CN114231556B - GmECT2在调控植物高度方面的应用 - Google Patents
GmECT2在调控植物高度方面的应用 Download PDFInfo
- Publication number
- CN114231556B CN114231556B CN202111336838.4A CN202111336838A CN114231556B CN 114231556 B CN114231556 B CN 114231556B CN 202111336838 A CN202111336838 A CN 202111336838A CN 114231556 B CN114231556 B CN 114231556B
- Authority
- CN
- China
- Prior art keywords
- sequence
- protein
- gmect2
- gly
- pro
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 230000001105 regulatory effect Effects 0.000 title abstract description 26
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 201
- 241000196324 Embryophyta Species 0.000 claims abstract description 108
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 89
- 244000068988 Glycine max Species 0.000 claims abstract description 30
- 235000010469 Glycine max Nutrition 0.000 claims abstract description 28
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 2
- 108020004414 DNA Proteins 0.000 claims description 56
- 239000002773 nucleotide Substances 0.000 claims description 55
- 125000003729 nucleotide group Chemical group 0.000 claims description 55
- 230000014509 gene expression Effects 0.000 claims description 44
- 239000013598 vector Substances 0.000 claims description 32
- 102000053602 DNA Human genes 0.000 claims description 29
- 150000007523 nucleic acids Chemical class 0.000 claims description 26
- 102000039446 nucleic acids Human genes 0.000 claims description 25
- 108020004707 nucleic acids Proteins 0.000 claims description 25
- 108020005004 Guide RNA Proteins 0.000 claims description 23
- 238000000034 method Methods 0.000 claims description 21
- 230000009261 transgenic effect Effects 0.000 claims description 18
- 239000000126 substance Substances 0.000 claims description 15
- 244000005700 microbiome Species 0.000 claims description 12
- 230000000694 effects Effects 0.000 claims description 10
- 239000012620 biological material Substances 0.000 claims description 9
- 239000003795 chemical substances by application Substances 0.000 claims description 8
- 230000035772 mutation Effects 0.000 claims description 8
- 230000008685 targeting Effects 0.000 claims description 7
- 230000002401 inhibitory effect Effects 0.000 claims description 6
- 238000002156 mixing Methods 0.000 claims description 4
- 108020001507 fusion proteins Proteins 0.000 claims description 2
- 102000037865 fusion proteins Human genes 0.000 claims description 2
- 230000001276 controlling effect Effects 0.000 abstract description 6
- 238000002474 experimental method Methods 0.000 abstract description 4
- 230000006872 improvement Effects 0.000 abstract description 3
- 238000003976 plant breeding Methods 0.000 abstract description 3
- 244000046052 Phaseolus vulgaris Species 0.000 description 28
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 28
- 239000007788 liquid Substances 0.000 description 21
- 241000589158 Agrobacterium Species 0.000 description 18
- 239000012634 fragment Substances 0.000 description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 16
- 230000001580 bacterial effect Effects 0.000 description 13
- XBGGUPMXALFZOT-UHFFFAOYSA-N glycyl-L-tyrosine hemihydrate Natural products NCC(=O)NC(C(O)=O)CC1=CC=C(O)C=C1 XBGGUPMXALFZOT-UHFFFAOYSA-N 0.000 description 13
- 210000004027 cell Anatomy 0.000 description 12
- 239000001963 growth medium Substances 0.000 description 12
- 150000001413 amino acids Chemical group 0.000 description 11
- 108091026890 Coding region Proteins 0.000 description 10
- 108010065920 Insulin Lispro Proteins 0.000 description 10
- 239000007787 solid Substances 0.000 description 10
- 238000010356 CRISPR-Cas9 genome editing Methods 0.000 description 9
- 108010093581 aspartyl-proline Proteins 0.000 description 9
- 230000033228 biological regulation Effects 0.000 description 9
- 238000012217 deletion Methods 0.000 description 9
- 230000037430 deletion Effects 0.000 description 9
- 108010087823 glycyltyrosine Proteins 0.000 description 9
- 230000006698 induction Effects 0.000 description 9
- 239000002609 medium Substances 0.000 description 9
- 239000013612 plasmid Substances 0.000 description 9
- 108010053725 prolylvaline Proteins 0.000 description 9
- 108010026333 seryl-proline Proteins 0.000 description 9
- 108091033409 CRISPR Proteins 0.000 description 8
- ICTZKEXYDDZZFP-SRVKXCTJSA-N Pro-Arg-Pro Chemical compound N([C@@H](CCCN=C(N)N)C(=O)N1[C@@H](CCC1)C(O)=O)C(=O)[C@@H]1CCCN1 ICTZKEXYDDZZFP-SRVKXCTJSA-N 0.000 description 8
- VMRFIKXKOFNMHW-GUBZILKMSA-N Val-Arg-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CO)C(=O)O)N VMRFIKXKOFNMHW-GUBZILKMSA-N 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 8
- 108010010096 glycyl-glycyl-tyrosine Proteins 0.000 description 8
- 108010051242 phenylalanylserine Proteins 0.000 description 8
- 241000894006 Bacteria Species 0.000 description 7
- 108010091092 arginyl-glycyl-proline Proteins 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 238000012163 sequencing technique Methods 0.000 description 7
- FTCGGKNCJZOPNB-WHFBIAKZSA-N Asn-Gly-Ser Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O FTCGGKNCJZOPNB-WHFBIAKZSA-N 0.000 description 6
- MGJLBZFUXUGMML-VOAKCMCISA-N Thr-Lys-Lys Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)O)N)O MGJLBZFUXUGMML-VOAKCMCISA-N 0.000 description 6
- 238000012258 culturing Methods 0.000 description 6
- 238000001514 detection method Methods 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- 108010037850 glycylvaline Proteins 0.000 description 6
- 210000001161 mammalian embryo Anatomy 0.000 description 6
- 238000012545 processing Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 230000009466 transformation Effects 0.000 description 6
- AXFMEGAFCUULFV-BLFANLJRSA-N (2s)-2-[[(2s)-1-[(2s,3r)-2-amino-3-methylpentanoyl]pyrrolidine-2-carbonyl]amino]pentanedioic acid Chemical compound CC[C@@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O AXFMEGAFCUULFV-BLFANLJRSA-N 0.000 description 5
- AAQGRPOPTAUUBM-ZLUOBGJFSA-N Ala-Ala-Asn Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(O)=O AAQGRPOPTAUUBM-ZLUOBGJFSA-N 0.000 description 5
- WNHNMKOFKCHKKD-BFHQHQDPSA-N Ala-Thr-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O WNHNMKOFKCHKKD-BFHQHQDPSA-N 0.000 description 5
- MUGAESARFRGOTQ-IGNZVWTISA-N Ala-Tyr-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)O)N MUGAESARFRGOTQ-IGNZVWTISA-N 0.000 description 5
- UVTGNSWSRSCPLP-UHFFFAOYSA-N Arg-Tyr Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccc(O)cc1)C(=O)O UVTGNSWSRSCPLP-UHFFFAOYSA-N 0.000 description 5
- XSGBIBGAMKTHMY-WHFBIAKZSA-N Asn-Asp-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O XSGBIBGAMKTHMY-WHFBIAKZSA-N 0.000 description 5
- KWQPAXYXVMHJJR-AVGNSLFASA-N Asn-Gln-Tyr Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 KWQPAXYXVMHJJR-AVGNSLFASA-N 0.000 description 5
- FAEIQWHBRBWUBN-FXQIFTODSA-N Asp-Arg-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CC(=O)O)N)CN=C(N)N FAEIQWHBRBWUBN-FXQIFTODSA-N 0.000 description 5
- KGHLGJAXYSVNJP-WHFBIAKZSA-N Asp-Ser-Gly Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O KGHLGJAXYSVNJP-WHFBIAKZSA-N 0.000 description 5
- 238000010354 CRISPR gene editing Methods 0.000 description 5
- UGPCUUWZXRMCIJ-KKUMJFAQSA-N Cys-Tyr-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CS)N UGPCUUWZXRMCIJ-KKUMJFAQSA-N 0.000 description 5
- HSHCEAUPUPJPTE-JYJNAYRXSA-N Gln-Leu-Tyr Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)NC(=O)[C@H](CCC(=O)N)N HSHCEAUPUPJPTE-JYJNAYRXSA-N 0.000 description 5
- MFORDNZDKAVNSR-SRVKXCTJSA-N Gln-Pro-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CCC(N)=O MFORDNZDKAVNSR-SRVKXCTJSA-N 0.000 description 5
- GHAXJVNBAKGWEJ-AVGNSLFASA-N Gln-Ser-Tyr Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O GHAXJVNBAKGWEJ-AVGNSLFASA-N 0.000 description 5
- BPQYBFAXRGMGGY-LAEOZQHASA-N Gly-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)CN BPQYBFAXRGMGGY-LAEOZQHASA-N 0.000 description 5
- HJARVELKOSZUEW-YUMQZZPRSA-N Gly-Pro-Gln Chemical compound [H]NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O HJARVELKOSZUEW-YUMQZZPRSA-N 0.000 description 5
- ZLCLYFGMKFCDCN-XPUUQOCRSA-N Gly-Ser-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CO)NC(=O)CN)C(O)=O ZLCLYFGMKFCDCN-XPUUQOCRSA-N 0.000 description 5
- UIQGJYUEQDOODF-KWQFWETISA-N Gly-Tyr-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=C(O)C=C1 UIQGJYUEQDOODF-KWQFWETISA-N 0.000 description 5
- FZKFYOXDVWDELO-KBPBESRZSA-N His-Gly-Tyr Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O FZKFYOXDVWDELO-KBPBESRZSA-N 0.000 description 5
- WSWAUVHXQREQQG-JYJNAYRXSA-N His-Tyr-Gln Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O WSWAUVHXQREQQG-JYJNAYRXSA-N 0.000 description 5
- 241000880493 Leptailurus serval Species 0.000 description 5
- ZYLJULGXQDNXDK-GUBZILKMSA-N Leu-Gln-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O ZYLJULGXQDNXDK-GUBZILKMSA-N 0.000 description 5
- ZFNLIDNJUWNIJL-WDCWCFNPSA-N Leu-Glu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ZFNLIDNJUWNIJL-WDCWCFNPSA-N 0.000 description 5
- KLSUAWUZBMAZCL-RHYQMDGZSA-N Leu-Thr-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(O)=O KLSUAWUZBMAZCL-RHYQMDGZSA-N 0.000 description 5
- OZTZJMUZVAVJGY-BZSNNMDCSA-N Leu-Tyr-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)N OZTZJMUZVAVJGY-BZSNNMDCSA-N 0.000 description 5
- WRODMZBHNNPRLN-SRVKXCTJSA-N Lys-Leu-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O WRODMZBHNNPRLN-SRVKXCTJSA-N 0.000 description 5
- SITLTJHOQZFJGG-UHFFFAOYSA-N N-L-alpha-glutamyl-L-valine Natural products CC(C)C(C(O)=O)NC(=O)C(N)CCC(O)=O SITLTJHOQZFJGG-UHFFFAOYSA-N 0.000 description 5
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 5
- IDUCUXTUHHIQIP-SOUVJXGZSA-N Phe-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O IDUCUXTUHHIQIP-SOUVJXGZSA-N 0.000 description 5
- GYEPCBNTTRORKW-PCBIJLKTSA-N Phe-Ile-Asp Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O GYEPCBNTTRORKW-PCBIJLKTSA-N 0.000 description 5
- KIZQGKLMXKGDIV-BQBZGAKWSA-N Pro-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 KIZQGKLMXKGDIV-BQBZGAKWSA-N 0.000 description 5
- XROLYVMNVIKVEM-BQBZGAKWSA-N Pro-Asn-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O XROLYVMNVIKVEM-BQBZGAKWSA-N 0.000 description 5
- HFNPOYOKIPGAEI-SRVKXCTJSA-N Pro-Leu-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 HFNPOYOKIPGAEI-SRVKXCTJSA-N 0.000 description 5
- AJNGQVUFQUVRQT-JYJNAYRXSA-N Pro-Pro-Tyr Chemical compound C([C@@H](C(=O)O)NC(=O)[C@H]1N(CCC1)C(=O)[C@H]1NCCC1)C1=CC=C(O)C=C1 AJNGQVUFQUVRQT-JYJNAYRXSA-N 0.000 description 5
- KWMZPPWYBVZIER-XGEHTFHBSA-N Pro-Ser-Thr Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KWMZPPWYBVZIER-XGEHTFHBSA-N 0.000 description 5
- GXWRTSIVLSQACD-RCWTZXSCSA-N Pro-Thr-Met Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@@H]1CCCN1)O GXWRTSIVLSQACD-RCWTZXSCSA-N 0.000 description 5
- LZHHZYDPMZEMRX-STQMWFEESA-N Pro-Tyr-Gly Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(O)=O LZHHZYDPMZEMRX-STQMWFEESA-N 0.000 description 5
- VEUACYMXJKXALX-IHRRRGAJSA-N Pro-Tyr-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(O)=O VEUACYMXJKXALX-IHRRRGAJSA-N 0.000 description 5
- YRBGKVIWMNEVCZ-WDSKDSINSA-N Ser-Glu-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O YRBGKVIWMNEVCZ-WDSKDSINSA-N 0.000 description 5
- XXXAXOWMBOKTRN-XPUUQOCRSA-N Ser-Gly-Val Chemical compound [H]N[C@@H](CO)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O XXXAXOWMBOKTRN-XPUUQOCRSA-N 0.000 description 5
- CKDXFSPMIDSMGV-GUBZILKMSA-N Ser-Pro-Val Chemical compound [H]N[C@@H](CO)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O CKDXFSPMIDSMGV-GUBZILKMSA-N 0.000 description 5
- XFTYVCHLARBHBQ-FOHZUACHSA-N Thr-Gly-Asn Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O XFTYVCHLARBHBQ-FOHZUACHSA-N 0.000 description 5
- QHUWWSQZTFLXPQ-FJXKBIBVSA-N Thr-Met-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCSC)C(=O)NCC(O)=O QHUWWSQZTFLXPQ-FJXKBIBVSA-N 0.000 description 5
- SGAOHNPSEPVAFP-ZDLURKLDSA-N Thr-Ser-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)NCC(O)=O SGAOHNPSEPVAFP-ZDLURKLDSA-N 0.000 description 5
- FKAPNDWDLDWZNF-QEJZJMRPSA-N Trp-Asp-Glu Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N FKAPNDWDLDWZNF-QEJZJMRPSA-N 0.000 description 5
- JWHOIHCOHMZSAR-QWRGUYRKSA-N Tyr-Asp-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 JWHOIHCOHMZSAR-QWRGUYRKSA-N 0.000 description 5
- AKLNEFNQWLHIGY-QWRGUYRKSA-N Tyr-Gly-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)O)N)O AKLNEFNQWLHIGY-QWRGUYRKSA-N 0.000 description 5
- HIINQLBHPIQYHN-JTQLQIEISA-N Tyr-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 HIINQLBHPIQYHN-JTQLQIEISA-N 0.000 description 5
- RWOKVQUCENPXGE-IHRRRGAJSA-N Tyr-Ser-Arg Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O RWOKVQUCENPXGE-IHRRRGAJSA-N 0.000 description 5
- AEOFMCAKYIQQFY-YDHLFZDLSA-N Tyr-Val-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 AEOFMCAKYIQQFY-YDHLFZDLSA-N 0.000 description 5
- UEOOXDLMQZBPFR-ZKWXMUAHSA-N Val-Ala-Asn Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](C(C)C)N UEOOXDLMQZBPFR-ZKWXMUAHSA-N 0.000 description 5
- MHAHQDBEIDPFQS-NHCYSSNCSA-N Val-Glu-Met Chemical compound CSCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)C(C)C MHAHQDBEIDPFQS-NHCYSSNCSA-N 0.000 description 5
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 5
- DVLWZWNAQUBZBC-ZNSHCXBVSA-N Val-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N)O DVLWZWNAQUBZBC-ZNSHCXBVSA-N 0.000 description 5
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 5
- 108010087924 alanylproline Proteins 0.000 description 5
- 239000003242 anti bacterial agent Substances 0.000 description 5
- 229940088710 antibiotic agent Drugs 0.000 description 5
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 5
- 238000005520 cutting process Methods 0.000 description 5
- 230000030279 gene silencing Effects 0.000 description 5
- 238000012226 gene silencing method Methods 0.000 description 5
- 108010048994 glycyl-tyrosyl-alanine Proteins 0.000 description 5
- 108010077515 glycylproline Proteins 0.000 description 5
- 108010028295 histidylhistidine Proteins 0.000 description 5
- 238000005286 illumination Methods 0.000 description 5
- 108010057821 leucylproline Proteins 0.000 description 5
- 108010090894 prolylleucine Proteins 0.000 description 5
- 239000008223 sterile water Substances 0.000 description 5
- 108010003137 tyrosyltyrosine Proteins 0.000 description 5
- YYSWCHMLFJLLBJ-ZLUOBGJFSA-N Ala-Ala-Ser Chemical compound C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O YYSWCHMLFJLLBJ-ZLUOBGJFSA-N 0.000 description 4
- CVGNCMIULZNYES-WHFBIAKZSA-N Ala-Asn-Gly Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O CVGNCMIULZNYES-WHFBIAKZSA-N 0.000 description 4
- SUHLZMHFRALVSY-YUMQZZPRSA-N Ala-Lys-Gly Chemical compound NCCCC[C@H](NC(=O)[C@@H](N)C)C(=O)NCC(O)=O SUHLZMHFRALVSY-YUMQZZPRSA-N 0.000 description 4
- KLALXKYLOMZDQT-ZLUOBGJFSA-N Ala-Ser-Asn Chemical compound C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC(N)=O KLALXKYLOMZDQT-ZLUOBGJFSA-N 0.000 description 4
- GHNDBBVSWOWYII-LPEHRKFASA-N Arg-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCCN=C(N)N)N)C(=O)O GHNDBBVSWOWYII-LPEHRKFASA-N 0.000 description 4
- ZATRYQNPUHGXCU-DTWKUNHWSA-N Arg-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCN=C(N)N)N)C(=O)O ZATRYQNPUHGXCU-DTWKUNHWSA-N 0.000 description 4
- GSUFZRURORXYTM-STQMWFEESA-N Arg-Phe-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CC=CC=C1 GSUFZRURORXYTM-STQMWFEESA-N 0.000 description 4
- QLSRIZIDQXDQHK-RCWTZXSCSA-N Arg-Val-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O QLSRIZIDQXDQHK-RCWTZXSCSA-N 0.000 description 4
- ZZXMOQIUIJJOKZ-ZLUOBGJFSA-N Asn-Asn-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(N)=O ZZXMOQIUIJJOKZ-ZLUOBGJFSA-N 0.000 description 4
- AYKKKGFJXIDYLX-ACZMJKKPSA-N Asn-Gln-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O AYKKKGFJXIDYLX-ACZMJKKPSA-N 0.000 description 4
- REQUGIWGOGSOEZ-ZLUOBGJFSA-N Asn-Ser-Asn Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)C(=O)N REQUGIWGOGSOEZ-ZLUOBGJFSA-N 0.000 description 4
- JWQWPRCDYWNVNM-ACZMJKKPSA-N Asn-Ser-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC(=O)N)N JWQWPRCDYWNVNM-ACZMJKKPSA-N 0.000 description 4
- SNAWMGHSCHKSDK-GUBZILKMSA-N Asp-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC(=O)O)N SNAWMGHSCHKSDK-GUBZILKMSA-N 0.000 description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- MFJAPSYJQJCQDN-BQBZGAKWSA-N Gln-Gly-Glu Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@@H](CCC(O)=O)C(O)=O MFJAPSYJQJCQDN-BQBZGAKWSA-N 0.000 description 4
- QSDKBRMVXSWAQE-BFHQHQDPSA-N Gly-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)CN QSDKBRMVXSWAQE-BFHQHQDPSA-N 0.000 description 4
- CIMULJZTTOBOPN-WHFBIAKZSA-N Gly-Asn-Asn Chemical compound NCC(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O CIMULJZTTOBOPN-WHFBIAKZSA-N 0.000 description 4
- FMNHBTKMRFVGRO-FOHZUACHSA-N Gly-Asn-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)CN FMNHBTKMRFVGRO-FOHZUACHSA-N 0.000 description 4
- JLJLBWDKDRYOPA-RYUDHWBXSA-N Gly-Gln-Tyr Chemical compound NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 JLJLBWDKDRYOPA-RYUDHWBXSA-N 0.000 description 4
- KMSGYZQRXPUKGI-BYPYZUCNSA-N Gly-Gly-Asn Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(N)=O KMSGYZQRXPUKGI-BYPYZUCNSA-N 0.000 description 4
- HHRODZSXDXMUHS-LURJTMIESA-N Gly-Met-Gly Chemical compound CSCC[C@H](NC(=O)C[NH3+])C(=O)NCC([O-])=O HHRODZSXDXMUHS-LURJTMIESA-N 0.000 description 4
- RHRLHXQWHCNJKR-PMVVWTBXSA-N Gly-Thr-His Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CC1=CN=CN1 RHRLHXQWHCNJKR-PMVVWTBXSA-N 0.000 description 4
- GNNJKUYDWFIBTK-QWRGUYRKSA-N Gly-Tyr-Asp Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O GNNJKUYDWFIBTK-QWRGUYRKSA-N 0.000 description 4
- JBCLFWXMTIKCCB-UHFFFAOYSA-N H-Gly-Phe-OH Natural products NCC(=O)NC(C(O)=O)CC1=CC=CC=C1 JBCLFWXMTIKCCB-UHFFFAOYSA-N 0.000 description 4
- IAYPZSHNZQHQNO-KKUMJFAQSA-N His-Ser-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CC2=CN=CN2)N IAYPZSHNZQHQNO-KKUMJFAQSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- YKRIXHPEIZUDDY-GMOBBJLQSA-N Ile-Asn-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YKRIXHPEIZUDDY-GMOBBJLQSA-N 0.000 description 4
- ZLFNNVATRMCAKN-ZKWXMUAHSA-N Ile-Ser-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)NCC(=O)O)N ZLFNNVATRMCAKN-ZKWXMUAHSA-N 0.000 description 4
- AOFYPTOHESIBFZ-KKUMJFAQSA-N Leu-His-His Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](Cc1cnc[nH]1)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O AOFYPTOHESIBFZ-KKUMJFAQSA-N 0.000 description 4
- DFXQCCBKGUNYGG-GUBZILKMSA-N Lys-Gln-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CCCCN DFXQCCBKGUNYGG-GUBZILKMSA-N 0.000 description 4
- VUTWYNQUSJWBHO-BZSNNMDCSA-N Lys-Leu-Tyr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O VUTWYNQUSJWBHO-BZSNNMDCSA-N 0.000 description 4
- VSJAPSMRFYUOKS-IUCAKERBSA-N Met-Pro-Gly Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O VSJAPSMRFYUOKS-IUCAKERBSA-N 0.000 description 4
- VYXIKLFLGRTANT-HRCADAONSA-N Met-Tyr-Pro Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N2CCC[C@@H]2C(=O)O)N VYXIKLFLGRTANT-HRCADAONSA-N 0.000 description 4
- BRDYYVQTEJVRQT-HRCADAONSA-N Phe-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O BRDYYVQTEJVRQT-HRCADAONSA-N 0.000 description 4
- CSYVXYQDIVCQNU-QWRGUYRKSA-N Phe-Asp-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O CSYVXYQDIVCQNU-QWRGUYRKSA-N 0.000 description 4
- UXQFHEKRGHYJRA-STQMWFEESA-N Phe-Met-Gly Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCSC)C(=O)NCC(O)=O UXQFHEKRGHYJRA-STQMWFEESA-N 0.000 description 4
- DRVIASBABBMZTF-GUBZILKMSA-N Pro-Ala-Met Chemical compound C[C@@H](C(=O)N[C@@H](CCSC)C(=O)O)NC(=O)[C@@H]1CCCN1 DRVIASBABBMZTF-GUBZILKMSA-N 0.000 description 4
- FKVNLUZHSFCNGY-RVMXOQNASA-N Pro-Ile-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 FKVNLUZHSFCNGY-RVMXOQNASA-N 0.000 description 4
- DRKAXLDECUGLFE-ULQDDVLXSA-N Pro-Leu-Phe Chemical compound CC(C)C[C@H](NC(=O)[C@@H]1CCCN1)C(=O)N[C@@H](Cc1ccccc1)C(O)=O DRKAXLDECUGLFE-ULQDDVLXSA-N 0.000 description 4
- MCWHYUWXVNRXFV-RWMBFGLXSA-N Pro-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 MCWHYUWXVNRXFV-RWMBFGLXSA-N 0.000 description 4
- SPLBRAKYXGOFSO-UNQGMJICSA-N Pro-Phe-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@@H]2CCCN2)O SPLBRAKYXGOFSO-UNQGMJICSA-N 0.000 description 4
- OYEDZGNMSBZCIM-XGEHTFHBSA-N Ser-Arg-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OYEDZGNMSBZCIM-XGEHTFHBSA-N 0.000 description 4
- BNFVPSRLHHPQKS-WHFBIAKZSA-N Ser-Asp-Gly Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)NCC(O)=O BNFVPSRLHHPQKS-WHFBIAKZSA-N 0.000 description 4
- OZPDGESCTGGNAD-CIUDSAMLSA-N Ser-Ser-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](N)CO OZPDGESCTGGNAD-CIUDSAMLSA-N 0.000 description 4
- ILZAUMFXKSIUEF-SRVKXCTJSA-N Ser-Ser-Phe Chemical compound OC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 ILZAUMFXKSIUEF-SRVKXCTJSA-N 0.000 description 4
- PMTWIUBUQRGCSB-FXQIFTODSA-N Ser-Val-Ala Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O PMTWIUBUQRGCSB-FXQIFTODSA-N 0.000 description 4
- 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 4
- WTMPKZWHRCMMMT-KZVJFYERSA-N Thr-Pro-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WTMPKZWHRCMMMT-KZVJFYERSA-N 0.000 description 4
- FWTFAZKJORVTIR-VZFHVOOUSA-N Thr-Ser-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O FWTFAZKJORVTIR-VZFHVOOUSA-N 0.000 description 4
- 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 4
- NHQVWACSJZJCGJ-FLBSBUHZSA-N Thr-Thr-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O NHQVWACSJZJCGJ-FLBSBUHZSA-N 0.000 description 4
- OGZRZMJASKKMJZ-XIRDDKMYSA-N Trp-Leu-Asp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N OGZRZMJASKKMJZ-XIRDDKMYSA-N 0.000 description 4
- NGALWFGCOMHUSN-AVGNSLFASA-N Tyr-Gln-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O NGALWFGCOMHUSN-AVGNSLFASA-N 0.000 description 4
- ISERLACIZUGCDX-ZKWXMUAHSA-N Val-Asp-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N ISERLACIZUGCDX-ZKWXMUAHSA-N 0.000 description 4
- ZHQWPWQNVRCXAX-XQQFMLRXSA-N Val-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](C(C)C)N ZHQWPWQNVRCXAX-XQQFMLRXSA-N 0.000 description 4
- UJMCYJKPDFQLHX-XGEHTFHBSA-N Val-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](C(C)C)N)O UJMCYJKPDFQLHX-XGEHTFHBSA-N 0.000 description 4
- 210000000349 chromosome Anatomy 0.000 description 4
- 239000012881 co-culture medium Substances 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000003209 gene knockout Methods 0.000 description 4
- 108010081551 glycylphenylalanine Proteins 0.000 description 4
- 108010036413 histidylglycine Proteins 0.000 description 4
- 108020004999 messenger RNA Proteins 0.000 description 4
- 108010005942 methionylglycine Proteins 0.000 description 4
- 230000002028 premature Effects 0.000 description 4
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 4
- 238000013518 transcription Methods 0.000 description 4
- 230000035897 transcription Effects 0.000 description 4
- 238000013519 translation Methods 0.000 description 4
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 4
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 3
- YXXPVUOMPSZURS-ZLIFDBKOSA-N Ala-Trp-Leu Chemical compound C1=CC=C2C(C[C@@H](C(=O)N[C@@H](CC(C)C)C(O)=O)NC(=O)[C@H](C)N)=CNC2=C1 YXXPVUOMPSZURS-ZLIFDBKOSA-N 0.000 description 3
- DNLQVHBBMPZUGJ-BQBZGAKWSA-N Arg-Ser-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CO)C(=O)NCC(O)=O DNLQVHBBMPZUGJ-BQBZGAKWSA-N 0.000 description 3
- GXMSVVBIAMWMKO-BQBZGAKWSA-N Asn-Arg-Gly Chemical compound NC(=O)C[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CCCN=C(N)N GXMSVVBIAMWMKO-BQBZGAKWSA-N 0.000 description 3
- JREOBWLIZLXRIS-GUBZILKMSA-N Asn-Glu-Leu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JREOBWLIZLXRIS-GUBZILKMSA-N 0.000 description 3
- DDPXDCKYWDGZAL-BQBZGAKWSA-N Asn-Gly-Arg Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N DDPXDCKYWDGZAL-BQBZGAKWSA-N 0.000 description 3
- QCVXMEHGFUMKCO-YUMQZZPRSA-N Asp-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC(O)=O QCVXMEHGFUMKCO-YUMQZZPRSA-N 0.000 description 3
- KZBUYRJDOAKODT-UHFFFAOYSA-N Chlorine Chemical compound ClCl KZBUYRJDOAKODT-UHFFFAOYSA-N 0.000 description 3
- AKJRHDMTEJXTPV-ACZMJKKPSA-N Glu-Asn-Ala Chemical compound C[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CCC(O)=O)C(O)=O AKJRHDMTEJXTPV-ACZMJKKPSA-N 0.000 description 3
- OCRQUYDOYKCOQG-IRXDYDNUSA-N Gly-Tyr-Phe Chemical compound C([C@H](NC(=O)CN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=C(O)C=C1 OCRQUYDOYKCOQG-IRXDYDNUSA-N 0.000 description 3
- 206010020649 Hyperkeratosis Diseases 0.000 description 3
- WINFHLHJTRGLCV-BZSNNMDCSA-N Lys-Tyr-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCCN)C(O)=O)CC1=CC=C(O)C=C1 WINFHLHJTRGLCV-BZSNNMDCSA-N 0.000 description 3
- 108010047562 NGR peptide Proteins 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- 238000012228 RNA interference-mediated gene silencing Methods 0.000 description 3
- 238000010459 TALEN Methods 0.000 description 3
- TWLMXDWFVNEFFK-FJXKBIBVSA-N Thr-Arg-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O TWLMXDWFVNEFFK-FJXKBIBVSA-N 0.000 description 3
- CDHQEOXPWBDFPL-QWRGUYRKSA-N Tyr-Gly-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CC1=CC=C(O)C=C1 CDHQEOXPWBDFPL-QWRGUYRKSA-N 0.000 description 3
- YODDULVCGFQRFZ-ZKWXMUAHSA-N Val-Asp-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O YODDULVCGFQRFZ-ZKWXMUAHSA-N 0.000 description 3
- 206010052428 Wound Diseases 0.000 description 3
- 208000027418 Wounds and injury Diseases 0.000 description 3
- 125000000539 amino acid group Chemical group 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 230000009368 gene silencing by RNA Effects 0.000 description 3
- 230000006801 homologous recombination Effects 0.000 description 3
- 238000002744 homologous recombination Methods 0.000 description 3
- 108010045397 lysyl-tyrosyl-lysine Proteins 0.000 description 3
- 108010064235 lysylglycine Proteins 0.000 description 3
- 230000011987 methylation Effects 0.000 description 3
- 238000007069 methylation reaction Methods 0.000 description 3
- 108091070501 miRNA Proteins 0.000 description 3
- 210000000056 organ Anatomy 0.000 description 3
- 230000004853 protein function Effects 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- 239000006228 supernatant Substances 0.000 description 3
- 210000001519 tissue Anatomy 0.000 description 3
- 244000153158 Ammi visnaga Species 0.000 description 2
- 235000010585 Ammi visnaga Nutrition 0.000 description 2
- 108020005544 Antisense RNA Proteins 0.000 description 2
- 241000233866 Fungi Species 0.000 description 2
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 2
- 108091027544 Subgenomic mRNA Proteins 0.000 description 2
- RYQUMYBMOJYYDK-NHCYSSNCSA-N Val-Pro-Glu Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(=O)O)C(=O)O)N RYQUMYBMOJYYDK-NHCYSSNCSA-N 0.000 description 2
- 108010017070 Zinc Finger Nucleases Proteins 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 238000000137 annealing Methods 0.000 description 2
- 244000052616 bacterial pathogen Species 0.000 description 2
- 238000007664 blowing Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 230000008632 circadian clock Effects 0.000 description 2
- 239000003184 complementary RNA Substances 0.000 description 2
- 238000004925 denaturation Methods 0.000 description 2
- 230000036425 denaturation Effects 0.000 description 2
- 238000004043 dyeing Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 238000010362 genome editing Methods 0.000 description 2
- 230000035784 germination Effects 0.000 description 2
- 108010049041 glutamylalanine Proteins 0.000 description 2
- 239000005556 hormone Substances 0.000 description 2
- 229940088597 hormone Drugs 0.000 description 2
- 230000002779 inactivation Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 238000002703 mutagenesis Methods 0.000 description 2
- 231100000350 mutagenesis Toxicity 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000001124 posttranscriptional effect Effects 0.000 description 2
- 238000011084 recovery 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
- 238000004904 shortening Methods 0.000 description 2
- 230000001954 sterilising effect Effects 0.000 description 2
- 229960005322 streptomycin Drugs 0.000 description 2
- 102000040650 (ribonucleotides)n+m Human genes 0.000 description 1
- ZBMRKNMTMPPMMK-UHFFFAOYSA-N 2-amino-4-[hydroxy(methyl)phosphoryl]butanoic acid;azane Chemical compound [NH4+].CP(O)(=O)CCC(N)C([O-])=O ZBMRKNMTMPPMMK-UHFFFAOYSA-N 0.000 description 1
- 108020003589 5' Untranslated Regions Proteins 0.000 description 1
- CKOMXBHMKXXTNW-UHFFFAOYSA-N 6-methyladenine Chemical compound CNC1=NC=NC2=C1N=CN2 CKOMXBHMKXXTNW-UHFFFAOYSA-N 0.000 description 1
- QDRGPQWIVZNJQD-CIUDSAMLSA-N Ala-Arg-Gln Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCC(N)=O)C(O)=O QDRGPQWIVZNJQD-CIUDSAMLSA-N 0.000 description 1
- CHFFHQUVXHEGBY-GARJFASQSA-N Ala-Lys-Pro Chemical compound C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N1CCC[C@@H]1C(=O)O)N CHFFHQUVXHEGBY-GARJFASQSA-N 0.000 description 1
- BTRULDJUUVGRNE-DCAQKATOSA-N Ala-Pro-Lys Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O BTRULDJUUVGRNE-DCAQKATOSA-N 0.000 description 1
- SAHQGRZIQVEJPF-JXUBOQSCSA-N Ala-Thr-Lys Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCCN SAHQGRZIQVEJPF-JXUBOQSCSA-N 0.000 description 1
- XCIGOVDXZULBBV-DCAQKATOSA-N Ala-Val-Lys Chemical compound CC(C)[C@H](NC(=O)[C@H](C)N)C(=O)N[C@@H](CCCCN)C(O)=O XCIGOVDXZULBBV-DCAQKATOSA-N 0.000 description 1
- 241000219194 Arabidopsis Species 0.000 description 1
- 101100010896 Arabidopsis thaliana ECT3 gene Proteins 0.000 description 1
- MCYJBCKCAPERSE-FXQIFTODSA-N Arg-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N MCYJBCKCAPERSE-FXQIFTODSA-N 0.000 description 1
- VBFJESQBIWCWRL-DCAQKATOSA-N Arg-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCNC(N)=N VBFJESQBIWCWRL-DCAQKATOSA-N 0.000 description 1
- DXQIQUIQYAGRCC-CIUDSAMLSA-N Arg-Asp-Gln Chemical compound C(C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N)CN=C(N)N DXQIQUIQYAGRCC-CIUDSAMLSA-N 0.000 description 1
- YSUVMPICYVWRBX-VEVYYDQMSA-N Arg-Asp-Thr Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O YSUVMPICYVWRBX-VEVYYDQMSA-N 0.000 description 1
- ZJEDSBGPBXVBMP-PYJNHQTQSA-N Arg-His-Ile Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O ZJEDSBGPBXVBMP-PYJNHQTQSA-N 0.000 description 1
- BSGSDLYGGHGMND-IHRRRGAJSA-N Arg-Phe-Cys Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N BSGSDLYGGHGMND-IHRRRGAJSA-N 0.000 description 1
- ISVACHFCVRKIDG-SRVKXCTJSA-N Arg-Val-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O ISVACHFCVRKIDG-SRVKXCTJSA-N 0.000 description 1
- LJUOLNXOWSWGKF-ACZMJKKPSA-N Asn-Asn-Glu Chemical compound C(CC(=O)O)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)N)N LJUOLNXOWSWGKF-ACZMJKKPSA-N 0.000 description 1
- DAPLJWATMAXPPZ-CIUDSAMLSA-N Asn-Asn-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC(N)=O DAPLJWATMAXPPZ-CIUDSAMLSA-N 0.000 description 1
- QNJIRRVTOXNGMH-GUBZILKMSA-N Asn-Gln-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC(N)=O QNJIRRVTOXNGMH-GUBZILKMSA-N 0.000 description 1
- DXVMJJNAOVECBA-WHFBIAKZSA-N Asn-Gly-Asn Chemical compound NC(=O)C[C@H](N)C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O DXVMJJNAOVECBA-WHFBIAKZSA-N 0.000 description 1
- GIQCDTKOIPUDSG-GARJFASQSA-N Asn-Lys-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCCN)NC(=O)[C@H](CC(=O)N)N)C(=O)O GIQCDTKOIPUDSG-GARJFASQSA-N 0.000 description 1
- OROMFUQQTSWUTI-IHRRRGAJSA-N Asn-Phe-Arg Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N OROMFUQQTSWUTI-IHRRRGAJSA-N 0.000 description 1
- QUMKPKWYDVMGNT-NUMRIWBASA-N Asn-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O QUMKPKWYDVMGNT-NUMRIWBASA-N 0.000 description 1
- QXNGSPZMGFEZNO-QRTARXTBSA-N Asn-Val-Trp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O QXNGSPZMGFEZNO-QRTARXTBSA-N 0.000 description 1
- KRXIWXCXOARFNT-ZLUOBGJFSA-N Asp-Ala-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@@H](N)CC(O)=O KRXIWXCXOARFNT-ZLUOBGJFSA-N 0.000 description 1
- ZCKYZTGLXIEOKS-CIUDSAMLSA-N Asp-Asp-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC(=O)O)N ZCKYZTGLXIEOKS-CIUDSAMLSA-N 0.000 description 1
- TVVYVAUGRHNTGT-UGYAYLCHSA-N Asp-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O TVVYVAUGRHNTGT-UGYAYLCHSA-N 0.000 description 1
- SVFOIXMRMLROHO-SRVKXCTJSA-N Asp-Asp-Phe Chemical compound OC(=O)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 SVFOIXMRMLROHO-SRVKXCTJSA-N 0.000 description 1
- KHBLRHKVXICFMY-GUBZILKMSA-N Asp-Glu-Lys Chemical compound N[C@@H](CC(=O)O)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCCCN)C(=O)O KHBLRHKVXICFMY-GUBZILKMSA-N 0.000 description 1
- PWAIZUBWHRHYKS-MELADBBJSA-N Asp-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CC(=O)O)N)C(=O)O PWAIZUBWHRHYKS-MELADBBJSA-N 0.000 description 1
- QPDUWAUSSWGJSB-NGZCFLSTSA-N Asp-Val-Pro Chemical compound CC(C)[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC(=O)O)N QPDUWAUSSWGJSB-NGZCFLSTSA-N 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- 241000195493 Cryptophyta Species 0.000 description 1
- FEJCUYOGOBCFOQ-ACZMJKKPSA-N Cys-Asp-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CS)N FEJCUYOGOBCFOQ-ACZMJKKPSA-N 0.000 description 1
- OXFOKRAFNYSREH-BJDJZHNGSA-N Cys-Ile-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)O)NC(=O)[C@H](CS)N OXFOKRAFNYSREH-BJDJZHNGSA-N 0.000 description 1
- 230000007067 DNA methylation Effects 0.000 description 1
- 101100214180 Drosophila melanogaster Ythdf gene Proteins 0.000 description 1
- 206010058314 Dysplasia Diseases 0.000 description 1
- 108090000790 Enzymes Proteins 0.000 description 1
- 102000004190 Enzymes Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 108700028146 Genetic Enhancer Elements Proteins 0.000 description 1
- KZEUVLLVULIPNX-GUBZILKMSA-N Gln-Asp-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CCC(=O)N)N KZEUVLLVULIPNX-GUBZILKMSA-N 0.000 description 1
- MADFVRSKEIEZHZ-DCAQKATOSA-N Gln-Gln-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CCC(=O)N)N MADFVRSKEIEZHZ-DCAQKATOSA-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
- SHAUZYVSXAMYAZ-JYJNAYRXSA-N Gln-Leu-Phe Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)NC(=O)[C@H](CCC(=O)N)N SHAUZYVSXAMYAZ-JYJNAYRXSA-N 0.000 description 1
- OZEQPCDLCDRCGY-SOUVJXGZSA-N Gln-Phe-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=CC=C2)NC(=O)[C@H](CCC(=O)N)N)C(=O)O OZEQPCDLCDRCGY-SOUVJXGZSA-N 0.000 description 1
- QFXNFFZTMFHPST-DZKIICNBSA-N Gln-Phe-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CCC(=O)N)N QFXNFFZTMFHPST-DZKIICNBSA-N 0.000 description 1
- BUAKRRKDHSSIKK-IHRRRGAJSA-N Glu-Glu-Tyr Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 BUAKRRKDHSSIKK-IHRRRGAJSA-N 0.000 description 1
- CXRWMMRLEMVSEH-PEFMBERDSA-N Glu-Ile-Asn Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O CXRWMMRLEMVSEH-PEFMBERDSA-N 0.000 description 1
- LKOAAMXDJGEYMS-ZPFDUUQYSA-N Glu-Met-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LKOAAMXDJGEYMS-ZPFDUUQYSA-N 0.000 description 1
- JWNZHMSRZXXGTM-XKBZYTNZSA-N Glu-Ser-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O JWNZHMSRZXXGTM-XKBZYTNZSA-N 0.000 description 1
- BKMOHWJHXQLFEX-IRIUXVKKSA-N Glu-Tyr-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CCC(=O)O)N)O BKMOHWJHXQLFEX-IRIUXVKKSA-N 0.000 description 1
- NPSWCZIRBAYNSB-JHEQGTHGSA-N Gly-Gln-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O NPSWCZIRBAYNSB-JHEQGTHGSA-N 0.000 description 1
- JSNNHGHYGYMVCK-XVKPBYJWSA-N Gly-Glu-Val Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O JSNNHGHYGYMVCK-XVKPBYJWSA-N 0.000 description 1
- QITBQGJOXQYMOA-ZETCQYMHSA-N Gly-Gly-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)CNC(=O)CN QITBQGJOXQYMOA-ZETCQYMHSA-N 0.000 description 1
- PAWIVEIWWYGBAM-YUMQZZPRSA-N Gly-Leu-Ala Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O PAWIVEIWWYGBAM-YUMQZZPRSA-N 0.000 description 1
- MIIVFRCYJABHTQ-ONGXEEELSA-N Gly-Leu-Val Chemical compound [H]NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O MIIVFRCYJABHTQ-ONGXEEELSA-N 0.000 description 1
- WMGHDYWNHNLGBV-ONGXEEELSA-N Gly-Phe-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 WMGHDYWNHNLGBV-ONGXEEELSA-N 0.000 description 1
- BMWFDYIYBAFROD-WPRPVWTQSA-N Gly-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)CN BMWFDYIYBAFROD-WPRPVWTQSA-N 0.000 description 1
- CSMYMGFCEJWALV-WDSKDSINSA-N Gly-Ser-Gln Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(N)=O CSMYMGFCEJWALV-WDSKDSINSA-N 0.000 description 1
- PYFHPYDQHCEVIT-KBPBESRZSA-N Gly-Trp-Gln Chemical compound [H]NCC(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(N)=O)C(O)=O PYFHPYDQHCEVIT-KBPBESRZSA-N 0.000 description 1
- KSOBNUBCYHGUKH-UWVGGRQHSA-N Gly-Val-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN KSOBNUBCYHGUKH-UWVGGRQHSA-N 0.000 description 1
- 239000004471 Glycine Substances 0.000 description 1
- RVKIPWVMZANZLI-UHFFFAOYSA-N H-Lys-Trp-OH Natural products C1=CC=C2C(CC(NC(=O)C(N)CCCCN)C(O)=O)=CNC2=C1 RVKIPWVMZANZLI-UHFFFAOYSA-N 0.000 description 1
- 108010034791 Heterochromatin Proteins 0.000 description 1
- YAALVYQFVJNXIV-KKUMJFAQSA-N His-Leu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC1=CN=CN1 YAALVYQFVJNXIV-KKUMJFAQSA-N 0.000 description 1
- CKRJBQJIGOEKMC-SRVKXCTJSA-N His-Lys-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O CKRJBQJIGOEKMC-SRVKXCTJSA-N 0.000 description 1
- 101000817237 Homo sapiens Protein ECT2 Proteins 0.000 description 1
- HUORUFRRJHELPD-MNXVOIDGSA-N Ile-Leu-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N HUORUFRRJHELPD-MNXVOIDGSA-N 0.000 description 1
- FFJQAEYLAQMGDL-MGHWNKPDSA-N Ile-Lys-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 FFJQAEYLAQMGDL-MGHWNKPDSA-N 0.000 description 1
- LRAUKBMYHHNADU-DKIMLUQUSA-N Ile-Phe-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)[C@@H](C)CC)CC1=CC=CC=C1 LRAUKBMYHHNADU-DKIMLUQUSA-N 0.000 description 1
- NJGXXYLPDMMFJB-XUXIUFHCSA-N Ile-Val-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N NJGXXYLPDMMFJB-XUXIUFHCSA-N 0.000 description 1
- LZDNBBYBDGBADK-UHFFFAOYSA-N L-valyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)C(C)C)C(O)=O)=CNC2=C1 LZDNBBYBDGBADK-UHFFFAOYSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- CQQGCWPXDHTTNF-GUBZILKMSA-N Leu-Ala-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCC(O)=O CQQGCWPXDHTTNF-GUBZILKMSA-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
- HQUXQAMSWFIRET-AVGNSLFASA-N Leu-Glu-Lys Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN HQUXQAMSWFIRET-AVGNSLFASA-N 0.000 description 1
- LXKNSJLSGPNHSK-KKUMJFAQSA-N Leu-Leu-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)O)N LXKNSJLSGPNHSK-KKUMJFAQSA-N 0.000 description 1
- WMIOEVKKYIMVKI-DCAQKATOSA-N Leu-Pro-Ala Chemical compound [H]N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O WMIOEVKKYIMVKI-DCAQKATOSA-N 0.000 description 1
- SQUFDMCWMFOEBA-KKUMJFAQSA-N Leu-Ser-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 SQUFDMCWMFOEBA-KKUMJFAQSA-N 0.000 description 1
- WSXTWLJHTLRFLW-SRVKXCTJSA-N Lys-Ala-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CCCCN)C(O)=O WSXTWLJHTLRFLW-SRVKXCTJSA-N 0.000 description 1
- LMVOVCYVZBBWQB-SRVKXCTJSA-N Lys-Asp-Lys Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCCCN LMVOVCYVZBBWQB-SRVKXCTJSA-N 0.000 description 1
- NRQRKMYZONPCTM-CIUDSAMLSA-N Lys-Asp-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O NRQRKMYZONPCTM-CIUDSAMLSA-N 0.000 description 1
- NDORZBUHCOJQDO-GVXVVHGQSA-N Lys-Gln-Val Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O NDORZBUHCOJQDO-GVXVVHGQSA-N 0.000 description 1
- GQZMPWBZQALKJO-UWVGGRQHSA-N Lys-Gly-Arg Chemical compound [H]N[C@@H](CCCCN)C(=O)NCC(=O)N[C@@H](CCCNC(N)=N)C(O)=O GQZMPWBZQALKJO-UWVGGRQHSA-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
- HVAUKHLDSDDROB-KKUMJFAQSA-N Lys-Lys-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O HVAUKHLDSDDROB-KKUMJFAQSA-N 0.000 description 1
- IPTUBUUIFRZMJK-ACRUOGEOSA-N Lys-Phe-Phe Chemical compound C([C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 IPTUBUUIFRZMJK-ACRUOGEOSA-N 0.000 description 1
- BOJYMMBYBNOOGG-DCAQKATOSA-N Lys-Pro-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O BOJYMMBYBNOOGG-DCAQKATOSA-N 0.000 description 1
- LUTDBHBIHHREDC-IHRRRGAJSA-N Lys-Pro-Lys Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(O)=O LUTDBHBIHHREDC-IHRRRGAJSA-N 0.000 description 1
- JMNRXRPBHFGXQX-GUBZILKMSA-N Lys-Ser-Glu Chemical compound NCCCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JMNRXRPBHFGXQX-GUBZILKMSA-N 0.000 description 1
- DLCAXBGXGOVUCD-PPCPHDFISA-N Lys-Thr-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DLCAXBGXGOVUCD-PPCPHDFISA-N 0.000 description 1
- ZNAPAUSAUBHENO-IHPCNDPISA-N Lys-Trp-His Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC3=CN=CN3)C(=O)O)NC(=O)[C@H](CCCCN)N ZNAPAUSAUBHENO-IHPCNDPISA-N 0.000 description 1
- HGAJNEWOUHDUMZ-SRVKXCTJSA-N Met-Leu-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O HGAJNEWOUHDUMZ-SRVKXCTJSA-N 0.000 description 1
- VQAYFKKCNSOZKM-IOSLPCCCSA-N N(6)-methyladenosine Chemical compound C1=NC=2C(NC)=NC=NC=2N1[C@@H]1O[C@H](CO)[C@@H](O)[C@H]1O VQAYFKKCNSOZKM-IOSLPCCCSA-N 0.000 description 1
- AUEJLPRZGVVDNU-UHFFFAOYSA-N N-L-tyrosyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CC1=CC=C(O)C=C1 AUEJLPRZGVVDNU-UHFFFAOYSA-N 0.000 description 1
- 108010066427 N-valyltryptophan Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- MDHZEOMXGNBSIL-DLOVCJGASA-N Phe-Ala-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N MDHZEOMXGNBSIL-DLOVCJGASA-N 0.000 description 1
- KAHUBGWSIQNZQQ-KKUMJFAQSA-N Phe-Asn-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 KAHUBGWSIQNZQQ-KKUMJFAQSA-N 0.000 description 1
- MSHZERMPZKCODG-ACRUOGEOSA-N Phe-Leu-Phe Chemical compound C([C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MSHZERMPZKCODG-ACRUOGEOSA-N 0.000 description 1
- WWPAHTZOWURIMR-ULQDDVLXSA-N Phe-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CC1=CC=CC=C1 WWPAHTZOWURIMR-ULQDDVLXSA-N 0.000 description 1
- ZJPGOXWRFNKIQL-JYJNAYRXSA-N Phe-Pro-Pro Chemical compound C([C@H](N)C(=O)N1[C@@H](CCC1)C(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=CC=C1 ZJPGOXWRFNKIQL-JYJNAYRXSA-N 0.000 description 1
- MVIJMIZJPHQGEN-IHRRRGAJSA-N Phe-Ser-Val Chemical compound CC(C)[C@@H](C([O-])=O)NC(=O)[C@H](CO)NC(=O)[C@@H]([NH3+])CC1=CC=CC=C1 MVIJMIZJPHQGEN-IHRRRGAJSA-N 0.000 description 1
- AGTHXWTYCLLYMC-FHWLQOOXSA-N Phe-Tyr-Glu Chemical compound C([C@H](N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N[C@@H](CCC(O)=O)C(O)=O)C1=CC=CC=C1 AGTHXWTYCLLYMC-FHWLQOOXSA-N 0.000 description 1
- HAAQQNHQZBOWFO-LURJTMIESA-N Pro-Gly-Gly Chemical compound OC(=O)CNC(=O)CNC(=O)[C@@H]1CCCN1 HAAQQNHQZBOWFO-LURJTMIESA-N 0.000 description 1
- FKLSMYYLJHYPHH-UWVGGRQHSA-N Pro-Gly-Leu Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(C)C)C(O)=O FKLSMYYLJHYPHH-UWVGGRQHSA-N 0.000 description 1
- CHYAYDLYYIJCKY-OSUNSFLBSA-N Pro-Thr-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O CHYAYDLYYIJCKY-OSUNSFLBSA-N 0.000 description 1
- 102100040437 Protein ECT2 Human genes 0.000 description 1
- 230000026279 RNA modification Effects 0.000 description 1
- 102000006382 Ribonucleases Human genes 0.000 description 1
- 108010083644 Ribonucleases Proteins 0.000 description 1
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 1
- 241000124033 Salix Species 0.000 description 1
- VAUMZJHYZQXZBQ-WHFBIAKZSA-N Ser-Asn-Gly Chemical compound OC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O VAUMZJHYZQXZBQ-WHFBIAKZSA-N 0.000 description 1
- CAOYHZOWXFFAIR-CIUDSAMLSA-N Ser-His-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CO)C(O)=O CAOYHZOWXFFAIR-CIUDSAMLSA-N 0.000 description 1
- LRWBCWGEUCKDTN-BJDJZHNGSA-N Ser-Lys-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O LRWBCWGEUCKDTN-BJDJZHNGSA-N 0.000 description 1
- LRZLZIUXQBIWTB-KATARQTJSA-N Ser-Lys-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O LRZLZIUXQBIWTB-KATARQTJSA-N 0.000 description 1
- SZRNDHWMVSFPSP-XKBZYTNZSA-N Ser-Thr-Gln Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CO)N)O SZRNDHWMVSFPSP-XKBZYTNZSA-N 0.000 description 1
- BEBVVQPDSHHWQL-NRPADANISA-N Ser-Val-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O BEBVVQPDSHHWQL-NRPADANISA-N 0.000 description 1
- 108091027967 Small hairpin RNA Proteins 0.000 description 1
- 108020004459 Small interfering RNA Proteins 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- JVTHIXKSVYEWNI-JRQIVUDYSA-N Thr-Asn-Tyr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O JVTHIXKSVYEWNI-JRQIVUDYSA-N 0.000 description 1
- LMMDEZPNUTZJAY-GCJQMDKQSA-N Thr-Asp-Ala Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O LMMDEZPNUTZJAY-GCJQMDKQSA-N 0.000 description 1
- YBXMGKCLOPDEKA-NUMRIWBASA-N Thr-Asp-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YBXMGKCLOPDEKA-NUMRIWBASA-N 0.000 description 1
- NIEWSKWFURSECR-FOHZUACHSA-N Thr-Gly-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CC(O)=O)C(O)=O NIEWSKWFURSECR-FOHZUACHSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- RFKVQLIXNVEOMB-WEDXCCLWSA-N Thr-Leu-Gly Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)O)N)O RFKVQLIXNVEOMB-WEDXCCLWSA-N 0.000 description 1
- YGCDFAJJCRVQKU-RCWTZXSCSA-N Thr-Pro-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)[C@@H](C)O YGCDFAJJCRVQKU-RCWTZXSCSA-N 0.000 description 1
- RVMNUBQWPVOUKH-HEIBUPTGSA-N Thr-Ser-Thr Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O RVMNUBQWPVOUKH-HEIBUPTGSA-N 0.000 description 1
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 1
- MHNHRNHJMXAVHZ-AAEUAGOBSA-N Trp-Asn-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)NCC(=O)O)N MHNHRNHJMXAVHZ-AAEUAGOBSA-N 0.000 description 1
- AWYXDHQQFPZJNE-QEJZJMRPSA-N Trp-Gln-Ser Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CO)C(=O)O)N AWYXDHQQFPZJNE-QEJZJMRPSA-N 0.000 description 1
- VMBBTANKMSRJSS-JSGCOSHPSA-N Trp-Glu-Gly Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O VMBBTANKMSRJSS-JSGCOSHPSA-N 0.000 description 1
- AYPAIRCDLARHLM-KKUMJFAQSA-N Tyr-Asn-Lys Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N)O AYPAIRCDLARHLM-KKUMJFAQSA-N 0.000 description 1
- IYHNBRUWVBIVJR-IHRRRGAJSA-N Tyr-Gln-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 IYHNBRUWVBIVJR-IHRRRGAJSA-N 0.000 description 1
- QFXVAFIHVWXXBJ-AVGNSLFASA-N Tyr-Ser-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(O)=O QFXVAFIHVWXXBJ-AVGNSLFASA-N 0.000 description 1
- HMPMGPISLMLHSI-JBACZVJFSA-N Tyr-Trp-Gln Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC3=CC=C(C=C3)O)N HMPMGPISLMLHSI-JBACZVJFSA-N 0.000 description 1
- RUCNAYOMFXRIKJ-DCAQKATOSA-N Val-Ala-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN RUCNAYOMFXRIKJ-DCAQKATOSA-N 0.000 description 1
- SDUBQHUJJWQTEU-XUXIUFHCSA-N Val-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](C(C)C)N SDUBQHUJJWQTEU-XUXIUFHCSA-N 0.000 description 1
- XTDDIVQWDXMRJL-IHRRRGAJSA-N Val-Leu-His Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](C(C)C)N XTDDIVQWDXMRJL-IHRRRGAJSA-N 0.000 description 1
- PQSNETRGCRUOGP-KKHAAJSZSA-N Val-Thr-Asn Chemical compound CC(C)[C@H](N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@H](C(O)=O)CC(N)=O PQSNETRGCRUOGP-KKHAAJSZSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000011543 agarose gel Substances 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 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 1
- 230000002902 bimodal effect Effects 0.000 description 1
- 230000004071 biological effect Effects 0.000 description 1
- 230000008827 biological function Effects 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 101150038500 cas9 gene Proteins 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 239000011248 coating agent Substances 0.000 description 1
- 238000000576 coating method Methods 0.000 description 1
- 239000002299 complementary DNA Substances 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 230000021040 cytoplasmic transport Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 230000018109 developmental process Effects 0.000 description 1
- 235000013399 edible fruits Nutrition 0.000 description 1
- 239000012636 effector Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 239000012877 elongation medium Substances 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 210000003608 fece Anatomy 0.000 description 1
- 239000003337 fertilizer Substances 0.000 description 1
- 230000037433 frameshift Effects 0.000 description 1
- 108010063718 gamma-glutamylaspartic acid Proteins 0.000 description 1
- 108010078144 glutaminyl-glycine Proteins 0.000 description 1
- 108010082286 glycyl-seryl-alanine Proteins 0.000 description 1
- 108010089804 glycyl-threonine Proteins 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010015792 glycyllysine Proteins 0.000 description 1
- 230000012010 growth Effects 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 210000004458 heterochromatin Anatomy 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000003780 insertion Methods 0.000 description 1
- 230000037431 insertion Effects 0.000 description 1
- 108010078274 isoleucylvaline Proteins 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 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 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 230000005087 leaf formation Effects 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 239000010871 livestock manure Substances 0.000 description 1
- 108010009298 lysylglutamic acid Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 230000000442 meristematic effect Effects 0.000 description 1
- 239000000178 monomer Substances 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 239000008188 pellet Substances 0.000 description 1
- 108010084572 phenylalanyl-valine Proteins 0.000 description 1
- 108010083476 phenylalanyltryptophan Proteins 0.000 description 1
- 239000013600 plasmid vector Substances 0.000 description 1
- 102000040430 polynucleotide Human genes 0.000 description 1
- 108091033319 polynucleotide Proteins 0.000 description 1
- 239000002157 polynucleotide Substances 0.000 description 1
- 230000032361 posttranscriptional gene silencing Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000011027 product recovery Methods 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 230000001737 promoting effect Effects 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000003259 recombinant expression Methods 0.000 description 1
- 230000009712 regulation of translation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 239000012883 rooting culture medium Substances 0.000 description 1
- 239000012882 rooting medium Substances 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 238000007790 scraping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000004055 small Interfering RNA Substances 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 230000007480 spreading Effects 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000001629 suppression Effects 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 108010072986 threonyl-seryl-lysine Proteins 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000009752 translational inhibition Effects 0.000 description 1
- 230000032258 transport Effects 0.000 description 1
- 108010078580 tyrosylleucine Proteins 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
-
- 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)
- C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
- C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
- Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
- Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biomedical Technology (AREA)
- Wood Science & Technology (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Virology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
本发明公开了GmECT2在调控植物高度方面的应用。GmECT2蛋白的氨基酸序列如序列表中序列1所示。本发明通过利用CRISPR‑Cas9系统对大豆GmECT2的基因进行编辑，得到四株GmECT2基因发生有效编辑的纯合突变体。实验发现四株GmECT2基因的纯合突变体植株高度明显矮于野生型(天隆一号)：在苗期(株龄10天)和株龄29天均表现出植株高度明显矮于野生型的表型。因此，GmECT基因在调控大豆株高中发挥重要的作用，当该基因发生突变，植株高度明显变矮，可应用于植物育种和品种改良。本发明为植株高度的调控手段提供了新的选择。
Description
技术领域
本发明属于生物技术领域，具体涉及GmECT2在调控植物高度方面的应用。
背景技术
RNA修饰是一种转录后水平的调控方式，广泛分布于mRNA、tRNA和rRNA等各类RNA上。其中6-甲基腺嘌呤(N6-methyladenosine，m6A)最常见并且研究较多。m6A通路中的效应因子包括三部分，添加和去除甲基化位点的“writers”和“erasers”以及识别甲基化位点的“readers”。转录本的m6A修饰水平受三者的动态调控。
ECT(Evolutionarily conserved c-terminal region)蛋白是植物基因组中识别甲基化位点的YTHDF蛋白。mRNA-protein研究发现ECT蛋白在体内具有与mRNA结合的能力，但是植物中YTH结构域蛋白的生物学功能和其他生物学特性仍是未知的。在对拟南芥进行研究的过程中发现，YTH结构域蛋白ECT2和ECT3具有读码能力，依赖完整的m6A结合位点来控制叶片形成的时间；定义正常的叶片形态；影响根系生长速率和方向；控制开花时间，导致开花时间变晚；影响花形态，造成发育异常；影响果实形态和大小。
发明内容
本发明所要解决的技术问题是如何调控植物植株的高度或如何降低植物植株的高度。
为了解决上述技术问题，本发明首先提供了蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质的应用。所述应用为下述任一种：
P1、所述蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在调控植物株高或降低植物株高中的应用；
P2、所述蛋白质或调控所述蛋白质编码基因表达的物质或调控所述蛋白质活性或含量的物质在植物育种或品质改良中的应用。
所述蛋白质可为如下A1)、A2)、A3)、A4)、A5)、A6)或A7)的蛋白质：
A1)氨基酸序列是序列表中序列1的蛋白质；
A2)氨基酸序列是序列表中序列10的蛋白质；
A3)氨基酸序列是序列表中序列12的蛋白质；
A4)氨基酸序列是序列表中序列14的蛋白质；
A5)氨基酸序列是序列表中序列16的蛋白质；
A6)将A1)、A2)、A3)、A4)或A5)所示的氨基酸序列经过一个以上氨基酸残基的取代和/或缺失和/或添加得到的且具有相同功能的由A1)、A2)、A3)、A4)或A5)衍生的或与A1)、A2)、A3)、A4)或A5)所示的蛋白质具有80％以上的同一性且具有相同功能的蛋白质；
A7)在A1)、A2)、A3)、A4)、A5)或A6)的N末端或/和C末端连接蛋白标签得到的融合蛋白质。
上文所述蛋白质可来源于大豆。
上文所述一个以上氨基酸残基具体可为十个以内的氨基酸残基。
上述应用中，调控所述蛋白质活性或含量的物质可为敲除所述蛋白质的编码基因的物质和/或调控所述蛋白质的编码基因表达的物质。
上述应用中，所述调控基因表达的物质可为进行如下6种调控中至少一种调控的物质：1)在所述基因转录水平上进行的调控；2)在所述基因转录后进行的调控(也就是对所述基因的初级转录物的剪接或加工进行的调控)；3)对所述基因的RNA转运进行的调控(也就是对所述基因的mRNA由细胞核向细胞质转运进行的调控)；4)对所述基因的翻译进行的调控；5)对所述基因的mRNA降解进行的调控；6)对所述基因的翻译后的调控(也就是对所述基因翻译的蛋白质的活性进行调控)。
上述应用中，所述调控基因表达可为抑制或降低所述基因表达，所述抑制或降低所述基因表达可通过基因敲除实现或通过基因沉默实现。
所述基因敲除(geneknockout)是指通过同源重组使特定靶基因失活的现象。基因敲除是通过DNA序列的改变使特定靶基因失活。
所述基因沉默是指在不损伤原有DNA的情况下使基因不表达或低表达的现象。基因沉默以不改变DNA序列为前提，使基因不表达或低表达。基因沉默可发生在两种水平上，一种是由于DNA甲基化、异染色质化以及位置效应等引起的转录水平的基因沉默,另一种是转录后基因沉默，即在基因转录后的水平上通过对靶标RNA进行特异性抑制而使基因失活,包括反义RNA、共抑制(co-suppression)、基因压抑(quelling)、RNA干扰(RNAi)和微小RNA(miRNA)介导的翻译抑制等。
上述应用中，所述调控基因表达的物质可为抑制或降低所述基因表达的试剂。所述抑制或降低所述基因表达的试剂可为敲除所述基因的试剂，如通过同源重组敲除所述基因的试剂，或通过CRISPR-Cas9敲除所述基因的试剂。所述抑制或降低所述基因表达的试剂可以包含靶向所述基因的多核苷酸，例如siRNA、shRNA、sgRNA、miRNA或反义RNA。
上述应用中，所述80％以上的同一性可为至少80％、81％、82％、83％、84％、85％、86％、87％、88％、89％、90％、91％、92％、93％、94％、95％、96％、98％或99％的同一性。
为了解决上述技术问题，本发明还提供了与上文所述蛋白质相关的生物材料的下述任一种应用：
Q1、所述生物材料在调控植物株高或降低植物株高中的应用；
Q2、所述生物材料在植物育种或品质改良中的应用。
所述生物材料可为下述B1)至B9)中的任一种：
B1)编码上文所述蛋白质的核酸分子；
B2)含有B1)所述核酸分子的表达盒；
B3)含有B1)所述核酸分子的重组载体、或含有B2)所述表达盒的重组载体；
B4)含有B1)所述核酸分子的重组微生物、或含有B2)所述表达盒的重组微生物、或含有B3)所述重组载体的重组微生物；
B5)含有B1)所述核酸分子的转基因植物细胞系、或含有B2)所述表达盒的转基因植物细胞系；
B6)含有B1)所述核酸分子的转基因植物组织、或含有B2)所述表达盒的转基因植物组织；
B7)含有B1)所述核酸分子的转基因植物器官、或含有B2)所述表达盒的转基因植物器官；
B8)抑制或降低上文所述蛋白质的编码基因的表达或上文所述蛋白质活性的核酸分子；
B9)含有B8)所述核酸分子的表达盒、重组载体、重组微生物或转基因植物细胞系。
上文所述核酸分子可为如下b1)或b2)或b3)或b4)或b5或b6或b7)所示的DNA分子：
b1)编码序列是序列表中序列2所示的DNA分子；
b2)编码序列是序列表中序列9所示的DNA分子；
b3)编码序列是序列表中序列11所示的DNA分子；
b4)编码序列是序列表中序列13所示的DNA分子；
b5)编码序列是序列表中序列15所示的DNA分子；
b6)与b1)、b2)、b3)、b4)或b5)限定的核苷酸序列具有90％或90％以上同一性，且编码上文所述蛋白质的DNA分子；
b7)在严格条件下与b1)、b2)、b3)、b4)、b5)或b6)限定的核苷酸序列杂交，且编码上文所述蛋白质的DNA分子。
上B8)所述核酸分子可为表达靶向上文A1)所述蛋白编码基因的gRNA的DNA分子或靶向上文A1)所述蛋白编码基因的gRNA。
所述靶向上文A1)蛋白编码基因的gRNA的靶序列对应于序列表序列2的第505-527位、第1070-1092位和/或第1188-1210位核苷酸。
术语“同一性”指与天然核酸序列的序列相似性。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。所述具有90％或90％以上同一性可为至少具有90％、至少具有95％、至少具有96％、至少具有97％、至少具有98％或至少具有99％的同一性。
上述生物材料中，B2)所述的含有核酸分子的表达盒，是指能够在宿主细胞中表达上述应用中所述蛋白质的DNA，该DNA不但可包括启动蛋白编码基因转录的启动子，还可包括终止蛋白编码基因转录的终止子。进一步，所述表达盒还可包括增强子序列。可用于本发明的启动子包括但不限于：组成型启动子，组织、器官和发育特异的启动子，和诱导型启动子。
可用现有的植物表达载体构建含有所述蛋白编码基因表达盒的重组表达载体。
上述生物材料中，所述重组微生物具体可为酵母，细菌，藻和真菌。
上文所述植物可为下述任一种：
D1)双子叶植物，
D2)豆目植物，
D3)豆科植物，
D4)大豆属植物，
D5)大豆。
为了解决上述技术问题，本发明还提供了一种降低植物株高的方法。所述方法包括通过抑制或降低植物中上文A1)所述的蛋白质的编码基因的表达量或上文A1)所述的蛋白质的活性来降低植物株高。
上述降低或抑制植物中上文所述蛋白编码基因的表达量可采用现有技术中的任何方式实现，以使基因产生缺失突变、插入突变或碱基变换突变，进而实现基因功能降低或丧失，具体可为化学诱变、物理诱变、RNAi、基因组定点编辑或同源重组等。
上述基因组定点编辑方法中，可采用锌指核酸酶(Zinc finger nuclease,ZFN)技术、类转录激活因子效应物核酸酶(Transcription activator-like effector nuclease,TALEN)技术或成簇的规律间隔的短回文重复序列及其相关系统(Clustered regularlyinterspaced short palindromic repeats/CRISPR associated,CRISPR/Cas9system)技术，以及其它能实现基因组定点编辑的技术。无论采取哪种方法，既可对上文所述蛋白的整个编码基因作为靶标，又可将调控上文所述蛋白编码基因表达的各个元件作为靶标，只要能实现基因功能丧失或降低即可。如可以将上文所述蛋白的编码基因的外显子或5’UTR等作为靶标。
上文所述方法还可包括向所述植物中导入降低或抑制上文所述的蛋白编码基因表达的物质。所述降低或抑制上文所述蛋白编码基因表达的物质可为如下c1)-c4)任一种物质：
c1)抑制或降低上文A1)所述蛋白编码基因表达的核酸分子；
c2)含有c1)所述核酸分子的表达盒；
c3)含有c1)所述核酸分子的重组载体、或含有c2)所述表达盒的重组载体；
c4)含有c1)所述核酸分子的重组微生物、或含有c2)所述表达盒的重组微生物、或含有c3)所述重组载体的重组微生物。
上述方法中，c1)所述的核酸分子可为表达靶向上文A1)所述蛋白编码基因的gRNA的DNA分子或靶向上文A1)所述蛋白编码基因的gRNA。
所述靶向所述A1)蛋白编码基因的gRNA的靶序列可对应于序列表中序列2的第505-527位、第1070-1092位和/或第1188-1210位核苷酸。
上文所述方法具体可为通过CRISPR/Cas9方法对上文所述A1)蛋白编码基因进行定点编辑，使植物中的上文所述A1)蛋白编码基因的表达量降低或被抑制，例如将表达Cas9蛋白和gRNA的重组质粒载体通过农杆菌的侵染实现外源基因向植物细胞的转移和整合。
上文所述方法中，所述抑制或降低植物中上文所述蛋白质的编码基因的表达可为将植物中的序列2所示的所述蛋白编码基因进行下述至少一种突变：
1)将植物中的序列2所示的所述蛋白编码基因突变为Gmect2-11-1-4-2，Gmect2-11-1-4-2是将序列表中序列2的第509-515位的7个核苷酸CCAGTGC缺失、保持序列表中序列2的其他核苷酸不变得到的DNA分子；
2)将植物中的序列2所示的所述蛋白编码基因突变为Gmect2-18-1-4-1，Gmect2-18-1-4-1是将序列表中序列2的1083-1090位的8个核苷酸TAATGGAC缺失、保持序列表中序列2的其他核苷酸不变得到的DNA分子；
3)将植物中的序列2所示的所述蛋白编码基因突变为Gmect2-20-2-6-4，Gmect2-20-2-6-4是将序列表中序列2的1200-1206位的7个核苷酸TAGGGCC缺失、保持序列表中序列2的其他核苷酸不变得到的DNA分子；
4)将植物中的序列2所示的所述蛋白编码基因突变为Gmect2-20-3-7-7-2，Gmect2-20-3-7-7-2是将序列表中序列2的1204-1205位的2个核苷酸之间插入了的1个核苷酸A、保持序列表中序列2的其他核苷酸不变得到的DNA分子。
上文所述方法中所述的蛋白质可为上文所述A1)中的蛋白质。
上述方法为改造后得到的植物可以是所有染色单体均被定点编辑的植物。
上文所述植物可为大豆。
上文所述的蛋白质和/或上文所述的生物材料也属于本发明的保护范围。
本发明通过利用CRISPR-Cas9系统对大豆GmECT2的基因进行编辑，得到四株GmECT2基因发生有效编辑的纯合突变体Gmect2-11-1-4-2、Gmect2-18-1-4-1、Gmect2-20-2-6-4和Gmect2-20-3-7-7-2。实验发现四株GmECT2基因的纯合突变体植株高度明显矮于野生型(天隆一号)：在苗期(株龄10天)和株龄29天均表现出植株高度明显矮于野生型的表型。因此，GmECT基因在调控大豆株高中发挥重要的作用，当该基因发生突变，植株高度明显变矮。本发明为植株高度的调控手段提供了新的选择。
附图说明
图1为Gmect2单突变体植株高度变矮的表型。A为所用CRISPR-Cas9载体pCas9-AtU6-sgRNA的载体示意图；B为GmECT2的基因结构示意图；C为Gmect2-11-1-4-2、Gmect2-18-1-4-1、Gmect2-20-2-6-4和Gmect2-20-3-7-7-2突变体编辑方式；D为Gmect2-11-1-4-2、Gmect2-18-1-4-1、Gmect2-20-2-6-4和Gmect2-20-3-7-7-2苗期(株龄10d)株高表型图；E为Gmect2-11-1-4-2、Gmect2-18-1-4-1、Gmect2-20-2-6-4和Gmect2-20-3-7-7-2株龄29d株高表型图；F为Gmect2-11-1-4-2、Gmect2-18-1-4-1、Gmect2-20-2-6-4和Gmect2-20-3-7-7-2苗期(株龄10d)株高统计数据，纵坐标为株高；G为Gmect2-11-1-4-2、Gmect2-18-1-4-1、Gmect2-20-2-6-4和Gmect2-20-3-7-7-2株龄29d时株高统计数据，纵坐标为株高，G图中的株高数据仅代表拍照植株的株高。
具体实施方式
下面结合具体实施方式对本发明进行进一步的详细描述，给出的实施例仅为了阐明本发明，而不是为了限制本发明的范围。以下提供的实施例可作为本技术领域普通技术人员进行进一步改进的指南，并不以任何方式构成对本发明的限制。
下述实施例中的实验方法，如无特殊说明，均为常规方法，按照本领域内的文献所描述的技术或条件或者按照产品说明书进行。下述实施例中所用的材料、试剂等，如无特殊说明，均可从商业途径得到。
本发明是实例中的共培养培养基、加入激素的液体诱导培养基、固体诱导培养基、固体伸长培养基和生根培养基记载于如下文献中：Paz,M.M.,J.C.Martinez,A.B.Kalvig,T.M.Fonger and K.Wang(2006)."Improved cotyledonary node method using analternative explant derived from mature seed for efficient Agrobacterium-mediated soybean transformation."Plant Cell Rep 25(3):206-213.
实施例1、CRISPR-Cas9重组载体GmECT2-gRNA的构建
大豆威廉82的GmECT2蛋白的氨基酸序列如序列表中序列1所示，其cDNA的CDS编码序列为序列表中序列2所示。
根据GmECT2蛋白的基因组序列设计靶向GmECT2的3个gRNA位点，相应靶位点的核苷酸序列如下：S11位点：5’-CCAACCAGTGCGCCATTCACACC-3’，对应于GmECT2基因的序列表中序列2的第505-527位核苷酸；S18位点：5’-ATGATTCTCGCACTAATGGACGG-3’，对应于GmECT2基因的序列表中序列2的第1070-1092位核苷酸；S20位点：5’-GAACAGAGGGCCTAGGGCCAAGG-3’，对应于GmECT2基因的序列表中序列2的第1188-1210位核苷酸。
1.U6-gRNA的获得
1.1引物序列用于扩增包含gRNA(guide RNA)序列的引物如下：
ECT2-F1：
5’-GCCACCACATGGATTGGTGTGAATGGCGCACTGGTGTTTTAGAGCTAGAAATAGC-3’；
ECT2-F2：
5’-GCCACCACATGGATTGTGATTCTCGCACTAATGGAGTTTTAGAGCTAGAAATAGC-3’；
ECT2-F3：
5’-GCCACCACATGGATTGAACAGAGGGCCTAGGGCCAGTTTTAGAGCTAGAAATAGC-3’；
gRNA-XbaI-R：
5’-ACGCGTTCTAGAAAAAAAAGCACCGACTCGGTGC-3’；
用于扩增U6启动子片段的引物序列为：
U6-Xbal-F：
5’-GCTTAGGCCTTCTAGAAAAATAAATGGTAAAATGTCAA-3’；
U6-R：5’-AATCCATGTGGTGGCACATTTC-3’。
1.2U6启动子和gRNA编码DNA片段扩增
1)反应体系：
2)反应程序：
预热98℃3min；变性98℃15s，退火56℃15s，延伸72℃30s(30-60s/kb)，35个循环；终延伸72℃5min；4℃保存。
以JRH0951载体(包含AtU6启动子序列及sgRNA序列)为模板，利用引物对U6-XbaI-F/U6-R扩增U6启动子片段(序列表中序列3的第17-321位)，分别利用引物ECT2-F1、ECT2-F2、ECT2-F3和gRNA-XbaI-R组成的引物对扩增含有不同gRNA的编码DNA的片段：ECT2-F1/gRNA-XbaI-R引物对扩增gRNA1的编码DNA(序列表中序列3的第322-344位)，ECT2-F2/gRNA-XbaI-R引物对扩增gRNA2的编码DNA(序列表中序列4的第322-344位)，ECT2-F3/gRNA-XbaI-R引物对扩增gRNA3的编码DNA(序列表中序列5的第322-344位)。
1.3、PCR产物回收和产物连接
琼脂糖凝胶回收试剂盒(购自Axygen公司)分别回收U6启动子片段和gRNA编码DNA片段，回收后，按照上述程序，其中Template变为U6启动子片段和gRNA的编码DNA片段，分别利用引物U6-XbaI-F/gRNA-XbaI-R组成的引物对对三种gRNA片段进行扩增，将U6启动子分别与三种gRNA的编码DNA片段连在一起，扩增产物回收得到3种U6-gRNA，即U6-gRNA1(序列表中序列3)、U6-gRNA2(序列表中序列4)和U6-gRNA3(序列表中序列5)。
2、CRISPR-Cas9重组载体GmECT2-gRNA的获得
2.1酶切
利用XbaI酶切CRISPR-Cas9载体pCas9-AtU6-sgRNA(本实验室保存，相关文献:Li,C.et al.A domestication-associated gene GmPRR3b regulates the circadian clockand flowering time in soybean.Mol Plant 13,745–759(2020).公众可从申请人处获得，仅用于重复本发明)，将酶切产物回收得到线性化的pCas9-AtU6-sgRNA载体。
反应体系：
反应条件：37℃水浴，30min。
2.2、In-fusion连接
所用试剂为ClontechHD Enzyme Premix。
将步骤1.3中回收得到的U6-gRNA1、U6-gRNA2、U6-gRNA3三种U6-gRNA片段分别与线性化的pCas9-AtU6-sgRNA载体进行连接，分别得到三种CRISPR-Cas9重组载体GmECT2-gRNA1、GmECT2-gRNA2和GmECT2-gRNA3。
1)反应体系：
In-fusion 0.5μL
DNA片段 1μL
线性化载体片段 1μL
2)反应条件：50℃，30min。
3、CRISPR-Cas9重组载体GmECT2-gRNA质粒扩繁
3.1重组载体转化大肠杆菌TOP10
1)从-80℃取出TOP10感受态细胞(康为世纪)置于冰盒中，待感受态细胞稍微融化后，取50μL到1.5mL EP管中，用移液枪将2.5μL CRISPR-Cas9重组载体GmECT2-gRNA加入其中，轻弹管壁使二者充分混匀，将其在冰中静置约30min；
2)将离心管置于42℃水浴锅中热激90s，然后迅速冰浴2min；
3)在每个离心管加入600μL不含抗生素的LB培养基，混匀后置于37℃摇床，200rpm振荡培养1h；
4)12000rpm瞬时离心，在超净工作台中，取出离心管，去除大部分上清LB培养基，留下约50μL，移液器吹打混匀；
5)将菌液均匀涂在加入卡那霉素抗生素的LB平板培养基上，待平板干了之后，倒置于37℃恒温箱中培养过夜。
3.2、阳性克隆的鉴定
用牙签挑取3.1中37℃恒温过夜平板上的单克隆，在含有抗生素的LB平板划线，然后将牙签在含有PCR反应混合物的管中轻轻搅动几下，使用引物35S-NOS-F1：5’-CGTTATTTATGAGATGGGTT-3’和LZL-S5A-R1：5’-TCTTGCCATACCATTTAG-3’进行菌落PCR鉴定。可以挑取多个单克隆，并在平板上做好标记，以增加获得阳性克隆的概率。之后将平板置于37℃过夜培养。
1)反应体系：(2×Taq MasterMix购自康为公司)
2)反应程序：
预热95℃2min；变性95℃30s，退火57℃30s，延伸72℃30s(2kb/min)，35个循环；终延伸72℃5min；12℃保存。
PCR产物经电泳检测，含有目的片段(607bp，序列6所示的DNA分子，或将序列6的第452-474位的核苷酸替换为序列4的第322-344位核苷酸并保持序列6的其它核苷酸不变得到的DNA分子，或将序列6的第452-474位的核苷酸替换为序列5的第322-344位核苷酸并保持序列6的其它核苷酸不变得到的DNA分子)的克隆，把对应平板上的划线，送菌液测序，测序结果含有序列表中的序列6所示的核苷酸，则对应的单克隆转化菌为阳性单克隆菌。阳性单克隆菌液为含有U6-gRNA1的重组载体GmECT2-gRNA1的阳性单克隆、含有U6-gRNA2重组载体GmECT2-gRNA2的阳性单克隆或含有U6-gRNA3的重组载体GmECT2-gRNA3的阳性单克隆。
3.3、质粒提取
对于测序正确的阳性单克隆，扩摇菌液，用试剂盒提取质粒，步骤如下：
1)取2mL培养过夜的菌液，12000g离心1min，弃尽上清。
2)加250μL Buffer S1(含有RNase)用移液器吹打悬浮细菌沉淀，一定要保证均匀。
3)加入250μL Buffer S2，温和地上下翻转数次混合均匀，使菌体充分裂解，直至形成透亮的溶液。此步骤不宜超过5min，以防质粒DNA被裂解。
4)加入350μL Buffer S3，温和并充分地混合数次，12000g离心10min。
5)吸取上清转移到制备管(置于2mL离心管中)，12000g离心1min，弃滤液。
6)将制备管放回2mL离心管中，加500μL Buffer W1，12000g离心1min，弃滤液。
7)将制备管放回离心管，加700μL Buffer W2，12000g离心1min，弃滤液；重复一次。
8)将制备管放回2mL离心管中，12000g离心1min。
9)将制备管移入干净的1.5mL离心管中，在吸附管膜中央加40μL ddH2O，室温静置1min。12000g离心1min，洗脱质粒DNA，分别得到三种纯化重组载体质粒GmECT2-gRNA1、GmECT2-gRNA2和GmECT2-gRNA3。
实施例2转基因大豆植株的获得
将实施例1中构建好的三种CRISPR-Cas9重组载体质粒GmECT2-gRNA1、GmECT2-gRNA2和GmECT2-gRNA3分别转化K599农杆菌感受态和EHA105农杆菌感受态，每一种重组载体质粒转化得到两种重组农杆菌，分别命名为K599/GmECT2-gRNA1和EHA105/GmECT2-gRNA1、K599/GmECT2-gRNA2和EHA105/GmECT2-gRNA2、K599/GmECT2-gRNA3和EHA105/GmECT2-gRNA3。
1、农杆菌感受态的制备与转化
1)农杆菌感受态的制备
挑取农杆菌K599(ZOMANBIO，货号ZC1506)、EHA105单菌落分别置于5mL含100μg/mL链霉素的LB液体培养基中和5mL含100μg/mL利福平(Rif)的LB液体培养基中(K599抗性为：100μg/mL链霉素；EHA105抗性为：100μg/mL利福平)。28℃培养过夜；取过夜培养菌液500μL接种于50mL LB含相应抗生素的液体培养基中，28℃培养到OD600nm约为0.5；冰上放置30min；4℃，5,000rpm离心10min，用15mL预冷的10mM CaCl2重悬农杆菌细胞，4℃，5,000rpm离心10min；用2mL预冷的10mM CaCl2重悬沉淀，冰上100μL/管分装，液氮速冻，-80℃保存。
2)农杆菌转化
取100μL K599或EHA105感受态细胞冰上解冻，加入1μg实施例1中得到的重组载体GmECT2-gRNA质粒(GmECT2-gRNA1、GmECT2-gRNA2或GmECT2-gRNA3)DNA混匀后，冰上放置30min，液氮速冻3-5min后立即放于37℃水浴5min，加入1mL无抗LB液体培养基，28℃，160rpm复苏3-5h后菌液均匀涂于含相应抗生素的固体培养基上。28℃倒置培养2～3d，挑单菌用PCR鉴定阳性克隆，引物为35S-NOS-F1：5’-CGTTATTTATGAGATGGGTT-3’和LZL-S5A-R1：5’-TCTTGCCATACCATTTAG-3’，阳性克隆菌落的PCR产物为含有607bp的目的片段(序列表中序列6所示的DNA分子，或将序列6的第452-474位的核苷酸替换为序列4的第322-344位核苷酸并保持序列6的其它核苷酸不变得到的DNA分子，或将序列6的第452-474位的核苷酸替换为序列5的第322-344位核苷酸并保持序列6的其它核苷酸不变得到的DNA分子)的单菌落，最终得到重组农杆菌K599/GmECT2-gRNA1和EHA105/GmECT2-gRNA1、K599/GmECT2-gRNA2和EHA105/GmECT2-gRNA2、K599/GmECT2-gRNA3和EHA105/GmECT2-gRNA3。
2、大豆发根检测CRISPR载体编辑效率
(1)种子消毒：挑选出健康，饱满，无病斑的大豆Tianlong 1(本实验室保存，相关文献：关文献:Li,C.et al.A domestication-associated gene GmPRR3b regulates thecircadian clock and flowering time in soybean.Mol Plant 13,745–759(2020).公众可从申请人处获得，仅用于重复本发明)种子。用4mL浓盐酸和100mL消毒水(花王Bleach)反应出的氯气对豆子进行灭菌，16-18小时左右，拿出，在超净工作台中吹干净氯气。
(2)种子催芽：将豆子均匀摆放在催芽培养基上，每皿摆6-8颗左右。(如果其中某一颗豆子染菌，则一板豆子都不要)温室中光照培养三天左右。
(3)切豆子，调菌液OD值：切子叶，取萌发4-7天(5-6天最好)的大豆种子，自下胚轴0.3-0.5cm处切下，将子叶一分为二切开，去除顶芽。取出摇好的重组农杆菌菌液(K599/GmECT2-gRNA1、K599/GmECT2-gRNA2或K599/GmECT2-gRNA3，OD600nm值＝0.6-0.8，一般切豆子前两天小摇，前一天晚上大摇，当天上午可以用)，离心(4000rpm，10min)，液体共培培养基重悬，使菌液OD600nm值＝0.6-0.8，将豆子中加入调好的菌液中，隔几分钟用手摇一次，共浸染15min，取出吹10min左右。
(4)共培养：在共培培养基中铺上已灭菌滤纸，然后将菌液浸染过的种子均匀的摆放在培养基中，黑暗条件下培养三天，温度26℃左右。
(5)诱导培养：暗培3天后，胚芽长长，用无菌水和加入激素的液体诱导培养基各洗4-5次，确保将农杆菌洗净。胚芽朝上斜插入固体诱导培养基中，放入温室中光照培养。在温室培养15天左右，有的豆瓣开始生根。
(6)取根检测：取豆瓣伤口处长出的根，2-3根/管，一般取3个重复，CTAB法提取DNA，进行PCR检测和测序，确定不同gRNA靶位点的编辑效果。检测S11靶位点位点的引物为MT11/13-F/R：5’-ATTATTGTGCAGGCTTTG-3’和5’-ATCTATCAGAAAAGATGGAC-3’；检测S18和S20靶位点的引物为MT18/20-F/R：5’-TGGTCCATCTTTTCTGATAG-3’和5’-CTGCCATCATTCACAGATA-3’，测序结果显示三个gRNA靶位点处的碱基均为双峰，表明三种CRISPR-Cas9重组载体GmECT2-gRNA1、GmECT2-gRNA2和GmECT2-gRNA3能够对三个靶位点发生有效编辑。
3、重组农杆菌转化大豆
(1)用浓盐酸和次氯酸钠反应出的氯气灭菌的豆子Tianlong 1(以下简称野生型或TL1)；大摇菌液，活化步骤1中得到的三种重组农杆菌EHA105/GmECT2-gRNA1、EHA105/GmECT2-gRNA2和EHA105/GmECT2-gRNA3。
(2)将豆子对半切开，去掉一部分胚尖，在豆子的分生区部分划伤口，泡在无菌水中。下午取出摇好的三种重组农杆菌菌液，离心(4000rpm，10min)，调菌，使菌液OD600nm值＝0.4-0.6，将豆子中的无菌水倒掉，分别加入调好的不同的三种重组农杆菌菌液，放在摇床中摇30min(28℃，200prm左右)，取出吹10min左右，平铺在共培培养基中暗培3天。
(3)暗培3天后，胚芽伸长，用无菌水和加入激素的液体诱导培养基进行脱菌处理，一般清洗4-5次，确保将农杆菌洗净。
(4)将长出的胚芽切掉，只保留3-4mm长度，胚芽朝下，有伤口的一面朝上斜插入固体诱导培养基中，放入温室中光照培养。
(5)在温室培养10天后，有的豆瓣开始出芽，有芽的从桩部将芽切掉并转到新的固体诱导培养基中，没长芽的扔掉。
(6)温室中培养10天后，将长芽的继代到新的固体诱导培养基中，没有长芽的豆瓣扔掉，温室培养10天。豆瓣在固体诱导培养基中一共培养30天。
(7)将长好的愈伤组织与豆瓣分离，扔掉豆瓣，把愈伤组织上黑色表面刮掉，转到固体伸长培养基中，每20天更换一次新的固体伸长培养基，一般继代3-4次，合计60-80天。
(8)愈伤在伸长培养的同时也在筛选，在筛选的过程中会有苗长出来。
(9)当苗长到超过100mL刻度的时候从愈伤上切下来，移到生根培养基中。
(10)苗子在培养基中培养20-30天左右，长得壮实有发达根系的苗子就可以开始放在背光处炼苗了，一般炼苗五天。
(11)每种一棵转基因苗就标上豆子品种，基因名称，生根日期和土培日期，以及培养人的名字。加入适量水，绿肥和缓释肥，盖上一层薄膜放在光照下，使其适应强光，3天后去膜。得到T0代转基因株系单株。
实施例3转基因阳性株系的鉴定
为了确定转基因阳性植株，取实施例2的T0代转基因株系单株的叶片，提取DNA，进行PCR检测。对于转基因CRISPR敲除突变体，检测其含有Basta抗性基因片段(序列表中序列7)、Cas9蛋白编码基因片段(序列表中序列8)和U6启动子(序列表中序列3的第17-321位核苷酸)。并对其gRNA所在位置进行PCR扩增和测序，扩增引物为MT11/13-F/R和MT18/20-F/R。
表1转基因阳性株系的鉴定引物
PCR检测结果和测序结果显示，T0代转基因大豆植株中发生基因编辑的植株为4株。将T0代基因编辑的植株分别自交2代，得到T2代基因编辑的植株(即由导入pGmECT2-gRNA得到的T0代转基因大豆植株自交2代得到的T2代基因编辑的植株)。之后按照上述PCR方法检测T2代基因编辑的植株的突变类型。共获得4个纯合突变体：Gmect2-11-1-4-2、Gmect2-18-1-4-1、Gmect2-20-2-6-4和Gmect2-20-3-7-7-2。4个纯合突变体的检测结果见图1(B为GmECT2基因结构示意图，C为具体编辑方式)。
Gmect2-11-1-4-2与野生型相比，对于GmECT2基因，2条同源染色体中GmECT2均突变为Gmect2-11-1-4-2基因，Gmect2-11-1-4-2基因是将大豆中的序列表中序列2所述蛋白编码基因突变为Gmect2-11-1-4-2，Gmect2-11-1-4-2是将序列表中序列2的第509-515位的7个核苷酸“CCAGTGC”缺失并保持序列表中序列2的其他核苷酸不变得到的DNA分子，核苷酸的缺失引起了移码，导致翻译提前终止，造成GmECT2蛋白功能缺失。
上述Gmect2-11-1-4-2突变体中突变后的Gmect2-11-1-4-2蛋白质的氨基酸序列如序列10所示，其编码序列如序列9所示。
Gmect2-18-1-4-1与野生型相比，对于GmECT2基因，2条同源染色体中GmECT2均突变为Gmect2-18-1-4-1基因，Gmect2-18-1-4-1基因是将大豆中的序列表中序列2所述蛋白编码基因突变为Gmect2-18-1-4-1，Gmect2-18-1-4-1是将序列表中序列2的第1083-1090位的8个核苷酸“TAATGGAC”缺失并保持序列表中序列2的其他核苷酸不变得到的DNA分子，核苷酸的缺失引起了移码，导致翻译提前终止，造成GmECT2蛋白功能缺失。
上述Gmect2-18-1-4-1突变体中突变后的Gmect2-18-1-4-1蛋白质的氨基酸序列如序列12所示，其编码序列如序列11所示。
Gmect2-20-2-6-4与野生型相比，对于GmECT2基因，2条同源染色体中GmECT2均突变为Gmect2-20-2-6-4基因，Gmect2-20-2-6-4基因是将大豆中的序列表中序列2所述蛋白编码基因突变为Gmect2-20-2-6-4，Gmect2-20-2-6-4是将序列表中序列2的第1200-1206位的7个核苷酸“TAGGGCC”缺失并保持序列表中序列2的其他核苷酸不变得到的DNA分子，核苷酸的缺失引起了移码，导致翻译提前终止，造成GmECT2蛋白功能缺失。
上述Gmect2-20-2-6-4突变体中突变后的Gmect2-20-2-6-4蛋白质的氨基酸序列如序列14所示，其编码序列如序列13所示。
Gmect2-20-3-7-7-2与野生型相比，对于GmECT2基因，2条同源染色体中GmECT2均突变为Gmect2-20-3-7-7-2，Gmect2-20-3-7-7-2基因是将大豆中的序列表中序列2所述蛋白编码基因突变为Gmect2-20-3-7-7-2，Gmect2-20-3-7-7-2是将序列表中序列2的第1204-1205位的2个核苷酸之间插入了1个核苷酸“A”并保持序列表中序列2的其他核苷酸不变得到的DNA分子，核苷酸的缺失引起了移码，导致翻译提前终止，造成GmECT2蛋白功能缺失。
上述Gmect2-20-3-7-7-2突变体中突变后的Gmect2-20-3-7-7-2蛋白质的氨基酸序列如序列16所示，其编码序列如序列15所示。
实施例4GmECT2基因敲除突变体大豆表型鉴定
将实施例3中得到的GmECT2基因的4个纯和突变体单株和野生型Tian-Long1大豆种子直接播种在中国农业科学院103温室(中日照，12h L、12h D)中，采用随机区组设计，3次重复。每个重复区随机设置5个小区，分别为TL1处理区、Gmect2-11-1-4-2处理区、Gmect2-18-1-4-1处理区、Gmect2-20-2-6-4处理区、和Gmect2-20-3-7-7-2处理区。各个处理区除播种的种子不同外，其它农业措施均相同。测定苗期(株龄10天)和成熟期(株龄29天)的株高，温室温度27℃、光照350μmol m-2s-1。待收获后，对种子进行拍照。
实验结果如图1所示，Gmect2-11-1-4-2、Gmect2-18-1-4-1、Gmect2-20-2-6-4和Gmect2-20-3-7-7-2突变体与野生型大豆相比，在苗期(株龄10天)和株龄29天均表现出植株高度明显矮于野生型P＜0.05(*)，P＜0.01(**)(图1中TL1)的表型，因此，GmECT基因在调控大豆株高中发挥重要的作用，当该基因发生突变，植株高度明显变矮。10d和29d两个不同阶段的两次株高统计和拍照为两次独立实验，Gmect2突变体均表现出株高变矮的表型。
因此，大豆GmECT2蛋白具有调控植株高度方面的功能，通过降低或抑制GmECT2蛋白的表达量，可以降低大豆植株的株高。
以上对本发明进行了详述。对于本领域技术人员来说，在不脱离本发明的宗旨和范围，以及无需进行不必要的实验情况下，可在等同参数、浓度和条件下，在较宽范围内实施本发明。虽然本发明给出了特殊的实施例，应该理解为，可以对本发明作进一步的改进。总之，按本发明的原理，本申请欲包括任何变更、用途或对本发明的改进，包括脱离了本申请中已公开范围，而用本领域已知的常规技术进行的改变。按以下附带的权利要求的范围，可以进行一些基本特征的应用。
序列表
<110> 中国农业科学院作物科学研究所
<120> GmECT2在调控植物高度方面的应用
<130> GNCSQ212347
<160> 16
<170> PatentIn version 3.5
<210> 1
<211> 708
<212> PRT
<213> 大豆（Glycine max）
<400> 1
Met Ala Thr Val Ala Asn Pro Ala Asp Gln Ala Thr Asp Leu Leu Gln
1 5 10 15
Lys Leu Ser Leu Glu Thr Gln Pro Lys Pro Leu Glu Ile Pro Glu Pro
20 25 30
Thr Lys Lys Ala Thr Gly Asn Gln Tyr Gly Ser Val Asp Ser Gly Asn
35 40 45
Ala Ala Asn Gly Gln Ile Gln Ser Tyr Asp Arg Ser Val Thr Pro Val
50 55 60
Leu Gln Asp Phe Ile Asp Pro Thr Met Cys Tyr Leu Pro Asn Gly Tyr
65 70 75 80
Pro Ser Thr Ala Tyr Tyr Tyr Gly Gly Tyr Asp Gly Thr Gly Asn Glu
85 90 95
Trp Asp Glu Tyr Ser Arg Tyr Val Asn Ser Glu Gly Val Glu Met Thr
100 105 110
Ser Gly Val Tyr Gly Asp Asn Gly Ser Leu Leu Tyr His His Gly Tyr
115 120 125
Gly Tyr Ala Pro Tyr Gly Pro Tyr Ser Pro Ala Gly Ser Pro Val Pro
130 135 140
Thr Met Gly Asn Asp Gly Gln Leu Tyr Gly Pro Gln His Tyr Gln Tyr
145 150 155 160
Pro Pro Tyr Phe Gln Pro Leu Thr Pro Thr Ser Ala Pro Phe Thr Pro
165 170 175
Thr Pro Ala Val Leu Pro Gln Gly Glu Val Ser Thr Ser Val Ala Ala
180 185 190
Asp Gln Lys Pro Leu Pro Val Asp Ala Ala Asn Gly Asn Ser Asn Gly
195 200 205
Val Ala Asn Gly Gly Asn Ala Lys Gly Asn Asn Ala Ala Ala Ser Ile
210 215 220
Lys Gln Ala Asn Gln Asn Ser Ser Phe Ser Ser Lys Ala Ser Asn Glu
225 230 235 240
Arg Val Thr Met Pro Gly Arg Gly Pro Thr Ser Gly Tyr Gln Asp Pro
245 250 255
Arg Phe Gly Phe Asp Gly Val Arg Ser Pro Ile Pro Trp Leu Asp Ala
260 265 270
Pro Leu Phe Ser Asp Gly Gln Pro Arg Pro Val Ser Ser Thr Thr Ile
275 280 285
Thr Ser Ser Ile Ser Gly Gly Asn Asn Thr Ala Ser Arg Asn Pro Thr
290 295 300
Phe Arg Pro Asn Ser Gln Phe Met Gly Leu His His Pro Arg Pro Met
305 310 315 320
Pro Ala Met Gly Ala Thr His Ser Phe Ile Asn Arg Met Tyr Pro Ser
325 330 335
Lys Leu Tyr Gly Gln Tyr Gly Asn Thr Val Arg Ser Gly Met Gly Tyr
340 345 350
Gly Thr His Gly Tyr Asp Ser Arg Thr Asn Gly Arg Ala Trp Leu Ala
355 360 365
Val Asp Ser Lys Tyr Lys Thr Arg Gly Arg Ser Gly Gly Tyr Phe Gly
370 375 380
Tyr Gly Asn Glu Asn Ala Asp Gly Leu Asn Glu Leu Asn Arg Gly Pro
385 390 395 400
Arg Ala Lys Gly Gly Lys Asn Gln Lys Gly Phe Ala Pro Thr Ile Leu
405 410 415
Ala Val Lys Gly Gln Thr Leu Pro Ala Thr Leu Gly Thr Asp Glu Glu
420 425 430
Lys Asp Lys Thr Ser Thr Ile Leu Glu Cys Asp Gln Tyr Asn Lys Ala
435 440 445
Asp Phe Pro Glu Glu Tyr Thr Asp Ala Lys Phe Phe Val Ile Lys Ser
450 455 460
Tyr Ser Glu Asp Asp Ile His Lys Ser Ile Lys Tyr Asn Val Trp Ala
465 470 475 480
Ser Thr Gln Asn Gly Asn Lys Lys Leu Asp Ala Ala Tyr Gln Glu Ala
485 490 495
Gln Gln Lys Pro Gly Gly Thr Pro Val Phe Leu Phe Phe Ser Val Asn
500 505 510
Thr Ser Gly Gln Phe Val Gly Leu Ala Glu Met Ile Gly Pro Val Asp
515 520 525
Phe Asn Lys Ser Val Glu Tyr Trp Gln Gln Asp Lys Trp Asn Gly Cys
530 535 540
Phe Pro Leu Lys Trp His Ile Val Lys Asp Val Pro Asn Asn Leu Leu
545 550 555 560
Arg His Ile Thr Leu Asp Asn Asn Glu Asn Lys Pro Val Thr Asn Ser
565 570 575
Arg Asp Thr Gln Glu Val Met Leu Glu Pro Gly Leu Lys Leu Ile Lys
580 585 590
Ile Phe Lys Glu Tyr Thr Ser Lys Thr Cys Ile Leu Asp Asp Phe Gly
595 600 605
Phe Tyr Glu Ala Arg Gln Lys Thr Ile Leu Glu Lys Lys Ala Lys Gln
610 615 620
Gln Phe Pro Lys Gln Val Trp Glu Gly Lys Pro Ala Asp Glu Lys Ile
625 630 635 640
Glu Ile Asn Gly Glu Val Asn Thr Gln Lys Ser Glu Val Ser Ser Glu
645 650 655
Leu Leu Lys Glu Ser Thr Leu Ala Glu Lys Asp Ser Asp Asp His Lys
660 665 670
Val Pro Glu Asn Gly Ser Ala Thr Lys Thr Gly Asp Ala Pro Lys Gly
675 680 685
Ala Lys Pro Val Val Pro Glu Ser Lys Ile Val Ala Asn Gly Val Val
690 695 700
Ser Asn Gly Val
705
<210> 2
<211> 2127
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 2
atggccaccg ttgctaatcc cgcggatcaa gcaactgatt tgctacagaa gctatcgtta 60
gaaactcagc ccaagccctt ggagattcct gagcctacca aaaaggccac tgggaatcag 120
tatggatcag ttgattcagg aaatgccgcg aatggccaga tccagtcgta tgatcggtct 180
gtaactcccg tgttgcagga ttttattgat cctactatgt gttacctccc aaatggttac 240
ccatctactg cctattatta cggtggttat gatggaactg gtaacgagtg ggatgagtat 300
tcaagatatg tgaattcgga aggagttgag atgacttcag gagtttatgg ggataatggg 360
tctctacttt atcaccatgg atatggatat gcgccttatg gcccctattc gccagcaggg 420
tctccagttc caaccatggg gaatgatggt cagttgtatg ggcctcaaca ctatcagtat 480
cctccatatt tccaaccgtt aacaccaacc agtgcgccat tcacacctac tcctgctgtc 540
ctacctcagg gtgaggtttc cacctctgta gctgctgatc aaaagcctct ccctgtcgat 600
gcagccaatg gaaattctaa tggtgttgcg aatggtggga atgcaaaagg taataatgct 660
gctgcttcta ttaaacaggc caatcagaat tcatctttta gttccaaagc atcgaatgaa 720
agggttacca tgccaggtcg gggtccaact tcaggttatc aggatccaag atttggtttt 780
gatggagtac gctcacctat cccctggcta gatgccccac tattttcaga tgggcagcca 840
aggcctgtaa gtagcacaac catcacttct tcgatatcag gtggcaacaa cactgcctca 900
aggaacccaa ctttccgccc taattctcag tttatgggct tgcaccaccc aagaccaatg 960
cctgccatgg gagccaccca tagcttcata aataggatgt acccaagcaa actatatggt 1020
caatatggga acactgtcag atctggaatg ggttatggta cacatgggta tgattctcgc 1080
actaatggac gggcttggtt agctgttgac agtaaataca aaactagggg aagaagtggt 1140
ggctactttg gctatggcaa tgagaacgca gatggtttga acgaactgaa cagagggcct 1200
agggccaagg gtggcaaaaa ccaaaaaggt tttgcaccaa ctattctggc agtcaaaggg 1260
cagactttgc ctgcaacttt aggcacagat gaagagaaag acaagactag taccattctg 1320
gaatgtgatc aatataacaa agctgatttt ccagaagaat atactgatgc caaatttttt 1380
gtcatcaagt cttacagcga ggatgatatc cataagagta ttaagtataa tgtgtgggct 1440
agtacacaaa atggcaacaa gaagcttgat gctgcatatc aggaggcaca gcagaaacct 1500
ggcggcaccc ctgtttttct ctttttctca gttaatacca gtgggcaatt tgtggggctt 1560
gctgagatga ttggtcctgt tgattttaac aagagtgttg agtattggca gcaagacaag 1620
tggaatggtt gctttcctct taagtggcac attgttaagg atgttcctaa caatttgttg 1680
aggcacatta ccttggacaa caatgagaac aaacctgtca ctaacagtag ggatacacaa 1740
gaggttatgt tggagcctgg gctgaaatta attaaaattt tcaaggaata taccagcaag 1800
acatgcattt tggatgattt tggcttctat gaggctcgtc agaaaactat tttggagaag 1860
aaagcaaagc aacaattccc aaagcaggta tgggaaggga aacctgctga tgagaagatt 1920
gagataaatg gggaagttaa tactcaaaaa tctgaagtta gctcggaatt gctcaaggag 1980
tctacccttg ctgagaagga tagtgatgac cacaaagttc ccgagaatgg atctgctaca 2040
aaaactggag atgccccaaa gggtgctaaa ccagttgttc ccgagagtaa gattgtagcc 2100
aatggggttg tttctaatgg tgtctaa 2127
<210> 3
<211> 438
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 3
gcttaggcct tctagaaaaa taaatggtaa aatgtcaaat caaaactagg ctgcagtatg 60
cagagcagag tcatgatgat actacttact acaccgattc ttgtgtgcag aaaaatatgt 120
taaaataatt gaatctttct ctagccaaat ttgacaacaa tgtacaccgt tcatattgag 180
agacgatgct tcttgtttgc tttcggtgga agctgcatat actcaacatt actccttcag 240
cgagttttcc aactgagtcc cacattgccc agacctaaca cggtattctt gtttataatg 300
aaatgtgcca ccacatggat tccaaccagt gcgccattca caccgtttta gagctagaaa 360
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 420
tttttttcta gaacgcgt 438
<210> 4
<211> 438
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 4
gcttaggcct tctagaaaaa taaatggtaa aatgtcaaat caaaactagg ctgcagtatg 60
cagagcagag tcatgatgat actacttact acaccgattc ttgtgtgcag aaaaatatgt 120
taaaataatt gaatctttct ctagccaaat ttgacaacaa tgtacaccgt tcatattgag 180
agacgatgct tcttgtttgc tttcggtgga agctgcatat actcaacatt actccttcag 240
cgagttttcc aactgagtcc cacattgccc agacctaaca cggtattctt gtttataatg 300
aaatgtgcca ccacatggat tatgattctc gcactaatgg acgggtttta gagctagaaa 360
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 420
tttttttcta gaacgcgt 438
<210> 5
<211> 438
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 5
gcttaggcct tctagaaaaa taaatggtaa aatgtcaaat caaaactagg ctgcagtatg 60
cagagcagag tcatgatgat actacttact acaccgattc ttgtgtgcag aaaaatatgt 120
taaaataatt gaatctttct ctagccaaat ttgacaacaa tgtacaccgt tcatattgag 180
agacgatgct tcttgtttgc tttcggtgga agctgcatat actcaacatt actccttcag 240
cgagttttcc aactgagtcc cacattgccc agacctaaca cggtattctt gtttataatg 300
aaatgtgcca ccacatggat tgaacagagg gcctagggcc aagggtttta gagctagaaa 360
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 420
tttttttcta gaacgcgt 438
<210> 6
<211> 607
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 6
cgttatttat gagatgggtt tttatgatta gagtcccgca attatacatt taatacgcga 60
tagaaaacaa aatatagcgc gcaaactagg ataaattatc gcgcgcggtg tcatctatgt 120
tactagatcg gaagcttagg ccttctagaa aaataaatgg taaaatgtca aatcaaaact 180
aggctgcagt atgcagagca gagtcatgat gatactactt actacaccga ttcttgtgtg 240
cagaaaaata tgttaaaata attgaatctt tctctagcca aatttgacaa caatgtacac 300
cgttcatatt gagagacgat gcttcttgtt tgctttcggt ggaagctgca tatactcaac 360
attactcctt cagcgagttt tccaactgag tcccacattg cccagaccta acacggtatt 420
cttgtttata atgaaatgtg ccaccacatg gccaaccagt gcgccattca caccgtttta 480
gagctagaaa tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc 540
gagtcggtgc ttttttttct agaacgcgtt gccgagcaca attgatcggc taaatggtat 600
ggcaaga 607
<210> 7
<211> 989
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 7
acagataaag ccacgcacat ttaggatatt ggccgagatt actgaatatt gagtaagatc 60
acggaatttc tgacaggagc atgtcttcaa ttcagcccaa atggcagttg aaatactcaa 120
accgccccat atgcaggagc ggatcattca ttgtttgttt ggttgccttt gccaacatgg 180
gagtccaaga ttctgcagtc aaatctcggt gacgggcagg accggacggg gcggtaccgg 240
caggctgaag tccagctgcc agaaacccac gtcatgccag ttcccgtgct tgaagccggc 300
cgcccgcagc atgccgcggg gggcatatcc gagcgcctcg tgcatgcgca cgctcgggtc 360
gttgggcagc ccgatgacag cgaccacgct cttgaagccc tgtgcctcca gggacttcag 420
caggtgggtg tagagcgtgg agcccagtcc cgtccgctgg tggcgggggg agacgtacac 480
ggtcgactcg gccgtccagt cgtaggcgtt gcgtgccttc caggggcccg cgtaggcgat 540
gccggcgacc tcgccgtcca cctcggcgac gagccaggga tagcgctccc gcagacggac 600
gaggtcgtcc gtccactcct gcggttcctg cggctcggta cggaagttga ccgtgcttgt 660
ctcgatgtag tggttgacga tggtgcagac cgccggcatg tccgcctcgg tggcacggcg 720
gatgtcggcc gggcgtcgtt ctgggctcat cgattcgatt tggtgtatcg agattggtta 780
tgaaattcag atgctagtgt aatgtattgg taatttggga agatataata ggaagcaagg 840
ctatttatcc atttctgaaa aggcgaaatg gcgtcaccgc gagcgtcacg cgcattccgt 900
tcttgctgta aagcgttgtt tggtacactt ttgactagcg aggcttggcg tgtcagcgta 960
tctattcaaa agtcgttaat ggctgcgga 989
<210> 8
<211> 601
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 8
cagctcgtcc aaacctacaa tcagctcttt gaggaaaacc caattaatgc ttcaggcgtc 60
gacgccaagg cgatcctgtc tgcacgcctt tcaaagtctc gccggcttga gaacttgatc 120
gctcaactcc cgggcgaaaa gaagaacggc ttgttcggga atctcattgc actttcgttg 180
gggctcacac caaacttcaa gagtaatttt gatctcgctg aggacgcaaa gctgcagctt 240
tccaaggaca cttatgacga tgacctggat aaccttttgg cccaaatcgg cgatcagtac 300
gcggacttgt tcctcgccgc gaagaatttg tcggacgcga tcctcctgag tgatattctc 360
cgcgtgaaca ccgagattac aaaggccccg ctctcggcga gtatgatcaa gcgctatgac 420
gagcaccatc aggatctgac ccttttgaag gctttggtcc ggcagcaact cccagagaag 480
tacaaggaaa tcttctttga tcaatccaag aacggctacg ctggttatat tgacggcggg 540
gcatcgcagg aggaattcta caagtttatc aagccaattc tggagaagat ggatggcaca 600
g 601
<210> 9
<211> 2120
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 9
atggccaccg ttgctaatcc cgcggatcaa gcaactgatt tgctacagaa gctatcgtta 60
gaaactcagc ccaagccctt ggagattcct gagcctacca aaaaggccac tgggaatcag 120
tatggatcag ttgattcagg aaatgccgcg aatggccaga tccagtcgta tgatcggtct 180
gtaactcccg tgttgcagga ttttattgat cctactatgt gttacctccc aaatggttac 240
ccatctactg cctattatta cggtggttat gatggaactg gtaacgagtg ggatgagtat 300
tcaagatatg tgaattcgga aggagttgag atgacttcag gagtttatgg ggataatggg 360
tctctacttt atcaccatgg atatggatat gcgccttatg gcccctattc gccagcaggg 420
tctccagttc caaccatggg gaatgatggt cagttgtatg ggcctcaaca ctatcagtat 480
cctccatatt tccaaccgtt aacaccaagc cattcacacc tactcctgct gtcctacctc 540
agggtgaggt ttccacctct gtagctgctg atcaaaagcc tctccctgtc gatgcagcca 600
atggaaattc taatggtgtt gcgaatggtg ggaatgcaaa aggtaataat gctgctgctt 660
ctattaaaca ggccaatcag aattcatctt ttagttccaa agcatcgaat gaaagggtta 720
ccatgccagg tcggggtcca acttcaggtt atcaggatcc aagatttggt tttgatggag 780
tacgctcacc tatcccctgg ctagatgccc cactattttc agatgggcag ccaaggcctg 840
taagtagcac aaccatcact tcttcgatat caggtggcaa caacactgcc tcaaggaacc 900
caactttccg ccctaattct cagtttatgg gcttgcacca cccaagacca atgcctgcca 960
tgggagccac ccatagcttc ataaatagga tgtacccaag caaactatat ggtcaatatg 1020
ggaacactgt cagatctgga atgggttatg gtacacatgg gtatgattct cgcactaatg 1080
gacgggcttg gttagctgtt gacagtaaat acaaaactag gggaagaagt ggtggctact 1140
ttggctatgg caatgagaac gcagatggtt tgaacgaact gaacagaggg cctagggcca 1200
agggtggcaa aaaccaaaaa ggttttgcac caactattct ggcagtcaaa gggcagactt 1260
tgcctgcaac tttaggcaca gatgaagaga aagacaagac tagtaccatt ctggaatgtg 1320
atcaatataa caaagctgat tttccagaag aatatactga tgccaaattt tttgtcatca 1380
agtcttacag cgaggatgat atccataaga gtattaagta taatgtgtgg gctagtacac 1440
aaaatggcaa caagaagctt gatgctgcat atcaggaggc acagcagaaa cctggcggca 1500
cccctgtttt tctctttttc tcagttaata ccagtgggca atttgtgggg cttgctgaga 1560
tgattggtcc tgttgatttt aacaagagtg ttgagtattg gcagcaagac aagtggaatg 1620
gttgctttcc tcttaagtgg cacattgtta aggatgttcc taacaatttg ttgaggcaca 1680
ttaccttgga caacaatgag aacaaacctg tcactaacag tagggataca caagaggtta 1740
tgttggagcc tgggctgaaa ttaattaaaa ttttcaagga atataccagc aagacatgca 1800
ttttggatga ttttggcttc tatgaggctc gtcagaaaac tattttggag aagaaagcaa 1860
agcaacaatt cccaaagcag gtatgggaag ggaaacctgc tgatgagaag attgagataa 1920
atggggaagt taatactcaa aaatctgaag ttagctcgga attgctcaag gagtctaccc 1980
ttgctgagaa ggatagtgat gaccacaaag ttcccgagaa tggatctgct acaaaaactg 2040
gagatgcccc aaagggtgct aaaccagttg ttcccgagag taagattgta gccaatgggg 2100
ttgtttctaa tggtgtctaa 2120
<210> 10
<211> 187
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 10
Met Ala Thr Val Ala Asn Pro Ala Asp Gln Ala Thr Asp Leu Leu Gln
1 5 10 15
Lys Leu Ser Leu Glu Thr Gln Pro Lys Pro Leu Glu Ile Pro Glu Pro
20 25 30
Thr Lys Lys Ala Thr Gly Asn Gln Tyr Gly Ser Val Asp Ser Gly Asn
35 40 45
Ala Ala Asn Gly Gln Ile Gln Ser Tyr Asp Arg Ser Val Thr Pro Val
50 55 60
Leu Gln Asp Phe Ile Asp Pro Thr Met Cys Tyr Leu Pro Asn Gly Tyr
65 70 75 80
Pro Ser Thr Ala Tyr Tyr Tyr Gly Gly Tyr Asp Gly Thr Gly Asn Glu
85 90 95
Trp Asp Glu Tyr Ser Arg Tyr Val Asn Ser Glu Gly Val Glu Met Thr
100 105 110
Ser Gly Val Tyr Gly Asp Asn Gly Ser Leu Leu Tyr His His Gly Tyr
115 120 125
Gly Tyr Ala Pro Tyr Gly Pro Tyr Ser Pro Ala Gly Ser Pro Val Pro
130 135 140
Thr Met Gly Asn Asp Gly Gln Leu Tyr Gly Pro Gln His Tyr Gln Tyr
145 150 155 160
Pro Pro Tyr Phe Gln Pro Leu Thr Pro Ser His Ser His Leu Leu Leu
165 170 175
Leu Ser Tyr Leu Arg Val Arg Phe Pro Pro Leu
180 185
<210> 11
<211> 2119
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 11
atggccaccg ttgctaatcc cgcggatcaa gcaactgatt tgctacagaa gctatcgtta 60
gaaactcagc ccaagccctt ggagattcct gagcctacca aaaaggccac tgggaatcag 120
tatggatcag ttgattcagg aaatgccgcg aatggccaga tccagtcgta tgatcggtct 180
gtaactcccg tgttgcagga ttttattgat cctactatgt gttacctccc aaatggttac 240
ccatctactg cctattatta cggtggttat gatggaactg gtaacgagtg ggatgagtat 300
tcaagatatg tgaattcgga aggagttgag atgacttcag gagtttatgg ggataatggg 360
tctctacttt atcaccatgg atatggatat gcgccttatg gcccctattc gccagcaggg 420
tctccagttc caaccatggg gaatgatggt cagttgtatg ggcctcaaca ctatcagtat 480
cctccatatt tccaaccgtt aacaccaacc agtgcgccat tcacacctac tcctgctgtc 540
ctacctcagg gtgaggtttc cacctctgta gctgctgatc aaaagcctct ccctgtcgat 600
gcagccaatg gaaattctaa tggtgttgcg aatggtggga atgcaaaagg taataatgct 660
gctgcttcta ttaaacaggc caatcagaat tcatctttta gttccaaagc atcgaatgaa 720
agggttacca tgccaggtcg gggtccaact tcaggttatc aggatccaag atttggtttt 780
gatggagtac gctcacctat cccctggcta gatgccccac tattttcaga tgggcagcca 840
aggcctgtaa gtagcacaac catcacttct tcgatatcag gtggcaacaa cactgcctca 900
aggaacccaa ctttccgccc taattctcag tttatgggct tgcaccaccc aagaccaatg 960
cctgccatgg gagccaccca tagcttcata aataggatgt acccaagcaa actatatggt 1020
caatatggga acactgtcag atctggaatg ggttatggta cacatgggta tgattctcgc 1080
acgggcttgg ttagctgttg acagtaaata caaaactagg ggaagaagtg gtggctactt 1140
tggctatggc aatgagaacg cagatggttt gaacgaactg aacagagggc ctagggccaa 1200
gggtggcaaa aaccaaaaag gttttgcacc aactattctg gcagtcaaag ggcagacttt 1260
gcctgcaact ttaggcacag atgaagagaa agacaagact agtaccattc tggaatgtga 1320
tcaatataac aaagctgatt ttccagaaga atatactgat gccaaatttt ttgtcatcaa 1380
gtcttacagc gaggatgata tccataagag tattaagtat aatgtgtggg ctagtacaca 1440
aaatggcaac aagaagcttg atgctgcata tcaggaggca cagcagaaac ctggcggcac 1500
ccctgttttt ctctttttct cagttaatac cagtgggcaa tttgtggggc ttgctgagat 1560
gattggtcct gttgatttta acaagagtgt tgagtattgg cagcaagaca agtggaatgg 1620
ttgctttcct cttaagtggc acattgttaa ggatgttcct aacaatttgt tgaggcacat 1680
taccttggac aacaatgaga acaaacctgt cactaacagt agggatacac aagaggttat 1740
gttggagcct gggctgaaat taattaaaat tttcaaggaa tataccagca agacatgcat 1800
tttggatgat tttggcttct atgaggctcg tcagaaaact attttggaga agaaagcaaa 1860
gcaacaattc ccaaagcagg tatgggaagg gaaacctgct gatgagaaga ttgagataaa 1920
tggggaagtt aatactcaaa aatctgaagt tagctcggaa ttgctcaagg agtctaccct 1980
tgctgagaag gatagtgatg accacaaagt tcccgagaat ggatctgcta caaaaactgg 2040
agatgcccca aagggtgcta aaccagttgt tcccgagagt aagattgtag ccaatggggt 2100
tgtttctaat ggtgtctaa 2119
<210> 12
<211> 366
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 12
Met Ala Thr Val Ala Asn Pro Ala Asp Gln Ala Thr Asp Leu Leu Gln
1 5 10 15
Lys Leu Ser Leu Glu Thr Gln Pro Lys Pro Leu Glu Ile Pro Glu Pro
20 25 30
Thr Lys Lys Ala Thr Gly Asn Gln Tyr Gly Ser Val Asp Ser Gly Asn
35 40 45
Ala Ala Asn Gly Gln Ile Gln Ser Tyr Asp Arg Ser Val Thr Pro Val
50 55 60
Leu Gln Asp Phe Ile Asp Pro Thr Met Cys Tyr Leu Pro Asn Gly Tyr
65 70 75 80
Pro Ser Thr Ala Tyr Tyr Tyr Gly Gly Tyr Asp Gly Thr Gly Asn Glu
85 90 95
Trp Asp Glu Tyr Ser Arg Tyr Val Asn Ser Glu Gly Val Glu Met Thr
100 105 110
Ser Gly Val Tyr Gly Asp Asn Gly Ser Leu Leu Tyr His His Gly Tyr
115 120 125
Gly Tyr Ala Pro Tyr Gly Pro Tyr Ser Pro Ala Gly Ser Pro Val Pro
130 135 140
Thr Met Gly Asn Asp Gly Gln Leu Tyr Gly Pro Gln His Tyr Gln Tyr
145 150 155 160
Pro Pro Tyr Phe Gln Pro Leu Thr Pro Thr Ser Ala Pro Phe Thr Pro
165 170 175
Thr Pro Ala Val Leu Pro Gln Gly Glu Val Ser Thr Ser Val Ala Ala
180 185 190
Asp Gln Lys Pro Leu Pro Val Asp Ala Ala Asn Gly Asn Ser Asn Gly
195 200 205
Val Ala Asn Gly Gly Asn Ala Lys Gly Asn Asn Ala Ala Ala Ser Ile
210 215 220
Lys Gln Ala Asn Gln Asn Ser Ser Phe Ser Ser Lys Ala Ser Asn Glu
225 230 235 240
Arg Val Thr Met Pro Gly Arg Gly Pro Thr Ser Gly Tyr Gln Asp Pro
245 250 255
Arg Phe Gly Phe Asp Gly Val Arg Ser Pro Ile Pro Trp Leu Asp Ala
260 265 270
Pro Leu Phe Ser Asp Gly Gln Pro Arg Pro Val Ser Ser Thr Thr Ile
275 280 285
Thr Ser Ser Ile Ser Gly Gly Asn Asn Thr Ala Ser Arg Asn Pro Thr
290 295 300
Phe Arg Pro Asn Ser Gln Phe Met Gly Leu His His Pro Arg Pro Met
305 310 315 320
Pro Ala Met Gly Ala Thr His Ser Phe Ile Asn Arg Met Tyr Pro Ser
325 330 335
Lys Leu Tyr Gly Gln Tyr Gly Asn Thr Val Arg Ser Gly Met Gly Tyr
340 345 350
Gly Thr His Gly Tyr Asp Ser Arg Thr Gly Leu Val Ser Cys
355 360 365
<210> 13
<211> 2120
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 13
atggccaccg ttgctaatcc cgcggatcaa gcaactgatt tgctacagaa gctatcgtta 60
gaaactcagc ccaagccctt ggagattcct gagcctacca aaaaggccac tgggaatcag 120
tatggatcag ttgattcagg aaatgccgcg aatggccaga tccagtcgta tgatcggtct 180
gtaactcccg tgttgcagga ttttattgat cctactatgt gttacctccc aaatggttac 240
ccatctactg cctattatta cggtggttat gatggaactg gtaacgagtg ggatgagtat 300
tcaagatatg tgaattcgga aggagttgag atgacttcag gagtttatgg ggataatggg 360
tctctacttt atcaccatgg atatggatat gcgccttatg gcccctattc gccagcaggg 420
tctccagttc caaccatggg gaatgatggt cagttgtatg ggcctcaaca ctatcagtat 480
cctccatatt tccaaccgtt aacaccaacc agtgcgccat tcacacctac tcctgctgtc 540
ctacctcagg gtgaggtttc cacctctgta gctgctgatc aaaagcctct ccctgtcgat 600
gcagccaatg gaaattctaa tggtgttgcg aatggtggga atgcaaaagg taataatgct 660
gctgcttcta ttaaacaggc caatcagaat tcatctttta gttccaaagc atcgaatgaa 720
agggttacca tgccaggtcg gggtccaact tcaggttatc aggatccaag atttggtttt 780
gatggagtac gctcacctat cccctggcta gatgccccac tattttcaga tgggcagcca 840
aggcctgtaa gtagcacaac catcacttct tcgatatcag gtggcaacaa cactgcctca 900
aggaacccaa ctttccgccc taattctcag tttatgggct tgcaccaccc aagaccaatg 960
cctgccatgg gagccaccca tagcttcata aataggatgt acccaagcaa actatatggt 1020
caatatggga acactgtcag atctggaatg ggttatggta cacatgggta tgattctcgc 1080
actaatggac gggcttggtt agctgttgac agtaaataca aaactagggg aagaagtggt 1140
ggctactttg gctatggcaa tgagaacgca gatggtttga acgaactgaa cagagggcca 1200
agggtggcaa aaaccaaaaa ggttttgcac caactattct ggcagtcaaa gggcagactt 1260
tgcctgcaac tttaggcaca gatgaagaga aagacaagac tagtaccatt ctggaatgtg 1320
atcaatataa caaagctgat tttccagaag aatatactga tgccaaattt tttgtcatca 1380
agtcttacag cgaggatgat atccataaga gtattaagta taatgtgtgg gctagtacac 1440
aaaatggcaa caagaagctt gatgctgcat atcaggaggc acagcagaaa cctggcggca 1500
cccctgtttt tctctttttc tcagttaata ccagtgggca atttgtgggg cttgctgaga 1560
tgattggtcc tgttgatttt aacaagagtg ttgagtattg gcagcaagac aagtggaatg 1620
gttgctttcc tcttaagtgg cacattgtta aggatgttcc taacaatttg ttgaggcaca 1680
ttaccttgga caacaatgag aacaaacctg tcactaacag tagggataca caagaggtta 1740
tgttggagcc tgggctgaaa ttaattaaaa ttttcaagga atataccagc aagacatgca 1800
ttttggatga ttttggcttc tatgaggctc gtcagaaaac tattttggag aagaaagcaa 1860
agcaacaatt cccaaagcag gtatgggaag ggaaacctgc tgatgagaag attgagataa 1920
atggggaagt taatactcaa aaatctgaag ttagctcgga attgctcaag gagtctaccc 1980
ttgctgagaa ggatagtgat gaccacaaag ttcccgagaa tggatctgct acaaaaactg 2040
gagatgcccc aaagggtgct aaaccagttg ttcccgagag taagattgta gccaatgggg 2100
ttgtttctaa tggtgtctaa 2120
<210> 14
<211> 424
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 14
Met Ala Thr Val Ala Asn Pro Ala Asp Gln Ala Thr Asp Leu Leu Gln
1 5 10 15
Lys Leu Ser Leu Glu Thr Gln Pro Lys Pro Leu Glu Ile Pro Glu Pro
20 25 30
Thr Lys Lys Ala Thr Gly Asn Gln Tyr Gly Ser Val Asp Ser Gly Asn
35 40 45
Ala Ala Asn Gly Gln Ile Gln Ser Tyr Asp Arg Ser Val Thr Pro Val
50 55 60
Leu Gln Asp Phe Ile Asp Pro Thr Met Cys Tyr Leu Pro Asn Gly Tyr
65 70 75 80
Pro Ser Thr Ala Tyr Tyr Tyr Gly Gly Tyr Asp Gly Thr Gly Asn Glu
85 90 95
Trp Asp Glu Tyr Ser Arg Tyr Val Asn Ser Glu Gly Val Glu Met Thr
100 105 110
Ser Gly Val Tyr Gly Asp Asn Gly Ser Leu Leu Tyr His His Gly Tyr
115 120 125
Gly Tyr Ala Pro Tyr Gly Pro Tyr Ser Pro Ala Gly Ser Pro Val Pro
130 135 140
Thr Met Gly Asn Asp Gly Gln Leu Tyr Gly Pro Gln His Tyr Gln Tyr
145 150 155 160
Pro Pro Tyr Phe Gln Pro Leu Thr Pro Thr Ser Ala Pro Phe Thr Pro
165 170 175
Thr Pro Ala Val Leu Pro Gln Gly Glu Val Ser Thr Ser Val Ala Ala
180 185 190
Asp Gln Lys Pro Leu Pro Val Asp Ala Ala Asn Gly Asn Ser Asn Gly
195 200 205
Val Ala Asn Gly Gly Asn Ala Lys Gly Asn Asn Ala Ala Ala Ser Ile
210 215 220
Lys Gln Ala Asn Gln Asn Ser Ser Phe Ser Ser Lys Ala Ser Asn Glu
225 230 235 240
Arg Val Thr Met Pro Gly Arg Gly Pro Thr Ser Gly Tyr Gln Asp Pro
245 250 255
Arg Phe Gly Phe Asp Gly Val Arg Ser Pro Ile Pro Trp Leu Asp Ala
260 265 270
Pro Leu Phe Ser Asp Gly Gln Pro Arg Pro Val Ser Ser Thr Thr Ile
275 280 285
Thr Ser Ser Ile Ser Gly Gly Asn Asn Thr Ala Ser Arg Asn Pro Thr
290 295 300
Phe Arg Pro Asn Ser Gln Phe Met Gly Leu His His Pro Arg Pro Met
305 310 315 320
Pro Ala Met Gly Ala Thr His Ser Phe Ile Asn Arg Met Tyr Pro Ser
325 330 335
Lys Leu Tyr Gly Gln Tyr Gly Asn Thr Val Arg Ser Gly Met Gly Tyr
340 345 350
Gly Thr His Gly Tyr Asp Ser Arg Thr Asn Gly Arg Ala Trp Leu Ala
355 360 365
Val Asp Ser Lys Tyr Lys Thr Arg Gly Arg Ser Gly Gly Tyr Phe Gly
370 375 380
Tyr Gly Asn Glu Asn Ala Asp Gly Leu Asn Glu Leu Asn Arg Gly Pro
385 390 395 400
Arg Val Ala Lys Thr Lys Lys Val Leu His Gln Leu Phe Trp Gln Ser
405 410 415
Lys Gly Arg Leu Cys Leu Gln Leu
420
<210> 15
<211> 2128
<212> DNA
<213> 人工序列（Artificial Sequence）
<400> 15
atggccaccg ttgctaatcc cgcggatcaa gcaactgatt tgctacagaa gctatcgtta 60
gaaactcagc ccaagccctt ggagattcct gagcctacca aaaaggccac tgggaatcag 120
tatggatcag ttgattcagg aaatgccgcg aatggccaga tccagtcgta tgatcggtct 180
gtaactcccg tgttgcagga ttttattgat cctactatgt gttacctccc aaatggttac 240
ccatctactg cctattatta cggtggttat gatggaactg gtaacgagtg ggatgagtat 300
tcaagatatg tgaattcgga aggagttgag atgacttcag gagtttatgg ggataatggg 360
tctctacttt atcaccatgg atatggatat gcgccttatg gcccctattc gccagcaggg 420
tctccagttc caaccatggg gaatgatggt cagttgtatg ggcctcaaca ctatcagtat 480
cctccatatt tccaaccgtt aacaccaacc agtgcgccat tcacacctac tcctgctgtc 540
ctacctcagg gtgaggtttc cacctctgta gctgctgatc aaaagcctct ccctgtcgat 600
gcagccaatg gaaattctaa tggtgttgcg aatggtggga atgcaaaagg taataatgct 660
gctgcttcta ttaaacaggc caatcagaat tcatctttta gttccaaagc atcgaatgaa 720
agggttacca tgccaggtcg gggtccaact tcaggttatc aggatccaag atttggtttt 780
gatggagtac gctcacctat cccctggcta gatgccccac tattttcaga tgggcagcca 840
aggcctgtaa gtagcacaac catcacttct tcgatatcag gtggcaacaa cactgcctca 900
aggaacccaa ctttccgccc taattctcag tttatgggct tgcaccaccc aagaccaatg 960
cctgccatgg gagccaccca tagcttcata aataggatgt acccaagcaa actatatggt 1020
caatatggga acactgtcag atctggaatg ggttatggta cacatgggta tgattctcgc 1080
actaatggac gggcttggtt agctgttgac agtaaataca aaactagggg aagaagtggt 1140
ggctactttg gctatggcaa tgagaacgca gatggtttga acgaactgaa cagagggcct 1200
agggaccaag ggtggcaaaa accaaaaagg ttttgcacca actattctgg cagtcaaagg 1260
gcagactttg cctgcaactt taggcacaga tgaagagaaa gacaagacta gtaccattct 1320
ggaatgtgat caatataaca aagctgattt tccagaagaa tatactgatg ccaaattttt 1380
tgtcatcaag tcttacagcg aggatgatat ccataagagt attaagtata atgtgtgggc 1440
tagtacacaa aatggcaaca agaagcttga tgctgcatat caggaggcac agcagaaacc 1500
tggcggcacc cctgtttttc tctttttctc agttaatacc agtgggcaat ttgtggggct 1560
tgctgagatg attggtcctg ttgattttaa caagagtgtt gagtattggc agcaagacaa 1620
gtggaatggt tgctttcctc ttaagtggca cattgttaag gatgttccta acaatttgtt 1680
gaggcacatt accttggaca acaatgagaa caaacctgtc actaacagta gggatacaca 1740
agaggttatg ttggagcctg ggctgaaatt aattaaaatt ttcaaggaat ataccagcaa 1800
gacatgcatt ttggatgatt ttggcttcta tgaggctcgt cagaaaacta ttttggagaa 1860
gaaagcaaag caacaattcc caaagcaggt atgggaaggg aaacctgctg atgagaagat 1920
tgagataaat ggggaagtta atactcaaaa atctgaagtt agctcggaat tgctcaagga 1980
gtctaccctt gctgagaagg atagtgatga ccacaaagtt cccgagaatg gatctgctac 2040
aaaaactgga gatgccccaa agggtgctaa accagttgtt cccgagagta agattgtagc 2100
caatggggtt gtttctaatg gtgtctaa 2128
<210> 16
<211> 430
<212> PRT
<213> 人工序列（Artificial Sequence）
<400> 16
Met Ala Thr Val Ala Asn Pro Ala Asp Gln Ala Thr Asp Leu Leu Gln
1 5 10 15
Lys Leu Ser Leu Glu Thr Gln Pro Lys Pro Leu Glu Ile Pro Glu Pro
20 25 30
Thr Lys Lys Ala Thr Gly Asn Gln Tyr Gly Ser Val Asp Ser Gly Asn
35 40 45
Ala Ala Asn Gly Gln Ile Gln Ser Tyr Asp Arg Ser Val Thr Pro Val
50 55 60
Leu Gln Asp Phe Ile Asp Pro Thr Met Cys Tyr Leu Pro Asn Gly Tyr
65 70 75 80
Pro Ser Thr Ala Tyr Tyr Tyr Gly Gly Tyr Asp Gly Thr Gly Asn Glu
85 90 95
Trp Asp Glu Tyr Ser Arg Tyr Val Asn Ser Glu Gly Val Glu Met Thr
100 105 110
Ser Gly Val Tyr Gly Asp Asn Gly Ser Leu Leu Tyr His His Gly Tyr
115 120 125
Gly Tyr Ala Pro Tyr Gly Pro Tyr Ser Pro Ala Gly Ser Pro Val Pro
130 135 140
Thr Met Gly Asn Asp Gly Gln Leu Tyr Gly Pro Gln His Tyr Gln Tyr
145 150 155 160
Pro Pro Tyr Phe Gln Pro Leu Thr Pro Thr Ser Ala Pro Phe Thr Pro
165 170 175
Thr Pro Ala Val Leu Pro Gln Gly Glu Val Ser Thr Ser Val Ala Ala
180 185 190
Asp Gln Lys Pro Leu Pro Val Asp Ala Ala Asn Gly Asn Ser Asn Gly
195 200 205
Val Ala Asn Gly Gly Asn Ala Lys Gly Asn Asn Ala Ala Ala Ser Ile
210 215 220
Lys Gln Ala Asn Gln Asn Ser Ser Phe Ser Ser Lys Ala Ser Asn Glu
225 230 235 240
Arg Val Thr Met Pro Gly Arg Gly Pro Thr Ser Gly Tyr Gln Asp Pro
245 250 255
Arg Phe Gly Phe Asp Gly Val Arg Ser Pro Ile Pro Trp Leu Asp Ala
260 265 270
Pro Leu Phe Ser Asp Gly Gln Pro Arg Pro Val Ser Ser Thr Thr Ile
275 280 285
Thr Ser Ser Ile Ser Gly Gly Asn Asn Thr Ala Ser Arg Asn Pro Thr
290 295 300
Phe Arg Pro Asn Ser Gln Phe Met Gly Leu His His Pro Arg Pro Met
305 310 315 320
Pro Ala Met Gly Ala Thr His Ser Phe Ile Asn Arg Met Tyr Pro Ser
325 330 335
Lys Leu Tyr Gly Gln Tyr Gly Asn Thr Val Arg Ser Gly Met Gly Tyr
340 345 350
Gly Thr His Gly Tyr Asp Ser Arg Thr Asn Gly Arg Ala Trp Leu Ala
355 360 365
Val Asp Ser Lys Tyr Lys Thr Arg Gly Arg Ser Gly Gly Tyr Phe Gly
370 375 380
Tyr Gly Asn Glu Asn Ala Asp Gly Leu Asn Glu Leu Asn Arg Gly Pro
385 390 395 400
Arg Asp Gln Gly Trp Gln Lys Pro Lys Arg Phe Cys Thr Asn Tyr Ser
405 410 415
Gly Ser Gln Arg Ala Asp Phe Ala Cys Asn Phe Arg His Arg
420 425 430
Claims (3)
1.与蛋白质相关的生物材料在降低植物株高中的应用；
所述蛋白质是如下A1）或A2）的蛋白质：
A1）氨基酸序列是序列表中序列1的蛋白质；
A2）在A1）的N末端或/和C末端连接蛋白标签得到的融合蛋白质；
所述生物材料为下述B1）或B2）：
B1）抑制或降低所述蛋白质的编码基因的表达或所述蛋白质活性的核酸分子；
B2）含有B1）所述核酸分子的表达盒、重组载体、重组微生物或转基因植物细胞系；
所述核酸分子为表达靶向所述蛋白编码基因的gRNA的DNA分子；
所述gRNA的靶序列对应于序列表中序列2的第505-527位、第1070-1092位和/或第1188-1210位核苷酸；
所述植物为大豆。
2.一种降低植物株高的方法，包括通过抑制或降低植物中权利要求1中所述的蛋白质的编码基因的表达量来降低植物株高；所述植物为大豆；
所述方法包括向所述植物中导入降低或抑制权利要求1中所述的蛋白编码基因表达的物质；所述降低或抑制权利要求1中所述蛋白编码基因表达的物质为如下c1）-c4）任一种物质：
c1）抑制或降低权利要求1中所述蛋白编码基因表达的核酸分子；
c2）含有c1）所述核酸分子的表达盒；
c3）含有c1）所述核酸分子的重组载体、或含有c2）所述表达盒的重组载体；
c4）含有c1）所述核酸分子的重组微生物、或含有c2）所述表达盒的重组微生物、或含有c3）所述重组载体的重组微生物；
c1）所述的核酸分子为表达靶向权利要求1中所述蛋白编码基因的gRNA的DNA分子；
所述gRNA的靶序列对应于序列表中序列2的第505-527位、第1070-1092位和/或第1188-1210位核苷酸。
3.根据权利要求2所述的方法，所述抑制或降低植物中权利要求1中所述蛋白质的编码基因的表达量为将植物中的序列2所示的所述蛋白编码基因进行下述至少一种突变：
1）将植物中的序列2所示的所述蛋白编码基因突变为Gmect2-11-1-4-2，Gmect2-11-1- 4-2是将序列表中序列2的第509-515位的7个核苷酸CCAGTGC缺失、保持序列表中序列2的其他核苷酸不变得到的DNA分子；
2）将植物中的序列2所示的所述蛋白编码基因突变为Gmect2-18-1-4-1，Gmect2-18-1- 4-1是将序列表中序列2的1083-1090位的8个核苷酸TAATGGAC缺失、保持序列表中序列2的其他核苷酸不变得到的DNA分子；
3）将植物中的序列2所示的所述蛋白编码基因突变为Gmect2-20-2-6-4，Gmect2-20-2- 6-4是将序列表中序列2的1200-1206位的7个核苷酸TAGGGCC缺失、保持序列表中序列2的其他核苷酸不变得到的DNA分子；
4）将植物中的序列2所示的所述蛋白编码基因突变为Gmect2-20-3-7-7-2，Gmect2-20- 3-7-7-2是将序列表中序列2的1204-1205位的2个核苷酸之间插入了的1个核苷酸A、保持序列表中序列2的其他核苷酸不变得到的DNA分子。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111336838.4A CN114231556B (zh) | 2021-11-12 | 2021-11-12 | GmECT2在调控植物高度方面的应用 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111336838.4A CN114231556B (zh) | 2021-11-12 | 2021-11-12 | GmECT2在调控植物高度方面的应用 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114231556A CN114231556A (zh) | 2022-03-25 |
| CN114231556B true CN114231556B (zh) | 2024-03-01 |
Family
ID=80749140
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111336838.4A Active CN114231556B (zh) | 2021-11-12 | 2021-11-12 | GmECT2在调控植物高度方面的应用 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114231556B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110506118A (zh) * | 2017-02-02 | 2019-11-26 | 杜克大学 | 用于控制基因表达的组合物和方法 |
-
2021
- 2021-11-12 CN CN202111336838.4A patent/CN114231556B/zh active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110506118A (zh) * | 2017-02-02 | 2019-11-26 | 杜克大学 | 用于控制基因表达的组合物和方法 |
Non-Patent Citations (3)
| Title |
|---|
| Genbank accession number：XP_014631212.1;无;Genbank;1 * |
| 无.Genbank accession number：XP_014631212.1.Genbank.2021,1. * |
| 水稻m6A结合蛋白的筛选和功能研究;孙策;中国优秀硕士论文全文数据库农业科技辑(第1期);D047-50 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114231556A (zh) | 2022-03-25 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110117320B (zh) | 棉花GhCAL-D07基因在促进植物开花中的应用 | |
| CN112226455B (zh) | 一种水稻籽粒粒长和粒重相关蛋白及其编码基因与应用 | |
| CN107475210B (zh) | 一种水稻白叶枯病抗性相关基因OsABA2及其应用 | |
| CN112626080B (zh) | 一种控制大豆-根瘤菌匹配性的r基因及其蛋白质和应用 | |
| CN113637684A (zh) | 一种水稻类病斑突变体基因scyl2及其应用 | |
| CN110819639B (zh) | 烟草低温早花相关基因NtDUF599及其应用 | |
| CN114107370A (zh) | 利用Cas12i在大豆中进行基因编辑的方法 | |
| CN113637060B (zh) | 大豆GmSPA3a/3b蛋白及其相关生物材料在调控植物开花和株高中的应用 | |
| CN108997487B (zh) | 抗逆相关蛋白z76在调控植物抗逆性中的应用 | |
| CN116515888A (zh) | GmMTAs蛋白在调控大豆株高中的应用 | |
| CN113046377B (zh) | 一种雄性不育基因MsGAL及其应用 | |
| CN114231556B (zh) | GmECT2在调控植物高度方面的应用 | |
| CN111499709B (zh) | 水稻穗粒数相关的rgn1蛋白及其编码基因与应用 | |
| CN116064568A (zh) | 紫花苜蓿MsASG166基因及在提高植物耐旱中的用途 | |
| CN109097388B (zh) | GmCOP1a和/或GmCOP1b在调控植物株高方面的应用 | |
| CN112553224B (zh) | 组蛋白去乙酰化酶基因OsHDT701在延长植物种子寿命中的应用 | |
| CN114958866B (zh) | 调控大豆分枝数的基因及其用途 | |
| CN110922459B (zh) | SlSNAT1蛋白及其相关生物材料在调控植物种子耐老化性能中的应用 | |
| CN115011631B (zh) | 调控玉米苗期抗旱性的蛋白及其编码基因和应用 | |
| CN114516908B (zh) | 水稻粒形调控蛋白hos59及其编码基因和应用 | |
| CN113136388B (zh) | 水稻OsMAPKKK5基因在改良水稻的株高和粒型方面的应用 | |
| CN114230649B (zh) | 水稻分蘖力相关的Tn1蛋白及其相关生物材料与应用 | |
| CN116789785B (zh) | 长雄蕊野生稻高产、高光效基因FarL1及其应用 | |
| CN116444637A (zh) | 调控植物叶片衰老和产量相关蛋白GmWRKY100及其编码基因与应用 | |
| CN115385992A (zh) | 石果衣真菌ndpk-2蛋白及其编码基因在调控植物高度和非生物胁迫抗性中的应用 |
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 |