CN114058639B - 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 - Google Patents
利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 Download PDFInfo
- Publication number
- CN114058639B CN114058639B CN202111272218.9A CN202111272218A CN114058639B CN 114058639 B CN114058639 B CN 114058639B CN 202111272218 A CN202111272218 A CN 202111272218A CN 114058639 B CN114058639 B CN 114058639B
- Authority
- CN
- China
- Prior art keywords
- gene
- rice
- oswall
- amylose content
- vector
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 102
- 235000007164 Oryza sativa Nutrition 0.000 title claims abstract description 82
- 235000009566 rice Nutrition 0.000 title claims abstract description 82
- 229920000856 Amylose Polymers 0.000 title claims abstract description 58
- 238000000034 method Methods 0.000 title claims abstract description 35
- 238000010362 genome editing Methods 0.000 title claims abstract description 30
- 238000005516 engineering process Methods 0.000 title abstract description 20
- 240000007594 Oryza sativa Species 0.000 title description 75
- 239000013598 vector Substances 0.000 claims abstract description 43
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 26
- 150000001413 amino acids Chemical class 0.000 claims abstract description 25
- 229930024421 Adenine Natural products 0.000 claims abstract description 15
- GFFGJBXGBJISGV-UHFFFAOYSA-N Adenine Chemical compound NC1=NC=NC2=C1N=CN2 GFFGJBXGBJISGV-UHFFFAOYSA-N 0.000 claims abstract description 15
- 229960000643 adenine Drugs 0.000 claims abstract description 15
- 230000008685 targeting Effects 0.000 claims abstract description 10
- 241000209094 Oryza Species 0.000 claims abstract 11
- 230000014509 gene expression Effects 0.000 claims description 33
- 230000035772 mutation Effects 0.000 claims description 31
- 108091033409 CRISPR Proteins 0.000 claims description 16
- 240000002582 Oryza sativa Indica Group Species 0.000 claims description 8
- 108091027544 Subgenomic mRNA Proteins 0.000 claims description 7
- 238000009395 breeding Methods 0.000 claims description 7
- 230000001488 breeding effect Effects 0.000 claims description 7
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 5
- 230000006872 improvement Effects 0.000 claims description 5
- 108020004707 nucleic acids Proteins 0.000 claims description 5
- 102000039446 nucleic acids Human genes 0.000 claims description 5
- 150000007523 nucleic acids Chemical class 0.000 claims description 5
- 101710163270 Nuclease Proteins 0.000 claims description 4
- 240000000111 Saccharum officinarum Species 0.000 claims description 3
- 235000007201 Saccharum officinarum Nutrition 0.000 claims description 3
- 230000009471 action Effects 0.000 claims description 3
- 238000010453 CRISPR/Cas method Methods 0.000 claims description 2
- 208000031753 acute bilirubin encephalopathy Diseases 0.000 abstract description 18
- 230000009466 transformation Effects 0.000 abstract description 10
- 230000009261 transgenic effect Effects 0.000 abstract description 10
- 239000000463 material Substances 0.000 abstract description 8
- 230000008859 change Effects 0.000 abstract description 7
- 238000005259 measurement Methods 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 17
- 108020004414 DNA Proteins 0.000 description 13
- 108700028369 Alleles Proteins 0.000 description 9
- 238000001514 detection method Methods 0.000 description 9
- 108020001507 fusion proteins Proteins 0.000 description 9
- 102000037865 fusion proteins Human genes 0.000 description 9
- 108020005004 Guide RNA Proteins 0.000 description 8
- 238000012216 screening Methods 0.000 description 8
- 108091026890 Coding region Proteins 0.000 description 7
- 238000010354 CRISPR gene editing Methods 0.000 description 6
- 101710169336 5'-deoxyadenosine deaminase Proteins 0.000 description 5
- 102000055025 Adenosine deaminases Human genes 0.000 description 5
- 241000589158 Agrobacterium Species 0.000 description 5
- 206010020649 Hyperkeratosis Diseases 0.000 description 5
- 230000000694 effects Effects 0.000 description 5
- 230000006870 function Effects 0.000 description 5
- 230000002068 genetic effect Effects 0.000 description 5
- 102000053602 DNA Human genes 0.000 description 4
- 108090000790 Enzymes Proteins 0.000 description 4
- 238000012300 Sequence Analysis Methods 0.000 description 4
- 102000004190 Enzymes Human genes 0.000 description 3
- 108700026244 Open Reading Frames Proteins 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 238000011161 development Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 239000002773 nucleotide Substances 0.000 description 3
- 125000003729 nucleotide group Chemical group 0.000 description 3
- 238000000926 separation method Methods 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- KTXKIYXZQFWJKB-VZFHVOOUSA-N Ala-Thr-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(O)=O KTXKIYXZQFWJKB-VZFHVOOUSA-N 0.000 description 2
- 108700004991 Cas12a Proteins 0.000 description 2
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 2
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 2
- 108700024394 Exon Proteins 0.000 description 2
- SWQALSGKVLYKDT-UHFFFAOYSA-N Gly-Ile-Ala Natural products NCC(=O)NC(C(C)CC)C(=O)NC(C)C(O)=O SWQALSGKVLYKDT-UHFFFAOYSA-N 0.000 description 2
- 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 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- YOKVEHGYYQEQOP-QWRGUYRKSA-N Leu-Leu-Gly Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O YOKVEHGYYQEQOP-QWRGUYRKSA-N 0.000 description 2
- JHNOXVASMSXSNB-WEDXCCLWSA-N Lys-Thr-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JHNOXVASMSXSNB-WEDXCCLWSA-N 0.000 description 2
- 240000008467 Oryza sativa Japonica Group Species 0.000 description 2
- VMLONWHIORGALA-SRVKXCTJSA-N Ser-Leu-Leu Chemical compound CC(C)C[C@@H](C([O-])=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]([NH3+])CO VMLONWHIORGALA-SRVKXCTJSA-N 0.000 description 2
- 238000010459 TALEN Methods 0.000 description 2
- IMULJHHGAUZZFE-MBLNEYKQSA-N Thr-Gly-Ile Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H]([C@@H](C)CC)C(O)=O IMULJHHGAUZZFE-MBLNEYKQSA-N 0.000 description 2
- 108010043645 Transcription Activator-Like Effector Nucleases Proteins 0.000 description 2
- 108700019146 Transgenes Proteins 0.000 description 2
- 108010005233 alanylglutamic acid Proteins 0.000 description 2
- 230000037429 base substitution Effects 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 230000033228 biological regulation Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 238000006243 chemical reaction Methods 0.000 description 2
- 230000005782 double-strand break Effects 0.000 description 2
- 238000003209 gene knockout Methods 0.000 description 2
- UYTPUPDQBNUYGX-UHFFFAOYSA-N guanine Chemical compound O=C1NC(N)=NC2=C1N=CN2 UYTPUPDQBNUYGX-UHFFFAOYSA-N 0.000 description 2
- HNDVDQJCIGZPNO-UHFFFAOYSA-N histidine Natural products OC(=O)C(N)CC1=CN=CN1 HNDVDQJCIGZPNO-UHFFFAOYSA-N 0.000 description 2
- 230000001404 mediated effect Effects 0.000 description 2
- 238000010369 molecular cloning Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 230000006780 non-homologous end joining Effects 0.000 description 2
- 238000004321 preservation Methods 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 238000003753 real-time PCR Methods 0.000 description 2
- 230000001172 regenerating effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 235000019640 taste Nutrition 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- COEXAQSTZUWMRI-STQMWFEESA-N (2s)-1-[2-[[(2s)-2-amino-3-(4-hydroxyphenyl)propanoyl]amino]acetyl]pyrrolidine-2-carboxylic acid Chemical compound C([C@H](N)C(=O)NCC(=O)N1[C@@H](CCC1)C(O)=O)C1=CC=C(O)C=C1 COEXAQSTZUWMRI-STQMWFEESA-N 0.000 description 1
- 101150033839 4 gene Proteins 0.000 description 1
- 101710197633 Actin-1 Proteins 0.000 description 1
- 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 1
- JBVSSSZFNTXJDX-YTLHQDLWSA-N Ala-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)N JBVSSSZFNTXJDX-YTLHQDLWSA-N 0.000 description 1
- JBGSZRYCXBPWGX-BQBZGAKWSA-N Ala-Arg-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CCCN=C(N)N JBGSZRYCXBPWGX-BQBZGAKWSA-N 0.000 description 1
- UHMQKOBNPRAZGB-CIUDSAMLSA-N Ala-Glu-Met Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CCSC)C(=O)O)N UHMQKOBNPRAZGB-CIUDSAMLSA-N 0.000 description 1
- CWEAKSWWKHGTRJ-BQBZGAKWSA-N Ala-Gly-Met Chemical compound [H]N[C@@H](C)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O CWEAKSWWKHGTRJ-BQBZGAKWSA-N 0.000 description 1
- NBTGEURICRTMGL-WHFBIAKZSA-N Ala-Gly-Ser Chemical compound C[C@H](N)C(=O)NCC(=O)N[C@@H](CO)C(O)=O NBTGEURICRTMGL-WHFBIAKZSA-N 0.000 description 1
- CCDFBRZVTDDJNM-GUBZILKMSA-N Ala-Leu-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O CCDFBRZVTDDJNM-GUBZILKMSA-N 0.000 description 1
- MFMDKJIPHSWSBM-GUBZILKMSA-N Ala-Lys-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O MFMDKJIPHSWSBM-GUBZILKMSA-N 0.000 description 1
- MDNAVFBZPROEHO-DCAQKATOSA-N Ala-Lys-Val Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(O)=O MDNAVFBZPROEHO-DCAQKATOSA-N 0.000 description 1
- MDNAVFBZPROEHO-UHFFFAOYSA-N Ala-Lys-Val Natural products CC(C)C(C(O)=O)NC(=O)C(NC(=O)C(C)N)CCCCN MDNAVFBZPROEHO-UHFFFAOYSA-N 0.000 description 1
- BDQNLQSWRAPHGU-DLOVCJGASA-N Ala-Phe-Cys Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)O)N BDQNLQSWRAPHGU-DLOVCJGASA-N 0.000 description 1
- CNQAFFMNJIQYGX-DRZSPHRISA-N Ala-Phe-Glu Chemical compound OC(=O)CC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 CNQAFFMNJIQYGX-DRZSPHRISA-N 0.000 description 1
- RUXQNKVQSKOOBS-JURCDPSOSA-N Ala-Phe-Ile Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O RUXQNKVQSKOOBS-JURCDPSOSA-N 0.000 description 1
- ADSGHMXEAZJJNF-DCAQKATOSA-N Ala-Pro-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](C)N ADSGHMXEAZJJNF-DCAQKATOSA-N 0.000 description 1
- XWFWAXPOLRTDFZ-FXQIFTODSA-N Ala-Pro-Ser Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O XWFWAXPOLRTDFZ-FXQIFTODSA-N 0.000 description 1
- JNLDTVRGXMSYJC-UVBJJODRSA-N Ala-Pro-Trp Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](Cc1c[nH]c2ccccc12)C(O)=O JNLDTVRGXMSYJC-UVBJJODRSA-N 0.000 description 1
- SYIFFFHSXBNPMC-UWJYBYFXSA-N Ala-Ser-Tyr Chemical compound C[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N SYIFFFHSXBNPMC-UWJYBYFXSA-N 0.000 description 1
- 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 1
- FSXDWQGEWZQBPJ-HERUPUMHSA-N Ala-Trp-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)N[C@@H](CC(=O)O)C(=O)O)N FSXDWQGEWZQBPJ-HERUPUMHSA-N 0.000 description 1
- OTOXOKCIIQLMFH-KZVJFYERSA-N Arg-Ala-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCCN=C(N)N OTOXOKCIIQLMFH-KZVJFYERSA-N 0.000 description 1
- RWWPBOUMKFBHAL-FXQIFTODSA-N Arg-Asn-Cys Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CS)C(O)=O RWWPBOUMKFBHAL-FXQIFTODSA-N 0.000 description 1
- MSILNNHVVMMTHZ-UWVGGRQHSA-N Arg-His-Gly Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)NCC(O)=O)CC1=CN=CN1 MSILNNHVVMMTHZ-UWVGGRQHSA-N 0.000 description 1
- INXWADWANGLMPJ-JYJNAYRXSA-N Arg-Phe-Arg Chemical compound NC(=N)NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CCCNC(N)=N)C(O)=O)CC1=CC=CC=C1 INXWADWANGLMPJ-JYJNAYRXSA-N 0.000 description 1
- MNBHKGYCLBUIBC-UFYCRDLUSA-N Arg-Phe-Phe Chemical compound C([C@H](NC(=O)[C@H](CCCNC(N)=N)N)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CC=CC=C1 MNBHKGYCLBUIBC-UFYCRDLUSA-N 0.000 description 1
- AOJYORNRFWWEIV-IHRRRGAJSA-N Arg-Tyr-Asp Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CC(O)=O)C(O)=O)CC1=CC=C(O)C=C1 AOJYORNRFWWEIV-IHRRRGAJSA-N 0.000 description 1
- FXGMURPOWCKNAZ-JYJNAYRXSA-N Arg-Val-Phe Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 FXGMURPOWCKNAZ-JYJNAYRXSA-N 0.000 description 1
- 239000004475 Arginine Substances 0.000 description 1
- 102100024630 Asc-type amino acid transporter 1 Human genes 0.000 description 1
- ACRYGQFHAQHDSF-ZLUOBGJFSA-N Asn-Asn-Asn Chemical compound NC(=O)C[C@H](N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O ACRYGQFHAQHDSF-ZLUOBGJFSA-N 0.000 description 1
- OWUCNXMFJRFOFI-BQBZGAKWSA-N Asn-Gly-Met Chemical compound [H]N[C@@H](CC(N)=O)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O OWUCNXMFJRFOFI-BQBZGAKWSA-N 0.000 description 1
- KSZHWTRZPOTIGY-AVGNSLFASA-N Asn-Tyr-Gln Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N)O KSZHWTRZPOTIGY-AVGNSLFASA-N 0.000 description 1
- CBWCQCANJSGUOH-ZKWXMUAHSA-N Asn-Val-Ala Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](C)C(O)=O CBWCQCANJSGUOH-ZKWXMUAHSA-N 0.000 description 1
- WQAOZCVOOYUWKG-LSJOCFKGSA-N Asn-Val-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)O)NC(=O)[C@H](CC(=O)N)N WQAOZCVOOYUWKG-LSJOCFKGSA-N 0.000 description 1
- SDHFVYLZFBDSQT-DCAQKATOSA-N Asp-Arg-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CC(=O)O)N SDHFVYLZFBDSQT-DCAQKATOSA-N 0.000 description 1
- 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 1
- ILJQISGMGXRZQQ-IHRRRGAJSA-N Asp-Arg-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O ILJQISGMGXRZQQ-IHRRRGAJSA-N 0.000 description 1
- ATYWBXGNXZYZGI-ACZMJKKPSA-N Asp-Asn-Gln Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O ATYWBXGNXZYZGI-ACZMJKKPSA-N 0.000 description 1
- BUVNWKQBMZLCDW-UGYAYLCHSA-N Asp-Asn-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BUVNWKQBMZLCDW-UGYAYLCHSA-N 0.000 description 1
- VILLWIDTHYPSLC-PEFMBERDSA-N Asp-Glu-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O VILLWIDTHYPSLC-PEFMBERDSA-N 0.000 description 1
- UMHUHHJMEXNSIV-CIUDSAMLSA-N Asp-Leu-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H](N)CC(O)=O UMHUHHJMEXNSIV-CIUDSAMLSA-N 0.000 description 1
- RXBGWGRSWXOBGK-KKUMJFAQSA-N Asp-Lys-Tyr Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O RXBGWGRSWXOBGK-KKUMJFAQSA-N 0.000 description 1
- JJQGZGOEDSSHTE-FOHZUACHSA-N Asp-Thr-Gly Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O JJQGZGOEDSSHTE-FOHZUACHSA-N 0.000 description 1
- IHZFGJLKDYINPV-XIRDDKMYSA-N Asp-Trp-His Chemical compound C([C@H](NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)NC(=O)[C@H](CC(O)=O)N)C(O)=O)C1=CN=CN1 IHZFGJLKDYINPV-XIRDDKMYSA-N 0.000 description 1
- VHUKCUHLFMRHOD-MELADBBJSA-N Asp-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC(=O)O)N)C(=O)O VHUKCUHLFMRHOD-MELADBBJSA-N 0.000 description 1
- GIKOVDMXBAFXDF-NHCYSSNCSA-N Asp-Val-Leu Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(O)=O GIKOVDMXBAFXDF-NHCYSSNCSA-N 0.000 description 1
- QOJJMJKTMKNFEF-ZKWXMUAHSA-N Asp-Val-Ser Chemical compound OC[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC(O)=O QOJJMJKTMKNFEF-ZKWXMUAHSA-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
- PRXCTTWKGJAPMT-ZLUOBGJFSA-N Cys-Ala-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O PRXCTTWKGJAPMT-ZLUOBGJFSA-N 0.000 description 1
- BVFQOPGFOQVZTE-ACZMJKKPSA-N Cys-Gln-Ala Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O BVFQOPGFOQVZTE-ACZMJKKPSA-N 0.000 description 1
- KABHAOSDMIYXTR-GUBZILKMSA-N Cys-Glu-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CS)N KABHAOSDMIYXTR-GUBZILKMSA-N 0.000 description 1
- LBOLGUYQEPZSKM-YUMQZZPRSA-N Cys-Gly-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CS)N LBOLGUYQEPZSKM-YUMQZZPRSA-N 0.000 description 1
- ZXCAQANTQWBICD-DCAQKATOSA-N Cys-Lys-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CS)N ZXCAQANTQWBICD-DCAQKATOSA-N 0.000 description 1
- 230000004543 DNA replication Effects 0.000 description 1
- 230000007018 DNA scission Effects 0.000 description 1
- 102100035102 E3 ubiquitin-protein ligase MYCBP2 Human genes 0.000 description 1
- 108010042407 Endonucleases Proteins 0.000 description 1
- 102000004533 Endonucleases Human genes 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 206010064571 Gene mutation Diseases 0.000 description 1
- JESJDAAGXULQOP-CIUDSAMLSA-N Gln-Arg-Ser Chemical compound C(C[C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CCC(=O)N)N)CN=C(N)N JESJDAAGXULQOP-CIUDSAMLSA-N 0.000 description 1
- IKFZXRLDMYWNBU-YUMQZZPRSA-N Gln-Gly-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N IKFZXRLDMYWNBU-YUMQZZPRSA-N 0.000 description 1
- FGYPOQPQTUNESW-IUCAKERBSA-N Gln-Gly-Leu Chemical compound CC(C)C[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CCC(=O)N)N FGYPOQPQTUNESW-IUCAKERBSA-N 0.000 description 1
- QQAPDATZKKTBIY-YUMQZZPRSA-N Gln-Gly-Met Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)NCC(=O)N[C@@H](CCSC)C(O)=O QQAPDATZKKTBIY-YUMQZZPRSA-N 0.000 description 1
- WZZSKAJIHTUUSG-ACZMJKKPSA-N Glu-Ala-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O WZZSKAJIHTUUSG-ACZMJKKPSA-N 0.000 description 1
- UTKUTMJSWKKHEM-WDSKDSINSA-N Glu-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCC(O)=O UTKUTMJSWKKHEM-WDSKDSINSA-N 0.000 description 1
- DYFJZDDQPNIPAB-NHCYSSNCSA-N Glu-Arg-Val Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(O)=O DYFJZDDQPNIPAB-NHCYSSNCSA-N 0.000 description 1
- HNVFSTLPVJWIDV-CIUDSAMLSA-N Glu-Glu-Gln Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O HNVFSTLPVJWIDV-CIUDSAMLSA-N 0.000 description 1
- MUSGDMDGNGXULI-DCAQKATOSA-N Glu-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CCC(O)=O MUSGDMDGNGXULI-DCAQKATOSA-N 0.000 description 1
- PXXGVUVQWQGGIG-YUMQZZPRSA-N Glu-Gly-Arg Chemical compound OC(=O)CC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCN=C(N)N PXXGVUVQWQGGIG-YUMQZZPRSA-N 0.000 description 1
- VMKCPNBBPGGQBJ-GUBZILKMSA-N Glu-Leu-Asn Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCC(=O)O)N VMKCPNBBPGGQBJ-GUBZILKMSA-N 0.000 description 1
- YKBUCXNNBYZYAY-MNXVOIDGSA-N Glu-Lys-Ile Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O YKBUCXNNBYZYAY-MNXVOIDGSA-N 0.000 description 1
- ILWHFUZZCFYSKT-AVGNSLFASA-N Glu-Lys-Leu Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O ILWHFUZZCFYSKT-AVGNSLFASA-N 0.000 description 1
- VJVAQZYGLMJPTK-QEJZJMRPSA-N Glu-Trp-Asp Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)O)NC(=O)[C@H](CCC(=O)O)N VJVAQZYGLMJPTK-QEJZJMRPSA-N 0.000 description 1
- OVSKVOOUFAKODB-UWVGGRQHSA-N Gly-Arg-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCN=C(N)N OVSKVOOUFAKODB-UWVGGRQHSA-N 0.000 description 1
- FIQQRCFQXGLOSZ-WDSKDSINSA-N Gly-Glu-Asp Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O FIQQRCFQXGLOSZ-WDSKDSINSA-N 0.000 description 1
- UFPXDFOYHVEIPI-BYPYZUCNSA-N Gly-Gly-Asp Chemical compound NCC(=O)NCC(=O)N[C@H](C(O)=O)CC(O)=O UFPXDFOYHVEIPI-BYPYZUCNSA-N 0.000 description 1
- XPJBQTCXPJNIFE-ZETCQYMHSA-N Gly-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)CN XPJBQTCXPJNIFE-ZETCQYMHSA-N 0.000 description 1
- TVDHVLGFJSHPAX-UWVGGRQHSA-N Gly-His-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CN=CN1 TVDHVLGFJSHPAX-UWVGGRQHSA-N 0.000 description 1
- SWQALSGKVLYKDT-ZKWXMUAHSA-N Gly-Ile-Ala Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](C)C(O)=O SWQALSGKVLYKDT-ZKWXMUAHSA-N 0.000 description 1
- UESJMAMHDLEHGM-NHCYSSNCSA-N Gly-Ile-Leu Chemical compound NCC(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(C)C)C(O)=O UESJMAMHDLEHGM-NHCYSSNCSA-N 0.000 description 1
- CCBIBMKQNXHNIN-ZETCQYMHSA-N Gly-Leu-Gly Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)NCC(O)=O CCBIBMKQNXHNIN-ZETCQYMHSA-N 0.000 description 1
- FXGRXIATVXUAHO-WEDXCCLWSA-N Gly-Lys-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CCCCN FXGRXIATVXUAHO-WEDXCCLWSA-N 0.000 description 1
- OQQKUTVULYLCDG-ONGXEEELSA-N Gly-Lys-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCCN)NC(=O)CN)C(O)=O OQQKUTVULYLCDG-ONGXEEELSA-N 0.000 description 1
- DHNXGWVNLFPOMQ-KBPBESRZSA-N Gly-Phe-His Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)O)NC(=O)CN DHNXGWVNLFPOMQ-KBPBESRZSA-N 0.000 description 1
- 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 1
- WOAMZMXCLBBQKW-KKUMJFAQSA-N His-Cys-Tyr Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)O)NC(=O)[C@H](CS)NC(=O)[C@H](CC2=CN=CN2)N)O WOAMZMXCLBBQKW-KKUMJFAQSA-N 0.000 description 1
- QYOGJYIRKACXEP-SLBDDTMCSA-N Ile-Asn-Trp Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)N QYOGJYIRKACXEP-SLBDDTMCSA-N 0.000 description 1
- JQLFYZMEXFNRFS-DJFWLOJKSA-N Ile-Asp-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N JQLFYZMEXFNRFS-DJFWLOJKSA-N 0.000 description 1
- KUHFPGIVBOCRMV-MNXVOIDGSA-N Ile-Gln-Leu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(C)C)C(=O)O)N KUHFPGIVBOCRMV-MNXVOIDGSA-N 0.000 description 1
- QHGBCRCMBCWMBJ-UHFFFAOYSA-N Ile-Glu-Ala-Lys Natural products CCC(C)C(N)C(=O)NC(CCC(O)=O)C(=O)NC(C)C(=O)NC(C(O)=O)CCCCN QHGBCRCMBCWMBJ-UHFFFAOYSA-N 0.000 description 1
- MTFVYKQRLXYAQN-LAEOZQHASA-N Ile-Glu-Gly Chemical compound [H]N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O MTFVYKQRLXYAQN-LAEOZQHASA-N 0.000 description 1
- ZXIGYKICRDFISM-DJFWLOJKSA-N Ile-His-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CC(=O)N)C(=O)O)N ZXIGYKICRDFISM-DJFWLOJKSA-N 0.000 description 1
- UDBPXJNOEWDBDF-XUXIUFHCSA-N Ile-Lys-Val Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C(C)C)C(=O)O)N UDBPXJNOEWDBDF-XUXIUFHCSA-N 0.000 description 1
- 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 1
- JNLSTRPWUXOORL-MMWGEVLESA-N Ile-Ser-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N JNLSTRPWUXOORL-MMWGEVLESA-N 0.000 description 1
- WLRJHVNFGAOYPS-HJPIBITLSA-N Ile-Ser-Tyr Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)O)N WLRJHVNFGAOYPS-HJPIBITLSA-N 0.000 description 1
- HQLSBZFLOUHQJK-STECZYCISA-N Ile-Tyr-Arg Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CCCN=C(N)N)C(=O)O)N HQLSBZFLOUHQJK-STECZYCISA-N 0.000 description 1
- 229930010555 Inosine Natural products 0.000 description 1
- UGQMRVRMYYASKQ-KQYNXXCUSA-N Inosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CO)O[C@H]1N1C2=NC=NC(O)=C2N=C1 UGQMRVRMYYASKQ-KQYNXXCUSA-N 0.000 description 1
- 108091092195 Intron Proteins 0.000 description 1
- FADYJNXDPBKVCA-UHFFFAOYSA-N L-Phenylalanyl-L-lysin Natural products NCCCCC(C(O)=O)NC(=O)C(N)CC1=CC=CC=C1 FADYJNXDPBKVCA-UHFFFAOYSA-N 0.000 description 1
- UGTHTQWIQKEDEH-BQBZGAKWSA-N L-alanyl-L-prolylglycine zwitterion Chemical compound C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UGTHTQWIQKEDEH-BQBZGAKWSA-N 0.000 description 1
- SENJXOPIZNYLHU-UHFFFAOYSA-N L-leucyl-L-arginine Natural products CC(C)CC(N)C(=O)NC(C(O)=O)CCCN=C(N)N SENJXOPIZNYLHU-UHFFFAOYSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- JKGHDYGZRDWHGA-SRVKXCTJSA-N Leu-Asn-Leu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O JKGHDYGZRDWHGA-SRVKXCTJSA-N 0.000 description 1
- VQPPIMUZCZCOIL-GUBZILKMSA-N Leu-Gln-Ala Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C)C(O)=O VQPPIMUZCZCOIL-GUBZILKMSA-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
- POZULHZYLPGXMR-ONGXEEELSA-N Leu-Gly-Val Chemical compound CC(C)C[C@H](N)C(=O)NCC(=O)N[C@@H](C(C)C)C(O)=O POZULHZYLPGXMR-ONGXEEELSA-N 0.000 description 1
- WXUOJXIGOPMDJM-SRVKXCTJSA-N Leu-Lys-Asn Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(O)=O WXUOJXIGOPMDJM-SRVKXCTJSA-N 0.000 description 1
- VCHVSKNMTXWIIP-SRVKXCTJSA-N Leu-Lys-Ser Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O VCHVSKNMTXWIIP-SRVKXCTJSA-N 0.000 description 1
- JDBQSGMJBMPNFT-AVGNSLFASA-N Leu-Pro-Val Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(O)=O JDBQSGMJBMPNFT-AVGNSLFASA-N 0.000 description 1
- JIHDFWWRYHSAQB-GUBZILKMSA-N Leu-Ser-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@H](C(O)=O)CCC(O)=O JIHDFWWRYHSAQB-GUBZILKMSA-N 0.000 description 1
- SVBJIZVVYJYGLA-DCAQKATOSA-N Leu-Ser-Val Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O SVBJIZVVYJYGLA-DCAQKATOSA-N 0.000 description 1
- GZRABTMNWJXFMH-UVOCVTCTSA-N Leu-Thr-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O GZRABTMNWJXFMH-UVOCVTCTSA-N 0.000 description 1
- KCXUCYYZNZFGLL-SRVKXCTJSA-N Lys-Ala-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H](CC(C)C)C(O)=O KCXUCYYZNZFGLL-SRVKXCTJSA-N 0.000 description 1
- GAOJCVKPIGHTGO-UWVGGRQHSA-N Lys-Arg-Gly Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCCNC(N)=N)C(=O)NCC(O)=O GAOJCVKPIGHTGO-UWVGGRQHSA-N 0.000 description 1
- GJJQCBVRWDGLMQ-GUBZILKMSA-N Lys-Glu-Ala Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C)C(O)=O GJJQCBVRWDGLMQ-GUBZILKMSA-N 0.000 description 1
- RFQATBGBLDAKGI-VHSXEESVSA-N Lys-Gly-Pro Chemical compound C1C[C@@H](N(C1)C(=O)CNC(=O)[C@H](CCCCN)N)C(=O)O RFQATBGBLDAKGI-VHSXEESVSA-N 0.000 description 1
- CANPXOLVTMKURR-WEDXCCLWSA-N Lys-Gly-Thr Chemical compound C[C@@H](O)[C@@H](C(O)=O)NC(=O)CNC(=O)[C@@H](N)CCCCN CANPXOLVTMKURR-WEDXCCLWSA-N 0.000 description 1
- ATNKHRAIZCMCCN-BZSNNMDCSA-N Lys-Lys-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](CCCCN)N ATNKHRAIZCMCCN-BZSNNMDCSA-N 0.000 description 1
- QQPSCXKFDSORFT-IHRRRGAJSA-N Lys-Lys-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCCN QQPSCXKFDSORFT-IHRRRGAJSA-N 0.000 description 1
- AFLBTVGQCQLOFJ-AVGNSLFASA-N Lys-Pro-Arg Chemical compound NCCCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O AFLBTVGQCQLOFJ-AVGNSLFASA-N 0.000 description 1
- IEIHKHYMBIYQTH-YESZJQIVSA-N Lys-Tyr-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CCCCN)N)C(=O)O IEIHKHYMBIYQTH-YESZJQIVSA-N 0.000 description 1
- WDTLNWHPIPCMMP-AVGNSLFASA-N Met-Arg-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O WDTLNWHPIPCMMP-AVGNSLFASA-N 0.000 description 1
- OBVHKUFUDCPZDW-JYJNAYRXSA-N Met-Arg-Phe Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 OBVHKUFUDCPZDW-JYJNAYRXSA-N 0.000 description 1
- NSGXXVIHCIAISP-CIUDSAMLSA-N Met-Asn-Gln Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O NSGXXVIHCIAISP-CIUDSAMLSA-N 0.000 description 1
- GPAHWYRSHCKICP-GUBZILKMSA-N Met-Glu-Glu Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O GPAHWYRSHCKICP-GUBZILKMSA-N 0.000 description 1
- FYRUJIJAUPHUNB-IUCAKERBSA-N Met-Gly-Arg Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCCNC(N)=N FYRUJIJAUPHUNB-IUCAKERBSA-N 0.000 description 1
- UNPGTBHYKJOCCZ-DCAQKATOSA-N Met-Lys-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](C)C(O)=O UNPGTBHYKJOCCZ-DCAQKATOSA-N 0.000 description 1
- CIDICGYKRUTYLE-FXQIFTODSA-N Met-Ser-Ala Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O CIDICGYKRUTYLE-FXQIFTODSA-N 0.000 description 1
- KZNQNBZMBZJQJO-UHFFFAOYSA-N N-glycyl-L-proline Natural products NCC(=O)N1CCCC1C(O)=O KZNQNBZMBZJQJO-UHFFFAOYSA-N 0.000 description 1
- 108010079364 N-glycylalanine Proteins 0.000 description 1
- 238000012408 PCR amplification Methods 0.000 description 1
- CSDMCMITJLKBAH-SOUVJXGZSA-N Phe-Glu-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CC2=CC=CC=C2)N)C(=O)O CSDMCMITJLKBAH-SOUVJXGZSA-N 0.000 description 1
- 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 1
- XALFIVXGQUEGKV-JSGCOSHPSA-N Phe-Val-Gly Chemical compound OC(=O)CNC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CC1=CC=CC=C1 XALFIVXGQUEGKV-JSGCOSHPSA-N 0.000 description 1
- 101150096292 Ppme1 gene Proteins 0.000 description 1
- FYQSMXKJYTZYRP-DCAQKATOSA-N Pro-Ala-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1 FYQSMXKJYTZYRP-DCAQKATOSA-N 0.000 description 1
- GRIRJQGZZJVANI-CYDGBPFRSA-N Pro-Arg-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H]1CCCN1 GRIRJQGZZJVANI-CYDGBPFRSA-N 0.000 description 1
- 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 1
- FKKHDBFNOLCYQM-FXQIFTODSA-N Pro-Cys-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CS)C(=O)N[C@@H](C)C(O)=O FKKHDBFNOLCYQM-FXQIFTODSA-N 0.000 description 1
- BRJGUPWVFXKBQI-XUXIUFHCSA-N Pro-Leu-Ile Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O BRJGUPWVFXKBQI-XUXIUFHCSA-N 0.000 description 1
- INDVYIOKMXFQFM-SRVKXCTJSA-N Pro-Lys-Gln Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(=O)N)C(=O)O INDVYIOKMXFQFM-SRVKXCTJSA-N 0.000 description 1
- KDBHVPXBQADZKY-GUBZILKMSA-N Pro-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@H]1NCCC1 KDBHVPXBQADZKY-GUBZILKMSA-N 0.000 description 1
- GOMUXSCOIWIJFP-GUBZILKMSA-N Pro-Ser-Arg Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O GOMUXSCOIWIJFP-GUBZILKMSA-N 0.000 description 1
- SXJOPONICMGFCR-DCAQKATOSA-N Pro-Ser-Lys Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCCCN)C(=O)O SXJOPONICMGFCR-DCAQKATOSA-N 0.000 description 1
- QKDIHFHGHBYTKB-IHRRRGAJSA-N Pro-Ser-Phe Chemical compound N([C@@H](CO)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C(=O)[C@@H]1CCCN1 QKDIHFHGHBYTKB-IHRRRGAJSA-N 0.000 description 1
- DYJTXTCEXMCPBF-UFYCRDLUSA-N Pro-Tyr-Phe Chemical compound C1C[C@H](NC1)C(=O)N[C@@H](CC2=CC=C(C=C2)O)C(=O)N[C@@H](CC3=CC=CC=C3)C(=O)O DYJTXTCEXMCPBF-UFYCRDLUSA-N 0.000 description 1
- 108020005067 RNA Splice Sites Proteins 0.000 description 1
- 108010025216 RVF peptide Proteins 0.000 description 1
- SWSRFJZZMNLMLY-ZKWXMUAHSA-N Ser-Asp-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O SWSRFJZZMNLMLY-ZKWXMUAHSA-N 0.000 description 1
- OJPHFSOMBZKQKQ-GUBZILKMSA-N Ser-Gln-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](N)CO OJPHFSOMBZKQKQ-GUBZILKMSA-N 0.000 description 1
- UGTZYIPOBYXWRW-SRVKXCTJSA-N Ser-Phe-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC(O)=O)C(O)=O UGTZYIPOBYXWRW-SRVKXCTJSA-N 0.000 description 1
- ADJDNJCSPNFFPI-FXQIFTODSA-N Ser-Pro-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@@H]1CCCN1C(=O)[C@@H](N)CO ADJDNJCSPNFFPI-FXQIFTODSA-N 0.000 description 1
- SIEBDTCABMZCLF-XGEHTFHBSA-N Ser-Val-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O SIEBDTCABMZCLF-XGEHTFHBSA-N 0.000 description 1
- MTCFGRXMJLQNBG-UHFFFAOYSA-N Serine Natural products OCC(N)C(O)=O MTCFGRXMJLQNBG-UHFFFAOYSA-N 0.000 description 1
- 101150081875 Slc7a10 gene Proteins 0.000 description 1
- 229920002472 Starch Polymers 0.000 description 1
- ZUXQFMVPAYGPFJ-JXUBOQSCSA-N Thr-Ala-Lys Chemical compound C[C@@H](O)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@H](C(O)=O)CCCCN ZUXQFMVPAYGPFJ-JXUBOQSCSA-N 0.000 description 1
- XPNSAQMEAVSQRD-FBCQKBJTSA-N Thr-Gly-Gly Chemical compound C[C@@H](O)[C@H](N)C(=O)NCC(=O)NCC(O)=O XPNSAQMEAVSQRD-FBCQKBJTSA-N 0.000 description 1
- MPUMPERGHHJGRP-WEDXCCLWSA-N Thr-Gly-Lys Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)O)N)O MPUMPERGHHJGRP-WEDXCCLWSA-N 0.000 description 1
- MSIYNSBKKVMGFO-BHNWBGBOSA-N Thr-Gly-Pro Chemical compound C[C@H]([C@@H](C(=O)NCC(=O)N1CCC[C@@H]1C(=O)O)N)O MSIYNSBKKVMGFO-BHNWBGBOSA-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
- 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 1
- IEZVHOULSUULHD-XGEHTFHBSA-N Thr-Ser-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O IEZVHOULSUULHD-XGEHTFHBSA-N 0.000 description 1
- NDZYTIMDOZMECO-SHGPDSBTSA-N Thr-Thr-Ala 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)C(O)=O NDZYTIMDOZMECO-SHGPDSBTSA-N 0.000 description 1
- OENGVSDBQHHGBU-QEJZJMRPSA-N Trp-Glu-Asn Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O OENGVSDBQHHGBU-QEJZJMRPSA-N 0.000 description 1
- YTYHAYZPOARHAP-HOCLYGCPSA-N Trp-Lys-Gly Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)NCC(=O)O)N YTYHAYZPOARHAP-HOCLYGCPSA-N 0.000 description 1
- GAYLGYUVTDMLKC-UWJYBYFXSA-N Tyr-Asp-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=C(O)C=C1 GAYLGYUVTDMLKC-UWJYBYFXSA-N 0.000 description 1
- QAYSODICXVZUIA-WLTAIBSBSA-N Tyr-Gly-Thr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(O)=O QAYSODICXVZUIA-WLTAIBSBSA-N 0.000 description 1
- HSBZWINKRYZCSQ-KKUMJFAQSA-N Tyr-Lys-Asp Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O HSBZWINKRYZCSQ-KKUMJFAQSA-N 0.000 description 1
- MWUYSCVVPVITMW-IGNZVWTISA-N Tyr-Tyr-Ala Chemical compound C([C@@H](C(=O)N[C@@H](C)C(O)=O)NC(=O)[C@@H](N)CC=1C=CC(O)=CC=1)C1=CC=C(O)C=C1 MWUYSCVVPVITMW-IGNZVWTISA-N 0.000 description 1
- YFOCMOVJBQDBCE-NRPADANISA-N Val-Ala-Glu Chemical compound C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N YFOCMOVJBQDBCE-NRPADANISA-N 0.000 description 1
- OVLIFGQSBSNGHY-KKHAAJSZSA-N Val-Asp-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](C(C)C)N)O OVLIFGQSBSNGHY-KKHAAJSZSA-N 0.000 description 1
- COSLEEOIYRPTHD-YDHLFZDLSA-N Val-Asp-Tyr Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 COSLEEOIYRPTHD-YDHLFZDLSA-N 0.000 description 1
- PFMAFMPJJSHNDW-ZKWXMUAHSA-N Val-Cys-Asn Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)N)C(=O)O)N PFMAFMPJJSHNDW-ZKWXMUAHSA-N 0.000 description 1
- WDIGUPHXPBMODF-UMNHJUIQSA-N Val-Glu-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N WDIGUPHXPBMODF-UMNHJUIQSA-N 0.000 description 1
- VXDSPJJQUQDCKH-UKJIMTQDSA-N Val-Ile-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](C(C)C)N VXDSPJJQUQDCKH-UKJIMTQDSA-N 0.000 description 1
- SYSWVVCYSXBVJG-RHYQMDGZSA-N Val-Leu-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](C(C)C)N)O SYSWVVCYSXBVJG-RHYQMDGZSA-N 0.000 description 1
- MJFSRZZJQWZHFQ-SRVKXCTJSA-N Val-Met-Val Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C(C)C)C(=O)O)N MJFSRZZJQWZHFQ-SRVKXCTJSA-N 0.000 description 1
- GBIUHAYJGWVNLN-AEJSXWLSSA-N Val-Ser-Pro Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N1CCC[C@@H]1C(=O)O)N GBIUHAYJGWVNLN-AEJSXWLSSA-N 0.000 description 1
- GBIUHAYJGWVNLN-UHFFFAOYSA-N Val-Ser-Pro Natural products CC(C)C(N)C(=O)NC(CO)C(=O)N1CCCC1C(O)=O GBIUHAYJGWVNLN-UHFFFAOYSA-N 0.000 description 1
- QHSSPPHOHJSTML-HOCLYGCPSA-N Val-Trp-Gly Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)NCC(=O)O)N QHSSPPHOHJSTML-HOCLYGCPSA-N 0.000 description 1
- AOILQMZPNLUXCM-AVGNSLFASA-N Val-Val-Lys Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CCCCN AOILQMZPNLUXCM-AVGNSLFASA-N 0.000 description 1
- XNLUVJPMPAZHCY-JYJNAYRXSA-N Val-Val-Phe Chemical compound CC(C)[C@H]([NH3+])C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C([O-])=O)CC1=CC=CC=C1 XNLUVJPMPAZHCY-JYJNAYRXSA-N 0.000 description 1
- 241000746966 Zizania Species 0.000 description 1
- 235000002636 Zizania aquatica Nutrition 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 108010076324 alanyl-glycyl-glycine Proteins 0.000 description 1
- 108010024078 alanyl-glycyl-serine Proteins 0.000 description 1
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 1
- 108010047495 alanylglycine Proteins 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- ODKSFYDXXFIFQN-UHFFFAOYSA-N arginine Natural products OC(=O)C(N)CCCNC(N)=N ODKSFYDXXFIFQN-UHFFFAOYSA-N 0.000 description 1
- 108010084758 arginyl-tyrosyl-aspartic acid Proteins 0.000 description 1
- 108010062796 arginyllysine Proteins 0.000 description 1
- 108010077245 asparaginyl-proline Proteins 0.000 description 1
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 1
- 108010093581 aspartyl-proline Proteins 0.000 description 1
- 108010047857 aspartylglycine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000012512 characterization method Methods 0.000 description 1
- 210000000349 chromosome Anatomy 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 239000013256 coordination polymer Substances 0.000 description 1
- 230000003247 decreasing effect Effects 0.000 description 1
- 238000012217 deletion Methods 0.000 description 1
- 230000037430 deletion Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 230000004069 differentiation Effects 0.000 description 1
- 230000011559 double-strand break repair via nonhomologous end joining Effects 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 235000013312 flour Nutrition 0.000 description 1
- 239000012634 fragment Substances 0.000 description 1
- 230000004927 fusion Effects 0.000 description 1
- 238000003198 gene knock in Methods 0.000 description 1
- 238000012239 gene modification Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 108010049041 glutamylalanine Proteins 0.000 description 1
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 1
- 108010023364 glycyl-histidyl-arginine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010037850 glycylvaline Proteins 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 239000000833 heterodimer Substances 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 230000006801 homologous recombination Effects 0.000 description 1
- 238000002744 homologous recombination Methods 0.000 description 1
- 230000006698 induction Effects 0.000 description 1
- 208000015181 infectious disease Diseases 0.000 description 1
- 229960003786 inosine Drugs 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 108010044311 leucyl-glycyl-glycine Proteins 0.000 description 1
- 108010090333 leucyl-lysyl-proline Proteins 0.000 description 1
- 108010073472 leucyl-prolyl-proline Proteins 0.000 description 1
- 108010000761 leucylarginine Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 108010017391 lysylvaline Proteins 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000030648 nucleus localization Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 230000002018 overexpression Effects 0.000 description 1
- 108010051242 phenylalanylserine Proteins 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 230000001124 posttranscriptional effect Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
- 108010031719 prolyl-serine Proteins 0.000 description 1
- 108010070643 prolylglutamic acid Proteins 0.000 description 1
- 108010090894 prolylleucine Proteins 0.000 description 1
- 230000004853 protein function Effects 0.000 description 1
- 230000013120 recombinational repair Effects 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- 230000008439 repair process Effects 0.000 description 1
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 1
- 238000002798 spectrophotometry method Methods 0.000 description 1
- 235000019698 starch Nutrition 0.000 description 1
- 239000008107 starch Substances 0.000 description 1
- 238000003860 storage Methods 0.000 description 1
- 108010061238 threonyl-glycine Proteins 0.000 description 1
- 230000001131 transforming effect Effects 0.000 description 1
- IBIDRSSEHFLGSD-UHFFFAOYSA-N valinyl-arginine Natural products CC(C)C(N)C(=O)NC(C(O)=O)CCCN=C(N)N IBIDRSSEHFLGSD-UHFFFAOYSA-N 0.000 description 1
- 108010073969 valyllysine 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)
- C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
- C12N15/8202—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation by biological means, e.g. cell mediated or natural vector
- C12N15/8205—Agrobacterium mediated transformation
-
- 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]
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
- C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/22—Ribonucleases RNAses, DNAses
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/78—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y305/00—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5)
- C12Y305/04—Hydrolases acting on carbon-nitrogen bonds, other than peptide bonds (3.5) in cyclic amidines (3.5.4)
- C12Y305/04004—Adenosine deaminase (3.5.4.4)
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Biomedical Technology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Nutrition Science (AREA)
- Medicinal Chemistry (AREA)
- Virology (AREA)
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明提供一种利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法。通过构建腺嘌呤碱基转化工具ABEs载体靶向OsWaxy基因特定序列，以水稻为转化材料，使OsWaxy蛋白的单个或多个氨基酸突变，突变体的直链淀粉含量测定结果显示，OsWaxy蛋白单个或多个氨基酸突变能够导致突变体内胚乳直链淀粉含量发生显著改变，说明通过单碱基基因编辑技术改变OsWaxy蛋白的单个或多个氨基酸能够显著改良水稻的直链淀粉含量。本发明提供的非转基因纯合OsWaxy基因突变体将成为非常有价值的种质资源，可用于改良水稻品种的直链淀粉含量，为利用单碱基基因编辑技术快速改良水稻品种的直链淀粉含量提供有效策略。
Description
技术领域
本发明涉及基因编辑技术领域，具体地说，涉及一种利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法。
背景技术
随着人民群众生活水平的日益提高，对大米的消费逐渐转向优质化和专业化。目前，行业内公认的影响大米食味品质的最重要因素为直链淀粉含量，它与米饭质地的硬度、凝聚性和粘度等特性密切相关。在优质化方面，在农业部新标准NY/T 593-2013食用稻品质定级指标中，籼稻和粳稻的一级米都要求直链淀粉含量在 13％-18％之间，籼稻二级米要求在13％-20％，而三级米则在13％-22％之间。因此，直链淀粉含量是国家评价优质食用稻的重要关键性指标。与之对应的，保证直链淀粉的含量在13％-20％之间也是水稻优质化育种方向的重要目标。而随着大米消费专业化的发展，对大米直链淀粉含量的需求更加多样化，如专门用于做米粉的大米，或专门供糖尿病患者或高危人群食用的高抗性淀粉大米，甚至是南北方人民口感的差异，都要求在水稻育种中，能够快速精准调整直链淀粉含量，开发能够调整不同直链淀粉含量的生物技术。因此，开发精准调整直链淀粉含量的生物技术手段具有重要的商业价值。
OsWaxy基因位于第6染色体的短臂，早在1990年就已被克隆，是决定直链淀粉合成的关键酶(Wang et al.,1990)。过表达或降低OsWaxy基因表达将导致贮藏器官中 OsWaxy基因酶活和直链淀粉含量明显上升或下降。通过突变体实验也发现，缺失 OsWaxy基因将获得直链淀粉缺失糯稻。OsWaxy基因表达调控的机制目前还不完全清楚。除基因结构发生改变，影响其表达和蛋白功能外，可能受多个水平、多种因子的协同调控。OsWaxy基因在自然界存在多种等位基因，在非糯品种中，OsWaxy基因分化为OsWaxya和OsWaxyb两种等位基因，其中，野生稻全为OsWaxya，籼稻以OsWaxya为主，直链淀粉含量较高；粳稻基本为OsWaxyb，直链淀粉含量较低。序列分析表明，与OsWaxya相比，OsWaxyb的第1内含子5'端剪切处发生由GT→TT的突变，导致第1内含子剪接效率降低及剪接不正常，从而使其转录本含量降低(Hirano et al.,1998；Isshiki et al.,1998)。此外，OsWaxy基因在栽培水稻品种中存在多个等位基因，不同等位基因可能造成该基因表达值或酶活有显著差异，从而导致栽培水稻各个品种中直链淀粉含量差异很大。Wxin突变点位于第6外显子第62位碱基由A突变为C，导致编码的酪氨酸突变为丝氨酸，Wxin中该位点的突变可能改变了基因酶活，使得含有该等位基因品种直链淀粉含量降低。Wxmq编码区域有两个位置发生突变，分别导致了第4外显子编码的第158位氨基酸由精氨酸突变为组氨酸，,5外显子编码的第191位氨基酸由酪氨酸突变为组氨酸，这两处突变显著降低了该品种中直链淀粉含量。可见，无论是人工突变还是自然突变产生的基因突变，OsWaxy基因编码区的点突变有可能使基因活性降低或基因表达值变化、从而影响直链淀粉的合成。
基因编辑技术自诞生以来就成为生命科学领域的有力工具，最新一代的 CRISPR/Cas9由于其高效性和便捷性逐渐成为科研人员关注的焦点，在CRISPR/Cas9 系统中，向导RNA(guide RNA,gRNA)引导Cas9蛋白在基因组的靶向位点进行精准切割造成DNA双链断裂(double strand break,DSB)，宿主细胞利用自身的非同源末端连接(nonhomologous end-joining,NHEJ)或基于同源重组(homologous end recombination repair,HDR)进行修复。在研究基因功能时，通常会用到基因敲除(knock out)、敲入(knock in)、碱基置换(basesubstitution)等，在作物育种中，很多突变是单个核苷酸或者单个氨基酸突变造成的，因此相较于基因敲除和敲入，基于单碱基水平的精准修饰更具发展前景。2017年，David Liu团队经过7轮的蛋白质分子进化与改造，突破性地开发出腺嘌呤碱基编辑器(Adenine baseeditors，ABEs)。通过融合表达野生型以及突变型的腺苷脱氨酶TadA和nCas9组成ABE，ABE通过sgRNA的引导在靶向位置将腺嘌呤脱氨变成肌苷(inosine,I)，随后通过DNA复制，DNA聚合酶将I识别为G，从而实现非常高效的腺嘌呤到鸟嘌呤的转换(Gaudelli et al.,2017)。在OsWaxy基因功能研究过程中，除了模仿自然界中已知的变异外，利用单碱基基因编辑技术能够在基因编码区创制新的点突变，这些点突变位点大多数在自然界中不存在，因此，单碱基编辑技术能够创制大量的OsWaxy基因的新等位基因，研究这些新等位基因的功能，不仅是解析稻米品质变异的重要途径，更是稻米品质改良的重要基础。
发明内容
本发明的目的是提供一种利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法。
为了实现本发明目的，第一方面，本发明提供一种靶向OsWaxy基因的单碱基基因编辑载体，所述载体包含腺嘌呤碱基编辑器和靶向OsWaxy基因的特异性核酸酶。
本发明中，OsWaxy基因编码的蛋白在NCBI上的参考序列编号为 XP_015644490.1。
所述特异性核酸酶可选自CRISPR/Cas9、CRISPR/Cas12a、TALEN、Meganuclease(归巢核酸内切酶)和ZFN等中的任一种。
优选地，所述载体是基于CRISPR/Cas9的腺嘌呤碱基编辑载体(ABEs)(图1)，所述载体至少包含第一表达盒和第二表达盒；
其中，ABEs载体的第一表达盒为ecTadA-ecTadA*7.10-nCas9表达盒；所述第一表达盒包含的核酸构建体ecTadA-ecTadA*7.10-nCas9的序列如SEQ ID NO:3所示。
第二表达盒为sgRNA表达盒，且sgRNA作用位点位于OsWaxy基因的外显子上。
更优选地，sgRNA作用位点的DNA序列选自如下①～⑤中的任一个：
①5′-CATCGACCATCCGTCATTCC-3′；
②5′-ATCCACAACATCTCCTACCA-3′；
③5′-TCGGCAGGCTGGAGGAACAG-3′；
④5′-GACACTGGAGTTGATTACAA-3′；
⑤5′-TCGTCAACGGCATGGACGTC-3′。
优选地，所述第一表达盒由甘蔗Ubi4启动子驱动，所述第二表达盒由水稻U3启动子驱动。
第二方面，本发明提供所述靶向OsWaxy基因的单碱基基因编辑载体在水稻育种和品种改良中的应用。
其中，育种目的为改良水稻直链淀粉含量。
第三方面，本发明提供利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法，所述方法包括将所述靶向OsWaxy基因的单碱基基因编辑载体导入水稻中。
优选地，所述水稻为籼稻。
第四方面，本发明提供OsWaxy蛋白突变体，所述突变体包含如下a～f突变中的任一种：
a.OsWaxy蛋白第166位氨基酸由D到G的突变；
b.OsWaxy蛋白第167位氨基酸由H到R的突变；
c.OsWaxy蛋白第265位氨基酸由N到S的突变；
d.OsWaxy蛋白第191位氨基酸由Y到H的突变；
e.OsWaxy蛋白第353位氨基酸由N到D的突变；
f.OsWaxy蛋白第353位氨基酸由N到S的突变。
本发明中，OsWaxy蛋白在NCBI上的参考序列编号为XP_015644490.1。
第五方面，本发明提供一种改良水稻直链淀粉含量的方法，所述方法包括：利用基因工程手段，在水稻基因组中引入突变，使其编码的OsWaxy蛋白包含D166G、 H167R、Y191H、N265S、N353D或N353S突变位点。
本发明的目的还可以采用以下的技术措施来进一步实现。
本发明提供一种通过修饰水稻OsWaxy基因改良水稻直链淀粉含量的方法，通过对所述水稻OsWaxy基因进行腺嘌呤碱基编辑从而改良直链淀粉含量，包括如下步骤：
(1)根据OsWaxy基因编码区设计靶位点；
(2)构建腺嘌呤碱基转化工具(Adenine base editors，ABEs)载体，将拟改良水稻品种的愈伤组织作为遗传转化的受体材料，用农杆菌介导法将单碱基编辑载体导入愈伤组织细胞，并再生成水稻植株；
(3)对OsWaxy基因编辑体的基因型检测和分析，选取单个或多个氨基酸发生改变的编辑体进行性状分析，考察直链淀粉含量。
所述方法是基于特异性核酸酶的基因编辑技术，序列特异性核酸酶选自 CRISPR/Cas9、CRISPR/Cas12a、TALEN、Meganuclease和ZFN。
其中，水稻OsWaxy基因由SEQ ID NO:1所示的核苷酸序列组成，或编码SEQ ID NO:2所示的氨基酸序列，或将SEQ ID NO:2所示的氨基酸序列经过一个或几个氨基酸残基的取代和/或缺失和/或添加且具有相同功能的其衍生的蛋白质。
本发明通过单碱基编辑改良水稻直链淀粉含量的方法，gRNA靶位点的选择。靶位点选在水稻OsWaxy基因的编码区内。
OsWaxy基因修饰结果为，该基因编码的蛋白质在编辑靶位点区域发生一个或多个氨基酸突变。
本发明还提供所述方法在创制水稻改良种质资源中的应用。
借由上述技术方案，本发明至少具有下列优点及有益效果：
本发明提供的利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法。通过构建腺嘌呤碱基编辑器ABEs载体靶向OsWaxy基因特定序列，以水稻为转化材料，使OsWaxy蛋白的单个或多个氨基酸突变，突变体的直链淀粉含量测定结果显示，OsWaxy蛋白单个或多个氨基酸突变能够导致突变体内胚乳直链淀粉含量发生显著改变，说明通过单碱基基因编辑技术改变OsWaxy蛋白的单个或多个氨基酸能够显著改良水稻的直链淀粉含量。本发明提供的非转基因纯合OsWaxy基因突变体将成为非常有价值的种质资源，可以利用它们改良水稻品种的直链淀粉含量，本发明为利用单碱基基因编辑技术快速改良水稻品种的直链淀粉含量提供有效策略。
附图说明
图1为本发明较佳实施例中腺嘌呤碱基编辑载体示意图。ABEs载体包含由Ubi4 启动子驱动的ecTadA-ecTadA*7.10-nCas9融合蛋白表达盒，以及由水稻U3启动子驱动的gRNA表达盒。NLS为细胞核定位信号，linker为融合蛋白中连接不同蛋白的肽段， nos为终止子。
具体实施方式
本发明提供一种以CAS9和腺苷脱氨酶融合蛋白的单碱基编辑技术创制OsWaxy 基因突变体改良水稻直链淀粉含量的方法，最终获得直链淀粉含量得以改良的优质水稻品种。
本发明采用如下技术方案：
本发明提供一种以CAS9和腺苷脱氨酶融合蛋白的单碱基编辑技术创制OsWaxy基因突变体来改良水稻直链淀粉含量的方法，该方法适应于所有含有功能型OsWaxy基因的水稻品种，包括以下步骤：
(1)拟改良水稻品种OsWaxy基因的序列分析，在基因编码区合适位点设计靶标；
(2)构建单碱基编辑载体，ABEs载体中含有编码区的一个靶点，将拟改良水稻品种的愈伤组织作为遗传转化的受体材料，用农杆菌介导法将单碱基编辑载体导入愈伤组织细胞，并再生成水稻植株；
(3)对OsWaxy基因编辑体的基因型检测和分析，选取获得了单个或多个氨基酸突变的编辑体进行传代分离；
(4)经过传代分离、鉴定筛选，得到编辑纯合且非转基因的OsWaxy基因突变体，鉴定其直链淀粉含量。
具体方法如下：
(1)水稻中OsWaxy基因的检测；
(2)ABEs载体的构建；
(3)将籼稻R1618(X35)的愈伤组织作为遗传转化的受体材料，用农杆菌介导法将单碱基编辑载体导入，鉴定转基因植株OsWaxy基因编辑情况，获得具有单个或多个氨基酸变化的OsWaxy等位基因基因型。经过传代分离、鉴定筛选，得到编辑纯合且非转基因的株系，最终获得直链淀粉含量显著变化的改良品系。
所述OsWaxy基因为编码OsWaxy蛋白的基因。
所述OsWaxy基因编码的OsWaxy蛋白的氨基酸序列如SEQ ID NO:2所示。
所述OsWaxy基因为：
1)编码区如序列表中SEQ ID NO:1所示的DNA分子；或
2)与1)限定的DNA序列杂交且编码OsWaxy蛋白的DNA分子；或
3)与1)限定的DNA序列至少具有70％以上同源性且编码所述OsWaxy功能蛋白的DNA分子。
所述水稻中OsWaxy基因的表达是通过对水稻中OsWaxy基因进行基因编辑实现的。所述基因编辑是借助单碱基编辑系统实现的。
所述腺嘌呤碱基编辑系统中，融合蛋白ecTadA-ecTadA*7.10-nCas9的表达由甘蔗Ubiquitin4启动子(Ubi4)驱动，其中ecTadA为一种大肠杆菌(Escherichia coli)tRNA来源的腺苷脱氨酶，ecTadA*7.10为经过七轮的定向进化和改造得到的腺苷脱氨酶ecTadA 突变体，nCas9只具备DNA一条链上切割活性，不能造成DNA链断裂，只能造成切口，该融合蛋白在单一多肽链中将野生型非催化性TadA单体、进化的TadA*单体和Cas9切口酶整合到了一起形成异二聚体，具有较强的A/T→G/C转换活性。ABEs载体中gRNA 表达盒由水稻U3启动子驱动。gRNA1的靶标序列为：CATCGACCATCCGTCATTCC。 gRNA2的靶标序列为：ATCCACAACATCTCCTACCA。gRNA3的靶标序列为： TCGGCAGGCTGGAGGAACAG。gRNA4的靶标序列为： GACACTGGAGTTGATTACAA。gRNA5的靶标序列为：TCGTCAACGGCATGGACGTC。
以下实施例用于说明本发明，但不用来限制本发明的范围。若未特别指明，实施例均按照常规实验条件，如Sambrook等分子克隆实验手册(Sambrook J&Russell DW,Molecular Cloning:a Laboratory Manual,2001)，或按照制造厂商说明书建议的条件。
以下实施例中转化的受体材料为籼稻R1618(X35)，其OsWaxy基因类型为 OsWaxya类型。籼稻R1618购自中国种子集团有限公司。
水稻种子R1618现已保藏于中国典型培养物保藏中心，地址：中国武汉，武汉大学，邮编430072，保藏编号CCTCC NO:P202118，保藏日期2021年10月25日。
实施例1水稻OsWaxy基因的序列分析及基因编辑靶点设计
水稻OsWaxy基因的序列如SEQ ID NO:1所示。序列分析显示，该基因含有14个外显子和13个内含子，本发明设计的三个靶序列在X35材料中均位于OsWaxy基因上外显子中，其中靶序列1在OsWaxy基因序列第2011-2030位碱基，靶序列2在OsWaxy 基因上的第2683-2702位碱基，靶序列3在OsWaxy基因上的第3351-3370位碱基，靶序列4在OsWaxy基因序列第2171-2190位碱基，靶序列5在OsWaxy基因上的第3069-3088 位碱基。
实施例2打靶载体构建及水稻遗传转化
本实施例采用基因编辑技术为单碱基编辑技术，所使用的载体参照(Gaudelli etal.,2017)等人的方法，由中国种子集团生命科学技术中心自主设计，所有载体原始骨架为pCambia1305.1，筛选标记为CP4基因，，ABEs载体包含Ubi4启动子驱动的ecTadA-ecTadA*7.10-nCas9融合蛋白表达盒(SEQ ID NO:3)，以及水稻U3启动子驱动的gRNA表达盒。利用Asc1和Pme1将含有OsWaxy基因靶标的gRNA表达盒(U3启动子驱动)组装入ABEs，挑选出正确的阳性克隆，并通过测序确认gRNA表达盒序列正确。将编辑载体转入农杆菌菌株EHA105(本实验室保存菌株)中。根据编码区序列，共设计了五个靶标，将含有靶标1(CATCGACCATCCGTCATTCC)的ABE载体命名为pzz000671，靶标2(ATCCACAACATCTCCTACCA)的ABE载体命名为pzz000685，靶标3(TCGGCAGGCTGGAGGAACAG)的ABE载体命名为pzz000684，靶标4(GACACTGGAGTTGATTACAA)的ABE载体命名为pzz000672，靶标5(TCGTCAACGGCATGGACGTC)的ABE载体命名为pzz000743(该载体靶标PAM序列为NG，因此融合蛋白中nCas9换成相应的nCas9-NG，SEQ ID NO:4)。将灭菌好的水稻种子放在诱导培养基上于28℃暗培养30天诱导愈伤组织。愈伤组织每2周继代1 次。农杆菌侵染水稻愈伤组织及筛选、分化流程参照Nishimura等的文献报道。待分化出2-3cm的小苗后，选取单株小苗转入生根盒进行生根培养。
实施例3 T0代水稻转化阳性且完成目标编辑的事件筛选和鉴定
将实施例2中的再生植株送至温室种植，取再生的E0代小苗叶片，通过CTAB法提取植物基因组DNA。DNA样品用荧光定量PCR方法进行阳性检测，选择筛选标记基因CP4作为检测对象，设计约200bp大小的扩增引物，引物序列为，csp356： CAGCACAGGTTAAGTCTG；csp357：GTCTGTCTCAACGGTAAG。在荧光定量PCR 仪上进行扩增和荧光值检测，选择水稻ACTIN1基因作为内参基因，引物序列为 csp106：TGCTATGTACGTCGCCATCCAG；csp107：AATGAGTAACCACGCTCCGTCA；根据RQ值筛选出转基因阳性的植株(CP4基因 RQ值>0.1，结果未示出)。在待编辑区两侧设计引物，pzz000671和pzz000672载体编辑植株编辑位点检测引物序列为(F:CGTTGTGGCTGAGGTAGGA；R: GATCCTAGGAGCCTCGAGTG)，pzz000685、pzz000684和pzz000743载体编辑植株编辑位点检测引物序列为(F:TTCCTTCCGGTTTGTTACTGA；R:TTGGCATATCGTGCAAGTGT)。利用Q5高保真DNA聚合酶进行PCR扩增，获得约 1kb大小DNA片段，进行一代测序，选取目标区域发生了单个或多个氨基酸变化的杂合或纯合植株，移入温室，收取T1代种子。
实施例4 OsWaxy基因编辑T1代纯合非转基因水稻植株的筛选和鉴定
选取目标编辑事件T1代种子(约100粒)，进行发芽、育苗，提取幼苗的DNA进行转基因成分检测及编辑位点PCR检测，从中筛选出非转基因及编辑位点纯合植株。转基因成分检测方法请参见《基因编辑后代材料GMO检测流程》，其是根据中国国家标准农业部953号公告6-2007号公告GB/T 19495-2004转基因产品检测标准要求进行。编辑位点纯合植株指利用一代测序确认，所有突变位点均为纯合的植株。结果显示，pzz000671载体转化植物共获得两种纯合突变体，分别为D166G和H167R， pzz000685载体转化植物共获得十种纯合突变体，分别为H264R、N265D、N265S、 I266V、H264R&N265D、H264R&N265S、H264R&I266V、N265G、N265D&I266V、 H264R&I266V；pzz000684载体转化植物共获得一种纯合突变体R408G；pzz000672 载体转化植物共获得一种纯合突变体Y191H；pzz000743载体转化植物共获得两种纯合突变体，分别为N353D和N353S，突变核苷酸序列如表1所示。
表1单碱基编辑水稻株系OsWaxy基因序列变化及直链淀粉含量测定
实施例5突变体直链淀粉含量的表型鉴定
将实施例4中的非转基因纯合目标编辑株系移栽入温室，收获种子后进行直链淀粉含量测定。直链淀粉含量测定方法参照农业部标NY/T 2639-2014《稻米直链淀粉的测定-分光光度法》，测定结果显示，OsWaxy基因编码区发生氨基酸单个或多个点突变后，直链淀粉含量发生显著改变，其中，D166G、H167R、Y191H、N265S、N353D 以及N353S几种突变体的直链淀粉含量获得显著改善，由野生型的29.4％下调至 10％-23％(表1)。
本发明将单碱基基因编辑技术加以巧妙运用，利用其单碱基编辑功能来实现基因的定点突变，并筛选出特定突变类型。该方法该系统简单易行、成本较低、效率高。本发明利用单碱基编辑技术对OsWaxy基因结构的定点编辑，成功获得了直链淀粉含量降低的籼稻品种，也为利用单碱基基因编辑技术快速改良水稻品种直链淀粉含量提供新思路。
虽然，上文中已经用一般性说明及具体实施方案对本发明作了详尽的描述，但在本发明基础上，可以对之做一些修改或改进，这对本领域技术人员而言是显而易见的。因此，在不偏离本发明精神的基础上所做的这些修改或改进，均属于本发明要求保护的范围。
参考文献:
1、Gaudelli,N.M.,Komor,A.C.,Rees,H.A.,Packer,M.S.,Badran,A.H.,Bryson,D.I.,Liu,D.R.Programmable base editing of A·T to G·C in genomic DNA withoutDNA cleavage.Nature 2017,551:464-471.
2、Hiro-Yuki Hirano,Mitsugu Eiguchi,Yoshio Sano.A single base changealtered the regulation of the Waxy gene at the posttranscriptional levelduring the domestication of rice.Molecular Biology and Evolution,1998,15(8):978-987
3、Masayuki Isshiki,Kazuko Morino,Midori Nakajima,Ron J.Okagaki,SusanR.Wessler,Takeshi Izawa,Ko Shimamoto. A naturally occurring functional alleleof the rice waxy locus has a GT to TT mutation at the 5'splice site of thefirst intron.The Plant Journal,1998,15(1):133-138
4、Zong-yang Wang,Zhi-liang Wu,Yan-yan Xing,Fei-gin Zheng,Xiao-Ii Guo,Wei-guo Zhang and Meng-min Hong. Nucleotide sequence of rice waxygene.Nucleic Acids Research,1990,18(19):5898。
序列表
<110> 中国种子集团有限公司
<120> 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法
<130> KHP211117902.4
<160> 4
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5035
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 1
accattcctt cagttctttg tctatctcaa gacacaaata actgcagtct ctctctctct 60
ctctctctct ctctctctct ctctgcttca cttctctgct tgtgttgttc tgttgttcat 120
caggaagaac atctgcaagt tatacatata tgtttataat tctttgtttc ccctcttatt 180
cagatcgatc acatgcatct ttcattgctc gtttttcctt acaagtagtc tcatacatgc 240
taatttctgt aaggtgttgg gctggaaatt aattaattaa ttaattgact tgccaagatc 300
catatatatg tcctgatatt aaatcttcgt tcgttatgtt tggttaggct gatcaatgtt 360
attctagagt ctagagaaac acacccaggg gttttccaac tagctccaca agatggtggg 420
ctagctgacc tagatttgaa gtctcactcc ttataattat tttatattag atcattttct 480
aatattcgtg tcttttttta ttctagagtc tagatcttgt gttcaactct cgttaaatca 540
tgtctctcgc cactggagaa acagatcagg agggtttatt ttgggtatag gtcaaagcta 600
agattgaaat tcacaaatag taaaatcaga atccaaccaa ttttagtagc cgagttggtc 660
aaaggaaaat gtatatagct agatttattg ttttggcaaa aaaaaatctg aatatgcaaa 720
atacttgtat atctttgtat taagaagatg aaaataagta gcagaaaatt aaaaaatgga 780
ttatatttcc tgggctaaaa gaattgttga tttggcacaa ttaaattcag tgtcaaggtt 840
ttgtgcaaga attcagtgtg aaggaataga ttctcttcaa aacaatttaa tcattcatct 900
gatctgctca aagctctgtg catctccggg tgcaacggcc aggatattta ttgtgcagta 960
aaaaaatgtc atatccccta gccacccaag aaactgctcc ttaagtcctt ataagcacat 1020
atggcattgt aatatatatg tttgagtttt agcgacaatt tttttaaaaa cttttggtcc 1080
tttttatgaa cgttttaagt ttcactgtct ttttttttcg aattttaaat gtagcttcaa 1140
attctaatcc ccaatccaaa ttgtaataaa cttcaattct cctaattaac atcttaattc 1200
atttatttga aaaccagttc aaattctttt aggctcacca aaccttaaac aattcaattc 1260
agtgcagaga tcttccacag caacagctag acaaccacca tgtcggctct caccacgtcc 1320
cagctcgcca cctcggccac cggcttcggc atcgccgaca ggtcggcgcc gtcgtcgctg 1380
ctccgccacg ggttccaggg cctcaagccc cgcagccccg ccggcggcga cgcgacgtcg 1440
ctcagcgtga cgaccagcgc gcgcgcgacg cccaagcagc agcggtcggt gcagcgtggc 1500
agccggaggt tcccctccgt cgtcgtgtac gccaccggcg ccggcatgaa cgtcgtgttc 1560
gtcggcgccg agatggcccc ctggagcaag accggcggcc tcggtgacgt cctcggtggc 1620
ctcccccctg ccatggctgt aagcacacac aaacttcgat cgctcgtcgt cgctgaccgt 1680
cgtcgtcttc aactgttctt gatcatcgca ttggatggat gtgtaatgtt gtgttcttgt 1740
gttctttgca ggcgaatggc cacagggtca tggtgatctc tcctcggtac gaccagtaca 1800
aggacgcttg ggataccagc gttgtggctg aggtaggagc atatgcgtga tcagatcatc 1860
acaagatcga ttagctttag atgatttgtt acatttcgca agattttaac ccaagttttt 1920
gtggtgcaat tcattgcaga tcaaggttgc agacaggtac gagagggtga ggtttttcca 1980
ttgctacaag cgtggagtcg accgtgtgtt catcgaccat ccgtcattcc tggagaaggt 2040
ggagtcatca ttagtttacc ttttttgttt ttactgaatt attaacagtg catttagcag 2100
ttggactgag cttagcttcc actggtgatt tcaggtttgg ggaaagaccg gtgagaagat 2160
ctacggacct gacactggag ttgattacaa agacaaccag atgcgtttca gccttctttg 2220
ccaggtcagt gattacttct atctgatgat ggttggaagc atcacgagtt taccatagta 2280
tgtatggatt cataactaat tcgtgtattg atgctacctg caggcagcac tcgaggctcc 2340
taggatccta aacctcaaca acaacccata cttcaaagga acttatggtg agttacaatt 2400
gatctcaaga tcttataact ttcttcgaag gaatccatga tgatcagact aattccttcc 2460
ggtttgttac tgacaacagg tgaggatgtt gtgttcgtct gcaacgactg gcacactggc 2520
ccactggcga gctacctgaa gaacaactac cagcccaatg gcatctacag gaatgcaaag 2580
gtctatgctt gttcttgcca taccaactca aatctgcatg cacactgcat tctgttcaga 2640
aactgactgt ctgaatcttt ttcactgcag gttgctttct gcatccacaa catctcctac 2700
cagggccgtt tcgctttcga ggattaccct gagctgaacc tctccgagag gttcaggtca 2760
tccttcgatt tcatcgacgg gtatgagtaa gattctaaga gtaacttact gtcaattcgc 2820
catatatcga ttcaatccaa gatccttttg agctgacaac cctgcactac tgtccatcgt 2880
tcaaatccgg ttaaatttca ggtatgacac gccggtggag ggcaggaaga tcaactggat 2940
gaaggccgga atcctggaag ccgacagggt gctcaccgtg agcccgtact acgccgagga 3000
gctcatctcc ggcatcgcca ggggatgcga gctcgacaac atcatgcggc tcaccggcat 3060
caccggcatc gtcaacggca tggacgtcag cgagtgggat cctagcaagg acaagtacat 3120
caccgccaag tacgacgcaa ccacggtaag aacgaatgca ttcttcacaa gatatgcaat 3180
ctgaattttc tttgaaaaag aaattatcat ctgtcacttc ttgattgatt ctgacaaggc 3240
aagaatgagt gacaaatttc aggcaatcga ggcgaaggcg ctgaacaagg aggcgttgca 3300
ggcggaggcg ggtcttccgg tcgacaggaa aatcccactg atcgcgttca tcggcaggct 3360
ggaggaacag aagggccctg acgtcatggc cgccgccatc ccggagctca tgcaggagga 3420
cgtccagatc gttcttctgg tataatataa tacactacaa gacacacttg cacgatatgc 3480
caaaaattca gaacaaattc agtggcaaaa aaaaaactcg aatattaggg aaggacctaa 3540
taatatcaaa taattagaag gggtgaggct ttgaacccag atcgtctagt ccaccacctt 3600
gtggagttag ccggaagacc tctgagcatt tctcaattca gtggcaaatg atgtgtataa 3660
ttttgatccg tgtgtgtttc agggtactgg aaagaagaag ttcgagaagc tgctcaagag 3720
catggaggag aagtatccgg gcaaggtgag ggccgtggtg aagttcaacg cgccgcttgc 3780
tcatctcatc atggccggag ccgacgtgct cgccgtcccc agccgcttcg agccctgtgg 3840
actcatccag ctgcagggga tgagatacgg aacggtatac aatttccatc tatcaattcg 3900
attgttcgat ttcatctttg tgcaatgcaa tgcaattgca aatgcaaatg catgatgatt 3960
ttccttgttg atttctccag ccctgtgctt gcgcgtccac cggtgggctc gtggacacgg 4020
tcatcgaagg caagactggt ttccacatgg gccgtctcag cgtcgacgta agcctataca 4080
tttacataac aatcagatat gacacatcct aataccgata agtcggtaca ctactacaca 4140
tttacatggt tgctggttat atggtttttt tggcagtgca aggtggtgga gccaagcgac 4200
gtgaagaagg tggcggccac cctgaagcgc gccatcaagg tcgtcggcac gccggcgtac 4260
gaggagatgg tcaggaactg catgaaccag gacctctcct ggaaggtata aattacgaaa 4320
caaatttaac ccaaacatat actatatact ccctccgctt ctaaatattc aacgccgttg 4380
tcttttttaa atatgtttga ccattcgtct tattaaaaaa attaaataat tataaattct 4440
tttcctatca tttgattcat tgttaaatat acttatatgt atacatatag ttttacatat 4500
ttcataaaat tttttgaaca agacgaacgg tcaaacatgt gctaaaaagt taacggtgtc 4560
gaatattcag aaacggaggg agtataaacg tcttgttcag aagttcagag attcacctgt 4620
ctgatgctga tgatgattaa ttgtttgcaa catggatttc aggggcctgc gaagaactgg 4680
gagaatgtgc tcctgggcct gggcgtcgcc ggcagcgcgc cggggatcga aggcgacgag 4740
atcgcgccgc tcgccaagga gaacgtggct gctccttgaa gagcctgaga tctacatatg 4800
gagtgattaa ttaatatagc agtatatgga tgagagacga atgaaccagt ggtttgtttg 4860
ttgtagtgaa tttgtagcta tagccaatta tataggctaa taagtttgat gttgtactct 4920
tctgggtgtg cttaagtatc ttatcggacc ctgaatttat gtgtgtggct tattgccaat 4980
aatattaagt aataaagggt ttattatatt attatatatg ttatattata cttcc 5035
<210> 2
<211> 609
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Ser Ala Leu Thr Thr Ser Gln Leu Ala Thr Ser Ala Thr Gly Phe
1 5 10 15
Gly Ile Ala Asp Arg Ser Ala Pro Ser Ser Leu Leu Arg His Gly Phe
20 25 30
Gln Gly Leu Lys Pro Arg Ser Pro Ala Gly Gly Asp Ala Thr Ser Leu
35 40 45
Ser Val Thr Thr Ser Ala Arg Ala Thr Pro Lys Gln Gln Arg Ser Val
50 55 60
Gln Arg Gly Ser Arg Arg Phe Pro Ser Val Val Val Tyr Ala Thr Gly
65 70 75 80
Ala Gly Met Asn Val Val Phe Val Gly Ala Glu Met Ala Pro Trp Ser
85 90 95
Lys Thr Gly Gly Leu Gly Asp Val Leu Gly Gly Leu Pro Pro Ala Met
100 105 110
Ala Ala Asn Gly His Arg Val Met Val Ile Ser Pro Arg Tyr Asp Gln
115 120 125
Tyr Lys Asp Ala Trp Asp Thr Ser Val Val Ala Glu Ile Lys Val Ala
130 135 140
Asp Arg Tyr Glu Arg Val Arg Phe Phe His Cys Tyr Lys Arg Gly Val
145 150 155 160
Asp Arg Val Phe Ile Asp His Pro Ser Phe Leu Glu Lys Val Trp Gly
165 170 175
Lys Thr Gly Glu Lys Ile Tyr Gly Pro Asp Thr Gly Val Asp Tyr Lys
180 185 190
Asp Asn Gln Met Arg Phe Ser Leu Leu Cys Gln Ala Ala Leu Glu Ala
195 200 205
Pro Arg Ile Leu Asn Leu Asn Asn Asn Pro Tyr Phe Lys Gly Thr Tyr
210 215 220
Gly Glu Asp Val Val Phe Val Cys Asn Asp Trp His Thr Gly Pro Leu
225 230 235 240
Ala Ser Tyr Leu Lys Asn Asn Tyr Gln Pro Asn Gly Ile Tyr Arg Asn
245 250 255
Ala Lys Val Ala Phe Cys Ile His Asn Ile Ser Tyr Gln Gly Arg Phe
260 265 270
Ala Phe Glu Asp Tyr Pro Glu Leu Asn Leu Ser Glu Arg Phe Arg Ser
275 280 285
Ser Phe Asp Phe Ile Asp Gly Tyr Asp Thr Pro Val Glu Gly Arg Lys
290 295 300
Ile Asn Trp Met Lys Ala Gly Ile Leu Glu Ala Asp Arg Val Leu Thr
305 310 315 320
Val Ser Pro Tyr Tyr Ala Glu Glu Leu Ile Ser Gly Ile Ala Arg Gly
325 330 335
Cys Glu Leu Asp Asn Ile Met Arg Leu Thr Gly Ile Thr Gly Ile Val
340 345 350
Asn Gly Met Asp Val Ser Glu Trp Asp Pro Ser Lys Asp Lys Tyr Ile
355 360 365
Thr Ala Lys Tyr Asp Ala Thr Thr Ala Ile Glu Ala Lys Ala Leu Asn
370 375 380
Lys Glu Ala Leu Gln Ala Glu Ala Gly Leu Pro Val Asp Arg Lys Ile
385 390 395 400
Pro Leu Ile Ala Phe Ile Gly Arg Leu Glu Glu Gln Lys Gly Pro Asp
405 410 415
Val Met Ala Ala Ala Ile Pro Glu Leu Met Gln Glu Asp Val Gln Ile
420 425 430
Val Leu Leu Gly Thr Gly Lys Lys Lys Phe Glu Lys Leu Leu Lys Ser
435 440 445
Met Glu Glu Lys Tyr Pro Gly Lys Val Arg Ala Val Val Lys Phe Asn
450 455 460
Ala Pro Leu Ala His Leu Ile Met Ala Gly Ala Asp Val Leu Ala Val
465 470 475 480
Pro Ser Arg Phe Glu Pro Cys Gly Leu Ile Gln Leu Gln Gly Met Arg
485 490 495
Tyr Gly Thr Pro Cys Ala Cys Ala Ser Thr Gly Gly Leu Val Asp Thr
500 505 510
Val Ile Glu Gly Lys Thr Gly Phe His Met Gly Arg Leu Ser Val Asp
515 520 525
Cys Lys Val Val Glu Pro Ser Asp Val Lys Lys Val Ala Ala Thr Leu
530 535 540
Lys Arg Ala Ile Lys Val Val Gly Thr Pro Ala Tyr Glu Glu Met Val
545 550 555 560
Arg Asn Cys Met Asn Gln Asp Leu Ser Trp Lys Gly Pro Ala Lys Asn
565 570 575
Trp Glu Asn Val Leu Leu Gly Leu Gly Val Ala Gly Ser Ala Pro Gly
580 585 590
Ile Glu Gly Asp Glu Ile Ala Pro Leu Ala Lys Glu Asn Val Ala Ala
595 600 605
Pro
<210> 3
<211> 5325
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
atgtcagagg tggagttctc tcacgagtac tggatgaggc atgcgctgac cctggcaaag 60
agggcctggg atgagaggga ggtgccagtg ggagccgtgc tcgtccacaa caatcgcgtg 120
atcggagagg gatggaatag gccaattgga aggcatgacc caacagcaca tgccgagatc 180
atggcactca ggcagggcgg cctggtcatg cagaattacc ggctgattga tgcgaccctc 240
tacgtgacac tggagccatg cgtcatgtgc gcgggagcca tgatccattc aaggattgga 300
agggtggtct tcggagcaag ggatgccaag acaggagcag ccggaagcct catggatgtg 360
ctgcaccatc ctggcatgaa ccatcgcgtc gagatcacag agggaattct ggcggatgag 420
tgcgcggccc tcctgtccga tttcttccgg atgaggcgcc aggagatcaa ggcccagaag 480
aaggcccagt ccagcacaga ttccggcggc tcatctggcg gctcctcagg atcagagacc 540
cctggcacat cagagtctgc gacaccagaa tcctctggcg gctccagcgg cggctcatct 600
gaggtggagt tcagccatga atactggatg aggcatgcac tcacactcgc aaagagggcc 660
agggatgagc gggaagtccc agtgggagcc gtgctggtcc tgaacaatcg cgtgatcggc 720
gagggatgga acagggcaat tggcctccac gacccaacag cccatgccga gatcatggcc 780
ctgcgccagg gcggcctggt catgcaaaat taccgcctca ttgatgccac cctctacgtg 840
acattcgaac cgtgcgtcat gtgcgccggc gcgatgattc actcccgcat cggcagggtg 900
gtcttcggcg tgaggaatgc aaagacagga gcggccggat ccctcatgga cgtgctgcac 960
taccctggca tgaatcacag ggtcgagatc acagagggca ttctggccga cgaatgcgcg 1020
gccctcctgt gctacttctt ccggatgccg aggcaggtct tcaatgccca gaagaaggcc 1080
cagtccagca cagattccgg cggctcatct ggcggctcct caggatcaga gacccctggc 1140
acatcagagt ctgcgacacc agaatcctct ggcggctcaa gcggcggctc cgacaagaag 1200
tactccatcg gcctcgccat cggcaccaac tccgtgggct gggccgtgat caccgacgag 1260
tacaaggtgc cgtccaagaa gttcaaggtg ctcggcaaca ccgaccgcca ctccatcaag 1320
aagaacctca tcggcgccct cctcttcgac tccggcgaga ccgccgaggc cacccgcctc 1380
aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta cctccaggag 1440
atcttctcca acgagatggc caaggtggac gactccttct tccaccgcct cgaggagtcc 1500
ttcctcgtgg aggaggacaa gaagcacgag cgccacccga tcttcggcaa catcgtggac 1560
gaggtggcct accacgagaa gtacccgacc atctaccacc tccgcaagaa gctcgtggac 1620
tccaccgaca aggccgacct ccgcctcatc tacctcgccc tcgcccacat gatcaagttc 1680
cgcggccact tcctcatcga gggcgacctc aacccggaca actccgacgt ggacaagctc 1740
ttcatccagc tcgtgcagac ctacaaccag ctcttcgagg agaacccgat caacgcctcc 1800
ggcgtggacg ccaaggccat cctctccgcc cgcctctcca agtcccgccg cctcgagaac 1860
ctcatcgccc agctcccggg cgagaagaag aacggcctct tcggcaacct catcgccctc 1920
tccctcggcc tcaccccgaa cttcaagtcc aacttcgacc tcgccgagga cgccaagctc 1980
cagctctcca aggacaccta cgacgacgac ctcgacaacc tcctcgccca gatcggcgac 2040
cagtacgccg acctcttcct cgccgccaag aacctctccg acgccatcct cctctccgac 2100
atcctccgcg tgaacaccga gatcaccaag gccccgctct ccgcctccat gatcaagcgc 2160
tacgacgagc accaccagga cctcaccctc ctcaaggccc tcgtgcgcca gcagctcccg 2220
gagaagtaca aggagatctt cttcgaccag tccaagaacg gctacgccgg ctacatcgac 2280
ggcggcgcct cccaggagga gttctacaag ttcatcaagc cgatcctcga gaagatggac 2340
ggcaccgagg agctcctcgt gaagctcaac cgcgaggacc tcctccgcaa gcagcgcacc 2400
ttcgacaacg gctccatccc gcaccagatc cacctcggcg agctccacgc catcctccgc 2460
cgccaggagg acttctaccc gttcctcaag gacaaccgcg agaagatcga gaagatcctc 2520
accttccgca tcccgtacta cgtgggcccg ctcgcccgcg gcaactcccg cttcgcctgg 2580
atgacccgca agtccgagga gaccatcacc ccgtggaact tcgaggaggt ggtggacaag 2640
ggcgcctccg cccagtcctt catcgagcgc atgaccaact tcgacaagaa cctcccgaac 2700
gagaaggtgc tcccgaagca ctccctcctc tacgagtact tcaccgtgta caacgagctc 2760
accaaggtga agtacgtgac cgagggcatg cgcaagccgg ccttcctctc cggcgagcag 2820
aagaaggcca tcgtggacct cctcttcaag accaaccgca aggtgaccgt gaagcagctc 2880
aaggaggact acttcaagaa gatcgagtgc ttcgactccg tggagatctc cggcgtggag 2940
gaccgcttca acgcctccct cggcacctac cacgacctcc tcaagatcat caaggacaag 3000
gacttcctcg acaacgagga gaacgaggac atcctcgagg acatcgtgct caccctcacc 3060
ctcttcgagg accgcgagat gatcgaggag cgcctcaaga cctacgccca cctcttcgac 3120
gacaaggtga tgaagcagct caagcgccgc cgctacaccg gctggggccg cctctcccgc 3180
aagctcatca acggcatccg cgacaagcag tccggcaaga ccatcctcga cttcctcaag 3240
tccgacggct tcgccaaccg caacttcatg cagctcatcc acgacgactc cctcaccttc 3300
aaggaggaca tccagaaggc ccaggtgtcc ggccagggcg actccctcca cgagcacatc 3360
gccaacctcg ccggctcccc ggccatcaag aagggcatcc tccagaccgt gaaggtggtg 3420
gacgagctcg tgaaggtgat gggccgccac aagccggaga acatcgtgat cgagatggcc 3480
cgcgagaacc agaccaccca gaagggccag aagaactccc gcgagcgcat gaagcgcatc 3540
gaggagggca tcaaggagct cggctcccag atcctcaagg agcacccggt ggagaacacc 3600
cagctccaga acgagaagct ctacctctac tacctccaga acggccgcga catgtacgtg 3660
gaccaggagc tcgacatcaa ccgcctctcc gactacgacg tggaccacat cgtgccgcag 3720
tccttcctca aggacgactc catcgacaac aaggtgctca cccgctccga caagaaccgc 3780
ggcaagtccg acaacgtgcc gtccgaggag gtggtgaaga agatgaagaa ctactggcgc 3840
cagctcctca acgccaagct catcacccag cgcaagttcg acaacctcac caaggccgag 3900
cgcggcggcc tctccgagct cgacaaggcc ggcttcatca agcgccagct cgtggagacc 3960
cgccagatca ccaagcacgt ggcccagatc ctcgactccc gcatgaacac caagtacgac 4020
gagaacgaca agctcatccg cgaggtgaag gtgatcaccc tcaagtccaa gctcgtgtcc 4080
gacttccgca aggacttcca gttctacaag gtgcgcgaga tcaacaacta ccaccacgcc 4140
cacgacgcct acctcaacgc cgtggtgggc accgccctca tcaagaagta cccgaagctc 4200
gagtccgagt tcgtgtacgg cgactacaag gtgtacgacg tgcgcaagat gatcgccaag 4260
tccgagcagg agatcggcaa ggccaccgcc aagtacttct tctactccaa catcatgaac 4320
ttcttcaaga ccgagatcac cctcgccaac ggcgagatcc gcaagcgccc gctcatcgag 4380
accaacggcg agaccggcga gatcgtgtgg gacaagggcc gcgacttcgc caccgtgcgc 4440
aaggtgctct ccatgccgca ggtgaacatc gtgaagaaga ccgaggtgca gaccggcggc 4500
ttctccaagg agtccatcct cccgaagcgc aactccgaca agctcatcgc ccgcaagaag 4560
gactgggacc cgaagaagta cggcggcttc gactccccga ccgtggccta ctccgtgctc 4620
gtggtggcca aggtggagaa gggcaagtcc aagaagctca agtccgtgaa ggagctcctc 4680
ggcatcacca tcatggagcg ctcctccttc gagaagaacc cgatcgactt cctcgaggcc 4740
aagggctaca aggaggtgaa gaaggacctc atcatcaagc tcccgaagta ctccctcttc 4800
gagctcgaga acggccgcaa gcgcatgctc gcctccgccg gcgagctcca gaagggcaac 4860
gagctcgccc tcccgtccaa gtacgtgaac ttcctctacc tcgcctccca ctacgagaag 4920
ctcaagggct ccccggagga caacgagcag aagcagctct tcgtggagca gcacaagcac 4980
tacctcgacg agatcatcga gcagatctcc gagttctcca agcgcgtgat cctcgccgac 5040
gccaacctcg acaaggtgct ctccgcctac aacaagcacc gcgacaagcc gatccgcgag 5100
caggccgaga acatcatcca cctcttcacc ctcaccaacc tcggcgcccc ggccgccttc 5160
aagtacttcg acaccaccat cgaccgcaag cgctacacct ccaccaagga ggtgctcgac 5220
gccaccctca tccaccagtc catcaccggc ctctacgaga cccgcatcga cctctcccag 5280
ctcggcggcg acagcggcgg ctcaccgaag aagcgccgcc gcgtg 5325
<210> 4
<211> 5325
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
atgtcagagg tggagttctc tcacgagtac tggatgaggc atgcgctgac cctggcaaag 60
agggcctggg atgagaggga ggtgccagtg ggagccgtgc tcgtccacaa caatcgcgtg 120
atcggagagg gatggaatag gccaattgga aggcatgacc caacagcaca tgccgagatc 180
atggcactca ggcagggcgg cctggtcatg cagaattacc ggctgattga tgcgaccctc 240
tacgtgacac tggagccatg cgtcatgtgc gcgggagcca tgatccattc aaggattgga 300
agggtggtct tcggagcaag ggatgccaag acaggagcag ccggaagcct catggatgtg 360
ctgcaccatc ctggcatgaa ccatcgcgtc gagatcacag agggaattct ggcggatgag 420
tgcgcggccc tcctgtccga tttcttccgg atgaggcgcc aggagatcaa ggcccagaag 480
aaggcccagt ccagcacaga ttccggcggc tcatctggcg gctcctcagg atcagagacc 540
cctggcacat cagagtctgc gacaccagaa tcctctggcg gctccagcgg cggctcatct 600
gaggtggagt tcagccatga atactggatg aggcatgcac tcacactcgc aaagagggcc 660
agggatgagc gggaagtccc agtgggagcc gtgctggtcc tgaacaatcg cgtgatcggc 720
gagggatgga acagggcaat tggcctccac gacccaacag cccatgccga gatcatggcc 780
ctgcgccagg gcggcctggt catgcaaaat taccgcctca ttgatgccac cctctacgtg 840
acattcgaac cgtgcgtcat gtgcgccggc gcgatgattc actcccgcat cggcagggtg 900
gtcttcggcg tgaggaatgc aaagacagga gcggccggat ccctcatgga cgtgctgcac 960
taccctggca tgaatcacag ggtcgagatc acagagggca ttctggccga cgaatgcgcg 1020
gccctcctgt gctacttctt ccggatgccg aggcaggtct tcaatgccca gaagaaggcc 1080
cagtccagca cagattccgg cggctcatct ggcggctcct caggatcaga gacccctggc 1140
acatcagagt ctgcgacacc agaatcctct ggcggctcaa gcggcggctc cgacaagaag 1200
tactccatcg gcctcgccat cggcaccaac tccgtgggct gggccgtgat caccgacgag 1260
tacaaggtgc cgtccaagaa gttcaaggtg ctcggcaaca ccgaccgcca ctccatcaag 1320
aagaacctca tcggcgccct cctcttcgac tccggcgaga ccgccgaggc cacccgcctc 1380
aagcgcaccg cccgccgccg ctacacccgc cgcaagaacc gcatctgcta cctccaggag 1440
atcttctcca acgagatggc caaggtggac gactccttct tccaccgcct cgaggagtcc 1500
ttcctcgtgg aggaggacaa gaagcacgag cgccacccga tcttcggcaa catcgtggac 1560
gaggtggcct accacgagaa gtacccgacc atctaccacc tccgcaagaa gctcgtggac 1620
tccaccgaca aggccgacct ccgcctcatc tacctcgccc tcgcccacat gatcaagttc 1680
cgcggccact tcctcatcga gggcgacctc aacccggaca actccgacgt ggacaagctc 1740
ttcatccagc tcgtgcagac ctacaaccag ctcttcgagg agaacccgat caacgcctcc 1800
ggcgtggacg ccaaggccat cctctccgcc cgcctctcca agtcccgccg cctcgagaac 1860
ctcatcgccc agctcccggg cgagaagaag aacggcctct tcggcaacct catcgccctc 1920
tccctcggcc tcaccccgaa cttcaagtcc aacttcgacc tcgccgagga cgccaagctc 1980
cagctctcca aggacaccta cgacgacgac ctcgacaacc tcctcgccca gatcggcgac 2040
cagtacgccg acctcttcct cgccgccaag aacctctccg acgccatcct cctctccgac 2100
atcctccgcg tgaacaccga gatcaccaag gccccgctct ccgcctccat gatcaagcgc 2160
tacgacgagc accaccagga cctcaccctc ctcaaggccc tcgtgcgcca gcagctcccg 2220
gagaagtaca aggagatctt cttcgaccag tccaagaacg gctacgccgg ctacatcgac 2280
ggcggcgcct cccaggagga gttctacaag ttcatcaagc cgatcctcga gaagatggac 2340
ggcaccgagg agctcctcgt gaagctcaac cgcgaggacc tcctccgcaa gcagcgcacc 2400
ttcgacaacg gctccatccc gcaccagatc cacctcggcg agctccacgc catcctccgc 2460
cgccaggagg acttctaccc gttcctcaag gacaaccgcg agaagatcga gaagatcctc 2520
accttccgca tcccgtacta cgtgggcccg ctcgcccgcg gcaactcccg cttcgcctgg 2580
atgacccgca agtccgagga gaccatcacc ccgtggaact tcgaggaggt ggtggacaag 2640
ggcgcctccg cccagtcctt catcgagcgc atgaccaact tcgacaagaa cctcccgaac 2700
gagaaggtgc tcccgaagca ctccctcctc tacgagtact tcaccgtgta caacgagctc 2760
accaaggtga agtacgtgac cgagggcatg cgcaagccgg ccttcctctc cggcgagcag 2820
aagaaggcca tcgtggacct cctcttcaag accaaccgca aggtgaccgt gaagcagctc 2880
aaggaggact acttcaagaa gatcgagtgc ttcgactccg tggagatctc cggcgtggag 2940
gaccgcttca acgcctccct cggcacctac cacgacctcc tcaagatcat caaggacaag 3000
gacttcctcg acaacgagga gaacgaggac atcctcgagg acatcgtgct caccctcacc 3060
ctcttcgagg accgcgagat gatcgaggag cgcctcaaga cctacgccca cctcttcgac 3120
gacaaggtga tgaagcagct caagcgccgc cgctacaccg gctggggccg cctctcccgc 3180
aagctcatca acggcatccg cgacaagcag tccggcaaga ccatcctcga cttcctcaag 3240
tccgacggct tcgccaaccg caacttcatg cagctcatcc acgacgactc cctcaccttc 3300
aaggaggaca tccagaaggc ccaggtgtcc ggccagggcg actccctcca cgagcacatc 3360
gccaacctcg ccggctcccc ggccatcaag aagggcatcc tccagaccgt gaaggtggtg 3420
gacgagctcg tgaaggtgat gggccgccac aagccggaga acatcgtgat cgagatggcc 3480
cgcgagaacc agaccaccca gaagggccag aagaactccc gcgagcgcat gaagcgcatc 3540
gaggagggca tcaaggagct cggctcccag atcctcaagg agcacccggt ggagaacacc 3600
cagctccaga acgagaagct ctacctctac tacctccaga acggccgcga catgtacgtg 3660
gaccaggagc tcgacatcaa ccgcctctcc gactacgacg tggaccacat cgtgccgcag 3720
tccttcctca aggacgactc catcgacaac aaggtgctca cccgctccga caagaaccgc 3780
ggcaagtccg acaacgtgcc gtccgaggag gtggtgaaga agatgaagaa ctactggcgc 3840
cagctcctca acgccaagct catcacccag cgcaagttcg acaacctcac caaggccgag 3900
cgcggcggcc tctccgagct cgacaaggcc ggcttcatca agcgccagct cgtggagacc 3960
cgccagatca ccaagcacgt ggcccagatc ctcgactccc gcatgaacac caagtacgac 4020
gagaacgaca agctcatccg cgaggtgaag gtgatcaccc tcaagtccaa gctcgtgtcc 4080
gacttccgca aggacttcca gttctacaag gtgcgcgaga tcaacaacta ccaccacgcc 4140
cacgacgcct acctcaacgc cgtggtgggc accgccctca tcaagaagta cccgaagctc 4200
gagtccgagt tcgtgtacgg cgactacaag gtgtacgacg tgcgcaagat gatcgccaag 4260
tccgagcagg agatcggcaa ggccaccgcc aagtacttct tctactccaa catcatgaac 4320
ttcttcaaga ccgagatcac cctcgccaac ggcgagatcc gcaagcgccc gctcatcgag 4380
accaacggcg agaccggcga gatcgtgtgg gacaagggcc gcgacttcgc caccgtgcgc 4440
aaggtgctct ccatgccgca ggtgaacatc gtgaagaaga ccgaggtgca gaccggcggc 4500
ttctccaagg agtccatccg cccgaagcgc aactccgaca agctcatcgc ccgcaagaag 4560
gactgggacc cgaagaagta cggcggcttc gtctccccga ccgtggccta ctccgtgctc 4620
gtggtggcca aggtggagaa gggcaagtcc aagaagctca agtccgtgaa ggagctcctc 4680
ggcatcacca tcatggagcg ctcctccttc gagaagaacc cgatcgactt cctcgaggcc 4740
aagggctaca aggaggtgaa gaaggacctc atcatcaagc tcccgaagta ctccctcttc 4800
gagctcgaga acggccgcaa gcgcatgctc gcctccgccc gcttcctcca gaagggcaac 4860
gagctcgccc tcccgtccaa gtacgtgaac ttcctctacc tcgcctccca ctacgagaag 4920
ctcaagggct ccccggagga caacgagcag aagcagctct tcgtggagca gcacaagcac 4980
tacctcgacg agatcatcga gcagatctcc gagttctcca agcgcgtgat cctcgccgac 5040
gccaacctcg acaaggtgct ctccgcctac aacaagcacc gcgacaagcc gatccgcgag 5100
caggccgaga acatcatcca cctcttcacc ctcaccaacc tcggcgcccc gcgcgccttc 5160
aagtacttcg acaccaccat cgaccgcaag gtgtaccgct ccaccaagga ggtgctcgac 5220
gccaccctca tccaccagtc catcaccggc ctctacgaga cccgcatcga cctctcccag 5280
ctcggcggcg acagcggcgg ctcaccgaag aagcgccgcc gcgtg 5325
Claims (6)
1.靶向OsWaxy基因的单碱基基因编辑载体，其特征在于，所述载体包含腺嘌呤碱基编辑器和靶向OsWaxy基因的特异性核酸酶；
OsWaxy基因编码的蛋白在NCBI上的参考序列编号为XP_015644490.1；
所述载体是基于CRISPR/Cas9的腺嘌呤碱基编辑载体，所述载体至少包含第一表达盒和第二表达盒；
其中，第一表达盒为ecTadA-ecTadA*7.10-nCas9r表达盒；所述第一表达盒包含的核酸构建体ecTadA-ecTadA*7.10-nCas9的序列如SEQ ID NO:3所示；
第二表达盒为sgRNA表达盒，且sgRNA作用位点位于OsWaxy基因的外显子上；
sgRNA作用位点的DNA序列为5′-TCGGCAGGCTGGAGGAACAG-3′。
2.根据权利要求1所述的载体，其特征在于，所述第一表达盒由甘蔗Ubi4启动子驱动，所述第二表达盒由水稻U3启动子驱动。
3.权利要求1或2所述载体在水稻育种和品种改良中的应用，其中，育种目的为改良水稻直链淀粉含量。
4.利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法，其特征在于，所述方法包括将权利要求1或2所述载体导入水稻中。
5.根据权利要求4所述的方法，其特征在于，所述水稻为籼稻。
6.根据权利要求4所述的方法，其特征在于，突变OsWaxy基因编码的突变体包含OsWaxy蛋白第408位氨基酸由R到G的突变。
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111272218.9A CN114058639B (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
| CN202311402604.4A CN117551688A (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
| CN202311406682.1A CN117587064A (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111272218.9A CN114058639B (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
Related Child Applications (2)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311406682.1A Division CN117587064A (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
| CN202311402604.4A Division CN117551688A (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114058639A CN114058639A (zh) | 2022-02-18 |
| CN114058639B true CN114058639B (zh) | 2023-11-07 |
Family
ID=80236078
Family Applications (3)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311406682.1A Pending CN117587064A (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
| CN202111272218.9A Active CN114058639B (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
| CN202311402604.4A Pending CN117551688A (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
Family Applications Before (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311406682.1A Pending CN117587064A (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
Family Applications After (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202311402604.4A Pending CN117551688A (zh) | 2021-10-29 | 2021-10-29 | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 |
Country Status (1)
| Country | Link |
|---|---|
| CN (3) | CN117587064A (zh) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652439A (zh) * | 2018-12-27 | 2019-04-19 | 宜春学院 | 利用CRISPR/Cas9介导的腺嘌呤碱基编辑系统改良水稻稻瘟病广谱抗性的方法 |
| CN109706185A (zh) * | 2019-02-01 | 2019-05-03 | 国家卫生计生委科学技术研究所 | 基于碱基编辑系统突变起始密码子实现基因敲除的方法及应用 |
| KR20190122595A (ko) * | 2018-04-20 | 2019-10-30 | 기초과학연구원 | 식물의 염기 교정용 유전자 구조체, 이를 포함하는 벡터 및 이를 이용한 염기 교정 방법 |
| CN110714010A (zh) * | 2019-11-28 | 2020-01-21 | 袁隆平农业高科技股份有限公司 | 一种通过基因编辑降低水稻直链淀粉含量的方法及其专用sgRNA |
| CN111197034A (zh) * | 2020-01-08 | 2020-05-26 | 江苏省农业科学院 | 基于基因编辑技术的Wx突变型蛋白及其基因在植物育种中的应用 |
| CN112048497A (zh) * | 2019-06-06 | 2020-12-08 | 中国科学院脑科学与智能技术卓越创新中心 | 一种新型的单碱基编辑技术及其应用 |
| CN112266420A (zh) * | 2020-10-30 | 2021-01-26 | 华南农业大学 | 一种植物高效胞嘧啶单碱基编辑器及其构建与应用 |
| CN112538492A (zh) * | 2020-12-14 | 2021-03-23 | 安徽省农业科学院水稻研究所 | 一种识别PAM序列为NRTH的SpCas9n变体及相应碱基编辑系统 |
| CN112980839A (zh) * | 2019-12-17 | 2021-06-18 | 中国种子集团有限公司 | 创制水稻高直链淀粉型新种质的方法及其应用 |
-
2021
- 2021-10-29 CN CN202311406682.1A patent/CN117587064A/zh active Pending
- 2021-10-29 CN CN202111272218.9A patent/CN114058639B/zh active Active
- 2021-10-29 CN CN202311402604.4A patent/CN117551688A/zh active Pending
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| KR20190122595A (ko) * | 2018-04-20 | 2019-10-30 | 기초과학연구원 | 식물의 염기 교정용 유전자 구조체, 이를 포함하는 벡터 및 이를 이용한 염기 교정 방법 |
| CN109652439A (zh) * | 2018-12-27 | 2019-04-19 | 宜春学院 | 利用CRISPR/Cas9介导的腺嘌呤碱基编辑系统改良水稻稻瘟病广谱抗性的方法 |
| CN109706185A (zh) * | 2019-02-01 | 2019-05-03 | 国家卫生计生委科学技术研究所 | 基于碱基编辑系统突变起始密码子实现基因敲除的方法及应用 |
| CN112048497A (zh) * | 2019-06-06 | 2020-12-08 | 中国科学院脑科学与智能技术卓越创新中心 | 一种新型的单碱基编辑技术及其应用 |
| CN110714010A (zh) * | 2019-11-28 | 2020-01-21 | 袁隆平农业高科技股份有限公司 | 一种通过基因编辑降低水稻直链淀粉含量的方法及其专用sgRNA |
| CN112980839A (zh) * | 2019-12-17 | 2021-06-18 | 中国种子集团有限公司 | 创制水稻高直链淀粉型新种质的方法及其应用 |
| CN111197034A (zh) * | 2020-01-08 | 2020-05-26 | 江苏省农业科学院 | 基于基因编辑技术的Wx突变型蛋白及其基因在植物育种中的应用 |
| CN112266420A (zh) * | 2020-10-30 | 2021-01-26 | 华南农业大学 | 一种植物高效胞嘧啶单碱基编辑器及其构建与应用 |
| CN112538492A (zh) * | 2020-12-14 | 2021-03-23 | 安徽省农业科学院水稻研究所 | 一种识别PAM序列为NRTH的SpCas9n变体及相应碱基编辑系统 |
Non-Patent Citations (2)
| Title |
|---|
| "A route to de novo domestication of wild allotetraploid rice";Hong Yu 等;《CELL》;第184卷(第5期);第e6页，附件6，表S6，图S7 * |
| Hong Yu 等."A route to de novo domestication of wild allotetraploid rice".《CELL》.2021,第184卷(第5期),第e6页，附件6，表S6，图S7. * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114058639A (zh) | 2022-02-18 |
| CN117551688A (zh) | 2024-02-13 |
| CN117587064A (zh) | 2024-02-23 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| WO2018086623A1 (en) | A method for base editing in plants | |
| CN107267527A (zh) | 雄性育性的保持方法及其应用 | |
| CN110592135A (zh) | 一种CRISPR/Cas9编辑水稻香味基因Badh2的方法 | |
| CA3114913A1 (en) | Genome editing to increase seed protein content | |
| CN114540369B (zh) | OsBEE1基因在提高水稻产量中的应用 | |
| CN111996177A (zh) | 一种玉米waxy基因突变体及其分子标记和应用 | |
| CN113862265A (zh) | 一种改良水稻粒型和外观品质的方法 | |
| CN113832179A (zh) | ZmELF3.1蛋白及其功能缺失突变体在调控作物雄穗分支数中的应用 | |
| CN114058639B (zh) | 利用单碱基基因编辑技术突变OsWaxy基因改良水稻直链淀粉含量的方法 | |
| CN112980839B (zh) | 创制水稻高直链淀粉型新种质的方法及其应用 | |
| CN113817033B (zh) | ZmELF3.1蛋白及其功能缺失突变体在调控作物气生根数目或层数的应用 | |
| CN115466747B (zh) | 糖基转移酶ZmKOB1基因及其在调控玉米雌穗结实性状或发育上的应用 | |
| CN114395580A (zh) | 用于控制玉米株高的基因 | |
| CN110079535B (zh) | 玉米ZmPIF3s突变型蛋白、其编码基因及其在育种上的应用 | |
| CN107075526A (zh) | 具有工程内源基因的植物 | |
| CN113151295A (zh) | 水稻温敏雄性不育基因OsFMS1及其应用 | |
| WO2018228348A1 (en) | Methods to improve plant agronomic trait using bcs1l gene and guide rna/cas endonuclease systems | |
| CN110951772A (zh) | 水稻OsPPR2-1基因在构建自然条件下育性提高的植株中的应用 | |
| CN114644692B (zh) | 一种定点突变创制旱敏感玉米种质的方法及其应用 | |
| CN113462661B (zh) | 从玉米中分离的siz1蛋白及其编码基因和在品种改良中的应用 | |
| CN113999871B (zh) | 创制矮杆直立株型的水稻种质的方法及其应用 | |
| CN110734484B (zh) | Nrt2_5蛋白在调控植物苞叶宽度中的应用 | |
| CN112359134B (zh) | 一种提高水稻单倍体诱导效率的分子标记引物及其应用 | |
| US20230193309A1 (en) | Method for obtaining wheat with increased resistance to powdery mildew | |
| US20240167046A1 (en) | Inducible mosaicism |
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 |