CN109678941B - PgSND1-like基因在促进石榴籽粒硬度形成的用途 - Google Patents
PgSND1-like基因在促进石榴籽粒硬度形成的用途 Download PDFInfo
- Publication number
- CN109678941B CN109678941B CN201811630966.8A CN201811630966A CN109678941B CN 109678941 B CN109678941 B CN 109678941B CN 201811630966 A CN201811630966 A CN 201811630966A CN 109678941 B CN109678941 B CN 109678941B
- Authority
- CN
- China
- Prior art keywords
- pgsnd1
- gene
- pomegranate
- leu
- ser
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
- 235000014360 Punica granatum Nutrition 0.000 title claims abstract description 35
- 241000219991 Lythraceae Species 0.000 title claims abstract description 33
- 230000015572 biosynthetic process Effects 0.000 title claims abstract description 29
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 29
- 230000001737 promoting effect Effects 0.000 title claims abstract description 8
- 239000002773 nucleotide Substances 0.000 claims abstract description 8
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 8
- 239000013604 expression vector Substances 0.000 claims description 8
- 239000013612 plasmid Substances 0.000 claims description 6
- 239000003795 chemical substances by application Substances 0.000 claims 1
- 229920005610 lignin Polymers 0.000 abstract description 22
- 230000009261 transgenic effect Effects 0.000 abstract description 17
- 238000003786 synthesis reaction Methods 0.000 abstract description 11
- 230000002018 overexpression Effects 0.000 abstract description 6
- 101100438273 Arabidopsis thaliana CAN1 gene Proteins 0.000 abstract description 4
- 241000219194 Arabidopsis Species 0.000 description 18
- 108020004414 DNA Proteins 0.000 description 18
- 230000014509 gene expression Effects 0.000 description 8
- 238000012163 sequencing technique Methods 0.000 description 7
- 238000001514 detection method Methods 0.000 description 6
- 238000000034 method Methods 0.000 description 6
- 108090000790 Enzymes Proteins 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 5
- 239000000047 product Substances 0.000 description 5
- 238000011160 research Methods 0.000 description 5
- 238000011144 upstream manufacturing Methods 0.000 description 5
- 241000219195 Arabidopsis thaliana Species 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- 238000010367 cloning Methods 0.000 description 4
- 239000013599 cloning vector Substances 0.000 description 4
- 235000013399 edible fruits Nutrition 0.000 description 4
- 229930027917 kanamycin Natural products 0.000 description 4
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 4
- 229960000318 kanamycin Drugs 0.000 description 4
- 229930182823 kanamycin A Natural products 0.000 description 4
- 230000037361 pathway Effects 0.000 description 4
- 239000002244 precipitate Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 239000013598 vector Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 4
- 241000589158 Agrobacterium Species 0.000 description 3
- 241000589155 Agrobacterium tumefaciens Species 0.000 description 3
- 102000004190 Enzymes Human genes 0.000 description 3
- 101000617805 Homo sapiens Staphylococcal nuclease domain-containing protein 1 Proteins 0.000 description 3
- 239000012880 LB liquid culture medium Substances 0.000 description 3
- 238000012408 PCR amplification Methods 0.000 description 3
- 244000294611 Punica granatum Species 0.000 description 3
- 102100021996 Staphylococcal nuclease domain-containing protein 1 Human genes 0.000 description 3
- 108091023040 Transcription factor Proteins 0.000 description 3
- 102000040945 Transcription factor Human genes 0.000 description 3
- 108010093581 aspartyl-proline Proteins 0.000 description 3
- 239000002299 complementary DNA Substances 0.000 description 3
- 238000012258 culturing Methods 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000007788 liquid Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- 230000001105 regulatory effect Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 230000009466 transformation Effects 0.000 description 3
- OPIFSICVWOWJMJ-AEOCFKNESA-N 5-bromo-4-chloro-3-indolyl beta-D-galactoside Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1OC1=CNC2=CC=C(Br)C(Cl)=C12 OPIFSICVWOWJMJ-AEOCFKNESA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 108010067661 Caffeate O-methyltransferase Proteins 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- 241000588724 Escherichia coli Species 0.000 description 2
- BUZMZDDKFCSKOT-CIUDSAMLSA-N Glu-Glu-Glu Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BUZMZDDKFCSKOT-CIUDSAMLSA-N 0.000 description 2
- UMZHHILWZBFPGL-LOKLDPHHSA-N Glu-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CCC(=O)O)N)O UMZHHILWZBFPGL-LOKLDPHHSA-N 0.000 description 2
- KFZMGEQAYNKOFK-UHFFFAOYSA-N Isopropanol Chemical compound CC(C)O KFZMGEQAYNKOFK-UHFFFAOYSA-N 0.000 description 2
- PJYSOYLLTJKZHC-GUBZILKMSA-N Leu-Asp-Gln Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@H](C(O)=O)CCC(N)=O PJYSOYLLTJKZHC-GUBZILKMSA-N 0.000 description 2
- PPGBXYKMUMHFBF-KATARQTJSA-N Leu-Ser-Thr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PPGBXYKMUMHFBF-KATARQTJSA-N 0.000 description 2
- 102000003960 Ligases Human genes 0.000 description 2
- 108090000364 Ligases Proteins 0.000 description 2
- GMJDSFYVTAMIBF-FXQIFTODSA-N Pro-Ser-Asp Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(O)=O)C(O)=O GMJDSFYVTAMIBF-FXQIFTODSA-N 0.000 description 2
- 239000011543 agarose gel Substances 0.000 description 2
- KOSRFJWDECSPRO-UHFFFAOYSA-N alpha-L-glutamyl-L-glutamic acid Natural products OC(=O)CCC(N)C(=O)NC(CCC(O)=O)C(O)=O KOSRFJWDECSPRO-UHFFFAOYSA-N 0.000 description 2
- 150000001413 amino acids Chemical group 0.000 description 2
- 238000010804 cDNA synthesis Methods 0.000 description 2
- JMFRWRFFLBVWSI-NSCUHMNNSA-N coniferol Chemical compound COC1=CC(\C=C\CO)=CC=C1O JMFRWRFFLBVWSI-NSCUHMNNSA-N 0.000 description 2
- 239000012634 fragment Substances 0.000 description 2
- 230000002068 genetic effect Effects 0.000 description 2
- 108010055341 glutamyl-glutamic acid Proteins 0.000 description 2
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 description 2
- 230000035772 mutation Effects 0.000 description 2
- 235000016709 nutrition Nutrition 0.000 description 2
- 230000035764 nutrition Effects 0.000 description 2
- 239000012188 paraffin wax Substances 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 108010031719 prolyl-serine Proteins 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000007787 solid Substances 0.000 description 2
- 238000010186 staining Methods 0.000 description 2
- 230000008719 thickening Effects 0.000 description 2
- 108010061238 threonyl-glycine Proteins 0.000 description 2
- LZFOPEXOUVTGJS-ONEGZZNKSA-N trans-sinapyl alcohol Chemical compound COC1=CC(\C=C\CO)=CC(OC)=C1O LZFOPEXOUVTGJS-ONEGZZNKSA-N 0.000 description 2
- 230000001131 transforming effect Effects 0.000 description 2
- 102000007469 Actins Human genes 0.000 description 1
- 108010085238 Actins Proteins 0.000 description 1
- 229920000936 Agarose Polymers 0.000 description 1
- ZIWWTZWAKYBUOB-CIUDSAMLSA-N Ala-Asp-Leu Chemical compound [H]N[C@@H](C)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(O)=O ZIWWTZWAKYBUOB-CIUDSAMLSA-N 0.000 description 1
- XAGIMRPOEJSYER-CIUDSAMLSA-N Ala-Cys-Lys Chemical compound C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CCCCN)C(=O)O)N XAGIMRPOEJSYER-CIUDSAMLSA-N 0.000 description 1
- DHBKYZYFEXXUAK-ONGXEEELSA-N Ala-Phe-Gly Chemical compound OC(=O)CNC(=O)[C@@H](NC(=O)[C@@H](N)C)CC1=CC=CC=C1 DHBKYZYFEXXUAK-ONGXEEELSA-N 0.000 description 1
- 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 1
- 108700017187 Arabidopsis ATAF1 Proteins 0.000 description 1
- 101100239718 Arabidopsis thaliana NAC012 gene Proteins 0.000 description 1
- 101100079138 Arabidopsis thaliana NAC098 gene Proteins 0.000 description 1
- IIABBYGHLYWVOS-FXQIFTODSA-N Arg-Asn-Ser Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(O)=O IIABBYGHLYWVOS-FXQIFTODSA-N 0.000 description 1
- UAOSDDXCTBIPCA-QXEWZRGKSA-N Arg-Ile-Gly Chemical compound CC[C@H](C)[C@@H](C(=O)NCC(=O)O)NC(=O)[C@H](CCCN=C(N)N)N UAOSDDXCTBIPCA-QXEWZRGKSA-N 0.000 description 1
- OTZMRMHZCMZOJZ-SRVKXCTJSA-N Arg-Leu-Glu Chemical compound [H]N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(O)=O)C(O)=O OTZMRMHZCMZOJZ-SRVKXCTJSA-N 0.000 description 1
- FIQKRDXFTANIEJ-ULQDDVLXSA-N Arg-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)[C@H](CCCN=C(N)N)N FIQKRDXFTANIEJ-ULQDDVLXSA-N 0.000 description 1
- IGFJVXOATGZTHD-UHFFFAOYSA-N Arg-Phe-His Natural products NC(CCNC(=N)N)C(=O)NC(Cc1ccccc1)C(=O)NC(Cc2c[nH]cn2)C(=O)O IGFJVXOATGZTHD-UHFFFAOYSA-N 0.000 description 1
- RATVAFHGEFAWDH-JYJNAYRXSA-N Arg-Phe-Met Chemical compound CSCC[C@@H](C(=O)O)NC(=O)[C@H](CC1=CC=CC=C1)NC(=O)[C@H](CCCN=C(N)N)N RATVAFHGEFAWDH-JYJNAYRXSA-N 0.000 description 1
- HJRBIWRXULGMOA-ACZMJKKPSA-N Asn-Gln-Asp Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(O)=O HJRBIWRXULGMOA-ACZMJKKPSA-N 0.000 description 1
- XVAPVJNJGLWGCS-ACZMJKKPSA-N Asn-Glu-Asn Chemical compound C(CC(=O)O)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)N)N XVAPVJNJGLWGCS-ACZMJKKPSA-N 0.000 description 1
- BZMWJLLUAKSIMH-FXQIFTODSA-N Asn-Glu-Glu Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCC(O)=O)C(O)=O BZMWJLLUAKSIMH-FXQIFTODSA-N 0.000 description 1
- GNKVBRYFXYWXAB-WDSKDSINSA-N Asn-Glu-Gly Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O GNKVBRYFXYWXAB-WDSKDSINSA-N 0.000 description 1
- WONGRTVAMHFGBE-WDSKDSINSA-N Asn-Gly-Gln Chemical compound C(CC(=O)N)[C@@H](C(=O)O)NC(=O)CNC(=O)[C@H](CC(=O)N)N WONGRTVAMHFGBE-WDSKDSINSA-N 0.000 description 1
- PTSDPWIHOYMRGR-UGYAYLCHSA-N Asn-Ile-Asn Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(O)=O PTSDPWIHOYMRGR-UGYAYLCHSA-N 0.000 description 1
- ACKNRKFVYUVWAC-ZPFDUUQYSA-N Asn-Ile-Lys Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)N)N ACKNRKFVYUVWAC-ZPFDUUQYSA-N 0.000 description 1
- ZVUMKOMKQCANOM-AVGNSLFASA-N Asn-Phe-Gln Chemical compound [H]N[C@@H](CC(N)=O)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CCC(N)=O)C(O)=O ZVUMKOMKQCANOM-AVGNSLFASA-N 0.000 description 1
- UGIBTKGQVWFTGX-BIIVOSGPSA-N Asp-Asn-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC(=O)O)N)C(=O)O UGIBTKGQVWFTGX-BIIVOSGPSA-N 0.000 description 1
- PDECQIHABNQRHN-GUBZILKMSA-N Asp-Glu-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@@H](N)CC(O)=O PDECQIHABNQRHN-GUBZILKMSA-N 0.000 description 1
- ICZWAZVKLACMKR-CIUDSAMLSA-N Asp-His-Ser Chemical compound OC(=O)C[C@H](N)C(=O)N[C@H](C(=O)N[C@@H](CO)C(O)=O)CC1=CN=CN1 ICZWAZVKLACMKR-CIUDSAMLSA-N 0.000 description 1
- OEDJQRXNDRUGEU-SRVKXCTJSA-N Asp-Leu-His Chemical compound N[C@@H](CC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)O OEDJQRXNDRUGEU-SRVKXCTJSA-N 0.000 description 1
- NVFSJIXJZCDICF-SRVKXCTJSA-N Asp-Lys-Lys Chemical compound C(CCN)C[C@@H](C(=O)N[C@@H](CCCCN)C(=O)O)NC(=O)[C@H](CC(=O)O)N NVFSJIXJZCDICF-SRVKXCTJSA-N 0.000 description 1
- VMVUDJUXJKDGNR-FXQIFTODSA-N Asp-Met-Asn Chemical compound CSCC[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)O)NC(=O)[C@H](CC(=O)O)N VMVUDJUXJKDGNR-FXQIFTODSA-N 0.000 description 1
- KACWACLNYLSVCA-VHWLVUOQSA-N Asp-Trp-Ile Chemical compound [H]N[C@@H](CC(O)=O)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KACWACLNYLSVCA-VHWLVUOQSA-N 0.000 description 1
- XGIAHEUULGOZHH-GUBZILKMSA-N Cys-Arg-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CS)N XGIAHEUULGOZHH-GUBZILKMSA-N 0.000 description 1
- SMEYEQDCCBHTEF-FXQIFTODSA-N Cys-Pro-Ala Chemical compound [H]N[C@@H](CS)C(=O)N1CCC[C@H]1C(=O)N[C@@H](C)C(O)=O SMEYEQDCCBHTEF-FXQIFTODSA-N 0.000 description 1
- RZSYLLSAWYUBPE-UHFFFAOYSA-L Fast green FCF Chemical compound [Na+].[Na+].C=1C=C(C(=C2C=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C=2C(=CC(O)=CC=2)S([O-])(=O)=O)C=CC=1N(CC)CC1=CC=CC(S([O-])(=O)=O)=C1 RZSYLLSAWYUBPE-UHFFFAOYSA-L 0.000 description 1
- 108091092584 GDNA Proteins 0.000 description 1
- RRYLMJWPWBJFPZ-ACZMJKKPSA-N Gln-Asn-Asp Chemical compound C(CC(=O)N)[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)O)C(=O)O)N RRYLMJWPWBJFPZ-ACZMJKKPSA-N 0.000 description 1
- MAGNEQBFSBREJL-DCAQKATOSA-N Gln-Glu-Lys Chemical compound C(CCN)C[C@@H](C(=O)O)NC(=O)[C@H](CCC(=O)O)NC(=O)[C@H](CCC(=O)N)N MAGNEQBFSBREJL-DCAQKATOSA-N 0.000 description 1
- VZRAXPGTUNDIDK-GUBZILKMSA-N Gln-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)N)N VZRAXPGTUNDIDK-GUBZILKMSA-N 0.000 description 1
- ZEEPYMXTJWIMSN-GUBZILKMSA-N Gln-Lys-Ser Chemical compound NCCCC[C@@H](C(=O)N[C@@H](CO)C(O)=O)NC(=O)[C@@H](N)CCC(N)=O ZEEPYMXTJWIMSN-GUBZILKMSA-N 0.000 description 1
- XUMFMAVDHQDATI-DCAQKATOSA-N Gln-Pro-Arg Chemical compound NC(=O)CC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCN=C(N)N)C(O)=O XUMFMAVDHQDATI-DCAQKATOSA-N 0.000 description 1
- LPIKVBWNNVFHCQ-GUBZILKMSA-N Gln-Ser-Leu Chemical compound [H]N[C@@H](CCC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(C)C)C(O)=O LPIKVBWNNVFHCQ-GUBZILKMSA-N 0.000 description 1
- BYKZWDGMJLNFJY-XKBZYTNZSA-N Gln-Ser-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@H](CO)NC(=O)[C@H](CCC(=O)N)N)O BYKZWDGMJLNFJY-XKBZYTNZSA-N 0.000 description 1
- RDPOETHPAQEGDP-ACZMJKKPSA-N Glu-Asp-Ala Chemical compound [H]N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C)C(O)=O RDPOETHPAQEGDP-ACZMJKKPSA-N 0.000 description 1
- SJPMNHCEWPTRBR-BQBZGAKWSA-N Glu-Glu-Gly Chemical compound OC(=O)CC[C@H](N)C(=O)N[C@@H](CCC(O)=O)C(=O)NCC(O)=O SJPMNHCEWPTRBR-BQBZGAKWSA-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
- YPHPEHMXOYTEQG-LAEOZQHASA-N Glu-Val-Asp Chemical compound OC(=O)C[C@@H](C(O)=O)NC(=O)[C@H](C(C)C)NC(=O)[C@@H](N)CCC(O)=O YPHPEHMXOYTEQG-LAEOZQHASA-N 0.000 description 1
- CLODWIOAKCSBAN-BQBZGAKWSA-N Gly-Arg-Asp Chemical compound NC(N)=NCCC[C@H](NC(=O)CN)C(=O)N[C@@H](CC(O)=O)C(O)=O CLODWIOAKCSBAN-BQBZGAKWSA-N 0.000 description 1
- CQZDZKRHFWJXDF-WDSKDSINSA-N Gly-Gln-Ala Chemical compound OC(=O)[C@H](C)NC(=O)[C@H](CCC(N)=O)NC(=O)CN CQZDZKRHFWJXDF-WDSKDSINSA-N 0.000 description 1
- NZOAFWHVAFJERA-OALUTQOASA-N Gly-Phe-Trp Chemical compound [H]NCC(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H](CC1=CNC2=C1C=CC=C2)C(O)=O NZOAFWHVAFJERA-OALUTQOASA-N 0.000 description 1
- YXTFLTJYLIAZQG-FJXKBIBVSA-N Gly-Thr-Arg Chemical compound NCC(=O)N[C@@H]([C@H](O)C)C(=O)N[C@H](C(O)=O)CCCN=C(N)N YXTFLTJYLIAZQG-FJXKBIBVSA-N 0.000 description 1
- 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 1
- MDBYBTWRMOAJAY-NHCYSSNCSA-N His-Asn-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)N)NC(=O)[C@H](CC1=CN=CN1)N MDBYBTWRMOAJAY-NHCYSSNCSA-N 0.000 description 1
- NELVFWFDOKRTOR-SDDRHHMPSA-N His-Gln-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCC(=O)N)NC(=O)[C@H](CC2=CN=CN2)N)C(=O)O NELVFWFDOKRTOR-SDDRHHMPSA-N 0.000 description 1
- IDQNVIWPPWAFSY-AVGNSLFASA-N His-His-Gln Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(N)=O)C(O)=O IDQNVIWPPWAFSY-AVGNSLFASA-N 0.000 description 1
- SKOKHBGDXGTDDP-MELADBBJSA-N His-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CC2=CN=CN2)N SKOKHBGDXGTDDP-MELADBBJSA-N 0.000 description 1
- LVXFNTIIGOQBMD-SRVKXCTJSA-N His-Leu-Ser Chemical compound [H]N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(O)=O LVXFNTIIGOQBMD-SRVKXCTJSA-N 0.000 description 1
- PNDMHTTXXPUQJH-RWRJDSDZSA-N Ile-Glu-Thr Chemical compound N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CCC(=O)O)C(=O)N[C@@H]([C@H](O)C)C(=O)O PNDMHTTXXPUQJH-RWRJDSDZSA-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
- HYLIOBDWPQNLKI-HVTMNAMFSA-N Ile-His-Gln Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CN=CN1)C(=O)N[C@@H](CCC(=O)N)C(=O)O)N HYLIOBDWPQNLKI-HVTMNAMFSA-N 0.000 description 1
- YSGBJIQXTIVBHZ-AJNGGQMLSA-N Ile-Lys-Leu Chemical compound CC[C@H](C)[C@H](N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(O)=O YSGBJIQXTIVBHZ-AJNGGQMLSA-N 0.000 description 1
- FHPZJWJWTWZKNA-LLLHUVSDSA-N Ile-Phe-Pro Chemical compound CC[C@H](C)[C@@H](C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N2CCC[C@@H]2C(=O)O)N FHPZJWJWTWZKNA-LLLHUVSDSA-N 0.000 description 1
- SVZFKLBRCYCIIY-CYDGBPFRSA-N Ile-Pro-Arg Chemical compound CC[C@H](C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O SVZFKLBRCYCIIY-CYDGBPFRSA-N 0.000 description 1
- COLNVLDHVKWLRT-QMMMGPOBSA-N L-phenylalanine Chemical compound OC(=O)[C@@H](N)CC1=CC=CC=C1 COLNVLDHVKWLRT-QMMMGPOBSA-N 0.000 description 1
- 241000880493 Leptailurus serval Species 0.000 description 1
- POJPZSMTTMLSTG-SRVKXCTJSA-N Leu-Asn-Lys Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CCCCN)C(=O)O)N POJPZSMTTMLSTG-SRVKXCTJSA-N 0.000 description 1
- WGNOPSQMIQERPK-GARJFASQSA-N Leu-Asn-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N1CCC[C@@H]1C(=O)O)N WGNOPSQMIQERPK-GARJFASQSA-N 0.000 description 1
- WGNOPSQMIQERPK-UHFFFAOYSA-N Leu-Asn-Pro Natural products CC(C)CC(N)C(=O)NC(CC(=O)N)C(=O)N1CCCC1C(=O)O WGNOPSQMIQERPK-UHFFFAOYSA-N 0.000 description 1
- LOLUPZNNADDTAA-AVGNSLFASA-N Leu-Gln-Leu Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(C)C)C(O)=O LOLUPZNNADDTAA-AVGNSLFASA-N 0.000 description 1
- CQGSYZCULZMEDE-SRVKXCTJSA-N Leu-Gln-Pro Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CCC(N)=O)C(=O)N1CCC[C@H]1C(O)=O CQGSYZCULZMEDE-SRVKXCTJSA-N 0.000 description 1
- CQGSYZCULZMEDE-UHFFFAOYSA-N Leu-Gln-Pro Natural products CC(C)CC(N)C(=O)NC(CCC(N)=O)C(=O)N1CCCC1C(O)=O CQGSYZCULZMEDE-UHFFFAOYSA-N 0.000 description 1
- OGUUKPXUTHOIAV-SDDRHHMPSA-N Leu-Glu-Pro Chemical compound CC(C)C[C@@H](C(=O)N[C@@H](CCC(=O)O)C(=O)N1CCC[C@@H]1C(=O)O)N OGUUKPXUTHOIAV-SDDRHHMPSA-N 0.000 description 1
- HMDDEJADNKQTBR-BZSNNMDCSA-N Leu-His-Tyr Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CC1=CC=C(O)C=C1)C(O)=O HMDDEJADNKQTBR-BZSNNMDCSA-N 0.000 description 1
- UCNNZELZXFXXJQ-BZSNNMDCSA-N Leu-Leu-Tyr Chemical compound CC(C)C[C@H](N)C(=O)N[C@@H](CC(C)C)C(=O)N[C@H](C(O)=O)CC1=CC=C(O)C=C1 UCNNZELZXFXXJQ-BZSNNMDCSA-N 0.000 description 1
- MVHXGBZUJLWZOH-BJDJZHNGSA-N Leu-Ser-Ile Chemical compound [H]N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O MVHXGBZUJLWZOH-BJDJZHNGSA-N 0.000 description 1
- KNKHAVVBVXKOGX-JXUBOQSCSA-N Lys-Ala-Thr Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(O)=O KNKHAVVBVXKOGX-JXUBOQSCSA-N 0.000 description 1
- JYXBNQOKPRQNQS-YTFOTSKYSA-N Lys-Ile-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O JYXBNQOKPRQNQS-YTFOTSKYSA-N 0.000 description 1
- DIBZLYZXTSVGLN-CIUDSAMLSA-N Lys-Ser-Ser Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(O)=O DIBZLYZXTSVGLN-CIUDSAMLSA-N 0.000 description 1
- RPWTZTBIFGENIA-VOAKCMCISA-N Lys-Thr-Leu Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(O)=O RPWTZTBIFGENIA-VOAKCMCISA-N 0.000 description 1
- YCUSPBPZVJDMII-YUMQZZPRSA-N Met-Gly-Glu Chemical compound CSCC[C@H](N)C(=O)NCC(=O)N[C@H](C(O)=O)CCC(O)=O YCUSPBPZVJDMII-YUMQZZPRSA-N 0.000 description 1
- RKIIYGUHIQJCBW-SRVKXCTJSA-N Met-His-Glu Chemical compound [H]N[C@@H](CCSC)C(=O)N[C@@H](CC1=CNC=N1)C(=O)N[C@@H](CCC(O)=O)C(O)=O RKIIYGUHIQJCBW-SRVKXCTJSA-N 0.000 description 1
- PHKBGZKVOJCIMZ-SRVKXCTJSA-N Met-Pro-Arg Chemical compound CSCC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCCNC(N)=N)C(O)=O PHKBGZKVOJCIMZ-SRVKXCTJSA-N 0.000 description 1
- BJPQKNHZHUCQNQ-SRVKXCTJSA-N Met-Pro-Val Chemical compound CC(C)[C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CCSC)N BJPQKNHZHUCQNQ-SRVKXCTJSA-N 0.000 description 1
- AJHCSUXXECOXOY-UHFFFAOYSA-N N-glycyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)CN)C(O)=O)=CNC2=C1 AJHCSUXXECOXOY-UHFFFAOYSA-N 0.000 description 1
- BQVUABVGYYSDCJ-UHFFFAOYSA-N Nalpha-L-Leucyl-L-tryptophan Natural products C1=CC=C2C(CC(NC(=O)C(N)CC(C)C)C(O)=O)=CNC2=C1 BQVUABVGYYSDCJ-UHFFFAOYSA-N 0.000 description 1
- 102000003992 Peroxidases Human genes 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 108010002747 Pfu DNA polymerase Proteins 0.000 description 1
- LGBVMDMZZFYSFW-HJWJTTGWSA-N Phe-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](CC1=CC=CC=C1)N LGBVMDMZZFYSFW-HJWJTTGWSA-N 0.000 description 1
- YYRCPTVAPLQRNC-ULQDDVLXSA-N Phe-Arg-Lys Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](N)CC1=CC=CC=C1 YYRCPTVAPLQRNC-ULQDDVLXSA-N 0.000 description 1
- XMPUYNHKEPFERE-IHRRRGAJSA-N Phe-Asp-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@@H](N)CC1=CC=CC=C1 XMPUYNHKEPFERE-IHRRRGAJSA-N 0.000 description 1
- ILGCZYGFYQLSDZ-KKUMJFAQSA-N Phe-Ser-His Chemical compound N[C@@H](Cc1ccccc1)C(=O)N[C@@H](CO)C(=O)N[C@@H](Cc1cnc[nH]1)C(O)=O ILGCZYGFYQLSDZ-KKUMJFAQSA-N 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N Phenol Chemical compound OC1=CC=CC=C1 ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 108700023158 Phenylalanine ammonia-lyases Proteins 0.000 description 1
- 108700001094 Plant Genes Proteins 0.000 description 1
- 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 1
- VWXGFAIZUQBBBG-UWVGGRQHSA-N Pro-His-Gly Chemical compound C([C@@H](C(=O)NCC(=O)[O-])NC(=O)[C@H]1[NH2+]CCC1)C1=CN=CN1 VWXGFAIZUQBBBG-UWVGGRQHSA-N 0.000 description 1
- YXHYJEPDKSYPSQ-AVGNSLFASA-N Pro-Leu-Arg Chemical compound NC(N)=NCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@@H]1CCCN1 YXHYJEPDKSYPSQ-AVGNSLFASA-N 0.000 description 1
- MCWHYUWXVNRXFV-RWMBFGLXSA-N Pro-Leu-Pro Chemical compound CC(C)C[C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@@H]2CCCN2 MCWHYUWXVNRXFV-RWMBFGLXSA-N 0.000 description 1
- MHHQQZIFLWFZGR-DCAQKATOSA-N Pro-Lys-Ser Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CO)C(O)=O MHHQQZIFLWFZGR-DCAQKATOSA-N 0.000 description 1
- POQFNPILEQEODH-FXQIFTODSA-N Pro-Ser-Ala Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H](CO)C(=O)N[C@@H](C)C(O)=O POQFNPILEQEODH-FXQIFTODSA-N 0.000 description 1
- IURWWZYKYPEANQ-HJGDQZAQSA-N Pro-Thr-Glu Chemical compound [H]N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCC(O)=O)C(O)=O IURWWZYKYPEANQ-HJGDQZAQSA-N 0.000 description 1
- 241001083505 Punica Species 0.000 description 1
- 238000002123 RNA extraction Methods 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- NRCJWSGXMAPYQX-LPEHRKFASA-N Ser-Arg-Pro Chemical compound C1C[C@@H](N(C1)C(=O)[C@H](CCCN=C(N)N)NC(=O)[C@H](CO)N)C(=O)O NRCJWSGXMAPYQX-LPEHRKFASA-N 0.000 description 1
- MMAPOBOTRUVNKJ-ZLUOBGJFSA-N Ser-Asp-Ser Chemical compound C([C@@H](C(=O)N[C@@H](CO)C(=O)O)NC(=O)[C@H](CO)N)C(=O)O MMAPOBOTRUVNKJ-ZLUOBGJFSA-N 0.000 description 1
- HVKMTOIAYDOJPL-NRPADANISA-N Ser-Gln-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](C(C)C)C(O)=O HVKMTOIAYDOJPL-NRPADANISA-N 0.000 description 1
- BKZYBLLIBOBOOW-GHCJXIJMSA-N Ser-Ile-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(O)=O)C(O)=O BKZYBLLIBOBOOW-GHCJXIJMSA-N 0.000 description 1
- XUDRHBPSPAPDJP-SRVKXCTJSA-N Ser-Lys-Leu Chemical compound CC(C)C[C@@H](C(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](N)CO XUDRHBPSPAPDJP-SRVKXCTJSA-N 0.000 description 1
- KJKQUQXDEKMPDK-FXQIFTODSA-N Ser-Met-Asp Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(O)=O)C(O)=O KJKQUQXDEKMPDK-FXQIFTODSA-N 0.000 description 1
- WNDUPCKKKGSKIQ-CIUDSAMLSA-N Ser-Pro-Gln Chemical compound OC[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CCC(N)=O)C(O)=O WNDUPCKKKGSKIQ-CIUDSAMLSA-N 0.000 description 1
- PYTKULIABVRXSC-BWBBJGPYSA-N Ser-Ser-Thr Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(O)=O PYTKULIABVRXSC-BWBBJGPYSA-N 0.000 description 1
- VGQVAVQWKJLIRM-FXQIFTODSA-N Ser-Ser-Val Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)N[C@@H](C(C)C)C(O)=O VGQVAVQWKJLIRM-FXQIFTODSA-N 0.000 description 1
- ZSDXEKUKQAKZFE-XAVMHZPKSA-N Ser-Thr-Pro Chemical compound C[C@H]([C@@H](C(=O)N1CCC[C@@H]1C(=O)O)NC(=O)[C@H](CO)N)O ZSDXEKUKQAKZFE-XAVMHZPKSA-N 0.000 description 1
- 108700005078 Synthetic Genes Proteins 0.000 description 1
- 108010006785 Taq Polymerase Proteins 0.000 description 1
- IRKWVRSEQFTGGV-VEVYYDQMSA-N Thr-Asn-Arg Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCCNC(N)=N)C(O)=O IRKWVRSEQFTGGV-VEVYYDQMSA-N 0.000 description 1
- VIBXMCZWVUOZLA-OLHMAJIHSA-N Thr-Asn-Asn Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)N)C(=O)N[C@@H](CC(=O)N)C(=O)O)N)O VIBXMCZWVUOZLA-OLHMAJIHSA-N 0.000 description 1
- NOWXWJLVGTVJKM-PBCZWWQYSA-N Thr-Asp-His Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(=O)O)C(=O)N[C@@H](CC1=CN=CN1)C(=O)O)N)O NOWXWJLVGTVJKM-PBCZWWQYSA-N 0.000 description 1
- RRRRCRYTLZVCEN-HJGDQZAQSA-N Thr-Leu-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O RRRRCRYTLZVCEN-HJGDQZAQSA-N 0.000 description 1
- NCXVJIQMWSGRHY-KXNHARMFSA-N Thr-Leu-Pro Chemical compound C[C@H]([C@@H](C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@@H]1C(=O)O)N)O NCXVJIQMWSGRHY-KXNHARMFSA-N 0.000 description 1
- MROIJTGJGIDEEJ-RCWTZXSCSA-N Thr-Pro-Pro Chemical compound C[C@@H](O)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N1[C@H](C(O)=O)CCC1 MROIJTGJGIDEEJ-RCWTZXSCSA-N 0.000 description 1
- FYBFTPLPAXZBOY-KKHAAJSZSA-N Thr-Val-Asp Chemical compound [H]N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(O)=O)C(O)=O FYBFTPLPAXZBOY-KKHAAJSZSA-N 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- BRBCKMMXKONBAA-KWBADKCTSA-N Trp-Ala-Ala Chemical compound C1=CC=C2C(C[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](C)C(O)=O)=CNC2=C1 BRBCKMMXKONBAA-KWBADKCTSA-N 0.000 description 1
- DVAAUUVLDFKTAQ-VHWLVUOQSA-N Trp-Asp-Ile Chemical compound CC[C@H](C)[C@@H](C(=O)O)NC(=O)[C@H](CC(=O)O)NC(=O)[C@H](CC1=CNC2=CC=CC=C21)N DVAAUUVLDFKTAQ-VHWLVUOQSA-N 0.000 description 1
- GBEAUNVBIMLWIB-IHPCNDPISA-N Trp-Ser-Phe Chemical compound C([C@H](NC(=O)[C@H](CO)NC(=O)[C@H](CC=1C2=CC=CC=C2NC=1)N)C(O)=O)C1=CC=CC=C1 GBEAUNVBIMLWIB-IHPCNDPISA-N 0.000 description 1
- UIRVSEPRMWDVEW-RNXOBYDBSA-N Trp-Tyr-Phe Chemical compound C1=CC=C(C=C1)C[C@@H](C(=O)O)NC(=O)[C@H](CC2=CC=C(C=C2)O)NC(=O)[C@H](CC3=CNC4=CC=CC=C43)N UIRVSEPRMWDVEW-RNXOBYDBSA-N 0.000 description 1
- MXKUGFHWYYKVDV-SZMVWBNQSA-N Trp-Val-Val Chemical compound CC(C)[C@H](NC(=O)[C@@H](NC(=O)[C@@H](N)Cc1c[nH]c2ccccc12)C(C)C)C(O)=O MXKUGFHWYYKVDV-SZMVWBNQSA-N 0.000 description 1
- ADBDQGBDNUTRDB-ULQDDVLXSA-N Tyr-Arg-Leu Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC(C)C)C(O)=O ADBDQGBDNUTRDB-ULQDDVLXSA-N 0.000 description 1
- GFHYISDTIWZUSU-QWRGUYRKSA-N Tyr-Asn-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CC(N)=O)C(=O)NCC(O)=O GFHYISDTIWZUSU-QWRGUYRKSA-N 0.000 description 1
- XKDOQXAXKFQWQJ-SRVKXCTJSA-N Tyr-Cys-Asp Chemical compound C1=CC(=CC=C1C[C@@H](C(=O)N[C@@H](CS)C(=O)N[C@@H](CC(=O)O)C(=O)O)N)O XKDOQXAXKFQWQJ-SRVKXCTJSA-N 0.000 description 1
- XGZBEGGGAUQBMB-KJEVXHAQSA-N Tyr-Pro-Thr Chemical compound C[C@H]([C@@H](C(=O)O)NC(=O)[C@@H]1CCCN1C(=O)[C@H](CC2=CC=C(C=C2)O)N)O XGZBEGGGAUQBMB-KJEVXHAQSA-N 0.000 description 1
- ZPFLBLFITJCBTP-QWRGUYRKSA-N Tyr-Ser-Gly Chemical compound [H]N[C@@H](CC1=CC=C(O)C=C1)C(=O)N[C@@H](CO)C(=O)NCC(O)=O ZPFLBLFITJCBTP-QWRGUYRKSA-N 0.000 description 1
- AZSHAZJLOZQYAY-FXQIFTODSA-N Val-Ala-Ser Chemical compound CC(C)[C@H](N)C(=O)N[C@@H](C)C(=O)N[C@@H](CO)C(O)=O AZSHAZJLOZQYAY-FXQIFTODSA-N 0.000 description 1
- DJEVQCWNMQOABE-RCOVLWMOSA-N Val-Gly-Asp Chemical compound CC(C)[C@@H](C(=O)NCC(=O)N[C@@H](CC(=O)O)C(=O)O)N DJEVQCWNMQOABE-RCOVLWMOSA-N 0.000 description 1
- SSYBNWFXCFNRFN-GUBZILKMSA-N Val-Pro-Ser Chemical compound CC(C)[C@H](N)C(=O)N1CCC[C@H]1C(=O)N[C@@H](CO)C(O)=O SSYBNWFXCFNRFN-GUBZILKMSA-N 0.000 description 1
- PZTZYZUTCPZWJH-FXQIFTODSA-N Val-Ser-Ser Chemical compound CC(C)[C@@H](C(=O)N[C@@H](CO)C(=O)N[C@@H](CO)C(=O)O)N PZTZYZUTCPZWJH-FXQIFTODSA-N 0.000 description 1
- 230000004913 activation Effects 0.000 description 1
- 108010005233 alanylglutamic acid Proteins 0.000 description 1
- 108010011559 alanylphenylalanine Proteins 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 108010092854 aspartyllysine Proteins 0.000 description 1
- 108010068265 aspartyltyrosine Proteins 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- LZFOPEXOUVTGJS-UHFFFAOYSA-N cis-sinapyl alcohol Natural products COC1=CC(C=CCO)=CC(OC)=C1O LZFOPEXOUVTGJS-UHFFFAOYSA-N 0.000 description 1
- 230000000295 complement effect Effects 0.000 description 1
- 229940119526 coniferyl alcohol Drugs 0.000 description 1
- 238000007405 data analysis Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 230000000249 desinfective effect Effects 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000001976 enzyme digestion Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011536 extraction buffer Substances 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 108010080575 glutamyl-aspartyl-alanine Proteins 0.000 description 1
- 108010010147 glycylglutamine Proteins 0.000 description 1
- 108010050848 glycylleucine Proteins 0.000 description 1
- 108010081551 glycylphenylalanine Proteins 0.000 description 1
- 108010084389 glycyltryptophan Proteins 0.000 description 1
- 238000003306 harvesting Methods 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 108010085325 histidylproline Proteins 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000006872 improvement Effects 0.000 description 1
- 230000036512 infertility Effects 0.000 description 1
- 108010044374 isoleucyl-tyrosine Proteins 0.000 description 1
- 108010003700 lysyl aspartic acid Proteins 0.000 description 1
- 108010054155 lysyllysine Proteins 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 239000002609 medium Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 239000004570 mortar (masonry) Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 230000001590 oxidative effect Effects 0.000 description 1
- 108040007629 peroxidase activity proteins Proteins 0.000 description 1
- COLNVLDHVKWLRT-UHFFFAOYSA-N phenylalanine Natural products OC(=O)C(N)CC1=CC=CC=C1 COLNVLDHVKWLRT-UHFFFAOYSA-N 0.000 description 1
- 108010072637 phenylalanyl-arginyl-phenylalanine Proteins 0.000 description 1
- 108010073025 phenylalanylphenylalanine Proteins 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 108010079317 prolyl-tyrosine Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000022532 regulation of transcription, DNA-dependent Effects 0.000 description 1
- 238000003757 reverse transcription PCR Methods 0.000 description 1
- 238000005096 rolling process Methods 0.000 description 1
- OARRHUQTFTUEOS-UHFFFAOYSA-N safranin Chemical compound [Cl-].C=12C=C(N)C(C)=CC2=NC2=CC(C)=C(N)C=C2[N+]=1C1=CC=CC=C1 OARRHUQTFTUEOS-UHFFFAOYSA-N 0.000 description 1
- 108010048397 seryl-lysyl-leucine Proteins 0.000 description 1
- JXOHGGNKMLTUBP-HSUXUTPPSA-N shikimic acid Chemical compound O[C@@H]1CC(C(O)=O)=C[C@@H](O)[C@H]1O JXOHGGNKMLTUBP-HSUXUTPPSA-N 0.000 description 1
- JXOHGGNKMLTUBP-JKUQZMGJSA-N shikimic acid Natural products O[C@@H]1CC(C(O)=O)=C[C@H](O)[C@@H]1O JXOHGGNKMLTUBP-JKUQZMGJSA-N 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000002002 slurry Substances 0.000 description 1
- 239000002689 soil Substances 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 108010003137 tyrosyltyrosine Proteins 0.000 description 1
- 108010027345 wheylin-1 peptide Proteins 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Genetics & Genomics (AREA)
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Molecular Biology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Biochemistry (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Physics & Mathematics (AREA)
- Microbiology (AREA)
- Plant Pathology (AREA)
- Cell Biology (AREA)
- Botany (AREA)
- Gastroenterology & Hepatology (AREA)
- Medicinal Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
本发明涉及PgSND1‑like基因在促进石榴籽粒硬度形成的用途，所述核苷酸序列由SEQ ID NO：1所示的核苷酸序列组成。本发明的PgSND1‑ like过量表达转基因拟南芥能明显增加次生壁木质素的合成，石榴籽粒硬度与石榴种皮木质素含量呈正相关关系，说明PgSND1‑like可能参与石榴籽粒硬度的形成。
Description
技术领域
本发明涉及一种PgSND1-like基因在促进石榴籽粒硬度形成的用途，属于植物基因用途领域。
背景技术
石榴（Punica granatum L.），属于石榴科石榴属落叶果树，在阿富汗、伊朗、中国等地广泛栽培。我国石榴品种资源丰富，具有丰富的遗传多样性。石榴果实中营养保健价值高，被誉为超级水果。软籽石榴核软可食，深受大家的重视和市场的欢迎。研究表明石榴籽粒硬度性状可能受到多个基因的调控，同时也受到光照等环境因素的影响。张永明和曹丹琴等人指出石榴籽粒硬度和种皮木质素含量呈正相关关系。
木质素是由松柏醇、香豆醇及芥子醇三种单体通过氧化聚合而形成的一种复杂多酚类物质。研究表明：木质素的合成途径包括莽草酸途径、苯丙氨酸途径和木质素特异合成途径。人们利用分子生物学手段对木质素生物合成关键酶基因进行了研究，主要有苯丙氨酸解氨酶（PAL）、香豆酸-3-羟基化酶（C3H）、咖啡酸-3-O-甲基转移酶（COMT）、咖啡酰COA3-O-甲基转移酶（CC O AOMT）、4-香豆酸辅酶A连接酶（4CL）、阿魏酸-5-羟化酶（F5H）、过氧化物酶等。木质素的生物合成不仅受到这些酶基因的调控，同时还受到转录因子的调控。研究表明，MYB、 NAC、WRKY等转录因子在木质素生物合成中起重要的调控作用。1997年，Aida等人发现在矮牵牛NAM 基因、拟南芥ATAF1/2 和 CUC2 基因编码蛋白的N 端都包含一段保守的氨基酸序列，取三基因首字母命名为NAC，所以后来就把含有NAC结构域的蛋白统称为NAC转录因子。Zhong和Ye 等2007年指出SND1/NST3是控制次生壁合成代谢的一种主要转录调控开关，调控下游MYB转录因子，继而调控次生壁合成基因的表达。已有研究发现：过表达SND1 可激活次生壁生物合成基因的表达，导致次生壁的增厚。
但是未见涉及石榴籽粒硬度的基因有关方面的研究，因此分析研究石榴软籽性状的形成机理，能够加快石榴育种进程。
发明内容
本发明提供了一种PgSND1-like基因及其用途，通过过量表达PgSND1-like基因，拟南芥能明显增加次生壁木质素的合成，进而提高了石榴籽粒硬度。
具体的本发明的内容包括以下几个方面：
一种促进石榴籽粒硬度形成的核苷酸序列，其特征在；所述核苷酸序列由SEQ IDNO ： 1 所示的核苷酸序列组成，其具体基因序列如下：
ATGCCACGAGACATGAATCTGTCCATAAATGGTCAGTCCCAGGTTCCCCCAGGCTTCCGCTTCCATCCGACCGAAGAAGAGCTCCTCCACTATTACCTGAGGAAGAAAGTCGCCCAGGAGAAGATCGACCTCGATGTGATCCGCGAAGTTGATCTCAACAAGCTCGAGCCGTGGGATATTCAAGAGAAGTGCAGAATAGGGTCGACGCCACAAAATGATTGGTACTTCTTCAGTCACAAGGACAAGAAGTACCCGACAGGGACTCGGACAAACCGAGCGACTGCAGCCGGATTCTGGAAGGCGACAGGGCGCGACAAGATAATCTACAGCGGGTTCAGAAGGATTGGGTTGCGGAAGACCCTGGTGTTTTACAAGGGGCGTGCTCCACATGGACAGAAGTCGGACTGGATCATGCACGAATACCGGCTAGACGAGACTCCAAGCATTGATCCGAGTGCTCTGAATCCTACAGTCGATCAGTTGAGTACAGAAGAAGGATGGGTAGTATGCCGAGTTTTTCGGAAGAAGAACTTCCAGAAAACCCTAGACCACAGTCCCAAGAGCTCATCCTCCACCTCCATGGACTCAAAACTCATCCACAACCTCAGCACCACAAACAACCTTAATATTAACAACATCAAAGACAATCCTAGCAGTGTCCTGGACCAGATTCTCCTCTACATGGGGGAAAGAAACAGTGCGTGCAAGATCGAGACTAGTCAACCCCGAATGCCTGTCACACTCGACCCTTCCGACTACTGCGACACAGTCTCCAGCATCAAGCTTATTCACCAGCCTCTGCCCGACCTCCACGAACGGTTCATGCACCTCCCGAGGCTCGAGTGCCCAGCAACCCTCCCTTCCATCTTCCCCACACCACCACTTGATCAACACAACGTCCATCAGCCGTTCGATGAGTTGACTGATCATAATCAAGATCAGTCAACGCAGTCACTCACAGTTGGAGACTGGGCTGCGTTCGACCGGCTTGTTGCTTCCCAGCTTAACGGCCAAGCAGAGACACCAAGCGACTCGGCCTTTGGACTCCAGCTAGCCAATGAAGAAGAGGAGGAAGAAGAAGAAGAAGATGCCAATGAAGGGCAGGTCCCATCGATTCCGAGGTTCAGAATAAGCAGGCCTCATCATCAGAATCTGCAACCGTACAACGGGAACGAAAACGCCGATTTGTGGAGCTTCACCAAGTCTTCATCGCCGCAGCCATCTGACCCCTTACGCCACTTATCGGTATAA
其蛋白质序列如下：
MPRDMNLSINGQSQVPPGFRFHPTEEELLHYYLRKKVAQEKIDLDVIREVDLNKLEPWDIQEKCRIGSTPQNDWYFFSHKDKKYPTGTRTNRATAAGFWKATGRDKIIYSGFRRIGLRKTLVFYKGRAPHGQKSDWIMHEYRLDETPSIDPSALNPTVDQLSTEEGWVVCRVFRKKNFQKTLDHSPKSSSSTSMDSKLIHNLSTTNNLNINNIKDNPSSVLDQILLYMGERNSACKIETSQPRMPVTLDPSDYCDTVSSIKLIHQPLPDLHERFMHLPRLECPATLPSIFPTPPLDQHNVHQPFDELTDHNQDQSTQSLTVGDWAAFDRLVASQLNGQAETPSDSAFGLQLANEEEEEEEEEDANEGQVPSIPRFRISRPHHQNLQPYNGNENADLWSFTKSSSPQPSDPLRHLSV
本发明涉及一种表达载体，所述的表达载体含有PgSND1-like的核苷酸序列， 所述载体可以通过例如将上述核苷酸序列插入克隆载体或表达载体而得到，或者可以通过人工合成得到。
进一步，优选地：所述的表达载体，所述的表达载体为PBI121质粒。
本发明也提供了一种重组细胞，所述重组细胞可以通过将含有本发明所述的载体转化至宿主细胞而得到。适于构建本发明所述重组细胞的宿主细胞包括但不限于，例如：根癌农杆菌细胞LBA4404、EHA105、GV3101 等。
本发明的核苷酸序列和/或载体和/或重组细胞在促进石榴籽粒硬度形成的用途。
本发明的有益效果：
PgSND1-like过量表达转基因拟南芥能明显增加次生壁木质素的合成，石榴籽粒硬度与种皮木质素含量呈正相关关系，说明它可能参与石榴籽粒硬度的形成。
附图说明
为了更清楚地说明本发明实施例或现有技术中的技术方案，下面将对实施例或现有技术描述中所需要使用的附图作简单地介绍，显而易见地，下面描述中的附图仅仅是本发明的一些实施例，对于本领域普通技术人员来讲，在不付出创造性劳动的前提下，还可以根据这些附图获得其他的附图。
图1为系统进化树；
图2为不同株系的表达水平结果；
图3为种植实验结果；
图4为石蜡切片染色结果；
图5为转基因株系和野生型株系木质素含量变化结果；
图6为四个次生壁木质素合成基因在转基因主茎中表达量的变化结果。
具体实施方式
下面将结合本发明实施例中的附图，对本发明实施例中的技术方案进行清楚、完整地描述，显然，所描述的实施例仅仅是本发明一部分实施例，而不是全部的实施例。基于本发明中的实施例，本领域普通技术人员在没有做出创造性劳动前提下所获得的所有其他实施例，都属于本发明保护的范围。
一、总RNA提取及第一链cDNA合成
用RNA提取试剂盒提取三白硬籽石榴60天籽粒总RNA，具体操作步骤参照其说明书。测量其浓度，并跑琼脂糖电泳检测RNA的质量。 接下来进行cDNA第一链的合成。具体步骤如下：Total RNA 500ng, Oligo(dT)18 1uL, 2xReaction Mix 10uL, TransScript RTEnzyme Mix 1uL, 补RNase-free water 至20uL。42℃孵育30min, 85℃加热5s。-20℃保存。提取的总RNA作如下分析：经分析所述PgSND1-like基因的CDS全长1251bp, 编码416个氨基酸，具有保守的A、B、C、D、E五个亚基组成的NAC结构域，如图1所示，系统进化分析表明其与拟南芥SND1亲缘关系较近。
二、基因CDS全长序列克隆及测序
根据参考基因组中PgSND1- like(突尼斯)已知序列，NCBI Primer Blast设计克隆引物，以三白硬籽石榴花后60天籽粒cDNA为模板，Pfu高保真酶进行扩增该基因CDS全长。
上游引物：5’-GGGTCTAGAATGCCACGAGACATGAAT-3’，
下游引物：5’-CTTGGATCCTTATACCGATAAGTGGCG-3’。
PCR扩增采用高保真Pfu DNA聚合酶进行扩增。反应体系：5×Pfu Fly Buffer,10uL；2.5mM dNTP，4μL；上游引物1 μL；下游引物1 μL； Pfu高保真酶1 μL；模板cDNA 1 μL；补ddH2O至50μL。
PCR反应程序为：95℃ 2min；95℃ 20s，58℃ 20s，72℃ 90s，30个循环；72℃5min；4℃ 保存。
利用琼脂糖凝胶回收试剂盒回收目的片段，将回收后的PCR产物连接到PEASYBlunt Simple Cloning 载体上，转化到大肠杆菌DH5α感受态中。
克隆反应体系
（1）加入
| component | Volume |
| PCR Product | 0.5~4uL |
| PEASY –T1 Cloning Vector | 1uL |
（2）轻轻混合，室温反应5min。反应结束后，将离心管置于冰上。
转化
（1）加连接产物于50uL DH5α感受态中（在感受态细胞刚刚解冻时加入连接产物），轻弹混匀，冰浴20~30分钟。
（2）42℃水浴热激30s，立即置于冰上2分钟。
（3）加250uL LB培养基，200rpm、37度培养1h。
（4）取8 uL 500mM IPTG和40 uL 20mg/mL X-gal 混合，均匀地涂在准备好的平板上，37℃培养箱中放置30分钟。
（5）待IPTG和X-gal 被吸收后，取200uL 菌液均匀地涂在平板上，在37℃培养箱中过夜培养。
挑取白色单菌落进行PCR检测 。
测序：将检测为阳性的单克隆摇成菌液，送去公司测序。用M13 Forward Primer和M13 Reverse Primer 进行测序。测序结果：序列全长1251bp, 与公司大数据分析碱基序列比对没有发生碱基突变。
三、过量表达载体的构建，具体步骤如下：
1、提取携带有PgSND1-like基因的PEASY –T1 Cloning Vector质粒，以此质粒为模板，利用上述克隆引物进行PCR扩增，琼脂糖凝胶回收得到PgSND1-like基因片段；
2、利用限制性内切酶BamHI/XbaI对PBI121载体进行酶切；
3、T4快速连接酶连接PgSND1-like基因片段与PBI121载体10min；
4 、将连接产物转化到大肠杆菌感受态DH5α中；
5 、将其涂布于含卡那霉素的LB培养基中，37度培养箱，倒置培养过夜。
6、挑取单克隆，进行PCR检测。
7、接种阳性克隆，提取质粒，进行跑胶、测序检验。
测序结果：没有发生碱基突变。
四、表达载体导入农杆菌
1、农杆菌转化
从-80℃冰箱取出100uL农杆菌GV3101感受态细胞，加1ug重组质粒，冰浴5min，液氮速冻5min，放置于37℃水浴锅5min，冰浴5min，然后加入1mL LB液体培养基；28℃，220rpm震荡培养2-4h，将菌液涂于含有Kan和Rif的LB固体平板上。28℃培养箱倒置培养2-3d，观察结果。挑取单菌落进行PCR检测。
2、拟南芥的遗传转化
挑取携带有目的基因的农杆菌单菌落接种于含有卡那霉素的LB液体培养基中进行活化，然后将活化过的菌液转接到新鲜的200mL含有卡那霉素的LB液体培养基中，28℃摇床，220 rpm，待OD值达到0.8~1时，5000 rpm，10 min，收集沉淀，用160 mL MS悬浮，加入silwet2.77 60uL，将带有花苞的拟南芥倒置在菌液中30s，用保鲜袋罩住，2天后去袋子，正常培养。
五、转基因拟南芥植株获得
收获转基因拟南芥T0代种子，消毒后铺于含50mg/mL卡那霉素的MS培养基上, 培养一周后，将筛选得到的绿苗移栽到营养钵中生长，两周后，提取其叶片基因组DNA进行PCR检测。。
采用以下方法提取DNA，步骤如下：
1、70%乙醇擦洗拟南芥叶片，保持叶片清洁。
2、吸取600uL gDNA提取buffer 置于研钵，加入叶片，快速研磨。吸取研磨液置于新的EP管中，12000 rpm，4℃离心，10 min。
3、取上清到一个新的EP管中，加入等体积酚和氯仿进行抽提，12000 rpm，4℃离心，5 min。
4、吸取上清到一个新的EP管中，然后加入等体积的异丙醇，混匀，12000 rpm，4℃，5min。
5、倒掉上清，向EP管中加入1mL 75% 乙醇清洗沉淀，12000 rpm, 4℃，5 min。
6弃上清，将EP管倒置在一个干净的滤纸上，晾干，加入约30 uL水，溶解沉淀。
利用TaqDNA聚合酶进行PCR扩增，引物选用克隆引物，上游引物：5’-GGGTCTAGAATGCCACGAGACATGAAT-3’，
下游引物：5’- CTTGGATCCTTATACCGATAAGTGGCG -3’。扩增反应程序为：95℃5min；95℃ 30s，58℃ 30s，72℃ 90s，32个循环；72℃ 10min，4℃保存。
对于PCR检测基因组DNA为阳性的株系进行了RNA的提取，RNA提取采用传统的Trizol法，并进行逆转录成cDNA。然后进行了半定量分析和定量分析，分析不同株系的表达水平（图2 ）。
引物选用PgSND1-like表达引物：
上游引物: 5’-TCAACGCAGTCACTCACAGTT-3’；
下游引物: 5’-GATGAAGACTTGGTGAAGCTC-3’
内参引物Actin:
上游引物：5’-GAAATCACAGCACTTGCACC-3’；
下游引物：5’-AAGCCTTTGATCTTGAGAGC-3’
六、将野生型拟南芥种子和T3代转基因拟南芥种子点种于MS固体培养基上，一周后移栽到土里面，一个月后观察转基因拟南芥植株表型，发现转基因拟南芥出现叶片变小且上卷的表型，并且其荚果变小且有部分不育现象，表明PgSND1-like基因可能参与到次生壁的加厚过程（图3）。
七、对野生型拟南芥和转基因拟南芥生长六周的花序主茎做石蜡切片，并进行番红固绿染色。结果如下图4所示：表明转基因株系拟南芥木质素合成增加，次生壁加厚。
八、对野生型拟南芥和转基因拟南芥生长六周的花序主茎进行木质素进行测量，结果表明转基因株系拟南芥木质素合成明显高于野生型拟南芥（图5）。
九、提取花序主茎基部的RNA，逆转录为cDNA，进行RT-PCR。检测CCoAOMT、PAL1、4CL、F5H四个个次生壁木质素合成基因在转基因主茎中表达量的变化，结果发现这些次生壁木质素合成基因的表达水平相对于野生型有明显的提高，说明PgSND1-like可能参与正调控次生壁木质素的合成(图6)。引物如下：
AtPAL1-up: TAGATTCGTGAGGGAAGAGCT
AtPAL1-dn: CCACTTCACAGACAATCATTTGG
AtCCoAOMT-up: TCGTTGATGCTGACAAAGACA
AtCCoAOMT-dn: ACTGATCCGACGGCAGATAG
At4CL-up: GGTTACCTCAACAATCCGGCA
At4CL-dn：CAAATGCAACAGGAACTTCAC
AtF5H-up：GGTCTCTTGTAACGTTGGTA
AtF5H-dn：AGATCATTACGGGCCTTCAC
综合以上结果：PgSND1-like过量表达转基因拟南芥能明显增加拟南芥次生壁木质素的合成，石榴籽粒硬度与石榴种皮木质素含量呈正相关关系，说明它可能参与石榴籽粒硬度的形成。
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、改进等，均应包含在本发明的保护范围之内。
序列表
<110> 中国农业科学院郑州果树研究所
<120> PgSND1-like基因在促进石榴籽粒硬度形成的用途
<160> 16
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1251
<212> DNA
<213> 石榴(Punica granatum L)
<400> 1
atgccacgag acatgaatct gtccataaat ggtcagtccc aggttccccc aggcttccgc 60
ttccatccga ccgaagaaga gctcctccac tattacctga ggaagaaagt cgcccaggag 120
aagatcgacc tcgatgtgat ccgcgaagtt gatctcaaca agctcgagcc gtgggatatt 180
caagagaagt gcagaatagg gtcgacgcca caaaatgatt ggtacttctt cagtcacaag 240
gacaagaagt acccgacagg gactcggaca aaccgagcga ctgcagccgg attctggaag 300
gcgacagggc gcgacaagat aatctacagc gggttcagaa ggattgggtt gcggaagacc 360
ctggtgtttt acaaggggcg tgctccacat ggacagaagt cggactggat catgcacgaa 420
taccggctag acgagactcc aagcattgat ccgagtgctc tgaatcctac agtcgatcag 480
ttgagtacag aagaaggatg ggtagtatgc cgagtttttc ggaagaagaa cttccagaaa 540
accctagacc acagtcccaa gagctcatcc tccacctcca tggactcaaa actcatccac 600
aacctcagca ccacaaacaa ccttaatatt aacaacatca aagacaatcc tagcagtgtc 660
ctggaccaga ttctcctcta catgggggaa agaaacagtg cgtgcaagat cgagactagt 720
caaccccgaa tgcctgtcac actcgaccct tccgactact gcgacacagt ctccagcatc 780
aagcttattc accagcctct gcccgacctc cacgaacggt tcatgcacct cccgaggctc 840
gagtgcccag caaccctccc ttccatcttc cccacaccac cacttgatca acacaacgtc 900
catcagccgt tcgatgagtt gactgatcat aatcaagatc agtcaacgca gtcactcaca 960
gttggagact gggctgcgtt cgaccggctt gttgcttccc agcttaacgg ccaagcagag 1020
acaccaagcg actcggcctt tggactccag ctagccaatg aagaagagga ggaagaagaa 1080
gaagaagatg ccaatgaagg gcaggtccca tcgattccga ggttcagaat aagcaggcct 1140
catcatcaga atctgcaacc gtacaacggg aacgaaaacg ccgatttgtg gagcttcacc 1200
aagtcttcat cgccgcagcc atctgacccc ttacgccact tatcggtata a 1251
<210> 2
<211> 416
<212> PRT
<213> 石榴(Punica granatum L)
<400> 2
Met Pro Arg Asp Met Asn Leu Ser Ile Asn Gly Gln Ser Gln Val Pro
1 5 10 15
Pro Gly Phe Arg Phe His Pro Thr Glu Glu Glu Leu Leu His Tyr Tyr
20 25 30
Leu Arg Lys Lys Val Ala Gln Glu Lys Ile Asp Leu Asp Val Ile Arg
35 40 45
Glu Val Asp Leu Asn Lys Leu Glu Pro Trp Asp Ile Gln Glu Lys Cys
50 55 60
Arg Ile Gly Ser Thr Pro Gln Asn Asp Trp Tyr Phe Phe Ser His Lys
65 70 75 80
Asp Lys Lys Tyr Pro Thr Gly Thr Arg Thr Asn Arg Ala Thr Ala Ala
85 90 95
Gly Phe Trp Lys Ala Thr Gly Arg Asp Lys Ile Ile Tyr Ser Gly Phe
100 105 110
Arg Arg Ile Gly Leu Arg Lys Thr Leu Val Phe Tyr Lys Gly Arg Ala
115 120 125
Pro His Gly Gln Lys Ser Asp Trp Ile Met His Glu Tyr Arg Leu Asp
130 135 140
Glu Thr Pro Ser Ile Asp Pro Ser Ala Leu Asn Pro Thr Val Asp Gln
145 150 155 160
Leu Ser Thr Glu Glu Gly Trp Val Val Cys Arg Val Phe Arg Lys Lys
165 170 175
Asn Phe Gln Lys Thr Leu Asp His Ser Pro Lys Ser Ser Ser Ser Thr
180 185 190
Ser Met Asp Ser Lys Leu Ile His Asn Leu Ser Thr Thr Asn Asn Leu
195 200 205
Asn Ile Asn Asn Ile Lys Asp Asn Pro Ser Ser Val Leu Asp Gln Ile
210 215 220
Leu Leu Tyr Met Gly Glu Arg Asn Ser Ala Cys Lys Ile Glu Thr Ser
225 230 235 240
Gln Pro Arg Met Pro Val Thr Leu Asp Pro Ser Asp Tyr Cys Asp Thr
245 250 255
Val Ser Ser Ile Lys Leu Ile His Gln Pro Leu Pro Asp Leu His Glu
260 265 270
Arg Phe Met His Leu Pro Arg Leu Glu Cys Pro Ala Thr Leu Pro Ser
275 280 285
Ile Phe Pro Thr Pro Pro Leu Asp Gln His Asn Val His Gln Pro Phe
290 295 300
Asp Glu Leu Thr Asp His Asn Gln Asp Gln Ser Thr Gln Ser Leu Thr
305 310 315 320
Val Gly Asp Trp Ala Ala Phe Asp Arg Leu Val Ala Ser Gln Leu Asn
325 330 335
Gly Gln Ala Glu Thr Pro Ser Asp Ser Ala Phe Gly Leu Gln Leu Ala
340 345 350
Asn Glu Glu Glu Glu Glu Glu Glu Glu Glu Asp Ala Asn Glu Gly Gln
355 360 365
Val Pro Ser Ile Pro Arg Phe Arg Ile Ser Arg Pro His His Gln Asn
370 375 380
Leu Gln Pro Tyr Asn Gly Asn Glu Asn Ala Asp Leu Trp Ser Phe Thr
385 390 395 400
Lys Ser Ser Ser Pro Gln Pro Ser Asp Pro Leu Arg His Leu Ser Val
405 410 415
<210> 3
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 3
gggtctagaa tgccacgaga catgaat 27
<210> 4
<211> 27
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 4
cttggatcct tataccgata agtggcg 27
<210> 5
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
tcaacgcagt cactcacagt t 21
<210> 6
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 6
gatgaagact tggtgaagct c 21
<210> 7
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gaaatcacag cacttgcacc 20
<210> 8
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
aagcctttga tcttgagagc 20
<210> 9
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
tagattcgtg agggaagagc t 21
<210> 10
<211> 23
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
ccacttcaca gacaatcatt tgg 23
<210> 11
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 11
tcgttgatgc tgacaaagac a 21
<210> 12
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 12
actgatccga cggcagatag 20
<210> 13
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 13
ggttacctca acaatccggc a 21
<210> 14
<211> 21
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 14
caaatgcaac aggaacttca c 21
<210> 15
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 15
ggtctcttgt aacgttggta 20
<210> 16
<211> 20
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 16
agatcattac gggccttcac 20
Claims (4)
1. 序列号SEQ ID NO：1的PgSND1-like基因在促进石榴籽粒硬度形成的用途。
2.一种促进石榴籽粒硬度形成的PgSND1-like基因，其特征在；所述基因的核苷酸序列由SEQ ID NO ：1组成。
3.一种表达载体，所述的表达载体含有权利要求2所述的PgSND1-like的基因。
4.根据权利要求3所述的一种表达载体，其特征在于：所述的表达载体为PBI121质粒。
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811630966.8A CN109678941B (zh) | 2018-12-29 | 2018-12-29 | PgSND1-like基因在促进石榴籽粒硬度形成的用途 |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811630966.8A CN109678941B (zh) | 2018-12-29 | 2018-12-29 | PgSND1-like基因在促进石榴籽粒硬度形成的用途 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109678941A CN109678941A (zh) | 2019-04-26 |
| CN109678941B true CN109678941B (zh) | 2022-03-15 |
Family
ID=66190987
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201811630966.8A Expired - Fee Related CN109678941B (zh) | 2018-12-29 | 2018-12-29 | PgSND1-like基因在促进石榴籽粒硬度形成的用途 |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN109678941B (zh) |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1782079A (zh) * | 2004-12-01 | 2006-06-07 | 中国农业科学院作物育种栽培研究所 | 一种小麦籽粒硬度相关基因及其编码蛋白与应用 |
-
2018
- 2018-12-29 CN CN201811630966.8A patent/CN109678941B/zh not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1782079A (zh) * | 2004-12-01 | 2006-06-07 | 中国农业科学院作物育种栽培研究所 | 一种小麦籽粒硬度相关基因及其编码蛋白与应用 |
Non-Patent Citations (1)
| Title |
|---|
| 石榴种皮木质素含量及PgSND1基因的表达与初步功能分析;夏小丛等;《果树学报（增刊）》;20171231;摘要，第78-79页桥连段 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN109678941A (zh) | 2019-04-26 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN106868021B (zh) | 控制水稻种子大小基因OsNAC1及其应用 | |
| CN107674873B (zh) | 小麦热激转录因子基因TaHsfA2i及其编码蛋白与应用 | |
| CN114591969B (zh) | 一种抗旱基因CrWRKY57及其在植物抗旱改良中的应用 | |
| CN108707623B (zh) | 一种草莓顶端分生组织相关基因FvMYB17及其应用 | |
| CN103183731B (zh) | 石斛DnMYB类转录因子、编码基因、载体、工程菌及应用 | |
| CN101698854A (zh) | 转盐芥cbf1基因提高玉米、小麦抗旱耐盐性的应用 | |
| CN111072762B (zh) | 一种毛竹衰老相关nap转录因子及其编码基因与应用 | |
| CN117286150A (zh) | 三七病程相关蛋白1基因PnPR1-3及其应用 | |
| CN103172716B (zh) | 植物抗热基因及其应用 | |
| CN108218969B (zh) | 甘薯花青素转运相关蛋白IbGSTF4及其编码基因与应用 | |
| CN114591409B (zh) | TaDTG6蛋白在提高植物抗旱性中的应用 | |
| CN109678941B (zh) | PgSND1-like基因在促进石榴籽粒硬度形成的用途 | |
| CN112029747B (zh) | 一种唐古特白刺NtSOS2基因及其表达蛋白和应用 | |
| CN110791506B (zh) | 一种唐古特白刺NtCIPK11基因及其表达蛋白和应用 | |
| CN108017696B (zh) | 菊花CmTFL1c基因及其应用 | |
| CN110760522B (zh) | Ak209基因及编码蛋白与抗逆增产的应用 | |
| CN104673803B (zh) | 基因甲基化在调控基因表达方面的应用 | |
| CN107177602B (zh) | 与植物耐旱相关的NtDR1基因及其应用 | |
| CN110628811A (zh) | 一种菊花CmSVP基因的应用 | |
| CN109628468A (zh) | 一种春兰CgWRKY53基因及其应用 | |
| CN114717245B (zh) | MsbHLH35基因及其编码蛋白在调控紫花苜蓿产量和耐渍性中的应用 | |
| CN111876430B (zh) | 光皮桦nfya2基因及其在提高植物对氮素吸收中的应用 | |
| CN115011607B (zh) | 芝麻育性调控基因及其表达载体和应用 | |
| CN110699362B (zh) | Afp5基因及其应用 | |
| CN113817038B (zh) | 来源于小豆的蛋白VaVPAC及其编码基因在增强烟草抗旱性方面的应用 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20220315 |