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
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- 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
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- 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
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- IGWDEVSBEKYORK-UHFFFAOYSA-N isoimperatorin Chemical compound O1C(=O)C=CC2=C1C=C1OC=CC1=C2OCC=C(C)C IGWDEVSBEKYORK-UHFFFAOYSA-N 0.000 description 12
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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.
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