CN104878025B - 一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用 - Google Patents
一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用 Download PDFInfo
- Publication number
- CN104878025B CN104878025B CN201510297824.4A CN201510297824A CN104878025B CN 104878025 B CN104878025 B CN 104878025B CN 201510297824 A CN201510297824 A CN 201510297824A CN 104878025 B CN104878025 B CN 104878025B
- Authority
- CN
- China
- Prior art keywords
- csugt84a22
- genes
- acid
- based transferase
- encoding proteins
- 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
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 63
- GDVRUDXLQBVIKP-HQHREHCSSA-N 1-O-galloyl-beta-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC(=O)C1=CC(O)=C(O)C(O)=C1 GDVRUDXLQBVIKP-HQHREHCSSA-N 0.000 title claims abstract description 25
- 229920000296 Glucogallin Polymers 0.000 title claims abstract description 25
- 102000004357 Transferases Human genes 0.000 title claims abstract description 22
- 108090000992 Transferases Proteins 0.000 title claims abstract description 22
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 21
- PFTAWBLQPZVEMU-DZGCQCFKSA-N (+)-catechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@@H]2O)=CC=C(O)C(O)=C1 PFTAWBLQPZVEMU-DZGCQCFKSA-N 0.000 claims abstract description 17
- ADRVNXBAWSRFAJ-UHFFFAOYSA-N catechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3ccc(O)c(O)c3 ADRVNXBAWSRFAJ-UHFFFAOYSA-N 0.000 claims abstract description 16
- 235000005487 catechin Nutrition 0.000 claims abstract description 16
- 229950001002 cianidanol Drugs 0.000 claims abstract description 16
- 150000002148 esters Chemical class 0.000 claims abstract description 14
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 3
- 241000894006 Bacteria Species 0.000 abstract description 19
- 239000013612 plasmid Substances 0.000 abstract description 14
- 102000007056 Recombinant Fusion Proteins Human genes 0.000 abstract description 13
- 108010008281 Recombinant Fusion Proteins Proteins 0.000 abstract description 13
- 240000003152 Rhus chinensis Species 0.000 abstract description 12
- 235000014220 Rhus chinensis Nutrition 0.000 abstract description 12
- -1 acyl β D glucosides Chemical class 0.000 abstract description 9
- HSCJRCZFDFQWRP-RDKQLNKOSA-N UDP-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)OC1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-RDKQLNKOSA-N 0.000 abstract description 7
- 230000009261 transgenic effect Effects 0.000 abstract description 5
- 230000001851 biosynthetic effect Effects 0.000 abstract description 2
- 238000012407 engineering method Methods 0.000 abstract description 2
- 239000002773 nucleotide Substances 0.000 abstract description 2
- 125000003729 nucleotide group Chemical group 0.000 abstract description 2
- 238000011160 research Methods 0.000 abstract description 2
- LNTHITQWFMADLM-UHFFFAOYSA-N gallic acid Chemical compound OC(=O)C1=CC(O)=C(O)C(O)=C1 LNTHITQWFMADLM-UHFFFAOYSA-N 0.000 description 13
- PCMORTLOPMLEFB-ONEGZZNKSA-N sinapic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-ONEGZZNKSA-N 0.000 description 12
- 239000000758 substrate Substances 0.000 description 12
- JMSVCTWVEWCHDZ-UHFFFAOYSA-N syringic acid Chemical compound COC1=CC(C(O)=O)=CC(OC)=C1O JMSVCTWVEWCHDZ-UHFFFAOYSA-N 0.000 description 12
- NGSWKAQJJWESNS-UHFFFAOYSA-N 4-coumaric acid Chemical compound OC(=O)C=CC1=CC=C(O)C=C1 NGSWKAQJJWESNS-UHFFFAOYSA-N 0.000 description 10
- 239000002253 acid Substances 0.000 description 10
- QAIPRVGONGVQAS-DUXPYHPUSA-N trans-caffeic acid Chemical compound OC(=O)\C=C\C1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-DUXPYHPUSA-N 0.000 description 10
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 9
- 102000004190 Enzymes Human genes 0.000 description 8
- 108090000790 Enzymes Proteins 0.000 description 8
- 150000001272 acylglucoses Chemical class 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- XMOCLSLCDHWDHP-IUODEOHRSA-N epi-Gallocatechin Chemical compound C1([C@H]2OC3=CC(O)=CC(O)=C3C[C@H]2O)=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-IUODEOHRSA-N 0.000 description 8
- 239000000047 product Substances 0.000 description 8
- 238000000746 purification Methods 0.000 description 8
- 239000000243 solution Substances 0.000 description 8
- WBYWAXJHAXSJNI-VOTSOKGWSA-M .beta-Phenylacrylic acid Natural products [O-]C(=O)\C=C\C1=CC=CC=C1 WBYWAXJHAXSJNI-VOTSOKGWSA-M 0.000 description 6
- WBYWAXJHAXSJNI-SREVYHEPSA-N Cinnamic acid Chemical compound OC(=O)\C=C/C1=CC=CC=C1 WBYWAXJHAXSJNI-SREVYHEPSA-N 0.000 description 6
- 244000269722 Thea sinensis Species 0.000 description 6
- 238000004458 analytical method Methods 0.000 description 6
- 238000006243 chemical reaction Methods 0.000 description 6
- 229930016911 cinnamic acid Natural products 0.000 description 6
- 235000013985 cinnamic acid Nutrition 0.000 description 6
- 150000001875 compounds Chemical class 0.000 description 6
- 229930003935 flavonoid Natural products 0.000 description 6
- 235000017173 flavonoids Nutrition 0.000 description 6
- 150000002215 flavonoids Chemical class 0.000 description 6
- 229940074391 gallic acid Drugs 0.000 description 6
- 235000004515 gallic acid Nutrition 0.000 description 6
- WBYWAXJHAXSJNI-UHFFFAOYSA-N methyl p-hydroxycinnamate Natural products OC(=O)C=CC1=CC=CC=C1 WBYWAXJHAXSJNI-UHFFFAOYSA-N 0.000 description 6
- PCMORTLOPMLEFB-UHFFFAOYSA-N sinapinic acid Natural products COC1=CC(C=CC(O)=O)=CC(OC)=C1O PCMORTLOPMLEFB-UHFFFAOYSA-N 0.000 description 6
- 230000001954 sterilising effect Effects 0.000 description 6
- 238000003756 stirring Methods 0.000 description 6
- YIBXWXOYFGZLRU-UHFFFAOYSA-N syringic aldehyde Natural products CC12CCC(C3(CCC(=O)C(C)(C)C3CC=3)C)C=3C1(C)CCC2C1COC(C)(C)C(O)C(O)C1 YIBXWXOYFGZLRU-UHFFFAOYSA-N 0.000 description 6
- WMBWREPUVVBILR-WIYYLYMNSA-N (-)-Epigallocatechin-3-o-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=C(O)C=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-WIYYLYMNSA-N 0.000 description 5
- ACEAELOMUCBPJP-UHFFFAOYSA-N (E)-3,4,5-trihydroxycinnamic acid Natural products OC(=O)C=CC1=CC(O)=C(O)C(O)=C1 ACEAELOMUCBPJP-UHFFFAOYSA-N 0.000 description 5
- NGSWKAQJJWESNS-ZZXKWVIFSA-M 4-Hydroxycinnamate Natural products OC1=CC=C(\C=C\C([O-])=O)C=C1 NGSWKAQJJWESNS-ZZXKWVIFSA-M 0.000 description 5
- DFYRUELUNQRZTB-UHFFFAOYSA-N Acetovanillone Natural products COC1=CC(C(C)=O)=CC=C1O DFYRUELUNQRZTB-UHFFFAOYSA-N 0.000 description 5
- 235000009024 Ceanothus sanguineus Nutrition 0.000 description 5
- WMBWREPUVVBILR-UHFFFAOYSA-N GCG Natural products C=1C(O)=C(O)C(O)=CC=1C1OC2=CC(O)=CC(O)=C2CC1OC(=O)C1=CC(O)=C(O)C(O)=C1 WMBWREPUVVBILR-UHFFFAOYSA-N 0.000 description 5
- 108700023372 Glycosyltransferases Proteins 0.000 description 5
- 240000003553 Leptospermum scoparium Species 0.000 description 5
- 235000015459 Lycium barbarum Nutrition 0.000 description 5
- 235000006468 Thea sinensis Nutrition 0.000 description 5
- 235000004883 caffeic acid Nutrition 0.000 description 5
- 229940074360 caffeic acid Drugs 0.000 description 5
- 239000000969 carrier Substances 0.000 description 5
- QAIPRVGONGVQAS-UHFFFAOYSA-N cis-caffeic acid Natural products OC(=O)C=CC1=CC=C(O)C(O)=C1 QAIPRVGONGVQAS-UHFFFAOYSA-N 0.000 description 5
- 238000006911 enzymatic reaction Methods 0.000 description 5
- 229940030275 epigallocatechin gallate Drugs 0.000 description 5
- 230000006698 induction Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 4
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 4
- XMOCLSLCDHWDHP-UHFFFAOYSA-N L-Epigallocatechin Natural products OC1CC2=C(O)C=C(O)C=C2OC1C1=CC(O)=C(O)C(O)=C1 XMOCLSLCDHWDHP-UHFFFAOYSA-N 0.000 description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 4
- HSCJRCZFDFQWRP-JZMIEXBBSA-N UDP-alpha-D-glucose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1OP(O)(=O)OP(O)(=O)OC[C@@H]1[C@@H](O)[C@@H](O)[C@H](N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-JZMIEXBBSA-N 0.000 description 4
- HSCJRCZFDFQWRP-UHFFFAOYSA-N Uridindiphosphoglukose Natural products OC1C(O)C(O)C(CO)OC1OP(O)(=O)OP(O)(=O)OCC1C(O)C(O)C(N2C(NC(=O)C=C2)=O)O1 HSCJRCZFDFQWRP-UHFFFAOYSA-N 0.000 description 4
- 241000219095 Vitis Species 0.000 description 4
- 235000009754 Vitis X bourquina Nutrition 0.000 description 4
- 235000012333 Vitis X labruscana Nutrition 0.000 description 4
- 235000014787 Vitis vinifera Nutrition 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000007795 chemical reaction product Substances 0.000 description 4
- 238000001514 detection method Methods 0.000 description 4
- DZYNKLUGCOSVKS-UHFFFAOYSA-N epigallocatechin Natural products OC1Cc2cc(O)cc(O)c2OC1c3cc(O)c(O)c(O)c3 DZYNKLUGCOSVKS-UHFFFAOYSA-N 0.000 description 4
- 238000004128 high performance liquid chromatography Methods 0.000 description 4
- 238000000589 high-performance liquid chromatography-mass spectrometry Methods 0.000 description 4
- IYRMWMYZSQPJKC-UHFFFAOYSA-N kaempferol Chemical compound C1=CC(O)=CC=C1C1=C(O)C(=O)C2=C(O)C=C(O)C=C2O1 IYRMWMYZSQPJKC-UHFFFAOYSA-N 0.000 description 4
- 239000012160 loading buffer Substances 0.000 description 4
- 239000006228 supernatant Substances 0.000 description 4
- 230000009182 swimming Effects 0.000 description 4
- 229910021642 ultra pure water Inorganic materials 0.000 description 4
- 239000012498 ultrapure water Substances 0.000 description 4
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-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
- 241000588724 Escherichia coli Species 0.000 description 3
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 3
- 239000012530 fluid Substances 0.000 description 3
- 230000014509 gene expression Effects 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 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 3
- MWDZOUNAPSSOEL-UHFFFAOYSA-N kaempferol Natural products OC1=C(C(=O)c2cc(O)cc(O)c2O1)c3ccc(O)cc3 MWDZOUNAPSSOEL-UHFFFAOYSA-N 0.000 description 3
- 238000000034 method Methods 0.000 description 3
- 235000015097 nutrients Nutrition 0.000 description 3
- 238000004321 preservation Methods 0.000 description 3
- 239000011347 resin Substances 0.000 description 3
- 229920005989 resin Polymers 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- 235000013616 tea Nutrition 0.000 description 3
- HUXWBFFRUHXOBU-CPRQJHDUSA-N (E,5R,6S,7R,8R)-1-(3,4-dihydroxyphenyl)-5,6,7,8,9-pentahydroxynon-1-ene-3,4-dione Chemical compound C(\C=C\C1=CC(O)=C(O)C=C1)(=O)C(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO HUXWBFFRUHXOBU-CPRQJHDUSA-N 0.000 description 2
- IOHPVZBSOKLVMN-UHFFFAOYSA-N 2-(2-phenylethyl)benzoic acid Chemical compound OC(=O)C1=CC=CC=C1CCC1=CC=CC=C1 IOHPVZBSOKLVMN-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
- 229920000856 Amylose Polymers 0.000 description 2
- 241000219194 Arabidopsis Species 0.000 description 2
- POOVYWIYTSHEES-TVKJYDDYSA-N C(C=CC1=CC=CC=C1)(=O)O[C@H]1[C@H](O)O[C@@H]([C@H]([C@@H]1O)O)CO Chemical compound C(C=CC1=CC=CC=C1)(=O)O[C@H]1[C@H](O)O[C@@H]([C@H]([C@@H]1O)O)CO POOVYWIYTSHEES-TVKJYDDYSA-N 0.000 description 2
- UBSCDKPKWHYZNX-UHFFFAOYSA-N Demethoxycapillarisin Natural products C1=CC(O)=CC=C1OC1=CC(=O)C2=C(O)C=C(O)C=C2O1 UBSCDKPKWHYZNX-UHFFFAOYSA-N 0.000 description 2
- 102000051366 Glycosyltransferases Human genes 0.000 description 2
- 108700020482 Maltose-Binding protein Proteins 0.000 description 2
- 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 description 2
- 244000223014 Syzygium aromaticum Species 0.000 description 2
- 235000016639 Syzygium aromaticum Nutrition 0.000 description 2
- 125000002252 acyl group Chemical group 0.000 description 2
- AVKUERGKIZMTKX-NJBDSQKTSA-N ampicillin Chemical compound C1([C@@H](N)C(=O)N[C@H]2[C@H]3SC([C@@H](N3C2=O)C(O)=O)(C)C)=CC=CC=C1 AVKUERGKIZMTKX-NJBDSQKTSA-N 0.000 description 2
- 229960000723 ampicillin Drugs 0.000 description 2
- 238000013459 approach Methods 0.000 description 2
- 235000019658 bitter taste Nutrition 0.000 description 2
- WQSDYZZEIBAPIN-UHFFFAOYSA-N caffeoylglucose Natural products OC1C(O)C(O)C(CO)OC1OC(=O)C=CC1=CC=C(O)C(O)=C1 WQSDYZZEIBAPIN-UHFFFAOYSA-N 0.000 description 2
- 150000001720 carbohydrates Chemical class 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 150000001765 catechin Chemical class 0.000 description 2
- 238000010367 cloning Methods 0.000 description 2
- 239000002299 complementary DNA Substances 0.000 description 2
- 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 2
- 238000010586 diagram Methods 0.000 description 2
- 230000029087 digestion Effects 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 239000012149 elution buffer Substances 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 229930182470 glycoside Natural products 0.000 description 2
- 150000002338 glycosides Chemical class 0.000 description 2
- 235000008777 kaempferol Nutrition 0.000 description 2
- UXOUKMQIEVGVLY-UHFFFAOYSA-N morin Natural products OC1=CC(O)=CC(C2=C(C(=O)C3=C(O)C=C(O)C=C3O2)O)=C1 UXOUKMQIEVGVLY-UHFFFAOYSA-N 0.000 description 2
- 235000019508 mustard seed Nutrition 0.000 description 2
- 229910052757 nitrogen Inorganic materials 0.000 description 2
- 150000007965 phenolic acids Chemical class 0.000 description 2
- 230000009465 prokaryotic expression Effects 0.000 description 2
- 238000010839 reverse transcription Methods 0.000 description 2
- 239000011780 sodium chloride Substances 0.000 description 2
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 2
- 238000004659 sterilization and disinfection Methods 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- 238000012795 verification Methods 0.000 description 2
- DGVVWUTYPXICAM-UHFFFAOYSA-N β‐Mercaptoethanol Chemical compound OCCS DGVVWUTYPXICAM-UHFFFAOYSA-N 0.000 description 2
- LSHVYAFMTMFKBA-TZIWHRDSSA-N (-)-epicatechin-3-O-gallate Chemical compound O([C@@H]1CC2=C(O)C=C(C=C2O[C@@H]1C=1C=C(O)C(O)=CC=1)O)C(=O)C1=CC(O)=C(O)C(O)=C1 LSHVYAFMTMFKBA-TZIWHRDSSA-N 0.000 description 1
- FTVWIRXFELQLPI-ZDUSSCGKSA-N (S)-naringenin Chemical compound C1=CC(O)=CC=C1[C@H]1OC2=CC(O)=CC(O)=C2C(=O)C1 FTVWIRXFELQLPI-ZDUSSCGKSA-N 0.000 description 1
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 239000004382 Amylase Substances 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 108010065511 Amylases Proteins 0.000 description 1
- 240000007087 Apium graveolens Species 0.000 description 1
- 235000015849 Apium graveolens Dulce Group Nutrition 0.000 description 1
- 235000010591 Appio Nutrition 0.000 description 1
- 101100316026 Arabidopsis thaliana UGGT gene Proteins 0.000 description 1
- 244000080767 Areca catechu Species 0.000 description 1
- 235000006226 Areca catechu Nutrition 0.000 description 1
- 240000004385 Centaurea cyanus Species 0.000 description 1
- 235000005940 Centaurea cyanus Nutrition 0.000 description 1
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 1
- 102000000340 Glucosyltransferases Human genes 0.000 description 1
- 108010055629 Glucosyltransferases Proteins 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 description 1
- 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 description 1
- 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 description 1
- 235000004347 Perilla Nutrition 0.000 description 1
- 244000124853 Perilla frutescens Species 0.000 description 1
- 240000007377 Petunia x hybrida Species 0.000 description 1
- 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 description 1
- 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 description 1
- 244000062793 Sorghum vulgare Species 0.000 description 1
- 239000007983 Tris buffer Substances 0.000 description 1
- 102100040363 UDP-glucose:glycoprotein glucosyltransferase 1 Human genes 0.000 description 1
- 240000001519 Verbena officinalis Species 0.000 description 1
- 235000018718 Verbena officinalis Nutrition 0.000 description 1
- 235000004282 Vitis labrusca Nutrition 0.000 description 1
- 244000068697 Vitis rotundifolia Species 0.000 description 1
- 235000004305 Vitis rotundifolia Nutrition 0.000 description 1
- 235000017242 Vitis vulpina Nutrition 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 238000001042 affinity chromatography Methods 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 238000000137 annealing Methods 0.000 description 1
- 235000019606 astringent taste Nutrition 0.000 description 1
- 238000000889 atomisation Methods 0.000 description 1
- 230000001580 bacterial effect Effects 0.000 description 1
- 230000006696 biosynthetic metabolic pathway Effects 0.000 description 1
- 235000020279 black tea Nutrition 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 235000013353 coffee beverage Nutrition 0.000 description 1
- 230000009514 concussion Effects 0.000 description 1
- 235000007336 cyanidin Nutrition 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 230000007850 degeneration Effects 0.000 description 1
- 238000004925 denaturation Methods 0.000 description 1
- 230000036425 denaturation Effects 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 238000001962 electrophoresis Methods 0.000 description 1
- 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 description 1
- 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 description 1
- 235000011797 eriodictyol Nutrition 0.000 description 1
- 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 description 1
- 239000013604 expression vector Substances 0.000 description 1
- 239000000284 extract Substances 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 229930182830 galactose Natural products 0.000 description 1
- 239000007789 gas Substances 0.000 description 1
- 229930182478 glucoside Natural products 0.000 description 1
- 150000008131 glucosides Chemical class 0.000 description 1
- 239000000937 glycosyl acceptor Substances 0.000 description 1
- 230000013595 glycosylation Effects 0.000 description 1
- 238000006206 glycosylation reaction Methods 0.000 description 1
- 238000009630 liquid culture Methods 0.000 description 1
- 239000003550 marker Substances 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 235000019713 millet Nutrition 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 230000003020 moisturizing effect Effects 0.000 description 1
- 239000012452 mother liquor Substances 0.000 description 1
- 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 description 1
- 235000007743 myricetin Nutrition 0.000 description 1
- 229940116852 myricetin Drugs 0.000 description 1
- WGEYAGZBLYNDFV-UHFFFAOYSA-N naringenin Natural products C1(=O)C2=C(O)C=C(O)C=C2OC(C1)C1=CC=C(CC1)O WGEYAGZBLYNDFV-UHFFFAOYSA-N 0.000 description 1
- 235000007625 naringenin Nutrition 0.000 description 1
- 229940117954 naringenin Drugs 0.000 description 1
- 235000019520 non-alcoholic beverage Nutrition 0.000 description 1
- 239000005426 pharmaceutical component Substances 0.000 description 1
- 150000002989 phenols Chemical class 0.000 description 1
- 238000013081 phylogenetic analysis Methods 0.000 description 1
- 150000008442 polyphenolic compounds Chemical class 0.000 description 1
- 235000013824 polyphenols Nutrition 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000002243 precursor Substances 0.000 description 1
- 238000001742 protein purification Methods 0.000 description 1
- 235000005875 quercetin Nutrition 0.000 description 1
- 229960001285 quercetin Drugs 0.000 description 1
- 238000005070 sampling Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000006250 specific catalysis Methods 0.000 description 1
- 230000003595 spectral effect Effects 0.000 description 1
- 239000007921 spray Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 1
- 239000012137 tryptone Substances 0.000 description 1
- 210000005239 tubule Anatomy 0.000 description 1
- 238000004704 ultra performance liquid chromatography Methods 0.000 description 1
- 238000002525 ultrasonication Methods 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Landscapes
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明公开了一种没食子酰葡萄糖基转移酶CsUGT84A22基因,所述CsUGT84A22基因具有如SEQ ID NO:1所示的核苷酸序列,所述基因的编码蛋白具有如SEQ ID NO:2所示的氨基酸序列;与现有技术相比,本发明首次克隆并验证了形成1‑O‑没食子酰‑β‑D‑葡糖苷相关的尿苷二磷酸葡萄糖依赖的没食子酰葡萄糖基转移酶CsUGT84A22基因的功能,并提供了含有该CsUGT84A22基因的重组质粒、转基因工程菌和重组蛋白,为通过生物工程方法大量合成1‑O‑没食子酰‑β‑D‑葡糖苷,进一步开展酯型儿茶素生物合成调控研究奠定基础。
Description
技术领域
本发明涉及的是分子生物学领域,尤其涉及的是一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用。
背景技术
茶叶是世界三大无酒精饮料之一。儿茶素(黄烷-3-醇),作为茶叶中主要的保健和药理成分,是茶叶中主要存在的茶多酚,约占茶鲜叶干重的12–24%,也是红茶浸提物中典型的苦涩味化合物,尤其是表没食子儿茶素没食子酸酯(简称EGCG)在茶汤中含量最高且对茶叶涩味贡献最大,并赋予茶叶苦涩的口感。
儿茶素类化合物是2-苯基苯并吡喃的衍生物,属于类黄酮化合物中的黄烷-3-醇类。根据C环上3位是否连接没食子基团,儿茶素类化合物可分为酯型儿茶素(主要包括表没食子儿茶素(ECG)、表儿茶素没食子酸酯(EGCG)),和非酯型儿茶素(主要包括儿茶素(C)、没食子儿茶素(GC)、表没食子儿茶素(EC)和表没食子儿茶素(EGC))。其中,如附图1所示,酯型儿茶素的合成包括两步酶催化反应,涉及尿苷二磷酸葡萄糖依赖的没食子酰葡萄糖基转移酶(UGGT)和没食子酰基转移酶(ECGT)。
迄今为止,茶树中编码尿苷二磷酸葡萄糖依赖的没食子酰葡萄糖基转移酶的基因还没有得到验证。
尿苷二磷酸葡萄糖依赖的没食子酰葡萄糖基转移酶的催化产物,1-O-没食子酰-β-D-葡糖苷(βG),是合成酯型儿茶素的底物,但是至今没有市场商品产物。为了得到1-O-没食子酰-β-D-葡糖苷(βG),可从茶树鲜叶中提取或利用化学合成得到,但这两种方法均存在方法繁琐、成本高、得率少的的缺点。
发明内容
本发明的目的在于克服现有技术的不足,提供了一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用,以提供一种能够大量合成1-O-没食子酰-β-D-葡糖苷(βG)的途径,为酯型儿茶素合成的工程化奠定基础。
本发明是通过以下技术方案实现的:
本发明提供了一种没食子酰葡萄糖基转移酶CsUGT84A22基因,具有如SEQ ID NO:1所示的核苷酸序列,该基因是从茶树鲜叶中分离获得的,为一种尿苷二磷酸葡萄糖依赖的没食子酰葡萄糖基转移酶CsUGT84A22基因。
本发明还提供了上述没食子酰葡萄糖基转移酶CsUGT84A22基因在制备酯型儿茶素中的应用。
本发明还提供了上述没食子酰葡萄糖基转移酶CsUGT84A22基因的编码蛋白,所述编码蛋白具有如SEQ ID NO:2所示的氨基酸序列。
本发明还提供了上述没食子酰葡萄糖基转移酶CsUGT84A22基因的编码蛋白在制备酯型儿茶素中的应用。
本发明还提供了一种含有上述没食子酰葡萄糖基转移酶CsUGT84A22基因的重组质粒。
所述重组质粒为将上述没食子酰葡萄糖基转移酶CsUGT84A22基因连接到pMal-c2X载体的多克隆位点中构建获得,命名为pMal-c2X-CsUGT84A22。
本发明还提供了一种转基因工程菌,所述转基因工程菌含有上述重组质粒,或其基因组中整合有外源的上述没食子酰葡萄糖基转移酶CsUGT84A22基因序列。
所述转基因工程菌为含有上述重组质粒,或其基因组中整合有外源的上述没食子酰葡萄糖基转移酶CsUGT84A22基因序列的大肠杆菌Novablue(DE3)菌株。
本发明相比现有技术具有以下优点:本发明提供了一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用,首次克隆并验证了形成1-O-没食子酰-β-D-葡糖苷(βG)相关的尿苷二磷酸葡萄糖依赖的没食子酰葡萄糖基转移酶CsUGT84A22基因的功能,本发明还提供了含有该CsUGT84A22基因的重组质粒、转基因工程菌和重组蛋白,为通过生物工程方法大量合成1-O-没食子酰-β-D-葡糖苷(βG),进一步开展酯型儿茶素生物合成调控研究奠定基础。
附图说明
图1为酯型儿茶素生物合成途径示意图;
图2为pMal-c2X载体的质粒图谱;
图3为CsUGT84A22基因与已知功能糖基转移酶基因的进化关系图;
图4为CsUGT84A22基因的编码蛋白与已知功能糖基转移酶蛋白序列同源性分析结果图;
图5为CsUGT84A22重组蛋白(rCsUGT84A22)的SDS-PAGE蛋白电泳分析图;其中,M为蛋白Marker;1为重组质粒诱导前;2为重组质粒诱导后;3为诱导后破碎后上清;4为诱导后破碎后沉淀;5为纯化后蛋白;
图6为rCsUGT84A22酶促反应产物的HPLC图谱;其中,图6(A~G)分别表示利用rCsUGT84A22催化没食子酸、丁香酸、肉桂酸、对香豆酸、咖啡酸、阿魏酸和芥子酸,形成对应的酶活产物没食子酰葡萄糖(1-O-没食子酰-β-D-葡糖苷)、丁香酰葡萄糖、肉桂酰葡萄糖、香豆酰葡萄糖、咖啡酰葡萄糖、阿魏酰葡萄糖和芥子酰葡萄糖的HPLC图谱;
图7为rCsUGT84A22酶促反应产物的HPLC-MS质谱分析结果图;其中,图7(A~G)分别表示利用rCsUGT84A22催化形成的酶活产物没食子酰葡萄糖(1-O-没食子酰-β-D-葡糖苷)、丁香酰葡萄糖、肉桂酰葡萄糖、香豆酰葡萄糖、咖啡酰葡萄糖、阿魏酰葡萄糖和芥子酰葡萄糖一级和二级质谱图;
图8为rCsUGT84A22催化各酚酸类化合物的相对活性的柱状图。
具体实施方式
下面通过实施例详细描述本发明,本领域的普通技术人员可以理解,下述实施例仅是用于举例说明的目的,并非限制本发明,本发明的保护范围由权利要求所界定,实施例中未注明具体条件者,按照常规条件或制造商建议的条件进行,所用试剂或仪器未注明生产厂商者,均为可以通过市购获得的常规产品。
实施例1
一、材料
1、茶树品种:农抗早(Camellia sinensis(L.)O.Kuntze.var.sinensis cultivarNongkangzao),采集茶树鲜叶,迅速用液氮冷冻,储存于-80℃冰箱中备用;
2、pMal-c2X载体:其质粒图谱如图2所示;
3、大肠杆菌Novablue(DE3)表达宿主菌:购于上海北诺生物科技有限公司;
4、LB培养基:称取10g的NaCl,5g的酵母提取物,10g的胰蛋白胨,加入950mL去超纯水搅拌溶解,用1mol/L的NaOH调pH至7.0,加水定容至1000mL,高压蒸汽灭菌15min,即获得LB液体培养基,LB固体培养基为在LB液体培养基中加入15g的琼脂粉即可;
5、质量浓度为40%的半乳糖溶液:称取40g半乳糖,加入超纯水溶解搅拌均匀,定容至100mL,110℃灭菌10min;
5、质量比为40%的葡萄糖溶液:称取40g葡萄糖,加入超纯水溶解搅拌均匀,定容至100mL,110℃灭菌10min;
6、氨苄青霉素母液(Amp+,100mg/mL):称取1g氨苄青霉素Amp,溶于10mL灭菌水,过滤除菌,分装小管,-20℃保存;
7、1mol/L的IPTG(异丙基硫代-β-D-半乳糖苷):称取2.383 g IPTG,溶于灭菌超纯水,定容至10mL,过滤除菌,分装并于-20℃保存;
8、蛋白纯化缓冲液:包括上柱缓冲液和洗脱缓冲液,:
上柱缓冲液:称取0.37g的EDTA,11.67g的NaCl,2.42g的Tris,0.15g的二硫苏糖醇DTT于足量纯水中,搅拌使其充分混匀;用稀盐酸调其PH至7.4,定容至1L,即得上柱缓冲液;
洗脱缓冲液:1L上柱缓冲液中加入3.60g麦芽糖,溶解搅拌均匀;
9、100mM的pH7.5的Tris-HCL缓冲溶液:称取1.1214gTris加水至90mL搅拌溶解均匀,加稀HCL调pH至7.5,补水定容至100mL;
10、体积比为1%的乙酸:用移液管量取10mL色谱级乙酸溶液于1L容量瓶中,用超纯水定容至1L。
二、CsUGT84A22基因的克隆:
1、设计带有表达载体pMal-c2X载体的多克隆酶切位点的特异引物,其引物序列如SEQ ID NO:3和SEQ ID NO:4所示:
SEQ ID NO:3:正向引物:5’-TCTAGAATGGGCTCTGAATCACTTGTCC-3’
SEQ ID NO:4:反向引物:5’-CTGCAGTTAAACAACAGTAGTAGTTGTG-3’;
2、按照TaKaRa RNAiso试剂盒和RNAiso Plus试剂盒说明书,提取茶树品种农抗早鲜叶RNA,并反转录为cDNA;
3、以反转录产物cDNA为模板,用SEQ ID NO:3和SEQ ID NO:4引物进行扩增,扩增程序为94℃预变性30s,94℃变性10s,62℃退火20s,72℃延伸95s,30个循环,72℃继续延伸10min,获得的PCR产物置于16℃保存。
4、将PCR产物利用PCR纯化试剂盒纯化,并连接到pMD19-T Simple Vector后进行菌落PCR验证,获得阳性菌落,提取菌落质粒,获得含有CsUGT84A22基因的pMD19-T simple载体,同时将菌液送至深圳华大公司进行测序。
三、CsUGT84A22基因的功能预测分析
本发明中,为了预测编码茶树尿苷二磷酸葡萄糖依赖的没食子酰葡萄糖基转移酶CsUGT84A22基因功能,我们通过生物信息学软件,利用MEGA软件邻位法(neighbor-joiningmethod,NJ)将CsUGT84A22基因与已知功能的糖基转移酶基因进行系统进化树分析,其中,已知功能的糖基转移酶基因包括:美洲葡萄VlRSgt(ABH03018.1)、葡萄VvgGT1(AEW31187.1)、葡萄VvgGT2(AEW31188.1)、葡萄VvgGT3(NP_001267849.1)、矮牵牛PhA5GT(BAA89009.1)、夏堇ThA5GT(BAC54093.1)、马鞭草GhA5GT(Q9ZR25.1)、紫苏PfA5GT(Q9ZR27.1)、拟南芥AtF3G7GT(NP_181217.1)、甘草GeIF7GT(BAC78438.1)拟南芥AtF7GT(NP_567955.1)、黄芩SbF7GT(BAA83484.1),获得如图3所示的进化树,图中可看出CsUGT84A22基因被分到酯形成的糖基转移酶进化支,且与VvgGT1,VvgGT2和VvgGT3的亲缘关系比较近。
利用MEGA软件对CsUGT84A22基因的编码蛋白,VvgGT1基因的编码蛋白,VvgGT2基因的编码蛋白和VvgGT3基因的编码蛋白同源性进行分析,获得如图4所示的结果,图中可看出,CsUGT84A22基因的编码蛋白与已知功能的葡萄的三条VvgGT1,VvgGT2和VvgGT3基因的编码蛋白的一致性达到92%以上。
综上所述,CsUGT84A22基因被预测为具有苯甲酸类化合物糖酯化功能。
四、CsUGT84A22基因的原核表达及功能验证
本实施例中所用到的原核表达和及其功能验证技术手段为本领域的普通技术人员常用或完全可以理解技术手段。
1、将含有CsUGT84A22基因的T载体用Xba Ⅰ和Sal I进行双酶切,酶切产物连接到pMal-c2X载体的多克隆位点中,获得pMal-c2X-CsUGT84A22重组质粒;
2、将pMal-c2X-CsUGT84A22重组质粒转化到大肠杆菌Novablue(DE3)表达宿主菌中,接种到100μL的LB液体培养基,37℃,180r/min下培养45~60min;取100μL的菌液涂布于含100μg/mL Amp+的LB平板上,37℃倒置培养;
3、经过菌落PCR验证,挑取阳性菌落,接种至含2g/L的100mL的灭菌的LB液体培养基中,37℃,200r/min下震荡培养,直至OD600≈0.6,获得转基因的工程菌;
4、在上述转基因的工程菌中加入IPTG至终浓度为1mmol/L,37℃过夜培养,收集菌体,加入10mL上柱缓冲溶液,充分悬浮菌体,置于-20℃过夜,将菌体置于冰上解冻,待解冻后置于超声破碎仪中以15%功率超声破碎10min,12000rpm离心收集上清液;利用直链淀粉树脂亲和柱纯化重组蛋白(affinity chromatography on an amylase resin,NewEngland Biolabs,MA,USA),利用本领域常用的SDS-PAGE方法检测蛋白表达和纯化效果,结果如图5所示。
图5中可看出,pMal-c2X-CsUGT84A22重组质粒转化表达宿主菌Novablue(DE3),诱导表达后,与诱导前(泳道1)相比,该基因在诱导后(泳道2)有重组蛋白的表达,且重组蛋白条带的大小与预测的一致,加上42.5kDa麦芽糖结合蛋白(MBP)重组标签后,在70kd到100kd之间有明显的重组蛋白条带;诱导后菌体经超声破碎离心后,上清中有可溶性重组蛋白(泳道3),可用于进一步纯化分析;上清蛋白经直链淀粉树脂柱纯化后,得到较纯的重组蛋白rCsUGT84A22(泳道4),纯化的蛋白可用于进一步的酶学分析。
五、CsUGT84A22重组蛋白的酶学活性检测分析
以酚酸类化合物作为底物,对CsUGT84A22重组蛋白的酶活进行检测,反应体系为50μL,在100mM,pH=5.5的MES缓冲溶液中加入2.5mM的UDP-葡萄糖、0.5mM酚酸类化合物(没食子酸、丁香酸、肉桂酸、对香豆酸、咖啡酸、阿魏酸或芥子酸),6μg纯化后的CsUGT84A22重组蛋白和0.1%的β-巯基乙醇。
以类黄酮类化合物作为底物,对CsUGT84A22重组蛋白的酶活进行检测,反应体系为50μL,在100mM pH7.5的Tris-HCL缓冲溶液中加入5mM的UDP-葡萄糖或UDP-半乳糖作为糖基供体,200μM潜在的类黄酮化合物(山奈酚、槲皮素、杨梅素、山奈素、柚皮素、圣草酚、芹菜素、儿茶素或矢车菊色素)作为糖基受体、5-10μg的纯化后的CsUGT84A22重组蛋白和0.1%的β-巯基乙醇。
所有酶反应体系,30℃水浴30min后加入等体积的甲醇终止反应,矢车菊为底物的反应体系例外,需加入20μL的5%盐酸终止反应。反应均以空载蛋白作为对照。
酶反应产物经产物标准品结合HPLC-MS进行鉴定。
HPLC-MS检测条件如下:维特斯HSS T3色谱柱(Waters ACQUITY UPLC HSS T3,150mm×2.1mm,1.7tzm);柱温为30℃;流速为1mL/min;进样体积为5μL;流动相A为含1%(v/v)乙酸溶液;流动相B为100%乙腈溶液;对于苯甲酸衍生物(没食子酸、丁香酸、肉桂酸、对香豆酸、咖啡酸、阿魏酸和芥子酸)的检测,HPLC梯度程序设置为:0~10min,1%~10%B;10~17min,10~12%B;17~19min,12~1%B;对于类黄酮化合物的检测,HPLC梯度程序设置为:0~5min,10~15%B;5~15min,15~40%B;15~20min,40~60%B;20~25min,60~80%B;25~30min,80~10%B;光谱检测波长扫描范围为200~550nm。在化合物的MS定性识别中,采用ESI电喷雾离子源,负离子模式;毛细管电压为3.5kV,离子源温度为350℃,雾化气(氮气)流速为6L/min,化合物检测扫描质荷比范围设置为m/z 100~1000,碰撞电压为45V。
利用HPLC-MS对酶促反应产物进行检测,获得如图6和图7所示的结果,结果表明当以UDP-葡萄糖作为糖供体时,rCsUGT84A22可特异性的催化酚酸类底物(没食子酸、丁香酸、肉桂酸、对香豆酸、咖啡酸、阿魏酸和芥子酸),并于糖苷结合形成相应的酚酸酯葡萄糖,而类黄酮化合物作为底物时,并未检测到糖苷化产物的峰出现。另外,UDP-半乳糖作为糖供体时,CsUGT84A22对于类黄酮底物或酚酸类底物均未检测到任何酶活性。
在UDP-葡萄糖充足的情况下,分别以没食子酸、丁香酸、肉桂酸、对香豆酸、咖啡酸、阿魏酸和芥子酸这7种酚酸类化合物作为底物,加入纯化后的rCsUGT84A22,在pH5.5条件下检测rCsUGT84A22对各酚酸类化合物底物的酶特异性。结果如图8所示,图中可看出,rCsUGT84A22对没食子酸活性最高,并生成没食子酰葡萄糖(1-O-没食子酰-β-D-葡糖苷,βG),此化合物是茶树酯型儿茶素即茶饮料苦涩味化合物EGCG生物合成的直接前体底物。以没食子酸活性为100%,rCsUGT84A22对酚酸类化合物底物的催化活性大小顺序依次为没食子酸>对香豆酸>芥子酸>咖啡酸>阿魏酸>丁香酸>肉桂酸。
Claims (1)
1.一种没食子酰葡萄糖基转移酶CsUGT84A22基因的编码蛋白在制备酯型儿茶素中的应用,其特征在于,所述没食子酰葡萄糖基转移酶CsUGT84A22基因的编码蛋白的氨基酸序列如下所示:
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510297824.4A CN104878025B (zh) | 2015-06-01 | 2015-06-01 | 一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN201510297824.4A CN104878025B (zh) | 2015-06-01 | 2015-06-01 | 一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN104878025A CN104878025A (zh) | 2015-09-02 |
CN104878025B true CN104878025B (zh) | 2017-11-03 |
Family
ID=53945701
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN201510297824.4A Expired - Fee Related CN104878025B (zh) | 2015-06-01 | 2015-06-01 | 一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN104878025B (zh) |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108070576A (zh) * | 2018-02-05 | 2018-05-25 | 安徽农业大学 | 一种茶树糖基转移酶突变体及其在植物害虫防御中的应用 |
CN113667655B (zh) * | 2021-08-24 | 2023-03-14 | 云南农业大学 | 一种仙茅糖基转移酶Co84A-471基因及在制备苔黑酚葡萄糖苷中的应用 |
CN115807034B (zh) * | 2023-02-08 | 2023-05-26 | 中国农业科学院北京畜牧兽医研究所 | Ugt84a1基因在调控花青素合成上的应用 |
CN116790562A (zh) * | 2023-06-28 | 2023-09-22 | 安徽农业大学 | 一种水解单宁合成酶和编码基因及其应用 |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101292028A (zh) * | 2005-10-20 | 2008-10-22 | 青森县 | 新型芳香族酰基转移酶基因 |
JP2013176361A (ja) * | 2012-02-06 | 2013-09-09 | Suntory Holdings Ltd | チャ由来モノテルペン配糖体化酵素及びその利用方法 |
CN103987840A (zh) * | 2011-08-08 | 2014-08-13 | 国际香料香精公司 | 用于香草醛或香草醛β-D-葡萄糖苷的生物合成的组合物和方法 |
CN104585374A (zh) * | 2015-01-06 | 2015-05-06 | 湖南中医药大学 | 一种含红景天苷的茶叶的制备方法及应用 |
-
2015
- 2015-06-01 CN CN201510297824.4A patent/CN104878025B/zh not_active Expired - Fee Related
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN101292028A (zh) * | 2005-10-20 | 2008-10-22 | 青森县 | 新型芳香族酰基转移酶基因 |
CN103987840A (zh) * | 2011-08-08 | 2014-08-13 | 国际香料香精公司 | 用于香草醛或香草醛β-D-葡萄糖苷的生物合成的组合物和方法 |
JP2013176361A (ja) * | 2012-02-06 | 2013-09-09 | Suntory Holdings Ltd | チャ由来モノテルペン配糖体化酵素及びその利用方法 |
CN104585374A (zh) * | 2015-01-06 | 2015-05-06 | 湖南中医药大学 | 一种含红景天苷的茶叶的制备方法及应用 |
Also Published As
Publication number | Publication date |
---|---|
CN104878025A (zh) | 2015-09-02 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
CN104878025B (zh) | 一种没食子酰葡萄糖基转移酶CsUGT84A22基因及其编码蛋白和应用 | |
Wang et al. | Molecular characterization of the C‐glucosylation for puerarin biosynthesis in Pueraria lobata | |
Wang et al. | Dissection of the general two-step di-C-glycosylation pathway for the biosynthesis of (iso) schaftosides in higher plants | |
Guimarães et al. | Characterization of α-galactosidases from germinating soybean seed and their use for hydrolysis of oligosaccharides | |
Koirala et al. | Methylation and subsequent glycosylation of 7, 8-dihydroxyflavone | |
CN105002193B (zh) | 一种黄酮醇3-O-葡萄糖基转移酶CsUGT78A14基因及其编码蛋白和应用 | |
CN108486136B (zh) | 黄酮醇3-O-葡萄糖基转移酶MdUGT71B1基因及其编码蛋白和应用 | |
CN109750071A (zh) | 一种生物催化合成莱鲍迪苷m的方法 | |
CN105087612A (zh) | 黄酮醇多位点葡萄糖基转移酶CsUGT73A20基因及其编码蛋白和应用 | |
BR112019018361B1 (pt) | Método de produção de uma composição de glicosídeos de esteviol compreendendo rebaudiosídeo d4 | |
Wang et al. | Identification of three (iso) flavonoid glucosyltransferases from Pueraria lobata | |
Hua et al. | Expression patterns of an isoflavone reductase-like gene and its possible roles in secondary metabolism in Ginkgo biloba | |
CN110184247A (zh) | 紫花苜蓿褪黑素合成基因MsASMT及其在调控植物褪黑素和黄酮类物质合成中的应用 | |
KR102028625B1 (ko) | 신규 당 전이 효소 유전자 및 그의 사용 | |
Si et al. | Functional analysis of Flavanone 3-hydroxylase (F3H) from Dendrobium officinale, which confers abiotic stress tolerance | |
CN105087613A (zh) | 黄酮醇7-O-葡萄糖基转移酶CsUGT75L12基因及其编码蛋白和应用 | |
Yao et al. | Genome-wide analysis of UGT gene family identified key gene for the biosynthesis of bioactive flavonol glycosides in Epimedium pubescens Maxim. | |
CN106916838A (zh) | 催化UDP‑鼠李糖生物合成的基因CsRHMb及其编码蛋白和应用 | |
Qiu et al. | Orientin and vitexin production by a one-pot enzymatic cascade of a glycosyltransferase and sucrose synthase | |
Nagashima et al. | cDNA cloning and expression of isoflavonoid-specific glucosyltransferase from Glycyrrhiza echinata cell-suspension cultures | |
Liang et al. | A uridine diphosphate-glycosyltransferase GFUGT88A1 derived from edible mushroom Grifola frondosa extends oligosaccharide chains | |
CN102174454A (zh) | 一种表达重组蔗糖磷酸化酶的大肠杆菌工程菌 | |
Nakatsuka et al. | UDP-glucose: 3-deoxyanthocyanidin 5-O-glucosyltransferase from Sinningia cardinalis | |
CN105063067B (zh) | 一种黄酮醇3-O-半乳糖基转移酶CsUGT78A15基因及其编码蛋白和应用 | |
Yang et al. | Synthesis of icariin in tobacco leaf by overexpression of a glucosyltransferase gene from Epimedium sagittatum |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
C06 | Publication | ||
PB01 | Publication | ||
EXSB | Decision made by sipo to initiate 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: 20171103 Termination date: 20180601 |