CN116694678B - Application of radix angelicae transcription factor AdNAC in improving radix angelicae coumarin content - Google Patents
Application of radix angelicae transcription factor AdNAC in improving radix angelicae coumarin content Download PDFInfo
- Publication number
- CN116694678B CN116694678B CN202310606877.4A CN202310606877A CN116694678B CN 116694678 B CN116694678 B CN 116694678B CN 202310606877 A CN202310606877 A CN 202310606877A CN 116694678 B CN116694678 B CN 116694678B
- Authority
- CN
- China
- Prior art keywords
- adnac
- radix angelicae
- coumarin
- content
- gene
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- ZYGHJZDHTFUPRJ-UHFFFAOYSA-N coumarin Chemical compound C1=CC=C2OC(=O)C=CC2=C1 ZYGHJZDHTFUPRJ-UHFFFAOYSA-N 0.000 title claims abstract description 55
- 235000001671 coumarin Nutrition 0.000 title claims abstract description 28
- 229960000956 coumarin Drugs 0.000 title claims abstract description 27
- 108091023040 Transcription factor Proteins 0.000 title claims abstract description 9
- 102000040945 Transcription factor Human genes 0.000 title claims abstract description 9
- 241000213006 Angelica dahurica Species 0.000 claims abstract description 34
- 108090000623 proteins and genes Proteins 0.000 claims abstract description 32
- 239000002773 nucleotide Substances 0.000 claims abstract description 3
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 3
- 238000000034 method Methods 0.000 claims description 4
- GOLORTLGFDVFDW-UHFFFAOYSA-N 3-(1h-benzimidazol-2-yl)-7-(diethylamino)chromen-2-one Chemical compound C1=CC=C2NC(C3=CC4=CC=C(C=C4OC3=O)N(CC)CC)=NC2=C1 GOLORTLGFDVFDW-UHFFFAOYSA-N 0.000 abstract description 3
- 241000219195 Arabidopsis thaliana Species 0.000 abstract description 2
- 238000010353 genetic engineering Methods 0.000 abstract description 2
- 241000196324 Embryophyta Species 0.000 description 27
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 15
- IGWDEVSBEKYORK-UHFFFAOYSA-N isoimperatorin Chemical compound O1C(=O)C=CC2=C1C=C1OC=CC1=C2OCC=C(C)C IGWDEVSBEKYORK-UHFFFAOYSA-N 0.000 description 12
- BGEBZHIAGXMEMV-UHFFFAOYSA-N 5-methoxypsoralen Chemical compound O1C(=O)C=CC2=C1C=C1OC=CC1=C2OC BGEBZHIAGXMEMV-UHFFFAOYSA-N 0.000 description 8
- OLOOJGVNMBJLLR-UHFFFAOYSA-N imperatorin Chemical compound C1=CC(=O)OC2=C1C=C1C=COC1=C2OCC=C(C)C OLOOJGVNMBJLLR-UHFFFAOYSA-N 0.000 description 8
- XKVWLLRDBHAWBL-UHFFFAOYSA-N imperatorin Natural products CC(=CCOc1c2OCCc2cc3C=CC(=O)Oc13)C XKVWLLRDBHAWBL-UHFFFAOYSA-N 0.000 description 8
- 150000004775 coumarins Chemical class 0.000 description 7
- ZPSAEGUNYMEKBP-UHFFFAOYSA-N 5-Isopentenyloxy-psoralen Natural products CC(C)C=COc1c2C=CC(=O)Oc2cc3occc13 ZPSAEGUNYMEKBP-UHFFFAOYSA-N 0.000 description 6
- BEYIWVKWKJROGZ-UHFFFAOYSA-N Alloimperatorin Natural products O1C(=O)C=CC2=C1C(O)=C1OC=CC1=C2OCC=C(C)C BEYIWVKWKJROGZ-UHFFFAOYSA-N 0.000 description 6
- KGGUASRIGLRPAX-UHFFFAOYSA-N Meranzin hydrate Natural products C1=CC(=O)OC2=C(CC(O)C(C)(C)O)C(OC)=CC=C21 KGGUASRIGLRPAX-UHFFFAOYSA-N 0.000 description 6
- 239000013612 plasmid Substances 0.000 description 6
- 230000014509 gene expression Effects 0.000 description 5
- 239000007788 liquid Substances 0.000 description 5
- 230000002018 overexpression Effects 0.000 description 5
- 241000125175 Angelica Species 0.000 description 4
- DBMJZOMNXBSRED-UHFFFAOYSA-N Bergamottin Natural products O1C(=O)C=CC2=C1C=C1OC=CC1=C2OCC=C(C)CCC=C(C)C DBMJZOMNXBSRED-UHFFFAOYSA-N 0.000 description 4
- 235000001287 Guettarda speciosa Nutrition 0.000 description 4
- 230000001580 bacterial effect Effects 0.000 description 4
- 229960002045 bergapten Drugs 0.000 description 4
- KGZDKFWCIPZMRK-UHFFFAOYSA-N bergapten Natural products COC1C2=C(Cc3ccoc13)C=CC(=O)O2 KGZDKFWCIPZMRK-UHFFFAOYSA-N 0.000 description 4
- 238000006243 chemical reaction Methods 0.000 description 4
- YQBNJPACAUPNLV-UHFFFAOYSA-N peucedanin Chemical compound O1C(=O)C=CC2=C1C=C1OC(C(C)C)=C(OC)C1=C2 YQBNJPACAUPNLV-UHFFFAOYSA-N 0.000 description 4
- 102000004169 proteins and genes Human genes 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 210000004556 brain Anatomy 0.000 description 3
- 238000010367 cloning Methods 0.000 description 3
- 238000010586 diagram Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 3
- 238000003209 gene knockout Methods 0.000 description 3
- 238000003208 gene overexpression Methods 0.000 description 3
- 238000004128 high performance liquid chromatography Methods 0.000 description 3
- 238000012986 modification Methods 0.000 description 3
- 230000004048 modification Effects 0.000 description 3
- 239000000243 solution Substances 0.000 description 3
- 239000011550 stock solution Substances 0.000 description 3
- 230000017260 vegetative to reproductive phase transition of meristem Effects 0.000 description 3
- 108091033409 CRISPR Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- XDROKJSWHURZGO-UHFFFAOYSA-N angelicin Chemical compound C1=C2OC=CC2=C2OC(=O)C=CC2=C1 XDROKJSWHURZGO-UHFFFAOYSA-N 0.000 description 2
- 230000003110 anti-inflammatory effect Effects 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 238000010276 construction Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 239000013604 expression vector Substances 0.000 description 2
- 239000000499 gel Substances 0.000 description 2
- 208000015181 infectious disease Diseases 0.000 description 2
- 230000002401 inhibitory effect Effects 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- 230000006798 recombination Effects 0.000 description 2
- 238000005215 recombination Methods 0.000 description 2
- QZAYGJVTTNCVMB-UHFFFAOYSA-N serotonin Chemical compound C1=C(O)C=C2C(CCN)=CNC2=C1 QZAYGJVTTNCVMB-UHFFFAOYSA-N 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000000341 volatile oil Substances 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- CFNMUZCFSDMZPQ-GHXNOFRVSA-N 7-[(z)-3-methyl-4-(4-methyl-5-oxo-2h-furan-2-yl)but-2-enoxy]chromen-2-one Chemical compound C=1C=C2C=CC(=O)OC2=CC=1OC/C=C(/C)CC1OC(=O)C(C)=C1 CFNMUZCFSDMZPQ-GHXNOFRVSA-N 0.000 description 1
- 241000589158 Agrobacterium Species 0.000 description 1
- 244000275105 Angelica sylvestris Species 0.000 description 1
- 235000014429 Angelica sylvestris Nutrition 0.000 description 1
- 208000019901 Anxiety disease Diseases 0.000 description 1
- 241000208173 Apiaceae Species 0.000 description 1
- 206010006187 Breast cancer Diseases 0.000 description 1
- 208000026310 Breast neoplasm Diseases 0.000 description 1
- 102000003915 DNA Topoisomerases Human genes 0.000 description 1
- 108090000323 DNA Topoisomerases Proteins 0.000 description 1
- 230000004568 DNA-binding Effects 0.000 description 1
- 241000238631 Hexapoda Species 0.000 description 1
- 206010058467 Lung neoplasm malignant Diseases 0.000 description 1
- RJQXTJLFIWVMTO-TYNCELHUSA-N Methicillin Chemical compound COC1=CC=CC(OC)=C1C(=O)N[C@@H]1C(=O)N2[C@@H](C(O)=O)C(C)(C)S[C@@H]21 RJQXTJLFIWVMTO-TYNCELHUSA-N 0.000 description 1
- QXKHYNVANLEOEG-UHFFFAOYSA-N Methoxsalen Chemical compound C1=CC(=O)OC2=C1C=C1C=COC1=C2OC QXKHYNVANLEOEG-UHFFFAOYSA-N 0.000 description 1
- 241000244206 Nematoda Species 0.000 description 1
- 241001254606 Peucedanum praeruptorum Species 0.000 description 1
- 241000083879 Polyommatus icarus Species 0.000 description 1
- 238000011529 RT qPCR Methods 0.000 description 1
- 241000318927 Shrimp white spot syndrome virus Species 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 108010059993 Vancomycin Proteins 0.000 description 1
- PCWZKQSKUXXDDJ-UHFFFAOYSA-N Xanthotoxin Natural products COCc1c2OC(=O)C=Cc2cc3ccoc13 PCWZKQSKUXXDDJ-UHFFFAOYSA-N 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000000202 analgesic effect Effects 0.000 description 1
- 230000000845 anti-microbial effect Effects 0.000 description 1
- 230000000259 anti-tumor effect Effects 0.000 description 1
- 239000003146 anticoagulant agent Substances 0.000 description 1
- 229940127219 anticoagulant drug Drugs 0.000 description 1
- 239000003443 antiviral agent Substances 0.000 description 1
- 230000036506 anxiety Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000001045 blue dye Substances 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 238000004587 chromatography analysis Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000007865 diluting Methods 0.000 description 1
- 230000000857 drug effect Effects 0.000 description 1
- 230000008451 emotion Effects 0.000 description 1
- 150000002148 esters Chemical class 0.000 description 1
- 238000010812 external standard method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- 239000003292 glue Substances 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 230000036541 health Effects 0.000 description 1
- 238000002347 injection Methods 0.000 description 1
- 239000007924 injection Substances 0.000 description 1
- 150000002596 lactones Chemical class 0.000 description 1
- 201000005202 lung cancer Diseases 0.000 description 1
- 208000020816 lung neoplasm Diseases 0.000 description 1
- 229960004469 methoxsalen Drugs 0.000 description 1
- SQBBOVROCFXYBN-UHFFFAOYSA-N methoxypsoralen Natural products C1=C2OC(=O)C(OC)=CC2=CC2=C1OC=C2 SQBBOVROCFXYBN-UHFFFAOYSA-N 0.000 description 1
- 229960003085 meticillin Drugs 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 239000011259 mixed solution Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 230000000144 pharmacologic effect Effects 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- MLMVLVJMKDPYBM-UHFFFAOYSA-N pseudoisopsoralene Natural products C1=C2C=COC2=C2OC(=O)C=CC2=C1 MLMVLVJMKDPYBM-UHFFFAOYSA-N 0.000 description 1
- ZCCUUQDIBDJBTK-UHFFFAOYSA-N psoralen Chemical class C1=C2OC(=O)C=CC2=CC2=C1OC=C2 ZCCUUQDIBDJBTK-UHFFFAOYSA-N 0.000 description 1
- 239000013558 reference substance Substances 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 238000012163 sequencing technique Methods 0.000 description 1
- 229940076279 serotonin Drugs 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000013589 supplement Substances 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 230000009466 transformation Effects 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- MYPYJXKWCTUITO-LYRMYLQWSA-N vancomycin Chemical compound O([C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=C2C=C3C=C1OC1=CC=C(C=C1Cl)[C@@H](O)[C@H](C(N[C@@H](CC(N)=O)C(=O)N[C@H]3C(=O)N[C@H]1C(=O)N[C@H](C(N[C@@H](C3=CC(O)=CC(O)=C3C=3C(O)=CC=C1C=3)C(O)=O)=O)[C@H](O)C1=CC=C(C(=C1)Cl)O2)=O)NC(=O)[C@@H](CC(C)C)NC)[C@H]1C[C@](C)(N)[C@H](O)[C@H](C)O1 MYPYJXKWCTUITO-LYRMYLQWSA-N 0.000 description 1
- 229960003165 vancomycin Drugs 0.000 description 1
- MYPYJXKWCTUITO-UHFFFAOYSA-N vancomycin Natural products O1C(C(=C2)Cl)=CC=C2C(O)C(C(NC(C2=CC(O)=CC(O)=C2C=2C(O)=CC=C3C=2)C(O)=O)=O)NC(=O)C3NC(=O)C2NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(CC(C)C)NC)C(O)C(C=C3Cl)=CC=C3OC3=CC2=CC1=C3OC1OC(CO)C(O)C(O)C1OC1CC(C)(N)C(O)C(C)O1 MYPYJXKWCTUITO-UHFFFAOYSA-N 0.000 description 1
- 239000013598 vector Substances 0.000 description 1
- -1 warfarin Chemical class 0.000 description 1
- PJVWKTKQMONHTI-UHFFFAOYSA-N warfarin Chemical compound OC=1C2=CC=CC=C2OC(=O)C=1C(CC(=O)C)C1=CC=CC=C1 PJVWKTKQMONHTI-UHFFFAOYSA-N 0.000 description 1
- 229960005080 warfarin Drugs 0.000 description 1
- 230000004580 weight loss Effects 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
- C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
-
- 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
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
Abstract
The invention belongs to the technical field of genetic engineering, and relates to an application of angelica dahurica transcription factor AdNAC in improving the content of angelica dahurica coumarin, which comprises the following steps: the AdNAC gene in the arabidopsis thaliana is knocked out, so that the content of the coumarin in the radix angelicae dahuricae can be greatly improved, and the nucleotide sequence of the AdNAC gene is shown as SEQ ID NO. 1.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to application of angelica dahurica transcription factor AdNAC in improving the content of angelica dahurica coumarin.
Background
The radix angelicae resources are widely distributed, the medicinal value is high, and the application history is long. The Chinese medicinal radix Angelicae Dahuricae is dried root of radix Angelicae Dahuricae Angelica dahurica (Fisch. Ex Hoffm.) or radix Angelicae Dahuricae Angelica dahurica (Fisch. Ex Hoffm.) of Umbelliferae, and is one of common Chinese medicinal materials.
The effective components of radix Angelicae Dahuricae are coumarin, and the rest are volatile oil, polysaccharide, amino acid and microelements. Currently, there are thousands of natural coumarin compounds. Du Xingxu the main active ingredients of coumarin compounds in radix Angelicae Dahuricae root include imperatorin, isoimperatorin, hydrated oxydecursin, radix Angelicae sinensis brain, bycichlin, xanthotoxin, bergapten, oxydecursin, etc., wherein the content of imperatorin is the largest. Wherein, according to the rule of Chinese pharmacopoeia 2020 edition, the mass of the angelica dahurica medicinal material is not less than 0.08 percent. Through Song Junna et al, it was found that the place where the coumarin component of dahurian angelica root accumulated was mainly located in the oil tube in the phloem of root.
The Lei Yu tut et al prove that the leukoangelicin and the isoimperatorin have obvious influence on the anti-inflammatory analgesic pharmacological experimental results; the leaf balance, the rhizoma coptidis and the like summarize that coumarin has remarkable anti-inflammatory effect; wu Longhuo et al comprehensively illustrate the antitumor and antimicrobial effects of simple coumarins; dong Wei et al found that imperatorin, isoimperatorin, oxydecursin and isopsoralen had inhibitory effect on breast cancer MDA-MB-231 cell proliferation; konkoiova and the like find that the tacrine-coumarin derivatives have the function of completely inhibiting the activity of topoisomerase I and can inhibit the growth of lung cancer cells; sokol and other newly synthesized coumarin derivatives show good drug effects on methicillin-resistant staphylococcus aureus and vancomycin-resistant strains; scan and the like find that the novel coumarin derivative N2905 has good treatment effect on WSSV infection and has potential of becoming a novel antiviral drug; liu Jia and the like have found that plants are protected against damage by microorganisms, nematodes, phytophagous insects and the like by synthesis of furocoumarin compounds. As coumarin compounds belong to lactones, have similar effects as ester essential oil, can positively influence serotonin secretion, relieve anxiety and inspire spirit, and especially, the coumarin has great help in inspiring emotion and improving depression. Coumarin compounds have fluorescent properties and are often used as polychromatic fluorescent targets and experimental blue dyes. Most of the anticoagulants used clinically at present are coumarin compounds such as warfarin, biscoumarin, and non-olefinic coumarin. In addition, the angelica dahurica also has wide application in aspects of food flavoring, health care products, daily chemical industry and the like.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides an application of a radix angelicae transcription factor AdNAC in improving the content of coumarin in radix angelicae.
The aim of the invention is realized by the following technical scheme:
An application of angelica dahurica transcription factor AdNAC in improving the coumarin content of angelica dahurica, wherein the application method comprises the following steps: knocking out AdNAC genes in the arabidopsis thaliana, wherein the nucleotide sequence of the AdNAC genes is as follows:
ATGGAGGAAAGTGATATCAAGGTGCAACATGACAATAGTGAATATGAGGCAGGCCTGCAAATTGAAGAAAGTATCGACAGAATTGAAACTTCTCAAGTGCGTGTTGACGACATGAAATTATTGCCCGGCTATCGGTTTCATCCATTTGATTATGAACTAGTAGTTCATTACTTGTGGAACAAGGTGAACAAACAGCCTCTCCCTCACAATAAGATCGTGGAACTTAAACAGCTTTACAAGTATCATCCAGAGGAAATTACAAAAACAGACCAGGGATTGGTAGAGAATGAGTGGTACTTTTTCACAGAGACGGAGAATGTGCAACTGGTGATGGTTACTGGAAAGCCACTGAAGATGAAGAAACGGTGTATTATAAAGGTGTTGCCGTTGGACATAGGAAGGAATTTGTGTGTTATCGAGGAAAAGCTTTTCCGCCAAAAGGAGACGAGACGAACTGGATCTTGCATGAATTTACAGTCACTGCATGTCCAAGTACCTGTAATTGTCAAGAGGACACAAGACTAG
the amino acid sequence of AdNAC protein is as follows:
MEESDIKVQHDNSEYEAGLQIEESIDRIETSQVRVDDMKLLPGYRFHPFDYELVVHYLWNKVNKQPLPHNKIVELKQLYKYHPEEITKTDQGLVENEWYFFTETENVQLVMVTGKPLKMKKRCIIKVLPLDIGRNLCVIEEKLFRQKETRRTGSCMNLQSLHVQVPVIVKRTQD
the above proteins have 5 conserved domains, as follows:
A domain: LLPGYRFHPFDYELVVHYLWNK A
B domain: IVELKQLYKYHPEEIT A
C domain: EWYFFTETENVQLVMVTGKP A
D domain: LKMKKRCIIKVLPLDIGRNLCVIEEKL A
E domain: QVPVIVKRTQD A
The beneficial effects of the invention are as follows: the invention provides an application of angelica dahurica transcription factor AdNAC in improving the content of angelica dahurica coumarin, and the content of the angelica dahurica coumarin can be greatly improved by knocking out AdNAC.
Drawings
FIG. 1 shows a homology analysis of a protein encoded by AdNAC gene;
FIG. 2 is a AdNAC gene over-expression plant identification;
FIG. 3 is an ACT glue pattern;
FIG. 4 is a diagram showing the detection and alignment of AdNAC gene sequences;
FIG. 5 is a graph showing statistics of coumarin content in gene-edited radix Angelicae Dahuricae and control roots and leaves; (a) coumarin content in the root; (b) coumarin content in leaves.
Detailed Description
The technical solution of the present invention will be described in further detail with reference to the accompanying drawings, but the scope of the present invention is not limited to the following.
EXAMPLE 1 cloning of Angelica dahurica flowering Gene AdNAC20
The candidate genes were designed with software PRIMER PREMIER (forward primer TGATTATGGAGGAAAGTGA and reverse primer TCCGAATGTTGTTTATGG), and were synthesized by the company. The gene was amplified using the common blue enzyme of the root of the heaven. The reaction procedure was set up according to the instructions for the enzyme Tian Gen blue. The bands were detected by 1% agarose gel electrophoresis and recovered by cutting. Coli was transformed, plated, cultured and sequenced according to the Opt Vector Kit of pClone007,007 of Opt. Plasmid extraction (OMEGA kit) was performed on the correct strain, and the strain was stored at 20 ℃. AdNAC 20A 20 was obtained by cloning and sequencing from Dahurian Angelica root No. 2.
EXAMPLE 2 homologous analysis of protein encoded by Angelica dahurica flowering Gene AdNAC20
Sequence homology alignment using DNAMAN software showed (fig. 1), that AdNAC gene had NAC complete domains a-E, D, E of DNA binding domain was an important domain as transcription factor, and that the low conservation of D domain of AdNAC020 sequence was presumed to represent that the gene may have different functions in dahurian angelica root than other crops.
Example 3 Angelica dahurica flowering gene AdNAC plant expression vector and bacterial liquid
The construction method of the gene overexpression recombinant plasmid pCAMBIA3301-AdNAC comprises the following steps: and carrying out recombination reaction on the over-expression vector (modified pCAMBIA 3301-35S-eGFP-kana) and the target gene by adopting the nupraise ClonExpress II One Step Cloning Kit to obtain the constructed recombinant plasmid pCAMBIA 3301-AdNAC.
The construction method of the gene knockout recombinant plasmid Cas9-AdNAC20 comprises the following steps: the constructed recombinant plasmid Cas9-AdNAC is obtained by carrying out recombination reaction by using Cas 9/gRNA-glufosinate-ammonium carrier (Catalog. No. VK 005-15) of Beijing-wei Shang Lide limited company and a target gene.
Conversion to GV3101: and respectively converting the recombinant plasmids into GV3101 agrobacterium competence (Shanghai Weidi biotechnology Co., ltd.) by using a heat shock method to obtain pCAMBIA 3301-AdNAC-GV 3101 bacterial liquid and Cas 9-AdNAC-GV 3101 bacterial liquid, and screening positive bacterial liquid for infection through monoclonal identification.
EXAMPLE 4 functional study of the gene AdNAC of Angelica dahurica in Angelica dahurica
(1) Identification of over-expressed radix Angelicae Dahuricae
RNA is extracted from wild angelica dahurica plants (WT) of Sichuan angelica No. 2 and 20 angelica dahurica plants (OE) leaves of AdNAC gene over-expression, and the expression condition of AdNAC gene in the angelica dahurica plants is quantitatively analyzed by qRT-PCR fluorescence. As can be seen from FIG. 2, after AdNAC genes are transferred, the gene expression quantity of most plants is increased, and the gene expression quantity is improved within the range of 0-103.01 times; the gene expression amounts of OE-6, OE-7, OE-9, OE-10, OE-14, OE-17 and OE-19 are 103.01 times, 40.72 times, 5.46 times, 31.63 times, 18.79 times, 38.13 times and 22.99 times respectively, which are very obviously different from those of control plants.
The results show that 7 AdNAC gene over-expressed radix angelicae plants are obtained, and the transformation efficiency is 35%. The gene AdNAC of dahurian angelica root is integrated in the genome of dahurian angelica root, and the over-expression of the gene can be normally expressed in the genetically transformed plant of dahurian angelica root.
(2) Identification of Gene knockout Angelica dahurica
Extracting DNA for identification. On the premise of guaranteeing the DNA quality (ACT gel diagram, figure 3), carrying out carrier identification (Cas 9 carrier primer identification gel diagram, figure 3), and primarily judging that the transfer AdNAC gene is successful; then, the gene sequence of AdNAC is detected and compared (figure 4), and finally, the gene knockout mutant of the angelica dahurica can be confirmed to be successfully obtained.
EXAMPLE 5HPLC coumarin content determination
In order to study the influence of AdNAC gene on coumarin content after the change of angelica dahurica, the wild type containing empty "Chuangzhi No. 2" is used as a control, the root at the same root position and the leaf at the same leaf position of the over-expression plant OE-NAC20 and the knockout mutant plant OV-NAC20 angelica dahurica are respectively taken, and HPLC is adopted to detect the coumarin content (repeated three times).
The HPLC detection method is as follows:
the content of seven coumarin components is determined by high performance liquid chromatography, the content of coumarin is quantitatively determined by an external standard method, and the purpose of distinguishing seven angelica furocoumarins is achieved by gradient elution. By using high performance liquid chromatograph parameters: agilent1100PLATISIL DAD C chromatography column (4.6 mm. Times.250 mm,5 μm); mobile phase: acetonitrile (A), ultrapure water (B), flow rate 1mL/min; the sample injection amount is 20 mu L; a wavelength of 300nm; gradient elution (0-8 min, 5-20% A; 8-40 min, 20-55% A; 40-55 min, 55-95% A; 55-58 min, 95-5%A); column temperature was 30 ℃. After the sample is chopped, the sample is crushed, sieved by a 100-mesh sieve and put into a glass dryer for standby. About 0.1g of radix Angelicae Dahuricae powder was precisely weighed by an ISO9001 electronic balance (SARTORIUS), and shaken well in a 50mL centrifuge tube with 20mL methanol. Ultrasonic extracting for 60min, adding methanol to supplement weight loss, shaking, and filtering.
Preparing a reference substance solution: 1.07mg of oxidized peucedanin, 3.42mg of isoimperatorin, 3.43mg of imperatorin, 1.13mg of bergapten, 1.30mg of hydrated oxidized peucedanin, 3.23mg of Bai Danggui mg of white angelica brain were dissolved in 5mL of methanol solution, respectively. And mixing the stock solutions in proportion, diluting the stock solutions to the volume by using methanol, and preserving the stock solutions at the temperature of 4 ℃. The mixed solution was diluted with methanol to 1, 2, 4, 8, 16, 20, 40, 100, 200 times for use, respectively.
The results show that the average total coumarin content in the roots of the control plants is 18.75mg/g DW, the average total coumarin content in the roots of the over-expression plants OE-NAC20 is 17.01mg/g DW, and the average total coumarin content in the roots of the knockout mutant plants OV-NAC20 is 21.8mg/g DW. Wherein, the average content of imperatorin in roots of control plants is 3.54mg/g DW, the average content of imperatorin in roots of OV-NAC20 is obviously different from that of control plants is 4.84mg/g DW, and the average content of imperatorin in roots of OE-NAC20 is not obviously different from that of control plants; the average content of isoimperatorin in roots of control plants is 9.43mg/g DW, the average content in OV-NAC20 roots is remarkably different from that of control plants by 11.20mg/g DW, and the average content in OE-NAC20 roots is remarkably different from that of control plants by 7.77mg/g DW; the average content of bergapten, angelicabrain, hu Suzai before oxidation was not significantly different before and after the change of gene AdNAC. (as in FIG. 5 a)
The average total coumarin content in the leaves of the control plant is 21.32mg/g DW, the average total coumarin content in the leaves of the over-expression plant OE-NAC20 is 20.77mg/g DW, and the average total coumarin content in the leaves of the knock-out mutant plant OV-NAC20 is obviously different from the control of 29.05mg/g DW. Wherein, the average content of imperatorin in leaves of a control plant is 5.80mg/g DW, the average content in OV-NAC20 leaves is 8.55mg/g DW with the control, and the average content in OE-NAC20 leaves is 3.25mg/g DW with the control; the average content of isoimperatorin in leaves of a control plant is 9.15mg/g DW, the average content in OV-NAC20 leaves is obviously different from that of the control plant by 12.00mg/g DW, and the average content in OE-NAC20 leaves is obviously different from that of the control plant by 10.20mg/g DW; the average content of bergapten in leaves of control plants is 2.23mg/g DW, the average content in OV-NAC20 leaves is significantly different from control by 4.30mg/g DW, and the average content in OE-NAC20 leaves is significantly different from control by 3.09mg/g DW; the average content of the white angelica brain and the oxidized peucedanum praeruptorum in the leaves is not obviously different before and after the gene AdNAC is changed. (as in FIG. 5 b)
The results show that AdNAC gene expression in root system and leaf of dahurian angelica root can raise coumarin synthesis and accumulation effectively.
The foregoing is merely a preferred embodiment of the invention, and it is to be understood that the invention is not limited to the form disclosed herein but is not to be construed as excluding other embodiments, but is capable of numerous other combinations, modifications and environments and is capable of modifications within the scope of the inventive concept, either as taught or as a matter of routine skill or knowledge in the relevant art. And that modifications and variations which do not depart from the spirit and scope of the invention are intended to be within the scope of the appended claims.
Claims (1)
1. The application of the angelica dahurica transcription factor AdNAC in improving the content of angelica dahurica coumarin is characterized in that the application method comprises the following steps: knocking out AdNAC genes in the angelica dahurica, wherein the nucleotide sequence of the AdNAC genes is shown as SEQ ID NO. 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310606877.4A CN116694678B (en) | 2023-05-26 | 2023-05-26 | Application of radix angelicae transcription factor AdNAC in improving radix angelicae coumarin content |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202310606877.4A CN116694678B (en) | 2023-05-26 | 2023-05-26 | Application of radix angelicae transcription factor AdNAC in improving radix angelicae coumarin content |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN116694678A CN116694678A (en) | 2023-09-05 |
| CN116694678B true CN116694678B (en) | 2024-04-30 |
Family
ID=87836610
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202310606877.4A Active CN116694678B (en) | 2023-05-26 | 2023-05-26 | Application of radix angelicae transcription factor AdNAC in improving radix angelicae coumarin content |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN116694678B (en) |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101062069A (en) * | 2007-06-18 | 2007-10-31 | 石任兵 | Whole coumarins extract from root of dahuriae angelica and the preparing method thereof |
| CN101637487A (en) * | 2008-07-29 | 2010-02-03 | 成都中医药大学 | Application of angelica dahurica coumarin extract in preparing 5-hydroxytryptamine regulator drug |
| CN110824029A (en) * | 2018-08-13 | 2020-02-21 | 成都中医药大学 | UPLC and one-test-multiple-evaluation method based detection of content of coumarins in radix angelicae medicinal material |
| KR20220076565A (en) * | 2020-11-30 | 2022-06-08 | 서울대학교산학협력단 | Composition for preventing or treating influenza virus infection comprising Angelica dahurica, Curcuma longa and Resina Pini extract |
-
2023
- 2023-05-26 CN CN202310606877.4A patent/CN116694678B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101062069A (en) * | 2007-06-18 | 2007-10-31 | 石任兵 | Whole coumarins extract from root of dahuriae angelica and the preparing method thereof |
| CN101637487A (en) * | 2008-07-29 | 2010-02-03 | 成都中医药大学 | Application of angelica dahurica coumarin extract in preparing 5-hydroxytryptamine regulator drug |
| CN110824029A (en) * | 2018-08-13 | 2020-02-21 | 成都中医药大学 | UPLC and one-test-multiple-evaluation method based detection of content of coumarins in radix angelicae medicinal material |
| KR20220076565A (en) * | 2020-11-30 | 2022-06-08 | 서울대학교산학협력단 | Composition for preventing or treating influenza virus infection comprising Angelica dahurica, Curcuma longa and Resina Pini extract |
Non-Patent Citations (4)
| Title |
|---|
| De novo transcriptome assembly of Angelica dahurica and characterization of coumarin biosynthesis pathway genes;Liqiong Zhao等;Gene;20210730;第791卷(第30期);全文 * |
| 基于高通量转录组测序的白芷差异表达基因分析;吴萍;王晓宇;李青苗;郭俊霞;张松林;方清茂;;西南农业学报;20200228(02);全文 * |
| 川白芷抑菌机理研究及香豆素生物合成关键基因的挖掘;刘洋;中国优秀硕士学位论文全文数据库;20210115;D047-588 * |
| 白芷和石菖蒲配伍对大鼠脑缺血损伤的保护作用;刘江红;王世祥;黄开远;李文彦;张志超;;西北药学杂志;20200510(03);全文 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN116694678A (en) | 2023-09-05 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ma et al. | AP2/ERF transcription factor, Ii049, positively regulates lignan biosynthesis in Isatis indigotica through activating salicylic acid signaling and lignan/lignin pathway genes | |
| Deng et al. | Molecular cloning, functional analysis of three cinnamyl alcohol dehydrogenase (CAD) genes in the leaves of tea plant, Camellia sinensis | |
| Hao et al. | Cloning, molecular characterization and functional analysis of a putative R2R3-MYB transcription factor of the phenolic acid biosynthetic pathway in S. miltiorrhiza Bge. f. alba | |
| CN109337915A (en) | Albumen and the application of sesame drought resisting and resistant gene of salt SiMYB75 and its coding | |
| US20210400965A1 (en) | Novel Use of Prodigiosin in Resisting Potyvirus | |
| CN104894143A (en) | Method for increasing content of tanshinone in salvia miltiorrhiza bunge hairy roots | |
| Zhao et al. | Genetic transformation system for woody plant Tripterygium wilfordii and its application to product natural celastrol | |
| CN109536509A (en) | Sesame drought resisting, moisture-proof and resistant gene of salt SiNAC56 and its coding albumen and application | |
| CN109306353A (en) | Sesame anti-drought gene SiSAM1 and its application | |
| Li et al. | Cloning and functional characterization of two cinnamate 4-hydroxylase genes from Pyrus bretschneideri | |
| Kim et al. | Overexpression of cinnamate 4-hydroxylase and 4-coumaroyl CoA ligase prompted flavone accumulation in Scutellaria baicalensis hairy roots | |
| Rui-Fang et al. | The phenylalanine ammonia-lyase gene family in Isatis indigotica Fort.: molecular cloning, characterization, and expression analysis | |
| Bai et al. | The ethylene response factor SmERF8 regulates the expression of SmKSL1 and is involved in tanshinone biosynthesis in Saliva miltiorrhiza hairy roots | |
| CN116694678B (en) | Application of radix angelicae transcription factor AdNAC in improving radix angelicae coumarin content | |
| Xia et al. | Structural and interactions analysis of a transcription factor PnMYB2 in Panax notoginseng | |
| CN107190015A (en) | Corn glycosyltransferase gene UFGT2 is improving the application in plant in flavones content | |
| Dong et al. | Transcriptome analysis of low-temperature-induced breaking of garlic aerial bulb dormancy | |
| Zhou et al. | Functional characterization of serotonin n-acetyltransferase genes (SNAT1/2) in melatonin biosynthesis of Hypericum perforatum | |
| CN108517323B (en) | Salvia miltiorrhiza AP2 transcription factor SmERF128 coding sequence, cloning method and application | |
| CN113637680B (en) | Application of salvia miltiorrhiza transcription factor SmbHLH124 in improving hairy root yield traits of salvia miltiorrhiza | |
| CN105385695A (en) | Tanshinone biosynthesis inhibiting factor gene SmJAZ3 and encoding protein and application thereof | |
| CN111154772B (en) | Pear sugar transport gene PbSWEET4 and application thereof | |
| CN114891810A (en) | Application of salvia miltiorrhiza SmSnRK2.7 gene in improvement of tanshinone content | |
| CN107365736A (en) | A kind of inhibitor of plant root tip stem cell Asymmetric division regulatory pathway and its application | |
| Li et al. | Identification and expression analysis of 4-Coumarate: Coenzyme A ligase gene family in Dryopteris Fragrans |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant |