CN114032229B - 高等植物acs双酶活关键结构域替换制备得到的单酶突变体 - Google Patents
高等植物acs双酶活关键结构域替换制备得到的单酶突变体 Download PDFInfo
- Publication number
- CN114032229B CN114032229B CN202111320329.2A CN202111320329A CN114032229B CN 114032229 B CN114032229 B CN 114032229B CN 202111320329 A CN202111320329 A CN 202111320329A CN 114032229 B CN114032229 B CN 114032229B
- Authority
- CN
- China
- Prior art keywords
- leu
- acs
- ser
- glu
- arg
- 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
- 230000000694 effects Effects 0.000 title claims abstract description 55
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 34
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 34
- 241000196324 Embryophyta Species 0.000 title claims abstract description 25
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 36
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 33
- 102000018120 Recombinases Human genes 0.000 claims abstract description 26
- 108010091086 Recombinases Proteins 0.000 claims abstract description 26
- 108010076637 carbon-sulfur lyase Proteins 0.000 abstract description 23
- 102000028406 carbon-sulfur lyase Human genes 0.000 abstract description 23
- VGGSQFUCUMXWEO-UHFFFAOYSA-N Ethene Chemical compound C=C VGGSQFUCUMXWEO-UHFFFAOYSA-N 0.000 abstract description 18
- 239000005977 Ethylene Substances 0.000 abstract description 18
- 238000000034 method Methods 0.000 abstract description 8
- 230000004345 fruit ripening Effects 0.000 abstract description 7
- 238000010353 genetic engineering Methods 0.000 abstract description 5
- 230000008569 process Effects 0.000 abstract description 5
- 238000009395 breeding Methods 0.000 abstract description 4
- 230000001488 breeding effect Effects 0.000 abstract description 4
- 101100433502 Arabidopsis thaliana ACS7 gene Proteins 0.000 description 29
- 235000018102 proteins Nutrition 0.000 description 28
- 239000012634 fragment Substances 0.000 description 26
- LCTONWCANYUPML-UHFFFAOYSA-N Pyruvic acid Chemical compound CC(=O)C(O)=O LCTONWCANYUPML-UHFFFAOYSA-N 0.000 description 22
- 238000006243 chemical reaction Methods 0.000 description 18
- 239000013598 vector Substances 0.000 description 13
- 108020004999 messenger RNA Proteins 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- 229940107700 pyruvic acid Drugs 0.000 description 11
- 108010030526 1-aminocyclopropanecarboxylate synthase Proteins 0.000 description 10
- 241000195887 Physcomitrella patens Species 0.000 description 10
- 235000001014 amino acid Nutrition 0.000 description 10
- 150000001413 amino acids Chemical group 0.000 description 10
- PAJPWUMXBYXFCZ-UHFFFAOYSA-N 1-aminocyclopropanecarboxylic acid Chemical compound OC(=O)C1(N)CC1 PAJPWUMXBYXFCZ-UHFFFAOYSA-N 0.000 description 9
- 241000219195 Arabidopsis thaliana Species 0.000 description 9
- 230000015572 biosynthetic process Effects 0.000 description 9
- 238000005259 measurement Methods 0.000 description 9
- LEVWYRKDKASIDU-QWWZWVQMSA-N D-cystine Chemical compound OC(=O)[C@H](N)CSSC[C@@H](N)C(O)=O LEVWYRKDKASIDU-QWWZWVQMSA-N 0.000 description 8
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 8
- 229960001570 ademetionine Drugs 0.000 description 8
- 230000003321 amplification Effects 0.000 description 8
- 229960003067 cystine Drugs 0.000 description 8
- 238000003199 nucleic acid amplification method Methods 0.000 description 8
- NGVDGCNFYWLIFO-UHFFFAOYSA-N pyridoxal 5'-phosphate Chemical compound CC1=NC=C(COP(O)(O)=O)C(C=O)=C1O NGVDGCNFYWLIFO-UHFFFAOYSA-N 0.000 description 8
- 210000004027 cell Anatomy 0.000 description 7
- 210000000349 chromosome Anatomy 0.000 description 7
- 239000002773 nucleotide Substances 0.000 description 7
- 125000003729 nucleotide group Chemical group 0.000 description 7
- 238000003786 synthesis reaction Methods 0.000 description 7
- 241000219194 Arabidopsis Species 0.000 description 6
- 102000016928 DNA-directed DNA polymerase Human genes 0.000 description 6
- 108010014303 DNA-directed DNA polymerase Proteins 0.000 description 6
- ITVBKCZZLJUUHI-HTUGSXCWSA-N Glu-Phe-Thr Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)O)C(O)=O ITVBKCZZLJUUHI-HTUGSXCWSA-N 0.000 description 6
- YBAFDPFAUTYYRW-UHFFFAOYSA-N N-L-alpha-glutamyl-L-leucine Natural products CC(C)CC(C(O)=O)NC(=O)C(N)CCC(O)=O YBAFDPFAUTYYRW-UHFFFAOYSA-N 0.000 description 6
- FTVRVZNYIYWJGB-ACZMJKKPSA-N Ser-Asp-Glu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O FTVRVZNYIYWJGB-ACZMJKKPSA-N 0.000 description 6
- 108010005233 alanylglutamic acid Proteins 0.000 description 6
- 108010077245 asparaginyl-proline Proteins 0.000 description 6
- 239000007795 chemical reaction product Substances 0.000 description 6
- 239000002299 complementary DNA Substances 0.000 description 6
- 108010054155 lysyllysine Proteins 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 101100063510 Notophthalmus viridescens DLX3 gene Proteins 0.000 description 5
- -1 aromatic amino acids Chemical class 0.000 description 5
- 108091036078 conserved sequence Proteins 0.000 description 5
- VPZXBVLAVMBEQI-UHFFFAOYSA-N glycyl-DL-alpha-alanine Natural products OC(=O)C(C)NC(=O)CN VPZXBVLAVMBEQI-UHFFFAOYSA-N 0.000 description 5
- 239000013642 negative control Substances 0.000 description 5
- 239000000243 solution Substances 0.000 description 5
- 230000035882 stress Effects 0.000 description 5
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 4
- 102000008300 Mutant Proteins Human genes 0.000 description 4
- 108010021466 Mutant Proteins Proteins 0.000 description 4
- 238000003556 assay Methods 0.000 description 4
- 239000013604 expression vector Substances 0.000 description 4
- 230000004927 fusion Effects 0.000 description 4
- 230000035772 mutation Effects 0.000 description 4
- 235000007682 pyridoxal 5'-phosphate Nutrition 0.000 description 4
- 239000011589 pyridoxal 5'-phosphate Substances 0.000 description 4
- 229960001327 pyridoxal phosphate Drugs 0.000 description 4
- 238000012163 sequencing technique Methods 0.000 description 4
- 238000011144 upstream manufacturing Methods 0.000 description 4
- YWWATNIVMOCSAV-UBHSHLNASA-N Ala-Arg-Phe Chemical compound NC(=N)NCCC[C@H](NC(=O)[C@@H](N)C)C(=O)N[C@H](C(O)=O)CC1=CC=CC=C1 YWWATNIVMOCSAV-UBHSHLNASA-N 0.000 description 3
- PXKLCFFSVLKOJM-ACZMJKKPSA-N Ala-Asn-Glu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O PXKLCFFSVLKOJM-ACZMJKKPSA-N 0.000 description 3
- JYEBJTDTPNKQJG-FXQIFTODSA-N Ala-Asn-Met Chemical compound C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCSC)C(=O)O)N JYEBJTDTPNKQJG-FXQIFTODSA-N 0.000 description 3
- PCIFXPRIFWKWLK-YUMQZZPRSA-N Ala-Gly-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)CNC(=O)[C@H](C)N PCIFXPRIFWKWLK-YUMQZZPRSA-N 0.000 description 3
- XUCHENWTTBFODJ-FXQIFTODSA-N Ala-Met-Ala Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](C)C(O)=O XUCHENWTTBFODJ-FXQIFTODSA-N 0.000 description 3
- OEVCHROQUIVQFZ-YTLHQDLWSA-N Ala-Thr-Ala Chemical compound C[C@H](N)C(=O)N[C@@H]([C@H](O)C)C(=O)N[C@@H](C)C(O)=O OEVCHROQUIVQFZ-YTLHQDLWSA-N 0.000 description 3
- YCTIYBUTCKNOTI-UWJYBYFXSA-N Ala-Tyr-Asp Chemical compound C[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(=O)O)C(=O)O)N YCTIYBUTCKNOTI-UWJYBYFXSA-N 0.000 description 3
- REWSWYIDQIELBE-FXQIFTODSA-N Ala-Val-Ser Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CO)C(O)=O REWSWYIDQIELBE-FXQIFTODSA-N 0.000 description 3
- QAODJPUKWNNNRP-DCAQKATOSA-N Arg-Glu-Arg Chemical compound NC(N)=NCCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O QAODJPUKWNNNRP-DCAQKATOSA-N 0.000 description 3
- FRMQITGHXMUNDF-GMOBBJLQSA-N Arg-Ile-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N FRMQITGHXMUNDF-GMOBBJLQSA-N 0.000 description 3
- SSZGOKWBHLOCHK-DCAQKATOSA-N Arg-Lys-Asn Chemical compound NC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCCN=C(N)N SSZGOKWBHLOCHK-DCAQKATOSA-N 0.000 description 3
- UZSQXCMNUPKLCC-FJXKBIBVSA-N Arg-Thr-Gly Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(O)=O UZSQXCMNUPKLCC-FJXKBIBVSA-N 0.000 description 3
- 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 3
- XEOXPCNONWHHSW-AVGNSLFASA-N Arg-Val-His Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@H](CCCN=C(N)N)N XEOXPCNONWHHSW-AVGNSLFASA-N 0.000 description 3
- 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 3
- PIWWUBYJNONVTJ-ZLUOBGJFSA-N Asn-Asp-Asn Chemical compound C([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)C(=O)N PIWWUBYJNONVTJ-ZLUOBGJFSA-N 0.000 description 3
- ZMUQQMGITUJQTI-CIUDSAMLSA-N Asn-Leu-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(O)=O ZMUQQMGITUJQTI-CIUDSAMLSA-N 0.000 description 3
- YUOXLJYVSZYPBJ-CIUDSAMLSA-N Asn-Pro-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O YUOXLJYVSZYPBJ-CIUDSAMLSA-N 0.000 description 3
- GXAJEYWSNDOXFA-UHFFFAOYSA-N Asp Thr His Gly Chemical compound OC(=O)CC(N)C(=O)NC(C(O)C)C(=O)NC(C(=O)NCC(O)=O)CC1=CN=CN1 GXAJEYWSNDOXFA-UHFFFAOYSA-N 0.000 description 3
- ZLGKHJHFYSRUBH-FXQIFTODSA-N Asp-Arg-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(O)=O)C(O)=O ZLGKHJHFYSRUBH-FXQIFTODSA-N 0.000 description 3
- PXLNPFOJZQMXAT-BYULHYEWSA-N Asp-Asp-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC(O)=O PXLNPFOJZQMXAT-BYULHYEWSA-N 0.000 description 3
- KESWRFKUZRUTAH-FXQIFTODSA-N Asp-Pro-Asp Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CC(O)=O)C(O)=O KESWRFKUZRUTAH-FXQIFTODSA-N 0.000 description 3
- UXRVDHVARNBOIO-QSFUFRPTSA-N Asp-Val-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](C(C)C)NC(=O)[C@H](CC(=O)O)N UXRVDHVARNBOIO-QSFUFRPTSA-N 0.000 description 3
- YZFCGHIBLBDZDA-ZLUOBGJFSA-N Cys-Asp-Ser Chemical compound [H]N[C@@H](CS)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O YZFCGHIBLBDZDA-ZLUOBGJFSA-N 0.000 description 3
- XELISBQUZZAPQK-CIUDSAMLSA-N Cys-His-Cys Chemical compound C1=C(NC=N1)C[C@@H](C(=O)N[C@@H](CS)C(=O)O)NC(=O)[C@H](CS)N XELISBQUZZAPQK-CIUDSAMLSA-N 0.000 description 3
- TWTWUBHEWQPMQW-ZPFDUUQYSA-N Gln-Ile-Arg Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O TWTWUBHEWQPMQW-ZPFDUUQYSA-N 0.000 description 3
- QDXMSSWCEVYOLZ-SZMVWBNQSA-N Gln-Leu-Trp Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CCC(=O)N)N QDXMSSWCEVYOLZ-SZMVWBNQSA-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
- MLCPTRRNICEKIS-FXQIFTODSA-N Glu-Asn-Gln Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(O)=O MLCPTRRNICEKIS-FXQIFTODSA-N 0.000 description 3
- AFODTOLGSZQDSL-PEFMBERDSA-N Glu-Asn-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N AFODTOLGSZQDSL-PEFMBERDSA-N 0.000 description 3
- LXAUHIRMWXQRKI-XHNCKOQMSA-N Glu-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CCC(=O)O)N)C(=O)O LXAUHIRMWXQRKI-XHNCKOQMSA-N 0.000 description 3
- JRCUFCXYZLPSDZ-ACZMJKKPSA-N Glu-Asp-Ser Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CO)C(O)=O JRCUFCXYZLPSDZ-ACZMJKKPSA-N 0.000 description 3
- INGJLBQKTRJLFO-UKJIMTQDSA-N Glu-Ile-Val Chemical compound CC(C)[C@@H](C(O)=O)NC(=O)[C@H]([C@@H](C)CC)NC(=O)[C@@H](N)CCC(O)=O INGJLBQKTRJLFO-UKJIMTQDSA-N 0.000 description 3
- FBEJIDRSQCGFJI-GUBZILKMSA-N Glu-Leu-Ser Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O FBEJIDRSQCGFJI-GUBZILKMSA-N 0.000 description 3
- HRBYTAIBKPNZKQ-AVGNSLFASA-N Glu-Lys-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CCC(O)=O HRBYTAIBKPNZKQ-AVGNSLFASA-N 0.000 description 3
- QXPRJQPCFXMCIY-NKWVEPMBSA-N Gly-Ala-Pro Chemical compound C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)CN QXPRJQPCFXMCIY-NKWVEPMBSA-N 0.000 description 3
- YYPFZVIXAVDHIK-IUCAKERBSA-N Gly-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)CN YYPFZVIXAVDHIK-IUCAKERBSA-N 0.000 description 3
- 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 3
- 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 3
- LLZXNUUIBOALNY-QWRGUYRKSA-N Gly-Leu-Lys Chemical compound NCC(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCCCN LLZXNUUIBOALNY-QWRGUYRKSA-N 0.000 description 3
- FXLVSYVJDPCIHH-STQMWFEESA-N Gly-Phe-Arg Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O FXLVSYVJDPCIHH-STQMWFEESA-N 0.000 description 3
- JPVGHHQGKPQYIL-KBPBESRZSA-N Gly-Phe-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@@H](NC(=O)CN)CC1=CC=CC=C1 JPVGHHQGKPQYIL-KBPBESRZSA-N 0.000 description 3
- POJJAZJHBGXEGM-YUMQZZPRSA-N Gly-Ser-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)CN POJJAZJHBGXEGM-YUMQZZPRSA-N 0.000 description 3
- WCORRBXVISTKQL-WHFBIAKZSA-N Gly-Ser-Ser Chemical compound NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O WCORRBXVISTKQL-WHFBIAKZSA-N 0.000 description 3
- 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 3
- HUFUVTYGPOUCBN-MBLNEYKQSA-N Gly-Thr-Ile Chemical compound [H]NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O HUFUVTYGPOUCBN-MBLNEYKQSA-N 0.000 description 3
- LKJCZEPXHOIAIW-HOTGVXAUSA-N Gly-Trp-Lys Chemical compound C1=CC=C2C(=C1)C(=CN2)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)CN LKJCZEPXHOIAIW-HOTGVXAUSA-N 0.000 description 3
- BAYQNCWLXIDLHX-ONGXEEELSA-N Gly-Val-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)CN BAYQNCWLXIDLHX-ONGXEEELSA-N 0.000 description 3
- 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 3
- OBTMRGFRLJBSFI-GARJFASQSA-N His-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O OBTMRGFRLJBSFI-GARJFASQSA-N 0.000 description 3
- TWROVBNEHJSXDG-IHRRRGAJSA-N His-Leu-Val Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O TWROVBNEHJSXDG-IHRRRGAJSA-N 0.000 description 3
- WNQKUUQIVDDAFA-ZPFDUUQYSA-N Ile-Gln-Met Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CCSC)C(=O)O)N WNQKUUQIVDDAFA-ZPFDUUQYSA-N 0.000 description 3
- QRTVJGKXFSYJGW-KBIXCLLPSA-N Ile-Glu-Cys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H](CS)C(=O)O)N QRTVJGKXFSYJGW-KBIXCLLPSA-N 0.000 description 3
- 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 3
- SAEWJTCJQVZQNZ-IUKAMOBKSA-N Ile-Thr-Asn Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(=O)N)C(=O)O)N SAEWJTCJQVZQNZ-IUKAMOBKSA-N 0.000 description 3
- ZGKVPOSSTGHJAF-HJPIBITLSA-N Ile-Tyr-Ser Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CO)C(=O)O)N ZGKVPOSSTGHJAF-HJPIBITLSA-N 0.000 description 3
- YWCJXQKATPNPOE-UKJIMTQDSA-N Ile-Val-Glu Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(=O)O)C(=O)O)N YWCJXQKATPNPOE-UKJIMTQDSA-N 0.000 description 3
- UYODHPPSCXBNCS-XUXIUFHCSA-N Ile-Val-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC(C)C UYODHPPSCXBNCS-XUXIUFHCSA-N 0.000 description 3
- QSXSHZIRKTUXNG-STECZYCISA-N Ile-Val-Tyr Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](C(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 QSXSHZIRKTUXNG-STECZYCISA-N 0.000 description 3
- NTRAGDHVSGKUSF-AVGNSLFASA-N Leu-Arg-Arg Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCCN=C(N)N)C(O)=O NTRAGDHVSGKUSF-AVGNSLFASA-N 0.000 description 3
- XYUBOFCTGPZFSA-WDSOQIARSA-N Leu-Arg-Trp Chemical compound C1=CC=C2C(C[C@H](NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@@H](N)CC(C)C)C(O)=O)=CNC2=C1 XYUBOFCTGPZFSA-WDSOQIARSA-N 0.000 description 3
- HFBCHNRFRYLZNV-GUBZILKMSA-N Leu-Glu-Asp Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HFBCHNRFRYLZNV-GUBZILKMSA-N 0.000 description 3
- 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 3
- PDQDCFBVYXEFSD-SRVKXCTJSA-N Leu-Leu-Asp Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O PDQDCFBVYXEFSD-SRVKXCTJSA-N 0.000 description 3
- QNBVTHNJGCOVFA-AVGNSLFASA-N Leu-Leu-Glu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CCC(O)=O QNBVTHNJGCOVFA-AVGNSLFASA-N 0.000 description 3
- IEWBEPKLKUXQBU-VOAKCMCISA-N Leu-Leu-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O IEWBEPKLKUXQBU-VOAKCMCISA-N 0.000 description 3
- HVHRPWQEQHIQJF-AVGNSLFASA-N Leu-Lys-Glu Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O HVHRPWQEQHIQJF-AVGNSLFASA-N 0.000 description 3
- LVTJJOJKDCVZGP-QWRGUYRKSA-N Leu-Lys-Gly Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)NCC(O)=O LVTJJOJKDCVZGP-QWRGUYRKSA-N 0.000 description 3
- UCBPDSYUVAAHCD-UWVGGRQHSA-N Leu-Pro-Gly Chemical compound CC(C)C[C@H](N)C(=O)N1CCC[C@H]1C(=O)NCC(O)=O UCBPDSYUVAAHCD-UWVGGRQHSA-N 0.000 description 3
- XFIHDSBIPWEYJJ-YUMQZZPRSA-N Lys-Ala-Gly Chemical compound OC(=O)CNC(=O)[C@H](C)NC(=O)[C@@H](N)CCCCN XFIHDSBIPWEYJJ-YUMQZZPRSA-N 0.000 description 3
- IWWMPCPLFXFBAF-SRVKXCTJSA-N Lys-Asp-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O IWWMPCPLFXFBAF-SRVKXCTJSA-N 0.000 description 3
- 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 3
- IMDJSVBFQKDDEQ-MGHWNKPDSA-N Lys-Tyr-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=C(C=C1)O)NC(=O)[C@H](CCCCN)N IMDJSVBFQKDDEQ-MGHWNKPDSA-N 0.000 description 3
- YNOVBMBQSQTLFM-DCAQKATOSA-N Met-Asn-Leu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O YNOVBMBQSQTLFM-DCAQKATOSA-N 0.000 description 3
- KQBJYJXPZBNEIK-DCAQKATOSA-N Met-Glu-Arg Chemical compound CSCC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@H](C(O)=O)CCCNC(N)=N KQBJYJXPZBNEIK-DCAQKATOSA-N 0.000 description 3
- MYAPQOBHGWJZOM-UWVGGRQHSA-N Met-Gly-Leu Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CC(C)C MYAPQOBHGWJZOM-UWVGGRQHSA-N 0.000 description 3
- AFFKUNVPPLQUGA-DCAQKATOSA-N Met-Leu-Ala Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C)C(O)=O AFFKUNVPPLQUGA-DCAQKATOSA-N 0.000 description 3
- 108091028043 Nucleic acid sequence Proteins 0.000 description 3
- XWBJLKDCHJVKAK-KKUMJFAQSA-N Phe-Arg-Gln Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCCN=C(N)N)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N XWBJLKDCHJVKAK-KKUMJFAQSA-N 0.000 description 3
- LJUUGSWZPQOJKD-JYJNAYRXSA-N Phe-Arg-Val Chemical compound CC(C)[C@H](NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)Cc1ccccc1)C(O)=O LJUUGSWZPQOJKD-JYJNAYRXSA-N 0.000 description 3
- HNURHHFOINNTPL-IHPCNDPISA-N Phe-Cys-Trp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC2=CNC3=CC=CC=C32)C(=O)O)N HNURHHFOINNTPL-IHPCNDPISA-N 0.000 description 3
- OWCLJDXHHZUNEL-IHRRRGAJSA-N Phe-Cys-Val Chemical compound [H]N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CS)C(=O)N[C@@H](C(C)C)C(O)=O OWCLJDXHHZUNEL-IHRRRGAJSA-N 0.000 description 3
- RJYBHZVWJPUSLB-QEWYBTABSA-N Phe-Gln-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC1=CC=CC=C1)N RJYBHZVWJPUSLB-QEWYBTABSA-N 0.000 description 3
- PMKIMKUGCSVFSV-CQDKDKBSSA-N Phe-His-Ala Chemical compound C[C@@H](C(=O)O)NC(=O)[C@H](CC1=CN=CN1)NC(=O)[C@H](CC2=CC=CC=C2)N PMKIMKUGCSVFSV-CQDKDKBSSA-N 0.000 description 3
- RTUWVJVJSMOGPL-KKUMJFAQSA-N Phe-Met-Glu Chemical compound CSCC[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)N RTUWVJVJSMOGPL-KKUMJFAQSA-N 0.000 description 3
- XQSREVQDGCPFRJ-STQMWFEESA-N Pro-Gly-Phe Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC1=CC=CC=C1)C(O)=O XQSREVQDGCPFRJ-STQMWFEESA-N 0.000 description 3
- FJLODLCIOJUDRG-PYJNHQTQSA-N Pro-Ile-His Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)NC(=O)[C@@H]2CCCN2 FJLODLCIOJUDRG-PYJNHQTQSA-N 0.000 description 3
- FXGIMYRVJJEIIM-UWVGGRQHSA-N Pro-Leu-Gly Chemical compound OC(=O)CNC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 FXGIMYRVJJEIIM-UWVGGRQHSA-N 0.000 description 3
- HATVCTYBNCNMAA-AVGNSLFASA-N Pro-Leu-Met Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCSC)C(O)=O HATVCTYBNCNMAA-AVGNSLFASA-N 0.000 description 3
- WXWDPFVKQRVJBJ-CIUDSAMLSA-N Ser-Asn-His Chemical compound C1=C(NC=N1)C[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CO)N WXWDPFVKQRVJBJ-CIUDSAMLSA-N 0.000 description 3
- SQBLRDDJTUJDMV-ACZMJKKPSA-N Ser-Glu-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(N)=O)C(O)=O SQBLRDDJTUJDMV-ACZMJKKPSA-N 0.000 description 3
- OHKFXGKHSJKKAL-NRPADANISA-N Ser-Glu-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](C(C)C)C(O)=O OHKFXGKHSJKKAL-NRPADANISA-N 0.000 description 3
- 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 3
- YUJLIIRMIAGMCQ-CIUDSAMLSA-N Ser-Leu-Ser Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O YUJLIIRMIAGMCQ-CIUDSAMLSA-N 0.000 description 3
- XNXRTQZTFVMJIJ-DCAQKATOSA-N Ser-Met-Leu Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(O)=O XNXRTQZTFVMJIJ-DCAQKATOSA-N 0.000 description 3
- JJUNLJTUIKFPRF-BPUTZDHNSA-N Ser-Met-Trp Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC1=CNC2=CC=CC=C21)C(=O)O)NC(=O)[C@H](CO)N JJUNLJTUIKFPRF-BPUTZDHNSA-N 0.000 description 3
- 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 3
- WLJPJRGQRNCIQS-ZLUOBGJFSA-N Ser-Ser-Asn Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O WLJPJRGQRNCIQS-ZLUOBGJFSA-N 0.000 description 3
- 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 3
- TYVAWPFQYFPSBR-BFHQHQDPSA-N Thr-Ala-Gly Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)NCC(O)=O TYVAWPFQYFPSBR-BFHQHQDPSA-N 0.000 description 3
- GUZGCDIZVGODML-NKIYYHGXSA-N Thr-Gln-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O GUZGCDIZVGODML-NKIYYHGXSA-N 0.000 description 3
- VTVVYQOXJCZVEB-WDCWCFNPSA-N Thr-Leu-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O VTVVYQOXJCZVEB-WDCWCFNPSA-N 0.000 description 3
- ZSPQUTWLWGWTPS-HJGDQZAQSA-N Thr-Lys-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(O)=O)C(O)=O ZSPQUTWLWGWTPS-HJGDQZAQSA-N 0.000 description 3
- XKWABWFMQXMUMT-HJGDQZAQSA-N Thr-Pro-Glu Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(O)=O)C(O)=O XKWABWFMQXMUMT-HJGDQZAQSA-N 0.000 description 3
- JAWUQFCGNVEDRN-MEYUZBJRSA-N Thr-Tyr-Leu Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC1=CC=C(C=C1)O)C(=O)N[C@@H](CC(C)C)C(=O)O)N)O JAWUQFCGNVEDRN-MEYUZBJRSA-N 0.000 description 3
- SLOYNOMYOAOUCX-BVSLBCMMSA-N Trp-Phe-Arg Chemical compound [H]N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SLOYNOMYOAOUCX-BVSLBCMMSA-N 0.000 description 3
- YGKVNUAKYPGORG-AVGNSLFASA-N Tyr-Asp-Glu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O YGKVNUAKYPGORG-AVGNSLFASA-N 0.000 description 3
- WTTRJMAZPDHPGS-KKXDTOCCSA-N Tyr-Phe-Ala Chemical compound C[C@H](NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@@H](N)Cc1ccc(O)cc1)C(O)=O WTTRJMAZPDHPGS-KKXDTOCCSA-N 0.000 description 3
- MDXLPNRXCFOBTL-BZSNNMDCSA-N Tyr-Ser-Tyr Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O MDXLPNRXCFOBTL-BZSNNMDCSA-N 0.000 description 3
- SRWWRLKBEJZFPW-IHRRRGAJSA-N Val-Cys-Phe Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)O)N SRWWRLKBEJZFPW-IHRRRGAJSA-N 0.000 description 3
- IJGPOONOTBNTFS-GVXVVHGQSA-N Val-Lys-Glu Chemical compound [H]N[C@@H](C(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCC(O)=O)C(O)=O IJGPOONOTBNTFS-GVXVVHGQSA-N 0.000 description 3
- QRVPEKJBBRYISE-XUXIUFHCSA-N Val-Lys-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CCCCN)NC(=O)[C@H](C(C)C)N QRVPEKJBBRYISE-XUXIUFHCSA-N 0.000 description 3
- 108010069020 alanyl-prolyl-glycine Proteins 0.000 description 3
- 108010047495 alanylglycine Proteins 0.000 description 3
- 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 3
- 108010013835 arginine glutamate Proteins 0.000 description 3
- 108010001271 arginyl-glutamyl-arginine Proteins 0.000 description 3
- 108010068380 arginylarginine Proteins 0.000 description 3
- 108010069205 aspartyl-phenylalanine Proteins 0.000 description 3
- 108010038633 aspartylglutamate Proteins 0.000 description 3
- 108010057083 glutamyl-aspartyl-leucine Proteins 0.000 description 3
- 108010008237 glutamyl-valyl-glycine Proteins 0.000 description 3
- 108010050848 glycylleucine Proteins 0.000 description 3
- 108010081551 glycylphenylalanine Proteins 0.000 description 3
- 108010045383 histidyl-glycyl-glutamic acid Proteins 0.000 description 3
- 108010092114 histidylphenylalanine Proteins 0.000 description 3
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 3
- 108010060857 isoleucyl-valyl-tyrosine Proteins 0.000 description 3
- 108010077158 leucinyl-arginyl-tryptophan Proteins 0.000 description 3
- 108010051673 leucyl-glycyl-phenylalanine Proteins 0.000 description 3
- 108010057821 leucylproline Proteins 0.000 description 3
- 108010064235 lysylglycine Proteins 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 108010016686 methionyl-alanyl-serine Proteins 0.000 description 3
- 108010005942 methionylglycine Proteins 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108010069117 seryl-lysyl-aspartic acid Proteins 0.000 description 3
- 108010026333 seryl-proline Proteins 0.000 description 3
- 108010038745 tryptophylglycine Proteins 0.000 description 3
- 238000012795 verification Methods 0.000 description 3
- 108010010888 1-aminocyclopropane-1-carboxylic acid oxidase Proteins 0.000 description 2
- WUUGFSXJNOTRMR-IOSLPCCCSA-N 5'-S-methyl-5'-thioadenosine Chemical compound O[C@@H]1[C@H](O)[C@@H](CSC)O[C@H]1N1C2=NC=NC(N)=C2N=C1 WUUGFSXJNOTRMR-IOSLPCCCSA-N 0.000 description 2
- IETUUAHKCHOQHP-KZVJFYERSA-N Ala-Thr-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@H](C)N)[C@@H](C)O)C(O)=O IETUUAHKCHOQHP-KZVJFYERSA-N 0.000 description 2
- OVZLLFONXILPDZ-VOAKCMCISA-N Leu-Lys-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(O)=O OVZLLFONXILPDZ-VOAKCMCISA-N 0.000 description 2
- SXJGROGVINAYSH-AVGNSLFASA-N Phe-Gln-Asp Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)N[C@@H](CCC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N SXJGROGVINAYSH-AVGNSLFASA-N 0.000 description 2
- OWQXAJQZLWHPBH-FXQIFTODSA-N Pro-Ser-Asn Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(O)=O OWQXAJQZLWHPBH-FXQIFTODSA-N 0.000 description 2
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 description 2
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 description 2
- 241000592344 Spermatophyta Species 0.000 description 2
- 102000003929 Transaminases Human genes 0.000 description 2
- 108090000340 Transaminases Proteins 0.000 description 2
- MVYRJYISVJWKSX-KBPBESRZSA-N Tyr-His-Gly Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CC2=CN=CN2)C(=O)NCC(=O)O)N)O MVYRJYISVJWKSX-KBPBESRZSA-N 0.000 description 2
- 108010068265 aspartyltyrosine Proteins 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 230000001276 controlling effect Effects 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 238000001976 enzyme digestion Methods 0.000 description 2
- 230000034659 glycolysis Effects 0.000 description 2
- 230000006872 improvement Effects 0.000 description 2
- OOYGSFOGFJDDHP-KMCOLRRFSA-N kanamycin A sulfate Chemical compound OS(O)(=O)=O.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 OOYGSFOGFJDDHP-KMCOLRRFSA-N 0.000 description 2
- 229960002064 kanamycin sulfate Drugs 0.000 description 2
- LWJROJCJINYWOX-UHFFFAOYSA-L mercury dichloride Chemical compound Cl[Hg]Cl LWJROJCJINYWOX-UHFFFAOYSA-L 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 230000002438 mitochondrial effect Effects 0.000 description 2
- 230000008121 plant development Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000000746 purification Methods 0.000 description 2
- 239000012264 purified product Substances 0.000 description 2
- 239000011535 reaction buffer Substances 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 230000000241 respiratory effect Effects 0.000 description 2
- 238000002798 spectrophotometry method Methods 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 230000004102 tricarboxylic acid cycle Effects 0.000 description 2
- MQHUHNALGOSWPX-QIFMNYRTSA-N (2r)-2-amino-3-[[(2r)-2-amino-2-carboxyethyl]disulfanyl]propanoic acid;(2r)-2-amino-3-sulfanylpropanoic acid Chemical compound SC[C@H](N)C(O)=O.OC(=O)[C@@H](N)CSSC[C@H](N)C(O)=O MQHUHNALGOSWPX-QIFMNYRTSA-N 0.000 description 1
- CVWXLIFTNILJFS-YHYXMXQVSA-N (2z)-2-[(2,4-dinitrophenyl)hydrazinylidene]propanoic acid Chemical compound OC(=O)C(/C)=N\NC1=CC=C([N+]([O-])=O)C=C1[N+]([O-])=O CVWXLIFTNILJFS-YHYXMXQVSA-N 0.000 description 1
- 101710194665 1-aminocyclopropane-1-carboxylate synthase Proteins 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- 101100161153 Arabidopsis thaliana ACS10 gene Proteins 0.000 description 1
- 101100161154 Arabidopsis thaliana ACS11 gene Proteins 0.000 description 1
- 101100161155 Arabidopsis thaliana ACS12 gene Proteins 0.000 description 1
- MBOAPAXLTUSMQI-JHEQGTHGSA-N Gly-Glu-Thr Chemical compound [H]NCC(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H]([C@@H](C)O)C(O)=O MBOAPAXLTUSMQI-JHEQGTHGSA-N 0.000 description 1
- CKLJMWTZIZZHCS-REOHCLBHSA-N L-aspartic acid Chemical compound OC(=O)[C@@H](N)CC(O)=O CKLJMWTZIZZHCS-REOHCLBHSA-N 0.000 description 1
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- CGOMLCQJEMWMCE-STQMWFEESA-N Phe-Arg-Gly Chemical compound NC(N)=NCCC[C@@H](C(=O)NCC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 CGOMLCQJEMWMCE-STQMWFEESA-N 0.000 description 1
- UUHXBJHVTVGSKM-BQBZGAKWSA-N Pro-Gly-Asn Chemical compound [H]N1CCC[C@H]1C(=O)NCC(=O)N[C@@H](CC(N)=O)C(O)=O UUHXBJHVTVGSKM-BQBZGAKWSA-N 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 102100026115 S-adenosylmethionine synthase isoform type-1 Human genes 0.000 description 1
- 108050008511 S-adenosylmethionine synthases Proteins 0.000 description 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical class [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 1
- 239000005708 Sodium hypochlorite Substances 0.000 description 1
- KZSYAEWQMJEGRZ-RHYQMDGZSA-N Thr-Leu-Val Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](C(C)C)C(O)=O KZSYAEWQMJEGRZ-RHYQMDGZSA-N 0.000 description 1
- 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 1
- 230000006578 abscission Effects 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 235000003704 aspartic acid Nutrition 0.000 description 1
- OQFSQFPPLPISGP-UHFFFAOYSA-N beta-carboxyaspartic acid Natural products OC(=O)C(N)C(C(O)=O)C(O)=O OQFSQFPPLPISGP-UHFFFAOYSA-N 0.000 description 1
- 238000010504 bond cleavage reaction 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
- 230000001149 cognitive effect Effects 0.000 description 1
- 238000010276 construction Methods 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 230000009977 dual effect Effects 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000004817 gas chromatography Methods 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 230000037353 metabolic pathway Effects 0.000 description 1
- 229930182817 methionine Natural products 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 210000000056 organ Anatomy 0.000 description 1
- 239000003375 plant hormone Substances 0.000 description 1
- 239000013612 plasmid Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000008057 potassium phosphate buffer Substances 0.000 description 1
- 229940076788 pyruvate Drugs 0.000 description 1
- 238000009790 rate-determining step (RDS) Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000010496 root system development Effects 0.000 description 1
- 230000007226 seed germination Effects 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000000967 suction filtration Methods 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/88—Lyases (4.)
-
- 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
- C12N15/70—Vectors or expression systems specially adapted for E. coli
-
- 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
- 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
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y404/00—Carbon-sulfur lyases (4.4)
- C12Y404/01—Carbon-sulfur lyases (4.4.1)
- C12Y404/01014—1-Aminocyclopropane-1-carboxylate synthase (4.4.1.14)
Landscapes
- Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Physics & Mathematics (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明适用于基因工程技术领域，提供了高等植物ACS双酶活关键结构域替换制备得到的单酶突变体，所述单酶突变体为重组酶R10或重组酶R12；其中，所述重组酶R10的蛋白质序列如SEQ ID NO.2所示，重组酶R12的蛋白质序列如SEQ ID NO.3所示。本发明不仅鉴定了影响高等植物ACS蛋白ACS和C‑S裂解酶活性的关键结构域，更为利用基因工程方法控制乙烯参与的多项生命过程比如果实成熟等提供了关键靶点，在抗逆作物分子育种中也具有广阔的应用前景和重要的经济价值。
Description
技术领域
本发明属于基因工程技术领域，尤其涉及高等植物ACS双酶活关键结构域替换制备得到的单酶突变体。
背景技术
乙烯是最古老的植物激素之一，对高等植物生长发育的诸多方面，包括种子萌发、细胞伸长、根系发育、叶片伸展、开花与果实成熟、器官衰老与脱落等都起着重要的调节作用。同时，乙烯在植物对生物与非生物逆境响应与抵抗过程中的作用也不可或缺。在种子植物中，乙烯的生物合成主要经过三步重要的催化反应：第一步，甲硫氨酸在 S-腺苷甲硫氨酸合酶的作用下生成 S-腺苷甲硫氨酸（SAM）；第二步，SAM在ACC合酶（ACC synthase， ACS）的催化下发生γ位的C-S健断裂和α，γ位C-C环化生成1-氨基环丙烷-1-羧酸（ACC），第三步，5′-甲硫腺苷（MTA）、ACC在ACC氧化酶（ACC oxidase，ACO）的作用下转化为乙烯。其中，ACS催化SAM形成ACC是种子植物乙烯合成的限速步骤，ACS也被称为乙烯合成的限速酶。
高等植物的 ACS 由多基因家族编码，其成员的氨基酸序列包含7个保守域（BOX1-BOX7），属于磷酸吡哆醛（Pyridoxal-5’-Phosphate，PLP）依赖型蛋白酶的 α 超家族（superfamily）。除ACS外，这个家族的其他亚族还包括氨基转移酶和C-S裂解酶等。模式植物拟南芥的 ACS 家族成员共有 12 个，其中 ACS1-2，ACS4-9 和 ACS11编码乙烯合成关键酶 ACS，ACS10 和 ACS12 编码以天冬氨酸和芳香族氨基酸为底物的氨基转移酶，而 ACS3是一个没有功能的假基因。长期以来，人们一直一致认为乙烯合成关键酶 ACS是单酶，只能催化SAM形成ACC，目前还没有报道对于乙烯合成关键酶 ACS活性的其他方面的研究。但是，申请人发现，高等植物中的乙烯合成关键酶ACS，包括拟南芥AtACS7，除了具有传统认知的ACS活性外，还能以胱氨酸为底物催化生成铵根离子和丙酮酸（图1），表明其也具有C-S裂解酶活性。丙酮酸是糖酵解的终产物和线粒体三羧酸循环的能量底物，是维持呼吸代谢的非常重要的代谢物，在植物抵抗多种逆境胁迫中发挥重要作用。高等植物ACS蛋白双酶活性的发现，使之成为促进植物发育、果实成熟以及抵抗逆境抗性两种不同而又紧密关联的生命过程的关键交叉点，对其双酶活性的平衡和调控是利用生物技术手段进行作物改造的重要靶标。
发明内容
本发明实施例的目的在于提供高等植物ACS双酶活关键结构域替换制备单酶突变体，旨在为利用生物技术手段平衡和调控高等植物ACS双酶活性进而为进行作物改造提供重要靶标。
本发明实施例是这样实现的：
高等植物ACS蛋白保守域BOX2的保守序列为F-Q-D-Y-H-G-[LM]-[PSKL],该保守域对ACS蛋白ACS活性的维持至关重要。例如，如果将位于拟南芥第四号染色体13275200-13277188位置区间的ACS7基因组中13275695-13275718位、或者其对应cDNA序列中第396-419位编码BOX2结构域的24bp碱基TTTCAAGACTACCACGGTCTCAAA,替换成小立碗藓三号染色体18432861-18432884位的24bp碱基TATGGAAATTTCCGAGGAGGCGAG，或将其相应mRNA序列中第396-419位编码BOX2结构域的24bp碱基UUUCAAGACUACCACGGUCUCAAA，替换成小立碗藓mRNA序列相应位置的24bp碱基UAUGGAAAUUUCCGAGGAGGCGAG，可以使ACS7基因组此位置区间所编码的氨基酸由FQDYHGLK变成YGNFRGGE, 重组后的蛋白被命名为R6。活性测定发现，与拟南芥野生型ACS7蛋白相比，R6重组酶丧失ACS活性，仅具有C-S裂解酶活性（图2）。
高等植物ACS蛋白保守域BOX6的保守序列为M-S-S-F-[GT]-L-[VI]-S-S-Q-T-Q，该保守域对ACS蛋白ACS活性的维持也至关重要。例如，如果将位于拟南芥第四号染色体13275200-13277188位置区间的ACS7基因组中13276533-13276568位或者其相应cDNA序列中第1038-1073位编码BOX6结构域的36bp碱基ATGTCGAGCTTCACGCTTGTCTCGTCTCAGACACAA替换成小立碗藓三号染色体18434808-18434843位的36bp碱基ATGGGCATGTTTGCAGCAGTTTCAAACGACACTCAG，或将其相应mRNA序列中第1038-1073位编码BOX6结构域的36bp碱基AUGUCGAGCUUCACGCUUGUCUCGUCUCAGACACAA替换成小立碗藓mRNA序列相应位置的36bp碱基AUGGGCAUGUUUGCAGCAGUUUCAAACGACACUCAG，可以使ACS7基因组此位置区间所编码的氨基酸由MSSFTLVSSQTQ变成MGMFAAVSNDTQ, 重组后的蛋白被命名为R10。活性测定发现，与拟南芥野生型ACS7蛋白相比，R10重组酶丧失ACS活性，仅具有C-S裂解酶活性（图2）。
高等植物ACS蛋白的保守域BOX4的保守序列为T-N-P-S-N-P-L-G-[TA]-[TAMVIF]，申请人发现，该保守域对ACS蛋白C-S裂解酶活性的维持至关重要。例如，如果将位于拟南芥第四号染色体13275200-13277188位置区间的ACS7基因组中13276239-13276268位或其对应cDNA序列中第744-773位编码BOX4保守序列的30bp碱基ACCAACCCATCGAACCCATTAGGGGCGACG替换成小立碗藓三号染色体18433956-18433985之间的30bp碱基ACTAATCCTGGGAACCCATTGGGCACTCTT，或将其对应mRNA序列中第744-773位编码BOX4保守序列的30bp碱基ACCAACCCAUCGAACCCAUUAGGGGCGACG替换成小立碗藓mRNA相应序列的30bp碱基ACUAAUCCUGGGAACCCAUUGGGCACUCUU，可以使ACS7基因组此位置区间所编码的氨基酸由TNPSNPLGAT变成 TNPGNPLGTL, 重组后的蛋白被命名为R12。活性测定发现，与拟南芥野生型ACS7蛋白相比，R12重组酶丧失C-S裂解酶活性，仅具有ACS活性（图2）
重组酶R6的蛋白质序列如由SEQ ID NO.1所示，重组酶R10的蛋白质序列如由SEQID NO.2所示，重组酶R12的蛋白质序列如由SEQ ID NO.3所示。
进一步的技术方案，所述重组酶R6、R10和R12的制备方法，包括以下步骤：
步骤（1）构建含有编码高等植物的突变型ACS7的核苷酸序列的载体，
利用基因合成的方法，合成ACS7基因的上下游引物以及包含突变位点信息的引物序列如下：
R6-AF: 5’-GGATCCATGGGTCTTCCTCTAATGATGGAGAGATCATCAAAC -3’
R6-AR：5’- CTCGCCTCCTCGGAAATTTCCATACAATGCGTTTTCAC-3’
R6-BF：5’-TATGGAAATTTCCGAGGAGGCGAGACTTTCAGACAAGCC-3’
R6-BR: 5’-GCGGCCGCTCAAAACCTCCTTCGTCGGT-3’
R10-AF: 5’-GGATCCATGGGTCTTCCTCTAATGATGGAGAGA -3’
R10-AR：5’- CTGAGTGTCGTTTGAAACTGCTGCAAACATGCCCATCCTTCTCGCTGTCCGA-3’
R10-BF：5’- ATGGGCATGTTTGCAGCAGTTTCAAACGACACTCAGCATATGCTGGCTTCTA-3’
R10-BR: 5’ - GCGGCCGCTCAAAACCTCCTTCGT-3’
R12-AF: 5’- GGATCCATGGGTCTTCCTCTAATGATGGAGAGATCATCAAACAACAACA-3’
R12-AR：5’- AAGAGTGCCCAATGGGTTCCCAGGATTAGTTATGAGCACTCCTCGGAC -3’
R12-BF：5’-ACTAATCCTGGGAACCCATTGGGCACTCTTGTCCAAAAGAAGGTTCTAG-3’
R12-BR: 5’ - GCGGCCGCTCAAAACCTCCTTCGTCGGTCCATG -3’；
步骤（2）将测序验证无误的载体，取20 ng转化到BL21感受态细胞中，涂布在含有硫酸卡那霉素的LB平板上，37℃培养过夜，第二挑取单克隆；
步骤（3）培养并收集转化后细胞，按照His-Trap FF column说明书提取纯化各ACS7单酶活性重组酶。
进一步的技术方案，在所述步骤（1）中，基因合成的方法包括以下步骤：
i. 利用PCR首先对含有突变位点的上下游片段进行扩增，以R6-AF与R6-AR为引物扩增出R6-A片段，以R6-BR与R6-BF为引物扩增出R6-B片段；用R10-AF与R10-AR扩增出R10-A片段；用R10 -BR与R10-BF扩增出R10-B片段；以R12-AF与R12-AR扩增出R12-A片段，以R12-BR与R12-BF扩增出R12-B片段；
ii. 以A、B片段互为引物通过融合PCR分别初步扩增获得R6、R10和R12 目的片段，然后分别以该步骤获得的纯化产物为模板，以HiFi Hot Start为DNA聚合酶，以R6-AF和R6-BR、R10-AF和R10-BR、R12-AF和R12-BR为引物通过融合PCR分别获得R6、R10和R12目标产物；
iii. 将上述PCR产物纯化后，按照pMD_18-T Vector Cloning Kit 试剂盒将产物连接到pMD_18-T载体；经测序验证正确后，用BamH I和Not I双酶切pMD_18-T载体和表达载体pET28a后将目标片段连接至pET28a，获得R6、R10和R12单酶活性重组酶核苷酸序列的表达载体。
进一步的技术方案，在步骤i中，扩增的反应体系为：在50 μl反应体系中，以含有野生型ACS7基因的质粒为模板，10 μM 的引物各1 μl，以HiFi Hot Start为DNA聚合酶。扩增的条件为：95℃ 5 min；98℃ 20s； 58℃ 15s；72 ℃ 2 min；72℃ 10 min；共25个循环。
进一步的技术方案，在步骤ii中，扩增的反应体系为：在50 μl反应体系中，分别根据A和B片段的浓度和长度，按照总体积15 μl原则，计算A和B片段各自应加入的体积，以HiFi Hot Start为DNA聚合酶。扩增的条件为：95℃ 5 min；98℃ 20s； 58℃ 15s；72℃ 2min；72℃ 10 min；共13个循环。
进一步的技术方案，ACS蛋白ACS和C-S裂解酶双酶活性测定的操作步骤如下：
a. ACS活性的测定；将纯化的拟南芥 ACS7 突变体蛋白或相应的负对照、ACS 反应缓冲液和 S-腺苷甲硫氨酸一起孵育，用100 mM HgCl2终止反应，然后加入新鲜配制的ACC assay solution将生成的ACC转化为乙烯，利用气相色谱仪分析计算乙烯生成量；
b. C-S裂解酶活性的测定：将纯化的拟南芥 ACS7突变体蛋白或相应的负对照和底物一起孵育，反应结束后加入氯仿抽提蛋白；对于反应产物丙酮酸的测定，采用分光光度法检测丙酮酸与 2,4-二硝基苯肼反应生成丙酮酸-2,4-二硝基苯腙的量；对于反应产物铵根离子的测定，则将上述抽提蛋白后的反应液抽滤，用氨基酸分析仪检测分析。
进一步的技术方案，包括所述的重组酶R6、R10和R12序列应用于果实成熟和抗逆作物育种方面。
本发明实施例提供的重组酶R12仅具有ACS活性，而重组酶R6和R10只具有C-S裂解酶活性。本发明不仅鉴定了影响ACS7 高等植物ACS蛋白 ACS和C-S裂解酶活性的重要结构域，更为利用基因工程方法控制乙烯参与的多项生命过程比如果实成熟等提供了关键靶点。同时，ACS蛋白C-S裂解酶单酶活性重组酶能够催化丙酮酸生成并参与细胞内的多种重要代谢路径，在抗逆作物分子育种中也具有广阔的应用前景和重要的经济价值。
附图说明
图1为本发明拟南芥AtACS7同时具有ACS和C-S裂解酶双重活性的示意图；
图2为本发明R6、R10 重组酶只具有C-S裂解酶单酶活性，R12重组酶只有ACS活性的示意图。
具体实施方式
为了使本发明的目的、技术方案及优点更加清楚明白，以下结合附图及实施例，对本发明进行进一步详细说明。应当理解，此处所描述的具体实施例仅仅用以解释本发明，并不用于限定本发明。
以下结合具体实施例对本发明的具体实现进行详细描述。
首先，本发明所述三种拟南芥ACS7单酶活性重组酶的制备方法，包括如下步骤：
（1）构建含有编码所述拟南芥突变型ACS7的核苷酸序列的载体
利用基因合成的方法，合成ACS7基因的上下游引物以及包含突变位点信息的引物序列如下：
R6-AF: 5’-GGATCCATGGGTCTTCCTCTAATGATGGAGAGATCATCAAAC -3’
R6-AR：5’- CTCGCCTCCTCGGAAATTTCCATACAATGCGTTTTCAC-3’
R6-BF：5’-TATGGAAATTTCCGAGGAGGCGAGACTTTCAGACAAGCC-3’
R6-BR: 5’-GCGGCCGCTCAAAACCTCCTTCGTCGGT-3’
R10-AF: 5’-GGATCCATGGGTCTTCCTCTAATGATGGAGAGA -3’
R10-AR：5’- CTGAGTGTCGTTTGAAACTGCTGCAAACATGCCCATCCTTCTCGCTGTCCGA-3’
R10-BF：5’- ATGGGCATGTTTGCAGCAGTTTCAAACGACACTCAGCATATGCTGGCTTCTA-3’
R10-BR: 5’ - GCGGCCGCTCAAAACCTCCTTCGT-3’
R12-AF: 5’- GGATCCATGGGTCTTCCTCTAATGATGGAGAGATCATCAAACAACAACA-3’
R12-AR：5’- AAGAGTGCCCAATGGGTTCCCAGGATTAGTTATGAGCACTCCTCGGAC -3’
R12-BF：5’-ACTAATCCTGGGAACCCATTGGGCACTCTTGTCCAAAAGAAGGTTCTAG-3’
R12-BR: 5’ - GCGGCCGCTCAAAACCTCCTTCGTCGGTCCATG -3’；
利用PCR首先对含有突变位点的上下游片段进行扩增，以R6-AF与R6-AR为引物扩增出R6-A片段，以R6-BR与R6-BF为引物扩增出R6-B片段；用R10-AF与R10-AR扩增出R10-A片段；用R10 -BR与R10-BF扩增出R10-B片段；以R12-AF与R12-AR扩增出R12-A片段，以R12-BR与R12-BF扩增出R12-B片段。扩增反应体系为，在50 μl反应体系中，以含有野生型ACS7基因的质粒为模板，10 μM 的引物各1 μl，以HiFi Hot Start（北京普凯瑞生物科技有限公司，货号KK2601）为DNA聚合酶。扩增条件为，95℃ 5 min；98℃ 20s； 58℃ 15s；72 ℃ 2 min；72℃ 10 min; 共25个循环。
以A、B片段互为引物分别通过融合PCR分别初步扩增获得R6、R10和R12 目的片段。扩增反应体系为，在50 μl反应体系中，分别根据A和B片段的浓度和长度，按照总体积15 μl原则，计算A和B片段各自应加入的体积，以HiFi Hot Start为DNA聚合酶。扩增条件为，95℃5 min；98℃ 20s； 58℃ 15s；72℃ 2 min；72℃ 10 min; 共13个循环。然后，分别以该步骤获得的纯化产物为模板，以HiFi Hot Start为DNA聚合酶，以R6-AF和R6-BR、R10-AF和R10-BR、R12-AF和R12-BR为引物通过融合PCR分别获得R6、R10和R12目标产物。
将上述PCR产物纯化后，按照pMD_18-T Vector Cloning Kit 试剂盒将产物连接到pMD_18-T载体。经测序验证正确后，用BamH I和Not I双酶切pMD_18-T载体和表达载体pET28a后将目标片段连接至pET28a，获得R6、R10和R12单酶活性重组酶核苷酸序列的表达载体。
（2）将测序验证无误的载体，取20 ng转化到BL21感受态细胞中，涂布在含有硫酸卡那霉素的LB平板上，37度培养过夜，第二挑取单克隆。
（3）培养并收集转化后细胞，按照His-Trap FF column (GE Healthcare，货号17-5255-01)说明书提取纯化各ACS7单酶活性重组酶。
其次，本发明所涉及的ACS蛋白ACS和C-S裂解酶双酶活性测定方法如下。其中ACS活性的测定方法为：将纯化的拟南芥 ACS7 突变体蛋白或相应的负对照（转化空载体pET28a 菌株的相同流程蛋白提取物）、ACS 反应缓冲液（50 mM EPPS，pH8.5，10 μM PLP，2mM DTT）和 S-腺苷甲硫氨酸（SAM）一起孵育，用 100 mM HgCl2 终止反应，然后加入新鲜配制的 ACC assay solution [饱和 NaOH:次氯酸钠=2:1(v:v)]将生成的 ACC 转化为乙烯，利用气相色谱仪（Agilent 7890A）分析计算乙烯生成量。C-S裂解酶活性测定的基本反应体系为：将纯化的拟南芥 ACS7突变体蛋白或相应的负对照、底物如 L-胱氨酸（L-cystine）、75 mM磷酸钾缓冲液 pH7.6和100 μM PLP一起孵育，反应结束后加入氯仿抽提蛋白。对于反应产物丙酮酸的测定，采用分光光度法检测丙酮酸与 2,4-二硝基苯肼反应生成丙酮酸-2,4-二硝基苯腙的量。对于反应产物铵根离子（NH4+）的测定，则将上述抽提蛋白后的反应液抽滤，用氨基酸分析仪（A300，MembraPure GmbH）检测分析。
如图1所示，A．氨基酸分析仪测定结果显示拟南芥AtACS7可以以胱氨酸为底物催化C-S裂解反应，造成反应体系中胱氨酸含量的显著下降并生成铵根离子；B. 化学显色法证明ACS7可催化底物胱氨酸发生C-S键断裂生成丙酮酸。同时GC法检测证明AtACS7具有正常的ACS活性。His-ACL1蛋白作为C-S裂解酶催化反应的正对照，以空载体pET28a为负对照（control）。
在拟南芥中，将位于第四号染色体13275200-13277188位置区间的ACS7基因组中13275695-13275718位、或其对应cDNA序列中第396-419位编码BOX2结构域的24bp碱基TTTCAAGACTACCACGGTCTCAAA,替换成小立碗藓三号染色体18432861-18432884位的24bp碱基TATGGAAATTTCCGAGGAGGCGAG，或将其相应mRNA序列中第396-419位编码BOX2结构域的24bp碱基UUUCAAGACUACCACGGUCUCAAA，替换成小立碗藓mRNA序列相应位置的24bp碱基UAUGGAAAUUUCCGAGGAGGCGAG，可以使ACS7基因组此位置区间所编码的氨基酸由FQDYHGLK变成YGNFRGGE, 重组后的蛋白被命名为R6。活性测定发现，与拟南芥野生型ACS7蛋白相比，R6重组酶丧失ACS活性，仅具有以胱氨酸为底物的C-S裂解酶活性（如图2）；
在拟南芥中，将位于第四号染色体13275200-13277188位置区间的ACS7基因组中13276533-13276568位或其相应cDNA序列中第1038-1073位编码BOX6结构域的36bp碱基ATGTCGAGCTTCACGCTTGTCTCGTCTCAGACACAA替换成小立碗藓三号染色体18434808-18434843位的36bp碱基ATGGGCATGTTTGCAGCAGTTTCAAACGACACTCAG，或将其相应mRNA序列中第1038-1073位编码BOX6结构域的36bp碱基AUGUCGAGCUUCACGCUUGUCUCGUCUCAGACACAA替换成小立碗藓mRNA序列相应位置的36bp碱基AUGGGCAUGUUUGCAGCAGUUUCAAACGACACUCAG，可以使ACS7基因组此位置区间所编码的氨基酸由MSSFTLVSSQTQ变成MGMFAAVSNDTQ, 重组后的蛋白被命名为R10。活性测定发现，与拟南芥野生型ACS7蛋白相比，R10重组酶丧失ACS活性，仅具有以胱氨酸为底物的C-S裂解酶活性（如图2）；
在拟南芥中，将位于第四号染色体13275200-13277188位置区间的ACS7基因组中13276239-13276268位或其对应cDNA序列中第744-773位编码BOX4保守序列的30bp碱基ACCAACCCATCGAACCCATTAGGGGCGACG替换成小立碗藓三号染色体18433956-18433985之间的30bp碱基ACTAATCCTGGGAACCCATTGGGCACTCTT，或将其对应mRNA序列中第744-773位编码BOX4保守序列的30bp碱基ACCAACCCAUCGAACCCAUUAGGGGCGACG替换成小立碗藓mRNA相应序列的30bp碱基ACUAAUCCUGGGAACCCAUUGGGCACUCUU，可以使ACS7基因组此位置区间所编码的氨基酸由TNPSNPLGAT变成 TNPGNPLGTL, 重组后的蛋白被命名为R12。活性测定发现，与拟南芥野生型ACS7蛋白相比，R12重组酶丧失以胱氨酸为底物的C-S裂解酶活性，仅具有ACS活性（如图2）。
本发明不仅鉴定了影响高等植物ACS蛋白 ACS和C-S裂解酶活性的重要结构域，更为利用基因工程方法控制乙烯参与的多项生命过程比如果实成熟等提供了关键靶点，同时，重组酶R6和R10作为C-S裂解酶能够催化丙酮酸生成并参与细胞内的多种重要代谢路径，在抗逆作物分子育种中也具有广阔的应用前景和重要的经济价值。因此申请保护利用各种不同技术手段针对所有高等植物中ACS同源蛋白相应于拟南芥ACS7蛋白中BOX2、BOX4和BOX6结构域的调节和利用。
高等植物中的乙烯合成关键酶ACS，包括拟南芥AtACS7，除了具有传统认知的ACS活性外，还能以胱氨酸为底物催化生成铵根离子和丙酮酸，表明其也具有C-S裂解酶活性。丙酮酸是糖酵解的终产物和线粒体三羧酸循环的能量底物，是维持呼吸代谢的非常重要的代谢物，在植物抵抗多种逆境胁迫中发挥重要作用。高等植物ACS蛋白双酶活性的发现，使之成为促进植物发育、果实成熟以及抵抗逆境抗性两种不同而又紧密关联的生命过程的关键交叉点，对其双酶活性的平衡和调控是利用生物技术手段进行作物改造的重要靶标。
以上所述仅为本发明的较佳实施例而已，并不用以限制本发明，凡在本发明的精神和原则之内所作的任何修改、等同替换和改进等，均应包含在本发明的保护范围之内。
序列表
<110> 南开大学
<120> 高等植物ACS双酶活关键结构域替换制备单酶突变体及应用
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 1
Met Gly Leu Pro Leu Met Met Glu Arg Ser Ser Asn Asn Asn Asn Val
1 5 10 15
Glu Leu Ser Arg Val Ala Val Ser Asp Thr His Gly Glu Asp Ser Pro
20 25 30
Tyr Phe Ala Gly Trp Lys Ala Tyr Asp Glu Asn Pro Tyr Asp Glu Ser
35 40 45
His Asn Pro Ser Gly Val Ile Gln Met Gly Leu Ala Glu Asn Gln Val
50 55 60
Ser Phe Asp Leu Leu Glu Thr Tyr Leu Glu Lys Lys Asn Pro Glu Gly
65 70 75 80
Ser Met Trp Gly Ser Lys Gly Ala Pro Gly Phe Arg Glu Asn Ala Leu
85 90 95
Tyr Gly Asn Phe Arg Gly Gly Glu Thr Phe Arg Gln Ala Met Ala Ser
100 105 110
Phe Met Glu Gln Ile Arg Gly Gly Lys Ala Arg Phe Asp Pro Asp Arg
115 120 125
Ile Val Leu Thr Ala Gly Ala Thr Ala Ala Asn Glu Leu Leu Thr Phe
130 135 140
Ile Leu Ala Asp Pro Asn Asp Ala Leu Leu Val Pro Thr Pro Tyr Tyr
145 150 155 160
Pro Gly Phe Asp Arg Asp Leu Arg Trp Arg Thr Gly Val Lys Ile Val
165 170 175
Pro Ile His Cys Asp Ser Ser Asn His Phe Gln Ile Thr Pro Glu Ala
180 185 190
Leu Glu Ser Ala Tyr Gln Thr Ala Arg Asp Ala Asn Ile Arg Val Arg
195 200 205
Gly Val Leu Ile Thr Asn Pro Ser Asn Pro Leu Gly Ala Thr Val Gln
210 215 220
Lys Lys Val Leu Glu Asp Leu Leu Asp Phe Cys Val Arg Lys Asn Ile
225 230 235 240
His Leu Val Ser Asp Glu Ile Tyr Ser Gly Ser Val Phe His Ala Ser
245 250 255
Glu Phe Thr Ser Val Ala Glu Ile Val Glu Asn Ile Asp Asp Val Ser
260 265 270
Val Lys Glu Arg Val His Ile Val Tyr Ser Leu Ser Lys Asp Leu Gly
275 280 285
Leu Pro Gly Phe Arg Val Gly Thr Ile Tyr Ser Tyr Asn Asp Asn Val
290 295 300
Val Arg Thr Ala Arg Arg Met Ser Ser Phe Thr Leu Val Ser Ser Gln
305 310 315 320
Thr Gln His Met Leu Ala Ser Met Leu Ser Asp Glu Glu Phe Thr Glu
325 330 335
Lys Tyr Ile Arg Ile Asn Arg Glu Arg Leu Arg Arg Arg Tyr Asp Thr
340 345 350
Ile Val Glu Gly Leu Lys Lys Ala Gly Ile Glu Cys Leu Lys Gly Asn
355 360 365
Ala Gly Leu Phe Cys Trp Met Asn Leu Gly Phe Leu Leu Glu Lys Lys
370 375 380
Thr Lys Asp Gly Glu Leu Gln Leu Trp Asp Val Ile Leu Lys Glu Leu
385 390 395 400
Asn Leu Asn Ile Ser Pro Gly Ser Ser Cys His Cys Ser Glu Val Gly
405 410 415
Trp Phe Arg Val Cys Phe Ala Asn Met Ser Glu Asn Thr Leu Glu Ile
420 425 430
Ala Leu Lys Arg Ile His Glu Phe Met Asp Arg Arg Arg Arg Phe
435 440 445
<210> 2
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 2
Met Gly Leu Pro Leu Met Met Glu Arg Ser Ser Asn Asn Asn Asn Val
1 5 10 15
Glu Leu Ser Arg Val Ala Val Ser Asp Thr His Gly Glu Asp Ser Pro
20 25 30
Tyr Phe Ala Gly Trp Lys Ala Tyr Asp Glu Asn Pro Tyr Asp Glu Ser
35 40 45
His Asn Pro Ser Gly Val Ile Gln Met Gly Leu Ala Glu Asn Gln Val
50 55 60
Ser Phe Asp Leu Leu Glu Thr Tyr Leu Glu Lys Lys Asn Pro Glu Gly
65 70 75 80
Ser Met Trp Gly Ser Lys Gly Ala Pro Gly Phe Arg Glu Asn Ala Leu
85 90 95
Phe Gln Asp Tyr His Gly Leu Lys Thr Phe Arg Gln Ala Met Ala Ser
100 105 110
Phe Met Glu Gln Ile Arg Gly Gly Lys Ala Arg Phe Asp Pro Asp Arg
115 120 125
Ile Val Leu Thr Ala Gly Ala Thr Ala Ala Asn Glu Leu Leu Thr Phe
130 135 140
Ile Leu Ala Asp Pro Asn Asp Ala Leu Leu Val Pro Thr Pro Tyr Tyr
145 150 155 160
Pro Gly Phe Asp Arg Asp Leu Arg Trp Arg Thr Gly Val Lys Ile Val
165 170 175
Pro Ile His Cys Asp Ser Ser Asn His Phe Gln Ile Thr Pro Glu Ala
180 185 190
Leu Glu Ser Ala Tyr Gln Thr Ala Arg Asp Ala Asn Ile Arg Val Arg
195 200 205
Gly Val Leu Ile Thr Asn Pro Ser Asn Pro Leu Gly Ala Thr Val Gln
210 215 220
Lys Lys Val Leu Glu Asp Leu Leu Asp Phe Cys Val Arg Lys Asn Ile
225 230 235 240
His Leu Val Ser Asp Glu Ile Tyr Ser Gly Ser Val Phe His Ala Ser
245 250 255
Glu Phe Thr Ser Val Ala Glu Ile Val Glu Asn Ile Asp Asp Val Ser
260 265 270
Val Lys Glu Arg Val His Ile Val Tyr Ser Leu Ser Lys Asp Leu Gly
275 280 285
Leu Pro Gly Phe Arg Val Gly Thr Ile Tyr Ser Tyr Asn Asp Asn Val
290 295 300
Val Arg Thr Ala Arg Arg Met Gly Met Phe Ala Ala Val Ser Asn Asp
305 310 315 320
Thr Gln His Met Leu Ala Ser Met Leu Ser Asp Glu Glu Phe Thr Glu
325 330 335
Lys Tyr Ile Arg Ile Asn Arg Glu Arg Leu Arg Arg Arg Tyr Asp Thr
340 345 350
Ile Val Glu Gly Leu Lys Lys Ala Gly Ile Glu Cys Leu Lys Gly Asn
355 360 365
Ala Gly Leu Phe Cys Trp Met Asn Leu Gly Phe Leu Leu Glu Lys Lys
370 375 380
Thr Lys Asp Gly Glu Leu Gln Leu Trp Asp Val Ile Leu Lys Glu Leu
385 390 395 400
Asn Leu Asn Ile Ser Pro Gly Ser Ser Cys His Cys Ser Glu Val Gly
405 410 415
Trp Phe Arg Val Cys Phe Ala Asn Met Ser Glu Asn Thr Leu Glu Ile
420 425 430
Ala Leu Lys Arg Ile His Glu Phe Met Asp Arg Arg Arg Arg Phe
435 440 445
<210> 3
<211> 447
<212> PRT
<213> 人工序列(Artificial Sequence)
<400> 3
Met Gly Leu Pro Leu Met Met Glu Arg Ser Ser Asn Asn Asn Asn Val
1 5 10 15
Glu Leu Ser Arg Val Ala Val Ser Asp Thr His Gly Glu Asp Ser Pro
20 25 30
Tyr Phe Ala Gly Trp Lys Ala Tyr Asp Glu Asn Pro Tyr Asp Glu Ser
35 40 45
His Asn Pro Ser Gly Val Ile Gln Met Gly Leu Ala Glu Asn Gln Val
50 55 60
Ser Phe Asp Leu Leu Glu Thr Tyr Leu Glu Lys Lys Asn Pro Glu Gly
65 70 75 80
Ser Met Trp Gly Ser Lys Gly Ala Pro Gly Phe Arg Glu Asn Ala Leu
85 90 95
Phe Gln Asp Tyr His Gly Leu Lys Thr Phe Arg Gln Ala Met Ala Ser
100 105 110
Phe Met Glu Gln Ile Arg Gly Gly Lys Ala Arg Phe Asp Pro Asp Arg
115 120 125
Ile Val Leu Thr Ala Gly Ala Thr Ala Ala Asn Glu Leu Leu Thr Phe
130 135 140
Ile Leu Ala Asp Pro Asn Asp Ala Leu Leu Val Pro Thr Pro Tyr Tyr
145 150 155 160
Pro Gly Phe Asp Arg Asp Leu Arg Trp Arg Thr Gly Val Lys Ile Val
165 170 175
Pro Ile His Cys Asp Ser Ser Asn His Phe Gln Ile Thr Pro Glu Ala
180 185 190
Leu Glu Ser Ala Tyr Gln Thr Ala Arg Asp Ala Asn Ile Arg Val Arg
195 200 205
Gly Val Leu Ile Thr Asn Pro Gly Asn Pro Leu Gly Thr Leu Val Gln
210 215 220
Lys Lys Val Leu Glu Asp Leu Leu Asp Phe Cys Val Arg Lys Asn Ile
225 230 235 240
His Leu Val Ser Asp Glu Ile Tyr Ser Gly Ser Val Phe His Ala Ser
245 250 255
Glu Phe Thr Ser Val Ala Glu Ile Val Glu Asn Ile Asp Asp Val Ser
260 265 270
Val Lys Glu Arg Val His Ile Val Tyr Ser Leu Ser Lys Asp Leu Gly
275 280 285
Leu Pro Gly Phe Arg Val Gly Thr Ile Tyr Ser Tyr Asn Asp Asn Val
290 295 300
Val Arg Thr Ala Arg Arg Met Ser Ser Phe Thr Leu Val Ser Ser Gln
305 310 315 320
Thr Gln His Met Leu Ala Ser Met Leu Ser Asp Glu Glu Phe Thr Glu
325 330 335
Lys Tyr Ile Arg Ile Asn Arg Glu Arg Leu Arg Arg Arg Tyr Asp Thr
340 345 350
Ile Val Glu Gly Leu Lys Lys Ala Gly Ile Glu Cys Leu Lys Gly Asn
355 360 365
Ala Gly Leu Phe Cys Trp Met Asn Leu Gly Phe Leu Leu Glu Lys Lys
370 375 380
Thr Lys Asp Gly Glu Leu Gln Leu Trp Asp Val Ile Leu Lys Glu Leu
385 390 395 400
Asn Leu Asn Ile Ser Pro Gly Ser Ser Cys His Cys Ser Glu Val Gly
405 410 415
Trp Phe Arg Val Cys Phe Ala Asn Met Ser Glu Asn Thr Leu Glu Ile
420 425 430
Ala Leu Lys Arg Ile His Glu Phe Met Asp Arg Arg Arg Arg Phe
435 440 445
Claims (1)
1.高等植物ACS双酶活关键结构域替换制备得到的单酶突变体，其特征在于，所述单酶突变体为重组酶R10或重组酶R12；
其中，所述重组酶R10的蛋白质序列如SEQ ID NO.2所示，重组酶R12的蛋白质序列如SEQ ID NO.3所示。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111320329.2A CN114032229B (zh) | 2021-11-09 | 2021-11-09 | 高等植物acs双酶活关键结构域替换制备得到的单酶突变体 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111320329.2A CN114032229B (zh) | 2021-11-09 | 2021-11-09 | 高等植物acs双酶活关键结构域替换制备得到的单酶突变体 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN114032229A CN114032229A (zh) | 2022-02-11 |
| CN114032229B true CN114032229B (zh) | 2023-11-17 |
Family
ID=80143657
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111320329.2A Active CN114032229B (zh) | 2021-11-09 | 2021-11-09 | 高等植物acs双酶活关键结构域替换制备得到的单酶突变体 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN114032229B (zh) |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2007234585A1 (en) * | 2003-06-23 | 2007-12-13 | Pioneer Hi-Bred International, Inc. | Engineering single-gene-controlled staygreen potential into plants |
| CN103333885A (zh) * | 2003-06-23 | 2013-10-02 | 先锋高级育种国际公司 | 将单基因控制的保绿潜力工程化进植物中 |
| CN112251392A (zh) * | 2020-10-26 | 2021-01-22 | 天津科技大学 | 一种生产麦角硫因的基因工程菌株及其应用 |
-
2021
- 2021-11-09 CN CN202111320329.2A patent/CN114032229B/zh active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2007234585A1 (en) * | 2003-06-23 | 2007-12-13 | Pioneer Hi-Bred International, Inc. | Engineering single-gene-controlled staygreen potential into plants |
| CN103333885A (zh) * | 2003-06-23 | 2013-10-02 | 先锋高级育种国际公司 | 将单基因控制的保绿潜力工程化进植物中 |
| CN112251392A (zh) * | 2020-10-26 | 2021-01-22 | 天津科技大学 | 一种生产麦角硫因的基因工程菌株及其应用 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN114032229A (zh) | 2022-02-11 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pathirana et al. | Alfalfa root nodule phosphoenolpyruvate carboxylase: characterization of the cDNA and expression in effective and plant-controlled ineffective nodules | |
| AU2014360597A1 (en) | Compositions and methods for making (R)-Reticuline and precursors thereof | |
| KR20200026261A (ko) | 엑토인-생산 효모 | |
| CN111304228B (zh) | 一种橡胶树线粒体型己糖激酶基因及其编码蛋白和应用 | |
| CN115427580A (zh) | 用于改善依克多因产生的经修饰的微生物和方法 | |
| CN108130334B (zh) | 柳枝稷s-腺苷甲硫氨酸合成酶基因sams1调控木质素合成的应用 | |
| CN114032229B (zh) | 高等植物acs双酶活关键结构域替换制备得到的单酶突变体 | |
| CN112553285B (zh) | 一种ω-转氨酶的应用及生物酶法去消旋化制备L-草铵膦的方法 | |
| Filetici et al. | Sequence of the GLT1 gene from Saccharomyces cerevisiae reveals the domain structure of yeast glutamate synthase | |
| Brauner et al. | Pea seedling aminoaldehyde dehydrogenase: primary structure and active site residues | |
| CN114107268B (zh) | 高等植物acs双酶活关键位点突变制备得到的单酶活突变体 | |
| CN109337884B9 (zh) | 一种丙酮酸激酶基因及其应用 | |
| CN112251451A (zh) | 一种编码GA20ox-氧化酶的基因及其应用 | |
| CN112080479A (zh) | 一种17β-羟基类固醇脱氢酶突变体及其应用 | |
| CN116926092B (zh) | 一种泛酸激酶基因RkPank及其应用 | |
| CN105087516B (zh) | 一种adp-葡萄糖焦磷酸化酶突变体及其筛选方法与应用 | |
| CN106636028A (zh) | 具有抗咪唑啉酮类除草剂活性的水稻蛋白质、其编码基因及应用 | |
| CN113846085B (zh) | 一种具有双酶活性的蛋白质及其用途 | |
| Tassoni et al. | Cloning, functional identification and structural modelling of Vitis vinifera S-adenosylmethionine decarboxylase | |
| CN108374002B (zh) | 一种脆江蓠磷酸葡萄糖变位酶的蛋白及其编码基因和用途 | |
| CN113699173A (zh) | HbACLB-1基因在提高原核表达菌生长速率、研究橡胶树产胶能力中的应用 | |
| CN111304227B (zh) | 一种橡胶树叶绿体型己糖激酶基因及其编码蛋白和应用 | |
| CN108085326B (zh) | 一种柳枝稷腺苷高半胱氨酸在改变木质素单体和提高细胞壁降解效率方面的应用 | |
| CN116348606A (zh) | 一种以双醋瑞因为供体的酶促合成乙酰辅酶a的方法 | |
| WO2013039378A1 (en) | A method for regulating cytosolic isoprenoid biosynthesis in hevea brasiliensis |
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 |