CN113755504A - Artemisia apiacea mediator AaMED25 gene and application thereof - Google Patents

Artemisia apiacea mediator AaMED25 gene and application thereof Download PDF

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CN113755504A
CN113755504A CN202111154091.0A CN202111154091A CN113755504A CN 113755504 A CN113755504 A CN 113755504A CN 202111154091 A CN202111154091 A CN 202111154091A CN 113755504 A CN113755504 A CN 113755504A
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aamed25
gene
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artemisinin
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张芳源
苟玉琴
苏菲
王诗艺
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Southwest University
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Abstract

The invention discloses a sweet wormwood intermediate AaMED25 gene and application thereof, wherein the nucleotide sequence of AaMED25 gene is shown as SEQ ID NO.3, the coded amino acid sequence is shown as SEQ ID NO.3, the content of artemisinin can be improved by passing through the AaMED25 gene in sweet wormwood, and the AaMED25 gene plays a key role in the regulation and control process, so the AaMED25 gene can be used for improving the content of artemisinin.

Description

Artemisia apiacea mediator AaMED25 gene and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to an artemisia apiacea mediator AaMED25 gene and application of an artemisia apiacea mediator AaMED25 gene.
Background
Artemisinin (artemisinin) is one of the drugs which have completely independent intellectual property rights and are internationally approved in China, has very remarkable curative effect on chloroquine-type malaria and cerebral malaria, and the world health organization recommends artemisinin combination therapy as a first choice method for treating malaria. A key factor that currently limits the supply of artemisinin is that the artemisinin content in the artemisia apiacea plants is too low to meet the therapeutic needs of the patients, especially in developing countries. Therefore, how to improve the yield of the artemisinin becomes a significant problem in the production of the artemisinin and a research hotspot at home and abroad. ABA and JA are more studied hormones capable of regulating and controlling the biosynthesis of plant secondary metabolism. Researchers found that artemisinin biosynthesis was induced by abscisic acid (ABA) and methyl jasmonate (MeJA) (tying et al 2009; Maes et al 2011), related transcription factors have also been cloned and functionally characterized, but very little has been studied about the interaction of ABA and JA signals in regulating artemisinin biosynthesis. Related studies in fungi, animals, humans and plants have found that the mediator gene has a key central role in the signal transduction process, being a "bridge" for the transfer of transcription factors to the transcription initiation complex. However, whether the mediator gene can influence the yield of the artemisinin in the artemisia apiacea is not reported.
Disclosure of Invention
In view of the above, an object of the present invention is to provide an Artemisia annua mediator AaMED25 gene; the second purpose of the invention is to provide a recombinant expression vector containing the Artemisia apiacea midbody AaMED25 gene; the invention also aims to provide the application of the Artemisia apiacea mediator AaMED25 gene in the over-expression of Artemisia apiacea to improve the content of artemisinin. The fourth purpose of the invention is to provide the application of the recombinant expression vector in transforming the sweet wormwood to improve the artemisinin content.
In order to achieve the purpose, the invention provides the following technical scheme:
1. the sweet wormwood intermediate AaMED25 gene is characterized in that: the method is characterized in that: the nucleotide sequence of the MED25 gene is shown in SEQ ID NO. 3.
2. A recombinant expression vector containing the artemisia apiacea intermediate AaMED25 gene.
Preferably, the recombinant expression vector is connected to the BamHI and XhoI cleavage sites of the pHB vector by a sequence shown in SEQ ID NO. 3.
3. The application of the Artemisia apiacea mediator AaMED25 gene in the improvement of artemisinin content through overexpression in Artemisia apiacea.
4. The recombinant expression vector is used for transforming the sweet wormwood to improve the artemisinin content.
5. A method for increasing the content of artemisinin in sweet wormwood herb comprises the steps of over-expressing an artemisinin mediator AaMED25 gene in sweet wormwood herb, and screening transgenic positive plants to obtain the sweet wormwood herb with increased artemisinin content.
Preferably, the method for over-expressing the artemisinin mediator AaMED25 gene in the artemisia apiacea is to perform agrobacterium-mediated transformation on a recombinant expression vector containing the artemisinin mediator AaMED25 gene, and screen a transgenic plant after regeneration.
Preferably, the recombinant expression vector is obtained by connecting a sequence shown in SEQ ID NO.3 into BamHI and XhoI enzyme cutting sites of the pHB vector.
The invention has the beneficial effects that: the invention discloses a sweet wormwood intermediate AaMED25 gene, which can remarkably improve the content of artemisinin after being over-expressed in sweet wormwood, so that the sweet wormwood intermediate AaMED25 gene can be used for improving the content of artemisinin.
Drawings
In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:
FIG. 1 is a diagram showing the detection of gene expression level of transgenic plants (A: the relative expression level of AaMED25 gene in the interference AaMED25 transgenic Artemisia annua and wild type Artemisia annua; B: the relative expression level of AaMED25 gene in the overexpression AaMED25 transgenic Artemisia annua and wild type Artemisia annua).
FIG. 2 is a diagram showing the content of artemisinin in AaMED25 transgene (A: the content of artemisinin in AaMED25 gene in AaMED25 transgene Artemisia annua and wild type Artemisia annua; B: the content of artemisinin in AaMED25 gene in over-expression AaMED25 transgene Artemisia annua and wild type Artemisia annua).
Detailed Description
The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.
Example 1 cloning of the Artemisia apiacea mediator AaMED25 Gene
Extracting Artemisia apiacea RNA, inverting the Artemisia apiacea RNA into cDNA, and designing primers for amplifying the upstream and downstream of AaMED25 according to the Artemisia apiacea genome sequence:
an upstream primer 5'-atggcggcggataagaaa-3' (SEQ ID NO. 1); a downstream primer: 5'-tcagtttatgaagcttccacctgacat-3' (SEQ ID NO. 2);
then taking the reversed cDNA as a template, and carrying out PCR amplification by using the upstream primer and the downstream primer of AaMED25 under the condition of pre-denaturation at 94 ℃ for 5 minutes; denaturation at 94 ℃ for 30 seconds, annealing at 52 ℃ for 30 seconds, and extension at 72 ℃ for 150 seconds for 32 cycles; extending for 2 minutes at 72 ℃, storing at 4 ℃, carrying out PCR amplification, recovering the amplification product, and sequencing to obtain the AaMED25 gene sequence, wherein the nucleotide sequence is shown as SEQ ID NO.3, and the coded amino acid is shown as SEQ ID NO. 4.
Example 2 construction of plant expression vector for Artemisia Annua mediator MED25 Gene (AaMED25)
A. Construction of AaMED25-pHB
Analyzing the restriction enzyme cutting site of the coding region and the multiple cloning site on pHB vector according to the sequence of the cloned AaMED25 gene, selecting BamHI and XhoI as restriction enzyme cutting sites for vector construction, introducing the BamHI restriction enzyme cutting site at the upstream of the full length of AaMED25, introducing the XhoI restriction enzyme cutting site at the downstream of AaMED25, and using the following primers:
AaMED25-F-BamH1：5’-atggcggcggataagaaacag-3’(SEQ ID NO.5)；
AaMED25-R-XhoI：5’-aggtggaagcttcataaacct-3’(SEQ ID NO.6)；
AaMED25 was amplified using the pair of primers, and then the amplified AaMED25 sequence and pHB were digested simultaneously with the endonucleases BamHI and XhoI, followed by T4 ligation.
B. Construction of AaMED25-pBin19
Selecting a specific segment of 200bp on the AaMED25 genome as an interference segment, and designing a pair of primers, wherein the specific primers are as follows: AaMED 25-RI-F: 5'-gcttgtgtgttgaaggta-3' (SEQ ID NO. 7); AaMED 25-RI-R: 5'-cactgtcagttatatgcc-3' (SEQ ID NO. 8).
HindIII and XbaI enzyme cutting sites are respectively introduced into the 5' ends of the upstream and downstream primers, and the fragment is positively cloned to an interference vector pHANNIBAL; and respectively introducing KpnI and XhoI enzyme cutting sites into the 5' ends of the upstream primer and the downstream primer, reversely cloning the fragment onto an interference vector pHANNIBAL introduced with a forward fragment, digesting the vector by SacI and SpeI, recovering an interference expression frame containing a forward and reverse AaMED25 fragment, and connecting the fragment to a pBin19 large skeleton of a plant expression vector digested by SacI and XbaI by T4 ligase to finally form the RNAi vector of AaMED 25.
Example 3 obtaining of transgenic Artemisia annua
The preparation method of the sweet wormwood seedling comprises the following specific steps:
(1) taking a proper amount of sweet wormwood seeds, putting the sweet wormwood seeds into a 1.5mL Ep tube, adding 1mL of 75% alcohol by volume fraction, soaking for 1min, then sucking off the alcohol, and washing for 3-5 times by using sterile water;
(2) adding 1mL of 10% sodium hypochlorite solution and 10 μ L of 0.1% Triton-100 solution, mixing by vortex, sterilizing for 10min, and washing with sterile water for 3-5 times;
(3) sucking 1mL of sterile water, sucking the seeds into a pipette together, uniformly coating the seeds on 1/2MS culture medium, sucking off excess water, and drying; vernalizing at 4 deg.C for 2-3 days, and culturing in light culture room. The culture conditions were: temperature: 25 ℃; the photoperiod: 16h light/8 h dark; the illumination intensity is as follows: 80-220 mu mol.m-2S-1, humidity 60%) for about one week, and cutting the first true leaf pair at the base of the petiole for genetic transformation of Artemisia annua.
Genetic transformation of sweet wormwood:
(1) transforming the constructed over-expression vector (pHB-AaMED25) into Agrobacterium EHA 105;
(2) screening on a resistance plate (Rif + Kan), and picking monoclonal bacterial plaques for PCR detection;
(3) and (3) amplification culture: adding 50mL YEP (Rif + Kan) solution into a 250mL triangular flask, adding 100 μ L positive bacteria solution, culturing at 28 deg.C and 200rpm overnight;
(4) when the bacterial liquid concentration of the engineering bacteria is cultured until the OD 600 is about 0.6, 4000rpm is carried out, centrifugation is carried out for 5min, and the bacteria are collected. The thallus precipitate is re-suspended with 1/2MS liquid culture medium to OD 600 of 0.3-0.5, cultured at 28 deg.c and 200rpm for 30 min;
(5) infecting the cultured bacterial solution with Artemisia apiacea leaf explant for 20min, flatly spreading on an MS plate (MS + NAA 0.1mg/L +6-BA 1.0mg/L), dark culturing at 25 deg.C for 2d, transferring to a differentiation plate (MS + NAA +6-BA + hygromycin) after 2 d; the screening culture medium is replaced every 10 days, and after one month, a large number of cluster buds grow from the differentiation culture substrate;
(6) shearing off the cluster buds, and sequentially transferring the cluster buds to a strong seedling culture medium and a rooting culture medium until a complete plant is formed;
(7) and (4) transplanting the plants of the southernwood which have roots and are identified as positive plants into soil for growing.
And screening 3 plants of the transgenic southernwood with the interference expression and 3 plants of the transgenic southernwood with the overexpression according to the results. And respectively detecting the expression quantity of the AaMED25 genes of the interference expression transgenic plant and the overexpression transgenic plant by fluorescent quantitative PCR. The primers used for detection were as follows:
AaMED25-qF：5’-atatgatgggtggtgtcggt-3’(SEQ ID NO.9)；
AaMED25-qR：5’-gtgttcgcatacctgggact-3’(SEQ ID NO.10)。
the detection results are shown in FIG. 1. The result shows that the expression level of the AaMED25 gene in the interference expression transgenic plant is reduced, and the expression level of the AaMED25 gene in the overexpression transgenic plant is increased.
Example 4 overexpression of AaMED25 and interference with artemisinin determination in Artemisia annua plants
Extracting artemisinin:
(1) collecting aerial part tissue of herba Artemisiae Annuae, baking in oven at 40 deg.C to completely dry, and grinding into powder;
(2) preheating petroleum ether in a constant-temperature water bath kettle at 60 ℃;
(3) weighing 0.2g of powder in a 50mL Ep tube, adding 25mL of petroleum ether, and carrying out 80Hz ultrasound for 40 min;
(3) filtering with filter paper, and collecting filtrate in a 50mL small beaker; then 25mL of petroleum ether is used for cleaning residues on the filter paper, and the filtrate is collected again by the same beaker;
(4) the liquid was transferred to a 100mL rotary evaporation flask and rotary evaporated in a water bath at 50 ℃ under reduced pressure until petroleum ether was completely evaporated.
(5) Adding 1mL of chromatographic grade methanol into a rotary evaporation bottle, and carrying out ultrasonic treatment for 1 min; transferring the heavy suspension liquid into a 1.5mL Ep tube, and centrifuging at 12000rpm for 10 min; filtering the supernatant with 0.22 μm filter membrane, and measuring artemisinin content of the filtrate with high performance liquid chromatography combined with evaporative light scattering detection (HPLC-ELSD) by the following specific method:
the HPLC instrument was SPD20A system controller, Evaporative Light-Scattering Detector (ELSD). Using a Waters C18 chromatography column, mobile phase: acetonitrile and water in a volume ratio of 60% to 40%, and a flow rate of: 1 mL/min; the ELSD detection system is water alias 2420, the temperature of the drift tube of the evaporative light scattering detector is 40 ℃, and the carrier gas pressure is 5 bar; the standard sample was injected in 20. mu.L, each sample was injected in 20. mu.L, and the injection was repeated 3 times. The peak time of the artemisinin standard is 9.703min, the retention time of the sample is 9.673min, the content of artemisinin in the sample is calculated according to the concentration and peak area of the standard, and then the dry weight of the artemisinin powder is divided by the dry weight of the artemisinin powder, so that the content of artemisinin in the dry weight of the sample is calculated, and the result is shown in fig. 2. The results show that there was some increase, some decrease and no significant difference in artemisinin yield in the interfering expression transgenic plants. In the over-expression transgenic plants, the yield of artemisinin is improved, and the difference is obvious.
The above-mentioned embodiments are merely preferred embodiments for fully illustrating the present invention, and the scope of the present invention is not limited thereto. The equivalent substitution or change made by the technical personnel in the technical field on the basis of the invention is all within the protection scope of the invention. The protection scope of the invention is subject to the claims.
Sequence listing
<110> university of southwest
<120> Artemisia apiacea mediator AaMED25 gene and application thereof
<160> 10
<170> SIPOSequenceListing 1.0
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tcagtttatg aagcttccac ctgacat 27
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<213> Artemisia annua (Artemisia annua)
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atggcggcgg ataagaaaca gctgattttg tgtgttgaag gtactgctgc tttgggtcct 60
tattggcgga ctattctttc tgattatctt gataaagtta tcaggtcttt ctgtgataac 120
gagactttga aatcacctgg gactattgtt gagctcgctc tcattgtatt caactctcat 180
ggatgttaca gttcttgctt ggtacgacgg agtggctgga ctagaaacgt tgactatttt 240
ttcgagtggc tatcggctat acacttttct ggtggcggtt tttgtgacgc cgcaattgca 300
gagggtcttg gtgaagcttt aatgatgttt cctgtaaatg cggcacaaaa tcaacataat 360
atcggtgttc aaaggcattg catacttgtt gctgcaagta atccatatcc attgccgaca 420
ccagtttacc gaccaccaat acaaaaaacg gagccgactg ataatactga ggcacagtca 480
gaaagcaggc tatcagatgc tgaaaccatc gctaaaacat ttccacagtg tcccgtctca 540
ctgtcagtta tatgcccgaa acagcttcca aaattaaaag caatctataa tgcgggaaaa 600
aggaacccat cagcaggcga acctactatt gatgtggtga aaaatccaca ttatcttgtg 660
ctaatatcgg aaacatttat ggaagctcgt gcggctttaa gtcgatcagg cattacaact 720
ttgccctctc agagtcctat aaaagttgat caaagttcag ttcctccggt atcagggcca 780
cctccaactt cagttccacc agtaaatgga tctatgatga gccgtcaacc agttcctgta 840
ggaagtgttc ctcccacaac tgttaaagtt gaaccaacca cagttccttc aatgccacct 900
gcaccagtac ccgtaccagc gccctcgttt cagcatgttc cgccagttgc tcgtcccacc 960
tcgcaaggaa tcccgacaat gcaaacttct tcaccgttat ctgtttctca aggtatgcta 1020
tcaaataacg atactgtgat gcaagatatg aaaccaaatg tcactggtat gcaacaaccg 1080
gcacgtcctg ctggtcccgt aaacgtcagc atactcaata atctttcaca agcacggttg 1140
atgaacaacg ggacatcgat ggggataccg tcaatcggcg ggaatccgat ggctatgcat 1200
atgtccaaca tgatatctag tggaatggcc tcaactgtac ccgtttctca aaccgtgatt 1260
tcatcgggtc aaccgggtat tgccccaatt tccggaacgg tgcagagtac ggtaccagta 1320
ccaagttcat ttacttcaac cacttcaaac atgactggga gtccgagcca accattgggt 1380
aatcttcaag gaagtgtcgg aatgggtcaa cccgtttcgg gaatcagcca aggaaatctt 1440
ccaggcactg gctcgcaaat ggtgcaaagt ggaatgggaa tgaatcagaa tatgatgggt 1500
ggtgtcggtc agggtcaatc gggtatgacg ggagtgggaa cgggaacggg aaccggttca 1560
ggacctggga tgatgccgac acctggcatt ggtcagcaag tcccaggtat gcgaacactc 1620
ggtgtaaata acaatacggc tgctaatgct ggtcttccgc aacaaacatc gggtggtggt 1680
gcactgcaat cagcacagtc aaaatatgtt aaagtttggg aggggaactt atctgggcag 1740
cgacaaggtc aacctgtgtt tattaccaga ttagaagggt accggagtgc atcagcttct 1800
gaatcgcttg ctgctaattg gccacctaca atgcaaatag ttcggcttat atctcaagat 1860
cacatgaata acaagcaata tgttggaaaa gcagattttc tagtttttcg cgcaatgaat 1920
cagcatggat ttcttggaca attgcaagaa aagaagcttt gtgcagtcat acagttgcca 1980
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caacaaccaa tgcaacaaca gccgatgcaa caacagccta tgcaacaaca gcaacagccg 2280
caacaaccga tgcaacagca acagcagcaa ccacagatgg ttgggacagg gatgaaccaa 2340
ggttatgttc aaggtccagg gggccgaaca caaatggtgc cccaggggca agtttcttcg 2400
cagggcccgc aaagcatgtc aggtggaagc ttcataaact ga 2442
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<213> Artemisia annua (Artemisia annua)
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Met Ala Ala Asp Lys Lys Gln Leu Ile Leu Cys Val Glu Gly Thr Ala
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Ala Leu Gly Pro Tyr Trp Arg Thr Ile Leu Ser Asp Tyr Leu Asp Lys
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Val Ile Arg Ser Phe Cys Asp Asn Glu Thr Leu Lys Ser Pro Gly Thr
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Ile Val Glu Leu Ala Leu Ile Val Phe Asn Ser His Gly Cys Tyr Ser
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Ser Cys Leu Val Arg Arg Ser Gly Trp Thr Arg Asn Val Asp Tyr Phe
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Phe Glu Trp Leu Ser Ala Ile His Phe Ser Gly Gly Gly Phe Cys Asp
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Ala Ala Ile Ala Glu Gly Leu Gly Glu Ala Leu Met Met Phe Pro Val
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Asn Ala Ala Gln Asn Gln His Asn Ile Gly Val Gln Arg His Cys Ile
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Leu Val Ala Ala Ser Asn Pro Tyr Pro Leu Pro Thr Pro Val Tyr Arg
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Pro Pro Ile Gln Lys Thr Glu Pro Thr Asp Asn Thr Glu Ala Gln Ser
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Glu Ser Arg Leu Ser Asp Ala Glu Thr Ile Ala Lys Thr Phe Pro Gln
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Cys Pro Val Ser Leu Ser Val Ile Cys Pro Lys Gln Leu Pro Lys Leu
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Lys Ala Ile Tyr Asn Ala Gly Lys Arg Asn Pro Ser Ala Gly Glu Pro
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Thr Ile Asp Val Val Lys Asn Pro His Tyr Leu Val Leu Ile Ser Glu
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Thr Phe Met Glu Ala Arg Ala Ala Leu Ser Arg Ser Gly Ile Thr Thr
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Leu Pro Ser Gln Ser Pro Ile Lys Val Asp Gln Ser Ser Val Pro Pro
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Val Ser Gly Pro Pro Pro Thr Ser Val Pro Pro Val Asn Gly Ser Met
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Met Ser Arg Gln Pro Val Pro Val Gly Ser Val Pro Pro Thr Thr Val
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Lys Val Glu Pro Thr Thr Val Pro Ser Met Pro Pro Ala Pro Val Pro
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Val Pro Ala Pro Ser Phe Gln His Val Pro Pro Val Ala Arg Pro Thr
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Ser Gln Gly Ile Pro Thr Met Gln Thr Ser Ser Pro Leu Ser Val Ser
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Gln Gly Met Leu Ser Asn Asn Asp Thr Val Met Gln Asp Met Lys Pro
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Asn Val Thr Gly Met Gln Gln Pro Ala Arg Pro Ala Gly Pro Val Asn
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Val Ser Ile Leu Asn Asn Leu Ser Gln Ala Arg Leu Met Asn Asn Gly
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Thr Ser Met Gly Ile Pro Ser Ile Gly Gly Asn Pro Met Ala Met His
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Met Ser Asn Met Ile Ser Ser Gly Met Ala Ser Thr Val Pro Val Ser
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Gln Thr Val Ile Ser Ser Gly Gln Pro Gly Ile Ala Pro Ile Ser Gly
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Thr Val Gln Ser Thr Val Pro Val Pro Ser Ser Phe Thr Ser Thr Thr
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Ser Asn Met Thr Gly Ser Pro Ser Gln Pro Leu Gly Asn Leu Gln Gly
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Ser Val Gly Met Gly Gln Pro Val Ser Gly Ile Ser Gln Gly Asn Leu
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Pro Gly Thr Gly Ser Gln Met Val Gln Ser Gly Met Gly Met Asn Gln
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Asn Met Met Gly Gly Val Gly Gln Gly Gln Ser Gly Met Thr Gly Val
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Gly Thr Gly Thr Gly Thr Gly Ser Gly Pro Gly Met Met Pro Thr Pro
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Gly Ile Gly Gln Gln Val Pro Gly Met Arg Thr Leu Gly Val Asn Asn
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Asn Thr Ala Ala Asn Ala Gly Leu Pro Gln Gln Thr Ser Gly Gly Gly
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Ala Leu Gln Ser Ala Gln Ser Lys Tyr Val Lys Val Trp Glu Gly Asn
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Leu Ser Gly Gln Arg Gln Gly Gln Pro Val Phe Ile Thr Arg Leu Glu
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Gly Tyr Arg Ser Ala Ser Ala Ser Glu Ser Leu Ala Ala Asn Trp Pro
595 600 605
Pro Thr Met Gln Ile Val Arg Leu Ile Ser Gln Asp His Met Asn Asn
610 615 620
Lys Gln Tyr Val Gly Lys Ala Asp Phe Leu Val Phe Arg Ala Met Asn
625 630 635 640
Gln His Gly Phe Leu Gly Gln Leu Gln Glu Lys Lys Leu Cys Ala Val
645 650 655
Ile Gln Leu Pro Ser Gln Thr Leu Leu Leu Ser Val Ser Asp Lys Ala
660 665 670
Cys Arg Leu Ile Gly Met Leu Phe Pro Gly Asp Met Val Val Phe Lys
675 680 685
Pro Gln Ile Ser Gly Gln Gln Gln Pro Gln Gln Leu Gln Pro Gln Gln
690 695 700
His Pro Gly Gln Met Gln Pro Gln Gln Gln Gln Met Gln His Met Gln
705 710 715 720
Gln Gln Gln Gln Gln Ile Pro Pro Leu Gln Gln Gln Gln Gln Gln Met
725 730 735
Gln Gln Pro Ser Gln Gln Pro Met Gln Gln Gln Pro Met Gln Gln Gln
740 745 750
Pro Met Gln Gln Gln Gln Gln Pro Gln Gln Pro Met Gln Gln Gln Gln
755 760 765
Gln Gln Pro Gln Met Val Gly Thr Gly Met Asn Gln Gly Tyr Val Gln
770 775 780
Gly Pro Gly Gly Arg Thr Gln Met Val Pro Gln Gly Gln Val Ser Ser
785 790 795 800
Gln Gly Pro Gln Ser Met Ser Gly Gly Ser Phe Ile Asn
805 810
<210> 5
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
atggcggcgg ataagaaaca g 21
<210> 6
<211> 21
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
aggtggaagc ttcataaacc t 21
<210> 7
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
gcttgtgtgt tgaaggta 18
<210> 8
<211> 18
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
cactgtcagt tatatgcc 18
<210> 9
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
atatgatggg tggtgtcggt 20
<210> 10
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 10
gtgttcgcat acctgggact 20

Claims (8)

1. The sweet wormwood intermediate AaMED25 gene is characterized in that: the method is characterized in that: the nucleotide sequence of the AaMED25 gene is shown in SEQ ID NO. 3.
2. A recombinant expression vector comprising the artemisia apiacea intermediate AaMED25 gene of claim 1.
3. The recombinant expression vector of claim 2, wherein: the recombinant expression vector is connected to BamHI and XhoI enzyme cutting sites of the pHB vector by a sequence shown in SEQ ID NO. 3.
4. The use of the Artemisia apiacea interposer AaMED25 gene in Artemisia apiacea for increasing artemisinin content through overexpression.
5. Use of the recombinant expression vector of claim 2 or 3 to transform Artemisia annua for increasing artemisinin content.
6. The method for improving the content of artemisinin in the sweet wormwood herb is characterized by comprising the following steps: over-expressing the artemisinin mediator AaMED25 gene in the artemisia apiacea, and screening transgenic positive plants to obtain the artemisia apiacea with the increased artemisinin content.
7. The method of claim 6, wherein the method comprises the steps of: the method for over-expressing the artemisinin mediator AaMED25 gene in the sweet wormwood herb is to perform agrobacterium-mediated transformation on a recombinant expression vector containing the artemisinin mediator AaMED25 gene, and screen a transgenic plant after regeneration.
8. The method of claim 7, wherein the amount of artemisinin in Artemisia annua is increased by: the recombinant expression vector is obtained by connecting a sequence shown in SEQ ID NO.3 to BamHI and XhoI enzyme cutting sites of a pHB vector.
CN202111154091.0A 2021-09-29 2021-09-29 Artemisia apiacea mediator AaMED25 gene and application thereof Pending CN113755504A (en)

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CN112375767A (en) * 2020-12-04 2021-02-19 衡阳师范学院 Artemisia apiacea WRKY transcription factor AaWRKY4 gene and application
CN112662678A (en) * 2020-12-08 2021-04-16 上海交通大学 Sweet wormwood MYB transcription factor AaMYB15 and application thereof
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CN107475265A (en) * 2017-09-26 2017-12-15 上海交通大学 Nucleotide sequence, carrier and the method for improving content of artemisinin in sweet wormwood
CN108530525A (en) * 2018-04-27 2018-09-14 上海交通大学 A kind of sweet wormwood bZIP class transcription factor AaABF3 and its application
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN116334109A (en) * 2022-07-29 2023-06-27 西南大学 Application of overexpression of AaMAPK6 gene in sweet wormwood herb in improving artemisinin content and method
CN116334109B (en) * 2022-07-29 2024-04-05 西南大学 Application of overexpression of AaMAPK6 gene in sweet wormwood herb in improving artemisinin content and method

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Application publication date: 20211207