CN101921735A - Encoding gene of Saussurea involucrate flavanonol-4-reductase and application thereof - Google Patents
Encoding gene of Saussurea involucrate flavanonol-4-reductase and application thereof Download PDFInfo
- Publication number
- CN101921735A CN101921735A CN2010102263446A CN201010226344A CN101921735A CN 101921735 A CN101921735 A CN 101921735A CN 2010102263446 A CN2010102263446 A CN 2010102263446A CN 201010226344 A CN201010226344 A CN 201010226344A CN 101921735 A CN101921735 A CN 101921735A
- Authority
- CN
- China
- Prior art keywords
- sequence
- protein
- smdfr
- encoding gene
- dna
- 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.)
- Granted
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 85
- 241000133134 Saussurea Species 0.000 title abstract 4
- 102000004169 proteins and genes Human genes 0.000 claims abstract description 25
- 240000004808 Saccharomyces cerevisiae Species 0.000 claims abstract description 13
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 7
- YEDFEBOUHSBQBT-UHFFFAOYSA-N 2,3-dihydroflavon-3-ol Chemical compound O1C2=CC=CC=C2C(=O)C(O)C1C1=CC=CC=C1 YEDFEBOUHSBQBT-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229930003939 flavanonol Natural products 0.000 claims abstract description 6
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract 4
- 241000196324 Embryophyta Species 0.000 claims description 31
- 108020004414 DNA Proteins 0.000 claims description 27
- 241000894006 Bacteria Species 0.000 claims description 25
- 229930003935 flavonoid Natural products 0.000 claims description 21
- 235000017173 flavonoids Nutrition 0.000 claims description 21
- HVQAJTFOCKOKIN-UHFFFAOYSA-N flavonol Natural products O1C2=CC=CC=C2C(=O)C(O)=C1C1=CC=CC=C1 HVQAJTFOCKOKIN-UHFFFAOYSA-N 0.000 claims description 20
- 235000011957 flavonols Nutrition 0.000 claims description 20
- 238000000034 method Methods 0.000 claims description 20
- 150000007946 flavonol Chemical class 0.000 claims description 19
- BEWYHVAWEKZDPP-UHFFFAOYSA-N bornane Chemical compound C1CC2(C)CCC1C2(C)C BEWYHVAWEKZDPP-UHFFFAOYSA-N 0.000 claims description 18
- 239000012634 fragment Substances 0.000 claims description 17
- 235000018102 proteins Nutrition 0.000 claims description 17
- REFJWTPEDVJJIY-UHFFFAOYSA-N Quercetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC=C(O)C(O)=C1 REFJWTPEDVJJIY-UHFFFAOYSA-N 0.000 claims description 16
- 239000013604 expression vector Substances 0.000 claims description 16
- 150000002215 flavonoids Chemical class 0.000 claims description 15
- 125000003729 nucleotide group Chemical group 0.000 claims description 15
- 239000002773 nucleotide Substances 0.000 claims description 14
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 13
- -1 ketone compounds Chemical class 0.000 claims description 12
- 239000013598 vector Substances 0.000 claims description 12
- 230000009261 transgenic effect Effects 0.000 claims description 11
- CXQWRCVTCMQVQX-LSDHHAIUSA-N (+)-taxifolin Chemical compound C1([C@@H]2[C@H](C(C3=C(O)C=C(O)C=C3O2)=O)O)=CC=C(O)C(O)=C1 CXQWRCVTCMQVQX-LSDHHAIUSA-N 0.000 claims description 10
- JMGZEFIQIZZSBH-UHFFFAOYSA-N Bioquercetin Natural products CC1OC(OCC(O)C2OC(OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5)C(O)C2O)C(O)C(O)C1O JMGZEFIQIZZSBH-UHFFFAOYSA-N 0.000 claims description 10
- IKMDFBPHZNJCSN-UHFFFAOYSA-N Myricetin Chemical compound C=1C(O)=CC(O)=C(C(C=2O)=O)C=1OC=2C1=CC(O)=C(O)C(O)=C1 IKMDFBPHZNJCSN-UHFFFAOYSA-N 0.000 claims description 10
- XCGZWJIXHMSSQC-UHFFFAOYSA-N dihydroquercetin Natural products OC1=CC2OC(=C(O)C(=O)C2C(O)=C1)c1ccc(O)c(O)c1 XCGZWJIXHMSSQC-UHFFFAOYSA-N 0.000 claims description 10
- IVTMALDHFAHOGL-UHFFFAOYSA-N eriodictyol 7-O-rutinoside Natural products OC1C(O)C(O)C(C)OC1OCC1C(O)C(O)C(O)C(OC=2C=C3C(C(C(O)=C(O3)C=3C=C(O)C(O)=CC=3)=O)=C(O)C=2)O1 IVTMALDHFAHOGL-UHFFFAOYSA-N 0.000 claims description 10
- PCOBUQBNVYZTBU-UHFFFAOYSA-N myricetin Natural products OC1=C(O)C(O)=CC(C=2OC3=CC(O)=C(O)C(O)=C3C(=O)C=2)=C1 PCOBUQBNVYZTBU-UHFFFAOYSA-N 0.000 claims description 10
- 235000007743 myricetin Nutrition 0.000 claims description 10
- 229940116852 myricetin Drugs 0.000 claims description 10
- FDRQPMVGJOQVTL-UHFFFAOYSA-N quercetin rutinoside Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 FDRQPMVGJOQVTL-UHFFFAOYSA-N 0.000 claims description 10
- 235000005493 rutin Nutrition 0.000 claims description 10
- IKGXIBQEEMLURG-BKUODXTLSA-N rutin Chemical compound O[C@H]1[C@H](O)[C@@H](O)[C@H](C)O[C@@H]1OC[C@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](OC=2C(C3=C(O)C=C(O)C=C3OC=2C=2C=C(O)C(O)=CC=2)=O)O1 IKGXIBQEEMLURG-BKUODXTLSA-N 0.000 claims description 10
- ALABRVAAKCSLSC-UHFFFAOYSA-N rutin Natural products CC1OC(OCC2OC(O)C(O)C(O)C2O)C(O)C(O)C1OC3=C(Oc4cc(O)cc(O)c4C3=O)c5ccc(O)c(O)c5 ALABRVAAKCSLSC-UHFFFAOYSA-N 0.000 claims description 10
- 229960004555 rutoside Drugs 0.000 claims description 10
- XETQTCAMTVHYPO-UHFFFAOYSA-N Isocamphan von ungewisser Konfiguration Natural products C1CC2C(C)(C)C(C)C1C2 XETQTCAMTVHYPO-UHFFFAOYSA-N 0.000 claims description 9
- 229930006742 bornane Natural products 0.000 claims description 9
- 239000003205 fragrance Substances 0.000 claims description 9
- 230000008521 reorganization Effects 0.000 claims description 9
- ZVOLCUVKHLEPEV-UHFFFAOYSA-N Quercetagetin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=C(O)C(O)=C(O)C=C2O1 ZVOLCUVKHLEPEV-UHFFFAOYSA-N 0.000 claims description 8
- HWTZYBCRDDUBJY-UHFFFAOYSA-N Rhynchosin Natural products C1=C(O)C(O)=CC=C1C1=C(O)C(=O)C2=CC(O)=C(O)C=C2O1 HWTZYBCRDDUBJY-UHFFFAOYSA-N 0.000 claims description 8
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 claims description 8
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 claims description 8
- 235000005875 quercetin Nutrition 0.000 claims description 8
- 229960001285 quercetin Drugs 0.000 claims description 8
- MZSGWZGPESCJAN-MOBFUUNNSA-N Melitric acid A Natural products O([C@@H](C(=O)O)Cc1cc(O)c(O)cc1)C(=O)/C=C/c1cc(O)c(O/C(/C(=O)O)=C/c2cc(O)c(O)cc2)cc1 MZSGWZGPESCJAN-MOBFUUNNSA-N 0.000 claims description 7
- XADJWCRESPGUTB-UHFFFAOYSA-N apigenin Natural products C1=CC(O)=CC=C1C1=CC(=O)C2=CC(O)=C(O)C=C2O1 XADJWCRESPGUTB-UHFFFAOYSA-N 0.000 claims description 7
- 235000008714 apigenin Nutrition 0.000 claims description 7
- KZNIFHPLKGYRTM-UHFFFAOYSA-N apigenin Chemical compound C1=CC(O)=CC=C1C1=CC(=O)C2=C(O)C=C(O)C=C2O1 KZNIFHPLKGYRTM-UHFFFAOYSA-N 0.000 claims description 7
- 229940117893 apigenin Drugs 0.000 claims description 7
- SBHXYTNGIZCORC-ZDUSSCGKSA-N eriodictyol Chemical compound C1([C@@H]2CC(=O)C3=C(O)C=C(C=C3O2)O)=CC=C(O)C(O)=C1 SBHXYTNGIZCORC-ZDUSSCGKSA-N 0.000 claims description 7
- TUJPOVKMHCLXEL-UHFFFAOYSA-N eriodictyol Natural products C1C(=O)C2=CC(O)=CC(O)=C2OC1C1=CC=C(O)C(O)=C1 TUJPOVKMHCLXEL-UHFFFAOYSA-N 0.000 claims description 7
- 235000011797 eriodictyol Nutrition 0.000 claims description 7
- SBHXYTNGIZCORC-UHFFFAOYSA-N eriodyctiol Natural products O1C2=CC(O)=CC(O)=C2C(=O)CC1C1=CC=C(O)C(O)=C1 SBHXYTNGIZCORC-UHFFFAOYSA-N 0.000 claims description 7
- 238000013467 fragmentation Methods 0.000 claims description 7
- 238000006062 fragmentation reaction Methods 0.000 claims description 7
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 7
- 238000003259 recombinant expression Methods 0.000 claims description 7
- 230000002441 reversible effect Effects 0.000 claims description 7
- 229930002878 anthoxanthin Natural products 0.000 claims description 6
- 150000004637 anthoxanthins Chemical class 0.000 claims description 6
- ZONYXWQDUYMKFB-UHFFFAOYSA-N SJ000286395 Natural products O1C2=CC=CC=C2C(=O)CC1C1=CC=CC=C1 ZONYXWQDUYMKFB-UHFFFAOYSA-N 0.000 claims description 5
- 229930003949 flavanone Natural products 0.000 claims description 5
- 150000002208 flavanones Chemical class 0.000 claims description 5
- 235000011981 flavanones Nutrition 0.000 claims description 5
- 230000008034 disappearance Effects 0.000 claims description 4
- 241001233957 eudicotyledons Species 0.000 claims description 4
- 108091028043 Nucleic acid sequence Proteins 0.000 claims description 3
- 102000053602 DNA Human genes 0.000 claims description 2
- 241000977585 Saussurea medusa Species 0.000 abstract description 24
- 239000002299 complementary DNA Substances 0.000 abstract description 23
- 108090000790 Enzymes Proteins 0.000 abstract description 15
- 102000004190 Enzymes Human genes 0.000 abstract description 15
- 238000012216 screening Methods 0.000 abstract description 7
- 238000006911 enzymatic reaction Methods 0.000 abstract description 5
- 238000002474 experimental method Methods 0.000 abstract description 3
- 238000000338 in vitro Methods 0.000 abstract 1
- 241001145025 Saussurea involucrata Species 0.000 description 27
- 230000000692 anti-sense effect Effects 0.000 description 20
- 206010020649 Hyperkeratosis Diseases 0.000 description 15
- 239000000047 product Substances 0.000 description 14
- 239000013612 plasmid Substances 0.000 description 13
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 12
- 239000007788 liquid Substances 0.000 description 12
- 238000006555 catalytic reaction Methods 0.000 description 10
- 239000000758 substrate Substances 0.000 description 10
- 230000014759 maintenance of location Effects 0.000 description 9
- 239000000243 solution Substances 0.000 description 9
- VEVZSMAEJFVWIL-UHFFFAOYSA-O cyanidin cation Chemical compound [O+]=1C2=CC(O)=CC(O)=C2C=C(O)C=1C1=CC=C(O)C(O)=C1 VEVZSMAEJFVWIL-UHFFFAOYSA-O 0.000 description 8
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000000284 extract Substances 0.000 description 7
- 239000000523 sample Substances 0.000 description 7
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 6
- NWBJYWHLCVSVIJ-UHFFFAOYSA-N N-benzyladenine Chemical compound N=1C=NC=2NC=NC=2C=1NCC1=CC=CC=C1 NWBJYWHLCVSVIJ-UHFFFAOYSA-N 0.000 description 6
- 150000001413 amino acids Chemical group 0.000 description 6
- 230000008859 change Effects 0.000 description 6
- 238000010790 dilution Methods 0.000 description 6
- 239000012895 dilution Substances 0.000 description 6
- 230000000694 effects Effects 0.000 description 6
- 230000006870 function Effects 0.000 description 6
- JPFCOVZKLAXXOE-XBNSMERZSA-N (3r)-2-(3,5-dihydroxy-4-methoxyphenyl)-8-[(2r,3r,4r)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-3,4-dihydro-2h-chromen-4-yl]-3,4-dihydro-2h-chromene-3,5,7-triol Chemical compound C1=C(O)C(OC)=C(O)C=C1C1[C@H](O)CC(C(O)=CC(O)=C2[C@H]3C4=C(O)C=C(O)C=C4O[C@@H]([C@@H]3O)C=3C=CC(O)=CC=3)=C2O1 JPFCOVZKLAXXOE-XBNSMERZSA-N 0.000 description 5
- 229920001991 Proanthocyanidin Polymers 0.000 description 5
- 238000000246 agarose gel electrophoresis Methods 0.000 description 5
- 238000001514 detection method Methods 0.000 description 5
- 239000003814 drug Substances 0.000 description 5
- 229930003944 flavone Natural products 0.000 description 5
- 235000011949 flavones Nutrition 0.000 description 5
- 150000002500 ions Chemical class 0.000 description 5
- 239000003550 marker Substances 0.000 description 5
- 229940081969 saccharomyces cerevisiae Drugs 0.000 description 5
- 239000000287 crude extract Substances 0.000 description 4
- 235000007336 cyanidin Nutrition 0.000 description 4
- 150000002213 flavones Chemical class 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 239000002609 medium Substances 0.000 description 4
- 235000015097 nutrients Nutrition 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- 239000000126 substance Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- DKVBOUDTNWVDEP-NJCHZNEYSA-N teicoplanin aglycone Chemical compound N([C@H](C(N[C@@H](C1=CC(O)=CC(O)=C1C=1C(O)=CC=C2C=1)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)OC=1C=C3C=C(C=1O)OC1=CC=C(C=C1Cl)C[C@H](C(=O)N1)NC([C@H](N)C=4C=C(O5)C(O)=CC=4)=O)C(=O)[C@@H]2NC(=O)[C@@H]3NC(=O)[C@@H]1C1=CC5=CC(O)=C1 DKVBOUDTNWVDEP-NJCHZNEYSA-N 0.000 description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 241000589158 Agrobacterium Species 0.000 description 3
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 3
- 108090000854 Oxidoreductases Proteins 0.000 description 3
- 102000004316 Oxidoreductases Human genes 0.000 description 3
- 108091081024 Start codon Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 230000003110 anti-inflammatory effect Effects 0.000 description 3
- 230000017531 blood circulation Effects 0.000 description 3
- 210000004027 cell Anatomy 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 239000002131 composite material Substances 0.000 description 3
- 239000000470 constituent Substances 0.000 description 3
- 230000029087 digestion Effects 0.000 description 3
- 108010060641 flavanone synthetase Proteins 0.000 description 3
- 210000001161 mammalian embryo Anatomy 0.000 description 3
- 238000001819 mass spectrum Methods 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 230000009467 reduction Effects 0.000 description 3
- JQXXHWHPUNPDRT-WLSIYKJHSA-N rifampicin Chemical compound O([C@](C1=O)(C)O/C=C/[C@@H]([C@H]([C@@H](OC(C)=O)[C@H](C)[C@H](O)[C@H](C)[C@@H](O)[C@@H](C)\C=C\C=C(C)/C(=O)NC=2C(O)=C3C([O-])=C4C)C)OC)C4=C1C3=C(O)C=2\C=N\N1CC[NH+](C)CC1 JQXXHWHPUNPDRT-WLSIYKJHSA-N 0.000 description 3
- 229960001225 rifampicin Drugs 0.000 description 3
- 238000012360 testing method Methods 0.000 description 3
- 238000013519 translation Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 2
- PRPINYUDVPFIRX-UHFFFAOYSA-N 1-naphthaleneacetic acid Chemical compound C1=CC=C2C(CC(=O)O)=CC=CC2=C1 PRPINYUDVPFIRX-UHFFFAOYSA-N 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 241000701489 Cauliflower mosaic virus Species 0.000 description 2
- 241000620209 Escherichia coli DH5[alpha] Species 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- ACFIXJIJDZMPPO-NNYOXOHSSA-N NADPH Chemical compound C1=CCC(C(=O)N)=CN1[C@H]1[C@H](O)[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OC[C@@H]2[C@H]([C@@H](OP(O)(O)=O)[C@@H](O2)N2C3=NC=NC(N)=C3N=C2)O)O1 ACFIXJIJDZMPPO-NNYOXOHSSA-N 0.000 description 2
- 238000012408 PCR amplification Methods 0.000 description 2
- 240000005373 Panax quinquefolius Species 0.000 description 2
- 235000003140 Panax quinquefolius Nutrition 0.000 description 2
- DYUQAZSOFZSPHD-UHFFFAOYSA-N Phenylpropyl alcohol Natural products CCC(O)C1=CC=CC=C1 DYUQAZSOFZSPHD-UHFFFAOYSA-N 0.000 description 2
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 2
- 229930006000 Sucrose Natural products 0.000 description 2
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 2
- 230000003321 amplification Effects 0.000 description 2
- 235000010208 anthocyanin Nutrition 0.000 description 2
- 229930002877 anthocyanin Natural products 0.000 description 2
- 239000004410 anthocyanin Substances 0.000 description 2
- 150000004636 anthocyanins Chemical class 0.000 description 2
- 230000001093 anti-cancer Effects 0.000 description 2
- 230000003115 biocidal effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000000872 buffer Substances 0.000 description 2
- 239000007795 chemical reaction product Substances 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000010411 cooking Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 238000013016 damping Methods 0.000 description 2
- 238000013461 design Methods 0.000 description 2
- 239000013024 dilution buffer Substances 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000003623 enhancer Substances 0.000 description 2
- ZEACOKJOQLAYTD-UHFFFAOYSA-N flavan-3,3',4,4',5,5',7-heptol Chemical compound OC1C(O)C2=C(O)C=C(O)C=C2OC1C1=CC(O)=C(O)C(O)=C1 ZEACOKJOQLAYTD-UHFFFAOYSA-N 0.000 description 2
- 239000012530 fluid Substances 0.000 description 2
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 2
- YQYJSBFKSSDGFO-FWAVGLHBSA-N hygromycin A Chemical compound O[C@H]1[C@H](O)[C@H](C(=O)C)O[C@@H]1Oc1ccc(\C=C(/C)C(=O)N[C@@H]2[C@@H]([C@H]3OCO[C@H]3[C@@H](O)[C@@H]2O)O)cc1O YQYJSBFKSSDGFO-FWAVGLHBSA-N 0.000 description 2
- 230000001939 inductive effect Effects 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 230000004060 metabolic process Effects 0.000 description 2
- 239000004570 mortar (masonry) Substances 0.000 description 2
- 229930027945 nicotinamide-adenine dinucleotide Natural products 0.000 description 2
- 238000003199 nucleic acid amplification method Methods 0.000 description 2
- 230000037361 pathway Effects 0.000 description 2
- 230000000144 pharmacologic effect Effects 0.000 description 2
- 230000001737 promoting effect Effects 0.000 description 2
- 238000011084 recovery Methods 0.000 description 2
- 201000003068 rheumatic fever Diseases 0.000 description 2
- 239000005720 sucrose Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- DQFBYFPFKXHELB-VAWYXSNFSA-N trans-chalcone Chemical compound C=1C=CC=CC=1C(=O)\C=C\C1=CC=CC=C1 DQFBYFPFKXHELB-VAWYXSNFSA-N 0.000 description 2
- 230000005026 transcription initiation Effects 0.000 description 2
- 238000012546 transfer Methods 0.000 description 2
- 239000005631 2,4-Dichlorophenoxyacetic acid Substances 0.000 description 1
- 108010011170 Ala-Trp-Arg-His-Pro-Gln-Phe-Gly-Gly Proteins 0.000 description 1
- 102000016912 Aldehyde Reductase Human genes 0.000 description 1
- 108010053754 Aldehyde reductase Proteins 0.000 description 1
- 206010001928 Amenorrhoea Diseases 0.000 description 1
- 206010003210 Arteriosclerosis Diseases 0.000 description 1
- 241000193830 Bacillus <bacterium> Species 0.000 description 1
- 101100275473 Caenorhabditis elegans ctc-3 gene Proteins 0.000 description 1
- 101100433727 Caenorhabditis elegans got-1.2 gene Proteins 0.000 description 1
- 206010007247 Carbuncle Diseases 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 102100021391 Cationic amino acid transporter 3 Human genes 0.000 description 1
- 241001594971 Centaurea maculosa Species 0.000 description 1
- 241000501711 Centaurium Species 0.000 description 1
- 206010065559 Cerebral arteriosclerosis Diseases 0.000 description 1
- LZZYPRNAOMGNLH-UHFFFAOYSA-M Cetrimonium bromide Chemical compound [Br-].CCCCCCCCCCCCCCCC[N+](C)(C)C LZZYPRNAOMGNLH-UHFFFAOYSA-M 0.000 description 1
- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 108091026890 Coding region Proteins 0.000 description 1
- 108020004705 Codon Proteins 0.000 description 1
- 206010011224 Cough Diseases 0.000 description 1
- 101150002919 DFR gene Proteins 0.000 description 1
- 230000003682 DNA packaging effect Effects 0.000 description 1
- 108010067770 Endopeptidase K Proteins 0.000 description 1
- YQYJSBFKSSDGFO-UHFFFAOYSA-N Epihygromycin Natural products OC1C(O)C(C(=O)C)OC1OC(C(=C1)O)=CC=C1C=C(C)C(=O)NC1C(O)C(O)C2OCOC2C1O YQYJSBFKSSDGFO-UHFFFAOYSA-N 0.000 description 1
- XZWYTXMRWQJBGX-VXBMVYAYSA-N FLAG peptide Chemical compound NCCCC[C@@H](C(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](CCCCN)NC(=O)[C@@H](NC(=O)[C@@H](N)CC(O)=O)CC1=CC=C(O)C=C1 XZWYTXMRWQJBGX-VXBMVYAYSA-N 0.000 description 1
- 108030006800 Flavanone 4-reductases Proteins 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 229930182566 Gentamicin Natural products 0.000 description 1
- CEAZRRDELHUEMR-URQXQFDESA-N Gentamicin Chemical compound O1[C@H](C(C)NC)CC[C@@H](N)[C@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](NC)[C@@](C)(O)CO2)O)[C@H](N)C[C@@H]1N CEAZRRDELHUEMR-URQXQFDESA-N 0.000 description 1
- 206010019468 Hemiplegia Diseases 0.000 description 1
- 108010093488 His-His-His-His-His-His Proteins 0.000 description 1
- 101000911390 Homo sapiens Coagulation factor VIII Proteins 0.000 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- GRRNUXAQVGOGFE-UHFFFAOYSA-N Hygromycin-B Natural products OC1C(NC)CC(N)C(O)C1OC1C2OC3(C(C(O)C(O)C(C(N)CO)O3)O)OC2C(O)C(CO)O1 GRRNUXAQVGOGFE-UHFFFAOYSA-N 0.000 description 1
- OFZBQQUVMQGHDJ-UHFFFAOYSA-N Leucofisetinidin Natural products OC1C(O)C2=CC=C(O)C=C2OC1C1=CC=C(O)C(O)=C1 OFZBQQUVMQGHDJ-UHFFFAOYSA-N 0.000 description 1
- 108060001084 Luciferase Proteins 0.000 description 1
- 201000005505 Measles Diseases 0.000 description 1
- 208000019255 Menstrual disease Diseases 0.000 description 1
- 208000019695 Migraine disease Diseases 0.000 description 1
- 101710135898 Myc proto-oncogene protein Proteins 0.000 description 1
- 102100038895 Myc proto-oncogene protein Human genes 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical class CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 240000002853 Nelumbo nucifera Species 0.000 description 1
- 235000006508 Nelumbo nucifera Nutrition 0.000 description 1
- 235000006510 Nelumbo pentapetala Nutrition 0.000 description 1
- 206010028980 Neoplasm Diseases 0.000 description 1
- 108700026244 Open Reading Frames Proteins 0.000 description 1
- 229910019142 PO4 Inorganic materials 0.000 description 1
- 208000002193 Pain Diseases 0.000 description 1
- 229940096437 Protein S Drugs 0.000 description 1
- 239000013614 RNA sample Substances 0.000 description 1
- 101000702488 Rattus norvegicus High affinity cationic amino acid transporter 1 Proteins 0.000 description 1
- 108091006230 SLC7A3 Proteins 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- GAMYVSCDDLXAQW-AOIWZFSPSA-N Thermopsosid Natural products O(C)c1c(O)ccc(C=2Oc3c(c(O)cc(O[C@H]4[C@H](O)[C@@H](O)[C@H](O)[C@H](CO)O4)c3)C(=O)C=2)c1 GAMYVSCDDLXAQW-AOIWZFSPSA-N 0.000 description 1
- 101710150448 Transcriptional regulator Myc Proteins 0.000 description 1
- 108700019146 Transgenes Proteins 0.000 description 1
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical class OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 1
- 108010028230 Trp-Ser- His-Pro-Gln-Phe-Glu-Lys Proteins 0.000 description 1
- 108090000848 Ubiquitin Proteins 0.000 description 1
- 210000001015 abdomen Anatomy 0.000 description 1
- 229930013930 alkaloid Natural products 0.000 description 1
- 150000003797 alkaloid derivatives Chemical class 0.000 description 1
- 230000036592 analgesia Effects 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000003712 anti-aging effect Effects 0.000 description 1
- 230000002929 anti-fatigue Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 230000002155 anti-virotic effect Effects 0.000 description 1
- 239000003963 antioxidant agent Substances 0.000 description 1
- 230000003078 antioxidant effect Effects 0.000 description 1
- 235000006708 antioxidants Nutrition 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 208000011775 arteriosclerosis disease Diseases 0.000 description 1
- 230000003416 augmentation Effects 0.000 description 1
- 238000010170 biological method Methods 0.000 description 1
- 230000003570 biosynthesizing effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 239000007844 bleaching agent Substances 0.000 description 1
- 244000309464 bull Species 0.000 description 1
- 210000004899 c-terminal region Anatomy 0.000 description 1
- 238000001354 calcination Methods 0.000 description 1
- 229940027138 cambia Drugs 0.000 description 1
- 239000012159 carrier gas Substances 0.000 description 1
- 210000003169 central nervous system Anatomy 0.000 description 1
- 239000000460 chlorine Substances 0.000 description 1
- 229910052801 chlorine Inorganic materials 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000004567 concrete Substances 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 230000008602 contraction Effects 0.000 description 1
- 230000001186 cumulative effect Effects 0.000 description 1
- 230000001934 delay Effects 0.000 description 1
- KXZOIWWTXOCYKR-UHFFFAOYSA-M diclofenac potassium Chemical compound [K+].[O-]C(=O)CC1=CC=CC=C1NC1=C(Cl)C=CC=C1Cl KXZOIWWTXOCYKR-UHFFFAOYSA-M 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000005611 electricity Effects 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 230000009088 enzymatic function Effects 0.000 description 1
- 238000003810 ethyl acetate extraction Methods 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 230000000763 evoking effect Effects 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 150000002212 flavone derivatives Chemical class 0.000 description 1
- 150000002216 flavonol derivatives Chemical class 0.000 description 1
- 238000007710 freezing Methods 0.000 description 1
- 230000008014 freezing Effects 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 238000002309 gasification Methods 0.000 description 1
- 239000008273 gelatin Substances 0.000 description 1
- 229920000159 gelatin Polymers 0.000 description 1
- 235000019322 gelatine Nutrition 0.000 description 1
- 235000011852 gelatine desserts Nutrition 0.000 description 1
- 229960002518 gentamicin Drugs 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 239000001963 growth medium Substances 0.000 description 1
- 101150054900 gus gene Proteins 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 239000004009 herbicide Substances 0.000 description 1
- 235000008216 herbs Nutrition 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 102000057593 human F8 Human genes 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- GRRNUXAQVGOGFE-NZSRVPFOSA-N hygromycin B Chemical compound O[C@@H]1[C@@H](NC)C[C@@H](N)[C@H](O)[C@H]1O[C@H]1[C@H]2O[C@@]3([C@@H]([C@@H](O)[C@@H](O)[C@@H](C(N)CO)O3)O)O[C@H]2[C@@H](O)[C@@H](CO)O1 GRRNUXAQVGOGFE-NZSRVPFOSA-N 0.000 description 1
- 229940097277 hygromycin b Drugs 0.000 description 1
- 238000005286 illumination Methods 0.000 description 1
- 230000007365 immunoregulation Effects 0.000 description 1
- 230000008676 import Effects 0.000 description 1
- 201000001881 impotence Diseases 0.000 description 1
- 230000001976 improved effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000002401 inhibitory effect Effects 0.000 description 1
- 230000000968 intestinal effect Effects 0.000 description 1
- 201000005851 intracranial arteriosclerosis Diseases 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- 229930027917 kanamycin Natural products 0.000 description 1
- 229960000318 kanamycin Drugs 0.000 description 1
- SBUJHOSQTJFQJX-NOAMYHISSA-N kanamycin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CN)O[C@@H]1O[C@H]1[C@H](O)[C@@H](O[C@@H]2[C@@H]([C@@H](N)[C@H](O)[C@@H](CO)O2)O)[C@H](N)C[C@@H]1N SBUJHOSQTJFQJX-NOAMYHISSA-N 0.000 description 1
- 229930182823 kanamycin A Natural products 0.000 description 1
- 125000000468 ketone group Chemical group 0.000 description 1
- 101150044508 key gene Proteins 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 210000004072 lung Anatomy 0.000 description 1
- 239000002932 luster Substances 0.000 description 1
- 230000002101 lytic effect Effects 0.000 description 1
- LTYOQGRJFJAKNA-VFLPNFFSSA-N malonyl-coa Chemical compound O[C@@H]1[C@H](OP(O)(O)=O)[C@@H](COP(O)(=O)OP(O)(=O)OCC(C)(C)C(O)C(=O)NCCC(=O)NCCSC(=O)CC(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 LTYOQGRJFJAKNA-VFLPNFFSSA-N 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 230000001404 mediated effect Effects 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 230000002175 menstrual effect Effects 0.000 description 1
- 238000000520 microinjection Methods 0.000 description 1
- 206010027599 migraine Diseases 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 210000003205 muscle Anatomy 0.000 description 1
- 230000035772 mutation Effects 0.000 description 1
- 239000006199 nebulizer Substances 0.000 description 1
- 239000013642 negative control Substances 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- 238000004806 packaging method and process Methods 0.000 description 1
- 230000036407 pain Effects 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 description 1
- 239000010452 phosphate Substances 0.000 description 1
- 210000002826 placenta Anatomy 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 239000013641 positive control Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000002360 preparation method Methods 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 230000004223 radioprotective effect Effects 0.000 description 1
- 229940047431 recombinate Drugs 0.000 description 1
- 239000012492 regenerant Substances 0.000 description 1
- 230000008929 regeneration Effects 0.000 description 1
- 238000011069 regeneration method Methods 0.000 description 1
- 230000000452 restraining effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 238000010839 reverse transcription Methods 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 229930184338 saussureoside Natural products 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 241000894007 species Species 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 150000003432 sterols Chemical class 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- 230000002103 transcriptional effect Effects 0.000 description 1
- 230000014621 translational initiation Effects 0.000 description 1
- 238000011144 upstream manufacturing Methods 0.000 description 1
- 210000004291 uterus Anatomy 0.000 description 1
- 235000013311 vegetables Nutrition 0.000 description 1
- 239000013603 viral vector Substances 0.000 description 1
- VHBFFQKBGNRLFZ-UHFFFAOYSA-N vitamin p Natural products O1C2=CC=CC=C2C(=O)C=C1C1=CC=CC=C1 VHBFFQKBGNRLFZ-UHFFFAOYSA-N 0.000 description 1
- 239000000341 volatile oil Substances 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
Images
Classifications
-
- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
- Y02P20/00—Technologies relating to chemical industry
- Y02P20/50—Improvements relating to the production of bulk chemicals
- Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses an encoding gene of Saussurea involucrate flavanonol-4-reductase and applications thereof. The invention provides a protein which is flavanonol-4-reductase and is named as SmDFR. The enzyme is the following protein 1) or following protein 2): 1) protein composed of the amino acid sequence shown in the sequence 2 of the sequence table; 2) protein which is the derivative of the protein 1) by replacing and/or losing and/or adding one or several amino acid residues to the amino acid sequence of the sequence 2 and has the same function. The experiments of the invention show that the cloned gene formed by constructing a Saussurea involucrate cDNA library, using the library and degenerate primer to screening out the encoding gene of Saussurea medusa SmDFR and performing yeast expression and in vitro enzymatic reaction, is the functional encoding gene of flavanonol-4-reductase. The related flavanonol synthetic protein of the invention and the encoding gene of the protein have important value for solving the shortage problem of the wild resources of Saussurea involucrate.
Description
Technical field
The present invention relates to a kind of saussurea involucrata flavanonol-4-reductase encoding gene and its application, relate in particular to a kind of saussurea involucrata flavanonol-4-reductase and encoding gene and application.
Background technology
Snow Lotus Herb has another name called saussurea involucrata, and the composite family hieracioides belongs to.Be the famous and precious medicinal plant of a class commonly used among the people, belong to per nnial herb.Generally be distributed on the high mountain flowstone beach of height above sea level more than 4,000 meters.In " territory, northwest note " and " the little knowledge in garden mutually ", just relevant for the record of saussurea involucrata.Has dispelling cold and removing dampness, functions such as promoting blood circulation to restore menstrual flow, strong muscle is supporing yang, anti-inflammatory, analgesia, contraction uterus, the rheumatic arthritis that is used for the treatment of among the people delays senility, arteriosclerosis, termination of pregnancy, diseases such as women's cold and pain in the lower abdomen, amenorrhoea, retention of placenta, measles without adequate eruption, lung cold cough, impotence.Snow Lotus Herb thallophyta kind is more, though all can be used as medicine on an equal basis according to the record of Chinese medicine document, its quality has the branch of quality.According to relevant, the sales volume maximum is Xinjiang Tianshan saussurea involucrata (snow lotus) (S.involucrata Kar.et Kir.) and Saussurea medusa (S.medusa Maxim) in above several saussurea involucrata.And Saussurea medusa (Saussurea medusa Maxim.) is the former plant of Tibetan medicine " saussurea involucrata " medicinal material, is distributed in ground such as China Qinghai, Gansu, Tibet.Saussurea medusa is the rare medicinal herbs in the Tibetan medicine, has effects such as dispelling cold and removing dampness, promoting blood circulation and removing obstruction in channels, anticancer, anti-inflammatory and antifatigue, can treat multiple diseases such as rheumatic arthritis, menoxenia, carbuncle pyogenic infections from tumour or sore and high mountain incompatibility.
Contain multiple compositions such as flavones, alkaloid, lactone, sterol, volatile oil, polysaccharide in the saussurea involucrata, its effective constituent is mainly flavonoid compound.The pharmacological action of flavonoid compound comprises anticancer, antitumor, resisting cardiovascular disease, kobadrin, anti-inflammatory, analgesic activity, immunoregulation effect, the effect of female hormone sample, antibiotic and antivirus action, anti-oxidant, anti-aging effects, radioprotective.As the promote blood circulation saussurea involucrata TONGMAI KOUFUYE of ball, Herba Saussureae Involueratae injection and treatment cerebral arteriosclerosis and ishemic stroke of the saussurea involucrata that is used for treating hemiplegia, all be quality standard with the flavonoid content.Especially the flavonols pharmacological action is remarkable for the saussurea involucrata flavones, as the Saussurea medusa flavone component central nervous system is had restraining effect; The Herba Saussureae Involueratae injection treatment migraine effect that with the flavonoid compound is effective constituent is remarkable.
The biosynthesizing of flavonoid compound realizes by phenylpropyl alcohol alkanes biosynthetic pathway.Produce phenyl styryl ketone (chalkone) beginning by 1 molecule 4-coumaric acyl-CoA and 3 molecule malonyl--CoA under chalcone synthase (CHS) catalysis, chalcone synthase is to guide phenylpropyl alcohol alkanes pathways metabolism into flavonoid synthetic first most critical enzyme.Flavanonol-4-reductase (DFR) is first key gene that leads to anthocyanin and proanthocyanidin in the flavonoid route of synthesis.Provide hydrogen DFR 4 ketone group reduction of flavanonol can be formed hydroxyl by NADPH and generate colourless proanthocyanidin, colourless proanthocyanidin instability is further generated anthocyanin and proanthocyanidin by cyanidin(e) aglycon synthetic enzyme (ANS) and colourless proanthocyanidin reductase enzyme enzyme catalysiss such as (LCR).Flavanonol-4-reductase (DFR) and flavonol synthetic enzyme (FLS) competition flavanonol substrate.
Summary of the invention
The purpose of this invention is to provide flavanonol-4-reductase albumen and encoding gene thereof and application.
A kind of protein provided by the invention is flavanonol-4-reductase, derives from Saussurea medusa (Saussureamedusa Maxim), and called after SmDFR, this enzyme are following 1) or 2) protein:
1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
2) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) deutero-protein.
Wherein, sequence 2 is made up of 342 amino-acid residues in the sequence table, the replacement of described one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.
In order to make 1) in SmDFR be convenient to purifying, label as shown in table 1 on proteinic N-terminal that can the aminoacid sequence shown in the sequence 2 is formed in by sequence table or C-terminal connect.
The sequence of table 1. label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (being generally 5) | RRRRR |
| Poly-His | 2-10 (being generally 6) | HHHHHH |
| FLAG | 8 | DYKDDDDK |
| Strep-tag?II | 8 | WSHPQFEK |
| c- |
10 | EQKLISEEDL |
Above-mentioned 2) but in the SmDFR synthetic, also can synthesize its encoding gene earlier, carry out biology again and express and to obtain.Above-mentioned 2) encoding gene of the SmDFR in can be by lacking sequence in the sequence table 1 codon of one or several amino-acid residue in the dna sequence dna shown in the 5 ' terminal 61-1089, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in the table 1.
The proteic encoding gene of SmDFR also belongs to protection scope of the present invention.
Described encoding gene is following 1), 2), 3) or 4) or 5) shown in gene:
1) dna molecular shown in the sequence 1 in the sequence table;
2) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 61-1089 position Nucleotide;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 56th-1129 Nucleotide;
4) under stringent condition with 1) or 2) or 3) dna molecule hybridize that limits and dna molecular with identical function;
5) with 1) or 2) or 3) dna sequence dna that limits has 90% homology at least and have the dna molecular of identical function.
Gene in the described step 5) is with 1) or 2) or 3) gene homology more than 95% is preferably arranged.
Sequence 1 in the sequence table is the coding region by 1166 based compositions from 5 ' terminal 61-1089 position, the protein of sequence 2 in the code sequence tabulation.Above-mentioned stringent condition can be at 6 * SSC, in the solution of 0.5%SDS, 68 ℃ of hybridization down, uses 2 * SSC then, and 0.1%SDS and 1 * SSC, 0.1%SDS respectively wash film once.
The recombinant vectors, transgenic cell line, reorganization bacterium or the expression cassette that contain above-mentioned SmDFR encoding gene also belong to protection scope of the present invention.
Described recombinant vectors is for inserting the recombinant vectors that described encoding gene obtains between the Hind of carrier pYES2 III and EcoR I site.
Described reorganization bacterium is for importing the reorganization bacterium that the host bacterium obtains with described recombinant vectors.
Described host bacterium is yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
Available existing plant expression vector construction contains the recombinant expression vector of antisense SmDFR encoding gene.Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other plant expression vector of deriving.Conventional biological methods such as the plant expression vector that carries SmDFR encoding gene of the present invention can lead by Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity, agriculture bacillus mediated are transformed in vegetable cell or the tissue.
When using the SmDFR encoding gene to make up the recombinant plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) etc., they can use separately or be used in combination with other plant promoter; In addition, when using SmDFR encoding gene of the present invention to make up plant expression vector, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.
For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can in plant, express enzyme or the gene (gus gene, luciferase genes etc.) of luminophor, antibiotic marker thing (gentamicin marker, kantlex marker etc.) or the anti-chemical reagent marker gene (as anti-weedkiller gene) etc. that can produce colour-change with resistance as adding.
The application of above-mentioned protein s mDFR in external vat yellow ketone compounds, or the application of above-mentioned encoding gene SmDFR in external vat yellow ketone compounds also is the scope of protection of the invention; Described flavonoid compound is preferably the pure and mild 3-of flavanone and removes hydroxyl dihydro anthoxanthin alcohol; Described flavanonol is preferably dihydroquercetin, and described 3-goes hydroxyl dihydro anthoxanthin alcohol to be preferably eriodictyol.
Another object of the present invention provides the method for the transgenic plant of cultivating flavonol and/or the raising of flavonoid content.
Method provided by the invention is with the afunction of the described proteic encoding gene in the purpose plant, obtains transgenic plant; Flavonol alcohol and/or flavonoid content are higher than described purpose plant in the described transgenic plant;
With the method for the afunction of the described proteic encoding gene in the purpose plant by realizing to wherein changing recombinant expression vector over to; Described recombinant expression vector is to insert dna fragmentation to obtain in the multiple clone site of pCAMBIA1302 carrier, and described dna fragmentation is connected in sequence by the reverse fragment and the Nos terminator of CaMV 35S promoter, described proteic encoding gene;
The reverse fragment of the described proteic encoding gene of described claim 1 is the reverse complemental fragment from dna fragmentation shown in 5 ' the terminal 56-1129 position Nucleotide of sequence 1 in the sequence table.
The sequence of described CaMV 35S promoter is a 1-364 position Nucleotide among the genbank V00140.1; Described Nos terminator promoter sequence, 1-318 position Nucleotide among the genbank AJ007623.1.
Described purpose plant is a dicotyledons, and described dicotyledons is preferably Herba Saussureae Involueratae;
Described flavonol is rutin, myricetin, the luxuriant and rich with fragrance alcohol of camphane and/or Quercetin, and described flavonoid is an apigenin.
Of the present invention experimental results show that, by making up saussurea involucrata cDNA library, and utilize this library to screen the encoding gene of Saussurea medusa SmDFR by degenerated primer, the gene that process yeast expression and external enzymatic reaction are cloned into is the encoding gene that the flavanonol-4-reductase of function is arranged, utilize special primer to clone the encoding gene of SmDFR by PCR, and made up the recombinant expression vector that contains SmDFR, obtained antisense SmDFR Herba Saussureae Involueratae.The measurement result of antisense SmDFR Herba Saussureae Involueratae flavonol shows that antisense SmDFR Herba Saussureae Involueratae is compared with the Herba Saussureae Involueratae that changes empty carrier, and flavonol content such as rutin and myricetin are significantly improved.Explanation imports antisense SmDFR in the saussurea involucrata, can obtain the saussurea involucrata that flavonol content raises.Flavonol synthesis associated protein of the present invention and encoding gene thereof have important value to solving saussurea involucrata wild resource problem of shortage.
Description of drawings
Fig. 1 is the external catalysis dihydroquercetin of Saussurea medusa SmDFR
Fig. 2 is the external catalysis eriodictyol of Saussurea medusa SmDFR
Fig. 3 is the conversion and the transgenic plant regeneration of Herba Saussureae Involueratae embryo callus subculture
Fig. 4 is that antisense transgene saussurea involucrata PCR detects
Fig. 5 is the HPLC collection of illustrative plates of flavonoid in the Herba Saussureae Involueratae.
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The clone and the vivoexpression of embodiment 1, saussurea involucrata flavanonol-4-reductase gene (SmDFR)
1. the structure in saussurea involucrata cDNA library
Build storehouse used Saussurea medusa 1-15 days callus red colour system, induce by the following method: with Saussurea medusa (Saussurea medusa Maxim; Xie H H, Wang T, Matsuda H, et al.2005.Bioactiveconstituents from Chinese natural medicines XV
.1)Inhibitory effect on aldosereductase and structures of Saussureosides A and B from Saussurea medusa.ChemPharm Bull, 53 (11): 1416-1422; The public can obtain from Institute of Botany, Chinese Academy of Sciences.) stem and blade make explant, containing 0.2mg/L benzyladenine, 2mg/L naphthylacetic acid, 30g/L sucrose, induce callus on the MS substratum of pH value 5.8, and in the MS substratum that contains 2mg/L naphthylacetic acid, 0.5mg/L benzyladenine and 30g/L sucrose, 25 ± 1 ℃, cultivation, subculture expand numerous acquisition red colour system callus under 16h illumination/8h dark.
Take by weighing each 50mg of red colour system callus that cultivated 1-14 days respectively, amount to the 700mg biased sample in mortar, extract total RNA with TRIZOL Reagent (available from GIBCO company).Get the total RNA sample of 1-3 μ l, the synthetic first chain cDNA of the PolyT primer of the CDSIII restriction enzyme site that (CLONTECH) carries with SMART cDNALibrary Construction Kit (Catlog#:K1051-1).Adopt LD PCR (long distance PCR) to increase with cDNA first chain to obtain double-stranded cDNA.
Add the Taq enzyme among the protease K digesting removal cDNA, cut cDNA with Sfi I enzyme again, (Catlog#:636076 Clontech) carries out size separation to cDNA to use CHROMASPIN-400 then.Collect the cDNA of size＞500bp, the cDNA that collects is connected with λ TripIEx2 Vector (Promega), after the DNA that connects is packed by packaging protein Packagene Lambda DNA Packaging System (available from Promega company), it is 0.01% that the adding gelatin makes final concentration, the DMSO final concentration is 7%, can be constant in 1 year titre of-70 ℃ of following preservations.The library is divided into 20 Ya Wenku, avoids freezing repeatedly molten, each Ya Wenku comes from when original amplified library the regenerant of different culture dish, and it is formed may be different, can preserve, and so just finished the saussurea involucrata phage library and made up.
2. from saussurea involucrata cDNA library screening saussurea involucrata flavanonol-4-reductase gene cDNA
According to the conservative region (69aa altogether) of the gene nucleotide series of the DFR of the nearly source species (being mainly composite family) that landed on the Genbank, with the synthetic upstream and downstream part degenerated primer of software DNAMAN4.0 (U.S. Lynnon Biosoft company) design:
DFRgl：5’-GAG?AAT?GAA?GT(A/G)AT(A/C/T)AA(A/G)CC-3’，
DFRg2：5’-AAA?ATA?CAT?CCA?TCC(A/G/C/T)GT?CAT-3’。
It is template that each Ya Wenku gets 1 μ L, is primer with DFRg1, DFRg2, and 25 μ L reaction systems prior to 95 ℃ of heating 10min lytic phages, add the pcr amplification mixture, carry out pcr amplification.
10×PCR?Buffer 2.5μl
dNTPs 0.5μl
Taq (Beijing ancient cooking vessel state) 0.2 μ l
Primer?DFRg1 0.5μl
Primer?DFRg2 0.5μl
Model 3.0μl
ddH
2O 17.8μl
The PCR program:
95 ℃ of pre-sex change 10min of template (phage of 1 * λ dilution buffer liquid wash-out);
94 ℃, 4min (heat start PCR)
72℃， 10min
Choose the strongest positive pond of signal, make 10 times of gradient dilutions to 10
6Doubly, it is template that each grade dilution is got 1 μ L, carries out PCR by same program and detects, and determines the maximum dilution multiple N (can detect the Method of Limited Dilution multiple of positive signal) in positive pond at the corresponding levels when next stage screens.The elementary positive pond of choosing is diluted by its maximum dilution multiple N, get 1 μ L dilution ehec infection and 10 9cm LB flat boards of shop system, cultivate also recovery eluate.Picking growth and separate good single plaque from the flat board places damping fluid, and the vortex concussion is centrifugal slightly, in 4 ℃ of refrigerator overnight.Getting 3 μ L eluates is template, and PCR program augmentation detection obtains positive monoclonal, and the positive colony note is made pTripIEX2-SmDFR.
3.PCR method clone saussurea involucrata flavanonol-4-reductase gene SmDFR full-length cDNA
Phage vector goal gene when building the storehouse inserts segmental two ends and designs following two primers:
VEC1：5’-AAG?CGC?GCC?ATT?GTG?TTG-3’，
VEC2：5’-AAG?TGA?GCT?CGA?ATT?GCG?G-3’，
The PCR reaction conditions:
10×PCR?Buffer 2.5μl
dNTPs 0.5μl
Taq (Beijing ancient cooking vessel state) 0.2 μ l
Primer?VEC1 0.5μl
Primer?VEC2 0.5μl
Model 3.0μl
ddH
2O 17.8μl
The PCR program:
95 ℃ of pre-sex change 10min of template (phage of 1 * λ dilution buffer liquid wash-out);
94 ℃, 4min (heat start PCR);
72℃， 10min
PCR product electrophoresis detection, the clone obtains the fragment of size greater than 1000bp, and with this PCR product order-checking, this PCR product has sequence 1 in the sequence table (cDNA of gene) as a result.With the unnamed gene of this PCR product is SmDFR, and with the albumen called after SmDFR of this genes encoding, this proteic aminoacid sequence is the sequence 2 in the sequence table.The open reading frame of SmDFR is the 61-1089 position Nucleotide of sequence 1.
To adopting the BLAST similarity analysis, by relatively learning, aminoacid sequence after SmDFR full length cDNA sequence and the cDNA translation is very high with the plant similarity that belongs to composite family together, wherein the similarity of DFR gene in the spot Minor centaury (Centaurea maculosa) and aminoacid sequence is the highest, reaches 92%.Show that the SmDFR of being cloned into is the encoding gene of flavanonol-4-reductase from Saussurea medusa.
5.SmDFR vivoexpression and enzymatic reaction product identify
1) structure of pYES2-SmDFR expression vector
Extract the RNA of Saussurea medusa, reverse transcription becomes cDNA, is template with cDNA, and with primer SmDFR-Yf and SmDFR-Yr amplification, the recognition site of Hind III and EcoRI is introduced at the two ends of primer SmDFR-Yf and SmDFR-Yr respectively, and the sequence of primer is as follows:
SmDFR-Yf：5’-TCC?AAG?CTT?ACAACATGGTACAAGATTCTC-3’
SmDFR-Yr：5’-GCG?GAA?TTC?AGTACAATGAGACATAAATGT-3’
The PCR product detects through agarose gel electrophoresis, reclaim the also dna fragmentation of purifying 1092bp, cut this fragment with Hind III and EcoRI enzyme, fragment after agarose gel electrophoresis recovery enzyme is cut and pYES2 carrier (Invitrogen through same double digestion, Cat.no:v82520) connect, obtain recombinant plasmid pYES2-SmDFR.The recombinant plasmid pYES2-SmDFR transformed into escherichia coli DH-5 α that obtains, choose single bacterium colony with the 5ml liquid LB culture medium culturing extraction plasmid that spends the night through amicillin resistance screening, identify with HindIII and EcoRI digested plasmid DNA, obtain the positive pYES2-SmDFR of fragment of 1092bp, positive pYES2-SmDFR is sent to order-checking, sequencing result is: this positive plasmid contains the 56th-1129 Nucleotide (containing SmDFR) from 5 ' end of sequence 1 in the ordered list, proves positive pYES2-SmDFR.
2) pYES2-SmDFR transformed saccharomyces cerevisiae (Saccharomyces cerevisiae) INVSc1 and protein induce are expressed
(Invitrogen, Cat.no:C81000), bacterium INVSc1/pYES2-SmDFR obtains recombinating to adopt the LiAC method to change yeast saccharomyces cerevisiae (Saccharomycescerevisiae) INVSc1 over to the positive pYES2-SmDFR of above-mentioned acquisition.The plasmid that will extract from the reorganization bacterium is a template, obtain purpose fragment (1092bp with primer SmDFR-Yf and SmDFR-Yr amplification checking, sequence 1 from 5 ' the 56th terminal-1129 Nucleotide), the bacterium called after positive of should the recombinate bacterium INVSc1/pYES2-SmDFR that recombinates.
Should carry out protein semi-lactosi abduction delivering by positive reorganization bacterium INVSc1/pYES2-SmDFR,, extract the yeast total protein behind the granulated glass sphere broken wall, obtain the SmDFR protein crude extract according to the pYES2 product description of Invitrogen company.
Adopt above-mentioned method that empty carrier pYES2 carrier is changed among yeast saccharomyces cerevisiae (Saccharomycescerevisiae) INVSc1, the INVSc1/pYES2 of acquisition, same abduction delivering obtains the protein crude extract of empty carrier.
3) external enzymatic reaction
With dihydroquercetin (Dihydroquercetin, CAS24198-97-8, SIGMA-ALDRICH T4512-25MG) and eriodictyol (3-Deoxydihydroquercetin, CAS552-58-9, the inferior training in Shanghai bio tech ltd) be substrate, reaction solution cumulative volume 500 μ l, comprising:
100mM Tris-HCl damping fluid (pH=7.5) 370 μ l
SmDFR protein crude extract
(0.15 μ g μ l
-1, by above-mentioned acquisition.) 70μl
10mM NADPH (is dissolved in 100mM Tris-HCl, pH=7.5) 50 μ l
10 μ g μ l
-1Substrate (being dissolved in methyl alcohol) 10 μ l
1. reaction solution is placed 30 ℃ of water-baths of 1.5ml centrifuge tube to be incubated 4h.
2. reaction solution merges mutually with 400 μ l ethyl acetate extractions, twice, twice supernatant ethyl acetate and transfers in the new centrifuge tube uncovered being positioned in 50 ℃ of baking ovens the ethyl acetate evaporate to dryness.
3. residue is with 50 μ l propyl carbinols: (95 ℃ are boiled 5min to hydrochloric acid for 95:5, the v/v) dissolving again of enzymatic preparation, are used for HPLC-MS and identify.
4) HPLC-MS of external enzymatic reaction product identifies
Identify product with Agilent 1100 HPLC/MSD Trap VL.Chromatographic column: ZORBAX Eclipse XDB-C18,4.6 * 150mm, 5 μ m (Agilent Co, USA), 30 ℃ of column temperatures, sample size 10 μ l, flow velocity 1ml min
-1Moving phase: solution A acetonitrile, solution B pH 3.0 trifluoroacetic acids (TFA) water.Elution program: 0-20min:A 90%-10%, B 10%-90%.Detect wavelength 214nm.
Mass spectrum parameter: electron spray(ES) (ESI) ion trap, 325 ℃ of gasification temperatures, carrier gas (N
2) flow velocity 10.01min
-1, nebulizer gas pressure 40psi, ends of the earth radio frequency amplitude (octopole RF amplitude) 150vpp, skim 1 voltage34V, skim 2 voltage 6V, capillary exit 107V, cap exit offset 73V, MSn collision energy 40%.Analyze and adopt positive ion preference pattern (m/z M+H
+), full ion scan, ion range of choice m/z50-1000.
The result as depicted in figs. 1 and 2, A is the total ion figure behind the external catalysis dihydroquercetin of Saussurea medusa SmDFR among Fig. 1, the arrow indication is a substrate; B. the product Cyanidin aglycon (arrow indication) (m/z 287) of Sheng Chenging; C. the mass-spectrogram of Cyanidin aglycon.Total ion figure among Fig. 2 behind the external catalysis eriodictyol of A. Saussurea medusa SmDFR, the arrow indication is a substrate; B. the product of Sheng Chenging (arrow indication) (m/z 271); C. the mass-spectrogram of product.Show that from the peak sequence of the passable product of Fig. 1 and mass spectrum the substrate dihydroquercetin has been generated the Cyanidin aglycon by chemical catalysis external again after by the SmDFR reduction, dihydroquercetin is a kind of of flavonol; Show that from the peak sequence and the mass spectrum of the product of Fig. 2 the substrate eriodictyol has been generated (-)-Leucofisetinidin by chemical catalysis external again after by the SmDFR reduction, eriodictyol is that 3-goes a kind of of hydroxyl dihydro anthoxanthin alcohol.Show that SmDFR can generate corresponding leucoanthocyanidin by external catalysis flavanonol substrate, SmDFR has the activity and the flavanone reductase enzyme (Flavanone4-reductase of flavanonol-4-reductase, it is FNR) active that (dihydroquercetin is the most frequently used substrate of flavanonol-4-reductase, SmDFR can just show that it has the flavanonol-4-reductase function by the catalysis dihydroquercetin, SmDFR also may have flavanone reductase enzyme function in addition, remove hydroxyl dihydro anthoxanthin alcohol with 3-, be that flavanone is a substrate, generate an other compounds.)。
Protein crude extract with the empty carrier of above-mentioned acquisition serves as that contrast detects, and the result is not for there being activity.
Embodiment 2 uses antisense Saussurea medusa flavanonol-4-reductase gene and improves Herba Saussureae Involueratae flavonol content
1.pCAMBIA1301-the structure of antisense SmDFR expression vector
The cDNA of the Saussurea medusa that obtains with embodiment is a template, the recognition site of BamHI and Sac I is introduced at the two ends of primer DFRa1 and DFRa2 respectively, and the sequence of primer DFRa1 and DFRa2 is as follows: DFRa1:5 '-TAC gag ctc ACAACA TGG TAC AAG ATT CTC-3 '; DFRa2:5 '-GCG gga tcc AGT ACA ATG AGA CAT AAATGT-3 ';
The PCR product detects through agarose gel electrophoresis, reclaim and purifying DNA fragment, cut this fragment with Sac I and BamHI enzyme, the fragment that agarose gel electrophoresis reclaims after enzyme is cut is connected with pBI121 (Clontech) carrier of the same double digestion of warp, SmDFR cDNA oppositely is inserted between pBI121 support C aMV 35S promoter and the Nos terminator, obtains recombinant plasmid pBI121-antisense SmDFR.With recombinant plasmid pBI121-antisense SmDFR transformed into escherichia coli DH-5 α, kantlex screening recon, extract plasmid and identify that with Sac I and BamHI double digestion positive recombinant is sent to order-checking, and the result is for obtaining containing the reverse segmental positive recombinant of SmDFR cDNA.
PBI121-antisense SmDFR carrier is cut through EcoRI and PstI enzyme, antisense SmDFR is downcut together with CaMV 35S promoter and Nos terminator, product detects through agarose gel electrophoresis, reclaim and purifying fragment (2100bp), with this fragment and pCAMBIA1302 (the Cambia Institute that cuts through EcoRI and PstI enzyme, Austrilia) carrier connects, and obtains recombinant expression vector pCAMBIA1302-antisense SmDFR.
The sequence of described CaMV 35S promoter is a 1-364 position Nucleotide among the genbank V00140.1; Described Nos terminator promoter sequence, 1-318 position Nucleotide among the genbank AJ007623.1.
With recombinant plasmid transformed intestinal bacteria DH-5 α, kantlex and hygromycin selection recon extract plasmid and cut evaluation with EcoRI and PstI enzyme.With the frozen-thawed method pCAMBIA1302-antisense SmDFR is imported Agrobacterium EHA105 (Invitrogen), obtain recon, with kantlex, Totomycin and Rifampin screening recon, with single bacterium colony is template, CaMV 35S primer (5 '-CCCACTATCCTTCG-3 ') and DFRa1 are that primer PCR is identified, obtain the positive single bacterium colony of fragment of 1100bp, called after EHA105/pCAMBIA1302-antisense SmDFR.
2. the Herba Saussureae Involueratae embryo callus subculture induces
Herba Saussureae Involueratae (Saussurea involucrata Kar.et Kir., Yi T, Chen H B, Zhao Z Z, etal.2009.Identification and determination of the major constituents in thetraditional uighur medicinal plant Saussurea involucrata by LC-DAD-MS.Chromatographia, 69:537-542; The public can obtain from Institute of Botany, Chinese Academy of Sciences.) aseptically process of seed: the Herba Saussureae Involueratae seed is degerming 20min in 0.3% chlorine bleach liquor, aseptic water washing 5-6 time, the Herba Saussureae Involueratae seed of pouring out after the sterilized water degerming is carefully peelled off kind of a skin with tweezers and inoculating needle, place MS+5mg/L 2,4-D+0.5mg/L6-BA substratum on evoked callus, callus growth is good back in MS+3mg/L 2, succeeding transfer culture on the substratum of 4-D+0.5mg/L 6-BA.The Herba Saussureae Involueratae rataria was cultivated on inducing culture 40 days, and per 20 days subcultures once can grow the yellowish callus of color and luster.
3. the acquisition of antisense SmDFR Herba Saussureae Involueratae
Single bacterium colony of picking EHA105/pCAMBIA1302-antisense SmDFR, it is inoculated in the YEB liquid nutrient medium (contains Kan (kantlex, Kanamycin) 100mg/L, Hyg (Totomycin, Hygromycin B) 50mg/L, Rif (Rifampin, Rifampicin) 50mg/L), 28 ℃ of shaking culture are spent the night.Agrobacterium (EHA105/pCAMBIA1302-antisense SmDFR) the bacterium liquid of drawing 200 μ l incubated overnight (contains Kan100mg/L in 20ml YEB liquid nutrient medium, Hyg50mg/L, Rif50mg/L), 28 ℃ of quick oscillation incubation growth are to logarithmic phase (OD600=0.6), about 3-4h.The centrifugal 5min of room temperature 6000rpm collects thalline, uses MS
0Liquid nutrient medium is washed the thalline of once collecting, and guarantees not contain microbiotic in the substratum, then thalline is suspended in 10ml MS again
0Stand-by in the liquid nutrient medium.
Select well-grown Herba Saussureae Involueratae callus, take out with the tweezers after the calcination, place ready bacterium liquid, contaminate 30min, pour out bacterium liquid, callus moves to MS+3mg/L 2, and dark the cultivation (measured too much for preventing bacterium in 48 hours on the 4-D+0.5mg/L 6-BA substratum, disturb the later stage degerming, can put one deck filter paper) in media surface.At last material is moved to MS+3mg/L 2, screening and culturing on the 4-D+0.5mg/L 6-BA+Hyg40mg/L+Cef 400mg/L substratum.Can see after 20 days that green callus grows, the well-grown green callus that filters out is inoculated on the substratum of MS+1mg/L 6-BA+0.1mg/LNAA+Hyg40mg/L and induce 17 strain antisense SmDFR Herba Saussureae Involueratae seedlings.
Herba Saussureae Involueratae is seen shown in Figure 3 from embryo callus subculture to the process that differentiates seedling.
Adopt identical method that empty carrier pCAMBIA1302 is changed in the Herba Saussureae Involueratae, obtain to change the empty carrier Herba Saussureae Involueratae.
4. the detection of transgenosis Herba Saussureae Involueratae
The antisense SmDFR Herba Saussureae Involueratae seedling of above-mentioned acquisition is extracted genomic dna with the CTAB method, with the genomic dna is template, with CaMV 35S primer and DFRa1 is whether primer PCR detection Saussurea medusa SmDFR cDNA has been incorporated in the Herba Saussureae Involueratae genome, detected result as shown in Figure 4, C+ wherein: positive control is a template with pCAMBIA1302-antisense SmDFR; C-: negative control is a template with wild-type Herba Saussureae Involueratae genomic dna; M:2000PlusDNA Marker; A3, A5, A6, A8, A11, A12, A13, A16, A22, A23, A24, A26, A27, A30, A24, A36, A39 are T0 for antisense SmDFR Herba Saussureae Involueratae seedling, are template with T0 for antisense SmDFR Herba Saussureae Involueratae genomic dna.As can be seen from the figure, obtain the positive antisense SmDFR of 17 strains Herba Saussureae Involueratae seedling, show that SmDFRcDNA has been incorporated in the Herba Saussureae Involueratae genome.
5. the detection of transgenosis Herba Saussureae Involueratae flavonol content
1) gets and obtain antisense SmDFR Herba Saussureae Involueratae seedling system behind the antisense SmDFR Herba Saussureae Involueratae seedling subculture, the 10d sampling, each is four bottles, from every bottle, select the antisense SmDFR Herba Saussureae Involueratae seedling of robust growth, remove yellow leaf of cured leaf leaf and bottom callus, choose about tissue cultured seedling fresh weight 100mg for every bottle, put into mortar at the liquid nitrogen grind into powder, add 100 μ l, 80% methyl alcohol (v/v) according to every 10mg fresh weight blade, change in the 2ml centrifuge tube, fully mixing extracts 24h under the room temperature, stirs centrifuge tube up and down once every several hours therebetween.
2) with under the extracting solution room temperature 12, the centrifugal 10min of 000rpm carefully collects supernatant liquor in the new centrifuge tube, and the precipitation of gained is used again under half 80% methyl alcohol (v/v) room temperature of former extracting liquid volume and extracted 24h.
3) under the room temperature, the centrifugal 10min of 12000rpm collects supernatant, and twice supernatant liquor merged, and detects with being used for HPLC behind the 0.22 μ m membrane filtration.
HPLC detect parameters: chromatographic column: ZORBAX Eclipse XDB-C18,2.1 * 150mm, 5 μ m (Agilent Co, USA), 30 ℃ of column temperatures, sample size 5 μ l, flow velocity 0.25ml min
-1Moving phase: solution A 0.20% phosphate aqueous solution, solution B methyl alcohol.Elution program: 0-3min, 95%A, 5%B; 3-20min, A 95%-65%, B 5%-35%; 20-30min, A 65%-60%, B 35%-40%; 30-38min:A 60%, and B 40%; 38-45min:A 60%-80%, B 40%-20%; 45-49min:A 80%, and B 20%; 49-50min:A 80%-95%, B 20%-5%; 50-70min:A 95%, B5%.Detect wavelength 270nm.
Under as above chromatographic condition, flavonoid standard substance (apigenin SIGMA-ALDRICH 460475; The luxuriant and rich with fragrance pure SIGMA-ALDRICH K1003 of camphane; Quercetin SIGMA-ALDRICH Q0125; Myricetin M6760; Rutin SIGMA-ALDRICHR5143) retention time is shown in Fig. 5 A.The retention time of various flavonol is respectively rutin 27.6min in the test sample, myricetin 29.2min, Quercetin 32.3min, the luxuriant and rich with fragrance pure 34.7min of camphane; The retention time of the apigenin in the flavonoid (derivative of the luxuriant and rich with fragrance alcohol of camphane) is 35.1min in addition.
To change empty carrier Herba Saussureae Involueratae and wild-type (Herba Saussureae Involueratae) is contrast, and experiment repeats four times, results averaged.
The result as shown in Figure 5, A wherein: flavonoid standard substance; B:SmDFR-A39 (being numbered the antisense SmDFR Herba Saussureae Involueratae seedling strain system of A39); C: wild-type contrast.The retention time of SmDFR-A39 is shown in Fig. 5 B, and the retention time of various flavonol is respectively rutin 27.5min in the test sample, myricetin 28.9min, Quercetin 32.8min, the luxuriant and rich with fragrance pure 34.4min of camphane; The retention time of the apigenin in the flavonoid (derivative of the luxuriant and rich with fragrance alcohol of camphane) is 35.3min in addition.The retention time of wild-type contrast is shown in Fig. 5 C, and the retention time of various flavonol is respectively rutin 27.6min in the test sample, myricetin 29.1min, Quercetin 31.7min.
Show rutin among the SmDFR-A39 among the figure, the content of flavonol such as myricetin and/or flavonoid significantly raises than the wild-type contrast, concrete calculating as can be known, the rutin of wild-type contrast is 25.16 ± 6.68 μ g/g fresh weights, myricetin is 22.43 ± 4.57 μ g/g fresh weights, the luxuriant and rich with fragrance alcohol of camphane is 9.23 ± 0.49 μ g/g fresh weights, Quercetin is 0.78 ± 1.35 μ g/g fresh weight, apigenin is 3.68 ± 1.19 μ g/g fresh weights, and the rutin of SmDFR-A39 is 71.15 ± 17.64 μ g/g fresh weights, myricetin is 54.30 ± 9.81 μ g/g fresh weights, the luxuriant and rich with fragrance alcohol of camphane is 13.41 ± 4.07 μ g/g fresh weights, Quercetin is 10.76 ± 2.71 μ g/g fresh weights and apigenin 16.47 ± 12.07 μ g/g fresh weights.
The result who changes the contrast of empty carrier Herba Saussureae Involueratae and wild-type does not have marked difference.
Show that importing antisense Saussurea medusa SmDFR gene makes flavonol content increase in the Herba Saussureae Involueratae.Effective gene in the flavones pathways metabolism being imported in the Herba Saussureae Involueratae, and then generate high yield flavones transgenic seedling, carry out artificial culture, will be the effective way that solves the shortage of saussurea involucrata wild resource.
Claims (9)
1. a protein is following 1) or 2) protein:
1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;
2) with the aminoacid sequence of sequence 2 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and have identical function by 1) deutero-protein.
2. the described proteinic encoding gene of claim 1.
3. according to claim 1 or 2 described encoding genes, it is characterized in that: described encoding gene is following 1), 2), 3) or 4) or 5) shown in gene:
1) dna molecular shown in the sequence 1 in the sequence table;
2) in the sequence table sequence 1 from the dna molecular shown in 5 ' the terminal 61-1089 position Nucleotide;
3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 56th-1129 Nucleotide;
4) under stringent condition with 1) or 2) or 3) dna molecule hybridize that limits and dna molecular with identical function;
5) with 1) or 2) or 3) dna sequence dna that limits has 90% homology at least and have the dna molecular of identical function.
4. the recombinant vectors, transgenic cell line, reorganization bacterium or the expression cassette that contain claim 2 or 3 described encoding genes.
5. recombinant vectors according to claim 4 or reorganization bacterium is characterized in that: described recombinant vectors is for inserting the recombinant vectors that claim 2 or 3 described encoding genes obtain in the multiple clone site of carrier pYES2;
Described reorganization bacterium is for importing the reorganization bacterium that the host bacterium obtains with the described recombinant vectors of claim 4;
Described host bacterium is preferably yeast saccharomyces cerevisiae (Saccharomyces cerevisiae).
6. the application of the described protein of claim 1 in external vat yellow ketone compounds, or claim 2 or the application of 3 described encoding genes in external vat yellow ketone compounds; Described flavonoid compound is preferably the pure and mild 3-of flavanone and removes hydroxyl dihydro anthoxanthin alcohol; Described flavanonol is preferably dihydroquercetin, and described 3-goes hydroxyl dihydro anthoxanthin alcohol to be preferably eriodictyol.
7. a transgenic plant method of cultivating flavonol and/or the raising of flavonoid content is with the afunction of the described proteic encoding gene of claim 1 in the purpose plant, obtains transgenic plant; Flavonol and/or flavonoid content are higher than described purpose plant in the described transgenic plant.
8. method according to claim 7 is characterized in that: with the method for the afunction of the described proteic encoding gene of claim 1 in the purpose plant by realizing to wherein changing recombinant expression vector over to; Described recombinant expression vector is to insert dna fragmentation to obtain in the multiple clone site of pCAMBIA1302 carrier, and described dna fragmentation is connected in sequence by the reverse fragment and the Nos terminator of CaMV 35S promoter, the described proteic encoding gene of claim 1;
The reverse fragment of the described proteic encoding gene of described claim 1 is the reverse complemental fragment from dna fragmentation shown in 5 ' the terminal 56-1129 position Nucleotide of sequence 1 in the sequence table.
9. method according to claim 8 is characterized in that: described purpose plant is a dicotyledons, and described dicotyledons is preferably Herba Saussureae Involueratae; Described flavonol is rutin, myricetin, the luxuriant and rich with fragrance alcohol of camphane and/or Quercetin; Described flavonoid is an apigenin.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102263446A CN101921735B (en) | 2010-07-06 | 2010-07-06 | Encoding gene of Saussurea involucrate flavanonol-4-reductase and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2010102263446A CN101921735B (en) | 2010-07-06 | 2010-07-06 | Encoding gene of Saussurea involucrate flavanonol-4-reductase and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN101921735A true CN101921735A (en) | 2010-12-22 |
| CN101921735B CN101921735B (en) | 2012-06-27 |
Family
ID=43336952
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2010102263446A Expired - Fee Related CN101921735B (en) | 2010-07-06 | 2010-07-06 | Encoding gene of Saussurea involucrate flavanonol-4-reductase and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101921735B (en) |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181452A (en) * | 2011-03-28 | 2011-09-14 | 四川农业大学 | Goat TNNT3 (human fast skeletal troponin T) gene and method for cloning complete sequence of gene coding region of goat TNNT3 gene |
| CN102181451A (en) * | 2011-03-28 | 2011-09-14 | 四川农业大学 | Goat TNNC1 gene and method for obtaining complete coding sequence of goat TNNC1 gene through cloning |
| CN103897993A (en) * | 2014-03-04 | 2014-07-02 | 安徽农业大学 | Saccharomyces cerevisiae gene engineering strain, construction method of same and method for producing eriodictyol or quercetin |
| CN105837671A (en) * | 2016-05-06 | 2016-08-10 | 山东农业大学 | Flavonol adjusting and controlling protein MsMYB22 obtained from functional apples and coding gene and application thereof |
| CN105296510B (en) * | 2015-12-07 | 2016-08-24 | 湖南农业大学 | Herba Artemisiae annuae flavanone alcohol oxidase gene AaDHFO2 and encoding proteins thereof and application |
| CN107557384A (en) * | 2017-09-12 | 2018-01-09 | 黔南民族师范学院 | A kind of genetic conversion system for inducing plant to downgrade and its structure and application |
| CN113755460A (en) * | 2021-09-10 | 2021-12-07 | 浙江华睿生物技术有限公司 | Flavone reductase for preparing dihydroquercetin |
| CN117004628A (en) * | 2023-08-07 | 2023-11-07 | 中国科学院青岛生物能源与过程研究所 | Saussurea involucrata flavonol synthase gene and its coding product and use |
-
2010
- 2010-07-06 CN CN2010102263446A patent/CN101921735B/en not_active Expired - Fee Related
Non-Patent Citations (4)
| Title |
|---|
| 《中国农学通报》 20091105 侯夫云等 植物花青素合成酶的研究进展 , 第21期 2 * |
| 《应用基础与工程科学学报》 19970630 颜华，李毅，宋云，邵莉，梁晓文，陈章良 二羟基黄酮醇还原酶基因的克隆、全序列分析及在大肠杆菌中的表达 , 第02期 2 * |
| 《现代化工》 19981220 孙淑香 二氢黄酮醇的合成 , 第12期 2 * |
| > 20070611 Lu,X. EF600682 1 1-4 , 2 * |
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102181452A (en) * | 2011-03-28 | 2011-09-14 | 四川农业大学 | Goat TNNT3 (human fast skeletal troponin T) gene and method for cloning complete sequence of gene coding region of goat TNNT3 gene |
| CN102181451A (en) * | 2011-03-28 | 2011-09-14 | 四川农业大学 | Goat TNNC1 gene and method for obtaining complete coding sequence of goat TNNC1 gene through cloning |
| CN103897993A (en) * | 2014-03-04 | 2014-07-02 | 安徽农业大学 | Saccharomyces cerevisiae gene engineering strain, construction method of same and method for producing eriodictyol or quercetin |
| CN105296510B (en) * | 2015-12-07 | 2016-08-24 | 湖南农业大学 | Herba Artemisiae annuae flavanone alcohol oxidase gene AaDHFO2 and encoding proteins thereof and application |
| CN105837671A (en) * | 2016-05-06 | 2016-08-10 | 山东农业大学 | Flavonol adjusting and controlling protein MsMYB22 obtained from functional apples and coding gene and application thereof |
| CN105837671B (en) * | 2016-05-06 | 2019-02-12 | 山东农业大学 | Obtained from the flavonols modulin MsMYB22 and its encoding gene of functional form apple and application |
| CN107557384A (en) * | 2017-09-12 | 2018-01-09 | 黔南民族师范学院 | A kind of genetic conversion system for inducing plant to downgrade and its structure and application |
| CN107557384B (en) * | 2017-09-12 | 2020-09-01 | 黔南民族师范学院 | Genetic transformation system for inducing plant dwarfing and construction and application thereof |
| CN113755460A (en) * | 2021-09-10 | 2021-12-07 | 浙江华睿生物技术有限公司 | Flavone reductase for preparing dihydroquercetin |
| CN117004628A (en) * | 2023-08-07 | 2023-11-07 | 中国科学院青岛生物能源与过程研究所 | Saussurea involucrata flavonol synthase gene and its coding product and use |
| CN117004628B (en) * | 2023-08-07 | 2024-01-23 | 中国科学院青岛生物能源与过程研究所 | Saussurea involucrata flavonol synthase gene and its coding product and use |
Also Published As
| Publication number | Publication date |
|---|---|
| CN101921735B (en) | 2012-06-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101921735B (en) | Encoding gene of Saussurea involucrate flavanonol-4-reductase and application thereof | |
| Shi et al. | Targeted metabolic engineering of committed steps improves anti-cancer drug camptothecin production in Ophiorrhiza pumila hairy roots | |
| CN109295080B (en) | Application of rhizoma panacis majoris beta-balsamol synthetase gene Pj beta-AS | |
| CN109055399B (en) | Gene sequence related to flavone composition in scutellaria baicalensis and application thereof | |
| CN101220353B (en) | Glycyrrhiza uralensis chalcone synthetase, encoding gene and application thereof | |
| CN112724217B (en) | Sweet wormwood MYB transcription factor AaMYB108 and application thereof | |
| CN113549630B (en) | Ginseng PgJAZ1 gene, method for improving protopanaxatriol saponin based on gene and application | |
| Yao et al. | Hairy root induction and polysaccharide production of medicinal plant Callerya speciosa Champ. | |
| CN105441462B (en) | A kind of Radix Notoginseng transcription factor gene PnERF1 and its application | |
| CN114645061B (en) | SmMYB76 gene and application thereof in improving salvianolic acid content in salvia miltiorrhiza bunge | |
| CN114634939A (en) | PgJMT1 gene for regulating synthesis of methyl jasmonate in ginseng and application thereof | |
| CN104561056B (en) | The '-hydroxylase gene AaF3HY of artemisia annua flavanones 3 and its encoding proteins and application | |
| CN103834685A (en) | Carrier capable of simultaneously improving rosmarinic acid and salvianolic acid B content of red sage root and use thereof | |
| Xia et al. | Structural and interactions analysis of a transcription factor PnMYB2 in Panax notoginseng | |
| KR20020013508A (en) | Multiple component rna vector system for expression of foreign sequences | |
| CN117143852A (en) | Histone deacetylase SmHDA6 and application of encoding gene thereof in regulation and control of salvianolic acid accumulation | |
| CN101942420A (en) | Protein related to synthesis of flavone compounds, coding gene and application thereof | |
| CN112301038B (en) | Ginseng WRKY64-04 gene and application thereof | |
| CN102021181B (en) | Application of paddy gene KT488 in improvement on stress resistance of plants | |
| Yang et al. | Establishment of recombinant Catharanthus roseus stem cells stably overexpressing ORCA4 for terpenoid indole alkaloids biosynthesis | |
| CN106636142A (en) | Clone identification and application of 2-oxoglutarate-dependent dioxygenase (2OGD-5) gene participating in tanshinone synthesis | |
| CN101693739A (en) | Chlorogenic acid synthesis associated protein and encoding gene thereof and application | |
| CN114058632A (en) | Gene PnCOX11 and application thereof in regulating and controlling synthesis of notoginsenoside | |
| CN104531753B (en) | The method that cotransformation Sps, Hmgr and Dxs gene cultivate bud Gaoqing punt-pole cellulose content sweet wormwood | |
| CN105950644A (en) | Chalcone isomerase gene of asparaguses, protein encoded by chalcone isomerase gene and application of chalcone isomerase gene |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| DD01 | Delivery of document by public notice |
Addressee: Guan Jianli Document name: Notification that Application Deemed not to be Proposed |
|
| ASS | Succession or assignment of patent right |
Owner name: NANJING QIANPENG BIOTECHNOLOGY CO., LTD. Free format text: FORMER OWNER: PLANT INST., CHINESE ACADEMY OF SCIENCES Effective date: 20140725 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| COR | Change of bibliographic data |
Free format text: CORRECT: ADDRESS; FROM: 100093 HAIDIAN, BEIJING TO: 210042 NANJING, JIANGSU PROVINCE |
|
| TR01 | Transfer of patent right |
Effective date of registration: 20140725 Address after: 210042, Room 305, room 3, building 699-8, Xuanwu Avenue, Nanjing, Jiangsu 2, China Patentee after: Nanjing Qian Peng Biological Technology Co. Ltd. Address before: Study on China Plant Science in Beijing city 100093 Haidian District Xiangshan No. 20 Nanxin Village Development Center Patentee before: Plant Inst., Chinese Academy of Sciences |
|
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20120627 Termination date: 20160706 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |