac aatgcagtag gtttcttaga ggcttggaag 840
atacctggag ttgcgccctt tgctctgtgc ctcttcttct ccaaattggt tgcttacacc 900
ttcttgtatt ggctaccgtt ctacatcagt catacaccta ttggcggtga gtacctctca 960
gatgctttgg ctggcagcct atcaacaatc tttgatgttg gaggcgtgtt gggtggagtc 1020
cttgctggtc acatctcgga tcgcttaaat gcacgagcgg ttacagctgc cagcttcatg 1080
tactgcgcga tacctgccct cttcctatac cgcacatacg gcagcatgtc gataatgtgg 1140
aacatttgcc tcatgttcat caccgggatg ttcgtcaatg gtccttatgc cctaatcaca 1200
actgcagtgt cagctgacct tggcactcac agctcattga atggaaattc ccgggcattg 1260
gctactgtga cagccatcat tgacgggacc gggtctgttg gcgccgccat tgggccattg 1320
ctgacaggct acatctcgtc gagtagttgg agcgctgtgt tcacgatgct tatggcagca 1380
gctctccttg ctgggctcct cttgacacaa cttgtgtgca gtgaactaaa aggaaaggcg 1440
acctccaatg cgagcaagga tgtcgccgat gctcaaggta cctactcaga tgaggtgtaa 1500
<210> 3
<211> 499
<212> PRT
<213> 水稻(Oryza sativa L.)
<400> 3
Met Ala His Ser His Glu Met Thr Ser Arg Lys Pro Pro Gly Ile Arg
1 5 10 15
Leu Phe Gly Gly Ile Thr Val Leu Arg Thr Tyr Gln Thr Leu Val Leu
20 25 30
Val Leu Thr Phe Val Ala Tyr Thr Cys Phe His Met Thr Arg Lys Ile
35 40 45
Pro Ser Ile Val Lys Ser Val Leu Asp Pro Gln Thr Lys Leu Gly Ser
50 55 60
Ser Pro Trp Gly Arg Leu His Thr Lys Asn Thr Leu Asn Ile Gly Trp
65 70 75 80
Leu Pro Phe Asn Thr Ile Asp Gly Ser Ala Leu Leu Gly Glu Ile Asp
85 90 95
Val Ala Phe Leu Ala Val Tyr Ser Val Gly Met Phe Phe Ala Gly His
100 105 110
Leu Gly Asp Arg Met Asp Leu Arg Ile Phe Leu Thr Ile Gly Met Phe
115 120 125
Gly Thr Ala Val Phe Thr Ala Leu Phe Gly Ala Gly Tyr Trp Leu Asn
130 135 140
Ile His Asn Phe Tyr Tyr Phe Leu Val Ile Gln Met Ile Ala Gly Leu
145 150 155 160
Phe Gln Ala Ile Gly Trp Pro Ser Val Val Ala Ile Val Gly Asn Trp
165 170 175
Phe Gly Lys Ser Lys Arg Gly Leu Ile Met Gly Ile Trp Asn Ala His
180 185 190
Thr Ser Val Gly Asn Ile Ser Gly Ser Leu Leu Ala Ala Phe Leu Leu
195 200 205
Lys Phe Gly Trp Gly Trp Ser Phe Ala Ile Pro Ser Leu Ile Met Val
210 215 220
Ala Val Gly Leu Leu Val Phe Val Phe Leu Pro Val Ser Pro Glu Val
225 230 235 240
Met Glu Ile Asp Ile Asp Asp Gly Glu Ile Ser Ser Val Lys Asp Thr
245 250 255
Thr Lys Glu Pro Leu Leu Glu Pro Gly Gln Glu Val Lys His Asn Ala
260 265 270
Val Gly Phe Leu Glu Ala Trp Lys Ile Pro Gly Val Ala Pro Phe Ala
275 280 285
Leu Cys Leu Phe Phe Ser Lys Leu Val Ala Tyr Thr Phe Leu Tyr Trp
290 295 300
Leu Pro Phe Tyr Ile Ser His Thr Pro Ile Gly Gly Glu Tyr Leu Ser
305 310 315 320
Asp Ala Leu Ala Gly Ser Leu Ser Thr Ile Phe Asp Val Gly Gly Val
325 330 335
Leu Gly Gly Val Leu Ala Gly His Ile Ser Asp Arg Leu Asn Ala Arg
340 345 350
Ala Val Thr Ala Ala Ser Phe Met Tyr Cys Ala Ile Pro Ala Leu Phe
355 360 365
Leu Tyr Arg Thr Tyr Gly Ser Met Ser Ile Met Trp Asn Ile Cys Leu
370 375 380
Met Phe Ile Thr Gly Met Phe Val Asn Gly Pro Tyr Ala Leu Ile Thr
385 390 395 400
Thr Ala Val Ser Ala Asp Leu Gly Thr His Ser Ser Leu Asn Gly Asn
405 410 415
Ser Arg Ala Leu Ala Thr Val Thr Ala Ile Ile Asp Gly Thr Gly Ser
420 425 430
Val Gly Ala Ala Ile Gly Pro Leu Leu Thr Gly Tyr Ile Ser Ser Ser
435 440 445
Ser Trp Ser Ala Val Phe Thr Met Leu Met Ala Ala Ala Leu Leu Ala
450 455 460
Gly Leu Leu Leu Thr Gln Leu Val Cys Ser Glu Leu Lys Gly Lys Ala
465 470 475 480
Thr Ser Asn Ala Ser Lys Asp Val Ala Asp Ala Gln Gly Thr Tyr Ser
485 490 495
Asp Glu Val
<210> 4
<211> 10107
<212> DNA
<213> 人工序列
<220>
<223>
<400> 4
aattcaattc ggtccccaga ttagcctttt caatttcaga aagaatgcta acccacagat 60
ggttagagag gcttacgcag caggtctcat caagacgatc tacccgagca ataatctcca 120
ggaaatcaaa taccttccca agaaggttaa agatgcagtc aaaagattca ggactaactg 180
catcaagaac acagagaaag atatatttct caagatcaga agtactattc cagtatggac 240
gattcaaggc ttgcttcaca aaccaaggca agtaatagag attggagtct ctaaaaaggt 300
agttcccact gaatcaaagg ccatggagtc aaagattcaa atagaggacc taacagaact 360
cgccgtaaag actggcgaac agttcataca gagtctctta cgactcaatg acaagaagaa 420
aatcttcgtc aacatggtgg agcacgacac acttgtctac tccaaaaata tcaaagatac 480
agtctcagaa gaccaaaggg caattgagac ttttcaacaa agggtaatat ccggaaacct 540
cctcggattc cattgcccag ctatctgtca ctttattgtg aagatagtgg aaaaggaagg 600
tggctcctac aaatgccatc attgcgataa aggaaaggcc atcgttgaag atgcctctgc 660
cgacagtggt cccaaagatg gacccccacc cacgaggagc atcgtggaaa aagaagacgt 720
tccaaccacg tcttcaaagc aagtggattg atgtgatatc tccactgacg taagggatga 780
cgcacaatcc cactatcctt cgcaagaccc ttcctctata taaggaagtt catttcattt 840
ggagagaaca cgggggacga gctgagctcg gtacccgggg atcctctaga gtcgacctgc 900
aggttcgagt attatggcat tgggaaaact gtttttcttg taccatttgt tgtgcttgta 960
atttactgtg ttttttattc ggttttcgct atcgaactgt gaaatggaaa tggatggaga 1020
agagttaatg aatgatatgg tccttttgtt cattctcaaa ttaatattat ttgttttttc 1080
tcttatttgt tgtgtgttga atttgaaatt ataagagata tgcaaacatt ttgttttgag 1140
taaaaatgtg tcaaatcgtg gcctctaatg accgaagtta atatgaggag taaaacacta 1200
agcttggcac tggccgtcgt tttacaacgt cgtgactggg aaaaccctgg cgttacccaa 1260
cttaatcgcc ttgcagcaca tccccctttc gccagctggc gtaatagcga agaggcccgc 1320
accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg aatgctagag cagcttgagc 1380
ttggatcaga ttgtcgtttc ccgccttcag tttaaactat cagtgtttga caggatatat 1440
tggcgggtaa acctaagaga aaagagcgtt tattagaata acggatattt aaaagggcgt 1500
gaaaaggttt atccgttcgt ccatttgtat gtgcatgcca accacagggt tcccctcggg 1560
atcaaagtac tttgatccaa cccctccgct gctatagtgc agtcggcttc tgacgttcag 1620
tgcagccgtc ttctgaaaac gacatgtcgc acaagtccta agttacgcga caggctgccg 1680
ccctgccctt ttcctggcgt tttcttgtcg cgtgttttag tcgcataaag tagaatactt 1740
gcgactagaa ccggagacat tacgccatga acaagagcgc cgccgctggc ctgctgggct 1800
atgcccgcgt cagcaccgac gaccaggact tgaccaacca acgggccgaa ctgcacgcgg 1860
ccggctgcac caagctgttt tccgagaaga tcaccggcac caggcgcgac cgcccggagc 1920
tggccaggat gcttgaccac ctacgccctg gcgacgttgt gacagtgacc aggctagacc 1980
gcctggcccg cagcacccgc gacctactgg acattgccga gcgcatccag gaggccggcg 2040
cgggcctgcg tagcctggca gagccgtggg ccgacaccac cacgccggcc ggccgcatgg 2100
tgttgaccgt gttcgccggc attgccgagt tcgagcgttc cctaatcatc gaccgcaccc 2160
ggagcgggcg cgaggccgcc aaggcccgag gcgtgaagtt tggcccccgc cctaccctca 2220
ccccggcaca gatcgcgcac gcccgcgagc tgatcgacca ggaaggccgc accgtgaaag 2280
aggcggctgc actgcttggc gtgcatcgct cgaccctgta ccgcgcactt gagcgcagcg 2340
aggaagtgac gcccaccgag gccaggcggc gcggtgcctt ccgtgaggac gcattgaccg 2400
aggccgacgc cctggcggcc gccgagaatg aacgccaaga ggaacaagca tgaaaccgca 2460
ccaggacggc caggacgaac cgtttttcat taccgaagag atcgaggcgg agatgatcgc 2520
ggccgggtac gtgttcgagc cgcccgcgca cgtctcaacc gtgcggctgc atgaaatcct 2580
ggccggtttg tctgatgcca agctggcggc ctggccggcc agcttggccg ctgaagaaac 2640
cgagcgccgc cgtctaaaaa ggtgatgtgt atttgagtaa aacagcttgc gtcatgcggt 2700
cgctgcgtat atgatgcgat gagtaaataa acaaatacgc aaggggaacg catgaaggtt 2760
atcgctgtac ttaaccagaa aggcgggtca ggcaagacga ccatcgcaac ccatctagcc 2820
cgcgccctgc aactcgccgg ggccgatgtt ctgttagtcg attccgatcc ccagggcagt 2880
gcccgcgatt gggcggccgt gcgggaagat caaccgctaa ccgttgtcgg catcgaccgc 2940
ccgacgattg accgcgacgt gaaggccatc ggccggcgcg acttcgtagt gatcgacgga 3000
gcgccccagg cggcggactt ggctgtgtcc gcgatcaagg cagccgactt cgtgctgatt 3060
ccggtgcagc caagccctta cgacatatgg gccaccgccg acctggtgga gctggttaag 3120
cagcgcattg aggtcacgga tggaaggcta caagcggcct ttgtcgtgtc gcgggcgatc 3180
aaaggcacgc gcatcggcgg tgaggttgcc gaggcgctgg ccgggtacga gctgcccatt 3240
cttgagtccc gtatcacgca gcgcgtgagc tacccaggca ctgccgccgc cggcacaacc 3300
gttcttgaat cagaacccga gggcgacgct gcccgcgagg tccaggcgct ggccgctgaa 3360
attaaatcaa aactcatttg agttaatgag gtaaagagaa aatgagcaaa agcacaaaca 3420
cgctaagtgc cggccgtccg agcgcacgca gcagcaaggc tgcaacgttg gccagcctgg 3480
cagacacgcc agccatgaag cgggtcaact ttcagttgcc ggcggaggat cacaccaagc 3540
tgaagatgta cgcggtacgc caaggcaaga ccattaccga gctgctatct gaatacatcg 3600
cgcagctacc agagtaaatg agcaaatgaa taaatgagta gatgaatttt agcggctaaa 3660
ggaggcggca tggaaaatca agaacaacca ggcaccgacg ccgtggaatg ccccatgtgt 3720
ggaggaacgg gcggttggcc aggcgtaagc ggctgggttg tctgccggcc ctgcaatggc 3780
actggaaccc ccaagcccga ggaatcggcg tgacggtcgc aaaccatccg gcccggtaca 3840
aatcggcgcg gcgctgggtg atgacctggt ggagaagttg aaggccgcgc aggccgccca 3900
gcggcaacgc atcgaggcag aagcacgccc cggtgaatcg tggcaagcgg ccgctgatcg 3960
aatccgcaaa gaatcccggc aaccgccggc agccggtgcg ccgtcgatta ggaagccgcc 4020
caagggcgac gagcaaccag attttttcgt tccgatgctc tatgacgtgg gcacccgcga 4080
tagtcgcagc atcatggacg tggccgtttt ccgtctgtcg aagcgtgacc gacgagctgg 4140
cgaggtgatc cgctacgagc ttccagacgg gcacgtagag gtttccgcag ggccggccgg 4200
catggccagt gtgtgggatt acgacctggt actgatggcg gtttcccatc taaccgaatc 4260
catgaaccga taccgggaag ggaagggaga caagcccggc cgcgtgttcc gtccacacgt 4320
tgcggacgta ctcaagttct gccggcgagc cgatggcgga aagcagaaag acgacctggt 4380
agaaacctgc attcggttaa acaccacgca cgttgccatg cagcgtacga agaaggccaa 4440
gaacggccgc ctggtgacgg tatccgaggg tgaagccttg attagccgct acaagatcgt 4500
aaagagcgaa accgggcggc cggagtacat cgagatcgag ctagctgatt ggatgtaccg 4560
cgagatcaca gaaggcaaga acccggacgt gctgacggtt caccccgatt actttttgat 4620
cgatcccggc atcggccgtt ttctctaccg cctggcacgc cgcgccgcag gcaaggcaga 4680
agccagatgg ttgttcaaga cgatctacga acgcagtggc agcgccggag agttcaagaa 4740
gttctgtttc accgtgcgca agctgatcgg gtcaaatgac ctgccggagt acgatttgaa 4800
ggaggaggcg gggcaggctg gcccgatcct agtcatgcgc taccgcaacc tgatcgaggg 4860
cgaagcatcc gccggttcct aatgtacgga gcagatgcta gggcaaattg ccctagcagg 4920
ggaaaaaggt cgaaaaggtc tctttcctgt ggatagcacg tacattggga acccaaagcc 4980
gtacattggg aaccggaacc cgtacattgg gaacccaaag ccgtacattg ggaaccggtc 5040
acacatgtaa gtgactgata taaaagagaa aaaaggcgat ttttccgcct aaaactcttt 5100
aaaacttatt aaaactctta aaacccgcct ggcctgtgca taactgtctg gccagcgcac 5160
agccgaagag ctgcaaaaag cgcctaccct tcggtcgctg cgctccctac gccccgccgc 5220
ttcgcgtcgg cctatcgcgg ccgctggccg ctcaaaaatg gctggcctac ggccaggcaa 5280
tctaccaggg cgcggacaag ccgcgccgtc gccactcgac cgccggcgcc cacatcaagg 5340
caccctgcct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg cagctcccgg 5400
agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt cagggcgcgt 5460
cagcgggtgt tggcgggtgt cggggcgcag ccatgaccca gtcacgtagc gatagcggag 5520
tgtatactgg cttaactatg cggcatcaga gcagattgta ctgagagtgc accatatgcg 5580
gtgtgaaata ccgcacagat gcgtaaggag aaaataccgc atcaggcgct cttccgcttc 5640
ctcgctcact gactcgctgc gctcggtcgt tcggctgcgg cgagcggtat cagctcactc 5700
aaaggcggta atacggttat ccacagaatc aggggataac gcaggaaaga acatgtgagc 5760
aaaaggccag caaaaggcca ggaaccgtaa aaaggccgcg ttgctggcgt ttttccatag 5820
gctccgcccc cctgacgagc atcacaaaaa tcgacgctca agtcagaggt ggcgaaaccc 5880
gacaggacta taaagatacc aggcgtttcc ccctggaagc tccctcgtgc gctctcctgt 5940
tccgaccctg ccgcttaccg gatacctgtc cgcctttctc ccttcgggaa gcgtggcgct 6000
ttctcatagc tcacgctgta ggtatctcag ttcggtgtag gtcgttcgct ccaagctggg 6060
ctgtgtgcac gaaccccccg ttcagcccga ccgctgcgcc ttatccggta actatcgtct 6120
tgagtccaac ccggtaagac acgacttatc gccactggca gcagccactg gtaacaggat 6180
tagcagagcg aggtatgtag gcggtgctac agagttcttg aagtggtggc ctaactacgg 6240
ctacactaga aggacagtat ttggtatctg cgctctgctg aagccagtta ccttcggaaa 6300
aagagttggt agctcttgat ccggcaaaca aaccaccgct ggtagcggtg gtttttttgt 6360
ttgcaagcag cagattacgc gcagaaaaaa aggatctcaa gaagatcctt tgatcttttc 6420
tacggggtct gacgctcagt ggaacgaaaa ctcacgttaa gggattttgg tcatgcattc 6480
taggtactaa aacaattcat ccagtaaaat ataatatttt attttctccc aatcaggctt 6540
gatccccagt aagtcaaaaa atagctcgac atactgttct tccccgatat cctccctgat 6600
cgaccggacg cagaaggcaa tgtcatacca cttgtccgcc ctgccgcttc tcccaagatc 6660
aataaagcca cttactttgc catctttcac aaagatgttg ctgtctccca ggtcgccgtg 6720
ggaaaagaca agttcctctt cgggcttttc cgtctttaaa aaatcataca gctcgcgcgg 6780
atctttaaat ggagtgtctt cttcccagtt ttcgcaatcc acatcggcca gatcgttatt 6840
cagtaagtaa tccaattcgg ctaagcggct gtctaagcta ttcgtatagg gacaatccga 6900
tatgtcgatg gagtgaaaga gcctgatgca ctccgcatac agctcgataa tcttttcagg 6960
gctttgttca tcttcatact cttccgagca aaggacgcca tcggcctcac tcatgagcag 7020
attgctccag ccatcatgcc gttcaaagtg caggaccttt ggaacaggca gctttccttc 7080
cagccatagc atcatgtcct tttcccgttc cacatcatag gtggtccctt tataccggct 7140
gtccgtcatt tttaaatata ggttttcatt ttctcccacc agcttatata ccttagcagg 7200
agacattcct tccgtatctt ttacgcagcg gtatttttcg atcagttttt tcaattccgg 7260
tgatattctc attttagcca tttattattt ccttcctctt ttctacagta tttaaagata 7320
ccccaagaag ctaattataa caagacgaac tccaattcac tgttccttgc attctaaaac 7380
cttaaatacc agaaaacagc tttttcaaag ttgttttcaa agttggcgta taacatagta 7440
tcgacggagc cgattttgaa accgcggtga tcacaggcag caacgctctg tcatcgttac 7500
aatcaacatg ctaccctccg cgagatcatc cgtgtttcaa acccggcagc ttagttgccg 7560
ttcttccgaa tagcatcggt aacatgagca aagtctgccg ccttacaacg gctctcccgc 7620
tgacgccgtc ccggactgat gggctgcctg tatcgagtgg tgattttgtg ccgagctgcc 7680
ggtcggggag ctgttggctg gctggtggca ggatatattg tggtgtaaac aaattgacgc 7740
ttagacaact taataacaca ttgcggacgt ttttaatgta ctgaattaac gccgaattaa 7800
ttcgggggat ctggatttta gtactggatt ttggttttag gaattagaaa ttttattgat 7860
agaagtattt tacaaataca aatacatact aagggtttct tatatgctca acacatgagc 7920
gaaaccctat aggaacccta attcccttat ctgggaacta ctcacacatt attatggaga 7980
aactcgagct tgtcgatcga cagatccggt cggcatctac tctatttctt tgccctcgga 8040
cgagtgctgg ggcgtcggtt tccactatcg gcgagtactt ctacacagcc atcggtccag 8100
acggccgcgc ttctgcgggc gatttgtgta cgcccgacag tcccggctcc ggatcggacg 8160
attgcgtcgc atcgaccctg cgcccaagct gcatcatcga aattgccgtc aaccaagctc 8220
tgatagagtt ggtcaagacc aatgcggagc atatacgccc ggagtcgtgg cgatcctgca 8280
agctccggat gcctccgctc gaagtagcgc gtctgctgct ccatacaagc caaccacggc 8340
ctccagaaga agatgttggc gacctcgtat tgggaatccc cgaacatcgc ctcgctccag 8400
tcaatgaccg ctgttatgcg gccattgtcc gtcaggacat tgttggagcc gaaatccgcg 8460
tgcacgaggt gccggacttc ggggcagtcc tcggcccaaa gcatcagctc atcgagagcc 8520
tgcgcgacgg acgcactgac ggtgtcgtcc atcacagttt gccagtgata cacatgggga 8580
tcagcaatcg cgcatatgaa atcacgccat gtagtgtatt gaccgattcc ttgcggtccg 8640
aatgggccga acccgctcgt ctggctaaga tcggccgcag cgatcgcatc catagcctcc 8700
gcgaccggtt gtagaacagc gggcagttcg gtttcaggca ggtcttgcaa cgtgacaccc 8760
tgtgcacggc gggagatgca ataggtcagg ctctcgctaa actccccaat gtcaagcact 8820
tccggaatcg ggagcgcggc cgatgcaaag tgccgataaa cataacgatc tttgtagaaa 8880
ccatcggcgc agctatttac ccgcaggaca tatccacgcc ctcctacatc gaagctgaaa 8940
gcacgagatt cttcgccctc cgagagctgc atcaggtcgg agacgctgtc gaacttttcg 9000
atcagaaact tctcgacaga cgtcgcggtg agttcaggct ttttcatatc tcattgcccc 9060
cccggatctg cgaaagctcg agagagatag atttgtagag agagactggt gatttcagcg 9120
tgtcctctcc aaatgaaatg aacttcctta tatagaggaa ggtcttgcga aggatagtgg 9180
gattgtgcgt catcccttac gtcagtggag atatcacatc aatccacttg ctttgaagac 9240
gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga 9300
ccactgtcgg cagaggcatc ttgaacgata gcctttcctt tatcgcaatg atggcatttg 9360
taggtgccac cttccttttc tactgtcctt ttgatgaagt gacagatagc tgggcaatgg 9420
aatccgagga ggtttcccga tattaccctt tgttgaaaag tctcaatagc cctttggtct 9480
tctgagactg tatctttgat attcttggag tagacgagag tgtcgtgctc caccatgtta 9540
tcacatcaat ccacttgctt tgaagacgtg gttggaacgt cttctttttc cacgatgctc 9600
ctcgtgggtg ggggtccatc tttgggacca ctgtcggcag aggcatcttg aacgatagcc 9660
tttcctttat cgcaatgatg gcatttgtag gtgccacctt ccttttctac tgtccttttg 9720
atgaagtgac agatagctgg gcaatggaat ccgaggaggt ttcccgatat taccctttgt 9780
tgaaaagtct caatagccct ttggtcttct gagactgtat ctttgatatt cttggagtag 9840
acgagagtgt cgtgctccac catgttggca agctgctcta gccaatacgc aaaccgcctc 9900
tccccgcgcg ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag 9960
cgggcagtga gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt 10020
tacactttat gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca 10080
caggaaacag ctatgaccat gattacg 10107
Claims (13)
1.蛋白质OsVPE2的应用,为X1)或X2):
X1)调控水稻液泡无机磷输出能力;
X2)调控水稻液泡里的无机磷浓度;
所述蛋白质OsVPE2的氨基酸序列是序列表中序列3所示的蛋白质;
所述调控为过表达蛋白质OsVPE2时,水稻液泡无机磷输出能力提高和/或水稻液泡里的无机磷浓度降低;
所述调控为抑制蛋白质OsVPE2时,水稻液泡无机磷输出能力降低和/或水稻液泡里的无机磷浓度增加。
2.如权利要求1所述的应用,其特征在于:所述水稻为水稻品种石狩白毛。
3.编码权利要求1中所述蛋白质OsVPE2的核酸分子的应用,为X1)或X2):
X1)调控水稻液泡无机磷输出能力;
X2)调控水稻液泡里的无机磷浓度;
所述调控为表达所述核酸分子时,水稻液泡无机磷输出能力提高和/或水稻液泡里的无机磷浓度降低;
所述调控为抑制所述核酸分子的表达时,水稻液泡无机磷输出能力降低和/或水稻液泡里的无机磷浓度增加。
4.如权利要求3所述的应用,其特征在于:所述核酸分子为序列表中序列2所示的DNA分子。
5.如权利要求3或4所述的应用,其特征在于:所述水稻为水稻品种石狩白毛。
6.一种培育转基因水稻甲的方法,包括如下步骤:提高出发水稻中权利要求1中所述蛋白质OsVPE2的表达量和/或活性,得到转基因水稻甲;与出发水稻相比,转基因水稻甲的液泡无机磷输出能力提高和/或液泡里的无机磷浓度降低。
7.如权利要求6所述的方法,其特征在于:所述水稻为水稻品种石狩白毛。
8.一种培育转基因水稻乙的方法,包括如下步骤:抑制出发水稻中权利要求1中所述蛋白质OsVPE2的表达量和/或活性,得到转基因水稻乙;与出发水稻相比,转基因水稻乙的液泡无机磷输出能力降低和/或液泡里的无机磷浓度增加。
9.如权利要求8所述的方法,其特征在于:所述水稻为水稻品种石狩白毛。
10.一种水稻育种方法,包括如下步骤:增加水稻中权利要求1中所述蛋白质OsVPE2的含量和/或活性,从而提高液泡无机磷输出能力和/或降低液泡里的无机磷浓度。
11.如权利要求10所述的方法,其特征在于:所述水稻为水稻品种石狩白毛。
12.一种水稻育种方法,包括如下步骤:降低水稻中权利要求1中所述蛋白质OsVPE2的含量和/或活性,从而降低液泡无机磷输出能力和/或增加液泡里的无机磷浓度。
13.如权利要求12所述的方法,其特征在于:所述水稻为水稻品种石狩白毛。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811318436.XA CN109232726B (zh) | 2018-11-07 | 2018-11-07 | 蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201811318436.XA CN109232726B (zh) | 2018-11-07 | 2018-11-07 | 蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN109232726A CN109232726A (zh) | 2019-01-18 |
CN109232726B true CN109232726B (zh) | 2020-11-24 |
Family
ID=65077108
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201811318436.XA Expired - Fee Related CN109232726B (zh) | 2018-11-07 | 2018-11-07 | 蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN109232726B (zh) |
Families Citing this family (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN109970868B (zh) * | 2019-03-29 | 2020-11-20 | 中国农业科学院农业资源与农业区划研究所 | 一种通过ptc基因操作提高藻类总磷和多聚磷酸含量的方法及应用 |
Family Cites Families (6)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2003000898A1 (en) * | 2001-06-22 | 2003-01-03 | Syngenta Participations Ag | Plant genes involved in defense against pathogens |
EP1402042A2 (en) * | 2001-06-22 | 2004-03-31 | Syngenta Participations AG | Abiotic stress responsive polynucleotides and polypeptides |
US8569575B2 (en) * | 2006-12-21 | 2013-10-29 | Basf Plant Science Gmbh | Plants having enhanced yield-related traits and a method for making the same |
CN101381730A (zh) * | 2008-10-24 | 2009-03-11 | 华中农业大学 | 基因OsPT2在控制磷酸盐吸收转运中的用途 |
CN102115754A (zh) * | 2010-12-16 | 2011-07-06 | 南京农业大学 | 一种水稻磷酸盐转运蛋白基因ORYsa;Pht1;4的应用 |
JP6993660B2 (ja) * | 2016-08-31 | 2022-02-15 | 国立大学法人広島大学 | 形質転換体、形質転換体の製造方法、および、当該形質転換体を用いた還元型リン化合物の有無の検出方法 |
-
2018
- 2018-11-07 CN CN201811318436.XA patent/CN109232726B/zh not_active Expired - Fee Related
Non-Patent Citations (1)
Title |
---|
High-Yield Expression and Functional Analysis of Escherichia coli Glycerol-3-phosphate Transporter;Manfred Auer等;《Biochemistry》;20010605;第40卷(第22期);第6628-66356页 * |
Also Published As
Publication number | Publication date |
---|---|
CN109232726A (zh) | 2019-01-18 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN110551752B (zh) | xCas9n-epBE碱基编辑***及其在基因组碱基替换中的应用 | |
CN108203714B (zh) | 一种棉花基因的编辑方法 | |
KR100785946B1 (ko) | 식물 형질전환 벡터 | |
CN107119063B (zh) | 一种提高蛹虫草中虫草素含量的方法 | |
CN110656114B (zh) | 一种烟草色素合成相关的基因及其应用 | |
CN109912720B (zh) | 一种蛛丝蛋白的设计合成方法和纺丝 | |
CN110283840A (zh) | 陆地棉基因组的精确高效编辑方法 | |
CN110760538B (zh) | 一种创制枯萎病抗性西瓜种质材料的方法 | |
CN106834338B (zh) | 拟南芥基因rem16的表达载体及其在调控植株开花期中的应用 | |
CN112126658A (zh) | 植物过表达荧光素酶报告基因重组载体及构建方法、应用 | |
CN109321576A (zh) | 一种无腺体低棉酚棉花种质的创制方法 | |
CN109232726B (zh) | 蛋白质OsVPE2在调控植物液泡无机磷输出能力中的应用 | |
CN109485707B (zh) | 蛋白质OsVPE1在调控植物液泡无机磷输出能力中的应用 | |
CN115058439A (zh) | 云锦杜鹃samt基因过表达载体及其构建方法和应用 | |
CN107142274A (zh) | 一种在蛹虫草中过表达天蚕素抗菌肽的方法 | |
KR101608078B1 (ko) | 이산화탄소 유래 숙신산 생산을 위한 균주 및 이를 이용한 이산화탄소 유래 숙신산 생산 방법 | |
TW201210471A (en) | Method for enhancing thermotolerance of plant and applicatibility of transgenic plant | |
CN113122516B (zh) | 一种植物epsps突变体及其在植物中的应用 | |
CN109337925B (zh) | 一种以黄花蒿悬浮细胞系为受体的转AaADS基因提高黄花蒿中青蒿素含量的方法 | |
CN107058368A (zh) | 一种在蛹虫草中过表达菌丝霉素抗菌肽的方法 | |
CN113281521B (zh) | 用于植物应激颗粒相关蛋白快速鉴定的Gateway双元质粒载体、其构建方法及应用 | |
CN111394385A (zh) | 一种快速鉴定水稻双向启动子的方法 | |
CN108841862A (zh) | 一种含有ha蛋白融合标签的植物表达质粒载体及其载体的构建方法 | |
CN107815435A (zh) | 具有增强的纤维素生产能力的葡糖醋杆菌 | |
CN114591996B (zh) | 一种凝结芽孢杆菌h-1的表达载体及其构建方法与应用 |
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 | ||
CF01 | Termination of patent right due to non-payment of annual fee | ||
CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20201124 |